Department of Chemistry, Vanderbilt University, Nashville, TN, 37235, USA.
Department of Chemistry, Vanderbilt University, Nashville, TN, 37235, USA.
Anal Biochem. 2021 Jan 1;612:114020. doi: 10.1016/j.ab.2020.114020. Epub 2020 Nov 15.
Rapid diagnostic tests (RDTs) are critical to the success of malaria elimination campaigns. These tests are rapid, user-friendly, and field-deployable to resource-limited regions. However, RDTs demonstrate poor sensitivity because they can only tolerate a small (5 μL) volume of blood, which limits the amount of protein biomarker delivered to the test. We have developed the Antibody-free Dual-biomarker Rapid Enrichment Workflow (AnDREW) for purifying histidine-rich protein 2 (HRP2) and Plasmodium lactate dehydrogenase (PLDH) from large volume (150 μL) blood samples. We used Zn(II)NTA and aptamer-conjugated magnetic beads to capture HRP2 and PLDH, respectively. Both biomarkers were then eluted into RDT-compatible volumes using ethylene diamine tetraacetic acid (EDTA). We optimized both bead conjugates individually by enzyme-linked immunosorbent assays (ELISAs) and then combined the optimized capture and elution assays for both biomarkers to produce the AnDREW. The AnDREW-enhanced RDTs exhibited a 11-fold and 9-fold improvement in analytical sensitivity for detection of HRP2 and PLDH, respectively, when compared to unenhanced RDTs. Moreover, the limit of detection for PLDH was improved 11-fold for the AnDREW-enhanced RDTs (3.80 parasites/μL) compared to unenhanced RDTs (42.31 parasites/μL). Importantly, the AnDREW utilizes a pan-specific PLDH aptamer and improves upon existing methods by eluting both biomarkers without complexed antibodies.
快速诊断检测（RDT）对于疟疾消除运动的成功至关重要。这些检测方法快速、易于使用且可在资源有限的地区进行现场部署。然而，RDT 的灵敏度较差，因为它们只能耐受小体积（5μL）的血液，这限制了递送到检测的蛋白质生物标志物的量。我们开发了无抗体双生物标志物快速富集工作流程（AnDREW），用于从小体积（150μL）的血液样本中纯化组氨酸丰富蛋白 2（HRP2）和疟原乳酸脱氢酶（PLDH）。我们分别使用 Zn(II)NTA 和适配体偶联的磁珠捕获 HRP2 和 PLDH。然后使用乙二胺四乙酸（EDTA）将两种生物标志物分别洗脱到适合 RDT 的体积中。我们通过酶联免疫吸附测定（ELISA）分别对两种珠结合物进行了优化，然后将优化后的捕获和洗脱测定组合用于两种生物标志物，以产生 AnDREW。与未增强的 RDT 相比，AnDREW 增强的 RDT 检测 HRP2 和 PLDH 的分析灵敏度分别提高了 11 倍和 9 倍。此外，与未增强的 RDT 相比，AnDREW 增强的 RDT 对 PLDH 的检测限提高了 11 倍（3.80 个寄生虫/μL）。重要的是，AnDREW 利用泛特异性 PLDH 适配体，并通过不使用复合抗体洗脱两种生物标志物来改进现有的方法。
