RU2242762C2 - Product and method for multiple-discipline serumal immunoanalysis - Google Patents

Abstract

FIELD: immunochemistry.

SUBSTANCE: the present innovation deals with immunochemical methods for analyzing human and animal sera to be applied for simultaneous detection of specific antibodies (AB) to some markers of infectious, parasitic, allergic and somatic diseases in the field of medicine, veterinary science, biotechnology, ecological trials and scientific researches. The innovation deals with an article being a bottom plate (strip) as a comb with teeth of 5-12 mm width to which one should spread the antigens tested. With the help of this bottom plate one should perform the method of immunochemical assay. The innovation enables to cheapen, simplify and considerably accelerate the procedure of serological testing due to simultaneous (in one and the same analysis) obtaining the data on the presence of AB to some markers of different diseases in a tested sample, decrease energy and labor expenses necessary to fulfill the analysis.

EFFECT: higher efficiency.

13 cl,3 ex, 3 tbl

Description

The invention relates to immunochemical methods for the analysis of human and animal blood products and can be used to simultaneously detect specific antibodies (AT) in the studied samples for a number of pathogens of infectious, parasitic, allergic and oncogenic diseases in medicine, veterinary medicine, environmental research and scientific experiments.

The essence of this identification is the formation of specific complexes between the AT of the test sample and the known antigens, in a certain order, discretely fixed on a dense substrate of the analytical device. The resulting complexes are then detected using a secondary immunoreagent specific for the AT of the analyzed serum (anti-species AT, staphylococcal proteins A and G, etc.) 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.

Many variants of the implementation of solid-phase immunoassay are known, however, all the test systems created to date for determining AT are monospecific, i.e. detect AT to one or more antigenically close agents. This is largely due to the characteristics of antigens used as primary immunoreagents.

By origin, antigens can be natural, recombinant and synthetic. Until recently, only natural antigens derived from natural biological agents were used in immunochemistry. The properties of such antigens (immunological, sorption, electrochemical, etc.) varied significantly depending on the species and strain differences of the source of the antigen, the method and environment of its cultivation, the methods used for inactivation, isolation, purification and conservation. A variety of antigens necessitated a careful selection of the components of the test system and the conditions for the analysis in each case.

The successes of molecular biology in recent years have made possible the large-scale production of recombinant antigens, which are increasingly being introduced into the practice of immunoassay every year. Such popularity of recombinant antigens, along with safety of work and relatively low material and labor costs, is explained by their high specificity and purity, providing high quality indicators of immunodiagnostics.

On the other hand, recombinant technologies make it possible to standardize antigens by unifying the methods of their production and purification, which, in turn, can make it possible to unify the composition of components and analysis conditions in test systems of different specificities and create diagnostics for simultaneous multidisciplinary analysis of clinical samples.

The technology for producing natural antigens has also progressed significantly recently. The use of high-quality components in the production of material and effective means of isolating and purifying antigens from it in some cases make the products obtained compatible (including with recombinant proteins) and suitable for use in multidisciplinary analysis. However, so far multidisciplinary analysis has not been implemented.

The closest technical solution is the test system "Orgenics immunocomb HIV 1 + 2 bi-spot" company "Orgenics", Israel [1, prototype]. This test system is one of the options for dot immunoassay (dot, blot, spot overlay immunoassay) on the surface of a non-porous carrier. Its working element (solid phase) is presented in the form of a flat plastic comb, on each tooth of which HIV 1 and 2 antigens are applied as separate spots, as well as human immunoglobulins - a control to check the operation of the conjugate. Another element of the test system is a plastic container divided into separate cells according to the number of comb teeth (horizontal row) and the number of working solutions used in the system (vertical row). Each horizontal row of cells is filled with a specific solution: a solution for diluting a sample, a washing solution, a solution of a conjugate of a secondary immunoreagent labeled with alkaline phosphatase, a washing solution, a solution of a substrate for the manifestation of alkaline phosphatase, washing solution. In the analysis, a certain volume of the studied sera is introduced into the first cells of the series and, then, the comb moves sequentially at certain intervals along the rows of cells. After removal from the last row, the result of the analysis of the presence of blue spots at the sites of antigen application and control is visually taken into account. The system is fully equipped with the components necessary for analysis and is suitable for working in off-laboratory conditions.

The disadvantage of this prototype is that the system allows you to identify only two related strains of the human immunodeficiency virus.

An object of the invention is to obtain products for multi-parameter immunoassay of sera and the use of this device for immunoassay.

The problem is solved by using the proposed device, on the teeth of the comb or individual strips of which several antigens are discretely applied (different infectious and parasitic diseases, different markers of somatic, allergic or oncogenic pathology) that can efficiently bind specific ATs under the same analysis conditions. The set of antigens on the device may be different depending on the tasks of immunodiagnostics. Immunoassay involves the simultaneous (in one analysis) identification of the presence of specific antibodies to several (up to several tens) antigens corresponding to various infectious, parasitic, autoimmune or oncological diseases.

A set of antigens may include a selection of several antigens of the same profile (for example, sexually transmitted infection pathogens antigens, transfusion control pathogens antigens, or animal infectious and parasitic pathogens antigens) or be relatively universal, including dozens of medical or veterinary antigens profile.

The selection of antigens for such a system can be carried out in a tablet version of ELISA with the same conditions for sorption of antigen and statement of analysis. In the same way, the concentration of antigen can be selected for sorption on a solid substrate. Application of antigen can be in the form of metered aliquots of no more than 1 μl or in the form of an electrospray. In the latter embodiment, the antigen can be applied in the form of spots or lines of certain shapes, which further increases the reliability of accounting for the results. For weakly sorbing substrate materials, effective variants of covalent binding of the antigen or its binding to the substrate by means of bifunctional agents can be used.

After antigen fixation, free areas of the substrate (strip) are blocked by one or more natural or synthetic polymers, such as bovine serum albumin, casein, polyethylene glycol, etc.

The analysis includes sequential incubation of the product:

1) in the dilution of the analyzed serum or blood plasma;

2) in a washing solution;

3) in a solution of a secondary immunoreagent (anti-species antibodies or staphylococcal protein A or G) associated with an easily distinguishable label (enzyme or sol of an inorganic catalyst);

4) in a washing solution;

5) in the substrate for the manifestation of the label;

6) in a washing solution.

The results are taken into account visually or using a scanner and subsequent computer analysis of the image. In the latter case, densitometric analysis of the results is possible with obtaining approximate quantitative characteristics of the content of the analyzed AT in the test sample.

The present invention involves the use as a secondary immunoreagent label of both enzymes (peroxidase, alkaline phosphatase, etc.) and highly dispersed (particle diameter in the range of 0.005-0.1 μm) sols of inorganic catalysts (gold, silver, selenium, palladium, etc. ) The sols can be associated with a secondary immunoreagent (immunosol) sorption or covalently. When using porous substrates, sols can be associated with a larger (particle diameter in the range 0.1–10 μm) carrier having a low density (latex, coal), and with an immunoreagent [2]. In the latter case, stable immunosuspension is obtained, the particles of which, by virtue of their size, do not penetrate into the pores of a dense substrate, which greatly facilitates the washing procedure (RF Patent No. 2133469). The manifestation of the label is carried out in one of the known substrates, providing detection sensitivity.

The analysis of the prior art by the applicant, including a search by patent and scientific and technical sources of information, and the identification of sources containing information about analogues of the claimed invention, allowed to establish that the applicant did not find an analogue characterized by features identical (identical) to all essential features of the claimed invention.

Therefore, the claimed invention meets the criteria of "novelty" and "inventive step".

The following are examples of the implementation of the proposed technical solution.

In trial experiments using:

Antigens:

1) a mixture of recombinant proteins (p17 and p41) Treponema pallidum (family Spirochaetaceae);

2) recombinant cytomegalovirus (CMV) antigen (Herpesvirdae family);

3) the recombinant antigen of the Crimean Congo virus hemorrhagic fever (WCCGL) (family Bunuaviridae);

4) recombinant antigen Toxoplasma gondii (class Sporozoa);

5) corpuscular antigen of Chlamidia psitacci (family Chlamudiaceae);

6) herpes simplex virus (HSV) corpuscular antigen (Herpesvirdae family);

7) smallpox vaccine virus (BOB) corpuscular antigen (Poxviridae family);

8) Brucella suis corpuscular antigen.

Solutions:

1) FSB - 0.1 M sodium phosphate buffer pH 7.2-7.4, with 0.15 M NaCl;

2) FSB-T with 0.05% 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) RBD-K - a solution for the dilution of conjugates and immunosols - FSB-T 6 with 0.05% casein;

6) RBD-S - a solution for diluting serums - FSB, pH 9.6, with 0.1% tween 20 and 5% CBD;

7) the substrate for the manifestation of peroxidase is a commercial preparation “Peroxidase substrate Kit DAB" (Cat. SK-41100), manufactured by "Vector lab. Inc. ", Burllingem, CA, USA;

8) PX-a / IgG — conjugate of peroxidase with MA against human IgG — commercial preparation of the Kapel company, Moscow;

9) Au-SpA - colloidal gold (average particle diameter 15 nm), adsorption bound to staphylococcal protein A [3], the initial immunosol had an OD of ₅₂₀ = 2.0 OE;

10) Ag-a / IgG - colloidal silver (average particle diameter of 10 nm), sorption associated with antibodies against human IgG [4];

11) “physical developer” for enhancing the signal of the gold and silver immunosols — an aqueous solution containing 0.5% citric acid, 0.2% metol and 0.2% silver nitrate [4, 5].

Example 1. MANUFACTURE AND APPLICATION OF THE DEVICE FOR MULTI-PROFILE ANALYSIS OF SERUMS WITH USE OF POLYSTYRENE SUBSTRATE AS A MATERIAL

The manufacture of the device.

Option A.

White polystyrene (food packaging) is cut into strips (strips) of size 8 × 60 mm, the upper layer of plastic is scraped off with a scalpel. On a fresh plastic surface in two rows along the strip at intervals of 5-7 mm, antigens are applied at a concentration of 50-100 μg / ml per KB, aliquots of 2 μl, and dried at room temperature. The length of the working part of the strip (plot with applied antigens) is about 20-25 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. The strips are rinsed with distilled water and dried at room temperature.

Option B.

The same as in option A, polystyrene strips were incubated for 2 hours at room temperature in a 2% solution of glutaraldehyde in 0.01 M potassium phosphate buffer, pH 7.0, and dried in air. Further operations for applying antigens and blocking the strip are carried out similarly to those described in option A.

Device application

Strips with the applied antigens in an upright position are incubated for 30 min at 37 ° C in a dilution of 1/40 of the tested sera on RBD-C, washed three times by immersion for 1 min in PBS-T, incubated for 30 min at 37 ° C in Ag-a / IgG (1:20 dilution on RBD-K) or HRP / IgG conjugate (working dilution on RBD-K). Wash three times by immersion for 1 min in FSB-T and rinse the glass with distilled water. Bound sols are shown by immersing strips for 10 15 min in the “physical developer”, and the bound conjugate - 20 min by a substrate for peroxidase. The developed strips are glued onto white paper and passed through a scanner. The scanned results are transferred to a computer and, using a special program, processed and printed.

The results obtained in comparison with the immunoperoxidase test on monospecific test systems are shown in table 1.

Example 2. MANUFACTURE AND APPLICATION OF THE DEVICE FOR MULTI-PROFILE ANALYSIS OF SERUMS WITH

USING GLASS SUBSTRATE AS A MATERIAL

Receiving device.

Fat-free coverslips for microscopy are boiled for 4 hours in 6N hydrochloric acid, washed with water and dried. Glass is boiled for 12 hours in a 10% solution of 3-ethoxylpropyl-1-amine in anhydrous toluene, washed with acetone and dried. Apply to certain points 2 μl of a 10% aqueous solution of glutaraldehyde, incubated for 12 hours at 20 ° C and 100% relative humidity (in a humid chamber), washed with water, acetone and dried. 2 μl of solutions of different antigens with a concentration of 0.5-1 mg / ml in phosphate buffer, pH 7.5, are applied to the points activated with glutaraldehyde and kept for 12 hours at 20 ° C in a humid chamber. Glass is washed with water, dried. Incubate the glass in a 0.2% solution of casein on phosphate buffer, pH 7.5, 1 h at 20 ° C, rinse with water, and dry.

Device application.

Glasses coated with antigens are incubated for 30 min at 37 ° C in the test sera (1/20 dilution on RBD-C, washed twice by immersion for 1 min in PBS-T, incubated for 30 min at 37 ° C in Au-SpA immunosol solution (dilution 1/50 on RBD-K). Wash twice by immersion for 1 min in FSB-T and rinse the glasses with distilled water. Bound gold is revealed by immersing the glasses for 10 min in a “physical developer." The developed strips are glued onto white paper and passed through a scanner. Scanned results are transferred to a computer and using special Social programs are processed and printed.

The results of a multiparametric dot analysis in comparison with a monospecific tablet ELISA are shown in table 2.

Example 3. OBTAINING AND APPLICATION OF THE DEVICE FOR MULTI-PROFILE ANALYSIS OF SERUMS WITH USE OF THE SUBSTANCE OF POROUS MEMBRANES AS A MATERIAL

Receiving device.

Millipore nitrocellulose membranes with a pore diameter of 0.45 μm (type HA) are cut in the form of strips 7-8 mm wide and 25 mm long, antigens at a concentration of 50-100 μg / ml per KB are applied in two rows along the strip at intervals of 5 -7 mm aliquots of 5 μl and dried at room temperature. Strips are immersed in a 0.2% casein solution on FSB and incubated for 1 h at 37 ° C. The strips are rinsed with distilled water and dried at room temperature.

Device application.

Strips with deposited antigens are incubated for 30 min at 37 ° C in a dilution of 1/40 of the studied sera on RBD-C, washed three times for 5 minutes on a shaker in PBS-T, incubated for 30 min at 37 ° C in Ag-a / IgG immunosol ( 1:20 dilution on RBD-K) or HRP / IgG conjugate (working dilution on RBD-K). Wash three times for 5 minutes on a shaker in FSB-T and once in distilled water. Bound sols are shown by immersion of strips for 10-15 minutes in the “physical developer”, and bound conjugate for 20 minutes in a substrate for peroxidase. The developed strips are glued onto white paper and passed through a scanner. Scanned results are transferred to a computer and processed and printed using a special program.

The results obtained in comparison with the immunoperoxidase test on monospecific test systems are shown in table 3.

The proposed product and method of immunoassay involve a complete set of components and can be used both for mass screening of sera 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.

LIST OF REFERENCES

1. HIV 1 + 2 bi-spot Cat: 432102920922.

2. RF patent No. 2133469 “Marker for surface blot immunological and hybridization analysis on porous media”, G 01 N 33/53.

3. 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 Czechoslovak Biochemical Society of the Czechoslovak Academy of Sciences, Prague, May 29 ^th -Jine 3 ^rd , 1988.

4. Poltavchenko A.G., Karavaev B.C., Tuzikov F.V. The use of silver sols as markers of immunoassay on microtiter tablets // ZHEMI, 1998, No. 2, pp. 108-111.

5. Poltavchenko A.G., Lavrinenko I.A., Kostrovsky V.G. et al. The use of silver immunosols for the detection of HIV antibodies in microtiter plates // Vopr. Virusol., 1997, No. 3, pp. 120-123.

Claims (13)

1. The product for immunochemical analysis, which is a plastic substrate (strip) in the form of a comb with an antigen adsorbed on a limited area of its surface and blocked by natural or synthetic polymers, areas free of antigen, characterized in that on the surface of the substrate with teeth 5- wide 12 mm each were applied in separate spots as all the antigens used in the analysis that are able to efficiently bind specific antibodies under the same analysis conditions. 2. The product according to claim 1, characterized in that the substrate material is glass or porous membranes. 3. The product according to claim 1, characterized in that the deposition of antigens on the substrate is made by the dispensing product in the form of limited spots. 4. The product according to claim 1, characterized in that the deposition of antigens on the substrate is carried out in a spray state (electrospray) in the form of limited spots or lines of a certain shape. 5. The product according to claim 1, characterized in that the fixation of antigens on its surface is due to covalent bonding or by means of bifunctional agents. 6. The product according to claim 1, characterized in that all of the antigens fixed on the substrate or part of them are represented by recombinant proteins. 7. The product according to claim 1, characterized in that all of the antigens fixed on the substrate or part of them are natural proteins. 8. The product according to claim 1, characterized in that all of the antigens fixed on the substrate or part of them are represented by synthetic proteins. 9. The method of immunochemical analysis, including incubating a strip with a fixed antigen in the dilution of the analyzed serum, washing the strip, incubating the strip in a solution of conjugate of secondary antibodies with an enzyme label, washing the strip, incubating the strip in a substrate solution for developing a label, washing the strip, visual, washing the strip, visual characterized in that the analysis is performed using the product described in claims 1-8, and several (more than 3) types of antibodies with different specificity are determined in serum at the same time. 10. The method according to claim 9, characterized in that as the conjugate use sols of inorganic catalysts: gold, or silver, or selenium, or palladium, associated with a secondary immunoreagent, or anti-species antibodies, or staphylococcal proteins A or G. 11. The method according to claim 9, characterized in that as the conjugate use the immunosols described in clause 10, associated with macrocarriers (latex, ground coal, soot, aerosil) having a density of 1.0-1.5 g / cm ³ and particle sizes in the range of 0.05-10 microns. 12. The method according to claim 9, characterized in that as a substrate for the manifestation of the catalytic labels using one of the known compositions of the "physical developers", including reducing agents and silver salts. 13. The method according to claim 9, characterized in that the calculation of the results is carried out using a scanner followed by computer image analysis.