Characterization Of Monoclonal Antibodies Against Sulfadiazine And Its Application In Immunoassay For Residues Detection

Sulfadiazine (SD) is a kind of synthetic antibacterial medicine, and was used widely in stockbreeding field due to the virtues of wide range, lower cost, and convenience to use. It, simultaneously, threats human health by residue in animal's body and transfer in anima-derived foods by means of food cycle.Manny detection assays of SD residues have been developed such as high performance liquid chromatography (HPLC), gas chromatography (GC), Hyphenated Method, Capillary Electrophoresis (CE) and immunochemistry. The immunological analytical technique, which has many merits of sensitivity, specificity, rapidity and convenience, has been generally used in determination of drug residues in animal food. This study aimed at immunological rapid determination of SD residues in animal food, including the synthesis of SD artificial antigen, preparation and special properties of SD monoclonal antibody (SD mAb) and reagent kits, the preparation and quality of immunological gold-labeled strip.Basis on analysis of molecular structure and immunogenicity of SD, the diazotization was used to conjugate SD to bovine serum albumin (BSA) and obtained artificial immunoantigen (BSA-SD), the coating antigen OVA-SD was obtained in the same way. BSA-SD and OVA-SD was synthesized successfully by identification with ultraviolet and SDS-PAGE, antiserum of BALB/c mice immunized with BSA-SD had been identified by indirect ELISA.After immunized BALB/c mice with BSA-SD, one hybridoma lines of 6B5-E4 with high affinity and high specificity was screened by cell fusion. SD mAb was produced by intraperitoneally inject cell line into prostane sensitized BALB/c mice. The mAb had a good sensitivity with 50% inhibitive concentration (IC50) of 1.155μg/L and 0.8% cross-reactivity (CR) to sulfamerazine (SM1) and little or no CR to other compounds by competitive ELISA.A competitive ELISA kit with 0.25μg/L of sensitivity and 0～4μg/L of linear detection range for determination of SD (SD-Kit) were developed with SD mAb of 6B5-E4. The average recoveries of SD in urine, milk and meat were 118.5%, 81.05% and 88.4% respectively. A rapid test strip of SD (SD-Strip) was developed with SD mAb of 6B5-E4. and its determination limit was 2μg/L by BioDot-TSR3000, 10μg/L by eyes. The sensitivity, specificity, simplicity and reliability of SD-Kit and SD Strip were proved to be useful for the rapid determination of SD residues in animal food.