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| | Binding Inhibition Assay |
| Description: | The ability of test substances to inhibit the binding of a human B cell line, RPMI-8866, which is known to express α4β7 integrin, to MAdCAM-1 was measured. To 96-well microtiter plates, recombinant mouse MAdCAM-1/Fc (R&D systems) solution (1 ug/mL) diluted with buffer A (carbonate buffer, pH 9.6) was added at 50 uL/well, followed by incubation at 4° C. overnight. After washing once with PBS, Block Ace (Snow Brand Milk Products Company, Limited) was added at 150 uL/well, followed by incubation at room temperature for 2 hours. After removal, washing was conducted once with PBS.100 uL of each test substance diluted with a binding buffer (DMEM containing 40 mM HEPES, 0.2% BSA, and 4 mM MnCl2) to various concentrations and 100 uL of RPMI-8866 cells (2x106 cell/mL) were added to plates coated with MAdCAM-1/Fc (5x105 cells/well) with human serum being contained at a final concentration of 50%, followed by incubation at 30° C. for 15 minutes to 60 minutes. After the cells were bound to each well, unbound cells were removed by washing with PBS. To the plates, buffer C (PBS containing 1.5% Triton X-100) was added at 50 uL/well to lyse the bound RPMI-8866 cells. To 30 uL of the cell lysate, 30 uL of Substrate Buffer (Promega, CytoTox 96 Non-Radioactive Cytotoxicity Assay) was added, and the reaction was allowed to proceed at room temperature in a dark place for 30 minutes. To each well, 30 uL of Stop Solution (Promega, CytoTox 96 Non-Radioactive Cytotoxicity Assay) was added, and the absorbance at 490 nm was measured by using a plate reader. Here, by the obtained absorbance, the activity of the lactate dehydrogenase (LDH) dissolved into the supernatant of each well was detected. In other words, the absorbance was proportional to the number of the RPMI-8866 cells bound to MAdCAM-1 and remaining on the plate. The test was duplicated. The binding ratios of the cells at various concentrations were determined, with the absorbance of a well containing no test substance taken as 100%. Then, the concentration IC50, at which 50% binding inhibition was achieved, was calculated. |
| Affinity data for this assay | |
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