- By:
- Davern, Sandra M; Noubissi, Felicite; McBride, Amber A; Leppert, Hannah; Millet, Larry; Wang, Xiaofei
- Journal Name:
- Scientific Reports
- Page Number:
- 8945
- Volume:
- 11
- Issue Number:
- 1
- Publication Date:
- June 4, 2025
- View DOI Listing:
- https://doi.org/10.1038/s41598-021-88296-3
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.
