Anti-RSV glycoprotein F Recombinant Antibody (Motavizumab) (CAT#: TAB-709)

Figure 1 Assessing the stability of pre-F during FI-RSV production.

(a) Dot blots demonstrate motavizumab recognition of antigenic site II, AM14 recognition of quaternary antigenic site V, and D25 and 5C4 recognition of antigenic site Ø after RSV is incubated at 37 °C with a 0.025% concentration of formalin in 24-hour intervals. Average antibody binding of virions is reported relative to antibody binding of a pre-F protein loading control with error bars representing standard deviation of three experiments. Significant differences between populations are calculated by One-Way ANOVA and noted graphically (ns, not significant; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001). Recognition of antigenic site II, present on both pre-F and post-F surfaces, is preserved after treatment with high temperature and a dilute concentration of formalin. In contrast, dot blots demonstrate pre-F-specific mAbs AM14, D25 and 5C4 rapidly lose reactivity against virions incubated at 37 °C with a dilute concentration of formalin. (b) Percentage of cells infected by RSV incubated at 37 °C with a 0.025% concentration formalin in 24-hour intervals by flow cytometry (Chen 2010).

Killikelly, A. M., Kanekiyo, M., & Graham, B. S. (2016). Pre-fusion F is absent on the surface of formalin-inactivated respiratory syncytial virus. Scientific reports, 6, 34108.

Figure 2 AM14 is a prefusion-specific neutralizing antibody.

(A) Neutralization of laboratory strains and clinical isolates of RSV. Red bars are geometric means. (B) Binding of AM14 and motavizumab IgGs to immobilized RSV F proteins was measured using a Luminex system. (C) Binding of AM14 Fab to immobilized prefusion RSV F was measured by surface plasmon resonance. Best fit of the data to a 1:1 binding model is shown in red.

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 1 Anti-RSV glycoprotein F Recombinant Antibody (TAB-709) in SDS-PAGE

SDS-PAGE analysis of TAB-709 in non-reduced (Lane 1, 1.5 μg) and β-mercaptoethanol-reduced (Lane 2, 1.5 μg) conditions. Gel stained for 30 minutes with Coomassie Blue. As a result of different reduced proteins (Heavy chain and Light chain) migrate as about 25 kDa and 50 kDa, respectively.

Figure 2 Anti-RSV glycoprotein F Recombinant Antibody (TAB-709) in SEC-HPLC

The purity of TAB-709 was greater than 95% as determined by SEC-HPLC.

Column: 3 µm, 7.8 x 300 nm
Mobile phase: 150 mM Sodium Phosphate Buffer, pH 7.0
Detection: UV 280 nm
Injection: 10 µl

Figure 3 Anti-RSV glycoprotein F Recombinant Antibody (TAB-709) in WB

Western blot analysis of TAB-709 was performed by loading Human respiratory syncytial virus (RSV) (A2) Fusion glycoprotein / RSV-F Protein (His Tag) onto a 12% Tris-HCl polyacrylamide gel. Proteins were transferred to a CN membrane and blocked with 5% skim milk for at least one hour. Membranes were probed with TAB-709 and HRP Goat Anti-Human IgG as a secondary antibody. Chemiluminescent detection was performed.
Lane 1: Reduced antigen (1 μg)

Figure 4 Anti-RSV glycoprotein F Recombinant Antibody (TAB-709) in DB

Dot Blot analysis of TAB-709 was performed by coating with Human respiratory syncytial virus (RSV) (A2) Fusion glycoprotein / RSV-F Protein (His Tag).

TAB-709 incubation concentration: 2 μg/mL.
HRP-conjugated goat anti-Human IgG as a secondary antibody (1: 2000)

Figure 5 Anti-RSV glycoprotein F Recombinant Antibody (TAB-709) in ELISA

ELISA analysis of TAB-709 was performed by coating with Human respiratory syncytial virus (RSV) (A2) Fusion glycoprotein / RSV-F Protein (His Tag). Then blocked with BSA, and incubated with TAB-709. 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.