Determination of Binding Constant and Stoichiometry for Antibody-Antigen Interaction with Surface Plasmon Resonance

Abstract

A surface plasmon resonance (SPR) biosensor technology has recently been applied biochemically and clinically to the study of immunologic recognition and the evaluation of binding parameters for various interactions between antibodies (Abs) and antigens (Ags) at liquid-solid interface. The simple interaction between hapten and Ab fragment, e.g., variable single-chain fragment and antigenbinding fragment, can be described sufficiently by a 1:1 stoichiometry in SPR. However, the determination of the binding constant of an anti-protein Ab is usually complicated by the multivalence of the protein Ag. The SPR-based method enables direct determination of binding constants for a variety of specific Ab-Ag interactions in real-time. It also allows estimation of the binding stoichiometry and binding ratio for low-, intermediate-, and high-affinity Ab-Ag interaction systems. The present review is designed to indicate the theoretical background of SPR-based biosensor technology as well as to present the great variety of measurement modes of interaction kinetics that can be performed with these techniques. Quantitative aspects of the Ab-Ag interaction kinetics are reviewed, focusing especially on mono-and multi-valent Ab-Ag interaction modes using a SPR biosensor. Four model binding systems developed recently for use with SPR biosenser are described with principles and examples: (i) one to one interaction mode, (ii) nonequivalent two-site interaction mode, (iii) multiple equivalent-site interaction mode and (iv) multisite interaction mode. This article closes with two descriptions of the determinations of the binding stoichiometry and maximum binding ratio of Ab-Ag interactions.

Kinetic analysis of IgM monoclonal antibodies for determination of dengue sample concentration using SPR technique

“…The association and dissociation rate constants are defined as k a and k d for formation of Ag_Ab complex respectively. 22 In an ideal situation, the Ab transport to the chip surface and its transport on the linker layer would not affect the binding kinetics. This occurs when the flow rate is quickly compared with binding.…”

“…In addition, the measured forward and backward rate constants approach to the constants of binding interaction kinetics. 22 According to mentioned conditions, the complex formation rate can be depicted as…”

“…The variable R and t represent the values acquired through the dissociation curve. 22 As a description of SPR method functionally, the incident laser light is reflected inside the prism which is usually coated with a thin gold layer from the outside. At a critical incident angle, an electron resonant oscillation appears on the surface of gold layer which results in sudden decrease of reflected light intensity.…”

“…These binding information may lead to calculation of their topology and concentration measurement of antigens which are active biologically. 21,22 Different experimental studies have been done to ease the computation of binding parameters, such as, the kinetic parameter for the interaction between Ags and Abs or the dissociation constant. Among these approaches, separation of free reactants and bound reactants are more preferred.…”

“…In addition, after completion of first experiment, the SPR biosensors can be applied to finding the mechanism of binding interaction and its stoichiometry. 22,25,26 Biacore configuration and SPR chip construction Biacore 3000 from GE Healthcare is a real-time system for biomolecular interaction analysis using SPR technique. [27][28][29] With this method it is possible to monitor the formation and dissociation of biomolecular complexes on chip surfaces.…”