Research paper Methods for comprehensive identification of membrane proteins recognized by a large number of monoclonal antibodies Gene Kurosawa a , Mariko Sumitomo b , Yasushi Akahori a , Kazuki Matsuda b , Chiho Muramatsu b , Akihiko Takasaki c , Yoshitaka Iba a , Keiko Eguchi b , Miho Tanaka d , Kazuhiro Suzuki e , Miwa Morita a , Noriko Sato f , Mototaka Sugiura d , Atsushi Sugioka g , Nobuhiro Hayashi c , Yoshikazu Kurosawa a, ⁎ a Division of Antibody Project, Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi 470-1192, Japan b 21st Century Center of Excellence (COE) Research Center, Fujita Health University, Toyoake, Aichi 470-1192, Japan c Department of Biological Polymer Science, Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi 470-1192, Japan d Department of Internal Medicine, School of Medicine, Fujita Health University, Toyoake, Aichi 470-1192, Japan e Institute for Antibodies, Ltd., Toyoake, Aichi 470-1192, Japan f Department of Urology, School of Medicine, Fujita Health University, Toyoake, Aichi 470-1192, Japan g Department of Surgery, School of Medicine, Fujita Health University, Toyoake, Aichi 470-1192, Japan article info abstract Article history: Received 15 April 2009 Received in revised form 2 September 2009 Accepted 8 September 2009 Available online 18 September 2009 In order to isolate monoclonal antibodies (mAbs) that bind to tumor-associated antigens (Ags) we developed the following strategy. Using the phage-display Ab library we isolated a large number of mAbs that bind to the surface of human tumor cells. The mAbs were individually screened by immunostaining, and clones that preferentially and strongly stained the malignant cells were chosen. Thereafter, the Ags recognized by the mAbs were identified. For identification of the Ags by MS candidate molecules had to be purified either by immunoprecipitation or by affinity chromatography. We isolated several hundred mAbs that showed cancer-specific staining patterns. In order to identify the Ags that were recognized by the numerous mAbs within a short time we developed two methods. Using the GFC [ grouping of clones by flow cytometry (FCM)] method many Abs could be grouped by comparing the staining patterns of FCM. Members in each group turned out to bind to the same molecule in many cases. After a candidate Ag was revealed, the polypeptide corresponding to its extracellular portion was prepared and used for identification of clones that bound to the Ag among all the mAbs by SITE (simultaneous identification of clones through three dimensional ELISA) method. Both methods can be generally applicable to various kinds of membrane proteins and the mAbs against them. © 2009 Elsevier B.V. All rights reserved. Keywords: Phage antibody library Tumor-associated antigen Therapeutic antibody Flow cytometry Enzyme-linked immunosorbent assay 1. Introduction The beneficial effects of several monoclonal antibodies (mAbs) for the treatment of patients with breast cancer and non-Hodgkins lymphoma (Carter et al., 1992; Reff et al. 1994) suggest that mAbs are likely to become very important therapeutic agents also against other forms of cancers. However, for the majority of the human cancers useful therapeutic Abs are not yet available because of our lack of knowledge of which antigens (Ags) are likely to become effective targets (Adams and Weiner, 2005). Therefore, we Journal of Immunological Methods 351 (2009) 1–12 Abbreviations: aa, amino acid; Ab, antibody; Ag, antigen; ELISA, enzyme- linked immunosorbent assay; FCM, flow cytometry; GFC, grouping of clones by flow cytometry; ICOS, isolation of Ag/Ab complexes through organic solvent; IP, immunoprecipitation; mAb, monoclonal Ab; MS, mass spec- trometry; rt, room temperature; scFv, single chain Fv; SITE, simultaneous identification of clones through three dimensional ELISA; TAA, tumor- associated antigen; 3D, three dimensional. ⁎ Corresponding author. Tel.: +81 562 93 9287; fax: +81 562 93 8835. E-mail address: kurosawa@fujita-hu.ac.jp (Y. Kurosawa). 0022-1759/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2009.09.003 Contents lists available at ScienceDirect Journal of Immunological Methods journal homepage: www.elsevier.com/locate/jim