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A Simple and Efficient Genetic Immunization Protocol for the Production of Highly Specific Polyclonal and Monoclonal Antibodies against the Native Form of Mammalian Proteins
We have generated polyclonal and monoclonal antibodies by genetic immunization over the last two decades. In this paper, we present our most successful methodology acquired over these years and present the animals in which we obtained the highest rates of success. The technique presented is convenie...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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MDPI
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7582275/ https://www.ncbi.nlm.nih.gov/pubmed/32992862 http://dx.doi.org/10.3390/ijms21197074 |
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author | Pelletier, Julie Agonsanou, Hervé Manica, Fabiana G. Lavoie, Elise Salem, Mabrouka Luyindula, Patrick Babou Kammoe, Romuald Brice Sévigny, Jean |
author_facet | Pelletier, Julie Agonsanou, Hervé Manica, Fabiana G. Lavoie, Elise Salem, Mabrouka Luyindula, Patrick Babou Kammoe, Romuald Brice Sévigny, Jean |
author_sort | Pelletier, Julie |
collection | PubMed |
description | We have generated polyclonal and monoclonal antibodies by genetic immunization over the last two decades. In this paper, we present our most successful methodology acquired over these years and present the animals in which we obtained the highest rates of success. The technique presented is convenient, easy, affordable, and generates antibodies against mammalian proteins in their native form. This protocol requires neither expensive equipment, such as a gene gun, nor sophisticated techniques such as the conjugation of gold microspheres, electroporation, or surgery to inject in lymph nodes. The protocol presented uses simply the purified plasmid expressing the protein of interest under a strong promoter, which is injected at intramuscular and intradermal sites. This technique was tested in five species. Guinea pigs were the animals of choice for the production of polyclonal antibodies. Monoclonal antibodies could be generated in mice by giving, as a last injection, a suspension of transfected cells. The antibodies detected their antigens in their native forms. They were highly specific with very low non-specific background levels, as assessed by immune-blots, immunocytochemistry, immunohistochemistry and flow cytometry. We present herein a detailed and simple procedure to successfully raise specific antibodies against native proteins. |
format | Online Article Text |
id | pubmed-7582275 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-75822752020-10-28 A Simple and Efficient Genetic Immunization Protocol for the Production of Highly Specific Polyclonal and Monoclonal Antibodies against the Native Form of Mammalian Proteins Pelletier, Julie Agonsanou, Hervé Manica, Fabiana G. Lavoie, Elise Salem, Mabrouka Luyindula, Patrick Babou Kammoe, Romuald Brice Sévigny, Jean Int J Mol Sci Technical Note We have generated polyclonal and monoclonal antibodies by genetic immunization over the last two decades. In this paper, we present our most successful methodology acquired over these years and present the animals in which we obtained the highest rates of success. The technique presented is convenient, easy, affordable, and generates antibodies against mammalian proteins in their native form. This protocol requires neither expensive equipment, such as a gene gun, nor sophisticated techniques such as the conjugation of gold microspheres, electroporation, or surgery to inject in lymph nodes. The protocol presented uses simply the purified plasmid expressing the protein of interest under a strong promoter, which is injected at intramuscular and intradermal sites. This technique was tested in five species. Guinea pigs were the animals of choice for the production of polyclonal antibodies. Monoclonal antibodies could be generated in mice by giving, as a last injection, a suspension of transfected cells. The antibodies detected their antigens in their native forms. They were highly specific with very low non-specific background levels, as assessed by immune-blots, immunocytochemistry, immunohistochemistry and flow cytometry. We present herein a detailed and simple procedure to successfully raise specific antibodies against native proteins. MDPI 2020-09-25 /pmc/articles/PMC7582275/ /pubmed/32992862 http://dx.doi.org/10.3390/ijms21197074 Text en © 2020 by the authors. 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 (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Technical Note Pelletier, Julie Agonsanou, Hervé Manica, Fabiana G. Lavoie, Elise Salem, Mabrouka Luyindula, Patrick Babou Kammoe, Romuald Brice Sévigny, Jean A Simple and Efficient Genetic Immunization Protocol for the Production of Highly Specific Polyclonal and Monoclonal Antibodies against the Native Form of Mammalian Proteins |
title | A Simple and Efficient Genetic Immunization Protocol for the Production of Highly Specific Polyclonal and Monoclonal Antibodies against the Native Form of Mammalian Proteins |
title_full | A Simple and Efficient Genetic Immunization Protocol for the Production of Highly Specific Polyclonal and Monoclonal Antibodies against the Native Form of Mammalian Proteins |
title_fullStr | A Simple and Efficient Genetic Immunization Protocol for the Production of Highly Specific Polyclonal and Monoclonal Antibodies against the Native Form of Mammalian Proteins |
title_full_unstemmed | A Simple and Efficient Genetic Immunization Protocol for the Production of Highly Specific Polyclonal and Monoclonal Antibodies against the Native Form of Mammalian Proteins |
title_short | A Simple and Efficient Genetic Immunization Protocol for the Production of Highly Specific Polyclonal and Monoclonal Antibodies against the Native Form of Mammalian Proteins |
title_sort | simple and efficient genetic immunization protocol for the production of highly specific polyclonal and monoclonal antibodies against the native form of mammalian proteins |
topic | Technical Note |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7582275/ https://www.ncbi.nlm.nih.gov/pubmed/32992862 http://dx.doi.org/10.3390/ijms21197074 |
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