Determination of relative binding affinity of influenza virus N9 sialidases with the Fab fragment of monoclonal antibody NC41 using biosensor technology

Abstract

The relative binding affinities of influenza virus N9 sialidase from tern and whale with thc Fab fragment of monoclonal antibody NC41 were determined using biosensor technology (Pharmacia BIAcoreTM). Thc 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 tern and whale N9 sialidase epitopes to which the NC41 Fab binds, the apparent binding constant for the interaction with tern N9 sialidase was ^I 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" M-' s-' compared to 4.3 X lo4 M-' s-') and the dissociation rate constants for the whale N9-sialidase-Fab complex were = 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 ratc rollowed 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.Influema virus can cause a highly infectious respiratory illness which affects not only humans but also animals and birds. Two surface glycoproteins, haemagglutinin and sialidasc, arc cmbcddcd in the outcr lipid bilayer of Lhe influenza virion. Both of these glycoproteins are recognized by the host immune system and they undergo structural change from strain to strain resulting in antigenic variation [l] allowing viruses to evade previously established immunity to earlier strains. Based on serological cross-reactivity, nine subtypes of sialidase have been identified [ 2 ] , with sequence differences of up to 50% occurring between them [3]. Of these nine subtypes, the three-dimensional structures of two subtypes, N2 and N9, have been determined by X-ray crystallography 14-61. The three-dimensional structures of the two complexes between N9 sialidase and the Fab fragments of monoclonal antibodies NC41 [7] and NClO [S] have been determined by X-ray crystallography. The only other threedimensional structures determined for complexes between Fab fragments of monoclonal antibodies and protein antigens are the three structures which involve lysozyme bound to different Fab fragments 19-1 11 and the complex between Abbreviations. R, resonance; buffer A: 10 mM Hcpcs, 0.15 M NaC1, 3.4 rnM EDTA, 0.05% surfactant P20, pH 7.4.anti-lysozyme D1.3 Fab and the anti-idiotypic Fab E225 [ l a . The affinity constant for binding of lysozyme to at least one of its Fab ant...

Recombinant anti‐sialidase single‐chain variable fragment antibody

“…8) showed that both the mode of attachment and the binding interaction between the scFv and the sialidases are very similar to that in the parent Fab/sialidase complex. This is consistent with binding studies which showed comparable affinity for the scFv and Fab [28,311. When the structures of antibody D1.3 Fv and Fab in complex with lysozyme were compared [42] there was an rms difference between the positions of Ca atoms in the antibody variable domains of 0.039 nm, compared with 0.069nm in this study.…”

“…Sialidase is a homotetramer with circular fourfold symmetry and both of these complexes show four Fab molecules bound to the tetrameric antigen [24,261. The protein sequences of the tern and whale N9 sialidases differ at 14 residues [27] but these substitutions have no effect on the Fabfsialidase interface [25] although they may influence the apparent binding constant for the interaction with NC41 Fab [28].…”

The Interleukin-6 (IL-6) Partial Antagonist (Q159E,T162P)IL-6 Interacts with the IL-6 Receptor and gp130 but Fails to Induce a Stable Hexameric Receptor Complex

“…Analysis of the . Such a phenomenon has been noted previously by others (26,27) and may be due to rebinding of the analyte to the immobilized ligand during dissociation (28,29). To investigate whether rebinding of sIL-6R to (QT)IL-6 was affecting the k d , excess ligand was injected during the dissociation phase, as detailed under "Experimental Procedures."…”

A naturally occurring NAR variable domain binds the Kgp protease from Porphyromonas gingivalis

“…Kgp at a concentration of 50 Wg/ml in 10 mM sodium acetate bu¡er, pH 4.5, was immobilised onto a CM5 sensor chip via amine groups using the Amine Coupling kit (BIAcore AB) [16]. The immobilisation was performed at 25 ‡C and 5 Wl/min £ow rate.…”