High-throughput affinity ranking of antibodies using surface plasmon resonance microarrays.
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.
Wassaf
Dina
D
Dyax Corp., Cambridge, MA 02139, USA.
Kuang
Guannan
G
Kopacz
Kris
K
Wu
Qi-Long
QL
Nguyen
Que
Q
Toews
Mark
M
Cosic
Janja
J
Jacques
Judith
J
Wiltshire
Steve
S
Lambert
Jeremy
J
Pazmany
Csaba C
CC
Hogan
Shannon
S
Ladner
Robert C
RC
Nixon
Andrew E
AE
Sexton
Daniel J
DJ
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*
D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D019151:Peptide Library; D020840:Tissue Kallikreins