State Key Laboratory of Virology, Department of Immunology, Hubei Province Key Laboratory of Allergy and Immunology, Wuhan University School of Basic Medical Sciences, Donghu Road 165#, Wuhan 430071, Hubei Province, China.
State Key Laboratory of Virology, Department of Immunology, Hubei Province Key Laboratory of Allergy and Immunology, Wuhan University School of Basic Medical Sciences, Donghu Road 165#, Wuhan 430071, Hubei Province, China; Department of Laboratory Medicine, Wuhan Medical Treatment Center, Wuhan, China.
J Infect. 2014 Dec;69(6):569-80. doi: 10.1016/j.jinf.2014.05.015. Epub 2014 Jun 23.
The development of effective Mycobacterial antigen diagnostic reagents remains a high priority. The 6-kDa early secreted antigenic target (ESAT6) and 10-kDa culture filtrate protein (CFP10) are secreted early by virulent Mycobacterium tuberculosis (M. tb) and are not present in the non-virulent Bacillus Calmette-Guerin (BCG). In this study, we used a Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technique to screen for a functional ssDNA aptamer "antibody" that specifically bound to ESAT6-CFP10 (CE) protein. The selected single ssDNA aptamers (CE24 and CE15) demonstrated the highest specificity and binding affinity to CFP10 (CE24: Kd = 3.75 × 10(-7) M) and ESAT6 (CE15: Kd = 1.6 × 10(-7) M). We further detected CFP10 and ESAT6 proteins in serum samples from active pulmonary tuberculosis (TB) patients, extrapulmonary TB patients and healthy donors by using an enzyme-linked oligonucleotide assay (ELONA). The results showed that the sensitivity and specificity were 100% and 94.1% (using CE24 aptamer-based ELONA) and 89.6% and 94.1% (using CE15 aptamer-based ELONA), respectively. A good correlation was observed between aptamer-based ELONA and T-SPOT TB assay. Thus, our study suggests that CE24 and CE15 have potentially broad applications as early antigen diagnostic agents not only for active pulmonary TB, extrapulmonary TB, but also possibly for latent TB infection and TB with immune-deficiency.
开发有效的分枝杆菌抗原诊断试剂仍然是当务之急。6kDa 早期分泌抗原靶标（ESAT6）和 10kDa 培养滤液蛋白（CFP10）是由毒力结核分枝杆菌（M.tb）早期分泌的，而不存在于非毒力卡介苗（BCG）中。在这项研究中，我们使用系统进化的配体指数富集（SELEX）技术筛选针对 ESAT6-CFP10（CE）蛋白的功能性 ssDNA 适体“抗体”。所选的单个 ssDNA 适体（CE24 和 CE15）对 CFP10（CE24：Kd=3.75×10(-7) M）和 ESAT6（CE15：Kd=1.6×10(-7) M）具有最高的特异性和结合亲和力。我们进一步通过酶联寡核苷酸检测法（ELONA）检测了来自活动性肺结核（TB）患者、肺外 TB 患者和健康供体的血清样本中的 CFP10 和 ESAT6 蛋白。结果显示，基于 CE24 适体的 ELONA 的灵敏度和特异性分别为 100%和 94.1%，基于 CE15 适体的 ELONA 的灵敏度和特异性分别为 89.6%和 94.1%。基于适体的 ELONA 与 T-SPOT TB 检测之间存在良好的相关性。因此，我们的研究表明，CE24 和 CE15 具有作为早期抗原诊断剂的潜在广泛应用，不仅可用于活动性肺结核、肺外肺结核，还可能用于潜伏性 TB 感染和免疫缺陷性 TB。
