Development And Application Of Immunoassay Kits Based On Monoclonal Antiboty For Detection Of Ractopamine

Ractopamine is one of the β-adrenergic agonists, which can redistribute nutrition, promote protein synthesis, enhance lean meat to fat ration and improve feed conversion efficiency. However, accumulative residue of ractopamine in food producing animals can cause symptoms of food poisoning in human. Considering that the illegal use of ractopamine in livestock production would be harmful for human after consumption of meat products containing residual ractopamine, ractopamine was limited or banned to be utilized as growth promoters in livestock breeding. Our research aimed to prepare monoclonal antibodies against ractopamine for establishment the cELISA method and colloidal gold strips for detection the residual ractopamine.In this study ractopamine was conjugated with keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) based on mannish reaction, and the synthesized antigens were identified by ultraviolet radiation, with the concentrations of3.6mg/ml and6.2mg/ml respectively. The spleen cells of BALB/c mice immunized by RCT-KLH were fused with SP2/0-Ag-14myeloma cells using PEG4000. The RCT-BSA was used for the coating antigen for indirect enzyme linked immunosorbent assay (ELISA) to screen hybridoma cells, and limited dilution method was performed to subclone the positive clones. Nearly10cell lines including5C3and3A10, which stably secreted monoclonal antibodies against ractopamine, were obtained. The indirect ELISA titers of ascites fluids of5C3and3A10were1:5x105. In addition, competitive indirect ELISA (ciELISA) was established based on the monoclonal antibodies, and the IC50detected with ciELISA was3.98ng/ml for ractopamine, whereas the McAb had no cross-reactivity with either clenbuterol or salbutamol.Next, an indirect competitive ELISA kit was developed using the monoclonal antibody5C3and manipulation of the kit was investigated thoroughly. The optimal condition for the kit was as following:Coating at37℃for3h, then blocking another3h at37℃with1% gelatin, besides, the optimal concentration for coated antigen was1:1600, while the optimal working dilution titer of McAb was1:8000. The linear range of ciELISA to detect ractopamine was0.5ng/ml～100ng/ml and minimum detectable concentration was0.5ng/ml. The minimal amount of ractopamine to be detected from samples of pig fodder, liver, urine were0.01mg/kg,0.01mg/kg and0.5ng/ml, respectively.We further developed a colloidal gold strip to detect ractopamine using ractopamine-OVA conjugated antigen and monoclonal antibody specific to ractopamine labeled with colloidal gold. Results indicated that the strip has high sensitivity and specificity with detecting limitation of10ng/mL of ractopamine and no cross-reactivity to clenbuterol and salbutamol. The minimal amount of ractopamine to be detected from samples of pig fodder, tissue and urine were0.05mg/kg,0.05mg/kg and lOng/ml, respectively.In summary, we developed immunoassay kits including an ELISA kit and colloidal gold strips to detect residual ractopamine and the kits have been used in clinical detection by some govern departments. Clinical results indicated that the kits may replace the use of similar kits from foreign companies and have a promising future in application.