Digital immunoassays enable the detection of protein biomarkers with very low concentrations, but the analysis stringently requires single-bead encapsulation. Low bead density has been adopted to minimize multiple-bead encapsulations, but the trade-off is the low droplet effectiveness (∼10%) in droplet-based assays. Here we report the method of inclusive droplet digital ELISA (iddELISA) that embraces all types of encapsulations by factoring in their varied “on–off” probabilities in the statistical inference. We derived the statistical model, optimized the bead encapsulation and immunoreaction, and developed an image analysis pipeline for accurate droplet and bead recognition, showing that approximately 40% of the droplets could be used in the analysis. Using the detection of SARS-CoV-2 nucleocapsid protein as a demonstration, the iddELISA achieved a limit of detection of 0.71 fg/mL, which was much lower than conventional ELISA as well as droplet digital ELISA. By effectively incorporating multiple bead encapsulations, the iddELISA simplified the digital immunoassay while improving the counting efficiency and sensitivity, representing a unique concept in digital immunoassays.

中文翻译：

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采用泊松封装统计量改进液滴式数字免疫测定


数字免疫检测能够检测浓度非常低的蛋白质生物标志物，但分析严格需要单珠封装。采用低微珠密度来最大限度地减少多微珠封装，但代价是基于微滴的检测中的低液滴有效性 （∼10%）。在这里，我们报告了包含液滴数字 ELISA （iddELISA） 的方法，该方法通过在统计推断中考虑它们不同的“开-关”概率来包含所有类型的封装。我们推导出了统计模型，优化了微珠封装和免疫反应，并开发了一个用于准确液滴和微珠识别的图像分析管道，表明大约 40% 的液滴可用于分析。以 SARS-CoV-2 核衣壳蛋白检测为示范，iddELISA 的检测限达到 0.71 fg/mL，远低于常规 ELISA 和液滴数字 ELISA。通过有效地结合多个微珠封装，iddELISA 简化了数字免疫测定，同时提高了计数效率和灵敏度，代表了数字免疫测定中的独特概念。