Development Of ELISA Methods And Kits For Rapid Detection Of Sulfadiazine And Sulfonamides Residues In Edible Animal Products

The research established two competitive direct ELISA (CdELISA) methods to detect sulfadiazine (SD) and sulfonamides (SAs) residues in edible animal products. And on the base of the two methods two ELISA Kits were developed respectively. The results are as follow:1. After resuscitation, screening, ascites production and purification of the hybridoma line named SD-4 that conserved in our laboratory, anti-SD monoclonal antibody was prepared. A Coated antigen SD-OVA was synthesized by conjugating SD to ovalbumin (OVA) with the method of diazotization and coupling with the mol ratio of SD/OVA as 2/1 in the reaction. Horseradish peroxidase (HRP) labeled antigen (SD-OVA-HRP) was developed by the method of periodates oxidation with the mol ratio of SD-OVA/HRP as 1/2 in the reaction. The direct ELISA method was selected by competitive ELISA. After comparation the sensitivity, coating time and stability between the general coating method and microwave method, the microwave coating method was established. The equation of calibration curve was y=12.05x+2.2538 with the R²= 0.995 and IC₅₀=41.7ng/mL in the concentration range from 1ng/mL to 729ng/mL. The cross-reactivities for sulfamonomethoxine(SMM), sulfametoxydiazine(SMD), sulfadimethoxine(SDM), sulfamethazine(SM2), sulfaquinoxaline(SQ) and sulfamethoxazole (SMZ) were 1.7%, 3.1%, <0.01%, <0.01%, <0.01% and 3.4% respectively.The extraction method of SD from pork, chicken, chicken liver, egg and milk was established, and the ELISA kit for SD residue detection was developed after that. The limit of quantitation in pork, chicken and chicken liver were all 20μg/kg with the recovery from 70% to 110%, and coefficient of variation (CV) in different lot below 26%. The limit of quantitation in egg and milk were both 5μg/kg with the recovery from 60% to 120%, and the interassay variability was below 32%.It is shown by compared with imported SD ELISA kit that our kit with the same accuracy and precision but not as sensitive as the kit made in UK.In animal experiment, chickens for muscle production or egg production were feed with forage mixed with SD in the concentration of 500 mg/kg for 7days or 400 mg/kg for 5days respectively, and the samples of successive 5 d or 7 d after using SD were detected by our kit and HPLC or imported kit at the same time. The results of our kit were accord with HPLC in detecting SD residue in chicken, and consistent with the imported kit in detecting SD residue in egg.All the results indicated that our ELISA kit can meet the need of screening SD residue in pork, chicken, chicken liver, egg and milk. The research work of kit development was accomplished basically.