Mouse Anti-α4β7 Recombinant Antibody (clone mAb ACT-1) (CAT#: PABL-746)

Figure 1 Binding of mAb ACT-1, HIV-1 gp120, and cyclic V2 peptides to RMPI8866 cells.

(A) RPMI8866 cells (0.5 x106) were incubated with 1 μg APC labeled ACT-1 (α4β7 specific antibody). Antibody binding was measured by flow cytometry and is presented as a histogram. Over 99% of the RPMI8866 cells expressed α4β7 integrin. Unstained cells (grey shading) and isotype control antibody (solid line) were used as controls. (A and C) Histograms show the binding of CHO-expressed HIV-1 gp120 (2.5 μg, blue line panel B), biotin labeled cyclic V2 TH023 peptide (1μg and 5 μg; black and orange lines, respectively; panel C), and biotin labeled cyclic V2 TH023 -fully scrambled peptide (SCR; 1 μg and 5 μg; blue line and green line; panel C) to RMPI8866 cells. Polyclonal mouse anti-gp145 antibody followed by goat anti-mouse-Texas Red was used to visualize the gp120 protein binding while the V2 peptides were visualized with neutrAvidin-PE. Cells treated with only anti-mouse-Texas Red (red line, panel B) and unstained cells (red line, panel C) were used as controls in addition to the fully scrambled V2 peptide (blue and green line, panel C). Representative histograms are shown for panels A, B, and C (n = 2).

Peachman, K. K., Karasavvas, N., Chenine, A. L., McLinden, R., Rerks-Ngarm, S., Jaranit, K., ... & Michael, N. L. (2015). Identification of new regions in HIV-1 gp120 variable 2 and 3 loops that bind to α4β7 integrin receptor. PLoS One, 10(12), e0143895.

Figure 2 RPMI8866 cell titration curves and inhibition of binding of α4β7 integrin receptor to MAdCAM-1.

(A) The top of the Fig shows a schematic representation of the experiment. Varying numbers of RPMI8866 cells in media were added to 96-well U bottom plates followed by the addition of AlamarBlue1 dye. Cell numbers ranged from 800–400,000 cells per well. The plate was incubated at 37°C in a CO2 incubator. Fluorescence was measured for 8 hours at 1 hour intervals using a M2 plate reader (excitation 560 nm; emission 590 nm). The data are plotted as relative fluorescence units as a function of time. The average ±
S.D. of six replicates for each time point are shown. (B) Schematic of the assay set up. RMPI8866 cells were incubated with media only (solid circles) or with ACT-1 (0.5 μg/well; open circles, left panel) and then added to MAdCAM-1 (natural ligand of α4β7 integrin; 0.2 μg/well) coated plates. The plates were washed and 100 μl of media and 10 μl of AlamarBlue1 dye were added to each well. Fluorescence was measured immediately after the addition of the dye (time 0) and then at 2 hours intervals for 8 hours. The percent inhibition at the 8 hour time point is shown. The data are plotted as relative fluorescence units as a function of time and represent the average ± SEM of 12 experiments done in triplicate.

Peachman, K. K., Karasavvas, N., Chenine, A. L., McLinden, R., Rerks-Ngarm, S., Jaranit, K., ... & Michael, N. L. (2015). Identification of new regions in HIV-1 gp120 variable 2 and 3 loops that bind to α4β7 integrin receptor. PLoS One, 10(12), e0143895.

Figure 2 On gut α4β7high CD4+ T cells α4β7 colocalizes with CD4 and CCR5.

Freshly isolated gut cells obtained from biopsies taken from healthy donors were stained with the α4β7 mAb Act-1 (red), the CD4 mAb OKT4 (purple), and the CCR5 mAb 2D7 (green) and viewed under a confocal microscope. Unstained and individual stains of a representative cell are presented along with digitally defined regions of colocalization between α4β7 and CD4 (yellow) and α4β7, CD4 and CCR5 (blue). This cell is representative of greater that 60 cells analyzed from four donors.

Cicala, C., Martinelli, E., McNally, J. P., Goode, D. J., Gopaul, R., Hiatt, J., ... & Patel, N. (2009). The integrin α4β7 forms a complex with cell-surface CD4 and defines a T-cell subset that is highly susceptible to infection by HIV-1. Proceedings of the National Academy of Sciences, 106(49), 20877-20882.

Figure 1 Mouse Anti-α4β7 Recombinant Antibody (PABL-746) in ELISA

ELISA analysis of PABL-746 was performed by coating with human α4β7 protein (His Tag).