A sensitive and selective direct competitive enzyme-linked immunosorbent assay for fast detection of Sudan I in food samples.

Sudan I, a synthetic azo dye, is considered to be a genotoxic carcinogen and is prohibited in foodstuffs for any purpose at any level worldwide. In this study, a sensitive and specific direct competitive enzyme-linked immunosorbent assay (dc-ELISA) for fast detection of Sudan I in food samples was developed for the first time. The monoclonal antibody against Sudan I was used as capture protein, while horseradish peroxidase labeled Sudan I conjugate prepared by the periodate method via ovalbumin (OVA) as a bridge was used as enzyme tracer. The standard curve of dc-ELISA for Sudan I was constructed in the range 0.1-100 ng mL⁻¹ and the assay time was within 80 min. Sensitivity was 2.6 ng mL⁻¹ and the limit of detection was 0.08 ng mL⁻¹. Cross-reactivity values of the assay with Sudan II, III and IV were 5.78%, 1.72% and 0.64%; no cross-reactivity was found with six other edible colorants. The assay was tolerated to 30% of methanol and 10% of acetonitrile without significant loss of IC₅₀. Recoveries of spiked Sudan I in five different samples including chilli powder, tomato sauce, hotpot seasoning and chilli sauce I and II were within 88.4-113.2% and the intra-assay relative standard deviation was less than 14%. The dc-ELISA was confirmed by conventional high-performance liquid chromatography and the correlation coefficient of the two methods was 0.9902. The proposed dc-ELISA method provides an alternative method for sensitive, specific and fast determination of Sudan I in food samples.