title: Quantitative measurement of binding kinetics in sandwich assay using a fluorescence detection fiber-optic biosensor.
abstract: Fiber-optic biosensors have been studied intensively because they are very useful and important tools for monitoring biomolecular interactions. Here we describe a fluorescence detection fiber-optic biosensor (FD-FOB) using a sandwich assay to detect antibody-antigen interaction. In addition, the quantitative measurement of binding kinetics, including the association and dissociation rate constants for immunoglobulin G (IgG)/anti-mouse IgG, is achieved, indicating 0.38 x 10(6) M(-1) s(-1) for k(a) and 3.15 x 10(-3) s(-1) for k(d). These constants are calculated from the fluorescence signals detected on fiber surface only where the excited evanescent wave can be generated. Thus, a confined fluorescence-detecting region is achieved to specifically determine the binding kinetics at the vicinity of the interface between sensing materials and uncladded fiber surface. With this FD-FOB, the mathematical deduction and experimental verification of the binding kinetics in a sandwich immunoassay provide a theoretical basis for measuring rate constants and equilibrium dissociation constants. A further measurement to study the interaction between human heart-type fatty acid-binding protein and its antibody gave the calculated kinetic constants k(a), k(d), and K(D) as 8.48 x 10(5) M(-1) s(-1), 1.7 x 10(-3) s(-1), and 2.0 nM, respectively. Our study is the first attempt to establish a theoretical basis for the florescence-sensitive immunoassay using a sandwich format. Moreover, we demonstrate that the FD-FOB as a high-throughput biosensor can provide an alternative to the chip-based biosensors to study real-time biomolecular interaction.
authors.affiliation: Department of Life Sciences and Institute of Genome Sciences, National Yang Ming University, Taipei, Taiwan.
authors.forename: Chao-Hsiung
authors.initials: CH
authors.lastname: Lin
authors.forename: Hsaing-Yin
authors.initials: HY
authors.lastname: Chen
authors.forename: Chih-Jen
authors.initials: CJ
authors.lastname: Yu
authors.forename: Pen-Li
authors.initials: PL
authors.lastname: Lu
authors.forename: Chu-Hsin
authors.initials: CH
authors.lastname: Hsieh
authors.forename: Bao-Yu
authors.initials: BY
authors.lastname: Hsieh
authors.forename: Ying-Feng
authors.initials: YF
authors.lastname: Chang
authors.forename: Chien
authors.initials: C
authors.lastname: Chou
mesh_terms: D000906:Antibodies; D000937:Antigen-Antibody Reactions; D015374:Biosensing Techniques / Q000379:methods*; D050556:Fatty Acid-Binding Proteins / Q000276:immunology; D005453:Fluorescence; D006321:Heart; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D007700:Kinetics; D009206:Myocardium / Q000737:chemistry; D055100:Optical Fibers
chemical_list: D000906:Antibodies; D050556:Fatty Acid-Binding Proteins