Real-time association rate constant measurement using combination tapered fiber-optic biosensor (CTFOB) dip-probes

This document reports a novel method of measuring association rate constant (k(a)) for antibody-antigen interaction using evanescent wave-based combination tapered fiber-optic biosensor (CTFOB) dip-probes. The method was demonstrated by measuring association rate constant for bovine serum albumin (BSA) and anti-BSA antibody interaction. "Direct method" was used for detection; goat anti-BSA "capture" antibodies were immobilized on the probe surfaces while the antigen (BSA) was directly labeled with Alexa 488 dye. The probes were subsequently submerged in 3 nM Labeled BSA in egg albumin (1 mg/ml). The fluorescence signal recorded was proportional to BSA anti-BSA conjugates and continuous signal was acquired suing a fiber optic spectrometer (Ocean Optics, Inc.). A 476 nm diode laser was use as an excitation source. Association constant was estimated from a plot of signal as a function of time. Measured association rate constant k(a) for the binding of BSA with anti-BSA at room temperature is (8.33 ± 0.01) ×10(4)M(-1)s(-1).