When cells are exposed to radiation, DNA is damaged, leading to the accumulation of phosphorylated histone H2AX (H2AX-P) at the site of damage. In our preliminary studies, we have found that assays using monoclonal antibodies against H2AX-P (mAbs/H2AX-P) may be able to discriminate between clinically significant levels of radiation exposures. Therefore, we hypothesize that mAbs/H2AX-P assays can be developed that quickly measure the amount of previous radiation exposure, and these assays can be stock-piled for future use if there is a large scale radiation exposure.

Specific aims.
1) Determine if mAbs/H2AX-P display dose-dependent increases after in vitro irradiation of human peripheral blood cells. During the first 1.5 years, in vitro studies will examine the amount of DNA damage in lymphocytes and other peripheral blood cells using mAbs/H2AX-P based immunocytochemistry and flow cytometry assays, which are currently available in our laboratory (see Preliminary Studies). The purpose of the studies are to determine if these assays can discriminate between clinically significant γ-irradiation exposures (0.15 Gy, 2 Gy, 4 Gy, 6 Gy, 9 Gy, and 12 Gy) at various time points after exposure (3, 8, and 24 hours).

2) Determine whether assays using mAbs/H2AX-P can be automated to facilitate high throughput dosimetry screening. During the later part of the second year, we will determine if these assays can be modified for high throughput screening using either flow cytometry or automated image acquisition analyses.