Monoclonal Antibody-based Immunoassays for the Phytotoxin Coronatine

Abstract

Coronatine (COR) is composed of two structural components, coronafacic acid (CFA) and the amino acid coronamic acid (CMA), which are joined by an amide bond. Monoclonal antibodies (MAbs) were prepared against COR and used in competitive ELISAs. MAbs were secreted by hybridoma cell lines that were prepared from mice immunized with two different COR analogues conjugated to ovalbumin (OVA). The first hapten was prepared by reduction of the keto-oxygen on the cyclopentane ring of the CFA moiety of COR and subsequent reaction of the hydroxyl group with succinic or glutaric anhydride, which liberated a terminal carboxyl. This carboxyl group was coupled to carrier proteins using active ester chemistry. For preparation of the second hapten, a CMA analogue was synthesized containing a protected methylsulphydryl function at the 2-position of the cyclopropane ring in place of the 2-ethyl group of CMA, and the carboxylic acid was protected as a methyl ester. The hapten was then prepared by reaction of coronafacoyl chloride with the amine function of the CMA analogue. Following deprotection, the hapten was conjugated to OVA and used to immunize mice. Hybridoma cell lines secreting high affinity MAbs 11B8 (hapten 1) and 11G8 (hapten 2) were isolated. MAb 11B8 predominantly recognized the coronamyl amide features of coronatine whereas 11G8 recognized the coronafacoyl amide of COR. Immunoassays were developed using each of the antibodies. COR could be quantitated in the range 0.5-40 ng ml−1 (limit of detection 100 pg ml−1) and 3-100 ng ml−1 (limit of detection 800 pg ml−1) for 11B8 and 11G8 respectively.