IMPROVED DIRECT COMPETITIVE ENZYME-LINKED-IMMUNOSORBENT-ASSAY FOR CYCLOPIAZONIC ACID IN CORN, PEANUTS, AND MIXED FEED

An improved direct competitive enzyme-linked immunosorbent assay (dc-E LISA) was developed for the analysis of cyclopiazonic acid (CPA) in co rn, peanuts, and mixed feed. Two new approaches were used for the prep aration of enzyme markers; one involved coupling CPA-bovine serum albu min (CPA-BSA) conjugate to horseradish peroxidase (HRP) using either t he glutaraldehyde (GA) or the periodate (PI) method, and the other inv olved conjugating CPA carboxymethyl oxime (CPA-CMO) derivative to an e thylenediamine-modified HRP using a water-soluble carbodiimide (WSC) m ethod. The concentrations causing 50% binding inhibition of the labele d HRP to the antibody by CPA in the dc-ELISAs using markers prepared b y PI, GA, and WSC methods (IC50) were 0.42, 0.68, and 0.93 ng/mL, resp ectively. The dc-ELISAs using CPA-BSA-HRP prepared by either PI or GA method were more effective and subsequently used for the analysis of C PA in earn, peanuts, and mixed feed. Pour extraction solvent systems w ith 70-80% of methanol in different buffers at pH 7.4-8.5 showed no ad verse effects on the dc-ELISA, and sample extracts after dilutions cou ld be directly used in the assay. Using CTA-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 60 0 ng/g (ppb), respectively. The mean analytical recoveries (200-5000 p pb range) for CPA added to the corn, mixed feed, and peanuts were foun d to be 97.6, 92, and 93%, respectively.