Department of Chemistry, Vanderbilt University, Station B 351822, Nashville, TN 37235, USA.
Analyst. 2021 Feb 21;146(4):1207-1215. doi: 10.1039/d0an01725f. Epub 2020 Dec 24.
Tuberculosis (TB) is one of the top ten causes of death globally, despite being treatable. The eradication of TB disease requires, amongst others, diagnostic tests with high specificity and sensitivity that will work at the point of care (POC) in low-resource settings. The TB surface glycolipid antigen, mannose-capped lipoarabinomannan (ManLAM) currently serves as the only POC molecular diagnostic biomarker suitable for use in low cost immunoassays. Here, we demonstrate the high affinity and exceptional specificity of microvirin-N (MVN), a 14.3 kDa cyanobacterial lectin, toward H37Rv TB ManLAM and utilize it to develop a novel on-bead ELISA. MVN binds to ManLAM with sub-picomolar binding affinity, but does not bind to other variants of LAM expressed by non-pathogenic mycobacteria - a level of binding specificity and affinity that current commercially available anti-LAM antibodies cannot achieve. An on-bead ELISA was subsequently developed using MVN-functionalized magnetic beads which allows for the specific capture of ManLAM from human urine with a limit of detection (LOD) of 1.14 ng mL and no cross-reactivity when tested with PILAM, a variant of LAM found on non-pathogenic mycobacteria.
结核病（TB）是全球十大死因之一，尽管可治疗。要消灭结核病，除其他外，需要在资源匮乏环境中具有高特异性和敏感性的诊断检测，可在护理点（POC）使用。结核分枝杆菌表面糖脂抗原甘露糖封端脂阿拉伯甘露聚糖（ManLAM）目前是唯一适合用于低成本免疫测定的 POC 分子诊断生物标志物。在这里，我们证明了微病毒-N（MVN），一种 14.3 kDa 的蓝细菌凝集素，对 H37Rv 结核分枝杆菌 ManLAM 具有高亲和力和卓越的特异性，并利用它开发了一种新型的珠上 ELISA。MVN 与 ManLAM 的结合亲和力达到皮摩尔级，而不与非致病性分枝杆菌表达的其他 LAM 变体结合 - 这种结合特异性和亲和力是当前市售的抗 LAM 抗体无法达到的。随后使用 MVN 功能化的磁性珠开发了珠上 ELISA，可特异性捕获人尿液中的 ManLAM，检测限（LOD）为 1.14ng/mL，与非致病性分枝杆菌上发现的 LAM 变体 PILAM 无交叉反应。
