结核分枝杆菌特异性38kD多肽抗原提纯及其血清学诊断价值的研究

目的提纯结核分枝杆菌特异38kD多肽抗原,建立基于38kD多肽抗原(P38)的斑点金免疫渗滤法(DIGFA)检测结核病人血清标本,评价38kD多肽用于结核病血清学诊断抗原的实际价值。方法采用超声破碎、SDS-PAGE、切胶浸泡回收法提取结核分枝杆菌H37Rv株的38kD多肽抗原。建立以38kD多肽为包被抗原的DIGFA(P38-DIGFA),并检测147例临床确诊的活动性肺结核病人血清标本。同时建立以结核分枝杆菌脂阿拉伯甘露糖(LAM)为包被抗原的DIGFA(LAM-DIGFA)作为对照。结果结核分枝杆菌H37Rv株38kD多肽抗原提取物经SDS-PAGE后仅显示为单一的条带,并可与临床确诊的肺结核病人阳性血清标本呈阳性免疫印迹反应。P38-DIGFA和LAM-DIGFA检测肺结核病人血清标本的总阳性率分别为84.4%(124/147)和76.2%(112/147),其中59份痰涂片及培养均阳性的肺结核病人血清标本P38-DIGFA和LAM-DIGFA阳性率分别为88.1%(52/59)和84.7%(50/59),88份痰涂片及培养均阴性的肺结核病人血清标本P38-DIGFA和LAM-DIGFA阳性率分别为81.8%(72/88)和70.5%(62/88),60例气管炎病人血清标本阳性率分别为13.3%(8/60)和6.7%(4/60),120例健康人血清标本阳性率分别为5.8%(7/120)和5.0%(6/120)。各P38-DIGFA和LAM-DIGFA检测阳性率之间均无显著性差异。结论结核分枝杆菌38kD多肽作为抗原用于结核病血清学检测时,其敏感性和特异性与LAM相似,可应用于研发结核病血清学诊断试剂盒。

Purification of the 38 kD peptide antigen of Mycobacterium tuberculosis and its value in the serological diagnosis for tuberculosis

The 38 kD peptide had been confirmed as the specific antigen of Mycobacterium tuberculosis. In the present study, this peptide was purified from M.tuberculosis H37Rv strain by using ultrasonic breakage, SDS-PAGE and immersion recovery from gel and established the DIGFA assay with 38 kD peptide used as the coating antigen (P38-DIGFA). The P38-DIGFA assay was used to detect the specific antibodies in serum samples of 147 cases of patients with active tuberculosis. Meanwhile, the DIGFA assay with LAM as the coating antigen (LAM-DIGFA) was applied as control. It was demonstrated that the 38 kD peptide extracted from the H37Rv strain showed just only a single band in gel after SDS-PAGE, and the positive immunoblot reaction could be detected with the positive serum samples of clinically diagnosed patients with tuberculosis. The total positive detection rates in 147 serum samples of patients with tuberculosis using P38-DIGFA and LAM-DIGFA assay were 84.4% (124/147) and 76.2% (112/147) respectively. For serum samples from 59 patients with positive direct smear and cultivation results and 88 patients with negative direct smear and cultivation results, the positive detection rates were 88.1% (52/59), 84.7% (50/59) and 81.8% (72/88), 70.5% (62/88) respectively. However, in 60 serum samples from patients with tracheitis and 120 serum samples from healthy adults, the positive rates of these two assays were 13.3% (8/60), 6.7% (4/60) and 5.8% (7/120), 5.0% (6/120) respectively. No significant difference in the positive rates between P38-DIGFA and LAM-DIGFA assay could be demonstrated. The results observed in the present study lead to such a conclusion that the sensitivity and specificity of P38-DIGFA are closed to those of LAM-DIGFA assay, thus suggesting to the potential use of the 38 kD peptide as the antigen for further development of serological diagnosis kits.