Modified cardiolipin and method for using the same

Field The present disclosure relates to modified cardiolipin compositions and their use, specifically methods of immobilizing modified cardiolipin on solid supports, and related immunoassays (such as ELISA) and immunoassay devices (test strips, influx (test strips, influx). With respect to (such as flow-through) devices, or lateral flow devices), these assays and devices are useful, for example, in detecting anti-lipoid antibodies and / or diagnosing diseases (such as syphilis).

Cross-reference of related applications This application claims the benefit of US Patent Provisional Application No. 60 / 737,901 filed on November 18, 2005, which is incorporated herein by reference in its entirety.

Approval of government support The present invention relates to the National Centers for HTV, STD, and TB Prevention, AIDS, STD, and TB of the Centers for Disease Control and Prevention, which is an organization of the US government. It was conducted by the Division of AIDS, STD, and TB Laboratory Research, and the Laboratory Reference and Research Branch.

background Syphilis is a sexually transmitted disease (STD) caused by the spirochete Treponema pallidum. Over 100,000 cases of adult syphilis are reported worldwide each year. In addition, the disease is congenitally transmitted and affects 3,000 or more infants each year. The course of syphilis infection is long-standing and can lead to a variety of clinical findings characterized by four stages.

The first stage of syphilis infection occurs 10-100 days after bacterial infection and is characterized by the appearance of one or more chancroids (typically red, bloodless, painless ulcers less than 1 cm in diameter). .. This chancroid can appear on the genitals or other parts of the body. Chancroid lasts for 3-6 weeks, heals without treatment, leaving small scars. Infected individuals are contact contagious during this stage.

The second stage of syphilis infection is characterized by rash-like skin lesions that can cover part or all of the body. This skin lesion is generally painless and appears 1 to 6 months after the onset of early chancroid. Skin lesions can resemble warts, small pustules or ulcers. If left untreated, they heal in 2-12 weeks without scarring. Fever, sore throat, weakness, weight loss, swelling of the lymph nodes, and partial loss of eyelashes and / or eyebrows can also occur during this period of infection. In addition, the symptoms may progress to meningeal vascular syphilis, which is characterized by inflammation of the meninges that line the brain and spinal cord and / or changes in the cerebrovascular system. Infected individuals are also contact contagious in Phase 2.

The next stage of the disease is latent syphilis or a hidden stage. During this stage, the infected person appears to be recovering and is asymptomatic. This stage lasts for life in approximately two-thirds of those who have not been treated for syphilis. In the first year of latency, a recurrence of stage 2 symptoms can occur. Infected individuals are not contact-infectious during this incubation period, except during recurrence; however, children born to children of latently infected mothers within 4 years of the appearance of early chancroids suffer from congenital syphilis. There is a risk of being infected.

Stage 3 or late syphilis is the final stage of untreated infection. This stage may appear as early as 1 year after infection or at any time thereafter, but 10-20 years is the most common. Benign syphilis, characterized by lesions called gumma, can occur in the bones, skin, and internal organs. Death is rare, but severe deformity and pain can occur. Cardiovascular syphilis is characterized by aortic aneurysms and other cardiovascular disorders and often results in death. Not only does neurological involvement manifest as a late symptomatology, but it can also occur in the early stages of syphilis. In late-stage illness, neurosyphilis can be asymptomatic, or the patient has serious neurological problems such as possible dementia, psychosis, movement disorders, blindness, hearing loss, or even death. sell.

The immune response in syphilis includes (i) Treponema antibodies specific for Treponema pallidum antigens, and (ii) lipoid materials released from damaged host cells, lipoprotein-like materials and possibly cardiolipin released from Treponema. Acknowledged, with the production of anti-lipoid antibodies. A pillar of syphilis screening and diagnosis is serological testing of one or both of these two antibodies.

Anti-lipoid antibody tests (often referred to as "non-treponema tests") are typically based on antigens composed of natural cardiolipin, cholesterol and lecithin. Widely used non-treponema tests (eg, sexually transmitted disease laboratory (VDRL) test and rapid plasma reagin (RPR) test) microscopically (eg,) the formation of cotton-like masses composed of antigen-antibody complexes. , VDRL test) or microscopically (eg, RPR test). Non-treponema tests have the advantages of being widely available, inexpensive and convenient to perform on large numbers of specimens. In addition, the titer of anti-lipoid antibody diminishes with successful treatment of syphilis and eventually disappears in most patients, whereas the titer of treponema antibody remains high over the long term or even for life. Non-treponema trials are considered to be a better choice for treatment monitoring or reinfection trials.

The Treponema test is based on an antigen derived from Treponema pallidum and includes Treponema pallidum particle aggregation (TP-PA), fluorescent treponema antibody absorption test (FTA-ABS) and enzyme immunoassay. Treponema tests are primarily used to verify reactivity in non-treponema tests. The Treponema pallidum test can also be used to confirm the clinical impression of syphilis for which the non-treponema pallidum test is non-reactive. The Treponema test is technically more difficult, time consuming, and costly to perform, and the test remains responsive for long periods of time or for life in approximately 85% of those who successfully treat syphilis. It seems to be so that it cannot be used to monitor treatment.

Each of the above antibody tests is performed using serum samples obtained in the clinical setting and sent to the laboratory for analysis. Therefore, test results are usually not available days after the samples are collected. Due to the often difficult follow-up of patients with STDs, rapid development of point-of-care trials is needed to help clinicians make decisions, preferably on the day of the trial.

Immunoassay devices (such as test strips, influx devices, or lateral flow devices) that provide rapid, field results are available for qualitatively testing serum levels of Treponema antibody (such as test strips, influx devices, or lateral flow devices). For example, DiaSys Corporation; ACON Laboratories, Inc .; Biokit, SA; Genix Technology; Standard Diagnostics; Cortez Diagnostics, Inc .; and Phoenix Bio-Tech Corp). However, similar tests for anti-lipoid antibodies have shown that, at least one, the hydrophobic antigen of the anti-lipoid antibody (eg, cardiolipin) resists attachment to a solid support, which is one element of the immunoassay device. , Was even more difficult to develop.

According to some experts, detection of syphilis appears to be further facilitated by a combination of non-treponema and treponema tests for screening and diagnostic purposes. This is a method proposed by World Health Organization, Treponemal Infections, Technical Report Series 674, Geneva: WHO, 1982 (Non-Patent Document 1). An easy-to-use, rapid, point-of-care trial that can detect both non-treponema and treponema antibodies at the same time will help meet this long-standing urgent need.

<p><nplcit num="1"><text>World Health Organization, Treponemal Infections, Technical Report Series 674, Geneva: WHO, 1982</text></nplcit></p>

Overview Efforts to develop non-solution immune assays for non-treponema tests (or combinations of non-treponema and treponema tests) have made antigens specifically recognized by anti-lipoid antibodies, such as cardiolipin, like nitrocellulose strips. However, it has been frustrated because it is difficult to attach it to a solid substrate. Due to the very small size of cardiolipin molecules, the localization of the molecules on the substrate is inadequate. The non-polarity of the fatty acid side chains of cardiolipin also gives the molecule a high degree of hydrophobicity, making it difficult to attach cardiolipin to polar surfaces such as nitrocellulose. By conjugating cardiolipin to a larger molecule (such as a protein), the size of the molecule can be increased, but such conjugation resulted in the loss of cardiolipin's antigenicity.

The present disclosure maintains cardiolipin (or lipoid antigens containing cardiolipin modified as described herein, phosphatidylcholine (also referred to as "lecithin"), and cholesterol while maintaining the antigenicity and specificity of the antigen against anti-lipoid antibodies. ) Provides a method for reliably adhering to a solid substrate (such as a microporous membrane or a multiwell plate). Using the methods described herein, it is possible to create cardiolipin-attached molecular complexes that can attach to a variety of solid supports in the future. This ability to attach the immunogenic cardiolipin complex makes it useful for non-solution based immunoassays such as ELISA and rapid, field-testing immunoassay devices for non-treponema antibodies. Will be possible. In certain embodiments, the disclosed immunoassay device also incorporates the Treponema antigen recognized by Treponema pallidum, so the device conveniently simultaneously detects both non-treponema and treponema antibodies.

To give a specific example, the fatty acid side chain of cardiolipin is oxidized to provide at least one terminal carboxyl group, which in turn is a permeable substrate for a solid support (multiwell plate or lateral flow strip). Crosslinks to polypeptides (such as BSA) that are easily (or already attached) to (such as).

The aforementioned and other features and advantages will become even more apparent from the detailed description of some of the embodiments that proceed in connection with the accompanying drawings.

<figref num="1">A digital image of the oxidation stage of cardiolipin is shown. (A) Unmodified cardiolipin in t-butanol; (B) Cardiolipin reaction mixture after addition of m-sodium permanganate and potassium permanganate; (C) Cardiolipin reaction mixture after addition of sodium bisulfite; and ( D) A two-phase solution obtained after centrifugation of the mixture (C), where some oxidized forms of cardiolipin are found primarily in the upper t-butanol phase.</figref><figref num="2">A digital image of a thin layer chromatograph of a cardiolipin preparation is shown. (A) Unmodified cardiolipin in t-butanol (see Figure 1A); (B) Cardiolipin oxide preparation; (D1) Upper t-butanol phase after cardiolipin oxidation reaction (see Figure 1D); and (D2) The lower aqueous phase after the cardiolipin oxidation reaction (see Figure 1D). The direction of movement of each sample is indicated by an arrow.</figref><figref num="3">The illustrated oxidation reaction of an exemplary cardiolipin molecule containing four 18-carbon, di-unsaturated fatty acid chains is shown. The cardiolipin reaction product shown in the figure has either one or four oxidized fatty acid side chains. In practice, the oxidation reaction product will also include cardiolipin oxide species in which any two or three fatty acid chains are oxidized to carboxyl groups.</figref><figref num="4">Demonstrates a schematic reaction useful for covalently linking cardiolipin oxide to amine-containing adherent molecules such as proteins. In the example shown in the figure, the attached molecule is BSA.</figref><figref num="5">Figure 3 shows a digital image of the dot blot assay used to determine the antigenicity of the cardiolipin oxide-BSA conjugate (Preparation 5 in Table 2).</figref><figref num="6">Shown are digital images of five different physical aspects of the lateral flow device that can be used in the disclosed method. The embodiments of the apparatus shown in (A), (B) and (E) are configured such that each can be immersed or partially submerged in a sample or a solution containing the sample. The embodiment of the apparatus shown in (C) and (D) is configured to receive a large amount of sample (or a solution containing the sample) as a droplet into the sample inlet.</figref><figref num="7">It is a perspective view of the physical aspect of the side flow device which cut out a part of a housing and showed the basic component of a device and its relationship with each other.</figref><figref num="8">A digital image of a thin layer chromatograph of a cardiolipin-lecithin mixture is shown. (A) Unmodified cardiolipin and unmodified lecithin mixture in t-butanol; (B) Oxidized cardiolipin / lecithin mixture; (C) Upper post-oxidized cardiolipin / lecithin mixture, t-butanol phase; and (D) The lower, aqueous phase of the cardiolipin / lecithin mixture after oxidation. The direction of movement of each sample is indicated by an arrow.</figref><figref num="9">The chemical structures of four typical natural phosphatidylcholine (lecithin) molecules are shown. For L-α-phosphatidylcholine (heart, bovine), the molecular formula is C₄₂H₈₀NO₈It is P, has a molecular weight of 758.07, a molecular weight (isotope) of 757.562157, and a composition percentage of C 66.55% H 10.64% N 1.85% O 16.88% P 4.09%. For L-α-phosphatidylcholine, plasmalogen (heart, bovine), the molecular formula is C₄₂H₈₀NO₇It is P, has a molecular weight of 742.07, a molecular weight (isotope) of 741.567242, and a composition percentage of C 67.98% H 10.87% N 1.89% O 15.09% P 4.17%. For L-α-phosphatidylcholine, ether (heart, bovine), the molecular formula is C₄₂H₈₂NO₇It is P, has a molecular weight of 744.09, a molecular weight (isotope) of 743.582893, and a composition percentage of C 67.80% H 11.11% N 1.88% O 15.05% P 4.16%.</figref><figref num="10">Shown are digital images of the dot blot assay used to determine the antigenicity of the six cardiolipin / lecithin-BSA or cardiolipin / lecithin-KLH conjugates. The amount of each antigen applied to each dot in the assay is shown in the table below the blot.</figref>

Detailed explanation I. Introduction As shown in FIG. 3, cardiolipin contains a central immunogenic glycerol moiety with a fatty acid side chain. This specification discloses a method for preparing an oxidized cardiolipin that can be linked to a polypeptide for attachment to a substrate while retaining the ability of a central glycerol moiety that is immunoreactive with an anti-lipoid antibody. In aspects of the disclosure, cardiolipin is permanganate (m-sodium periodate) to oxidize at least one of the fatty acid side chains of cardiolipin so as to provide terminal carboxyl groups for one or more of the side chains. To stop the oxidation of cardiolipin to retain the immunogenicity of the central glycerol moiety and to the central glycerol moiety after reacting with (such as) and permanganate (such as potassium permanganate). A reducing agent (such as hydrogen sulfite) is added to reduce the formed β-ketone to β-hydroxynate groups. In an aspect of the disclosure, cardiolipin is reacted with periodate before it is reacted with permanganate. In certain embodiments, the oxidation reaction with cardiolipin is carried out in an alcohol solvent under an argon atmosphere. Examples of suitable alcohol solvents are one or more of t-butanol, ethanol, propanol, or methanol. The molar ratio of sodium m-sodium permanganate to cardiolipin is about 4: 1 to about 5: 1, and the molar ratio of potassium permanganate to cardiolipin is about 0.5: 1 to about 1: 1. In some examples, the hydrogen sulfite is sodium bisulfite.

The carboxyl groups of cardiolipin oxide can be activated to covalently attach the polypeptide carriers to at least one of the carboxyl groups. Carboxyl activation, in certain cases, cardiolipin oxide with carbodiimides such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and succinimides such as N-hydroxysuccinimide (NHS). It is achieved by reacting. The polypeptide (eg, bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), or avidin), or biotin is then covalently attached to at least one of the activated carboxyl groups.

Also disclosed herein are methods for preparing oxidized cardiolipin that is immunoreactive with anti-lipoid antibodies and can be linked to a polypeptide for attachment to a substrate. Such methods use cardiolipin under an argon atmosphere in a t-butanol solvent to oxidize cardiolipin and to provide terminal carboxyl groups in one or more of the side chains with sodium permanganate and Including the step of reacting with potassium permanganate. Then, in order to retain the immunogenicity of the central glycerol moiety, to stop the oxidation of cardiolipin, and to reduce the β-ketone formed in the central glycerol moiety to the β-hydroxyl group, an aqueous solution. Sodium bisulfite in is added to the cardiolipin suspension. The terminal carboxyl group of cardiolipin oxide is then activated by reacting EDC and then NHS with cardiolipin oxide, followed by covalently conjugating the protein carrier to at least one of the carboxyl groups.

Oxidized cardiolipin conjugated to a polypeptide carrier is then applied in one application to create a lateral flow chromatography apparatus by attaching the cardiolipin-polypeptide conjugate to a lateral flow substrate, such as a nitrocellulose strip. Can be used to The ability to provide cardiolipin conjugated to a polypeptide ensures that cardiolipin can adhere to a solid substrate. Such a substrate can also contain a Treponema antigen that reacts with an antibody against Treponema pallidum. The presence of both cardiolipin-polypeptide conjugates and treponema antigens on (or in) the same solid support conveniently allows rapid clinical trials for both anti-lipoid and anti-treponema antibodies in the same biological specimen. ..

Also disclosed herein are immunoassay devices (such as lateral flow devices or influx devices) for determining the presence and / or amount of anti-lipoid antibody in a fluid sample. These devices were typically provided with a sample application region and an immobilized cardiolipin-polypeptide conjugate, the conjugate of which has a specific binding affinity for a mobile phase antilipoid antibody. Includes a separate cardiolipin capture area. Any liquid applied during the sample application area (such as a biological sample of fluid) will flow along the flow path from the sample application area to the cardiolipin capture area. This flow path can lead to at least one downstream resorbable pad associated with the immunoassay device that acts as a liquid reservoir. The formation of a complex between the anti-lipoid antibody and the immobilized cardiolipin oxide can be detected to determine the presence and / or amount of anti-lipoid antibody in the fluid sample.

In some embodiments of the lateral flow device, the conjugate pad is located in the flow path from the sample application area to the cardiolipin capture area. The conjugate pad contains a detection reagent for mobility or mobility against the anti-lipoid antibody, so the flow of liquid through the pad transfers the detection reagent to the cardiolipin capture region. The formation of a complex containing a detection reagent, an anti-lipoid antibody (analyte), and immobilized cardiolipin provides a visible or otherwise detectable indicator of the presence of the anti-lipoid antibody in a biological specimen. In another embodiment (including a lateral flow device or inflow device), the detection reagent is not fed to the conjugate pad, but instead is applied to the substrate or sample, for example from a developer container. To.

Examples of detection reagents include enzymes, colloidal gold particles, colored latex particles, silver particles adsorbed on proteins, iron particles adsorbed on proteins, copper particles adsorbed on proteins, and selenium adsorbed on proteins. One or more of the particles, the sulfur particles adsorbed on the protein, the tellurium particles adsorbed on the protein, the carbon particles adsorbed on the protein, or the protein-conjugated diesac (dye sac), labeled cardiolipin oxide or Included are labeled anti-human antibodies.

In some embodiments of the immunoassay device (such as a lateral flow device or inflow device), the Treponema pallidum capture region is also included in the flow path from the sample application region. Such a treponema capture region may be, for example, an immobilized treponema antigen having a specific binding affinity for a mobile phase anti-Treponema pallidum antibody or a specific binding affinity for a mobile phase Treponema pallidum organism or a Treponema pallidum antigen. Immobilized anti-treponema pallidum antibody with. The lateral flow device can also have a treponema antibody or antigen-specific mobility or mobility detection reagent in the conjugate pad. The detection reagent for the Treponema antibody or antigen may be in the same or different pad as the detection reagent for the anti-lipoid antibody. In certain embodiments, the detection reagents specific for the anti-treponema antibody are labeled (eg, gold-conjugated) protein A, labeled (eg, gold-conjugated) protein G, or labeled (eg, gold-conjugated) anti-human. Contains antibodies. In another embodiment, the detection reagent for the mobile Treponema pallidum antigen can be a labeled (eg, gold-conjugated) anti-Treponema antigen antibody.

The disclosed immunoassay device is a subject-derived living body by application of a biological sample to the device and detection of the formation of a complex between an anti-lipoid antibody, oxidized cardiolipin, and a detection reagent in a capture region. It can be used as a method for diagnosing syphilis in a subject by analyzing the sample. Detection of complex formation in the capture area detects anti-lipoid antibodies associated with syphilis infection. In an embodiment in which the device includes a conjugate pad in the flow path from the sample application area to the cardiolipin capture area, the complex detected comprises a mobility or mobility detection reagent. In other embodiments where the detection reagent is applied to the device from an external source, the complex detected comprises an externally applied detector.

In a particularly advantageous embodiment of the method, the lateral flow device can detect both anti-lipoid antibody (which is an indicator of active infection) and anti-treponema antibody (which verifies the reactivity of non-treponema tests). In the embodiment of the device comprising the treponema antigen, the method comprises detecting the formation of a complex between the anti-treponema pallidum antibody in the capture region, the immobilized treponema antigen and the detection reagent. Similar to the detection reagents used for cardiolipin, the detection reagents for Treponema pallidane antigen may be provided in the device or applied from an external source.

Also disclosed herein are kits for the diagnosis of syphilis. These kits include the disclosed immunoassay device and instructions for applying the biological sample to the sample application area or device. The kit can also include instructions for interpreting the results of the test.

The disclosed immunoassay device is also subject-derived by application of a biological sample to the device and detection of the formation of a complex between the anti-lipoid antibody, oxidized cardiolipin, and the detection reagent in the capture region. Can be used as a method for diagnosing wheal in a subject by analysis of a biological sample of. Detection of complex formation in the capture area detects anti-lipoid antibodies associated with lupus. In some cases, one or more cofactors (β)₂-A presence (such as glycoprotein I) is present for the detection of lupus (eg, added to the sample).

II. Abbreviations and terms BSA bovine serum albumin EDC 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide ELISA enzyme-linked immunoadsorption assay HPLC high pressure liquid chromatograph KLH Keyhole Limpet Hemocyanin LFD lateral flow device MES N-morpholinoethan sulfonic acid NHS N-Hydroxysulfosuccinimide NMR nuclear magnetic resonance PEG polyethylene glycol PVA polyvinyl alcohol PVP polyvinylpyrrolidone SDS sodium dodecyl sulfate TLC thin layer chromatography

Unless otherwise noted, technical terms are used in accordance with conventional usage. Definitions of common terms in molecular biology are given by Benjamin Lewin, Genes V, 1994 (ISBN 0-19-854287-9), published by Oxford University Press; Kendrew et al., Published by Blackwell Science Ltd. ( Ed.), The Encyclopedia of Molecular Biology, 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Published by VCH Publishers, Inc., Molecular Biology and Biotechnology: a Comprehensive Desk Reference, It can be found in 1995 (ISBN 1-56081-569-8).

The following description of a particular term is provided to aid in the study of various aspects of the invention.

Carboxyl group activation: Carboxyl activation is a drug, such as carbodiimide (eg, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide) for forming nucleolytic reagent-reactive derivatives, such as O-urea derivatives. It refers to the reaction of the carboxyl group due to. Nucleophile-reactive derivatives react easily with nucleophiles, (i) ether ligation with alcohol groups, (ii) ether ligation with acids and alcohols or phenols, and (iii) peptide binding with acids and amines. Can be used to form.

Catalytic auxiliary nucleophilic reagents such as 1-hydroxybenzotriazole, N-hydroxysuccinimide, N-hydroxysulfosuccinimide, and N-hydroxy-5-norben-endo-2,3-dicarbodiimide. Can be used to assist in carbodiimide-mediated activation of carboxyl groups, reduce possible side reactions, including succinimide, and increase the reaction rate when active esters are used.

For example, by reacting the terminal carboxyl group of one or more fatty acid side chains of cardiolipine oxide with 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, in some cases continuing, such as N-hydroxysuccinimide, It can be activated by reaction with a catalytic auxiliary nucleophile.

Analytical material: Atomic, molecular, molecular groups, or compounds of natural or synthetic origin (eg, drugs, hormones, enzymes, proliferations) that can be specifically bound to the aspects of cardiolipin described herein and are to be detected or measured. Factor antigens, antibodies, haptens, lectins, apoproteins, cofactors). Analysts can include, but are not limited to, antibodies, drugs, hormones, antigens, haptens, lectins, apoproteins, or cofactors. In some embodiments, the analyte comprises an antibody, such as an anti-lipoid antibody or an anti-cardiolipin antibody, produced in response to infection by Treponema pallidum. In other embodiments, the analytes are (i) autoimmune diseases such as ulcer, (ii) various venous and arterial thrombosis including cerebral infarction, (iii) deep vein thrombosis, (iv) thrombocytopenia. Includes antilipoid antibodies produced in response to either disease, (v) pulmonary embolism, or (vi) recurrent fetal loss due to cerebral infarction.

antibody: A protein consisting of one or more polypeptides that is substantially encoded by an immunoglobulin gene or fragment of an immunoglobulin gene. The recognized immunoglobulin genes include κ, λ, α, γ, δ, ε and μ constant region genes, as well as a myriad of immunoglobulin variable region genes. Light chains are classified as either κ or λ. Heavy chains are classified into γ, μ, α, δ, or ε, which define the immunoglobulin classes IgG, IgM, IgA, IgD and IgE, respectively.

The basic immunoglobulin (antibody) structural unit is generally a tetramer. Each tetramer is composed of the same two pairs of polypeptide chains, each pair having one "light" chain (about 25 kD) and one pair of "heavy" chains (about 50-70 kD). .. The N-terminus of each strand defines a variable region of about 100-110 or more amino acids that are primarily involved in antigen recognition. "Variable light chain" (V_L) And "Variable Heavy Chain" (V_H) Refers to these light and heavy chains, respectively.

Antibodies are naturally produced in plants and animals in response to antigens presented to the immune system. Naturally produced antibodies can be found, for example, in animal sera. For example, a person infected with Treponema pallidum produces antibodies against at least Treponema pallidum antigen, and antibodies (ie, anti-lipoids) to lipoid material resulting from Treponema infection, eg, from host cells damaged by the infection. Can produce antibodies).

Antibodies can exist as complete immunoglobulins or as several well-characterized fragments produced by digestion with various peptidases or by recombinant DNA methods. For example, pepsin digests the antibody under a disulfide link in the hinge region and F (ab)'₂That is, V by itself due to disulfide bonds_H-C_HIt produces a dimer of Fab, a light chain linked to 1. F (ab)'₂Is reduced under mild conditions to cleave the disulfide linkage in the hinge region, thereby F (ab)'₂The dimer can be converted to a Fab'monomer. The Fab'monomer is essentially a Fab with a portion of the hinge region (see Fundamental Immunology, WE Paul, ed., Raven Press, NY, 1993). It should be understood that while certain antibody fragments are defined by complete antibody digestion, Fab'fragments or other antibody fragments can be newly synthesized either chemically or by utilizing recombinant DNA methods. As such, the term antibody as used herein also includes antibody fragments that are either produced by modification of the whole antibody or newly synthesized by recombinant DNA methods.

The anti-lipoid antibody used in some aspects of the methods and devices disclosed herein may be of any origin, but is most likely found in the serum of a subject. ..

The antibody may be monoclonal or polyclonal. By way of example only, monoclonal antibodies can be prepared from mouse hybridomas according to Kohler and Milstein's classical method (Nature 256: 495-497, 1975) or derivative methods thereof. Briefly, mice are repeatedly inoculated with 2-3 μg of the selected analyte compound (or fragment thereof) for 2-3 weeks. In some cases, it may be beneficial to use an adjuvant or carrier molecule to increase the immunogenicity and / or stability of the analyte in the animal system. Mice are then slaughtered and spleen antibody-producing cells are isolated. Spleen cells are fused with mouse myeloma cells by polyethylene glycol and excess unfused cells are destroyed by system proliferation in selective medium (HAT medium) containing aminopterin. The successfully fused cells are diluted and a certain amount of dilution is placed in the wells of a microtiter plate where the culture continues to grow. The antibody-producing clone was originally Engvall (Meth. Enzymol. Identification by detection of antibodies in well supernatants by immunoassay procedures, such as ELISA, and derivative methods thereof, described by 70: 419-439, 1980). Selected positive clones may be grown and their monoclonal antibody products collected for use. Detailed procedures for the production of monoclonal antibodies are described in Harlow and Lane (Antibodies, A Laboratory Manual, CSHL, New York, 1988).

antigen: Chemical or biochemical compounds, compositions, structures, determinants, antigens or theirs that can stimulate the production of antibodies or T cell responses in animals, including compositions that are injected or absorbed into animals. Part. Antigens react with certain humoral or cell-mediated immunity products, including those induced by heterologous immunogens. The term "antigen" includes all relevant antigenic epitopes.

Anti-lipoid antibody: Antibodies (such as IgM or IgG) produced by the immune system of a subject (such as humans) in response to lipoid antigens present in diseased conditions, such as infections. For example, the term contemplates lipoid materials released from host cells as a result of Treponema pallidum infection, and anti-lipoid antibodies produced in response to lipoprotein-like materials, and optionally cardiolipin released from treponemas. Anti-lipoid antibodies also vary, including (i) autoimmune diseases such as lupus (Harris et al., Clin. Rheum. Dis., 11: 591-609, 1985), (ii) cerebral infarction. Viral and arterial thrombosis (Harris et al., Clin. Exp. Rheumatol., 2: 47-51, 1984), (iii) Deep venous thrombosis (Mueh et al., Ann. Intern. Med., 92: 156-159, 1980), (iv) Thrombosis (Harris et al., Clin. Rheum. Dis., 11: 591-609, 1985), (v) Pulmonary embolism (Anderson and Ali, Ann. Rheum. Dis., 43: 760-763, 1984), or (vi) Recurrent fetal loss due to placental infarction Lipoid antigens, including (Derue et al., J. Obstet. Gynaecol., 5: 207-209, 1985), may also be produced in response to diseases other than treponema, in which they are also present.

Cardiolipin is one of the well-known specific binding partners of anti-lipoid antibodies formed in response to disease conditions, such as Treponema pallidum infection. Natural cardiolipin has traditionally been used in agglutination tests, often in combination with cholesterol and lecithin, for example to detect anti-lipoid antibodies in the serum of patients infected with Treponema pallidum.

Argon atmosphere: An atmosphere that substantially contains argon gas, created for the purpose of conducting a chemical reaction. The argon atmosphere is sufficient to flush the reaction vessel with argon gas, driving away the atmosphere present in the vessel at that time, and then maintaining the atmosphere in the reaction vessel that is substantially containing argon gas. By maintaining the gas flow, it can be created in any closed container useful for conducting a chemical reaction. Atmosphere is at least about 25% Argon, at least about 30% Argon, at least about 40% Argon, at least about 50% Argon, at least about 60% Argon, at least about 70% Argon, at least about 75% Argon, at least about 80% Argon, When at least about 85% argon, at least about 90% argon, at least about 92% argon, at least about 95% argon, at least about 98% argon, or at least about 99% argon, the atmosphere substantially comprises argon gas.

Attached molecule or protein: Any polypeptide or other molecule (eg, atom, molecule, group of molecules, protein, etc.) that can be directly or indirectly linked to the cardiolipin described herein to facilitate the attachment of cardiolipin to a solid support. Polymers, or compounds of natural or synthetic origin). In some embodiments, the link between the adherent molecule and cardiolipin is formed via a carboxyl group in cardiolipin oxide, and / or optionally a linking group may be utilized.

Suitable adherent molecules are albumin, hemocyanin, tyroglobulin and derivatives thereof, especially polypeptides and proteins such as bovine serum albumin (BSA), synthetic proteins MAPS, IgY, streptavidin, avidin, and keyhole limpet hemocyanin (KLH). , Or peptides, but are not limited to these. Substances derived from other polypeptides or non-proteins are known to those of skill in the art. Adherent molecules typically have a molecular weight of at least 50,000 Daltons, preferably greater than 60,000 Daltons. Adherent molecules often contain reactive groups to facilitate covalent conjugation to cardiolipin oxide. The amine group of an amino acid is often used in this way. Adherent molecules lacking such groups can often be reacted with the appropriate chemicals to produce them.

Binding affinity: A term that refers to the strength of a molecule's bond to another molecule at a site on the molecule. If a particular molecule binds to or specifically binds to another particular molecule, then these two molecules are said to exhibit a binding affinity for each other. The binding affinity is related to the binding and dissociation constants of the molecular pair, but it is not critical to the present invention that these constants are measured or determined. Rather, the affinities used herein to describe the methods of description and the intermolecular interactions of the devices are broadly the apparent affinities observed in empirical studies (unless otherwise specified). ), To compare the relative strength at which one molecule (eg, an antibody or other specific binding partner) binds to two other molecules (eg, the analyte and the analyte tracker conjugate). Can be used. The concepts of binding affinity, binding constant, and dissociation constant are well known.

Binding domain: The molecular structure associated with the portion of the receptor that binds to the ligand. More specifically, the binding domain is a natural or synthetic polypeptide, or nucleic acid encoding such a polypeptide, either alone or in combination with other domains, of the desired ligand / receptor. It can refer to the amino acid sequence of a polypeptide corresponding to a specific (binding domain) region of a protein, which exhibits the same or similar binding properties as those of a body binding pair. Neither the specific sequence nor the specific boundaries of such domains are critically important as long as binding activity is demonstrated. Similarly, when used in such a context, binding properties necessarily include a set of affinities, binding strengths and specificities, and combinations thereof, as long as binding activity is shown.

Biological sample: Whole blood, plasma, serum, tears, mucus, saliva, urine, pleural fluid, spinal fluid, gastric juice, sweat, semen, vaginal discharge, sputum, ulcers and / or other surface rashes, blisters, fluids from abscesses, And / or any sample that can be obtained directly or indirectly from the subject, including extracts of tissues, cells or organs. The biological sample may also be a laboratory research sample, such as a cell culture supernatant. Samples are collected or obtained using methods well known to those of skill in the art.

Carbodiimide: Structure R₁-N = C = NR₂Compound, here R₁And R₂Is an independent hydrogen or hydrocarbyl group. Specific examples include HN = C = NH, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide; N, N'-dicyclohexyl-carbodiimide; and diisopropylcarbodiimide.

Cardiolipin: It contains both natural cardiolipin and any modified cardiolipin that has structural similarities to natural cardiolipin and is capable of specifically binding to anti-lipoid antibodies.

Cardiolipin capture area: A capture area where cardiolipin is immobilized as a capture reagent.

Capture reagent: Auxiliary, such as (i) sandwich assay, etc., or (ii) competitive assay, etc., detection reagent or analyte, or (iii) indirect assay, etc., which is itself specific to the analyte. An unlabeled specific binding partner that is specific to a specific binding partner. As used herein, the "auxiliary specific binding partner" is a specific binding partner that binds to the specific binding partner of the analyte. For example, the auxiliary specific binding partner can include an antibody specific for another antibody, such as a goat anti-human antibody. The "capture area" is the area of the lateral flow device on which the capture reagent is immobilized. The lateral flow device can have a plurality of capture regions, such as a "first capture region", a "second capture region", and the like. In many cases, different capture reagents can be immobilized in the first, second, or other capture regions. Multiple capture regions may have arbitrary orientation with respect to each other on the lateral flow substrate; For example, the first capture area may be distal or proximal to the second (or other) capture area, and vice versa. Alternatively, the first and second (or other) capture regions are oriented perpendicular to each other so that the two (or more) capture regions form a cross or plus sign or other symbol. May be good.

Conjugate: When used in the form of a verb, the term "conjugate" means a covalent coupling of one molecule (eg, cardiolipin oxide) to another (eg, protein). Such coupling can be achieved by chemical means, optionally with linking groups. When used in the form of a noun, the term "conjugate" means a coupled molecular complex formed by conjugation.

Detect or detect: Quantitatively or quantitatively measuring the presence of an analyte under investigation (eg, anti-lipoid antibody and / or anti-Syphilis treponema antibody). "Detecting the formation of a complex" is any method suitable for observing a particular label associated with a detection reagent; for example, a visual observation of a colored (or otherwise visible) label. , Detecting a complex containing a detection reagent by measurement or visual detection of a fluorescent label, chemiluminescent label or radioactive label.

Detection reagent (or detection reagent): A specific binding partner conjugated to a label. The detection reagent can be, for example, a labeled analyte-specific binding member (such as the gold conjugate's cardiolipin oxide-attached molecular complex) or a labeled auxiliary specific binding member (enzyme conjugate goat antibody). Includes (like human antibodies).

Epitope (or antigenic determinant): Sites on the surface of an antigen molecule to which a single antibody molecule binds; in general, an antigen has several or many different antigenic determinants and reacts with antibodies of many different specificities.

Immunogenicity: A property that can elicit an immune response in an organism. For example, if an anti-lipoid antibody has the ability to bind to an epitope present on cardiolipin oxide, cardiolipin oxide retains immunogenicity.

Sign: Any molecule attached to an analyte, analyte analog, detection reagent, or binding partner that can be detected spectroscopically, photochemically, biochemically, immunochemically, electrically, optically or by chemical means. Or the composition. Examples of labeling are disclosed, including enzymes, colloidal gold particles, and colored latex particles (US Pat. Nos. 4,275,149; 4,313,734; 4,373,932, respectively, which are incorporated herein by reference; And No. 4,954,452). Further examples of useful labels include, but are not limited to, radioisotopes, cofactors, ligands, chemical luminescent agents or fluorescent agents, silver particles adsorbed on proteins, iron particles adsorbed on proteins, proteins. Examples include copper particles adsorbed on the protein, selenium particles adsorbed on the protein, sulfur particles adsorbed on the protein, tellurium particles adsorbed on the protein, carbon particles adsorbed on the protein, and protein-conjugated disac. Adhesion of a compound (eg, a detection reagent) to a label is through covalent bonds, adsorption processes, hydrophobic and / or electrostatic bonds, or a combination of these bonds and interactions, as in chelates and the like. May also include / or linking groups.

Lateral flow device: An analyzer in the form of test strips used in lateral flow chromatography, the fluid of test samples suspected of containing an analyte is made of this strip (paper, nitrocellulose, and hygroscopic materials such as cellulose. Flows through (often due to capillary action). The test fluid and any suspended analyte can flow along the strip to the detection zone, where the analyte (if present) interacts with the detector to allow the analyte to be present, absent and / or Indicates the amount.

A number of lateral flow analyzers are disclosed, each of which is incorporated by reference in U.S. Pat. Nos. 4,313,734; 4,435,504; 4,775,636; 4,703,017; 4,740,468; 4,806,311; No. 4,806,312; No. 4,861,711; No. 4,855,240; No. 4,857,453; No. 4,943,522; No. 4,945,042; No. 4,496,654; No. 5,001,049; No. 5,075,078; 5,451,504; 5,424,193; 5,712,172; 6,555,390; and 6,368,876; European Patent 0810436; and International Publication 92/12428 (WO92 / 12428); International Publication Includes those shown in WO 94/01775; WO 95/16207; and WO 97/06439.

Many lateral flow devices are one-step lateral flow assays, in which case the biological fluid is applied to a hygroscopic strip (although a non-hygroscopic material is used, and a surfactant is applied to the material). (May be hygroscopic) and move along the strip until the liquid comes into contact with a specific binding partner that interacts with the assay in the liquid. Once the analyte interacts with the binding partner, a signal (such as a fluorescent dye or otherwise visible dye) suggests that the interaction has occurred. Multiple separate binding partners can be placed on the strip (eg, in parallel lines) to detect multiple analytes in the liquid. The test strip may incorporate a control indicator, which signals that the test was adequately performed without a positive signal indicating the presence (or absence) of the analyte on the strip. Is obtained.

Lecithin (also known as phosphatidylcholine): Lecithin is the common name for phosphatidylcholine. Phosphatidylcholine is a glycerophospholipid, which is usually the most abundant phospholipid in animals and plants. It is an important building block of the membrane bilayer and is also the major phospholipid that circulates in plasma. Phosphatidylcholine is a neutral or zwitterionic phospholipid over the pH range from strong acids to strong alkalis. Phosphatidylcholine contains two fatty acid side chains, which can have variable chemical structures, both naturally and synthetically modified. Figure 9 shows the chemical structures of four typical natural phosphatidylcholine molecules.

As used herein, the term "lecithin" includes both natural lecithin and any modified synthetic lecithin that has structural similarities to natural lecithin. For example, fatty acids in synthetic lecithin can include:<img file="JP2011237443A_D0001.tif" />

Linkage group: A chemical arm between two compounds, eg, a compound and a label (eg, an analyte and a label). Each of the reactants must contain a reactive group to achieve the required chemical structure. Typical combinations of such groups are amino with carboxyl to form an amide link; amino with an alkyl halide to form a carboxy or alkylamino with hydroxy to form an ester link; with a thiol to form a disulfide. Thiols; or thiols with maleimides or alkyl halides to form thioethers. Hydroxy, carboxyl, amino and other functional groups can be introduced by known methods if they are not present in the natural compound.

Similarly, a wide variety of linking groups can be utilized. The structure of the linkage should be a stable covalent linkage formed to attach the two compounds to each other (eg, label the analyte). In some cases, the linking group may be designed to be either hydrophilic or hydrophobic to enhance the desired binding properties of, for example, the modified ligand and its cognate receptors. The covalent linkage should be stable to the solution conditions in which the linking compound is provided. Examples of linking groups are 1 to 20 carbons and 0 to 10 heteroatoms (NH, O, S), which may be branched or straight. Without limiting the above, it is clear that only chemically compatible combinations of atoms constitute the linking group. For example, amides, esters, thioethers, thiol esters, ketos, hydroxyls, carboxyls, ether groups in combination with carbon-carbon bonds are specific examples of chemically compatible linking groups.

Operable or continuous contact: The two solid components are in direct or indirect contact with the aqueous liquid in a manner that allows the aqueous liquid to flow from one of the two components to the other virtually uninterrupted by capillarity or otherwise. If so, it is in operable contact. Direct or continuous contact means that the two elements are in physical contact, for example end-to-end or front-to-back. When the two components are in direct contact, they can overlap with an overlap of about 0.5 mm to about 3 mm. However, these components may be arranged end-to-end. "Indirect contact" means that the two elements are not physically in contact, but are bridged by one or more conductors. Operable contacts can also be referred to as "fluid conductive" or "fluid continuous" contacts.

Oxidized cardiolipin (or modified cardiolipin): Cardiolipin that has been chemically modified as described herein. For example, cardiolipin oxide is a mixture of chemically related molecules in which at least one of the four fatty acid chains in non-oxidized cardiolipin is oxidatively cleaved to give rise to a terminal carboxyl group. In some embodiments, cardiolipin oxide has the chemical structure shown in FIG.

Reducing agent: Any reduction that reduces the β-ketone group resulting from cardiolipin oxidation to the corresponding β-hydroxyl without causing substantial degradation of other oxidized cardiolipin functional groups, such as fatty acid ester groups and / or phosphate esters. Agent. Those skilled in the art can select suitable reducing agents from those taught, for example, by Larock, Comprehensive Organic Transformations, 2nd Edition, New York: John Wiley &amp; Sons, 1999. Specific examples of reducing agents include sodium bisulfite, dimethyl sulfide, and sodium cyanoborohydride (NaBH).₃CN), sodium borohydride (NaBH)₄), Sodium triacetoxyborohydride (NaBH (OAc))₃), Morphorin borane, potassium triisopropoxyhydrogenated, t-butylamine borane, dimethylamine borane, pyridineborane, triethylamine borane, trimethylamine borane.

Sample application area: The area where the fluid sample is introduced into the immunochromatography test strip, such as the immunochromatography test strip present in the lateral flow device. As an example, the sample can be introduced into the sample application area by external application, such as with a dropper or other applicator. As another example, the sample application area can be submerged directly in the sample, for example when the test strip is immersed in a container holding the sample. As another example, the sample can be poured or exuded over the sample application area.

Solid support (or substrate): Any material that is insoluble or can be made insoluble by subsequent reactions. Numerous and various solid supports are known to those skilled in the art and, but are not limited to, nitrocellulose, reaction tray well walls, multi-well plates, test tubes, polystyrene beads, magnetic beads, membranes, microparticles. Includes (like latex particles), and sheep (or other animal) red blood cells. Any suitable porosity that is porous enough to allow access by the detection reagent and has surface affinity suitable for immobilizing capture reagents (eg, cardiolipin oxide or cardiolipin oxide-adherent molecular complex). Sexual materials are contemplated by this term. For example, the porous structure of nitrocellulose has excellent absorbency and adsorptivity for a wide variety of reagents, such as trapping reagents. Nylon has similar properties and is equally suitable. Microporous structures, such as materials that have a gel structure in the hydrated state, are useful.

Further examples of useful solid supports include natural polymeric carbohydrates and synthetically modified, cross-linked or substituted derivatives thereof such as agar, agarose, cross-linked alginic acid, substituted and cross-linked guar gum, cellulose esters, especially nitrates and Esters with carboxylic acids, mixed cellulose esters, and cellulose ethers; natural polymers containing nitrogen, such as proteins and derivatives, including crosslinked or modified gelatin; natural hydrocarbon weights, such as latex and rubber. Combined; Containing synthetic polymers that can be prepared to have a suitable porous structure, such as polyethylene, polypropylene, polystyrene, polyvinyl chloride, polyvinyl acetate and partially hydrolyzed derivatives thereof, polyacrylamide, polymethacrylate. , Vinyl polymers, copolymers and ternary polymers of the above condensation polymers, such as polyesters, polyamides, and other polymers, such as polyurethanes or polyepoxides; Sulfates or carbonates of alkaline earth metals and magnesiums, such as barium sulfate, calcium sulfate, calcium carbonate, alkaline silicates and porous inorganic materials such as alkaline earth metals, aluminum and magnesium; and aluminum or silicon. Oxides or hydrates such as clay, alumina, talc, kaolin, zeolite, silica gel, or glass (these materials can be used as filters with the above copolymer materials); and mixtures of the above classes and Examples thereof include a copolymer, for example, a graft copolymer obtained by initiating the polymerization of a synthetic polymer on an existing natural polymer.

It is contemplated that the porous solid support, such as nitrocellulose described above, is preferably in the form of a sheet or strip. The thickness of such sheets or strips is in a wide range, for example, from about 0.01 to 0.5 mm, from about 0.02 to 0.45 mm, from about 0.05 to 0.3 mm, from about 0.075 to 0.25 mm, from about 0.1. It can vary from 0.2 mm, or from about 0.11 to 0.15 mm. The pore size of such sheets or strips can also vary over a wide range, for example, from about 0.025 to 15 microns, or more specifically from about 0.1 to 3 microns; However, pore size is not intended to be a limiting factor in the selection of solid supports. When applied, the flow velocity of the solid support also falls within a wide range, for example, from about 12.5 to 90 seconds / cm (ie, 50 to 300 seconds / 4 cm), from about 22.5 to 62.5 seconds / cm (ie). That is, from 90 to 250 seconds / 4 cm), from about 25 to 62.5 seconds / cm (ie 100 to 250 seconds / 4 cm), from about 37.5 to 62.5 seconds / cm (ie 150 to 250 seconds / 4 cm). Can vary from, or about 50 to 62.5 seconds / cm (ie, 200 to 250 seconds / 4 cm). In a specific embodiment of the apparatus described herein, the flow velocity is about 62.5 s / cm (ie, 250 s / 4 cm). In another specific embodiment of the device described herein, the flow velocity is about 37.5 seconds / cm (ie 150 seconds / 4 cm).

The surface of the solid support can be activated by a chemical process that causes a covalent link of the agent (eg, capture reagent) to the support. However, but not limited to, any other suitable method, including, but not limited to, ionic interactions, hydrophobic interactions, covalent interactions, etc., can be applied to the solid support with the agent (eg, capture reagent). Can be used to immobilize. The particular force that results in immobilization of the agent on the solid phase is not important to the methods and devices described herein.

The solid phase can be selected for its inherent ability to attract and immobilize agents, such as capture reagents. Alternatively, the solid phase can carry factors that have the ability to attract and immobilize agents, such as capture reagents. This factor can include, for example, a charged material that is reversely charged to the capture reagent itself or to the charged material conjugated to the capture reagent. In another embodiment, the specific binding member can be immobilized on the solid phase to immobilize its binding partner (eg, capture reagent). Therefore, in this example, the specific binding member allows indirect binding of the capture reagent to the solid phase material.

Unless otherwise physically constrained, the solid support may be used in any suitable shape, such as film, sheet, strip, or plate, or it may be of paper, glass, plastic film, or fabric. It may be coated or bound or laminated with a suitable inert carrier such as.

A "lateral flow substrate" is any solid support or substrate useful in a lateral flow device.

Specific binding partner (or binding partner): Members of molecular pairs that interact by specific, non-covalent interactions that depend on the three-dimensional structure of the molecules involved. Typical pairs of specific binding partners are antigen / antibody, hapten / antibody, hormone / receptor, nucleic acid chain / complementary nucleic acid chain, substrate / enzyme, inhibitor / enzyme, carbohydrate / lectin, biotin / (strept). Includes avidin, and virus / cell receptors.

The phrase "specifically binds to an analyte" or "specifically immunoreactive" when referring to an antibody is in the presence of a heterogeneous population of molecules, such as proteins and other biomolecules. Refers to a binding reaction that determines the presence of an analyte. Therefore, under certain immunoassay conditions, the specified antibody binds to a particular analyte and does not bind to other analytes present in the sample in significant amounts. Various immunoassay formats can be used to select antibodies that are immunoreactive specifically with a particular analyte. For example, solid phase ELISA immunoassays are routinely used to select monoclonal antibodies that are immunoreactive specifically with proteins. See Harlow and Lane, Antibodies, A Laboratory Manual, CSHP, New York (1988) for a description of immunoassay formats and conditions that can be used to determine specific immune reactivity.

Subject: A living multicellular organism, including vertebrates, a category that includes both humans and non-human mammals.

Treponema pallidum antigen: An antigen containing at least one antigenic determinant that specifically binds an anti-Syphilis treponema antibody. Numerous Treponema antigens have been reported in the art; see, for example, U.S. Pat. Nos. 6,479,248; 6,248,331; 5,681,934; 5,578,456; 4,868,118; and 4,740,467. Each of these is incorporated herein by reference. For example, polypeptides with at least the following apparent molecular weights have been reported as syphilis treponema antigens: 16-20 kDa, 18 kDa, 18-23 kDa, 25 kDa, 35 kDa, 37 kDa, 37-46 kDa, 38 kDa. , 39 kDa, 41 kDa, 43 kDa, 44 kDa, 46 kDa, 47 kDa, 58 kDa, 150 kDa; and 180 kDa (see US Pat. No. 4,846,118 for further details).

Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. The singular terms "one (a)", "one (an)" and "the" include multiple objects unless otherwise specified by the context. Similarly, the word "or" is intended to include "and" unless otherwise specified in the context. Therefore, "including A or B" means including A, or B, or A and B. It should be further understood that all base or amino acid sizes and all molecular weight or molecular weight values shown for nucleic acids or polypeptides are approximations and are given for illustration purposes. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, but suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, it is governed by this specification, including explanations of terms. Moreover, it is intended that the materials, methods, and examples are merely examples and are not limiting.

III. Cardiolipin Cardiolipin is diphosphatidylglycerol (specifically, 1,3-diphosphatidylglycerol), which has a backbone consisting of three molecules of glycerol linked by two phosphodiester bridges, as shown below. ::<img file="JP2011237443A_D0002.tif" />Each of the four hydroxyl groups in the outer glycerol moiety of cardiolipin is esterified with saturated or unsaturated fatty acid chains (typically 14-18 carbons in length). As used herein, the term "cardiolipin" contemplates 1,3-diphosphatidylglycerol having an arbitrary distribution of fatty acid side chains; provided that at least one fatty acid side chain is at least one C = C double bond. Is a condition of having. Therefore, the four fatty acid side chains of cardiolipin are of length (eg, about 14 to about 25 carbons, about 14 to about 22 carbons, about 14 to about 20 carbons, about 14 to about 18 carbons). Carbon, or about 14 to about 16 carbons) and / or saturation (eg, fully saturated to about 6 double bonds, fully saturated to about 4 doubles) Bonded or fully saturated (with about 2 double bonds) can change independently. Illustrative fatty acid side chains of cardiolipin are myristoylation (14: 0); palmitoyl (16: 0); stearoyl (18: 0); ole oil (18: 1); myristoylation oil (14: 1); palmitoyl Oil (16: 1); Petrocerinoyl (18: 1); Linole oil (18: 2); Linolenoyl (18: 3); Eicosenoyl (20: 1); Arachidonoyl (20: 4); Elcoil (22: 1); DHA (22: 6) Or include Nerbonoyle (24: 1) independently.

In some embodiments, cardiolipin is in its natural form. Natural cardiolipin is commercially available from several sources, such as Sigma Aldrich, Avanti Polar Lipids (Alabaster, AL), and Lee Laboratories (Grayson, GA). It can also be obtained, for example, by solvent extraction of bovine myocardial tissue, by precipitation, or by high pressure column chromatography. The fatty acid composition of natural cardiolipin is broadly similar to a wide variety of natural fatty acids such as palmitoyl (16: 0); stearoyl (18: 0); oleoyl (18: 1); and linoleoyl (18: 2). It is distributed. The most abundant fatty acid molecular species in the natural form of cardiolipin is 90% linoleic acid, followed by 5% oleic acid and 1% palmitic acid.

In other embodiments, cardiolipin is a non-natural form (also referred to as "synthetic cardiolipin"). Non-limiting examples of synthetic cardiolipin include, for example, tetraoleoyl cardiolipin, bis (dipalmitoyl D, L-α-glycerylphosphoryl) -1,3-glycerol benzyl ether disodium salt, bis (dipalmitoyl D, L-). α-Glycerylphosphoryl) -1,5-pentanediol disodium salt, bis (dipalmitoyl D, L-α-glycerylphosphoryl) -1,3 propanediol disodium salt, bis (dipalmitoyl D, L-α-glycerylphosphoryl) ) -1,4 Butanediol disodium salt, bis (dipalmitoyl D, L-α-glyceryl phosphoryl) -1,2 ethanediol disodium salt, bis (dipalmitoyl D, L-α-glyceryl phosphoryl) -methanediol Disodium salt, bis (dipalmitoyl D, L-α-glycerylphosphoryl) -1,3glycerol disodium salt, bis (benzylphosphoryl) -1,3-propanediol disodium salt, or D, L-α-di Palmitoylbis-phosphatidic acid can be mentioned.

The antigenic epitope of cardiolipin is believed to contain two phosphate and β-hydroxyl groups in the central glycerol moiety. This antigen epitope is present in both natural and synthetic cardiolipin. One or both of natural cardiolipin and synthetic cardiolipin may be oxidized as described herein as long as the anti-lipoid antibody specifically binds to its oxidized form.

IV. Oxidation of cardiolipin A. General Oxidative cleavage of the fatty acid side chain of cardiolipin is NaIO₄And KMnO₄, Or NaIO₄And ruthenium tetroxide, or HIO₄And KMnO₄Those skilled in the art will understand what can happen in the presence of oxidants, such as March, Advanced Organic Chemistry: Reactions, Mechanisms and Structure, Second Edition, New York: McGraw Hill Book Company, 1977, page See 1095). That is, the double bond present in the fatty acid side chain can eventually be oxidized to a carboxyl group, thereby releasing an alkylcarboxylic acid, such as malonic acid and / or caproic acid (shown in FIG. 3). Street). In a fully oxidized cardiolipin molecule, all four fatty acid side chains can be cleaved at the position of the first (most proximal) double bond to give rise to a fatty acid chain, each with a terminal carboxyl group (Figure). As shown in 3). Since natural cardiolipin can be heterogeneous with respect to the composition of its fatty acid side chains, a fully oxidized cardiolipin preparation is a mixture of oxidized species that reflects the heterogeneity of the fatty acid side chains in its non-oxidizing molecules. Conceivable. Incomplete oxidation of cardiolipin can lead to further heterogeneity. In that situation, a mixture of cardiolipin oxide species in which one, two or three of the fatty acid side chains are oxidized to carboxylic acid will result (eg, one terminal carboxyl group exemplifying incomplete oxidation). See Figure 3 for an example of a cardiolipin oxide molecule that has and another example of a fully oxidized cardiolipin oxide). It is contemplated herein that any or all of the cardiolipin oxide species described in this paragraph will be useful in the described compositions, methods, and devices.

B. NaIO₄And KMnO₄Oxidation of cardiolipin by Periodic acid (IO) is one of the useful methods for oxidizing cardiolipin.₄^-) And / or permanganate (MnO)₄^-) Is used as an oxidizing agent. Salts of these oxidants include any counterion that results in an electrically neutral compound. Periodic acid is typically NaIO₄Or HIO₄ In the form of periodate, such as (periodic acid), periodic acid is typically a salt (such as sodium or potassium salt), eg, KMnO.₄Is in the form of. In one specific embodiment, NaIO₄And KMnO₄Is used as an oxidant. To practice this oxidation method, cardiolipin obtained from any source can be added to any solvent such that cardiolipin is substantially dissolved. In terms of the hydrophobicity of cardiolipin, the use of polar organic solvents such as t-butanol, ethanol, methanol, propanol, acetone, dimethylformamide, or diethyl ether is preferred. Other non-polar organic solvents, such as chloroform, can be used to suspend cardiolipin; however, these non-polar organic solvents are less useful. This is because the other components of the oxidation reaction (described below) are less soluble in these solvents than, for example, polar organic or aqueous solvents.

The concentration of the cardiolipin solution can be any useful concentration within the higher range that still makes cardiolipin soluble in the solvent of choice. One of ordinary skill in the art can easily measure the saturation point of cardiolipin in any particular solvent. In the example where t-butanol is the solvent of choice, the cardiolipin concentrations are about 10 mg / ml, about 25 mg / ml, about 50 mg / ml, about 75 mg / ml, about 100 mg / ml, about 125 mg /. It can be in the range of about 10 mg / ml to 250 mg / ml, such as ml, about 150 mg / ml, about 200 mg / ml and about 250 mg / ml. If fine particles are present after dissolving cardiolipin in a solvent of choice, the solution may optionally be clarified by any technique known in the art, such as centrifugation or filtration.

In some embodiments, cardiolipin oxidation can be carried out in the absence of oxygen, for example in an argon, helium, or nitrogen atmosphere. Oxidation of cardiolipin is thought to occur in an oxygen-containing atmosphere, such as air, or in other atmospheres; however, the oxidizing molecules contained therein, such as carbonate, reduce the efficiency of the reaction. Sometimes. Nonetheless, any atmosphere in which the oxidation reaction of cardiolipin takes place is contemplated by this disclosure, regardless of the reaction efficiency.

NaIO sufficient to oxidatively cleave at least one cardiolipin fatty acid side chain₄And KMnO₄The amount of oxidant, such as, is then added to the suspended cardiolipin with constant stirring. In some examples, periodate and permanganate (NaIO) to oxidize cardiolipin₄And KMnO₄Like) is used together. In some of those examples, periodate (NaIO)₄Permanganate (like KMnO)₄Is added to the cardiolipin solution prior to the addition of). Oxidizing agents can be dissolved in any solvent in which they are soluble and can be mixed with cardiolipin solution. For example, NaIO₄And KMnO₄Can be dissolved in water.

NaIO₄NaIO used in the reaction when used in the method of oxidizing cardiolipin₄The amount of is not crucial. At a minimum, the amount is such that the reaction takes place in a reasonable amount of time under certain circumstances. At the other end of the spectrum, a significant molar excess of NaIO₄May be used in the oxidation reaction. For example, periodate (NaIO)₄The molar ratio of cardiolipin to cardiolipin is about 0.1: 1 to about 100: 1, about 0.5: 1 to about 50: 1, about 1: 1 to about 25: 1, about 2: 1 to about 15: 1. , About 2.5: 1 to about 10: 1, about 3: 1 to about 7.5: 1, or about 4: 1 to about 5: 1. In a specific embodiment, the molar ratio of sodium m-sodium periodate to cardiolipin is from about 4: 1 to 5: 1, or more specifically about 4.2: 1.

Permanganate (KMnO)₄The amount of permanganate used in the reaction is not critical when used in methods of oxidizing cardiolipin. At a minimum, the amount is such that the reaction takes place in a reasonable amount of time under certain circumstances. At the other end of the spectrum, a significant molar excess of permanganate may be used in the oxidation reaction. For example, permanganate (KMnO)₄The molar ratio to cardiolipin is about 0.01: 1 to about 100: 1, about 0.02: 1 to about 50: 1, about 0.1: 1 to about 25: 1, about 0.25: 1 to about 15: 1. , About 0.3: 1 to about 10: 1, about 0.4: 1 to about 7.5: 1, about 0.5: 1 to about 5: 1, about 0.6: 1 to about 2: 1, or about 0.7: 1 to about 1: Can be 1. In a specific embodiment, permanganate (KMnO)₄The molar ratio to cardiolipin (such as) is about 0.5: 1 to 1: 1, or more specifically about 0.75: 1.

Mixing of the oxidation reactants can be carried out at any temperature that does not prevent the reaction from taking place. Similarly, the reaction can proceed for any time sufficient to cause oxidative cleavage of at least one fatty acid side chain of cardiolipin. Each of these uncertainties is routine, as one of ordinary skill in the art understands that reaction time depends on several uncertainties, including reaction temperature, solvent type, and reactant and product concentrations. It can be easily optimized experimentally. For example, the oxidation reaction takes place at room temperature and proceeds for at least 24-48 hours.

The oxidation reaction of cardiolipin is carried out by any reducing agent capable of neutralizing the oxidizing agent present during the reaction and reducing any ketone formed at the β-carbon position of the central glycerol moiety to the corresponding β-hydroxyl group. Can be stopped. Sodium bisulfite (eg sodium bisulfite), dimethyl sulfide, sodium cyanoborohydride (NaBH)₃CN), sodium borohydride (NaBH)₄), Sodium triacetoxyborohydride (NaBH (OAc))₃), Morphorin borane, potassium triisopropoxyhydrogenated, t-butylamine borane, dimethylamine borane, pyridineborane, triethylamine borane, or trimethylamine borane, reducing agents are useful for this purpose. In some specific examples, the reducing agent is bisulfite, and in more specific examples, the reducing agent is sodium bisulfite.

The amount of reducing agent added to the reaction is not critical as long as the oxidizing agent is stopped. If the immunogenicity of cardiolipin oxide is adversely affected by the oxidation of the β-hydroxyl group of the β-hydroxyl group in the central glycerol portion of cardiolipin to ketones, then the amount of reducing agent is also due to the sufficient immunogenicity of cardiolipin oxide. An amount that restores the β-hydroxyl group. For example, the amount of sodium bisulfite sufficient to turn the reaction mixture virtually colorless is NaIO.₄And KMnO₄It can be added to the cardiolipin oxidation reaction product containing an oxidizing agent.

If present, the aqueous and organic phases in the cardiolipin oxidation reactant can be separated by any method known in the art, such as centrifugation. In one embodiment, the t-butanol phase predominantly containing cardiolipin oxide and the aqueous phase predominantly containing an alkylcarboxylic acid can be separated by centrifugation. Cardiolipin oxide is sufficiently hydrophobic that it can be expected to separate into the organic phase. However, if necessary, those skilled in the art may use methods known in the art, such as TLC, HPLC, NMR or gas chromatography, to determine whether the aqueous or organic phase contains cardiolipin oxide. Can be determined.

Optionally, cardiolipin oxide in solution may be dialyzed into a useful buffer, such as 10 mM phosphate buffer, pH 8.0, and lyophilized using methods well known in the art.

C. Antigenicity of cardiolipin oxide Oxidation has been shown to alter the antigenic properties of some phospholipids (see, eg, US Pat. No. 6,177,282). Anti-lipoid antibodies in patients with syphilis are thought to bind to two phosphate and β-hydroxyl groups in the central glycerol moiety (eg, Castro et al., Clin. Diagn. Lab. Immunol., 7 (4)). : 658-661, 2000). Therefore, in order to retain the antigenicity of cardiolipin, it is beneficial to maintain the configuration of the central glycerol moiety, for example, to detect anti-lipoid antibodies in syphilis serum. The antigenicity of cardiolipin oxide is by one of several widely used techniques (eg, ELISA, dot blot, enzyme immunoassay, fluorescence immunoassay) and / or as described herein. It can be tested (see, eg, Example 4). As shown in Example 4 (below), the method of oxidizing cardiolipin described herein maintains the antigenicity of cardiolipin.

D. Cardiolipin / Lecithin Mixture A mixture of cardiolipin and lecithin can be oxidized by the same method described above for cardiolipin alone; the only difference is that the starting material is a mixture of cardiolipin and lecithin. In the initial oxidation reaction, cardiolipin and lecithin are about 20: 1 to about 1: 1 or about 1: 1 to about 1:10, or about 10: 1 to about 1:10, or about 5: 1. Mix together in a weight ratio of ~ about 1: 5 or about 1: 1 ~ about 1: 5 (cardiolipin: lecithin). In certain examples, the cardiolipin: lecithin weight ratio is about 20: 1, about 10: 1, about 5: 1, about 2: 1, about 1: 1, about 1: 2, about 1: 3, about 1: 1. Includes 5, or about 1:10.

V. Cardiolipin Oxidation-Adherent Molecular Complex Cardiolipin oxide is a relatively small molecule. Therefore, it is beneficial to attach cardiolipin oxide to larger molecules (such as polypeptides) to facilitate the attachment of cardiolipin to the solid surface used in the methods and devices described herein. In some examples, the larger the molecule, the more easily it adheres to the substrate, such as the type of hygroscopic substrate used in lateral flow or inflow techniques. Derivatized cardiolipin oxide is more easily adsorbed and localized to porous substrates (such as nitrocellulose porous substrates) than very small cardiolipin or cardiolipin oxide molecules thereof. Derivatizing cardiolipin oxide by attaching a polypeptide moiety to it, its many in solid surface-based immunoassays, such as ELISA, side flow diagnostic strips and / or devices, and / or influx devices. Greatly enhances versatility and usefulness.

Suitable adherent molecules include albumin, hemocyanin, tyroglobulin and derivatives thereof, in particular bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH), avidin, streptavidin, and proteins, polypeptides or peptides such as biotin. .. Substances derived from other polypeptides or non-proteins are known to those of skill in the art.

Adherent molecules often contain reactive groups to promote covalent conjugation to cardiolipin oxide. The amine group of the amino acid can be used in this way. Adherent molecules lacking such groups can often be reacted with suitable chemicals to produce reactive groups. Examples of chemicals that can be used to generate useful reactive groups in the attached molecule are, but are not limited to, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), and N-. Contains hydroxysulfosuccinimide (NHS).

The provision of cardiolipin oxide herein provides at least one reactive carboxyl group that is not otherwise available in natural or synthetic cardiolipin. Thus, cardiolipin oxide can be linked to attached molecules by any means known in the art and / or via reactive groups produced by oxidation, as described herein. Such linkage may or may not be accompanied by additional linking groups. Many different methods can be used to provide a link between cardiolipin oxide and attached molecules.

Figure 4 schematically shows an example of a method that can be used to link oxidized cardiolipin to amine-containing adherent molecules (eg, proteins such as BSA, synthetic proteins MAPS, IgY, streptavidin, avidin or KLH). The reaction in FIG. 4 describes the carboxyl group produced by the oxidative cleavage of the cardiolipin fatty acid side chain as described herein by NHS (or sulfo-NHS, as shown in FIG. 4). ) And EDC can be used to illustrate that it can be modified to amine-reactive NHS esters. The NHS ester of cardiolipin oxide is then reacted with an amine group present in the adherent molecule, such as BSA, to form an amide link between the cardiolipin oxide and the adherent molecule. ..

Some aspects of the methods and devices herein utilize cardiolipin oxide or cardiolipin oxide-adhered molecular complexes immobilized on a solid phase. Any conventional method of immobilizing a substrate on a solid surface is contemplated in the present disclosure.

Suitable methods for immobilizing a cardiolipin oxide or cardiolipin oxide-adherent molecular complex to a solid phase include ionic interactions, hydrophobic interactions, covalent interactions and the like. The solid phase (see, eg, Section II) can be selected for its unique ability to attract and immobilize cardiolipin oxide or cardiolipin oxide-attached molecular complex. Alternatively, the solid phase can carry a factor capable of attracting and immobilizing cardiolipin oxide or cardiolipin oxide-attached molecular complex. This factor can be used, for example, for charged substances that are reversely charged to cardiolipin oxide or cardiolipin oxide-attached molecular complex, or to charged substances that are conjugated to, for example, cardiolipin oxide or cardiolipin oxide-attached molecular complex. On the contrary, it can contain a charged substance that is charged. Yet another option is that the factor may be any specific binding partner, such as avidin or streptavidin, which is immobilized on a solid phase and which, through a specific binding reaction, cardiolipin oxide. Alternatively, it has the ability to immobilize cardiolipin oxide-attached molecular complexes, such as biotinylated cardiolipin oxide.

In some aspects of the methods and devices herein, the cardiolipin oxide or cardiolipin oxide-adherent molecular complex may serve as a detection reagent and is therefore conjugated to a label. Any molecule or composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means can serve as a label. Examples of labeling are disclosed, including enzymes, colloidal gold particles, and colored latex particles (US Pat. Nos. 4,275,149; 4,313,734; 4,373,932; and each incorporated herein by reference. No. 4,954,452). Further examples of useful labels include, but are not limited to, cofactors, ligands, chemical luminescent agents or fluorescent agents, silver particles adsorbed on proteins, iron particles adsorbed on proteins, adsorbed on proteins. Examples include copper particles, protein-adsorbed selenium particles, protein-adsorbed sulfur particles, protein-adsorbed tellurium particles, protein-adsorbed carbon particles, and protein-conjugated dysacs.

Adhesion of the compound to the label may be through covalent bonds, adsorption processes, hydrophobic bonds and / or electrostatic bonds, or a combination of these bonds and interactions, as well as in chelates and / or. It may contain a linking group. Guidance on labeling methods and selection of labels suitable for a variety of purposes can be found, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989 and Ausubel et al., Current Protocols in Molecular Biology, Greene. Discussed in Publ. Assoc, and Wiley-Intersciences, 1998.

In some aspects of the method and apparatus, colloidal gold conjugates of cardiolipin oxide or colloidal gold conjugates of cardiolipin oxide-adherent molecular complexes are envisioned.

VI. Immunoassay device By the discovery herein of how to prepare cardiolipin-adherent molecular conjugates that can oxidize cardiolipin and immobilize it on solid supports (such as microporous membranes such as nitrocellulose, nylon or PVDF). Solid surface-based immunoassays (eg, EIA, ELISA, influx, dipstick, and lateral) for the detection of cardiolipin binding assays, such as anti-lipoid antibodies in biological samples from subjects infected with Treponema pallidum. Flow device) becomes possible. In some examples, the disclosed immunoassays allow the detection of the presence (or absence) of anti-lipoid antibodies in biological samples for the diagnosis of syphilis.

A. Typical immunoassay device formats and related information Immunoassay devices allow the execution of relatively inexpensive, disposable, membrane-based assays for the visual identification of the presence (absence) of an analyte in a liquid sample. Such devices are usually configured as independent dipsticks (eg, test strips) or as devices with some sort of housing. Typically, the immunoassay device can be used with only about 200 μl of liquid sample, and detection of the analyte in the sample can be completed (but not necessarily) within 2-5 minutes. In clinical assays, the sample can be urine, blood, serum, saliva, or other body fluid. In nonclinical studies, the sample may be a small solution prepared from soil, dust, plants, or food, and may also be applied directly to the membrane test strip. In most cases, auxiliary equipment is not needed to perform such tests, and such equipment can be easily used in clinics, laboratories, outdoors, and at home, even for inexperienced people. it can.

The immunoassay device routinely identifies or monitors physiological and pathological conditions (eg, infections, pregnancy, cancer, endocrine disorders) using different biological samples (eg, urine, serum, plasma, blood, saliva). Developed for, and for the analysis of environmental samples (eg, natural fluids and factory effluents), eg, for contamination. Many of these tests are based on highly specific interactions between specific binding pairs. Examples of such binding pairs include antigen / antibody, hapten / antibody, lectin / carbohydrate, apoprotein / cofactor and biotin / (streptavidin) avidin. In addition, many of these tests are one or more of the members of the binding pair attached to a mobile or immobile solid phase material such as latex beads, fiberglass, glass beads, cellulose strips or nitrocellulose membranes. (Eg, solid phase, lateral flow test strip, inflow test) is required (US Pat. No. 4,703,017; No. 4,743,560; No. 5,073,484).

One of the major categories of immunochromatographic assays is the "sandwich" assay. In general, sandwich immunochromatography procedures require mixing an assay to be assayed, eg, a sample that appears to contain an anti-lipoid antibody, with an antigen recognized by the assay, eg cardiolipin oxide. The antigen, i.e., the detection reagent, is mobile and is typically linked to a label or another signaling reagent, such as a stained latex, colloidal metal sol, or radioisotope. The mixture is then applied to a chromatographic medium containing a band or band of immobilized antigen recognized by the analyte antibody of interest. Chromatographic media are often in the form of strips that resemble dipsticks. When the molecular complex to be assayed and the detection reagent reach the band of immobilized antigen on the chromatography medium, binding occurs and the detection reagent complex is localized to that band. This indicates the presence of the molecule to be assayed. This technique can be used to obtain quantitative or semi-quantitative results.

Alternatively, a sandwich immunoassay requires mixing a sample of interest, eg, a sample that may contain an anti-lipoid antibody, with an antibody that recognizes the analyte, eg, Protein A or a goat anti-human secondary antibody. May be. The secondary antibody in this mixture is mobile, labeled (eg, with enzyme, colloidal gold, or the like) and serves as a detection reagent. As described in the Sandwich Immunoassay precedent, the analyte (if present) to be assayed is a chromatograph in which this mixture contains a band or band of immobilized antigen recognized by the assay assay. It can be detected when applied to a imaging medium.

Examples of sandwich immunoassays performed on test strips are described in US Pat. Nos. 4,168,146 and 4,366,241, each of which is incorporated herein by reference.

The solid phase immunoassay device provides highly sensitive detection of the analyte in the biofluid sample. The solid phase immunoassay apparatus incorporates a solid support to which one member of the ligand-receptor pair, usually an antibody, antigen, or hapten, is bound. Common early forms of solid supports were plates, test tubes, or polystyrene beads, which were known in the fields of radioimmunoassay and enzyme immunoassays. Recently, several porous materials such as nylon, nitrocellulose, cellulose acetate, fiberglass, and other porous polymers have been utilized as solid supports. In other common forms of membrane-based immunoassays, test strips (or dipsticks), as represented by some home pregnancy and ovulation detection kits, are suspected samples containing the study of interest. "Dip" in. Enzyme-labeled detection reagents are then added simultaneously or after the incubation time. The device is then washed and then placed in a second solution containing the substrate for the enzyme. Enzyme labeling, if present, causes the formation of colored products, either interacting with the substrate and depositing as a precipitate on the solid phase or causing a visible color change in the substrate solution. cause. European Patent A 0 125 118 describes an immunoassay for such sandwiched dipsticks. European Patent A 0 282 192 describes a dipstick device used in competitive assays.

The influx-type immunoassay device was designed, in part, to eliminate the need for incubation and washing steps associated with dipstick assays. The influx immunoassay device requires a capture reagent (such as cardiolipin oxide-adherent molecular complex) bound to a porous membrane or filter to which a liquid sample is added. As the liquid passes through the membrane, the target analyte (such as an anti-lipoid antibody) binds to the capture reagent. Sample addition is prior to (or at the same time) the addition of a detection reagent (such as gold-conjugated cardiolipin, labeled (eg, gold-conjugated) protein A or labeled (eg, gold-conjugated) anti-human IgG). Alternatively, the detection reagent may be placed on the membrane in a manner that allows the detection agent to be mixed with the sample, thereby labeling the analyte. Visual detection of the detection reagent is an indicator of the presence of the target analyte in the sample. Typical influx immunoassay devices are U.S. Pat. Nos. 4,246,339; 4,277,560; 4,632,901; 4,812,293; 4,920,046; and 5,279,935; Also described in US Patent Application Publication Nos. 20030049857 and 20040241876.

Migration assay devices typically incorporate reagents attached to colored labels within them, which allows for visible detection of assay results without the addition of additional material. See, for example, US Pat. No. 4,770,853; PCT Publication No. WO 88/08534 and European Patent No. A 0 299 428.

There are patents that disclose methods for a number of commercially available lateral flow tests and detection of large analytes (MW over 1,000 daltons). U.S. Pat. No. 5,229,073 describes a semi-quantitative competitive immunoassay lateral flow method for measuring plasma lipoprotein levels. This method utilizes multiple capture regions or capture lines containing antibodies immobilized to bind both labeled and free lipoproteins to produce semi-quantitative results.

U.S. Pat. No. 5,591,645 provides a chromatographic test strip having at least two portions. The first part contains a mobile tracker and the second part contains an immobilized binder capable of binding to the analyte. Further examples of lateral flow tests on large analytes are disclosed in the following patent documents: US Pat. Nos. 4,168,146; 4,366,241; 4,855,240; 4,861,711; and 5,120,643; European Patent No. 0296724; International Publication No. 97/06439; and International Publication No. 98/36278.

Lateral flow tests are also available for the detection of small analytes (MW 100-1,000 daltons). In general, testing of these small analytes will result in negative or indirect reporting results (ie, a decrease in signal with increasing analyte concentration), as illustrated by US Pat. No. 4,703,017. "Typical" competitive inhibition is required. However, several techniques have been developed to detect small analytes using lateral flow tests that result in positive or direct reporting results (ie, increased signal with increasing analyzer concentration). There is. These include, for example, US Pat. Nos. 5,451,504; 5,451,507; 5,798,273; and 6,001,658.

U.S. Pat. No. 5,451,504 provides a method with three specific (movement, trap, and detection) regions, each containing a different latex conjugate to produce a positive signal. The mobile zone contains a labeled antibody to bind to the analyte in the sample. In the trap area, the unbound labeled antibody is then trapped by the immobilized analyte analog. The detection area captures the labeled analyte-antibody complex.

U.S. Pat. No. 5,451,507 describes a two-band, non-contact immunoassay. The first band is non-diffusively bound to a component, eg, an analyte analog that is bound to a member of the signaling system or may become bound to a member of the signaling system. Has a reagent. The second band binds to the component only if the analyte to be tested is present. The distance that the component travels to the second band is directly related to the concentration of the analyte.

U.S. Pat. No. 5,798,273 discloses a lateral flow device that includes a capture area with an immobilized analyte analog and one or more readout zones for binding to the labeled analyte-analog. ing.

U.S. Pat. No. 6,001,658 discloses a test strip device with a diffusible, labeled binding partner that binds to an analyte, an immobilized analyte, and a detection region containing an immobilized antibody. ..

The devices described herein include strips of absorbent material (such as microporous membranes) as a whole, which in some cases are from different materials, each linked to another material in the band. It can also consist of adjacent and / or overlapping. In some examples, the absorbent strip can be immobilized on a supporting non-interfering material (such as non-woven polyester) to give the strip even higher stiffness, for example. The band within each strip selectively contains a specific binding partner and / or, for example, other reagents necessary for the detection and / or quantification of the particular analyte being tested against the anti-lipoid antibody. can do. Therefore, these bands can be considered as functional areas or functional areas within the test equipment.

Generally, a fluid sample (or a sample suspended in a fluid) is introduced into the strip at the position of the proximal end of the strip, for example by immersion or spotting. Samples are collected or obtained by methods well known to those of skill in the art. Samples containing the detected anti-lipoid antibody can be obtained from any biological source. Examples of biological sources include human or animal serum, plasma, urine, spinal fluid, saliva, fermented fluid, lymph, tissue culture and ascites. To optimize the results of the immunoassay, the sample may be diluted, purified, concentrated, filtered, dissolved, suspended or otherwise prior to the immunoassay. It may be manipulated in a way. Fluid travels distally through all functional areas of the strip. The final distribution of the fluid within the individual functional regions depends on the dimensions and adsorptive capacity of the material used.

In some embodiments, the porous solid support described above, such as nitrocellulose, is preferably in the form of a sheet or strip. The thickness of such sheets or strips is in a wide range, for example, from about 0.01 to 0.5 mm, from about 0.02 to 0.45 mm, from about 0.05 to 0.3 mm, from about 0.075 to 0.25 mm, from about 0.1. It can vary from 0.2 mm, or from about 0.11 to 0.15 mm. The pore size of such sheets or strips can also vary over a wide range, for example, from about 0.025 to 15 microns, or more specifically from about 0.1 to 3 microns; However, pore size is not intended to be a limiting factor in the selection of solid supports. When applied, the flow velocity of the solid support also falls within a wide range, for example, from about 12.5 to 90 seconds / cm (ie, 50 to 300 seconds / 4 cm), from about 22.5 to 62.5 seconds / cm (ie). That is, from 90 to 250 seconds / 4 cm), from about 25 to 62.5 seconds / cm (ie 100 to 250 seconds / 4 cm), from about 37.5 to 62.5 seconds / cm (ie 150 to 250 seconds / 4 cm). Can vary from, or about 50 to 62.5 seconds / cm (ie, 200 to 250 seconds / 4 cm). In a specific embodiment of the apparatus described herein, the flow velocity is about 62.5 s / cm (ie, 250 s / 4 cm). In another specific embodiment of the device described herein, the flow velocity is about 37.5 seconds / cm (ie 150 seconds / 4 cm).

Another common feature to be considered in the use of immunoassay devices is the formation of complexes between the analyte (such as anti-lipoid antibody) and the capture reagent (such as cardiolipin oxide-adherent molecular complex). Is a means for detecting. Detectives (also called detection reagents) serve this purpose. The detector may be incorporated into an immunoassay device (eg, contained in a conjugate pad, as described below) or applied to the device from an external source.

The detection agent may be a single reagent or a series of reagents that collectively serve the detection purpose. In some cases, the detection reagent is an analyte-specific, labeled binding partner (eg, gold-conjugated protein A in the case of antibody analytes, or gold-labeled in the case of human antibody analytes. Anti-human Ab (Fc), or gold-labeled cardiolipin oxide in the case of anti-lipoid antibody analytes). In other examples, the detection reagents collectively include an unlabeled first binding partner specific for the analyte and a labeled second binding partner specific for the first binding partner. In any case, the detection reagent is analyte - specifically detecting binding analyte capture reagent complex, and therefore, the detection reagent is catchingTrialthe other components that are localized to the agent or analyte capture region It is preferred that they do not bind substantially or react substantially with them. Such non-specific binding or reaction of the detector may result in false positives. Optionally, the detection reagent is in the case of a positive control molecule (labeled protein A detector, or labeled protein G detector, or labeled anti-human Ab (Fc)) present in the second capture region. May specifically recognize (such as non-specific human IgG).

B. Construction and design of inflow equipment The inflow device comprises a capture reagent (such as cardiolipin oxide-adhered molecular complex) immobilized on a solid support, typically a microtiter plate or membrane (eg, nitrocellulose, nylon or PVDF). .. Useful membrane features have been described so far; however, similar to the lateral flow assay, capillary elevation in the influx assay is due to the vertical movement of the sample through the membrane rather than across the membrane. It is useful to note that it is not a particularly important feature of the membrane. In a simple typical form, the membrane of the inflow device is arranged in functional or physical contact with the absorbent layer (see, eg, the description of "absorbent pad" below), which is located. , Acts as a reservoir for drawing fluid samples through the membrane. Optionally, after immobilization of the capture reagent, any protein binding site remaining on the membrane may be blocked (before or at the same time as sample administration) to minimize non-specific interactions.

In the operation of the inflow device, a fluid sample (such as a body fluid sample) is placed in contact with the membrane. Typically, the inflow device also includes a sample application area (or reservoir) for receiving and temporarily holding a fluid sample of the desired volume. The sample passes through the membrane matrix. In this process, the analyte in the sample (such as an anti-lipoid antibody) can specifically bind to an immobilized capture reagent (such as an oxidized cardiolipin-attached molecular complex). If detection of the analyte-capture reagent complex is desired, a detection reagent (such as Labeled Protein A, Labeled Protein G, Labeled Anti-Human IgG, or Labeled Cardiolipin) may be added with the sample, or the detection reagent may be added. The containing solution may be added after application of the sample. If the analyte is specifically bound by the capture reagent, a visual specimen due to the particular detection reagent can be observed on the surface of the membrane. Any wash step may be added at any time during the process, eg, after application of the sample and / or after adaptation of the detection reagents.

C. Construction and design of lateral flow device Lateral flow devices are generally known in the art. In short, a lateral flow device is, in essence, an analyzer that has a test strip that flows the fluid of a test sample suspected of containing the analyte of interest. The test fluid and any suspension analyte can flow along the strip into the detection zone, where the analyte (if any) interacts with the scavenger and detector, with or without the analyte. And / or indicate quantity.

A number of lateral flow analyzers are disclosed, each of which is incorporated by reference in U.S. Pat. Nos. 4,313,734; 4,435,504; 4,775,636; 4,703,017; 4,740,468; 4,806,311; No. 4,806,312; No. 4,861,711; No. 4,855,240; No. 4,857,453; No. 4,943,522; No. 4,945,042; No. 4,496,654; No. 5,001,049; No. 5,075,078; 5,451,504; 5,424,193; 5,712,172; 6,555,390; and 6,368,876; European Patent 0810436; and International Publication 92/12428 (WO92 / 12428); International Publication Includes those shown in WO 94/01775; WO 95/16207; and WO 97/06439.

Many lateral flow devices are one-step lateral flow assays, in which case the biofluid is a hygroscopic strip (although a non-hygroscopic material is used, and for example a surfactant is used as the material. (May be hygroscopic by application) Placed in the sample area above and moved along the strip until the liquid comes into contact with a specific binding partner that interacts with the assay in the liquid. Once the analyte interacts with the binding partner, a signal (such as a fluorescent dye or otherwise visible dye) suggests that the interaction has occurred. Multiple separate binding partners can be placed on the strip (eg, in parallel lines) to detect multiple analytes in the liquid. The test strip may incorporate a control indicator, which signals that the test was adequately performed without a positive signal indicating the presence (or absence) of the analyte on the strip. Is obtained.

Construction and design of lateral flow devices are available, for example, at the request of (800) 645-5476, Millipore Corporation, A Short Guide Developing Immunochromatographic Test Strips, 2nd Edition, pp. 1-40, 1999; and Schleicher. &amp; Schuell BioScience, Inc., 10 Optical Avenue, Keene, NH 03431, (603) Schleicher &amp; Schuell, Easy to Work with BioScience, Products and Protocols 2003, pp. 73-98, available at request at 352-3810. , 2003, 2003 are very well known in the art and both are incorporated herein by reference.

Lateral flow devices have a wide variety of physical forms that are equally well known in the art. Any physical form that supports and / or accommodates the basic components of a lateral flow device in an appropriate functional correlation is contemplated by the present disclosure. Figure 7 shows some examples of lateral flow devices. These examples demonstrate some of the physical aspects that may be useful in the construction of lateral flow devices.

The basic components of a particular embodiment of the lateral flow device are illustrated in FIG. 8, in which the long housing 10 includes a hygroscopic lateral flow strip 12 that substantially spans the entire length of the housing 10. The specific aspect of being shown is shown. The lateral flow strip 12 is divided into a proximal sample application pad 14 located below the sample inlet 15, an intermediate test result membrane 16, and a distal absorbent pad 18. The fluidized strip 12 is interrupted by a conjugate pad 20 containing a labeled conjugate (such as gold conjugate protein A, gold conjugate protein G, gold conjugate anti-human Ab). The flow path along the strip 12 proceeds from the proximal pad 14 through the conjugate pad 20 into the test result membrane 16 for final recovery at the absorbent pad 18. Selective binders (such as anchor antibody-lipoid antigen complex) are located on the proximal test line 22 in the test result membrane 16. The control line 24 is provided in the test result membrane 16 slightly distal to the test line 22.

In the operation of a particular embodiment of the lateral flow device illustrated in FIG. 8, a fluid sample containing an analyte of interest, such as an anti-lipoid antibody, is applied to the sample pad 14 through the sample inlet 15. In some examples, the sample can be applied as a droplet into the sample inlet 15, or, less preferably, by immersing the end of the device containing the sample inlet 15 into the sample. In other cases where the sample is whole blood, any developer A fluid) may be added to the blood sample to cause hemolysis of the red blood cells and, in some cases, an appropriate dilution of the whole blood sample. From the sample pad 14, the sample moves to the conjugate pad 20 due to, for example, capillarity. At the conjugate pad 20, the analyte of interest can bind to (or be bound to) a mobility or mobility detection reagent. For example, the anti-lipoid antibody analyte can bind to a labeled (eg, gold-conjugated) protein A or gold-conjugated cardiolipin detection reagent contained in the conjugate pad. The analyte complexed with the detection reagent can then flow to the test result membrane 16, where the complex is further immobilized on the proximal test line 22, a study-specific binding. Can interact with partners (such as cardiolipin oxide-attached molecular complex). In some examples, such as detection reagents (gold conjugate cardiolipin, labeled (eg gold conjugate) protein A, labeled (eg gold conjugate) protein G, labeled (eg gold conjugate) anti-human Ab. The anti-lipoid antibody complexed with) can further bind to an unlabeled, cardiolipin oxide-attached molecular complex immobilized on proximal test line 22. The formation of immune complexes between anti-lipoid antibodies, labeling (eg, gold conjugate) detection reagents, and immobilized cardiolipin-attached molecular complexes is visible at proximal test line 22. It can be detected by the appearance of the line, which is due to the accumulation of labels (eg, gold) in the local area of the proximal test line 22. The control line 24 can contain an immobilized, detection reagent-specific binding partner that can bind to the detection reagent in the presence or absence of the analyte. Such binding at control line 24 indicates proper execution of the test, even in the absence of the analyte of interest.

In another aspect of the lateral flow device, there may be a second test line located parallel to or perpendicular to (or in any other spatial relationship) with the test line 22 in the test result membrane 16. This particular aspect of operation can include (i) a second detection reagent specific for the second analyte, such as anti-Treponema antibody or Treponema pallidum organism or antigen, in the conjugate pad. , And (ii) are described in the preceding paragraph, further taking into account that the second test line contains a second specific binding partner that has an affinity for the second analyte in the sample. Similar to the one. For example, the second test line may contain an immobilized Treponema antigen that specifically binds the anti-Treponema antibody present in the sample, or the Treponema pallidum antigen or organism present in the sample. It may contain an immobilized anti-treponema pallidum antibody that specifically binds to.

Table 1 shows some of the materials that are useful for the components of the lateral flow device. However, one of ordinary skill in the art will rely on some uncertainties as the particular material used in a particular lateral flow device will depend on several uncertainties, including, for example, detected analytes, sample volumes, desirable flow rates and others. And can routinely select useful materials as appropriate.

(table 1)<img file="JP2011237443A_D0003.tif" />

1. <u style="single">Sample pad</u> A sample pad (such as sample pad 14 in FIG. 8) is any component of the lateral flow device that first receives the sample and can serve to remove particulates from the sample. Among the various materials that can be used to build the sample pad (see Table 1), a large bed capacity (eg 250 μl / cm)²) Is a factor of specific application, a cellulose sample pad may be beneficial. Sample pads can be treated with one or more release agents, such as buffers, salts, proteins, detergents, and detergents. Such release agents are used to promote resolubilization of conjugate pad components, for example, and block non-specific binding sites in other components of the lateral flow device, such as nitrocellulose membranes. Can be useful to do. Typical release agents are, for example, trehalose or glucose (1% -5%), PVP or PVA (0.5% -2%), Tween 20 or Triton X-100 (0.1% -1%), casein (1%). Includes ~ 2%), SDS (0.02% ~ 5%), and PEG (0.02% ~ 5%).

2. <u style="single">Membrane and applicable solution</u>:: Membrane types useful in lateral flow devices (such as nitrocellulose, nylon and PVDF), and considerations for applying capture reagents to such membranes have been discussed so far.

3. <u style="single">Conjugate pad</u> The conjugate pad (such as the conjugate pad 20 in FIG. 8) serves, among other things, to hold the detection reagent. In some embodiments, the detection reagent may be applied externally, eg, from a color developer container, in which case the lateral flow device need not include a conjugate pad (eg, US Pat. No. 4,740,468). See).

The detection reagents contained in the conjugate pad are typically released into solution by application of a test sample. The conjugate pad can be treated with a variety of substances to affect the release of the detection reagent into solution. For example, the conjugate pad can be treated with PVA or PVP (0.5% ~ 2%) and / or Triton X-100 (0.5%). Other release agents include, but are not limited to, hydroxypropyl methylcellulose, SDS, Brij and β-lactose. A mixture of two or more release agents can be used in any given application. In certain disclosure embodiments, the detection reagent in the conjugate pad 20 is gold conjugated cardiolipin oxide, labeled protein A, labeled protein G, or labeled anti-human IgG.

4. <u style="single">Absorbent pad</u> The use of absorbent pads 18 in the lateral flow device is optional. The absorbent pad serves to increase the total amount of sample that enters the device. This volume increase can be useful, for example, to flush unbound analytes from the membrane. Any of the various materials are useful in preparing absorbent pads, see, for example, Table 1. In some device embodiments, the absorbent pad can be paper (ie, cellulose fibers). One of ordinary skill in the art can select a paper absorbent pad based on, for example, its thickness, compression ratio, and bed capacity uniformity. The volume uptake of the produced absorbent can be adjusted by varying the dimensions (usually length) of the absorbent pad.

D. Antigen-coated microtiter plate Other common solid surface-based immunoassays are various forms of immunoabsorption assays, such as enzyme-linked immunosorbent assays (or ELISAs). These assays typically require an antigen applied to the wells of a microtiter plate (eg, cardiolipin-oxidized molecular complex). In this assay, a test sample (eg, serum or blood) that may contain an analyte of interest (eg, an anti-lipoid antibody) is subjected to an immobilized binding partner (eg, eg, serum or blood) specific for the analyte of interest. Place in the wells of a microtiter plate containing cardiolipin oxide-adherent molecular complex). The analyte specifically binds to the immobilized antigen; The unbound material is then washed away, leaving primarily the analyte-antigen complex bound to the plate. This complex can be detected in a variety of ways, as described in detail above. One of the advantages of the microtiter plate format is that multiple samples can be tested simultaneously (with controls) in one or more different wells on the same plate, thus allowing for fast mass processing analysis of large numbers of samples. Is what you can do.

E. Antigen combination Each of the immunoassays and / or immunoassay devices (eg, ELISA, dipsticks, influx devices or lateral flow devices) discussed herein is, in some embodiments, an analyte of interest (eg, a side fluid device). For example, the format can be defined to detect multiple assays by the addition of a capture reagent specific for (treponema antigen). For example, a particular well of a microtiter plate can contain a capture reagent specific for another analyte of interest. In some aspects of the immunoassay device, a second, third or higher capture region may be included containing capture reagents specific for other analytes of interest.

In certain embodiments, an immunoassay device is required to simultaneously detect anti-lipoid antibody and Treponema or anti-Treponema antibody in a fluid sample (eg, human serum). Such combination devices further include (a) immobilized Treponema antigens that can be specifically bound by anti-Treponema antibodies, or (b) immobilized anti-Syphilis that specifically binds to migratory Treponema antigens. Includes a Treponema capture region containing Treponema antibody. As used herein, a "treponema antigen" is an antigen that contains at least one antigenic determinant that specifically binds to an anti-Syphilis treponema antibody. Numerous Treponema antigens have been reported in the art; for example, U.S. Pat. Nos. 6,479,248; 6,248,331; 5,681,934; 5,578,456; 4,868,118; And see No. 4,740,467. For example, polypeptides with at least the following apparent molecular weights have been reported as syphilis treponema antigens: 16-20 kDa, 18 kDa, 18-23 kDa, 25 kDa, 35 kDa, 37 kDa, 37-46 kDa, 38 kDa. , 39 kDa, 41 kDa, 43 kDa, 44 kDa, 46 kDa, 47 kDa, 58 kDa, 150 kDa; and 180 kDa (see US Pat. No. 4,846,118 for further details).

Treponema antigens and anti-treponema antibodies are polypeptides; therefore, when used as capture reagents, these molecules can attach directly to solid supports (eg, nitrocellulose, nylon or PVDF). Nevertheless, it is believed that the treponema antigen or anti-treponema pallidum antibody can be immobilized (directly or indirectly) on a solid support by any available method.

The detection reagent can be used to detect the formation of a complex between the Treponema pallidum capture reagent and a Treponema-specific analyte (eg, Treponema, Treponema antigen, or anti-Treponema antibody). In some embodiments, the detection reagent (such as anti-human Ab) is a bound treponema-specific assay (eg, human anti-treponema antibody) and a bound anti-lipoid antibody assay (eg, human anti-lipoid). Antibodies) can be detected specifically. In other examples, two separate detection reagents are envisioned for the specific detection of bound treponema-specific analytes (eg, anti-treponema antibody or treponema antigen) or bound anti-lipoid antibody analytes. ..

The behavior of immunoassay devices useful for performing concurrent and non-treponema tests is substantially similar to the devices described elsewhere herein. One of the specific features of the combination device is that the fluid sample applied to the sample application region communicates with each of the anti-lipoid antibody capture region and to the Treponema pallidum capture region (eg, flowing through or within them). It is possible to flow).

In other immunoassay and immunoassay device aspects, a combination of an antigen containing cardiolipin oxide (eg, cardiolipin oxide-adherent molecular complex) and other antigens specific for anti-lipoid antibody; eg cardiolipin, lecithin and cholesterol. Requires an immobilized lipoid antigen that contains. Immobilization of lipoid antigens is described in detail in PCT / US2006 / 024117, which is incorporated herein by reference in its entirety. For example, lipoid antigens, including cardiolipin (eg, natural or synthetic cardiolipin), lecithin and cholesterol, are contacted with a population of lipoid antigen-specific Fab fragments to yield a lipoid antigen-Fab complex, which results in this complex. Can be easily attached to a solid support in combination with an antigen containing cardiolipin oxide.

VII. Kit Disclosed herein are kits used to detect anti-lipoid antibodies in a sample (eg, a biological sample). Such kits can also be used, for example, in the diagnosis of diseases in which the presence of anti-lipoid antibodies is a minor disease (eg, syphilis or lupus). Certain aspects of the disclosed kits are generally portable, for detecting anti-lipoid antibodies without the need for laboratory facilities, such as in point-of-care facilities, and / or diseases (such as syphilis). ) Provides a convenient, short-term, and / or cost-effective method for diagnosing.

The kit includes one or more immunoassay devices (and / or antigen-coated microtiter plates) and boxes, bags, small bags, large plastic containers (molded plastic or other transparent) disclosed herein. Includes means of transport, such as packaging), wrappers (eg, sealed or sealable plastic, paper, or metal packaging), or other containers. In some examples, the kit components are contained in a single packaging unit, such as a box or other container, which has a compartment in which one or more components of the kit can be placed. Can be done. In other examples, the kit may include, for example, one or more biological samples to be tested, positive and / or negative control samples or solutions (eg, positive control serum containing anti-lipoid antibody or anti-treponema antibody). Distillers (eg, phosphate buffer, or saline buffer), detection reagents (eg, for external application to kit devices), detection reagents, substrate reagents for enzyme visualization (eg, 5-bromo-phosphate) 4-Chloro-3-indrill, dimethylformamide solution of nitroblue tetrazolium), and / or one or more containers capable of holding wash solution (eg, Tris buffer, phosphate buffer, or distilled water), eg vials, Including tubes and the like.

Other kit aspects include syringes, finger-prick devices, rubbing alcohol cotton, square gauze, cotton balls, bandages, latex gloves, incubation trays with varying numbers of troughs, adhesive plate sealers, data reporting sheets. Including, they may be useful in the handling, recovery and / or processing of biological samples. The kit also includes any tools useful for introducing the sample into the sample chamber of the immunoassay device, including, for example, droppers, Dispo-pipettes, capillaries, rubber balls (eg for capillaries), etc. May be included in. In yet another aspect of the kit, it may include a disposal means for disposing of the immunoassay device used and / or other articles used in the device (such as patient samples). Such disposal means can include, but are not limited to, containers capable of containing spills from waste materials, such as plastic, metal or other impermeable bags, boxes or containers.

In some examples, the disclosed kit will include instructions for use with an immunoassay device or antigen-coated plate. The instructions for use provide instructions on how to apply the sample to the test equipment or plate, the amount of time required or recommended to wait for the results to become apparent, and details on how to decipher and interpret the test results. Can be provided. Such instructions may also include standards, such as standard tables, graphs, or figures for comparison of test results. These standards are optional and a standard curve that correlates the information needed to quantify the analyte using the test equipment, eg, signal intensity or number of signal lines, and thus the amount of analyte present in the sample. May include.

<p> The following examples are given to illustrate certain specific features and / or embodiments. These examples should not be construed as limiting the invention to the particular features or aspects described.</p><p>Example 1 Oxidation of cardiolipin This example describes the oxidation of double bonds in unsaturated fatty acids of unmodified cardiolipin to carboxyl groups.</p><p> Approximately 100 mg of lyophilized cardiolipin (Alabaster, AL) was dissolved in 2 ml of t-butanol and placed in a vial under an argon atmosphere. m-sodium periodate (NaIO)₄) 60 mg was dissolved in 600 μl of distilled water and added dropwise to the cardiolipin suspension with constant stirring. Immediately after that, permanganate (KMnO) dissolved in 400 μl of distilled water₄) NaIO with continuous stirring of 8 mg₄It was added dropwise to the reaction mixture. The mixture discolored (Fig. 1B) and was mixed at room temperature for 24-48 hours.</p><p> The degree of oxidation of cardiolipin was qualitatively measured by thin layer chromatography (TLC). Chloroform: methanol: ammonium hydroxide solution (61.9: 30.9: 7.1 by volume) Approximately 10 ml was placed in a 100 ml beaker. The folded filter paper was placed in a beaker and allowed to saturate with solvent.</p><p> NaIO₄/ KMnO₄1 μl of the reaction mixture and 1 μl of a 20 mg / ml cardiolipin t-butanol (control) solution were placed 1 cm from the end of a 7.5 cm × 2.5 cm strip of TLC silica gel. The strip was placed against the filter paper in the beaker and the solvent was transferred to the top of the strip.</p><p> After removing the strip from the solvent, it was evaporated to dryness. The strips were then moistened by simply immersing them in 50 ml of 5% ethanol containing at least 10 ml of Molybdenum Blue (Spray Reagent; Alltech; stock number 18213). The strips were dry heated with the help of a hair dryer to develop color in the stain.</p><p> NaIO as soon as cardiolipin oxidation is complete₄/ KMnO₄The reaction of the reaction mixture was stopped by adding 80 mg of sodium bisulfite in 200 μl of distilled water with constant stirring. The colored mixture then became colorless (compare Figures 1B and 1C). The stopped reaction mixture was centrifuged at 1000 xg for 5 minutes. After centrifugation, two visible phases were observed (Fig. 1D).</p><p> Optionally, the supernatant t-butanol phase was placed in an evaporation round bottom flask and dried under vacuum. Alternatively, the t-butanol phase is dialyzed against 10 mM phosphate buffer pH 8.0, or another buffer, and the alcohol solvent is used in the next use of the cardiolipin oxide contained therein, eg, immunizing. It may be replaced with a solution suitable for use in chromatographic assays or, for example, for conjugation to adherent molecules.</p><p> As shown in FIG. 2, unmodified cardiolipin moves primarily to the top of the TLC strip in lane A, as indicated by the gray dot farthest from the origin. As described in Section IV, cardiolipin naturally contains heterogeneous molecular populations. Since linoleic acid makes up approximately 90% of the fatty acid side chains in cardiolipin, the predominantly linoleic acid-containing cardiolipin molecule (and closely related cardiolipin morphology) is represented by the gray dots in lane A, Figure 2. It is likely to be done. The low abundance cardiolipin morphology is likely to have different mobility and can occupy the very small smear observed in lane A in the direction of movement.</p><p> As mentioned above, the oxidation of cardiolipin oxidizes the alkene, cleaves the fatty acid side chain and introduces a carboxyl group into one or more of the fatty acid side chains. The carboxyl groups present in cardiolipin oxide interact more strongly with the silica substrate of the TLC strip, which slows the movement of cardiolipin oxide along the strip. Therefore, in cardiolipin oxide preparations (see Figure 2, lanes B and D1), smears are more pronounced in the direction of travel (compare lane A with lanes B and D1). In addition, oxidative cleavage of the fatty acid side chains results in alkylcarboxylic acids, which have low mobility on TLC strips under these conditions. As shown in FIGS. 2, Lanes B and D2, these carboxylic acids are considered to be represented by white points (due to dye exclusion) having the same spread as the starting point. After the cardiolipin oxidation described in this example, the oxidized form of cardiolipin was found predominantly in the t-butanol phase (Fig. 2, shown in lane D1), whereas alkylcarboxylic acids were predominantly found in the aqueous phase. Found (see Figure 2, Lane D2).</p><p> The upper t-butanol phase (alcohol phase) (see Figure 2, lane D1) contained cardiolipin oxide, as determined by reactivity with syphilis serum (see, eg, Example 4). In contrast, the lower aqueous phase (see Figure 2, lane D2) appeared to predominantly contain a mixture of malonic acid and caproic acid based on TLC results.</p><p>Example 2 Activation of cardiolipin oxide and conjugation with attached molecules This example demonstrates several methods of conjugating BSA or KLH with cardiolipin oxide using EDC and NHS.</p><p>A. First method After complete evaporation of the t-butanol phase described in Example 1, the dry cardiolipin oxide preparation was suspended in 2 ml of N, N-formamide to a concentration of approximately 25 mg / ml. 10 mg of EDC and 10 mg of NHS were dissolved in 1 ml of distilled water. The EDC / NHS solution was then added to the formamide solution containing cardiolipin oxide. The resulting mixture was stirred at room temperature for 1 hour. As will be appreciated by those skilled in the art, EDC and NHS will convert the carboxyl groups in cardiolipin oxide to amine-reactive NHS esters.</p><p> Up to 40-fold molar excess of BSA or KLH was added to the EDC / NHS / cardiolipin oxide mixture and the mixture was stirred at room temperature for 1 hour. Preferably, approximately 1 ml of 5 mg / ml KLH or BSA solution was added. As the amount of BSA or KLH in the reaction mixture increases (eg, up to 10 mg / ml or more), the protein component crosslinks itself, which increases the likelihood that a precipitate will appear. .. In this event, it may be necessary to separate the precipitate by centrifugation and discard it.</p><p> The reaction mixture was clarified as needed and then dialyzed against two exchanges of 10 mM phosphate buffer, pH 8.0 1 liter. The preparation was concentrated by membrane filtration (Centricon filter; Millipore) to approximately 10 mg / ml cardiolipin oxide. This cardiolipin oxide solution can be stored at 2-8 ° C or -20 ° C until required.</p><p>B. Second law As described in Example 1, after complete evaporation of the t-butanol phase, the dry cardiolipin oxide preparation was suspended in 2 ml of N, N-formamide to a concentration of approximately 25 mg / ml. This formamide mixture is then dialyzed against two 10 mM phosphate buffer, pH 8.0 1 liter exchanges prior to the addition of the EDC / NHS solution, as described in Method 1 of this example. did. The steps after the addition of the EDC / NHS solution are the same as those described in Method 1.</p><p>C. Third law The cardiolipin oxide preparation was prepared by dialyzing the t-butanol phase described in Example 1 against 10 mM phosphate buffer, pH 8.0 and then lyophilizing. Lyophilized cardiolipin oxide was reconstituted in water to approximately 25 mg / ml for use in subsequent reactions. Then, 10 mg of EDC and 10 mg of NHS dissolved in 1 ml of distilled water were added to the cardiolipin oxide solution with stirring at room temperature for 1 hour. The steps after the addition of the EDC / NHS solution are the same as those described in Method 1.</p><p>Example 3 Biotinlation of cardiolipin oxide This example describes biotinylation of cardiolipin oxide.</p><p> 10 mg of cardiolipin oxide (see Example 1) was dissolved in 1 ml of 100 mM N-morpholinoetan sulfonic acid (MES). 20 mg / ml biotin-PEO-amine 100 μl was added to the cardiolipin oxide solution. Immediately afterwards, 25 μl of the newly prepared 1 mg / ml EDC solution was added to the biotin / cardiolipin mixture and the solution was mixed at room temperature for 2 hours. The reaction mixture was then dialyzed against two exchanges of 10 mM phosphate buffer, pH 8.0. The biotinylated product can be concentrated by lyophilization and then reconstituted with distilled water to the desired concentration, eg 10 mg / ml.</p><p>Example 4 Antigenicity of cardiolipin preparation This example demonstrates that the cardiolipin oxide prepared in Example 1 retains antigenicity when tested against syphilis sera using two immunoassays.</p><p>A. RPR inhibition test 100 μl of syphilis serum (lot 50 L) was mixed with 50 μl of cardiolipin-BSA or cardiolipin-KLH oxide preparation (approximately 2.5-5 mg / ml) as described in Table 2. If the cardiolipin oxide preparation retains antigenicity, the anti-lipoid antibody present in serum should specifically bind to the cardiolipin oxide preparation, which determines the number of anti-lipoid antibody binding sites available. It should work to reduce. In other words, serum is considered to be "deprived" of some or all of the anti-lipoid antibody binding sites. Serum stripped of the anti-lipoid antibody binding site will be less reactive when tested in conventional RPR studies.</p><p> (Table 2) Cardiolipin oxide preparation<img file="JP2011237443A_D0004.tif" /></p><p> Deprived (and control) sera were tested in the RPR study according to Chapter 10 of the Manual of Tests for Syphilis, 9th Edition, Washington, DC: American Public Health Association, 1998.</p><p> As shown in Table 3, control syphilis serum supplemented with 50 μl of 5 mg / ml unconjugated BSA was added and reacted with the RPR test antigen at a serum dilution of 1: 8. Incubation of syphilis sera with each of the cardiolipin preparations 1-12 (described in Table 2) inhibited the sero-reactivity with the RPR test antigen at least 2-fold. This result suggests that each of the cardiolipin preparations reacted to some extent with the anti-lipoid antibody present in the test syphilis serum, thereby inhibiting the reaction of the stripped serum with the RPT test antigen. For example, preincubation of syphilis test sera with cardiolipin preparations 2, 11, and 12 completely inhibited the reactivity of the sera in the RPR test.</p><p> (Table 3) Results of RPR inhibition test<img file="JP2011237443A_D0005.tif" />^*See Table 2 for a description of cardiolipin preparations. R = strong positive reaction R_(-) = Positive reaction Rm = weak reaction Rm_(-) = Very weak reaction N = negative reaction</p><p>B. Immunoblot test Dot blot assays were performed using the Immuno-Blot® Assay Kit (BioRad) with goat anti-human IgG alkaline phosphatase according to the manufacturer's instructions. As shown in Figure 5, cardiolipin oxide-BSA preparation 5 (see Table 2) contains syphilis serum (lot 00) and a wide range of antigens (7.5-120 μg) and antibodies (1: 10-1: 640 dilutions). Reacted over concentration. Relatively, Preparation 5 did not react with normal human sera (ie, non-syphilitic sera) at all over the same range of antigen and antibody concentrations.</p><p> Figure 5 also shows that serum lot 00 (1: 640 dilution) reacts with the whole-cell Treponema pallidum preparation, confirming that the serum donor was infected with Treponema pallidum. Become.</p><p>Example 5 Preparation of Oxidized Cardiolipin Gold Conjugate This example demonstrates the conjugation of cardiolipin oxide-BSA or cardiolipin oxide-KLH with colloidal gold, and the determination of conditions useful for the preparation of such gold conjugates. This colloidal gold preparation can be used as a conjugate in a lateral flow strip, such as the lateral flow strip described in connection with FIG.</p><p>A. Determining pH useful for cardiolipin oxide / colloidal gold conjugation reaction Approximately 25 ml of 10 mM phosphate buffer was placed in a 50 ml beaker and adjusted to pH 5.0 with 0.2 M phosphate. Two 0.5 ml aliquots of pH 5.0 buffer were transferred to two 12 x 75 mm tubes, one labeled "test" and another labeled "control". Then, the pH of the phosphate buffer remaining in the beaker was continuously adjusted to 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, and 10.0 with 0.2 M potassium carbonate. At each pH, two 0.5 ml aliquots were transferred to "test" and "control" tubes as described for the pH 5.0 sample.</p><p> 5 mg / ml cardiolipin-BSA preparation 6 μL or 5 mg / ml cardiolipin-KLH preparation (30 μg) prepared as described in Example 2 in each of the test and control tubes. It was added and mixed well.</p><p> 40 nm Colloidal Gold (1% Solution) (British Biocell International, London, England) Place approximately 25 ml in a separate 50 ml beaker and pH 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 , 9.0, 9.5, and 10.0 were produced as a series of "control" and "test" colloidal gold samples as described above.</p><p> 1 ml of colloidal gold at each pH was added to "test" and "control" cardiolipin solutions with corresponding pH. The gold / cardiolipin solution was thoroughly mixed and incubated at room temperature for 20 minutes. Then 200 μL of 2 M NaCl was added to the set of tubes labeled Test and 200 μL of distilled water was added to the set of tubes labeled Control. The contents of both sets of tubes were incubated for 30 minutes at room temperature.</p><p> Optical density (OD) at 580 nm for each "test" sample₅₈₀) Was measured against the corresponding "control" sample. Lowest OD₅₈₀The pH of the sample with was determined to be the preferred pH for the formation of gold conjugates of the cardiolipin oxide-BSA or cardiolipin oxide-KLH preparation.</p><p> Colloidal gold particles have a surface that is negatively charged by a layer of anions adsorbed on the surface of the gold particles during the manufacturing process. Oxidized cardiolipin-BSA or oxidized cardiolipin-KLH Proteins, such as BSA or KLH in preparations, are attracted to negatively charged gold particles through ionic, hydrophobic, and coordinate interactions. These interactions underlie the formation of colloidal gold-protein conjugates. At the pI of the protein conjugated to the gold particles (ie, the pH at which the protein has a net zero charge), the conjugate is considered to be the most stable.</p><p> Adding NaCl to unconjugated colloidal gold particles destroys the layer of negatively charged ions adsorbed on the surface of the gold. As a result, the gold particles dissociate and eventually the gold ions (ie, Au).⁺) Is released into the solution. OD this free gold ion₅₈₀Can be measured with. In contrast, protein-gold conjugates (eg, cardiolipin oxide-BSA-gold conjugate or cardiolipin oxide-KLH-gold conjugate) are at the pI of the protein component of the conjugate (eg, at the pI of cardiolipin oxide-BSA). Or resistant to destruction by NaCl (at pI of cardiolipin oxide-KLH). Therefore, in this example, the lowest OD₅₈₀Select the pH of the sample with.</p><p> Since the molar ratio of cardiolipin to adherent molecules in each cardiolipin oxide-attached molecule (eg, cardiolipin oxide-BSA or cardiolipin oxide-KLH) preparation can be different, the pI of each such preparation can also be different. Therefore, for each cardiolipin oxide-attached molecule preparation produced, it is beneficial to determine a useful pH, as described in this example.</p><p>B. Determining Cardiolipin-BSA or Cardiolipin-KLH Concentrations Useful for Colloidal Gold Conjugation Reactions 100 μl of distilled water was added to each of a series of 11 12 × 75 mm test tubes. Then a series of 1 mg / ml solutions of either cardiolipin-BSA or cardiolipin-KLH 1 μl, 2 μl, 5 μl, 7 μl, 10 μl, 15 μl, 25 μl, 50 μl, 75 μl or 100 μl. Added to the corresponding tube. A series of 11 tubes was used as a control. Each tube was mixed well and incubated for 5 minutes at room temperature. At this point, the solution in each tube was red in color. 500 μl of 10% NaCl solution was then added to each tube with shaking and these tubes were again incubated for 5 minutes at room temperature. The color of the solution in each tube was visually observed to change somewhat from red to blue after the addition of NaCl. The minimum amount of cardiolipin-attached molecules useful for stabilizing gold conjugates was determined to be the lowest concentration of cardiolipin-attached molecules in the blue solution after NaCl addition.</p><p> Higher concentrations of cardiolipin oxide-attached molecular complex are useful, but less preferred. This is because the excess cardiolipin-adhered molecular complex can form a layer on top of the cardiolipin-adhered molecular complex already bound to the gold particles, for example, resulting in weak binding to the gold particles. ..</p><p> Similarly, lower concentrations of cardiolipin oxide-attached molecular complex may be useful, but less preferred. This is because unconjugated gold particles can provide background "noise" in other applications of the gold-conjugated cardiolipin-adhesive complex. Alternatively, unconjugated gold particles may be separated from the gold-conjugated cardiolipin-attached molecular complex using methods generally known in the art, such as centrifugation or filtration.</p><p>C. Small doses of cardiolipin-BSA or cardiolipin-KLH gold-conjugate For the preparation of specific cardiolipin-BSA or cardiolipin oxide-KLH, determine the useful pH and useful cardiolipin oxide-BSA or cardiolipin oxide-KLH concentration as described above. If the useful pH is pH 8.0 or higher, add 5 ml of 10 mM borate buffer to the amount of lyophilized cardiolipin oxide-attached molecular complex required to achieve a useful concentration; then , Adjust the pH to a useful pH. If the useful pH is pH 8.0 or lower, add 5 ml of 10 mM phosphate buffer to the amount of lyophilized cardiolipin oxide-attached molecular complex required to achieve a useful concentration; Then adjust the pH to a useful pH. Then add 10 ml of 40 nm colloidal gold (1% solution adjusted to a useful pH) and mix well. Incubate the mixture at room temperature for 20 minutes. Then add 1.6 ml of 10% BSA to the final concentration of 1% BSA and incubate for an additional 20 minutes at room temperature. Centrifuge the reaction mixture at 6500 xg for 10 minutes and remove the supernatant. Resuspend the pellet in 0.5 ml of resuspension buffer (150 mM NaCl, 20 mM Trizma base, 10% sucrose, 5% trehalose, 0.1% BSA, 0.05% sodium azide). Resuspended pellets containing gold-conjugated cardiolipin-BSA or -KLH are for a variety of purposes, including, but not limited to, as reagents for detecting anti-lipoid antibodies in lateral flow devices. Can be used.</p><p>Example 6 Adhesion of cardiolipin-adherent molecular complex to nitrocellulose This example describes a typical method for attaching a cardiolipin-protein complex to a solid surface, in this case nitrocellulose.</p><p> For the preparation of specific cardiolipin-BSA or cardiolipin-KLH oxide, a useful pH is determined as described above. Freeze-dried cardiolipin oxide-BSA or cardiolipin oxide-KLH complex in 10 mM sodium acetate buffer (useful pH 4.0-5.6) or 10 mM phosphate buffer (useful pH 7.0-9.0) ) To resuspend. Adjust the solution to a useful pH with 2 M acetic acid for a pH value of 4.0 to 5.6 or 1 M mono- or di-sodium phosphate for a pH value of 7.0 to 9.0. Ethanol (0.5%) may be optionally added to the cardiolipin oxide solution to reduce the viscosity of the solution and thus improve reagent use. Then, using the Matrix 1600 Reagent Dispensing Module (Kinematic Automation, Twain Harte, CA) according to the manufacturer's instructions, apply cardiolipin oxide-BSA or cardiolipin oxide-KLH solution to nitrocellulose.</p><p> After application of the cardiolipin oxide-BSA or cardiolipin oxide-KLH preparation to nitrocellulose, the membrane is, for example, at 37 ° C for 30 minutes prior to use in a lateral flow device, followed by 2 hours in a vacuum desiccator. Should be dried.</p><p>Example 7 Detection of anti-lipoid antibody in human serum by cardiolipin oxide-protein conjugate capture reagent In this example, the anti-lipoid antibody in syphilis serum is detected by the cardiolipin-protein conjugate capture reagent immobilized on the nitrocellulose membrane in cooperation with the mobile cardiolipin-protein-gold conjugate detection reagent. Demonstrate what you can do.</p><p> 1 μl of each of preparations 1-17 (as described in Table 2) was applied to a separate nitrocellulose membrane as described in Example 6 and set aside. Colloidal gold conjugates were prepared using cardiolipin oxide preparation 6 (see Table 2) as described in Example 5.</p><p> Syphilis serum (lot 00) or non-reactive (normal, human) serum was diluted 1:10 and placed in an appropriate number of separate wells in a 40-well microtiter plate. Next, 3 μl of gold-conjugate preparation 6 (for detection reagents) was added to each well. Nitrocellulose strips containing each of the immobilized preparations 1-17 (see Table 2) were then placed in wells containing a solution of this antibody and detection reagent. The solution in the wells flowed up the strip by capillary action. Each immobilized cardiolipin oxide preparation was tested against both syphilis and non-reactive (ie, control) sera.</p><p> As shown in Table 4, many of the cardiolipin oxide preparations immobilized in or on nitrocellulose have immobilized cardiolipin oxide capture reagents, at least one anti-lipoid antibody, and cardiolipin oxide gold conjugate detection. It showed a positive reaction showing a "sandwich" complex with the reagents. Reactivity was measured from 0 (negative) to 4+, with 4+ showing the strongest signal observed on the nitrocellulose strip.</p><p> (Table 4)<img file="JP2011237443A_D0006.tif" /></p><p>Example 8 Oxidation of a mixture of cardiolipin and lecithin This example describes the oxidation of a mixture of cardiolipin and lecithin and demonstrates that the oxidation mixture of cardiolipin and lecithin functions at least as well as cardiolipin oxide alone in the assays described in Examples 4-7.</p><p> Cardiolipin and lecithin were mixed together in a weight ratio of 1: 1, 1: 3, and 1: 5 to cardiolipin lecithin. The mixture was then oxidized as described in Example 1. As shown in Figure 8, Lane A, the mixture of unmodified cardiolipin and unmodified lecithin decomposes into two points on the TLC strip. Unmodified cardiolipin moves to the top of the TLC strip, as indicated by the gray dot farthest from the origin (see also Figure 2, Lane A). Unmodified lecithin moves slightly later in the TLC place and is shown as a gray dot approximately midway between cardiolipin and the origin.</p><p> As mentioned above, oxidation under the conditions of Example 1 oxidizes the alkene, cleaves the fatty acid side chain and introduces a carboxyl group into one or more of the fatty acid side chains of cardiolipin and lecithin. The carboxyl groups present in the oxide molecule interact more strongly with the silica substrate of the TLC strip, which slows the transfer of the oxidized form along the strip. Therefore, in the oxidized mixture of cardiolipin and lecithin, significant smears are observed in the direction of migration (see Figure 8, Lanes B and C). In addition, oxidative cleavage of the fatty acid side chains results in alkylcarboxylic acids, which have low mobility on TLC strips under these conditions. As is particularly apparent in FIG. 8, Lane D, these carboxylic acids are believed to be represented by white points (due to dye exclusion) that have the same spread as the starting point. After the oxidation of the cardiolipin / lecithin mixture described in this example, the oxidized form of cardiolipin was found predominantly in the t-butanol phase (Fig. 8, shown in Lane C), whereas the alkylcarboxylic acid was water. Mainly found in the phase (see Figure 8, Lane D).</p><p>A. BSA and KLH conjugations of cardiolipin oxide / lecithin mixture An oxidative mixture of cardiolipin and lecithin was conjugated to either BSA or KLH as described in Method 3 of Example 2. Cardiolipin / lecithin preparations conjugated to BSA or KLH are listed in Table 5.</p><p> (Table 5) Cardiolipin / lecithin mixture<img file="JP2011237443A_D0007.tif" /></p><p>B. RPR inhibition test of cardiolipin oxide / lecithin mixture The antigenicity of the cardiolipin / lecithin preparations described in Table 5 was tested in an RPR inhibition test as described in Example 3. As shown in Table 6, each of the cardiolipin / lecithin preparations completely inhibited the reactivity of syphilis serum in conventional RPR tests.</p><p> (Table 6) Results of RPR inhibition test of cardiolipin / lecithin mixture<img file="JP2011237443A_D0008.tif" />R = strong positive reaction R_(-) = Positive reaction Rm = weak reaction Rm_(-) = Very weak reaction N = negative reaction</p><p> This result demonstrates that the cardiolipin oxide / lecithin mixture deprives (ie, removes) anti-lipoid antibodies from syphilis sera very efficiently, thus demonstrating that such sera are non-reactive in conventional RPR studies. Will be done.</p><p>C. Immune dot test of cardiolipin oxide / lecithin mixture Dot blot assays were performed using the Immuno-Blot® Assay Kit (BioRad) with goat anti-human IgG alkaline phosphatase according to the manufacturer's instructions. As shown in FIG. 10, a cardiolipin / lecithin oxide mixture in the range of 375 ng to 21 μg per dot reacted with syphilis serum (lot 94265; 1:50 dilution). Relatively, none of the test doses of the oxidized cardiolipin / lecithin preparation showed a significant response to normal human serum (ie, non-syphilitic serum).</p><p> Preparations in which cardiolipin and lecithin are oxidized (by weight) in a 1: 1 ratio and then conjugated to either BSA or KLH (ie, 1: 1 BSA and 1: 1 KLH) are best with syphilis serum. It was reactive. Only 375 ng of 1: 1 BSA preparation and 656 ng of 1: 1 KLH preparation bound to human antibody present in syphilis serum in this assay.</p><p>Example 9 Detection of anti-lipoid antibody in human serum with protein A or anti-human antibody capture reagent In this example, the anti-lipoid antibody present in human serum is immobilized on a nitrocellulose membrane in cooperation with a mobile cardiolipin-oxidized protein-gold conjugate detection reagent, a protein A or anti-human antibody capture reagent. Demonstrate that it can be detected by.</p><p> Whole blood is collected from human subjects who may be at risk of producing serum-level anti-lipoid antibodies. Such conditions include, for example, Treponema pallidum infection (ie, syphilis) or lupus. Serum is separated from whole blood by methods known in the art. Whether the serum, for example, denatures or otherwise affects the specific binding of anti-lipoid antibodies present in saline or serum, or otherwise interferes with the desired method. It can be diluted with other solutions that do not.</p><p> Serum samples are applied to nitrocellulose strips containing a mobile or mobile, labeled cardiolipin oxide-protein conjugate detection reagent. Such conjugates may be, for example, gold-labeled cardiolipin oxide-BSA, or gold-labeled cardiolipin oxide-KLH conjugate, or gold-labeled BSA-conjugated mixture of cardiolipin oxide and lecithin oxide, or gold-labeled. Can be a KLH conjugate mixture of cardiolipin oxide and lecithin oxide.</p><p> The anti-lipoid antibody present in the serum sample binds to the detection reagent cardiolipin and onto the portion of the nitrocellulose strip on which the protein A or anti-human antibody capture reagent is immobilized (eg, by capillary action or lateral flow). Let's flow (by force). Anti-lipoid antibodies complexed with cardiolipin detection reagents will be captured by protein A or anti-human antibody capture reagents. The accumulation of the detection reagent complex in this way provides a detectable signal (eg, a visible signal) on the nitrocellulose in the region where the capture reagent is immobilized.</p><p> The appearance of the descriptive detectable signal suggests that anti-lipoid antibodies are present in serum samples and may be useful in diagnosing certain conditions, such as syphilis or lupus.</p><p>Example 10 Detection of anti-lipoid antibody in human serum by enzyme-linked immunoassay In this example, the anti-lipoid antibody present in human serum is immobilized on a microtiter plate for performing an enzyme-linked immunoassay by using BSA or KLH-conjugated cardiolipin oxide as a capture reagent. Demonstrate that it can be detected. Alternatives to BSA and KLH include synthetic proteins MAPS, IgY, streptavidin, and avidin. The wells of a 96-well microtiter plate were coated with 100 μl (10 μg / ml) of a solution containing 10 μg / ml cardiolipin oxide-BSA or cardiolipin oxide-KLH complex. The solution was dried at 37 ° C overnight. Wells were then blocked with at least 200 μl of 1% casein in Tris Phosphate Buffered Saline (TPBS) pH 7.2 for 2 hours at room temperature. Well TPBS 200 Washed once with μl. Each well was then added with 100 μl of human serum (either from a control or Treponema pallidum infected individual) diluted 1:20, 1:40, 1:80 or 1: 160 in 1% casein TPBS. Microtiter plates were incubated at room temperature for 60 minutes, followed by 3 washes with 200 μl TBST. 100 μl of conjugated goat anti-human antibody to horseradish peroxidase (HRP) diluted 1: 3000 in 1% casein TPBS was added to each well at room temperature for 45 minutes. Microtiter plates were washed 3 times with TPBS prior to the addition of 100 μl TMB substrate per well. In the presence of HRP, the TMB substrate discolors. The HRP enzyme-substrate reaction was stopped by the addition of 2 M sulfuric acid. The solution in each well was measured spectroscopically at 450 nm.</p><p> As shown in Tables 7 and 8, anti-lipoid antibodies present in human syphilis serum (reactive) bind to cardiolipin-BSA or cardiolipin-KLH complexes immobilized in wells of microtiter plates. did. However, no significant binding of control (non-reactive) sera to the same immobilized antigen was observed.</p><p> (Table 7) Enzyme-linked immunoassay with oxidized cardiolipin-BSA antigen<img file="JP2011237443A_D0009.tif" />Two sets of tests (results shown) were performed on each data point. The syphilis sera used in this dataset have a titer of 1:64 as measured by the RPR test.</p><p> (Table 8) Enzyme-linked immunoassay with cardiolipin oxide-BSA antigen<img file="JP2011237443A_D0010.tif" />Two sets of tests (results shown) were performed on each data point. The syphilis sera used in this dataset have a titer of 1: 128 as measured by the RPR test.</p><p> Although the present disclosure has been described with an emphasis on a particular aspect, variations of the particular aspect may be used and the disclosure is otherwise not as specifically described herein. It will be apparent to those skilled in the art that it is intended to be practiced. Features, properties, compounds, chemical moieties, or examples described in connection with a particular aspect, aspect, or example of the invention are applicable to any other aspect, aspect, or example of the invention. Should be understood as a thing. Accordingly, this disclosure includes all modifications contained within the spirit and scope of the disclosure as defined by the claims.</p>