Development of Monoclonal Antibodies Against Cry1Ab Protein from<i>Bacillus thuringiensis</i>and Their Application in an ELISA for Detection of Transgenic Bt-Maize

Abstract

Several transgenic insect resistant maize varieties, commonly referred to as Bt-maize, express the gene for the Bacillus thuringiensis toxin Cry1Ab. As a method for the determination of Bt-maize, a sandwich-type ELISA for Cry1Ab was established. Monoclonal antibodies were developed by immunization of mice with trypsin-activated Cry1Ab. The secondary antibody was labeled with horseradish peroxidase, and 3,3',5,5'-tetramethylbenzidine (TMB) was used as chromogene. The detection limit was 0.4 ng ml m 1 of Cry1Ab. No (Cry1Ac, Cry1C, Cry2A, Cry3A) or only little (Cry9C) cross-reactivity with other Cry proteins was observed. Furthermore, the assay was applied to protein extracts from maize powder containing different amounts of Bt-maize from three different lines (Bt-176, Bt-11 and MON810). Using 100 mg maize per sample and Tris-borate buffer for rapid protein extraction, the minimum detectable mass fraction was 0.10% for Bt-11 and 0.23% for MON810. In contrast, Bt-176 maize could not be detected due to the fact that the Cry1Ab gene is not expressed in seed tissue of Bt-176 maize.