Biological validation of enzyme-linked immunosorbent assays for detection of Cry proteins in the environment

As the use of transgenic crops expressing genes for the production of insecticidal crystalline (Cry) proteins increases, concerns that these proteins may have adverse effects on non-target organisms persist. Accurately detecting and quantifying these proteins in various environmental matrices is essential for determining possible exposure of non-target organisms to these proteins and any significance of the exposure. Enzyme-linked immunosorbent assays (ELISAs) have been widely used for detection of Cry proteins in the environment.  However, their results are not typically validated biologically to ensure that only bioactive Cry proteins are being detected; therefore, accurate concentrations of the bioactive protein may not be properly represented in ELISA results. This could potentially lead to overly conservative risk assessments and unnecessary regulation. Thus, in order to properly study Cry proteins in environmental matrices, standardized methods of detection that can be biologically validated are needed. This research project will improve methods of detection for Cry proteins in environmental matrices by developing a framework for biological validation of ELISA procedures. The first objective was to develop a laboratory model system capable of degrading Cry1Ab to mimic protein degradation occurring in the environment. Chymotrypsin was shown to be capable of degrading Cry1Ab and generating fragments. The second objective was to perform ELISAs and bioassays on the solutions of fragmented protein generated by chymotrypsin to determine if the fragments were still detectable by ELISAs and/or retained any bioactivity. A decrease in detectability by ELSIA and a decrease in bioactivity were observed as the quantity of fragments increased.