A highly specific enzyme-linked immunosorbent assay for the detection of Cry1Ac insecticidal crystal protein in transgenic WideStrike cotton.

A highly selective enzyme-linked immunosorbent assay (ELISA) has been developed for the quantitative detection of the Cry1Ac protein expressed in transgenic cotton. Two Cry1Ac-specific monoclonal antibodies (MAb), Kbt and 158E6, were developed and selected to form a sandwich format ELISA. The MAb Kbt was used as a capture antibody, and 158E6 was conjugated with horseradish peroxidase and served as a detection antibody. The assay was optimized and validated with different cotton matrices. Tissues were extracted with phosphate-buffered saline containing 0.05% Tween 20 and 1% polyvinylpyrrolidone. The extract was then treated with trypsin to truncate full-length Cry1Ac into the core toxin for quantitation. The resulting assay has good accuracy and precision with a validated limit of quantitation ranging from 0.1 to 0.375 mug/g dry weight of cotton tissues. This assay is highly specific for Cry1Ac protein and has no cross-reactivity with the nontarget proteins tested such as Cry1Ab and Cry1F.