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A competitive enzyme-linked immunosorbent assay (ELISA) was developed to quantify the amount of d-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 d-exotoxin. The d-exotoxin was coupled to several proteins via glutaraldehyde, diazotization, and periodate procedures. The antibodies used in the assay were obtained from antisera raised against d-exotoxin linked to keyhole limpet hemocyanin, and the ELISA coating antigens consisted of d-exotoxin-bovine serum albumin conjugates. Both homologous and heterologous ELISA systems were examined. The homologous systems were not useful because the free d-exotoxin did not inhibit antibody binding to the solid phase. The heterologous systems yielded the most sensitive assays, but antisera obtained from all of the immunogens were used successfully in developing ELISAs for d-exotoxin. With these sensitive ELISAs, d-exotoxin was detected in samples of commercial formulations at levels as low as 0.1 ng/mL. A good correlation was observed when culture media was fortified with d-exotoxin and analyzed in a blind fashion with both HPLC and ELISA. These data suggest that the ELISA could be a valuable tool for detecting and quantifying d-exotoxin.

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