title: Exploring the dynamic range of the kinetic exclusion assay in characterizing antigen-antibody interactions.
abstract: Therapeutic antibodies are often engineered or selected to have high on-target binding affinities that can be challenging to determine precisely by most biophysical methods. Here, we explore the dynamic range of the kinetic exclusion assay (KinExA) by exploiting the interactions of an anti-DKK antibody with a panel of DKK antigens as a model system. By tailoring the KinExA to each studied antigen, we obtained apparent equilibrium dissociation constants (K(D) values) spanning six orders of magnitude, from approximately 100 fM to 100 nM. Using a previously calibrated antibody concentration and working in a suitable concentration range, we show that a single experiment can yield accurate and precise values for both the apparent K(D) and the apparent active concentration of the antigen, thereby increasing the information content of an assay and decreasing sample consumption. Orthogonal measurements obtained on Biacore and Octet label-free biosensor platforms further validated our KinExA-derived affinity and active concentration determinations. We obtained excellent agreement in the apparent affinities obtained across platforms and within the KinExA method irrespective of the assay orientation employed or the purity of the recombinant or native antigens.
authors.affiliation: Rinat, Pfizer Inc., South San Francisco, California, USA.
authors.forename: Christine
authors.initials: C
authors.lastname: Bee
authors.forename: Yasmina N
authors.initials: YN
authors.lastname: Abdiche
authors.forename: Donna M
authors.initials: DM
authors.lastname: Stone
authors.forename: Sierra
authors.initials: S
authors.lastname: Collier
authors.forename: Kevin C
authors.initials: KC
authors.lastname: Lindquist
authors.forename: Alanna C
authors.initials: AC
authors.lastname: Pinkerton
authors.forename: Jaume
authors.initials: J
authors.lastname: Pons
authors.forename: Arvind
authors.initials: A
authors.lastname: Rajpal
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000915:Antibody Affinity; D001681:Biological Assay / Q000379:methods*; D015374:Biosensing Techniques; D002138:Calibration; D045744:Cell Line, Tumor; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000276:immunology*; D051379:Mice; D051381:Rats
chemical_list: D000911:Antibodies, Monoclonal; C501126:DKK1 protein, human; D036341:Intercellular Signaling Peptides and Proteins