Enzyme-Linked Immunosorbent Assay of Ochratoxin A in Wheat

Abstract

An enzyme-linked immunosorbent assay (ELISA) was developed for the quantitation of ochratoxin A amended to wheat. Ochratoxin A conjugated to horseradish peroxidase (HRP) was used as an enzyme marker in the assay. At toxin levels below 30 ppb, a cleanup treatment was necessary for ELISA. Among 3 cleanup methods tested (solvent partition, Sep-Pak cartridge treatment, solvent partition and cartridge treatment), reverse phase cartridge treatment was the most simple and effective. In the analysis, ochratoxin A was extracted from wheat with methanol. The methanolic extract was diluted with water to a final 10-15% methanol content, and then passed through a cartridge. Ochratoxin A was eluted from the cartridge with 85% methanol which was then concentrated. The final solution, in 0.1M, pH 7.5 sodium phosphate-Tween 20 buffer and 5% methanol, was then subjected to ELISA. ELISA allowed minimal detection of the toxin in wheat at the 1-2 ppb level after cleanup. Recoveries of toxin added to wheat samples in the 1.0-30 ppb range were 85-90% with standard deviations of 10-15%.