Development of a flow-through enzyme immunoassay and application in screening green coffee samples for ochratoxin A with confirmation by high-performance liquid chromatography

A flow-through enzyme immunoassay has been developed for the screening of g reen coffee bean samples for ochratoxin A (OA) and was later used in a surv ey on OA in green coffee from different countries. The test has a sensitivi ty of 8 ng/g, and calculated recoveries ranged from 70 to 89% and from 86 t o 95% for spiked and naturally contaminated samples, respectively. There we re no significant differences in within-day and between-day assay performan ce (P>0.05). Green coffee samples (15 Arabica and 7 Robusta) received from an international coffee trader were analyzed for intrinsic fungal contamina tion, screened for OA, and subsequently confirmed by high-performance liqui d chromatography (HPLC). All 22 samples were contaminated by fungal species of the genus Aspergillus, while Penicillium species were isolated from a m ere 13.6% of the total number of samples. Isolates were tested for their ab ility to produce OA, and only 3.9% were positive. There was no correlation between occurrence of OA-producing isolates and levels of OA in contaminate d samples. Results of the screening procedure showed that 4 of the 22 sampl es were contaminated with 8 ng/g or higher. The HPLC method confirmed that the OA levels ranged from 27 to 168 ng/g. A fifth sample, which was shown t o be negative during screening, had an OA concentration of 4 ng/g. There we re no false negatives or positives recorded, and the flow-through enzyme im munoassay results correlated with those obtained by HPLC.