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 also increase. Accurately detecting these proteins in various environmental matrices is essential for determining possible exposure of non-target organisms to these proteins. 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 will be to develop a laboratory model system to degrade Cry1Ab. This will mimic degradation occurring in the environment and produce solutions of Cry1Ab fragments. In the second objective, aliquots of these solutions will be analyzed using ELISAs and bioassays. The results of the ELISAs and bioassays will be compared to determine anomalies in the data, i.e. if the fragments are detectable by ELISA but have no bioactivity and vice versa. Finally, specific guidelines for biological validation of ELISAs will be established.