RU2611359C1 - Method and set for determination of cholera toxin production and differentiation of epidemiologically significant strains of cholera vibrios of classical and el tor biovars - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of the inventions relates to medicine and deals with method of determination of cholera toxin production and differentiation of epidemiologically significant strains of cholera vibrios, which includes preparation of tested sample, identification of cholera toxin by ELISA method, which provides introduction of antigens in holes of tablet, incubation and registration of results. Group of the inventions also deals with set for determination of cholera toxin production and differentiation of epidemiologically significant strains of cholera vibrios, realising claimed method.

EFFECT: group of the inventions ensures creation of highly specific, sensitive, simple and available for reproduction, without application of additional equipment, biologically safe, financially not costly (low cost price), executable for short amount of time method for determination of cholera toxin, produced by strains of cholera causative agent of classical and el tor biovars in conditions in vitro and in vivo, and differentiation of toxigenic (epidemiologically significant) strains of V cholerae.

4 cl, 3 ex, 3 tbl

Description

The invention relates to medical microbiology and can be used in laboratory diagnostics to determine the presence of Vibrio cholerae classic and eltor biovar strains producing cholera toxin and differentiate them by epidemic significance, identify toxigenic strains of cholera vibrios in human clinical material.

One of the most important components of cholera epidemiological surveillance is the timely identification and identification of toxigenic (epidemiologically significant) strains of cholera vibrios. The effectiveness of such studies depends on the availability of highly sensitive and specific drugs for the laboratory diagnosis of cholera.

A known method for identifying epidemiologically significant cultures of V. cholerae eltor and V. cholerae 0139 by their adhesive ability, including incubating the test culture with subsequent preparation of microscopic preparations and determining the adhesive ability by the number of vibrioes adhered to the substrate represented by human blood group II, is carried out by the reaction to glass. According to the results of the reaction, differentiation into toxigenic (epidemiologically significant) and atoxigenic is carried out, however, this can be done only in isolated pure culture, but not in biological material [1].

A known method and kit for the detection and determination of the biotype, serogroup and toxigenicity of the cholera pathogen using the multilocus polymerase chain reaction method, which provides the determination of serogroup, biovar, as well as toxigenicity (epidemic significance) by identifying the genetic marker of the toxin - ctxA gene, but does not allow to determine cholera toxin (XT) production [2].

Known designed on the basis of monoclonal antibodies (MCAs) specific for XT, the diagnostic test strip V. cholerae tox +, designed to detect toxigenic strains of V. cholerae. The sensitivity of the immunochromatographic (THEM) test strips is 10 ⁸ -10 ⁹ mk / ml and depends on the quality of the antibodies used and the concentration of antigen in the biomaterial. However, the proposed approach will not allow the determination of toxigenic strains of cholera vibrios in the clinical material from a person with their content below 10 ⁸ m.k./ ml (from vibration carriers, after taking antibacterial drugs). The production of their strips is economically costly, and their cost in the end is quite high [3].

The literature describes a test system based on cholera toxin-specific MCAs in a plate version of an enzyme-linked immunosorbent assay (ELISA) and in the format of hydrogel biochips made using polymerization immobilization technology. The detection limit of the toxin reaches 0.2 ng / ml in the ELISA plate format and 0.44 ng / ml in the microarray format. The presented test system was tested on samples containing a commercial preparation of cholera toxin (Sigma, USA). The use of modern biomicrochip technology gives accurate results in a short time. However, the sophisticated cooking technology along with the high cost of analysis and the use of special software limits the possibility of their use by practical laboratories. As the test samples used milk, meat broth, water containing pure XT preparation, however, data on the study of the effectiveness of the method in the study of strains of V. cholerae, in particular, Eltor biovar and clinical material are not presented. This is important, since strains of cholera vibrios of the biovar eltor under normal conditions do not induce the release of XT outside the cell [4].

There is a method of enzyme-linked immunosorbent assay for the determination of XT production by V. cholerae strains, which includes preparing the test sample, introducing GM1 sensitized gangliosides into the wells of the tablet, incubating the sample at 37 ° C for 2 hours, washing with buffer solution, introducing conjugate, followed by incubation and taking into account the results of optical density (OD).

Sample preparation is to induce the release of cholera toxin from the cells of the classical vibrios and Eltor biovars. Cultivation is carried out in different environments, depending on the biovar affiliation. The cholera vibrio of the classical biovar is cultivated with constant stirring in Craig medium at a temperature of 30 ° C for 48 hours, and the cholera vibrio biovar eltor is cultured without shaking in AKI at 37 ° C for 20 hours or Craig medium at a temperature of 30 ° C in within 48 hours [5]. The disadvantage of the described method is the need for preliminary determination of membership in a biovar and, depending on it, the use of different environments and conditions for the induction of XT. In addition, with this method of detecting XT, it is necessary to use for the study of expensive reagents: GM1 gangliosides, polyclonal serum against XT, the duration of the sample preparation stage and ELISA. All this does not allow us to consider this method as promising for use in laboratories at the territorial level (laboratories of especially dangerous infections of the Center for Hygiene and Epidemiology in the constituent entities of the Russian Federation, regional centers for monitoring the pathogen of pathogenicity groups II-IV).

The literature describes the cultivation of cholera vibrio to induce cholera toxin from the cells of the cholera vibrio classic and eltor biovars. For this, a pathogen culture in the volume of one complete bacterial loop No. 2 is seeded in a test tube with 10 ml of AKI broth (bacto-peptone - 1.5%, yeast extract - 0.4%, sodium bicarbonate - 0.3%, sodium chloride - 0 , 5%) and incubated at a temperature of 30 ° C for 3.5-4 hours, and then transfer the contents of the tube in full to a bottle with 250 ml of AKI broth and incubate with constant stirring for 17-19 hours at a temperature of 30 ° C [6]. However, the cultivation of vibrios according to the presented scheme is time-consuming, takes place in a large volume of nutrient medium using additional equipment that provides constant swaying for a long time, which is associated with a high risk of biological hazard, since the broth culture contains no less than 5-6 hours 1 × 10 ⁹ MK / ml of the pathogen.

The technical task of the invention is to develop a method and an enzyme-linked immunosorbent assay system for determining the presence of the classic Vibrio cholerae strains, the cholera toxin biovar strain and differentiating them by epidemic significance, determining the toxigenic cholera vibrio strains in human clinical material.

The technical result of the invention is the creation of a highly specific, sensitive, simple and accessible to reproduce, without the use of additional equipment, biologically safe, not financially expensive (low cost), performed in less time the method for determining cholera toxin produced by classical and eltor cholera pathogen strains biovars in vitro and in vivo, and differentiation of toxigenic (epidemiologically significant) strains of V. cholerae. An additional technical result consists in the possibility of conducting studies on a different number of samples.

The technical result is achieved by a method that includes the preparation of the test sample, the detection of ELISA cholera toxin, which includes the addition of antigens to the wells of the tablet, incubation and recording of the results. According to the invention, the cholera toxin is induced by strains of cholera vibrios when cultivated primarily in a glass tube in 10 ml AKI broth at a temperature (37 ± 1) ° C for 2.5 hours and again in 5-10 ml of AKI broth in a plastic tube with a maximum slope to form a thin layer of a liquid medium temperature (37 ± 1) ° C for 14 hours followed by detection of a cholera toxin by a sandwich ELISA, in which the test sample and conjugate are introduced into the wells of a tablet sensitized with monoclonal antibodies specific for the B subunit of cholera toxin of isotype G2a, which is a monoclonal antibody specific for the B subunit of cholera toxin of isotype G1, labeled with horseradish peroxidase and according to the OP differentiation of strains is carried out, while if the value of OPOb≥OPcr (K-) × 2 - the strain produces cholera toxin and refers to t ksigennym, and when the value OPobr <SPRI (K) × - strain does not produce cholera toxin under the nontoxigenic.

The detection of ELISA cholera toxin is carried out using the kit components, while the sensitization of the ELISA plate wells and the preparation of the peroxidase immunoglobulin conjugate are carried out using MICA specific for different cholera toxin epitopes. Monoclonal antibodies specific for the B subunit of cholera toxin of isotypes G1 and G2a are produced by the hybridomas Mus musculus XT 3E5 and Mus musculus XT 2E5, respectively, which were obtained at the Microbe Russian Research Anti-Plague Institute of Rospotrebnadzor and deposited in the State Collection of Pathogenic Microorganisms and Cellular Organisms crops "GKPM-Obolensk" under the number H-43, under the number H-44.

The technical result is achieved by a kit, which is a kit containing at least one 96-well collapsible strip plate with sequentially applied onto its surface MCA specific for the b-subunit of cholera toxin of isotype G2a and lactalbumin; as well as containers containing a portion of AKI medium, conjugate of MAB specific for the B subunit of cholera toxin of isotype G1 with horseradish peroxidase; conjugate dilution reagents: glycerin, lactalbumin, Tween; reagents for the preparation of the substrate mixture: citric acid, sodium citrate, 2,2'-azino-bis- (3-ethylbenzthiazoline-6-sulfonic acid), urea peroxide; a negative control sample, which is a phosphate-buffered saline; positive control sample, which is a lyophilized cholera toxin.

Some components of the kit, such as reagents for preparing the working solution of the conjugate, substrate mixture, positive control sample, are packaged in duplicates, which allows the study to be carried out both on the minimum number of strips - 3, and on the entire tablet - 12.

The kit includes 12 components:

tablet of 12 strips of 8 wells polystyrene, for enzyme immunoassay, single use (Costar, USA), sensitized with monoclonal immunoglobulins to XT at a concentration of 10 μg / ml, followed by blocking with lactalbumin. The wells of the tablet are transparent, without visible traces of the substance;

sample of AKI medium (cream-colored powder, after dissolution - light yellow liquid) 12.12 g in 2 tubes;

phosphate-buffered saline (FSB) (white tablet, after dissolution - a clear liquid), 1 tablet in 5 packets;

Skimmed milk powder (lactalbumin) (cream-colored powder, after dissolution - white liquid), 0.0113 g in 4 tubes;

peroxidase immunoglobulin monoclonal conjugate (MKAHT conjugate) (amorphous mass of white color, which is dried monoclonal immunoglobulin XT conjugated with horseradish peroxidase, after dissolution - yellow liquid) in 0.02 ml in 1 tube;

glycerin (viscous clear liquid) of 0.025 ml in 1 tube;

Tween-20 (a viscous, slightly yellowish liquid), 0.002 ml in 4 tubes;

citric acid (white powder, after dissolution - a clear liquid) of 0.0225 g in 4 tubes;

sodium citrate (white powder, after dissolution - a clear liquid), 0.025 g in 4 tubes;

ABTS (2,2'-azino-bis- (3-ethylbenzthiazo-line-6-sulfonic acid), diammonium salt (pale green powder, after dissolution - pale green liquid), 0.001625 g in 4 tubes ;

hydroperite (white powder, after dissolution - a clear liquid) at 0.067 g in 4 tubes;

positive control sample (PKO-XT) (amorphous mass of white color, which is a dried cholera toxin at a concentration of 200 ng, after dissolution - a clear liquid) 0.1 ml in 4 tubes.

The method is as follows.

A portion of AKI medium from one tube is dissolved in 500 ml of distilled water and transferred to 50 glass tubes of 10 ml each (used for primary growth). A portion of AKI medium from the second tube is dissolved in 500 ml of distilled water and poured into 50 or 100 ml into vials. AKI broth tubes and vials are sterilized at 120 ° C for 30 minutes. Prepared tubes and bottles with AKI broth are stored in plastic bags in a refrigerator at a temperature of 6 to 8 ° C for the entire shelf life of the test system (6 months).

Before starting the study, the required number of glass tubes is removed from 10 ml of AKI broth, corresponding to the number of studied strains of cholera vibrios or samples of biological material. Prepare the same number of disposable Falcon type centrifuge tubes with a volume of 15 ml or 50 ml for secondary growth. To do this, from bottles with AKI broth, 5 ml of broth is added to Falcon centrifuge tubes of 15 ml or 10 ml of broth to Falcon centrifuge tubes of 50 ml.

To prepare the test sample — induction of XT by V. cholerae strains of both biovars under in vitro conditions, the studied vibrio agar cultures in the volume of three colonies (2-3 mm in diameter) or 1/2 loop (number 2) are seeded in a glass tube with 10 ml AKI for primary growth and incubated for 2.5 hours at a temperature of (37 ± 1) ° C. Then, 1.0 ml from the grown culture is transferred into centrifuge tubes with a 15 ml cap containing 5 ml of AKI broth, or 2.0 ml each into centrifuge tubes with a 50 ml cap containing 10 ml of AKI broth. The prepared tubes are placed in a thermostat so that they are practically horizontal, but without touching the tube cover with culture fluid, and secondary growth is carried out at a temperature of (37 ± 1) ° C for 14 hours.

When examining clinical material from humans, suspended samples at the rate of 1 g of sample per 10 ml of 0.9% sodium chloride solution can be examined immediately after their additional dilution by 10 times or after cultivation in AKI medium, similarly to the analysis of pathogen cultures.

Before the start of the study, all components requiring dissolution are diluted and stored throughout the shelf life of the test system (6 months) under appropriate temperature conditions.

To dilute the components, the FSB is prepared. For this, the FSB tablet is dissolved in 100 ml of purified water. The solution is stored in the refrigerator at a temperature of (5 ± 3) ° C for 3 days.

To prepare a concentrated conjugate solution, 20 μl of purified water are added to the test tube containing lyophilized MCA 2E5 labeled with horseradish peroxidase and completely dissolved. Then gently mix and transfer the entire volume to a test tube containing glycerin, mix thoroughly. The resulting concentrated conjugate solution was stored at minus (18 ± 2) ° C for 6 months.

Immediately before the study, working dilutions of the conjugate and substrate mixture, as well as a positive control sample, are prepared. One tube with each component is designed for studies with 3 strips.

2.7 ml of prepared FSB are added to a test tube with a sample of skimmed milk powder (lactalbumin) and mixed thoroughly until a homogeneous mass is obtained for 1-2 minutes. The solution is stored in the refrigerator at a temperature of (5 ± 3) ° C for 3 days.

In a test tube containing Tween-20, add 0.375 ml of FSB and mix thoroughly, avoiding the formation of bubbles. The solution is stored in the refrigerator at a temperature of plus (5 ± 3) ° C for 3 days.

In a test tube with a hinge of citric acid add 2 ml of purified water. The solution is stored at a temperature of plus (5 ± 3) ° C for 3 days.

1.7 ml of purified water is added to a test tube with a portion of sodium citrate. The solution is stored in the refrigerator at a temperature of (5 ± 3) ° C for 3 days.

0.15 ml of purified water is added to a test tube containing lyophilized ABTS (2,2'-azino-bis- (3-ethylbenzthiazo-line-6-sulfonic acid). The solution is stored in a refrigerator at a temperature of (5 ± 3) ° C within 3 days.

0.2 ml of purified water is added to a test tube with a sample of hydroperite and mixed thoroughly until complete dissolution, obtaining a 33% hydrogen peroxide solution. Store in the refrigerator at a temperature of (5 ± 3) ° C for 3 days.

0.1 ml of purified water is added to a test tube with a lyophilized positive control sample and the dry substance is completely dissolved. The solution is stored in the refrigerator at a temperature of (5 ± 3) ° C for 1 day. If necessary, for longer storage (up to 6 months), the solution can be placed in the freezer at a temperature of (18 ± 2) ° C.

Preparation of working solutions of the conjugate and substrate mixture for 3 strips is carried out ex tempore. The resulting solutions are not stored.

Conjugate working solution: 0.3 ml Tween-20 solution is added to a test tube with a lactalbumin solution and mixed thoroughly. Just before adding to the wells in the prepared solution add 0.02 ml of a concentrated conjugate solution and mix thoroughly again.

The working solution of the substrate mixture: the substrate mixture is prepared immediately before entering into the wells. For this, the contents of the tube with sodium citrate are introduced into the test tube with citric acid in full, 0.1 ml of ABTS solution and 0.004 ml of hydroperite solution are added.

If it is necessary to study more strips, the preparation of reagents is carried out similarly, using other tubes with lactalbumin, Tween-20, citric acid, sodium citrate, ABTS, and hydroperite.

Select the required number of strips for research. 100 μl from tubes with cultures of V. cholerae after secondary growth or native material, positive and negative controls, are successively introduced into the wells of strips in duplicates.

The plate is incubated at a temperature of (37 ± 1) ° C for (40 ± 5) minutes. At the end of the incubation, the tablet is removed from the thermostat, the contents of the wells are taken using a multichannel dispenser and carefully dumped into a container for waste liquids with a disinfectant, avoiding the formation of droplets (aerosol).

The wells of the plate are washed 3 times, for this, 250 μl of FSB are added, then the contents of the wells are removed using a multi-channel dispenser and carefully dumped into a waste liquid container with a disinfectant, avoiding the formation of droplets (aerosol).

100 μl of prepared conjugate working solution are added to the wells. The plate is incubated at a temperature of (37 ± 1) ° C for (40 ± 5) minutes. Next, the wells of the plate are washed 6 times, as described above.

100 μl of the working solution of the substrate mixture are added to all wells and kept at room temperature from + 18 ° C to + 25 ° C for (12 ± 2) ° C min, after which the results are recorded.

The differentiation of toxigenic (epidemiologically significant) strains of cholera vibrios, as well as the assessment of clinical material, is carried out on the basis of a positive or negative analysis result. If positive (OPob≥OP (K-) × 2) - cholera toxin was detected in the sample, the studied culture of cholera vibrio was toxigenic (epidemiologically significant) or toxigenic strains of the cholera pathogen were detected in the samples of clinical material. If negative (OPobr <OPsr (K-) × 2) - no cholera toxin was detected in the sample, the studied culture of cholera vibrio was nontoxigenic (epidemiologically safe), or toxigenic strains of the cholera causative agent were not detected in the samples of clinical material.

Thus, the selected medium, XT induction conditions, and ELISA with monoclonal antibodies to the XT B subunit of isotype G2a, isotype G1 allow the detection of XT production of both biovars.

Example 1. The determination of the presence of XT in strains of cholera vibrios using the claimed set on 3 strips.

Tubes for XT induction in cholera vibrio strains were prepared as described above, using 15 ml centrifuge tubes for secondary growth.

The research material was 11 strains of cholera vibrios, 2 of which were toxigenic V. cholerae of the classical biovar, 6 were toxigenic V. cholerae of the biovar eltor, 2 were non-toxigenic V. cholerae of the biovar eltor, and 1 strain of E. coli O157. All cultures were obtained from the State collection of pathogenic bacteria FKUZ RosNIPCHI "Microbe". Strains of cholera vibrios and Escherichia coli were grown at 37 ° C in LB broth for 4 hours, followed by plating on LB agar, pH 7.6 and incubation in an incubator at 37 ° C for 18-24 hours. Pre-cultivation of the test cultures were performed as described above.

In a semi-automatic dispenser, 100 μl in succession was added to the two wells of the sensitized, blocked plate of the test samples. As a positive control, 100 μl of XT solution was added at a concentration of 200 ng, and 100 μl of FSB as a negative control.

The plate was incubated at a temperature of (37 ± 1) ° C for (40 ± 5) minutes. At the end of the incubation, the tablet was removed from the thermostat, the contents of the wells were taken using a multi-channel dispenser and carefully dumped into a container for waste liquids with a disinfectant, avoiding the formation of droplets (aerosol). The wells of the plate were washed 3 times, for this, 200 μl of FSB was added, then the contents from the wells were removed using a multi-channel dispenser and carefully dumped into a waste liquid container with a disinfectant, avoiding the formation of droplets (aerosol).

Preparation of working solutions of the substrate mixture, the conjugate and the positive control sample was carried out on 3 strips, as indicated previously.

100 μl of prepared conjugate working solution were added to the wells. The plate was incubated at a temperature of (37 ± 1) ° C for (40 ± 5) minutes. Next, the wells of the plate were washed six times, as described above. 100 μl of substrate mixture were added to all wells and kept at room temperature from + 18 ° C to + 25 ° C for (12 ± 2) min. The reaction was taken into account photometrically at a wavelength of 405 nm by a color change in the wells. For this, the arithmetic mean value was calculated in the wells with a negative control sample — OPsr (K-). The result of the analysis was considered positive if OPOb≥Opsr (K-) × 2. The result of the analysis was considered negative if OPOb <Opsr (K-) × 2 (OPOb - optical density in the well with the analyzed sample).

In the study of toxigenic isolates of V. cholerae cholerae 569 B, M-41, V. cholerae eltor P-3122, M-1509, M-879, M-1298, M-1328, P-18899 by ELISA using the claimed kit in all In cases, a positive response was obtained, which indicates the presence of cholera toxin in these strains. Whereas, when studying non-toxigenic cultures of V. cholerae eltor KM-26, M-1262 and E. coli O157, a negative result was recorded, which confirms the absence of cholera toxin in these strains. The results of the determination of XT production by V. cholerae strains in the “sandwich” variant of enzyme-linked immunosorbent assay using monoclonal antibodies are shown in table 1.

Thus, the claimed method provides a determination of the production of XT strains of V. cholerae classical and eltor biovars.

Example 2. The definition of XT in clinical material. (Model experiment)

We studied stool samples artificially contaminated with toxigenic strains of V. cholerae P-3122 (Eltor Biovar) and M-1509 (Biovar Eltor) at a concentration of 1 × 10 ⁹ to 1 × 10 ⁴ MK./ml. The strains were pre-grown in AKI medium to induce XT in them (imitation of XT production by vibrios in vivo). Also, stool samples containing a non-toxigenic culture of V. cholerae KM-26 were prepared.

1 g of feces was added to the tubes and 0.1 ml of culture liquid was added after the secondary growth of the V. cholerae P-3122, M-1509 and KM-26 strains at a concentration of 1 × 10 ⁹ to 1 × 10 ⁴ m.k. / ml . To the prepared samples, 10 ml of 0.9% sodium chloride solution was added and thoroughly mixed, settled for 10-15 minutes to settle large particles. The supernatant was taken and diluted 10 times and immediately introduced 100 μl into the wells of the sensitized tablet, and 100 μl were seeded in a glass tube with 10 ml of AKI medium for primary growth, after which the preparation of the test sample was performed as described in example 1.

ELISA was carried out according to example 1. It was shown that in all cases a positive response in ELISA was detected only in samples containing toxigenic strains of V. cholerae, regardless of their concentration and belonging to the classical or eltor biovars. The analysis results were not affected by the additional enrichment of samples in AKI medium, which indicates the possibility of using ELISA to study native stool samples for the presence of XT. The results of the determination of XT production in the clinical material in the “sandwich” variant of enzyme-linked immunosorbent assay using monoclonal antibodies are shown in table 2.

The inventive set is arranged in such a way that allows you to conduct research on the minimum number of strips - 3, and on the tablet as a whole - 12.

Example 3. Determination of the presence of XT in strains of cholera vibrios using the claimed set on 6 strips.

Tubes for XT induction in cholera vibrio strains were prepared as described above, using 15 ml centrifuge tubes for secondary growth.

The study material was 23 strains of cholera vibrios, of which: 2 — toxigenic V. cholerae of the classical biovar, 10 — toxigenic V. cholerae of the biovar eltor, 7 — non-toxigenic V. cholerae of the biovar eltor, 1 strain — V. cholerae O139 (ctx A + ), 1 strain is V. cholerae O139 (ctx A-), 1 strain is V. cholerae not O1 not 0139 (ctx A +), 1 strain is V. cholerae not O1 not O139 (ctx A-). All cultures were obtained from the State collection of pathogenic bacteria FKUZ RosNIPCHI "Microbe". Strains of cholera vibrios were grown by the method described in example 1. Pre-cultivation of the studied cultures, sensitization, blocking the tablet for ELISA was performed as described above.

The introduction of the test samples, incubation, washing the tablet is performed as described in example 1.

Preparation of working solutions of the conjugate and substrate mixture for 6 strips is carried out ex tempore. The resulting solutions are not stored.

Conjugate working solution: 2 tubes with a lactalbumin solution are combined and 0.6 ml of Tween-20 solution is added and mixed thoroughly. Just before adding to the wells in the prepared solution add 0.04 ml of a concentrated conjugate solution and mix thoroughly again.

The working solution of the substrate mixture: the substrate mixture is prepared immediately before entering into the wells. To do this, 2 tubes with citric acid contribute the full contents of the combined 2 tubes containing sodium citrate, add 0.2 ml of ABTS solution and 0.008 ml of hydroperite solution.

Further analysis steps were performed as described in example 1.

When studying toxigenic isolates by ELISA using the claimed kit in all cases, a positive response was obtained, which indicates the presence of cholera toxin in these strains. When studying non-toxigenic cultures, a negative result was registered, which confirms the absence of cholera toxin in these strains. The results of the determination of XT production by V. cholerae strains in the “sandwich” variant of enzyme-linked immunosorbent assay using monoclonal antibodies are presented in table 3.

Thus, the inventive method provides for the determination of cholera toxin production by V. cholerae classical and Eltor biovar strains, as well as in clinical material.

Literature

1. Patent RU 2332460. A method for detecting epidemically significant cholera vibrios Vibrio eltor and Vibrio cholerae O139 by their adhesive ability, publ. 08/27/2008.

2. Patent RU 2360972. A method for detecting and determining the biotype, serogroup and toxigenicity of the causative agent of cholera and a kit for its implementation, publ. 07/10/2009.

3. Baranova E.V. Development of an immunochromatographic test for the detection of toxigenic strains of Vibrio cholerae / E.V. Baranova, P.V. Soloviev, V.V. Agafonova et al. // Actual. prob. warning and liquidity. consequences of emergency. situations in the field of a dignity. well-being.

4. Petrova E.E. Obtaining and characterization of monoclonal antibodies to cholera toxin / E.E. Petrova, R.L. Komaleva, O.E. Lakhtin et al. // Bioorganic chemistry. - 2009. - T. 35, No. 3. - S. 357-367.

5. Detection of Cholera Toxin: Laboratory Methods for the Diagnosis of Vibrio cholerae Centers for Disease Control and Prevention. - Atlanta, JA, 1994 .-- P. 62-88.

6. G. Jonson, J. Holingren, AM Svennerholm Analisis of expression of toxin-coregulated pili in classical and eltor V. cholerae O1 in vivo and in vitro // Inf. Immun. - 1992 - V. 0160 No. 10 - P. 4278-4284.

Claims (4)

1. The method of determining the production of cholera toxin and differentiation of epidemically significant strains of cholera vibrios, including the preparation of the test sample, detection of cholera toxin by ELISA, which involves the introduction of antigens into the wells of the tablet, incubation and recording of results, characterized in that the induction of cholera toxin by strains of cholera vibrios is carried out at cultivation primarily in a glass tube in 10 ml of AKI broth at a temperature of (37 ± 1) ° C for 2.5 hours and secondly in 5-10 ml of AKI broth in a plastic tube with a maximum slope for the formation of a thin layer of a liquid medium at a temperature of (37 ± 1) ° C for 14 hours, followed by the detection of cholera toxin a “sandwich” enzyme-linked immunosorbent assay, in which the wells are sensitized with monoclonal antibodies specific for the cholera B subunit isotype G2a toxin, make the test sample and conjugate, which is a monoclonal antibody specific for the B subunit of cholera toxin G1, labeled with horseradish peroxidase, and optical density rovodyat differentiation of strains wherein if OPobr≥OPsr value (K) × 2 - strain produces cholera toxin, and relates to a toxigenic, and when the value OPobr <SPRI (K) × 2 - strain does not produce XT under the nontoxigenic. 2. The method according to p. 1, characterized in that the test sample is a strain of cholera vibrios or clinical material from patients. 3. The kit for determining the production of cholera toxin and differentiation of epidemiologically significant strains of cholera vibrios, carrying out the method according to claim 1, is a kit containing at least one 96-well collapsible stripped plate with monoclonal antibodies sequentially applied to the surface of the wells, specific to the B subunit of cholera toxin of isotype G2a and lactalbumin; as well as containers containing a portion of AKI medium, a conjugate of monoclonal antibodies specific for the B subunit of cholera toxin of isotype G1 with horseradish peroxidase; conjugate dilution reagents: glycerin, lactalbumin, Tween; reagents for the preparation of the substrate mixture: citric acid, sodium citrate, 2,2'-azino-bis- (3-ethylbenzthiazoline-6-sulfonic acid), urea peroxide; a negative control sample, which is a phosphate-buffered saline; positive control sample, which is a lyophilized cholera toxin. 4. The kit according to claim 3, characterized in that the reagents for preparing the working solution of the conjugate, substrate mixture, and positive control sample are packaged in duplicates, which allows the study to be performed on the minimum number of strips - 3, and on the entire tablet - 12.