Anti-HIV gp160 V3-glycan antibody [DH270.6] - BSA and Azide free

• FuncS

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Functional Studies - Anti-HIV gp160 V3-glycan antibody [DH270.6] - BSA and Azide free (AB324727)

Neutralization methods :

Env-pseudotyped virus neutralization assays completed were measured as a function of reductions in luciferase (Luc) reporter gene expression after a single round of infection in TZM-bl cells (Montefiori 2009; Li 2005). TZM-bl cells (also called JC57BL-13) were obtained from the NIH AIDS Research and Reference Reagent Program, as contributed by John Kappes and Xiaoyun Wu. Briefly, a pre-titrated dose of virus was incubated with serial dilutions of antibodies in duplicate for 1hr at 37°C in 96-well flat-bottom culture plates, followed by addition of freshly trypsinized cells. One set of control wells received cells + virus (virus control) and another set received cells only (background control). After 48-72hrs of incubation, cells were lysed and measured for luminescence using the Britelite Luminescence Reporter Gene Assay System (PerkinElmer Life Sciences). ID50 and ID80 neutralization titers are the concentration at which relative luminescence units (RLU) were reduced by 50% or 80% compared to virus control wells after subtraction of background RLUs from cell controls.  Maximum Percent Inhibition (MPI) is the percent neutralization at the lowest dilution of antibody tested.

Values are the antibody concentration (ug/ml) which relative luminescence unit (RLUs) were reduced 50% compared to virus control and max percent inhibition (MPI).

Neutralization assay confirm that the anti-HIV gp160 V3-glycan (ab324727) broadly neutralizing the different HIV strains.

Negative control : SVA-MLV : Simian Virus A (SVA) lentivirus pseudotyped with Murine Leukemia Virus (MLV) envelope, Q23.17 : HIV-1 Env variant Q23.17, JR-FL : HIV-1 isolate JR-FL, CH848.10.17 : HIV-1 envelope (Env) protein variant A, CH848.10.17.DT : HIV-1 envelope (Env) protein variant B.

• Flow Cyt

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Flow Cytometry - Anti-HIV gp160 V3-glycan antibody [DH270.6] - BSA and Azide free (AB324727)

Flow cytometric analysis of 293-F (human embryonic kidney epithelial cell) transfected with HIV gp160 variant A (CH848.10.17.DS.DT.gp160) (Right) 293-F transfected with HIV gp160 variant B (CH848.10.17.DS.gp160) (Left) cells labelling HIV gp160 with ab324727 at 1/500 dilution (0.2ug) (Red) compared with a isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Goat anti-Human IgG (PE, 0.5mg/ml) at 1/200 dilution was used as the secondary antibody.

Gated on viable cells.
Cells were stained with an irrelevant antibody (black line) or ab324727 (red line) at 1 : 500 dilution. Secondary antibody an anti-human IgG conjugate to PE (2.5ug/mL).
Data kindly provided by the Duke Human Vaccine Institute.
HIV gp160 variant A and B are different constructs stabilizing the transmembrane gp160 as described in this paper (PMID : 35294883).

• I-ELISA

Supplier Data

Indirect ELISA - Anti-HIV gp160 V3-glycan antibody [DH270.6] - BSA and Azide free (AB324727)

Indirect ELISA analysis of ab324727 at 10000-0 ng/ml. The Secondary antibody used was HRP conjugated goat anti-human secondary antibody at 1 : 15000 dilution dilution.

Antigen : engineered HIV-1 Envelope (Env) trimer variant A (CH848.10.17.DS.DT.SOSIP).

Antigen concentration : 2000 ng/ml

Data kindly provided by the Duke Human Vaccine Institute. engineered HIV-1 Envelope (Env) trimer variant A and B are engineered HIV-1 Envelope (Env) trimer specifically designed with stabilizing mutations as described in this paper PMID : 35294883.

• I-ELISA

Supplier Data

Indirect ELISA - Anti-HIV gp160 V3-glycan antibody [DH270.6] - BSA and Azide free (AB324727)

Indirect ELISA analysis of ab324727 at 10000-0 ng/ml. The Secondary antibody used was HRP conjugated goat anti-human secondary antibody at 1 : 15000 dilution dilution.

Antigen : engineered HIV-1 Envelope (Env) trimer variant A (CH848.10.17.DS.SOSIP).

Antigen concentration : 2000 ng/ml

Data kindly provided by the Duke Human Vaccine Institute. Engineered HIV-1 Envelope (Env) trimer variant A and B are engineered HIV-1 Envelope (Env) trimer specifically designed with stabilizing mutations as described in this paper PMID : 35294883.