Characterization of monoclonal antibodies to a crystal protein of Bacillus thuringiensis subsp. kurstaki.

Ten monoclonal antibodies were produced against a k-1-type crystal protein of Bacillus thuringiensis subsp. kurstaki. Eight of the antibodies belong to the immunoglobulin G1 (IgG1) subclass, with pI values ranging from 5.5 to 8.6, one could be assigned to the IgG2b subclass, and one could be assigned to the IgM class. Competitive antibody-binding assays and analysis of antibody specificity indicated that the 10 antibodies recognized at least nine distinct antigenic determinants. Eight antibodies bound to both protoxin and toxin, whereas the other two interacted with protoxin only. One antibody completely inhibited the biological activity of the delta-endotoxin, five antibodies reduced it by 15 to 82%, and four antibodies did not affect it at all. Based on cross-reaction studies, homologies and differences in the crystal protein structures of different B. thuringiensis subspecies were revealed. All of the monoclonal antibodies strongly cross-reacted with crystal proteins from strains of B. thuringiensis subsp. tolworthi, B. thuringiensis subsp. galleriae, B. thuringiensis subsp. dendrolimus, B. thuringiensis subsp. sotto, and B. thuringiensis subsp. subtoxicus. Some antibodies interacted only weakly with crystal proteins from strains of B. thuringiensis subsp. morrisoni and B. thuringiensis subsp. entomocidus, and some of these did not interact with B. thuringiensis subsp. kenyae and B. thuringiensis subsp. darmstadiensis. No cross-reaction was found with the parasporal inclusion protein of B. thuringiensis subsp. israelensis. Studies with the monoclonal antibodies also disclosed that crystal proteins from strains of the same subspecies can exhibit substantial differences in antigenic structure. In particular, striking strain-specific differences in the protoxins of B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. thuringiensis were observed.

Specificities of monoclonal antibodies against the activated delta-endotoxin of Bacillus thuringiensis var. thuringiensis.

Eight hybrid cell lines secreting monoclonal antibodies directed against the activated delta-endotoxin of Bacillus thuringiensis var. thuringiensis were grown in BALB/c mice. Ascites fluids were collected, and the antibodies were purified by antigen-affinity chromatography. The specificity of each monoclonal antibody for the toxin and protoxin was established by the indirect enzyme-linked immunosorbent assay. All the antibodies consisted of gamma 1 heavy and kappa light chains. They were reactive with both the native toxin and the protoxin. In contrast to specific goat antiserum, they failed, however, to bind to heat and sodium dodecyl sulfate denatured antigen. These eight cloned cell lines gave rise to five kinds of antibodies distinguished by isoelectric focusing. Competitive antibody binding studies revealed that these five antibodies recognize at least four distinct antigenic determinants of the native toxin and the protoxin. Two of the epitopes are unrelated, whereas three antibodies compete for binding to their antigenic determinants. In the bioassay with larvae of Pieris brassicae, one antibody was found to block the toxin and protoxin activity completely. A second inhibited it partially, whereas the other three antibodies did not affect it at all.