盐酸克仑特罗单克隆抗体的制备及特性鉴定

Preparation and characterization of monoclonal antibody to clenbuterol

目的:制备抗盐酸克仑特罗(clenbuterol,CL)的单克隆抗体并进行免疫学特性鉴定。方法:用重氮化法将盐酸克仑特罗偶联于载体牛血清白蛋白(BSA),形成的结合抗原BSA-CL作为免疫原免疫BALB/c小鼠,同时将CL与卵清白蛋白(OVA)偶联制备检测原(OVA-CL)。应用杂交瘤技术建立分泌抗盐酸克仑特罗单克隆抗体的杂交瘤细胞株,体内诱生腹水并进行纯化。用竞争性ELISA法测定抗体的亲和常数及交叉反应性。结果及结论:获得了18株稳定分泌抗盐酸克仑特罗单克隆抗体的杂交瘤细胞株,挑选其中E9-C11、C11-E3、E7-G8三株高亲和力的细胞株进行免疫学特性鉴定。三株抗体的亚类均为IgG1。细胞上清的间接ELISA效价分别为1∶1 280、1∶1 000、1∶800,三株腹水效价都约为1×10-6。亲和常数分别为2.4×10-8mol/L、2.83×10-8mol/L、3.28×10-8mol/L。E9-C11单抗对CL的IC50为6.1035μg/L;对沙丁胺醇的IC50大于781.25μg/L,交叉反应性小于0.78%;对肾上腺素、去甲肾上腺素、异丙肾上腺素、莱克多巴胺及部分维生素和抗生素均无交叉反应性。蛋白免疫印迹分析证明三株单抗特异性均很高,可用于与其相关的免疫检测或应用研究。

Objective：To produce and identify monoclonal antibodies against clenbuterol（CL）. Methods：CL-conjugated antigens （BSA-CL and OVA-CL） were produced by coupling diazotized CL with bovine serum albumin （BSA） and ovalbumin（OVA） , respectively, Hybridoma cell lines were established for specificity to CL by using the conventional hy- bridoma technique. Results and Conclusion： Eighteen hybridoma cell lines were established. The ELISA titer of hybridoma supematants of three clones in the 18 lines were 1：1 280 in E9-C11,1：1 000 in Cll-E3,and 1： 800 in E7-G8. The ascites titers of all three clones were 1 × 10^-6. The affinity constant was 2.4 × 10^ -8 moL/L ,2.83 × 10^-8 moL/L ,and 3.28 ×10-^8 moL/L, respectively, and all three McAb belonged to IgGT subclass. McAb of E9-Cll indicated high sensitivity with an IC5o of 6. 1035 μ g/L to CL, higher than 781.25 la, g/L to salbuterol, and showed lower than O. 78% cross activity towards salbuterol, no cross reactivity towards epinephrine, norepinephrine, isoprenaline, leukodopamine,antibiotics and vitamin. Furthermore, protein blotting assay showed that these McAb had high specificity to CL and can be used to detect CL residues.