双抗原夹心胶体金免疫层析法快速检测结核分枝杆菌抗体的研究

A study of double antigen sandwich colloidal gold immunochromatography rapid detection forMycobacterium tuberculosis antibody

目的 建立一种简易、快速和准确的双抗原夹心胶体金免疫层析法，用于检测结核病患者血清中的抗MTB抗体。方法采用胶体金标记相对分子质量分别为6000、16000和38000的重组MTB早期分泌抗原靶蛋白（ESAT），成为重组MTB融合蛋白ESAT-6-16-38，研制出双抗原胶体金免疫层析检测卡，对浙江省德清县疾病预防控制中心2007--2009年临床确诊的结核病患者血清163份（其中肺结核痰MTB阳性57份、痰MTB阴性64份和肺外结核42份）和对照血清573份（其中体检者血清224份、急性肺炎和支气管炎患者血清217份、肺吸虫患者血清132份）样本进行检测，并与MTB蛋白芯片抗体法的检测结果进行比较。检出率的比较采用X。检验。结果双抗原法和蛋白芯片法检测结核病患者血清的阳性符合率分别为73．O％（120／163）和72．4％（118／163），差异无统计学意义（x。=0．062，P〉0．05）；检测对照血清的阴性检出率分别为93．9％（538／573）和92．0％（527／573），差异无统计学意义（x。=0．635，P〉0．05）；未见与肺吸虫患者血清有交叉反应，双抗原法对痰MTB阳性血清的检出率高达87．7％（50／57）。结论重组ESAT-6和ESAT．16双抗原胶体金免疫层析检测卡具有较高的敏感度和特异度，与蛋白芯片法的检测结果相似，且方法简便、快速，有很好的重现性和稳定性。

Objective To establish a simple, fast and accurate double antigen colloidal gold immunochromatographie technique for detecting Mycobacterium tuberculosis antibody from tuberculosis patients. Methods The fusion protein ESAT-6-16-38 was constructed by using gene cloning technique for the 6, 16 and 38 kDa early secreted antigenic target from Mycobacterium tuberculosis. The ESAT-6-16-38 fusion protein was marked to colloidal gold to establish the double antigen colloidal gold immunochromatographic assay. Serum samples from 163 patients with tuberculosis, including 57 sputum- positive cases, 64 sputum-negative cases, and 42 cases with extrapulmonary tuberculosis, were collected during 2007and 2009 from the Disease Prevention and Control Center of Deqing County. In addition, 573 controls （224 healthy volunteers, 217 patients with acute pneumonia and bronchitis, 132 patients with paragonimiasis） were recruited for comparison. Mycobacterium tuberculosis specific antibodies were detected by using immnnochromatographic and protein chip technique. Detection rate was compared with Chi-square test. Results Among the 163 tuberculosis patients, the positive rates of immunochromatographic detection and protein chip were 73.0% （ 120/163 ） and 72. 4% （ 118/163 ） respectively; the difference was not statistically significant （ X^2 = 0. 062, P 〉 0.05 ） . Among the 573 controls, the negative rates of immunochromatographic detection and protein chip were 93.9% （ 538/573 ） and 92. 0% （ 527/573 ） respectively; the difference was not statistically significant （ X^2 = 0. 635, P 〉 0. 05 ）. There was no cross reaction in the paragonimiasis patients. The positive rate of the immunochromatographie assay was as high as 87. 7% （50/57） in the sputum-positive patients. Conclusions The double antigen immunochromatographie technique is an easy to operate, rapid, highly sensitive, specific, and reproducible method for thedetection of Mycobacterium tuberculosis antibodies.