title: Rapid affinity measurement of protein-protein interactions in a microfluidic platform.
abstract: A rapid screening method has been developed to determine binding affinities for protein-ligand interactions using the Gyrolab workstation, a commercial microfluidic platform developed to accurately and precisely quantify proteins in solution. This method was particularly suited for assessing the high-affinity interactions that have become typical of therapeutic antibody-antigen systems. Five different commercially available antibodies that bind digoxin and a digoxin-bovine serum albumin (BSA) conjugate with high affinity were rigorously evaluated by this method and by the more conventional kinetic exclusion assay (KinExA) method. Binding parameter values obtained using Gyrolab were similar to those recovered from KinExA. However, the total experimental time for 20 binding affinity titrations, with each titration covering 12 data points in duplicate, took approximately 4h by the Gyrolab method, which reduced the experimental duration by more than 10-fold when compared with the KinExA method. This rapid binding analysis method has significant applications in the screening and affinity ranking selection of antibodies from a very large pool of candidates spanning a wide range of binding affinities from the low pM to μM range.
authors.affiliation: Department of Pharmacokinetics and Drug Metabolism, Amgen Inc., Thousand Oaks, CA 91320, USA. hsalimi@amgen.com
authors.forename: Hossein
authors.initials: H
authors.lastname: Salimi-Moosavi
authors.forename: Palaniswami
authors.initials: P
authors.lastname: Rathanaswami
authors.forename: Surendran
authors.initials: S
authors.lastname: Rajendran
authors.forename: Mike
authors.initials: M
authors.lastname: Toupikov
authors.forename: John
authors.initials: J
authors.lastname: Hill
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000378:metabolism*; D000936:Antigen-Antibody Complex; D002417:Cattle; D004077:Digoxin / Q000276:immunology / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D051379:Mice; D044085:Microfluidics; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D012710:Serum Albumin, Bovine / Q000276:immunology / Q000378:metabolism*
chemical_list: D000906:Antibodies; D000936:Antigen-Antibody Complex; D008024:Ligands; D012710:Serum Albumin, Bovine; D004077:Digoxin