DEVELOPMENT OF A DOUBLE-SANDWICH ELISA SPECIFIC FOR THE DETECTION OF CLOSTRIDIUM PERFRINGENS BETA-TOXIN.

Abstract

Clostridium perfringens, one of the most ubiquitous microbial pathogens, is responsible for a variety of disease in both man and animals. The great diversity of clinical manifestations of C. perfringens-mediated afflictions is attributable to the fact that the organism produces a number of different potent protein toxins, the major ones being alpha(α)-toxin, beta(β)-toxin, epsilon(ε)-toxin, iota(ι)-toxin, as well as an enterotoxin. Because C. perfringens is a commensal organism in the gastrointestinal tract of both man and animals, assays which detect a specific virulence factor (toxin) are necessary for accurate clinical characterization of C. perfringens-caused illnesses. This thesis describes the development of a specific assay, an ELISA, for the detection of beta-toxin produced by C. perfringens serotypes B and C. The development of this assay involved: a) procurement of a polyspecific antiserum against C. perfringens type C culture supernate for use as a "capture" antibody; b) production of a beta-toxin-specific antiserum by immunization of rabbits using inactivated, purified beta-toxin; and 3) use of a peroxidase conjugated anti-rabbit-IgG antibody as a signal reagent. The ELISA had a linear dose-response range of 8 - 80 ng/mL, and a sensitivity limit of 5 ng/ml. The specificity of the ELISA for beta-toxin is contingent upon the specificity of the rabbit antiserum for this antigen. This specificity was demonstrated with western blots of the rabbit serum versus crude preparations of bacterial culture growth. Once optimized, this specific ELISA was employed to monitor the presence of beta-toxin in experiments designed to: a) optimize the culture conditions for beta-toxin production by C. perfringens type C; and b) purify this toxin from crude preparations of C. perfringens type C cultures using techniques such as ion-exchange, metal-affinity, and immunoaffinity chromatography. Once a purified beta-toxin preparation is obtained and its concentration determined, it can be used both as a calibration standard for the ELISA and as an immunogen for generation of greater quantities of specific antiserum. One of the purification techniques tested, immunoaffinity chromatography with a low-affinity monoclonal antibody obtained from a recently developed hybridoma cell line, produced promising results in terms of beta-toxin yield.