Broad specificity indirect competitive immunoassay for determination of nitrofurans in animal feeds

A generic hapten of nitrofurans was synthesized by derivatization of 5-nitrofurfural with diamine, and the hapten was coupled to carrier protein to prepare different immunogens and coating antigens by using diazotization method and glutaraldehyde method. The obtained novel polyclonal antibodies from immunized rabbits showed broad cross reactivity among seven nitrofurans. After assessment of four coating antigen/antibody combinations, an indirect competitive immunoassay was developed to simultaneously detect the seven nitrofurans in animal feeds. The limits of detection for these analytes were in the range of 5-16 μg kg⁻¹ depending on the compound. Recoveries from nitrofurans fortified blank feeds at levels of 30 and 100 ng g ⁻¹ were in a range of 82.6-108.4% with coefficients of variation lower than 11.4%. The immunoassay was further validated by a HPLC method and the two methods showed good correlation (r = 0.9924). Therefore, the proposed immunoassay could be used as a practical method to monitor the illicit use of nitrofurans in animal feeds.