Many protein biomarkers occur at very low concentrations in biofluids like blood and saliva, and ultrasensitive detection methods are required in order to measure them. Approaches such as digital enzyme-linked immunosorbent assays (ELISA) and single molecule arrays (Simoa) have been developed to accurately quantitate protein concentrations as low as attomolar levels. Although these techniques are being implemented in research and clinical laboratories to develop ultrasensitive clinical diagnostic assays, the size and cost of the instruments required to run these digital assays have precluded them from being implemented into point-of-care diagnostic formats. Here, we report the development of a simplified digital ELISA format that is more amenable to point-of-care technologies, referred to as catalyzed reporter deposition digital ELISA (CARD-dELISA). On-bead signal generation using the CARD tyramide signal amplification technique is combined with bead immobilization in fibrin hydrogels for single molecule counting in a simplified workflow format. CARD-dELISA allows for ultrasensitive protein detection (IL-6: ∼1 fM) with a dynamic range similar to the conventional Simoa assay. We use CARD-dELISA to measure IL-6 in saliva samples and show good agreement with conventional Simoa.

中文翻译：

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使用酪酰胺信号放大和纤维蛋白水凝胶的简化数字酶联免疫吸附测定

许多蛋白质生物标记物以非常低的浓度存在于血液和唾液等生物流体中，因此需要超灵敏的检测方法才能对其进行测量。已经开发了诸如数字酶联免疫吸附测定（ELISA）和单分子阵列（Simoa）之类的方法，以准确定量低至attomolar水平的蛋白质浓度。尽管正在研究和临床实验室中实施这些技术以开发超灵敏的临床诊断检测方法，但是运行这些数字检测所需的仪器的尺寸和成本使得它们无法实施为即时诊断格式。在这里，我们报告了简化的数字ELISA格式的发展，这种格式更适合即时医疗技术，称为催化报告物沉积数字ELISA（CARD-dELISA）。使用CARD酪酰胺信号放大技术产生的磁珠信号与固定在纤维蛋白水凝胶中的磁珠结合在一起，以简化的工作流程格式进行单分子计数。CARD-dELISA可以进行超灵敏的蛋白检测（IL-6：〜1 fM），其动态范围与传统的Simoa分析相似。我们使用CARD-dELISA测量唾液样品中的IL-6，并与常规Simoa表现出良好的一致性。