Human Anti-HIV-1 Env Recombinant Antibody (clone C11) (CAT#: PABL-144)

Figure 1 Binding of N12-i3 to EGFP-CEM-NKr-CCR5-SNAP cells infected with BaL infectious molecular clone (IMC)

EGFP-CEM-NKr-CCR5-SNAP were infected with IMC BaL virus and evaluated 5-days post-infection for (A) intracellular p24, cell surface levels of CD4 and (B) the binding of: N12-i3, Cluster A mAbs A32, N5-i5 and C11, and Synagis. Panel A shows the gating strategy for EGFP-CEM-NKr-CCR5-SNAP Bal-infected (Blue gates) and Mock (Red gate) cells, previously defined as GFP+ and L/D dye- cells. For Bal-infected cells two gates were delineated: p24+/CD4- and p24+low/CD4+; one gate was delineated for Mock p24-/CD4+. (C) ADCC using the RFADCC method. The curves shown are normalized for plateau cytotoxicity values using C11.

Tolbert, W. D., Gohain, N., Alsahafi, N., Van, V., Orlandi, C., Ding, S.,... & Pazgier, M. (2017). Targeting the late stage of HIV-1 entry for antibody-dependent cellular cytotoxicity: structural basis for Env epitopes in the C11 region. Structure,25(11), 1719-1731.

Figure 2 Exposure of A32-like and C11-like epitopes at the surface of HIV-1 infected cells and HIV-1 virions

(A) CEM.NKr cells were infected with the infectious HIV-1NL-4.3 ADA/GFP molecular clone; 48h post-infection cells were stained with Alexa Fluor 647-conjugated mAbs: 17b, A32, N5-i5, N12-i3 and C11 alone, in the presence of the CD4 mimetic M48U1 (100nM) or together with M48U1 and co-receptor binding site 17b (5µg/ml). Data are the averages of four independent experiments. (B) Binding to HIV-1JR-FL Env trimers expressed at the surface of 293T cells. At 48-hours post-transfection, the exposure of Cluster A epitopes was assessed with Alexa Fluor 647 (AF647)-conjugated A32, N12-i3, or C11 antibodies in the presence or absence of 17b, the 17b Fab-fragment, or the Fab-2 fragment with or without CD4mimetic-M48U1 (100nM). Data are the averages from four independent experiments. (C) Effect of M48U1 on the binding of Cluster A mAbs to BaL pseudovirus particles in solution as determined by the FCS assay. The relative fraction of mAb that adopts a lower diffusion coefficient as a result of virion binding is shown. All measurements were performed in triplicate.

Tolbert, W. D., Gohain, N., Alsahafi, N., Van, V., Orlandi, C., Ding, S.,... & Pazgier, M. (2017). Targeting the late stage of HIV-1 entry for antibody-dependent cellular cytotoxicity: structural basis for Env epitopes in the C11 region. Structure,25(11), 1719-1731.

Figure 1 Human Anti-HIV-1 Env Recombinant Antibody (PABL-144) in WB

Western blot analysis was performed with 1 µg (lane 1), 2 µg(lane 2) recombinant HIV-1 Env 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 PABL-144 at a dilution of 1:500 overnight at 4°C on a rocking platform. Then probed with Goat anti-Human IgG HRP secondary antibody at a dilution of 1:2,000 for one hour. Chemiluminescent was detected.

Figure 2 Human Anti-HIV-1 Env Recombinant Antibody (PABL-144) in Dot Blot

Dot Blot analysis of PABL-144 was performed by coating with HIV-1 Env antigen.
PABL-144 incubation concentration: 4 ng/μL.
The secondary antibody: HRP-Anti-Human IgG (H+L)

Figure 3 Human Anti-HIV-1 Env Recombinant Antibody (PABL-144) in ELISA

ELISA analysis of PABL-144 was performed by coating with recombinant HIV-1 Env antigen. Then blocked with BSA, and incubated with PABL-144. The HRP-conjugated goat anti-human IgG was used 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 Human Anti-HIV-1 Env Recombinant Antibody (PABL-144) in SDS-PAGE

SDS-PAGE analysis of PABL-144 in non-reduced (Lane 1) and reduced (Lane 2) conditions.

Figure 5 Human Anti-HIV-1 Env Recombinant Antibody (PABL-144) in HPLC

HPLC analysis of PABL-144 shows the purity is>95%.