Development Of A Chemiluminescent Enzyme-Linked Immunosorbent Assay For Sulfadiazine Residues In Pig Muscle

With the rapid development of breed, the food safety problems in edible animal products have provoked considerable debates, especially for drug residue, it is one of the most important problems. SAs are common veterinary drugs, if the proper withdrawal periods are not observed before slaughtering or milking of medicated animals, foodstuffs (for example, meat and milk) derived from these animals may be contaminated with residual sulfonamides. The detection methods for SD mainly include physical and chemical methods, such as high performance liquid chromatography and chromatography-mass spectrometry. These methods are difficult to meet practical needs for their low efficiency, high cost and complicate sample pretreatment. However, the immunological method has been used extensively in food safety and inspection for its high sensitive, convenient and low cost. This study aimed to develop the anti-SD McAb based on the SD artificial antigen; and establish a CLEIA for SD.A. SD was coupled with carrier protein BSA, OVA by diazotization and glutaraldehyde methods, then immunized BALB/c mice with immunogen SD-BSA. The SD polyclonal antibodies were prepared, the effects of the antigen synethesis methods on the anti-serum were studied. The results proved that diazotization method was better than glutaraldehyde. Three separate immunogens SD-BSA with conjugation ratio 9.0:1,8.0:1 and 3.7:1 were prepared by diazotization method. The highest titer of anti-SD antibodies for mice were got by immunizing SD-BSA with conjugation ratio 8.0:1.B. Based on the polyclonal antibodies, McAb against SD were produced from a stable hybridoma cell line,1F6, generated by fusion of SP/20 myeloma cells with spleen cells isolated from a BALB/c mouse immunized with SD-BSA of conjugation ratio 8.0:1 prepared by diazotization method. The 1F6 McAb belong to IgG 2b (kappa chain). The titer of ascitic fluid was 1:1.28×106 by indirect ELISA. The association constant was 5.5 x 109 L/mol. And it showed cross-reactivity of 60.3% to STZ, while no or little cross-reactivity to SP, SM2, SMD, SMM, SMZ, SQX, PABA, SN, SMT, p-ASA.C. A CLEIA for SD residues in pig muscle has been developed based on the McAb. The concentration causing 50% inhibition of the binding of the antibodies to SD-OVA by free SD was 37.6 ng/mL, the detection limit was 5.1 ng/mL, and the linear working range was 6.4-219.8 ng/mL. The recovery ratio of SD (40,80, 140μg/kg) added to pig muscle ranged from 71.0% to 96.3%. The intra-assay coefficient of variation were between 0.9% and 7.4%, while the inter-assay coefficient of variation were between 7.6% and 10.5%.60 pig muscle samples were analyzed by CLEIA, and the results show good correlation to HPLC (R2=0.9386).