Detection of Receptor Activation in FFPE Specimens Figure 3. Epitope mapping of c-MET antibodies by peptide scanning. A. Peptide ELISA. A series of 43 overlapping peptides, each 14–15 amino-acid residues in length, spanning the complete intracellular domain of c-MET (K955-S1370), were evaluated to map the binding epitope of c- MET antibodies. Peptides were spotted on microtiter plates and ELISA assays were performed using standard protocols. The binding properties of peptide 28 and 43 localized the binding sites of c-MET antibody 3D4 and c-MET antibodies CVD13 and SP44, respectively. The sequence of peptide #28 and #43 are shown (inset). B. A schematic representing the c-MET receptor and binding sites for c-MET antibodies. doi:10.1371/journal.pone.0015932.g003 that potentiates cell signaling [5]. Aberrant HGF/c-MET signaling via ligand dependent (both paracrine and autocrine) and ligand independent mechanisms is well documented in several human cancers [4,5] and multiple therapeutic agents targeting this pathway are in clinical development [4–6]. c-MET phosphoryla- tion has been reported in Non-Small Cell Lung (NSCLC) carcinoma, Head and Neck Squamous Cell Carcinoma (HNSCC), and other carcinomas [7–9]. Like most receptor kinases, improved methods for reliable measurement of c-MET receptor activation are needed. In this report, we describe the development of assays to detect and quantify ligand-receptor complexes as surrogate measure of receptor activation. Using an antibody proximity-based technol- ogy (VeraTag), we describe a novel approach for the detection and quantification of the HGF-c-MET ligand-receptor complex in FFPE specimens including cell lines and human carcinoma tissues. To our knowledge, this is the first report describing detection and quantification of ligand-receptor complexes in the FFPE format. Additionally, we also describe the development of assays to detect and quantify total c-MET and HGF levels in FFPE specimens. Our approach demonstrates the potential of antibody proximity assays to provide reliable measurements of aberrant receptor activation in FFPE specimens. Materials and Methods H441, H226, A549, H2170, MCF7, H661, Ln229, U87MG, Ln18, U138, U118, and H596 cell lines were obtained from ATCC (American Type culture collection, Manassas, VA) and maintained in DMEM/F12 (50:50) containing 10% fetal bovine serum (Invitrogen, Carlsbad, CA), 1% penicillin-streptomycin, 2 mM L-Glutamine (CellGro, Manassas, VA). Antibodies used in this study are as follows: Mouse anti-c-Met (clone 3D4) (Invitrogen, Carlsbad, CA), Rabbit anti-c-Met (CVD13) (Invitro- gen, Carlsbad, CA), Anti-c-MET (clone SP44) (Spring Biosciences, Pleasanton, CA), Human HGF affinity chromatography purified Anti-human HGF (R&D systems, Minneapolis, MN), Anti-human HGF Ab-2 (clone SBF5) (Thermo Scientific, Fremont, CA), PLoS ONE | www.plosone.org 4 January 2011 | Volume 6 | Issue 1 | e15932