High-resolution characterization of antibody fragment/antigen interactions using Biacore T100.
A Biacore T100 optical biosensor was used to characterize the binding kinetics of a panel of antigen binding fragments (Fabs) directed against the PcrV protein from Pseudomonas aeruginosa. PcrV protein forms part of the type III secretion system complex of this opportunistic pathogen. We demonstrate that the biosensor response data for each Fab collected from three different surface densities of the antigen could be fit globally to a simple 1:1 interaction model. Importantly, we found that the Fabs with the slowest dissociation rate provided the best protection in cell cytotoxicity studies. To further characterize the Fab interactions, binding data were automatically acquired at different temperatures and under different buffer conditions. The comprehensive characterization of these Fabs shows how Biacore T100 can be used to complement protein therapeutic discovery programs from basic research to the selection of therapeutic candidates.
Papalia
Giuseppe A
GA
Center for Biomolecular Interaction Analysis, University of Utah, Salt Lake City, UT 84132, USA.
Baer
Mark
M
Luehrsen
Kenneth
K
Nordin
Helena
H
Flynn
Peter
P
Myszka
David G
DG
D000818:Animals; D000937:Antigen-Antibody Reactions / Q000276:immunology*; D000942:Antigens, Bacterial / Q000096:biosynthesis* / Q000737:chemistry / Q000276:immunology; D001427:Bacterial Toxins / Q000096:biosynthesis* / Q000737:chemistry / Q000276:immunology; D015374:Biosensing Techniques / Q000379:methods*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis* / Q000276:immunology*; D051379:Mice; D052899:Pore Forming Cytotoxic Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology*; D011550:Pseudomonas aeruginosa / Q000276:immunology / Q000472:pathogenicity*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology; D012107:Research Design
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D007140:Immunoglobulin Fab Fragments; D052899:Pore Forming Cytotoxic Proteins; D011994:Recombinant Proteins; C118546:antigen V, Pseudomonas