Development Of Anti-Clenbuterol Hydrochloride Myosin Monoclonal Antibodies And Study Their Application In Immunise Diagnosis

Clenbuterol Hydrochloride (CL), one of theβ-adrenergic agonists, when animals eated it, they can significantly increase the growth rate, so that the relative increase in lean meat. But its easy to precipitate in vivo, human consumption of animal products containing the drug residue, may lead to food poisoning, severe cases leading to death. Therefore, CL was banned in food production applications. In order to strict supervision and control of CL abuse, the researchers developed a namber of test methods.The experiments which focus on the development of monoclonal antibodies against clenbuterol launched a four-level research work, for the basic research of immunological detection methods.Experimental content and the results are as follows:(1) Preparation of CL complete antigen:Conjugates of (CL) to protein bovine serum albumin (BSA) and ovalbumin (OVA) were prepared via diazo coupling to form immunogen BSA-CL and OVA-CL.Identification by uv scanning and SDS-PAGE, which shows the peak changed, before and after coupling of carrier protein OVA and BSA, SDS-PAGE results showed that the molecular weight conjugates of molecular weight greater than the carrier protein. And with the CL-BSA immunized mice to produce antibodies, using CL-OVA-coated elisa plate, Detection of antibodies can be obtained free of CL specific binding reaction occurs.(2)CL-BSA was prepared as antigen to immunize BALB/C mice. Using hybridoma technique, the hybridoma cells were obtained by fusing spleen cells of BALB/C mice to myeloma cells SP2/O and cultured in the HAT selective medium. Designated 7 CL-McAbs(3)Establishment of competitive CL ELISA method:ELISA optimal reaction conditions were:the CL-OVA concentration of coating antigen is 0.4μg/mL, that of antibody is 1:600, the best conditions for coated overnight at 4℃16h, the best blocking agent for the 2% BSA prepared with pH7.4 PBS,37℃competition is about 30min, linear range 0-8ng/ml, sensitivity was 0.2ng/ml.(4)Dewelopment of colloidal gold immunochromatography for detection of CL Nanocolloidal gold particles were prepared and labeled to an Anti- Clenbuterol monoclonal antibody. CL was conjugated to bovine serum albumin (BSA) and dispersed on a nitrocellulose membrane to be the test line (T). The more analyte present in the sample, the more effectively it would compete with the CLEN - BSA for binding to the limited amount of gold- labeled anti-Clenbuterol monoclonal antibody. The presence or absence of a colored band on the test line indicated a negative or positive result. Result When measuring the water sample spiked with CL, the minimum detection concentration could reach 0.5 ng/ml.