RU2703282C1 - Method for producing a diagnosticum for detecting a toxin of a cholera vibrio recovered from environmental objects - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to microbiology, and can be used to produce a diagnosticum for quantitative determination of toxin of cholera vibrio recovered from environmental objects. That is ensured by producing experimental antitoxic immune rabbit serum by immunizing rabbits weighing 2.5–3.0 kg by administering a pure cholera toxin preparation in dose of 100 mcg of protein subcutaneously into the inguinal lymph node and intravenously into a marginal ear vein in amount of 3 injections with interval of 14 days, then 14 days after the last injection, rabbits are bleeding, and blood is prepared with serums to determine antibody titre. Further, immunoglobulins are recovered from rabbit serums, for this purpose physiological saline is prepared in advance, buffered with disubstituted sodium phosphate and monosubstituted potassium phosphate at pH 7.2 (PBS), then preparing an aqueous solution of methanol, mixing 6 ml of methanol with 8 ml of distilled water, the mixture is cooled, after which the obtained serum in amount of 4 ml is combined with 2 ml of buffered saline (PBS), placed in centrifuge cups, put on ice and poured into them whey at temperature 0 °C, then buffered physiological solution is added, mixed, methanol water is poured in with 14 ml, while reducing the temperature to -5° C, after which sleeve with mixture is put in refrigerator for 30–40 minutes, and then centrifuged at 2,000 rpm for 20 minutes at zero temperature, obtained supernatant is drained, and the precipitate is suspended in ½ PBS from the initial volume of whey is determined by the method of Lowry, resulting in sensitin. Polymer support is prepared using an anionic polymerization method of a monomer – acrylic aldehyde – in an aqueous-alkaline medium to obtain monodisperse particles of a spherical shape with diameter of 1.0±0.1 mcm, which are stained with thianine and washed with distilled water. Polymeric carrier is then sensitized with immunoglobulins to the cholera toxin; for this purpose, the deposited carrier residue is suspended in a borate buffer (pH 8.4), immunoglobulins are added thereto and allowed to contact while stirring for 2 hours at 20 °C, as a result, aldehyde reaction groups on the surface of particles specifically interact with amino groups of protein molecules of immunoglobulins. After that, suspension is cooled to 4–5 °C and held at this temperature for 16–18 hours. Free aldehyde groups are blocked by adding 0.5 % solution of gelatase to the suspension in buffered saline (pH 7.0–7.1) and incubation for 2 hours at room temperature. Suspension of diagnosticum is centrifuged for 10 minutes at 6,000 rpm and washed three times with buffered physiological solution (pH 7.0–7.1) from excess gelatine, and final sediment is suspended in 0.1 % solution of gelatase in buffered physiologic saline (pH 7.0–7.1). Diagnosticum is lyophilized by suspending preparation in 20 ml of 3 % gelatose-sucrose medium, then the suspension is poured into ampoules of 1 ml, frozen in liquid nitrogen with subsequent vacuum drying, gradually raising temperature to 22–23 °C for a day.

EFFECT: use of the given method enables producing a diagnosticum for quantitative determination of cholera vibrio toxin, recovered from environmental objects, in order to detect epidemic dangerous toxigenic strains of cholera vibrio.

3 cl, 3 tbl, 2 ex

Description

The alleged invention relates to medical microbiology and can be used in laboratory clinical diagnostics of especially dangerous infections to quantify the toxin production of cholera vibrio when it is isolated from environmental objects during monitoring, followed by an assessment of its epidemic potential.

Currently, the problem of studies of cholera toxin remains relevant. The U.S. Centers for Disease Control and Prevention provides a list of cholera toxin research methods. This is an enzyme immunoassay, an immunofluorescence method using a ganglioside-GM1-containing DASS system, a coagglutination reaction with coagglutinating diagnostics, latex agglutination with anti-XT serum, polymerase chain reaction, DNA sequencing. (1) Developed by Taiwan sor, based on the combined use of GM1 liposomes and antibodies to XT in the flow system with the registration of a specific fluorescence signal.

A biological microchip is known for detecting and multi-parameter analysis of cholera antibodies, including cholera toxin. (2).

According to the adopted scheme for laboratory diagnosis of cholera, a number of methodological approaches are used to determine the epidemic significance of cholera vibrios: determining hemolytic activity in the Greig test, sensitivity to ctx- and ctx + cholera diagnostic bacteriophages, detecting a cholerogenic effect on a biological model (sucker rabbits), detection of ctxAB and tcpA genes by polymerase chain reaction [3,].

However, staging a biological sample is time-consuming and time-consuming, the use of bacteriophages is difficult due to the increase in recent years the number of vibrio strains resistant to them. The determination of genes encoding enterotoxin synthesis does not always indicate the presence of expression of the toxin itself. Therefore, the development and improvement of methods for the detection of cholera toxin in vitro is still relevant.

In microbiological practice, enzyme-linked immunosorbent assay, polymerase chain reaction or biological test on a model of rabbits is most often used to assess the toxicity of cholera vibrios. The use of bioassay animals gives mixed results, since some non-toxigenic strains can give a picture of a toxic reaction.

Polymerase chain reaction is an expensive study, it requires special equipment, often imported, and specialists with training in molecular biology.

Enzyme-linked immunosorbent assay for the determination of cholera toxin, includes several stages of the study, requires special reagents, equipment and trained personnel. In addition, this method also requires significant costs, which limits its use in mass epidemiological surveys.

The WHO and CDC laboratories have proposed a method for determining cholera toxin in an enzyme-linked immunosorbent assay, where GM1 gangliosides act as a binding ligand [4].

The essence of the method is to use a “sandwich” variant of enzyme-linked immunosorbent assay, where specific components of the system are serum or monoclonal antibodies to cholera toxin, monosialo-gangsiid, as well as second monoclonal antispecies antibodies labeled with horseradish peroxidase to identify the antigen-antibody complex.

Another variant of an enzyme immunoassay for the detection of cholera toxin was proposed by Mikheeva EA (5)

The disadvantages of these methods are their multi-stage nature, the results of the study are affected by the quality of adsorption of MCA or monosialogan-hyosides in the wells of the tablet, the quality of reusable washes, and also the need for instrumentation, which is often imported, in trained personnel and financial support. The enzyme immunoassay system includes 12 components.

At the same time, there are diagnostic preparations for the determination of biological agents (antigens or antibodies) based on polymer colored carriers in a simple one-step and very economical bulk agglomeration reaction. The volumetric immunosuspension agglomeration reaction (RAW), carried out using latex diagnosticums, in fact, is an analogue of RNGA, an accessible and technically simple method, because It does not require special equipment, highly qualified personnel and can be used when conducting research in the field.

The closest in technical essence is the method for detecting atoxigenic hemolytic strains of cholera vibrio (6), which consists in the stage-by-stage immunization of rabbits with lipase Pseudomonas spp. strains of cholera vibrios exhibit the ability to produce lipase when interacting with antilipase antibodies immobilized on a polymer carrier and at the same time give a positive reaction, confirming the atoxigenicity of the studied strains.

However, the known method does not allow to detect toxigenic strains of cholera vibrio isolated from environmental objects, to determine the concentration of cholera toxin produced by them to assess the epidemic danger, since the antibodies and antigens used are not directly related to the ability of cholera vibrio to produce toxin.

The technical task of the invention is the creation of a diagnostic product capable of detecting and determining the amount of cholera vibrio toxin in the process of its isolation from environmental objects and assessing its epidemic hazard.

The problem is achieved in that the method for producing a diagnosticum for the quantitative determination of cholera toxin from environmental objects includes the following stages:

a) experimental antitoxic immune rabbit serums are obtained by immunization of rabbits weighing 2.5-3.0 kg with the introduction of a preparation of pure cholera toxin in a dose of 100 μg of protein subcutaneously in the inguinal lymph node and intravenously into the marginal ear vein in the amount of 3x injections with an interval of 14 days, then 14 days after the last injection, rabbits are bled, and serum is prepared from the blood, followed by determination of antibody titer;

ЗФР от исходного объема сыворотки, определяют количество белка по методу Лоури, получая в результате сенситин;b) isolate immunoglobulins from rabbit serums, for this, physiological saline, buffered with disubstituted sodium phosphate and monosubstituted potassium phosphate at pH 7.2 (PBS), is preliminarily prepared, then an aqueous methanol solution is prepared by mixing 6 ml of methanol with 8 ml of distilled water, the mixture is cooled after which the resulting serum in an amount of 4 ml is combined with 2 ml of buffered saline (PBS) is mixed, placed in a centrifuge cups are put on ice add methanol water 14 ml while lowering the temperature about -5 ° C, after which the glass with a mixture put in a refrigerator for 30-40 minutes and then centrifuged at 2000 rev. / min for 20 minutes at zero temperature, the resulting supernatant was discarded and the pellet resuspended in

PBS from the initial serum volume, the amount of protein is determined by the Lowry method, resulting in sensitin;

c) a polymer carrier is prepared using the method of anionic polymerization of an acrylic aldehyde monomer in an aqueous alkaline medium to obtain spherical monodisperse particles with a diameter of 1.0 ± 0.1 microns, which are stained with tianine and washed with distilled water;

d) the polymer carrier is sensitized with cholera toxin by immunoglobulins, for this, the precipitate of the washed carrier is suspended in borate buffer (pH 8.4), immunoglobulins are added to it and left to contact with stirring for 2 hours at 20 ° С, as a result, the aldehyde reaction groups on particle surfaces specifically interact with the amino groups of protein molecules of immunoglobulins. After this, the suspension is cooled to 4-5 ° C. and maintained at this temperature for 16-18 hours.

e) block free aldehyde groups by introducing into suspension a 0.5% gelatose solution in buffered saline solution (pH 7.0-7.1) and incubating for 2 hours at room temperature with constant stirring on an electromagnetic stirrer;

e) the diagnosticum suspension is centrifuged for 10 minutes at 6000 rpm. and washed three times with buffered saline (pH 7.0-7.1) from excess gelatose, and the final precipitate was suspended in a 0.1% gelatine solution in buffered saline (pH 7.0-7.1).

g) determine the sensitivity of the drug using a pure cholera toxin preparation, for this, 50 μl of normal rabbit serum are added to the wells of the tablet, then 50 μl of a cholera toxin solution with a protein content of 100 μg / ml is added to the first well and titrated twice to 12 wells inclusive moreover, from the last 12 wells, 50 μl of liquid are removed, 13 and 14 wells, into which only 50 μl of normal rabbit serum are added to control for spontaneous agglutination of the diagnosticum, then 25 μl of the suspension are added to all wells of the rows stickum, after 2.5 hours of incubation at room temperature, the results of the reaction are recorded visually, with the last well, namely the 10th, giving a positive result - a blue agglomerate lining the entire bottom of the hole, the dilution of which contains 100 ng protein / ml, which corresponds to the sensitivity of the diagnostic drug, and the specificity of the drug is determined in the reaction of volumetric agglomeration (RAO);

h) lyophilization of the diagnosticum is carried out by suspending the drug in 20 ml of a 3% gelatin-sucrose medium, then the suspension is poured into 1 ml ampoules, frozen in liquid nitrogen, followed by vacuum drying, gradually raising the temperature to 22-23 ° C during the day.

In this case, the volumetric agglomeration reaction is carried out in tablets by adding 50 μl of 1% normal rabbit serum to the wells, then 50 μl of the test substance is added to the first well of each row and titrated twice to the end of the row, 50 μl is removed from the last well, only any two wells are added normal rabbit serum to control spontaneous agglutination of the diagnosticum, then 25 μl of the diagnosticum are added to all wells and left at room temperature, RAO results are taken into account after 2-2.5 hours, with a positive result etsya uniform color sinter lining the entire bottom of the wells, with well-rounded edges, and in the negative case, and in the negative control, a compact ring or a point in the center of the hole.

In addition, the concentration of cholera toxin in a liquid nutrient medium is determined for the cultivation of cholera vibrio, into which the toxin is produced, for this the reaction is performed according to the RAO method, but in the wells of a tablet with a diluting liquid, the NCC titrates the liquid nutrient medium twice after culturing the cholera excitance in it, then add 25 μl of immunoglobulin diagnosticum, the last well with a positive reaction fixes the value of the last dilution of the culture fluid, therefore, the concentration of cholera t Xin calculated by the formula:

C = P × H

where C is the desired concentration of cholera toxin;

P is the last dilution of the test fluid in which a positive result was recorded;

H is the sensitivity of the diagnosticum in ng / ml.

The method is as follows.

At the first stage, a diagnostic preparation is prepared according to the following technology:

a) Experimental hyperimmune rabbit serums are obtained using a pure cholera toxin preparation obtained by the authors from the toxigenic strain V. cholerae 569B O1 of the serogroup from the collection of the Museum of Living Cultures with the center of human pathogens of the Rostov-on-Don Institute of Antiplague.

To select immunizing doses, determine the protein concentration according to Lowry. Then, male Californian rabbits weighing 2.5 to 3.0 kg were selected for immunization. Immunization is carried out by administering a cholera toxin preparation in a dose of 100 μg of protein subcutaneously into the inguinal lymph node and intravenously into the marginal ear vein in the amount of 3 injections with an interval of 14 days. 14 days after the last injection, rabbits are bled, and serum is prepared from the blood, followed by determination of antibody titer. The resulting hyperimmune serum is packaged and stored frozen at -30 ° C.

b) Isolate immunoglobulins from rabbit hyperimmune sera using the methanol method.

ЗФР от исходного объема сыворотки, определяют количество белка по методу Лоури, получая в результате сенситин;To do this, a 0.9% NaCl solution is preliminarily prepared, disubstituted sodium phosphate and monosubstituted potassium phosphate are added at pH 7.2 (PBS), then an aqueous methanol solution is prepared by mixing 6 ml of methanol with 8 ml of distilled water, the mixture is cooled. After that, centrifuge glasses are placed on ice, 4 ml of the obtained serum are poured into them, 2 ml of PBS are added, stirred and 14 ml of methanol water are poured, lowering the temperature to -5 ° C. The glass with the mixture is placed in the refrigerator for 30-40 minutes, and then centrifuged at 2 000 rpm./min for 20 minutes at zero temperature. The resulting supernatant is drained, and the precipitate is suspended in

PBS from the initial serum volume, the amount of protein is determined by the Lowry method, resulting in sensitin;

c) A polymer carrier is prepared using the method of anionic polymerization of a monomer — acrylic aldehyde — in an aqueous alkaline medium to obtain spherical monodisperse particles with a diameter of 1.0 ± 0.1 microns, which are stained with tianine and washed with distilled water;

g) Immobilization of sensitin on the carrier.

To immobilize sensitin, the precipitate of the polyacrolein colored carrier is suspended in borate buffer (pH 8.4), cholera immunoglobulins are added to it and left to contact with stirring for 2 hours at 20 ° C. As a result, aldehyde reaction groups on the surface of the particles specifically interact with the amino groups of protein molecules of immunoglobulins. After this, the suspension is cooled to 4-5 ° C. and incubated at this temperature for 16-18 hours.

e) Block free aldehyde groups by introducing into suspension a 0.5% gelatose solution in buffered saline (pH 7.0-7.1) and incubating for 2 hours at room temperature with constant stirring. As a result of stabilization of gelatosis, it allows blocking reaction groups on the surface of particles that are not bound in the sensitization process with immunoglobulins.

f) The diagnosticum suspension is centrifuged for 10 minutes at 6000 rpm. and washed three times with buffered saline (pH 7.0-7.1) from excess gelatose, and the final precipitate was suspended in a 0.1% gelatine solution in buffered saline (pH 7.0-7.1).

g) The sensitivity of the preparation is determined using a preparation of pure cholera toxin, for this, 50 μl of normal rabbit serum are added to the wells of the tablet, then 50 μl of a cholera toxin solution with a protein content of 100 μg / ml is added to the first well and titrated up to 12 wells twice moreover, from the last 12 wells, 50 μl of liquid, 13 and 14 wells are removed, into which only 50 μl of normal rabbit serum is added to control for spontaneous agglutination of the diagnosticum. After that, 25 μl of diagnosticum suspension is added to all wells of the rows, after 2.5 hours of incubation at room temperature, and the results of the reaction are recorded visually, while the last well, namely the 10th, giving a positive result, a blue agglomerate lining the bottom wells, the dilution of which corresponds to 100 ng protein / ml, determines the sensitivity of the diagnostic drug, i.e. the minimum concentration of cholera toxin that a diagnostic drug can detect, which allows, using a quantitative expression of sensitivity (H), to determine the concentration of cholera toxin in the studied objects after isolation of toxigenic cholera vibrio.

The specificity of the drug is determined in a similar way in the bulk agglomeration (RAO) reaction using non-toxigenic strains of cholera vibrio and heterologous strains of intestinal pathogens.

h) Lyophilization of the diagnosticum is carried out by suspending the preparation in 20 ml of a 3% gelatin-sucrose medium, then the suspension is poured into 1 ml ampoules, frozen in liquid nitrogen, followed by vacuum drying, gradually raising the temperature to 22-23 ° C during the day.

Methodology for the formulation of the reaction.

The volumetric agglomeration reaction is carried out in tablets by adding 50 μl of 1% normal rabbit serum to the wells, then 50 μl of the test substance is added to the first well of each row and titrated twice to the end of the row, 50 μl is removed from the last well. Only normal rabbit serum is added to any two wells to control spontaneous agglutination of the diagnosticum. Then, 25 μl of the diagnosticum is added to all wells and left at room temperature, the RAO results are taken into account after 2-2.5 hours, with a positive result, a uniform colored agglomerate is fixed that lines the entire bottom of the hole with clearly defined edges, and in the negative case in the negative control, a compact ring or a dot in the center of the hole is formed.

1) Determination of the specific sensitivity of the immunoglobulin antitoxic diagnosticum in the reaction of bulk agglomeration (RAO) according to the above technology.

For the study, a cholera toxin solution with a protein concentration of 100 μg / ml is prepared, and 50 μl of 1% normal rabbit serum are added to row A to wells of a 96-well immunological plate. Then, 50 μl of a cholera toxin solution is pipetted into the first well of row A with a micropipette-dispenser and titrated, i.e. make two serial dilutions of 12 holes. In the other two wells of rows 1 and 2 of row B, only 50 μl of normal rabbit serum was added. 25 μl of diagnosticum in working dilution (1 ml of diagnosticum + 6 ml of PBS) are added to all 14 wells of rows A and B. Two wells of row B contain only NKS and diagnosticum and serve as a negative control for the absence of spontaneous agglomeration. The contents of the wells are mixed by shaking and left at room temperature for 2.5 hours.

The reaction results are taken into account visually according to the formation of blue agglomerate. For a positive result, a colored blue agglomerate is taken, evenly lining the entire bottom of the hole. In the negative case and the control of the diagnosticum, a compact ring or “point” is formed in the center of the well. The last well, in which a positive result is recorded, corresponds to the level of specific sensitivity (H) of the investigated diagnosticum, the protein concentration in which is 100ng / ml and is the sensitivity of the diagnosticum, i.e. the minimum concentration of cholera toxin that a diagnostic drug can detect, which allows using a quantitative expression of sensitivity to determine the concentration of cholera toxin in the studied objects after isolation of toxigenic cholera vibrio.

2) Determine the concentration of cholera toxin in a liquid nutrient medium for the cultivation of cholera vibrio, into which the toxin is produced, to do this, put the reaction according to the RAO method, but in the wells of a tablet with a diluting liquid, the NCC titrates the liquid nutrient medium twice after culturing the cholera pathogen in it, then add 25 μl of immunoglobulin diagnostic product, while the last well with a positive reaction fixes the value of the last dilution of the culture fluid, therefore, the concentration of cholerae oh toxin is calculated by the formula:

C = P × H

where C is the desired concentration of cholera toxin;

P is the last dilution of the test fluid in which a positive result was recorded;

H is the sensitivity of the diagnosticum in ng / ml.

Example 1. Determine the amount of toxin in the test material of museum broth cultures of toxigenic strains of cholera vibrio taken from the collection of the Museum of Living Cultures with the center of human pathogenic vibrios of the Rostov-on-Don Institute of Plague.

Row C - is planted in the NCC V. cholerae 12214;

Row D - planted in NCC V. cholerae 1310;

The results of the determination of toxin production using diagnostic polymer antitoxic cholera immunoglobulin diagnosticum in RAO.

Accounting for the results of the reaction after 2.5 hours.

V.cholerae 12214 - a positive reaction is recorded at a dilution of 1: 512 and the amount of toxin is determined by the formula:

C = P × H - 512 × 100 ng = 51.2 μg / ml

V.cholerae 1310 - a positive reaction is recorded at a 1:32 dilution and the amount of toxin according to the formula

C = P × H will be 32 × 100ng = 3.2 μg / ml

Conclusion: since strain V. cholerae 12214 produces 51.2 μg / ml of toxin, and strain V. cholerae 1310 - 3.2 μg / ml of toxin, strain V. cholerae 12214 is more toxic and epidemiologically significant than strain V. cholerae 1310.

Example 2. It differs from example 1 in that the studied material is broth cultures of non-toxigenic strains of cholera vibrio from the collection of the Museum of Living Cultures with the center of human pathogenic vibrios of the Rostov-on-Don Antiplague Institute

A number of L- are parted in the NCC V. cholerae 18963;

A number of F- are rastitrazhivaetsya in the NCC V. cholerae 15031;

The results of the determination of toxin production using diagnostic polymer antitoxic cholera immunoglobulin diagnosticum in RAO.

Accounting for the results of the reaction after 2.5 hours.

The absence of reaction was recorded in these rows, i.e. A “button” was formed in the center of the hole, which indicates the absence of toxin production by these cholera strains.

Conclusion: the studied cultures of V.cholerae 18963 and V.cholerae l5031 are nontoxigenic, i.e. not producing cholera toxin and not representing an epidemic threat.

Example 3

Toxin production and the amount of toxin produced are determined by cholera vibrio cultures with unknown toxigenicity isolated from the aquatic environment.

It differs from example 2 in that the studied material is a cholera vibrio culture isolated from water sources during monitoring after bacteriological identification.

A number of G-grafted in the NQF broth culture of cholera vibrio No. 1 from a water source;

A number of Н- is broth culture of cholera vibrio No. 2 from a water source;

The results of determining the toxin production of cholera vibrios using a diagnostic polymer antitoxic cholera immunoglobulin diagnosticum in RAO.

Accounting for the results of the reaction after 2.5 hours.

A negative reaction is recorded in row G, which indicates that the selected cholera culture is not toxigenic.

. In the N series, a positive reaction at a dilution of 1: 8 is recorded, which indicates a low level of toxigenicity of this culture and the amount of toxin will be:

C = P × H 8 × 100ng = 0.800 μg / m

Conclusion: thus, the culture of cholera vibrio isolated from water source No. 1 is non-toxic, safe in the epidemic respect.

Vibrio cholerae isolated from water source No. 2 has a weak toxin production, however, this source must be continued to be monitored.

Thus, the above examples of the study of toxin production of various cultures of cholera vibrio show the effectiveness of the proposed method.

Using the proposed method allows to determine the toxin production of cholera vibrio in a simple one-stage agglomeration volumetric reaction by the polymer immunoglobulin diagnosticum developed by the authors and makes it possible to solve problems not only in scientific research, but also in epidemiological monitoring of environmental objects in order to identify epidemiologically dangerous toxigenic strains of cholera vibrio, greatly simplifying and shortening the time of bacteriological analysis.

Information sources

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

2. Patent No. 2528099, cl. GO1N 33/53, 10.27.2013, “Biological microchip for detection and multi-parameter analysis of cholera antibodies”,

// Determination of gene determinants of cholera enterotoxin by polymerase chain reaction. // SV. Balakhonov, B.C. Ganin, L.Ya. Urbanovich, Journal of Infectious Pathology. - 1998. - N 4. - S. 52-54.

4. // A practical guide to the lab. the diagnosis dangerous inf. diseases. // Ed. Acad. RAMS G.G. Onishchenko, Acad. RAMS V.V. Kutyreva. - Ed. 2nd. -M.: 30 “Shiko”, 2013. -560 p.

5. Mikheeva E. A

Construction of a diagnostic enzyme immunoassay test system for the identification of toxigenic strains of cholera vibrio. The dissertation for the degree of candidate of medical sciences, RosNIPCHI "Microbe" G. Saratov, 2017

6. Patent No. 2261444, class G01N 35/50 of March 22, 2004, “A method for detecting atoxigenic hemolytic strains of cholera vibrios”

1.11 Cholera: assessment of the epidemiological situation in the world and Russia in 2006-2015, forecast for 2016 / / CB. Titova, E.A. Moskvitina, V.D. Kruglikov A.V. Samorodova. G. Tyuleneva, E.V. Monakhova, R.V. Pisanov, A.S. Vodopyanov, new, I.V. Arkhangelsk, SM. Ivanova, T.V. Kovaleva, CO. Vodopyanov, Probl. especially dangerous. infections. - 2016. - Issue. 1. - S. 20-27.

8. // Construction of a pseudotuberculosis species-specific polymer diagnosticum / G.L. Karbyshev, D.I. Simakova, L.V. Larionova et al. // By Mater, Scientific Pract. conf. “Modern Aspects of Surveillance and Prevention of Particularly Dangerous and Natural Focal Diseases”, dedicated to the 75th anniversary of the Federal State Health Institution of Research and Development of Biology and Natural Sciences of Siberia and the Far East. - Irkutsk, 2009. - p. 122-123, as well as patent No.),

9. Larionova L.V., Chernikova A.A., Arkhangelskaya I.V., Simakova D.I., Narkevich A.N., Karbyshev G.L., Terentyev A.N. // Development of drugs for the comprehensive serological diagnosis of cholera in humans // Metabolism during damage adaptation. Materials of the X interuniversity conference with international participation. - Rostov-on-Don. - 2011 - C 101 - 103.),

10. Narkevich A.N., Karbyshev G.L., Terentyev A.N., Larionova L.V., Kochetkova A.P., Shelokhovich A.I., Natalich A.N., Simakova D.I., Sokirkina O.G. // Design of a set of polymer immunoglobulin diagnosticums for serological identification of L. pneumophila // Act. prob. diseases common to humans and animals: Mater, All-Russian. scientific-practical conf. from the international participation. (Stavropol, 2012 .-- pp. 138-139.).

Claims (15)

1. A method of obtaining a diagnosticum for determining the toxin of cholera vibrio isolated from environmental objects, comprising the following stages: a) receive experimental antitoxic immune rabbit serums by immunizing rabbits weighing 2.5-3.0 kg by administering a pure cholera toxin preparation in a dose of 100 μg of protein subcutaneously into the inguinal lymph node and intravenously into the marginal ear vein in an amount of 3 injections with an interval of 14 days, then 14 days after the last injection, rabbits are bled, and serum is prepared from the blood, followed by determination of antibody titer; b) isolate immunoglobulins from rabbit serums, for this, physiological saline, buffered with disubstituted sodium phosphate and monosubstituted potassium phosphate at pH 7.2 (PBS), is preliminarily prepared, then an aqueous methanol solution is prepared by mixing 6 ml of methanol with 8 ml of distilled water, the mixture is cooled after which the resulting serum in an amount of 4 ml is combined with 2 ml of buffered saline (PBS), mixed, placed in centrifuge glasses, put on ice, 14 ml of methanol water is added, while lowering the temperature y to -5 ° C, after which the glass with a mixture put in a refrigerator for 30-40 minutes and then centrifuged at 2000 rev / min for 20 minutes at zero temperature, the resulting supernatant was discarded and the pellet resuspended in

PBS from the initial volume of serum, the amount of protein is determined by the Lowry method, resulting in sensitin; c) a polymer carrier is prepared using the method of anionic polymerization of a monomer — acrylic aldehyde — in an aqueous alkaline medium to obtain monodisperse spherical particles with a diameter of 1.0 + 0.1 microns, which are stained with tianine and washed with distilled water; d) the polymer carrier is sensitized with cholera toxin by immunoglobulins, for this, the precipitate of the washed carrier is suspended in borate buffer (pH 8.4), immunoglobulins are added to it and left to contact with stirring for 2 hours at 20 ° С, as a result, the aldehyde reaction groups on particle surfaces interact specifically with the amino groups of protein molecules of immunoglobulins, after which the suspension is cooled to 4-5 ° C and kept at this temperature for 16-18 hours; e) block free aldehyde groups by introducing into suspension a 0.5% gelatose solution in buffered saline (pH 7.0-7.1) and incubating for 2 hours at room temperature with constant stirring on an electromagnetic stirrer; f) the diagnosticum suspension is centrifuged for 10 minutes at 6000 rpm and washed three times with buffered saline (pH 7.0-7.1) from excess gelatose, and the final precipitate is suspended in a 0.1% gelatine solution in physiologically buffered saline solution (pH 7.0-7.1); g) determine the sensitivity of the drug using a pure cholera toxin preparation, for this, 50 μl of normal rabbit serum are added to the wells of the tablet, then 50 μl of cholera toxin solution with a protein content of 100 μg / ml is added to the first well of the first row and titrated twice to 12 wells inclusive, moreover, from the last 12 wells, 50 μl of liquid are removed, 13 and 14 wells into which only 50 μl of normal rabbit serum are added to control for spontaneous agglutination of the diagnosticum, then 25 μl of the diagnosis suspension is added to all wells of the rows stickum, after 2.5 hours of incubation at room temperature, the results of the reaction are recorded visually, while the last well, namely the 10th, giving a positive result, is a blue agglomerate lining the entire bottom of the well, the dilution of which contains 100 ng protein / ml, which corresponds to the sensitivity of the diagnostic drug, and the specificity of the drug is determined in the bulk agglomeration reaction (RAW); h) lyophilization of the diagnosticum is carried out by suspending the drug in 20 ml of a 3% gelatin-sucrose medium, then the suspension is poured into 1 ml ampoules, frozen in liquid nitrogen, followed by vacuum drying, gradually raising the temperature to 22-23 ° C during the day. 2. The method according to p. 1, characterized in that the volumetric agglomeration reaction is carried out in tablets by adding 50 μl of 1% normal rabbit serum to the wells, then 50 μl of the test substance are added to the first well of each row and titrated twice to the end of the row, from the last 50 μl wells are removed, only normal rabbit serum is added to any two wells to control spontaneous agglutination of the diagnosticum, then 25 μl of the diagnosticum are added to all wells and left at room temperature, RAO results are taken into account after 2-2.5 hours, with a positive result, a uniform colored agglomerate is fixed, lining the entire bottom of the well, with clearly defined edges, and in the negative case and in the negative control, a compact ring or a dot in the center of the well is formed. 3. The method according to p. 2, characterized in that the concentration of cholera toxin in the liquid nutrient medium is determined for culturing the cholera vibrio into which the toxin is produced. For this, the reaction is performed according to the RAO method, but in the wells of the dilution liquid plate, the NKS is twice titrated with the liquid nutrient medium after culturing the cholera pathogen in it, then 25 μl of immunoglobulin diagnosticum is added, and the last well with a positive reaction fixes the value of the last dilution of the culture fluid, therefore cholera toxin concentration is calculated by the formula: C = P × H where C is the desired concentration of cholera toxin; P is the last dilution of the test fluid in which a positive result was recorded; H is the sensitivity of the diagnosticum in ng / ml.