Macromolecular binding and kinetic analysis with optically sectioned planar format assays

Abstract

Real-time analysis of macromolecular interactions and competitive binding of ligands to receptors on surfaces are not typically performed using fluorescence intensity methods due to background interference from solution fluorescence. Separation-free optically sectioned planar format assays (OSPFAs) with confocal detection remove this problem. We report OSPFAs for indicator displacement and kinetic assessment of binding. A commercial androgen receptor binding domain indicator displacement assay adapted as an OSPFA yielded an IC₅₀ of 6.5 nM for testosterone with Z′ = 0.77. These measured IC₅₀ and Z′ values are in ranges suitable for drug screening applications with Z′ > 0.5 indicating good to excellent screenability. An OSPFA was applied to study the rate of antibody binding to a sandwich immunoassay on a planar surface. Langmuir fits provided forward rate constants in the range 2 × 10³ M⁻¹ s⁻¹ to 6 × 10⁴ M⁻¹ s⁻¹ and reverse constants 1 × 10⁻⁴ s⁻¹ to 4 × 10⁻³ s⁻¹ which cover a useful range for characterising probe–target interactions. This work demonstrates the suitability of OSPFAs for investigating kinetics and binding interactions using fluorescence under wash-free conditions. OSPFAs are an alternative to other separation-free methods such as acoustic, surface plasmon resonance, ellipsometry, fluorescence polarisation and other related methods. OSPFAs should make practical dynamic binding studies for small and large molecules including cases where the reaction under investigation results in no appreciable mass change on a surface.