title: Biosensor-based estimation of kinetic and equilibrium constants.
abstract: The steady-state affinity constant for the interaction of glutathione S-transferase (GST) with anti-GST immunoglobulin (IgG) was determined by solution-phase equilibrium analysis. A Biacore concentration assay for the determination of free anti-GST IgG was employed giving a Kd value of 6.83 x 10(-10) M. A simple 1:1 solution-phase, equilibrium model approximated the data well. Furthermore, saturation studies showed a maximum occupation of approximately 50%. The choice of affinity-capture ligand, used to anchor anti-GST IgG to the hydrogel, influenced the interaction curves, as evidenced by contact-time-dependent dissociation-phase curves. This was apparent when performing the analysis on anti-mouse Fc-coated surfaces. When the interaction was conducted on a protein A-coated CM5 sensor chip, the interaction conformed well to ideal behavior and was selected for kinetic analysis of the GST interaction.
authors.affiliation: School of Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland.
authors.forename: J G
authors.initials: JG
authors.lastname: Quinn
authors.forename: R
authors.initials: R
authors.lastname: O'Kennedy
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D003911:Dextrans / Q000737:chemistry; D005982:Glutathione Transferase / Q000276:immunology*; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate / Q000737:chemistry; D007136:Immunoglobulins / Q000276:immunology*; D007700:Kinetics; D012680:Sensitivity and Specificity; D013205:Staphylococcal Protein A / Q000737:chemistry
chemical_list: D003911:Dextrans; D007136:Immunoglobulins; D013205:Staphylococcal Protein A; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate; D005982:Glutathione Transferase