title: High throughput solution-based measurement of antibody-antigen affinity and epitope binning.
abstract: Advances in human antibody discovery have allowed for the selection of hundreds of high affinity antibodies against many therapeutically relevant targets. This has necessitated the development of reproducible, high throughput analytical techniques to characterize the output from these selections. Among these characterizations, epitopic coverage and affinity are among the most critical properties for lead identification. Biolayer interferometry (BLI) is an attractive technique for epitope binning due to its speed and low antigen consumption. While surface-based methods such as BLI and surface plasmon resonance (SPR) are commonly used for affinity determinations, sensor chemistry and surface related artifacts can limit the accuracy of high affinity measurements. When comparing BLI and solution equilibrium based kinetic exclusion assays, significant differences in measured affinity (10-fold and above) were observed. KinExA direct association (k(a)) rate constant measurements suggest that this is mainly caused by inaccurate k(a) measurements associated with BLI related surface phenomena. Based on the kinetic exclusion assay principle used for KinExA, we developed a high throughput 96-well plate format assay, using a Meso Scale Discovery (MSD) instrument, to measure solution equilibrium affinity. This improved method combines the accuracy of solution-based methods with the throughput formerly only achievable with surface-based methods.
authors.affiliation: Protein Analytics, Adimab, LLC, Lebanon, NH, USA.
authors.forename: Patricia
authors.initials: P
authors.lastname: Estep
authors.forename: Felicia
authors.initials: F
authors.lastname: Reid
authors.forename: Claire
authors.initials: C
authors.lastname: Nauman
authors.forename: Yuqi
authors.initials: Y
authors.lastname: Liu
authors.forename: Tingwan
authors.initials: T
authors.lastname: Sun
authors.forename: Joanne
authors.initials: J
authors.lastname: Sun
authors.forename: Yingda
authors.initials: Y
authors.lastname: Xu
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000378:metabolism; D000915:Antibody Affinity; D000937:Antigen-Antibody Reactions / Q000276:immunology; D000941:Antigens / Q000276:immunology / Q000378:metabolism; D018604:Epitope Mapping / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D007700:Kinetics; D051379:Mice; D011485:Protein Binding; D012015:Reference Standards; D012996:Solutions
chemical_list: D000906:Antibodies; D000941:Antigens; D012996:Solutions
keywords: BLI; KD; MSD; affinity; antibody; epitope; high throughput; kinetic exclusion