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

Autor: Noubissi FK; Department of Biology, Jackson State University, Jackson, MS, USA., McBride AA; Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, USA., Leppert HG; Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, USA., Millet LJ; Center for Environmental Biotechnology, University of Tennessee, Knoxville, TN, USA., Wang X; Department of Biological Sciences, Tennessee State University, Nashville, TN, USA., Davern SM; Radioisotope Science and Technology Division, Oak Ridge National Laboratory, Oak Ridge, TN, USA. davernsm@ornl.gov.
Jazyk: angličtina
Zdroj: Scientific reports [Sci Rep] 2021 Apr 26; Vol. 11 (1), pp. 8945. Date of Electronic Publication: 2021 Apr 26.
DOI: 10.1038/s41598-021-88296-3
Abstrakt: 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.
Databáze: MEDLINE
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