Generation Of Anti-clenbuterol Monoclonal Antibodies And Development Of Enzyme Immunoassay For The Detection Of Clenbuterol Residues

This paper reported the preparation of CL-McAb and the development of enzyme immunoassay for the detection of clenbuterol residues.β2- agonist clenbuterol has been used as bronchiodilators in human medicine for over 30 years. Unfortunately, this agent has also been used illegally now as growth-promoter in livestock production. Its illegal use will led to toxic effects after human consumption of meat products. The analytical method for clenbuterol residues detection is urgently needed.Clenbuterol was a hapten. To prepare antigen, clenbuterol was conjugated to carrier proteins KLH, BSA and OVA by diazotization. CL-KLH and CL-BSA were used for the immunization of mice and CL-OVA was used as the coating antigen for the ELISA study. Both antiserums of CL-KLH and CL-BSA could bind specifically with CL. The anti-clenbuterol antibody titer of antiserum immunized with CL-KLH was higher than that with CL-BSA.Fore hybridoma secreting monoclonal antibodies against CL were achieved by injection of BALB/c mice with CL-KLH. These four McAbs were characterized for the cross reactivities, specificities, neutralizing abilities vs. CL and relative affinity. The titer degree of ascites fluid of these four McAb was 1 : 3×106~1 : 6×105. 1D6 showed 3.98% cross reaction with Salbutamol. Relative affinity showed 1D6>1H5>1H7>2F10.Clenbuterol was diazotized to be labelled with HRP. HRP-CL could bind with CL-McAb specifically. The samples was added in the ELISA plat wellswhich were coated with CL-McAb(lD6) and blocked with 10% rabbit serum. CL in the samples could bind with CL-McAb. After washing, the HRP-CL was added and incubated for 15min at 37℃. After the plat was washed again, OPD-H2O2 was added and the reaction was terminated by adding 2M H2SO4 in 15 min. The OD490nm values was read at 490nm in a microplat reader. The contraction of CL-McAb and dilution of HRP-CL were determined. The standard curve was obtained. This immunoassay provided a analytical method for the detection of clenbuterol residues and could be use in preparation of clenbuterol ELISA kit.