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A sandwich enzyme-linked immunosorbent assay for the Bacillus sphaericus binary toxin
S Jiaviriyaboonya1, R J Penfold, P L Rogers
1Department of Biotechnology, University of New South Wales, Sydney, 2052, Australia.
Current Microbiology
|April 29, 1998
Summary
Bacillus sphaericus (Bs) binary toxin was purified and used to develop a sensitive ELISA assay. This assay accurately quantifies mosquito larvicidal toxin levels, aiding in pest control research.
Area of Science:
- Microbiology
- Biochemistry
- Immunology
Background:
- Bacillus sphaericus (Bs) produces a binary toxin with potent mosquito larvicidal activity.
- Developing sensitive detection methods for this toxin is crucial for its application in mosquito control.
Purpose of the Study:
- To purify Bacillus sphaericus binary toxin from recombinant E. coli.
- To develop and validate a sandwich ELISA for quantifying the toxin.
- To assess the correlation between ELISA results and bioassays.
Main Methods:
- Purification of Bacillus sphaericus binary toxin from recombinant E. coli DH5alpha.
- Generation of polyclonal antibodies in sheep and mice against the purified toxin.
- Development of a sandwich ELISA using specific antibodies and an alkaline phosphatase conjugate.
- Larvicidal bioassays on Culex quinquefasciatus larvae.
Main Results:
- Purified binary toxin (BsEcAg) contained 51- and 42-kDa proteins, showing high toxicity to Culex quinquefasciatus larvae (LC50 = 9.22 ng/ml).
- A sensitive sandwich ELISA was established with a detection limit of 200 ng/ml for both recombinant and native toxin.
- A significant correlation was observed between toxin levels measured by ELISA and by bioassay.
Conclusions:
- The developed sandwich ELISA is a reliable and sensitive method for quantifying Bacillus sphaericus binary toxin.
- This assay can be effectively used to monitor toxin production in fermentation processes.
- The findings support the use of ELISA for quality control and research involving mosquito larvicides.