title: Comparison and validation of methods to quantify Cry1Ab toxin from Bacillus thuringiensis for standardization of insect bioassays.
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.
authors.affiliation: Department of Entomology, 202 Plant Industry Bldg., University of Nebraska, Lincoln, NE 68583-0816, USA.
authors.forename: André L B
authors.initials: AL
authors.lastname: Crespo
authors.forename: Terence A
authors.initials: TA
authors.lastname: Spencer
authors.forename: Emily
authors.initials: E
authors.lastname: Nekl
authors.forename: Marianne
authors.initials: M
authors.lastname: Pusztai-Carey
authors.forename: William J
authors.initials: WJ
authors.lastname: Moar
authors.forename: Blair D
authors.initials: BD
authors.lastname: Siegfried
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000032:analysis*; D001427:Bacterial Toxins / Q000032:analysis*; D001681:Biological Assay / Q000592:standards*; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004731:Endotoxins / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay; D006460:Hemolysin Proteins / Q000032:analysis*; D015151:Immunoblotting; D007928:Lethal Dose 50; D009036:Moths / Q000187:drug effects*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis