Cargando…

Effective preparation of a monoclonal antibody against human chromogranin A for immunohistochemical diagnosis

BACKGROUND: Human chromogranin A (CgA) is a ~ 49 kDa secreted protein mainly from neuroendocrine cells and endocrine cells. The CgA values in the diagnosis of tumor, and in the potential role in prognostic and predictive tumor as a biomarker. RESULTS: The synthesized gene of CgA coding area was clon...

Descripción completa

Detalles Bibliográficos
Autores principales: Zhang, Danping, Xie, Chengjie, Wang, Rongzhi, Yang, Qinghai, Chen, Huiling, Ling, Sumei, Wang, Shihua, Jia, Kunzhi
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5935939/
https://www.ncbi.nlm.nih.gov/pubmed/29728076
http://dx.doi.org/10.1186/s12896-018-0436-z
_version_ 1783320359335886848
author Zhang, Danping
Xie, Chengjie
Wang, Rongzhi
Yang, Qinghai
Chen, Huiling
Ling, Sumei
Wang, Shihua
Jia, Kunzhi
author_facet Zhang, Danping
Xie, Chengjie
Wang, Rongzhi
Yang, Qinghai
Chen, Huiling
Ling, Sumei
Wang, Shihua
Jia, Kunzhi
author_sort Zhang, Danping
collection PubMed
description BACKGROUND: Human chromogranin A (CgA) is a ~ 49 kDa secreted protein mainly from neuroendocrine cells and endocrine cells. The CgA values in the diagnosis of tumor, and in the potential role in prognostic and predictive tumor as a biomarker. RESULTS: The synthesized gene of CgA coding area was cloned and expressed as fusion protein CgA-His in procaryotic system. Then the purified CgA-His protein was mixed with QuickAntibody-Mouse5W adjuvant, and injected into mice. The CgA-His protein was also used as coating antigen to determine the antiserum titer. By screening, a stable cell line named 4E5, which can generate anti-CgA monoclonal antibody (mAb), was obtained. The isotype of 4E5 mAb was IgG(2b), and the chromosome number was 102 ± 4. Anti-CgA mAb was purified from ascites fluid, and the affinity constant reached 9.23 × 10(9) L/mol. Furthermore, the specificity of the mAb was determined with ELISA, western blot and immunohistochemistry. Results indicated that the mAb 4E5 was able to detect chromogranin A specifically and sensitively. CONCLUSIONS: A sensitive and reliable method was successfully developed for rapid production of anti-CgA mAb for immunohistochemistry diagnosis in this study, and the current study also provides conclusive guidelines for preparation of mAbs and implements in immunohistochemistry diagnosis.
format Online
Article
Text
id pubmed-5935939
institution National Center for Biotechnology Information
language English
publishDate 2018
publisher BioMed Central
record_format MEDLINE/PubMed
spelling pubmed-59359392018-05-11 Effective preparation of a monoclonal antibody against human chromogranin A for immunohistochemical diagnosis Zhang, Danping Xie, Chengjie Wang, Rongzhi Yang, Qinghai Chen, Huiling Ling, Sumei Wang, Shihua Jia, Kunzhi BMC Biotechnol Research Article BACKGROUND: Human chromogranin A (CgA) is a ~ 49 kDa secreted protein mainly from neuroendocrine cells and endocrine cells. The CgA values in the diagnosis of tumor, and in the potential role in prognostic and predictive tumor as a biomarker. RESULTS: The synthesized gene of CgA coding area was cloned and expressed as fusion protein CgA-His in procaryotic system. Then the purified CgA-His protein was mixed with QuickAntibody-Mouse5W adjuvant, and injected into mice. The CgA-His protein was also used as coating antigen to determine the antiserum titer. By screening, a stable cell line named 4E5, which can generate anti-CgA monoclonal antibody (mAb), was obtained. The isotype of 4E5 mAb was IgG(2b), and the chromosome number was 102 ± 4. Anti-CgA mAb was purified from ascites fluid, and the affinity constant reached 9.23 × 10(9) L/mol. Furthermore, the specificity of the mAb was determined with ELISA, western blot and immunohistochemistry. Results indicated that the mAb 4E5 was able to detect chromogranin A specifically and sensitively. CONCLUSIONS: A sensitive and reliable method was successfully developed for rapid production of anti-CgA mAb for immunohistochemistry diagnosis in this study, and the current study also provides conclusive guidelines for preparation of mAbs and implements in immunohistochemistry diagnosis. BioMed Central 2018-05-04 /pmc/articles/PMC5935939/ /pubmed/29728076 http://dx.doi.org/10.1186/s12896-018-0436-z Text en © The Author(s). 2018 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research Article
Zhang, Danping
Xie, Chengjie
Wang, Rongzhi
Yang, Qinghai
Chen, Huiling
Ling, Sumei
Wang, Shihua
Jia, Kunzhi
Effective preparation of a monoclonal antibody against human chromogranin A for immunohistochemical diagnosis
title Effective preparation of a monoclonal antibody against human chromogranin A for immunohistochemical diagnosis
title_full Effective preparation of a monoclonal antibody against human chromogranin A for immunohistochemical diagnosis
title_fullStr Effective preparation of a monoclonal antibody against human chromogranin A for immunohistochemical diagnosis
title_full_unstemmed Effective preparation of a monoclonal antibody against human chromogranin A for immunohistochemical diagnosis
title_short Effective preparation of a monoclonal antibody against human chromogranin A for immunohistochemical diagnosis
title_sort effective preparation of a monoclonal antibody against human chromogranin a for immunohistochemical diagnosis
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5935939/
https://www.ncbi.nlm.nih.gov/pubmed/29728076
http://dx.doi.org/10.1186/s12896-018-0436-z
work_keys_str_mv AT zhangdanping effectivepreparationofamonoclonalantibodyagainsthumanchromograninaforimmunohistochemicaldiagnosis
AT xiechengjie effectivepreparationofamonoclonalantibodyagainsthumanchromograninaforimmunohistochemicaldiagnosis
AT wangrongzhi effectivepreparationofamonoclonalantibodyagainsthumanchromograninaforimmunohistochemicaldiagnosis
AT yangqinghai effectivepreparationofamonoclonalantibodyagainsthumanchromograninaforimmunohistochemicaldiagnosis
AT chenhuiling effectivepreparationofamonoclonalantibodyagainsthumanchromograninaforimmunohistochemicaldiagnosis
AT lingsumei effectivepreparationofamonoclonalantibodyagainsthumanchromograninaforimmunohistochemicaldiagnosis
AT wangshihua effectivepreparationofamonoclonalantibodyagainsthumanchromograninaforimmunohistochemicaldiagnosis
AT jiakunzhi effectivepreparationofamonoclonalantibodyagainsthumanchromograninaforimmunohistochemicaldiagnosis