We report methods that improve the quantification of digital bead assays (DBA)─such as the digital enzyme-linked immunosorbent assay (ELISA)─that have found widespread use for high sensitivity measurement of proteins in clinical research and diagnostics. In digital ELISA, proteins are captured on beads, labeled with enzymes, individual beads are interrogated for activity from one or more enzymes, and the average number of enzymes per bead (AEB) is determined based on Poisson statistics. The widespread use of digital ELISA has revealed limitations to the original approaches to quantification that can lead to inaccurate AEB. Here, we have addressed the inaccuracy in AEB due to deviations from Poisson distribution in a digital ELISA for Aβ-40 by changing the AEB calculation from a fixed threshold between digital counting and average normalized intensity to a smooth, continuous combination of digital counting and intensity. We addressed issues with determining the average product fluorescence intensity from single enzymes on beads by allowing outlier, high intensity arrays to be removed from average intensities, and by permitting the use of a wider range of arrays. These approaches improved the accuracy of a digital ELISA for tau protein that was affected by aggregated detection antibodies. We increased the dynamic range of a digital ELISA for IL-17A from AEB ∼25 to ∼130 by combining long and short exposure images at the product emission wavelength to create virtual images. The methods reported will significantly improve the accuracy and robustness of DBA based on imaging─such as single molecule arrays (Simoa)─and flow detection.

中文翻译：

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提高数字磁珠分析的准确性、稳定性和动态范围

我们报告了改进数字微珠测定 (DBA) 定量的方法——例如数字酶联免疫吸附测定 (ELISA)——已在临床研究和诊断中广泛用于蛋白质的高灵敏度测量。在数字 ELISA 中，蛋白质被捕获在珠子上，用酶标记，询问单个珠子的一种或多种酶的活性，并根据泊松统计确定每个珠子的平均酶数 (AEB)。数字 ELISA 的广泛使用揭示了原始量化方法的局限性，可能导致 AEB 不准确。这里，我们通过将 AEB 计算从数字计数和平均归一化强度之间的固定阈值更改为数字计数和强度的平滑、连续组合，解决了 Aβ-40 数字 ELISA 中泊松分布偏差导致的 AEB 不准确问题。我们通过允许从平均强度中移除离群值、高强度阵列，并允许使用更广泛的阵列，解决了确定珠子上单个酶的平均产物荧光强度的问题。这些方法提高了受聚集检测抗体影响的 tau 蛋白数字 ELISA 的准确性。我们通过组合产品发射波长的长曝光和短曝光图像来创建虚拟图像，将 IL-17A 数字 ELISA 的动态范围从 AEB ∼25 增加到∼130。