抗结核分枝杆菌脂阿拉伯甘露聚糖ELISA的建立及其临床应用

Establishment of ELISA for lipoarabinomannan from Mycobacterium tuberculosis and its clinical application

目的提取纯化结核分枝杆菌(MTB)脂阿拉伯甘露聚糖(LAM),建立抗LAM抗体(LAM-IgG)检测的酶联免疫吸附试验(ELISA)。方法 MTB菌体彻底破碎后,去除蛋白、脂质、核酸,得到粗制的脂多糖。经Sephacryl-100凝胶过滤层析分离纯化,得到LAM。以该LAM作为间接ELISA的包被抗原来检测血清中的LAM抗体。结果所得LAM经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)银染,与对照标准品大小一致,为37 000。115例肺结核患者LAM抗体阳性率71.3%;92例非结核肺部疾病患者假阳性率11.9%;188名健康体检者假阳性率3.2%。敏感性71.3%,特异性93.9%。结论本研究成功提取到LAM抗原,此抗原可作为间接ELISA的包被抗原用于结核抗体的检测。

Objective To extract and purify lipoarabinomannan （LAM）from Mycobacterium tuberculosis （MTB） and establish a method of enzyme-linked immunosorbent assay （ELISA） for the detection of anti-LAM antibody （LAM- lgG）. Methods The MTB was crashed. The protein, nucleic acid and lipids were removed, and crude lipopolysaccharides were prepared. The crude lipopolysaccbarides were purified by Sephamyl-100 gel filtration, and LAM was obtained. The LAM was used as coating antigen to detect LAM-IgG by indirect ELISA. Results The relative molecular weight of the prepared LAM was determined by silver staining of sodium dodecyl sulfate polyacrylamide gel electrophoresis （SOS-PAGE）. The result was approximately 37 000, consistent with the reference material. The positive rate of LAM-IgG was 71.3% in 115 pulmonary tuberculosis patients. The false positive rates were 11.9% in 92 other non-tuberculosis lung disease patients and 3.2% in 189 healthy subjects. The sensitivity and the specificity were 71.3% and 93.9%. Conclusions LAM antigen is prepared successfully and can be used as coating antigen in indirect ELISA to detect anti-MTB antibody.