抗结核分枝杆菌特异抗原单克隆抗体的制备和特异荧光染色检测技术的研究  

Preparation of the Monodonal Antibody against the Specific Antigen of Mycobacterium Tuberculosis and Establishing the Specific Immunofluorescent Assay

作　　者：张选民[1] 魏云芳[2] 曾令城[1] 刘志广[2] 王西临[1] 赵秀芹[2] 张媛媛[2] 万康林[2] 

机构地区：[1]西安市疾病预防控制中心,陕西710054 [2]传染病预防控制国家重点实验室中国疾病预防控制中心传染病预防控制所,北京102206

出　　处：《中国预防医学杂志》2008年第11期942-945,共4页Chinese Preventive Medicine

基　　金：国家专项经费"公共卫生突发应急反应机制运行项目";西安市社会攻关项目"提高结核病发现率新技术的研究"(GG06165)

摘　　要：目的对结核分枝杆菌的蛋白抗原进行初步分析，制备抗结核分枝杆菌的特异单克隆抗体，探讨特异性免疫荧光法在痰标本结核分枝杆菌检查上应用的可行性。方法采用丙烯酰胺凝胶电泳和蛋白免疫印迹技术对结核分枝杆菌的蛋白抗原进行初步分析，确定特异、免疫原性强的蛋白抗原，制备抗特异蛋白抗原的高效价单克隆抗体，采用饱和硫酸铵粗提，Sephadex G-50层析纯化；同时制备高效价的抗结核分枝杆菌免疫血清。用异硫氰酸荧光素（FITC）标记，初步应用于临床痰涂片的检测。结果获得4株针对结核分枝杆菌38×10^3蛋白抗原的单克隆抗体，ELISA检测小鼠腹水单克隆抗体效价达到1：6400～1：12800。建立了直接荧光抗体染色方法，60份临床疑似肺结核病人痰标本，用抗酸染色法、结核分枝杆菌免疫血清和单克隆抗体免疫荧光法三种方法检测，阳性率分别为：36．7％（22／60）、58．3％（35／60）和56．7％（34／60），其中，抗酸染色阳性22份标本，两种免疫荧光法检查均为阳性，符合率100％。结论成功制备出抗结核分枝杆菌38×10^3蛋白的单克隆抗体。免疫荧光法检查痰标本结核分枝杆菌阳性率大大高于抗酸染色，具有良好的应用前景。Objective To analyze protein antigens of Mycobacterium tuberculosis （M.tuberculosis） preliminarily; prepare the monoclonal antibody （McAb） against the specific antigen of M. tuberculosis and probe into the feasibility with specific immunofluorescence technique on determination of the sputum M. tuberculosis. Methods Protein antigens of M. tuberculosis were analyzed preliminarily with sodium dodecyl sulfate -polyacrylamide gel electrophoresis （SDS -PAGE） and Western blotting （WB） for identification of the specific protein antigens. The monoclonal antibody against the specific antigen of M. tuberculosis was prepared with the McAb technique. The McAb was purified by the saturated solution of ammonium sulfate and column chro- matography with Sephadex G-50. Immuno-sera against M. tuberculosis were prepared with conventional rabbits. The immuno-sera and McAb were marked with fluorescein isothiocyanate （FITC）. Sputum was smeared and firmed then added with fluores- cence antibody, observed with fluorescence microscope. Results Four cell strains produced high - titer McAb against 38 kD protein antigen of M. tuberculosis have been obtained. The titer in mice ascites was from 1：6 400 to 1：12 800 by enzyme - linked immunosorbent assay （ELISA）. With the immuno -sera and McAb marked with FITC the specific direct immunofluorescent as- say （DFA） has been set up. There were 60 sputums of clinical suspect pulmonary tuberculosis cases in all were detected by acid -fast stain, the immuno-sera DFA and McAb DFA, of which the positive- rate of M. tuberculosis was 36. 7% （22/60）, 58.3% （35/60） and 56.7% （34/60） respectively. Among these detecting, positive of sputums with acid - fast stain were all positive in immunofluorescence tests, the coincidence-rate was 100%. Conclusion A high - titer McAb against 38 kD protein antigen of 31. tuberculosis has been prepared successfully. The positive - rate of the immuno - sera DFA and McAb DFA tested M. tuberculosis in sputums was much higher than that o

关 键 词：结核病 结核分枝杆菌 单克隆抗体 直接免疫荧光抗体法 

分 类 号：R392.1[医药卫生—免疫学]