Re-Evaluating the Conventional Wisdom about Binding Assays. Issue 8 (August 2020)

• Record Type: Journal Article
• Authors: Wilson, Brandon D.; Soh, H. Tom
• Is Part Of: Trends in biochemical sciences. Volume 45:Issue 8(2020)
• Page Start: 639
• Page End: 649
• Publication Date: 2020-08
• Subjects: binding assays -- affinity reagent -- digital detection -- Langmuir isotherm -- specificity; Biochemistry -- Periodicals; 572
• DOI: 10.1016/j.tibs.2020.04.005
• Languages: English
• ISSNs: 0968-0004

Abstract

Abstract : Analytical technologies based on binding assays have evolved substantially since their inception nearly 60 years ago, but our conceptual understanding of molecular recognition has not kept pace. Contemporary technologies, such as single-molecule and digital measurements, have challenged, or even rendered obsolete, core concepts behind conventional binding assay design. Here, we explore the fundamental principles underlying molecular recognition systems, which we consider in terms of signals generated through concentration-dependent shifts in equilibrium. We challenge certain orthodoxies related to binding-based detection assays, including the primary importance of a low dissociation constant ( K D ) and the extent to which this parameter constrains dynamic range and limit of detection. Lastly, we identify key principles for designing binding assays that are optimally suited for a given detection application. Highlights: Binding assays utilize concentration-dependent shifts in equilibrium to achieve target quantification; binding assays, such as ELISAs, are indispensable in the diagnostic armamentarium. Binding assays have been primarily characterized by the dissociation constant ( K D ) of the affinity reagent; there are many other parameters that are important in describing the performance of an assay, such as limit of detection (LOD) and detection range. Long-held heuristics used to describe binding assays fail to describe the results of many modern detection technologies, such as digital and single-molecule readouts, and we argue that these heuristics are unnecessarily restrictive. This opinion article does not aim to negate the existing conventional wisdom about molecular recognition, but rather to point out that stringent adherence to prior conventions limits what could otherwise be achieved with contemporary detection technologies.