Detection and quantification of γ-H2AX using a dissociation enhanced lanthanide fluorescence immunoassay-Reference-Cited by-同舟云学术

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

Published:2021-04-26 Issue:1 Volume:11 Page:
ISSN:2045-2322
Container-title:Scientific Reports
language:en
Short-container-title:Sci Rep

Author:

Noubissi Felicite K.,McBride Amber A.,Leppert Hannah G.,Millet Larry J.,Wang Xiaofei,Davern Sandra M.

Abstract

AbstractPhosphorylation 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.

Publisher

Springer Science and Business Media LLC

Subject

Multidisciplinary

Link

http://www.nature.com/articles/s41598-021-88296-3.pdf

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