Preparation And The Preliminary Application Of The Monoclonal Antibodies Against Ractopamine

Ractopamine, one of theβ2-adrenergic agonists, which is used to cure the bronchitis of human, could promote growth, redistribute the nourishment in livestock production when used in large dose. However large dose of the drug will make human or animal toxicosis. So, the use of Ractopamine have banned as growth promoters in livestock in our country. This research aimed to prepare monoclonal antibodies against Ractopamine for establishment the cELISA method to detect the remains in food.In this study, Rac was linked with carriers though carboxyl which was reconstructed from the hydroxyl of the micromolecule, and the carries included KLH, BSA and OVA as soon as HPR. The recombination was identified by ultraviolet radiation. The consistence of the linked antigens was respectively 3.6mg/ml, 6.2 mg/ml, 4.4 mg/ml and 1.2 mg/ml. Six-week-old BALB/c mice were immunized with Rac-KLH and Rac-BSA respectively. The spleen cells of immunized mice were fused with Sp2/0 myeloma cells using PEG1500. Indirect ELISA was used to screen hybridoma cells with the Rac-OVA and limited dilution method was performed to subclone positive clones. Four cell lines, 1F7, 2G9, 4D2 and 5A9, wich could secrete McAbs were obtained. Though the method of indirect compete ELISA, 2G9 and 4D2 were confirm to be specifically against Rac, the IC50 of them were 1000ng/ml and 2ng/ml and they were IgM and IgG1 subtypes respectively. The ELISA titers of ascitic fluids were 10×28 and 10×210 respectively. The results of specificity test showed that the McAb had no cross-reactivity towards Clenbuterol and two carriers. As results above, 2G9 and 4D2 could be used as biomaterial to set up detecting method of Rac.The ELISA plate was coated with the McAb of 4D2, and the optimal density of the McAb and Rac-HRP were determined by the test of square matrix. Based on the above, direct competition ELISA was established for detecting the remains of Rac, .and the Rac samples ranging from 10ng/ml-100ng/ml.According to the detect results of the standard preparation, this method could be established as screening means of the Rac-remains. Sensitivity and specificity need be further elevated, and condition of clinical sample treat should be explored for more.