Baf358

Capture antibodies for Axl, Met, EGFR, and Her2 were purchased from R&D Systems (MAB154, MAB3581, AF231, and MAB1129 respectively). Biotinylated detection antibodies for Axl, Met, EGFR and Her2 were purchased from R&D Systems (BAF154, BAF358, BAF231, and BAF1129 respectively). Streptavidin Phycoerythrin (SAPE) was purchased from Biorad (cat. no. 171304501). Assay diluent and washing buffer used in all steps was 0.1% BSA + 0.1% Tween20 in 1× PBS.
100 μL MagPix beads (Luminex Corp.) were centrifuged at 10,000×g for 2 min and the supernatant was discarded. EDC (N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide, Sigma) and S-NHS (N-hydroxysulfosuccinimide, Pierce) were dissolved in diH₂0 to 50 mg/mL. Beads were incubated with 80 μL activation buffer (100 mM NaH₂PO₄ pH 6.3), 10 μL EDC, and 10 μL S-NHS for 20 min dark at room temperature shaking at ~ 900 rpm. The mixture was centrifuged at 10,000×g for 2 min and the supernatant was discarded. Antibodies were diluted to 0.1 mg/mL in 100 μL coupling buffer (50 mM HEPES, pH 7.4) and incubated with the bead suspension, overnight shaking, at 4 °C. The following day beads were washed 3× and resuspended in 1 mL 1% BSA + 1% Tween20 in 1x PBS and stored at 4 °C.

Met, phosphotyrosyl-Met, and soluble Met ectodomain (sMet) proteins were measured in tissue and plasma samples as described previously [22 (link)]. Briefly, streptavidin-coated 96-well plates were blocked, washed with PBS and coated with a biotin-tagged, affinity-purified human Met ectodomain-specific capture antibody (BAF358, R&D Systems). Wells were washed again before adding sample or sMet standard (358-MT, R&D Systems) for 1 h with shaking. After washing with PBS, detection antibody (AF276, R&D Systems) labeled with MSD-Sulfo-Tag (Meso Scale Discovery, Gaithersburg, MD) was added for 1 h with shaking. Wells were washed with PBS before adding read buffer and plates were read in a Meso Scale Discovery SectorImager 2400. Tissue samples were extracted with ice-cold detergent containing buffer with physical disruption with 0.5mm glass beads and shaking in a bead beater (Mini Bead Beater 8, BioSpec Products, Inc., Bartlesville, OK) as described previously [26 (link)]. Assays were performed blinded to study endpoint.

Normally voided urine samples were stored at −80°C prior to analysis. Thawed samples were processed by centrifugation, ultrafiltration and pH adjustment to 7.0 prior to sMet quantitation as described previously [12 (link)]. Briefly, streptavidin-coated 96-well plates were blocked, washed with PBS and coated with a biotin-tagged, affinity-purified human Met ectodomain-specific capture antibody (BAF358, R&D Systems). Wells were washed again before adding sample or sMet standard (358-MT, R&D Systems) for 1 h with shaking. After washing with PBS, detection antibody (AF276, R&D Systems) labeled with MSD-Sulfo-Tag (Meso Scale Discovery) was added for 1 h with shaking. Wells were washed with PBS before adding read buffer and plates were read in a MSD Sector Imager 2400. Assays were performed blinded to study endpoint.