Development of Kinetic Ligand-binding Assays Using a Fiber Optic Sensor

Abstract

Ligand-binding assays are ubiquitous in biochemical research and clinical determinations. In most common assay methods, binding proceeds until an equilibrium condition has been obtained, which leads to relatively long incubation times. The requirement for separating bound from free reagents in the reaction mixture before signal detection precludes direct observation of binding events. To reduce the duration of an assay and to facilitate kinetic analysis, methods based on evanescent field technology have recently been used in assay development. These include fiber optic fluorometric sensors (1)(2)(3) and surface plasmon resonance (4). Specifically, evanescent field technology is based on the observation that when light travels through a waveguide at angles approaching the critical angle for total internal reflection, an evanescent field is produced on the surface of the waveguide. This field falls off exponentially with distance from the surface and is exquisitely sensitive to the refractive indices of the waveguide surface and the medium in which the surface resides. In surface plasmon resonance, this sensitivity to refractive index is used to measure the amount of substance that binds to the waveguide surface. In evanescent field fluorometry, the field stimulates fluorophores, which become attached to the surface through specific binding interactions (2). We describe here the use of evanescent fiber optic fluorometric sensors to characterize ligand binding of IgG and monovalent Fab, both specific for estrone-1-glucuronide (E1g), and of the human estrogen receptor α (hER-α) to fibers bearing E1g or the specific estrogen response element (ERE) and demonstrate the determination of apparent association and dissociation binding constants. Fused silica optical fibers obtained from Polymicro Technologies were cleaved in 11.5-cm lengths, and the cladding was removed from a 7.0-cm portion of the fiber using Fluorinert™ (3 M). To assess the binding kinetics of IgG, Fab, and hER-α, fibers were sensitized by …