Publications & Posters

Digital Enzyme-linked Immunosorbent Assays With Sub-attomolar Detection Limits Based On Low Numbers Of Capture Beads Combined With High Efficiency Bead Analysis


Kan CW, Tobos CI, Rissin DM, Wiener AD, Meyer RE, Svancara DM, Comperchio A, Warwick C, Millington R, Collier N and Duffy DC

Lab Chip. 2020 May 11

DOI: 10.1039/d0lc00267d


We report the development of digital enzyme-linked immunosorbent assays (ELISAs) based on single molecule arrays (Simoa) with improved sensitivities over conventional digital ELISA, enabling detection of proteins at sub-attomolar concentrations. The improvements in sensitivity were based on using fewer beads to capture the target proteins (≤5000 vs. ∼500 000 beads) that increased the ratio of molecules to beads, and increasing the fraction of beads that were analyzed (bead read efficiency) from ∼5% to ∼50%. Bead read efficiency was increased by: a) improving the loading of beads into arrays of microwells by combining capillary and magnetic forces in a method called magnetic-meniscus sweeping (MMS); b) using a centrifugal washer to minimize bead loss during the assay; and, c) improved optics and image analysis to enable the analysis of more microwells. Using this approach, we developed an assay for IL-17A with a limit of detection (LOD) of 0.7 aM, 437-fold more sensitive than standard digital ELISA. A digital ELISA with improved sensitivity was used to measure IL-17A in 100 serum and plasma samples with 100% detectability, compared to 51% for standard digital ELISA. Low numbers of capture beads yielded improved LODs for IL-12p70 (0.092 aM), p24 (9.1 aM), and interferon alpha (45.9 aM). IL-4 and PSA showed no improvements in sensitivity using fewer beads, primarily due to low antibody loading on beads and increased non-specific binding, respectively. The results were consistent with a kinetic model of binding that showed that combining capture antibodies with high on-rates with high antibodies per bead yields the greatest improvement in sensitivity.