Determination of relative binding affinity of influenza virus N9 sialidases with the Fab fragment of monoclonal antibody NC41 using biosensor technology.
The relative binding affinities of influenza virus N9 sialidase from term and whale with the Fab fragment of monoclonal antibody NC41 were determined using biosensor technology (Pharmacia BIAcoreTM). The apparent association and dissociation rate constants were measured in real time for the interaction of the Fab with both sialidases, the Fab being immobilised on the sensor surface. Although three-dimensional structural studies have shown that there are no apparent structural differences between the term and whale N9 sialidase epitopes to which the NC41 Fab binds, the apparent binding constant for the interaction with tern N9 sialidase was approximately 2.4-fold higher than that with whale N9 sialidase. The kinetic analysis showed that the association rate constant for the binding of whale N9 sialidase was higher than that for tern N9 sialidase (12.0 x 10(4) M-1 s-1 compared to 4.3 x 10(4) M-1 s-1) and the dissociation rate constants for the whale N9-sialidase-Fab complex were approximately 6-fold higher than for the tern N9-sialidase-Fab complex. Furthermore, kinetic analysis of the dissociation reaction showed that it was composed of two stages, an initial, faster rate followed by a late, slower rate. The values of the relative affinity constants calculated using the initial dissociation rate constant were similar to the values measured at equilibrium in the BIAcore and those determined in true solution equilibrium studies using sedimentation equilibrium. The late, slower, dissociation rate constant yielded affinity constants significantly higher than those obtained by true solution methods.
Gruen
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LC
Commonwealth Scientific and Industrial Research Organisation, Division of Biomolecular Engineering, Parkville, Victoria, Australia.
Kortt
A A
AA
Nice
E
E
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D015374:Biosensing Techniques; D001717:Birds / Q000382:microbiology; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C; D009439:Neuraminidase / Q000276:immunology / Q000378:metabolism*; D009975:Orthomyxoviridae / Q000201:enzymology*; D014907:Whales / Q000382:microbiology
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D009439:Neuraminidase