Influence of mass transfer and surface ligand heterogeneity on quantitative BIAcore binding data. Analysis of the interaction of NC10 Fab with an anti-idiotype Fab'.
The interaction of monovalent Fab fragments of NC10, an antiviral neuraminidase antibody, and the anti-idiotype antibody 3-2G12 has been used as a model system to demonstrate experimentally the influence of non-ideal binding effects on BIAcore binding data. Because the association rate constant for these two molecules was found to be relatively high (about 5 x 10(5) M-1 S-1), mass transfer was recognised as a potential source of error in the analysis of the interaction kinetics. By manipulation of the flow rate and the surface density of the immobilised ligand, however, the magnitude to this error was minimised. In addition, the application of site-specific immobilisation procedures was found to improve considerably the correlation of experimental binding data to the ideal 1:1 kinetic model such that the discrepancy between experimental and fitted curves was within the noise range of the instrument. Experiments performed to measure the equilibrium constant (KD) in solution resulted in a value of similar magnitude to those obtained from the ratio of the kinetic rate constants, even those measured with a heterogeneous ligand or with a significant mass transfer component. For this system, the experimental complexities introduced by covalent immobilisation did not lead to large errors in the KD values obtained using the BIAcore.
Kortt
A A
AA
CSIRO, Division of Biomolecular Engineering, Parkville, Victoria, Australia.
Gruen
L C
LC
Oddie
G W
GW
D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000276:immunology / Q000378:metabolism*; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D000937:Antigen-Antibody Reactions; D001426:Bacterial Proteins; D015374:Biosensing Techniques; D001710:Biotin / Q000031:analogs & derivatives; D007140:Immunoglobulin Fab Fragments / Q000276:immunology / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D009439:Neuraminidase / Q000276:immunology; D011485:Protein Binding; D013438:Sulfhydryl Compounds
D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D007140:Immunoglobulin Fab Fragments; D013438:Sulfhydryl Compounds; C066114:biotin-streptavidin complex; D001710:Biotin; D009439:Neuraminidase