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.

Details

Title
Detection and quantification of γ-H2AX using a dissociation enhanced lanthanide fluorescence immunoassay
Author
Noubissi, Felicite K. 1 ; McBride, Amber A. 2 ; Leppert, Hannah G. 2 ; Millet, Larry J. 3 ; Wang, Xiaofei 4 ; Davern, Sandra M. 5 

 Jackson State University, Department of Biology, Jackson, USA (GRID:grid.257990.0) (ISNI:0000 0001 0671 8898) 
 Oak Ridge National Laboratory, Biosciences Division, Oak Ridge, USA (GRID:grid.135519.a) (ISNI:0000 0004 0446 2659) 
 University of Tennessee, Center for Environmental Biotechnology, Knoxville, USA (GRID:grid.411461.7) (ISNI:0000 0001 2315 1184) 
 Tennessee State University, Department of Biological Sciences, Nashville, USA (GRID:grid.280741.8) (ISNI:0000 0001 2284 9820) 
 Oak Ridge National Laboratory, Radioisotope Science and Technology Division, Oak Ridge, USA (GRID:grid.135519.a) (ISNI:0000 0004 0446 2659) 
Publication year
2021
Publication date
2021
Publisher
Nature Publishing Group
e-ISSN
20452322
Source type
Scholarly Journal
Language of publication
English
ProQuest document ID
2706508643
Copyright
© This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply 2021. This work is published under http://creativecommons.org/licenses/by/4.0/ (the “License”). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.