Objective To analyze protein antigens of Mycobacterium tuberculosis (M.tuberculosis) preliminarily;pre- pare 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 pro- tein antigen of M.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 of classical acid-fast stain.

The monoclonal antibodies against the 38 kD antigen of Mycobacterium tuberculosis were prepared and these antibodies after purification and labeled with FITC were used to establish the direct immunofluorescent antibody assay for the detection of M. tuberculosis in sputum specimens of patients. Under this circumstance, monoclonal antibodies against M.tuberculosis H37Rv strain was prepared by routine procedure with positive hybridoma clones selected by ELISA and identified with immunoblotting. And these monoclonal antibodies were purified with ammonia sulfate precipitation and Sephadex G-50 chromatography. In this way, 4 hybridoma cell lines producing monoclonal antibodies had been selected, and the titer of antibodies ranged from 1∶6 400 to 1∶12 800 demonstrated by ELISA assay. As confirmed by Western blot assay, all these 4 antibodies could bind specifically with 38 kD protein of M.tuberculosis and appearance strong bands of immune reaction. The positive rate M.tuberculosis in 41 sputum specimens of patients with tuberculosis direct by immunofluorscent antibody assay was 90.24%, significantly higher than that of acid-fast staining method (P0.05). These results suggest that this method of testing should be valuable to the preliminary clinical diagnosis of tuberculosis.

目的:制备抗结核分支杆菌的特异单克隆抗体,经纯化标记荧光后,建立直接荧光抗体染色方法用于痰标本的检测.方法:采用常规方法制备单克隆抗体.分泌单克隆抗体阳性杂交瘤细胞株的筛选采用ELISA法,确认采用免疫印迹法.单克隆抗体的纯化采用饱和硫酸铵粗提,Sephadex G-50层析纯化.单抗纯化后采用透析法标记异硫氰酸荧光素,初步应用于临床痰涂片的检测. 结果:经筛选获得4株单克隆抗体杂交瘤细胞株.ELISA检测小鼠腹水单克隆抗体效价达到1:6 400～1:12 800.免疫印迹实验表明4株单抗均为抗结核杆菌 38 KD 蛋白单抗,其中两株产生较强的免疫反应条带.单抗纯化后标记异硫氰酸荧光素,建立直接荧光抗体染色方法,对41份结核病患者的痰标本进行检测,阳性率为 90.24%,与抗酸染色法比较,直接荧光抗体染色方法敏感性明显高于抗酸染色法(P<0.05).结论:制备抗结核杆菌 38 KD 蛋白的单克隆抗体,并建立直接荧光抗体染色方法,初步应用于临床痰涂片检测中,对于辅助诊断结核病具有一定价值.

某私营印刷厂使用"三无"含苯稀料,车间内未安装任何通风排毒设施,职工缺乏自我防护意识,导致慢性重度苯中毒发生.

结核病是一种严重危害人类健康的慢性呼吸道传染病,其病原体是绔核分枝杆菌.据WHO估计全球约有20亿人感染,其中5%-10%会发展成活动性肺结核,每年新病例达800-1,000万,约300万人死亡[1].