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Production of monoclonal antibodies against GPCR using cell-free synthesized GPCR antigen and biotinylated liposome-based interaction assay
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, a...
Autores principales: | , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group
2015
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4462149/ https://www.ncbi.nlm.nih.gov/pubmed/26061673 http://dx.doi.org/10.1038/srep11333 |
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author | Takeda, Hiroyuki Ogasawara, Tomio Ozawa, Tatsuhiko Muraguchi, Atsushi Jih, Pei-Ju Morishita, Ryo Uchigashima, Motokazu Watanabe, Masahiko Fujimoto, Toyoshi Iwasaki, Takahiro Endo, Yaeta Sawasaki, Tatsuya |
author_facet | Takeda, Hiroyuki Ogasawara, Tomio Ozawa, Tatsuhiko Muraguchi, Atsushi Jih, Pei-Ju Morishita, Ryo Uchigashima, Motokazu Watanabe, Masahiko Fujimoto, Toyoshi Iwasaki, Takahiro Endo, Yaeta Sawasaki, Tatsuya |
author_sort | Takeda, Hiroyuki |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-4462149 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | Nature Publishing Group |
record_format | MEDLINE/PubMed |
spelling | pubmed-44621492015-06-12 Production of monoclonal antibodies against GPCR using cell-free synthesized GPCR antigen and biotinylated liposome-based interaction assay Takeda, Hiroyuki Ogasawara, Tomio Ozawa, Tatsuhiko Muraguchi, Atsushi Jih, Pei-Ju Morishita, Ryo Uchigashima, Motokazu Watanabe, Masahiko Fujimoto, Toyoshi Iwasaki, Takahiro Endo, Yaeta Sawasaki, Tatsuya Sci Rep Article 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. Nature Publishing Group 2015-06-10 /pmc/articles/PMC4462149/ /pubmed/26061673 http://dx.doi.org/10.1038/srep11333 Text en Copyright © 2015, Macmillan Publishers Limited http://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ |
spellingShingle | Article Takeda, Hiroyuki Ogasawara, Tomio Ozawa, Tatsuhiko Muraguchi, Atsushi Jih, Pei-Ju Morishita, Ryo Uchigashima, Motokazu Watanabe, Masahiko Fujimoto, Toyoshi Iwasaki, Takahiro Endo, Yaeta Sawasaki, Tatsuya Production of monoclonal antibodies against GPCR using cell-free synthesized GPCR antigen and biotinylated liposome-based interaction assay |
title | Production of monoclonal antibodies against GPCR using cell-free synthesized GPCR antigen and biotinylated liposome-based interaction assay |
title_full | Production of monoclonal antibodies against GPCR using cell-free synthesized GPCR antigen and biotinylated liposome-based interaction assay |
title_fullStr | Production of monoclonal antibodies against GPCR using cell-free synthesized GPCR antigen and biotinylated liposome-based interaction assay |
title_full_unstemmed | Production of monoclonal antibodies against GPCR using cell-free synthesized GPCR antigen and biotinylated liposome-based interaction assay |
title_short | Production of monoclonal antibodies against GPCR using cell-free synthesized GPCR antigen and biotinylated liposome-based interaction assay |
title_sort | production of monoclonal antibodies against gpcr using cell-free synthesized gpcr antigen and biotinylated liposome-based interaction assay |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4462149/ https://www.ncbi.nlm.nih.gov/pubmed/26061673 http://dx.doi.org/10.1038/srep11333 |
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