Recombinant Human Anti-RSV Antibody (D25) (CAT#: PABL-322)

Figure 1 Internalization of Fab fragments.

RSV-infected HEp-2 cells were incubated with RSV F-specific MAbs D25, AM14, 5C4, and MPE8 or corresponding monomeric Fab fragments at the same concentration for 90 min to induce internalization. Afterwards the cells were fixed, permeabilized, and stained with AF488 human anti-goat IgG or AF488 chicken anti-mouse IgG (green). Nuclei were visualized with DAPI (blue). The amount of internalized vesicles was quantified in 50 positive cells.

Leemans, A., De Schryver, M., Van der Gucht, W., Heykers, A., Pintelon, I., Hotard, A. L.,... & Broadbent, L. (2017). Antibody-induced internalization of the human respiratory syncytial virus fusion protein. Journal of virology, 91(14), e00184-17.

Figure 2 N426D disrupts binding of AM14 to prefusion F. Relative binding of D25, 101F, MPE8 and AM14 to cell surface-expressed prefusion F (grey) or prefusion F containing the AM14 escape mutation, N426D (white) was measured by flow cytometry. Data were normalized to motavizumab binding. Binding of D25, 101F and MPE8 to N426D was comparable to wild-type prefusion F, whereas AM14 binding to N426D was reduced four-fold.

Gilman, M. S., Moin, S. M., Mas, V., Chen, M., Patel, N. K., Kramer, K.,... & Beaumont, T. (2015). Characterization of a prefusion-specific antibody that recognizes a quaternary, cleavage-dependent epitope on the RSV fusion glycoprotein.PLoS pathogens, 11(7), e1005035.

Figure 3 Binding of antibodies (A) AM14, (B) D25 to uncleaved monomeric RSV F (open black triangles), cleaved monomeric RSV F (black circles), uncleaved postfusion RSV F (open blue triangles), cleaved postfusion RSV F (blue circles), uncleaved prefusion RSV F (open red triangles) and cleaved prefusion RSV F (red circles) was measured by ELISA.

Gilman, M. S., Moin, S. M., Mas, V., Chen, M., Patel, N. K., Kramer, K.,... & Beaumont, T. (2015). Characterization of a prefusion-specific antibody that recognizes a quaternary, cleavage-dependent epitope on the RSV fusion glycoprotein.PLoS pathogens, 11(7), e1005035.

Figure 4 AM14 stabilizes RSV F trimer in the absence of the foldon trimerization motif. Size-exclusion chromatography profiles from a Superose 6 column are shown for AM14 Fab or D25 Fab complexed with prefusion RSV F containing the foldon trimerization motif (black and grey, respectively) and for AM14 Fab or D25 Fab co-expressed with RSV F ectodomain without foldon (red and blue, respectively).

Gilman, M. S., Moin, S. M., Mas, V., Chen, M., Patel, N. K., Kramer, K.,... & Beaumont, T. (2015). Characterization of a prefusion-specific antibody that recognizes a quaternary, cleavage-dependent epitope on the RSV fusion glycoprotein.PLoS pathogens, 11(7), e1005035.

Figure 5 RSV neutralization by antibodies. Palivizumab is the FDA-approved prophylactic antibody that prevents severe RSV disease.

McLellan, J. S., Chen, M., Leung, S., Graepel, K. W., Du, X., Yang, Y.,... & Kumar, A. (2013). Structure of RSV fusion glycoprotein trimer bound to a prefusion-specific neutralizing antibody. Science, 340(6136), 1113-1117.

Figure 6 ELISA measuring antibody binding to postfusion F glycoprotein.

McLellan, J. S., Chen, M., Leung, S., Graepel, K. W., Du, X., Yang, Y.,... & Kumar, A. (2013). Structure of RSV fusion glycoprotein trimer bound to a prefusion-specific neutralizing antibody. Science, 340(6136), 1113-1117.

Figure 7 Highly effective RSV-neutralizing antibodies target a site at the membrane-distal apex of the prefusion F trimer.

(A) The ability of antibodies to block D25 binding to RSV-infected cells was measured as a function of antibody concentration. (B) Analysis of RSV F-Fab complexes by negative stain electron microscopy: Reprojection of a 12 Å slice through the crystal structure of RSV F + D25 Fab filtered to 10 Å resolution (left). A slice was used to emphasize visibility of the F glycoprotein cavity. Aligned average of 263 particles of RSV F + D25 Fab (middle left). Aligned average of 550 particles of RSV F + AM22 Fab (middle -right). Aligned average of 171 particles of RSV F + 5C4 Fab (right). Scale bar is 50 Å. (C) Fusion inhibition and (D) attachment inhibition activity for antibodies targeting antigenic site Ø and F-specific antibodies targeting other antigenic sites. For the attachment-inhibition assay, heparin was used as a positive control.

McLellan, J. S., Chen, M., Leung, S., Graepel, K. W., Du, X., Yang, Y.,... & Kumar, A. (2013). Structure of RSV fusion glycoprotein trimer bound to a prefusion-specific neutralizing antibody. Science, 340(6136), 1113-1117.

Figure 1 Anti-RSV Antibody (PABL-322) in ELISA

ELISA analysis of PABL-322 was performed by coating with Recombinant F(ΔTM)(Strain A2)(RSV). Secondary antibody: Goat Anti-Human-IgG-HRP

Figure 2 Anti-RSV Antibody (PABL-322) in WB

Western blot analysis of PABL-322 was performed with Recombinant F(ΔTM)(Strain A2)(RSV).
Secondary antibody: Goat Anti-Human-IgG-HRP
Lane 1: Antigen (3ul)
Lane 2: Antigen (4ul)

Figure 3 Anti-RSV Antibody (PABL-322) in ELISA

ELISA analysis of PABL-322 was performed by coating with Human respiratory syncytial virus (RSV) (A2) Fusion glycoprotein / RSV-F Protein (His Tag) (11049-V08B). Then blocked with BSA and incubated with Anti-RSV Antibody (D25) (PABL-322) at a starting concentration of 1 ng/mL. The HRP-conjugated goat anti-human IgG as a secondary antibody. Detection was performed using TMB substrate and stopped with sulfuric acid. The absorbances were read on a spectrophotometer at 450 nm.

Figure 4 Anti-RSV Antibody (PABL-322) in WB

Western blot analysis was performed with Human respiratory syncytial virus (RSV) (A2) Fusion glycoprotein / RSV-F Protein (His Tag) (11049-V08B) onto a 12% Tris-HCl polyacrylamide gel. Proteins were transferred to a CN membrane and blocked with 5% skim milk for at least 1 hour. Membranes were probed with Anti-RSV Antibody (D25) (PABL-322) at a dilution of 1:1000 overnight at 4°C on a rocking platform. Then probed with Goat anti-Human IgG HRP secondary antibody at a dilution of 1:8,000 for one hour. Chemiluminescent was detected.
Lane 1: Reducing antigen (0.1 μg)
Lane 2: Reducing antigen (0.3 μg)
Lane 3: Reducing antigen (0.6 μg)

Figure 5 Recombinant Human Anti-RSV Antibody (D25) (PABL-322) in HPLC

The purity of PABL-322 was greater than 90% as determined by SEC-HPLC.

Figure 6 Recombinant Human Anti-RSV Antibody (D25) (PABL-322) in SDS-PAGE

SDS-PAGE analysis of PABL-322 in reduced (Lane 1) and non-reduced (Lane 2) conditions. Gel stained for 30 minutes with Coomassie Blue. As a result, different β-mercaptoethanol-reduced proteins (Heavy chain and Light chain) migrate as about 50 kDa and 25 kDa, respectively.

Figure 7 Recombinant Human Anti-RSV Antibody (D25), FITC (PABL-322-FITC) in SDS-PAGE

SDS-PAGE analysis of PABL-322-FITC (Lane 2) and PABL-322 (Lane 1) was performed in non-reduced conditions (Left). Antibodies went through fluorescence detection directly by the imaging system in the UV wavelength 200 nm-400 nm (Right).
Lane 1: PABL-322, Non-Reduced (1.5 μg)
Lane 2: PABL-322-HRP, Non-Reduced (1.5 μg)

Figure 8 Recombinant Human Anti-RSV Antibody (D25), FITC (PABL-322-FITC) in Absorption Spectroanalysis

Absorption Spectroanalysis of PABL-322-FITC was performed with the full-wavelength multi-mode microplate reader.
A280: 1.86
A495: 1.656

Figure 9 Recombinant Human Anti-RSV Antibody (D25), HRP (PABL-322-HRP) in SDS-PAGE

SDS-PAGE analysis of PABL-322-HRP (Lane 2) and PABL-322 (Lane 1) was performed in non-reduced conditions. Antibodies were transferred to an NC membrane and went through colorimetric detection directly by the imaging system.
Lane 1: PABL-322, Non-Reduced (0.2 μg)
Lane 2: PABL-322-HRP, Non-Reduced (0.2 μg)

Figure 10 Recombinant Human Anti-RSV Antibody (D25), HRP (PABL-322-HRP) in Absorption Spectroanalysis

Absorption Spectroanalysis of PABL-322-HRP was performed with the full-wavelength multi-mode microplate reader.
A280: 1.966
A403: 1.857