RU2184970C1 - Method of multiple-analyte immunoassay - Google Patents

Abstract

FIELD: immunology, screening. SUBSTANCE: at the bottom of holes in microtitration plate one should immobilize the first biospecific components to different analytes under testing as discrete microareas, the total area of which is less or equal to that of a dry blood spot. Buffer : conjugate mixture with the second, either labeled or unlabeled, biospecific components to the certain analytes is applied right onto a dry blood spot. At the volume equivalent to its sorption capacity a dry blood spot is removed out of a hole. Reaction of biospecific binding with labeled Pt copro- or uroporphyrine by biospecific components is conducted in discrete microareas, holes are washed out, dried. Emission of label's phosphorescence is detected by subsequently scanning microareas by a focused laser ray. EFFECT: higher efficiency in providing improved sensitivity of an assay. 6 cl, 7 tbl, 16 dwg _

Description

 The invention relates to immunological methods of analysis and can be used for mass screening of congenital malformations in newborns, monitoring the immune and hormonal status, reproductive function of women and some infectious diseases.

 Diagnosis of diseases such as congenital hypothyroidism, cystic fibrosis, adrenogenital hyperplasia, phenylketonuria and galactosemia of newborns in the first days and months of their life provides the possibility of timely therapeutic correction of development. Prenatal diagnostics for predicting the risk of Down syndrome, neonatal and prenatal screening for a number of infectious diseases (toxoplasmosis, viral hepatitis, herpes viruses, etc.) are important for reducing the number of newborns with congenital anomalies. For these purposes, clinical material is used in the form of dry blood stains, which can last a very long time (years) without a noticeable decrease in activity and can be easily delivered from remote regions to specialized centers. However, when using clinical material in the form of dry blood spots in neonatal diagnostics, problems arise associated with a limited amount of test material and the need to identify several analytes from one sample of a dry blood spot.

 A known method for determining the analyte in a blood sample (EP 0460620, class MKI G 01 N 33/76). The method is designed to determine thyrotropin thyroxine and free thyroxine in a dry blood sample by fluorometric immunoassay. The method is carried out in the wells of microtiter plates and is suitable for screening congenital hypothyroidism from neonatal blood samples on filter paper. A dry blood stain is either dissolved in a buffer and then used as a sample, or, preferably, a dry stain is used directly in wells prepared in advance for immunoassay, and the analyte is eluted into the wells simultaneously with the immunological reaction. Horseradish peroxidase with antibodies against the analyte is used as a conjugate, and 3-hydroxypropionic acid as a fluorogenic substrate. The method is convenient for analysis in microtiter plates, accurate, fast, and can be easily automated.

 The fluorogenic substrates used in the method do not allow to obtain sufficient sensitivity when detecting low concentrations of analyte in the sample. In addition, when measuring fluorescence at a wavelength of 405 nm (excitation of 320 nm), the background fluorescence of solid-state polymeric materials (plate wells) has a great influence on the measured signals. The method is not suitable for multi-analytic analysis.

 A known method of immunoassay and a reagent for detecting hormones in a biological sample (US patent 5089423, class NCI 436-518). The method is intended to detect alpha-fetoprotein, human chorionic gonadotropin, ferritin, thyrotropin and other hormones in a blood sample. The method uses the immobilization of immunoreactive substances, for example, antibodies, on the solid phase, the reaction of complementary immunoreactive substances with substances immobilized on the solid phase. In this case, complementary immunoreactive substances are associated with a part of the ligand, forming fluorescent chelate labels with metal ions of lanthanides. After the implementation of the immunological binding reaction, immunoreactive substances that are not bound to immunoreactive substances immobilized on the solid phase are washed off and the solid phase is dried. Fluorescence of chelate marks at the bottom of wells on complementary bound immunoreactive substances is measured in the time resolution mode in the presence of metal ions stably bound to chelates. The method eliminates the need for dissociation of labeled complexes formed on the solid phase, which greatly simplifies the analysis procedure. Signal amplification is achieved due to the fact that avidin and streptavidin have many marker regions on their surface, which makes it possible to obtain the effect of light amplification. Drying the solid phase before measuring fluorescence eliminates the problem of absorption or scattering of the excitation flux and emission of radiation by the residual amount of reagents in the well.

The method is difficult to implement due to the large number of operations and cannot be used to detect low analyte concentrations in the sample, since a high level of the solid phase background upon excitation of Eu chelates in the region of 360 nm limits the signal / background ratio. In this method, the threshold level of luminescence detection of Eu ions is not higher than 10 ^-12 M.

The closest is a method for detecting several analytes in a dry blood sample (Jong-Juan, at all. Simaltaneous Quadruple-Label Fluorometric Immunoassay of Thyroid-Stimulating Hormone, 17α-Hydroxyprogesterone, Immunoreactive Trypsin, and Creative Kinase MM Isoenzyme in Dried Blood Spots. B: CLIN. CHEM., Vol. 38, 10, 1992, p. 2038-2043). The method is used to simultaneously detect four analytes in a dry blood sample: thyrotropin (TSH), 17α-OH-progesterone, immunoreactive trypsin (JRT) and creatine kinase MM (CK-MM). The method uses immunoreagents labeled with four different lanthanide ions: Eu ³⁺ , Sm ³⁺ , Tb ³⁺ and Dy ³⁺ . The analysis is carried out in the wells of a microtiter strip, the surface of which is coated with a mixture of monoclonal against β-TSH, JRT and CK-MM and polyclonal against 17α-OH progesterone. Coated strips are blocked and stabilized during incubation with bovine serum albumin, dried and stored at 4 ^o C.

An immune response is carried out in the wells of microtiter strips prepared for analysis in the presence of a standard dry blood spot. After incubation and washing, the bound Eu ³⁺ , Sm ³⁺ , Tb ^3+, and Dy ³⁺ dissociate into the well volume by adding an enhancement solution and shaking for 4 minutes. The fluorescence intensity of each ion is measured sequentially on a time-resolved fluorimeter Arcus model 1230 (Wallac OY, Turku, Finland) equipped with narrow-band interference filters: for Eu ³⁺ (613 nm), Tb ³⁺ (545 nm), Sm ³⁺ (644 nm) and Dy ³⁺ (573 nm) and a broadband filter to generate excitation radiation (250-400 nm).

 When using a fluorimetric detection system with time resolution, spatial and temporal resolution of the labels and selecting them in accordance with the sensitivity required for each analyte, the method allows the simultaneous detection of four or more analytes in one sample. To achieve the necessary sensitivity in the method, the re-complexing of lanthanide metal ions from one chelate complex (with low luminescence parameters) to another, providing a high level of quantum yield of luminescence, is carried out. However, this operation complicates the method and does not allow the measurement of the luminescence signal labels directly from the surface of the solid phase. The disadvantage of this method is the need to select labels in accordance with the required sensitivity for each analyte and the difficulties arising from the cross emission of the emission of labels, which limits the possibilities for choosing labels, i.e. limits the number of analytes detected in the sample.

 The objective is to create a multi-analytic, simple, sensitive method for analyzing samples of dry blood stains suitable for mass screening of the population.

 The technical result achieved by using the invention is to increase the sensitivity by improving the signal / background ratio and low antibody consumption in multi-analytic analysis.

 The technical result is achieved by the invention.

 The essence of the invention lies in the fact that in the method of multi-analyte immunoassay, which includes immobilizing the first biospecific components to the analytes on the surface of the microtiter plate wells, extracting the analytes from the dried blood spot by adding a buffer and conjugate with the second biospecific components to the analytes to be determined in the well, carrying out the reaction biospecific binding of reagents and detection of emission of label emission associated with the analyte by fluorimetry with by time resolution, the first biospecific components to various antibodies are immobilized at the bottom of the microtiter plate wells in the form of discrete microregions, the total area of which is less than or equal to the area of the dry blood spot, a mixture of buffer and conjugate with second, labeled or unlabeled, biospecific components for the determined analytes is applied directly to dry blood stain in a volume equivalent to its sorption capacity, remove a dry blood stain from a well, a biospecific binding reaction with labeled Pt copro - or uroporphyrin with biospecific components is carried out in discrete microregions of the immobilized first biospecific components, the wells are washed, dried, and the phosphorescence emission of the label associated with various determined analytes is detected by sequentially scanning the microregions with a focused laser beam.

 The invention also consists in the fact that antibodies to the analytes to be determined are used as the first biospecific components.

 The invention also consists in the fact that conjugates of carrier proteins with analytes are used as the first biospecific components.

 The essence of the invention also lies in the fact that the second biospecific components consist of conjugates with antibodies to detectable analytes labeled with Pt copro or uroporphyrin.

 The essence of the invention lies in the fact that the second biospecific components consist of a complex of a conjugate of second antibodies to detectable analytes with biotin and streptavidin (or avidin) labeled with Pt copro- or uroporphyrin.

 The essence of the invention also lies in the fact that the second biospecific components consist of second antibodies and conjugates of antispecies antibodies to the second antibodies labeled with Pt copro- or uroporphyrin.

 The invention also lies in the fact that the cross-sectional area of the scanning laser beam is less than the area of the microplates with the first antibodies immobilized.

 The authors are not aware of technical solutions having the same combination of features as the combination of features of the claimed invention. Therefore, the claimed invention meets the criterion of novelty.

 The claimed invention differs from the prototype by new features and other performance of the known features. Water-soluble Pt complexes of copro- or uroporphyrins, phosphorescent at room temperature, used as immunoreagent taps, have luminescence intensities comparable to those of rare-earth metals (Eu, for example), can luminesce for a long time on the solid phase, and allow the signal to be read directly from discrete zones of binding of antibodies to the desired analytes without the operation of their dissociation into the volume of the reaction vessel (well), which allows you to simultaneously identify many analytes at using a single label. Using the indicated volume of the mixture of the extracting solution (buffer and conjugate) avoids the masking effect of blood cells on the measured signal, which, together with other signs, makes it possible to increase the concentration of the desired analytes in discrete zones and, therefore, increase the sensitivity of the method. Therefore, the present invention meets the criteria of the prior art.

 The invention can be used in healthcare, in particular in pediatrics and neonatology, for the timely detection of congenital malformations of children with the aim of timely corrective therapy. Therefore, the present invention meets the criterion of industrial applicability.

 In FIG. 1 shows a diagram of the experiment in examples 1, 2 (direct method of analysis); figure 2 shows a diagram of the experiment in example 3 (indirect method of analysis); figure 3 shows a diagram of the experiment in example 4 (analysis method with streptavidin-avidin); in FIG. 4-7 shows the calibration curves for determining the concentration of T4 (tyropsin), AFP (alpha-fetoprotein), hCG (human chorionic gonadotropin) and TSH (thyrotropic hormone), respectively, as in example 1, 2; on Fig-11 shows the calibration curves for determining the concentration of T4, AFP, hCG and TSH, respectively, as in example 3; in FIG. 12-15 shows calibration curves for determining the concentration of T4, AFP, hCG and TSH, respectively, as in example 4; on Fig shows the dependence of the luminescence of the label on the volume of the extraction buffer at various concentrations of TSH: curve 9 - concentration of TTg 250 μI / ml; curve 10 - TSH concentration of 25 μMU / ml.

 In FIG. 1-3 depict: a microtiter plate 1 well, analyte 2 being determined, antibody 3 immobilized on the solid phase (bottom of the well), conjugates of the second antibodies with Pt coproporphyrin 4, bovine serum albumin conjugate with analyte 6; conjugates of second antibodies with biotin 7; conjugate of streptavidin with Pt coproporphyrin 8.

 The method is as follows.

 Example 1. Detection of four analytes from a sample of a dry blood spot, the label material is Pt coproporphyrin.

A solid-phase carrier (wells of a microtiter 96-well microplate) is treated with the first biospecific component — specific antibodies to each of the desired analytes. To do this, 1 μl of the first antibodies to each of the desired analytes is introduced into the bottom of the holes using a microdoser (Labsystems OY) in the form of discrete zones. The concentration of antibodies is 5 μg / ml. The distribution of antibodies in the form of separate zones is carried out so that the distance between the zones does not exceed 1 mm and the total area occupied by sensitized antibodies does not exceed the area occupied by the dry spot (from 3 to 5 mm). Antibodies are incubated for 2 hours at 37 ^{° C.} and then excess unbound antibodies are removed by washing with water 3 times. After that, to exclude the possibility of nonspecific binding, the bottom and walls of the wells are treated with a blocking 15 mM phosphate buffer solution containing bovine serum albumin at a concentration of 1%. BSA blocking is carried out for 1-2 hours at room temperature, then the BSA solution is removed, the microplate is dried and used for analysis.

 A paper disk with a dry blood stain (disk diameter from 3 to 5 mm) is introduced into the well of the prepared microplate. A mixture of a buffer solution in the amount of 5 μl containing Tween 20 - 0.05%, Triton X-100 - 0.1%, Tris-Hcl - 15 mm, BSA - 1%, and specific antibody conjugates are applied to the surface of the disk for extraction of analytes. thyrotropin, alpha-fetoprotein, choriogonadotropin and thyroxine with Pt coproporphyrin at a concentration of 1 μg per ml each (second biospecific components). The label / protein ratio in the conjugate is 5: 1-4: 1. The conjugates are obtained by treating Pt with coproporphyrin with carbodiimide and succinimide to activate it, followed by adding antibodies to the reaction mixture (see 9. De Haas RR et al., Platinum porphyrins as phosphorescent label for time-resolved microscopy, J. Histochem. & Cytochem., 45, (1997) 1279-1292). To obtain conjugates, commercial antibody preparations with known characteristics are used (Monoclonal antibodies to hormones were obtained according to TU SSC GosNIIIBP for the corresponding sets of TU9398-334-05031637-97-TTG, TU9398-065-05031637-01-AFP, TU-9398-066-05031637 -01-HCG, TU 79398-067-05031637-01 - T4, Boehringer Manheim chorionic gonadotropin, company number 253065, thyrotropin cat. Number 252948, AFP - DAKO Finbio, price list 2000, cat.X0900, monoclonal antibodies: T4 - ICN cat. 631021, TTG - DAKO Finbio, price list 2000, cat. M3503, hCG - ICN cat. 10810, 63011, AFP - ICN cat. 692031).

 Incubate the disc for 2 hours. Then, a blood stain disk is removed with a Wallrem OY Diskremove company (Finland), the wells are washed three times with an aqueous solution, dried until water is completely removed, and the phosphorescence signal of the label from the bottom of the hole is recorded on a time-resolved phosphorimeter (IFI-02, Russia) with stroboscopic excitation a light source with a wavelength of 532 nm and emission with a maximum of 645 nm. The delay time between the acts of excitation and the reception of phosphorescence is 40 μs, the time of registration of the phosphorescence gate is 100 μs. A phosphorimeter provides registration of a phosphorescence signal from each of the four zones (microregions) during the detection of four analytes, while the area of the probe light beam is deliberately smaller than the analyzed microregion with the desired analyte, which eliminates the influence of the signals of neighboring zones on each other. The experimental data for determining the concentration of the desired analytes are shown in table 1. The experimental design is shown in figure 1.

 As can be seen from example 1, the consumption of antibodies used for sensitization of the solid phase is reduced to 5-10 ng, which is almost 50-100 times less than in the prototype.

 Example 2. Detection of four analytes from a sample of a dry blood spot, the label material is Pt uroporphyrin.

 The method is carried out in the same way as in example 1. Pt uroporphyrin is used as a label for the second antibodies (second biospecific component). Label loading in conjugates varies between 6: 1 ... 4: 1. The experimental data for determining the concentration of the desired analytes are shown in table 2.

 Example 3. Detection of four analytes from a sample of a dry blood spot using, as a second biospecific component, a system of second labeled antibodies and a conjugate of antispecies antibodies with Pt coproporphyrin.

The solid-phase carrier (wells of a microtiter 96-well microplate) is treated analogously to Example 1 with specific antibodies to each of the desired analytes. To do this, 1 μl of antibodies to each of the desired analytes is added as separate zones to the bottom of the wells using a microdoser (Labsystems OY). The concentration of antibodies is 5 μg / ml. The distribution of antibodies in the form of separate zones is carried out so that the distance between the zones does not exceed 1 mm and the total area occupied by sensitized antibodies does not exceed the area occupied by the dry spot (from 3 to 5 mm). Antibodies are incubated for 2 hours at 37 ^{° C.} and then excess unbound antibodies are removed by washing with water 3 times. After that, to exclude the possibility of nonspecific binding, the bottom and walls of the wells are treated with a blocking 15 mM phosphate buffer solution containing bovine serum albumin at a concentration of 1%. BSA blocking is carried out for 1-2 hours at room temperature, then the BSA solution is removed, the microplate is dried and used for analysis.

 A paper disk with a dry blood stain (disk diameter from 3 to 5 mm) is introduced into the well of the prepared microplate. A mixture of a buffer solution in the amount of 5 μl containing Tween 20 - 0.05%, Tween 60 - 0.05%, Triton X-100 - 0.1%, Tris-Hcl -15 mM, BSA is applied to the surface of the disk for extraction of analytes - 1%, and specific antibodies (to thyrotropin, alpha-fetoprotein, choriogonadotropin, thyroxine) at a concentration of 1 μg per ml each. For analysis, commercial hormone preparations and rabbit antibodies to the above hormones are used (Sigma catalog).

 Disc incubate for 1 hour. Then, a blood stain disk is removed with a Wallrem OY Diskremove (Finland), then 20 μl of a conjugate of anti-mouse anti-rabbit immunoglobulin antibodies (Sigma catalog) with Pt coproporphyrin at a concentration of 1 μg per ml are added to the wells. The label / protein ratio in the conjugate is 5: 1-4: 1. The conjugates are prepared by treating Pt of coproporphyrin with carbodiimide and succinimide to activate it, followed by adding antibodies to the reaction mixture (see 9. De Haas RR et al., Platinum porphyrins as phosphorescent label for time-resolved microscopy, J. Histochem. & Cytochem., 45 , (1997) 1279-1292). To obtain conjugates using commercial antibody preparations with known characteristics. The conjugate is incubated in the well for 30 minutes and then the wells are washed three times with an aqueous solution, dried until the water is completely removed, and the phosphorescence signal of the mark from the bottom of the well is recorded on a time-resolved phosphorimeter (IFI-02, Russia) with stroboscopic excitation by a light source with a wavelength of 532 nm and emission with a maximum of 645 nm. The delay time between the acts of excitation and the reception of phosphorescence is 40 μs, the time of registration of the phosphorescence gate is 100 μs. A phosphorimeter provides registration of a phosphorescence signal from each of the 4 zones, while the area of the probe beam must be deliberately smaller than the analyzed area with the desired analyte, which excludes the influence of the signal of the neighboring zone with another analyte. The experimental data for determining the concentration of the desired analytes are given in table. 3. The experimental design is shown in FIG. 2.

 Example 4. Detection of four analytes from a sample of a dry blood spot using, as the second biospecific components, a complex of second antibodies labeled with biotin and a streptavidin conjugate with a label, the label material is Pt coproporphyrin.

 Processing of the solid-phase carrier (the bottom of the 96-well microplate well) with specific antibodies (the first biospecific components) and treatment with blocking buffer solution are carried out as described in Example 3. Then, a paper disk with a dry blood stain is introduced into the well of the prepared microplate. A mixture of buffer solution in the amount of 5 μl containing Tween 20 - 0.05%, Tween 60 - 0.05%, Triton X-100 - 0.1%, Tris-Hcl - 15 mM, BSA is applied to the surface of the disk for extraction of analytes - 1%, and conjugates of biotinylated murine monoclonal antibodies to the desired analytes. Antibody conjugates with biotin are prepared using activated biotin derivatives (Biotin Hydroxysuccinimide ICN, cat. 62601). As antibodies, commercial antibodies are used in accordance with TU SSC GosNIIBP for the corresponding sets of TU9398-334-05031637-97-TTG, TU9398-065-05031637-01-AFP, TU-9398-066-05031637-01 -HGCh, TU-79398-067 -05031637-01 - T4: T4 - ICN ca1. 631021, TTG - DAKO Finbio, price list 2000 cat. M3503. hCG - ICN ca1. 10810, 63011, AFP - ICN cat. 692031. Antibody conjugates mixed with buffer are added at a concentration of 1 μg / ml. After incubation and removal of the dry blood stain, conjugates of streptavidin or avidin with a luminescent label (second biospecific component) in a volume of 10 μl with a concentration of 100 ng / ml are added to the wells. Pt copro- or uroporphyrin are used as a luminescent label. The conjugates are obtained by treating Pt of coproporphyrin with carbodiimide or succinimide to activate it, followed by streptavidin or avidin in the reaction mixture. De Haas R.R. et al., Platinum porphyrins as phosphorescent label for time-resolved microscopy, J. Histochem. & Cytochem., 45, (1997) 1279-1292). The load of the luminescent label in streptavidin conjugates varies from 6: 1 to 4: 1.

 Spend incubation for 30 minutes. Then the wells are washed 3 times with distilled water, dried and a long-term luminescence signal is recorded. The experimental data for determining the concentration of the desired analytes are shown in table 4. The experimental design is shown in FIG. 3.

 The use of indirect variants of immunophosphorescence analysis using the second biospecific components in the form of complexes (specific specific and anti-species antibodies or the streptavidin-biotin system) is advisable if obtaining direct conjugates with the first antibodies specific for the desired analyte is difficult, because leads to a decrease in their specific activity or, if these antibodies are available in very limited quantities and cannot be used to obtain conjugates.

 Example 5. Obtaining calibration curves.

 A quantitative assessment of the analyte content in the studied dry blood spot is carried out according to calibration curves constructed according to experimental data obtained in the study of dry blood samples (as described in examples 1-4) with a given concentration of analytes in them. The experimental data for constructing the calibration curves are shown in Table 5, the calibration curves in FIG. 4-7 - to determine the concentration of analytes in example 1; table 6, the calibration curves in Fig.8-11 - to determine the concentration of analytes in example 3; in table 7, the calibration curves in FIGS. 12-15 are for determining the analyte concentration in Example 4. These curves are plotted for the case when Pt coproporphyrin is used as a luminescent label; for the case when Pt uroporphyrin is used as a luminescent label, the curves are plotted during similar experiments. As follows from the calibration curves, the proposed method provides an increase in the sensitivity of the analysis (lowering the threshold level of detection) in comparison with the known method.

 Example 6. The choice of the optimal volume of extraction buffer.

 In FIG. Figure 16 shows the experimental dependences of the luminescence of the test sample on the volume of the extraction buffer at various concentrations of TSH hormone in the sample of a dry blood spot. As can be seen from curves 9, 10, the desorption efficiency decreases sharply with increasing buffer volume. Moreover, the decrease in the efficiency of desorption is the higher, the higher the concentration of the analyzed hormone (curve 9 in Fig. 16). This is due to the fact that during extraction with large volumes at the same time as hormones, blood cells are extracted, which shield the layer of specific antibodies and interfere with the passage of the immune response. When the buffer volume is reduced to 3-5 μl, free water is practically absent in the analyzed volume and the blood stains are not extracted from the material. In this regard, for a multicomponent analysis, a buer with a volume of not more than 5 μl is used. Reducing the volume of the buffer to 3-5 μl, in addition, allows you to increase the sensitivity of the analysis due to the fact that the concentration of the hormone increases and the interaction with antibodies of the solid phase improves.

 The use of copro or uroporphyrin as a Pt label allows measurements of luminescent signals in the longer wavelength region of the spectrum, which significantly reduces the effect of the short-wave fluorescence of the solid phase material on the measured signal, i.e. The signal-to-background ratio improves. In this case, the drying of the reaction material on the solid phase (bottom of the well) increases the quantum yield of the label by 30-50 times. The limited volume of the mixture of the buffer and the second biospecific component allows, due to the gradient distribution between the dry blood stain and the solid phase with the first biospecific components immobilized, only analytes to be extracted from the dry blood stain, which excludes the masking effect of blood cells. The combination of these features allows us to achieve a sensitivity (threshold level) sufficient to detect small amounts of analytes in microzones. Therefore, the method allows for multi-analytic analysis of a dry blood sample using a single label, which simplifies the method and solves the problem of detecting the number of analytes determined that arises in the prototype due to overlapping emission spectra of the labels.

Claims (7)

 1. A method of multi-analytic immunoassay, including immobilizing the first biospecific components to the analytes on the surface of the microtiter plate wells, extracting the analytes from the dried blood stain by adding a mixture of buffer and conjugate to the second biospecific components to the antibodies to be determined, carrying out the biospecific binding reaction of the reagents and children emission of a label associated with an analyte using a time-resolved fluorometry method, characterized in that the first biospecific components for the determined analytes are immobilized on the bottom of the microtiter plate wells in the form of discrete microregions, the total area of which is less than or equal to the area of the dry blood spot, a mixture of buffer and conjugate with the second, labeled or unlabeled, biospecific components for the determined analytes is applied directly to the dry blood spot in a volume equivalent to its sorption capacity, a dry blood stain is removed from the well, a biospecific binding reaction with Pt-labeled copro or uroporphyrin b The iospecific components are carried out in discrete microregions of the immobilized first biospecific components, the wells are washed, dried, and the phosphorescence emission of the label associated with various analytes determined is detected by sequentially scanning the microregions with a focused laser beam.  2. The method according to p. 1, characterized in that as the first biospecific components use antibodies to analytes being determined.  3. The method according to p. 1, characterized in that as the first biospecific components use conjugates of carrier proteins with analytes.  4. The method according to p. 1, characterized in that the second biospecific components consist of conjugates with antibodies to detectable analytes labeled with Pt copro or uroporphyrin.  5. The method according to p. 1, characterized in that the second biospecific components consist of conjugates of antibodies to determined analytes with biotin and streptavidin or avidin, Pt-labeled with copro or uroporphyrin.  6. The method according to p. 1, characterized in that the second biospecific components consist of second antibodies and a conjugate of antispecies antibodies to the second antibodies labeled with Pt copro- or uroporphyrin.  7. The method according to p. 1, characterized in that the cross-sectional area of the scanning laser beam is less than the area of micro-sites with the first antibodies immobilized.

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