Abstract
BACKGROUND: As a broad-spectrum drug against chicken coccidiosis, nicarbazine is widely used. The international community has made regulations and requirements on the residue limits of nicarbazine metabolites in chicken. The research reports on the detection methods of nicarbazine residues are mainly based on large-scale instruments such as high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry-mass spectrometry (LC/MS/MS) and so on. However, in the practical monitoring and detection application, the rapid, sensitive, efficient and accurate detection of nicarbazine residues is becoming more and more urgent. METHODS: This study aimed to establish an enzyme-linked immunosorbent assay (ELISA)-based method to detect nicarbazin drug residues with high sensitivity and specificity, and wide applicability. Artificial immunogens were prepared by molecular modification synthesis. Nuclear magnetic resonance (NMR) and ultraviolet analyses were conducted to confirm that the correct product was obtained. Monoclonal antibodies were acquired by immunizing mice and preparing hybridoma cells. RESULTS: In this study, 4,4'-dinitrocarbanilide (DNC), a metabolite of nicarbazine, was synthesized and modified to make it have immunogenicity. Fifteen healthy female mice of 6-8 weeks old were immunized in three groups. The successfully immunized mice were screened by serum titer. One mouse with the highest titer was fused and cloned three times, and four positive cell lines were obtained. Nine monoclonal antibodies were obtained from mouse ascites. The best matched antigens and antibodies were screened by an ELISA chessboard method. A detection method of nicarbazine ELISA kit was developed. Our prepared anti-nicarbazin monoclonal antibody had a half-maximal inhibitory concentration (IC(50)) of 0.825 ng/mL, and the curve range was 0.3-24.3 ng/mL. There was no cross reaction to other six common anti-coccidiosis drugs. The recovery results showed that the fortified recovery of the chicken and duck samples ranged from 74.4-111.7%, the test results of which all met the requirements for veterinary drug residue detection. CONCLUSIONS: This method, which uses a specific antibody against the nicarbazin metabolic product DNC, enables rapid quantitative detection. Our new ELISA-based method should facilitate the development of assays to monitor and detect agricultural and veterinary drug residues.