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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1:6 400～1:12 800。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 preliminary analyze the protein antigens of Mycobacterium tuberculosis and to prepare specific monoclonal antibodies against Mycobacterium tuberculosis and to explore the feasibility of using specific immunofluorescence in the detection of Mycobacterium tuberculosis in sputum samples. Methods The protein antigens of Mycobacterium tuberculosis were preliminarily analyzed by acrylamide gel electrophoresis and Western blotting to identify specific and immunogenic protein antigens and to prepare high titer monoclonal antibodies against specific protein antigens using saturated sulfuric acid Ammonium crude extract, Sephadex G-50 chromatographic purification; at the same time preparation of high titer anti-TB Mycobacterium immune serum. With fluorescein isothiocyanate (FITC) labeled, initially used in clinical sputum smear detection. Results Four monoclonal antibodies against Mycobacterium tuberculosis 38 × 10 ~ 3 protein were obtained. The titer of McAbs against mouse ascites was 1: 640 ~ 1: 12 800 by ELISA. A direct fluorescent antibody staining method was established. Sixty sputum samples from patients with suspected pulmonary tuberculosis were detected by antacid staining, Mycobacterium tuberculosis immune serum and monoclonal antibody immunofluorescence. The positive rates were 36.7% (22 /60),58.3%(35/60) and 56.7% (34/60) respectively. Among them, 22 samples were positive for acid-fast staining, and both of them were positive by immunofluorescence. The coincidence rate was 100%. Conclusion Monoclonal antibodies against Mycobacterium tuberculosis 38 × 10 ~ 3 were successfully prepared. The positive rate of Mycobacterium tuberculosis detected by immunofluorescence in sputum specimens was much higher than that of acid-fast staining, which had a good prospect of application.