Comparison and Validation of Methods To Quantify Cry1Ab Toxin from Bacillus thuringiensis for Standardization of Insect Bioassays
- 1 January 2008
- journal article
- research article
- Published by American Society for Microbiology in Applied and Environmental Microbiology
- Vol. 74 (1), 130-135
- https://doi.org/10.1128/aem.01855-07
Abstract
Standardization of toxin preparations derived from Bacillus thuringiensis (Berliner) used in laboratory bioassays is critical for accurately assessing possible changes in the susceptibility of field populations of target pests. Different methods were evaluated to quantify Cry1Ab, the toxin expressed by 80% of the commercially available transgenic maize that targets the European corn borer, Ostrinia nubilalis (Hübner). We compared three methods of quantification on three different toxin preparations from independent sources: enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulfate-polyacrylamide gel electrophoresis and densitometry (SDS-PAGE/densitometry), and the Bradford assay for total protein. The results were compared to those obtained by immunoblot analysis and with the results of toxin bioassays against susceptible laboratory colonies of O. nubilalis . The Bradford method resulted in statistically higher estimates than either ELISA or SDS-PAGE/densitometry but also provided the lowest coefficients of variation (CVs) for estimates of the Cry1Ab concentration (from 2.4 to 5.4%). The CV of estimates obtained by ELISA ranged from 12.8 to 26.5%, whereas the CV of estimates obtained by SDS-PAGE/densitometry ranged from 0.2 to 15.4%. We standardized toxin concentration by using SDS-PAGE/densitometry, which is the only method specific for the 65-kDa Cry1Ab protein and is not confounded by impurities detected by ELISA and Bradford assay for total protein. Bioassays with standardized Cry1Ab preparations based on SDS-PAGE/densitometry showed no significant differences in LC 50 values, although there were significant differences in growth inhibition for two of the three Cry1Ab preparations. However, the variation in larval weight caused by toxin source was only 4% of the total variation, and we conclude that standardization of Cry1Ab production and quantification by SDS-PAGE/densitometry may improve data consistency in monitoring efforts to identify changes in insect susceptibility to Cry1Ab.Keywords
This publication has 21 references indexed in Scilit:
- Rapid evolution and the cost of resistance toBacillus thuringiensisin greenhouse populations of cabbage loopers,Trichoplusia niProceedings Of The Royal Society B-Biological Sciences, 2003
- Interaction between Cry9Ca and two Cry1A δ-endotoxins fromBacillus thuringiensisin larval toxicity and binding to brush border membrane vesicles of the spruce budworm,Choristoneura fumiferanaClemensFEMS Microbiology Letters, 2002
- An analysis of Bacillus thuringiensisδ‐endotoxin action on insect‐midgut‐membrane permeability using a light‐scattering assayJBIC Journal of Biological Inorganic Chemistry, 1993
- Use of Sodium Dodecyl Sulfate—Polyacrylamide Gel Electrophoresis To Quantify Bacillus thuringiensis δ-EndotoxinsPublished by American Chemical Society (ACS) ,1990
- Insect Resistance to the Biological Insecticide Bacillus thuringiensisScience, 1985
- Clear background and highly sensitive protein staining with Coomassie Blue dyes in polyacrylamide gels: A systematic analysisElectrophoresis, 1985
- A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye BindingAnalytical Biochemistry, 1976
- A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye bindingAnalytical Biochemistry, 1976
- Probit AnalysisTechnometrics, 1973
- Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4Nature, 1970