Immunodiagnostic methods for detection of 5-enolpyruvylshikimate-3-phosphate synthase in Roundup Ready® soybeans

Abstract

We have developed immunological-based detection methods to support labeling of protein-containing food fractions derived from Roundup Ready® soybeans. Western blotting and enzyme linked immunosorbent assay (ELISA) procedures were developed to measure the 5-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) protein derived from the Agrobacterium sp. strain CP4 in the major processed fractions derived from Roundup Ready soybean. Expression of the CP4 EPSPS protein confers tolerance to Roundup® herbicide. The western blotting method utilizes a polyclonal goat anti-CP4 EPSPS antibody which specifically binds to CP4 EPSPS followed by detection of bound goat antibody with biotinylated Protein-G. Detection of this complex is accomplished using horseradish-peroxidase (HRP) labeled NeutrAvidin™ and signal development by enhanced chemiluminesence. Data from western blot analyses of these fractions establish that stable epitopes remain after the seed has been subjected to processing conditions typically employed by the food industry, thereby enabling development of an ELISA method. The ELISA for measurement of CP4 EPSPS is a triple antibody sandwich procedure utilizing a monoclonal capture antibody and a polyclonal detection antibody followed by a third biotin labeled monoclonal anti-rabbit antibody. Sandwich formation is detected using HRP labeled NeutrAvidin™ with color development using TMB substrate. In the sandwich ELISA, the immunological activity of CP4 EPSPS was reduced by the extraction method required to solubilize CP4 EPSPS protein from processed fractions. Sensitivity of the CP4 EPSPS ELISA was sufficient to detect CP4 EPSPS protein in processed soybean fractions that contained 2% Roundup Ready soybean mixed with conventional processed soybean fractions, thereby making the ELISA an acceptable method to assess CP4 EPSPS protein in processed soybean fractions. Data on sensitivity, accuracy, precision and specificity, established that the western blot and ELISA methods are appropriate for compliance with the EC Novel Foods Regulation.