Generation of High Affinity Monoclonal Antibody and Development of Cielisa Kit for Rapid Detection of Ractopamine

Mixed anhydride(MA) was used to conjugate ractopamine(RAC) to bovine serum albumin(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 fusion.A ciELISA kit for detection of RAC(RAC-Kit) was developed with RAC mAb and its traits were tested.The results indicated that BSA-RAC was synthesized successfully and its conjugation ratio of RAC to BSA was about 24.5∶1.Three hybridoma lines were filtered and the best one was 4D8,its affinity constant(Ka) was 1.65×1010L/mol.The detection limit of RAC-Kit was 0.5ng/ml and its detection range was 0.5 to 151ng/ml.The recoveries of RAC spiked in feed were 87.2% and in swine urine were 89.4%.The precision and accuracy of the assay as determined by inter-assay and intra-assay coefficient variation were less than 15%,and had 9.7% cross-reactivity(CR%) to dobutamine,and little or no CR to other compounds.The validity of RAC-Kit in 4℃ was in 180d.