An indirect competitive enzyme linked immunosorbent assay (ic-ELISA) using monoclonal antibody was developed to determine furazolidone metabolite. Made 3-{[(4-carboxyphenyl) methylene]amino}-2-oxazolidinone (CPAOZ) was conjugated with Ovalbumin (OVA) using N-hyduoxysuccinimide ester method to obtain CPAOZ-OVA. Then the influences of several physicochemical factors such as dilution ratios of CPAOZ-OVA and monoclonal antibody, blocking solution formulation, competitive reaction time, incubation time of HRP-IgG, chromogenic reaction time were optimized and the sensitivity, specificity, accuracy and precision of the method were evaluated. In the optimized system, the dilution ratios of CPAOZ-OVA and monoclonal antibody were 800-fold and 6400-fold respectively; blocking solution contained 1% skimmed milk; competitive reaction time was 60 min; incubation time for HRP-IgG was 45 min; chromogenic reaction time was 15 min. Based on the established standard curve, the linear equation for ELISA was set as Y=-0.253X+1.336(R2=0.995). IC50 was 2.015 ng/mL and linearity range was 0.131-30.911 ng/mL. The recovery rate of negative pork samples was 84.8%-90.3%. The coefficients of variation in intra-assay and inter-assay were 3.3%-4.1% and 4.6%-6.7% respectively. The method was proved to have excellent specificity, accuracy, precision and reproducibility, thus could be applied to rapid detect AOZ content in animal-derived food.
