Method and device for reveal of very small quantities of particles

UKRAINE

(19) and A (11) 85560 (13) C2

(51) IPC (2009)

Ο01Ν 21/82 (2006.01) S01Y 21 / 59S01Y 33/543

MINISTRY OF EDUCATION SCIENCE OF UKRAINE

STATE DEPARTMENT OF INTELLECTUAL PROPERTY

DESCRIPTION

TO THE INVENTORY PATENT

(54) METHOD AND DEVICE FOR DETERMINING VERY SMALL NUMBER OF PARTS

1

(21) a200602261

(22) 09.09.2004

(24) Feb 10, 2009

(86) PCT / ЕР2004 / 052109, 09.09.2004

(31) 103 44 924.8

(32) Sep 25, 2003

(33) ΕΕ

(46) Feb 10, 2009, No.3, 2009

(72) CONCEPT OF CONSTANTINE

(73) KONTANTIN'S ODES, VERNER FREDRICH

(56) of 5534441 A 09.07.1996

from 5100805 and 31.03.1992

(57) 1. A method of detecting small amounts of particles by recording a precipitate antigen-antibody, which includes:

- preparation of a test fluid, containing the main particles with a certain maximum size, with particles having at least two sites of antibody binding;

- preparation of a fluid containing an antibody, in which the particles have basically a certainmaximum size;

- contacting a test fluid with a fluid containing an antibody, resulting in a reactive fluid, wherein the antibody in the presence of particles with at least two binding sitesantibodies can form a precipitate antigen-antibody;

- transmission of a light beam through a reaction fluid;

- registration of the signal by measuring the extinction photoconductor at the edge of the light-dark world-light cone formed by passing laser beam through the measuring chamber with an action fluid, and the signal strength depends on the magnitude and number of anticipated anti-antibody precipitates.

2. The method of claim 1, wherein the sensitivity of the detecting particles is in the femto-iatto-molar range.

3. A method according to any one of the preceding claims, characterized in that the stage of preparation of a test fluid containing substantially particle with a certain maximum size includes:

a) preparation of a fluid, addition of a sample of doping and separation of particles, the size of which

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Exaggerates a certain value, to obtain a test fluide, which contains mainly only particles with a certain maximum size, or

B) the preparation of a fluid containing mainlyparticles with a certain maximum size, and the addition of a sample to a fluid containing mainlyparticles with a certain maximum size, forobtaining a test fluid contained in the main particle with a certain maximum size.

4. The method according to any one of the preceding claims, characterized in that the separation of particles with a meter over a certain maximum value is carried out by filtration, the filter having pore sizes of 20-450 nm, preferably 100-300 nm, in particular 200 nm.

5. The method of any one of the preceding claims, characterized in that as an antibody, at least two monoclonal antibodies or one polyclonal antibody are used.

6. The method of any one of the preceding claims, wherein the antibody is selected from the group consisting of immunoglobulin C or an immunoglobulin M.

7. A method according to any one of the preceding claims, characterized by performing a numerical or numerical registration of the number of particles.

8. A method according to any of the preceding claims, characterized in that at a constant concentration of antibodies, the growth of the measurement signal is directly related to the concentration of antigens.

9. Machine-readable data carrier containing the program code written thereon for implementing a method according to any of the preceding claims. 1-8 with the use of a computer or network device, or an analytic device for registration.

10. A set for the qualitative and / or quantitative detectioncertain particle, with a certain particle has at least two sites of antibody binding, whilethe kit contains:

- at least one antibody, capable of specifically binding to a specific particle, and

- at least one fluid suitable for acceptance of the test,

- as well as a device for the registration of small quantitiesparticles, which contains:

- laser

iA (11) 85560 (13) C2

σ>

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- measuring chamber and

- photodetector, made with the ability to measure extinction on the edge of light-dark light

a cone formed by the passage of a laser beam through a measuring chamber containing particles in the fluid.

The invention relates to a method and apparatus for detecting very small amounts of particles by means of the registration of the reaction products of antigen-attityl, and the method has a very high sensitivity - even the dofemot or atomolar range.

It is known from the prior art that various methods for detecting very small amounts of particles are known, for example, nephelometric and turbidimetric methods, as well as a method known as UI_Z (Supposit-IidI-5sayegipd = dynamic scattering of light).

In nephelometric and turbidimetric ways, the Tindall effect (TupSaII) is used, according to which, when illuminating a small particle, there is a wide-angle diffused light. It is possible to register light scattering by measuring the attenuation of the intensity of the incident light beam after passing through the scattering medium or by determining the intensity of the deflection to the side light.In the first case, talk about the method of turbidimetry or measure the absorption of light, and in the second case - about the actual nephelometry or tindallome t-rhythm

Another approach offers a method called the method of dynamic light scattering (Supatys-Iidpius-5sayegipd = JUBZ). In this method, we consider only one (or several) points on the light-emitting sphere, covering the particle, and evaluate the additional modulation of the brightness caused by the Brownian motion of the molecules. By focusing on one single observation volume, they try to make noise suppressing scattered light from several particles. As a result, the particle flies through a small illuminated volume very quickly, so the optoelectronic circuit must recognize significant fluctuation frequencies. Processing such signals is very costly, so this system can be applied for quantitative analysis only conditionally.

In addition, known methods of detection have other disadvantages. The first is to mention that, although the sensitivity of detecting the described means in recent years has increased significantly, in the nary-intensive industries there is an urgent need for ways of detecting with a higher sensitivity. In addition, known inequalities in the methods of detection of the need-for-appearance of still relatively large quantities of pro-material, which in particular in medical and technical applications may be associated with a significantload for the subject.

Therefore, the object of the invention is to develop a method for detecting small amounts of particles, which has a higher sensitivity than techniques of the state of the art.

According to the invention, this problem is solved by the detection of small amounts of particles by the registration of an antigen-antibody precipitate, which comprises: preparing a test fluid containing substantially particles of a certain maximum size, with particles having at least two antibody binding sites; cooking

the fluid containing the antibody, which containsin the particle of a certain maximum size; contacting the test fluid with the fluid containing the antibody, resulting in a reaction fluid, wherein the antibody in the presence of a particle with at least two binding sites of the antibodies can form a precipitate antigen antibody; transmission of light beam through reaction fluid; Signal recording by measuring the extinction photoconductor on the interworld-dark light cone formed when the laser beam passes through a measuring chamber with a reactive fluid, and the signal depends on the magnitude and quantity of the formed anticipant anti-antibody precipitates.

In addition, the object of the invention is solved in a device for recording small quantities of a part-piece, which comprises: a light source, a measuring chamber and a photodetector, designed to carry out measurement of extinction at the edge of the light-dark light light cone formed during the passage of a laser beam through the measuring chamber, in which a fluid with particles is placed. Rose-sowing forward light forms a cone, on the border of light-darkness which is set photodetector. The laser and the photodetector are located mainly on the same axis, however with such a displacement that the laser beam passes past the photodetector very close.

A brief description of the figures

The figures are schematically shown in:

1 a-C Processes in the formation of precipitate antigen antibody using a bivalent antitum.

2a. The result of the registration using an appropriate inventive method, wherein the test fluide containing the particle was tested after filtration with a pore size of 200nm (antibody addition). The signals correspond to the parts whose diameter is smaller than the filter's pores. On the other hand, the size of the particles is delayed, the number of particles in the y-axis.

FIG. 2b is the result of the registration, and a reaction mixture obtained after a single injection of a separate injection by a filtered filter with a pore size of 200 nm of solutions of the test fluide and the fluid with the antibody was investigated. The reaction mixture was reacted in this reaction and received a microprecipitate whose diameter was greater than that of the used filter. On the x-axis, the size of particles is set aside, the number of particles in the y-axis.

FIG. 3 is an arrangement for implementing an appropriately-derived method. FIG.

During numerous experiments that led to this invention, it was possible to develop a method, the sensitivity of which is 1000 times greater than the sensitivity of comparable methods of the state of the art. Such a significant increase in sensitivity is based on the changed physical method of registration, in which the registration of the signal is carried out by measuring the pho-

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the receiver of extinction at the edge of the light-dark light cone formed during the passage of the laser beam through the measuring chamber with the reaction fluid in conjunction with the analytical preparation of the sample, coordinated with that method of measurement.

In addition, this invention reduces the volume of the measuring chamber by about 30-50 times. While the methods of the prior art require a volume of a measuring chamber in the range of 1.6 ml, in the corresponding inventive method, measuring chambers may be used in the range of microliters ( close-up 40μl). This provides a significant advantage, since each sample to obtain a uniform matrix, which has, for example, the same transparency, viscosity, etc., should be diluted; in comparison with the prior art technique for the inventive method, the sample should be diluted less.

In addition, the inventive method provides for a reduction in the required amount of material for the test. While the methods of the state of the art require more than 100 μl, the amount of the material of the sample, which is many times smaller (about 3.5 μL), is sufficient for the appropriate inventive means.

The term "particle" in the sense of this application denotes any three-dimensional structure having a coefficient refraction, different from the refractive index of the carrier medium.

The term "precipitate" in the sense of this application describes the process of formation of an antigen-antibody complex in the reaction of soluble antigens with specific antibodies, which has a less solubility in the solvent used than the antigen used or antibody used, which initially leads to clouding of the reaction mixture, and then to the sedimentation of this complex-su antigen-antibody.

An appropriate inventive method makes it possible to detect small amounts of particles.

For example, in the case of low molecular weight gin wines, that is, substances with a molecular weight of up to 500 mg / mole, when using the appropriate inventive method, the limit value of detection may reach the range of femto and atthograms per liter, whereas for conventional methods it lies in the diagonal of micro- , nano-pyrograms per liter. In the case of substances with a molecular weight in the range of over 500g / mole, the detection limit is higher; for example, in the case of substances with a molecular weight of 150000 g / mol (eg, idO-antibody) the boundary of the detection is about 300 femtog / l. It means that the test fluid may contain parts in the range of femto or atomol per liter.

At the first partial stage of the corresponding wine-making process, the preparation of the test fluide is carried out, which contains mainly particles with a certain maximum size.

This can be done in two ways. Consistently, with the first option for this, first prepare a fluid, which contains mainly only particles with a certain maximum size, and then to the fluid-do a sample containing substantially the parts with a certain maximum size is added. According to the second option, a fluid, a sample is first prepared

add to the fluid, and then separate the time-series, which prevails a certain size.

The maximum particle size in the testfluid or in other fluids, containing in the main only particles of a certain maximum size, can be selected depending on the desired application. When using many common antibodies, separations of particles larger than 20-450 nm, preferably more than 100-300 nm, in particular larger than 200 nm, can be isolated. Such separation can, for example, be effected by means of filters with the corresponding pore size: 20-450 nm, preferably 100-300 nm, in particular, 200 nm, or other known specialist way. Ifagglutination is considered approximately as a plane, thendetermine the size of the filter for the numbermolecules, which contains the detectable reaction products, is manifested in a quadratic dependence. For example, if instead of a 200-nanometer filter used 100-nanometer, then it is necessary more than a quarter of the required at a 200-nanometer filter of antigen-antibody molecules that have to react with each other to obtain an identifiable result. In addition to this, for example, when using 25-nanometer filters, it is possible to detect antigens-antigens trimers. However, when using filters with such a large pore size, work very carefully, since already a small number of molecules can lead to a suitable reaction for registration.

In order for the reaction fluid to occur, the particles in the test fluid must have at least two sites of antibody binding and act as antigen. Figures 1a-1b schematically explain that foreign substances or times, such as bacteria, viruses, toxins, proteins, act as antigens and react with the antibody against the key-lock principle (FIG. 1b). In this case, the equivalent antibody, for example, an immunogen globulin O (iDO) antibody, binds two antigens (FIG. 1c). As each antigen can bind several antibodies, there is a crosslinking (precipitate) that is registered by the appropriate inventive method (FIG. 10 ) For antibodies with higher valence, pre-piation is performed in a similar manner. In the case when the antigen used has a greater number of antibody binding sites, the cross-linking reaction is even better.

A suitable inventive method is suitable for any antigens having at least two months of antibody binding. The investigated particles must have a size of more than 10 nm, and at the same time they are smaller than the selected maximum size of time. Molecules smaller than 10 nm can act as gaptens. The gaps are incomplete antigens, i.e., their molecular size is not sufficient to induce an immune response or cause agglutination. These low molecular weight substances, although highly specific at the site of antibody binding, however, are exited from these binding sites in an insufficient manner so that the second antibody could join them. They block the antibody, preventing the possibility of cross-linking reactions. Larger antigens, for example, bacteria, before measurement, should be crushed by a known chemical chemist or

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by physical means, for example, by processing ultrasound, acids, alkalis, surfactants. In this case, the advantage is that thus dozens of fragments can be obtained from a single bacterium, and no longer needs to agglutinate individual bakteriaries among themselves. The resulting fragments, for example, superficial proteins, in turn, represent smaller antigens and may cause a specificly measured reaction with suitable cinnamate thymol.

In addition, the antigen should be mainly soluble in the used buffer solution and should have a low propensity to adsorb the wall of the devices and filters used.

In addition, the inventive method also encompasses the preparation of a fluid containing an anti-body.

In the method of the invention, in principle, all desired antibodies may be used. Anti-bodies that are particularly well-proven in order to implement the inventive method include, for example, bivalent immunoglobulin C (IDC) ordifferent immunoglobulin M (IdM). In this way, a known antibody can be obtained with a certain specificity. In addition, other antibodies -a belonging to another class of antibodies can also be used, depending on the type of particles to be detected. In this case, the antibodies may be monoclonal or poly-clonal. When using monoclonal anti-antibodies, two monoclonal antibodies, targeting different antigens, can be detected to effect precipitation. As has already been said in the form of a antigen,

In the implementation of the invention, it is unwanted, an excessive excess of antigen-yeer antibodies may be avoided in various ways, for example, by preparing series of dilutions. Otherwise, an excessive excess of antigens or anti-titils could lead to inhibition of precipitation (to the so-called "phenomenon prosesis), because a susceptible excess of antibodies, each epitope (connecting place of the antigen) monovalently binds to only one single antibody and cross-linking is not possible, or because with too muchexperienced antigens often The trimers of an isode molecule of an antibody and two antigen molecules are buried.

As a fluid, in principle, any gas or any liquid can be used in any way to prepare a sample fluids and to prepare a fluid that contains antibodies. Prevailing fluid is a fluid. Often, as a liquid, it refers to water, or known in the art, buffer solutions, such as RVP (RPPP), in particular, when the biochemical reaction is based on the method of analysis. However, in principle, in the case of a liquid, one can also refer to another transparent liquid, for example, of liquid hydrocarbons, acids or alkalis.

In a subsequent step of the invention, the test fluid is contacted with the antibody-containing fluids, wherein the antibody in the presence of an antigen can form a precipitate antigen antibody, which can now be detected using an appropriate device of the invention.

In addition, often of considerable interest is the determination of the presence of haptens in the test. Gaptans are incomplete antigens, that is, they are too small to be able to bind more than one antibody. Haptens may include pharmaceuticals, drugs, pesticides, toxic substances, steroid hormones or mycotoxins. The gaps are specific for antibodies. However, they only block the paratopes of the antibody; a chain re-action can not take place. The minimum size for immunogene (total antigen) is from 5 to 10kDa, that is, more than 30 amino acid residues, that is, over 3nm in length, because starting from this size, at least two antibody molecules bind to this antigen with the corresponding epitopes and cause a chain reaction that is many times leads to a precipitate.

The detection of haptens can be carried out, for example, by hydrophilic macromolecule multiparticulates (ITM) or the like, known to those skilled in the art. Hydrophilic macromolecule multipowers are known in the art and contain a hydrophilic macromolecule, for example, al-boomine. By this hydrophilic macromolecule, with the help of the well-known technology of the spacers chemically joined the same or different molecules of the hapten-niv. In the case where a hydrophilic macromolecular multispeiser has at least two identical hapten molecules, it can be used for prescription. Such a molecule can be used for an antibody detection test, which is based on the substitution reaction. Thus, in the case of haptens, the measurement principle is based on the reaction of the assimilation, which will be described below for an example, and only the peaks of reactions in negative samples are considered,

If a blood sample or saliva should be tested for the presence of a hapten, for example, cocaine, then a diluted drop of blood or saliva should be added with a cocaine-specific antibody. In about a minute, a small amount of synthetically obtained cocaine iteM, that is, a hydrophilic macromolecule, is bound via a spacer to cocaine molecules.

If the sample contains cocaine, it acts as a hapten and blocks the site of binding of antibodies. Further addition of cocaine ITM illustrates the lack of effect. One way, if the cocaine sample was absent, then the sites of binding of antibodies are not blocked and the addition of cocaine ITM leads to the formation of a chain, which in the form of particle growth can be confirmed by the appropriate inventive method, for example, using the O-MAP device described below.

According to the invention, the method is light

beam, in particular a coherent light beam,

for example, a laser beam, passed through

the test fluid. Below is a corresponding invention

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the driving mode is described with reference to the laser light beam, however, this does not rule out the use of other, known to a specialist suitable light source. For example, a laser beam may be produced by the inventive device described below, which is also referred to as a device for quantitatively measuring the atomic molecular weight of the products of precipitation (O-MAP = Cyapiyuidea tevigetepisoiotiaag pyracillioptera-rhubisii). However, alternatively, other devices may be used which may be developed by a person on the basis of disclosure of the invention.

The laser sends a ray through the investigated fluid and determines the number of particles present in it and their size. In this case, by means of photoreceiving, measurements of extinction on the edge of the light-dark light cone formed when the laser beam passes through a measuring chamber containing the reaction flux (and the photodetector can have an adjustable amplification of the signal as well as an adjustable working point) , that is, the device works as a "molecularphotoelement". The following device O-MARzdaten can detect particles with sizes from 20nm to 5μm, however, based on this measurement it is not possible to conclude on the structure or composition of the particles. The laser and photodetector are placed almost on the same axis. The laser beam is only slightly shifted from the photodetector. Distortion before the light forms a cone,

Each particle, which is in the fluid of the entire edge of the measuring chamber and falls into the laser beam, causes the signal, and the number of signals corresponds to the statistical distribution of the part-knock in the measuring chamber. When the particles, even the smallest, pass through the light-cone in the focus of the laser, they obscure it, give skachoktini. Change of primary brightness (without caused by shadow particles) is measured. A high-speed computer, for example, RapidIt, can distinguish up to 10,000 particle passes per second. Smaller parts move faster than larger ones; the passage time is an immediate measure for the size of the time.

In the case of simultaneous hit a few hours to the laser beam is measured the largest largest. Based on the various velocities with which the particles cross the laser beam, with the help of a well-known specialist of the Stokes-Einstein equation (ZOOKEV-EIPVIIiP), one can determine the size of the particles. In this measurement, lessvalue has an absolute particle size, than change in the size of particles (caused by the growth of a part-noc). The selected filter serves as a measure for determining the origin of particle growth. The larger the frames, the larger must be precipitated to differ from the "main noise". In case of pore size of the filter portions 100-200nm for the formation of the data for the measurement of the signal are suitable only some grown particles. Since the particles are rosized-statistically divided, the growth of particles is always located in the focus of the laser, placed all-edge of the measuring chamber.

The rate at which particles are crossed by a laser beam are recorded by measuring the time during which the particle passes through the beam (from the beginning of the change of brightness to the end).

It turned out that the cleaner the rozhen, the more likely it is to stay one particle in the laser beam, and therefore the dirtier solution, the more the signals are superimposed, which leads to a decrease in sensitivity.

Signal strength depends on the size and number of antibiotic antigens.

At a constant concentration of attityl, the growth of a measurable signal is directly related to the concentration of antigens.

The corresponding inventive detection can be carried out, for example, as follows: all fluids to be investigated, through a filter with a certain pore size, are injected into the measuring chamber, so that in the study of the test fluids or fluids containing antibodies, for each separate treatment, only signals arise fromparticles smaller than a certain size. In the corresponding inventive method, the antibodies used, and the antigens used, have sizes smaller than a certain limit value, thus, less than the pore size of the filtrate used for separation (for example, less than 200 nm), due to which they were not separated during the filtration.

Fig. 2 shows the results of a test fluid on the basis of a MIaCI solution. On these Figures along the x-axis, the size of the particles is set aside, along the axis in - the number of particles. As can be seen from Fig. 2a, only particles in the solution are present up to a certain maximum size, and the amount of particles is shrinking with increasing size. A similar result of the study is obtained for a filtered fluid, which contains mainly only parts of a certain size (not shown).

After simultaneous, separate introduction of pro-bluyu and fluid with antibodies in the measuring chamber there is a reaction to the formation of microprecipitates, the size and amount of which are then recorded.

Such a measurement result for an example is shown in FIG. 2b, which shows the appearance of a portion whose dimensions are predominantly of a predetermined boundary size, for example 200 nm. Thus, the formation of larger particles indicates that the reaction occurred. If the reaction to the formation of larger particles does not occur, this means that the test fluid does not contain this antigen.

Measurements can be initiated at any time after the reaction chamber is filled with a test fluid and a fluid with an antibody. According to an expedient form of this embodiment, the measurement begins only after 60 seconds after filling the measuring chamber, since during the injection of fluids, light fluctuations in flows can lead to convective inhomogeneities. The weakening of the measuring signal coming after it enables an approximate quantitative registration or evaluation of different concentrations of the antigen. At the same time, the initial concentration of both participants in the reaction plays a decisive role

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role. The higher these initial concentrations, the more important is the time interval to the measured reduction of the maximum concentration.

When implementing the invention, it is advisable to observe the highest purity of used fluids and filter the filter with pore sizes, for example, 200 nm, in order to avoid or minimize the effect of the main noise in registration. In addition, materials that are not manufactured or used in the method of the device should be as small as possible particles. For example, the measuring chamber can be made of polytetrafluoroethylene (PTFE). To avoid the effects of particles (for example, measuring chamber) particles exposed during a three-time period, it is necessary to observe the shortest possible time of measurement.

In addition, the concentrations of antigens or antibodies used should be so low that during the reaction, the antigen-antibody did not produce too much or too large pre-cypitates, because otherwise, several particles can simultaneously get into the beam, which would greatly impede determination of travel time. The highest sensitivity of the inventive method is in the femto and atmolar range.

A particular advantage of the inventive method is that it also enables quantitative or semi-quantitative analysis.

To assess the quantitative registration may beused two methods. In accordance with the first method, estimates are determined by the area (E) under the measuring curve at moments of time H and 12 (for example, 11 = 120 seconds, and 12 = 180 seconds), and inequation

Ee = (Eti + E (2): 2

get a well-approximated value Εν for the space from which the number-not the value of the number of particles can then be obtained. With these very niche concentrations, the growth of particles is linear, that is, for example, doubling of the concentration leads to a doubling of the area of ​​the measuring signal.

An alternative and / or supplementing method for quantifying the conclusion is based on conducting solutions until the kinetic boundary of the bimolecular reaction is reached. At concentrations up to 100 atoms, the free path lengths of the reaction particles are too large (> 100 μm), and at the pre-set time of measurement, there is no precipitate that is suitable for registration. At concentrations of more than 10 nanometers begins stereotyping due to the excessive number of large quantities of reaction products; In addition, the above explanation of the phenomenon of prose is manifested. When the concentration of solution 10micromol freelong mileage of the particle is already only100nm.

The determined degree of dilution in this physical terminal point depends only on the temperature andconvection, since the viscosity at high dilutions, for example, in RVS, does not play a role. If these offensive parameters are stable, then the degree of expansion of the initial solutions is directly to the extent of concentration of solutions.

Exact quantitative determination of the concentration of samples in the implementation of an appropriate inventive method can be performed using a standard solution with a known concentration of the corresponding protein, and the application of this method to the home-specialist.

In accordance with another embodiment of the method, continuous and / or multiple test sample measurements of the fluid in the measuring chamber may be performed. Due to this you can in particular set the final reaction point and create average values ​​of measurements. It is expedient to carry out measurements for a carrier fluid free of particles with diameters above a certain value, prior to introduction into the chamber of an antibody and / or sample; the result of this measurement can be used as a reference value for measuring precipitate.

In the implementation of the invention, it is possible to dispense relatively small amounts of fluids and therefore the costs of removing from the fluid used substances whose particle size exceeds a certain value may be reduced. To remove such substances, the fluide used, for example, by means of valve devices, may be omitted because of a filter-rung shunt that separates these substances. The filtering of the fluid used may be carried out before entering a sample through a predetermined time interval to ensure that it no longer contains substances that will interfere with the measurement. Materials that have particles with dimensions over a certain set value can be removed from the sample using filters integrated into the fluid inlets or installed in front of them.

According to another embodiment of the method, a non-drying fluid after the addition of an antibody that has supported or not confirmed the presence of an antigen is again filtered prior to the introduction of another antibody. Due to this, materials that may be interrupted by measurement may be removed from the carrier fluid. In addition, thus, from the used fluid, the detected reaction product may also be removed to analyze possible further components of the sample.

The appropriate inventive method is suitable for numerous applications in the field of water analysis (display of components and harmful substances), food technologies (detection of microorganisms, constituents of substances) or biological or medical tests (for example, the detection of DNA or RNA sequences, bacteria or allergens (tests for allergies)). In addition, possible applications in the field of hydrophilic macromolecular multispeakers, DNA probes and quantitative polymerase chain reaction (PCR). In particular, an appropriate inventive method can also be used to detect the infectious prion protein RgR^Cc when conducting the tests of VSE (Lompe vropdiUpti

epasporoiI = bovine spongiform encephalopathy

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thia). Since the inventive method is capable of detecting extremely small amounts of this prion protein, it is suitable for detecting an infectious Prong protein PgR^& in the blood

In addition, the corresponding inventive measurement method of O-MAP provides a quick and easy way to test the adsorption behavior of various proteins on different surfaces. Thus, non-destructive control of high-polished surfaces may be carried out on the "value of protein adsorption", and the deviation from the given value indicates the defects of the surface. In this way, defects on the surface of a thin metal film deposited by the vapor deposition method may simply and quickly be detected.

In addition, a complete immobilization of a certain protein on the surface can be tested, which has a large value, for example, in the field of DNA analytics and biochip technology.

Another possibility of using an appropriately derivative method may be based on the ability to determine the impact of chemicals on the surface. In particular, Do / After tests can be performed to detect changes in surface treatment, since the pro-etched surface has a larger area than is not pro-etched, and therefore will be able to adsorb a larger number of antigenic materials, for example, proteane. In this case, for example, the amount of protein remaining in the solution may be determined.

Another possibility of application is based on the fact that the inventive method can also be used to check the condition of the internal surfaces of the hoses and pipes.

After the steps have elapsed, the proper way of anticipating the antigen-antibody can be dissolved, for example, by the K proteinase, of substances that are not destroyed by the K proteinase (for example, prions and substances that do not contain amino acids) and subjected to the next stages of processing the study.

An important advantage of the appropriate invention is the ability to implement a quantitative registry. In addition, the method can be carried out quite automatically, which provides significant cost savings.

In addition, the inventive method allows the detection of pathogens in diseases in the fluid or the body, in particular in the blood, in very small quantities. This has a significant advantage, when a very small amount of blood should be taken, for example, one drop (about 10-20 microns) of a finger or ear of the ear without the involvement of a doctor or an experienced nurse. Such tests can be carried out, for example, in pharmacy in houses for the elderly or in rehabilitation institutions; they simply and quickly conclude that there is an infection with a certain bacterium. A particular advantage is that both the sample taken and the implementation of an appropriate inventive method with the use of an appropriate invention device does not require an executor of medical education or high qualification.

substances, such as drugs or medicines.

Whereas in the case of the sale of measuring devices, the ratio between antigens and anti-titiles can not differ more than 2-3 times, using the O-MAP method, measurement is possible even when the ratio of interantigens and antibodies differs from ideal one hundred times. (At the same concentration, the antibody-neutral concentration of the antigen may be reduced-up to 1% or increased to 10,000%. This is also true for the antibody at a constant concentration of an-tigena). This is an interesting deviation from the "Heidelberg curve" ("NeiSeijierdeg-Kip / e") in the femto or atmolar range caused by certain spatial constraints of the molecules, making it superfluous in the course of the formation of a series ofdevelopments.

Particularly suitable inventive method for studying accumulated samples, which is particularly interesting for studies of preserved blood (for example, from the point of view of the immunodeficiency virus or hepatitis virus) and test specimens in the EEZ. With the use of the method of the invention, about 50 measurements per hour can be performed, which gives at least 4800 studies a day or more than 80,000 measurements per year for an 8-10-hour day. In the case of 10-100 accumulated samples, only one meter measuring device per year can be tested for 80000-800 00 samples.

When studying canned blood it can be added one time, for example, 10 different antibodies (against 10 different diseases) to determine whether this blood is suitable for use. In the case of a zytic reaction, this blood should be discarded, because all-one - what it is infected: an immunodeficiency virus or hepatitis. When it is necessary to determine which pathogen reacts positively, antibodies may be added separately. If, for example, in case of 100 canned blood vials simultaneously apply 10 different antibodies, then with the help of the whole one, which lasts about 60 seconds, it is possible to determine whether all 100 vials are suitable for use.

A measurement method for which such a small amount of the product is sufficient and which has a high sensitivity in the femto or atmolar range is still not known from the prior art and is a significant progress.

Additionally, the present invention includes a computer software product having software coding software written on a computer-readable medium for executing, respectively, an output method for performing a computer program product on a computer, network device, or other device, in particular analytical device for registration. In addition, this guideline includes a computer software product, which is software code and can be downloaded from the server for the implementation of the appropriate inventive way when performing a computer software product on a computer, network device or other device, in particular, analytical device

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for registration (for example, described in this application device for registration).

In addition, the present invention relates to a device for detecting small amounts of particles, which is also referred to as a device for the quantitative measurement of atomologic amounts of products of precipitation (O-MAP = Cyapiyayeuye tevigetepisiotioaggheriyiaopy-rhobisiv).

Such a device contains a light source, in which the laser is used predominantly as a light source.

The measuring chamber of such a device must be made of material that does not emit time and allows the passage of the light beam, in particular the laser beam. Suchmaterials are known to a specialist. For example, a measuring flax can be made of polytetrafluoroethylene (PTFE). The measuring chamber has a volume of 100 μl, preferably 30-50 μl, in particular 40 μl.

The corresponding inventive device comprises a photodetector having adjustable sig-pole amplification and an adjustable working point.

The photodetector can be selected from the group consisting of thermal detectors, photodiodes, in particular photoconductivity detectors, photovoltaic detectors, avalanche diodes, diode arrays, photomopoints.

An appropriate inventive device for registration allows detection of particles in the size from 20nm to 5μm.

Additionally, the present invention is also available on-the-spot for the qualitative and / or quantitative detection of certainparticles, for example, proteins or hormones, in which case certain particles have at least two sites of binding of antibodies. The kit contains the device described above for registration, at least one antibody that can be specific to a particular particle, and at least one fluid for receiving the sample. This set can be designed specifically for certain user needs and contains mutually agreed components and a corresponding description for the user-wach, which by this set can immediately simply detect the part. For example, such a kit can be used to detect a particular pathogen in a small amount of blood or for a description of the surface of the study.

Exhaustive description of FIG. 3

In the following, the invention is explained in more detail with the help of the example given, for example, a schematic illustration of an apparatus for implementing, respectively, a derivative method that does not limit the volume of wine-stroke.

Apparatus 1 for changing samples 1 can be injected into mixers 2 and 3 as various antigenic solutions (for example, blood samples), as well as various solutions with antibodies, so that one test is consistently investigated on the availability of various possible agents. Mixers 2 and 3 for solutions containing antigens and antibodies, respectively, can serve not only for breeding the appropriate solutions, but also for washing the measuring chamber with a buffer solution (RVZ) or solution with the antibody.

Filters 4 are variable and have pore sizes, for example, 200nm. The valve 5 can be executed in such a way that the solutions can be separately or in turn fed to the measuring chamber 6, with the continuous, single or multiple samples, detecting the particles.

Pump 7 is a compact vacuum suction pump that pumps out all solutions. In my work, I am in agreement with the valve 5, and after measuring the content of the measuring chamber to the vessel 8 for waste is measured. It can contain disinfectant sterile imaging fluid, which immediately makes non-harmful all possible hazardous substances.

In the case when the antibody is injected with the antigen and the test is reacted with the formation of the anti-anti-antibody precipitate, they become particles with sizes exceeding certain values. These precipitates cause a signal that is clearly different from signals that are possible in a carrier fluid of time, having dimensions smaller than a certain value. As a result, these reaction products are uniquely recognized by the device for detectingparticles. The fact of the discovery suggests that some substance will flourish, and in certain circumstances, in accordance with the methods described above, it may be defined as its content.

In the case when after the injection of the first antibody with the antibody, the registration device did not detect particles with dimensions larger than the established value, or when it is assumed that another antigen that does not enter into a specific reaction with the antibody used can be absorbed into the solution with another antibody. Prior to this, possibly detectable precipitates may be removed from the measuring chamber by flushing.

The following examples serve to explain the invention. The scope of legal protection of the givenproperty is by no means limited to the subject of these examples.

Examples

Example 1: Blood testing for co-spongiform encephalopathy (VZE) The number of cattle in Germany is close to 15 million. Every year in Germany, 2 to 3 million WHO tests (EDZZL and ShevEgypVioI) are performed on the brain of dead animals. High-speed VZE-testing (EDZZL or ShevEgyp VIA) lasts 6-8 hours. With the use of the inventive method of the pathogen PVE in a living animal, it can be detected with only one drop of blood for two minutes, with only insignificant amounts of expenditure.

Example 2: Examination of canned blood on the presence of a new pathogen of the Kreutzfeld-Jacob disease (Cerebrospellum; pSbC)

In order to protect the population from the possible risk of transfusion of pSbK through blood, a number of different measures were proposed. The most promising success would be to test each individual donor for the on-the-spot infection, as for hepatitis or CHD. However, the causative agent is in the body of the patient in very small quantities. Reliable testing at the technical level is absent.

The appropriate inventive method is able to detect

an agent in blood or in blood products.

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Example 3: Application in Food Processing

Mycotoxins are a strong poisonous product of decay of some mushrooms. Mycotoxins are a hapten-we and therefore, using the appropriate inventive method, their detection and quantitative analysis can be carried out.

In screening surveys, for example, the entire ship's cargo of coffee, tea, flour or chips, it is enough to carry out a qualitative testing, which can be carried out in place in a couple of minutes.

In order to establish whether the animal was illicitly fed using hormones, a sample of meat should be checked for the presence of about 15 different hormones. Hormones are also hap-tena, so using the appropriate wine-making method can be carried out their detection and quantitative analysis.

For the testing of pregnancy and for the study of thyroid gland, a check on the presence of hormones is also required.

Since large slaughterhouses process 2000-3000 head of cattle per month, to detect unauthorized feeding, you need to run about 50-100 sample samples. At this time, hormonal testing of a piece of meat (on the presence of about 15 different hormones) costs 600 euros. Therefore, the slaughterhouses cost only 5-10 selective-

per month, which is clearly not enough to detect unauthorized feeding. At the entrance to the negotiations with large slaughterers, there was a growing interest in a 10-fold increase in the number of cheap test runs (about 60-70 euros for testing a piece of meat) based on the corresponding inventive method. Normal testing on the presence of hormones can not offer the sameprices.

Example 4: Identification of plant protection products

Tubulin monomers are isolated small protein balls, which, through the reaction of carrier methylphenidia, develop in long chains. Inhibitory other toxins slow down, and even stop, this growth of chains. Separate chains are weaved in kanato-like structures, the so-called protofilamen-ti. These protofilaments, in turn, are linked by microtubules, which play a decisive role in the solids. By inhibiting the development of monomers in the chain affect the division of cells and destroy pests.

Conversely, this method makes it possible to detect residues of plant protection products in foods and thus provide increased consumer protection. Using the method of the invention, it is possible to carry out indirect measurements in the course of the described above chain or inhibition thereof.

FIG 2a

FIG 2b

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Computer layout S. Litvinenko Signature Circulation 28 copies.

Ministry of Education and Science of Ukraine

State Department of Intellectual Property, st. Uritskogo, 45, Kyiv, Ukraine, 03680

State Enterprise "Ukrainian Institute of Industrial Property", st. Glazunova, 1, m. Kiv - 42, 01601