Development And Evaluation Of Monoclonal Antibodies Against Phosphatidylethanolamine Binding Protein 1 In Pancreatic Cancer Patients

Phosphatidylethanolamine binding protein 1(PEBP1), also known as Raf kinase inhibitor protein (RKIP), is a member of the phosphatidylethanolamine-binding protein (PEBP) family, which is a widely expressed protein in a variety of different species. It plays a pivotal modulatory role in several intracellular signaling cascades. PEBP1 binds to Raf-1, and blocks MAPK signaling pathway. Also it has been demonstrated to regulate G-protein-coupled receptor signaling pathway and NF-κB signaling. PEBP1 plays a critical role in several physiological processes including membrane biosynthesis, spermatogenesis, neural development and apoptosis. Additionally, recent researches show PEBP1 is a novel metastasis suppressor, which suppresses metastasis in prostate cancer, breast cancer, colorectal cancer, gastrointestinal stromal tumors, melanoma cancer and epithelial ovarian cancer. But the expression and the role of PEBP1 in pancreatic cancer are unknown. Our lab has expressed and purified PEBP1 protein and the purity was above 90%. The aim of this study was to generate monoclonal antibodies (mAb) against human PEBP1 and to establish a Sandwich ELISA method for potential clinical applications across mutiple cancer types including pancreatic cancer. The study was designed as following:1. Monoclonal antibody production and characteration2. Establishment of Sanwich ELISA to detect PEBP1 native protein.3. Application of the antibody in pancreatic cancer patients Materials and Methods1. Monoclonal antibody production and characteration:Mouse mAbs against PEBP1 were produced by injecting BALB/c mice i.p. with puri?ed native and denatured (boiled in SDS sample buffer) PEBP1 protein (20μg/mouse). Spleen cells were isolated from the sacri?ced mice and then were fused with OUR-1 myeloma cells using standard techniques four days after the ?nal injections. Four clones with the highest OD values in capture ELISA were picked after expanded. The mAbs were purified through a protein-G column. After obtain mAbs, we evaluated the activities of them. We chose the pancreatic cancer cell lines, and characterized using ELISA, Western Blot analysis.2. Establishment of Sanwich ELISA to detect PEBP1 native protein.To screen for a pair of mAbs with optimal binding af?nity to soluble PEBP1, ForteBio's Octet system was used. They can be used in clinical diagnosis of early stage pancreatic cancer.3. Application of the antibody in Pancreatic cancer patientsPEBP1 has been considered as a suppressor of metastasis and a prognostic marker in many kinds of cancer. Here, human pancreatic tissue array was purchased. We use the mAbs to detect the level of PEBP1 in normal pancreatic tissue and pancreatic cancer tissue.Results1. A panel of monoclonal antibodies against PEBP1 with high specificity and affinity was generated and characterized using ELISA, Western Blot analysis, immunoflurescent staing. Clones 4A11, 4F10, 7F12 and 8E2 were subcloned as a result of their reactivities with PEBP1 in different applications. 7F12 detects denatured PEBP1 in Western Blot analysis.2. mAb pair 4F10 and 8E2 has the optimal binding affinity for soluble PEBP1 detection in sandwich ELISA. They were able to detect the ng/ml level of soluble PEBP1 in a sandwich ELISA. MAbs 4F10 and 8E2 are a pair that can be used in a sandwich ELISA detection system for rapid and early detection of pancreatic cancer and other cancers in which PEBP1 has been implicated. Further studies are needed to evaluate sensitivity and speci?city of PEBP1 ELISA in well-annotated clinical patient sample sets (serum, plasmas or pancreatic juice).3. MAb 4A11 detected a high level expression of PEBP1 in normal pancreatic tissue, and cancer adjacent normal pancreatic tissue in a pancreatic tissue microarray (TMA) comprising 80 human tissue cores. Pancreatic cancer tissues show a no or very weak staining intensity of PEBP1. PEBP1 expression in pancreatic cancer was not associated with pTMN stage, differentiation grade and pathologic diagnosis.ConclusionsOur results suggest that PEBP1 overexpresses in normal pancreas but signi?cantly decreases its expression in pancreatic cancer tissues. Anti-PEBP1 mAbs 4A11, 4F10, 7F12, and 8E2 are potential clinical diagnostic agents for pancreatic cancer.