RU2796940C1 - Method for diagnosing coronavirus enteritis in horses by enzyme immunoassay (options) - Google Patents

Abstract

FIELD: medicine; veterinary virology.

SUBSTANCE: group of inventions can be used to diagnose equine coronavirus enteritis by enzyme immunoassay. Coronavirus immunoglobulin 0.1 cm³ is introduced to the wells of the plate. It is incubated in a thermostat for 1.5 hours at 36–37°C followed by 18 h at 4°C. The test samples of horse feces are introduced into the washed wells of the plate, 0.1 cm³ each, incubated for 1.5 hours at a temperature of 36–37°C. Next, the enzyme-linked immunosorbent conjugate is introduced in the amount of 0.1 cm³, it is incubated for 1.5 hours at a temperature of 36–37°C, adding a substrate mixture consisting of a solution of 5-aminosalicylic acid and hydrogen peroxide with a volume of 0.1 cm³. When registering a brown coloration of the product, coronavirus enteritis is diagnosed in horses. In the analysis of blood serum samples of horses, the enzyme-linked immunosorbent conjugate is isolated by affinity chromatography on a column with immunosorbent cyanogen bromide sepharose.

EFFECT: method provides the possibility of diagnosing equine coronavirus enteritis by developing reliable methods for diagnosing equine coronavirus enteritis by enzyme immunoassay.

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Description

The group of the invention relates to the field of veterinary virology and can be used by veterinarians to diagnose equine coronavirus enteritis.

The leading role in the etiology of diarrhea in young farm animals is played by rota-coronaviruses, diarrhea virus - diseases of the mucous membranes, and to a lesser extent other infectious agents. These diseases are widespread both in the Russian Federation and abroad and cause tangible economic damage to livestock farms, consisting of losses from death, especially at an early age, funds spent on the treatment of sick animals and a shortage of products from recovered animals. Every year, half of the losses of young animals are accounted for by gastrointestinal diseases at an early age. In connection with the foregoing, the problem of diagnosing and combating these infections is relevant, [Veterinary Journal 2005, No. 5, C21]

State of the art

A known method for the diagnosis of viral gastrointestinal infections by enzyme immunoassay using a diagnostic kit. The globulin fraction was isolated from hyperimmune sera by salting out with a half-saturated solution of ammonium sulphate, followed by ion-exchange chromatography on DEAE-cellulose. The conjugates were prepared using horseradish peroxidase with RZ-3 purity.

Conjugation of antibodies with the enzyme was carried out according to the method of Wilson et al. by covalent binding of sodium periodate-modified peroxidase with immunoglobulins isolated from hyperimmune anti-rotavirus serum (conjugate for the detection of rotavirus antigen) or with immunoglobulins of bovine rabbit serum (conjugate for the detection of antibodies in blood sera and colostrum cattle). [Proceedings of VIEV, 1984, No. 60. pp. 63-66]

Also known is a method for detecting the diarrhea virus antigen, for which specific immunoglobulins included in the diagnostic kit are added to the wells of the tablet by 0.1 cm ³ and adsorbed on their surface. The solution containing the test antigen is added to the wells by 0.1 cm and incubated with antibodies sensitized on the surface of the carrier for 1.5 hours at 37°C. Unbound antigen is removed by washing. Then add ELISA conjugate in a volume of 0.1 cm ³ . In positive cases, the conjugate binds to the antigen fixed on the sensitized surface, forming an antibody-antigen-conjugate complex. The excess conjugate is removed by washing and a substrate indicator solution is added to the wells, which changes its color according to the principle of a redox reaction, in proportion to the amount of enzyme conjugated with antibodies. The amount of enzyme is proportional to the amount of antigen fixed on the sensitized surface of the plate wells. [Reports of the Russian Agricultural Academy of Agricultural Sciences. 1998, no. No. 3. S. 25]. When studying the state of the art, we did not reveal any literature data on the diagnosis of coronavirus enteritis in horses.

The technical problem is the need to expand the arsenal of methods for diagnosing equine coronovirus enteritis

Disclosure of the essence of the invention

The technical result is to expand the arsenal of methods for diagnosing equine coronovirus enteritis by developing reliable methods for diagnosing equine coronavirus enteritis by enzyme immunoassay.

The proposed method for diagnosing coronavirus enteritis in horses by enzyme immunoassay has two options:

Option number 1. A method for diagnosing coronavirus enteritis in horses by enzyme immunoassay by detecting coronavirus antigen in fecal samples from sick horses by enzyme immunoassay. The method includes adding coronovirus immunoglobulin 0.1 cm ³ into the washed wells of the tablet, incubating them in a thermostat for 1.5 hours at 36-37°C, introducing test samples from horses into the wells of the tablet 0.1 cm, their incubation in for 1.5 hours at a temperature of 36-37°C, then the introduction of an enzyme-linked immunosorbent conjugate in the amount of OD cm ³ , its incubation for 1.5 hours at a temperature of 36-37°C, the introduction of a substrate mixture consisting of a solution of 5-aminosalicylic acid and hydrogen peroxide with a volume of 0.1 cm ³ and taking into account the reaction by the changed color of the reaction product.

The result of the reaction is taken into account after 30-40 minutes by changing the color of the resulting reaction product from brown to light brown.

- intense brown staining, brown staining - a positive result;

- light brown staining - doubtful result;

- staining is absent or is at the level of a negative control - a negative result.

Option number 2. Method for diagnosing equine coronavirus enteritis by enzyme immunoassay by serological diagnosis of equine coronavirus enteritis by enzyme immunoassay. The method includes introducing coronovirus antigen in 0.1 cm ³ into the washed wells of the tablet, incubating them in a thermostat for 1.5 hours at 36-37°C, introducing the test samples of horse blood sera into the wells of the tablet in 0.1 cm ³ , their incubation for 1.5 hours at a temperature of 36-37 ° C, then adding to the washed wells of the tablet in a volume of 0.1 cm ³ of an anti-species immunoenzyme conjugate isolated by affinity chromatography on a column with immunosorbent cyanogen bromide sepharose, its incubation for 1.5 hours at a temperature of 36-37°C, introducing a substrate mixture consisting of a solution of 5-aminosalicylic acid and hydrogen peroxide with a volume of 0.1 cm ³ and taking into account the reaction by changing the color of the reaction product.

The result of the reaction is taken into account after 30 minutes by changing the color of the resulting reaction product from brown to light brown.

- intense brown staining, brown staining - a positive result;

- light brown staining - doubtful result;

- staining is absent or is at the level of a negative control - a negative result.

Both methods of diagnosis can be carried out both separately and together to increase the reliability of the results obtained detection of coronavirus antigen in samples from horses

The implementation of the invention is illustrated by the following examples:

Example No. 1 Detection of coronavirus antigen in samples from sick animals.

To make a diagnosis, the laboratory received 5 samples of feces from foals.

The reaction is carried out on a tablet sensitized with coronavirus immunoglobulins. To do this, immunoglobulins of 0.1 cm ³ are added to all wells of the tablet. The tablet is covered with a lid and incubated in a thermostat for 1.5 hours at a temperature of 36-37°C, and then 18 hours at 4°C. The immunoglobulin solution is removed and the wells of the SPBR plate are washed three times.

At the next stage of the reaction, 0.1 cm 3 of phosphate buffer solution is added to all wells of the tablet with an automatic pipette. In the first wells of rows with coronavirus immunoglobulins, 0.1 cm3 of the test sample is added, pipetted, and 0.1 ^cm3 is transferred to the next well, etc. To control the reaction, 0.1 cm ³ of the coronavirus antigen is added in parallel and it is also titrated as a sample. In the same way make a negative antigen. The tablet is incubated in a thermostat for 1.5 hours at 36-37°C, then it is washed from unbound antigens. In the washed wells of the tablet, 0.1 cm 3, an enzyme immunoassay conjugate, consisting of anticoronavirus immunoglobulins labeled with horseradish peroxidase, is added. The tablet is incubated in a thermostat at 36-37°C for 1.5 hours and again washed three times with a phosphate buffer solution. Then, 0.1 cm of the substrate mixture consisting of a solution of 5-aminosalicylic acid (1.0 cm ³ ) and hydrogen peroxide (5 mg) is added to the wells. The result of the reaction is taken into account after 30-40 minutes by changing the color of the resulting reaction product from brown to light brown.

- intense brown staining, brown staining - a positive result;

- light brown staining - doubtful result;

- staining is absent or is at the level of a negative control - a negative result.

In the study of 5 samples of feces from foals for coronavirus enteritis by enzyme immunoassay, the following was established. The coronavirus antigen was found in 4 samples in titers from 1:16 to 1:256.

The results of the study of 5 fecal samples of foals for the presence of a specific coronavirus antigen by ELISA are presented in the table.

The proposed method will find application in the examination of horses in farms that are unfavorable for gastrointestinal infections in order to diagnose coronavirus enteritis.

Example No. 2 Serological diagnosis of equine coronavirus enteritis by enzyme immunoassay

To make a diagnosis, the laboratory received 5 blood serum samples from mares and 5 blood serum samples from foals.

When examining horse blood sera for the presence of antibodies to coronavirus by ELISA, a tablet sensitized with coronavirus antigen is used. In each well contribute antigen 0.1 cm ³ . The tablet is covered with a lid and incubated in a thermostat for 1.5 hours at a temperature of 36-37°C, and then for 18 hours at 4°C, and then the wells of the plate are washed from unbound antigens as follows. The tablet is freed from the contents by sharp shaking and washed with SPBS three times, each time completely filling the wells with buffer and shaking out the contents.

At the second stage of the reaction, 0.1 cm ³ of 0.01M phosphate buffer solution is added to all wells of the tablet with an automatic pipette. 0.1 cm ³ of the test serum is added to the first wells of the rows with applied coronavirus antigens, pipetted and 0.1 cm ³ are transferred to the next well, etc. To control the reaction, 0.1 cm ³ of specific homologous serum is added in parallel and it is also titrated as a sample. Negative serum is added and titrated in the same way. The tablet is incubated in a thermostat for 1.5 hours at 36°C, then it is washed from unbound antibodies. In the washed wells of the tablet contribute 0.1 cm anti-species enzyme-linked immunosorbent conjugate, consisting of horse serum globulins labeled with horseradish peroxidase. Specific antibodies against horse IgG are isolated by affinity chromatography on an immunosorbent column (cyanogen bromide sepharose). The column is filled with immunosorbent, washed with 100 cm ³ of 0.1 M phosphate buffer, pH 7.4 with 0.1 M NaCl (PBS) and monospecific rabbit serum containing antibodies to horse IgG is applied. The immunosorbent is washed with PBS until the protein disappears at the outlet of the column. Specific antibodies to horse IgG adsorbed on the immunosorbent column are eluted with 0.1 M glycine buffer, pH 2.5, with 0.1 M NaCl fractions of 3.0 cm ³ . The eluted solution is rapidly alkalized to neutral pH.

The amount of protein in the fractions is determined spectrophotometrically at λ=280 nm. Fractions containing the maximum amount of protein are combined, dialyzed against PBS for 14-18 hours at 4°C.

The specificity and titer of the isolated specific antibodies are checked by the immunoprecipitation reaction. The isolated specific antibodies are used to obtain an enzyme-linked immunosorbent conjugate.

The method allows to increase the specificity; specific antibodies against horse IgG bind only to horse IgG and do not bind to IgG of other animal species. To stabilize the conjugate, add BSA (1%), glycerol (50%) and store in small portions at 4°C. Chemical stabilization is included in the list of requirements for ELISA test systems reagents for their implementation in the countries of the Customs Union. The tablet is incubated in a thermostat at 36°C for 1.5 hours and again washed three times with a phosphate buffer solution with tween-20. Then, 0.1 ml of a substrate mixture consisting of a solution of 5-aminosalicylic acid (1.0 cm ³ ) and hydrogen peroxide (5 mg) is added to the wells. The result of the reaction is taken into account after 30 minutes by changing the color of the resulting reaction product from brown to light brown.

- intense brown staining, brown staining - a positive result;

- light brown staining - doubtful result;

- staining is absent or is at the level of a negative control - a negative result.

The results of the study of 5 blood serum samples of mares and 5 blood serum samples of foals for the presence of specific antibodies to coronavirus by ELISA are presented in the table.

It follows from the data in the table that antibodies to coronavirus in the blood sera of foals were detected in 2 samples in titers from 1:100 to 1:200, in the sera of mares also in 2 samples in titers from 1:200 to 1:400. The proposed method for the serological diagnosis of coronavirus enteritis in horses has no domestic analogues.

This test system can be used to assess the immune status of livestock, to control the antigenic activity of the corresponding components of mono- and polyvalent vaccines.

High sensitivity, specificity, ease of setting and the possibility of automating the research processes by enzyme immunoassay allow conducting mass examinations of animals in a short time to determine the spread of these infections. Accelerated diagnosis allows you to start timely treatment and preventive measures and, thereby, improve and preserve the livestock.

The developed method can solve the problem of diagnosing equine coronavirus enteritis with great efficiency.

Claims (2)

1. A method for diagnosing coronavirus enteritis in horses by enzyme immunoassay, including the introduction of coronavirus immunoglobulin 0.1 cm ³ into the wells of the tablet, their incubation in a thermostat for 1.5 hours at 36-37°C, and then 18 hours at 4°C , introducing the test samples of horse feces into the washed wells of the tablet by 0.1 cm ³ , their incubation for 1.5 hours at a temperature of 36-37 ° C, then introducing the enzyme immunoassay conjugate in the amount of 0.1 cm ³ , its incubation for 1 ,5 h at a temperature of 36-37°C, the introduction of a substrate mixture consisting of a solution of 5-aminosalicylic acid and hydrogen peroxide with a volume of 0.1 cm ³ , and when registering a brown color of the product, coronavirus enteritis is diagnosed in horses. 2. A method for diagnosing coronavirus enteritis in horses by enzyme-linked immunosorbent assay, including introducing coronavirus antigen 0.1 cm ³ into the wells of the tablet, incubating them in a thermostat for 1.5 hours at 36-37°C, and then 18 hours at 4°C , the introduction of the tested samples of blood serum of horses into the washed wells of the tablet at 0.1 cm ³ , their incubation for 1.5 hours at a temperature of 36-37 ° C, then the introduction into the washed wells of the tablet in a volume of 0.1 cm ³ of the anti-species enzyme-linked immunosorbent conjugate isolated by affinity chromatography on a column with Sepharose bromide immunosorbent, its incubation for 1.5 hours at a temperature of 36-37°C, the introduction of a substrate mixture consisting of a solution of 5-aminosalicylic acid and hydrogen peroxide with a volume of 0.1 cm ³ , and when registering a brown coloration of the product, coronavirus enteritis is diagnosed in horses.