Page 333 - 2014 Printable Abstract Book
P. 333
The ability to quickly identify and characterize a broad panel of radiation-responsive plasma
proteins from animal models and radiotherapy patients is essential to achieve optimal sensitivity and
selectivity of the test for radiation biodosimetry. While many of these proteins are known from
publications, their verification and validity are often limited by the availability of high quality affinity
reagents and assays, especially for the non-human primate (NHP). We have recently developed a
quantitative label-free proteomics strategy employing liquid chromatography with online tandem mass
spectrometry (LC-MS/MS) and have demonstrated its utility in the rapid profiling of protein biomarkers
from murine, NHP and radiotherapy patient samples as a function of radiation dose and post-irradiation
time. Specifically, sample processing methods were optimized for the enrichment of low-abundance
proteins necessary for the discovery and quantification of tissue-specific biomarkers, and novel
candidates were identified by immunodepleting up to 14 most abundant plasma proteins and followed
by extensive multidimensional chromatographic fractionation. Preliminary verifications of these novel
markers and comparisons across species models were achieved by either ELISA analysis or isotope-dilution
LC-MS/MS when commercial antibodies were unavailable. As a result, unique protein isoforms that are
species independent were discovered, e.g. serine-protease inhibitor form A3N linking results from the
murine model to the NHP model. Further analysis of plasma samples from patients undergoing treatment
with total-body irradiation by the same methods provides insights into differences and similarities in the
radiation of humans and animal models. Our results are not only useful for radiation biodosimetry, but
also shed light on the mechanisms of radiation damage and the development of animal models for
countermeasures. Acknowledgement. This project has been funded in whole or in part with Federal funds
from the Biomedical Advanced Research and Development Authority (BARDA), Office of the Assistant
Secretary for Preparedness and Response, Office of the Secretary, Department of Health and Human
Services, under Contract No. HHSSO10020100007C to SRI International.
(PS6-16) Development and Performance of a Rapid, High Throughput, Gene Expression Based
2
1
1
Biodosimetric Assay. Bob Terbrueggen, PhD ; Majid Abedi, PhD ; Nelson Chao, MD, MBA ; John P. Chute,
1
2
1
2
2
1
MD ; Holly Dressman, PhD ; Ed Iversen, PhD ; Bill Coty, PhD ; Aviva Jacobs, PhD ; Tim Holzer, PhD ; Joe
3
2
2
3
2
Lucas, PhD ; Joel Ross, PhD ; Muriel Brengues, PhD ; Gary Phillips, MS ; and Frederic Zenhausern ,
2
1
DxTerity Diagnostics, Rancho Dominguez, CA ; Duke University Medical Center, Durham, NC ; and Center
3
for Applied Nanobioscience and Medicine, Phoenix, AZ
Following a mass scale nuclear event caused by detonation of a nuclear weapon or a nuclear
accident, the rapid and accurate biodosimetry of thousands of potentially affected individuals will be
essential for effective medical management of the crisis. Currently, health care providers lack an accurate,
high throughput biodosimetric assay that is suitable for the testing of large numbers of potential radiation
victims. Here, we describe the development and performance of a genomic assay suitable for the high-
throughput determination of individualized levels of radiation exposure for up to 7 days post-exposure.
The multi-gene assay was developed by cross-correlating the time dependent (6 hours to 7 days post
exposure) RNA gene expression of peripheral blood collected from in-vivo irradiated mice, in-vivo
irradiated non-human primates (NHPs), ex vivo irradiated human peripheral blood, and human cancer
patients undergoing total body irradiation (TBI) in preparation for bone marrow transplantation. Gene
orthologues between mice, humans, and NHPs were mapped. Genes that showed similar response
profiles to radiation exposure in all species were determined and used to develop a multi-gene predictor
capable of highly accurate prediction of human radiation status (irradiated vs non-irradiated) and
331 | P a g e
proteins from animal models and radiotherapy patients is essential to achieve optimal sensitivity and
selectivity of the test for radiation biodosimetry. While many of these proteins are known from
publications, their verification and validity are often limited by the availability of high quality affinity
reagents and assays, especially for the non-human primate (NHP). We have recently developed a
quantitative label-free proteomics strategy employing liquid chromatography with online tandem mass
spectrometry (LC-MS/MS) and have demonstrated its utility in the rapid profiling of protein biomarkers
from murine, NHP and radiotherapy patient samples as a function of radiation dose and post-irradiation
time. Specifically, sample processing methods were optimized for the enrichment of low-abundance
proteins necessary for the discovery and quantification of tissue-specific biomarkers, and novel
candidates were identified by immunodepleting up to 14 most abundant plasma proteins and followed
by extensive multidimensional chromatographic fractionation. Preliminary verifications of these novel
markers and comparisons across species models were achieved by either ELISA analysis or isotope-dilution
LC-MS/MS when commercial antibodies were unavailable. As a result, unique protein isoforms that are
species independent were discovered, e.g. serine-protease inhibitor form A3N linking results from the
murine model to the NHP model. Further analysis of plasma samples from patients undergoing treatment
with total-body irradiation by the same methods provides insights into differences and similarities in the
radiation of humans and animal models. Our results are not only useful for radiation biodosimetry, but
also shed light on the mechanisms of radiation damage and the development of animal models for
countermeasures. Acknowledgement. This project has been funded in whole or in part with Federal funds
from the Biomedical Advanced Research and Development Authority (BARDA), Office of the Assistant
Secretary for Preparedness and Response, Office of the Secretary, Department of Health and Human
Services, under Contract No. HHSSO10020100007C to SRI International.
(PS6-16) Development and Performance of a Rapid, High Throughput, Gene Expression Based
2
1
1
Biodosimetric Assay. Bob Terbrueggen, PhD ; Majid Abedi, PhD ; Nelson Chao, MD, MBA ; John P. Chute,
1
2
1
2
2
1
MD ; Holly Dressman, PhD ; Ed Iversen, PhD ; Bill Coty, PhD ; Aviva Jacobs, PhD ; Tim Holzer, PhD ; Joe
3
2
2
3
2
Lucas, PhD ; Joel Ross, PhD ; Muriel Brengues, PhD ; Gary Phillips, MS ; and Frederic Zenhausern ,
2
1
DxTerity Diagnostics, Rancho Dominguez, CA ; Duke University Medical Center, Durham, NC ; and Center
3
for Applied Nanobioscience and Medicine, Phoenix, AZ
Following a mass scale nuclear event caused by detonation of a nuclear weapon or a nuclear
accident, the rapid and accurate biodosimetry of thousands of potentially affected individuals will be
essential for effective medical management of the crisis. Currently, health care providers lack an accurate,
high throughput biodosimetric assay that is suitable for the testing of large numbers of potential radiation
victims. Here, we describe the development and performance of a genomic assay suitable for the high-
throughput determination of individualized levels of radiation exposure for up to 7 days post-exposure.
The multi-gene assay was developed by cross-correlating the time dependent (6 hours to 7 days post
exposure) RNA gene expression of peripheral blood collected from in-vivo irradiated mice, in-vivo
irradiated non-human primates (NHPs), ex vivo irradiated human peripheral blood, and human cancer
patients undergoing total body irradiation (TBI) in preparation for bone marrow transplantation. Gene
orthologues between mice, humans, and NHPs were mapped. Genes that showed similar response
profiles to radiation exposure in all species were determined and used to develop a multi-gene predictor
capable of highly accurate prediction of human radiation status (irradiated vs non-irradiated) and
331 | P a g e
