ELISA-Based Reporter Assay to Study the Receptor-Ligand Interaction in BW Cells
Transcrição
To study receptor-ligand interactions, begin with a multi-well plate containing media with target cells expressing test ligands on their surface.
Add engineered mouse BW cells — a novel B-cell population expressing membrane-bound chimeric receptors. Chimeric receptors comprise a specific receptor's extracellular domain fused to the B-cell receptor's transmembrane and intracellular signaling domain.
Ligands on the target cells interact with the extracellular domain of BW cells' chimeric receptors, causing intracellular signaling domain activation in BW cells. This triggers downstream signaling and the production of cytokines — secretory reporter molecules that get released outside the BW cells, appearing in the media.
Centrifuge the plate. Transfer the cytokine-containing supernatant into an ELISA plate pre-coated with anti-cytokine antibodies. Cytokines interact with the antibodies' Fab regions, leading to their immobilization on the plate surface.
Treat the plate with a blocking solution to block non-specific binding sites. Add biotinylated anti-cytokine antibodies, which interact with pre-bound cytokines and form immune complexes.
Overlay the plate with an enzyme-conjugated streptavidin solution. Streptavidin binds with biotin on the immune complexes.
Treat the plate with a substrate. The enzyme-substrate interaction produces a colored product, imparting the same color to the solution. The colored solution confirms the presence of reporter molecules, indicating successful receptor-ligand interaction.
