Determination Of Sudan â…  By Indirect Competitive Enzyme Linked Immunosorbent Assay

Sudan I, one of the most widely used synthetic fat-soluble organic colorants, has been proved to be a potential carcinogen and forbitted as an additive used in foods. Enzyme linked immunoabsorbant assay (ELISA) is a simple, fast, highly sensitive and selective analytical method widly used in clinical detection, biological determination, food analysis and enviornment monitoring. In this study, a highly selective and sensitive indirect competitive ELISA for Sudan I was developed. Two hapten derivatives with different length of carboxylic spacer at the azobound para-position were synthesized and coupled to carrier proteins. The hapten-bovine serum albumin (BSA) conjugates were used as immunogens, while the hapten-ovalbumin (OV) conjugates were applied as coating antigens. The antisera obtained from four immunized rabbits were characterized in terms of sensitivity and specificity. At optimal experimental conditions, it was found that IC50 (the concentration of Sudan I inhibiting 50% of the highest signal) and limit of detection (LOD) of 7 pairs based on four antisera and two coating antigens were in the range of 0.3-2.0 ng/mL and 0.02-1.0 ng/mL, respectively. The most sensitive ELISA with the lowest IC50 of 0.3 ng/ml could be established with Sudan I-propionic acid-OV coating antigen and the antiserum which was obtained with the corresponding immunogen. The cross-reactivity values of the four antisera with Sudan II, III, and IV was estimated with 0.1-14.3%. No cross-reactivity was found with 6 edible colorants Sunset yellow, Amarant, Kermes, Indigotin, Bright blue and Lemon yellow, indicating high specificity for Sudan I. Six food samples were fortified with Sudan I and extracted by simple sample preparation. The methanolic extracts after dilution with methanol:water (5:95, v/v) were analyzed by the developed ELISA. Assay precision and accuracy was estimated by determination of 3 replicates. Acceptable recovery rates of 92.5-114% and intra-assay coefficients of variation of 5.9-24.8% were obtained. The data were validated by conventional HPLC method. As revealed, both methods were highly correlated (r=0.84, n=7) demonstrating the applicability of the developed ELISA for Sudan I analysis in food samples. In conclusion, a simple, fast, highly selective and sensitive ELISA for the determination of Sudan I has been successfully developed, which can be used in food surveillance for quantitative analysis of Sudan I in food samples.