Production of specific components of bacteria Clostridium perfringens for ELISA

The article describes a new method to produce components specific to Clostridium perfringens and hyperimmune serum for ELISA test. The antigen was produced by ultrasonification of CL.perfringens bacterial suspension containing 5 billion of each A, B, C, D serotypes per 1 cm3. The ul- trasonication was performed using ultrasonic disintegrator at 20 mHz frequency during 10-15 min at 4 C. It was followed by endotoxin sedimentation with ammonium sulfate and dialysis against tap water. Positive serum was produced by young calves hyperimmunization with corpuscular antigen and anatoxin from Cl. perfringens A, B, C, D serotypes. The 4-fold hyperimmunization was performed with 14-days intervals. Simultaneously the animals were introduced corpuscular antigen subcutaneously and anatoxin intravenously. The basic conditions for ELISA test (determining optimal and antigen absorption time in polysterol plates, appropriate exposure time of tested sera) were standardized. The most appropriate titration in animal blood sera was determined at 1:2500 conjugate solution and 5-8 mkg/cm3 absorbed antigen concentration. The most efficient mode of antigen fixation was was 18hours at 4C. Interlaboratory commission trial showed that ll the components of the test-system were active and specific. Specific antigen does not respond to heterogenic hyperimmune sera (salmonella, escherichia) while with homogenic sera (hyperimmune and blood sera sampled from vaccinated and infected with anaerobic enterotoxemy) it responses at high titers - 1:3200-1:12800.