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%. © 2012 Elsevier Ltd.
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