Preparation Ofantibody Against Nicarbazin And Development Of ELISA Detection Method

Nicarbazin is a globally recognized chemical synthetic anticoccidial old drug composed by an equimolar complex of DNC and HDP, with the peak period of function being at day four post infection and the role of drug targeting at the second generation of reproductive fission. At present, Nicarbazin is mainly used as feed additive for the prevention of chicken coccidiosis. Nicarbazin could impact the reproduction performance of hens, potentially inhibit the growth of younger chicken, and enhance the heat stress of chicken. Moreover, it is hazardous to human health due to the drug residues in the poultry meat and eggs. Thus, many countries have developed legislation that formulate the maximum limitation of Nicarbazin residue in poultry products or prohibit Nicarbazin being detected in the import and export poultry products.Immunoassay technology has been widely used for the rapid detection of veterinary drug residues based on its advantages, such as specific and sensitive, simple, and fast, etc. Currently, however, chromatographic technology is used in the detection of Nicarbazin residue in animal-derived food in China. Chromatographic technology is time-consuming and cumbersome, and will contaminate the environment and endanger human health due to a large number of organic solvents being used in the process. In this study, the artificial synthesis of complete antigen, monoclonal and polyclonal antibody preparation technology were used and the hybridoma cell lines secreting antibody of Nicarbazin was developed. Moreover, a blocking ELISA method was developed for detecting Nicarbazin by using polyclonal antibody. Thus, the present study provided a new technology for the rapid detection of Nicarbazin in poultry products.The DNC-mimicking coupled with BSA as immunogen and to OVA as a label in the ELISA. The carbodiimide method was used in the conjugate. Mice were immunized with 200μg·0.2mL^-1, five tims every 3 weeks. An indirect ELISA was established with anti-DNC serum. Results showed that the optimal coating concentration and anti-DNC serun was 8.33μg·mL^-1 and 1:4000, respectively. The working concentration anti-antibody markered enzyme was 1:5000.The same method as above was used in the DNC-mimicking coupled with HRP (carbodiimide reaction), Sephadex G-25 chromatography. UV spectrophotometer showed that the elution peak of the collected sample is obvious, the absorption peak at 280nm and 405nm, After SDS-PAGE and UV identification the couple was successful, which activity of the enzyme was 1: 2560, show that the coupling method is reliable, provide the basis of Competitive ELISA detection method.Fusion was carried between myeloma cells(SP2/0) and spleen cells from Balb/c mice immunized with PNA-BSA antigen. Screening with ELISA, clone cells by 3 limited dilutions, 2 strains of monoclonal antibodies against DNC were obtained named 2A8,2F5. Ascites titer was above 1:6400 and caryotype analysis of hybridoma of NCZ mAb was 95-98.Immunogen emulsions were injected PNA-BSA into five sites on the rabbits with 1 mg·kg^-1, four times every 2 week. The serum titer was 1:12800. Results showed the optimal coating concentration was 4.165μg·mL^-1, the optimal working concentration was 1:3200, the working concentration anti-antibody markered enzyme was 1:1000.The blocking ELISA results showed competitive inhibitory curve of pAb was y=-14.220x+31.693, R²=0.995. IC₅₀ of DNC-pAb against NCZ was 47.53ng·mL^-1. The detecting range was 0.004-10μg·mL^-1. The antibody for DNC was specific and displayed no cross-reacting with Sodium sulfa chlorpromazine,Toltrazuril,Amprolium Hydrochloride,sodium sulfaquinoxaline and Sulfamonomethoxine.Preparation of monoclonal antibodies, polyclonal antibodies anti Nicarbazin,the establishment and optimization of ELISA detection method in this study, provide a scientific basis for the rapid detection of nicarbazin in poultry products.