Rapid kinetic-based screening of human Fab fragments.
Human antibodies able to bind with high affinity and specificity to numerous targets have been successfully identified from Fab phage display libraries. A key step in the library selection screening process is the early characterization of library isolates in order to determine which of these isolates to pursue further. Here we describe a Biacore assay that allows isolated clones expressed as soluble Fab fragments in E. coli to be screened and ranked based on their affinity against the target. The assay takes advantage of our ability to measure Fab concentrations in crude bacterial extracts in Biacore using very high density Protein A chips. The procedure allows up to 100 clones per week to be screened and permits the identification of a small number of high-affinity Fabs from a large batch obtained following library selection or affinity maturation.
Dyax s.a., Building 22, Sart-Tilman, Liège, Belgium.
Mieke
M
Steukers
Jean-Michel
JM
Schaus
Reinoud
R
van Gool
Anne
A
Hoyoux
Pascale
P
Richalet
Daniel J
DJ
Sexton
Andrew E
AE
Nixon
Marc
M
Vanhove
D000915:Antibody Affinity / Q000276:immunology; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000032:analysis / Q000235:genetics / Q000276:immunology*; D007700:Kinetics; D019151:Peptide Library; D042810:Receptor, TIE-1 / Q000276:immunology*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D013205:Staphylococcal Protein A; D020349:Surface Plasmon Resonance / Q000379:methods
D007140:Immunoglobulin Fab Fragments; D019151:Peptide Library; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A; D042810:Receptor, TIE-1