We describe an approach for improving the sensitivity of Simoa immunoassays by at least 100-fold to sub-attomolar concentrations. In this approach, the ratio of molecules to beads—and, therefore, assay slope—was increased by using 100-fold fewer beads than conventional Simoa. High efficiency detection of low numbers of beads was achieved by loading beads into arrays of microwells using a magnetic field and meniscus forces. A Simoa immunoassay for IL-17A was developed with a limit of detection (LOD) of 0.7 aM, 437-fold more sensitive than the standard Simoa assay. This assay was used to measure IL-17A in 100 serum and plasma samples with 100% detectability, compared to 51% for the standard assay. The “100x” approach yielded improved LODs for IL-12p70 (0.092 aM), HIV p24 (9 aM), and interferon alpha (46 aM). Improved sensitivity requires capture antibodies with high on rates, so antibody engineering is needed for sub-attomolar detection of all proteins.
1. Describe the approach for increasing Simoa sensitivity using low numbers of capture beads and high efficiency bead analysis.
2. Illustrate “100x” approach for improving the limit of detection of several proteins (IL-17A, IL-12p70, HIVp24, and IFNa) to attomolar and sub-attomolar concentrations.
3. Demonstrate improvement of detectability and quantification of a cytokine in blood using a 100x Simoa assay.