title: High-throughput affinity ranking of antibodies using surface plasmon resonance microarrays.
abstract: A method was developed to rapidly identify high-affinity human antibodies from phage display library selection outputs. It combines high-throughput Fab fragment expression and purification with surface plasmon resonance (SPR) microarrays to determine kinetic constants (kon and koff) for 96 different Fab fragments in a single experiment. Fabs against human tissue kallikrein 1 (hK1, KLK1 gene product) were discovered by phage display, expressed in Escherichia coli in batches of 96, and purified using protein A PhyTip columns. Kinetic constants were obtained for 191 unique anti-hK1 Fabs using the Flexchip SPR microarray device. The highest affinity Fabs discovered had dissociation constants of less than 1 nM. The described SPR method was also used to categorize Fabs according to their ability to recognize an apparent active site epitope. The ability to rapidly determine the affinities of hundreds of antibodies significantly accelerates the discovery of high-affinity antibody leads.
authors.affiliation: Dyax Corp., Cambridge, MA 02139, USA.
authors.forename: Dina
authors.initials: D
authors.lastname: Wassaf
authors.forename: Guannan
authors.initials: G
authors.lastname: Kuang
authors.forename: Kris
authors.initials: K
authors.lastname: Kopacz
authors.forename: Qi-Long
authors.initials: QL
authors.lastname: Wu
authors.forename: Que
authors.initials: Q
authors.lastname: Nguyen
authors.forename: Mark
authors.initials: M
authors.lastname: Toews
authors.forename: Janja
authors.initials: J
authors.lastname: Cosic
authors.forename: Judith
authors.initials: J
authors.lastname: Jacques
authors.forename: Steve
authors.initials: S
authors.lastname: Wiltshire
authors.forename: Jeremy
authors.initials: J
authors.lastname: Lambert
authors.forename: Csaba C
authors.initials: CC
authors.lastname: Pazmany
authors.forename: Shannon
authors.initials: S
authors.lastname: Hogan
authors.forename: Robert C
authors.initials: RC
authors.lastname: Ladner
authors.forename: Andrew E
authors.initials: AE
authors.lastname: Nixon
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Sexton
mesh_terms: D001331:Automation; D001665:Binding Sites; D001666:Binding Sites, Antibody; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D000939:Epitopes / Q000032:analysis; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000302:isolation & purification; D020128:Inhibitory Concentration 50; D007700:Kinetics; D019151:Peptide Library; D040081:Protein Array Analysis / Q000379:methods; D020349:Surface Plasmon Resonance / Q000379:methods*; D020840:Tissue Kallikreins / Q000276:immunology*
chemical_list: D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D019151:Peptide Library; D020840:Tissue Kallikreins