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    Enzyme-Linked Immunosorbent Assay for Quantification of Bacillus Thuringiensis Var. Kurstaki Crystalline Protein in Some Commercial Formulations

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    The widespread use of microbial pesticides for forest insect control necessitates determination of the delta-endotoxin protein levels in commercial formulations to optimize their use-patterns in the field. As the analyte is biochemical and/or biological in nature, classical analytical procedures are often not applicable for this purpose, and new types of analytical methods need to be developed. A sandwich type enzyme-linked immunosorbent assay (ELISA) was developed to detect and quantify Bacillus thuringiensis var. kurstaki delta-endotoxin in nine commercial formulations that are being used for forest insect control in North America. Formulation ingredients (formulants) interfered with the toxin response in three of the nine formulations, and the total protein needed to be separated from formulants for ELISA. Total protein levels were consistently higher (about 40 to 60%), in all formulations than the delta-endotoxin content because of the presence of inactive proteins. Results of three types of force-feeding bioassays [using spruce budworm, Choristoneura fumiferana (Clemens.)] viz., with diluted formulations, formulation extracts, and redissolved protein precipitates, showed that the formulants contributed to bioactivity and had to be removed to assess toxicity of the BTK protein alone. The delta-endotoxin content of formulations correlated better with the LD50's (ng of total protein/larva required for 50% mortality) of the redissolved protein precipitates of formulations than with those of the diluted formulations and formulation extracts. As more microbial pesticides are being introduced for forest insect control, it is crucial that analytical methods are in place for quality control of commercial formulations, both from the standpoint of improved efficacy and environmental safety.


    Bacillus thuringiensis, formulations, enzyme-linked immunosorbent assay, immunoassay, delta-endotoxin assay, total protein assay, spruce budworm bioassay

    Author Information:

    Sundaram, KMS
    Research Scientist and Project Leader, Forestry Canada, Forest Pest Management Institute, Sault Ste. Marie, Ontario

    Sundaram, A
    Research Scientist and Project Leader, Forestry Canada, Forest Pest Management Institute, Sault Ste. Marie, Ontario

    Gee, SJ
    Research Associate, Pesticide Biotechnology Laboratory, Department of Entomology and Environmental Toxicology, University of California, Davis, California

    Harrison, RO
    Research Scientist, University of CaliforniaImmunosystems, Inc., DavisScarborough, CaliforniaMaine

    Hammock, BD
    Professor, University of California, Davis, California

    Committee/Subcommittee: E35.22

    DOI: 10.1520/STP13017S