Affinity ranking of antibodies using flow cytometry: application in antibody phage display-based target discovery.
Application of antibody phage display to the identification of cell surface antigens with restricted expression patterns is often complicated by the inability to demonstrate specific binding to a certain cell type. The specificity of an antibody can only be properly assessed when the antibody is of sufficient high affinity to detect low-density antigens on cell surfaces. Therefore, a robust and simple assay for the prediction of relative antibody affinities was developed and compared to data obtained using surface plasmon resonance (SPR) technology. A panel of eight anti-CD46 antibody fragments with different affinities was selected from phage display libraries and reformatted into complete human IgG1 molecules. SPR was used to determine K(D) values for these antibodies. The association and dissociation of the antibodies for binding to CD46 expressed on cell surfaces were analysed using FACS-based assays. We show that ranking of the antibodies based on FACS data correlates well with ranking based on K(D) values as measured by SPR and can therefore be used to discriminate between high- and low-affinity antibodies. Finally, we show that a low-affinity antibody may only detect high expression levels of a surface marker while failing to detect lower expression levels of this molecule, which may lead to a false interpretation of antibody specificity.
Geuijen
Cecilia A W
CA
Crucell Holland B.V., P.O. Box 2048, Leiden 2301 CA, The Netherlands.
Clijsters-van der Horst
Marieke
M
Cox
Freek
F
Rood
Pauline M L
PM
Throsby
Mark
M
Jongeneelen
Mandy A C
MA
Backus
Harold H J
HH
van Deventer
Els
E
Kruisbeek
Ada M
AM
Goudsmit
Jaap
J
de Kruif
John
J
D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D000915:Antibody Affinity; D015703:Antigens, CD / Q000276:immunology; D001435:Bacteriophages / Q000276:immunology*; D001666:Binding Sites, Antibody; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D051916:Membrane Cofactor Protein; D008562:Membrane Glycoproteins / Q000276:immunology; D051379:Mice; D019151:Peptide Library; D020349:Surface Plasmon Resonance
D000906:Antibodies; D015703:Antigens, CD; C497857:CD46 protein, human; C497859:Mcp protein, mouse; D051916:Membrane Cofactor Protein; D008562:Membrane Glycoproteins; D019151:Peptide Library