抗丁丙诺啡单克隆抗体的制备

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。7E6，6CA和3C2抗体的亲和常数分别为3．6×10^-9 mol／L，4．3×10^-9 mol／L和6．3×10^-9 mol／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。

Preparation and characteristics of anti-buprenorphine monoclonal antibodies

ObjectiveTo prepare anti-buprenorphine monoclonal antibodies through hybridoma cell line technique,and identify their immunological characteristics.MethodsAfter introduced an active carboxyl group,the hapten buprenorphine was linked to the blue blood(KLH) and bovine serum albumin(BSA) through condensation reaction to form a complete antigen.Immunized the Balb/c mice with the complete antigen,the hybridoma cell line which could secrete anti-buprenorphine monoclonal antibodies was acquired via cell fusion and screening.Then,the anti-buprenorphine monoclonal antibody was produced by intraperitoneal injection with the hybridoma cell.After purified with octoic acid-ammonium sulfate and affinity chromatography,the anti-buprenorphine monoclonal antibody was tested for the affinity and specificity by ELISA and Gold colloid immunochromatographic method.ResultsA total of three cell lines,named as 7E6,6G4 and 3C2 respectively,were finally obtained.For 7E6 and 6G4,the sensitivity for detecting buprenorphine was 10.0 ng/ml,and 3C2 was 20.0 ng/ml.The affinity constants of 7E6,6G4 and 3C2 antibodies were 3.6×10-9 mol/L,4.3×10-9 mol/L and 6.3×10-9 mol/L respectively.Except for 3C2 antibody with morphine,no any more cross reaction was observed for these antibodies with 40 control substances.ConclusionThe anti-buprenorphine monoclonal antibodies produced by hybridoma cell lines 7E6 and 6G4 are of high sensitivity and specificity.