Preparation Of Anti-clenbuterol Monoclonal Antibody

Clenbuterol, what usually called “lean” or “nutrient redistribution agent”, is aselective β-adrenergic receptor kinetin, can significantly increase the growth rate ofanimals and reduce the synthesis of fat of animals, which often used as feed additivesby unscrupulous traders to reap the economic benefits. After entering the body,Clenbuterol is easily to be absorbed. Due to longer half-life in vivo, residues is easilyaccumulated in animals. People absorb the residual clenbuterol of meat products willlead to different degrees of poisoning phenomenon. So this drug is forbidden duringprocess of food production.There are different ways to detect residual clenbuterol in food including physicaland chemical detection methods, immunological detection methods and biosensortechnology, among which ELISA technique has been rapid development and widelyused because of its high sensitivity，can be quantitative, ease of operation and low cost indetection of drug residues.As the molecular weight of Clenbuterol is only313.7, it belongs to a smallmolecule hapten, which only has reactogenicity and do not has immunogenicity. Sothe first step of this experiment would be synthesis of artificial antigen bydiazotization method. CL-BSA and CL-OVA was obtained by means of couplingClenbuterol small molecules and macromolecular carrier, as bovine serum albumin(BSA) and ovalbumin (OVA). The coupling ratio of CL-BSA and CL-OVA were27.4:1and12.0:1respectively, and the identification of artificial antigen wasconducted by SDS-PAGE gel electrophoresis and UV scanning methods subsequently.Results of the electrophoresis showed that molecular weight of artificial antigen wassignificantly higher than the carrier protein and the identification results of UVscanning show the UV absorption peak of the artificial antigen contains characteristicpeak of clenbuterol and macromolecular carrier protein, and there has been some shiftin the basis; thus results proved production of complete antigens.BALB/c mice were immunized with complete antigen CL-BSA. Post-immunization the indirect ELISA method was conducted to detect mouse polyclonalantiserum antibody (pAb) titers, and the determination of sensitivity of pAb used indirect competitive ELISA method. The measurement results showed that miceantiserum titer of1:12800can be achieved, and the determination of serum sensitivityexperiments also showed that mice produced antibodies against CL. The IC50of theantiserum was160ng/mL. The results illustrated the immunogenicity of the completeantigens were good and the preparations of the antigens were successful.NO.2BALB/c mouse,with high serum antibody titer and good sensitivity, wasselected for cell fusion. After fusing Splenocyte of mouse and myeloma NS0withPEG, hybridoma cells were obtained. And indirect ELISA and indirect competitiveELISA were used to screen the hybridoma cells. As a result, a anti-CL hybridoma cellline2E8, with a supernatant IC50of37.28ng/mL, was isolated.This study successfully prepared immunogen CL-BSA and coating antigenCL-OVA, and got a anti-CL hybridoma cell line, laying foundation for futurepreparation of anti-CL kits.