title: Determining the affinity and stoichiometry of interactions between unmodified proteins in solution using Biacore.
abstract: We describe a general Biacore method for measuring equilibrium binding affinities and stoichiometries for interactions between unmodified proteins and their unmodified ligands free in solution. Mixtures of protein and ligand are preequilibrated at different ratios in solution and then analyzed by Biacore using a sensor chip surface that detects only unbound analyte. Performing the Biacore analysis under mass transport limited conditions allows the concentration of unbound analyte to be determined from the initial velocity of binding. Plots of initial velocity versus the concentration of the varied binding partner are fitted to a quadratic binding equation to give the affinity and stoichiometry of binding. We demonstrate the method using soluble Her2 extracellular domain binding to monovalent, bivalent, and trivalent forms of an anti-Her2 antibody. The affinity we measured agrees with that obtained from conventional Biacore kinetic analysis, and the stoichiometries for the resulting 1:1, 1:2, and 1:3 complexes were confirmed by gel filtration with in-line light scattering. The method is applicable over an affinity range of approximately 100 pM to 1 μM and is particularly useful when there is concern that covalently modifying one or the other binding partner might affect its binding properties or where multivalency might otherwise complicate a quantitative analysis of binding.
authors.affiliation: Biogen Idec, Cambridge, MA 02142, USA. estevenday@gmail.com
authors.forename: Eric S
authors.initials: ES
authors.lastname: Day
authors.forename: Allan D
authors.initials: AD
authors.lastname: Capili
authors.forename: Christopher W
authors.initials: CW
authors.lastname: Borysenko
authors.forename: Mohammad
authors.initials: M
authors.lastname: Zafari
authors.forename: Adrian
authors.initials: A
authors.lastname: Whitty
mesh_terms: D000818:Animals; D002850:Chromatography, Gel / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000032:analysis / Q000378:metabolism*; D020349:Surface Plasmon Resonance / Q000379:methods*
chemical_list: D011506:Proteins; D018719:Erb-b2 Receptor Tyrosine Kinases
keywords: Affinity; Mass transport; Multivalency; Solution binding; Stoichiometry; Surface plasmon resonance