Cargando…
Epitope Mapping of Anti-Mouse CCR3 Monoclonal Antibodies Using Flow Cytometry
The CC chemokine receptor 3 (CCR3) is a receptor for CC chemokines, including CCL5/RANTES, CCL7/MCP-3, and CCL11/eotaxin. CCR3 is expressed on the surface of eosinophils, basophils, a subset of Th2 lymphocytes, mast cells, and airway epithelial cells. CCR3 and its ligands are involved in airway hype...
Autores principales: | , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2022
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9774254/ https://www.ncbi.nlm.nih.gov/pubmed/36546900 http://dx.doi.org/10.3390/antib11040075 |
_version_ | 1784855362838462464 |
---|---|
author | Tateyama, Nami Asano, Teizo Suzuki, Hiroyuki Li, Guanjie Yoshikawa, Takeo Tanaka, Tomohiro Kaneko, Mika K. Kato, Yukinari |
author_facet | Tateyama, Nami Asano, Teizo Suzuki, Hiroyuki Li, Guanjie Yoshikawa, Takeo Tanaka, Tomohiro Kaneko, Mika K. Kato, Yukinari |
author_sort | Tateyama, Nami |
collection | PubMed |
description | The CC chemokine receptor 3 (CCR3) is a receptor for CC chemokines, including CCL5/RANTES, CCL7/MCP-3, and CCL11/eotaxin. CCR3 is expressed on the surface of eosinophils, basophils, a subset of Th2 lymphocytes, mast cells, and airway epithelial cells. CCR3 and its ligands are involved in airway hyperresponsiveness in allergic asthma, ocular allergies, and cancers. Therefore, CCR3 is an attractive target for those therapies. Previously, anti-mouse CCR3 (mCCR3) monoclonal antibodies (mAbs), C(3)Mab-3 (rat IgG(2a), kappa), and C(3)Mab-4 (rat IgG(2a), kappa) were developed using the Cell-Based Immunization and Screening (CBIS) method. In this study, the binding epitope of these mAbs was investigated using flow cytometry. A CCR3 extracellular domain-substituted mutant analysis showed that C(3)Mab-3, C(3)Mab-4, and a commercially available mAb (J073E5) recognized the N-terminal region (amino acids 1–38) of mCCR3. Next, alanine scanning was conducted in the N-terminal region. The results revealed that the Ala2, Phe3, Asn4, and Thr5 of mCCR3 are involved in C(3)Mab-3 binding, whereas Ala2, Phe3, and Thr5 are essential to C(3)Mab-4 binding, and Ala2 and Phe3 are crucial to J073E5 binding. These results reveal the involvement of the N-terminus of mCCR3 in the recognition of C(3)Mab-3, C(3)Mab-4, and J073E5. |
format | Online Article Text |
id | pubmed-9774254 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-97742542022-12-23 Epitope Mapping of Anti-Mouse CCR3 Monoclonal Antibodies Using Flow Cytometry Tateyama, Nami Asano, Teizo Suzuki, Hiroyuki Li, Guanjie Yoshikawa, Takeo Tanaka, Tomohiro Kaneko, Mika K. Kato, Yukinari Antibodies (Basel) Article The CC chemokine receptor 3 (CCR3) is a receptor for CC chemokines, including CCL5/RANTES, CCL7/MCP-3, and CCL11/eotaxin. CCR3 is expressed on the surface of eosinophils, basophils, a subset of Th2 lymphocytes, mast cells, and airway epithelial cells. CCR3 and its ligands are involved in airway hyperresponsiveness in allergic asthma, ocular allergies, and cancers. Therefore, CCR3 is an attractive target for those therapies. Previously, anti-mouse CCR3 (mCCR3) monoclonal antibodies (mAbs), C(3)Mab-3 (rat IgG(2a), kappa), and C(3)Mab-4 (rat IgG(2a), kappa) were developed using the Cell-Based Immunization and Screening (CBIS) method. In this study, the binding epitope of these mAbs was investigated using flow cytometry. A CCR3 extracellular domain-substituted mutant analysis showed that C(3)Mab-3, C(3)Mab-4, and a commercially available mAb (J073E5) recognized the N-terminal region (amino acids 1–38) of mCCR3. Next, alanine scanning was conducted in the N-terminal region. The results revealed that the Ala2, Phe3, Asn4, and Thr5 of mCCR3 are involved in C(3)Mab-3 binding, whereas Ala2, Phe3, and Thr5 are essential to C(3)Mab-4 binding, and Ala2 and Phe3 are crucial to J073E5 binding. These results reveal the involvement of the N-terminus of mCCR3 in the recognition of C(3)Mab-3, C(3)Mab-4, and J073E5. MDPI 2022-12-02 /pmc/articles/PMC9774254/ /pubmed/36546900 http://dx.doi.org/10.3390/antib11040075 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Tateyama, Nami Asano, Teizo Suzuki, Hiroyuki Li, Guanjie Yoshikawa, Takeo Tanaka, Tomohiro Kaneko, Mika K. Kato, Yukinari Epitope Mapping of Anti-Mouse CCR3 Monoclonal Antibodies Using Flow Cytometry |
title | Epitope Mapping of Anti-Mouse CCR3 Monoclonal Antibodies Using Flow Cytometry |
title_full | Epitope Mapping of Anti-Mouse CCR3 Monoclonal Antibodies Using Flow Cytometry |
title_fullStr | Epitope Mapping of Anti-Mouse CCR3 Monoclonal Antibodies Using Flow Cytometry |
title_full_unstemmed | Epitope Mapping of Anti-Mouse CCR3 Monoclonal Antibodies Using Flow Cytometry |
title_short | Epitope Mapping of Anti-Mouse CCR3 Monoclonal Antibodies Using Flow Cytometry |
title_sort | epitope mapping of anti-mouse ccr3 monoclonal antibodies using flow cytometry |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9774254/ https://www.ncbi.nlm.nih.gov/pubmed/36546900 http://dx.doi.org/10.3390/antib11040075 |
work_keys_str_mv | AT tateyamanami epitopemappingofantimouseccr3monoclonalantibodiesusingflowcytometry AT asanoteizo epitopemappingofantimouseccr3monoclonalantibodiesusingflowcytometry AT suzukihiroyuki epitopemappingofantimouseccr3monoclonalantibodiesusingflowcytometry AT liguanjie epitopemappingofantimouseccr3monoclonalantibodiesusingflowcytometry AT yoshikawatakeo epitopemappingofantimouseccr3monoclonalantibodiesusingflowcytometry AT tanakatomohiro epitopemappingofantimouseccr3monoclonalantibodiesusingflowcytometry AT kanekomikak epitopemappingofantimouseccr3monoclonalantibodiesusingflowcytometry AT katoyukinari epitopemappingofantimouseccr3monoclonalantibodiesusingflowcytometry |