Method of multianalytic immune assay with using microparticles

FIELD: medicine.

SUBSTANCE: invention refers to biology and medicine, namely to immunodiagnosis. There is offered method of multianalytic immune assay based on immunochemical, genetic and other types of reactions of biospecific binding analyte and ligands. There are mixed various categories of microparticles coated with biospecific reagents for binding of various required analytes and marked with one or more fluorochromes in various concentrations emitting a long-living fluorescence. The analysed sample and biospecific developing reagent marked with a detecting fluorochrome with a short-living fluorescence with its excitation area being outside that of fluorochromes with long-living fluorescence are added to the particle mixture. It is followed with reaction for biospecific complex formation. The prepared biospecific complexes are deposited on a solid-phase carrier. The fluorescence emission of all fluorochromes is excited with emitters in two spectral ranges herewith measuring an amount of long-living fluorescence in a time resolution mode to identify the microparticle and an amount of short-living fluorescence of detecting fluorochrome for measuring concentration of required analytes. Thus the concentration ratio of long fluorescing fluorochromes in microparticles for detecting the same type of analyte is constant, and for determining different types of analytes, the concentration ratio differs at least twice.

EFFECT: possibility of simultaneous analysis of great array of microparticles on a solid phase, ie increased multiplexity of the analysis, and wide-range measurement of concentration of required analytes.

6 cl; 3 ex, 10 dwg

The invention relates to mnogayaleta analysis on the basis of immunohistochemistry, genetic and other types of reactions biospecific binding of the analyte with ligands for biological, medical and other purposes.

Methods multiplex (mnogoukladnogo) detection, identification and quantification of biological materials (cells, virions, nucleic acids, proteins, antigenic molecular complexes of low molecular weight biologically active compounds)found in low concentrations in the samples of complex composition, and height requirements: methods must be fast, to give unequivocal results, to be efficient in terms of using different complex of matrices, 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. The requirement multiplexes diagnostic tests using nucleotides, chemical compounds and proteomic synthetic structures (recombinant proteins, peptides and the like) is the possibility of parallel registration of tens and hundreds of interactions of different analytes in the analyzed sample.

The known method and compositions for the simultaneous detection of many who Nalimov in the sample (PCT application No. 01/073443, class MKI G01N 33/58). The invention can be used for rapid, accurate and automatic detection of pathogens, as well as for the diagnosis and prognosis of diseases. Search analytes (antigens) in the sample will detect and differentiate, using a variety of fluorescent latex particles associated with different binding molecules that are specific for the desired analytes (antigens). When the determination of multiple analytes each particle one category has a different fluorescent ("address") tag, and each antigen (analyte) revealed the same fluorescent label associated with reagents for their detection. Measurement of the fluorescence of the particles is carried out by determining the ratio of the fluorescence of one or more dyes. As fluorescent labels for microparticles using fluorescein, phycoerythrin, rhodamine, and the like, and for labeling antigens using a secondary fluorescent dyes (labels)that differ in the spectral range of excitation and spectral emissions from the fluorescent dyes used for labeling categories of particles. For detection of antigens (analytes) and estimates of their number register secondary fluorescence label, which is associated with biospecific reagents for detection of the analyte using the methods of running the CIT is metry.

The disadvantage of this method is the low performance caused by the use of methods of flow cytometry for detection of signals, and the limited sensitivity, so as not contacting microparticles reagents create a background luminescence.

There is a method of simultaneous analysis and detection of multiple analytes by the use of microparticles (PCT application No. 01/44812, IPC class G01N 33/53). In the invention, a method for preparation and use of the reagent containing several types of microparticles with a set of fluorochromes for detection of multiple analytes. One kind of particles differs from the other spectrum of emission, i.e. each particle contains various tags for the identification of analytes and different affinity ligands are immobilized on the microparticles. Sample microparticles are incubated under conditions and time necessary and sufficient for the formation of a biospecific complex. After incubation analyze the formed complexes under a microscope in order to identify categories of microparticles (analyte) and the concentration of the desired analyte fluorescence labels, with each particle analyzed sequentially.

The disadvantages of the method are the limited sensitivity due to the need for the operation of the dilution of the sample and the longer the beard the identification of the analyte under a microscope.

Known biospecific mnogokanalnyy method for studying biological samples (U.S. patent No. 5891738. class 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 (shown) reagents labeled secondary photoluminescent label, initiate biospecific reaction, the category of microparticles, i.e. the type of analytes is determined by the fluorescence intensity of the indicator labels associated with particles, and the concentration of analytes is determined by the fluorescence intensity of the secondary photoluminescent labels. As indicator substances use tetrapyrrole (porphyrin, chlorin, phthalocyanine and others) and cyanine substances, and as a photoluminescent label - phycoerythrin. When detecting examine each particle separately on microfluorimetry based on two-photon excitation method luminescence and confocal scheme of excitation and registration of luminescence.

The disadvantages of this method are the limited performance because of the need to analyze successively the signals from each of the second microparticles and the limited sensitivity due to background luminescence.

Know the use of microparticles with multiple fluorescent labels for detection of multiple analytes (PCT application No. 01/13120, IPC class G01N 33/58). The method can be used for studies of biological fluids and samples taken from the environment, water or air, industrial sources, waste water, and so the Method is based on the use of microparticles strictly fixed size, having on its surface one or more populations of fluorescently labeled nanoparticles. On microparticles or put several types of nanoparticles, is equal to the number of detected analytes or different concentrations of the same nanoparticles. Changing the number and proportion of different populations of nanoparticles, establish and distinguish a large number of discrete populations of particles-carriers with the same range of emission and accordingly identify a large number of desired analytes. The analysis is carried out in a flow cytometer, the processing speed of the signals is equal to 2000 particles per second.

The disadvantages of the method are limited multiplexes system due to the inability to distinguish spectral large number of particles, characterized by the ratio of the two fluorochromes in the particle, and poor performance caused by the use of methods of flow cytometry for analysis.

The known method p is Ogadenia immunoassay using two particles (U.S. patent No. 6096563, class NCI 436/523), the Method can be used for analysis of biological samples, and to determine various environmental pollutants. Detection is carried out with the naked eye or by a colorimeter, a Refractometer. 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 biospecific complex, which is retained on the membrane surface and is measured by the colorimeter. Covered and detecting particles may vary in color and size and are made of different materials. As covered and detecting particles commercially advantageous to use the latex.

The disadvantage of this method is limited multiplexes analysis and that the registration signal from the detecting molecules is colorimetrically, this severely limits the sensitivity analysis.

The closest is a multivariate method studies using different categories of particulate matter (EP No. 0617286, IPC class G01N 33/533). The method is designed to determine multiple analytes in one sample, in which for marking (tagging) categories of microparticles corresponding to different analytes and for determination of concentrations of detected analytes using labels (fluorochrome)with long-lived fluorescence. As fluorochromes use of rare earth metals, such as Eu, Sm, Tb, Dy. How is that different categories of the same size and properties of microparticles labeled with fluorescent molecules (markers) and covered with a biospecific (bioaffinity) reagents for binding of analytes, is mixed with a test sample and biospecific showing (detecting) the reagent is also labeled with a fluorescent label, conduct biospecific reaction, excite fluorescence of all fluorochromes and measure the emission of fluorescence to identify categories of microparticles and to determine the concentration of the desired analyte. The analysis is performed using the registration of fluorescent signals in the temporary permission. Microparticles and the detecting reagent have been labelled with fluorochromes that emit to koivumaa fluorescence at different wavelengths. Using different combinations of concentrations of fluorochromes for marking particles allows to identify a large number of analytes in a wide range of concentrations.

The disadvantage of this method is the interference signals from the fluorochromes used to specify the "address" (categories) of particles and fluorochromes used for detection of analytes in a sample, which does not allow to detect analytes in a wide range of concentrations of analytes. In addition, the disadvantage of this method is the necessity of using the same size and properties of the microparticles, which complicates and increases the cost of the technology of their production, and methods of measurement of signals used in the way that does not allow the simultaneous recording of fluorescence of the microparticles, which affects the performance of the method.

The task is to create rapid detection and quantitative detection of a large number of desired analytes in a wide dynamic range of the analyzed concentrations when performing immunochemical and clinical tests, the screening of biologically active compounds, genetic research, etc.

The technical result that is achievable with the use of the invention is to reduce the influence of fluorochromes, providing multiplexes analysis, and fluoro the Ohm, used for detection of the analyte, with a parallel measurement of the signals from an array of particles, which increases the dynamic range of measured concentrations of detected analytes.

The technical result is achieved by the invention.

The essence of the invention is achieved in that in the method mnogoukladnogo analysis using microparticles, comprising the mixing of different categories of microparticles coated with biospecific reagents for binding a variety of desired analytes and labeled with one or more fluorochromes in various concentrations, emitting long-lived fluorescence, adding to the mixture of microparticles of the sample and biospecific developing reagent, labeled by detecting fluorochromes, conducting the reaction for the formation of biospecific complexes, the excitation of the fluorochromes and the measurement mode time resolution number of long-lived emission of fluorescence to identify categories of microparticles and the amount of emission of fluorescence to measure the amount of desired analytes formed biospecific complexes precipitated on the solid phase carrier, the emission of fluorescence all excite fluorochromes sources of radiation in two spectral ranges, for tagging biospecific showing the reagent is used with a detector fluorochrome with short-lived fluorescence, the region of excitation of the fluorescence of which is located outside of the regions of fluorescence excitation of fluorochromes with long-lived fluorescence, the ratio of the concentrations of fluorochromes with long-lived fluorescence of the microparticles to determine one type of analytes constantly, and to determine different types of analytes, the concentration ratio varies at least twice.

The invention is also achieved by the fact that as detecting fluorochromes used phycoerythrin, So, NIRD750 NHS, NIRD782 NHS and other fluorochromes that emit short-lived fluorescence.

The invention is also achieved by the fact that for tagging categories of microparticles using long fluorescent complexes of ions of Eu, Sm, Tb, Dy, and other rare earth elements.

The invention is also achieved by the fact that for tagging categories of microparticles using long fluorescent metal complex coproporphyrin, uroporphyrin, protoporphyrin, tetraphenylporphyrin and other porphyrins and their derivatives with Central atoms of platinum, palladium, copper, zinc, aluminum and other metals having delayed fluorescence or phosphorescence at room temperature.

The invention is also achieved by the fact that for biospecific binding of analytes as p is oakleyi reagents use of polymer nanoparticles with a diameter of 40 to 150 nm, containing fluorochromes with a short decay time of fluorescence associated with molecules having high affinity with respect to desired analytes.

The invention is also achieved by the fact that different categories of microparticles using latex particles with a diameter of 0.5-5 microns.

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

Known methods of conducting mnogoukladnogo analysis using multiple categories of colored microparticles. In U.S. patent No. 5891738, class NCI 436/50 and No. 5028545, class NCI 435/501 and in PCT application 01/13120, class MKI G01N 33/58 described the ways in which to identify categories of microparticles and, respectively, of analytes using fluorescent substances (fluorochromes) with a short decay time of fluorescence, and detecting the analyte concentration using fluorescent substances with a long decay time of fluorescence, in particular chelates of lanthanide rare earth elements, europium, terbium, and others), or Vice versa. In the known methods mnogoukladnogo detection using either the number of kinds of particles equal to the number of detected analytes or different concentrations of the same particles. The implementation of the decrees of the data above methods requires either complex optical devices (spectrofluorimetry with two-photon excitation and measurement of 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, with multiple sources and multiple detectors signals. Multiplexes these methods is limited due to the overlap of the emission spectra of the fluorochromes used for marking categories of microparticles. In comparison with the known technical solutions in the proposed method, the above effect can be obtained by the proposed set of features. The method does not require the use of particles of a specific size as the category of each particle is determined only by the ratio of the fluorochromes, and not their absolute number, this allows you to create a set of particles with the ratio of concentrations in the range of four orders of magnitude, from 100:1 to 1:100. The proposed set of features allows you to increase multiplexes and performance analysis, simple and affordable way. Therefore, the proposed solution meets the criterion of "prior art".

The invention can be used for detection, identification and quantitative determination of an analyte that can specifically bind with ligands, in particular biological mA the materials, such as biologically active chemical compounds, peptides, proteins, antigens, microorganisms and nucleic acids. The method can be used for direct and competitive schemes immunoassay. In particular, the method can be used for serodiagnosis of infectious diseases, detection of low concentrations of hormones, microorganisms, their specific genes, species or serotypes in an isolated form or as contaminants of food, environment, forensic samples. The method allows for high-performance analysis with the use of industrially produced microplates, standard equipment for washing and preparation of samples for analysis on the basis of what can be created with simple devices, including portable, accessible element basis. Therefore, the claimed invention meets the criterion of "industrial applicability".

Figure 1 shows the spectra of excitation and emission of fluorochromes used to specify the category (address) microparticles, i.e. to detect the type of the analyte: 1-chelate complex Eu-fluorene, 2 - Pt-coproporphyrin, 3 - Pd-coproporphyrin.

Figure 2 shows the degree of overlap of the emission spectra of Eu-fluorene, Pt-coproporphyrin, Pd-coproporphyrin and NIRD782 NHS mode time resolution of the signal, the maximum excitation λ=380 nm, 100% p is inyat level signal fluorochrome, for which the wavelength is the maximum of the emission: 4 - Eu-fluoren; 5 - Pt-coproporphyrin; 6 - Pd-coproporphyrin; 7 - NIRD782 NHS. when the maxima of the excitation Eu-fluorene - 615 nm, Pt-coproporphyrin 645 nm, Pd-coproporphyrin 665 nm, NIRD782 NHS - 800 nm; the modes of registration: wavelength of 615 nm to the duration of the excitation pulse 600 microseconds, the delay time of the gate 300 microseconds, the duration of the gate 600 microseconds; the wavelength of 645 nm to the duration of the excitation pulse is 40 microseconds, the delay time of the strobe 40 microseconds, the duration of the gate - 80 microseconds; the wavelength of 665 nm is the duration of the excitation pulse 1000 microseconds, the delay time of the gate is 1000 microseconds, the duration of the strobe 1000 microseconds; wavelength 800 nm without temporary permissions.

Figure 3 shows the scheme for multiplex analysis using the universal conjugate with fluorochromes: 8 - incubation of the sample with a mixture of biotinylated antibodies and remove unbound reagents by filtration; 9 - incubation of the sample with the universal manifesting reagent (streptavidin labeled with CY-5 or NIRD782) and remove unbound reagents filtering; 10 - registration of the fluorescence signal from individual microparticles; 11 - membrane; 12 - particles with Pt-coproporphyrin and Unit-fluorenol to link 1-th analyte; 13 - ligands for detection of 1-th analyte;14 - microparticles with Pd-coproporphyrin and Pt-coproporphyrin for binding 2-th analyte; 15 - ligands for the detection of 2-th analyte; 16 - microparticles with Pd-coproporphyrin and Unit-fluorenol to bind the 3rd type of analyte; 17 - ligands for detection of 3-th analyte; 18 - molecule Pd-coproporphyrin in the microparticle; 19 - molecule Eu-fluorene in the microparticle; 20 - molecules Pt-coproporphyrin in the microparticle; 21 - molecules of the 1st of the analyte in the sample (antibodies of the desired analyte); 22 - molecule 2nd analyte in the sample; 23 - molecule 3rd of the analyte in the sample; 24 - molecules universal ligand for detection of analytes in a sample, labeled with Biotin; 25 - universal manifesting reagent streptavidin, labeled NIRD782 NHS, 26 impurity of the sample.

Figure 4 shows the results of the detection of antibodies to tick-borne encephalitis in 20 samples of human blood serum in comparison with the results of the immunoassay detection of antibodies using a commercial test. On the axis of ordinates indicate the optical density in ELISA tests, the abscissa axis is the intensity of the signal emission fluorescence detecting fluorochrome from the analyzed particles processed antigen to detect antibodies to tick-borne encephalitis.

Figure 5 shows the results of detection of antibodies to borellian 20 samples of human blood serum in comparison with the results of the immune the enzymatic determination using commercial tests. On the axis of ordinates indicate the optical density in ELISA tests, the abscissa axis is the intensity of the signal emission fluorescence detecting fluorochrome from the analyzed particles processed antigen to detect antibodies to the causative agent of Lyme-borreliose.

Figure 6 shows the results of detection of antibodies to syphilis in 20 samples of human blood serum in comparison with the results of the immunoassay determination using commercial tests. On the axis of ordinates indicate the optical density in ELISA tests, the abscissa axis is the intensity of the signal emission fluorescence detecting fluorochrome from the analyzed particles processed antigen to detect antibodies to syphilis.

7 shows the results of determination of thyroid stimulating hormone in 25 samples of blood sera of newborns compared with results determined using lanthanide immunofluorescent method using commercial test kits are used. On the y - axis the intensity of the signal emission fluorescence of europium ions in the system kits are used; on the x-axis is the intensity of the signal emission fluorescence detecting fluorochrome from the analyzed particles treated with antibodies to thyroid stimulating hormone.

On Fig shows the results of determination of thyroxine 25 about what Azzah blood sera of newborns compared with results determined using a commercial test method kits are used. On the y - axis the intensity of the signal emission fluorescence of europium ions in the system kits are used; on the x-axis is the intensity of the signal emission fluorescence detecting fluorochrome from the analyzed microparticles intended for the detection of thyroxine.

Figure 9 shows the results of determination of 17 hydroxyprogesterone in 25 samples of blood sera of newborns compared with results determined using a commercial test method kits are used. On the y - axis the intensity of the signal emission fluorescence of europium ions in the system kits are used; on the x-axis is the intensity of the signal emission fluorescence detecting fluorochrome from the analyzed microparticles intended for the detection of 17 hydroxyprogesterone.

Figure 10 shows the results of determination of immunoreactive trypsin in 25 samples of blood spots of newborns compared with results determined using a commercial test method kits are used. On the y - axis the intensity of the signal emission fluorescence of europium ions in the system kits are used; on the x-axis is the intensity of the signal emission fluorescence detecting fluorochrome from the analyzed microparticles intended for detection of immunoreactive trypsin.

The analysis on the example of the detection of antibodies in serodiagnostics research includes the following stages (see f the D.3): 8 - incubation of the sample with a mixture of biotinylated antibodies and remove unbound reagents by filtration; 9 - incubation of the sample with the universal manifesting reagent (streptavidin labeled with CY-5 or NIRD782 NHS) and remove unbound reagents filtering; 10 - registration of the fluorescence signal from individual microparticles. The registration is carried out on the membrane 11. To link 1-th analyte using microparticles 12 with a ratio of concentrations of two long fluorescent fluorochromes Pt-coproporphyrin/Eu-fluoren - 1:1. As ligands for detecting 1-th analyte is Saharsa-acetone antigen of the virus of tick-borne encephalitis 13. To detect a second analyte using microparticles 14 with a ratio of concentrations of two long fluorescent fluorochromes Pd-coproporphyrin/Pt-coproporphyrin - 1:1. As ligands for detecting 2-th analyte using peptide 15 C6. To identify a third type of analyte using microparticles 16 with a ratio of concentrations of two long fluorescent fluorochromes Pd-coproporphyrin/Eu-fluoren - 1:1. As ligands 17 for binding of antibodies to syphilis, a mixture of recombinant proteins. The ratio of molecules Pd-coproporphyrin - 18, Eu-fluorene 19, Pt-coproporphyrin 20 in the microparticle sets with regard to their molar ratio extinc the AI at the wavelength of excitation 380 nm and the quantum yield of emission (emission). To identify molecules of the first analyte 21, molecules of the second analyte 22 and molecules of the third analyte 23 in the sample using the biotinylated conjugate of rabbit antibodies against human immunoglobulins. Biotinylated antibodies 24 are a versatile reagent for the detection of all three analytes. The concentration of each identified analyte is determined using the universal manifesting reagent 25 streptavidin, labeled CY-5 or NIRD782. Unreacted components of the sample and impurities 26 is removed by filtering.

For conducting immunoassay using 96-cellular microtiter Board (Millipore, USA) with the bottom of the cell area 30-36 mm²made of polymer filtration membrane material (polypropylene, polymethylmethacrylate) with a pore diameter of less than the diameter of the microparticles is from 0.2 to 0.4 micron. Samples of sera at a dilution of 1:100 are mixed in equal volumes with a suspension of microparticles and placed in wells of a microplate with membrane filters Unipor company Biorad with a pore diameter of 0.2 μm. Incubated for 15 minutes, filtered through the porous bottom of the wells of the microplate and contribute conjugate specific antibodies with a fluorescent label su-5 or NIRD782 in the amount of 200 microliters per well. Incubated for 10 minutes, washed precipitated microparticles buffer solution, and then dried under a stream of air within 2-3 the minutes. The microplate with the processed samples samples are placed in a scanning fluorescence analyzer and measure the luminescence signals from the microparticles immobilized on a membrane filter bottom holes. The distribution of the signal from each of the microparticles recorded in the spectral and temporal intervals, optimal for the registration of the issue of each of the fluorochromes. Scanning the bottom of the microtiter wells are carried out with the permission of microprosodic 30×30 µm mode time resolution luminescence emitting long fluorescence (phosphorescence) in the range from 40 to 100 microseconds at a wavelength of maximum emission 645 nm for Pt-coproporphyrin; emitting long fluorescence in the range from 400 to 700 microseconds at a wavelength of 615 nm to chelate Unit and 665 nm for Pd-coproporphyrin. Summarize the fluorescent signals su-5 or NIRD782 from particles of one species and calculate the average signal per particle. As can be seen from figure 1, the region of excitation of long-fluorescent compounds (fluorochromes) is limited to a maximum wavelength of 600 nm, a spectral region of emission of these compounds is in the range 540-750 nm, and emission spectra of fluorescence overlap. The maximum emission of each of the fluorochromes is the fluorescence of other fluorochromes, the contribution of which can castigat the 1%. To determine the concentration of analytes in the proposed method used substances with a short decay time of fluorescence, spectral range of excitation and emission of which are outside the spectral range of excitation and emission of long fluorescent fluorochromes. The signals from the fluorochromes with a long fluorescence used to identify categories of microparticles (analyte), measured in the regime of temporary permissions constant decay time of emission. Along with these signals is measured signals of short-lived fluorescence fluorochrome used to identify the concentration of the analyte, which is excited at a different wavelength. This eliminates the influence of the emission fluorescence of fluorochromes with a long decay time of the fluorescence signals of the fluorescence emission of fluorochromes with a short decay time of fluorescence increases multiplexes analysis and extends the dynamic range of measured concentrations of desired analytes due to its ability to measure low concentrations. The emission signals of all fluorochromes from the whole array of micro-particles on the solid phase, measured simultaneously by scanning fluorescence analyzer. As fluorochromes with a short decay time of fluorescence can be used is phycoerythrin, So, NIRD782 NHS (N-Hydroxysuccinamide) and other fluorochromes, the maximum excitation and emission which is outside the range of excitation emission long fluorescent compounds used to create categories of microparticles. For example, NIRD782 NHS has a long-wavelength maximum of the excitation in the region of 780 nm and emission maximum at a wavelength of 800 nm, which excludes the possibility of excitation of fluorochromes with a long fluorescence used to specify the category of microparticles.

From figure 2 (position 4, 5, 6, 7) shows that the emission of fluorochromes used to detect the concentration of an analyte, does not affect the issue of fluorochromes used to address particles, and Vice versa. As a developing reagent can be used polymer nanoparticles with a diameter of 40 to 150 nm, including fluorochromes with a short decay time of fluorescence associated with molecules having high affinity with respect to desired analytes.

Example 1. Detection in blood serum samples of human antibodies to the causative agents of tick-borne encephalitis, Lyme disease, syphilis using the universal manifesting reagent

In the analysis, as fluorescence labels used Pt-coproporphyrin and Pd-coproporphyrin, as long fluorescent labels used chelate complexes of europium ions. For analysis of IP is result melamineformaldehyde microparticles CJSC "Immunoscreen (TU 2634-015-49941990-03) with a diameter of from 0.5 to 5 microns in a concentration of 3·10 ³the sample (0.1 to 0.3 ml)containing "address" long fluorescent fluorochromes used for coding categories microparticles, the spectral characteristics of the excitation and emission are presented in figure 1. Prepare a set of microparticles, each of which includes two of the three following fluorophores: the fluorophore 1 - chelate with europium ion-fluorene, the fluorophore 2 - platinum-coproporphyrin and the fluorophore 3 - palladium-coproporphyrin. For the detection of antibodies to the three above-mentioned analytes used three types of microparticles, characterized by the value included in them long fluorescent fluorophores. The first microparticles with a diameter of from 0.9 to 1.3 microns with the ratio of the luminosity (product of the quantum yield on the molar extinction coefficient at the wavelength of 380 nm) εφ_Eu/εφ_p1KP=1; the second microparticles with a diameter from 1.6 to 2.1 microns with the ratio of the luminosity εφ_PdKP/εφ_PtKP=1; third microparticles with diameters from 4.3 to 5 microns with the ratio of the luminosity εφ_PdKP/εφ_Eu=1. In the analysis used samples from donors with low content of specific antibodies, and 3 samples with high content of specific antibodies to antigens of tick-borne encephalitis, antigens of the causative agent of Lyme disease antigens to syphilis

Microparticles treated with specific antigens (recombinant proteins) to specific binding of immunoglobulins from samples of blood sera. Antigens immobilized on the surface of the microparticles according to the standard methods by adsorption or covalent binding through the carboxyl group of the polymer, unbound (free) antigens are removed by standard procedures, for example by deposition of microparticles by centrifugation or by the gel filtration method. The first category of microparticles process antigens for detection of antibodies to tick-borne encephalitis virus, the second category of antigens for detection of antibodies to borellian - the causative agent of Lyme disease, the third category - antigens to syphilis. For detection of specific antibodies in serodiagnostics research microparticles of the first category for the detection of the first analyte antibodies to tick-borne encephalitis virus - treated, Saharsa-acetone antigen of the virus of tick-borne encephalitis, microparticles of the second category for detecting the second analyte - antibody to borellian - treated C6 peptide, microparticles third category to identify the third analyte antibodies to syphilis is treated with a mixture of recombinant proteins. The prepared microparticles in the form of a mixture with a concentration of 5,000 particles to the each grade per 1 ml of suspension is used for the analysis of clinical samples. Individule antibodies (antibodies against immunogloblins person) marked by fluorochromes NIRD782, the wavelength of 780 nm excitation, the wavelength of 800 nm emission.

From the data of figure 4, 5, 6, it follows that the samples of sera containing high titers (concentrations) of antibodies against these pathogens, are many times higher fluorescence signal detecting fluorochromes, and the results of multiplex analysis of the proposed method correlates well with the results of the immunoassay determination using commercial tests, the antibodies of the proposed method is carried out in a 1000-fold range of variation of fluorescent signals, i.e. the concentration of analytes.

Example 2. Detection in blood serum samples of human antibodies to the causative agents of tick-borne encephalitis, Lyme disease and syphilis

The method of analysis is similar to that described in example 1. In the process, a mixture of particles of the same size as in example 1, but with a different ratio of fluorochromes for detection of antibodies to tick-borne encephalitis use of microparticles with a ratio εφ_Eu/εφ_ptKPp=1:100, to detect antibodies of Borelli use of microparticles with a ratio εφ_PdKP/εφ_PtKP=1:100, for detection of antibodies to syphilis pathogen using microparticles with a ratio of the φ _PdKP/εφ_Eu=1:50. As manifesting biospecific reagent used individule rabbit antibodies against human immunoglobulins, linked to nanoparticles with a diameter of from 40 to 150 nm, including the su-5, the wavelength of the excitation 710 nm, the wavelength of emission of 740 nm. The obtained results similar to the results shown in figure 4, 5, 6; a change in the ratio of fluorochromes 100 times did not lead to distortion of the results of the analysis, with the detected signal from the sample is increased proportionally to the increase in the content of fluorochrome SU-5. This suggests the possibility of creating a large number of combinations of multiple fluorochromes in a wide dynamic range of concentrations and increasing the reliability of the registration of low concentrations of analytes.

Example 3. Quantitative determination of markers of congenital hypothyroidism - thyrotropin and thyroxine, adrenogenital hyperplasia - 17 hydroxyprogesterone and cystic fibrosis - immunoreactive trypsin in samples from dried blood spot

For the analysis used a specially selected group of 25 blood samples of newborns, including 4 sample content of the above markers in several times higher than normal values for a group of healthy newborns. The samples were prepared by extraction of material from dry the blood stains, spotted on filter paper. For the detection of analytes microparticles prepared in accordance with example 1, is treated with specific reagents. To determine thyrotropin microparticles treated with commercial monoclonal antibodies. For the determination of thyroxine microparticles treated with BSA conjugated with thyroxine. For determination of 17 hydroxyprogesterone microparticles treated with BSA conjugated with 17-hydroxyprogesterone. For determination of immunoreactive trypsin microparticles treated with monoclonal antibodies specific to immunoreactive trypsin. Samples of serum are mixed in equal volumes with a suspension of microparticles contribute conjugates of antibodies to thyroid stimulating hormone, thyroxine, 17 hydroxyprogesterone and immunoreactive trypsin and the mixture is placed into the wells of the microplate with membrane filters Unipor company Biorad with a pore diameter of 0.2 μm. Incubated for 25 minutes, filtered through the porous bottom of the wells of the microplate and make a mixture of biotinylated conjugates specific antibodies with a fluorescent label NIRD782 in the amount of 200 microliters per well. Incubated for 10 minutes, washed precipitated microparticles buffer solution, and then dried under a stream of air. The microplate with the thus treated microparticles analyze the scan is the dominant fluorescence analyzer. To quantify the content of an analyte in a sample pre-built calibration graphs. For this purpose, serum samples with a known predetermined concentrations of the reactants.

Figure 7 presents the results of determination of thyroid stimulating hormone in 25 samples of blood sera of newborns compared with results determined using a commercial test method kits are used. On Fig presents the results of determination of thyroxine in 25 samples of blood sera compared with results determined using a commercial test method kits are used. Figure 9 presents the results of the determination of 17 hydroxyprogesterone in 25 samples of blood spots of newborns compared with results determined using a commercial test method kits are used. Figure 10 shows the test results of blood samples of newborns to determine trypsin compared with results determined using a commercial test method.

From the data 7, 8, 9 and 10 it follows that the results of multiplex analysis of the proposed method with the use of microparticles in a ratio of fluorochromes, changing in a wide range of concentrations, correlate well with the results immunosenescence lanthanide immunoassay using commercial tests.

1. The way mnogoukladnogo immunoassay with use the reattaching the microparticles, including the mixing of different categories of microparticles coated with biospecific reagents for binding a variety of desired analytes and labeled with one or more fluorochromes in various concentrations, emitting long-lived fluorescence, adding to the mixture of microparticles of the sample and biospecific developing reagent, labeled by detecting fluorochromes, conducting the reaction for the formation of biospecific complexes, the excitation of the fluorochromes and the measurement mode time resolution number of long-lived emission of fluorescence to identify categories of microparticles and the number of emission fluorescence detecting fluorochrome to measure the amount of desired analytes, wherein the formed biospecific complexes precipitated on the solid phase carrier, the emission of fluorescence of all excite fluorochromes sources of radiation in two spectral the ranges for tagging biospecific developing reagent is used with a detector fluorochrome with short-lived fluorescence, the region of excitation and emission fluorescence of which is located outside of the regions of excitation and emission fluorescence spectra of fluorochromes with long-lived fluorescence, the ratio of the concentrations of fluorochromes with long-lived fluorescence in ICRI the particles to determine one type of analyte constantly, for definitions of different types of analytes, the concentration ratio varies at least twice.

2. The way mnogoukladnogo immunoassay using microparticles according to claim 1, characterized in that the detecting fluorochromes used phycoerythrin, su 5, NIRD750 NHS, NIRD782 NHS and other fluorochromes having short-lived fluorescence.

3. The way mnogoukladnogo immunoassay using microparticles according to claim 1, characterized in that the marking categories of microparticles using long y complexes of ions of Eu, Tb, Sm, Dy and other rare earth elements.

4. The way mnogoukladnogo immunoassay using microparticles according to claim 1, characterized in that the marking categories of microparticles using long fluorescent metal complex coproporphyrin, uroporphyrin, protoporphyrin, tetraphenylporphyrin and other porphyrins and their derivatives with Central atoms of platinum, palladium, copper, zinc, aluminum and other metals having at room temperature delayed fluorescence or phosphorescence at room temperature.

5. The way mnogoukladnogo immunoassay using microparticles according to claim 1, characterized in that as a developing reagent for biospecific binding of analytes using polymer nanocalorimeter 40-150 nm, containing fluorochromes with a short decay time of fluorescence associated with molecules having high affinity with respect to desired analytes.

6. The way mnogoukladnogo immunoassay using microparticles according to claim 1, characterized in that the different categories of microparticles using latex particles with a diameter of 0.5 to 5 μm.