Synthesis of olaquindox metabolite, methyl-3-quinoxaline-2-carboxylic acid for development of an immunoassay

A novel synthesis method of methyl-3-quinoxaline-2-carboxylic acid (MQCA) and the preparation of its hapten were described. MQCA was synthesised involving in two steps with a high purity. For improving the sensitivity of detection, five different haptens were synthesised and corresponding immunogens and coating antigens were prepared. After comparing the sensitivity of immunoassay with different pairs of antibody and coating antigen, a specific immunoassay was obtained using an antibody raised against hapten (four-atom spacer arm) - BSA and a suitable coating antigen with a heterologous spacer arm group. The 50% inhibitory concentration of MQCA by indirect competitive enzyme-linked immunosorbent assay with optimised antibody and coating antigen was 3.84 ng/ml and the limit of detection was 0.25 ng/ml.