Anti-IAV HA Recombinant Antibody (12D1) (CAT#: MRO-276CT)

Figure 1 Binding of stalk-reactive antibodies to group 2 HAs.

Binding of stalk-reactive antibodies 12D1 and CR8020 and H3 antiserum to recombinant soluble Wisc05 HA without (w/o T4 trim. domain, black lines) or with (w/T4 trim. domain, red line) trimerization domain.

Krammer, F., Margine, I., Tan, G. S., Pica, N., Krause, J. C., & Palese, P. (2012). A carboxy-terminal trimerization domain stabilizes conformational epitopes on the stalk domain of soluble recombinant hemagglutinin substrates. PloS one, 7(8), e43603.

Figure 2 mAb 12D1 reacts with the LAH of HA2.

Lysates from 293T cells were transfected with pCAGGs-GFP or pCAGGs-GFP HA2 76–130, incubated with mAb 12D1, and pulled down with protein G beads. Pulled-down fractions were blotted with mAb 12D1 or rabbit anti-GFP. Arrows indicate location of the amino acid 76–130 region–GFP fusion protein. Anti-mouse HRP used to detect 12D1-binding reacts with the mouse Ig heavy and light chains within the pulled-down fraction.

Wang, T. T., Tan, G. S., Hai, R., Pica, N., Ngai, L., Ekiert, D. C., ... & Palese, P. (2010). Vaccination with a synthetic peptide from the influenza virus hemagglutinin provides protection against distinct viral subtypes. Proceedings of the National Academy of Sciences, 107(44), 18979-18984.

Figure 3 Anti-H3 mabs in microneutralization assay.

Neutralization of virus expressing the HA from either (A) A/Hong Kong/1/1968 virus or (B) A/Panama/2007/1999 virus. mAb XY102 is specific for A/HK/1968 virus. Purified mouse IgG was used for the negative control.

Wang, T. T., Tan, G. S., Hai, R., Pica, N., Petersen, E., Moran, T. M., & Palese, P. (2010). Broadly protective monoclonal antibodies against H3 influenza viruses following sequential immunization with different hemagglutinins. PLoS pathogens, 6(2), e1000796.

Figure 4 Activity of anti-H3 mabs in plaque reduction assay on MDCK cells.

mAb 12D1 neutralize all H3 viruses tested by plaque reduction assay but not representative H1, H4 or H7 viruses. Purified mouse IgG was used for the negative control. The plaque reduction assays were performed multiple times and with each new antibody preparation. Data shown are from a single representative experiment.

Wang, T. T., Tan, G. S., Hai, R., Pica, N., Petersen, E., Moran, T. M., & Palese, P. (2010). Broadly protective monoclonal antibodies against H3 influenza viruses following sequential immunization with different hemagglutinins. PLoS pathogens, 6(2), e1000796.

Figure 5 Treatment with anti-H3 mAbs diminishes lung damage associated with viral pneumonia caused by HK68/PR8 reassortant virus.

Mice treated with mAb 12D1. 40× magnification.

Wang, T. T., Tan, G. S., Hai, R., Pica, N., Petersen, E., Moran, T. M., & Palese, P. (2010). Broadly protective monoclonal antibodies against H3 influenza viruses following sequential immunization with different hemagglutinins. PLoS pathogens, 6(2), e1000796.

Figure 6 Red blood cell fusion assay.

Anti-H3 mabs inhibit low-pH induced fusion of HK/68 hemagglutinin with chicken red blood cells.

Wang, T. T., Tan, G. S., Hai, R., Pica, N., Petersen, E., Moran, T. M., & Palese, P. (2010). Broadly protective monoclonal antibodies against H3 influenza viruses following sequential immunization with different hemagglutinins. PLoS pathogens, 6(2), e1000796.

Figure 1 Anti-IAV HA Recombinant Antibody (MRO-276CT) in SDS-PAGE

SDS-PAGE analysis of MRO-276CT in non-reduced (Lane 1) and β-mercaptoethanol-reduced (Lane 2) conditions. Gel stained for 30 minutes with Coomassie Blue. As a result of different reduced proteins (Heavy chain and Light chain) migrate as about 50 kDa and 25 kDa, respectively. And non-reduced protein migrates as 180-245 kDa.

Figure 2 Anti-IAV HA Recombinant Antibody (MRO-276CT) in SEC-HPLC

SEC-HPLC analysis of MRO-276CT shows that the purity is more than 95%.

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-IAV HA Recombinant Antibody (MRO-276CT) in ELISA

ELISA analysis of MRO-276CT was performed by coating with Influenza A H3N2 (A/Perth/16/2009) Hemagglutinin / HA Protein (His Tag). Then blocked with BSA and incubated with MRO-276CT. The HRP-conjugated goat anti-Mouse IgG as a secondary antibody (1: 2000). Detection was performed using TMB substrate and stopped with sulfuric acid. The absorbances were read on a spectrophotometer at 450 nm.

Figure 4 Anti-IAV HA Recombinant Antibody (MRO-276CT) in ELISA

ELISA analysis of MRO-276CT was performed by coating with Influenza A H3N2 (A/Victoria/210/2009) Hemagglutinin / HA Protein (His Tag). Then blocked with BSA and incubated with MRO-276CT. The HRP-conjugated goat anti-Mouse IgG as a secondary antibody (1: 2000). Detection was performed using TMB substrate and stopped with sulfuric acid. The absorbances were read on a spectrophotometer at 450 nm.

Figure 5 Anti-IAV HA Recombinant Antibody (MRO-276CT) in WB

Western blot analysis of MRO-276CT was performed with Influenza A H3N2 (A/Perth/16/2009) Hemagglutinin / HA 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 MRO-276CT and HRP goat anti-Mouse IgG as a secondary antibody (1: 2000). Chemiluminescent detection was performed.

Lane 1: Reduced antigen (0.8 μg)

Figure 6 Anti-IAV HA Recombinant Antibody (MRO-276CT) in WB

Western blot analysis of MRO-276CT was performed with Influenza A H3N2 (A/Victoria/210/2009) Hemagglutinin / HA 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 MRO-276CT and HRP goat anti-Mouse IgG as a secondary antibody (1: 2000). Chemiluminescent detection was performed.

Lane 1: Reduced antigen (0.8 μg)

Figure 7 Anti-IAV HA Recombinant Antibody (MRO-276CT) in DB

Dot Blot analysis of MRO-276CT was performed by coating with Influenza A H3N2 (A/Perth/16/2009) Hemagglutinin / HA Protein (His Tag).

MRO-276CT incubation concentration: 2 μg/mL.
HRP-conjugated goat anti-Mouse IgG as a secondary antibody (1: 2000)

Figure 8 Anti-IAV HA Recombinant Antibody (MRO-276CT) in DB

Dot Blot analysis of MRO-276CT was performed by coating with Influenza A H3N2 (A/Victoria/210/2009) Hemagglutinin / HA Protein (His Tag).

MRO-276CT incubation concentration: 2 μg/mL.
HRP-conjugated goat anti-Mouse IgG as a secondary antibody (1: 2000)