Mouse Anti-HA Recombinant Antibody (clone HC19) (CAT#: HPAB-S0058-YC)

Figure 1 Affinities of IgG and Fab measured by using biolayer interferometry.

(A) Binding sensorgrams show binding of BHA to immobilized HC19 IgG (blue), HC19 Fab (green), FI6v3 IgG (red), and FI6v3 Fab (orange). (B) Association and dissociation constants yield similar KDs between HC19 IgG and Fab and between FI6v3 IgG and Fab. Values shown are the averages and standard deviations of data from experiments repeated in triplicates.

Williams, J. A., Gui, L., Hom, N., Mileant, A., & Lee, K. K. (2018). Dissection of epitope-specific mechanisms of neutralization of influenza virus by intact IgG and Fab fragments. Journal of Virology, 92(6), e02006-17.

Figure 2 Fluorescence fusion assay monitors antibody-mediated inhibition of HA function by tracking dequenching of the fluorescent dyes SRB.

Intact HC19 IgG could effectively inhibit both an early stage of fusion peptide-induced membrane disruption and the later stage of lipid mixing of HA-mediated membrane fusion, while the Fab fragment was effective only in inhibiting lipid mixing while permitting significant fusion peptide interactions with and disruption of membranes.

Williams, J. A., Gui, L., Hom, N., Mileant, A., & Lee, K. K. (2018). Dissection of epitope-specific mechanisms of neutralization of influenza virus by intact IgG and Fab fragments. Journal of Virology, 92(6), e02006-17.

Figure 3 Neutralization assay measuring TCID50.

Both HC19 and FI6v3 intact IgGs displayed strong neutralizing activity against X31 influenza virus, where 70% and> 95% neutralizations were achieved, respectively.

Williams, J. A., Gui, L., Hom, N., Mileant, A., & Lee, K. K. (2018). Dissection of epitope-specific mechanisms of neutralization of influenza virus by intact IgG and Fab fragments. Journal of Virology, 92(6), e02006-17.

Figure 4 Dynamic light scattering monitors the time and concentration-dependent aggregation of influenza virus X31 by HC19 IgG.

DLS detected HC19-mediated aggregate formation at IgG/HA ratios of>1:3. Negative-stain imaging further illustrated the IgG-mediated aggregation of virus with increasing concentrations of HC19. The HC19 Fab data support the conclusion that aggregation by IgG is attributed to bivalent interactions in the intact HC19 antibody.

Williams, J. A., Gui, L., Hom, N., Mileant, A., & Lee, K. K. (2018). Dissection of epitope-specific mechanisms of neutralization of influenza virus by intact IgG and Fab fragments. Journal of Virology, 92(6), e02006-17.

Figure 5 HC19 IgG-mediated cross-linking of HA on the surface of intact X31 influenza virus occurs between adjacent antigens on single particles and between antigens on separate particles, causing aggregation to occur.

Both the cross-linking of adjacent HA molecules and aggregation of the virus were observed in ~30 tomograms of samples collected at IgG/HA ratios of 1:3. The density between the two particles shown in Fig.C is consistent with 2 Fab layers with a thickness of ~7 nm, with the Fc fragment likely being localized in an adjacent x-y plane at a different z height. Further increasing the HC19/HA ratio to 1:2 led to extensive aggregation and coating of exposed HA by HC19.

Williams, J. A., Gui, L., Hom, N., Mileant, A., & Lee, K. K. (2018). Dissection of epitope-specific mechanisms of neutralization of influenza virus by intact IgG and Fab fragments. Journal of Virology, 92(6), e02006-17.