Detection and quantification of γ-H2AX using a dissociation enhanced lanthanide fluorescence immunoassay

Phosphorylation of the histone protein H2AX to form γ-H2AX foci directly represents DNA double-strand break formation. Traditional γ-H2AX detection involves counting individual foci within individual nuclei. The novelty of this work is the application of a time-resolved fluorescence assay using diss...

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Autores principales: Noubissi, Felicite K., McBride, Amber A., Leppert, Hannah G., Millet, Larry J., Wang, Xiaofei, Davern, Sandra M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2021
Materias:
Article
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8076281/
https://www.ncbi.nlm.nih.gov/pubmed/33903655
http://dx.doi.org/10.1038/s41598-021-88296-3
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author Noubissi, Felicite K.
McBride, Amber A.
Leppert, Hannah G.
Millet, Larry J.
Wang, Xiaofei
Davern, Sandra M.
author_facet Noubissi, Felicite K.
McBride, Amber A.
Leppert, Hannah G.
Millet, Larry J.
Wang, Xiaofei
Davern, Sandra M.
author_sort Noubissi, Felicite K.
collection PubMed
description Phosphorylation of the histone protein H2AX to form γ-H2AX foci directly represents DNA double-strand break formation. Traditional γ-H2AX detection involves counting individual foci within individual nuclei. The novelty of this work is the application of a time-resolved fluorescence assay using dissociation-enhanced lanthanide fluorescence immunoassay for quantitative measurements of γ-H2AX. For comparison, standard fluorescence detection was employed and analyzed either by bulk fluorescent measurements or by direct foci counting using BioTek Spot Count algorithm and Gen 5 software. Etoposide induced DNA damage in A549 carcinoma cells was compared across all test platforms. Time resolved fluorescence detection of europium as a chelated complex enabled quantitative measurement of γ-H2AX foci with nanomolar resolution. Comparative bulk fluorescent signals achieved only micromolar sensitivity. Lanthanide based immunodetection of γ-H2AX offers superior detection and a user-friendly workflow. These approaches have the potential to improve screening of compounds that either enhance DNA damage or protect against its deleterious effects.
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spelling pubmed-80762812021-04-27 Detection and quantification of γ-H2AX using a dissociation enhanced lanthanide fluorescence immunoassay Noubissi, Felicite K. McBride, Amber A. Leppert, Hannah G. Millet, Larry J. Wang, Xiaofei Davern, Sandra M. Sci Rep Article Phosphorylation of the histone protein H2AX to form γ-H2AX foci directly represents DNA double-strand break formation. Traditional γ-H2AX detection involves counting individual foci within individual nuclei. The novelty of this work is the application of a time-resolved fluorescence assay using dissociation-enhanced lanthanide fluorescence immunoassay for quantitative measurements of γ-H2AX. For comparison, standard fluorescence detection was employed and analyzed either by bulk fluorescent measurements or by direct foci counting using BioTek Spot Count algorithm and Gen 5 software. Etoposide induced DNA damage in A549 carcinoma cells was compared across all test platforms. Time resolved fluorescence detection of europium as a chelated complex enabled quantitative measurement of γ-H2AX foci with nanomolar resolution. Comparative bulk fluorescent signals achieved only micromolar sensitivity. Lanthanide based immunodetection of γ-H2AX offers superior detection and a user-friendly workflow. These approaches have the potential to improve screening of compounds that either enhance DNA damage or protect against its deleterious effects. Nature Publishing Group UK 2021-04-26 /pmc/articles/PMC8076281/ /pubmed/33903655 http://dx.doi.org/10.1038/s41598-021-88296-3 Text en © This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply 2021 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Article
Noubissi, Felicite K.
McBride, Amber A.
Leppert, Hannah G.
Millet, Larry J.
Wang, Xiaofei
Davern, Sandra M.
Detection and quantification of γ-H2AX using a dissociation enhanced lanthanide fluorescence immunoassay
title Detection and quantification of γ-H2AX using a dissociation enhanced lanthanide fluorescence immunoassay
title_full Detection and quantification of γ-H2AX using a dissociation enhanced lanthanide fluorescence immunoassay
title_fullStr Detection and quantification of γ-H2AX using a dissociation enhanced lanthanide fluorescence immunoassay
title_full_unstemmed Detection and quantification of γ-H2AX using a dissociation enhanced lanthanide fluorescence immunoassay
title_short Detection and quantification of γ-H2AX using a dissociation enhanced lanthanide fluorescence immunoassay
title_sort detection and quantification of γ-h2ax using a dissociation enhanced lanthanide fluorescence immunoassay
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8076281/
https://www.ncbi.nlm.nih.gov/pubmed/33903655
http://dx.doi.org/10.1038/s41598-021-88296-3
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