Study On An Enzyme-Linked Immunosorbent Assay For The Detection Of Heterocyclic Aromatic Amines

The enzyme-linked immunosorbent assay for the determination of heterocyclic aromatic amines(HAAs) in food had been developed and applied in the real food samples.As HAAs is a class of compounds with similar structure, a representative heterocyclic amine IQ (2-amino-3-methyl-imidazo[4,5-f]quinoline) with a relatively simple structure was selected to create a standard ELISA method..In order to obtain better antibody, we synthesized a hapten (PQA) with PQ (1H-pyrrolo[2,3-f]quinoline), a structural analogue of IQ. Immunogen (PQA-BSA),coating antigen (PQA-OVA) and enzyme-labelled antigen (PQA-HRP) were obtained via the active ester method.For the indirect competitive ELISA, the standard curve obtained the coating antigen incubation concentration was0.05μg per well, the blocking solution was0.5%skim milk powder, the antibody dilution was1:3500and the buffer was PBS (pH7.4). The assay sensitivity (IC50) was71±3μg L-1and the limit of detection (IC15) was3±0.5μg L-1. For the direct competitive ELISA, the antibody incubation concentration was0.5μg per well, e the dilution ratio of enzyme tracer was1:2000, the blocking solution was0.5%skim milk powder and the buffer was PBS(pH7.4). The sensitivity of this method (IC50) was50±0.3μg L-1, while the limit of detection (IC15) was3±0.3μg L-1. The antibody showed low cross-reactivity with other tested compounds.The recoveries of IQ from spiked beef, pork and dried fish floss were studied. The recoveries are89.76%-101.44%,89.79%-107.34%and84.96%-116.23%respectively, the coefficients of variation (CV) were less than20%. The linear regression equation between data obtained using the ELISA and HPLC was y=1.043x-0.029with a correlation coefficient (R2) of0.9917. It showed that the two methods had a good correlation.