(a) Detection of EGFR in cell extracts. Beads coated with different concentrations of EGF were used to capture EGFR from a cell lysate in a concentration-dependent manner. The ligand EGF was immobilized at 1.2 μM (diamonds) and 0.3 μM (circles) on different bead types. Both bead sets were mixed and incubated with different amounts of BT-20 cell lysates. Captured EGFR was detected using an anti-EGFR antibody and a labeled secondary antibody. (b) Specificity of capture activity. A BT-20 cell lysate (500 μg/mL) was coincubated with various concentrations of purified and unbiotinylated sEGF and beads coated with EGF-Biot (1.2 μM). The amount of captured EGF receptor was strongly reduced at the presence of sEGF. This indicates that the bead-bound EGF-EGFR complex is captured in a specific fashion by applying an anti-EGFR antibody followed by a labeled secondary antibody. (c) Correlation of ligand binding and sandwich immunoassay. A series of tissue lysates with known EGFR concentrations were analyzed with both, a ligand binding assay and an immunoassay using an antibody as capture reagent. In both assays, the same anti-EGFR detection molecule was used and the profiles obtained reveal a correlation of 0.94, which indicated a good concordance between the two tests.
