结核分枝杆菌脂多糖的提取及其检测血清结核抗体的研究

The extraction of Mycobacterium tuberculosis lipopolysaccharide and the detection of anti-lipopolysaccharide antibody in sera

目的提取结核分枝杆菌(MTB)脂多糖抗原,检测结核血清参考品,评价其在血清学诊断中的价值。方法将MTB菌体冰浴超声破碎,用酚/氯仿、氯仿/甲醇抽提除去蛋白和脂质,再用DNase I酶和RNase酶作用除去DNA和RNA,得到初步纯化的脂多糖即LPS-1,LPS-1在去污剂Triton-X114作用后,分成含有阿拉伯甘露糖(AM)的水相即LPS-2和含有脂阿拉伯甘露糖(LAM)和脂甘露糖(LM)的有机相即LPS-3,以三种多糖作为抗原,通过ELISA方法检测结核阳性和阴性血清参考品。结果LPS-1对结核血清参考品检测的敏感性为71.7%,特异性为96%,LPS-3的敏感性为43.9%(18/41),而特异性达到100%。结论脂多糖抗原检测血清结核抗体显示出较强的特异性,可望成为结核病血清学的诊断组合抗原之一。

Objective To obtain the lipopolysaccharide of Mycobacterium tuberculosis to detect TB refference sera, and to evaluate its potential value for serodiagnosis of tuberculosis. Methods Lyse Mycobacterium tuberculosis in ice by sonication, and then treat with phenol/chloroform, chloroform/methanol, DNase I and RNase. Remove proteins, lipide and nucleotides from the cellular glycans and lipopolysaccharide （LPS-I） by a step of dialysis. LPS-I was separated into a aqueous phase （LPS-2） containing arabinomannan （AM） and an amphipathic detergent-rich phase （LPS-3） containing lipoarabinomannan （LAM） and lipomannan （LM） by Triton- X114 phase separation technique. These LPSs were used as antigens to detect TB postive and negative refference sera by indirect ELISA method. Results The sensitivity and the specificity of LPS-I used as antigen were 71.7% and 96%, those of LPS-3 used as antigen were 43.9% and 100%, respectively. Conclusions Using LPS as antigen to detect anti-TB antibodies in sera had satisfactory the specificity. LPS might be selected as one of serodiagnostic antigens of tuberculosis.