Screening and kinetic analysis of recombinant anti-CEA antibody fragments.
Four different carcinoembryonic antigen (CEA)-binding antibody fragments were prepared using the genes of the variable regions of the T84 epitope-specific antibody 7F7 and phage display techniques. The genes were successfully cloned and expressed in the pCANTAB5 phage display vector to investigate the kinetic binding parameters of each synthesized construct. Single chain fragments, Fab fragments, and two diabodies were purified and compared in their CEA-binding properties with the parent IgG using surface plasmon resonance detection. The on-rates for all these molecules were in the same order of magnitude (about 1 x 10(5) M-1 s-1) whereas major differences were detected in the off-rates. IgG and diabodies had slow off-rates due to bivalent binding, while single chain and Fab fragments dissociated rather fast. We also present a method for the immobilization of large amounts of CEA on CM5 sensorchips. These high density surfaces can be used for observing mass transport limited binding of CEA-specific molecules and are convenient tools for screening and quality control.
Abraham
R
R
IGEN, Inc., Rockville, MD 20852, USA.
Buxbaum
S
S
Link
J
J
Smith
R
R
Venti
C
C
Darsley
M
M
D000915:Antibody Affinity / Q000276:immunology*; D015374:Biosensing Techniques; D002272:Carcinoembryonic Antigen / Q000276:immunology* / Q000378:metabolism; D003001:Cloning, Molecular; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology* / Q000378:metabolism; D007128:Immunoglobulin Fragments / Q000276:immunology; D007074:Immunoglobulin G / Q000276:immunology; D007700:Kinetics; D011485:Protein Binding / Q000276:immunology; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism
D002272:Carcinoembryonic Antigen; D007140:Immunoglobulin Fab Fragments; D007128:Immunoglobulin Fragments; D007074:Immunoglobulin G; D011994:Recombinant Proteins