title: Generation and characterization of monoclonal antibodies against human LGR6.
abstract: Leucine-rich repeat-containing G protein-coupled receptor 6 (LGR6) is a seven-pass transmembrane protein known to be a marker of stem cells in several organs. To deepen our understanding of the cell biology of LGR6-positive cells, including stem cells, we generated monoclonal antibodies (mAbs) against human LGR6. DNA immunization followed by whole-cell immunization with LGR6-expressing transfectants was performed to obtain mAbs that recognized the native form of LGR6. Hybridomas were screened by flow cytometry using LGR6-transfected cells. Because the molecules of LGR4, LGR5, and LGR6 are 50% homologous at the amino acid level, specificity of the mAbs was confirmed by transfectants expressing LGR4, LGR5, or LGR6. Three LGR6-specific mAbs were generated. Two of the three mAbs (designated 43A6 and 43D10) recognized the large N-terminal extracellular domain of LGR6, and competitively blocked the binding of R-spondin 1, which is known to be the ligand for LGR6. The other mAb, 43A25, recognized the seven-pass transmembrane domain of LGR6, and was able to be used for immunoblot analysis. In addition, mAbs 43A6 and 43D10 detected endogenous expression of LGR6 in cancer cell lines. We expect that our mAbs will contribute to widening our understanding of LGR6-positive cells in humans.
authors.affiliation: Forerunner Pharma Research Co., Ltd., Komaba Open Laboratory, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo, 153-8904, Japan.
authors.forename: Shin-Ichi
authors.initials: SI
authors.lastname: Funahashi
authors.affiliation: Forerunner Pharma Research Co., Ltd., Komaba Open Laboratory, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo, 153-8904, Japan.
authors.forename: Yasunori
authors.initials: Y
authors.lastname: Suzuki
authors.affiliation: Forerunner Pharma Research Co., Ltd., Komaba Open Laboratory, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo, 153-8904, Japan.
authors.forename: Kiyotaka
authors.initials: K
authors.lastname: Nakano
authors.affiliation: Forerunner Pharma Research Co., Ltd., Komaba Open Laboratory, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo, 153-8904, Japan.
authors.forename: Shigeto
authors.initials: S
authors.lastname: Kawai
authors.affiliation: Forerunner Pharma Research Co., Ltd., Komaba Open Laboratory, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo, 153-8904, Japan.
authors.forename: Masami
authors.initials: M
authors.lastname: Suzuki
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology* / Q000378:metabolism; D000915:Antibody Affinity / Q000276:immunology*; D000918:Antibody Specificity / Q000276:immunology*; D001667:Binding, Competitive; D002460:Cell Line; D045744:Cell Line, Tumor; D005434:Flow Cytometry; D006801:Humans; D006825:Hybridomas; D015151:Immunoblotting; D008807:Mice, Inbred BALB C; D011485:Protein Binding; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D019699:Thrombospondins / Q000378:metabolism; D000098907:R-Spondins
chemical_list: D000911:Antibodies, Monoclonal; D043562:Receptors, G-Protein-Coupled; D019699:Thrombospondins; C417544:LGR6 protein, human; C503243:RSPO1 protein, human; D000098907:R-Spondins
keywords: LGR6; R-spondin; flow cytometry; monoclonal antibody; stem cell marker