Analysis of the binding of the Fab fragment of monoclonal antibody NC10 to influenza virus N9 neuraminidase from tern and whale using the BIAcore biosensor: effect of immobilization level and flow rate on kinetic analysis.
The binding of the Fab fragment of monoclonal antibody NC10 to influenza virus N9 neuraminidase, isolated from tern and whale, was measured using an optical biosensor. Both neuraminidases, homotetramers of 190 kDa, were immobilized to avoid multivalent binding, and the binding of the monovalent NC10 Fab to immobilized neuraminidase was analyzed using the 1:1 Langmuir binding model. A contribution of mass transport to the kinetic constants was demonstrated at higher surface densities and low flow rates, and was minimized at low ligand densities and relatively high flow rates (up to 100 microl/min). Application of a global fitting algorithm to a 1:1 binding model incorporating a correction term for mass transport indicated that mass transport was minimized under appropriate experimental conditions; analysis of binding data with a mass transport component, using this model, yielded kinetic constants similar to those obtained with the 1:1 Langmuir binding model applied to binding data where mass transport had been minimized experimentally. The binding constant for binding of NC10 Fab to N9 neuraminidase from tern influenza virus (K(A) = 6.3 +/- 1.3 x 10(7) M(-1)) was about 15-fold higher than that for the NC10 Fab binding to N9 neuraminidase from whale influenza virus (K(A) = 4.3 +/- 0.7 x 10(6) M(-1)). This difference in binding affinity was mainly attributable to a 12-fold faster dissociation rate constant of the whale neuraminidase-NC10 Fab complex and may be due to either (i) the long-range structural effects caused by mutation of two residues distant from the binding epitope or (ii) differences in carbohydrate residues, attached to Asn(200), which form part of the binding epitope on both neuraminidases to which NC10 Fab binds.
Kortt
A A
AA
CRC for Diagnostic Technologies, 343 Royal Parade, Parkville, 3052, Australia. alec.kortt@molsci.csiro.au
Nice
E
E
Gruen
L C
LC
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D015374:Biosensing Techniques / Q000379:methods*; D001717:Birds; D007140:Immunoglobulin Fab Fragments / Q000276:immunology*; D007128:Immunoglobulin Fragments / Q000276:immunology*; D009980:Influenza A virus / Q000201:enzymology* / Q000276:immunology; D007700:Kinetics; D009439:Neuraminidase / Q000276:immunology* / Q000378:metabolism; D011485:Protein Binding; D014907:Whales
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D007128:Immunoglobulin Fragments; D009439:Neuraminidase