Production of the monoclonal antibody against Sudan 4 for multi-immunoassay of Sudan dyes in egg

Sudan dyes (Sudan 1, 2, 3, 4, Para red and Sudan red G) have been banned as food colorants due to their carcinogenicity. This study first synthesized a hapten of Sudan 4 and produced the monoclonal antibody for immunoassay of these red dyes. The hapten was synthesized by coupling 4-amino-3-methylbenzoic acid to o-toluidine to obtain an intermediate hapten and then the intermediate hapten was coupled to β-naphthol to obtain a tentative hapten. The generated monoclonal antibody showed broad specificity to the six Sudan dyes. After evaluation of different coating antigens, a heterologous competitive indirect immunoassay was developed to determine the six red dyes in egg yolk simultaneously. The crossreactivities for the six analytes were in a range of 64%–110% and the limits of detection were in a range of 0.08–0.2 ng/g depending on the compound. Intra- and inter-assay recoveries from the standards fortified blank samples ranged from 71.5% to 107.4% with coefficients of variation lower than 14.6%.