Human Anti-ITGAL Recombinant Antibody (clone AL-57) (CAT#: PABW-159)

Figure 1 SPR analysis of binding by the αL I domains to Abs AL-57 and MHM24.

The HA (K287C/K294C), IA (L161C/F299C), or low-affinity WT I domain was perfused onto immobilized Abs in the presence of 1 mM MgCl₂. The concentration series for MHM24 was 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, and 500 nM. For AL-57, the concentration series was 15.6, 31.3, 62.5, 125, and 250 nM for the HA I domain and 31.25, 62.5, 125, 250, and 500 nM for WT and IA I domains. In all cases, higher concentrations gave higher responses (except that differences are not visible for WT with AL-57 in Mg²⁺).

Shimaoka, M., Kim, M., Cohen, E. H., Yang, W., Astrof, N., Peer, D., ... & Springer, T. A. (2006). AL-57, a ligand-mimetic antibody to integrin LFA-1, reveals chemokine-induced affinity up-regulation in lymphocytes. Proceedings of the National Academy of Sciences, 103(38), 13991-13996.

Figure 2 SPR analysis of binding by the αL I domains to Abs AL-57 and MHM24.

The HA (K287C/K294C), IA (L161C/F299C), or low-affinity WT I domain was perfused onto immobilized Abs in the presence of 10 mM EDTA. The concentration series for MHM24 was 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, and 500 nM. For AL-57, the concentration series was 15.6, 31.3, 62.5, 125, and 250 nM for the HA I domain and 31.25, 62.5, 125, 250, and 500 nM for WT and IA I domains. In all cases, higher concentrations gave higher responses (except that differences are not visible for HA with AL-57 in EDTA).

Shimaoka, M., Kim, M., Cohen, E. H., Yang, W., Astrof, N., Peer, D., ... & Springer, T. A. (2006). AL-57, a ligand-mimetic antibody to integrin LFA-1, reveals chemokine-induced affinity up-regulation in lymphocytes. Proceedings of the National Academy of Sciences, 103(38), 13991-13996.

Figure 3 Acidic residue D101 of AL-57 is critical for binding.

Binding of parental and mutant AL-57 IgG (3 μg/ml) to the isolated, locked HA I domain expressed on K562 cells was examined by immunofluorescent flow cytometry in the presence of 1 mM Mg²⁺ (filled histograms) or 5 mM EDTA (open histograms). Binding of AL-57D101A in Mg²⁺ and EDTA is hardly distinguishable from background binding of control IgG.

Shimaoka, M., Kim, M., Cohen, E. H., Yang, W., Astrof, N., Peer, D., ... & Springer, T. A. (2006). AL-57, a ligand-mimetic antibody to integrin LFA-1, reveals chemokine-induced affinity up-regulation in lymphocytes. Proceedings of the National Academy of Sciences, 103(38), 13991-13996.

Figure 4 Binding of AL-57 IgG to WT LFA-1 activated by agonists as determined by immunofluorescent flow cytometry.

K562 transfectants expressing WT LFA-1 and IL-15-cultured human primary T lymphocytes were stained for 20 min at 37°C with 20 μg/ml AL-57, TS2/4, or isotype-matched human or mouse control IgG in Hepes-buffered saline containing 1 mM MgCl₂/1 mM CaCl₂, 5 mM MgCl₂/1 mM EGTA, or 1 mM MnCl₂. Cells were washed and stained with FITC-conjugated goat anti-human or anti-mouse Abs. Staining with AL-57 and TS2/4 is shown as solid lines, and background staining with control IgGs is shown as dashed lines. Numbers within the panels show the specific mean fluorescence intensity of AL-57 and TS2/4.

Shimaoka, M., Kim, M., Cohen, E. H., Yang, W., Astrof, N., Peer, D., ... & Springer, T. A. (2006). AL-57, a ligand-mimetic antibody to integrin LFA-1, reveals chemokine-induced affinity up-regulation in lymphocytes. Proceedings of the National Academy of Sciences, 103(38), 13991-13996.

Figure 5 The kinetics of LFA-1 activation after stimulation with CXCL-12.

Representative histograms for a time course of AL-57 epitope expression are shown. T lymphocytes were stimulated with 50 ng/ml CXCL-12 for the indicated time period. Cells were stained with Fab AL-57 or control Fab labeled with Alexa 488 for 2 min by adding Abs 2 min before the end of stimulation. Cells were then fixed and subjected to flow cytometry. Background staining with control Fab is shown in the open histograms. Numbers within the panels show specific mean fluorescence intensity values for Fab AL-57. At 0, 10, and 20 min, differences between AL-57 Fab and control Fab binding are hardly visible.

Shimaoka, M., Kim, M., Cohen, E. H., Yang, W., Astrof, N., Peer, D., ... & Springer, T. A. (2006). AL-57, a ligand-mimetic antibody to integrin LFA-1, reveals chemokine-induced affinity up-regulation in lymphocytes. Proceedings of the National Academy of Sciences, 103(38), 13991-13996.