Research paper Development and evaluation of monoclonal antibodies against phosphatidylethanolamine binding protein 1 in pancreatic cancer patients Xiaoting Wang a,1 , Shuangshuang Wang b,c,1 , Xiaojun Tang b , Aixia Zhang b , Tessa Grabinski c , Zhenying Guo b,c , Eric Hudson d , Bree Berghuis d , Craig Webb e , Ping Zhao c, ⁎, Brian Cao b,c a Jiangsu Institute of Parasitic Disease, 117 Mei Yuan Yang Xiang, Wuxi,Jiangsu Province 214064, China b Nanjing Medical University, Key Laboratory of Antibody Technology of Ministry of Health, 140 Hanzhong Road, Nanjing,Jiangsu Province 210029, China c Antibody Technology Laboratory, Van Andel Research Institute, 333 Bostwick Ave NE, Grand Rapids, MI 49503,USA d Laboratory of Analytical, Cellular and Molecular Microscopy, Van Andel Research Institute, 333 Bostwick Ave NE, Grand Rapids, MI 49503,USA e Program of Translational Medicine, Van Andel Research Institute, 333 Bostwick Ave NE, Grand Rapids, MI 49503,USA a r t i c l e i n f o a b s t r a c t Article history: Received 12 April 2010 Received in revised form 8 July 2010 Accepted 16 September 2010 Available online 1 October 2010 Phosphatidylethanolamine binding protein 1 (PEBP1), also known as Raf kinase inhibitor protein (RKIP), has been considered as a suppressor of metastasis and a prognostic marker in prostate cancer,breastcancer,gastrointestinalstromal tumors,melanoma,and epithelial ovarian cancer. In this report, recombinant PEBP1 was successfully expressed in an Escherichia coli system. A panel of monoclonal antibodies (mAbs) against PEBP1 with high speci ficity and affinity was generated and characterized using ELISA, western blot analysis, immuno fluorescent staining and immunohistochemical staining. PEBP1 expression in normal 293 cells and a few pancreatic cancer cell lines was detected with mAb 7F12 in western blot analysis. To screen for a pair of mAbs with optimal binding affinity to soluble PEBP1, ForteBio's Octet system was used. Sandwich ELISA with mAb pair 4F10 and 8E2 showed a linear correlation between absorbance and PEBP1 protein concentration over a range of 7 to 100 ng/ml. MAb 4A11 detected a high leve 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. In 69 valid cases, PEBP1 expression was significantly lower in tumor than in normal pancreas ( p = 8.40E-14) and adjacent normal tissue (p = 8.46E-17). PEBP1 expression in pancreatic cancer was not associated with pTMN stage,differentiation grade and pathologic diagnosis. In conclusion,our results suggest that PEBP1 overexpresses in normal pancreas but significantly decreases its expression in pancreatic cancer tissues. Anti-PEBP1 mAbs 4A11, 4F10,7F12,and 8E2 are potential clinical diagnostic agents for pancreatic cancer. © 2010 Elsevier B.V. All rights reserved. Keywords: PEBP1 Monoclonal antibody Pancreatic cancer Sandwich ELISA Tissue microarray 1. Introduction Pancreatic cancer is the sixth-highest cause of mortality from malignant tumors in Europe and the fourth-highest in the United States ( Li et al., 2004). In 2008, the estimated incidence of pancreatic cancer in the United States was 37,700 cases, and an estimated 34,300 patients died from the disease (Hidalgo, 2010). The dismal prognosis of pancreatic cancer is due to the late stage at which it is usually diagnosed, because pancreatic cancerpatients seldom exhibit disease-specific symptoms until late in the course of the disease (Jemal et al., 2007), and the high invasive and metastatic potential of pancreatic tumors result in a low rate of curative resections and a high frequency of relapse.Long-term survivalfrom pancreatic cancer is possible if the disease is identified at an early stage (Cleary et al., 2004).Currently, no single clinical Journal of Immunological Methods 362 (2010) 151–160 ⁎ Corresponding author. Tel.: + 1 616 234 5394; fax: +1 616 234 5395. E-mail address: ping.zhao@vai.org (P. Zhao). 1 These two authors contributed equally to this work. 0022-1759/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2010.09.026 Contents lists available at ScienceDirect Journal of Immunological Methods j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j i m