Immunodiagnostic methods for detection of 5-enolpyruvylshikimate-3-phosphate synthase in Roundup Ready (R) soybeans

We have developed immunological-based detection methods to support labeling of protein-containing food fractions derived from Roundup Ready(R) soybean s. Western blotting and enzyme linked immunosorbent assay (ELISA) procedure s were developed to measure the 5-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) protein derived from the Agrobacterium sp. strain CP4 in the m ajor processed fractions derived from Roundup Ready soybean. Expression of the CP4 EPSPS protein confers tolerance to Roundup(R) herbicide. The wester n blotting method utilizes a polyclonal goat anti-CP4 EPSPS antibody which specifically binds to CP4 EPSPS followed by detection of bound goat antibod y with biotinylated Protein-G. Detection of this complex is accomplished us ing horseradish-peroxidase (HRP) labeled NeutrAvidin(TM) and signal develop ment by enhanced chemiluminesence. Data from western blot analyses of these fractions establish that stable epitopes remain after the seed has been su bjected to processing conditions typically employed by the food industry, t hereby enabling development of an ELISA method. The ELISA for measurement o f CP4 EPSPS is a triple antibody sandwich procedure utilizing a monoclonal capture antibody and a polyclonal detection antibody followed by a third bi otin labeled monoclonal anti-rabbit antibody. Sandwich formation is detecte d using HRP labeled NeutrAvidin(TM) With color development using TMB substr ate. In the sandwich ELISA, the immunological activity of CP4 EPSPS was red uced by the extraction method required to solubilize CP4 EPSPS protein from processed fractions. Sensitivity of the CP3 EPSPS ELISA was sufficient to detect CP4 EPSPS protein in processed soybean fractions that contained 2% R oundup Ready soybean mixed with conventional processed soybean fractions, t hereby 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 approp riate for compliance with the EC Novel Foods Regulation. (C) 1999 Published by Elsevier Science Ltd. All rights reserved.