Theoretical framework and experimental validation of multiplexed analyte quantification using cross-reactive affinity reagents

Abstract

Gold standard immunoassays depend on specific affinity reagents for accurate molecular quantification. Any cross-reactivity of affinity reagents, wherein the reagent non-specifically binds to unintended molecules, can create false positive binding signals and result in inaccurate quantification of analytes. Mitigating cross-reactivity represents one of the greatest challenges in molecular diagnostics, and remains an unsolved problem. To instead overcome the effects of cross-reactivity, we present a mathematical framework that uses generalized binding equations and noise estimation to enable the use of multiple cross-reactive reagents for multiplexed molecular quantification. As a proof-of-concept, we experimentally demonstrate accurate quantification of a small molecule for which no specific affinity reagents are available, even at high concentrations of a cross-reactive molecule. Furthermore, this robust schema yields well-defined bounds of quantification that make it easier to assess the quality of assay results and predicts under which conditions assay performance is likely to break down. This work turns cross-reactive affinity reagents, which were previously a liability, into an asset for achieving accurate quantification of analytes.