抗丁丙诺啡单克隆抗体的制备

发布时间：2018-09-09 19:50

【摘要】：目的建立抗丁丙诺啡单克隆抗体的杂交瘤细胞株,制备高特异性的丁丙诺啡单克隆抗体,并对其免疫学特性进行鉴定。方法在丁丙诺啡的分子上连接活性羧基基团,通过缩合反应将丁丙诺啡半抗原连接于血蓝蛋白(KLH)和小牛血清白蛋白(BSA),形成完全抗原。以完全抗原免疫Balb/c小鼠,通过细胞融合,筛选等杂交瘤技术,建立稳定的分泌抗丁丙诺啡单克隆抗体的杂交瘤细胞株。通过腹腔注射杂交瘤细胞,诱导小鼠产生含有单抗的腹水。用辛酸-硫酸铵加亲和层析法纯化抗丁丙诺啡单克隆抗体。采用酶联免疫反应和胶体金膜层析实验测定丁丙诺啡单抗的特异性以及免疫反应动力学参数。结果共获得3株分泌抗丁丙诺啡单克隆抗体的杂交瘤细胞株,分别命名为7E6,6G4和3C2。7E6,6G4抗体灵敏度为10.0ng/ml,3C2抗体灵敏度为20.0ng/ml。7E6,6G4和3C2抗体的亲和常数分别为3.6×10-9mol/L,4.3×10-9mol/L和6.3×10-9mol/L。特异性测试结果表明7E6和6G4抗体与40种药物、毒品无任何交叉反应,而3C2抗体与吗啡有交叉反应。结论杂交瘤细胞株7E6和6G4产生的抗丁丙诺啡单克隆抗体具有很高的特异性和灵敏度。
[Abstract]:Objective to establish a hybridoma cell line with monoclonal antibody against buprenorphine and to prepare a high specific monoclonal antibody against buprenorphine and to identify its immunological characteristics. Methods the active carboxyl group of buprenorphine was attached to the molecule of buprenorphine, and the hemocyanin (KLH) and bovine serum albumin (BSA) (BSA), were linked to buprenorphine hapten by condensation to form a complete antigen. Balb/c mice were immunized with complete antigen. A stable hybridoma cell line secreting monoclonal antibody against buprenorphine was established by cell fusion and screening. Mice were induced to produce ascites containing monoclonal antibodies by intraperitoneal injection of hybridoma cells. Monoclonal antibodies against buprenorphine were purified by octanoic acid-ammonium sulfate and affinity chromatography. The specificity and kinetic parameters of buprenorphine monoclonal antibody were determined by enzyme linked immunosorbent assay (Elisa) and colloidal gold membrane chromatography. Results three hybridoma cell lines secreting monoclonal antibodies against buprenorphine were obtained and named as 7E6G4 and 3C2.7E6G4, respectively. The sensitivity of antibodies was 10.0 ng / ml ~ 3C2 and the affinity constants of 3C2 antibodies were 3.6 脳 10 ~ (-9) mol / L ~ (4.3 脳 10-9mol/L) and 6.3 脳 10 ~ (-9) mol 路L ~ (-1) 路L ~ (-1), respectively.The affinity constants of these two antibodies were 3.6 脳 10 ~ (-9) mol 路L ~ (-1) and 6.3 脳 10 ~ (-9) mol 路L ~ (-1). The specificity test showed that 7E6 and 6G4 antibodies had no cross reaction with 40 drugs, but 3C2 antibody had cross reaction with morphine. Conclusion Monoclonal antibodies against buprenorphine produced by hybridoma cell lines 7E6 and 6G4 have high specificity and sensitivity.
【作者单位】：上海市刑事科学技术研究所;
【基金】：“十一五”国家科技支撑计划项目基金(2006038022001)
【分类号】：D919

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