Multi-antigen rapid diagnostic tests (RDTs) are highly informative, simple, mobile, and inexpensive, making them valuable point-of-care (POC) diagnostic tools. However, these RDTs suffer from several technical limitations—the most significant being the failure to detect low levels of infection. To overcome this, we have developed a magnetic bead-based multiplex biomarker enrichment strategy that combines metal affinity and immunospecific capture to purify and enrich multiple target biomarkers. Modifying antibodies to contain histidine-rich peptides enables reversible loading onto immobilized metal affinity magnetic beads, generating a novel class of antibodies coined “Capture and Release” (CaR) antibody reagents. This approach extends the specificity of immunocapture to metal affinity magnetic beads while also maintaining a common trigger for releasing multiple biomarkers. Multiplex biomarker enrichment is accomplished by adding magnetic beads equipped with CaR antibody reagents to a large sample volume to capture biomarkers of interest. Once captured, these biomarkers are magnetically purified, concentrated, and released into a RDT-compatible volume. This system was tailored to enhance a popular dual-antigen lateral flow malaria RDT that targets Plasmodium falciparum histidine-rich protein-II (HRPII) and Plasmodium lactate dehydrogenase (pLDH). A suite of pLDH CaR antibody reagents were synthesized, characterized, and the optimal CaR antibody reagent was loaded onto magnetic beads to make a multiplex magnetic capture bead that simultaneously enriches pLDH and HRPII from Plasmodium falciparum parasitized blood samples. This system achieves a 17.5-fold improvement in the dual positive HRPII/pan-pLDH detection limits enabling visual detection of both antigens at levels correlating to 5 p/μL. This front-end sample processing system serves as an efficient strategy to improve the sensitivity of RDTs without the need for modifications or remanufacturing.

中文翻译：

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金属亲和力捕获和释放抗体试剂可产生多重生物标志物富集系统，可提高快速诊断测试的检测限

多抗原快速诊断测试（RDT）具有很高的信息量，简单，可移动且价格便宜，使其成为有价值的即时诊断（POC）诊断工具。但是，这些RDT受到若干技术限制-最重要的是无法检测到低水平的感染。为了克服这个问题，我们开发了一种基于磁珠的多重生物标志物富集策略，该策略结合了金属亲和力和免疫特异性捕获来纯化和富集多个目标生物标志物。将抗体修饰为包含富含组氨酸的肽，可以将其可逆地加载到固定的金属亲和力磁珠上，从而生成一类称为“捕获和释放”（CaR）抗体试剂的新型抗体。这种方法将免疫捕获的特异性扩展到金属亲和力磁珠，同时还保持了释放多种生物标志物的共同触发条件。多重生物标志物富集是通过将配备有CaR抗体试剂的磁珠添加到大量样品中以捕获感兴趣的生物标志物来实现的。一旦被捕获，这些生物标志物将被磁纯化，浓缩并释放到与RDT兼容的体积中。该系统经过量身定制，以增强针对性的流行的双抗原侧向流疟疾RDT恶性疟原虫组氨酸富集蛋白II（HRPII）和乳酸疟原虫脱氢酶（p LDH）。合成，表征了一组p LDH CaR抗体试剂，然后将最佳CaR抗体试剂加载到磁珠上，制成了多重磁捕获珠，可同时从恶性疟原虫寄生的血液样本中富集p LDH和HRPII 。该系统在双重阳性HRPII / pan- p方面实现了17.5倍的提高LDH检出限使得能够以5 p /μL的相关水平目测两种抗原。该前端样品处理系统可作为一种有效策略，无需修改或重新制造即可提高RDT的灵敏度。