Ultrasensitive time-resolved fluoroimmunoassay of ochratoxin A

In order to provide a rapid, selectivity and very high sensitivity method for the determination of ochratoxin A (OTA), an indirect competitive time-resolved fluoroimmunoassay(TRFIA) was used. Tests were performed in a 96-well microplate using the self-produced toxin-specific monoclonal antibodies 3G9, obtained from mice immunized with ochratoxin A - bovine serum albumin (OTA-BSA). In indirect TRFIA format, OTA-BSA was coated onto the microtitre plate and incubated with standard toxin and anti-OTA antibody. A goat antimice IgG- Eu3+ conjugate was used to enable detection. The suitability of the assay for quantification of OTA was also studied. Results showed ascitic fluids could be used at a dilution exceeding 1:10 000 and the OTA detection limit to be 0.03 mu g /L for indirect competitive TRFIA formats. The 80%, 50% and 20% inhibition binding effective dose (ED80 ED50 ED20) of OTA were (0.33 +/- 0.02) mu g/L, (1.44 +/- 0.08) mu g/L and (5.22 +/- 0.12) mu g/L. The assay range was 0.03 - 1 000 mu g /L. The cross reactivity with ochratoxin B was 3.7% and the antibodies did not react with aflatoxin B1, phenylalanine and BSA. The within-run and between-run CVs of the OTA- TRFIA were 3.7% and 5.3% respectively. The mean recoveries from OTA-free cereals spiked with 1 200 mu g/kg OTA of cereals sample were 94.2%. Both OTA- TRFIA and OTA-ELISA test were applied for the quantitative measurement of OTA in the same cereals, and the coefficient of correlation was 0.925. It was shown that the newly developed TRFIA could be applied to detect the OTA contamination in cereals. The OTA-TRFIA provides very high sensitivity and optimal range, and it will be useful to screen OTA contamination easily, simply and economically when the number of samples is large.