State Key Laboratory of Virology and Hubei Province Key Laboratory of Allergy and Immunology, Medical Research Institute, and Department of Immunology, Wuhan University School of Medicine, Donghu Road 165#, Wuhan 430071, Hubei Province, China; Department of Clinical Laboratory, the Second Hospital of Wuhu, Wuhu 241000, Anhui Province, China.
State Key Laboratory of Virology and Hubei Province Key Laboratory of Allergy and Immunology, Medical Research Institute, and Department of Immunology, Wuhan University School of Medicine, Donghu Road 165#, Wuhan 430071, Hubei Province, China; Wuhan Center for Clinical Laboratory, Puai Hospital, Tongji Medical College, Huazhong University of Science and Technology, China.
J Infect. 2016 May;72(5):573-86. doi: 10.1016/j.jinf.2016.01.014. Epub 2016 Feb 3.
The development of effective Mycobacterial antigen diagnostic reagents remains a high priority. Mannose-capped lipoarabinomannan (ManLAM) is a lipoglycan serving as a major cell wall component. ManLAM is also an early released antigen in the blood circulation system during Mycobacteria tuberculosis (M.tb) infection and is a perfect target antigen for TB diagnosis. In this study, ssDNA aptamers "antibodies" against ManLAM of the predominant clinical epidemic M.tb Beijing genotype strains were generated by the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technique. The selected single aptamer T9 demonstrated the highest specificity and binding affinity, with an equilibrium dissociation constant (Kd) of 668 ± 159 nmol/L. We further detected ManLAM antigens in serum and sputum samples from active pulmonary tuberculosis (aPTB) patients, extrapulmonary TB (EPTB) patients and healthy donors by using a T9 based enzyme-linked oligonucleotide assay (ELONA). The results showed that the specificity and sensitivity were 95.31% and 83.00% (for 100 aPTB serum samples), 98.70% and 92.71% (for 96 aPTB sputum samples), and 94.44% and 88.71% (for 62 EPTB serum samples), respectively. A good correlation was observed between the T9 aptamer-based ELONA and the clinical T-SPOT.TB. Thus, T9 based ELONA has potentials for diagnosis of TB, including inactive TB, smear-negative TB, EPTB, and TB with immunodeficiency, and assist the diagnosis of LTBI albeit it could not distinguish LTBI and active TB.
开发有效的分枝杆菌抗原诊断试剂仍然是当务之急。甘露糖封端的脂阿拉伯甘露聚糖 (ManLAM) 是一种糖脂，作为细胞壁的主要成分。ManLAM 也是分枝杆菌结核 (M.tb) 感染时血液循环系统中早期释放的抗原，是结核病诊断的理想靶抗原。在这项研究中，通过系统进化的配体指数富集 (SELEX) 技术，针对主要临床流行的 M.tb 北京基因型菌株的 ManLAM 产生了 ssDNA 适体“抗体”。筛选出的单适体 T9 表现出最高的特异性和结合亲和力，平衡解离常数 (Kd) 为 668±159nmol/L。我们进一步通过基于 T9 的酶联寡核苷酸测定法 (ELONA) 检测了活动性肺结核 (aPTB) 患者、肺外结核 (EPTB) 患者和健康供体的血清和痰标本中的 ManLAM 抗原。结果表明，对于 100 份 aPTB 血清样本，其特异性和敏感性分别为 95.31%和 83.00%，对于 96 份 aPTB 痰样本，其特异性和敏感性分别为 98.70%和 92.71%，对于 62 份 EPTB 血清样本，其特异性和敏感性分别为 94.44%和 88.71%。T9 基于适体的 ELONA 与临床 T-SPOT.TB 之间存在良好的相关性。因此，T9 基于 ELONA 具有诊断结核病的潜力，包括不活动性结核病、阴性涂片结核病、肺外结核病和免疫缺陷性结核病，并有助于诊断 LTBI，尽管它不能区分 LTBI 和活动性结核病。
