Abstract
Traditional enzyme-linked immunosorbent assay (ELISA), long the workhorse for specific target protein detection using microplate wells, is nearing its fundamental limit of sensitivity. New opportunities in health care call for in vitro diagnostic tests with ultrahigh sensitivity. Magnetic bead-based sandwich immunoassay formats have been developed that can reach unprecedented sensitivities, orders of magnitude better than are allowed for by the rate constants for a single ligand-receptor interaction. However, these ultrahigh sensitivity assays are vulnerable to a host of confounding factors, including nonspecific binding from background molecules and loss of low-abundance target to tube walls and during wash steps. Moreover, the optimization of workflow is often time-consuming and expensive. In this work, we present a simulation tool that allows users to graphically define arbitrary binding assays, including fully reversible first-order binding kinetics, timed addition of extra components, and timed wash steps. The tool is freely available as a user-friendly webapp. The framework is lightweight and fast, allowing for inexpensive simulation and visualization of arbitrarily complex assay schemes, including but not limited to digital immunoassays, DNA hybridization, and enzyme kinetics, for validation and optimization of assay designs without requiring any programming knowledge from the user. We demonstrate some of these capabilities and provide practical guidance on assay simulation design.