DEVELOPMENT OF AN ENZYME-LINKED-IMMUNOSORBENT-ASSAY FOR THE BETA-EXOTOXIN OF BACILLUS-THURINGIENSIS

A competitive enzyme-linked immunosorbent assay (ELISA) was developed to quantify the amount of beta-exotoxin from Bacillus thuringiensis in solution and to evaluate the ability of the antibodies to distinguish among various natural and synthetic compounds related to the beta-exo toxin. The beta-exotoxin was coupled to several proteins via glutarald ehyde, diazotization, and periodate procedures. The antibodies used in the assay were obtained from antisera raised against beta-exotoxin li nked to keyhole limpet hemocyanin, and the ELISA coating antigens cons isted of beta-exotoxin-bovine serum albumin conjugates. Both homologou s and heterologous ELISA systems were examined. The homologous systems were not useful because the free beta-exotoxin did not inhibit antibo dy binding to the solid phase. The heterologous systems yielded the mo st sensitive assays, but antisera obtained from all of the immunogens were used successfully in developing ELISAs for beta-exotoxin. With th ese sensitive ELISAs, beta-exotoxin was detected in samples of commerc ial formulations at levels as low as 0.1 ng/mL. A good correlation was observed when culture media was fortified with beta-exotoxin and anal yzed in a blind fashion with both HPLC and ELISA. These data suggest t hat the ELISA could be a valuable tool for detecting and quantifying b eta-exotoxin.