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.

Stability of Cry1Ab protein during long-term storage for standardization of insect bioassays

“…1). Crespo et al (2008) compared SDS-PAGE, ELISA and Bradford assays for estimating Cry1Ab concentrations in production batches and found that Bradford assays had the lowest coefficient of variation. However, this method did not allow determining whether a Cry1Ab protein in a production batch is really stable or degraded, because it is not possible to differentiate between active Cry1Ab proteins and its degradation products.…”

“…These batch-to-batch differences could be caused by different treatments during production of these batches and/or differences in the concentration of Cry1Ab depending on the protein determination methods. Crespo et al (2008) noted that different protein batches should be produced and measured with standardized methods in order to eliminate the difference in their activity. When using the same standardized production and quantification methods, bioassay of three different Cry1Ab batches produced in E. coli and B. thuringiensis showed no significant difference in LC 50 values (Crespo et al, 2008).…”

“…Crespo et al (2008) noted that different protein batches should be produced and measured with standardized methods in order to eliminate the difference in their activity. When using the same standardized production and quantification methods, bioassay of three different Cry1Ab batches produced in E. coli and B. thuringiensis showed no significant difference in LC 50 values (Crespo et al, 2008). Our study indicates that the choice of the storage temperature is a crucial factor concerning the bioactivity of Cry1Ab protein in long-term monitoring the susceptibility of a target insect.…”

“…Hence, the standardization of the bioassay methodology as well as the application of standardized Cry1Ab proteins is of greatest importance in order to obtain meaningful data. Recently, different standardized methods for quantifying Cry1Ab protein from independent sources were compared and validated (Crespo et al, 2008). It was concluded that standardization of production and quantification of Cry1Ab batches may improve data consistency in monitoring efforts, and will be essential for the identification of changes of the susceptibility of target pests to Cry1Ab protein.…”

Inter-laboratory comparison of Cry1Ab toxin quantification inMON 810maize by enzyme-immunoassay

“…ELISA methods are the method of choice for the analytical determination of Cry type lectins (Grothaus et al, 2006;Huber-Lukac, Luthy, & Braun, 1983;Walschus, Witt, & Wittmann, 2002;Wang et al, 2007), but other immunoanalytical formats, for example, dot blot (Tapp & Stotzky, 1995), lateral flow immunostrips (Ermolli et al, 2006a), microsphere-based immunoassay (Ermolli et al, 2006b;Fantozzi et al, 2007) or immunomagnetic electrochemical sensor (Volpe, Ammid, Moscone, Occhigrossi, & Palleschi, 2006) have also been reported. ELISAs are widely used for Cry toxin detection in microbial preparations (Crespo et al, 2008;Takahashi et al, 1998) and in GM plants or food (Adamczyk, Adam, & Hardee, 2001;Baumgarte & Tebbe, 2005;Bruns & Abel, 2003;Chen et al, 2009;Chilcutt & Tabashnik, 2004;Douville et al, 2005;Ezequiel, Reggiardo, Vallejos, & Permingeat, 2006;Harwood, Wallin, & Obrycki, 2005;Margarit, Reggiardo, Vallejos, & Permingeat, 2006;Mendelsohn, Kough, Zigfridais, & Matthewsm, 2003;Nguyen & Jehle, 2007;Sims & Berberich, 1996;Stave, 1999Stave, , 2002Xie & Shu, 2001;Zwahlen, Hilbeck, Gugerli, & Nentwig, 2003). Commercial ELISA kits are available and used frequently for detection of Cry toxins from B. thuringiensis or GM plants.…”