Abstract: With the aim of developing a generic enzyme-linked immunosorbent assay (ELISA) for the detection of heterocyclic amines, a new approach was used to prepare hapten, in which, methyl 4-chloro-4-oxobutyrate (MCO) was attached to the 2-amino group of 2-amino-3,4,8-trimethyl-3H-imidazo[4,5-f]quinoxaline (4,8-DiMeIQx). The antigen was obtained by a modified carbodiimide method. In pH buffer system at the appropriate pH, the hydrolysis of ester and the coupling step were completed simultaneously. Compared with the traditional method, the new method shortened the reaction time and cut down the cost effectively. The results of UV identification and polyclonal antibody titer indicated that the coupling was successfully achieved. And the immunogens produced polyclonal antibody for 4,8-DiMeIQx with a high titer of 1:450 000. High-affinity antibody against 4,8-DiMeIQx was optimized and a direct competitive ELISA assay was developed. It was shown that the analyte concentration giving 50% inhibition of color development (IC50) was 24.0 μg/L and that giving 15% inhibition of color development (IC15) was 1.8 μg/L for 4,8-DiMeIQx, and IC50 was 35.0 μg/L for 7,8-DiMeIQx, 30.0 μg/L for 8-MeIQx, 28.0 μg/L for IQx, and 40.0 μg/L for TriMeIQx, respectively, while almost no cross-reactivity (CR < 0.01%) with other heterocyclic amines having a structure different from that of 4,8-DiMeIQx, such as PhIP, IQ and MeIQ, was observed. The developed ELISA method was suitable for the rapid quantitative and qualitative determination of heterocyclic amines in processed meat products.

Key words: eterocyclic aromatic amines, hapten, antigen, polyclonal antibody, enzyme-linked immunosorbent assay (ELISA)