Development of an icELISA and Immunochromatographic Assay for Methyl-3-Quinoxaline-2-Carboxylic Acid Residues in Fish

Abstract

To evaluate residues of olaquindox (OLA) and its marker compound methyl-3-quinoxaline-2-carboxylic acid (MQCA) in aquatic products in the field, five types of hapten and corresponding antigens with exposure of different antigenic determinants were designed and synthesized. The optimal immunogen-coating combinations were obtained by antiserum detection and heterologous coating screening, successfully achieving a highly specific monoclonal antibody (mAb) against MQCA. Based on the highly specific mAb 1A11 showing no cross-reactivity with other analogs, an indirect competitive ELISA and an immunochromatographic strip for MQCA detection in fish were developed using the MQCA derivative MQCA-(NH2)4 coupled to KLH as the immunogen and MQCA-NH2 coupled to OVA as the heterologous coating antigen. The IC50 value of the indirect competitive enzyme-linked immunosorbent assay (icELISA) was 3.17 ng/mL, and the linear range was 0.58–17.29 ng/mL for MQCA under optimized concentrations of coating antigen and antibody of 0.03 and 0.3 μg/mL, respectively. The recoveries of MQCA by the developed icELISA in fish samples ranged from 81.6 to 90.7%. The cutoff value of the strip was 5 ng/mL, and the limit of detection was 0.25 ng/mL under optimal working conditions of 4 μL 0.1 M K2CO3 and 50 μL 0.2 mg/mL per 1 mL colloidal gold solution. The developed icELISA and immunochromatographic strip will be very useful tools for the primary screening of MQCA in fish.