摘要
目的制备抗结核分枝杆菌单克隆抗体,经纯化后标记荧光素,建立直接免疫荧光法用于痰标本的结核分枝杆菌检测。方法常规方法制备单克隆抗体。ELISA法筛选阳性杂交瘤细胞株,用免疫印迹法确认。单克隆抗体采用饱和硫酸铵粗提和SephadexG-50层析纯化。单抗纯化后采用直接法标记异硫氰酸荧光素,标记后的荧光抗体用于临床痰涂片结核分枝杆菌的检测。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∶6400—1∶12800。免疫印迹实验表明4株单抗均为抗结核分枝杆菌38kDa蛋白单抗,其中两株产生较强的免疫反应条带。采用异硫氰酸荧光素标记纯化的单抗,建立直接免疫荧光检测法,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,直接荧光检测法敏感性显著高于抗酸染色法(P<0.05)。结论应用抗结核分枝杆菌38kDa蛋白的单克隆抗体,建立直接免疫荧光检测法,应用于临床痰标本结核分枝杆菌检测,对于辅助诊断结核病具有一定价值。
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 （P〈0. 05）. These results suggest that this method of testing should be valuable to the preliminary clinical diagnosis of tuberculosis.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2007年第5期459-461,473,共4页
Chinese Journal of Zoonoses
关键词
结核分枝杆菌
单克隆抗体
直接免疫荧光检测
Mycobacterium tuberculosis
monoclonal antibody
direct immuno-fluorescent antibody assay
