A SANDWICH ENZYME-LINKED-IMMUNOSORBENT-ASSAY FOR THE BACILLUS-SPHAERICUS BINARY TOXIN

Bacillus sphaericus (Bs) binary toxin was purified from recombinant E. coli DH5 alpha harboring the recombinant plasmid pAR5, which carries a 3.6-kb DNA fragment of Bs 1593M encoding mosquito larvicidal activit y. The binary toxin preparation, designated BsEcAg, contained mainly 5 1- and 42-kDa toxin proteins and was toxic to 50% of Culex quinquefasc iatus larvae at a concentration of 9.22 ng toxin protein/ml. This prep aration was used to raise antibodies in sheep and mice. The sandwich E LISA used sheep antitoxin antibody as primary antibody (coating antibo dy), mouse antitoxin antibody as second antibody, and goat antimouse a ntibody as an alkaline phosphatase-conjugated detecting antibody. The assay sensitivity was 200 ng/ml for both BsEcAg and binary toxin antig en (BsAg) from Bs 2362 cells. There is a significant correlation betwe en toxin level determined by ELISA and bioassay. This procedure has al so been used to monitor toxin levels in batch fermentations of Bs 2362 .