title: Production of monoclonal antibodies against GPCR using cell-free synthesized GPCR antigen and biotinylated liposome-based interaction assay.
abstract: G-protein-coupled receptors (GPCRs) are one of the most important drug targets, and anti-GPCR monoclonal antibody (mAb) is an essential tool for functional analysis of GPCRs. However, it is very difficult to develop GPCR-specific mAbs due to difficulties in production of recombinant GPCR antigens, and lack of efficient mAb screening method. Here we describe a novel approach for the production of mAbs against GPCR using two original methods, bilayer-dialysis method and biotinylated liposome-based interaction assay (BiLIA), both of which are developed using wheat cell-free protein synthesis system and liposome technology. Using bilayer-dialysis method, various GPCRs were successfully synthesized with quality and quantity sufficient for immunization. For selection of specific mAb, we designed BiLIA that detects interaction between antibody and membrane protein on liposome. BiLIA prevented denaturation of GPCR, and then preferably selected conformation-sensitive antibodies. Using this approach, we successfully obtained mAbs against DRD1, GHSR, PTGER1 and T1R1. With respect to DRD1 mAb, 36 mouse mAbs and 6 rabbit mAbs were obtained which specifically recognized native DRD1 with high affinity. Among them, half of the mAbs were conformation-sensitive mAb, and two mAbs recognized extracellular loop 2 of DRD1. These results indicated that this approach is useful for GPCR mAb production.
authors.affiliation: Proteo-Science Center, Ehime University, Ehime 791-8577, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Takeda
authors.affiliation: Proteo-Science Center, Ehime University, Ehime 791-8577, Japan.
authors.forename: Tomio
authors.initials: T
authors.lastname: Ogasawara
authors.affiliation: Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, Toyama 930-0194, Japan.
authors.forename: Tatsuhiko
authors.initials: T
authors.lastname: Ozawa
authors.affiliation: Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, Toyama 930-0194, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Muraguchi
authors.affiliation: Abnova (Taiwan) Corporation, Taoyuan County 320, Taiwan.
authors.forename: Pei-Ju
authors.initials: PJ
authors.lastname: Jih
authors.affiliation: Cell-Free Sciences Co., Ltd., Ehime 791-8577, Japan.
authors.forename: Ryo
authors.initials: R
authors.lastname: Morishita
authors.affiliation: Graduate School of Medicine, Hokkaido University, Hokkaido 060-8638, Japan.
authors.forename: Motokazu
authors.initials: M
authors.lastname: Uchigashima
authors.affiliation: Graduate School of Medicine, Hokkaido University, Hokkaido 060-8638, Japan.
authors.forename: Masahiko
authors.initials: M
authors.lastname: Watanabe
authors.affiliation: Graduate School of Medicine, Nagoya University, Aichi 466-8550, Japan.
authors.forename: Toyoshi
authors.initials: T
authors.lastname: Fujimoto
authors.affiliation: Proteo-Science Center, Ehime University, Ehime 791-8577, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Iwasaki
authors.affiliation: Institute for the Promotion of Science and Technology, Ehime University, Ehime 791-8577, Japan.
authors.forename: Yaeta
authors.initials: Y
authors.lastname: Endo
authors.affiliation: Proteo-Science Center, Ehime University, Ehime 791-8577, Japan.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Sawasaki
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology*; D000915:Antibody Affinity / Q000276:immunology; D019921:Biotinylation; D002474:Cell-Free System; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D000939:Epitopes / Q000276:immunology; D006801:Humans; D008081:Liposomes / Q000276:immunology; D051379:Mice; D011817:Rabbits; D017447:Receptors, Dopamine D1 / Q000276:immunology*; D043562:Receptors, G-Protein-Coupled / Q000276:immunology*; D054440:Receptors, Ghrelin / Q000276:immunology*; D058306:Receptors, Prostaglandin E, EP1 Subtype / Q000276:immunology*; D011994:Recombinant Proteins / Q000138:chemical synthesis / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; C583260:DRD1 protein, human; D000939:Epitopes; D008081:Liposomes; C545635:PTGER1 protein, human; D017447:Receptors, Dopamine D1; D043562:Receptors, G-Protein-Coupled; D054440:Receptors, Ghrelin; D058306:Receptors, Prostaglandin E, EP1 Subtype; D011994:Recombinant Proteins; C431926:taste receptors, type 1