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A Novel System for the Efficient Generation of Antibodies Following Immunization of Unique Knockout Mouse Strains

BACKGROUND: We wished to develop alternate production strategies to generate antibodies against traditionally problematic antigens. As a model we chose butyrylcholinesterase (BChE), involved in termination of cholinergic signaling, and widely considered as a poor immunogen. METHODOLOGY/PRINCIPAL FIN...

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Autores principales: Hrabovska, Anna, Bernard, Véronique, Krejci, Eric
Formato: Texto
Lenguaje:English
Publicado: Public Library of Science 2010
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2944837/
https://www.ncbi.nlm.nih.gov/pubmed/20886120
http://dx.doi.org/10.1371/journal.pone.0012892
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author Hrabovska, Anna
Bernard, Véronique
Krejci, Eric
author_facet Hrabovska, Anna
Bernard, Véronique
Krejci, Eric
author_sort Hrabovska, Anna
collection PubMed
description BACKGROUND: We wished to develop alternate production strategies to generate antibodies against traditionally problematic antigens. As a model we chose butyrylcholinesterase (BChE), involved in termination of cholinergic signaling, and widely considered as a poor immunogen. METHODOLOGY/PRINCIPAL FINDINGS: Jettisoning traditional laborious in silico searching methods to define putative epitopes, we simply immunized available BChE knock-out mice with full-length recombinant BChE protein (having been produced for crystallographic analysis). Immunization with BChE, in practically any form (recombinant human or mouse BChE, BChE purified from human serum, native or denatured), resulted in strong immune responses. Native BChE produced antibodies that favored ELISA and immunostaining detection. Denatured and reduced BChE were more selective for antibodies specific in Western blots. Two especially sensitive monoclonal antibodies were found capable of detecting 0.25 ng of BChE within one min by ELISA. One is specific for human BChE; the other cross-reacts with mouse and rat BChE. Immunization of wild-type mice served as negative controls. CONCLUSIONS/SIGNIFICANCE: We examined a simple, fast, and highly efficient strategy to produce antibodies by mining two expanding databases: namely those of knock-out mice and 3D crystallographic protein-structure analysis. We conclude that the immunization of knock-out mice should be a strategy of choice for antibody production.
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spelling pubmed-29448372010-09-30 A Novel System for the Efficient Generation of Antibodies Following Immunization of Unique Knockout Mouse Strains Hrabovska, Anna Bernard, Véronique Krejci, Eric PLoS One Research Article BACKGROUND: We wished to develop alternate production strategies to generate antibodies against traditionally problematic antigens. As a model we chose butyrylcholinesterase (BChE), involved in termination of cholinergic signaling, and widely considered as a poor immunogen. METHODOLOGY/PRINCIPAL FINDINGS: Jettisoning traditional laborious in silico searching methods to define putative epitopes, we simply immunized available BChE knock-out mice with full-length recombinant BChE protein (having been produced for crystallographic analysis). Immunization with BChE, in practically any form (recombinant human or mouse BChE, BChE purified from human serum, native or denatured), resulted in strong immune responses. Native BChE produced antibodies that favored ELISA and immunostaining detection. Denatured and reduced BChE were more selective for antibodies specific in Western blots. Two especially sensitive monoclonal antibodies were found capable of detecting 0.25 ng of BChE within one min by ELISA. One is specific for human BChE; the other cross-reacts with mouse and rat BChE. Immunization of wild-type mice served as negative controls. CONCLUSIONS/SIGNIFICANCE: We examined a simple, fast, and highly efficient strategy to produce antibodies by mining two expanding databases: namely those of knock-out mice and 3D crystallographic protein-structure analysis. We conclude that the immunization of knock-out mice should be a strategy of choice for antibody production. Public Library of Science 2010-09-23 /pmc/articles/PMC2944837/ /pubmed/20886120 http://dx.doi.org/10.1371/journal.pone.0012892 Text en Hrabovska et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Hrabovska, Anna
Bernard, Véronique
Krejci, Eric
A Novel System for the Efficient Generation of Antibodies Following Immunization of Unique Knockout Mouse Strains
title A Novel System for the Efficient Generation of Antibodies Following Immunization of Unique Knockout Mouse Strains
title_full A Novel System for the Efficient Generation of Antibodies Following Immunization of Unique Knockout Mouse Strains
title_fullStr A Novel System for the Efficient Generation of Antibodies Following Immunization of Unique Knockout Mouse Strains
title_full_unstemmed A Novel System for the Efficient Generation of Antibodies Following Immunization of Unique Knockout Mouse Strains
title_short A Novel System for the Efficient Generation of Antibodies Following Immunization of Unique Knockout Mouse Strains
title_sort novel system for the efficient generation of antibodies following immunization of unique knockout mouse strains
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2944837/
https://www.ncbi.nlm.nih.gov/pubmed/20886120
http://dx.doi.org/10.1371/journal.pone.0012892
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