Detection and quantification of γ-H2AX using a dissociation enhanced lanthanide fluorescence immunoassay.
Sci Rep
; 11(1): 8945, 2021 04 26.
Article
em En
| MEDLINE
| ID: mdl-33903655
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Algoritmos
/
Histonas
/
Európio
/
Quebras de DNA de Cadeia Dupla
/
Fluorescência
Tipo de estudo:
Diagnostic_studies
Limite:
Humans
Idioma:
En
Ano de publicação:
2021
Tipo de documento:
Article