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

Abstract 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 u...

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Autores principales:	Felicite K. Noubissi, Amber A. McBride, Hannah G. Leppert, Larry J. Millet, Xiaofei Wang, Sandra M. Davern
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/b318484cefd641de873ac56966a7f9c7
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spelling	oai:doaj.org-article:b318484cefd641de873ac56966a7f9c72021-12-02T17:16:17ZDetection and quantification of γ-H2AX using a dissociation enhanced lanthanide fluorescence immunoassay10.1038/s41598-021-88296-32045-2322https://doaj.org/article/b318484cefd641de873ac56966a7f9c72021-04-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-88296-3https://doaj.org/toc/2045-2322Abstract 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.Felicite K. NoubissiAmber A. McBrideHannah G. LeppertLarry J. MilletXiaofei WangSandra M. DavernNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-12 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Felicite K. Noubissi Amber A. McBride Hannah G. Leppert Larry J. Millet Xiaofei Wang Sandra M. Davern Detection and quantification of γ-H2AX using a dissociation enhanced lanthanide fluorescence immunoassay
description	Abstract 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.
format	article
author	Felicite K. Noubissi Amber A. McBride Hannah G. Leppert Larry J. Millet Xiaofei Wang Sandra M. Davern
author_facet	Felicite K. Noubissi Amber A. McBride Hannah G. Leppert Larry J. Millet Xiaofei Wang Sandra M. Davern
author_sort	Felicite K. Noubissi
title	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_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_sort	detection and quantification of γ-h2ax using a dissociation enhanced lanthanide fluorescence immunoassay
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/b318484cefd641de873ac56966a7f9c7
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Detection and quantification of γ-H2AX using a dissociation enhanced lanthanide fluorescence immunoassay

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