Alpha Toxin Purification and Antibody Production Against Local Strain of Clostridium septicum NH2

Clostridium septicum has played a significant role as a causative agent of many acute fetal diseases in man and animals. Alpha- toxin is the main factor in the pathogenesis of C. septicum with hemolytic, necrotic and lethal activities.

The study was designed to evaluate alpha-toxin purification and antibody production rate against a local strain of C. septicum NH2 which could be applied in diagnosing kits, potency test of the vaccines, and other related applications.

Local strain of C. septicum NH2 was cultured in liver broth. Alpha-toxin in supernatant purified by three steps: the first step was done by 25% and 60% of ammoniums sulfate precipitation and continued by DEAE-Sephadex ion exchange chromatography, and finally finished in gel filtration on Sephadex G-50. Alpha-toxin was assayed in all steps and purification procedures were analyzed by SDS-PAGE. After immunization of rabbits with alpha- toxin and serum collection, immunoglobulin was separated by three purifying steps: ammoniums sulfate, ion exchange chromatography, and gel filtration. Serum purification process was evaluated by electrophoresis, double immunodiffusion (DID), single radial immunodiffusion (SRID), western blot, and SDS-PAGE. RESUTLS: SDS-PAGE

showed the alpha-toxin and anti-alpha-toxin were purified partially. Double immunodiffusion and single radial immunodiffusion methods detected the specific antibody. Heavy and light chains of anti-alpha-toxin separated by 2ME in electrophoresis reacted with 48 kDa alpha-toxin during the western blot without any reaction to other proteins in nitrocellulose paper.

The present study showed a modified protocol for C. septicum alpha-toxin and anti-alpha-toxin production. The purification method is more economical and faster than previously reported procedures, and anti-alpha-toxin production is  an advantage in detection of C. septicum infection