Ultrasensitive time-resolved fluoroimmunoassay of aflatoxins B1

The aim of this work is to provide a rapid, selective and very high sensitive method for the determination of aflatoxins B1(AFB1) using the indirect competitive time-resolved fluoroimmunoassay (TRFIA). On the indirect competitive assay, aflatoxins B1-horseradish peroxidase (AFB1-HRP) is bound on the surface of a microtiter plate. AFB1 containing samples or standards and a rabbit anti-AFB1 antibody are given in the wells of the microtiter plate. Solid-phase bound and free AFB1 compete for the antibody binding sites, the secondary antibody, labelled with Eu3+ is used to enable detection. The luminescent enhancement solution contained mainly 2-naphthoyltrifluoroacetone. Results showed the AFB1 detection limit to be 0.01 μg/L for indirect competitive TRFIA formats. The 80%, 50%, 20% inhibition binding effect dose (ED80, ED50, ED20) of AFB1 were (0.07 ± 0.01) μg/L, (0.63 ± 0.02) μg/L and (12.5 ± 0.47) μg/L, respectively. The assay range was 0.01-100 μg/L. The cross reactivity with aflatoxins B2 was 10% and antibodies did not react with aflatoxins G2, ochratoxim A, ochratoxim B and HRP. The within-run and between-run CVs of the AFB1-TRFIA were 4.1% and 6.8%, respectively. the mean recoveries of samples were 97.2%. Both TRFIA and ELISA tests were applied to the quantitative measurement of AFB1 in the same samples, and the coefficient of correlation was 0.917. The present studies clearly show that the newly developed TRFIA could be applied to detect the AFB1 contamination in samples. The AFB1-TRFIA provides very high sensitivity and optimal range, and it will be useful to screen AFB1 contamination easily, simply and deonomically when the number of samples is large. (authors)