Development and Validation of Radiation-Responsive Protein Bioassays for Biodosimetry Applications

摘要

The present need to assess radiation dose rapidly in mass-casualty and population-monitoring scenarios prompted an evaluation of potential protein biomarkers that can provide early diagnostic information after exposure. Using an ex vivo model system of human peripheral lymphocytes as well as an in vivo murine model, we demonstrated radiation-responsive changes in the expression of the proto-oncogene proteins rasp21, raf-1, and DNA repair protein p21Waf1Cip, each with a progressive time-and radiation-dose-dependent increase. In addition, we adopted a methodology to identify, optimize, and validate radiation- responsive molecular biomarkers that employs Luminex' technology, a microsphere- based multi-analyte assay system. This technology is based on the principles of the sandwich immunoassay and flow-cytometric analysis in a 96-well microtiter plate format. Current studies use reagents prepared in-house by conjugating capture antibodies to Luminex' microspheres, and biotin to detection antibodies. Preliminary results demonstrate that radiation-responsive changes in the level of GADD45 DNA repair protein occurred with a progressive time-and radiation-dose-dependent increase in the range of 0.15 to 6.0 Gy. A robotic analysis system for processing the blood protein bioassay was established at the Armed Forces Radiobiology Research Institute (AFRRI). The system consists of a Qiagen Biorobot-8000 for large-sample liquid handling to work in concert with the Luminex' platform. This robotic system provides 'proof of concept' for high-throughput isolation, etection, and quantification of blood protein biomarkers for radiation-dose assessment. Use of this sandwich immunoassay bioassay approach is compatible with field-deployable and hand-held diagnostic platforms.