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目的 :建立抗克伦特罗 (CL)单克隆抗体杂交瘤细胞系 ,制备CL单克隆抗体 ,并鉴定其免疫学特性。方法 :用重氮化法将半抗原CL偶联于载体蛋白BSA ,形成结合抗原BSA CL ;以BSA CL免疫BALB/C小鼠 ,应用杂交瘤技术建立分泌CL单克隆抗体细胞株 ;用体内诱生腹水法生产CL单抗 ,硫酸钠盐析法提纯。应用抗小鼠同种型抗体测定单抗的同种型 ,用竞争性ELISA和胶体金膜层析试验测定CL单抗的反应特异性和反应动力学。结果 :筛选出C 4G1、C 2H6、C 2D6和C 4C94株高敏感性和高特异性杂交瘤生产单抗 ,其单抗的间接ELISA效价分别为 5× 10 -5、3× 10 -4、6× 10 -4、2× 10 -5;同种型分别为IgG1/κ、IgG1/κ、IgG2a/κ、IgG2a/λ。C 4G1的亲和力常数K =1 6 8× 10 8L/mol,C 2H6的亲和力常数K =1 3× 10 8L/mol。经WestGold蛋白质印迹鉴定单抗与CL特异性结合。C 4G1单抗对CL的半数抑制浓度 (IC50 )为 7 94ng/ml,对沙丁胺醇 (Sb)的IC50 为 10 0 0ng/ml,对肾上腺素、去甲肾上腺素、异丙肾上腺素和来克多巴胺的IC50 均≥ 6 4 0 0ng/ml;C 4G1单抗与Sb的交叉反应性为0 79% ,与异丙肾上腺素的交叉反应性为 0 12 % ,而与肾上腺素、去甲肾上腺素、来克多巴胺、各类抗生素及多种维生素均无交叉反应性。结论 :用单克隆抗体杂交瘤技术
Objective: To establish a monoclonal antibody against clenbuterol (CL) hybridoma cell lines, preparation of monoclonal antibodies CL, and to identify their immunological properties. METHODS: The hapten CL was coupled to the carrier protein BSA by diazotization to form the binding antigen BSA CL. BALB / C mice were immunized with BSA CL and the monoclonal antibody secreting monoclonal antibody was established by hybridoma technique. Ascites produced CL monoclonal antibody, sodium sulfate salting purification. Isotypes of monoclonal antibodies were determined using anti-mouse isotype antibodies and the reaction specificity and reaction kinetics of the CL mAbs were determined using competitive ELISA and colloidal gold-film chromatography assays. Results: The monoclonal antibodies against C 4G1, C 2H6, C 2D6 and C 4C94 were screened for high sensitivity and high specificity. The indirect ELISA titers of their McAbs were 5 × 10 -5 and 3 × 10 -4 , 6 × 10 -4, 2 × 10 -5, respectively. The isotypes were IgG1 / κ, IgG1 / κ, IgG2a / κ, and IgG2a / λ, respectively. The affinity constant of C 4G1 is K = 168 × 10 8 L / mol, and the affinity constant of C 2H6 is K = 13 × 10 8 L / mol. Monoclonal antibodies were identified as specific for CL by WesternGold Western Blotting. The IC50 of C 4G1 monoclonal antibody against CL was 7 94 ng / ml, the IC50 against salbutamol (Sb) was 100 ng / ml, and the effects of epinephrine, norepinephrine, isoproterenol and leprodopamine The IC50 of the C 4G1 monoclonal antibody was 0 79% for cross-reactivity with Sb and 0 12% for cross-reactivity with isoproterenol, while it was not significantly different from that for epinephrine, norepinephrine, To Dopamine, all kinds of antibiotics and multivitamin no cross-reactivity. Conclusion: Monoclonal antibody hybridoma technology