High throughput evaluation of gamma-H2AX.
The DNA double-strand break (DSB) is the primary lethal lesion after therapeutic radiation. Thus, the development of assays to detect and to quantitate these lesions could have broad preclinical and clinical impact. Phosphorylation of histone H2AX to form gamma-H2AX is a known marker for irradiation-induced DNA DSBs. However, the first generation assay involves the use of immunofluorescent staining of gamma-H2AX foci. This assay is time consuming, operator dependent and is not scalable for high throughput assay development. Thus, we sought to develop a new assay using a high throughput electrochemiluminescent platform from Mesoscale Discovery Systems to quantify gamma-H2AX levels. The results show that our assay utilizes significantly less time and labor, has greater intra-assay reproducibility and has a greater dynamic range of gamma-H2AX versus irradiation dose.
Avondoglio
Dane
D
Radiation Oncology Branch, National Cancer Institute, National Cancer Institute, Bethesda, Maryland, USA. avondogd@mail.nih.gov
Scott
Tamalee
T
Kil
Whoon Jong
WJ
Sproull
Mary
M
Tofilon
Philip J
PJ
Camphausen
Kevin
K
D000818:Animals; D045744:Cell Line, Tumor; D053903:DNA Breaks, Double-Stranded / Q000528:radiation effects*; D004307:Dose-Response Relationship, Radiation; D055664:Electrochemical Techniques; D005455:Fluorescent Antibody Technique; D006657:Histones / Q000528:radiation effects*; D006801:Humans; D008163:Luminescent Measurements; D051379:Mice; D008819:Mice, Nude; D010766:Phosphorylation / Q000528:radiation effects; D011878:Radiotherapy / Q000009:adverse effects*
C468783:H2AX protein, human; D006657:Histones
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