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%.

Chemiluminescent and bioluminescent assays as innovative prospects for mycotoxin determination in food and feed

“…The detection threshold for the CL immunoassay was 20 pg ochratoxin A/tube. This sensitivity is similar to that obtained with ELISA for ochratoxin A analysis in wheat (33). Known quantities of ochratoxin A were added to corn samples which were analysed before and after this step.…”

Development of a quantitative and sensitive enzyme-linked immunosorbent assay for ochratoxin A using antibodies from the yolk of the laying hen

“…Ochratoxin A Extraction Procedure Used for Comparison of Recoveries. The methanol-based extraction and reversed-phase cartridge cleanup procedure, which was used for comparison, was a slight modification of that originally described by Lee and Chu (1984) for the determination of OA in wheat by ELISA. In brief, 5 g of diet sample was mixed with 15 mL of pure methanol and shaken for 30 min.…”

A monoclonal antibody to ochratoxin A

“…Although substrates are normally analysed for OA by thin layer chromatography (Nesheim et al 1973) and by high performance liquid chromatography (Nesheim 1984), radio-and enzyme-linked immunoassays have been developed with animal antisera (Lee & Chu 1984;Rousseau et al 1985). However, the antibodies employed have been made by conventional techniques and so far no monoclonal antibodies (McAb) have been described.…”