Method of serologic diagnosis of viral gastrointestinal infections in cattle by method of enzyme-immunoassay

FIELD: veterinary medicine.

SUBSTANCE: method includes the interaction of antigens with antibodies, with anti-species antibodies labeled with horseradish peroxidase, adding the substrate mixture and recording the results of the reaction on the colour intensity of the complex formed. In the reaction the plates are used with antigens of rotavirus, coronavirus, the virus of diarrhea preliminary sorbed on them, and after applying the test samples of serum 0.10-0.12 ml each. The plates were incubated for 1.5-2 h at 36-37C and washed. Then total anti-species immunosorbent conjugate is applied of 0.10-0.12 ml consisting of enzyme-labeled antibodies against globulins of blood serum of cattle and the results of the reaction are recorded.

EFFECT: invention provides simultaneous diagnosis of rotavirus, coronavirus enteritis, viral diarrhea in cattle, sensitivity, specificity, and ease of the assay, and the ability to automate the process of research.

2 ex

 

The present invention relates to the field of veterinary Virology, in particular to the development of a method for detection of antibodies against the viruses that cause gastrointestinal infections in cattle enzyme-linked immunosorbent assay.

A leading role in the etiology of diarrhea in young farm animals play Rota-coronavirus, virus diarrhea-diseases of the mucous membranes, to a lesser extent other infectious agents. These diseases are widely spread in the Russian Federation and abroad and put livestock farms tangible economic damage resulting from the loss from death, especially at an early age, funds spent on treatment of patients and loss of production from the animals recover. Every year half of the losses of juveniles accounted for gastrointestinal disease at an early age.

In view of the above problem diagnosis and control of these infections is important. With great efficiency to solve the problem of diagnostics allows a highly sensitive enzyme immunoassay.

There is a method of study of blood sera for antibodies to the causative agent of viral diarrhea by ELISA in epidemiological survey of livestock farms [1].

There is also known a method for serodiagnosis of transmissible gastroenteritis with the frost by ELISA, why in the wells of polystyrene tablet make specific antigen in 0.1 ml and adsorb on the surface of the hole. The next step in the wells microarrays make the control subjects and serum 0.1 ml and incubated for 1.5-2 h at 37C. Unbound antibodies are removed by repeated washing, and then added to each well individuai enzyme conjugate in a volume of 0.1 ml working dilution. In positive cases, the conjugate binds fixed on the sensitized surface antibodies, forming a complex of the antigen-antibody-conjugate. The excess conjugate is removed by washing and contribute to all wells of the substrate indicator solution, which changes color according to the principle of redox reaction, is proportional to the number of conjugated with antibodies to the enzyme. The amount of enzyme is proportional to the amount of antibody bound peroxidase fixed on the surface of the wells with antigen [2]. (Prototype).

The objective of our research was to develop a sensitive, effective way serodiagnosis of Rota-and coronavirus enteritis, viral diarrhea, bovine enzyme-linked immunosorbent assay and significantly reduce the time to diagnosis due to the simultaneous study of serum antibodies to these pathogens. Literature the data on the use of ELISA test for the study of blood sera for antibodies to Rota-, coronavirus, bovine missing.

The essence of the proposed method consists in the following. To the wells of one plate with pre-adsorbed rotavirus, coronavirus, diarrhoeal antigens automatic pipette make a test sample of blood plasma 0,10-0,12 ml in the wells with different antigens is incubated for 1.5-2 hours at a temperature of 37-38C, washed tablet, contribute 0,10-0,12 ml individuai enzyme conjugate, consisting of enzyme labeled antibodies to globulin in the blood serum of cattle, incubated under the same conditions, the tablet is washed and after adding substrate mixture undergo simultaneous analysis of responses to the company, coronavirus enteritis, viral diarrhea in cattle.

Example 1

In the first stage reaction in all wells automatic pipette contribute to 0.1 ml of 0,01M phosphate buffer solution. In the first wells of rows coated with antigens of rotavirus, coronavirus, virus diarrhoea contribute to 0.1 ml of test serum, pipeinput, and 0.1 ml is transferred into the next pit, etc. To control reactions in parallel contribute to 0.1 ml of specific homologous sera and also titrated them as a sample. Likewise contribute and titrate with negative serum. The tablet is incubated in the incubator for 1.5 hours at 36C, then washed it is from unbound antibodies. In the washed wells contribute to 0.1 ml individuai enzyme conjugate, consisting of globulin in the blood serum of cattle, labeled with horseradish peroxidase. Tablet incubated in an incubator at 36C for 1.5 h and again washed three times with phosphate-buffer solution with tween-20. Then in the wells contribute to 0.1 ml of substrate mixture consisting of a solution of 5-aminosalicylic acid and hydrogen peroxide. The result of the reaction into account in 30 minutes by a colour change of the formed reaction product from brown to light brown:

- intense brown staining, brown staining - positive result;

- light brown staining - equivocal result;

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

In the study of 97 blood serum samples of cattle from disadvantaged by gastrointestinal diseases farms antibodies to rotavirus was detected in 54 of animals, for coronavirus antibodies were detected in 47, and to the virus diarrhoea - 21 animal, which accounted for 55.6 per cent to 48.4 per cent, 21.6 per cent, respectively.

Example 2.

The reaction is carried out analogously to example 1, but the serum, conjugate and substrate mixture contribute to the volume of 0.12 ml and tablets incubated at 37C for 2 hours. Obtained similar results as in example 1.

The proposed method has no domestic analogues. Allows for diagnostics company, coronavirus enteritis, viral diarrhea in cattle by ELISA significantly reduce the time for setting-up the reaction and to obtain more reliable results when taking into account the reaction, as the research on these infections are carried out simultaneously on the same tablet with pre-adsorbed antigens Rota-and coronaviruses, virus diarrhoea in cattle using a General-enzyme conjugate under the same conditions. In addition, there are no published data about the use of enzyme or antibody-based test diagnostics when the Rota-, coronavirus enteritis of cattle.

The proposed method for serological diagnosis of Rota-and coronavirus enteritis, viral diarrhea cattle enzyme-linked immunosorbent assay tested by a Commission in September 2009 and has been tested in several veterinary laboratories with a positive result (FGI Central scientific-methodical vetebrate 2010, GU Kursk regional vetebrate 2010, Tverskaya interregional vetebrate 2011)

On the basis of the obtained test results, the Commission concluded that the method is sensitive and can detect these specific antic the La in the tested sera in much shorter time.

The proposed method will be applied for the inspection of cattle affected with gastrointestinal infections farms for the purpose of diagnosis Rota-, coronavirus enteritis, viral diarrhea cattle. High sensitivity, specificity, ease of setting reaction and the possibility of automation of the processes of research allows us to quickly carry out mass screening animals to determine the spread of these infections. Reducing the time to diagnosis is very important for disadvantaged by these infections farms, because Express diagnosis makes it possible to begin timely treatment and preventive measures and, thereby, improve and maintain livestock.

This test system can be used in the evaluation of the immune status of the population, to control the antigenic activity of the respective components of mono - and polyvalent vaccines.

Sources of information

1. Veterinary medicine, 4, 2004, p.20

2. Veterinary medicine, 5, 2005, p.17-20.

Method for the serological diagnosis of viral gastrointestinal infections bovine enzyme-linked immunosorbent assay, mainly Rota-, coronavirus enteritis, viral diarrhea in cattle, involving the interaction of antigens with antibodies, with antiv the annual antibodies, labeled with horseradish peroxidase, the addition of the substrate mixture and records the results on the intensity of the color formed complex, characterized in that the reaction using tablets with pre-adsorbed on them antigens Rota-coronavirus, virus diarrhea, and after application of the tested serum samples for 0,10-0,12 ml tablets incubated for 1.5-2 h at 36-37C, washed, then contribute 0,10-0,12 ml total individualo enzyme conjugate, consisting of enzyme labeled antibodies against globulin in the blood serum of cattle and conduct analysis of reaction.



 

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