OPTIMIZATION OF ELISA CONDITIONS FOR BLUETONGUE DIAGNOSTICS

Abstract

The article presents the results of research on optimizing the conditions for setup a domestic enzyme immunoassay kit (ELISA) designed to detect specific antibodies to the bluetongue virus in cattle and small cattle. The paper describes in detail the key stages of improving the technique, including the selection of antigen sorption conditions, optimization of reagent concentrations, temperature-time incubation modes and analysis parameters. It was found that the optimal concentration of recombinant VP7 antigen for sorption is 5 micrograms per well using 0.05 M carbonate-bicarbonate buffer (BB) and incubation for 20 ±2 hours at a temperature of 5±3 °C. These conditions ensure stable binding of the antigen to the antibodies and high sensitivity of the analysis.

Optimization of incubation temperature regimes has shown that the most effective analysis is at 37 ± 1 ° C, which helps to increase the rate of formation of immune complexes and enzyme activity. The use of biotinylated monoclonal antibodies and streptavidin-HRP conjugate in combination with a TMB substrate made it possible to achieve high specificity and sensitivity of the method. Stopping the enzymatic reaction using 0.5N hydrochloric acid solution proved its effectiveness, providing a clear visualization of the results.

The developed test system demonstrates a high potential for use in the diagnosis of bluetongue, which is especially important for the control and prevention of this disease in Kazakhstan. The introduction of this kit into veterinary practice not only reduces dependence on imported analogues, but also strengthens the country's diagnostic capabilities in the fight against particularly dangerous infectious diseases of animals. The results obtained emphasize the importance of further research in the field of improving diagnostic methods for infectious animal diseases, which corresponds to the strategic objectives of agricultural development and food security.