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Mixed anhydride(MA) was used to conjugate ractopamine(RAC)to BSA and obtained artificial antigen BSA-RAC identified by UV and SDS -PAGE. Balb/c mice were immunized with BSA-RAC and hybridoma lines that secrete RAC monoclonal antibody(mAb) were generated with cell fu-sion. A ciELISA kit for detection of RAC (RAC-Kit) was developed with RAC mAb and its performance were tested. The results indicated that BSA-RAC was successfully synthesized and its conjugation ratio of RAC to BSA was about 24.5:1. Three hybridoma lines were filtered and the best one was 4D8-3E11, its affinity constant (Ka) was 1.65×1010 L/mol. The limit of detection of RAC-Kit was 0.5 ng/ml and its detection range was 0.5-184 ng/ml. The mean recoveries of RAC spiked in feed were 85.6% and in swine urine were 88.6%. The precision and accuracy of the assay as determined by inter-assay and intra-assay coefficient variation were below 15%. It had 9.4% cross-reactivity (CR%) to dobutamine and little or no CR to other com-pounds. The validity of RAC-Kit in 4℃ was in 180 d.