Analysis of the binding of the Fab fragment of monoclonal antibody NC10 toinfluenza 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 vi rus N9 neuraminidase, isolated from tern and whale, was measured using an o ptical biosensor. Both neuraminidases, homotetramers of 190 kDa, were immob ilized to avoid multivalent binding, and the binding of the monovalent NC10 Fab to immobilized neuraminidase was analyzed using the 1:1 Langmuir bindi ng model. A contribution of mass transport to the kinetic constants was dem onstrated at higher surface densities and low flow rates, and was minimized at low ligand densities and relatively high how rates (up to 100 mu l/min) . Application of a global fitting algorithm to a 1:1 binding model incorpor ating a correction term for mass transport indicated that mass transport wa s minimized under appropriate experimental conditions; analysis of binding data with a mass transport component, using this model, yielded kinetic con stants similar to those obtained with the 1:1 Langmuir binding model applie d to binding data where mass transport had been minimized experimentally. T he binding constant for binding of NC10 Fab to N9 neuraminidase from tern i nfluenza virus (KA 6.3 +/- 1.3 x 10(7) M-1) was about 15-fold higher than t hat 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 m ainly attributable to a 12-fold faster dissociation rate constant of the wh ale neuraminidase-NC10 Fab complex and may be due to either (i) the long-ra nge structural effects caused by mutation of two residues distant from the binding epitope or (ii) differences in carbohydrate residues, attached to A sn(200), which form part of the binding epitope on both neuraminidases to w hich NC10 Fab binds. (C) 1999 Academic Press.