A novel method for artificial antigen synthesis and preparation of a polyclonal antibody for the sensitive determination of leucomalachite green in fish samples by enzyme-linked immunoassay

Abstract

A novel method for leucomalachite green (LMG) hapten synthesis was presented. This method could introduce a pure alkyl chain at the para-position of LMG to form a spacer arm for hapten preparation. It also could be expanded to prepare a series of haptens with different lengths of spacer arms by increasing the lengths of alkyl chains. This synthesis is easy to operate with high yield. In order to find out the best match coating antigens, three other haptens were synthesized. Polyclonal antibodies against LMG were developed by using the above synthesized antigens. The sensitivity and specificity were examined by indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) by optimizing the types of coating antigens and the concentrations of the antigens and antiserum. We demonstrate that the antibody developed from the hapten with a pure alkyl chain gives the best performance. We also reported that heterogenous coating antigen gives better sensitivity in this ELISA method development. The ELISA assay was further used to analyze the LMG residue in fish tissue with an IC₅₀ value of 2.2 ng mL⁻¹ and a limit of detection (LOD) of around 0.06 ng mL⁻¹. Recoveries ranged from 79.8–92.4%, indicating that the IC-ELISA could be used for effective and low-cost analysis of LMG in fish samples.