Immunoaffinity column as cleanup tool for a direct competitive enzyme-linked immunosorbent assay of cyclopiazonic acid in corn, peanuts, and mixed feed.
An immunoaffinity column (IAC) for cyclopiazonic acid (CPA) was prepared by coupling a CPA-specific monoclonal antibody to CNBr-activated sepharose 4B. A direct competitive enzyme-linked immunosorbent assay (dc-ELISA) was used to study the chromatographic behavior of a 0.2 mL gel column with a binding capacity of 4 micrograms CPA/column as well as to evaluate its efficacy as a cleanup tool for analysis of naturally occurring CPA. Sample extract either in buffer solution or in a solution containing up to 35% methanol could be loaded onto the column. After the column is washed with 5 mL deionized water and 5 mL 50% methanol, CPA could be quantitatively eluted with 2 mL 100% methanol. The column could be regenerated at least 10 times by washing with 10 mL equilibrating buffer and then storing in a cold room overnight before reuse. Recoveries of CPA added to corn, peanut, and mixed feed extracts in the range 10-200 ng/g were 88-105, 86-100, and 90-110%, respectively. Detection limits were 2.0, 4.4, and 4.7 ng/g for corn, mixed feed, and peanuts, respectively. Twenty-two peanut samples naturally contaminated with CPA were subjected to both IAC and solvent partition cleanup followed by dc-ELISA. Although a good correlation between data obtained from IAC-dc-ELISA and from SP-dc-ELISA (r = 0.75, p < 0.0001) was obtained, the slope of the linear regression was low (0.67), indicating loss during solvent partition cleanup. The overall data showed that the combination of IAC and dc-ELISA is an effective method for CPA analysis.