title: Methods for comprehensive identification of membrane proteins recognized by a large number of monoclonal antibodies.
abstract: 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.
authors.affiliation: Division of Antibody Project, Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi 470-1192, Japan.
authors.forename: Gene
authors.initials: G
authors.lastname: Kurosawa
authors.forename: Mariko
authors.initials: M
authors.lastname: Sumitomo
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Akahori
authors.forename: Kazuki
authors.initials: K
authors.lastname: Matsuda
authors.forename: Chiho
authors.initials: C
authors.lastname: Muramatsu
authors.forename: Akihiko
authors.initials: A
authors.lastname: Takasaki
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Iba
authors.forename: Keiko
authors.initials: K
authors.lastname: Eguchi
authors.forename: Miho
authors.initials: M
authors.lastname: Tanaka
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Suzuki
authors.forename: Miwa
authors.initials: M
authors.lastname: Morita
authors.forename: Noriko
authors.initials: N
authors.lastname: Sato
authors.forename: Mototaka
authors.initials: M
authors.lastname: Sugiura
authors.forename: Atsushi
authors.initials: A
authors.lastname: Sugioka
authors.forename: Nobuhiro
authors.initials: N
authors.lastname: Hayashi
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Kurosawa
mesh_terms: D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D000951:Antigens, Neoplasm / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D000954:Antigens, Surface / Q000276:immunology; D001671:Biochemistry / Q000379:methods; D020650:Combinatorial Chemistry Techniques; D015723:Gene Library; D006801:Humans; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D009369:Neoplasms / Q000235:genetics / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000235:genetics
chemical_list: D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D000954:Antigens, Surface; D008565:Membrane Proteins; D034741:RNA, Small Interfering