Method of multyanalite immunoassay with use of microparticles

FIELD: medicine.

SUBSTANCE: on surface of porous membrane apply the reactionary admixture containing analyte, the first binding molecules bound to detecting substance and specific to analyte, the investigated sample and the particles, not capable to pass through the pores of a membrane covered with the second binding molecules, also specific to analyte, incubate an admixture for formation of a biospecific complex, wash an admixture from not bound reagents and register in a regimen of the time permission phosphorescence signals in spectral ranges of the detecting substances corresponding to a constant of time of attenuation of these substances. Determine the required analyte on a parity of measured phosphorescence signals, thus use on two kinds of the first and second binding molecules, each kind of the first binding molecule is bound to two detecting is long luminescing substances, for example chelate of europium and platinaporphyrine which parity of concentration in each first binding molecule is chosen in advance and corresponds to defined analyte.

EFFECT: method allows finding out and quantitatively to detect low concentration biological analytes in assays at carrying out of researches on solid surface.

5 cl, 5 tbl, 3 dwg

The invention relates to immunoassay and can be used for detection and the detection of low concentrations of microorganisms, proteins and other biological analytes in an isolated form or as contaminants of food or the environment.

Methods screening of specific substances in low concentrations in the environment (bacteria, microorganisms, toxins and the like), and height requirements: the methods should be simple, quick, give unequivocal results, to be able to operate in harsh environmental conditions, to have the necessary level of sensitivity and specificity, be suitable for the determination of very low concentrations of biological analytes, results should be read quickly, visually or with a minimum number of simple equipment. Existing technologies bacterial detection require a large number of bacterial cells (10⁵-10⁸) at the final stage of analysis before detection. The increase in the number of cells is achieved by laboratory methods, including the procedure for selective enrichment and isolation, and requires a lot of time. For continuous monitoring of the external environment (air, water, soil) in cases of anthropogenic or biogenic accidents or biological terrorism is necessary is to provide for rapid and simultaneous detection of low concentrations of multiple biological analytes in site contamination.

Known indicator reagents and methods of their use (U.S. patent No. 5252459, NCI 435/6). As indicator reagents are used in the invention of latex particles with specific binding members (Biotin, avidin, and other), and as immunoreagents - various antibodies, haptens, antigens and the like, the Method consists of contacting a test sample with an indicator and linking reagents, the binding of the indicator reagent with the analyte, the target in the test sample, the detection of the indicator reagent and determining the presence or amount of analyte in the sample. The method can be used for visual examination of the results of the analysis of clinical samples.

The method cannot be used to determine low concentrations of the desired analytes due to insufficient sensitivity.

The known method and compositions for the simultaneous detection of multiple analytes in the sample (PCT application No. 01/073443, MKI G01N 33/58). The invention can be used for detection of pathogens, as well as for the diagnosis and prognosis of diseases. Search analytes in the sample will detect and differentiate, using a variety of fluorescent latex particles associated with different binding molecules that are specific to antigens; antigens have been labelled with a second fluorescent label and, fluorescence which is different from the fluorescent latex particles, and detects the formed complexes containing fluorescent microparticles binding molecules and labeled antigens. Measurement of the fluorescence of the particles is carried out by determining the ratio of the fluorescence of one or more dyes. When the determination of multiple analytes, each particle has its different from other fluorescent ("address") tag, and each antigen is labeled with the same fluorescent label. For detection of antigens, preferably using flow cytometry. As fluorescent labels for microparticles using fluorescein, phycoerythrin, rhodamine, and the like; for labeling antigens using fluorescent dyes that differ from the fluorescent dyes used for particles.

The disadvantage of this method is the need for multiple fluorochromes for tagging the address of the particles, which leads to the necessity of using multiple sources of excitation of luminescence and multiple detecting devices to determine the form of particles, i.e. the type of analyte, and determining the analyte concentration. For detection of the analyte using flow cytometry methods, which have low performance due to the fact that in the duct of each particle is investigated separately. Cu is IU, flow cytometry methods are carried out on expensive equipment and additionally require the use of complicated devices dynamic focusing of the studied stream.

Known biospecific mnogokanalnyy way analysis of biological samples (U.S. patent No. 5028545, NCI 435/501). In the process for determining multiple analytes using several categories of microparticles, each of which has a different number associated fluorescent substances with a short decay time of the fluorescence emission, each category of microparticles corresponding to a particular analyte, cover biospecific reagent, such as antibodies, various categories of particles placed in suspension, which adds a sample with the desired analytes and the mixture biospecific reagents labeled fluorescent label with a long decay time, bred suspension in order to reduce the concentration of labeled reagents, not contacting microparticles (to reduce background), at the same time excite the fluorescence of both fluorescent substances, particles identified by emission fluorescence with a short decay time, and the concentration of analytes - emission fluorescence with a long decay time. The suspension is analyzed by way of a quick scan under the microscope in oncol ditch, in which the slurry is pumped from the reaction tube.

The disadvantage of this method is the length of the holding analysis of the analyte under the microscope, because the size of the sample is limited and you want to analyze a lot of micro. In addition, the sensitivity and performance of the method is limited because of the need to use the transaction dilution of the sample.

Known biospecific mnogokanalnyy method for studying biological samples (U.S. patent No. 5891738, NCI 436/501). The method is based on the use of different categories of microparticles coated with different first specific analyte reagents that include one or more fluorescent indicators in one or more concentrations. In a suspension of microparticles add the sample to the desired analyte and the second biospecific reagents, labeled photoluminescent label, initiate biospecific reaction, diluted suspension in order to reduce the concentration of labeled reagents, not contacting microparticles, excite and detect the emission of luminescence indicator and photoluminescent labels, while the category of microparticles, respectively views of analytes is determined by the intensity of the two briefly luminescent fluorochromes indicator labels, and the concentration of an analyte is determined by intensive the spine antistokovskogo fluorescence photoluminescent labels. As indicator substances use tetrapyrrole (porphyrin, chlorin, phthalocyanine and others) and cyanine substances, and as a photoluminescent label - phycoerythrin. The study sample is carried out microfluorimetry using two-photon excitation method luminescence or confocal method of generating and recording the luminescence, which can significantly reduce background fluorescence.

The disadvantage of this method is the need for a research sample in suspension without the Department is not bound peroxidase labeled ligands, resulting in a large background fluorescent signal, which eliminates the devices with complex optical system, which increases the cost of equipment

Know the use of microparticles with multiple fluorescent labels for detection of multiple analytes (PCT application No. 01/13120, MKI G01N 33/58). In the proposed invention the microparticles having on its surface one or more populations of fluorescently labeled nanoparticles. Changing the amount and ratio of different populations of nanoparticles, can be installed and distinguish a large number of discrete populations of particles-carriers and, accordingly, the desired analytes with the same emission spectrum. The method can be used to study biological fluids, as well as treatment the samples, taken from the environment, water or air, industrial sources, waste water, etc. For detection of analytes mainly using flow cytometry.

The disadvantage of this method is the necessity of using methods of flow cytometry for detection of desired analytes, the shortcomings of which were analyzed previously.

A known method of distinguishing multiple subpopulations of different types of particles in one sample (U.S. patent No. 4717655, class MKI G01N 33/58). The method includes tagging of sensitive substances two or more marking agents by using different pre-selected ratio. Relationships can range from 0% to 100% of each agent, each agent has a different and high-quality set of marking characteristics. The method also includes mixing the various labeled substances with particles having specific receptors to variously labeled substances. As marking agents using fluorescein and rhodamine. Each particle analyzed to determine the relationship between two identifiable marking characteristics associated with each particle. The particle study conducted using methods flow cytometry.

The disadvantage of this method is the use of labeling agents having partially overlapping pectra issue, that limits the number of designated agents and increases background fluorescence.

The closest is a method of performing immunoassay using two particles (U.S. patent No. 6096563, NCI 436/523). The method is designed to detect a wide range of analytes and can be used for analysis of biological samples, and to determine various environmental pollutants. The method allows you to get immediate and reliable results about the presence of residual contamination at the place of pollution, in the field, at home. Detection is carried out with the naked eye or simple devices with a minimum number of operations, in liquid and on solid phase. The essence of the method lies in the fact that on the surface of the porous membrane is applied, the reaction mixture containing the first binding molecule specific for the analyte, the test sample and particles coated with a second binding molecules specific to the analyte, but not able to pass through the pores of the membrane. Then the membrane is applied detecting particles that may pass through the pores of the membrane, these particles are covered with a binding substance that binds to the detecting particles with the first binding molecules, such as second antibodies. In the presence of the analyte in the sample on the membrane surface is formed comp is CEN "detecting particles the first binding molecule - analyte - second binding molecule - coated particles, which are retained on the membrane surface and detected, for example, to the naked eye, a colorimeter, a Refractometer, etc. depending on the type label. Covered and detecting particles may vary in color and size and are made of different materials; the size of the second coated molecules preferably 3 μm; the size of the first coated molecules preferably 0.3 microns. As the membrane is an inert porous filter, which can be made of plastic, ceramic, glass, metal. As covered and detecting particles commercially advantageous to use the latex.

The disadvantage of this method is the need for multiple kinds of dyes for labeling of detecting particles, which entails a more complicated way as in visual and instrumental detection of the analyte in the sample.

The task is to create mnogoukladnogo detection and quantitative detection of concentrations of biological pathogens in samples from objects in the external environment, as well as other specially prepared materials, such as food products, clinical samples.

The technical result that is achievable with the use of the invention is the possibility of the simultaneous measurement of the luminescence signal of at least two fluorescent markers, related searched analytes on a solid surface, allowing you to explore multiple analytes with a single source of excitation of luminescence and one detector. In addition, all cells of the desired analytes associated with a solid surface are scanned simultaneously, which significantly reduces the time of analysis.

The technical result is achieved by the invention.

The invention consists in that in mnogayaleta the way for immunoassay, comprising coating the surface of the porous membrane of the reaction mixture containing the analyte, the first linking molecule that is associated with the detecting substance and specific to the analyte, the test sample and the particles unable to pass through the pores of the membrane, covered with a second binding molecules specific for the analyte, the incubation mixture for the formation of a biospecific complex, the laundering of the reaction mixture from unbound reagents and detection of the analyte in the sample by the light signal detecting substances associated with biospecific complex, use at least two types specific to desired analytes the first and second binding molecules, each type those and other molecules specific to only one of the desired analyte, each of the first binding molecule is bound at the very the very least two detecting long luminescense substances, the ratio of the concentrations in each of the first binding molecule is selected in advance and corresponds to the desired analyte is recorded in a temporary resolution the phosphorescence signals in the spectral ranges of emission detecting substances corresponding to the time constant of decay of these substances, and determine the desired analyte in the ratio of the measured fluorescence signal, while the spatial resolution of the detection system, Δ(μ²) is determined from the following equation

Δ≤S/10Nμ²where

S - area adsorption of particles on the membrane, μ²,

N is the given number of latex particles in the analyzed sample.

The technical result is also achieved by the fact that as the detecting substance use chelating agents with europium ion.

The technical result is also achieved by the fact that as the detecting substance use complexes coproporphyrin, uroporphyrin, protoporphyrin, tetraphenylporphyrin with metal Pt(II).

The technical result is achieved by the fact that as the first binding molecules using polymer nanoparticles with a diameter of from 40 to 150 nm, associated with molecules having high affinity with respect to desired analytes.

The technical result is also achieved by the fact that the quality of the ve particles, covered with the second binding molecules, use latex microparticles with a diameter of 0.5 to 10 microns.

In addition, the technical result is also achieved by the fact that the concentration ratio detecting substances of the first binding molecules constantly and differs from the ratio of concentrations for a different first binding molecule at least two times.

The authors do not know the way possessing claimed by a collection of characteristics, therefore, the claimed method meets the criterion of "novelty."

Known methods of conducting mnogoukladnogo analysis using microparticles. In U.S. patent No. 5028545, NCI 435/501 described the way in which to identify the types of particles, respectively, of an analyte using substances with a short decay time of fluorescence, and detecting the analyte concentration is the use of substances with a long decay time of fluorescence, in particular chelates of lantanides rare earth elements, europium, terbium, and others). The detection and quantitative measurement of different analytes is carried out by the research sample in a thin cell under a microscope and detection of each individual particle. In U.S. patent No. 5891738, NCI 436/501 described method of making mnogoukladnogo analysis to determine multiple analytes in the sample with ISOE what Itanium microparticles. For identification of microparticles using substances with a long decay time of the fluorescence emission, and for detecting the concentration of an analyte is a substance with a short decay time of fluorescence. The measurement is carried out by use of flow cytometry. In PCT application No. 01/13120, MKI G01N 33/58 described method of making mnogoukladnogo detection using many kinds of colored microparticles. Using either the number of kinds of particles equal to the number of detected analytes or different concentrations of the same particles. The analysis is carried out in a flow cytometer. In U.S. patent No. 4717655, MKI G01N 33/58 describes how to use the ratio of marking agents to determine the type and concentration of the detected particles (analytes), however, used by marking agents have overlapping emission spectra of fluorescence, which makes it impossible to determine a wide range of subpopulations of particles. In addition, due to the short decay time of fluorescence used marking agents, and the overlapping of the spectra of the emission fluorescence measurements recorded significant error due to the influence of background fluorescence. The implementation of the above methods requires either complex optical devices (spectrofluorimetry with two-photon wosb is the establishment and measurement of the fluorescence emission, using confocal optical systems, microscopes with CCD cameras), or expensive methods of flow cytometry with a complex system of dynamic focusing of the stream, multiple radiation sources and multiple detectors. In comparison with the known technical solutions, the proposed method through the use of a variety of ratios of marking agents with a long decay time of phosphorescence and use their temporal characteristics of attenuation in conjunction with a time resolution measurement of signals from particles and simultaneous scanning of the phosphorescence of all particles in the solid phase, and also by reducing the background fluorescence of impurities has made possible the rapid identification of a large number of analytes (particles) in the sample at low concentrations. However, the different combination of multiple fluorescence labels used for identification of analytes, and to measure their concentration, which further reduces the cost and simplifies the way. Therefore, the claimed invention meets the criterion of "prior art".

The invention can be used for detection, identification and quantification of materials that are able to bind to specific binding agents, in particular biological materials, such the AK antigens, microorganisms and nucleic acids. In particular, the method can be used to detect low concentrations of microorganisms specific genes, species or serotypes in an isolated form or as contaminants of food, environmental or forensic samples. The method allows for rapid analysis in the field, on the site of contamination, based on it can be created a simple device that is portable, simple elemental basis. Therefore, the claimed invention meets the criterion of "industrial applicability".

Figa - distribution of long-luminescent microparticles 1 on the membrane area of 1 mm²(fragment of the bottom of a microplate well) when analyzing samples with the plague pathogen at a concentration of 4000 ml/ml staphylococcal enterotoxin type b at a concentration of 10 ng/ml when measured signal intensity at a wavelength of 645 nm.

Figb - fragment of membrane area 330×330 microns: y-axis Z - signal intensity at a wavelength of 615 nm;

Figw - the same fragment; y-axis Z - signal intensity at a wavelength of 645 nm; 2 - peaks of luminescence, which is the ratio of 1/1 (615 nm/645 nm); 3 - peaks of luminescence, which is the ratio of 2/1 (615 nm/645 nm).

Figa - distribution of long-luminescent particles 4 IU the bran area of 1 mm ²(fragment of the bottom of a microplate well) when analyzing samples with Francisella tularensis in a concentration of 5000 ml/ml staphylococcal enterotoxin type b at a concentration of 50 ng/ml when measured signal intensity at a wavelength of 645 nm.

Figb - fragment of membrane area 330×330 microns; y-axis Z - signal intensity at a wavelength of 615 nm;

Figw - the same fragment; y-axis Z - signal intensity at a wavelength of 645 nm; 5 - peaks of luminescence, which is the ratio of 1/2 (615 nm/645 nm); 6 - peaks of luminescence, which is the ratio of 2/1 (615 nm/645 nm).

Figa - distribution of long-luminescent microparticles - 7 on the membrane area of 1 mm²(fragment of the bottom of a microplate well) when analyzing samples with botulinum toxin type a at a concentration of 10 ng/ml, the causative agent of anthrax 5000 spores/ml staphylococcal enterotoxin type b at a concentration of 100 ng/ml when measured signal intensity at a wavelength of 645 nm.

Figb - fragment of membrane area 330×330 microns; y-axis Z - signal intensity at a wavelength of 615 nm;

Figv the same fragment of the y-axis, Z - signal intensity at a wavelength of 645 nm; 8 - peaks of luminescence, which is the ratio of 1/2 (615 nm/645 nm); 9 - peaks of luminescence, which is the ratio of 1/4 (615 nm/65 nm); 10 - peaks of luminescence, which is a ratio of 4/1 (615 nm/645 nm).

For conducting immunoassay using 96-cellular microtiter plate with the bottom of the cell area 30-36 mm²made of filter membrane material with a pore diameter of less than the diameter of the microparticles with the second binding molecules. For adsorption of detected analytes (antigens) use latex microparticles with immobilized antibodies at a concentration of 10²particles with a diameter of 2.5 microns for each of the analytes. For washing and removing unbound components, the use of membrane filtration. As manifesting biospecific reagent using a second antibody (the first binding molecule), marked by at least two phosphorescent (detecting) labels, such as metal and chelating complex with europium ion. To detect, for example, four types of analytes using a mixture containing antibodies to all four analytes. The first binding molecules that are specific to different analytes, different pre-selected concentration ratio detecting these substances on their surface that allows the identification of immune complexes adsorbed on latex particles. Scanning the bottom of the cell microtiter card OS is p resolution microprosodic 30× 30 µm mode time resolution luminescence, emitting a long luminescence (phosphorescence) in the range from 50 to 300 μs, while the spatial resolution of the detection system, Δ(μ²)=900 μ²that provides performance ratio

Δ≤S/10N=36×10⁶/10×4×10²=9000 μ²

Detection and identification of the desired analyte carried out on the level and ratio of phosphorescence in two spectral ranges, 610-620 nm and 640-660 nm, which correspond to the maxima of the luminescence of europium ion (615 nm) in the composition of the chelate complex and Pt-coproporphyrin (645 nm).

The analytes that can be detected by the proposed method, refer to biological agents found in the environment (water, soil, waste and industrial waters), in food, biological samples, and represent viruses, pathogens, antigens, toxins, low-molecular biologically active compounds.

As the first (detecting) binding molecules can be used for the first antibody, fragments thereof, and other biological molecules that have the ability to biospecific binding with the target analyte.

As a second binding molecules can be used for the second antibody, fragments thereof, the other biological molecules, with the ability to biospecific binding with the target analyte. For the desired analytes containing several repetitive epitopes as the first (detection) and second binding molecules can be used the same molecule.

As the detecting substances associated with the first binding molecules can be used chelating ions of europium complexes coproporphyrin, uroporphyrin, protoporphyrin, tetraphenylporphyrin with metal Pt(II) and other substances having a long decay time of the luminescence.

The bottom of the wells of microtiter payment made in the form of filter (membrane) made of polymeric material with a pore size allowing to be unbound components of the biospecific reaction and impervious latex particles with associated complex of the analyte and first binding molecules with the detecting substances.

Example 1. The preparation of the first binding molecules containing the detecting substance on the basis of orthosilicate nanoparticles.

To determine in a sample of one of biopathogens, plague, anthrax, tularemia, staphylococcal enterotoxin type In preparing the conjugates of the first binding molecules in the form of long-luminescent orthosilicate nanoparticles with monoclonal antibodies to each of ISCO who's the bioagent.

Obtaining nanoparticles associated with Pt-coproporphyrin and chelate complexes of Eu ions, is performed by the method of S. Santra et al (Anal. Chem., 2001, 73, 4988-4993).

The nanoparticles get controlled hydrolysis of tetraethylorthosilicate (TEOS) in itoge-oil emulsion, atage-oil emulsion is prepared by mixing on a magnetic stirrer to 0.94 g of Triton X-100 (Serva, cat no.37240), 3.75 ml of cyclohexane (Sigma, cat. No-8456), 0.9 ml of n-hexanol (Aldrich, cat. No. n 1330-3).

As fluorescence labels used Pt-coproporphyrin obtained in accordance with U.S. Pat. Of the Russian Federation No. 1707539, class MKI G01N 33/532. As chelate complexes of europium ions used compounds based on bisβ-dicarbonyl derivative carbazole (RF patent No. 2296756, class MKI C07D 307/91). Upon receipt of the nanoparticles molar ratio of molecules chelate complex with respect to molecules Pt-porphyrin in the reaction mixture were established taking into account the required ratio of the number of molecules of europium and latinoporvida in the nanoparticles.

To retrieve the set of nanoparticles with a given approximate ratio: 1/1, 1/2, 1/4, 2/1 levels of phosphorescence signals at wavelengths of 615 nm/645 nm in the reaction mixture was injected on of 0.26 ml, respectively chelate Eu(III) and plantincorporated. For the above ratios of the signals of phosphorescence in the ratio of chelate Eu(III) and aqueous solution of Pt-coprop is hirin tetracylines salt, respectively: 1,5· 10^-2M /1,15·10^-2M; 1,5·10^-2M /2,3·10^-2M; 1,5·10^-2M /4,6·10^-2M; 3,0·10^-2M /1,15·10^-2M; 6,0·10^-2M /1,15·10^-2M Then in each reaction mixture were added 50 μl of TEOS (Sigma, cat. # T-3143) and initiated the process of polymerization by addition of 33 μl of 25% aqueous ammonia solution. When continuous mixing process continued for 24 hours at room temperature.

The resulting suspension of nanoparticles (with an average diameter of 60-70 nm and a density increase of 1.96 g/cm³) were washed by centrifugation at 8000 rpm, 15 min: twice in 50% ethanol and twice in bidistilled water. Finally nanoparticles resuspendable in 4 ml of bidistilled.

Fluorescent spectral characteristics of the suspension obtained nanoparticles was controlled by an LS-5B excitation wavelength λ_vasb380 nm, λ_emplantincorporated - 645 nm, λ_emchelate Eu(III) - 615 nm. All measurements were carried out in the presence of O₂. The supernatant under these conditions, even in the absence of O₂he gave a signal, not exceeding 0.3% of the signal level of the luminescence of the nanoparticles.

Nanoparticles containing in its composition detecting substances, after a preliminary modification of cyanogenmod then covalently linked molecules monoclonal the different antibodies. Each of the types of nanoparticles, characterized by the ratio of luminescent labels, was treated with only one type of binding molecules (antibodies) to one of the desired analytes by incubation with nanoparticles and subsequent washing with dialysis is not bound peroxidase antibodies.

To obtain the first binding molecules for the detection of the causative agent of plague, anthrax, tularemia, staphylococcal enterotoxin type b botulinum toxin type a used monoclonal antibodies produced by JSC VSC molecular diagnostics and therapy, LLC "Impact" (Sveshnikov p. g, I. Kisilev, E.S. Severin, Fingers M.A. in Molecular medicine, 2004, №4, p.67-72).

Nanoparticles from orthosilicate with the inclusion of platinum complexes of uroporphyrin, protoporphyrin and tetraphenylporphyrin received a manner similar to the above procedure.

For carrying out multiplex analysis using a mixture of conjugates of the first binding molecules by detecting particles in a concentration in a mixture of 10¹⁰nanoparticles for each of the search pathogens. The number of types of the first binding molecules in the mixture corresponds to the number of desired pathogens.

The molar ratio of the luminescent compound in the first binding molecules, nanoparticles with covalently linked antibodies to various pathogens is presented in table 1.

P is the iMER 2. The preparation of the first binding molecules containing the detecting substance on the basis of the nanoparticles from melamine-formaldehyde resin.

The nanoparticles were obtained by THE 2634-015-49941990-03 CJSC "IMMUNOSCREEN Moscow. To retrieve the set of nanoparticles with a given approximate ratio: 1/1, 1/2, 1/4, 2/1 levels of phosphorescence signals at wavelengths of 615 nm/645 nm in a 10 ml reaction mixture injected with 0.3 ml, respectively chelate Eu(III) and plantincorporated. For the above ratios of the signals of phosphorescence ratio of concentrations of chelate Eu(III) and aqueous solution of Pt-coproporphyrin tetracylines salt, respectively: 1,5·10^-2M /1,15·10^-2M; 1,5·10^-2M /2,3·10^-2M; 1,5·10^-2M /4,6·10^-2M; 3,0·10^-2M /1,15·10^-2M; 6,0·10^-2M /1,15·10^-2M. Nanoparticles with a diameter of 100-120 nm represent the polycondensation product of mono-, di - and trimethylol derivatives of melamine, formed by the interaction of melamine with formaldehyde. Detecting substances: Eu chelate(III) based on bisβ-dicarbonyl derivative carbazole and plantincorporated was introduced into the reaction mixture at the start of the polycondensation process. Their inclusion in the particles was controlled by the signal strength of the phosphorescence chelate of europium (λ_vasb380 nm, λ_emthat is 615 nm, C4; ˜ 320 ISS) and platinum-coproporphyrin (λ_vasb380 nm, λ_em- 645 nm, τ ˜ 80 μs). Fraction of nanoparticles with a diameter of 120-150 nm was obtained by filtering the total reaction mixture through the membrane filters Unipor company Biorad diameter of 0.2 and 0.1 micron.

Nanoparticles containing in its composition of the detecting substance, then the adsorption was associated with the molecules of monoclonal antibodies by the addition of 1 ml of a suspension of nanoparticles of 30 µg of antibodies specific to one of the bioagent in the volume of 10 µl. After 10 hours of incubation, unbound antibodies were removed by centrifugation as described in example 1. Each of the types of nanoparticles, characterized by the ratio of luminescent labels, was treated with only one type of binding molecules (antibodies) to one of the desired analytes by incubation with nanoparticles and subsequent washing with dialysis unbound antibodies. The molar ratio of the luminescent compound in the first binding molecules to different analytes in the melamine-formaldehyde nanoparticles are presented in table 2.

The nanoparticles of the melamine-formaldehyde resin with the inclusion of platinum complexes of uroporphyrin, protoporphyrin and tetraphenylporphyrin received a manner similar to the above procedure.

Example 3. Getting latex particles coated with a second binding molecules on the Snov, melamine-formaldehyde resin.

For the preparation of latex particles coated with a second binding molecules that are specific to one of the many designated pathogens used methylmethacrylic latex particles (manufactured by CJSC "IMMUNOSCREEN" Moscow, THE 2634-015-49941990-03) with a diameter of 2.5 microns.

Immobilization second binding molecules (antibodies to the target pathogens) was carried out by incubation of latex particles at a concentration of about 10¹⁰particles/ml in 20 mm phosphate buffer solution with mouse monoclonal antibodies to each of the search pathogens. The reaction mixture contained 50 μg/ml of antibody and 10¹⁰latex particles/ml Latex particles were incubated 4 hours at room temperature and freed from unreacted (free) antibodies by precipitation by centrifugation (2000 rpm) with subsequent resuspending in a solution of 20 mm phosphate buffer with bovine serum albumin in a concentration of 0.1%.

To conduct mnogoukladnogo analysis was prepared a mixture of latex particles coated with a second binding molecules to all of the target pathogens. With this purpose, mixed in equal parts with latex particles coated with antibodies to each of these pathogens at a concentration of 4000 particles/ml

Example 4. Simultaneous determination in samples of plague and staphylococcal enterotoxin is type C.

In a microplate well with a volume of 350 microliters with the bottom, made in the form of a membrane of a polymeric material (polypropylene) with a thickness of 10 microns with a pore diameter of 1.5 µm membrane production research Institute of crystallography, Russian Academy of Sciences), bring a water sample volume of 100 microliters containing cells of the plague pathogen (live vaccine plague production Stavropol NPCI) at a concentration of 4000 M.K./ml staphylococcal enterotoxin type b (supplier LLC "Impact") at a concentration of 10 ng/ml, then add 50 ál of the mixture of the first binding molecules associated with fluorescence labels, prepared as described in example 1 and 50 μl of a mixture of latex particles coated with a second binding molecules. The membrane is pre-treated for 30 minutes with 1% solution of bovine serum albumin to exclude non-specific adsorption of biological molecules. The sample is incubated for 15 minutes, filtered through the membrane and optionally washed by filtering a 3-fold volume of phosphate buffer, and then washed once with distilled water. The membrane is dried by blowing air through it for 1 minute. After that, the bottom of the wells scanned using a fluorescence scanner IFS (development of the FSUE ″GosNII BP") in sequential excitation laser diode private microzone area of up to 900μ². Ska is the key of the bottom of the wells perform a series of two spectral and timing modes: the first mode corresponds to the registration of the issue of the luminescence of europium ions - the maximum wavelength registration 615 nm, the delay time between the excitation pulse and registration of emission of 300 microseconds, the time measurement from 300 to 700 microseconds; the second mode corresponds to issue registration plantincorporated maximum wavelength registration 645 nm, delay time is 40 microseconds, the time measurement from 40 to 200 microseconds. When this register phosphorescence peaks above the background level of luminescence of the membrane is not less than 5 times. The results of the scan area of the membrane of the bottom of the wells of the microplate with square 1 mm²the wavelength of 645 nm presented on figa, where 1 signals (peaks) of the phosphorescence from the individual latex particles, bound on their surface the desired analytes. On figb shows a partial section of the same membrane area 330×330 microns, made in the form of three-coordinate grid indicating the signal intensity at a wavelength of 645 nm along the Z axis, where 2 signals (peaks) of phosphorescence, for which the ratio of 615 nm/645 nm is close to 1, and 3 signals (peaks) of phosphorescence, for which the ratio of 615 nm/645 nm is close to 2. On FIGU presents a fragment of the same plot, made in the form of three-coordinate grid indicating the signal intensity at a wavelength of 615 nm along the z axis.

The data presented in figa, 1B, 1C, it is seen that the signal of phosphorescence in videotechnik peaks were registered as at a wavelength of 645 nm, and at a wavelength of 615 nm. The ratio of signal 615 nm/645 nm for each of the microparticles is close to 1 or 2, which indicates the presence of plague and staphylococcal enterotoxin in the sample.

Similar results were obtained when analyzing samples containing mixtures and other concentrations of plague and staphylococcal enterotoxin C. the results of the analysis are presented in table 3. The increase in the concentration of Athena accompanied by an increase in the signal level of the microparticles, which adsorbed the desired antigen in complex with the first binding molecule and detecting substances.

From table 3 and figure 1, it follows that in the analyzed samples show a long luminescence in the form of individual peaks of luminescence, the ratio of the signals in the two spectral ranges corresponds to the ratio of the signals characteristic of the first detecting molecules to comname the microbe and staphylococcal enterotoxin type Century. And the number of "peaks"characteristic luminescence of the plague microbe, increases in proportion to its concentration, while the number of "peaks"corresponding to the toxin remains constant. This is because the toxin is detected only on solid media (second binding antibodies), and the plague microbe is delayed by member is not in conjunction with the second antibody, and directly in the form of individual cells or their associates. The larger scatter in the range of signals caused by adsorption on the particles of several cells as well as the formation of associates from multiple cells.

Example 5. Simultaneous determination in samples of tularemia and staphylococcal enterotoxin type Century.

In a microplate well with a volume of 350 µl with a bottom made in the form of a membrane of a polymeric material (polypropylene) with a thickness of 10 microns with a pore diameter of 1.5 microns, bring a water sample volume of 100 µl, containing cells of tularemia (diagnosticum tularemia for trovano-drip agglutination reaction Odessa enterprises on production of bactericidal drugs) at a concentration of 5000 M.K./ml and 50 ng/ml staphylococcal enterotoxin In, then add 50 ál of the mixture of the first binding molecules, labeled fluorescence labels prepared in accordance with example 2, and 50 μl of a mixture of latex particles coated with the second connecting the molecules. Membrane pre for 30 minutes, treated with 1% solution of bovine serum albumin to exclude non-specific adsorption of biological molecules. The sample is incubated for 10 minutes, filtered through a membrane, optionally washed by filtering a 3-fold volume of phosphate buffer and is continuously washed with distilled water. The membrane is dried by blowing air through it for 1 minute. After that, the bottom of the wells scan, as described in example 4. The results of the scan area of the membrane of the bottom of the wells of the microplate with square 1 mm²presented at Figo. On figb shows a partial section of the same membrane area 330×330 microns, made in the form of three-coordinate grid indicating the signal intensity at a wavelength of 645 nm in the z-axis On FIGU presents a fragment of the same plot, made in the form of three-coordinate grid indicating the signal intensity at a wavelength of 615 nm along the Z axis, where 5 - signals (peaks) of phosphorescence, for which the ratio of 615 nm/645 nm is close to 1/2, and 6 signals (peaks) of phosphorescence, for which the ratio of 615 nm/645 nm close to 2.

The data presented in figa, 2B, 2C, it is seen that the signal of phosphorescence in the form of individual peaks is registered as at a wavelength of 645 nm, and 615 nm. The ratio of signal 615 nm/645 nm for each of the microparticles is close to 1/2, or 2, indicating the presence of tularemia and enterotoxin in the sample.

Similar results were obtained when analyzing samples containing mixtures and other concentrations of tularemia and staphylococcal enterotoxin C. the results of the analysis are presented in table 4. The increase in the concentration of Athena accompanied INCR is the group of the signal level of the microparticles, which is adsorbed the desired antigen in complex with the first binding molecule and detecting substances.

The number of "peaks"characteristic luminescence of tularemia pathogen increases with increase in its concentration, while the number of "peaks"corresponding to the toxin remains constant. This is because the toxin is detected only on solid media (second binding antibodies), and tularemia microbe partially retained on the membrane in conjunction with the second antibody, and directly in the form of individual cells or their associates. The larger scatter in the range of signals caused by adsorption on the particles of several cells as well as the formation of associates from multiple cells.

Example 6. Simultaneous determination in samples of botulinum toxoid type a spores of anthrax and staphylococcal enterotoxin type Century.

In a microplate well with a volume of 350 microliters described in the previous examples, bring a water sample volume of 100 microliters containing staphylococcal enterotoxin type b at a concentration of 100 ng/ml botulinum toxoid type a at a concentration of 10 ng/ml (produced by Ufa NEWS), spores of Bacillus anthracis (STI vaccine production Stavropol NPCI) at a concentration of 5000/ml; then add 50 µl of the mixture is the first binding molecules, labeled fluorescence labels, and 50 μl of a mixture of latex particles coated with a second binding molecules. Further, the method is carried out as described in the previous examples. The results of the scan area of the membrane of the bottom of the wells of the microplate with square 1 mm²presented at Figo. On figb shows a partial section of the same membrane area 330×330 microns, made in the form of three-coordinate grid indicating the signal intensity at a wavelength of 645 nm in the z-axis On FIGU presents a fragment of the same plot, made in the form of three-coordinate grid indicating the signal intensity at a wavelength of 615 nm along the Z axis, where 7 - signals (peaks) of phosphorescence, for which the ratio of 615 nm/645 nm close to 1/4 and 8 signals (peaks) of phosphorescence, for which the ratio of 615 nm/645 nm close to 2/1, 9 signals (peaks) of phosphorescence, for which the ratio of 615 nm/645 nm close to 4/1. Table 5 presents the results of the determination of several analytes in the test sample.

The data presented in figa, 3B, 3C, it is seen that the signal of phosphorescence in the form of individual peaks is registered as at a wavelength of 645 nm and at a wavelength of 615 nm. The ratio of signal 615 nm/645 nm for each of the microparticles is close to 1/4, 2/1 or 4/1, which indicates the presence of anthrax, staphylococcal enterotoxin In and botuline the institutions toxin type a in the sample.

The ratio of signal levels of phosphorescence 615 nm / 645 nm for the first binding molecules (orthosilicate nanoparticles with monoclonal antibodies to various pathogens)

The ratio of signal levels of phosphorescence 615 nm / 645 nm for the first binding molecules (melamine-formaldehyde nanoparticles with monoclonal antibodies to various pathogens)

The results of the analysis of samples containing the causative agent of plague and staphylococcal enterotoxin b In the test

The results of the analysis of samples containing tularemia and staphylococcal enterotoxin In various concentrations.

Simultaneous determination in samples of botulinum toxoid type a spores of anthrax and staphylococcal enterotoxin type Century.

1. The way mnogoukladnogo immunoassay with the use of microparticles, comprising coating the surface of the porous membrane of the reaction mixture containing the analyte, the first linking molecule that is associated with the detecting substance and specific to the analyte, the test sample and the particles unable to pass through the pores of the membrane, covered with a second binding molecules specific for the analyte, the incubation mixture for the formation of a biospecific complex, the laundering of the reaction mixture from not bound peroxidase reagents and detection of the analyte in the sample is along the light signal detecting substances associated with biospecific complex, characterized in that use at least two types specific to desired analytes of the first and second binding molecules, each type those and other molecules specific to only one of the desired analyte, each of the first binding molecule associated with at least two long luminescense detecting substances, the ratio of the concentrations in each of the first binding molecule is selected in advance and corresponds to the desired analyte is recorded in a temporary resolution the phosphorescence signals in the spectral ranges of emission detecting substances corresponding to the time constant of decay of these substances, and determine the desired analyte in the ratio of fluorescence signals, while the spatial resolution of the detection system, Δ(μ²) is determined from the following equation:

Δ≤S/10Nμ²,

where S is the area of the adsorption of particles on the membrane, μ²;

N is the given number of latex particles in the analyzed sample.

2. The way mnogoukladnogo immunoassay using microparticles according to claim 1, characterized in that the detecting substance use chelating agents with europium ion.

3. The way mnogoukladnogo immunoassay with ispolzovaniem according to claim 1, characterized in that the detecting substance use complexes coproporphyrin, uroporphyrin, protoporphyrin, tetraphenylporphyrin with metal Pt(II).

4. The way mnogoukladnogo immunoassay using microparticles according to claim 1, characterized in that as the first binding molecules using polymer nanoparticles with a diameter of from 40 to 150 nm, associated with molecules having high affinity with respect to desired analytes.

5. The way mnogoukladnogo immunoassay using microparticles according to claim 1, characterized in that particles coated with a second binding molecules, use of latex particles with a diameter of 0.5-10 microns.