Application of Surface Plasmon Resonance toward Studies of Low-Molecular-Weight Antigen–Antibody Binding Interactions

doi:10.1006/meth.1999.0925

Methods

Volume 20, Issue 3, March 2000, Pages 319-328

https://doi.org/10.1006/meth.1999.0925 Get rights and content

Abstract

Methods for studying low-molecular-weight antigen–antibody binding interactions using surface plasmon resonance detection are presented. The experimental parameters most relevant to studies of low-molecular-weight antigen–antibody binding interactions are discussed. Direct kinetic analysis of the binding interactions is most informative, providing both apparent association and dissociation rate constants from which equilibrium constants can be calculated. Equilibrium analysis, including steady-state and solution affinity studies, offers an alternative approach to direct kinetic analysis when knowledge of the individual kinetic rate constants is not required or difficult to determine. The various methods are illustrated by studies of an anti-T₄ Fab fragment binding interaction with several thyroxine analogs. The methods utilized were dependent on the affinity of the interaction. The high-affinity anti-T₄ Fab fragment/l-T₄ binding interaction was evaluated using direct kinetic analysis. An intermediate affinity anti-T₄ Fab fragment/l-T₃ binding interaction was evaluated using a combination of direct kinetic analysis, steady-state analysis, and solution affinity analysis. The relatively weak anti-T₄ Fab fragment/l-T₂ binding interaction was evaluated using steady-state and solution affinity analysis protocols. Several thyroxine tracers that could not be immobilized to a biosensor surface were also evaluated via the solution affinity format. In cases where a given binding interaction was examined using multiple methods the results were comparable.

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Inter-peptide interactions mediate a broad range of biological phenomena, such as protein folding and peptide aggregation, and an extensive and in-depth understanding of inter-peptide and peptide–small molecule interactions could be beneficial for designing and selecting potential peptide drugs to the target antigens (Adamczyk et al., 2000; Abdiche et al., 2008).
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Although the application of biosensors to analyte detection is not difficult in itself, the complex nature of clinical samples complicates discrimination between specific and non-specific interactions. The use of specific, high affinity antibodies facilitates detection and increases assay sensitivity [6]. Adequate antibodies are not always available, however, and in some cases, specificity is incompatible with the recognition of multiple forms of a protein of interest.
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The optical detection technologies have been used in biosensors for the highly sensitive, real-time and label-free detection of biomolecules. In this report, a metal clad leaky waveguide (MCLW) was used as an immunosensing tools. We optimized the structure of the sensor chip for highly sensitive detection, followed by fabrication of the MCLW sensor chips and construction of a detection system for immunosensing. Titanium and SiO₂ films were deposited onto a BK7 substrate using an E-beam evaporator and the thickness of the films was 9 nm and 347 nm, respectively. The sensing response of the system to the bulk refractive index was calibrated using various concentrations of a glycerol solution. As a result, the MCLW sensor system was able to detect a change of ∼3.8 × 10⁻⁶ RIU in the refractive index of the ambient solution. In addition, immunosensing was achieved using the MCLW sensor for an antigen–antibody reaction in real time. The biotin-labeled antibody of human interleukin 5 (hIL5) was immobilized on the sensor surface containing modified with streptavidin. The hIL5 in a bio-solution was quantitatively analyzed in real time using the MCLW sensor system.

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Regular ArticleApplication of Surface Plasmon Resonance toward Studies of Low-Molecular-Weight Antigen–Antibody Binding Interactions

Abstract

J. Mol. Biol.

Methods: A Companion to Methods Enzymol.

J. Colloid Interface Sci.

Curr. Opin. Biotechnol.

Curr. Opin. Struct. Biol.

Curr. Opin. Biotechnol.

Curr. Opin. Biotechnol.

Anal. Biochem.

J. Immunol. Methods

Anal. Biochem.

Anal. Biochem.

Anal. Biochem.

Regular Article
Application of Surface Plasmon Resonance toward Studies of Low-Molecular-Weight Antigen–Antibody Binding Interactions