Improvement of conditions for setting up the indirect enzyme-related immunoassay method for diagnostics of bovine enzootic leukemia

Enzootic leukemia is the most common infectious disease of cattle, which causes significant economic damage, posing a threat to the extinction of the gene pool of the main valuable highly productive breeds. One of the leading factors reducing the effectiveness of health measures against this infection is insufficient diagnostics. Enzyme-linked immunosorbent assay (ELISA) is a highly sensitive serological test. The International Epizootic Bureau recommends using ELISA as one of the main methods for diagnosing enzootic leukemia in cattle. Due to its high sensitivity, specificity, and the possibility of automating the reaction, ELISA has shown high efficiency in eliminating leukemia in disadvantaged farms. The ELISA mechanism involves the interaction of an antigen with antibodies specific to it. The “antigen-antibody” complex is detected using auxiliary reaction components, such as a conjugate of anti-species antibodies labeled with an enzyme and a substrate with a chromogen. However, when setting up ELISA, false-positive and false-negative results may occur, the reduction of which with a simultaneous improvement in the diagnostic characteristics of the analysis are relevant. The purpose of these studies was to improve the conditions for setting up ELISA for the detection of specific antibodies against the causative agent of bovine leukemia. It was found that an aqueous solution of 2% glutaraldehyde increases the sorption activity of the tablet, the optimal concentration of the viral antigen obtained from a chronically infected culture of sheep embryo kidney cells for sensitization of the tablet is 5 micrograms/ml, the blocking solution is 0.5% intact sheep serum. This serum also has a stabilizing effect on the conjugate and eliminates non-specific interaction of antispecies antibodies with antibodies of the analyzed samples. The use of the shaking parameter of the tablet in a thermoshaker increases the positivity coefficient of the positive serum and, therefore, the sensitivity of the analysis. The optimized version of the ELISA showed high specificity and sensitivity.