ENZYME LINKED IMMUNOASSAY (ELISA) METHOD FOR DIAGNOSTICS OF ANIMALS ANAEROBIC ENTEROTOXEMIA
The research data on developing ELISA test-system to detect specific antibodies against bacteria Cl.Perfringens are presented. The strains were used as producton: № 28 (type A), LD-1 (type B), № 392 (type C), № 213 (type D). To obtain antigen the daily suspension of Cl.Perfringens containing 20 milliards of microbial cells in 1 cm3 (5 milliards of microbial cells in each serotype), was sounded on a ultrasound disintegrator of 20 mHertz for 15 minutes at 40°C, then Endotoxin was precipitated by ammonium sulfate and purified by differential solving of precipitation in 0,02 M phosphate buffer followed by dialysis against tap water. The positive test serum with antibody titers not below than 1:12800 against Cl. Perfringens antigens was obtained by hyperimmunization of cattle using corpuscular antigens and anatoxins. The standardization of the basic conditions for conducting ELISA reaction was carried out. The antigen sorbtion concentration was detected while simultaneous testing of different conjugant dilutions. The most acceptable animals blood serum titer index was detected at conjugate dilution 1:2500 and in sorbet antigen concentration of 5-8 mkg/cm3. The production testing of ELISA test-system was carried out in unwell for calves anaerobic enterotoxemia cattle breeding farms of the 3 regions of the Russian Federation. Altogether 677 blood serum samples were examined, including 240 - from anaerobic enterotoxemia animals, 389 samples - from vaccinated against anaerobic enterotoxemia and 48 - from clinically health animals in stationary well-being farm. The ELISA method was found to make it possible to detect specific antibodies against Cl.Perfringens bacteria in 97.7% vaccinated and 94,6 % anaerobic enterotoxemia animals. The positive results of laboratory and production test of the test-system enable to recommend it to be implemented it into veterinary practice of the Russian Federation.