RU2296995C2 - Method for multiple-discipline immunochemical detection of antigens in liquid samples - Google Patents

Abstract

FIELD: medicine, laboratory diagnostics.

SUBSTANCE: the present innovation deals with simultaneous detecting in the samples the specific antigens (AG) being the markers of infectious, parasitic, allergic and somatic diseases in veterinary science, medicine, biotechnology, epidemiological and ecological researches and scientific experiments. The method deals with simultaneous detecting several (by the number of antibodies applied onto a base layer) antigens being different by their specificity. Results should be registered either visually or due to scanning and subsequent computed analysis of the picture. The innovation enables to simplify and considerably accelerate immunochemical survey due to simultaneous (in one analysis) getting data on the presence of certain antigens in the sample under testing.

EFFECT: higher accuracy and efficiency of detection.

6 cl, 3 dwg, 3 ex, 3 tbl

Description

The invention relates to immunochemical methods of analysis and can be used to simultaneously detect specific antigens (AH) in the studied samples - markers of various infectious, parasitic, allergic, somatic and oncogenic diseases in medicine, veterinary medicine, environmental research and scientific experiments.

The essence of this identification is the specific immobilization of the antigens of the test sample with a set of known antibodies (primary immunoreagents), in a specific order, discretely fixed on a dense substrate of the analytical device. Immobilized antigens are processed by a mixture of antibodies specific to the entire spectrum of the analyzed antigens (secondary immunoreagent), as a result of which a triple complex is formed: the primary immunoreagent - antigen - secondary immunoreagent. The resulting complexes are then detected using the conjugate - antibodies specific for the secondary immunoreagent and associated with an easily identifiable label.

Of the variety of immunological analysis methods developed to date, solid-phase systems in which one of the components is associated with a dense substrate (matrix) are especially popular. This approach makes it easy to separate the formed complexes from unbound components by washing the matrix, which significantly reduces the time and simplifies the analysis procedure.

There are many options for implementing solid-phase immunoassay, however, all the test systems created to date for determining antigens are monospecific, i.e. allow the detection of antigens of one or more antigenically close agents. This is largely due to the properties of antibodies used as primary immunoreagents.

Antibody sources can be servae of convalescents (people and animals that have gained immunity as a result of an infection), vaccinated (people and animals that have gained immunity as a result of the introduction of weakened or killed infectious agents, as well as individual antigens isolated from microorganisms or obtained artificially ( by synthesis or by recombinant technologies)). Such sera are polyclonal i.e. contain a large number of antibodies of various specificity. The levels and spectrum of antibodies in them vary significantly depending on many factors (type and individual characteristics of the host organism, history, method of antigen administration, etc.). The use of polyclonal sera in immunoassay necessitated the use of complex, often affinity, methods for isolating the desired antibodies, a careful selection of the components of the test system and the conditions for the analysis in each case.

The successes of biotechnology in recent years have allowed the creation of hybridoma technologies capable of obtaining the required amounts of monoclonal (specific for one specific antigenic determinant) antibodies, as well as developing more effective methods for the isolation and purification of antibodies from polyclonal sera. This allowed, on the one hand, to significantly increase the specificity of the analysis, and on the other hand, to achieve physicochemical unification of antibodies with their immunological diversity, which makes them suitable for use in multidisciplinary analysis. However, so far multidisciplinary analysis has not been implemented.

Known test system "ImmunoComb HBs Ag 90" company "Orgenics", Israel [1], designed for high-quality detection of surface antigen of hepatitis B virus (HBs Ag). Its working device is presented in the form of a flat plastic comb, on each tooth of which anti-HBs Ag antibodies are applied as separate spots, as well as biotinylated bovine serum albumin control to test the conjugate. The method for detecting antigen in this test system is one of the variants of dot-immunoassay (dot (blot, spot) overlay immunoassay) on the surface of a non-porous carrier, including successive incubations of the device in: a pre-diluted sample; washing solution; a solution of a secondary immunoreagent labeled with biotin; washing solution; a solution of streptavidin conjugate with alkaline phosphatase; washing solution; substrate solution for the manifestation of alkaline phosphatase; washing solution. After analysis, the device is dried in air and visually take into account the results of the presence of blue spots at the sites of application of antigens and control. Such a device and method of analysis are suitable for performing studies in off-laboratory conditions, however, they allow only one antigen to be detected - a marker of viral hepatitis B infection.

The closest technical solution is the device and analysis method described in US Patent No. 5486452 [2, prototype]. An immunological analysis device is a porous substrate containing multiple limited regions of various antigens adsorbed on its surface, represented as dots, microdots, or lines arranged in a specific geometric order and obtained by applying aliquots of antigens to the substrate with metering devices. Antigen-free regions of the substrate are blocked by a non-specific protein. The immunoassay method is a dot immunoassay using antibodies or a complement protein conjugated to an isotopic, fluorescent or enzymatic (horseradish peroxidase) label. The basic scheme of immunoassay is similar to the scheme described in [1].

The device described in the description [2] has the following disadvantages:

1. A porous matrix, preferably made of nitrocellulose, with a sheet thickness of preferably 0.1 mm is used as a dense substrate of the device. Such matrices are fragile and have low mechanical strength, which greatly complicates their routine use. Another disadvantage of such substrates is the difficulty of removing unbound reaction components from the fine pores of the matrix (long, repeated washing with activation), which greatly complicates the technique and increases the likelihood of background phenomena.

2. In order to miniaturize the described device, the antigen adsorption zones are represented as spots with a diameter of less than 2 mm or lines with a width of less than 2 mm (obtained by applying aliquots of antigens in a volume of less than 1 μl), which involves the use of special readers, and will not allow for reliable direct visual recording of analysis results.

The analysis method described in [2] has disadvantages associated with the use of conjugates based on isotopic, fluorescent, or enzymatic (horseradish peroxidase) labels. Working with isotopes requires special safety conditions and cannot be widely used in routine analyzes. The use of fluorescent labels requires very expensive and therefore usually inaccessible to clinical practice equipment for instrumental recording of analysis results. Assays using horseradish peroxidase as a label in combination with substrates giving insoluble products (4-chloro-1-naphthol or 3,3-diaminobenzidine) are characterized by a relatively low sensitivity, which is approximately an order of magnitude lower than the sensitivity of the tablet version of the immunoperoxidase test using substrates, giving soluble colored products [3].

An object of the present invention is to provide a device and method for implementing multidisciplinary, simultaneous detection of several (more than three) antigens with high sensitivity and visual consideration of the results, suitable for routine use in clinical practice.

The problem is solved by using the proposed device for immunological determination, which is a flat non-porous substrate on which primary antibodies are discretely applied to different antigens (markers of infectious, parasitic, somatic, allergic or oncogenic pathologies) that are able to efficiently bind specific antigens under the same analysis conditions. The device can be made in the form of individual strips or combs, each tooth of which is an individual device.

Unlike [1], each device contains a set of several (more than 3) primary antibodies. Unlike [2], the device is made of non-porous plastic (preferably polystyrene). Such substrate materials firmly retain primary antibodies on their surface, are easily washed from unbound components and provide mechanical strength of the device (see Example 3). In contrast to [2], primary antibodies on the substrate of the proposed device are applied in aliquots of 2.5 to 3 μl in the form of dots (individual spots), which allows a reliable visual analysis result.

It is optimal to use white substrates on which it is easier to visually record color immunological signals. The shape and size of the device may vary depending on the number of primary immunoreagents and the design of their placement (see figure 1). It is important that each of the primary immunoreagents is strictly positioned on the device, since the results of multidisciplinary analysis are taken into account by the location of the color immunological signal on the substrate.

Antibodies on the substrate are applied in a saturating concentration (5 μg / ml). The set of antibodies on the device may be different depending on the tasks of immunodiagnostics. It may include a selection of several antibodies of the same profile (for example, antibodies to sexually transmitted infection pathogens; or antibodies to infectious and parasitic animal pathogens; or antibodies that can differentiate generic or strain differences of any one type of microorganism) or be relatively universal , including a number of antibodies to antigens of a medical or veterinary profile.

Monoclonal antibodies or affinity isolated antibodies from polyclonal sera can be used as the primary immunoreagent. Serum of various animals can be used as a source of primary immunoreagent. It is of fundamental importance that these sources differ (in the species of animals) from the source of the secondary immunoreagent and the primary antibodies do not interact with the conjugate used. The selection of antibodies for such a system can be carried out in the tablet version of the enzyme-linked immunosorbent assay (ELISA) with the same conditions for the adsorption of antibodies and analysis. In the same way, the concentration of antibodies can be selected for sorption on a solid support.

Along with primary specific antibodies, control samples can be immobilized on the device. As a positive control (control of conjugate operability), serum or immunoglobulins of the animal species that serves as a source of secondary antibodies can be applied at a specific location on the substrate. As a negative control (control of the specificity of the device), a blood serum of an animal serving as a source of primary polyclonal antibodies, or mouse ascites fluid (when using murine monoclonal antibodies as a primary immunoreagent), which does not contain specific spectrum antibodies, can be additionally applied to a specific area of the substrate detectable antigens. Other negative controls are sites free of antigens and controls. Like primary immunoreagents, control samples are applied in the form of strictly positioned limited spots with a diameter of 2 to 5 mm. The procedure for applying primary antibodies and controls is marked on the non-working part of the substrate and is given in the instructions for use.

After fixing the primary immunoreagents and control samples, free areas of the substrate are blocked by one or more natural or synthetic polymers, such as bovine serum albumin, casein, polyethylene glycol, etc., and, if necessary, can be treated with a stabilizer that slows down the degradation of antibodies on the substrate.

The proposed method of analysis is a dot-immunoassay performed according to the general scheme, which is fundamentally similar to that described in [1 and 2], which includes successive incubations of the device: (1) in the dilution of the analyzed sample; (2) in a washing solution; (3) in a solution of a secondary immunoreagent; (4) in a washing solution; (5) in a conjugate solution; (6) in a washing solution; (7) in the substrate for the development of the label; (8) in a washing solution.

In contrast to [1], the proposed method of immunoassay involves multidisciplinary simultaneous detection of the presence of several (more than three) specific antigens corresponding to various infectious, parasitic, autoimmune or oncological diseases. The multidisciplinary analysis is achieved by using the proposed device with several primary antibodies and using as a secondary immunoreagent a reaction mixture containing an excess of antibodies to the entire spectrum of antigens determined by the device. The present invention allows the use of a polyclonal serum, or a mixture of globulin fractions or sera of one animal species, or a mixture of monoclonal antibodies containing an excess of antibodies to the entire nomenclature of the determined antigens as a secondary immunoreagent. It is important that the sources of the secondary immunoreagent differ (in animal species) from the source of primary antibodies and interact with the conjugate used.

In contrast to [1 and 2], the proposed method of immunoassay involves the use of conjugates based on tertiary antibodies associated with catalytically active sols of gold or silver, as well as “physical developers” to enhance the immunological signal of the sols. The use of such conjugates and physical developers can significantly increase the sensitivity of the analysis compared to conjugates based on enzymes (see Examples 1 and 2). Monoclonal or polyclonal antibodies specific for secondary antibodies but not reacting with primary antibodies can be used as tertiary antibodies. used on the device. If anti-species antibodies to the Fc fragment of the immunoglobulins of the secondary immunoreagent are used as tertiary antibodies, then stages 3 and 5 in the above scheme can be combined into one, which significantly reduces the analysis time.

The present invention allows the use in the third stage of the above scheme of a mixture of ash conjugates based on polyclonal or monoclonal antibodies to individual antigens with the exception of stages 4 and 5.

The preparation of gold or silver sols (average particle diameter of 15 nm) and their binding to tertiary antibodies can be carried out by known methods [4, 5].

To enhance the signal of the ash conjugate in the proposed analysis method, it is proposed to use a physical developer containing a soluble silver salt and a reducing agent (preferably a developer containing: 0.2% metol and 0.2% silver nitrate, dissolved in 0.02 M citrate buffer solution, pH 3.5). The developer is prepared before use; it is stable for up to 20 min at room temperature [5].

After the analysis is performed, if one or more antigens are present in the test sample, colored spots are formed at the places of application of the corresponding primary antibodies to the substrate, taking into account their location on the device visually or using a scanner and subsequent computer image analysis.

The proposed multidisciplinary analysis may include: clinical samples of urine, blood serum, sputum, cerebrospinal fluid, transudates and exudates, extracts from tissue samples and feces, swabs and scrapings from the skin and mucous membranes, etc .; flushes, extracts and concentrates of environmental objects, food products, hygiene items, etc. Samples intended for analysis should be in the liquid phase with a neutral pH and should not contain coarse impurities and interfering with the test impurities.

The sensitivity of the proposed multidisciplinary analysis is not inferior to the sensitivity of commercially available plate monospecific immunoperoxidase tests (see examples 1 and 2).

The proposed device and method of immunoassay can be used both for mass screening of samples, and for individual analyzes. They can be used not only in equipped laboratories, but also (in medicine) at the patient’s bedside, in the doctor’s office, for self-diagnosis; (in ecology) in field experiments; (in veterinary medicine) in livestock farms and on a personal compound. The invention allows, due to the determination of a number of parameters in one analysis, to significantly reduce the time for examination of the patient. In addition, since one analysis of the proposed method is equivalent to several analyzes in monospecific systems and these analyzes are close in cost, the introduction of the invention into widespread practice can have a significant economic effect. Savings include not only the difference in the cost of test systems, but also the reduction in instrumentation and labor costs for the analysis.

Description of figures

Figure 1 - Examples of the overall design of a device for multidisciplinary analysis of antigens.

Figure 2 - Scheme of the application of primary antibodies (5 μg / ml, 3.0 μl) on the working part of the device: (a) ascitic mouse fluid that does not contain specific antibodies - negative control (K-); (b) human IgG - positive control (K +); monoclonal antibodies to pathogens: (c) Trichomonas vaginalis, (d) Mycoplasma hominis, (e) Neisseria gonorrhoeae, (f) Chlamydia trachomatis, (g) Ureaplasma urealyticum, (h) Gardnerella vaginalis and results of multidisciplinary washouts, performed using gold immunosols (for example 1, the numbers indicate the numbers of the samples in table 1).

Figure 3 - Scheme of the working part of the device for assessing the sensitivity of the method of multidisciplinary analysis of antigens (for example 2). The volume of application of antibodies is 2.5 μl, the concentration of antibodies is 5 μg / ml.

The following are examples of specific applications of the invention.

The following solutions are used in trial experiments.

1) FSB - 0.01 M sodium phosphate buffer, pH 7.2-7.4, with 0.15 M NaCl;

2) FSB-T - FSB with 0.1% tween-20;

3) CBD - a blocking solution concentrate containing E. coli lysate, a commercial preparation manufactured by Kapel, Moscow;

4) KB - 0.025 M Na-carbonate-bicarbonate buffer, pH 9.5;

5) BB - 0.01 M borate buffer, pH 8.0;

6) RBD-K - a solution for diluting the conjugate and immunosol - FSB-T with 0.05% casein, pH 7.2-7.4;

7) RBD-S - a solution for the dilution of serum - FSB-T with 5% CBD and 0.05% casein, pH 9.6;

8) a substrate for the manifestation of peroxidase - commercial preparation "Peroxidase substrate Kit DAB" (Cat. SK-4100), manufactured by "Vector Lab. Inc.", Burlingem, CA, USA;

9) PX-a / IgG-Hum — conjugate of peroxidase with monoclonal antibodies against human IgG — commercial preparation of the Kapel company, Moscow;

10) Au-a / IgG-Hum - colloidal gold (average particle diameter 15 nm), sorption associated with goat antibodies against human IgG [4];

11) Ag-a / IgG-Hum - colloidal silver (average particle diameter of 12 nm), sorption associated with goat antibodies against human IgG;

12) a “physical developer” for enhancing the signal of the gold and silver immunosols — 0.2% metol and 0.2% silver nitrate, 0.5% citric acid, pH 3.5 [5].

Example 1. The manufacture and use of a device for multidisciplinary analysis of vaginal swabs for the presence of pathogens of genitourinary infections

Device manufacturing

Washed with distilled water and dried synthetic paper “Polylith” of brand “G” is cut into strips (strips) of size 6 × 60 mm. Monoclonal antibodies to various pathogens of urogenital infections (see figure 2), as well as human serum IgG (K +) and mouse ascites fluid that does not contain specific antibodies (K) are applied in two rows along the strip at intervals of 5 mm on the plastic surface -) at a concentration of 5 μg / ml on BB, 3.0 μl aliquots, and dried for 10 hours at room temperature. The length of the working part of the strip (plot with applied antibodies) is about 20 mm, the free part of the strip is used as a handle. Strips for 2/3 of the length of the working part are immersed in a 0.2% casein solution on FSB and incubated for 1 h at 37 ° C. Rinse the strips of FSB-T and dry at room temperature.

Device application

Devices the working part is immersed in samples of vaginal swabs on physical. solution obtained from patients with a different spectrum of diseases and incubated for 60 min at 37 ° C. Washed three times by immersion for 1 min in FSB-T, incubated for 30 min at 37 ° C in 1/10 dilution on human RBR-C serum (serum mixture) containing (according to ELISA) IgG to detectable microorganisms in titers from 1/160 up to 1/640. Next, the devices are washed for 1 min in 3 shifts of FSB-T and incubated at 37 ° C for 30 min in Au-a / IgG-Hum immunosol or PX-a / IgG-Hum conjugate (working dilutions on RBD-K). Washed three times by immersion for 1 min in FSB-T and rinsed with distilled water. Bound sols are shown by immersing strips for 10-15 minutes in a “physical developer”, and a bound conjugate for 20 minutes in a peroxidase substrate. The analysis results are carried out visually. A positive result is considered to be a clearly distinguishable colored spot at the place of application of the corresponding antibodies to the device. At the same time, in accordance with the manufacturer's instructions, the same washes are analyzed in ELISA on monospecific test systems manufactured by ImDi-Spectrum, Novosibirsk.

The results are shown in table 1, and the scheme of application of primary antibodies to the device and a picture of the results obtained in the dot analysis using a gold immunosol are shown in figure 2.

Example 2. A comparative assessment of the sensitivity of the tablet immunoperoxidase test and multidisciplinary dot-analysis of antigens using immobilization of primary mono and polyclonal antibodies on polystyrene using a bifunctional agent - glutaraldehyde.

Receiving device.

White polystyrene UPS-0801 (TU-24-27-90) 0.5 mm thick is cut into strips (strips) of size 6 × 60 mm, the upper layer of plastic is scraped off.

Strips are incubated for 2 hours at room temperature in a 2% solution of glutaraldehyde in 0.01 M potassium phosphate buffer, pH 5.0, washed three times with 0.01 M potassium phosphate buffer, pH 7.0, with 0.1 M NCl and dried in air. Controls are applied, monoclonal antibodies to Chlamidia trachomatis and polyclonal affinity purified antibodies to the Crimean-Congo hemorrhagic fever virus (HCCGF) at a concentration of 5 μg / ml per KB, aliquots of 2.5 μl, in two rows along the strip at 5 mm intervals ( 3) and strips are incubated for 2 hours at 37 ° C in a humid chamber. FSB-T strips are washed twice. Further operations to block the strip are carried out similarly to the operations described in example 1.

Device application

Prepare a series of two-fold dilutions in physical. sample solution: vaginal washout No. 1 (see example 1) and VKKGL culture antigen. Devices are placed with the working part in test tubes with dilutions of samples and incubated for 1 h at 37 ° C. Washed three times by immersion for 1 min in FSB-T, incubated for 30 min at 37 ° C in 1/10 dilution on human RBR-C containing (according to ELISA) IgG to Chlamidia trachomatis in titer 1/320 and to WCCGL in titer 1/160. Next, the devices are washed for 1 min in 3 shifts of FSB-T and incubated for 30 min at 37 ° C in the Ag-a / IgG-Hum immunosol or ПХ-a / IgG-Hum conjugate (working dilutions on RBD-K). Washed three times by immersion for 1 min in FSB-T and rinsed with distilled water. Bound sols are shown by immersing strips for 10-15 minutes in a “physical developer”, and a bound conjugate for 20 minutes in a peroxidase substrate. Accounting for the results is carried out visually. A positive result is considered a clearly distinguishable dark spot at the place of application of the corresponding antibodies to the device. In parallel, in accordance with the manufacturer’s instructions, the same samples are analyzed in ELISA on monospecific test systems for the determination of Chlamidia trachomatis antigens (manufactured by ImDi, Novosibirsk) and VKKGL (produced by the Institute of Virology RAMS, Moscow).

The results are shown in table 2.

Example 3. Evaluation of the suitability of sheet synthetic materials for the manufacture of the substrate of the device for the multidisciplinary detection of antigens in liquid samples.

Receiving Devices

Samples of three categories of sheet synthetic materials (Table 3): porous matrices based on polyvinyl chloride and cellulose ethers, non-porous organic polymers (polystyrene, polyvinyl chloride and lavsan) and non-porous combined plastics combining organic and mineral components (synthetic paper) are washed with distilled water and cut into strips. Based on strips made from different materials, devices for multidisciplinary immunoassay of antigens are obtained as described in example 1.

Device use

On devices made of different materials, a multidisciplinary dot-immunoassay of vaginal washout 1 is carried out in parallel using gold immunosols and “physical manifestation” as described in Example 1. After the analysis, a visual comparative assessment of the color signal intensity on substrates of different materials is carried out . In this case, two parameters are evaluated:

- the efficiency of adsorption by the material of primary immunoreagents, which is judged by the intensity of a specific signal at the sites of control (K +), as well as primary antibodies to Trichomonas vaginalis, Chlamidia trachomatis and Mycoplasma hominis;

- the ability of the material to nonspecific binding of components or to spontaneous manifestation, which is judged by the presence of a background signal in non-sensitized areas of the device.

Suitable for the manufacture of the substrate devices are materials that provide good adsorption on itself of primary antibodies and do not provoke background signals.

The results are shown in table 3.

Information sources

1. www.orgenics.com; www.biograd.ru

2. US patent No. 5486452 "Devices and kits for immunological analysis". G 01 N 033/548.

3. Egorov A.M., Osipov A.P., Dzantiev B.B. and other Theory and practice of enzyme immunoassay. - M .: Higher. School, 1991 .-- 360 p.

4. Raska I. Electron microscopic immunocytoshemistry with colloidal gold. - Laboratory manual of the practical course organized by the Institute of Experimental Medicine, Czechoslovak Academy of Sciences in collaboration with the Czeshoslovak Biochemical Society of me Czechoslovak Academy of Sciences, Prague, May 29th-Jine 3 rd, 1988.

5. Poltavchenko A.G., Karavaev B.C., Tuzikov F.V. The use of silver sols as immunoassay markers on microtiter tablets // ZHEMI, 1998, N2, pp. 108-111.

Table 1 Comparative results of the detection of antigens of the causative agents of genitourinary infections in samples of vaginal swabs by monospecific plate ELISA and multidisciplinary dot-immunoassay with gold (Au-SpA) and peroxidase (PCh-a / IgG} conjugates. Test system Results of mono-specific ELISA Multidisciplinary Dot Immunoassay Results Conjugate HRP-a / IgG Au-spa HRP-a / IgG Flushing one 2 3 four 5 6 one 2 3 four 5 6 one 2 3 four 5 6 T. vaginalis + - - - - - + - - - - - ± - - - - - C.trachomatis + + - - + - + + - - + - + ± - - ± - M. hominis + - + - - - + - + - - - - - ± - - - U. urealyticum - - + - + - - - + - + - - - + - - - N. gonorrhoeae - - - + + - - - - + + - - - - ± + - G. vaginalis - + + + - - - + + + - - - - - + - - IgG Hum (K +) + + + + + + + + + + + + Ascitic fluid without specific antibodies (K-) - - - - - - - - - - - - + a clearly defined spot at the site of antibody loading (positive result), ± a poorly defined spot at the site of antibody application (weakly positive), - a clean background of the substrate at the site of antibody application (negative result)

table 2 Comparative results of the assessment of the sensitivity of detection of Chlamidia trachomatis (Chlamidia) in the vaginal washout and the Crimean-Congo hemorrhagic fever virus (VCCGL) in the culture fluid (QOL) using monospecific ELISA test systems and multidisciplinary dot immunoassay with gold (Au-a / IgG) and peroxidase (PCh-a / IgG) conjugates. (device diagram is shown in figure 2). Sample Sample Dilution The results of the monospecific ELISA tablet option Results of a multiparametric Dot - Immunoassay HRP-a / IgG Ag-a / IgG HRP-a / IgG Chlamidia WCCGL Chlamidia WCCGL TO- K + Chlamidia WCCGL TO- K + + - + - + - + - + K F 1/50 + - + - + - + - + 1/100 + - + - + - ± - + 1/200 ± - + - + - - - + 1/400 - - - - + - - - + 1/800 + + - - + + - - + Flushing 0 + + - - + + - - + 1/5 + + - - + ± - - + 1/10 - - - - + - - - + + a clearly defined spot at the site of application of antibodies (positive result), ± a poorly defined spot at the site of application of antibodies (weakly positive result), - a clean background of the substrate at the site of application of antibodies (negative result).

Table 3 The results of the assessment of the suitability of various sheet synthetic materials for the manufacture of the substrate of a device for multidisciplinary analysis of antigens (for example 3).
The number of “+” signs in the table is proportional to the level of the visual signal after analysis. Material characteristics Evaluation results Category Brand (TU) Source (manufacturer) Color Sorption Background Suitability Porous membranes HAWP Millipore, USA White ++++ - Unsuitable because of the fragility of the material and the complexity of washing Trans-Blot 162-0114 Bio-Rad, United States White ++++ - FPM-G1 VX
Plastopo. Zhmer St. Petersburg White ++ - Unsuitable Polystyrene UPPS-O.SOl (Tu-24-2 7-90) CJSC Kairos, Omsk White +++ - Suitable Substation (TU-10-24-27-90) LLC "Termahim" Novosibirsk White +++ - Suitable Polyethylene Terifthalate (Lavsan) Chemical plant Vladimir Transparent + + Unsuitable 3R96002 Type With Rank Xerox, UK Transparent + - Unsuitable 3R96002 Type C Chemically Modified Research Institute of Okhotsk, Novosibirsk Transparent +++ - Suitable Synthetic paper "POLILITH" Brand "R" Firm "Coast", St. Petersburg White ++++ ++ Unsuitable Brand “G” Firm “Bereg”, St. Petersburg White ++++ - The best material for the manufacture of the substrate

Claims (7)

1. The method of immunochemical analysis, including incubation of a strip with fixed antibodies in a liquid sample, washing, incubation in a solution of secondary antibodies to a detectable antigen, washing, incubation in a conjugate of labeled anti-species antibodies, washing, incubation in a substrate solution for the manifestation of the label, washing and accounting characterized in that on the surface of the substrate made of non-porous plastic, primary antibodies to various antigens are immobilized in the form of separate spots, additionally as a control of immunoglobulins of the animal species that serves as the source of the secondary antibodies are applied, the ascitic fluid of the mouse is applied as a negative control, the antibodies and the control are applied in the form of solutions with a protein concentration of 5 μg / ml in a volume of 2.5-3 μl, and secondary antibodies are used a mixture of polyclonal antibodies specific to all the antigens determined and differing in the source of production from primary antibodies sorbed on a substrate, conjugates based on catalytically active gold or silver sols are used with a particle diameter of 0.005-0.1 μm, associated with antibodies to detectable antigens, and “physical developers” are used as a substrate for the manifestation of the catalytic mark. 2. The method according to claim 1, characterized in that as the conjugate use sols associated with anti-species immunoglobulins specific for the Fc fragment of secondary antibodies. 3. The method according to claim 1, characterized in that they use conjugates based on catalytically active sols of gold or silver with an average particle diameter of 15 nm. 4. The method according to claim 1, characterized in that as physical developers apply a solution comprising 0.2% metol, 0.2% silver nitrate and 0.5% citric acid. 5. The method according to claim 1, characterized in that they use a substrate made of white polystyrene. 6. The method according to claim 1, characterized in that the substrate is made in the form of a flat "comb", on each tooth of which the antibodies used in the analysis are discretely applied. 7. The method according to claim 1, characterized in that the immobilized antibodies are monoclonal antibodies.

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