Detection and Quantification of γ-H2AX Using a Dissociation Enhanced Lanthanide Fluorescence Immunoassay

Overview

Journal Sci Rep

Specialty Science

Date 2021 Apr 27

PMID 33903655

Citations 10

Authors

Felicite K Noubissi

Amber A McBride

Hannah G Leppert

Larry J Millet

Xiaofei Wang

Sandra M Davern

Affiliations

Soon will be listed here.

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.

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