ES2550004T3

Highly sensitive system and methods for analysis of troponin

Abstract

A method for determining a cardiovascular disease in an individual comprising: (a) measuring the serum or plasma concentration of cardiac troponin from a blood sample of the individual; (b) compare the measured concentration to a predetermined threshold concentration representing the 99th percentile concentration of cardiac troponin in a group of normal individuals with a corresponding coefficient of variation (CV) of 10% or less, where the threshold concentration is less than 10 pg / mL; (c) determine at least one of the following in the individual when the concentration of cardiac troponin is greater than the threshold concentration: cardiac damage, myocardial damage, a previous cardiac event and cardiotoxicity.

ES2550004T3, drawing sheet 1 — Sheet 1 of 11

Term

0.5 yearsto projected expiry

Projected expiry 4 April 2027, counted from filing; an application has no term until it is granted.

17 claims: 8 independent, 9 dependent

1
ES 2 550 004 T3 Reivindicaciones 1. Un método para determinar una enfermedad cardiovascular en un individuo que comprende:(a) medir la concentración en suero o plasma de la troponina cardiaca a partir de una muestra de sangre del individuo;(b) comparar la concentración medida a una concentración umbral predeterminada que representa la concentración de 5 percentil 99° de la troponina cardiaca en un grupo de individuos normales con un coeficiente de variación (CV) correspondiente del 10% o menos, en donde la concentración umbral es menos de 10 pg/mL;(c) determinar al menos uno de los siguientes en el individuo cuando la concentración de troponina cardiaca es mayor que la concentración umbral: daño cardiaco, daño miocárdico, un evento cardiaco anterior y cardiotoxicidad.
2
El método de la reivindicación 1, en donde la determinación se realiza en una serie de muestras tomadas 10 periódicamente a intervalos de minutos, horas, días, semanas, meses o años.
3
El método de la reivindicación 2, en donde las muestras han sido tomadas durante un período de hasta 48 horas a intervalos entre 4 y 8 horas.
4
El método de la reivindicación 2, que comprende además determinar una velocidad de cambio de la concentración de dicha serie de muestras. 15
5
El método de la reivindicación 2, en donde un aumento en la concentración indica progreso y empeoramiento del daño cardiaco.
6
Un método de acuerdo con cualquiera de las reivindicaciones 1-5, en donde se ha tomado una muestra de sangre al menos una durante una prueba de estrés y después de una prueba de esfuerzo.
7
El método de cualquiera de las reivindicaciones precedentes, en donde, dicha troponina cardiaca se selecciona del 20 grupo que consiste en troponina I cardiaca y troponina T cardiaca.
8
El método de cualquiera de las reivindicaciones precedentes, en donde (c) comprende determinar AMI en el individuo cuando la concentración de troponina cardiaca es mayor que la concentración umbral, en donde la concentración umbral es menos de 9 pg/mL, menos de 8 pg/mL o menos de 7 pg/mL.
9
Un método para determinar la enfermedad cardiovascular en un individuo que comprende:25 (a) medir la concentración en suero o plasma de la troponina I cardiaca (cTnI) en una muestra de sangre del individuo;(b) comparar la concentración con una concentración umbral de 7 pg/mL, y (c) determinar al menos uno de los siguientes en el individuo cuando la concentración de cTnI es mayor que la concentración umbral: daño cardiaco, daño miocárdico, un evento cardiaco anterior y cardiotoxicidad.
10
El método de la reivindicación 9, en donde la determinación se realiza en una serie de muestras tomadas 30 periódicamente a intervalos de minutos, horas, días, semanas, meses o años.
11
El método de la reivindicación 10, en donde las muestras han sido tomadas durante un período de hasta 48 horas a intervalos entre 4 y 8 horas.
12
El método de la reivindicación 10, que comprende además determinar una velocidad de cambio de la concentración para dicha serie de muestras. 35
13
El método de la reivindicación 10, en donde un aumento en la concentración indica progreso y empeoramiento de daño cardiaco.
14
Un método de acuerdo con cualquiera de las reivindicaciones 9-13, en donde una muestra de sangre se ha tomado al menos una durante una prueba de estrés y después de una prueba de esfuerzo.
15
Un método de cualquiera de las reivindicaciones 1 y 9, que comprende además:40 (a) medir la concentración en suero o plasma de la troponina I cardiaca (cTnI) a partir de una muestra de sangre antes de realizar una prueba de esfuerzo en el individuo;ES 2 550 004 T3 (b) realizar la prueba de esfuerzo;(c) medir la concentración inducida por el estrés de cTnI a partir de muestras de sangre tomadas al menos una de durante y después de la prueba de esfuerzo;(d) comparar las concentraciones de cTnI antes y al menos una de durante y después de la prueba de esfuerzo una con 5 otra y con la concentración umbral.
16
El método de la reivindicación 15, en donde el individuo es un paciente que se está evaluando para al menos uno de un posible evento cardiaco y cardiotoxicidad.
17
El método de cualquiera de las reivindicaciones anteriores, que comprende además determinar un tratamiento para el individuo.

Independent claims17

511 paragraphs in 30 sections, as filed

IS 2 550 004 T3

DESCRIPTION

High sensitivity system and troponin analysis methods

Background of the invention

Each year in the United States, about six million people present to the emergency department with chest pain. Although only 15% to 20% of these patients are ultimately diagnosed with acute coronary syndrome (ACS), about half are admitted for evaluation. In contrast, 2% of patients with ACS are mistakenly discharged. As patients with ACS have a relatively high risk of adverse cardiovascular events in the short term, there is a clear need for precise objective tools with which they can be identified.

The markers currently used for cardiac damage suffer disadvantages that limit their clinical utility. Cardiac enzyme assays have served as the basis for determining whether or not there is damage to the heart muscle. Unfortunately, the standard creatine kinase-MB (CK-MB) assay is not reliable in excluding infarction until 10 to 12 hours after the onset of chest pain. Early diagnosis would have very specific advantages over fibrinolytic therapy and prioritization.

Summary of the invention

The invention provides a method for determining cardiovascular disease in an individual comprising: (a) measuring the serum or plasma concentration of cardiac troponin from a blood sample of the individual; (b) comparing the measured concentration to a predetermined threshold concentration representing the 99th percentile concentration of cardiac troponin in a group of normal individuals with a corresponding coefficient of variation (CV) of 10% or less, where the concentration threshold is less than 10 pg / mL; (c) determining at least one of the following in the individual when the cardiac troponin concentration is greater than the threshold concentration: cardiac damage, myocardial damage, a previous cardiac event, and cardiotoxicity. The invention also provides a method for determining cardiovascular disease in an individual comprising: (a) measuring the serum or plasma concentration of cardiac troponin I (cTnl) in a blood sample from the individual; (b) compare the concentration with a threshold concentration of 7 pg / mL, and (c) determine at least one of the following in the individual when the cTnI concentration is greater than the threshold concentration: cardiac damage, myocardial damage, a previous cardiac event, and cardiotoxicity.

In some cases, the specification provides a method for determining the presence or absence of a single troponin molecule or a fragment or complex thereof in a sample, including i) marking the molecule, fragment, or complex, if present, with a label; and ii) detecting the presence or absence of the tag, where detection of the presence of the tag indicates the presence of the single troponin molecule, fragment, or complex in the sample.

According to the methods of the invention, as specified in the claims, troponin is a cardiac isoform of troponin. In some embodiments of the methods of the invention, as specified in the claims, the troponin may be cardiac troponin I (cTnl) or cardiac troponin C (cTnC). In some embodiments of the methods of the invention, as specified in the claims, the troponin is cTnl. In some instances of the methods described herein, a single troponin molecule can be detected at a detection limit of less than about 100 pg / mL. In some instances of the methods described herein, a single troponin molecule can be detected at a detection level of less than about 20 pg / mL. In some instances of the methods described herein, the tag includes a fluorescent building block. In some cases, the fluorescent frame unit is capable of emitting at least about 200 photons when simulated by a laser light emitter at the excitation wavelength of the frame unit, when the laser is focused at a point of not less than approximately 5 microns in diameter containing the structural unit, and where the total energy directed at the point by the laser is no more than approximately 3 microjoules. In some instances of the methods described herein, the fluorescent backbone includes a molecule containing at least one substituted indolium ring system in which the 3-carbon substituent of the indolium ring contains a chemically reactive group or a group of conjugated substances. In some instances of the methods described herein, the fluorescent building block includes a dye. Examples of colorants include, but are not limited to, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 647, Alexa Fluor 680, and Alexa Fluor 700. In some instances of the methods described herein, the fluorescent building unit includes Alexa Fluor 647 . fifty In some cases, the fluorescent structural unit includes a molecule that contains at least one substituted indolium ring system in which the substituent at carbon 3 of the indolium ring contains a chemically reactive group or a group of conjugated substances. In some instances of the methods described herein, the tag further includes a binding partner of the troponin molecule, fragment, or complex. In some instances of the methods described herein, the binding partner includes an antibody specific for the troponin molecule, fragment, or complex. In some instances of the methods described herein, the antibody is specific for a specific region of the troponin molecule. In some cases of the methods described in this

In document ES 2 550 004 T3, the antibody is specific for a region comprising amino acids from 27-41 of cardiac troponin I. In some instances of the methods described herein, the antibody may be a polyclonal antibody. In some cases of the methods described herein, the antibody is a monoclonal antibody. In some instances of the methods described herein, the methods further include capturing troponin or troponin complex on a solid support. In some cases of the methods described herein, the solid support can be a microtiter plate or paramagnetic beads. In some instances of the methods described herein, the solid support includes a specific capture partner for troponin or the troponin complex that is bound to the solid support. In some cases of the methods described herein, the binding of the capture partner to the solid support is non-covalent. In some cases of the methods described herein, the binding of the capture partner to the solid support is covalent. In some instances of the methods described herein, the covalent attachment of the capture partner is such that the capture partner is attached to the solid support in a specific orientation. In some instances of the methods described herein, the specific orientation serves to maximize the specific binding of the troponin or troponin complex to the capture partner. In some instances of the methods described herein, the capture partner comprises an antibody. In some cases of the methods described herein, the antibody is a monoclonal antibody. In some instances of the methods described herein, the antibody is specific for amino acids 87-91 of cardiac troponin I. In some instances of the methods described herein, the antibody is specific for amino acids 41-49 of cardiac troponin I. According to the invention, the sample is a blood, serum or plasma sample. In some embodiments of the methods of the invention, the sample is a serum sample. In some instances of the methods described herein, the tag includes a fluorescent backbone, and step ii) includes passing the tag through a single molecule detector. In some instances of the methods described herein, the single molecule detector includes: a) a source of electromagnetic radiation to stimulate the fluorescent building block; b) a capillary flow cell to pass the fluorescent framework unit; c) a source of motive force to move the fluorescent frame unit in the capillary flow cell; d) a defined interrogation space within the capillary flow cell to receive the electromagnetic radiation emitted from the electromagnetic source; e) an electromagnetic radiation detector operatively connected to the interrogation space for measuring an electromagnetic characteristic of the stimulated fluorescent frame unit; and f) a microscope objective lens located between the interrogation space and the detector, where the lens is a high numerical aperture lens.

In some cases, the specification describes a method of determining a diagnosis, prognosis, or method of treatment in an individual that includes: i) determining a cardiac troponin concentration in a sample or determining cardiac troponin concentrations in a series of samples from the individual, where the concentration is determined by a cardiac troponin assay with a limit of detection for cardiac troponin in the sample less than about 50 pg / mL; and ii) determining a diagnosis, prognosis, or method of treatment in the individual, based on the concentration in the sample, or the concentrations in the series of samples. In some cases of the methods described in this document, step ii) includes an analysis such as comparing the concentration or the series of concentrations with a normal value for the concentration, comparing the concentration or the series of concentrations with a predetermined threshold level , comparing the concentration or series of concentrations with a reference value, and determining a rate of change of the concentration for the series of concentrations. In some cases of the methods described herein, step ii) includes comparing the troponin concentration in the sample with a predetermined threshold concentration, and determining a diagnosis, prognosis, or treatment method if the sample concentration is greater than the threshold level. In some cases of the methods described in this document, the threshold concentration is determined by determining a 99th percentile concentration of troponin in a group of normal individuals, and setting the 45th threshold concentration at the 99th percentile concentration. °. In some embodiments that fall within the scope of the methods of the invention as specified in the claims, at least one sample is taken during or after a cardiac stress test. In some embodiments of the methods of the invention as specified in the claims, cardiac troponin is selected from the group consisting of cardiac troponin I and cardiac troponin T. In some embodiments of the methods of the invention as specified in the claims, the cardiac troponin is cardiac troponin I. In some instances of the methods described herein, the cardiac troponin concentration is a concentration of total cardiac troponin. In some instances of the methods described herein, the cardiac troponin concentration is a concentration of a cardiac troponin complex, cardiac troponin fragment, phosphorylated cardiac troponin, oxidized cardiac troponin, or a combination thereof. In some cases of the methods described herein, the cardiac troponin concentration is compared to the total cardiac troponin. In some cases of the methods described herein, the diagnosis, prognosis, or method of treatment is a diagnosis, prognosis, or method of treatment of myocardial infarction. In some cases of the methods described herein, the diagnosis, prognosis, or treatment method comprises risk stratification for the risk level of myocardial infarction. In some cases of the methods described in this document, the concentration or series of 60 concentrations is determined at or near the time the individual presents to a healthcare professional with one or more symptoms indicative of myocardial ischemia or infarction or the possibility of it. In some cases, the symptom (s) may be chest pain, chest pressure, arm pain, abnormal EKG, enzyme levels

ES 2 550 004 T3 abnormal, or shortness of breath. In some cases, the concentration is determined by a method that includes the detection of single troponin molecules or complexes, or fragments thereof. In some cases, the methods described herein involve labeling the troponin or a troponin complex with a tag comprising a fluorescent building block. In some cases of the methods described herein, the fluorescent frame unit is capable of emitting at least about 200 photons when simulated by a laser light emitter at the excitation wavelength of the frame unit, when the laser is focuses on a 5 micron diameter spot that contains the structural unit, and where the total energy directed at the spot by the laser is no more than about 3 microjoules. In some instances of the methods described herein, the fluorescent building block includes a molecule containing at least one substituted indolium ring system in which the substituent on carbon 3 of the indole ring contains a chemically reactive group or a group of conjugated substances. In some cases of the methods described herein, the fluorescent structural unit includes a dye selected from the group consisting of Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 647, Alexa Fluor 680, or Alexa Fluor 700. In some cases of the methods described herein, the fluorescent framework unit comprises Alexa Fluor 647. In some embodiments of the methods described herein, the tag further comprises a binding partner for troponin. In some cases, the binding partner comprises an antibody specific for troponin. In some cases, the antibody is a polyclonal antibody. In some instances of the methods described herein, the methods further include capturing troponin or troponin complex on a solid support. In some cases of the methods described herein, the solid support can be a microtiter plate or paramagnetic beads. In some instances of the methods described herein, the solid support includes a specific capture partner for troponin or the troponin complex that is bound to the solid support. In some cases of the methods described herein, the binding of the capture partner to the solid support is non-covalent. In some cases of the methods described herein, the binding of the capture partner to the solid support is covalent. In some instances of the methods described herein, the covalent attachment of the capture partner is such that the capture partner is attached to the solid support in a specific orientation. In some instances of the methods described herein, the specific orientation serves to maximize the specific binding of the troponin or troponin complex to the capture partner. In some cases of the methods described in this document, step i) further involves the evaluation of another indicator for the individual, and step ii) involves determining a diagnosis, prognosis, or method of treatment in the individual, based on the troponin concentration and the evaluation of the other indicator of the marker no30 troponin in the sample, or in the concentrations in the series of samples. In some cases, the other indicator is a clinical indicator of myocardial infarction or ischemia. In some cases, the other indicator is the concentration of one or more non-troponin markers in the sample or series of samples. In some instances of the methods described herein, the one or more markers are markers of cardiac ischemia, or markers of inflammation and plaque instability. In some cases, the one or more markers of cardiac ischemia may be creatine 35 kinase (CK) and its myocardial band (MB) of the myocardial structural unit CK, aspartate aminotransferase, lactate dehydrogenase (LDH), α-hydroxybutyrate dehydrogenase, myoglobin , glutamate oxaloacetate transaminase, glycogen phosphorylase BB, unbound free fatty acids, heart fatty acid binding protein (H-FABP), ischemia-modified albumin, light chain myosin 1, or light chain myosin 2. In some instances of the methods described herein, the one or more markers include one or more specific markers of myocardial damage. In some embodiments of the methods of the invention, the diagnosis, prognosis, or method of treatment is a diagnosis, prognosis, or method of treatment of a condition other than myocardial infarction. In some embodiments, the condition is cardiac toxicity. In some embodiments, cardiac toxicity is associated with the administration of a drug to the individual. In some instances of the methods described herein, the condition is selected from the group consisting of acute rheumatic fever, amyloidosis, cardiac trauma (including contusion, ablation, rhythm, firing, cardioversion, catheterization, and cardiac surgery), reperfusion injury , congestive heart failure, end-stage renal failure, type II glycogen storage disease (Pompe disease), heart transplantation, hemoglobinopathy with transfusional hemosiderosis, hypertension, including gestational hypertension, hypotension, often with arrhythmias, hypothyroidism, myocarditis, pericarditis, post-operative non-cardiac surgery, pulmonary embolism, and sepsis.

The specification also describes the compositions.

In some cases the specification describes a composition for the detection of a troponin isoform including a binding partner with the troponin isoform attached to a fluorescent building block, where the fluorescent building unit is capable of emitting at least about 200 photons when simulated. by a laser light emitter at the excitation wavelength of the structural unit, when the laser is focused on a spot 55 of not less than about 5 microns in diameter that contains the structural unit, and where the total energy directed at the spot by the laser is not more than about 3 microjoules. In some instances of the compositions, the binding partner comprises an antibody to the troponin isoform. In some cases, the antibody is a polyclonal antibody. In some cases, the antibody is a monoclonal antibody. In some cases, the troponin isoform is a cardiac isoform. In some cases, the cardiac isoform is selected from the group consisting of 60 cTnI and cTnT. In some cases, the cardiac isoform is cTnI. In some cases, the antibody is specific for a specific region of the troponin molecule. In some cases, the antibody is specific for a region that

ES 2 550 004 T3 comprises amino acids 27-41 of cardiac troponin I. In some cases of the compositions, the fluorescent building block comprises a molecule comprising at least one substituted indole ring system in which the substituent at carbon 3 of the indole ring contains a chemically reactive group or a group of conjugated substances. In some cases, the fluorescent building unit includes a dye which may be Alexa 5 Fluor 488, Alexa Fluor 532, Alexa Fluor 647, Alexa Fluor 680, or Alexa Fluor 700. In some cases, the fluorescent building unit comprises Alexa Fluor 647.

Described herein is a composition comprising a set of standards for determining a concentration of a cardiac troponin, wherein at least one of the standards is at a cardiac troponin concentration of less than about 10 pg / mL.

Described herein is a kit containing a composition that includes an antibody to cardiac troponin bound to a fluorescent dye building block, wherein the building block is capable of emitting at least about 200 photons when simulated by a laser light emitter. at the excitation wavelength of the frame unit, when the laser is focused on a point not less than about 5 microns in diameter that contains the frame unit, and where the total energy directed at the point by the laser is no more than about 3 microjoules, where the composition is packaged in an appropriate package. In some cases of the kits, the cardiac troponin is cardiac troponin I or cardiac troponin T. In some cases, the cardiac troponin is cardiac troponin I. In some cases of kits, the kits also include instructions. In some instances of the kits, the kits further include a composition containing a capture antibody to cardiac troponin I bound to a solid support. In some cases, the solid support comprises a microtiter plate or paramagnetic microparticles. In some instances of the kits, the kits further include a component selected from the group consisting of wash buffer, assay buffer, elution buffer, and the calibrator diluent. Some cases of the kits also include a standard for cardiac troponin.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A and 1B. Schematic diagram of the configuration of the components of a single particle analyzer. Figure 3A shows an analyzer including an electromagnetic source and an electromagnetic detector; Figure 3B shows an analyzer that includes two electromagnetic sources and an electromagnetic detector.

Figures 2A and 2B. Schematic diagrams of a capillary flow cell for a single particle analyzer. Figure 4A shows the flow cell of an analyzer that includes an electromagnetic source; and Figure 4B shows the flow cell of an analyzer that includes two electromagnetic sources.

Figures 3A and 3B. Schematic diagrams showing the conventional (A) and confocal (B) positioning of the detector optics and the laser of a single particle analyzer. Figure 3A shows the configuration of an analyzer having an electromagnetic source and an electromagnetic detector; Figure 3B shows the configuration of an analyzer having two electromagnetic sources and two electromagnetic detectors.

Figure 4. Linearized standard curve for the range of cTnI concentrations.

Figure 5. Biological threshold (cutoff concentration) for cTnI is at a cTnI concentration of 7pg / mL, as established at the 99th percentile with a corresponding CV of 10%.

Figure 6. Correlation of the cTnI assay results determined using the specification analysis system with the standard measurements provided by the National Institute of Standards and Technology (R2 = 0.9999).

Figure 7. Detection of cTnI in serum samples from series of patients who presented to the emergency room with chest pain. Measurements made with the specification analysis system were compared to measurements made with a commercially available assay.

Figure 8. Distribution of normal biological concentrations of cTnI (Non-ischemia) and concentrations of cTnI in serum samples from patients with chest pain.

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description setting forth the illustrative embodiments, utilizing the principles of the invention and the accompanying drawings of which:

DETAILED DESCRIPTION

IS 2 550 004 T3

Resume

introduction

II. Cardiac troponin

III. Labels for cardiac troponin

A. Binding partners for troponin

1. Antibodies

two. Cross-reactive antibodies

B. Fluorescent building blocks to be used with binding partners

1. Colorants

two. Quantum dots

C. Fluorescent structural unit-bond partner compositions

IV. High sensitivity cardiac troponin analysis

A. Sample

B. Sample preparation

C. Detection of troponin and determination of concentration

V. Appropriate instruments and systems for high-sensitivity troponin analysis

A. Device / System

B. Single Particle Analyzer

1. Source of electromagnetic radiation

two. Capillary flow cell

3. Driving force

Four. Detectors

C. Sampling system

D. Sample preparation system

E. Sample recovery

SAW. Methods Using High Sensitivity Cardiac Troponin Assay

A. Samples

B. Determination of diagnosis, prognosis, or method of treatment

1. Acute myocardial infarction

two. Conditions other than AMI

to. Cardiac toxicity

C. Business methods

IS 2 550 004 T3

VII. Compositions

VIII. Kits

introduction

The specification describes compositions and methods for the highly sensitive detection of troponin, eg, cardiac troponin. The release into the blood of cardiac troponin isoforms, which are unique to cardiac muscle (cardiac troponin I and / or T) is indicative of cardiac muscle damage, and provides the basis for their use as diagnostic or prognostic markers. or to assist in determining treatment.

The troponin complex in muscle is made up of troponin I, C, and T. Troponin C exists as two isoforms, one from the slow-twitch heart muscle and one from the fast-twitch muscle; Because it is found in virtually all striated muscles, its use as a specific marker is limited. In contrast, troponin I and T are expressed as different isoforms in slow-twitch, fast-twitch, and cardiac muscle. The unique cardiac isoforms of troponin I and T allow them to be immunologically distinguished from the other skeletal muscle troponins. Therefore, the release into the blood of cardiac troponin I and T is indicative of damage to the cardiac muscle, and provides the basis for their use as diagnostic or prognostic markers, or to aid in determining treatment.

Because the level of troponin found in the circulation of healthy individuals is very low, and specific cardiac troponins do not come from extra-cardiac sources, troponins are highly sensitive and specific markers of cardiac injury. In addition to heart attack, a number of other conditions can cause damage to the heart muscle, and early detection of such damage would be helpful to clinicians. However, current methods of detection and quantification of cardiac troponin do not have sufficient sensitivity to detect the release of cardiac troponin into the blood, until the levels have reached abnormally high concentrations, for example, 0.1 ng / mL or higher. .

The methods and compositions described herein thus include methods and compositions for the highly sensitive detection and quantification of cardiac troponin, and compositions and methods for the diagnosis, prognosis and / or determination of treatment based on such highly sensitive detection and quantification. sensitive.

II. Cardiac troponin

When the two unique forms of cardiac troponin, cardiac troponin I (cTnI) and cardiac troponin (cTnT) are released into the blood from the heart muscle, several species of each can exist in the blood. These include various complexes of the two forms, with each other and / or with cardiac troponin C (cTnC). Furthermore, the two forms 35 are subject to virtually immediate proteolytic degradation, resulting in a variety of fragments.

Also, various phosphorylated and oxidized forms of troponins can exist in the blood. See, for example, US Patent No. 6,991,907. Unless otherwise specified, cardiac troponin, as used herein, encompasses all forms of cardiac troponin, including

In some cases, the specification describes methods and compositions for the detection and / or determination of the total cardiac troponin concentration, ie, the sum of all or a substantial part of the cardiac troponin in a sample, for example, sample of blood, serum, or plasma, if free, in complex, a proteolytic fragment, phosphorylated, oxidized, or otherwise modified. In some embodiments of the invention as specified in the claims, the cardiac troponin is cTnI, in others, it is cTnT, and, in still other embodiments of the invention as specified in the claims, the cardiac troponin is cTnI and cTnT. It will be appreciated that an absolute total measurement does not have to be achieved, as long as a constant ratio of the total is determined, which can be compared to the standard values. It will also be appreciated that whether a form of troponin is a minor constituent of total levels, low or no detection of that form will not appreciably affect total troponin measurements. Therefore, as used herein, total cardiac troponin refers to a measurement that is intended to measure all or substantially all forms of a particular cardiac troponin, eg, all 50 cTnI, or all cTnT, in a sample, where sample-to-sample consistency is such that clinically relevant conclusions can be drawn from comparisons of samples to standards, or comparison of one sample to another.

IS 2 550 004 T3

In some instances, the specification describes methods and compositions for the detection and / or determination of the concentration of one or more of the various forms of troponin in the sample as a separate entity, eg, cTnI in complex, free cTnI, cTnI cloudy (eg, oxidized or phosphorylated), or complex cTnT, free cTnT, cloudy cTnT (eg, oxidized or phosphorylated), and can generally provide such a concentration in the sample. In the latter cases, relationships or absolute values can be determined by the different entities. Therefore, in some cases, the specification describes methods of detecting and, usually, determining the concentration of, one or more forms of troponin in complex, or one or more fragments of troponin, or one or more oxidized forms or phosphorylated troponin. In some cases, more than one form is detected, and the concentrations of the various forms can be determined, for example, by performing multiplexed assays on a single sample for the different entities, or by performing separate assays in aliquots at from the same or similar samples. Relationships of the concentrations of the various forms can be obtained. For example, a ratio of the concentration of a particular form, eg, a fragment, complex, or modified form, of cardiac troponin to the concentration of total cardiac troponin can be determined. These relationships and / or absolute values can provide meaningful clinical information. For example, the relative fragment ratio of cardiac troponin can indicate the time elapsed since release into the blood and thus, indirectly, the length of time since, for example, a myocardial infarction. See, for example, US Patent No. 6,991,907.

III. Labels for cardiac troponin

In some cases, the specification describes methods and compositions that include labels for the highly sensitive detection and quantification of cardiac troponin.

One skilled in the art will recognize that many of the strategies can be used to tag target molecules to allow their detection or discrimination in a mixture of particles. Tags can be attached by any known means, including methods that utilize the non-specific or specific interactions of the tag and target. The labels can provide a detectable signal or affect the mobility of the particle in an electric field. 25 Furthermore, the marking can be carried out directly or through binding partners.

In some cases, the tag comprises a troponin binding partner attached to a fluorescent building block.

A. Binding partners for troponin

Any appropriate binding partner with the specificity required for the cardiac form of troponin to be detected can be used. For example, a specific binding partner can be used for all or substantially all forms of cTnI or a specific binding partner can be used for all or substantially all forms of cTnT; such binding partners generally bind to a region of cardiac troponin that is common to all or most of the different probable forms found in a sample. In some cases, a specific binding partner to one or more particular forms of cardiac troponin can be used, for example, a binding partner to a complex cTnI, free cTnI, cloudy cTnI (for example, oxidized or phosphorylated), or cTnT in complex, free cTnT, cloudy cTnT (eg, oxidized or phosphorylated). Binding partners are known in the art and include, for example, aptamers, lectins, and the receptors. A useful and versatile type of binding partner is an antibody.

1. Antibodies

In some cases, the binding partner is an antibody specific for cardiac troponin. The term antibody, as used herein, is a broad term and is used in its ordinary sense, including, without limitation, to refer to naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, antibodies to single chain, bifunctional, chimeric, and humanized antibodies, as well as antigen-binding fragments thereof. In some cases, the antibody is specific for cTnI. In some cases, the antibody is specific for 45 cTnT. In some cases, the label includes antibodies to both cTnI and cTnT. The antibody can be specific for all or substantially all forms of cardiac troponin; for example, all or substantially all forms of cTnI, or all or substantially all forms of cTnT. In some cases, an antibody specific to one or more particular forms of cardiac troponin can be used, for example, a binding partner for a complex cTnI, free cTnI, cloudy cTnI (eg, oxidized or phosphorylated), or cTnT in complex, free cTnT, cloudy cTnT (eg, oxidized or phosphorylated). Antibody mixtures are also encompassed by the specification, for example, mixtures of antibodies to cTnI and cTnT, or mixtures of antibodies to the various forms of troponin (free, complex, etc.), or mixtures of the mixtures.

It will be appreciated that the choice of epitope or region of troponin to which the antibody is raised will determine its specificity, for example, for total troponin for certain fragments, for troponin in complex, for

ES 2 550 004 T3 modified troponin, and the like. In some cases, the antibody is specific for a specific amino acid region of cardiac troponin. In some cases, the antibody is specific for amino acids 27-41 of human cardiac troponin I. Both monoclonal and polyclonal antibodies are useful as binding partners. In some cases, the antibody is a polyclonal antibody. In some cases, the antibody is a monoclonal antibody.

In some cases, the antibody is a polyclonal antibody specific for amino acids 27-41 of human cardiac troponin I. In some cases, this antibody is unaffected by heparin, phosphorylation, oxidation, and troponin complexation, and does not cross-react with skeletal muscle troponin I.

Methods for producing antibodies are well established. Cardiac specific sequences for troponin 10 I and troponin T are described in FEBS Lett. 270,57-61 (1990) and Genomics 21, 311-316 (1994). One skilled in the art will recognize that many procedures are available for the production of antibodies, for example, as described in Antibodies, A Laboratory Manual, Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988), Cold Spring Harbor, NY One skilled in the art will also appreciate that antibody-mimicking Fab fragments or binding fragments can also be prepared from genetic information by various procedures (Antibody 15 Engineering: A Practical Approach (Borrebaeck, C., ed.), 1995 , Oxford University Press, Oxford; J. Immunol. 149.39143920 (1992)). Methods for producing antibodies to the various forms of complex, fragment, phosphorylated, and oxidized troponins are described in US Patent Nos. 5,579,687; 6,991,907; and in United States Patent Application No. 20050164317. A synthetic peptide composed of 14 amino acids that mimics a specific sequence of cardiac troponin I and the methods used to prepare antibodies to the peptide are described in International Patent Application No. PCT / US94 / 05468. Monoclonal and polyclonal antibodies to complex and free cardiac troponins are also commercially available (HyTest, HyTest Ltd., Turku Finland; Abcam Inc., Cambridge, MA, USA, Life Diagnostics, Inc., West Chester, PA, uSa; Fitzgerald Industries International, Inc., Concord, MA 01742-3049 USA; BiosPacific, Emeryville, CA).

In some cases, the antibody is a mammalian antibody, eg, goat anti-cTnI polyclonal antibody. The antibody can be specific for specific regions of cTnI, eg, amino acids 27-41 of human cardiac troponin I. Capture binding partner pairs and detection binding partners, eg, capture and detection antibody pairs, can be used in the cases described herein. Therefore, in some cases, a heterogeneous assay protocol is used in which typically two binding partners are used, for example two antibodies. One binding partner is a capture partner, usually immobilized on a solid support, and the other binding partner is a detection binding partner, usually with a detectable tag attached. In some cases, the capture binding partner element of a pair is an antibody that is specific for all or substantially all forms of cardiac troponin. An example is an antibody, eg, a monoclonal antibody, specific for free cardiac troponin I (cTnI) aa 41-49 and cTnI that complexes with other troponin components. Preferably, this antibody is unaffected by troponin complex formation, heparin, phosphorylation, and oxidation, and is not cross-reactive with skeletal muscle troponin I. Therefore, the antibody is thought to bind to total cTnI. Another example is a monoclonal antibody, specific for cardiac troponin I (cTnI) aa 87-91 and does not cross-react with skeletal muscle troponin I. Such antibodies are available from BiosPacific, Emeryville, CA. Other pairs of antibodies are known or can be designed.

Cross-reactive antibodies. In some cases it is useful to use an antibody that cross-reacts with a variety of species, either as a capture antibody, a detection antibody, or both. Such cases include measuring the toxicity of the drug by determining, for example, the release of cardiac troponin into the blood as a marker of cardiac damage. A cross-reactive antibody allows toxicity studies to be done in one species, eg, a non-human species, and direct transfer of the results for studies or clinical observations of another species, eg, humans, using the same antibody or pair of antibodies in the assay reagents, thus decreasing the variability between assays. Thus, in some cases, one or more of the antibodies for use as a binding partner with the marker, eg, cardiac troponin, such as cardiac troponin I, may be a cross-reactive antibody.

In some cases, the antibody cross-reacts with the marker, eg, cardiac troponin, from at least two species selected from the group consisting of human, monkey, dog, and mouse. In some cases the antibody is cross-reactive with the marker, eg, cardiac troponin, from the entire group consisting of human, monkey, dog, and mouse.

B. Fluorescent building blocks to be used with binding partners

In some cases, the binding partner, eg, the antibody, binds to a fluorescent building block. The fluorescence of the framework unit will be sufficient to allow detection in a single molecule detector, such as the single molecule detectors described herein. A fluorescent structural unit, as that term is used herein, includes one or more fluorescent entities whose overall fluorescence is such that the structural unit can be detected in the single molecule detectors described herein. For

Therefore, a fluorescent structural unit may comprise a single entity (eg a quantum dot or fluorescent molecule) or a plurality of entities (eg a plurality of fluorescent molecules). It will be appreciated that when structural unit, as that term is used herein, refers to a group of fluorescent entities, eg, a plurality of fluorescent dye molecules, each individual entity 5 may be attached to the binding partner separately or the entities can be linked together as long as the entities as a group provide sufficient fluorescence to be detected.

Generally, structural unit fluorescence involves a combination of quantum efficiency and lack of sufficient photobleaching that the structural unit is detectable above background levels in a single molecule detector, with the consistency required for the level. desired detection, accuracy and precision of the assay. 10 For example, in some cases, the fluorescence of the fluorescent building block is such that it allows detection and / or quantification of troponin at a detection level of less than about 10, 5, 4, 3, 2, or 1 pg / mL and with a coefficient of variation of less than about 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% or less, for example, about 10% or less, in the instruments described in this document. In some cases, the fluorescence of the fluorescent structural unit is such that it allows the detection and / or quantification of troponin at a detection limit of less than approximately 5 pg / mL and with a coefficient of variation of less than approximately 10%. , in the instruments described in this document. Limit of detection, as that term is used herein, includes the lowest concentration at which a sample can be identified as containing a molecule of the substance of interest, eg, the first non-zero value. It can be defined by the variability of zeros and the slope of the standard curve. For example, the detection limit of an assay can be determined by running a standard curve, determining the value of zero from the standard curve, and adding 2 standard deviations to that value. A concentration of the substance of interest that produces a signal equal to this value is the lower limit of concentration detection.

On the other hand, the structural unit has properties that are consistent with its use in the test of choice. In some cases, the assay is an immunoassay, where the fluorescent building block is bound to an antibody; The structural unit must have properties such that it does not aggregate with other antibodies or proteins, or undergoes no further aggregation that is consistent with the required accuracy and precision of the assay. In some cases, the fluorescent building blocks that are preferred are fluorescent building units, eg, dye molecules that have a combination of 1) high absorption coefficient; 2) high quantum yield; 3) high photostability (low photobleaching); and 4) compatibility with the labeling of the biomolecule of interest (for example, protein) so that it can be analyzed using the analyzers and systems described herein (for example, it does not cause precipitation of the protein of interest, or precipitation of a protein to which the structural unit has been attached).

Fluorescent structural units, for example a single fluorescent dye molecule or a plurality of fluorescent dye molecules, which are useful in some cases described herein can be defined in terms of their photon emission characteristics when stimulated by the EM radiation. For example some cases use a fluorescent dye structural unit, for example, a single fluorescent dye molecule or a plurality of fluorescent dye molecules, which is capable of emitting an average of at least about 10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 350, 400, 500, 600, 700, 800, 900, or 1000, photons when simulated by a laser light emitter at the excitation wavelength of the structural unit, when the laser is focused on a spot not less than about 5 microns in diameter that contains the structural unit, and wherein the total energy directed at the spot by the laser is not more than about 3 microjoules. It will be appreciated that the total energy can be achieved by many different combinations of laser output power and duration of exposure time of the dye build unit. For example, a laser with an output power of 1 mW can be used for 3 ms, 3 mW for 1 ms, 6 mW for 0.5 ms, 12 mW for 0.25 ms, and so on.

In some cases they use a fluorescent dye structural unit, for example a single fluorescent dye molecule or a plurality of fluorescent dye molecules, which is capable of emitting an average of at least about 50 photons when simulated by a light emitter. laser at the excitation wavelength of the structural unit, when the laser is focused on a point not less than about 5 microns in diameter that contains the structural unit, and wherein the total energy directed at the point by the laser is no more than about 3 microjoules. Some cases use a fluorescent dye structural unit, for example a single fluorescent dye molecule or a plurality of fluorescent dye molecules, which is capable of emitting an average of at least about 100 photons when simulated by a laser light emitter. at the excitation wavelength of the frame unit, when the laser is focused on a point not less than about 5 microns in diameter that contains the frame unit, and wherein the total energy directed at the point by the laser is no more than about 3 microjoules. Some cases use a fluorescent dye structural unit, for example a single fluorescent dye molecule or a plurality of fluorescent dye molecules, which is capable of emitting an average of at least about 150 photons when simulated by a laser light emitter. at the excitation wavelength of the frame unit, when the laser is focused on a point not less than about 5 microns in diameter that contains the frame unit, and where the

ES 2 550 004 T3 total energy directed at the point by the laser is no more than about 3 microjoules. Some cases use a fluorescent dye structural unit, for example a single fluorescent dye molecule or a plurality of fluorescent dye molecules, which is capable of emitting an average of at least about 200 photons when simulated by a laser light emitter. at the excitation wavelength of the structural unit, when laser 5 is focused on a point not less than about 5 microns in diameter that contains the structural unit, and wherein the total energy directed at the point by the laser is no more than about 3 microjoules. Some cases use a fluorescent dye structural unit, for example a single fluorescent dye molecule or a plurality of fluorescent dye molecules, which is capable of emitting an average of at least about 300 photons when simulated by a laser light emitter. at the excitation wavelength of the frame unit, when the laser is focused on a point not less than about 5 microns in diameter that contains the frame unit, and wherein the total energy directed at the point by the laser is no more than about 3 microjoules. Some cases use a fluorescent dye structural unit, for example a single fluorescent dye molecule or a plurality of fluorescent dye molecules, which is capable of emitting an average of at least about 500 photons when simulated by a laser light emitter at the excitation wavelength of the frame unit, when the laser is focused on a point not less than about 5 microns in diameter that contains the frame unit, and wherein the total energy directed at the point by the laser is no more than about 3 microjoules.

In some cases, the fluorescent structural unit comprises an average of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 fluorescent entities, eg, fluorescent molecules. In some cases, the fluorescent structural unit 20 comprises an average of no more than about 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 fluorescent entities, eg, fluorescent molecules. In some cases, the fluorescent structural unit comprises an average of about 1 to 11, or about 2 to 10, or about 2 to 8, or about 2 to 6, or about 2 to 5, or about 2 to 4, or about 3 at 10, or about 3 to 8, or about 3 to 6, or about 3 to 5, or about 4 to 10, or about 4 to 8, or about 4 to 6, or about 2, 3, 4, 5, 6, or more than about 6 fluorescent entities. In some cases, the fluorescent building block comprises an average of about 2 to 8 fluorescent building units. In some cases, an average of about 2 to 6 fluorescent entities. In some cases, the fluorescent building block comprises an average of about 2 to 4 fluorescent entities. In some cases, the fluorescent building block comprises an average of about 3 to 10 30 fluorescent entities. In some cases, the fluorescent building block comprises an average of about 3 to 8 fluorescent entities. In some cases, the fluorescent building block comprises an average of about 3 to 6 fluorescent entities. By mean is meant that, in a given sample that is a representative sample of a group of labels described herein, where the sample contains a plurality of the fluorescent structural unit units of the binding partner, the molar ratio of the entity The particular fluorescent unit of which the fluorescent structural unit is comprised, with the binding partner, as determined by standard analytical methods, corresponds to the specified number or range of numbers. For example, in cases where the tag comprises a binding partner that is an antibody and a fluorescent structural unit that comprises a plurality of fluorescent dye molecules of a specific absorbance, a spectrophotometric assay in which one is diluted Label solution at an appropriate level 40 and the absorbance at 280 nm is taken to determine the molarity of the protein (antibody) and an absorbance is taken at, for example, 650 nm (for Alexa Fluor 647) to determine the molarity of the fluorescent dye molecule. The ratio of the latter molarity to the former represents the average number of fluorescent entities (dye molecules) in the fluorescent structural unit attached to each antibody.

1. Colorants

Some cases use fluorescent building blocks that comprise fluorescent dye molecules. Some cases use a fluorescent dye molecule that is capable of emitting an average of at least about 50 photons when simulated by a laser light emitter at the excitation wavelength of the molecule, where the laser is focused on a point of no less than about 5 microns in diameter that the molecule contains, and wherein the total energy directed at the point by the laser is no more than about 3 microjoules.

Some cases use a fluorescent dye molecule that is capable of emitting an average of at least about 75 photons, when simulated by a laser light emitter at the excitation wavelength of the molecule, where the laser is focused on one point. of no less than about 5 microns in diameter that the molecule contains, and wherein the total energy directed at the spot by the laser is no more than about 3 microjoules.

Some cases use a fluorescent dye molecule that is capable of emitting an average of at least approximately 100 photons when simulated by a laser light emitter at the excitation wavelength of the molecule, where the laser is focused on one point. of no less than about 5 microns in diameter that the molecule contains, and wherein the total energy directed at the spot by the laser is no more than about 3 microjoules.

Some cases use a fluorescent dye molecule that is capable of emitting an average of at least about 150 photons when simulated by a laser light emitter at the excitation wavelength of the 60 molecule, when the laser is focused on a point. not less than about 5 microns in diameter containing

ES 2 550 004 T3 the molecule, and wherein the total energy directed at the point by the laser is no more than about 3 microjoules. Some cases use a fluorescent dye molecule that is capable of emitting an average of at least about 200 photons when simulated by a laser light emitter at the excitation wavelength of the molecule, when the laser is focused on a point of no less than about 5 microns in diameter containing the molecule, and wherein the total energy directed at the spot by the laser is no more than about 3 microjoules.

A non-exhaustive list of useful fluorescent entities for use in the fluorescent building blocks described herein is given in Table 1, below. In some cases, the fluorescent entity is selected from the group consisting of Alexa Flour 488, 532, 647, 700, 750, fluorescein, B-phycoerythrin, allophycocyanin, PBXL-3, and Qdot 605.

TABLE 1

FLUORESCENT ENTITIES

<td>Colorant</td><td>Ex (nm)</td><td>E (M) -1</td><td>Em (nm)</td><td>Mw</td>

<td>Bimanous</td><td> 380</td><td> 5,700</td><td> 458</td><td> 282.31</td>

<td>Dapoxil</td><td> 373</td><td> 22,000</td><td> 551</td><td> 362.83</td>

<td>Dimethylamino coumarin-4-acetic acid</td><td> 375</td><td> 22,000</td><td> 470</td><td> 344.32</td>

<td>8-Anilino naphthalene-1-sulfonic acid</td><td> 372</td><td></td><td> 480</td><td></td>

<td>Blue waterfall</td><td> 376</td><td> 23,000</td><td> 420</td><td> 607.42</td>

<td>Alexa Fluor 405</td><td> 402</td><td> 35,000</td><td> 421</td><td> 1028.26</td>

<td>Blue waterfall</td><td> 400</td><td> 29,000</td><td> 420</td><td> 607.42</td>

<td>Yellow waterfall</td><td> 402</td><td> 24,000</td><td> 545</td><td> 563.54</td>

<td>Pacific Blue</td><td> 410</td><td> 46,000</td><td> 455</td><td> 339.21</td>

<td>PyMPO</td><td> 415</td><td> 26,000</td><td> 570</td><td> 582.41</td>

<td>Alexa 430</td><td> 433</td><td> 15,000</td><td> 539</td><td> 701.75</td>

<td>Alexa 488</td><td> 495</td><td> 73,000</td><td> 519</td><td> 643.41</td>

<td>Fluorescein</td><td> 494</td><td> 79,000</td><td> 518</td><td> 376.32</td>

<td>Oregon Green 488</td><td> 496</td><td> 76,000</td><td> 524</td><td> 509.38</td>

IS 2 550 004 T3

<td>Alexa Fluor 532</td><td> 531</td><td> 81,000</td><td> 554</td><td> 723.77</td>

<td>BODIPY 530/550</td><td> 534</td><td> 77,000</td><td> 554</td><td> 513.31</td>

<td>Rhodamine 6G</td><td> 525</td><td> 108,000</td><td> 555</td><td> 555.59</td>

<td>Alexa Fluor 610</td><td> 612</td><td> 138,000</td><td> 628</td><td></td>

<td>Alexa Fluor 633</td><td> 632</td><td> 159,000</td><td> 647</td><td>AC. 1200</td>

<td>Alexa Fluor 647</td><td> 650</td><td> 250,000</td><td> 668</td><td>AC. 1250</td>

<td>BODIPY 630/650</td><td> 625</td><td> 101,000</td><td> 640</td><td> 660.50</td>

<td>Alexa Fluor 660</td><td> 663</td><td> 110,000</td><td> 690</td><td></td>

<td>Alexa Fluor 680</td><td> 679</td><td> 184,000</td><td> 702</td><td></td>

<td>Alexa Fluor 700</td><td> 702</td><td> 192,000</td><td> 723</td><td></td>

<td>Alexa Fluor 750</td><td> 749</td><td> 240,000</td><td> 782</td><td></td>

<td>B-phycoerythrin</td><td> 546,565</td><td> 2,410,000</td><td> 575</td><td> 240,000</td>

<td>R-phycoerythrin</td><td> 480,546,565</td><td> 1,960,000</td><td> 578</td><td> 240,000</td>

<td>Allophycocyanin</td><td> 650</td><td> 700,000</td><td> 660</td><td> 700,000</td>

Atto-tec dyes

IS 2 550 004 T3

Dyomics Fluors

<td>Hashtag</td><td>Ex (nm)</td><td>Molar absorbance * [I • mol-1 • cm-1]</td><td>Em (nm)</td><td>molecular weight # [g</td>

IS 2 550 004 T3

<td>EVOblue-10</td><td> 651</td><td> 101.440</td><td> 664</td><td> 389.88</td>

<td>EVOblue-30</td><td> 652</td><td> 102.000</td><td> 672</td><td> 447.51</td>

<td>Quantum dots: 525,</td><td> 565, 585,</td><td> 605, 655, 705, 800</td><td></td><td></td>

Suitable dyes for use as described herein include modified carbocyanine dyes. Modification of carbocyanin dyes includes modification of an indole ring of the carbocyanin dye to allow a reactive group or conjugated substance at position number 3. Modification of the 5 indolium ring provides dye conjugates that are uniformly and substantially more fluorescent in proteins, nucleic acids, and other biopolymers than structurally similar carbocyanine dye-labeled conjugates attached through the nitrogen atom at the position. number one. In addition to having a more intense fluorescence emission than structurally similar dyes at practically identical wavelengths, and the decrease in artifacts in their absorption spectra on conjugation with biopolymers, the 10 modified carbocyanine dyes have higher photostability and higher absorbance ( extinction coefficients) at peak absorbance wavelengths than dyes of similar structure. Therefore, the modified carbocyanine dyes result in increased sensitivity in assays using the modified dyes and their conjugates. Preferred modified colorants include compounds having at least one substituted indole ring system in which the substituent at carbon 3 of the indole ring contains a chemically reactive group or a conjugated substance. Other coloring compounds include compounds that incorporate an aza-benzazolium ring moiety and at least one sulfonate moiety. Modified carbocyanine dyes that can be used to detect unique particles in various cases described herein are described in US Patent 6,977,305. Therefore, in some cases the labels described in this document use a fluorescent dye that includes a substituted indole ring system in which the substituent on carbon 3 of the indole ring contains a chemically reactive group or a group of conjugated substances.

In some cases, the tag comprises a fluorescent building block that includes one or more Alexa dyes (Molecular Probes, Eugene, OR). Alexa dyes are disclosed in US Patents 6,977,305; 6,974,874; 6,130,101; and 6,974,305. Some cases use a dye selected from the group consisting of Alexa 25 Fluor 647, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 555, Alexa Fluor 610, Alexa Fluor 680, Alexa Fluor 700, and Alexa

Fluor 750. Some cases use a dye selected from the group consisting of Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 647, Alexa Fluor 700 and Alexa Fluor 750. Some cases use the Alexa Fluor 647 molecule, which has a maximum absorption between about 650 and 660 nm and an emission maximum between about 660 and 670 nm. Alexa Fluor 647 dye is used alone or in combination with other Alexa Fluor dyes.

Furthermore, currently available organic fluorescent compounds can be improved by making them less hydrophobic by adding hydrophilic groups such as polyethylene. Alternatively, currently sulfonated organic fluorescent compounds like Alexa Fluor 647 dye can be made less acidic by making them zwitterionic. Particles such as antibodies that are labeled with the modified fluorescent compounds are less likely to bind non-specifically to surfaces and proteins in immunoassays, and therefore allow assays to have higher sensitivity and lower background. Methods for modifying and improving the properties of fluorescent dyes in order to increase the sensitivity of a system that detects single particles are known in the art. Preferably, the modification improves the Stokes shift, while maintaining a high quantum yield.

IS 2 550 004 T3

two. Quantum dots

In some cases, the fluorescent tag structural unit that is used to detect a molecule in a sample using the assay systems described herein is a quantum dot. Quantum dots (QD), also known as semiconductor nanocrystals or artificial atoms, are semiconductor crystals that contain anywhere between 100 to 1,000 electrons and range from 2-10 nm. Some QDs can be 10-20 nm in diameter. QDs have high quantum yields, which makes them particularly useful for optical applications. QDs are fluorophores that fluoresce into excitons, which can be considered the excited state of traditional fluorophores, but have a much longer lifespan of up to 200 nanoseconds. This property offers QD with low photobleaching. The energy level of the QDs can be controlled by changing the size and shape of the QD, and the depth of the potential of the QDs. One of the optical characteristics of small excitonic QDs is coloration, which is determined by the size of the spot. The larger the dot, the redder it is, or more toward the red end of the fluorescence spectrum. The smaller the dot, the bluer or more toward the blue end it is. The bandgap energy that determines the energy and therefore the color of fluorescent light is inversely proportional to the square of the size of the QD. Larger QDs have more energy levels and are closely spaced, allowing the QD to absorb photons that contain less energy, ie, those closer to the red end of the spectrum. Because the emission frequency of a point depends on the forbidden band, therefore, it is possible to control the output wavelength of a point with extreme precision. In some cases, the protein that is detected with the single particle analysis system is labeled with a QD. In some cases, the single particle analyzer is used to detect a protein labeled with a QD and the use of a filter to allow the detection of different proteins at different wavelengths. QDs have wide excitation and narrow emission properties that when used with color filtering require only a single electromagnetic source for multiplex analysis of multiple targets on a single sample to resolve individual signals. Therefore, in some cases, the analysis system comprises a continuous wave laser and particles that are each marked with a single QD. Colloidally prepared QDs are free-floating and can be attached to a variety of molecules through metal coordination functional groups. These groups include, but are not limited to, thiol, amine, nitrile, phosphine, phosphine oxide, phosphonic acid, carboxylic acids, or other ligands. By attaching appropriate molecules to the surface, quantum dots can be dispersed or dissolved in almost any solvent or incorporated into a variety of inorganic and organic films. Quantum dots (QD) can be coupled to streptavidin directly through a maleimide ester coupling reaction or to antibodies through a maleimide-thiol coupling reaction. This produces a material with a covalently bound biomolecule on the surface, which produces conjugates with high specific activity. In some cases, the protein that is detected with the single particle analyzer is marked with a quantum dot. In some cases the quantum dot is between 10 and 20 nm in diameter. In other cases, the quantum dot is between 2 and 10 nm in diameter. Useful quantum dots include QD 605, QD 610, QD 655, and QD 705. A particularly preferred quantum dot is QD 35 605.

C. Fluorescent structural unit-binding partner compositions (labels)

The labels described herein generally contain a binding partner, eg, antibody, bound to a fluorescent structural unit to provide the fluorescence required for detection and quantification in the instruments described herein. Any appropriate combination of binding partner 40 and fluorescent building block in the single molecule detectors described herein can be used as a tag as described herein. Some instances provide a tag for a cardiac troponin molecule, or fragment, complex, phosphorylated, or oxidized form thereof, where the tag includes an antibody to a cardiac troponin and a fluorescent building block. The antibody can be any antibody as described above, for example an antibody to cTnT or cTnI. In some cases, the antibody is an antibody to cTnI. In some cases, the antibody is specific for a specific region of cardiac troponin, eg, specific for amino acids 27-41 of human cTnI. Some instances provide compositions comprising a fluorescent building block bound to an anti-cTnI antibody, eg, a polyclonal antibody such as a goat polyclonal antibody designated G129C available from BiosPacific, Emeryville. A fluorescent structural unit can be attached such that the tag is capable of emitting an average of at least 50 about 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 350, 400, 500, 600, 700, 800, 900, or 1000, photons when simulated by a laser light emitter at the excitation wavelength of the frame unit, when the laser is focused on a point of not less than about 5 microns diameter containing the label, and wherein the total energy directed at the point by the laser is no more than about 3 microjoules. In some cases, the fluorescent framework unit may be a fluorescent framework unit that is capable of emitting an average of at least about 50, 100, 150, or 200 photons when simulated by a laser light emitter at the wavelength of structural unit excitation, where the laser is focused on a point not less than approximately 5 microns in diameter that contains the structural unit, and wherein the total energy directed at the point by the laser is no more than about 3 microjoules. The fluorescent building block may be a fluorescent building block that includes one or more dye molecules with a structure that includes a substituted indolium ring system in which the substituent at carbon 3 of the indole ring contains a group

ES 2 550 004 T3 chemically reactive or a group of conjugated substances. The label composition can include a fluorescent building block that includes one or more dye molecules selected from the group consisting of Alexa Fluor 488, 532, 647, 700, or 750. The label composition can include a fluorescent building unit that includes a or more dye molecules selected from the group consisting of Alexa Fluor 488, 532, 700, or 750. The tag composition can include a fluorescent building block that includes one or more dye molecules that are Alexa Fluor 488. The tag composition can include a fluorescent building unit that includes one or more dye molecules that are Alexa Fluor 555. The Label composition can include a fluorescent building block that includes one or more dye molecules that are Alexa Fluor 610. The tag composition can include a fluorescent building block that includes one or more dye molecules that are Alexa Fluor 647. The tag composition can include a fluorescent building block that includes one or more dye molecules that are Alexa Fluor 680. The The label composition can include a fluorescent building block that includes one or more dye molecules that are Alexa Fluor 700. The label composition can include a fluorescent building block that includes one or more dye molecules that are Alexa Fluor 750.

Some examples provide a composition for the detection of cardiac troponin I that includes an AlexFluor molecule, for example, an Alexa Fluor molecule selected from the groups described, such as an Alexa Fluor 647 molecule bound to an antibody, for example , a polyclonal goat anti-cTnI antibody, specific for amino acids 27-41 of human cTnI. Some cases provide a composition for cardiac troponin I detection that includes a mean of 1 to 11, or about 2 to 10, or about 2 to 8, or about 2 to 6, or about 2 to 5, or about 2 to 4 , or about 3 to 10, or about 3 to 8, or about 3 to 6, or about 3 to 5, or about 4 to 10, or about 4 to 8, or about 4 to 6, or about 2, 3, 4, 5, 6, or more than about 6 molecules of Alexa Fluor 647, molecule bound to an antibody, eg, a polyclonal goat anti-cTnI antibody, specific for amino acids 27-41 of human cTnI. Some cases provide a composition for the detection of cardiac troponin I that includes a mean of 1 to 11, or about 2 to 10, or about 2 to 8, or about 2 25 to 6, or about 2 to 5, or about 2 to 4, or about 3 to 10, or about 3 to 8, or about 3 to 6, or about 3 to 5, or about 4 to 10, or about 4 to 8, or about 4 to 6, or about 2, 3, 4, 5, 6, or more than about 6 Alexa Fluor 647 molecule molecules bound to an antibody, eg, a polyclonal goat anti-cTnI antibody, specific for amino acids 27-41 of human cTnI. Some cases provide a composition for the detection of cardiac troponin I that includes an average of about 2 to 10 molecules of Alexa Fluor 647, molecule bound to an antibody, for example, a polyclonal goat anti-cTnI antibody, specific for the amino acids 27-41 of human cTnI. Some cases provide a composition for the detection of cardiac troponin I that includes an average of about 2 to 8 molecules of Alexa Fluor 647, molecule bound to an antibody, for example, a polyclonal goat anti-cTnI antibody, specific for amino acids. 27-41 of human cTnI. Some cases 35 provide a composition for the detection of cardiac troponin I that includes an average of approximately 6 molecules of Alexa Fluor 647, a molecule linked to an antibody, for example, a polyclonal goat anticTnI antibody, specific for amino acids 27- 41 of human cTnI. Some cases provide a composition for the detection of cardiac troponin I that includes an average of about 2 to 4 molecules of Alexa Fluor 647, molecule bound to an antibody, for example, a polyclonal goat anti-cTnI antibody, specific for the 40 amino acids 27-41 of human cTnI. Some cases provide a composition for the detection of cardiac troponin I that includes an average of about 3 to 8 molecules of Alexa Fluor 647, molecule bound to an antibody, for example, a polyclonal goat anti-cTnI antibody, specific for amino acids. 27-41 of human cTnI. Some cases provide a composition for the detection of cardiac troponin I that includes an average of about 3 to 6 molecules of Alexa Fluor 647, a molecule bound to an antibody, for example, a polyclonal goat anti-cTnI antibody, specific for the amino acids 27-41 of human cTnI. Some cases provide a composition for the detection of cardiac troponin I that includes an average of about 4 to 8 molecules of Alexa Fluor 647, a molecule bound to an antibody, for example, a polyclonal goat anticTnI antibody, specific for amino acids 27- 41 of human cTnI.

The binding of the fluorescent building block, or fluorescent entities that make up the fluorescent building unit 50, with the binding partner, eg, antibody, can be by any appropriate means; such methods are well known in the art and exemplary methods are given in the Examples. In some cases, after binding of the fluorescent building block to the binding partner to form a tag for use in the methods described herein, and prior to use of the tag for labeling the protein of interest, it is useful to carry out a filtration step. For example, an antibody-dye tag can be filtered prior to use, eg, through a 0.2 micron filter, or any appropriate filter for removal of aggregates. Other reagents for use in the assays described herein can also be filtered, for example, through a 0.2 micron filter, or any appropriate filter. Without wishing to be bound by theory, it is believed that such filtration removes a portion of the aggregates from, for example, the antibody-dye tags. As such aggregates they will bind as a unit to the protein of interest, but in case of release in elution buffer that is likely to disaggregate, false positives may result; ie, multiple tags are detected from

ES 2 550 004 T3 of an aggregate that has been bound to a single protein molecule of interest. Regardless of theory, filtration has been found to reduce false positives in subsequent testing and improve accuracy and precision.

IV. High sensitivity cardiac troponin analysis

The specification provides a method for determining the presence or absence of a single cardiac troponin molecule or a fragment or complex thereof in a sample, by i) labeling the molecule, fragment, or complex, if present, with a hashtag; and ii) detecting the presence or absence of the tag, where detecting the presence of the tag indicates the presence of the single cardiac troponin molecule, fragment, or complex in the sample. As used herein, "cardiac troponin molecule" includes a molecule that contains substantially all of the naturally occurring amino acid sequence of the particular type of cardiac troponin, including post-translationally modified forms, eg, phosphorylated forms, as well as forms. oxidized or otherwise chemically altered. As used herein, a fragment of a molecule includes a cardiac troponin molecule that contains less than the entire naturally occurring amino acid sequence, including modifications as for the entire molecule. As used herein, a complex of a cardiac troponin molecule includes a cardiac troponin molecule or a fragment that associates with one or more other molecules or substances, for example, that associates with one or more other molecules of cardiac troponin. In some cases, the method is capable of detecting troponin at a detection limit of less than approximately 100, 80, 60, 50, 40, 30, 20, 15, 12, 10, 9, 8, 7, 6, 5 , 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 pg / mL. In some cases, the method is capable of detecting troponin at a detection limit of less than approximately 100 pg / mL. In some cases, the method is capable of detecting troponin at a detection limit of less than approximately 50 pg / mL. In some cases, the method is capable of detecting troponin at a detection limit of less than approximately 20 pg / mL. In some cases, the method is capable of detecting troponin at a detection limit of less than approximately 10 pg / mL. In some cases, the method is capable of detecting troponin at a detection limit of less than approximately 5 pg / mL. In some cases, the method is capable of detecting troponin at a detection limit of less than approximately 3 pg / mL. In some cases, the method is capable of detecting troponin at a detection limit of less than about 1 pg / mL. Detection limits can be determined by using the appropriate standard reference material from the National Institute of Standards and Technology, eg, cTnI standard.

The methods also provide methods for determining a cardiac troponin concentration in a sample by detecting unique troponin molecules in the sample. Detection of a single troponin molecule includes detecting the molecule directly or indirectly. In the case of indirect detection, tags that correspond to unique cardiac troponin molecules, for example, a tag that has bound to unique cardiac troponin molecules can be detected.

The types of cardiac troponin for detection are as described herein, eg, cTnT, cTnI, total cardiac troponin (eg, total cTnI or total cTnT) or free, complex, or cardiac troponin 35 fragments . In some embodiments, total cardiac troponin is detected and / or quantified. In some embodiments, total cTnT is detected. In some embodiments, total cTnI is detected and / or quantified.

A. Sample

The sample can be any appropriate sample. Usually the sample is a biological sample, for example, a biological fluid. Such fluids include, without limitation, exhaled air condensate (EBC), bronchoalveolar lavage fluid (BAL), blood, serum, plasma, urine, cerebrospinal fluid, pleural fluid, synovial fluid, peritoneal fluid, amniotic fluid, gastric fluid, lymphatic fluid, interstitial fluid, tissue homogenate, cell extracts, saliva, sputum, feces, physiological secretions, tears, mucus, sweat, milk, semen, seminal fluid, vaginal secretions, fluid from ulcers and other superficial eruptions, blisters and abscesses, and tissue extracts, including biopsies of normal, malignant and suspicious tissues or any other constituents of the body that may contain the target particle of interest. Other similar samples, such as cell or tissue culture or culture broth are also of interest.

In some embodiments, the sample is a blood sample. In some embodiments, the sample is a plasma sample. In some embodiments, the sample is a serum sample.

B. Sample preparation

In general, any method of sample preparation can be used to produce a tag corresponding to a cardiac troponin molecule to be measured, where the tag is detectable on the instruments described herein. As is known in the art, sample preparation in which a tag is added to one or more particles can be performed in a homogeneous or heterogeneous format. In some cases, the sample preparation is established in a homogeneous format. In the analysis system that uses a homogeneous format, the

ES 2 550 004 T3 unattached label is not removed from the sample. See, for example, United States Patent Application No. 11 / 048,660, published as US2006 / 0078998 A1. In some cases, the particle or particles of interest are labeled by the addition of labeled antibody or antibodies that bind to the particle or particles of interest.

In some cases, a heterogeneous assay format is used, where typically a step is employed to remove the unattached label. Such assay formats are well known in the art. A particularly useful assay format is a sandwich assay, eg, a sandwich immunoassay. In this format, the molecule of interest, eg, marker of a biological state, is captured, eg, on a solid support, using a capture binding partner. Unwanted molecules and other substances can then optionally be washed away, followed by attachment of a tag comprising a detection binding partner and a detectable tag, eg, fluorescent building block. Other washes remove the unbound tag, then the detectable tag is released, usually, though not necessarily yet attached to the detection binding partner. In alternative cases, the sample and tag are added to the capture binding partner without a wash in between, for example, at the same time. Other variations will be apparent to one skilled in the art.

In some cases, the method for detection of troponin particles uses a sandwich assay with 15 antibodies, eg, monoclonal antibodies as capture binding partners. The method comprises the binding of troponin molecules in a sample with a capture antibody that is immobilized on a binding surface, and the binding of the detection antibody to the troponin molecule to form a sandwich complex. The detection antibody comprises a detectable fluorescent tag, as described herein, which is detected, for example, using the single molecule analyzers described herein. Both the capture and detection antibodies specifically bind to troponin. Many examples of sandwich immunoassays are known, and some are described in US Patent No. 4,168,146 to Grubb et al. and United States Patent No. 4,366,241 to Tom et al. Other specific examples for cardiac troponin are described in the Examples.

The capture binding partner can be attached to a solid support, eg, a microtiter plate or paramagnetic beads. In some cases, the invention provides a binding partner for a cardiac troponin attached to a paramagnetic bead. Any appropriate binding partner that is specific for the type of cardiac troponin to be captured can be used. The binding partner can be an antibody, eg, a monoclonal antibody. The antibody may be specific for free cardiac troponin (cTnI or cTnT) or for complex-forming cardiac troponin, modified cardiac troponin, or cardiac troponin fragments, as described herein, or specific to all or virtually all of the forms of cardiac troponin, eg, cTnI or cTnT, likely to be found in the sample of interest. The production and sources of antibodies to cardiac troponin are described herein. Preferred antibodies for measuring total troponin are those that are not substantially affected by heparin, phosphorylation, oxidation, and troponin complexing, and that are not cross-reactive with skeletal muscle troponin, eg, troponin. I. In some cases, the antibody is specific for a specific region of a cardiac troponin. In some cases, the region includes amino acids 41-49 of human cardiac troponin I. In some cases, the region includes amino acids 87-91 of human cardiac troponin I. Such antibodies are well known in the art and are available from, for example, BiosPacific, Emeryville, CA. An example of a capture antibody useful in the cases of the invention is an antibody, for example a monoclonal antibody, that reacts with free cardiac troponin I (cTnI) aa of 41-49 and 40 cTnI to complex with other components of troponin. Preferably, this antibody is unaffected by heparin, phosphorylation, oxidation, and troponin complexing, and does not undergo cross-reaction with skeletal muscle I troponin. An exemplary antibody of this type is Monoclonal Antibody Clone Number A34650228P, available from BiosPacific, Emeryville, CA. Another example of a capture antibody useful in the cases of the invention is an antibody, for example a monoclonal antibody, that reacts with free cardiac troponin I (cTnI) aa 87-91 and cTnI that complexes with other components of troponin.

Preferably, this antibody is unaffected by heparin, phosphorylation, oxidation, and troponin complexing, and is not cross-reactive with skeletal muscle troponin I. An exemplary antibody of this type is Monoclonal Antibody Clone Number A34440228P, available from BiosPacific, Everyville, CA. It will be appreciated that the antibodies identified herein, as useful as a capture antibody may also be useful as detection antibodies, and vice versa.

The binding of the binding partner, eg, antibody, to the solid support can be covalent or non-covalent. In some cases, the bond is non-covalent. An example of a non-covalent bond well known in the art is biotin-avidin / streptavidin interactions. Therefore, in some cases, a solid support, for example a microtiter plate or a paramagnetic bead, binds to the capture binding partner, for example, the antibody, through covalent binding, for example, interactions biotin-avidin / streptavidin. In some cases, the bond is covalent. Therefore, in some cases, a solid support, eg, a microtiter plate or a paramagnetic bead, is attached to the capture binding partner, eg, the antibody, through covalent attachment. Covalent binding in which the orientation of the capture antibody is such that it is especially useful that the capture of the molecule of interest is optimized. For example, in some cases a solid support, for example, you can use a

ES 2 550 004 T3 microtiter plate or paramagnetic microparticle, wherein the binding of the binding partner, eg antibody, is an oriented binding, for example a covalent oriented binding.

An example protocol for targeted binding of an antibody to a solid support is as follows: IgG is dissolved in 0.1 M sodium acetate buffer, pH 5.5 to a final concentration of 1 mg / mL. An equal volume of ice cold 20 mM sodium periodate in 0.1 M sodium acetate, pH 5.5 is added. IgG is allowed to oxidize per hour on ice. Excess periodate reagent was quenched by adding 0.15 volume of 1M glycerol. Low molecular weight by-products of the oxidation reaction are removed by ultrafiltration. The oxidized IgG building block was diluted to an appropriate concentration (typically 0.5 micrograms IgG per mL) and reacted with hydrazine activated multi-well plates for at least two hours at room temperature. Unbound IgG is removed by washing the multiwell plate with borate buffered saline or other appropriate buffer. The plate can be dried during storage, if desired. A similar protocol can be followed for microbeads if the microbead material is appropriate for such binding.

In some cases, the solid support is a microtiter plate. In some cases, the solid support is a paramagnetic bead. An exemplary paramagnetic bead is C1 streptavidin (Dynal, 650.01-03). Other suitable beads 15 will be apparent to those skilled in the art. Methods for binding antibodies to paramagnetic beads are well known in the art. An illustration is given in the Examples.

The cardiac troponin of interest is contacted with the capture binding partner, eg, the capture antibody immobilized on a solid support. Some sample preparation can be used; for example, preparation of serum from blood samples or concentration procedures before the sample is contacted with the capture antibody. Protocols for binding of proteins in immunoassays are well known in the art and are included in the Examples.

The time allowed for bonding will vary depending on conditions; It will be apparent that shorter bonding times are desirable in some settings, especially a clinical setting. The use of, for example, paramagnetic beads can reduce the time required for bonding. In some cases, the time allowed for the binding of the protein of interest to the capture binding partner, eg, antibody, is less than about 12,

10, 8, 6, 4, 3, 2, or 1 hour, or less than about 60, 50, 40, 30, 25, 20, 15, 10, or 5 minutes. In some cases, the time allowed for the binding of the protein of interest to the capture binding partner, eg, antibody, is less than about 60 minutes. In some cases, the time allowed for the binding of the protein of interest to the capture binding partner, eg, antibody, is less than about 40 minutes. In some cases, the time allowed for the binding of the protein of interest to the capture binding partner, eg, antibody, is less than about 30 minutes. In some cases, the time allowed for the binding of the protein of interest to the capture binding partner, eg, antibody, is less than about 20 minutes. In some cases, the time allowed for the binding of the protein of interest to the capture binding partner, eg, antibody, is less than about 15 minutes. In some cases, the time allowed for binding of the protein of interest to the capture binding partner, eg, antibody, is less than about 10 minutes. In some cases, the time allowed for the binding of the protein of interest to the capture binding partner, eg, antibody, is less than about 5 minutes.

In some cases, after binding of the troponin particles to the capture binding partner, eg, capture antibody, particles that may have been nonspecifically bound, as well as other unwanted substances in the sample, are removed. washing, leaving substantially only specifically bound troponin particles. In other cases, no wash is used between sample and label additions; It will be appreciated that this further reduces the sample preparation time. Therefore, in some cases, the time allowed for the binding of the protein of interest to the capture binding partner, eg, antibody, and the binding of the tag to the protein of interest, is less than about 12, 10, 8, 6, 4, 3, 2, or 1 hour, or less than about 60, 50, 40, 30, 25, 20, 15, 10, or 5 minutes. In some cases, the time allowed for the binding of the protein of interest to the capture binding partner, eg, antibodies, and the binding of the tag to the protein of interest, is less than 60 minutes. In some cases, the time allowed for the binding of the protein of interest to the capture binding partner, eg, antibody, and the binding of the tag to the protein of interest, is less than 40 minutes.

In some cases, the time allowed for the binding of the protein of interest to the capture binding partner, eg, antibody, and the binding of the tag to the protein of interest, is less than about 30 minutes. In some cases, the time allowed for the binding of the protein of interest to the capture binding partner, eg, antibody, and the binding of the tag to the protein of interest, is less than about 20 minutes. In some cases, the time allowed for the binding of the protein of interest to the capture binding partner, eg, antibody, and the binding of the tag to the protein of interest, is less than 15 minutes. In some cases, the time allowed for the binding of the protein of interest to the capture binding partner, eg, antibody, and the binding of the tag to the protein of interest, is less than about 10 minutes. In some cases, the time allowed for the binding of the protein of interest to the capture binding partner, eg, antibody, and the binding of the tag to the protein of interest, is less than about 5 minutes.

IS 2 550 004 T3

Some immunoassay diagnostic reagents including capture and signal antibodies used to measure target analytes can be derived from animal sera. Endogenous human heterophile antibodies, or animal anti-human antibodies, which have the ability to bind immunoglobulins from other species, are present in the serum or plasma of more than 10% of patients. These circulating heterophile antibodies 5 can interfere with immunoassay measurements. In sandwich immunoassays, these heterophile antibodies can bridge the capture and detection (diagnostic) antibodies, thereby producing a false positive signal, or they can block the binding of diagnostic antibodies, thereby producing a false negative signal. In competitive immunoassays, heterophile antibodies can bind to test antibody and inhibit its binding to troponin. Separation of the troponin-antibody complex from free troponin can also be either blocked or increased, especially when antispecies antibodies are used in separation systems. Therefore, the impact of these heterophilic antibody interferences is difficult to predict. Therefore, it would be advantageous to block the binding of any heterophile antibody. In some cases, the immunoassay includes the step of dropping the sample of heterophile antibodies using one or more heterophile antibody blockers. The methods for the removal of heterophile antibodies from samples to be tested in immunoassays are 15 known and include: heating the sample in a buffer solution of sodium acetate, pH 5.0, for 15 minutes at 90 ° C and centrifugation at 1200g for 10 minutes, or heterophile antibodies can be precipitated using polyethylene glycol (PEG); immunoextraction of the interference of heterophilic immunoglobulins from the sample using protein A or protein G; or the addition of non-immune mouse IgG. The cases of the methods described in this document contemplate the preparation of the sample before analysis with the single molecule detector. The suitability of the pretreatment method can be determined. Biochemicals to minimize immunoassay interference caused by heterophile antibodies are commercially available. For example, a product called MAK33, which is an IgG1 monoclonal antibody to h-CK-MM, can be obtained from Boehringer Mannheim. The MAK33 plus product contains a combination of IgG1 and IgG1-Fab. PolyMAK33 contains IgG1-polymerized IgG1-Fab and Polymac 2b / 2a contains IgG2b-polymerized IgG2a-Fab. A second commercial source of biochemicals for neutralizing heterophile antibodies is an immunoglobulin inhibitor reagent available from Bioreclamation Inc., East Meadow, NY. This product is a preparation of immunoglobulins (IgG and IgM) from multiple species, mainly murine IgG2a, IgG2b, and IgG3 from Balb / c mice. In some cases the heterophile antibody can be immuno extracted from the sample using methods known in the art, for example dropping the sample of the heterophile antibody by binding the antibody that interferes with protein A or G. In some cases, the heterophile antibody it is neutralized using one or more heterophilic antibody blockers. Heterophilic blockers can be selected from the group consisting of heterophile anti-isotype antibody blockers, heterophile anti-idiotype antibody blockers, and heterophile anti-anti-idiotype antibody blockers. In some cases, a combination of heterophile antibody blockers can be used.

The label is added either with or after the addition of the sample and the wash. Protocols for the binding of antibody and other immunolabels to proteins and other molecules are well known in the art. If the step of tag binding is independent of capture binding, the time allowed for tag binding may be important, for example, in the clinical setting. In some cases, the time allowed for the binding of the protein of interest to the tag, for example, antibody-dye, is less than about 12, 10, 8, 6, 4, 3, 2, or 1 hour, or less. of approximately 60, 50, 40, 30, 25, 20, 15, 10, or 5 minutes. In some cases, the time allowed for binding of the protein of interest to the tag, eg, antibody-dye, is less than about 60 minutes. In some cases, the time allowed for the binding of the protein of interest to the tag, eg, antibody-dye, is less than about 40 minutes.

In some cases, the time allowed for the binding of the protein of interest to the tag, eg, dye antibody, is less than about 30 minutes. In some cases, the time allowed for the binding of the protein of interest to the tag, eg, antibody-dye, is less than about 20 minutes. In some cases, the time allowed for the binding of the protein of interest to the tag, eg, dye antibody, is less than about 15 minutes. In some cases, the time allowed for the binding of the protein of interest to the tag, eg, antibody-dye, is less than about 10 minutes. In some cases, the time allowed for the binding of the protein of interest to the tag, eg, dye antibody, is less than about 5 minutes. Excess label is removed by washing.

The tag is then eluted from the protein of interest. Preferred elution buffers are effective in releasing the tag without generating a significant background. It is also useful if the elution buffer is bacteriostatic. Elution buffers for use include a chaotropic agent, eg, urea or a guanidinium compound; a buffer solution, eg, borate-buffered saline; a carrier of protein, eg, an albumin, such as human, bovine, or fish albumin, or an IgG, to coat the capillary tube wall in the detection instrument; and a surfactant, eg, an ionic or non-ionic detergent, selected to produce a relatively low background, eg, Tween 20, Triton X-100, or SDS.

The buffer / label elution aliquot that is sampled from the single molecule detector is referred to as the running sample, to distinguish it from the original sample that was obtained from an individual.

IS 2 550 004 T3

In another example, the solid phase binding assay may employ a competitive binding assay format. One such method comprises a) competitive binding with a capture antibody immobilized on a binding surface i) a troponin particle in a sample and ii) a labeled analog of the troponin particle comprising a detectable label (the detection reagent ) and b) measure the amount of the label using a single particle analyzer. Another such method comprises a) competitive binding with an antibody having a detectable label (the detection reagent) i) a troponin particle in a sample and ii) an analog of the troponin particle that is immobilized on a binding surface (the capture reagent) and b) measure the amount of the label using a single particle analyzer. A "troponin analog" refers herein to a species that competes with troponin for binding with a capture antibody. Examples of competitive immunoassays are described in US Patent No. 4,235,601 to Deutsch et al., US Patent No.

US Patent No. 4,442,204 to Liotta, and US Patent No. 5,208,53 to Buechler et al.

C. Detection of troponin and determination of concentration

After elution, the tag is passed through a single molecule detector in, for example, the elution buffer. A sample in processing may contain no label, a single label, or a plurality of labels. The number of tags corresponds to or is proportional (if sample dilutions or structural units are used) to the number of cardiac troponin molecules captured during the capture step.

Any appropriate single molecule detector capable of detecting the tag used with the protein of interest can be used. Appropriate single molecule detectors are described herein. Typically, the detector will be part of a system that includes an autosampler for sampling the prepared samples, and optionally a retrieval system to retrieve the samples.

In some cases, the sample being processed is analyzed in a single molecule analyzer that uses a capillary flow system, and that includes a capillary flow cell, a laser to illuminate an interrogation space in the capillary through which it is made passing the processing sample, a detector for detecting radiation emitted from the interrogation space, and a source of motive force for moving a processing sample through the interrogation space. In some cases, the single molecule analyzer further comprises a microscope objective lens that collects the light emitted from the sample being processed as it passes through the interrogation space, for example, a high numerical aperture of the microscope objective. In some cases, the laser and detector are in a confocal configuration. In some cases, the laser is a continuous wave laser. In some cases, the detector is an avalanche photodiode detector. In some cases, the source of motive power is a pump to provide the pressure. Some cases provide an analysis system that includes a sampling system capable of automatically sampling a plurality of samples by providing liquid communication between a sample container and the interrogation space. In some cases, the interrogation space has a volume between approximately 0.001 and 500 pL, or between approximately 0.01 pL and 100 pL, or between approximately 0.01 pL and 10 pL, or between approximately 0.01 pL and 5 pL, or between approximately 0.01 pL. and 0.5 pL, 35 or between approximately 0.02 pL and approximately 300 pL, or between approximately 0.02 pL and approximately 50 pL or between approximately 0.02 pL and approximately 5 pL or between approximately 0.02 pL and approximately 0.5 pL or between approximately 0.02 pL and approximately 2 pL, or between approximately 0.05 pL and approximately 50 pL, or between approximately 0.05 pL and approximately 5 pL, or between approximately 0.05 pL and approximately 0.5 pL, or between about 0.05 pL and about 0.2 pL, or between about 0.1 40 pL and about 25 pL. In some cases, the interrogation space has a volume between approximately

0.004 pL and 100 pL. In some cases, the interrogation space has a volume between approximately 0.02 pL and 50 pL. In some cases, the interrogation space has a volume between approximately 0.001 pL and 10 pL. In some cases, the interrogation space has a volume between approximately 0.001 pL and 10 pL. In some cases, the interrogation space has a volume between approximately 0.01 pL and 5 pL. In some cases, the interrogation space has a volume between about 0.02 pL and about 5 pL. In some cases, the interrogation space has a volume between approximately 0.05 pL and 5 pL. In some cases, the interrogation space has a volume between approximately 0.05 pL and 10 pL. In some cases, the interrogation space has a volume between about 0.5 pL and about 5 pL. In some cases, the interrogation space has a volume between about 0.02 pL and about 0.5 pL.

In some cases, the single molecule detector used in the methods of the invention uses a capillary flow system, and includes a capillary flow cell, a continuous wave laser to illuminate an interrogation space in the capillary through which a passes the processing sample, a high-aperture numerical microscope objective lens that collects the light emitted from the processing sample as it passes through the interrogation space, an avalanche photodiode detector to detect radiation emitted from the interrogation space, and a pump to provide the pressure to move a processing sample through the interrogation space, where the interrogation space is between about 0.02 pL and about 50 pL. In some cases, the single molecule detector used in the methods of the invention uses a capillary flow system, and includes a capillary flow cell, a continuous wave laser to illuminate an interrogation space in the capillary through which the processing sample is passed through a high-precision microscope objective lens

ES 2 550 004 T3 numerical aperture that collects the light emitted from the sample being processed as it passes through the interrogation space where the lens has a numerical aperture of at least about 0.8, an avalanche photodiode detector to detect the radiation emitted from the interrogation space, and a pump to provide the pressure to move a sample being processed through the interrogation space, where the 5 interrogation space is between about 0.004 pL and about 100 pL. In some cases, the single molecule detector used in the methods of the invention uses a capillary flow system, and includes a capillary flow cell, a continuous wave laser to illuminate an interrogation space in the capillary through which the sample is passed in processing, a high numerical aperture microscope objective lens that collects the light emitted from the sample being processed as it passes through the interrogation space where the lens 10 has a numerical aperture of at least about 0.8, a photodiode detector at avalanche to detect radiation emitted from the interrogation space, and a pump to provide the pressure to move a processing sample through the interrogation space, where the interrogation space is between about 0.05 pL and about 10 pL. In some cases, the single molecule detector used in the methods of the invention uses a capillary flow system, and includes a capillary flow cell, a continuous wave laser to illuminate an interrogation space in the capillary through the which is the sample being processed, a high numerical aperture microscope objective lens that collects the light emitted from the sample being processed as it passes through the interrogation space where the lens has a numerical aperture of at least about 0.8, an avalanche photodiode detector for detecting radiation emitted from the interrogation space, and a pump for providing pressure to move a sample being processed through the interrogation space 20, where the interrogation space is between about 0.05 pL and about 5 pL. In some cases, the single molecule detector used in the methods of the invention uses a capillary flow system, and includes a capillary flow cell, a continuous wave laser to illuminate an interrogation space in the capillary through which the sample is passed in processing, a high numerical aperture microscope objective lens that collects the light emitted from the sample being processed as it passes through the interrogation space where the lens has a numerical aperture of at least about 0.8, a photodiode detector at avalanche to detect radiation emitted from the interrogation space, and a pump to provide the pressure to move a processing sample through the interrogation space, where the interrogation space is between about 0.5 pL and about 5 pL.

In some cases, the single molecule detector is capable of determining a concentration of a molecule of interest in a sample where the sample can vary in concentration over a range of at least approximately

100 times, or 1000 times, or 10,000 times, or 100,000 times, or 300.00 times, or 1,000,000 times, or 10,000,000 times, or 30,000,000 times.

In some cases, the methods of the invention utilize a single molecule detector capable of detecting a difference of less than about 50%, 40%, 30%, 20%, 15%, or 10% in the concentration of an analyte between 35 a first sample and a second sample entering the detector, when the volume of the first sample and said second sample entering the analyzer is less than about 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 10, 5, 4, 3, 2, or 1 ul, and wherein the analyte is present in a concentration of less than about 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 10, 5, 4, 3, 2, or 1 femtomolar. In some cases, the methods of the invention use a single molecule detector capable of detecting a difference of less than about 50% in the concentration of an analyte between a first sample and a second sample that are introduced into the detector, when the volume of the first sample and said second sample fed into the analyzer is less than about 100 ul, and wherein the analyte is present in a concentration of less than about 100 femtomolar. In some cases, the methods of the invention use a single molecule detector capable of detecting a difference of less than about 40% in the concentration of an analyte between a first sample and a second sample that is introduced into the detector, when the volume of the first sample and said second sample introduced into the analyzer is less than about 50 ul, and wherein the analyte is present in a concentration of less than about 50 femtomolar. In some cases, the methods of the invention use a single molecule detector capable of detecting a difference of less than about 20% in the concentration of an analyte between a first sample and a second sample that are introduced into the detector, when 50 the volume of the first sample and said second sample fed into the analyzer is less than about ul, and wherein the analyte is present in a concentration of less than about 20 femtomolar. In some cases, the methods of the invention use a single molecule detector capable of detecting a difference of less than about 20% in the concentration of an analyte between a first sample and a second sample that is introduced into the detector, when the volume of the first sample and said second sample 55 introduced into the analyzer is less than about 10 ul, and wherein the analyte is present in a concentration of less than about 10 femtomolar. In some cases, the methods of the invention use a single molecule detector capable of detecting a difference of less than about 20% in the concentration of an analyte between a first sample and a second sample that is introduced into the detector, when the volume of the first sample and said second sample introduced into the analyzer is less than about 60 µl, and wherein the analyte is present in a concentration of less than about 5 femtomolar.

IS 2 550 004 T3

The single molecule detector and systems are described in more detail below. Other instances of single molecule analyzers useful in the methods of the invention, such as detectors with more than one interrogation window, detectors utilize electrokinetic or electrophoretic flux, and the like, can be found in US Patent Application No. United States No. 11 / 048,660, published as US2006 / 0078998 A1.

The instrument can be washed between tests. A wash buffer can be used that maintains the salt and surfactant concentrations of the sample, in some cases to maintain capillary conditioning; ie, to keep the capillary surface relatively constant between samples to reduce variability.

A feature that contributes to the extremely high sensitivity of the instruments and methods of the invention is the method of detecting and counting tags, which, in some cases, are attached to single molecules to be detected or, more generally, correspond to a single molecule that is detected. In summary, the processing sample flowing through the capillary is effectively divided into a series of detection events, subjecting a given interrogation space of the capillary to EM radiation from a laser light emitter at a length of excitation wave appropriate to the fluorescent structural unit used in the label for a predetermined period of time, and the detection of photons emitted during that time. Each predetermined period of time is an interval. If the total number of photons detected in a given interval exceeds a predetermined threshold level, a detection event is recorded for that interval, ie, a tag has been detected. If the total number of photons is not at the predetermined threshold level, no detection event is recorded. In some cases, the concentration of the sample being processed is sufficiently dilute that, for a large percentage of detection events, the detection event represents only a tag that passes through the window, corresponding to a single molecule of interest in the original sample, that is, few detection events represent more than one label in a single interval. In some cases, other refinements are applied to allow higher concentrations of label in the sample being processed to be detected accurately, ie, concentrations in which the probability of two or more labels being detected as a single detection event 25 already it is not insignificant.

Although other times in the interval can be used, in some cases they are selected in the range of approximately 1 microsecond to approximately 5 ms. In some cases, the interval time is more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 250, 300, 400, 500, 600, 700, 750, 800, 900, 1000, 2000, 3000, 4000, or 5000 microseconds .. In some cases, the interval time is less than 30 about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 , 200, 250, 300, 400, 500, 600, 700, 750,

800, 900, 1000, 2000, 3000, 4000, or 5000 microseconds. In some cases, the interval time is approximately 1 to 1000 microseconds. In some cases, the interval time is approximately 1 to 750 microseconds. In some cases, the interval time is approximately 1 to 500 microseconds. In some cases, the interval time is approximately 1 to 250 microseconds. In some cases, the interval time is approximately 1 to 100 microseconds. In some cases, the interval time is approximately 1 to 50 microseconds. In some cases, the interval time is approximately 1 to 40 microseconds. In some cases, the interval time is approximately 1 to 30 microseconds. In some cases, the interval time is approximately 1 to 500 microseconds. In some cases, the interval time is approximately 1 to 20 microseconds. In some cases, the interval time is approximately 1 to 10 microseconds. In some cases, the interval time is approximately 1 to 500 microseconds. In some cases, the interval time is approximately 1 to 5 microseconds. In some cases, the interval time is approximately 5 to 500 microseconds. In some cases, the interval time is approximately 5 to 250 microseconds. In some cases, the interval time is approximately 5 to 100 microseconds. In some cases, the interval time is approximately 5 to 50 microseconds. In some cases, the interval time is approximately 5 to 20 45 microseconds. In some cases, the interval time is approximately 5 to 10 microseconds. In some cases, the interval time is approximately 10 to 500 microseconds. In some cases, the interval time is approximately 10 to 250 microseconds. In some cases, the interval time is approximately 10 to 100 microseconds. In some cases, the interval time is approximately 10 to 50 microseconds. In some cases, the interval time is approximately 10 to 30 microseconds. In some cases, the interval time is 50 approximately 10 to 20 microseconds. In some cases, the interval time is approximately 5 microseconds. In some cases, the interval time is approximately 5 microseconds. In some cases, the interval time is approximately 6 microseconds. In some cases, the interval time is approximately 7 microseconds. In some cases, the interval time is approximately 8 microseconds. In some cases, the interval time is approximately 9 microseconds. In some cases, the interval time is approximately 10 microseconds. In some cases, the interval time is approximately 11 microseconds. In some cases, the interval time is approximately 12 microseconds. In some cases, the interval time is approximately 13 microseconds. In some cases, the interval time is approximately 14 microseconds. In some cases, the interval time is approximately 5 microseconds.

In some cases, the interval time is approximately 15 microseconds. In some cases, the 60 interval time is approximately 16 microseconds. In some cases, the interval time is approximately 17

IS 2 550 004 T3 microseconds. In some cases, the interval time is approximately 18 microseconds. In some cases, the interval time is approximately 19 microseconds. In some cases, the interval time is approximately 20 microseconds. In some cases, the interval time is approximately 25 microseconds. In some cases, the interval time is approximately 30 microseconds. In some cases, the interval time is approximately 40 microseconds. In some cases, the interval time is approximately 50 microseconds. In some cases, the interval time is approximately 100 microseconds. In some cases, the interval time is approximately 250 microseconds. In some cases, the interval time is approximately 500 microseconds. In some cases, the interval time is approximately 750 microseconds. In some cases, the interval time is approximately 1000 microseconds.

In some cases, the background noise level is determined from the mean noise level, or the root mean square of the noise. In other cases, a typical noise value or a statistical value is chosen. In most cases, the noise is expected to follow a Poisson distribution. Therefore, in some cases, determining the concentration of a tag particle complex in a sample comprises determining the level of background noise.

Therefore, as a label flows through the capillary flow cell, it is irradiated by the laser beam to generate a burst of photons. The photons emitted by the tag are discriminated from the background light or background noise emission, considering that only the bursts of photons have energy above a predetermined threshold energy level that represents the amount of background noise that is present in the sample.Background noise generally comprises low frequency emission produced, for example, by the intrinsic fluorescence of unlabeled particles that are present in the sample, the buffer or diluent used in preparing the sample for analysis, Raman scattering and electronic noise. In some cases, the value assigned to the background noise is calculated as the average of the detected background noise signal in a plurality of intervals, which are measurements of the photon signals that are detected in an interrogation space during a period of predetermined time. Thus, in some cases, the background noise is calculated for each sample as a specific number for that sample.

Taking into account the value of the background noise, the threshold energy level can be assigned. As discussed above, the threshold value is determined to discriminate true signals (due to fluorescence from a tag) from background noise. Care must be taken in choosing a threshold value such that the number of false positive random noise signals is minimized, while the number of true signals that are rejected is also minimized. Methods for choosing a threshold value include determining a fixed value above the noise level and calculating a threshold value based on the distribution of the noise signal. In one case, the threshold is set at a fixed number of standard deviations above the background level. Assuming a Poisson distribution of the noise, using this method the number of false positive signals can be estimated during the time course of the experiment. In some cases, the threshold level is calculated as 4 sigma above the background noise. For example, given an average background noise level of 200 photons, the analysis system sets a threshold level of 4 ^ 200 above the average background / noise level of 200 photons, which is 256 photons. Therefore, in some cases, determining the concentration of a tag in a sample includes setting the threshold level above which photon signals represent the presence of a tag. In contrast, photon signals that have an energy level that is not greater than the threshold level indicate the absence of a label.

Many interval measurements are taken to determine the concentration of a sample, and the absence or presence of a label is confirmed for each interval measurement. Typically 60,000 or more measurements can be made in one minute (for example, in cases where the interval size is 1 ms for smaller interval sizes the number of measurements is proportionally greater, for example 6,000,000 measurements per minute for an interval size of 10 microseconds). Therefore, no single measurement is crucial and the method provides a high margin of error. Ranges that are determined to not contain a label (no ranges) are discounted 45 and only measurements made in the ranges that are determined to contain label (yes ranges) are counted to determine the concentration of the label in the sample at prosecution. Discounting measurements made in the non-intervals or intervals that are devoid of label, increases the signal-to-noise ratio and the accuracy of the measurements. Therefore, in some cases, determining the concentration of a label in a sample comprises detecting the interval measurements that reflect the presence of a label.

The signal-to-noise ratio or sensitivity of the analysis system can be increased by minimizing the time that background noise is detected, during a measurement of the range in which a particle-label complex is detected. For example, in a measurement of the interval lasting 1 millisecond, during which a tag particle complex is detected as it passes through an interrogation gap in 250 microseconds, 750 microseconds of the 1 millisecond are spent to detect the emission of background noise . The signal-to-noise ratio can be improved by decreasing the interval time. In some cases, the interval time is 1 millisecond. In other cases, the interval time is 750, 500, 250 microseconds, 100 microseconds, 50 microseconds, 25 microseconds, or 10 microseconds. Other times in the interval are as described in this document.

IS 2 550 004 T3

Other factors that affect the measurements are the brightness or penumbra of the fluorescent structural unit, the flow rate, and the laser power. Various combinations of the relevant factors that allow detection of the tag will be apparent to those skilled in the art. In some cases, the interval time is adjusted without changing the flow rate. It will be appreciated by those skilled in the art that as the interval time decreases, the laser power output directed to the interrogation space must increase to maintain a constant total energy applied to the interrogation space during the interval time. For example, if the interval time is decreased from 1000 microseconds to 250 microseconds, as a first approximation, the laser power output should be increased approximately four times. These settings allow for the detection of the same number of photons in about 250 ms as the number of photons counted during the 1000 ms, given the previous settings, and allow a faster analysis of the sample with less funds and therefore a greater sensitivity. Additionally, flow rates can be adjusted in order to speed up sample processing. These numbers are merely exemplary, and the skilled professional can adjust the parameters as necessary to achieve the desired result.

In some cases, the interrogation space encompasses the entire cross section of the sample stream.

When the interrogation space encompasses the entire cross section of the sample stream, only 15 the counted number of labels and the volume that passes through a cross section of the sample stream in a fixed length of time are needed to calculate the concentration of the label in the sample being processed. In some cases, the interrogation space can be defined to be less than the current cross-sectional area of the sample, for example, by the interrogation space which is defined by the size of the spot illuminated by the laser beam. In some cases, the interrogation space can be defined by adjusting the 20 apertures 306 (Figure 1A) or 358 and 359 (Figure 1B) of the analyzer and reducing the illuminated volume that is reflected by the objective lens to the detector. In cases where the interrogation space is defined to be smaller than the cross-sectional area of the sample stream, the concentration of the tag can be determined by interpolation of the signal emitted by the complex from a standard curve that is generated using one or more samples of known standard concentrations. In still other cases, the tag concentration can be determined by comparing the measured particles to an internal tag standard. In cases where a diluted sample is analyzed, the dilution factor is taken into account in calculating the concentration of the molecule of interest in the starting sample.

As discussed above, when the interrogation space encompasses the entire cross section of the sample stream, only the number of counted labels that passes through a cross section of the sample stream in a fixed length of time (interval) and the volume of the sample that was interrogated in the interval are needed to calculate the sample concentration. The total number of labels contained in the yes intervals is determined and related to the sample volume represented by the total number of intervals used in the analysis to determine the concentration of labels in the sample being processed. Therefore, in one case, determining the concentration of a label in a sample being processed comprises determining the total number of labels detected in each case and relating the total number of labels detected to the total volume of the sample that was analyzed. The total volume of the sample being analyzed is the volume of the sample that is passed through the capillary flow cell and through the interrogation space in a specified time interval. Alternatively, the concentration of the label complex in a sample is determined by interpolation of the signal emitted by the label in a number of intervals from a standard curve that is generated by determining the signal emitted by the labels in the same number of intervals. of standard samples containing known concentrations from the label.

In some cases, the number of individual labels that is detected in a range is related to the relative concentration of the particle in the sample being processed. At relatively low concentrations, for example at concentrations below about 10-16 M the number of labels is proportional to the photon signal that is detected in an interval. Therefore, at low concentrations of tag the photon signal is provided as a digital signal. At relatively high concentrations, for example at concentrations greater than about 10 -16 M, the proportionality of the photon signal with a tag is lost as the probability of two or more tags crossing the interrogation space at about the same time and that it is counted as one becomes meaningful. Therefore, in some cases, individual particles in a sample of a concentration greater than about 10-16 M are resolved by decreasing the time length of the interval measurement.

Alternatively, in other cases, the total of the photon signal that is emitted by a plurality of particles that are present in any range is detected. These examples allow single molecule detectors of the invention where the dynamic range is at least 3, 3.5, 4, 4.5, 5.5, 6, 6.5, 7, 7.5, 8, or more than 8 logs.

Dynamic range, as that term is used in this document, refers to the range of sample concentrations that can be quantified by the instrument without the need for dilution or other treatment to alter the concentration of successive samples of different concentrations, where concentrations are determined with an appropriate precision for the intended use. For example, if a microtiter plate contains a sample from a

ES 2 550 004 T3 femtomolar concentration 1 for an analyte of interest in one well, a sample of 10,000 femtomolar concentration for an analyte of interest in another well, and a sample of 100 femtomolar concentration for the analyte in a third well, thus, An instrument with a dynamic range of at least 4 logs and a lower limit of quantitation of 1 femtomolar is capable of accurately quantifying the concentration of all samples without the need for additional treatment to adjust the concentration, eg dilution. Precision can be determined by standard methods, for example, using a series of concentration standards spanning the entire dynamic range and the construction of a standard curve. The standard measures of fit of the resulting standard curve can be used as a measure of precision, for example, an r<sup>2</sup> greater than approximately 0.7, 0.75, 0.8, 0.85, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, or 0.99.

The increase in dynamic range is achieved by altering the way the data from the detector is analyzed, and / or by using an attenuator between the detector and the interrogation space. At the lower end of the range, where the sample being processed is sufficiently dilute such that each detection event, ie, each burst of photons above a threshold level in an interval (the event photons), probably represents only one label, the data is analyzed to count detection events as single molecules. That is, each interval is analyzed as a simple yes or no for the presence of the tag, as described above. For a more concentrated processing sample, where the probability that two or more labels occupying a single interval becomes significant, the number of photons of events in a significant number of intervals is found to be substantially greater than the expected number for a single label, for example, the number of event photons in a significant number of intervals corresponds to two times, three times, or more, than the number of photons 20 of events expected for a single label. For these samples, the instrument changes its data analysis method to one that integrates the total number of event photons for the sample intervals being processed. This total will be proportional to the total number of labels that were in all the intervals. For an even more concentrated processing sample, where many labels are present in most intervals, the background noise becomes a negligible portion of the total signal for each interval, and the instrument changes its data analysis method to one that counts the total photons per interval (including the background). A further increase in dynamic range can be achieved by using an attenuator between the flow cell and the detector, when concentrations are such that the intensity of light reaching the detector otherwise exceeds the detector's ability to count. photons accurately, ie, saturate the detector.

The instrument may include a data analysis system that receives input from the detector and determines the appropriate analysis method for the sample being run, and the output values based on that analysis. The data analysis system can prompt output instructions to use or not to use an attenuator, if an attenuator is included in the instrument.

Using such methods, the dynamic range of the instrument can be clearly increased. Therefore, in some cases, the instrument is capable of measuring sample concentrations over a dynamic range of more than 35 approximately 1000 (3 log), 10,000 (4 log), 100,000 (5 log), 350,000 (5.5 log) , 1,000,000 (6 log), 3,500,000 (6.5 log),

10,000,000 (7 log), 35,000,000 (7.5 log), or 100,000,000 (8 log). In some cases, the instrument is capable of measuring sample concentrations over a dynamic range of more than approximately 100,000 (5 log). In some cases, the instrument is capable of measuring sample concentrations over a dynamic range of more than approximately 1,000,000 (6 log). In some cases, the instrument is capable of measuring concentrations of the 40 samples over a dynamic range of more than approximately 10,000,000 (7 log). In some cases, the instrument is capable of measuring sample concentrations in a dynamic range from about 1-10 femtomolar to at least about 1000; 10,000; 100,000; 350,000; 1,000,000; 3,500,000; 10,000,000, or 35,000,000 femtomolar .. In some cases, the instrument is capable of measuring sample concentrations in a dynamic range from about 1-10 femtomolar to at least about 10,000 femtomolar. In some 45 cases, the instrument is capable of measuring sample concentrations in a dynamic range from about 1-10 femtomolar to at least about 100,000 femtomolar. In some cases, the instrument is capable of measuring sample concentrations in a dynamic range from approximately 110 femtomolar to at least approximately 1,000,000 femtomolar. In some cases, the instrument is capable of measuring sample concentrations in a dynamic range from about 1-10 femtomolar to at least about 10,000,000.

In some cases, an analyzer or analyzer system described herein is capable of detecting an analyte, for example, a biomarker at a detection limit of less than 1 nanomolar or 1 picomolar, or 1 femtomolar, or 1 attomolar, or 1 zeptomolar. In some cases, the analysis system or analyzer is capable of detecting a change in the concentration of the analyte, or of multiple analytes, for example a biomarker or biomarkers, from sample to sample of less than about 0.1, 1, 2, 5, 10, 20, 30, 40, 50, 60, or 80% when the biomarker is present at a concentration of less than 1 nanomolar or 1 picomolar, or 1 femtomolar, or 1 attomolar, or 1 zeptomolar, in the samples, and when the size of each of the samples is less than approximately 100, 50, 40, 30, 20, 10, 5, 2, 1, 0.1, 0.01, 0.001, or 0.0001 ul. In some cases, the analyzer or analyzer system is capable of detecting a change in analyte concentration from a first sample to a second sample of less than approximately

IS 2 550 004 T3

20%, when the analyte is present in a concentration of less than approximately 1 picomolar, and when the size of each of the samples is less than approximately 50 mL. In some cases, the analysis system or analyzer is capable of detecting a change in analyte concentration from a first sample to a second sample of less than about 20%, when the analyte is present in a concentration of less than 5%. approximately 100 femtomolar, and when the size of each of the samples is less than approximately mL. In some cases, the analysis system or analyzer is capable of detecting a change in analyte concentration from a first sample to a second sample of less than about 20%, when the analyte is present in a concentration of less than about 50 femtomolar, and when the size of each of the samples is less than approximately 50, uL. In some cases, the analysis system or analyzer is capable of detecting a change in analyte concentration from a first sample to a second sample of less than about 20%, when the analyte is present in a concentration of less than about 5 femtomolar, and when the size of each of the samples is less than about 50, uL. In some cases, the analysis system or analyzer is capable of detecting a change in analyte concentration from a first sample to a second sample of less than about 20%, when the analyte is present in a concentration of less than approximately 5 femtomolar, and when the size of each of the samples is less than approximately 5, uL. In some cases, the analysis system or analyzer is capable of detecting a change in analyte concentration from a first sample to a second sample of less than about 20%, when the analyte is present in a concentration of less than about 1 femtomolar, and when the size of each of the samples is less than approximately 5 mL.

V. Appropriate instruments and systems for high-sensitivity troponin analysis

The methods of the invention use highly sensitive analytical instruments, eg, single molecule detectors. Such single molecule detectors include examples as described below.

A. Device / System

In one aspect, the methods described herein utilize an assay system capable of detecting a single particle in a sample. In one case, the analysis system is capable of detecting a single particle of a fluorescently labeled particle wherein the analysis system detects the energy emitted by an excited fluorescent tag in response to exposure by a source of electromagnetic radiation when the particle Unique is present in a defined interrogation space within a capillary flow cell fluidly connected to the sampling system of the analysis system. In a further case of the analysis system, the single particle is moved through the interrogation space of the capillary flow cell by means of a motive force. In another case of the analysis system, an automatic sampling system can be included in the analysis system for the introduction of the sample into the analysis system. In another case of the analysis system, a sample preparation system can be included in the analysis system for the preparation of a sample. In a further case, the analysis system may contain a sample recovery system for the recovery of at least a portion of the sample after the analysis is complete.

In one case, the analysis system consists of a source of electromagnetic radiation to excite a single particle marked with a fluorescent tag. In one case, the electromagnetic radiation source of the analysis system is a laser. In a further case, the source of electromagnetic radiation is a continuous wave laser.

In a typical case, the source of electromagnetic radiation excites a fluorescent frame unit attached to a tag as the tag passes through the interrogation space of the capillary flow cell. In some cases, the fluorescent tag moiety includes one or more fluorescent dye molecules. In some cases, the fluorescent tag structural unit is a quantum dot. Any fluorescent building block as described in this document can be used on the label.

A tag is exposed to electromagnetic radiation when the tag passes through an interrogation space 45 located within the capillary flow cell. The interrogation space is generally fluidly connected to a sampling system. In some cases, the label passes through the interrogation space of the capillary flow cell due to a driving force to advance the label through the analysis system. The interrogation space is positioned in such a way that it receives the electromagnetic radiation emitted from the radiation source. In some cases, the sampling system is an automated sampling system capable of sampling a plurality of samples without the intervention of a human operator.

The tag passes through the interrogation space and emits a detectable amount of energy when excited by the source of electromagnetic radiation. In one embodiment, an electromagnetic radiation detector is operatively connected to the interrogation space. The electromagnetic radiation detector is capable of detecting the energy emitted by the tag, for example by the fluorescent structural unit of the tag.

IS 2 550 004 T3

In a further case of the analysis system, the system further includes a sample preparation mechanism, where a sample can be partially or completely prepared for analysis by the analysis system. In some cases of the analysis system, the sample is discarded after it is analyzed by the system. In other cases, the analysis system further includes a sample recovery mechanism whereby at least a part, or alternatively all or substantially all, of the sample can be recovered after analysis. In such a case, the sample can be returned to the sample source. In some cases, the sample can be returned to microtiter wells in a sample microtiter plate. In general, the analysis system also consists of a data acquisition system for the collection and notification of the detected signal.

B. Single Particle Analyzer

As shown in FIG. 1A, an example of an analysis system 300 is described herein. The analysis system 300 includes an electromagnetic radiation source 301, a mirror 302, a lens 303, a capillary flow cell 313, a microscopic objective lens 305, an aperture 306, a detector lens 307, a detector filter 308 , a single photon detector 309, and a processor 310 operatively connected to the detector.

In operation, the source 301 of electromagnetic radiation is aligned so that its output 311 is reflected off a front surface 312 of the mirror 302. The lens 303 focuses the beam 311 in a single interrogation space (an illustrative example of a space 314 question mark is shown in FIG. 2A) in capillary flow cell 313. The microscope objective lens 305 collects light from sample particles and images the beam over aperture 306. The aperture 306 affects the structural unit of light emitted by the sample in the interrogation space of the capillary flow cell 313 that can be collected. The detector lens 307 collects the light that passes through the aperture 306 and focuses the light onto an active area of the detector 309 after it passes through the detector filters 308. The detector filters 308 minimize aberrant noise signals due to scattering of light or ambient light while maximizing the signal emitted by the particle-bound excited fluorescent building block. Processor 310 processes the light signal from the particle according to the methods described in this document.

In one case, the microscope objective lens 305 has a high numerical aperture of the microscope objective. As used herein, a high numerical aperture lens includes a lens with a numerical aperture of equal to or greater than 0.6. Numerical aperture is a measure of the number of highly diffracted imaging light rays captured by the lens. A higher numerical aperture allows more and more oblique rays to enter the objective lens and therefore produce a more highly resolved image. Also, the brightness of an image increases with a higher numerical aperture. In the analysis system, you can use high numerical aperture lenses that are commercially available from a variety of vendors, and any lens that has a numerical aperture equal to or greater than about 0.6. In some cases, the lens has a numerical aperture of

<td>approximately</td><td> 0.6</td><td>to</td><td>approximately</td><td> 1.3.</td><td>On</td><td>Some</td><td>cases,</td><td>the</td><td>lens</td><td>have</td><td>a</td><td>opening</td><td>numerical</td><td>from</td>

<td>approximately</td><td> 0.6</td><td>to</td><td>approximately</td><td> 1.0.</td><td>On</td><td>Some</td><td>cases,</td><td>the</td><td>lens</td><td>have</td><td>a</td><td>opening</td><td>numerical</td><td>from</td>

<td>approximately</td><td> 0.7</td><td>to</td><td>approximately</td><td> 1.2.</td><td>On</td><td>Some</td><td>cases,</td><td>the</td><td>lens</td><td>have</td><td>a</td><td>opening</td><td>numerical</td><td>from</td>

<td>approximately</td><td> 0.7</td><td>to</td><td>approximately</td><td> 1.0.</td><td>On</td><td>Some</td><td>cases,</td><td>the</td><td>lens</td><td>have</td><td>a</td><td>opening</td><td>numerical</td><td>from</td>

<td>approximately</td><td> 0.7</td><td>to</td><td>approximately</td><td> 0.9.</td><td>On</td><td>Some</td><td>cases,</td><td>the</td><td>lens</td><td>have</td><td>a</td><td>opening</td><td>numerical</td><td>from</td>

<td>approximately</td><td> 0.8</td><td>to</td><td>approximately</td><td> 1.3.</td><td>On</td><td>Some</td><td>cases,</td><td>the</td><td>lens</td><td>have</td><td>a</td><td>opening</td><td>numerical</td><td>from</td>

<td>approximately</td><td> 0.8</td><td>to</td><td>approximately</td><td> 1.2.</td><td>On</td><td>Some</td><td>cases,</td><td>the</td><td>lens</td><td>have</td><td>a</td><td>opening</td><td>numerical</td><td>from</td>

<td>approximately</td><td>0.8 to</td><td colspan="3">about 1.0. On</td><td colspan="4">some cases, the lens</td><td>have</td><td colspan="2">An opening</td><td>numerical</td><td colspan="2">of at least</td>

about 0.6. In some cases, the lens has a numerical aperture of at least about 0.7. In some cases, the lens has a numerical aperture of at least about 0.8. In some cases, the lens has a numerical aperture of at least about 0.9. In some cases, the lens has a numerical aperture of at least about 1.0. In some cases, the aperture of the microscope objective lens 305 is approximately 1.25. In one case, where a 305 0.8 microscope objective lens is used, a Nikon 60X / 0.8 NA Achromat lens (Nikon, Inc., USA) can be used.

In some cases, the source 301 of electromagnetic radiation is a laser that emits light in the visible spectrum. In all cases, the source of electromagnetic radiation is adjusted such that the wavelength of the laser is adjusted so that it is of a wavelength sufficient to excite the fluorescent tag attached to the particle. In some cases, the laser is a continuous wave laser with a wavelength of 639 nm. In other cases, the laser is a continuous wave laser with a wavelength of 532 nm. In other cases, the laser is a continuous wave laser with a wavelength of 422 nm. In other cases, the laser is a continuous wave laser with a wavelength of 405 nm. Any continuous wave laser with an appropriate wavelength can be used to excite a fluorescent building block as used in the methods and compositions described herein.

In a single particle analysis system 300, as each particle passes through beam 311 from the source of electromagnetic radiation, the particle enters an excited state. When the particle relaxes from its excited state, a detectable burst of light is emitted. The excitation-emission cycle is repeated many times for each particle in the length of time it takes for it to pass through the beam allowing the analysis system 300

ES 2 550 004 T3 detects tens to thousands of photons for each particle as they pass through an interrogation space 314. Photons emitted by fluorescent particles are recorded by detector 309 (Figure 1A) with a time delay indicative of the time for the particle tag complex to pass through the interrogation space. The photon intensity is recorded by the detector 309 and the sampling time is divided into intervals, which are uniform, arbitrary time segments with freely selectable time channel widths. The number of signals contained in each evaluated interval. One or a combination of several statistical analysis methods are employed in order to determine when a particle is present. Such methods include determining the baseline noise of the analysis system and setting a signal intensity for the fluorescent tag at a statistical level above the baseline noise to eliminate false positive signals from the detector.

The source 301 of electromagnetic radiation is focused on a capillary flow cell 313 of the analysis system 300, where the capillary flow cell 313 is fluidly connected to the sample system. An interrogation space 314 is shown in FIG. 2A. The beam 311 of the continuous length electromagnetic radiation source 301 of FIG. 1A is optically focused to a specified depth within the capillary flow cell 313. Beam 311 is directed toward sample-filled capillary flow cell 313 at an angle perpendicular to capillary flow cell 313. Beam 311 is operated at a predetermined wavelength that is selected to excite a particular fluorescent tag, used to tag the particle of interest. The size or volume of the interrogation space 314 is determined by the diameter of the beam 311 along with the depth to which the beam 311 is centered. Alternatively, the interrogation space can be determined by analyzing a calibration sample of known concentration through the analysis system.

When single molecules are detected at the sample concentration, the beam size and depth of focus required for the detection of single molecules is fixed and thus defines the size of the interrogation space 314. The interrogation space 314 is configured such that, with an appropriate sample concentration, only one particle is present in the interrogation space 314 during each time interval during which time observations are made. It will be appreciated that the detection interrogation volume as defined by the beam is not perfectly spherical in shape, and generally has a bow tie shape. However, for the purpose of definition, interrogation gap volumes are defined herein as the volume encompassed by a sphere of diameter equal to the diameter of the focused spot of the beam. The focused point of the beam 311. In some cases, the diameter of the focused spot of the beam is from about 1 to about 5, 10, 15, or 20 microns, or about 5 to about 10, 15, or 20 microns, or about 10 to about 20 microns, or about 10 to about 15 microns. In some cases, the diameter of the focused spot of the beam is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 microns. In some cases, the diameter of the focused spot of the beam is approximately 5 microns. In some cases, the diameter of the focused spot of the beam is approximately 10 microns. In some cases, the diameter of the focused spot of the beam is approximately 12 microns. In some cases, the diameter of the focused spot of the beam is approximately 13 microns. In some cases, the diameter of the focused spot of the beam is approximately 14 microns. In some cases, the diameter of the focused spot of the beam is approximately 15 microns. In some cases, the diameter of the focused spot of the beam is approximately 16 microns. In some cases, the diameter of the focused spot of the beam is approximately 17 microns. In some cases, the diameter of the focused spot of the beam is approximately 18 microns. In some cases, the diameter of the focused spot of the beam is approximately 19 microns. In some cases, the diameter of the focused spot of the beam is approximately 20 microns.

In an alternative instance of the single particle analysis system, more than one source of electromagnetic radiation can be used to excite the particles labeled with fluorescent markers of different wavelengths. In another alternative case, more than one interrogation space can be used in the capillary flow cell. In another alternative case, multiple detectors can be used to detect emission wavelengths different from fluorescent labels. An illustration incorporating each of these alternative cases of an analysis system is shown in Figure 1B. These examples are shown in earlier United States Patent Application No. 11 / 048,660, published as US2006 / 078998 A1.

In some cases of the analysis system 300, a driving force is required to move a particle through the capillary flow cell 313 of the analysis system 300. In one case, the driving force can be a form of pressure. The pressure used to move a particle through the capillary flow cell can be generated by a pump. In some embodiments, a Scivex, Inc. HPLC pump can be used. In some cases where a pump is used as a driving force, the sample can pass through the capillary flow cell at a rate of 1 mL / min to about 20 mL / min, or about 5 mL / min to about 20 mL. / min. In some cases, the sample can pass through the capillary flow cell at a rate of approximately 5 mL / min. In some cases, the sample can pass through the capillary flow cell at a rate of approximately 10 mL / min. In some cases, the sample can pass through the capillary flow cell at a rate of approximately 15 mL / min. In some cases, the sample can pass through the capillary flow cell at a rate of approximately 20 mL / min. In some cases, an electrokinetic force can be used to move the particle to

ES 2 550 004 T3 through the analysis system. This method has been previously described in the earlier United States Patent Application No. 11 / 048,660, published as US2006 / 078998 A1.

In one case of the analysis system 300, the detector 309 of the analysis system detects the photons emitted by the fluorescent tag. In one case, the photon detector is a photodiode. In a further case, the detector is an avalanche photodiode detector. In some cases, the photodiodes can be silicon photodiodes with a detection wavelength of 190 nm and 1100 nm. When using germanium photodiodes, the wavelength of the detected light is between 400nm to 1700nm. In other cases, when using an indium gallium arsenide photodiode, the wavelength of the light detected by the photodiode is between 800 nm and 2600 nm. When lead sulfide photodiodes are used as detectors, the wavelength of the detected light is between 1000 nm and 3500 nm.

In some cases, the optics of the electromagnetic radiation source 301 and the optics of the detector 309 are arranged in a conventional optical configuration. In such a configuration, the source of electromagnetic radiation and the detector are aligned in different focal planes. The analysis system detector optics and laser configuration as shown in Figures 1A and 1B is that of a conventional optical configuration.

In some cases, the optics of the electromagnetic radiation source and the optics of the detector are arranged in a confocal optical configuration. In such a configuration, the source 301 of electromagnetic radiation and the detector 309 are aligned in the same focal plane. The confocal configuration makes the analyzer more robust because the source 301 of electromagnetic radiation and the optics of the detector 309 would not need to be realigned if the analysis system moves. This configuration also makes the use of the analyzer more simplified, as it eliminates the need to realign the components of the analysis system. The confocal configuration for analyzer 300 (Figure 1A) and analyzer 355 (Figure 1B) are shown in Figures 3A and 3B respectively. Figure 3A shows that beam 311 from an electromagnetic radiation source 301 is focused by microscope objective 315, to form an interrogation space 314 (Figure 2A) within capillary flow cell 313. To separate the fluorescent light from the laser light, a dichroic mirror 316 is used, which reflects the laser light, but allows the fluorescent light to pass through. The filter 317 that is placed in front of the detector removes any non-fluorescent light in the detector. In some cases, an analysis system configured in a confocal configuration may comprise two or more interrogation spaces. Such a method has been previously disclosed in US Patent Application No. 11 / 048,660, published as US2006 / 078998 A1.

The laser can be a tunable dye laser, such as a helium-neon laser. The laser can be configured to emit a wavelength of 632.8 nm. Alternatively, the wavelength of the laser can be configured to emit a wavelength of 543.5 nm or 1523 nm. Alternatively, the electromagnetic laser can be an argon ion laser. In such a case, the argon ion laser can be operated as a continuous gas laser at approximately 25 different wavelengths in the visible spectrum, the wavelength set between 408.9 and 686.1 nm, but at its optimum performance setting between 488 and 514.5 nm.

Source of electromagnetic radiation

In some cases, the analysis system of a chemiluminescent tag can be used. In such a case, it may not be necessary to use an EM source for particle detection. In another case, the extrinsic label or intrinsic feature of the particle is a feature or label that interacts with light, such as a fluorescent label or a light scattering label. In such a case, a source of EM radiation is used to illuminate the label and / or the particle. Sources of EM radiation are preferred for the excitation of fluorescent labels.

In some cases, the analysis system consists of a source 301 of electromagnetic radiation. Any number of radiation sources can be used in any analysis system 300. Multiple sources of electromagnetic radiation have been previously described in earlier United States Patent Application No. 11 / 048,660, published as US2006 / 0078998 A1. In some cases, all sources of continuous electromagnetic wave (EM) radiation emit electromagnetic radiation at the same wavelengths. In other cases, different sources emit different wavelengths of EM radiation.

In one case, the EM source (s) 301, 351, 352 are continuous wave lasers that produce wavelengths between 200 nm and 1000 nm. Such EM sources have the advantage of being small, durable, and relatively inexpensive. In addition, they generally have the ability to generate larger fluorescent signals than other light sources. Specific examples of suitable continuous wave EM sources include, but are not limited to: argon, krypton, helium-neon, helium-cadmium type 50 lasers, as well as, tunable diode lasers (red to infrared regions ), each with the possibility of frequency doubling. Lasers provide continuous illumination with no electronic accessories or mechanical devices, such as shutters, to interrupt their illumination. In a case where a continuous wave laser is used, a 3 mW source of electromagnetic radiation may be of sufficient energy to excite a fluorescent tag. A beam from a continuous wave laser of such an energy output can be between 2 to 5 pm in diameter. The time of exposure of the particle to the laser beam in order to be exposed to

IS 2 550 004 T3

3mW can be a time period of approximately 1 msec. In alternative cases, the exposure time to the laser beam can be equal to or less than about 500 msec. In an alternative case, the exposure time can be equal to or less than about 100 msec. In an alternative case, the exposure time can be equal to or less than about 50 msec. In an alternative case, the exposure time can be equal to or less than about 10 msec.

LEDs are another low-cost, highly reliable source of illumination. Recent advancements in ultra-bright LEDs and high-absorption, quantum-yield cross-sectional dyes support the applicability of LEDs to the detection of a single particle. Such lasers can be used alone or in combination with other light sources such as mercury arc lamps, elemental arc lamps, halogen lamps, arc discharges, plasma discharges, light emitting diodes, or a combination of these.

In other cases, the source of EM could be in the form of a pulse wave laser. In such a case, the size of the laser pulse is an important factor. In such a case, the size, the focus point, and the total energy emitted by the laser is important and must be of sufficient energy to be able to excite the fluorescent tag. When using a pulse laser, a longer pulse duration may be required. In some cases, a 2 nanosecond laser pulse can be used. In some cases a 5 nanosecond laser pulse can be used. In some cases, a pulse between 2 and 5 nanoseconds can be used.

The optimal laser intensity depends on the photobleaching characteristics of the individual dyes and the length of time required to traverse the interrogation space (including particle velocity, the distance between interrogation spaces if more than one is used and the size of the question mark (s)). To obtain maximum signal, it is desirable to illuminate the sample at the highest intensity that will not result in photobleaching of a high percentage of the dyes. The preferred intensity is such that no more than 5% of the colorants are bleached by the time the particle has passed through the interrogation space.

The laser power is adjusted based on the type of dye molecules that need to be stimulated and the length of time that the dye molecules are stimulated, and / or the speed with which the dye molecules pass through the cell. capillary flow. Laser power is defined as the speed at which energy is delivered by the beam and is measured in units of Joules / second, or Watts. It will be appreciated that the higher the output power of the laser, the shorter the time that the laser illuminates the particle can be, while providing a constant amount of energy for the interrogation space, as the particle passes through the space. Therefore, in some cases, the combination of laser illumination time and power is such that the total energy received by the interrogation space during the illumination time is more than about 0.1, 0.5, 1, 2, 3, 4 , 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 microjoules. In some cases, the combination of laser power and illumination time is such that the total energy received by the interrogation space during the illumination time is less than about 0.5, 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, or 110 microjoules. In some cases, the combination of laser power and illumination time is such that the total energy received by the interrogation space during the illumination time is between about 0.1 and 100 microjoules. In some cases, the combination of laser power and illumination time is such that the total energy received by the interrogation space during the illumination time is between about 1 and 100 microjoules. In some cases, the combination of laser power and illumination time is such that the total energy received by the interrogation space during the illumination time is between about 1 and 50 microjoules. In some cases, the combination of laser power and illumination time is such that the total energy received by the interrogation space during the illumination time is between about 2 and 50 microjoules. In some cases, the combination of laser power and illumination time is such that the total energy received by the interrogation space during the illumination time is between about 3 and 60 microjoules. In some cases, the combination of laser power and illumination time is such that the total energy received by the interrogation space during the illumination time is between about 3 and 50 microjoules. In some cases, the combination of laser power and illumination time is such that the total energy received by the interrogation space during the illumination time is between about 3 and 40 microjoules. In some cases, the combination of laser power and illumination time is such that the total energy received by the interrogation space during the illumination time is between about 3 and 30 microjoules. In some cases, the combination of laser power and illumination time is such that the total energy received by the interrogation space during the illumination time is approximately 1 microjoules. In some cases, the combination of laser power and illumination time is such that the total energy received by the interrogation space during the illumination time is approximately 3 microjoules. In some cases, the combination of laser power and illumination time is such that the total energy received by the interrogation space during the illumination time is approximately 5 microjoules. In some cases, the combination of laser power and illumination time is such that the total energy received by the interrogation space during the illumination time is approximately 10 microjoules. In some cases, the combination of laser power and illumination time is such that the total energy received by the interrogation space during the illumination time is approximately 15 microjoules. In some cases, the combination of laser power and illumination time is such that the total energy

ES 2 550 004 T3 received by the interrogation space during the illumination time is approximately 20 microjoules. In some cases, the combination of laser power and illumination time is such that the total energy received by the interrogation space during the illumination time is approximately 30 microjoules. In some cases, the combination of laser power and illumination time is such that the total energy received by the interrogation space during the illumination time is approximately 40 microjoules. In some cases, the combination of laser power and illumination time is such that the total energy received by the interrogation space during the illumination time is approximately 50 microjoules. In some cases, the combination of laser power and illumination time is such that the total energy received by the interrogation space during the illumination time is approximately 60 microjoules. In some cases, the combination of laser power and illumination time is such that the total energy received by the interrogation space during the illumination time is approximately 70 microjoules. In some cases, the combination of laser power and illumination time is such that the total energy received by the interrogation space during the illumination time is approximately 80 microjoules. In some cases, the combination of laser power and illumination time is such that the total energy received by the interrogation space during the illumination time is approximately 90 microjoules. In some cases, the combination of laser power and illumination time is such that the total energy received by the interrogation space during the illumination time is approximately 100 microjoules.

In some cases, the laser power output is set to at least about 1mW, 2mW, 3mW, 4mW, 5mW, 6mW, 7mW, 8mW, 9mW, 10mW, 13mW, 15mW , 20mW, 25mW, 30mW, 40mW, 50mW, 60mW, 70mW, 80mW, 90mW, 100mW, or more than 100mW. In some cases, the laser power output is set to 20 at least about 1 mW. In some cases, the laser power output is set to at least about 3 mW. In some cases, the laser power output is set to at least about 5 mW. In some cases, the laser power output is set to at least about 10 mW. In some cases, the laser power output is set to at least about 20 mW. In some cases, the laser power output is set to at least about 30 mW. In some cases, the laser's power output is set to at least about 40 mW. In some cases, the laser power output is set to at least about 50 mW. In some cases, the laser power output is set to at least about 60 mW. In some cases, the laser power output is set to at least about 90 mW.

The time that the laser illuminates the interrogation space can be set to no less than approximately 1, 2, 30 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 , 150, 200, 150, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 microseconds. The time that the laser illuminates the interrogation space can be set to no more than approximately 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 150, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1500, or 2000 microseconds. The time that the laser illuminates the interrogation space can be adjusted between approximately 1 and 1000 microseconds. The time that the laser illuminates the interrogation space can be adjusted between 5 and 500 microseconds. The time that the laser illuminates the interrogation space can be adjusted between approximately 5 and 100 microseconds. The time that the laser illuminates the interrogation space can be adjusted between approximately 10 and 100 microseconds. The time that the laser illuminates the interrogation space can be adjusted between approximately 10 and 50 microseconds. The time that the laser illuminates the interrogation space can be adjusted between approximately 10 and 20 microseconds. The time that the laser illuminates the interrogation space can be adjusted between 5 and 50 microseconds. The time that the laser illuminates the interrogation space can be adjusted between approximately 1 and 100 microseconds. In some cases, the time that the laser illuminates the interrogation space is approximately 1 microsecond. In some cases, the time that the laser illuminates the interrogation space is approximately 5 microseconds. In some cases, the time that the laser illuminates the interrogation space is about 10 microseconds. In some cases, the time that the laser illuminates the interrogation space is about 25 microseconds. In some cases, the time that the laser illuminates the interrogation space is about 50 microseconds. In some cases, the time that the laser illuminates the interrogation space is approximately 100 microseconds. In some cases, the time that the laser illuminates the interrogation space is approximately 250 microseconds. In some cases, the time that the laser illuminates the interrogation space is approximately 500 microseconds. In some cases, the time that the laser illuminates the interrogation space is approximately 1000 microseconds.

For example, the time that the laser illuminates the interrogation space can be set to 1 millisecond, 250 microseconds, 100 microseconds, 50 microseconds, 25 microseconds, or 10 microseconds with a laser that provides an output power of 3 mW, 4 mW, 5 mW, or more than 5 mW. In some cases, a label is illuminated with a laser that provides an output power of 3 mW and illuminates the label for about 1000 microseconds. In another 55 cases, a tag is illuminated for less than 1000 milliseconds with a laser that provides an output power of no more than approximately 20 mW. In other cases, the tag is illuminated with a laser power output of 20 mW for less than or equal to approximately 250 microseconds. In some cases, the tag is illuminated with a laser output power of approximately 5 mW for less than or equal to approximately 1,000 microseconds.

two. Capillary flow cell

IS 2 550 004 T3

The capillary flow cell is fluidly connected to the sample system. In one case, the interrogation space 314 of an analysis system is determined by the cross-sectional area of the corresponding beam 311 and by a segment of the beam within the field of view of the detector 309. In one case of the analysis system, the interrogation space 314 has a volume, as defined herein, between approximately 0.01 and 500 pL, or 5 between approximately 0.01 pL and 100 pL, or between approximately 0.01 pL and 10 pL, or between approximately 0.01 pL and 1 pL, or between approximately 0.01 pL and 0.5 pL, or between approximately 0.02 pL and approximately 300 pL, or between approximately 0.02 pL and approximately 50 pL or between approximately 0.02 PL and approximately 5 pL or between approximately 0.02 pL and approximately 0.5 pL or between approximately 0.02 pL and approximately 2 pL, or between approximately 0.05 pL and approximately 50 pL, or between approximately 0.05 pL and approximately 5 10 pL, or between approximately 0.05 pL and approximately 0.5 pL, or between approximately 0.05 pL and approximately

0.2 pL, or between about 0.1 pL and about 25 pL. In some cases, the interrogation space has a volume between approximately 0.01 pL and 10 pL. In some cases, the interrogation space 314 has a volume between about 0.01 pL and 1 pL. In some cases, the interrogation space 314 has a volume between about 0.02 pL and about 5 pL. In some cases, the interrogation space 314 has a volume between about 0.02 pL and about 0.5 pL. In some cases, the interrogation space 314 has a volume between about 0.05 pL and about 0.2 pL. Other volumes of usable interrogation space are as described in this document. It should be understood by one skilled in the art that the interrogation space 314 can be selected for maximum analyzer performance. Although very small interrogation spaces have been shown to minimize background noise, large interrogation spaces have the advantage that low concentration samples can be analyzed in a reasonable amount of time. In cases where two interrogation spaces 370 and 371 are used, volumes such as those described herein can be used for a single interrogation space 314.

In one case, the interrogation gaps are large enough to allow detection of particles at concentrations ranging from about 1000 femtomolar (fM) to about 1 zeptomolar (zM). In one case, the interrogation gaps are large enough to allow detection of particles at concentrations ranging from about 1000 fM to about 1 attomolar (aM). In one case, the interrogation gaps are large enough to allow detection of particles at concentrations ranging from about 10 fM to about 1 attomolar (aM). In many cases, the large interrogation gaps allow the detection of particles at concentrations of less than about 1 fM without additional pre-concentration techniques or devices. One skilled in the art will recognize that the most appropriate interrogation space size depends on the luminosity of the particles to be detected, the background signal level, and the concentration of the sample to be analyzed.

The size of the interrogation space 314 may be limited by the setting of the analyzer optics. In one case, the diameter of the beam 311 can be adjusted to vary the volume of the interrogation space 314. Otherwise, the field of view 35 of the detector 309 can be varied. Therefore, source 301 and detector 309 can be adjusted so that single particles will illuminate and are detected within interrogation space 314. In another case, the width of the aperture 306 (Figure 1A) that determines the field of view of the detector 309, is variable. This configuration allows the interrogation space to be altered, in near real time, to compensate for more or less concentrated samples, which guarantees a low probability that two or more particles are simultaneously within an interrogation space. Similar alterations can be made for two or more interrogation spaces 370 and 371.

In another case, the interrogation space can be defined by using a calibration sample of known concentration that is passed through the capillary flow cell before the actual sample is analyzed. When only a single particle is detected at a time in the calibration sample as the sample is passed through the capillary flow cell, the depth of focus along with the diameter of the beam from the source of electromagnetic radiation 45 determines the size of the interrogation space in the capillary flow cell.

Physical limitations to interrogation spaces can also be provided by a solid wall. In one case, the wall is one or more of the walls of a flow cell 313 (Figure 2A), when the sample liquid is contained within a capillary. In one case, the cell is made of glass, but other light-transparent substances in the range of about 200 to about 1,000 nm or higher can be used, such as quartz, fused silica, and organic materials such as Teflon, nylon. , plastics, such as polyvinyl chloride, polystyrene, and polyethylene, or any combination thereof. Although other cross-sectional shapes (eg, rectangular, cylindrical) can be used, in one case the capillary flow cell 313 has a square cross-section. In another example, the interrogation space may be defined at least in part by a channel (not shown) recorded on a chip (not shown). Similar considerations apply to cases where two interrogation spaces are used (370 and 371 in Fig. 2B).

The interrogation space is bathed in a liquid. In one case, the liquid is watery. In other cases, the liquid is non-aqueous or a combination of aqueous and non-aqueous liquids. In addition the liquid may contain agents for adjusting the pH, ionic composition, or sieving agents, such as soluble particulates or polymers or gels. Is contemplated

ES 2 550 004 T3 that valves or other devices may be present between the interrogation spaces to temporarily interrupt the connection of the liquid. Interrogation spaces temporarily interrupted are considered to be connected by liquid.

In another case of the invention, an interrogation space is the only interrogation space present within the flow cell 5 313 that is limited by the size of a laminar flow of sample material within a volume of diluent, also called flow covering. In these and other examples, the interrogation space can be defined by the cover flux alone or in combination with the dimensions of the illumination source or the detector's field of view. Cover flow can be configured in many ways, including: The sample material is the inner material in a concentric laminar flow, with the volume of the diluent on the outside; the volume of the diluent is on one side of the sample volume; the volume of diluent is on two sides of the sample material; the volume of diluent is on various sides of the sample material, but does not completely enclose the sample material; the volume of diluent completely surrounds the sample material; the volume of diluent completely surrounds the sample material concentrically; The sample material is the inner material in a discontinuous series of drops and the diluent volume completely surrounds each drop of sample material.

In some cases, the single molecule detectors of the invention comprise no more than one interrogation space. In some cases, multiple question spaces are used. Multiple question marks have been previously described in United States Patent Application No. 11 / 048,660, published as US2006 / 0078998 A1. One skilled in the art will recognize that in some cases the parser will contain 2, 3, 4, 5, 6, or more distinct interrogation spaces.

3. Driving force

In one case of the analysis system, the particles are moved through the interrogation space by a motive force. In some cases, the driving force for moving particles is pressure. In some cases, the pressure is supplied by a pump, and the air pressure source, a vacuum source, a centrifuge, or a combination thereof. In some cases, the driving force for moving particles is an electrokinetic force. The use of an electrokinetic force as a driving force has been previously disclosed in United States Patent Application No. 11 / 048,660, published as US2006 / 0078998 A1.

In one case, pressure can be used as a driving force to move the particles through the interrogation space of the capillary flow cell. In a further case, pressure is supplied to move the sample by means of a pump. Appropriate pumps are known in the art. In one case, pumps made for HPLC applications, such as those made by Scivax, Inc., can be used as the driving force. In other cases, pumps made for microfluidic applications can be used when pumping smaller volumes of sample. Such pumps are described in US Patent Nos. 5,094,594,5,730,187,6,033,628, and 6,533,553, which disclose devices that can pump volumes of liquid in the nanoliter or picoliter range. Preferably all materials within the pump that come into contact with the sample are made of highly inert materials, eg, polyetheretherketone (PEEK), fused silica, or sapphire.

A driving force is necessary to move the sample through the capillary flow cell to push the sample through the interrogation space for analysis. A driving force is also required to push a wash sample through the capillary flow cell after the sample has passed through. A motive force is also required to push the sample back out into a sample recovery container, when sample recovery is employed. Standard pumps come in a variety of sizes, and the appropriate size can be chosen to suit expected sample size and flow requirements. In some cases, separate pumps are used for sample analysis and for flushing the system. The test pump can have a capacity of approximately 0.000001 mL to approximately 10 mL, or approximately 0.001 mL to approximately 1 mL, or approximately 0.01 mL to approximately 0.2 mL, or approximately 0.005, 0.01, 0.05, 0.1, or 0.5 mL. The wash pumps can be of greater capacity than the analysis pumps. The wash pumps can have a volume of approximately 0.01 mL to approximately 20 mL, or approximately 0.1 mL to approximately 10 mL, or approximately 0.1 mL to approximately 2 mL, or approximately or approximately 0.05, 0.1, 0.5, 1, 5, or 10 mL. These pump sizes are illustrative only, and those skilled in the art will appreciate that the pump size can be chosen according to the application, sample size, viscosity of liquid to be pumped, tube dimensions. , flow rate, temperature, and other factors well known in the art. In some cases, the system pumps are driven by stepper motors, which are easy to control very precisely with a microprocessor.

In preferred cases, the wash and analysis pumps are used in series, with special check valves to control the direction of flow. The tubing is designed so that when the analysis pump draws the maximum sample, the sample does not reach the pump itself. This is accomplished by choosing the ID and length of the tubing.

ES 2 550 004 T3 between the analysis pump and the analysis capillary such that the volume of the tube is greater than the ejection volume of the analysis pump.

Four. Detectors

In one case, light (eg, light in the ultraviolet, visible or infrared range) emitted by a fluorescent tag after exposure to electromagnetic radiation is detected. Detector 309 (Figure 1A), or detectors (364, 365, Figure 1B), is capable of capturing the amplitude and duration of photon bursts from a fluorescent tag-structural unit complex, and further converting the amplitude and the duration of the burst from photons to electrical signals. Detection devices such as CCD cameras, video input module cameras, and Streak cameras can be used to produce images with contiguous signals. In another case, devices 10 such as a bolometer, a photodiode, a photodiode array, avalanche photodiodes, and photomultipliers that produce sequential signals can be used. Any combination of the aforementioned detectors can also be used. In one case, avalanche photodiodes are used for photon detection.

Using the specific optics between an interrogation space 314 (Figure 2A) and its corresponding detector 309 (Figure 1A), several distinctive characteristics of the emitted electromagnetic radiation 15 can be detected including: emission wavelength, emission intensity, size of burst, burst duration, and fluorescence polarization. In some cases, the detector 309 is a photodiode that is used in reverse bias. A reverse biased photodiode usually has a very high resistance. This resistance is reduced when light of an appropriate frequency shines at the P / N junction. Therefore, a reverse biased diode can be used as a detector by controlling the current run through it. Circuits based on this effect are more sensitive to light than those based on zero bias.

In one case of the analysis system, the photodiode may be an avalanche photodiode, which can be operated with much higher reverse bias than conventional photodiodes, thus allowing each photo-generated carrier to be multiplied by avalanche breakdown, resulting in in increasing range within the photodiode, which increases the effective responsiveness (sensitivity) of the device. The choice of photodiode is determined by the wavelength of energy or emission emitted by the fluorescently labeled particle. In some cases, the photodiode is a silicon photodiode that senses energy in the 190-1100nm range; otherwise, the photodiode is a germanium photodiode that detects energy in the range 800-1700 nm; otherwise, the photodiode is an indium gallium arsenide photodiode that detects energy in the range 800-2600 nm; In still other cases, the photodiode is a lead sulfide photodiode that detects energy in the range of less than 1000 nm to 3500 nm. In some 30 cases, the avalanche photodiode is a single photon detector designed to detect energy in the wavelength range 400 nm to 1100 nm. Single photon detectors are commercially available (eg, Perkin Elmer, Wellesley, MA).

In some cases the detector is an avalanche photodiode detector that detects energy between 300nm and 1700nm. In one case, silicon avalanche photodiodes can be used to detect wavelengths between 300 nm and 35 1100 nm. Indium Gallium Arsenic photodiodes can be used to detect wavelengths between 900nm and

1700 nm. In some cases, an analysis system may comprise at least one detector; in other cases, the analysis system may comprise at least two detectors, and each detector may be chosen and configured to detect light energy in a specific wavelength range. For example, two separate detectors can be used to detect particles that have been labeled with different labels, which upon excitation with an EM source will emit photons with energy in different spectra. In one case, an analysis system may comprise a first detector that can detect fluorescent energy in the 450-700 nm range, such as that emitted by a green dye (eg, Alexa 546); and a second detector that can detect fluorescent energy in the 620-780 nm range, such as that emitted by a far red dye (eg, Alexa 647). It can also be used, detectors for detecting fluorescent energy in the range of 400-600 nm such as that emitted by blue dyes (for example, Hoechst 33342), and for detecting energy in the range of 560- 700 nm, such as that emitted by the red dyes (Alexa 546 and Cy3).

A system comprising two or more detectors can be used to detect unique particles that are each tagged with two or more tags that emit light in different spectra. For example, two different detectors can detect an antibody that has been labeled with two different dye labels. Alternatively, an analysis system comprising two detectors can be used to detect particles of different types, each type is tagged with different dye molecules, or with a mixture of two or more dye molecules. For example, two different detectors can be used to detect two different types of antibodies that recognize two different proteins, each type being tagged with a different dye tag or with a mixture of two or more dye molecules from the tag. By varying the ratio of the two or more dye molecules on the label, two or more different particle types can be detected individually using two detectors. It is understood that three or more detectors can be used.

IS 2 550 004 T3

It should be understood by one skilled in the art that one or more detectors can be configured in each interrogation space, whether one or more interrogation spaces are defined within a flow cell, and that each detector can be configured to detect any of the characteristics of the emitted electromagnetic radiation listed above. The use of multiple detectors, eg, for multiple interrogation spaces, has been previously disclosed in US Patent Application No. 11 / 048,660, published as

US2006 / 0078998 A1. Once a particle is labeled to be detectable (or if the particle possesses an intrinsic characteristic that makes it detectable), any appropriate detection mechanism known in the art can be used, for example a CCD camera, a module camera video input, a Streak camera, a bolometer, a photodiode, a photodiode array, avalanche photodiodes, and photomultipliers that produce sequential signals, and combinations thereof. Different characteristics of electromagnetic radiation can be detected including: emission wavelength, emission intensity, burst size, burst duration, fluorescence polarization, and any combination thereof.

C. Sampling system

In a further case, the analysis system may include a sampling system to prepare the sample for introduction into the analysis system. The included sampling system is capable of automatically sampling a plurality of samples and providing liquid communication between a sample container and a first interrogation space.

In some cases, the analysis system of the invention includes a sampling system for introducing an aliquot of a sample into the single particle analyzer for analysis. Any mechanism that can introduce a sample can be used. Samples can be made using either a vacuum suction created by a pump or pressure applied to the sample that would push the liquid into the tube, or by any other mechanism that serves to introduce the sample into the sample tube. Generally, but not necessarily, the sampling system feeds a sample of known sample volume into the single particle analyzer; In some cases where the presence or absence of a particle or particles is detected, precise knowledge of the sample size is not critical. In preferred cases the sampling system provides automated sampling for a single sample or a plurality of samples. In cases where a sample of known volume is introduced into the system, the sampling system provides a sample for analysis of more than approximately 0.0001, 0.001, 0.01, 0.1, 1, 2, 5, 10, 20, 30, 40 , 50, 60, 70, 80, 90, 100, 150, 200, 500, 1000, 1500, or 2000 mL .. In some cases the sampling system provides a sample for analysis of less than approximately 2000, 1000, 500,200 , 100, 90, 80, 70, 60, 50, 40, 30, 20, 30 10, 5, 2, 1, 0.1, 0.01, or 0.001 mL. In some cases the sampling system provides a sample for analysis of between approximately 0.01 and 1500 mL, or approximately 0.1 and 1000 mL, or approximately 1 and 500 mL, or approximately 1 and 100 mL, or approximately 1 and 50 mL. , or approximately 1 and 20 mL. In some cases, the sampling system provides a sample for analysis of between approximately 5 mL and 200 mL, or approximately 5 mL and approximately 100 mL, or approximately 5 μ £ and 50 μ £. In some cases, the sampling system provides a sample for analysis of between approximately 10 μ £ and 200 mL, or between approximately 10 μ £ and 100 ml, or between approximately 10 mL and 50 mL. In some cases, the sampling system provides a sample for analysis between approximately 0.5 mL and approximately 50 mL.

In some cases, the sampling system provides a sample size that can vary from sample to sample. In these cases, the sample size can be any of the sample sizes described in this document, and they can be changed with each sample, or with sample sets, if desired.

Sample volume accuracy, and sample-to-sample volume precision of the sampling system, is required for the analysis at hand. In some cases, the precision of the sample volume is determined by the pumps used, usually represented by a CV of less than about 50, 40, 30, 20, 10, 5.4, 3, 2, 1, 0.5, 0.1, 0.05, or 0.01% of sample volume. In some cases, the sample-to-sample precision of sampling system 45 is represented by a CV of less than approximately 50, 40, 30, 20, 10, 5.4, 3, 2, 1, 0.5, 0.1, 0.05, or 0.01% sample volume. In some cases, the within-run precision of the sampling system is represented by a CV of less than approximately 10, 5, 1, 0.5, or 0.1%. In some cases, the intra-assay precision of the sampling system shows a CV of less than about 5%. In some cases, the interassay precision of the sampling system is represented by a CV of less than about 10, 5, or 1%. In some cases, the inter-assay precision of the sampling system shows a CV of less than about 5%.

In some cases, the sampling system provides sample transfer, the advantage that an additional wash step is not required between samples. Therefore, in some cases, the sample carryover is less than about 1, 0.5, 0.1, 0.05, 0.04, 0.03, 0.02, 0.01, 0.005, or 0.001%. In some cases, the sample carryover is less than about 0.02%. In some cases, the sample carryover is less than approximately 55 0.01%.

IS 2 550 004 T3

In some cases the sampler provides a sample loop. In these cases, multiple samples are taken into tubes sequentially and each is separated from the others by a buffer of buffer. Samples are generally read one after the other without intermediate drainage. Drainage is done once at the end of the loop. In cases where a buffer plug is used, the plug can be recovered by ejecting the buffer plug into a separate well of a microtiter plate.

The sampling system can be adapted for use with standard assay equipment, eg, a 96-well microtiter plate, or preferably a 384-well plate. In some cases the system includes a 96-well plate positioner and a mechanism to dip the sample tube in and out of the wells, for example, a mechanism that provides movement along the X, Y, and Y axes. Z. In some cases, the sampling system provides multiple sampling tubes from which samples can be stored and drawn from, when the test was started. In some cases, all the samples in the multiple tubes are analyzed in one detector. In other cases, multiple single-molecule detectors may be connected to the sample tubes. Samples can be prepared by steps that include operations performed on the sample in the plate wells prior to sampling by the sampling system, or the sample can be prepared within the analysis system, or some combination of both.

D. Sample preparation system

Sample preparation includes the steps necessary to prepare a raw sample for analysis. These steps may include, by way of example, one or more steps of: separation steps such as centrifugation, filtration, distillation, chromatography; concentration, cell lysis, pH alteration, buffer addition, diluent addition, reagent addition, heating or cooling, tag addition, tag binding, crosslinking with illumination, detachment of unbound tag, inactivation and / or elimination of interfering compounds and any other of the steps necessary for the sample to be prepared for analysis by the single particle analyzer. In some cases, the blood is treated to separate plasma or serum. Additional labeling, removal of unbound label, and / or dilution steps can also be performed on the serum or plasma sample.

In some cases, the analysis system includes a sample preparation system that performs some or all of the processes necessary to provide a sample ready for analysis by the single particle analyzer. This system can perform any or all of the steps indicated above for sample preparation. In some cases the samples are partially processed by the sample preparation system of the analysis system. Therefore, in some cases, a sample may be partially processed outside of the first analysis system. For example, the sample can be centrifuged first. The sample can then be partially processed inside the analyzer by a sample preparation system. Processing within the analyzer includes labeling the sample, mixing the sample with a buffer, and other processing steps that will be known to one in the art. In some cases, a blood sample is processed outside the test system to provide a serum or plasma sample, which is fed into the test system and further processed by a sample preparation system to label the particle. or particles of interest and optionally to remove the unbound tag. In other cases, sample preparation may include immunocapping the sample to remove particles that are not of interest or to remove particles that may interfere with analysis of the sample. In still other cases, the sample may be subjected to falling particles that can interfere with the analysis of the sample. For example, sample preparation can include knockdown of heterophile antibodies, which are known to interfere with immunoassays that use non-human antibodies to directly or indirectly detect a particle of interest. Similarly, other proteins that interfere with measurements of the particles of interest can be removed from the sample using antibodies that recognize the interfering proteins.

In some cases, the sample can be subjected to solid phase extraction before being tested and analyzed. For example, a serum sample that was tested for cAMP first can be subjected to solid phase extraction using a C18 column to which it is bound. Other proteins such as proteases, lipases, and phosphatases are washed from the column, and cAMP is eluted essentially free of proteins that can degrade or interfere with cAMP measurements. Solid phase extraction can be used to remove the basic matrix from a sample, which can decrease the sensitivity of the assay. In still other cases, the particles of interest present in a sample can be concentrated by drying or lyophilizing a sample and solubilizing the particles in a volume smaller than that of the original sample. For example, a sample of exhaled condensed breath (EBC) can be dried and resuspended in a small volume of an appropriate buffer to improve detection of the particle of interest.

In some cases the analysis system provides a sample preparation system that provides the complete preparation of the sample to be analyzed in the system, such as the complete preparation of a blood sample, a saliva sample, a urine sample. , a cerebrospinal fluid sample, a lymph sample, a sample

ES 2 550 004 T3 of BAL, an exhaled air condensate (EBC) sample, a biopsy sample, a forensic sample, a bioterrorism sample, and the like. In some cases the analysis system provides a sample preparation system that provides some or all of the sample preparation. In some cases, the initial sample is a blood sample that is further processed by the analysis system. In some cases, the sample is a serum or plasma sample that is further processed by the analysis system. The serum or plasma sample can be further processed, for example, by contacting it with a label that binds to a particle or particles of interest; the sample can then be used with or without removal of unattached label.

In some cases, sample preparation is carried out, either outside of the assay system or in the sample preparation component of the assay system, in one or more microtiter plates, such as a 96 10-well plate. . Reagent reservoirs, buffers, and the like may be in intermittent fluid communication with the wells of the plate by means of tubes or other appropriate structures, as are well known in the art. Samples can be prepared separately in 96-well plates or tubes. Sample isolation, tag bonding and, if necessary, tag separation steps, can be done on a plate. In some cases, the prepared particles are then released from the plate and the samples are moved into tubes for sampling in the sample analysis system. In some cases, all stages of sample preparation are performed on one plate and the analysis system acquires sample directly from the plate. Although this case is described in terms of a 96-well plate, it will be appreciated that any container can be used to hold one or more samples and appropriate for sample preparation. For example, standard 384 or 1536 well microtiter plates can be used. More generally, in some cases, the sample preparation system is capable of containing and preparing more than about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 500, 1000, 5000, or 10,000 samples. In some cases, samples can be taken from multiple samples for analysis on multiple analyzer systems. Therefore, in some cases, 2 samples, or more than about 2, 3, 4, 5, 7, 10, 15, 20, 50, or 100 samples are sampled from the sample preparation system and run in parallel in multiple sample analysis systems.

Microfluidic systems can also be used for sample preparation and as sample preparation systems that are part of analyzer systems, especially for samples suspected of containing high enough concentrations of particles that detection requires smaller samples. Microfluidic manipulation principles and techniques are known in the art. See, for example, US Patent Nos. 4,979,824; 5,770,029; 5,755,942; 5,746,901; 5,681,751; 5,658,413; 5,653,939; 5,653,859;

5,645,702; 5,605,662; 5,571,410; 5,543,838; 5,480,614, 5,716,825; 5,603,351; 5,858,195; 5,863,801; 5,955,028;

5,989,402; 6,041,515; 6,071,478; 6355,420; 6,495,104; 6,386,219; 6,606,609; 6,802,342; 6,749,734; 6,623,613; 6,554,744; 6,361,671; 6,143,152; 6,132,580; 5,274,240; 6,689,323; 6,783,992; 6,537,437; 6,599,436; 6,811,668 and published PCT patent application No. WO9955461 (A1). Samples can be prepared in series or in parallel, for use in a single or multiple analysis system.

Preferably, the sample comprises a buffer solution. The buffer can be mixed with the sample outside of the test system, or it can be provided by the sample preparation mechanism. While any appropriate buffer can be used, the preferred buffer has a low fluorescence background, is inert to the detectably labeled particle, can maintain the working pH, and, in cases where the driving force is electrokinetic, it has the appropriate ionic strength for electrophoresis. The concentration of the buffer solution can be any appropriate concentration, such as in the range of about 1 to about 200 mM. Any buffer system can be used as long as the solubility, function, and detection ability of the molecules of interest are provided. Preferably, for pump application, the buffer is selected from the group consisting of phosphate, glycine, acetate, citrate, acidulate, carbonate / bicarbonate, imidazole, triethanolamine, glycine amide, borate, MES, Bis-Tris, ADA, aces, PIPES, 45 MOPSO, Bis-Tris propane, BES, MOPS, TES, HEPES, DIPSO, MOBS, TAPSO, Trizma, HEPPSO, POPSO, TEA, EPPS, Tricina, Gly-Gly, Bicina, HEPBS, TAPS, AMPD, TABS, AMPSO, CHES, CAPSO, AMP, CAPS, and CABS. The buffer can also be selected from the group consisting of Gly-Gly, bicine, tricine, 2-morpholine ethanesulfonic acid (MES), 4-morpholine propanesulfonic acid (MOPS), and 2-amino-2-methyl-1-propanol hydrochloride ( AMP). A useful buffer is 2 mM Tris / borate at pH 8.1, but Tris / glycine and Tris / HCl are also acceptable.

Other buffer solutions are as described in this document.

Useful buffer solutions for electrophoresis are disclosed in US Patent Application No. 11 / 048,660, published as US2006 / 0078998 A1

E. Sample recovery

A very useful feature of the cases of the analyzers and analysis systems described in this document is that the sample can be analyzed without consuming it. This can be especially important when sample materials are limited. Sample retrieval also allows for further testing or retesting. The advantages of this feature for applications where the sample size is limited and / or where the

ES 2 550 004 T3 ability to retest the sample, for example clinical diagnostic, forensic, drug detection, and clinical diagnostic applications, will be apparent to those skilled in the art.

Therefore, in some cases, the analysis system described in this document provides a sample recovery system for sample recovery after analysis. In these cases, the system includes mechanisms and 5 methods by which the sample is introduced into the analyzer, analyzed and then returned, for example, by the same path, to the sample carrier, for example, the sample tube. . Because none of the samples is destroyed and since it does not enter any of the valves or other tubes, it remains uncontaminated. Also, because all materials in the sample path are highly inert, eg PEEK, fused silica, or sapphire, there is little contamination of the sample path. The use of stepper motor controlled pumps (particularly the analysis pump) allows precise control of the volumes withdrawn and driven back. This allows complete or near complete recovery of the sample with little or no dilution by the wash buffer.

Therefore, in some cases, more than about 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of the sample was recovered after analysis. In some cases, the recovered sample is undiluted. In some cases, the recovered sample is diluted less than approximately 1.5 times, 1.4 times, 1.3 times, 1.2 times, 1.1 times, 1.05 times, 1.01 times, 1,005 times, or 1,001 times.

For sample sampling and / or recovery, any mechanism can be used to transport a liquid sample from a sample container to the analyzer. In some cases the inlet end of the analysis capillary has been connected to a short length of tubing, for example PEEK tubing that can be immersed in a sample container, for example a test tube or sample well, or It can be kept above a 20 waste container. When washing, to clean the old sample from the apparatus, this tube is placed above the waste container to catch the wash residue. When a sample is drawn, the tube is inserted into the sample well or test tube. Typically the sample is drawn quickly, and then slowly ejected while observing the particles within the sample. Alternatively, in some cases, the sample is drawn slowly during at least part of the extraction cycle; the sample can be analyzed while slowly withdrawing. This can be followed by a quick return of the sample and a quick wash. In some cases, the sample can be analyzed both in the entry (introduction) and exit (extraction) cycles, which improves the counting statistics, for example, of small and diluted samples, as well as confirming the results, and the like . If you want to save the sample, it can be driven back to the same well as the sample it came from, or to another. If storing the sample is not desired, the tube is placed over the waste container.

SAW. Methods Using High Sensitivity Cardiac Troponin Assay

The methods of the present invention make it possible to measure cardiac troponin levels at concentrations much lower than previously measured. Although cardiac troponin is an accepted marker for cardiac muscle damage, its usefulness has been limited by the fact that, with current testing methods, it is only detectable after significant cardiac muscle damage has occurred, due to the 35 lack of sensitivity of current methods. The Joint European Society of Cardiology / American College of Cardiology committee for the Redefinition of Myocardial Infarction has recommended that an increase in cardiac troponin concentration is defined as an upper measure of the 99th percentile of the distribution of cardiac troponin concentrations in the reference group, a very low threshold. A total imprecision of (CV) at the decision limit of <10% is recommended. However, the analytical imprecision obtained with currently available immunoassays 40 for cardiac troponins is not uniform, especially in the low concentration range. Furthermore, the assays that are currently available lack sufficient sensitivity to detect troponin levels in non-clinical (normal) subjects, and a true baseline or defined troponin level has not been defined in a normal population. The assay systems described in this document have been shown to be capable of systematically detecting cTnI levels at concentrations of less than 10 pg / mL with a total imprecision of less than 10% (see examples). Therefore, the invention provides, as specified in the claims, methods for diagnosis, prognosis or treatment methods based on the high sensitivity detection of cardiac troponin in humans.

Some examples provide a method of determining a diagnosis, prognosis, or method of treatment in an individual by i) determining a cardiac troponin concentration in a sample or determining cardiac troponin concentrations in a series of samples from the individual, where the concentration is determined by a cardiac troponin assay with a detection limit for cardiac troponin in said sample of less than about 50, 40, 30, 10, 5, 4, 3, 2, or 1 pg / mL, for example, less than about 20 pg / mL; and ii) determining a diagnosis, prognosis, or method of treatment in said individual, based on the concentration in the sample, or the concentrations in the series of samples. The method for determining cardiac troponin concentration includes any appropriate method with the required sensitivity, for example, the methods described herein. In some cases, the methods use a method of determining a cardiac troponin concentration in the sample, where the method comprises detecting single troponin molecules or complexes, or fragments thereof.

IS 2 550 004 T3

In some cases, the threshold troponin concentration is determined by analyzing samples, eg, blood, serum, or plasma samples, from an apparently healthy population for cardiac troponin, eg, cardiac troponin I, and determining the level at which 80, 90, 95, 96, 97, 98, 99, 99.5, or 99.9% of the population fall below that level (concentration). This value is the threshold value. In some embodiments, as specified in the claims, the threshold value is set at the 99th percentile. In some cases, the test is performed using a method with a detection level for cardiac troponin of less than about 50, 20, 10, 5, or 1 pg / mL, for example, less than about 5 pg / mL.

In some cases, the invention provides a method of determining a diagnosis, prognosis, or method of treatment in an individual by comparing a value for a cardiac troponin concentration in a sample of the individual with a normal value or a range of the normal values for cardiac troponin, where the normal trough or range of normal values is determined by a cardiac troponin assay with a limit of detection for cardiac troponin in said sample of less than approximately 50, 40, 30, 10, 5, 4, 3, 2, or 1 pg / mL, for example, less than about 20 pg / mL; and ii) determining a diagnosis, prognosis, or method of treatment in said individual, based on the comparison.

In some embodiments, as specified in the claims, the cardiac troponin is cardiac troponin I or cardiac troponin T. In some embodiments, the cardiac troponin is cardiac troponin T. In some embodiments, the cardiac troponin is cardiac troponin I. The method can use the total troponin of, for example, total cTnI, or cTnT, or total cTnI + cTnT, as described herein, to determine a diagnosis, prognosis, or method of treatment. In some cases, the method may use the concentration of complex, free cardiac troponin 20, or cardiac troponin fragments, or a comparison of these (eg, a ratio), to determine a diagnosis, prognosis, or method of diagnosis. treatment.

A. Samples

The sample or a series of samples can be any appropriate sample; As specified in the claims, the sample (s) will be blood, serum or plasma. In some embodiments, the sample or a series of samples is 25 serum samples. The individual can be an animal, eg, mammal, eg, human.

A single sample can be taken, or a series of samples can be taken. If a series of samples is taken, they can be taken at any appropriate interval, for example intervals of minutes, hours, days, weeks, months, or years. In an acute clinical setting, a series of samples will generally be taken over the course of hours and days, with the samples separated by a matter of hours. When an individual is followed for longer periods, sample intervals can be months or years. The diagnosis, prognosis, or method of treatment can be determined from a single sample, or from one or more than a series of samples, or from changes in the series of samples, for example, an increase in concentration to a certain Speed may indicate a severe condition while increasing at a slower rate or no increase may indicate a relatively benign or less severe condition. The rate of change can be measured over the course of hours, days, weeks, months, or years. The rate of change in a given individual may, in some cases, be more relevant than an absolute value. In an acute setting, an extremely rapid rate of change, for example a spike, may indicate an impending, in-progress, or recent cardiac event. In other contexts, increasing values over a period of days, weeks, months, or years in an individual may indicate progress and worsening of heart damage, for example, heart damage due to a heart condition (for example, hypertrophy heart failure or congestive heart failure) or heart damage due to a non-cardiac condition (eg, toxicity from drug exposure).

In some embodiments, at least one sample is taken during or after a cardiac stress test. For example, one sample can be taken before the stress test, and one or more samples taken during the test. Deviations in cardiac troponin levels between the pre-test sample and the sample (s) taken during the test can provide diagnostic or prognostic information, for example, indicate the likelihood of artery disease. coronary or other pathology associated with the heart muscle. Other comparisons can be made, as well as comparisons of none of the samples to normal or threshold levels, or determination of a rate of change in cardiac troponin concentration in the samples, all of which can provide useful information regarding the heart and cardiovascular health, as well as other conditions as described in this document.

In some cases, at least one sample is taken at or near the time that individuals are present with a healthcare professional with one or more symptoms indicative of a condition that may involve heart damage. The contexts in which an individual may be present with a healthcare professional include, but are not limited to, ambulatory care, urgent care, critical care, intensive care, surveillance unit, hospital inpatient, non-patient. hospitalized, doctor's office, medical clinic, emergency response context, including an ambulance and health screening situations. In some cases, one or more samples are taken from the individual and tested for cardiac troponin locally, ie, in or near the environment in

ES 2 550 004 T3 where the samples are taken. For example, an individual presenting to a hospital may have one or more samples taken that are tested for cardiac troponin at the hospital. In some cases, one or more samples are taken from the individual and subjected to cardiac troponin analysis in a CLIA laboratory. In some cases, the individual shows one or more symptoms consistent with acute coronary syndrome. In some cases, individual 5 shows one or more symptoms consistent with AMI. These symptoms include, but are not limited to, chest pain, chest pressure, arm pain, abnormal EKG, abnormal enzyme levels, and shortness of breath.

B. Determination of diagnosis, prognosis, or method of treatment

In some cases, step ii) includes comparing said concentration or a series of concentrations with a normal value for said concentration, comparing said concentration or series of concentrations with a predetermined threshold level, comparing said concentration or series of concentrations with a reference value, or determine a rate of change of the concentration of said series of concentrations.

In some cases, step ii) comprises comparing said troponin concentration in said sample with a predetermined threshold concentration, and determining a diagnosis, prognosis, or treatment method if the sample concentration is greater than the threshold level. The threshold concentration can be determined, for example, by determining the 99th percentile concentration of troponin in a group of individuals, and setting said threshold concentration at said 99th percentile concentration. An example of this is given in the Examples.

Normal values, threshold values, rate of change, value relationships, and other useful diagnostic and prognostic indicators can be established by methods well known in the art. For example, these values can be determined by comparing samples from a study population and a control population, where the study population shows the biological state for which the diagnosis, prognosis, or method of treatment is desired and the control population does not present the biological status. In some cases, a longitudinal study can be performed, for example, the study population can be a subset of the control population that, over time, shows biological status. It will be appreciated that data from a plurality of studies can be used to determine a consensus value or range of values for normal, and for prognostic or diagnostic levels.

In developing the diagnostic or prognostic test, data for one or more potential markers can be obtained from a group of subjects. The group of subjects is divided into at least two groups, and preferably the first group and the second group each have approximately equal numbers of subjects. The first group includes subjects who have been confirmed to have a disease or, more generally, to be in a first state of condition. For example, this first group of patients may be those who have recently had an incidence of the disease, or they may be those with a specific type of disease, such as AMI. Confirmation of condition status can be done through a more rigorous and / or expensive test, such as MRI or CT. From now on, the subjects in this first group are known as sick. The second group of subjects is nothing more than those who do not fall into the first group. Subjects in this second group may be non-diseased; that is, 35 normal subjects. Alternatively, subjects in this second group can be selected for displaying a symptom or a constellation of symptoms that mimic the symptoms exhibited by diseased subjects. In yet another alternative, this second group may represent those who are at a different time of disease incidence. Preferably, data for the same group of markers is available for each patient. This group of markers can include all candidate markers that may be suspected of being relevant for the detection of a particular disease or condition. Known current relevance is not required. Examples of the compositions, methods, and systems described herein can be used to determine which of the candidate markers are most relevant to the diagnosis of the disease or condition. The levels of each marker in the two groups of subjects can be distributed over a wide range, for example, in the form of a Gaussian distribution. However, a distribution adjustment is not required.

1. Acute myocardial infarction

The methods of the invention are especially useful in the diagnosis, prognosis and / or selection of treatment in patients with suspected acute myocardial infarction (AMI). Serial or individual cardiac troponin measurements in patients with suspected AMI provide increasing prognostic information that improves prognosis and indicates early and appropriate therapeutic intervention to minimize the risk of adverse outcomes.

Therefore, the specification describes a method for diagnosing, predicting and / or preventing or treating AMI in an individual by analyzing a sample from the individual, for example, a blood sample, plasma sample, and / or serum sample. , for cardiac troponin, eg, cTnI, and detection of a cardiac troponin concentration in the sample at a detection limit of less than about 50, 40, 30, 20, 15, 10, 9, 8, 7, 6 , 5, 4, 3, 2, or 1 pg / mL, for example, less than approximately 20 pg / mL, where the cardiac troponin concentration in sample 55 indicates or predicts AMI. Cardiac troponin can be cTnI or cTnT, and can be troponin, total, or a measure of a

ES 2 550 004 T3 particular form, eg free, complex, or fragment; In some embodiments, a ratio of one or more forms of the troponin is used, as described herein. In some cases, the total cTnI is measured on the sample or a series of samples. In some cases, the total cTnT is measured on the sample or a series of samples. In some cases, total cTnI + cTnT is measured on the sample or a series of samples. In some cases, the cardiac troponin level is determined at or near the time the individual is present with a healthcare professional with symptoms indicative of AMI. These symptoms include, but are not limited to, chest pain, chest pressure, arm pain, abnormal EKG, abnormal enzyme levels, and shortness of breath.

In some cases, a series of measurements are taken, and an increase in the concentration of cardiac troponin in the samples indicates, predicts, or provides a basis for the prognosis of AMI. In some cases, an increase of more than 50%, more than 100%, more than 150%, more than 200%, more than 250%, more than 300%, more than 400%, or more than 500% of the value of reference indicates, predicts, or provides a basis for forecasting the AMI. In some cases, a cardiac troponin level above about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 pg / mL in a single sample indicates, predicts, or provides a basis for the forecast of AMI, regardless of the reference levels, if they are obtained. In some cases, a cardiac troponin level of about 1-10, or about 5-15, or about 10-50, about 10-200, about 10-100, or about 10-40, or about 15-50, or about 15-40, or about 20-200, about 20-150, about 20-100, about 20-50, or about 20-40, or about 20-30 pg / mL indicates, predicts, or provides a basis for AMI forecast.

In some cases the diagnosis or prognosis includes stratification for the individual, based on the concentration of cardiac troponin in the sample or a series of samples. Such stratification may be based on the concentration of cardiac troponin in individual samples, the presence of increases and / or the size of the increases from reference values in a series of samples, the ratios of the different forms of cardiac troponin. , the absolute values for the different forms of cardiac troponin, the rate of change in the concentration for cardiac troponin or for one or more forms of cardiac troponin in a series of samples, the change in ratios of the different forms of cardiac troponin over time in a series of samples, and any other information based at least in part on the cardiac troponin concentration in the sample or a series of samples. Stratification can be based on values obtained from normal and diseased subject populations, as described herein. The appropriate treatment can also be determined based on the stratification of the individual.

In some cases, the cardiac troponin concentration is determined in combination with one or more other markers, for example, markers of myocardial ischemia, myocardial infarction, or markers of stroke are considered, and the concentrations of each marker determine the diagnosis , prognosis, or method of treatment. As will be apparent to those skilled in the art, other clinical indications will generally also be considered, for example EKG, symptoms, history, and the like. Appropriate algorithms for diagnosis, prognosis, or treatment can be constructed based on combinations of such markers and clinical indications in combination with troponin levels.

Useful markers in combination with cardiac troponin in the methods of the invention include, but are not limited to, creatine kinase (CK) and its myocardial band (MB) of the myocardial building block CK, aspartate aminotransferase, lactate dehydrogenase (LDH) , α-hydroxybutyrate dehydrogenase, myoglobin, glutamate oxaloacetate transaminase, glycogen phosphorylase BB, unbound free fatty acids, heart fatty acid binding protein (H-FABP), ischemia-modified albumin, light chain myosin 1, light chain myosin 2. Markers of inflammation and plaque instability useful in combination with cardiac troponin in the methods of the invention include but are not limited to C-reactive protein, white blood cell count, soluble CD40 ligand, myeloperoxidase, monocyte chemoattractant protein-1, whole blood choline, and pregnancy-associated plasma protein A. Other markers of inflammation can be detected, and include combinations of IL-8, IL-1β, IL6, IL10, TNF, and IL-12p70, as well as other cytokines or markers that will be apparent to those of skill in the art.

In some cases, cardiac troponin, eg, cTnI, is jointly measured, eg, in the same sample, or in samples from the same individual taken at or near the same time, with a marker selected from the group consisting of creatine kinase. (CK) and its myocardial band (MB) of the myocardial structural unit CK, aspartate aminotransferase, lactate dehydrogenase (LDH), a hydroxybutyrate dehydrogenase, myoglobin, transaminase oxaloacetate glutamate, glycogen phosphorylase BB, unbound free fatty acids, heart fatty acid binding protein (H-FABP), ischemia-modified albumin, myosin light chain 1, and myosin light chain 2. In some cases cardiac troponin, eg, cTnI , is measured together with CK-MB, for example, in the same sample, or in samples from the same individual taken at or near the same time.

In some cases, cardiac troponin, alone or in combination with other clinical signs or markers, measured as described herein, is used to determine reinfarction. In some cases, cardiac troponin, alone or in combination with other clinical markers or signs, measured as described herein, is used to

ES 2 550 004 T3 determine the characteristics of an infarct, for example, the size, or the duration since the infarction. In the latter case, the troponin fragments produced by proteolysis in the blood can be compared with the total troponin, the higher the fragment ratio, the longer the time elapsed since the infarction.

two. Conditions other than AMI

The methods of the invention also include methods of diagnosis, prognosis and treatment based on the concentration of cardiac troponin in a sample that are useful in conditions other than AMI.

Many conditions include potential or actual heart damage, and the ability to measure cardiac troponin at the levels described herein allows early detection of such damage and early intervention. Knowledge of the cardiac troponin concentration as measured by the methods and compositions of the invention is useful in the diagnosis, prognosis, and determination of treatment for such conditions. Conditions include percutaneous coronary interventions, cardiac surgery, heart failure, acute rheumatic fever, amyloidosis, cardiac trauma (including contusion, ablation, rhythm, interruption, cardioversion, catheterization and cardiac surgery), reperfusion injury, cardiotoxicity of the therapy of the cancer, congestive heart failure, chronic kidney failure, type II glycogen storage disease (Pompe disease), heart transplantation, hemoglobinopathy with transfusion hemosiderosis, hypertension, including gestational hypertension, hypotension, often with arrhythmias, hypothyroidism, myocarditis, pericarditis, post-operative non-cardiac surgery, pulmonary embolism, and sepsis.

In these cases, troponin levels can be determined concomitantly with levels of marker (s) that are specific for non-cardiac disease or other clinical symptoms or signs of the disease; The concentration and / or marker (s) information regarding other clinical symptoms or signs is combined with the information regarding cardiac troponin concentrations, determined as described herein, to determine a diagnosis, prognosis, and / or or method of treatment. For example, the cases of the invention may employ, in addition to determining cardiac troponin concentration, determining the concentration of one or more of the aforementioned polypeptides, or other protein markers useful in diagnosis, prognosis, or differentiation of the disease. In some cases, a panel of markers for disease is provided, where the panel includes cardiac troponin concentration, as described herein, and at least one other marker for disease. The panel can include 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more or individual markers, including one or more cardiac troponins, eg, total cTnI. Analysis of an individual marker or marker subsets can be carried out by one of skill in the art to optimize clinical sensitivity or specificity in various clinical settings. These include, but are not limited to, outpatient care, urgent care, intensive care, intensive therapy, supervisory unit, hospital inpatient, outpatient, doctor's office, medical clinic, and health screening situations. Furthermore, one skilled in the art can use a single marker or a subset of the markers in combination with an adjustment of the diagnostic threshold in each of the aforementioned settings to optimize clinical sensitivity and specificity.

to. Cardiac Toxicity The methods of the invention are especially useful in determining and monitoring cardiac toxicity resulting from treatment, eg, cardiac toxicity from drug treatment. Thus, for example, the specification provides a method of evaluating the cardiac toxicity of a treatment by measuring cardiac troponin in an individual by i) determining a cardiac troponin 40 concentration in a sample or determining cardiac troponin concentrations. in a series of samples from the individual, where at least one of the samples is taken from the person during or after a time when the individual is receiving treatment, where the concentration (s) is determined by a cardiac troponin assay with a limit of detection for cardiac troponin in said sample less than about 50, 40, 30, 10, 5, 4.3.2, or 1 pg / mL, eg, less than about 20 pg / mL; and ii) evaluation of the degree of cardiac toxicity of the treatment based on said concentration or concentrations. In some cases, the treatment is a drug treatment. In some cases, the treatment is a non-drug treatment. The method for determining cardiac troponin concentration includes any appropriate method with the required sensitivity, for example, the methods described herein. In some cases, the methods use a method of determining a cardiac troponin concentration in the sample, where the method comprises detecting individual troponin molecules or complexes, or fragments thereof.

Especially useful are methods of determining cardiac toxicity using the cross-reactive antibodies described herein, ie, antibodies that react with troponin from at least two species, such as humans and other species, such as rat, dog, mouse, or monkey. Such antibodies can be used in animal studies of drug toxicity, where the individual for whom toxicity is assessed is, for example, a mammal, such as a rat, mouse, dog, monkey, or other animal used in such studies. Toxicity in various species can be directly compared when the antibody used in the assay is the same antibody, thus reducing variability.

IS 2 550 004 T3

It will be appreciated that the methods of the invention can be used in conjunction with specific drugs whose side effects include cardiotoxicity in order to control cardiac toxicity. Therefore, the specification provides methods for monitoring cardiac toxicity in an individual who is receiving a drug known to cause cardiac toxicity by determining the cardiac troponin concentration in one or more samples obtained from the individual, where the concentration o Concentrations are determined by a cardiac troponin assay with a limit of detection for cardiac troponin in said sample or samples of less than about 50, 40, 30, 10, 5, 4, 3, 2, or 1 pg / mL, for example, less than about 20 pg / mL; and ii) evaluating the degree of cardiac toxicity of drug treatment based on said concentration or concentrations. In some cases the method further includes a step iii) determining whether or not to continue with the drug treatment based on the evaluation of step ii). Drugs whose side effects include cardiac toxicity are well known in the art.

C. Business methods

Described herein are systems and methods (including commercial methods) for the establishment of cardiac troponin markers that can be used to diagnose, predict, or determine a method of treating a biological state or condition in an organism, the preparation of diagnostics based on such markers and marketing / marketing and service diagnostics using such diagnostics. The biological state can be acute myocardial infarction, or cardiac damage due to drug toxicity, or non-AMI states as described herein.

In one instance, the commercial methods herein comprise: the establishment of one or more cardiac troponin markers using a method comprising: establishing a range of 20 concentrations for said marker (s) in biological samples obtained from a first population by measuring the concentrations of the marker (s) in biological samples by detecting single molecules of the marker (s) at one level detection of less than about 50, 20, 10, 5, or 1 pg / mL; and the commercialization of the one or more markers established in the previous step, for example, in a diagnostic product. The diagnostic product herein may include one or more antibodies that specifically bind to the cardiac troponin marker and a fluorescent building block that is capable of emitting an average of at least about 200 photons when simulated by a light emitter. laser at the excitation wavelength of the structural unit, when the laser is focused on a point not less than about 5 microns in diameter that contains the structural unit, and wherein the total energy directed at the point by the laser is no more than about 3 microjoules.

In one instance, the commercial methods herein comprise: establishing a range of normal values for a cardiac troponin marker using a system comprising: the establishment of a range of concentrations for said cardiac troponin marker in biological samples obtained from a first population by measuring the marker concentrations of the biological samples by detecting single molecules of the marker at a detection level of less than about 50, 20, 10, 5, or 1 pg / mL; and 35 providing a diagnostic service to determine whether or not an organism has a state or condition of interest, eg, AMI, cardiac toxicity due to drug treatment, or a non-AMI condition. A diagnostic service in this document may be provided by a CLIA approved laboratory that is licensed in the company or business itself. The diagnostic services in this document may be provided directly to a health care provider, a disease care insurer, or a patient. Thus, the business methods in this document may generate income from the sale, for example, of diagnostic services or diagnostic products.

The business methods in this document also contemplate the provision of diagnostic services, for example, for healthcare providers, insurers, patients, etc. The business in this document may provide diagnostic services through a contract with a service laboratory or the establishment of a service laboratory (under the Clinical Laboratory Improvement Amendment (CLIA) or other regulatory approval).

Such a service laboratory can then perform the procedures disclosed herein to identify whether a cardiac troponin marker is found within a sample.

VII. Compositions

The specification describes compositions useful in the detection and quantification of cardiac troponin. The 50 compositions include cardiac troponin binding partners that are tagged with appropriate tags for detection by the methods of the invention, pairs of binding partners wherein one or both of the binding partners are tagged with appropriate tags for detection By means of the methods of the invention, solid supports to which associated capture binding are attached, in some cases also with detection binding partners.

Exemplary cases include a composition for the detection of cardiac troponin that includes a cardiac troponin binding partner bound to a fluorescent building block, where the fluorescent building block is capable of emitting an average of at least about 200 photons when is simulated by a laser light emitter at the

ES 2 550 004 T3 frame unit excitation wavelength, when the laser is focused on a point not less than about 5 microns in diameter that contains the frame unit, and where the total energy directed at the point by the laser is no more than about 3 microjoules. In some cases, the binding partner includes an antibody to cardiac troponin. In some cases, the antibody is a polyclonal antibody. In some cases, the antibody is a monoclonal antibody. In some cases, the antibody is a cross-reactive antibody, for example, an antibody that cross-reacts with cardiac troponin of at least two species, for example, at least two species selected from the group consisting of human, monkey, dog, and mouse. In some cases the antibody cross-reacts with cardiac troponins from all humans, monkeys, dogs, and mice. In some cases, cardiac troponin is selected from the group consisting of cTnI and cTnT. In some cases, the cardiac troponin is cTnI. In some cases, the cardiac troponin is cTnT. The antibody can be specific to a specific region of the troponin molecule, eg, specific for a region comprising amino acids 27-41 of cardiac troponin I. The fluorescent building block may contain one or more molecules comprising at least one substituted indole ring system wherein the substituent at carbon 3 of the indole ring contains a chemically reactive group or a conjugated substance group. The label composition may include a fluorescent building block that includes one or more dye molecules selected from the group consisting of Alexa Fluor 488, 532, 647, 700, or 750. The tag composition can include a fluorescent building block that includes one or more dye molecules selected from the group consisting of Alexa Fluor 488, 532, 700, or 750. The label composition can include a fluorescent building block that includes one or more molecules dye molecules that are Alexa Fluor 488. The label composition may include a fluorescent building block that includes one or more dye molecules that are Alexa Fluor 555. The tag composition can include a fluorescent building block that includes one or more dye molecules that are Alexa Fluor 610. The tag composition can include a fluorescent building unit that includes one or more dye molecules that are Alexa Fluor 647. The composition The tag may include a fluorescent building block that includes one or more dye molecules that are Alexa Fluor 680. The tag composition can include a fluorescent building block that includes one or more dye molecules that are Alexa Fluor 700. The tag composition can include a fluorescent building unit that includes one or more dye molecules that are Alexa Fluor 750.e

In some cases, the specification describes a composition that includes a set of standards for the determination of a concentration of a cardiac troponin, wherein at least one of the standards is at a concentration of cardiac troponin less than about 20, 15, 10, 5, 4, 3, 2, or 1 pg / mL. In some cases, the specification describes a composition that includes a set of standards for determining a concentration of a cardiac troponin, wherein at least one of the standards is at a cardiac troponin concentration of less than about 20 pg / mL. In some cases, the specification describes a composition that includes a set of standards for the determination of a concentration of a cardiac troponin, wherein at least one of the standards is at a concentration of cardiac troponin less than about 10 mg / mL. In some cases, the specification describes a composition that includes a set of standards for the determination of a concentration of a cardiac troponin, wherein at least one of the standards is at a cardiac troponin concentration of less than about 5 pg / mL. . In some cases, the specification describes a composition that includes a set of standards for determining a concentration of a cardiac troponin, wherein at least one of the standards is at a cardiac troponin concentration of less than about 40 µg / mL.

Other compositions are as described herein.

VIII. Kits

The specification further describes kits. The kits described herein include one or more compositions useful for the sensitive detection of cardiac troponin, as described herein, in appropriate packaging. In some cases the kits described herein provide labels, eg, binding partner such as an antibody that is specific for cardiac troponin, where the binding partner is bound to a fluorescent building block. In some cases the kits described herein provide pairs of binding partners, eg, antibody pairs, that are specific for cardiac troponin, where at least one of the binding partners is a tag for a cardiac troponin, as described. described in this document. In some cases, the binding partners, eg, antibodies, are provided in separate containers. In some cases, binding partners, eg, antibodies, are provided in the same container. In some cases, one of the binding partners, eg, antibody, is immobilized on a solid support, eg, a microtiter plate or a paramagnetic bead. In some of these cases, the other binding partner, eg, antibody, is labeled with a fluorescent building block as described herein.

The binding partners, eg, antibodies, solid supports, and fluorescent labels for the components of the kits can be any type of appropriate components as described herein.

IS 2 550 004 T3

Kits may additionally include reagents useful in the methods described herein, for example, buffers and other reagents used in reactions, washes, buffers, or other binding reagents for preconditioning the instrument in which the assays will be performed, and elution buffers or other reagents to run through the instrument.

Kits can include one or more standards, eg, the standards for use in the assays described herein, such as the highly purified standards of eg, recombinant, human cTnI or human cTnT, or various fragments, complexes, and similar, of the same. Kits may also include instructions.

EXAMPLES

The following examples are offered by way of illustration and not by way of limiting the remaining disclosure.

Unless otherwise specified, run samples in the Examples were analyzed on a single molecule detector (SMD) as described herein, with the following parameters: Laser: continuous wavelength gallium arsenite diode laser 639 nm wavelength (Blue Sky Research, Milpitas, CA), focused to a spot size of approximately 2 microns (0.004 pL interrogation space as defined herein); flow rate = 5 microliters / min through a 100 sq. micron ID fused silica capillary and 300 sq. micron OD; non-confocal lens configuration (see, for example, Fig. 1A); centering the 0.8 numerical aperture lens (Olympus); silicon avalanche photodiode detector (Perkin Elmer, Waltham, MA).

Example 1. Biomarker sandwich assays: cardiac troponin I (cTnI)

The Assay: The purpose of this assay was to detect the presence of cardiac Troponin I (cTnI) in human serum. The assay format was a two-step sandwich immunoassay based on a mouse monoclonal capture antibody and a goat polyclonal detection antibody. Ten microliters of sample are required. The working range of the assay is 0 - 900 pg / mL with a typical analytical limit of detection of 1 - 3 pg / mL. The essay requires approximately four hours of bench time to complete.

Materials: The following materials are used in the procedure described below: Assay Plate: Nunc Maxisorp, product 464718, 384 wells, clear, passively coated with monoclonal antibody, BiosPacific 25 A34440228P Lot # A0316 (5 pg / mL in carbonate 0.05 M sodium pH 9.6, overnight at room temperature);

it was blocked with 5% sucrose, 1% BSA in PBS, and stored at 4 ° C. For the standard curve, human cardiac troponin (BiosPacific Cat # J34000352) was used. The diluent for the standard concentrations was human serum that was immunocompromised for endogenous cTNI, aliquoted, and stored at -20 ° C. Dilution of the standards was done in a 96-well, conical, polypropylene, (Nunc product # 249 944). The following buffers and solutions were used: (a) Assay buffer: BBS with 1% BSA and 0.1% Triton X-100;

(b) passive blocking solution in assay buffer containing 2 mg / mL mouse IgG, (Equitech Bio);

mg / mL of goat IgG, (Equitech Bio); and 2 mg / mL of poly MAK33, Roche # 11939661; (c) detection antibody (Ab): the affinity of the purified goat polyclonal antibody to Peptide 3, (BiosPacific G129C), which was labeled with a fluorescent dye Alexa Fluor 647, and stored at 4 ° C; Detection Antibody Diluent: 50% Assay Buffer, 50% Passive Blocking Solution; Wash Buffer: Borate Buffer Saline Triton Buffer (BBST) (1.0 M borate, 15.0 M sodium chloride, 10% Triton X-100, pH 8.3); Elution buffer solution: BBS with 4 M urea, 0.02% Triton X-100 and 0.001% BSA.

Preparation of Alexa Fluor 647 Labeled Antibodies: Detection Antibody G-129-C Was Conjugated to Alexa

Fluor 647, first dissolving 100 ug of G-129-C in 400uL of coupling buffer 40 (0.1M NaHCO3). The antibody solution was then concentrated to 50 ul by transferring the solution onto a filter.

YM-30 and submitting the solution and filter to centrifugation. The YM-30 filter and the antibody were then washed three times by adding 400ul of the coupling buffer. The antibody was recovered by adding 50 µl to the filter, inverting the filter, and centrifuging for 1 minute at 5,000 x g. The resulting antibody solution was 1-2 ug / ul. Alexa Fluor 647 NHS ester was reconstituted, by adding 20 ul of DMSO to a vial of 45 Alexa Fluor 647, this solution was stored at -20 ° C for up to one month. 3uL of Alexa stock solution were added

Fluor 647 to the antibody solution, which are then mixed and incubated in the dark for one hour. After one hour, 7.5ul of 1M tris was added to the Alexa Fluor 647 antibody solution and mixed. The solution is ultrafiltered with YM-30 to remove low molecular weight components. The volume of the retentate, containing the Alexa Fluor 647 conjugated antibody, was adjusted to 200-400 µl by the addition of PBS. 3uL of 10% NaN3 50 were added to the solution, the resulting solution was transferred to an Ultrafree 0.22 centrifuge unit and centrifuged for 2 minutes at 12,000 x g. The filtrate containing the conjugated antibody was collected and used in the assays.

Procedure: cTnI standard and sample preparation and analysis:

IS 2 550 004 T3

The standard curve was prepared as follows: working standards (0 - 900 pg / mL) were prepared by serial dilutions of the stock solutions of cTnI in standard diluent or to achieve a range of cTnI concentrations between 1.2 pg / mL - 4.3 mg / mL.

10 mL of passive blocking solution and 10 mL of standard or sample were added to each well. Standards 5 were performed in quadruplicate. The plate was sealed with Axyseal sealing film, centrifuged for 1 min at 3000

RPM, and incubated for 2 hours at 25 ° C with shaking. The plate was washed five times, and spun until the rotor reached 3000 RPM in an inverted position on a paper towel. A 1 nM working dilution of detection antibody was prepared, and 20 mL of detection antibody was added to each well. The plate was sealed and centrifuged, and the assay was incubated for 1 hour at 25 ° C with shaking. 30 mL of 10 elution buffer was added per well, the plate was sealed and incubated for the X hour assay at 25 ° C. The plate was stored for up to hours at 4 ° C prior to analysis, or the sample was analyzed immediately.

For analysis, 20 μ £ were acquired per well at 40 mL / minute, and 5 μ £ were analyzed at 5 mL / minute. Data were analyzed based on a 4 sigma threshold. The raw signal was recorded against the concentration of the standards. A linear fit was made for the low concentration range, and a non-linear fit was made for the full standard curve. The limit of detection (LoD) was calculated as LOD = (3 x zero standard deviation) / slope of linear fit. Sample concentrations were determined from the appropriate equation (non-linear or linear) for the sample signal.

An aliquot was pumped into the analyzer. Individually labeled antibodies were measured during capillary flow by adjusting the interrogation volume such that the emission of only 1 fluorescent tag was detected in a defined space following laser excitation. With each signal representing a digital event, this configuration allows extremely high analytical sensitivities. Total fluorescent signal is determined as a sum of the individual digital events. Each molecule counted is a positive data point with hundreds of thousands of DMC events / sample. The detection limit of the cTnI assay of the invention was determined by the mean + 3 SD method.

Results: Data for a typical cTnI standard curve measured in quadruplicate using the assay protocol are shown in Table 2.

Table 2

Standard curve for cTnI

<td>cTnI (pg / mL)</td><td>Sign</td><td>Standard deviation</td><td>% CV</td>

IS 2 550 004 T3

The sensitivity of the assay system was tested in 15 assays and was routinely found to detect sub femtomol / L (FM) levels of calibrator, as shown by the data in Table 3. The precision was 10% to 4 and 12 pg / mL cTnI.

Table 3

Instrument Sensitivity

<td>Calibrator (FM)</td><td>Account sign</td><td>CV</td>

The linearized standard curve for the cTnI concentration ranges is shown in Figure 5.

The analytical limit of detection (LoD) was determined through 15 sequential tests. The LoD was the mean of the intra-assay determinations 0 std + 3 SD (n = 4). The mean LoD was 1.7 pg / mL (range 0.4 to 2.8 pg / mL).

Sample recovery was determined by analyzing serum samples that had been immunocapped for cTnI and boosted with known amounts of cTnI. Table 4 shows the sample recovery data for the analyzed system over 3 days.

Table 4

Sample Recovery

<td>Peak (pg / mL)</td><td>Recovery (average)</td><td>Standard deviation</td><td>% CV</td>

Assay linearity was determined in pooled human serum that was boosted with cTnI and diluted with standard diluent. The results in 56, show the dilutions and the% of the signal expected for the corresponding dilution.

Table 5

Linearity test

<td>Serum dilution</td><td>% of expected</td>

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These data show that the assay system of the invention enables the highly sensitive laser-induced immunoassay to be performed for sub-femtomolar concentrations of cTnI.

Example 2: Sandwich-Pearl based assays for TnI:

The assays described above use the same microtiter plate format where the plastic surface is used to immobilize the target molecules. The single particle analysis system is also compatible with assays performed in solution using microparticles or beads to achieve separation of bound from unbound entities.

Materials: MyOne Streptovidin C1 (MP) microparticles are obtained from Dynal (650.01-03, 10 mg / mL of stock). The buffers used in the assay include: 10X Borate Buffer Saline 10 Triton Buffer (BBST) (1.0 M borate, 15.0 M sodium chloride, 10% Triton X-100, pH 8.3); Assay buffer (2 mg / mL normal goat IgG, 2 mg / mL normal mouse IgG, and 0.2 mg / mL MAB-33-IgG-polymer in 0.1 M Tris (pH 8.1), 0.025 EDTA M, 0.15 M NaCl, 0.1% BSA, 0.1% Triton X-100, 0.1% NaNa and, stored at 4 ° C); and elution buffer solution (BBS with 4 M urea, 0.02% Triton X-100, and 0.001% BSA, stored at 2-8C). Antibodies used in the sandwich bead-based assay include: Bio-Ab (A34650228P (BiosPacific) with 1-2 15 biotins per IgG) and Det-AB (G-129-C (BiosPacific) conjugated to A647, 2-4 compounds fluorescent IgG). The standard is recombinant human cardiac troponin I (BiosPacific, cat # J34120352). The calibrating diluent is 30 mg / mL BSA in TBS wEDTA.

Coating the microparticles: 100 ul of the MP stock solution is placed in an Eppendorf tube. The PMs are washed three times with 100 ul of BBST wash buffer, by applying a magnet, removing the supernatant, removing the magnet, and resuspended in wash buffer. After washing the

MP are resuspended in 100 ul of assay buffer and 15 ug of Bio-Ab were added. The mixture is then incubated for one hour at room temperature with constant mixing. The PMs were washed five times with 1 mL wash buffer, as described above. After the PM washes, they are resuspended in 15 mL of assay buffer (or 100 ul to store at 4 ° C).

Standard and sample preparation: The standard is diluted with calibrator diluent to prepare the appropriate standard curve (typically 200 pg / mL to 0.1 pg / mL). Serum and frozen plasma samples need to be centrifuged for 10 minutes at room temperature at 13K rpm. The cleared serum / plasma is carefully removed to avoid the adoption of any possible pellets or floats and placed in new tubes. 50 ul of each standard or sample is pipetted into the appropriate wells.

Target capture: 150 ul MP (after resuspension to 15 mL in assay buffer + 400 mM NaCl) are added to each well. The mixture was incubated in JitterBug, 5 at room temperature for 1 hr.

Washing and Detection: The plate is placed on a magnet and the supernatant is removed after ensuring that all PMs are captured by the magnet. 250 ul of wash buffer is added after removing the plate from the magnet. The plate is then placed on the magnet and the supernatant is removed after ensuring that all PMs are captured by the magnet. 20 ul of Det-Ab are added per well (Det-Ab at 500 ng / mL is diluted in assay buffer + 400 mM NaCl)). The mixture was incubated in JitterBug, 5 at room temperature for 30 min.

Wash and Elution: The plate is placed on a magnet and washed three times with wash buffer. The supernatant is removed after ensuring that all PM are captured by the magnet and 250 ul of wash buffer was added. After the washes the samples are transferred to a new 96-well plate. Next, the new plate is placed on the magnet and the supernatant is removed after ensuring that all PMs are captured by the magnet. 250 ul of wash buffer is then added after removing the plate from the magnet. The plate is then placed over the magnet and the supernatant is removed after ensuring that all PMs are captured by the magnet. Then 20 ul of elution buffer are added and the mixture is incubated in JitterBug, 5 at room temperature for 30 min.

The PMs are filtered and transferred to the 384-well plate: The standard and samples are transferred to a 384-well filter plate placed on top of a 384-well assay plate. The plate was then centrifuged at room temperature at 3000 rpm with a plate rotor. The filter plate is removed and the appropriate calibrators are added. The board is covered and ready to be analyzed in SMD.

SMD: An aliquot is pumped into the analyzer. Individually labeled antibodies are measured during capillary flow by defining the interrogation volume in such a way that the emission of only 1 fluorescent molecule in a defined space is detected following laser excitation. Where each signal represents a digital event, this configuration allows extremely high analytical sensitivities. The total fluorescent signal is determined as a sum of the individual digital events. Each counted molecule is a positive data point with

ES 2 550 004 T3 Hundreds of thousands of DMC events / sample. The detection limit of the cTnI assay of the invention is determined by the mean 3 SD method.

Example 3. Range of cTnI concentration in a population of normal non-diseased subjects.

A reference range or normal range for human serum cTnI concentrations was established using 5 serum samples from 88 apparently healthy (non-diseased) subjects. A sandwich immunoassay was carried out as described in Example 1 and the number of signals or events was counted as described above using the single particle analysis system of the invention. The serum troponin I concentration was determined by correlating the signals detected by the analyzer with the standard curve as described above. All trials were performed in quadruplicate.

According to the current European and American Cardiology Societies (ESC / ACC) recommendations, troponin assays should accurately quantify the 99th percentile of normal values with an assay imprecision (CV) of less than 10%, with the in order to reliably distinguish between patients with ACS and patients without ischemic heart disease and stratify the risk of adverse cardiac events. The assay showed that the biological threshold (cutoff concentration) for TnI is at a TnI concentration of 7 g / mL, which is established at the 99th 15th percentile with a corresponding CV of 10% (Figure 5). A 10% CV level of the precision profile points at a TnI concentration of 4 and 12 pg / mL.

In addition, the assay correlates well with measurements of the troponin I standard, provided by the National Institute of Standards and Technology (Figure 6).

The assay of the invention is sufficiently sensitive and accurate to meet ESC / ACC requirements, and is the 20 most sensitive assay for cardiac troponin I compared to assays such as those described by Koerintervalo et al. (Ann Clin Biochem, 42 : 19-23 (2005) The assay of the invention has a sensitivity 10-20 times greater than that of currently available assays, which has determined that the biological threshold range is 111 to 333 pg / mL of cTnI.

Example 4. Detection of the early release of TnI in the circulation of patients with acute myocardial infarction (AMI)

Study 1: 47 serial samples were obtained from 18 patients who presented chest pain in the emergency department (ED). These patients all had elevated EKGs without ST, and were diagnosed with AMI. The cTnI concentration in the initial samples from the 18 patients was determined according to a commercial test at the time of admission to the emergency room that was <350 pg / mL (10% cut-off point), and 12 were < 100 pg / mL percentile (99%). These samples were subsequently analyzed with the same commercial assay, and were determined to test positive for cTnI. The same serum samples were also analyzed for TnI according to the assay of the invention as described in Examples 1 and 3, and the results were compared with the results obtained using the commercial assay.

Blood was drawn for the first time at the time the patient presented with chest pain (sample 1), and subsequently at intervals between 4-8 hours (sample 2 at 12 hours; sample 3 at 16 hours; sample 4 to 24 hours 35; sample 5 to 30 hours; sample 6 at 36 hours; sample 7 to 42 hours, and sample 8 at 48 hours). The serum was analyzed by the methods of the invention and by a current commercial method, and the results obtained are shown in Figure 7. The analyzer of the invention detects TnI at the time the patient presented with chest pain (sample 1), whereas the commercial assay first detects cTnI at a much later time (sample 6 at 36 hours). The TnI concentration in sample 3 exceeds the biological threshold level that was established using the analyzer of the invention (7 pg / mL, see figure 5), and indicated that sample 3 is positive for TnI to suggest the incidence of a cardiac event. The biological threshold for the commercial assay is between 111 and 333 pg / mL of TnI. Consequently, sample 3 would not have been considered to indicate a possible cardiac event.

Furthermore, the methods of the present invention allow much earlier diagnosis and possible intervention, based on cardiac troponin levels, as evidenced by the results for the first sample taken from all 45 patients. In the 3 cases that had initial values, from the commercial cTnI assay between 100 and 350 ng / mL, all were positive for cTnI by the analytical methods of the invention (ie, cTnI over 7 pg / mL). In the 12 cases that had initial commercial cTnI values of less than 100 pg / mL, 5 were determined to be positive for a cardiovascular event according to the assay of the invention (ie, cTnI over 7 pg / mL). Prospective use of the invention assay would have detected 53% more AMI cases than the current commercial trial when the 50 intake sample was evaluated.

Study 2: 50 additional serum samples, which were negative according to the commercial test, were tested using the analyzer and assay of the invention. The results are shown in Figure 8. Of the 50 samples, 36 were within 99% and were determined to be within the normal range established by the assay of the invention. Without

ES 2 550 004 T3 however, the remaining 14 samples were determined to be within the normal or non-diseased commercial range, tested above the biological threshold established by the invention.

Therefore, the high sensitivity cTnI assay of the invention allows the detection of myocardial damage in patients when serum cTnI levels are below the threshold values by commercially available technology. 5 The use of the highly sensitive and accurate cTnI assay of the invention allows detection of AMI earlier than with existing cTnI assays, thereby providing the opportunity for appropriate diagnosis and early medical intervention to improve outcome.

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789304P	United States of America	–	2006-04-04
78930406	United States of America	P	2006-04-04
793664P	United States of America	–	2006-04-19
79366406	United States of America	P	2006-04-19
808622P	United States of America	–	2006-05-26
80862206	United States of America	P	2006-05-26
861498P	United States of America	–	2006-11-28
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87298606	United States of America	P	2006-12-04

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US2005216313A1	United States of America	A1	2005-09-29
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WO2006036182A3	World Intellectual Property Organization (WIPO)	A3	2007-01-18
EP1805500A2	European Patent Office (EPO)	A2	2007-07-11
US2007214018A1	United States of America	A1	2007-09-13
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Highly sensitive system and methods for analysis of troponin

Abstract

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DESCRIPTION

BRIEF DESCRIPTION OF THE DRAWINGS

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Priority claims10

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