Development Of A ELISA For Detection Of Ractopamine Based On Monoclonal Antibodies

Ractopamine is one of theβ2-adrenergic agonists. The illegal use of Ractopamine in livestock production has led to symptoms of food poisoning after human consumption of meat products. So, the use of Ractopamine was banned or limited as growth promoters in livestock. Our research aimed to prepare monoclonal antibodies against Ractopamine for establishment the cELISA method for detection.In this study, hapten Rac was linked with a new carrier-cationised bovine serum albumin, and the synthesized antigen was identified by ultraviolet radiation. The consistence of the linked antigen is 0.70mg/ml. The spleen cells of BALB/c mice immunized by Rac-cBSA were fused with SP2/0-Ag-14 myeloma cells using PEG4000. Rac-OVA, as the coating antigen, was synthesized by mixed acid anhydride method. Indirect enzyme linked immunosorbent assay (ELISA) was used to screen hybridoma cells and limited dilution method was performed to subclone the positive clones. Two cell lines, 5D2 and 6B3, which secrete monoclonal antibodies against Ractopamine stably were obtained by the technology of hybridoma. Both of them were IgM subtypes. The ELISA titers of ascites fluids were 2×105 and 1×105. The sensitivity and specificity was identified by a competitive inhibition enzyme-linked immunosorbent assay (cELISA). The IC50 of 6B3 was 0.01ng/ml, and the IC50 of 5D2 was 0.1ng/ml. The McAbs have no cross-reactivity towards Clenbuterol and two carriers. The results showed that these two cell lines were good material to set up detecting method of Ractopamine.An indirect competitive ELISA mode was founded base on the monoclonal antibody. The best condition of coating is 37℃react 3h.and then block it in 37℃for 3h. The best concentration of coating antigen was 1:1600, the optional working dilution of McAb was 1:8000. The linear detecting range of calibration curves to detect Ractopamine was 0.1ng/ml-100ng/m1.The formula was Y=-0.4052X+0.0111, R2=0.9815. cELISA was a very promising method for Rac detection. The various factors and conditions for ELISA kits were to be explored,and the optimal reaction conditions need to be determined in further research.