莱克多巴胺单克隆抗体间接竞争ELISA检测方法的建立

Development of an indirect competitive ELISA based on monoclonal antibodies for detection of ractopamine

用混合酸酐法将莱克多巴胺(Rac)与匙孔蓝蛋白(KLH)、牛血清白蛋白(BSA)偶联,经紫外光谱扫描确定偶联成功,测得偶联物Rac-KLH浓度为3.6 mg/mL,Rac-BSA浓度为6.2 mg/mL。以偶联物Rac-KLH作为免疫原,免疫6～8周龄雌性BALB/c小鼠,取小鼠脾细胞和骨髓瘤细胞SP2/O-Ag-14进行融合。以Rac-BSA作为包被抗原,经间接ELISA筛选出阳性细胞株,再用有限稀释法进行多次亚克隆,获得稳定分泌单克隆抗体的杂交瘤细胞株1D9、1F3、2C11、5C3、3A10、4A8、6B7、6D5、7C11、7D3。其中单克隆抗体5C3和3A10间接ELISA效价1∶500 000,对莱克多巴胺的半数阻断浓度(IC50)均为3.98 ng/mL,对盐酸克伦特罗和沙丁醇胺的IC50均大于2 000 ng/mL;对盐酸克伦特罗和沙丁醇胺的交叉反应率均小于0.2%。间接竞争ELISA检测莱克多巴胺在0.5～100 ng/mL范围内为线性分布,确定的最低检测限为0.5 ng/mL。

Ractopamine was coupled with keyhole limpet hemocyanin （KLH） and bovine serum albumin （BSA） to prepare complete antigens Rac-KLH and Rac-BSA, respectively. The two antigens were identified by ultraviolet radiation, with the concentrations of 3.6 mg,/mL and 6. 2 mg/mL. The spleen cells of BALB/c mice immunized by Rac-KLH were fused with SP2/0-Ag-14 myeloma cells using PEG4000. The Rac-BSA was used as the coating antigen for indirect enzyme linked immunosorbent assay （ELISA） to screen hybridoma cells, and limit- ed dilution method was performed to subelone the positive clones. Ten cell lines, which secreted monoclonal antibodies against ractopamine stably, were obtained. The indirect ELISA titers of ascites fluids of 5C3 and 3A10 were 1 ×10^-5. Competitive indirect ELISA （ciELISA） was developed based on the monoelonal antibodies. The eiELISA had a good sensitivity with an IC50 of 4 ng/mL and less than 0.2% cross-reactivity to clenbuterol and salbutamol. The linear range of ciELISA to detect ractopamine was 1 - 100 ng/mL and the detection limit of the assay was 1 ng/mL.