Preparation And Preliminary Characterization Of Clenbuterol Monoclonal Antibody

In order to obtain hybridoma cell line producing monoclonal antibody against clenbuterol (CL), CL was diazotized and directly conjugated with three kinds of carrier protein, Human Sera Albumin (HSA), Bovine Sera Albumin (BSA) and Ovalbumin (OVA). The conjugates were identified by uv-visible recording spectrophotometer, which demonstrate that the artificial antigens were synthesized successfully.Using CL-HSA as an immunogen, antiserum was taken from Balb/c mice's tail vein and detected by double agarose immunodiffusion (DADT). After the fourth booster injection, the highest titer of antiserum is 1: 16. No significant difference in titer value and in specifity was observed between four doses (125 vs 100vs 75 vs 50ug per mouse) of immunogen and two breaks (2 vs 3 weeks). Two of the mice were scarified and antiserum was used to develop an indirect enzyme-linked immunosorbent assay (ELISA) with 2 g/ml CL-OVA as coating antigen for selecting the positive cloning wells.Stimulated splenic lymphocytes from the mouse giving best immunizing response were fused with SP2/0 myeloma cells to produce hybridomas. 21 stable positive wells were selected by ELISA and 3 highest positive wells of A-76, C-710 and C-611 were cloning. 3 stable hybrids CLA-76, CLC-710, CLC-611 producing monoclonal antibodies, of some subclass of IgG, were isolated after three cloning. The ELISA tilers of the ascites were 1.6 105, 1.24 l06, and 9.1 10s respectively, and the specificity of theMcAbs is measured by competitive inhibition enzyme linked immunosorbent assay, in which 1: 4 104 of ascites were used. The consentration of clenbuterol inhibiting 50% (ICso) for CLA-76, ILC-710, CLC-611 were 10 ng ml-1, 103 ng ml-11, 102 ng -ml-1. The McAbs specific for clenbuterol will be very useful for studying the immunoassay in the determination of clenbuterol residues in biological sample.