title: A sandwich enzyme-linked immunosorbent assay for the Bacillus sphaericus binary toxin.
abstract: Bacillus sphaericus (Bs) binary toxin was purified from recombinant E. coli DH5alpha harboring the recombinant plasmid pAR5, which carries a 3.6-kb DNA fragment of Bs 1593M encoding mosquito larvicidal activity. The binary toxin preparation, designated BsEcAg, contained mainly 51- and 42-kDa toxin proteins and was toxic to 50% of Culex quinquefasciatus larvae at a concentration of 9.22 ng toxin protein/ml. This preparation was used to raise antibodies in sheep and mice. The sandwich ELISA used sheep antitoxin antibody as primary antibody (coating antibody), mouse antitoxin antibody as second antibody, and goat antimouse antibody as an alkaline phosphatase-conjugated detecting antibody. The assay sensitivity was 200 ng/ml for both BsEcAg and binary toxin antigen (BsAg) from Bs 2362 cells. There is a significant correlation between toxin level determined by ELISA and bioassay. This procedure has also been used to monitor toxin levels in batch fermentations of Bs 2362.
authors.affiliation: Department of Biotechnology, University of New South Wales, Sydney, 2052, Australia.
authors.forename: S
authors.initials: S
authors.lastname: Jiaviriyaboonya
authors.forename: R J
authors.initials: RJ
authors.lastname: Penfold
authors.forename: P L
authors.initials: PL
authors.lastname: Rogers
mesh_terms: D000907:Antibodies, Bacterial / Q000276:immunology / Q000302:isolation & purification; D000917:Antibody Formation / Q000276:immunology; D000942:Antigens, Bacterial / Q000276:immunology / Q000302:isolation & purification; D001407:Bacillus / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D001427:Bacterial Toxins / Q000032:analysis* / Q000096:biosynthesis / Q000633:toxicity*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D007306:Insecticides / Q000378:metabolism / Q000633:toxicity; D009032:Mosquito Control / Q000379:methods; D011994:Recombinant Proteins / Q000032:analysis / Q000276:immunology; D012680:Sensitivity and Specificity
chemical_list: D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D007306:Insecticides; D011994:Recombinant Proteins; C046134:binB protein, Bacillus sphaericus