Improved Direct Competitive Enzyme-Linked Immunosorbent Assay for Cyclopiazonic Acid in Corn, Peanuts, and Mixed Feed

Abstract

An improved direct competitive enzyme-linked immunosorbent assay (dc-ELISA) was developed for the analysis of cyclopiazonic acid (CPA) in corn, peanuts, and mixed feed. Two new approaches were used for the preparation of enzyme markers; one involved coupling CPA−bovine serum albumin (CPA-BSA) conjugate to horseradish peroxidase (HRP) using either the glutaraldehyde (GA) or the periodate (PI) method, and the other involved conjugating CPA carboxymethyl oxime (CPA-CMO) derivative to an ethylenediamine-modified HRP using a water-soluble carbodiimide (WSC) method. The concentrations causing 50% binding inhibition of the labeled HRP to the antibody by CPA in the dc-ELISAs using markers prepared by PI, GA, and WSC methods (IC50) were 0.42, 0.68, and 0.93 ng/mL, respectively. The dc-ELISAs using CPA-BSA-HRP prepared by either PI or GA method were more effective and subsequently used for the analysis of CPA in corn, peanuts, and mixed feed. Four extraction solvent systems with 70−80% of methanol in different buffers at pH 7.4−8.5 showed no adverse effects on the dc-ELISA, and sample extracts after dilutions could be directly used in the assay. Using CPA-BSA-HRP prepared according to the GA method in the dc-ELISA, the detection limits of CPA in corn, mixed feed, and peanuts were estimated to be around 100, 300, and 600 ng/g (ppb), respectively. The mean analytical recoveries (200−5000 ppb range) for CPA added to the corn, mixed feed, and peanuts were found to be 97.6, 92, and 93%, respectively. Keywords: CPA; ELISA; immunoassay; corn; peanuts; mixed feed