Recombinant Mouse Anti-IAV HA Antibody (HC19) (CAT#: PABW-077)

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

Binding sensorgrams show binding of BHA to immobilized HC19 IgG (blue), HC19 Fab (green), FI6v3 IgG (red), and FI6v3 Fab (orange).

Williams, J. (2018). Mechanisms of antibody-mediated neutralization targeting viral glycoproteins (Doctoral dissertation).

Figure 2 Fluorescence fusion assay monitors antibody-mediated inhibition of HA function by tracking dequenching of the fluorescent dyes SRB, indicating membrane disruption, and DiD, indicating lipid mixing.

HC19 inhibition of membrane disruption is enhanced through bivalent interactions between IgG and HA on X31 virus, even at subsaturating concentrations, where aggregation was observed by DLS. Fab binding has little impact on membrane disruption inhibition.

Williams, J. (2018). Mechanisms of antibody-mediated neutralization targeting viral glycoproteins (Doctoral dissertation).

Figure 3 Fluorescence fusion assay monitors antibody-mediated inhibition of HA function by tracking dequenching of the fluorescent dyes SRB, indicating membrane disruption, and DiD, indicating lipid mixing.

Extents of inhibition of lipid mixing by IgG and Fab are similar, suggesting that bivalency may not play a large role in fusion inhibition for HC19.

Williams, J. (2018). Mechanisms of antibody-mediated neutralization targeting viral glycoproteins (Doctoral dissertation).

Figure 4 Neutralization assay measuring TCID50.

Antibody-mediated neutralization of influenza virus was evaluated by measuring the TCID50 of influenza virus. IgGs for both FI6v3 and HC19 displayed stronger neutralization than did the respective Fabs. While HC19 Fab could neutralize virus to some degree, FI6v3 Fab did not exhibit a significant ability to neutralize the virus (*, P> 0.05; **, P < 0.01).

Williams, J. (2018). Mechanisms of antibody-mediated neutralization targeting viral glycoproteins (Doctoral dissertation).

Figure 5 Dynamic light scattering monitors the time- and concentration-dependent aggregation of influenza virus X31 by HC19 IgG.

As the ratio of IgG/HA increases, HC19 aggregates virus particles, with peak aggregation occurring at 2:1 ratios.

Williams, J. (2018). Mechanisms of antibody-mediated neutralization targeting viral glycoproteins (Doctoral dissertation).

Figure 6 Dynamic light scattering monitors the time- and concentration-dependent aggregation of influenza virus X31 by HC19 IgG.

Negative-stain micrographs further illustrate the extent of aggregation as the ratio of IgG/HA increases.

Williams, J. (2018). Mechanisms of antibody-mediated neutralization targeting viral glycoproteins (Doctoral dissertation).

Figure 7 Dynamic light scattering monitors the time- and concentration-dependent aggregation of influenza virus X31 by HC19 IgG.Figure 8 HC19 IgG-mediated cross-linking of HA on the surface of intact X31 influenza occurs between adjacent antigens on single particles and between antigens on separate particles, causing aggregation to occur. Figure 9 HC19 IgG-mediated cross-linking of HA on the surface of intact X31 influenza occurs between adjacent antigens on single particles and between antigens on separate particles, causing aggregation to occur. Figure 10 HC19 IgG-mediated cross-linking of HA on the surface of intact X31 influenza occurs between adjacent antigens on single particles and between antigens on separate particles, causing aggregation to occur. Figure 1 Inhibition of wild-type and mutant NA activity by NC10. Figure 2 Inhibition of wild-type and mutant NA activity by NC10. Figure 3 ELISA measuring binding of mutant NAs to NC10. Figure 4 Correlation between A405 ELISA reading and amount of NA measured by enzyme activity using the fluorogenic substrate 4-methylumbelliferyl-Nacetylneuraminic acid.Figure 5 Scatchard plots of inhibition of wild-type and mutant NAs by NC10. Figure 6 Scatchard plots of the ELISA results to determine apparent K_d in the solid phase assay.Figure 1 Loss of mAb OPG2 binding following substitution of β3 Asp²¹⁷ or Glu²²⁰.Figure 1 Monoclonal antibodies 1H10 and 1A5 do not bind in the presence of prebound 2A8.Figure 1 Fluorescence microscopic images of LS174T cells. The relative mean fluorescence intensity in the tumor and liver regionsRepresentative images of dissected organs of athymic nude miceRepresentative images of dissected organs of athymic nude miceTissue-to-heart ratios for (B) the mice sacrificed 2 hours after injection and (C) the mice sacrificed 18 hours after injection.Figure 1 (A) HEK/CFP-HA-DAT cells were treated with 1 μM PMA or Me₂SO for 15 min,lysed, and DAT was precipitated with the HA11 antibody. Figure 2 (B) the recom-binant four-ubiquitin Lys63-linked, Lys48-linked, and linear chains wereelectrophoresed and blotted with the same antibodies that were used toprobe DAT immunoprecipitates in A.Figure 3 HEK/CFP-HA-DAT cells were treated with 1 µM PMA for 15 min, lysed and DAT was precipitated with the HA11 antibody. Figure 4 Quantification of experiments presented in Figure 3. Figure 1 Binding curves of monoclonal antibodies A20 (left column), and S67-27 (right column) against Kdo-BSA. Figure 2 Binding curves of monoclonal antibodies A20 (left column), and S67-27 (right column) against Ko-BSA. Figure 3 Binding curves of monoclonal antibodies A20 (left column), and S67-27 (right column) against Ko(2→4)Kdo-BSA. Figure 4 Binding curves of monoclonal antibodies A20 (left column), and S67-27 (right column) against Kdo(2→4)Ko-BSA. Figure 5 Binding curves of monoclonal antibodies A20 (left column), and S67-27 (right column) against Kdo(2→4)Kdo-BSA. Figure 6 Surface plasmon resonance of S67-27 binding to Kdo analogs.Figure 7 Surface plasmon resonance of S67-27 binding to Kdo analogs.Figure 8 Surface plasmon resonance of S67-27 binding to Kdo analogs.Figure 9 Surface plasmon resonance of S67-27 binding to Kdo analogs.Figure 10 Surface plasmon resonance of S67-27 binding to Kdo analogs.Figure 1 Effect of 17b on bNAb and non-bNAb binding to monomeric HXBc2 gp120 core by Sandwich ELISA assay. Figure 2 ELISA binding profiles of human CD4bs and non-CD4bs MAbs (left), and selected sera of Imm 3 and Imm 7 (right) from complex-GLA immunized rabbit group tested with CD4bs bNAb probes, RSC3/G367R and RSC3 Δ371I/P363N.Figure 3 The binding kinetics of CD4bs MAb b13 and representative rabbit MAbs 30-9 and 46-1 to HXBc2 core and RSC3 core. Figure 4 Surface plasmon resonance analysisFigure 1 Surface plasmon resonance analysis of S54-10 Fab binding to different Kdo antigensFigure 2 Surface plasmon resonance analysis of S54-10 Fab binding to different Kdo antigensFigure 3 Surface plasmon resonance analysis of S54-10 Fab binding to different Kdo antigensFigure 4 Surface plasmon resonance analysis of S54-10 Fab binding to different Kdo antigensFigure 1 Surface plasmon resonance analysis of S73-2 Fab binding to different Kdo antigensFigure 2 Surface plasmon resonance analysis of S73-2 Fab binding to different Kdo antigensFigure 3 Surface plasmon resonance analysis of S73-2 Fab binding to different Kdo antigensFigure 4 Surface plasmon resonance analysis of S73-2 Fab binding to different Kdo antigensFigure 1 SPR analysis of binding by the αL I domains to Abs AL-57 and MHM24. Figure 2 SPR analysis of binding by the αL I domains to Abs AL-57 and MHM24. Figure 3 Acidic residue D101 of AL-57 is critical for binding.

Digestion of IgG into monovalent Fab arms fails to aggregate influenza virus X31, emphasizing the complex binding that occurs between bivalent IgG and HA
presented on the virus surface. Bar = 200 nm.

Williams, J. (2018). Mechanisms of antibody-mediated neutralization targeting viral glycoproteins (Doctoral dissertation).

Figure 8 HC19 IgG-mediated cross-linking of HA on the surface of intact X31 influenza occurs between adjacent antigens on single particles and between antigens on separate particles, causing aggregation to occur.

(A and B) A 4.4-nm-thick computational slice through HC19 IgG-complexed virus at a ratio of 1:6 (IgG/HA). Black arrowheads point toward IgG-cross-linked HA trimers on the surface of individual particles, and expanded views of these interactions are shown in the insets.

Williams, J. (2018). Mechanisms of antibody-mediated neutralization targeting viral glycoproteins (Doctoral dissertation).

Figure 9 HC19 IgG-mediated cross-linking of HA on the surface of intact X31 influenza occurs between adjacent antigens on single particles and between antigens on separate particles, causing aggregation to occur.

A 4.4-nm-thick computational slice through HC19 IgG-complexed virus at a ratio of 1:3 (IgG/HA). The radial density average at the interface between two virus particles was measured so that the distribution of lipid and protein components could be assigned. The blue box indicates the measured area used for the radial density plot.

Williams, J. (2018). Mechanisms of antibody-mediated neutralization targeting viral glycoproteins (Doctoral dissertation).

Figure 10 HC19 IgG-mediated cross-linking of HA on the surface of intact X31 influenza occurs between adjacent antigens on single particles and between antigens on separate particles, causing aggregation to occur.

(D and E) Computational slices (8.0 nm thick) through HC19 IgG-complexed virus at a ratio of 1:2 (IgG/HA) illustrating extensive aggregation that occurs. Bar = 25 nm.

Williams, J. (2018). Mechanisms of antibody-mediated neutralization targeting viral glycoproteins (Doctoral dissertation).