【摘要】 目的　利用单克隆抗体技术分析分支杆菌多肽抗原的共性和个性 ,建立纯化单克隆抗体的最佳方法。方法　按常规方法制备单克隆抗体。分泌单克隆抗体阳性杂交细胞株的筛选采用ELISA法 ,确认采用免疫印迹法。 2 4种分支杆菌菌株均生长于改良罗氏培养基上 ,H3 7Rv株分别生长于改良罗氏培养基和苏通液体培养基上。单克隆抗体的纯化采用硫酸铵沉淀法、辛酸沉淀法和离子交换法。结果　经筛选获得 14株单克隆抗体杂交细胞瘤。其中 ,C2 、7C12 单克隆抗体所抗的 76 0 0 0、16 0 0 0抗原成份为分泌抗原 ,76 0 0 0抗原存在于 2 4种分支杆菌菌体中 ,为共同抗原。 16 0 0 0抗原仅存在于H3 7Rv ,H3 7Ra,牛分支杆菌 ,BCG株中。单克隆抗体免疫印迹结果显示 ,分支杆菌H3 7Rv株在不同培养条件下 ,其 40 0 0 0 / 380 0 0多肽抗原表达不同。单克隆抗体纯化以硫酸铵沉淀法回收率最高 ,离子交换法获得的单抗纯度最高 ,辛酸沉淀法可以有效地去除白蛋白 ,并具有简单、省时等优点。单克隆抗体的相对亲和力均在 10 -4 ～ 10 -7之间。结论　C2 、7C12 单克隆抗体均抗结核分支杆菌H3 7Rv株的分泌抗原成份。 40 0 0 0 / 380 0 0多肽抗原为结核分支杆菌H3 7Rv株固体培养菌体特有。单抗纯化结果显示离子交换层析法适用于科研分析 ,而辛酸更多还原

【Abstract】 Objective Using monoclonal antibody (McAb) technique to analyze polypeptide antigens of Mycobacterium strains and try to find out the best method for purification of McAb.Methods McAbs against H 37 Rv strain were produced by routine procedure. McAb producing hybridomas were detected by ELISA and immunoblots technique. 24 strains of mycobacterium were grown on Loewenstein Jensen medium for 1 to 3 weeks at 37℃. H 37 Rv strain were grown on Loewenstein Jensen medium or Sauton liquid medium. Ammonium sulfate, caprylic acid precipitation and ion exchange chromatography methods were used to purify McAbs against antigens of H 37 Rv strain.Results 14 McAb produing hybridomas were obtained. C 2 McAb reacted with all 24 strains of mycobacterium and 7C 12 only reacted with H 37 Rv, H 37 Ra, M. bovis ,BCG strains, C 2 and 7C 12 reacted with secreted proteins of H 37 Rv strain. 40 000/38 000 antigen of H 37 Rv strain was expressed differently in Loewenstein Jensen medium and Sauton liquid medium. Relative activity of all McAbs was between 10 4 ～10 7 .The results of purified McAb showed that ammonium sulfate precipitation method could have the highest rate of recovery. Ion exchange chromatography method could have the highest purity. Caprylic acid method can remove albumin effectively and the purification of McAbs in ascific fluid was simple and easy to perform.Conclusions Polypeptide antigens of H 37 Rv against by C 2, 7C 12 McAb were the secreted proteins. For purification of McAb, the results suggested that ion exchange chromatography method can be applied for analysis and caprylic acid method for a very large volume of ascitic fluid.更多还原