| Assay Method Information | |
| | Solid Phase Receptor Assay (SPRA) |
| Description: | The potency of compounds in blocking ligand binding to integrin a4b7 was determined by modification of previously described methods (Henderson et al, Nature Medicine, 2013). Briefly, purified human VCAM1 (R & D Systems) diluted to 5 μg/ml in TBS+ buffer (25 mM Tris pH 7.4, 137 mM NaCl, 2.7 mM KCl, 1 mM CaCl2), 1 mM MgCl2, 1 mM MnCl2) was added to wells of a 96-well transparent microtiter plate and incubated overnight at 4° C. Wells were washed 3 times with TBS+ and blocking buffer (TBS+ with 1% bovine serum albumin), the plate was incubated for 1 hr at 37° C., and then washed 3× with TBS+ buffer. Recombinant human a4b7 (R & D Systems) was diluted to 1 μg/ml in TBS+/0.1% bovine serum albumin. Test compounds were diluted into the integrin solution and added to the washed ligand-coated plate according to a standard template with each sample repeated in triplicate. After incubation for 2 hr at room temperature, the plate was washed 3× with 150 μl of TBS+ buffer. To each well, biotinylated anti-A4 antibody (R & D Systems) at 1 μg/ml in TBS+/0.1% BSA was added and the plate covered and incubated for 1 hr at room temperature. After washing the plate 3× with TBS+ buffer, streptavidin-conjugated horseradish peroxidase (R & D Systems) diluted in TBS+ blocking buffer was added to the wells and the plate incubated for 20 min at room temperature. The plate was washed 3× with TBS+ buffer followed by addition of 50 μl of TMB substrate (Sigma T4444). After incubation for 20 min at room temperature, plates were stopped with TMB stop solution (Sigma S5689) by colorimetric detection at 450 nm wavelength using a Tecan Safire II plate reader. |
| Affinity data for this assay | |
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