Results of studies on the development of an immuno-enzymed test system for diagnosing infectious enterotoxemia in animals

¹ Federal Center for Toxicological, Radiation and Biological Safety, Nauchnyi Gorodok-2, Kazan, 420075, Russia
² Kazan State Academy of Veterinary Medicine by N.E. Bauman, Sibirsky tract 35, Kazan city, 420029, Russia
³ Mari State University, Lenin Square 1, Yoshkar-Ola city, 424000, Russia

^* Corresponding author: gennady.spiridonov@bk.ru

Abstract

Research materials on the development of an enzyme-linked immunosorbent assay for the determination of specific antibodies to Cl. perfringens bacteria were presented. Strains of Cl. perfringens were used as production strains: No. 28 (type A), LD-1 (type B), No. 392 (type C), No. 213 (type D). To obtain antigen, a daily suspension of Cl perfringens bacteria, containing 20 billion microbial cells in 1 cm3 (5 billion microbial cells of each serotype), was sounded on an ultrasonic disintegrator at a frequency of 20 MHz for 15 minutes at 4 °C, then the endotoxin was precipitated with ammonium sulfate and purified by differential dissolution of the precipitate in 0.02 M phosphate buffer, followed by dialysis against tap water. Control positive serum with specific antibodies of at least 1:12800 to antigens of Cl. perfringens bacteria was obtained by hyperimmunization of cattle with corpuscular antigens and toxoids. The basic conditions of the ELISA reaction were standardized. Determination of the concentration of sorption of the antigen was carried out by parallel testing of various dilutions of the conjugate. The most acceptable titer of the blood serum of animals was found at a dilution of the conjugate of 1:2500 and a concentration of the sorbed antigen of 5-8 μg/cm3. The production test of the enzyme immunoassay test system was carried out in cattle farms in three regions of the Russian Federation, unfavorable for anaerobic enterotoxemia in calves.