WO2011127056A2

WO2011127056A2 - Biomarkers for assessing exposure to ionizing radiation and absorbed dose

Info

Publication number: WO2011127056A2
Application number: PCT/US2011/031253
Authority: WO
Inventors: Martin R. Johnson; Ashley Nicole Upton Filiano; Hassan Fathallah-Shaykh
Original assignee: The Uab Research Foundation
Priority date: 2010-04-05
Filing date: 2011-04-05
Publication date: 2011-10-13
Also published as: WO2011127056A3

Abstract

The present invention concerns radiation-responsive biomarkers capable of determining qualitative exposure status (yes/no. i.e., irradiated/non-irradiated) and distinguishing levels of exposure (quantitative absorbed dose determination), and materials and methods to profile the radiation-responsive biomarkers in blood samples to detect radiation exposure and/or to determine the dose of radiation exposure.

Description

DESCRIPTION

BIOMARKERS FOR ASSESSING EXPOSURE TO IONIZING RADIATION

AND ABSORBED DOSE GOVERNMENT SUPPORT

This invention was made with government support under U.S. Environmental Protection Agency (EPA) STAR Fellowship Assistance Agreement Number FP-91694801. The government has certain rights in the invention. BACKGROUND OF THE INVENTION

The worldwide expansion of nuclear energy as well as ongoing nuclear proliferation in developing, politically unstable countries raises the possibility of large-scale acute radiation exposure. The ability to rapidly triage exposed individuals and assess the extent of exposure is imperative to decrease morbidity and mortality through rationally directed medical intervention.

Significant exposure to ionizing radiation (IR) induces varying degrees of nausea, vomiting, and pancytopenia (1-3). Attempts have been made to exploit these symptoms for estimating dose of exposure. However, these methods lack sensitivity and high inter- individual variability limits their utility. On the molecular level, IR induces DNA double- stranded breaks directly or through the secondary generation of free radicals and reactive oxygen species (4, 5). Improper repair of damaged DNA results in chromosomal aberrations, especially dicentrics, whose frequency has been correlated to radiation dose (6). However, while cytogenetic analysis remains the current "gold standard" of retrospective radiation dose assessment, it is both time- and labor-intensive (7-5) and may not be amendable to mass exposure scenarios.

Previous studies have demonstrated that in addition to DNA damage, exposure of mammalian cells to IR initiates a complex series of signal transduction cascades related to maintaining genomic integrity through damage recognition, repair, induction of cell cycle checkpoints, senescence, and/or initiation of apoptosis. Recent microarray studies have identified transcriptional changes in lymphoid/myeloid cells (7-20), mice (21, 22), primary cells (23-26), ex vivo irradiated whole blood (27, 28) and blood from irradiated patients (22, 29) that could potentially be used for radiation dose assessment. Direct comparison of most array-based analyses remains difficult because these studies were performed using different array platforms, controls, experimental models, doses of irradiation, and time points. Limiting exclusion criteria to genes identified by at least two independent investigators in more than one model reveals consistent changes in a surprisingly small population of genes including ACTA2, BAX, BBC3, BTG2, CCNG1, CDKN1A, DDB2, FDXR, GADD45A, HSPE1, PCNA, PPM1D, TP53I3, TNFot, and XPC (Table IB). Furthermore, a dose- dependent induction of GADD45A (30, 31), DDB2 (15, 26, 29, 30), XPC (75, 26) and CDKN1A (8, 15, 26-29, 32) has been observed following irradiation in some contexts.

Published reports of global (microarray) human in vivo studies examining changes in gene expression following total body irradiation (TBI) remain limited. Two published studies involving a small number of patients collectively identified subsets of radiation- responsive genes that differed from in vitro or ex vivo models and emphasize that the effect of irradiation on gene expression profiles may be influenced by the cellular microenvironment within "whole animal" models (22, 29). More importantly, a profile of radiation response genes capable of distinguishing irradiated from non-irradiated blood samples with an accuracy of 90% was identified (22). Taken together, these studies support the hypothesis that there is no single cellular response to radiation and that examining the overall expression profile of multiple biodosimetry genes may be more useful than determining expression of individual genes.

BRIEF SUMMARY OF THE INVENTION

The present invention concerns radiation-responsive biomarkers capable of qualitatively determining exposure status (yes/no, i.e., irradiated/non-irradiated) and distinguishing levels of exposure (quantitative dose determination), and materials and methods to profile the radiation-responsive biomarkers in blood samples to detect radiation exposure and/or to determine the dose of radiation exposure.

The inventors obtained whole blood samples from cancer patients exposed to TBI and used them to identify potential biodosimetry genes. Time- and dose-dependent changes in the expression of these biodosimetry genes were subsequently examined in in vivo mouse models. An associative classification algorithm (weighted voting) was then utilized to identify molecular signatures capable of distinguishing irradiated from non-irradiated mice. Multiple linear regression analysis allowed for quantitative dose determination. The results show that gene expression analysis can be used for assessment of radiation exposure and incorporated into current biodosimetry protocols. Thus, the present invention relates to radiation exposure screening. The genes disclosed herein constitute biomarkers for detection and monitoring of radiation exposure. For example, one or more of the polypeptides disclosed herein, or nucleic acids that encode the polypeptides, can be used to detect, quantify, and monitor radiation exposure. These polypeptides and polynucleotides (referred to herein collectively as "biomarkers"), and agents that bind to them, can be used to detect, quantify, and monitor radiation in mammals, including humans. Biomarkers of the invention include Bbc3, Ccngl, Cdknla, Serpinel, and Tnfrsf10b.

In one aspect, the method of the invention comprises a method of identifying a mammalian subject exposed to ionizing radiation, comprising: detecting the presence of, and/or quantifying the level of, one or more biomarker nucleic acids or proteins in a blood sample or blood derived sample from the subject, wherein the presence of the biomarker, or a level or concentration of the biomarker above a pre-determined threshold is indicative of ionizing radiation exposure in the subject, and wherein the one or more biomarkers comprise or consist of one or more biomarkers among Bbc3 , Ceng 1 , Cdknl a, Serpine 1 , and Tnfrsf 1 Ob.

The method of the invention can be a qualitative assessment of radiation exposure (distinguishing irradiated versus non-irradiated), a quantitative (dose) assessment of radiation exposure (and, optionally, for two or more time points post-exposure i.e., time-dependent and/or dose-dependent), or both a qualitative and quantitative assessment.

In some embodiments, the method is a qualitative assessment of radiation exposure

(distinguishing irradiated versus non-irradiated samples), wherein a level or concentration of the biomarker above a pre-determined threshold is indicative of ionizing radiation exposure in the subject, and wherein a level or concentration of the biomarker below a pre-determined threshold is indicative of a lack of ionizing radiation exposure in the subject. In some embodiments, the one or more biomarkers comprise or consist of one or two from among Bbc3, Ccngl, and Cdknla. In some embodiments, the one or more biomarkers comprise or consist of Bbc3 and Ccngl . In some embodiments, the one or more biomarkers comprise or consist of Ccngl. In some embodiments, the one or more biomarkers comprise or consist of Ccngl and Cdknl .

In some embodiments, a level or concentration of a biomarker above a pre-determined threshold is indicative of ionizing radiation exposure in the subject within a certain period of time of obtaining a sample from the subject (e.g., within 5 hours to within 48 hours postexposure; see, for example, Table 4 - Prediction of Exposure in Mice (Non-irradiated vs. Irradiated)). In some embodiments, the one or more biomarkers comprise or consist of one or two from among Bbc3, Ccngl, and Cdknla within 5 hours to within 48 hours post-exposure. In some embodiments, the one or more biomarkers comprise or consist of Bbc3 and Ccngl within 23 hours post-exposure. In some embodiments, the one or more biomarkers comprise or consist of Ccngl within 5-12 hours post-exposure. In some embodiments, the one or more biomarkers comprise or consist of Ccngl and Cdknla within 48 hours post-exposure.

In some embodiments, the method is a quantitative (dose) assessment of radiation exposure, in which the actual dose of radiation exposure or a range of exposure is determined (e.g., 0 grays (Gy), 1 Gy, 2 Gy, 3 Gy, 4 Gy, 5 Gy, 6 Gy, 7 Gy, 8 Gy, and so forth). In some embodiments of the quantitative assessment, the one or more biomarkers comprise or consist of Ccngl and Cdknla (see, for example, Table 5 - Predicted Dose based on Multiple Linear Regression Analysis).

In some embodiments, the one or more biomarkers comprise or consist of or one or both biomarkers from among Ccngl and Tnfrsf10b, wherein a level or concentration of the biomarker(s) above a pre-determined threshold is indicative of ionizing radiation exposure of equal to or greater than 2 Gy in the subject (e.g. , within 5 hours, 12 hours, 24 hours, or 48 hours post-exposure), and wherein a level or concentration of the biomarker(s) below a predetermined threshold is indicative of ionizing radiation exposure of less than 2 Gy in the subject (see, for example, Table 6 - Class Prediction of Dosing Groups in Mice).

Another aspect of the invention is a method of identifying an expression profile in a sample from a mammalian subject that is indicative of the subject's exposure or non- exposure to ionizing radiation, comprising: providing a subject expression profile from the mammalian subject; providing one or more reference profiles, wherein each reference profile is associated with (i.e., representative of) exposure or non-exposure to ionizing radiation, wherein the subject expression profile and each reference profile has a value, wherien each value represents the expression level of one or more biomarkers selected from among Bbc3, Ccngl, Cdknla, Serpinel, and Tnfrsf10b; and identifying the reference profile most similar to the subject expression profile, to thereby identify an expression profile that is indicative of exposure or non-exposure to ionizing radiation. The most similar reference profile can be identified using bioinformatics methods known in the art, such as by weighting a comparison value for each value of each reference profile using a weight value associated with the corresponding biomarker from among Bbc3, Ccngl, Cdknla, Serpinel, and Tnfrsf10b. In some embodiments, the identified reference profile is associated with exposure to ionizing radiation, and the method further comprises treating the mammalian subject for radiation exposure. In some embodiments, the identified reference profile is associated with exposure to ionizing radiation, and the method further comprises verifying that the mammalian subject is suffering from radiation exposure. In some embodiments, the mammalian subject is human, wherein the identified reference profile is associated with exposure to ionizing radiation, and wherein the method further comprises informing the subject of the exposure or potential exposure to ionizing radiation. In some embodiments, the one or more reference profiles comprise a plurality of reference profiles. Optionally, for quantitative assessment of radiation dose, each reference profile of the plurality of reference profiles can be associated with exposure to a different dose of ionizing radiation, and/or a dose of ionizing radiation exposure after a duration of time. Thus, the method of identifying an expression profile can provide a qualitative assessment of radiation exposure (distinguishing irradiated versus non- irradiated), a quantitative (dose) assessment of radiation exposure (and, optionally, for two or more time points post-exposure, i.e., time-dependent and/or dose-dependent), or both a qualitative and quantitative assessment.

In some embodiments, the method of identifying an expression profile provides a qualitative assessment of radiation exposure (distinguishing irradiated versus non-irradiated samples). In some embodiments, the one or more biomarkers comprise or consist of one or two from among Bbc3, Ccngl, and Cdknla. In some embodiments, the one or more biomarkers comprise or consist of Bbc3 and Ccngl. In some embodiments, the one or more biomarkers comprise or consist of Ccngl. In some embodiments, the one or more biomarkers comprise or consist of Ccngl and Cdknl .

In some embodiments, one or more reference profiles are associated with ionizing radiation exposure in the subject within a certain period of time of obtaining a sample from the subject {e.g., within 5 hours to within 48 hours post-exposure; see, for example, Table 4 - Prediction of Exposure in Mice (Non-irradiated vs. Irradiated)). In some embodiments, the one or more biomarkers comprise or consist of one or two from among Bbc3, Ccngl, and Cdknla within 5 hours to within 48 hours post-exposure. In some embodiments, the one or more biomarkers comprise or consist of Bbc3 and Ccngl within 23 hours post-exposure. In some embodiments, the one or more biomarkers comprise or consist of Ccngl within 5-12 hours post-exposure. In some embodiments, the one or more biomarkers comprise or consist of Ccngl and Cdknla within 48 hours post-exposure.

In some embodiments, the one or more biomarkers comprise or consist of one or both biomarkers from among Ccngl and Tnfrsf10b, wherein a level or concentration of the biomarker(s) above a pre-determined threshold is indicative of ionizing radiation exposure of equal to or greater than 2 Gy in the subject (e.g., within 5 hours, 12 hours, 24 hours, or 48 hours post-exposure), and wherein a level or concentration of the biomarker(s) below a predetermined threshold is indicative of ionizing radiation exposure of less than 2 Gy in the subject (see, for example, Table 6 - Class Prediction of Dosing Groups in Mice).

In some embodiments of the methods of the invention (e.g. , the method of identifying a mammalian subject exposed to ionizing radiation, and the method of identifying an expression profile), radiation exposure is detected by screening for the presence or elevated levels of one or more polypeptides from among the biomarkers Bbc3, Ccngl, Cdknla, Serpinel, and Tnfrsf10b, or their encoding polynucleotides, in a blood sample or blood derived sample. Optionally, multiple samples can be obtained from the subject over time and evaluated using the methods of the invention.

Optionally, in the methods of the invention, the quantification (measurement of the level of biomarker polypeptides or encoding nucleic acids) can be used to determine the time or estimated time that the subject was previously exposed to ionizing radiation (i.e., a time- dependent assessment).

Optionally, in the methods of the invention, the subject's sample can be obtained and biomarker nucleic acid and/or protein levels determined by the subject (i.e., as a self- administered test or assay).

Optionally, the methods further comprise verifying that the subject is suffering from the radiation exposure detected (e.g., by assessing the presence of one or more signs or symptoms of radiation exposure, or detecting additional biomarkers of radiation exposure or radiation sickness, or by other confirmatory diagnostic procedures), verifying the amount of exposure, and/or treating the subject for the radiation exposure detected. In addition to a complete physical examination and monitoring clinical signs and symptoms (e.g., vomiting), diagnostic procedures for radiation exposure and/or dosimetry may include, for example, physical dosimetry badges, and laboratory tests such as chromosome aberration cytogenetics and absolute lymphocyte count. Treatments for radiation exposure include, but are not limited to, decontamination, treatment of damaged bone marrow with granulocyte colony- stimulating factor (GC-SF) such as filgrastim (Neupogen) and pegfilgrastim (Neulasta), transfusions of red blood cells or blood platelets, treatment for internal contamination by specific radionuclides uptake inhibitors or chelating agents such as potassium iodide, Prussian blue, or diethyl enetriamine pentaacetic acid (DTPA), supportive treatment for bacterial infections, headache, fever, diarrhea, nausea and vomiting, and dehydration, as well as end-of-life care.

The present invention also relates to kits that may be used for carrying out the methods of the invention. In some embodiments, the kit comprises an array probe (also referred to as a "probe array" or simply an "array") of nucleic acids attached to a substrate (e.g., solid support), wherein the nucleic acids of the array comprise or consist of one or more oligonucleotides that will hybridize to one or more target nucleic acid molecules of biomarkers from among Bbc3, Ccngl, Cdknla, Serpinel, and Tnfrsfl Ob (e.g., hybridizing to one or more target nucleic acid sequences encoding the one or more biomarker polypeptides, or hybridizing to a complement thereof). Preferably, the one or more oligonucleotides will hybridize to the nucleic acid molcules under stringent hybridization conditions. In some embodiments, the one or more biomarkers comprise or consist of one or two from among Bbc3, Ccngl, and Cdknla. In some embodiments, the one or more biomarkers comprise or consist of Bbc3 and Ccngl. In some embodiments, the one or more biomarkers comprise or consist of Ccngl . In some embodiments, the one or more biomarkers comprise or consist of Ccngl and Cdknl. In some embodiments, the one or more biomarkers comprise or consist of one or both biomarkers from among Ccngl and Tnfrsfl Ob. In some embodiments, the substrate has no more than 500 oligonucleotides attached to it. In some embodiments, the target nucleic acid sequence bound (hybridized) by each oligonucleotide is unique among a plurality of oligonucleotides attached to the substrate.

Other kits of the invention may comprise primers (e.g., one or more primer pairs) for amplifying target nucleic acid sequences of one or more biomarkers from among Bbc3, Ccngl, Cdknla, Serpinel, and Tnfrsf10b. In some embodiments, the one or more biomarkers comprise or consist of one or two from among Bbc3, Ccngl, and Cdknla. In some embodiments, the one or more biomarkers comprise or consist of Bbc3 and Ccngl . In some embodiments, the one or more biomarkers comprise or consist of Ccngl. In some embodiments, the one or more biomarkers comprise or consist of Ccngl and Cdknl . In some embodiments, the one or more biomarkers comprise or consist of one or both biomarkers from among Ccngl and TnfrsflOb.

In another aspect, the present invention relates to a device for the rapid detection of one or more of the biomarkers (Bbc3, Ccngl, Cdknla, Serpinel, and Tnfrsf10b) in a blood sample or blood derived sample. In some embodiments, the one or more biomarkers comprise or consist of one or two from among Bbc3, Ccngl, and Cdknla. In some embodiments, the one or more biomarkers comprise or consist of Bbc3 and Ccngl . In some embodiments, the one or more biomarkers comprise or consist of Ccngl. In some embodiments, the one or more biomarkers comprise or consist of Ccngl and Cdknl . In some embodiments, the one or more biomarkers comprise or consist of one or both biomarkers from among Ccngl and Tnfrsf10b.

Preferably, the device is a lateral flow device. In one embodiment, the device comprises an application zone for receiving a sample of blood; a labeling zone containing a binding agent that binds to a biomarker in the sample; and a detection zone where a biomarker-bound binding agent is retained to give a signal, wherein the signal given for a sample from a subject with a biomarker level lower than a threshold concentration is different from the signal given for a sample from a subject with a biomarker level equal to or greater than a threshold concentration.

In another aspect, the invention relates to a test for radiation biomarkers in blood, similar to currently available in-home diabetic testing equipment, such as blood glucose testing strips that involve collecting blood for the test, most often by pricking the fingertip with a lancet, and squeezing a drop of blood out of the finger. The finger is then pressed against the diabetic testing strip, allowing measurement of blood glucose either with an electronic blood glucose meter or testing strip color chart. Such tests can be used by subjects at home, at a place of employment, in a physicians' office, or at a patient's bedside, e.g., at a health care facility. In one embodiment, the test is a method for detecting and, optionally measuring, one or more radiation biomarkers comprising or consisting of Bbc3, Ccngl, Cdknla, Serpinel, and Tnfrsf10b in a blood sample or blood derived sample, comprising: (a) obtaining a sample of blood from a subject; (b) contacting the sample with a binding agent that binds to any biomarker in the sample; (c) separating biomarker-bound binding agent; (d) detecting a signal associated with the separated binding agent from (c); and (e) comparing the signal detected in step (d) with a reference signal which corresponds to the signal given by a sample from a subject with a biomarker level equal to a threshold concentration. The test can be a qualitative assessment of radiation exposure (distinguishing irradiated versus non- irradiated), a quantitative (dose) assessment of radiation exposure (and, optionally, for two or more time points post-exposure i.e. , time-dependent and/or dose-dependent), or both a qualitative and quantitative assessment. In some embodiments, the test is a qualitative assessment of radiation exposure (distinguishing irradiated versus non-irradiated samples), wherein a level or concentration of the biomarker above a pre-determined threshold is indicative of ionizing radiation exposure in the subject, and wherein a level or concentration of the biomarker below a pre-determined threshold is indicative of a lack of ionizing radiation exposure in the subject. In some embodiments, the one or more biomarkers comprise or consist of one or two from among Bbc3, Ccngl, and Cdknla. In some embodiments, the one or more biomarkers comprise or consist of Bbc3 and Ccngl. In some embodiments, the one or more biomarkers comprise or consist of Ccngl. In some embodiments, the one or more biomarkers comprise or consist of Ccngl and Cdknl . In some embodiments, the one or more biomarkers comprise or consist of one or both biomarkers from among Ccngl and Tnfrsf10b.

In some embodiments of the various aspects of the invention (e.g., methods, kits

(arrays, primers), devices), the biomarkers correspond to one or more nucleic acid sequences of SEQ ID NOs: 1-25, or polypeptides encoded by such nucleic acids. For example, in some embodiments, Bbc3 is SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; Ccngl is SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10; Cdknla is SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14; Serpinel is SEQ ID NO: 21 or SEQ ID NO: 22; and/or TnfrsflOb is SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25.

In the various aspects of the invention (e.g., methods, kits (arrays, primers), devices), the ionizing radiation exposure may occur in potentially any setting in which ionizing radiation exposure is known to occur or there is a risk of occurrence, e.g., medical treatment (e.g., patients receiving total body irradiation), industrial accident (e.g., nuclear power plant or laboratory), environmental exposure, terrorist-driven radiological and nuclear attacks (e.g., warfare).

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a patient treatment schema. Patients received fractionated total body irradiation (TBI) prior to bone marrow transplantation. TBI was administered in two daily 2 Gy fractions for 3 consecutive days (12 Gy total). Whole blood for gene expression analysis was collected prior to initiation of TBI and at three time points throughout the treatment regimen.

Figure 2 shows gene expression in healthy volunteers with no treatment (□) (n=9), and in patients at baseline (prior to irradiation) (a) (n=4), 5 hours (■) (n=4), 23 hours (■) (n=4), and 48 hours (■) (n=4) following the first dose of irradiation. The total body accumulated dose received at 5, 23, and 48 hours was 2 Gy, 4 Gy, and 8 Gy, respectively. Data are presented as mean ± SEM. Figure 3 shows expression of A) Bbc3, B) Ccngl, C) Cdknla, D) Serpinel, and E) Tnfrsf10b in mice at 0 hr (baseline), and 5, 12, 23, and 48 hours following mock (□) (n=14 at each time point) or total body irradiation at doses of 1 (ui), 2 (■), or 6 Gy («) (n=8 at each time point for each treated group). Significance was determined using One-way ANOVA followed by Tukey's Multiple Comparison test (*p<0.05, **p<0.01, ***p<0.001). Data are presented as mean ± SEM.

Figure 4 shows a comparison of actual dose vs. predicted dose in the A) training set and B) validation set of mice. Predicted dose was determined based on the multiple linear regression model developed using expression of Ccngl and Cdknla in the training set (y= - 0.7674 + 0.6343*Ccngl + 0.0804*Cdknla).

Figure 5 shows a flow diagram demonstrating how the materials and methods of the invention can be used to facilitate rapid triage of individuals in need of immediate medical intervention following a radiological event, thereby optimizing the use of available medical resources and to improve treatment and survival. The embodiment shown in Figure 5 utilizes gene expression analysis of radiation biomarkers of the invention to identify and differentiate subjects exposed to radiation from subjects not exposed to radiation, and further determining the absorbed dose of those exposed.

DETAILED DISCLOSURE OF THE INVENTION

Dose assessment following radiological disasters is imperative to decrease mortality through rationally directed medical intervention. The goal of the study described herein was to identify biomarkers capable of qualitative (non-irradiated/irradiated) and/or quantitative (dose) assessment of radiation exposure. Using real-time quantitative PCR, biodosimetry genes were identified in blood samples from cancer patients undergoing total body irradiation. Time (5, 12, 23, 48 hours) and dose (0-8 Gy) dependent changes in gene expression were examined in C57BL/6 mice. A training set was used to derive weighted voting classification algorithms (non-irradiated/irradiated) and continuous regression (dose assessment) models which were tested in a separate validation set of mice. Of eight biodosimetry genes identified in cancer patients (ACTA2, BBC3, CCNG1, CDKN1A, GADD45A, MDK, SERPINE1, TNFRSF10B), expression of Bbc3, Ccngl, Cdknla, Serpinel, and Tnfrsf10b was significantly (p<0.05) increased in irradiated mice. Ccngl and Cdknla expression segregated irradiated mice from controls with an accuracy, specificity and sensitivity of 96.3, 100.0, 94.4%, respectively, at 48 hours. Multiple linear regression analysis predicted doses for the 0, 1, 2, 4, 6, and 8 Gy treatment groups as 0.0±0.2, 1.6±1.0, 2.9±1.4, 5.1±2.0, 5.3±0.7, and 10.5±5.6 Gy, respectively. Results suggest that gene expression analysis could be incorporated into biodosimetry protocols for qualitative and quantitative assessment of radiation exposure.

The present invention relates to a method for the detection, quantification, and monitoring of radiation exposure, by detecting in a blood sample or blood derived sample from a subject at least one biomarker for radiation exposure identified herein, such as Bbc3, Ccngl, Cdknla, Serpinel, and Tnfrsf10b. The biomarkers may be detected and, optionally, measured using an agent that detects or binds to the biomarker protein (such as antibodies specifically reactive with the biomarker protein or a portion thereof), or an agent that detects or binds to encoding nucleic acids.

Figure 5 shows a flow diagram demonstrating how the materials and methods of the invention can be used to facilitate rapid triage of individuals in need of immediate medical intervention following a radiological event, thereby optimizing the use of available medical resources and to improve treatment and survival. Potential sources of high-dose radiation include, for example, an accident at a nuclear industrial facility, attack on a nuclear industrial facility, detonation of a small radioactive device, detonation of a conventional explosive device that disperses radioactive material (dirty bomb), and detonation of a standard nuclear weapon. In addition to detection of radiation exposure and dose determination from high- dose radiation from catastrophic events, the materials and methods of the invention can be used to monitor exposure to individuals before, during, and after radiation-dependent medical treatments and diagnostic procedures involving low or intermediate dose radiation or local or focused radiation (e.g., X-ray examinations or other imaging procedures, or radiation therapy for treatment of cancer), and the materials and methods of the invention may be used to detect radiation exposure and determine absorbed dose in these contexts as well. Radiation exposure by patients, as well as medical personnel can be monitored. Radiation exposure can also conceivably occur through the malfunctioning or defect of an implant, resulting in internal contamination, and detection of exposure and dose determination can be carried out in this instance as well.

The present invention concerns materials and methods for detecting a radiation exposure and measuring the dose of radiation exposure received by the subject from which a sample has been obtained. The polypeptides and/or nucleic acid molecules (e.g., D A or mRNA) encoding the polypeptides disclosed herein (Bbc3, Ccngl, Cdknla, Serpinel, and Tnfrsf10b) can be used as molecular markers for radiation exposure and biodosimetry. The biomarkers of the invention include, but are not limited to, Bbc3, Ccngl, Cdknla, Serpinel, and Tnfrsf10b. Protein and nucleotide sequences of biomarker proteins and nucleic acids encoding them can be found at numerous publicly available sequence databases including GenBank. In one embodiment, the biomarker is one or more polypeptides, and/or nucleic acids encoding the polypeptides, selected from one, two, three, four, or all five of Bbc3, Ccngl , Cdknl a, Serpinel , and Tnfrsfl Ob. In some embodiments, the one or more biomarkers comprise or consist of one or two from among Bbc3, Ccngl, and Cdknla. In some embodiments, the one or more biomarkers comprise or consist of Bbc3 and Ccngl. In some embodiments, the one or more biomarkers comprise or consist of Ccngl. In some embodiments, the one or more biomarkers comprise or consist of Ccngl and Cdknl. In some embodiments, the one or more biomarkers comprise or consist of one or both biomarkers from among Ccngl and Tnfrsfl Ob.

Radiation biomarkers are molecules such as polynucleotides or polypeptides found in the body the presence or absence (e.g., elevation or reduction) of which are associated with radiation exposure and, preferably, radiation dose determination (e.g., dose-dependent elevation or depletion). Measurement or identification of radiation biomarkers can be useful in patient diagnosis or clinical management of radiation exposure. They can be products of the blood cells themselves, or of the body in response to irradiation. As with other radiation biomarkers, the biomarkers described herein can be used for a variety of purposes, such as: screening a healthy population or a high risk population for radiation exposure; making a diagnosis of radiation exposure (such as radiation overdose); determining the prognosis of a subject; and monitoring the course in a subject in remission or while receiving surgery, radiation, chemotherapy, or other treatment. Thus, levels of these biomarkers in the blood can be used to detect and/or monitor the occurrence and extent of radiation exposure throughout a. course of treatment, for example, and can be used in predicting therapeutic and prognostic outcome. In addition to monitoring an individual's or population's exposure to radiation and the radiation dose received during acute or chronic radiation treatment, the radiation biomarkers can be used to monitor an individual or population's exposure following a known radiation exposure or suspected radiation exposure.

One aspect of the invention concerns a method for identifying a mammalian subject exposed to radiation, comprising detecting the presence of and/or quantifying the level of at least one biomarker selected from among Bbc3, Ccngl, Cdknla, Serpinel, and Tnfrsf10b in a blood sample or blood derived sample from the subject, wherein the presence of the biomarker, or a level (e.g., concentration) of the biomarker above a pre-determined threshold is indicative of radiation exposure in the subject. In some embodiments, the one or more biomarkers comprise or consist of one or two from among Bbc3, Ccngl, and Cdknla. In some embodiments, the one or more biomarkers comprise or consist of Bbc3 and Ccngl. In some embodiments, the one or more biomarkers comprise or consist of Ccngl . In some embodiments, the one or more biomarkers comprise or consist of Ccngl and Cdknl. In some embodiments, the one or more biomarkers comprise or consist of one or both biomarkers from among Ccngl and Tnfrsf10b.

In one embodiment of a method of the invention, the detecting comprises: (a) contacting a blood sample or blood derived with a binding agent (or binding agents) that binds the biomarker protein (or biomarker proteins) to form a complex (or complexes); and (b) detecting the complex(es); and optionally correlating the detected complexes) to the amount of biomarker protein(s) in the sample, wherein the presence of one or more biomarkers, or the presence of elevated levels biomarker protein(s), is indicative of ionizing radiation exposure. In a specific embodiment, the binding agent is an aptamer or peptide or antibody. In a specific embodiment, the binding agent for detecting of step (b) further comprises a label linked or incorporated onto the agent. In one embodiment, the detecting comprises using ELISA-based immunoenzymatic detection. In another embodiment, the detecting comprises using Western blotting or radioimmunoassays (RIA).

In another embodiment, the biomarker protein (or proteins) is detected using electrophoretic, chromatographic, or spectroscopy methods, or a combination thereof. In one embodiment, proteins can be identified using 2-D gel electrophoresis of a sample followed by digestion of the separated proteins and mass spectrometry of peptides. In another embodiment, proteins or peptides can be identified by amino acid sequencing all or a portion of the molecule. In a specific embodiment, the mass spectrometry is tandem mass spectrometry (MS/MS). In a further embodiment, the mass spectrometry is carried out using matrix-assisted laser desorption/ionization (MALDI), such as MALDI-TOF. In a still further embodiment, proteins or peptides are identified using liquid chromatography (LC) methods, such as high pressure LC (HPLC).

Antibodies against numerous biomarker proteins of the invention are commercially available. Antibodies can also be readily prepared using standard and routine procedures known in the art.

Optionally, the methods of the invention further comprise detecting and/or quantifying one or more additional biomarkers of radiation exposure in the same blood sample or blood derived sample, or a different blood sample or blood derived sample, or a same or different biological sample (e.g., tissue such as a biopsy), obtained from the same subject, before, during, or after said detecting of the biomarker(s) of the invention is carried out on the sample. In this way, one or more biomarkers of the invention can be used as part of a panel of biomarkers utilized in surveillance protocols for detecting radiation exposure and, optionally, radiation dose. For example, a panel of 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more markers could be utilized.

In some embodiments, the detecting is performed at two or more time points or intervals. For example, the detecting can be performed at various time points or intervals as part of monitoring of the subject before, during, or after radiation treatment (e.g., for treatment of cancer) or some other radiation exposure.

Optionally, the methods of the invention further comprise comparing the level of one or more radiation biomarkers in a blood sample, or blood derived sample, with the level of the radiation biomarker present in a normal control sample, wherein a higher level of biomarker in the test sample as compared to the level in the normal control sample is indicative of the presence of radiation exposure.

In some embodiments, the subject exhibits no signs or symptoms of radiation sickness or radiation poisoning at the time the detecting of the radiation biomarker(s) is carried out. In other embodiments, the subject exhibits one or more signs or symptoms of radiation exposure. Signs and symptoms of radiation sickness include, but are not limited to, signs and symptoms resulting from an acute absorbed dose of about 1 to 2 Gy, i.e., mild radiation sickness (e.g., nausea and vomiting within 24 to 48 hours, headache, fatigue, weakness); signs and symptoms resulting from an acute absorbed dose of about 2 to 3.5 Gy, i.e., moderate radiation sickness (e.g., nausea and vomiting within 12 to 24 hours, fever, hair loss, infections, vomiting blood, bloody stool, poor wound healing, any signs and symptoms associated with a lower absorbed dose); signs and symptoms resulting from an absorbed dose of about 3.5 to 5.5 Gy, i.e., severe radiation sickness (e.g., nausea and vomiting less than one hour after exposure to radiation, diarrhea, high fever, any signs and symptoms associated with a lower absorbed dose); and signs and symptoms resulting from an absorbed dose greater than about 5.5 to 8 Gy, i.e., very severe radiation sickness (e.g., nausea and vomiting less than 30 minutes after exposure to radiation, dizziness, disorientation, low blood pressure (hypotension), and any signs and symptoms associated with a lower absorbed dose).

The subject invention also concerns methods for prognostic evaluation of a subject having, or suspected of having, radiation exposure, comprising: a) determining the level of one or more radiation biomarkers of the present invention from among Bbc3, Ccngl, Cdknla, Serpinel, and Tnfrsf10b in a blood sample or blood derived sample obtained from the subject; b) comparing the level determined in step (a) to a level or range of the one or more radiation biomarkers known to be present in a blood sample or blood derived sample obtained from a normal subject not exposed to radiation; and c) determining the prognosis of the subject based on the comparison of step (b), wherein a high level of the one or more radiation biomarkers in step (a) indicates a higher absorbed dose of radiation and, therefore, a poorer prognosis. In one embodiment, the radiation biomarker is one or more polypeptides, and/or nucleic acids encoding the polypeptides, selected from Bbc3, Ccngl, Cdknla, Serpinel, and Tnfrsf10b. In some embodiments, the one or more biomarkers comprise or consist of one or two from among Bbc3, Ccngl, and Cdknla. In some embodiments, the one or more biomarkers comprise or consist of Bbc3 and Ccngl. In some embodiments, the one or more biomarkers comprise or consist of Ccngl . In some embodiments, the one or more biomarkers comprise or consist of Ccngl and Cdknl. In some embodiments, the one or more biomarkers comprise or consist of one or both biomarkers from among Ccngl and Tnfrsf10b.

The terms "detecting" or "detect" include assaying or otherwise establishing the presence or absence of the target radiation biomarker (one or more encoding nucleic acid sequences or gene products (polypeptides) of the invention (Bbc3, Ccngl, Cdknla, Serpinel, and Tnfrsf10b), subunits thereof, or combinations of agent bound targets, and the like, or assaying for, interrogating, ascertaining, establishing, or otherwise determining one or more factual characteristics of radiation sickness or poisoning. The term encompasses diagnostic, prognostic, and monitoring applications for one or more of the radiation biomarkers of the invention (Bbc3, Ccngl, Cdknla, Serpinel, and Tnfrsf10b) and, optionally, in combination with other radiation biomarkers. The term encompasses quantitative, semi-quantitative, and qualitative detection methodologies. In embodiments of the invention involving detection of one or more polypeptides (as opposed to nucleic acid molecules encoding the polypeptides), the detection method may be, for example, an ELISA-based method. Preferably, in the various embodiments of the invention, the detection method provides an output (i.e., readout or signal) with information concerning the presence, absence, or amount of the radiation biomarker(s) in a blood sample, or blood derived sample, from a subject. For example, the output may be qualitative (e.g., "positive" or "negative"; or "yes" or "no"; or "irradiated" or "non-irradiated"), or quantitative (e.g., a concentration such as nanograms per milliliter, or gray unit (Gy)), and/or unit of time post-exposure (e.g., 5 or 6 hours post-radiation exposure, 12 hours post-radiation exposure, 23 or 24 hours post-radiation exposure, 48 hours post- radiation exposure). In one embodiment, the invention relates to a method for detecting radiation exposure in a subject by quantitating one or more radiation biomarker polypeptides of the invention (Bbc3, Ccngl, Cdknla, Serpinel, and Tnfrsf10b), or their encoding nucleic acids (DNA or RNA), in a blood sample or blood derived sample from the subject, comprising (a) contacting (reacting) the sample with an antibody specific for the radiation biomarker polypeptide(s) which is directly or indirectly labeled with a detectable substance; and (b) detecting the detectable substance. In one embodiment, the radiation biomarker is one or more polypeptides, and/or nucleic acids encoding the polypeptides, selected from Bbc3, Ccngl, Cdknla, Serpinel, and Tnfrsf10b. In some embodiments, the one or more biomarkers comprise or consist of one or two from among Bbc3, Ccngl, and Cdknla. In some embodiments, the one or more biomarkers comprise or consist of Bbc3 and Ccngl . In some embodiments, the one or more biomarkers comprise or consist of Ccngl. In some embodiments, the one or more biomarkers comprise or consist of Ccngl and Cdknl. In some embodiments, the one or more biomarkers comprise or consist of one or both biomarkers from among Ccngl and Tnfrsf10b.

In one aspect, the invention relates to a method for diagnosing and/or monitoring radiation exposure in a subject by quantitating one or more radiation biomarker polypeptides of the invention in a blood sample or blood derived sample from the subject, comprising (a) reacting the sample with an antibody or antibodies specific for the biomarker or biomarkers which are directly or indirectly labeled with a detectable substance; and (b) detecting the detectable substance. In one embodiment, the biomarker is one or more polypeptides, and/or nucleic acids encoding the polypeptides, selected from Bbc3, Ccngl, Cdknla, Serpinel, and Tnfrsf10b. In some embodiments, the one or more biomarkers comprise or consist of one or two from among Bbc3, Ccngl, and Cdknla. In some embodiments, the one or more biomarkers comprise or consist of Bbc3 and Ccngl. In some embodiments, the one or more biomarkers comprise or consist of Ccngl . In some embodiments, the one or more biomarkers comprise or consist of Ccngl and Cdknl. In some embodiments, the one or more biomarkers comprise or consist of one or both biomarkers from among Ccngl and Tnfrsf10b.

Some embodiments of the methods of the invention involve (a) contacting a blood sample, or blood derived sample, from a subject with an antibody or antibodies specific for the biomarker or biomarker polypeptides of the invention which are directly or indirectly labeled with an enzyme; (b) adding a substrate for the enzyme wherein the substrate is selected so that the substrate, or a reaction product of the enzyme and substrate, forms fluorescent complexes; (c) quantitating the biomarker(s) in the sample by measuring fluorescence of the fluorescent complexes; and (d) comparing the quantitated levels to that of a standard. In one embodiment, the biomarker is one or more polypeptides, and/or nucleic acids encoding the polypeptides, selected from Bbc3, Ccngl, Cdknla, Serpinel, and Tnfrsf10b. In some embodiments, the one or more biomarkers comprise or consist of one or two from among Bbc3, Ccngl, and Cdknla. In some embodiments, the one or more biomarkers comprise or consist of Bbc3 and Ccngl. In some embodiments, the one or more biomarkers comprise or consist of Ccngl . In some embodiments, the one or more biomarkers comprise or consist of Ccngl and Cdknl. In some embodiments, the one or more biomarkers comprise or consist of one or both biomarkers from among Ccngl and Tnfrsf10b.

A specific embodiment of the invention comprises:

A method for detecting radiation exposure, comprising (a) incubating a blood sample or blood derived sample with a first antibody specific for at least one radiation biomarker polypeptide of the invention which is directly or indirectly labeled with a detectable substance, and a second antibody specific for the biomarker polypeptide which is immobilized;

(b) separating the first antibody from the second antibody to provide a first antibody phase and a second antibody phase;

(c) detecting the detectable substance in the first or second antibody phase thereby quantitating the biomarker in the sample; and

(d) comparing the quantitated biomarker with a standard.

A standard used in a method of the invention may correspond to biomarker levels obtained for samples from healthy control subjects, from subjects with benign disease, subjects with a known extent of radiation exposure, or from other samples of the subject, or other reference samples. Increased levels of a radiation biomarker as compared to the standard may be indicative of radiation exposure. In one embodiment, the biomarker is one or more polypeptides, and/or nucleic acids encoding the polypeptides, selected from Bbc3, Ccngl , Cdknl a, Serpinel , and Tnfrsfl Ob.

The invention also contemplates using the methods, devices, and kits described herein in conjunction with one or more additional radiation biomarkers. Therefore, the invention contemplates a method for analyzing a blood sample or blood derived sample, for the presence of a radiation biomarker of the invention (a polypeptide or encoding nucleic acid molecule selected from among Bbc3, Ccngl , Cdknla, Serpinel, and Tnfrsf10b) and analyzing the same sample, or another sample from the same subject, for other markers that are specific indicators of radiation exposure and/or radiation sickness or poisoning. The one or more additional biomarkers may be detected before, during, and/or after detection of the one or more radiation biomarkers of the invention is carried out. The methods, devices, and kits described herein may be modified by including agents to detect the additional markers, or nucleic acids encoding the markers.

In one embodiment, the one or more additional biomarker in the methods, devices, and kits described heren is one or more additional diagnostic and/or prognostic biomarkers for radiation exposure. In one embodiment, the additional radiation biomarker is one or more selected from Table IB herein. In another embodiment, the additional radiation biomarker is one or more disclosed in WO 2009/145830 (Chute et al), which is incorporated herein by reference in its entirety.

As indicated above, the present invention provides a method for detecting and/or quantifying radiation exposure in a subject by detecting and/or measuring a radiation biomarker of the invention in a blood sample or blood derived sample from the subject. In one embodiment, the method comprises contacting the sample with an antibody specific for the biomarker polypeptide which is directly or indirectly labeled with a detectable substance, and detecting the detectable substance.

The methods, devices, and kits of the invention can be used for the detection of either an over-abundance or an under-abundance of one or more radiation biomarkers relative to a non-exposure state, or relative to a known exposure state, or the presence of a modified (e.g., less than full length) radiation biomarker which correlates with a disorder state (e.g., radiation sickness or poisoning), or a progression toward a disorder state.

The methods, devices, and kits of the invention can be used in the detection and, optionally, determination of the absorbed radiation dose when the subject is symptomatic or asymptomatic of radiation exposure, and for monitoring and evaluating the prognosis of radiation sickness or poisoning progression and mortality. Depending upon the particular radiation biomarker of the invention, increased levels or decreased levels of detected biomarker blood sample or blood derived sample compared to a standard (e.g., levels for no or normal radiation exposure, levels for benign disorders, or other reference levels) may be indicative of extent of radiation exposure (dose and/or duration) and radiation sickness or poisoning progression and mortality.

The terms "sample", "biological sample", and the like refer to a type of material known to or suspected of expressing or containing a radiation biomarker, such as a blood sample or a sample derived from blood that has undergone separation, purification, or preparative steps. The test sample can be used directly as obtained from the source or following a pretreatment to modify the character of the sample. The sample can be derived from any biological source, such as tissues or extracts, including cells and physiological fluids, such as, for example, whole blood, plasma, serum, peritoneal fluid, ascites, and the like. The sample can be obtained from mammals, most preferably humans. The sample can be pretreated by any method and/or can be prepared in any convenient medium that does not interfere with the assay. The sample can be treated prior to use, such as preparing plasma from blood, diluting viscous fluids, applying one or more protease inhibitors to samples, and the like. Sample treatment can involve filtration, distillation, extraction, concentration, inactivation of interfering components, the addition of reagents, and the like.

The presence and quantity of a radiation biomarker of the invention or nucleic acids

(DNA or RNA) encoding the polypeptides may be detected in human or non-human mammalian blood samples or blood derived samples.

In several embodiments of the invention, the method described herein is adapted for determining the dose of radiation exposure by quantitating a biomarker of the invention in blood samples or blood derived samples from a subject. Preferably, the amount of biomarker quantitated in a sample from a subject being tested is compared to levels quantitated for another sample or an earlier sample from the subject, or levels quantitated for a control sample. Levels for control samples from healthy subjects may be established by prospective and/or retrospective statistical studies. Healthy subjects who have no clinically evident radiation exposure, or disease or abnormalities, may be selected for statistical studies. Diagnosis may be made by a finding of statistically different levels of one or more biomarkers compared to a control sample or previous levels quantitated for the same subject.

The radiation biomarkers of the invention include all homologs, naturally occurring allelic variants, isoforms and precursors of the human or non-human mammalian molecules. In general, naturally occurring allelic variants of human biomarkers will share significant sequence homology (70-90%) to other sequences. Allelic variants may contain conservative amino acid substitutions or will contain a substitution of an amino acid from a corresponding position in a homologue. Preferably, the homologs or variants of the biomarkers selected for detection will be appropriately matched to the species of the mammalian subject (species) from which the sample is obtained (human sequences within a human subject).

In some embodiments of the various aspects of the invention (e.g., methods, kits (arrays, primers), devices), the biomarkers correspond to one or more nucleic acid sequences of SEQ ID NOs: 1-25, or polypeptides encoded by such nucleic acids. For example, in some embodiments, Bbc3 is SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; Ccngl is SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10; Cdknla is SEQ ID NO: 1 1 , SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14; Serpinel is SEQ ID NO: 21 or SEQ ID NO: 22; and/or Tnfrsf10b is SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25.

The terms "subject", "individual", and "patient" are used interchangeably herein to refer to a mammal, which may be exposed to ionizing radiation. The subject may be male or female. The subject may be any age (adult, child, etc.). The term includes dogs, cats, and horses. The term also includes primates such as apes, chimps, monkeys, and humans.

Agents that are capable of detecting radiation biomarkers of the invention in the samples of subjects include those that interact or bind with the polypeptide or the nucleic acid molecule (e.g., nucleic acid encoding the polypeptide). Examples of such agents (also referred to herein as binding agents) include, but are not limited to, antibodies or fragments thereof that bind the polypeptide, polypeptide binding partners, and nucleic acid molecules that hybridize to the nucleic acid molecules encoding the polypeptides. Preferably, the binding agent is labeled with a detectable substance (e.g., a detectable moiety). The binding agent may itself function as a label.

Biomarker Antibodies

Antibodies specific for radiation biomarkers of the invention that are used in the methods, devices, and kits of the invention may be obtained from scientific or commercial sources. Alternatively, the isolated native polypeptides or recombinant polypeptides may be utilized to prepare antibodies, monoclonal or polyclonal antibodies, and immunologically active fragments (e.g., a Fab or (Fab)₂ fragment), an antibody heavy chain, an antibody light chain, humanized antibodies, a genetically engineered single chain F_v molecule (Ladne et al, U.S. Patent No. 4,946,778), or a chimeric antibody, for example, an antibody which contains the binding specificity of a murine antibody, but in which the remaining portions are of human origin. Antibodies, including monoclonal and polyclonal antibodies, fragments and chimeras, may be prepared using methods known to those skilled in the art. Preferably, antibodies used in the methods of the invention are reactive against biomarkers of the invention if they bind with a K_a of greater than or equal to 10⁷ M. In a sandwich immunoassay of the invention, mouse polyclonal antibodies and rabbit polyclonal antibodies can be utilized, for example.

In order to produce monoclonal antibodies, a host mammal is inoculated with a protein or peptide representing a radiation biomarker of the invention and then boosted. Spleens are collected from inoculated mammals a few days after the final boost. Cell suspensions from the spleens are fused with a tumor cell in accordance with the general method described by Kohler and Milstein {Nature, 1975, 256:495-497). In order to be useful, a peptide fragment must contain sufficient amino acid residues to define the epitope of the biomarker molecule being detected.

If the fragment is too short to be immunogenic, it may be conjugated to a carrier molecule. Some suitable carrier molecules include keyhole limpet hemocyanin and bovine serum albumin. Conjugation may be carried out by methods known in the art. One such method is to combine a cysteine residue of the fragment with a cysteine residue on the carrier molecule. The peptide fragments may be synthesized by methods known in the art. Some suitable methods are described by Stuart and Young in "Solid Phase Peptide Synthesis," Second Edition, Pierce Chemical Company (1984).

Purification of the antibodies or fragments can be accomplished by a variety of methods known to those skilled in the art including, precipitation by ammonium sulfate or sodium sulfate followed by dialysis against saline, ion exchange chromatography, affinity or immunoaffinity chromatography as well as gel filtration, zone electrophoresis, etc. (Goding in, Monoclonal Antibodies: Principles and Practice, 2d ed., pp. 104-126, Orlando, Fla., Academic Press). It is preferable to use purified antibodies or purified fragments of the antibodies having at least a portion of a biomarker binding region, including such as Fv, F(ab')2, Fab fragments (Harlow and Lane, 1988, Antibody Cold Spring Harbor) for the detection of the biomarker(s) in the blood samples or blood derived samples of subjects (e.g., subjects suspected of radiation exposure or at risk of radiation exposure).

For use in detection, dose determination, and/or monitoring of radiation exposure, the purified antibodies can be covalently attached, either directly or via linker, to a compound which serves as a reporter group to permit detection of the presence of the biomarker. A variety of different types of substances can serve as the reporter group, including but not limited to enzymes, dyes, radioactive metal and non-metal isotopes, fluorogenic compounds, fluorescent compounds, etc. Methods for preparation of antibody conjugates of the antibodies (or fragments thereof) of the invention useful for detection, monitoring are described in U.S. Patent Nos. 4,671,958; 4,741,900 and 4,867,973.

In one aspect of the invention, preferred binding epitopes may be identified from a known biomarker gene sequence and its encoded amino acid sequence and used to generate antibodies to the biomarker with high binding affinity. Also, identification of binding epitopes on the biomarker polypeptide can be used in the design and construction of preferred antibodies. For example, a DNA encoding a preferred epitope on a biomarker polypeptide may be recombinantly expressed and used to select an antibody which binds selectively to that epitope. The selected antibodies then are exposed to the sample under conditions sufficient to allow specific binding of the antibody to the specific binding epitope on the biomarker and the amount of complex formed then detected. Specific antibody methodologies are well understood and described in the literature. A more detailed description of their preparation can be found, for example, in Practical Immunology, Butt, W. R., ed., Marcel Dekker, New York, 1984.

The present invention also contemplates the detection of radiation biomarker antibodies. Thus, detection of antibodies to the radiation biomarkers of the invention in blood samples or blood derived samples of a subject may enable the detection of radiation exposure and dose determination, and is also contemplated within the scope of the invention.

Protein Binding Assays

Antibodies specifically reactive with the radiation biomarkers disclosed or their derivatives, such as enzyme conjugates or labeled derivatives, may be used to the detect radiation biomarkers in various biological samples, for example they may be used in any known immunoassays which rely on the binding interaction between an antigenic determinant of a protein and the antibodies. Examples of such assays are radioimmunoassays, enzyme immunoassay (e.g., ELISA), immunofluorescence, immunoprecipitation, latex agglutination, hemagglutination, and histochemical tests.

An antibody specific for a biomarker of the invention can be labeled with a detectable substance and localized in biological samples such as blood based upon the presence of the detectable substance. Examples of detectable substances include, but are not limited to, the following radioisotopes (e.g., ³H, ¹⁴C, ³⁵S, ¹²⁵I, ¹³¹I), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), luminescent labels such as luminol; enzymatic labels (e.g. , horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase, acetylcholinestease), biotinyl groups (which can be detected by marked avidin, e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or calorimetric methods), predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). Indirect methods may also be employed in which the primary antigen-antibody reaction is amplified by the introduction of a second antibody, having specificity for the antibody reactive against the biomarker. By way of example, if the antibody having specificity against a biomarker is a rabbit IgG antibody, the second antibody may be goat anti-rabbit gamma-globulin labeled with a detectable substance.

Methods for conjugating or labeling the antibodies discussed above may be readily accomplished by one of ordinary skill in the art. (See, for example, Imman, Methods In Enzymology, Vol. 34, Affinity Techniques, Enzyme Purification: Part B, Jakoby and Wichek (eds.), Academic Press, New York, p. 30, 1974; and Wilchek and Bayer, "The Avidin-Biotin Complex in Bioanalytical Applications," Anal. Biochem., 1988, 171:1-32, regarding methods for conjugating or labeling the antibodies with an enzyme or ligand binding partner).

Time-resolved fluorometry may be used to detect a signal. For example, the method described in Christopoulos T.K. and Diamandis E.P., Anal. Chem., 1992:64:342-346 may be used with a conventional time-resolved fluorometer.

Therefore, in accordance with an embodiment of the invention, a method is provided wherein an antibody to a radiation biomarker of the invention is labeled with an enzyme, a substrate for the enzyme is added wherein the substrate is selected so that the substrate, or a reaction product of the enzyme and substrate, forms fluorescent complexes with a lanthanide metal. A lanthanide metal is added and the biomarker is quantitated in the sample by measuring fluorescence of the fluorescent complexes. The antibodies specific for the biomarkers may be directly or indirectly labeled with an enzyme. Enzymes are selected based on the ability of a substrate of the enzyme, or a reaction product of the enzyme and substrate, to complex with lanthanide metals such as europium and terbium. Examples of suitable enzymes include alkaline phosphatase and beta-galactosidase. Preferably, the enzyme is alkaline phosphatase. The biomarker antibodies may also be indirectly labeled with an enzyme. For example, the antibodies may be conjugated to one partner of a ligand binding pair, and the enzyme may be coupled to the other partner of the ligand binding pair. Representative examples include avidin-biotin, and riboflavin-riboflavin binding protein. Preferably the antibodies are biotinylated, and the enzyme is coupled to streptavidin.

In an embodiment of the method, antibody bound to a radiation biomarker of the invention in a sample is detected by adding a substrate for the enzyme. The substrate is selected so that in the presence of a lanthanide metal {e.g., europium, terbium, samarium, and dysprosium, preferably europium and terbium), the substrate or a reaction product of the enzyme and substrate, forms a fluorescent complex with the lanthanide metal. Examples of enzymes and substrates for enzymes that provide such fluorescent complexes are described in U.S. Patent No. 5,312,922 to Diamandis. By way of example, when the antibody is directly or indirectly labeled with alkaline phosphatase, the substrate employed in the method may be 4-methylumbeliferyl phosphate, or 5-fluorpsalicyl phosphate. The fluorescence intensity of the complexes is typically measured using a time-resolved fiuorometer, e.g., a CyberFluor 615 Immoanalyzer (Nordion International, Kanata Ontario).

The sample, antibody specific for the radiation biomarker, or the biomarker itself, may be immobilized on a carrier. Examples of suitable carriers are agarose, cellulose, dextran, Sephadex, Sepharose, liposomes, carboxymethyl cellulose polystyrene, filter paper, ion-exchange resin, plastic film, plastic tube, glass beads, polyamine-methyl vinyl ether- maleic acid copolymer, amino acid copolymer, ethylene-maleic acid copolymer, nylon, silk, etc. The carrier may be in the shape of, for example, a tube, test plate, well, beads, disc, sphere, etc. The immobilized antibody may be prepared by reacting the material with a suitable insoluble carrier using known chemical or physical methods, for example, cyanogen bromide coupling.

In accordance with an embodiment, the present invention provides a mode for determining the presence and, optionally, the amount of radiation biomarker in an appropriate sample such as blood by measuring the biomarker(s) by immunoassay. It will be evident to a skilled artisan that a variety of immunoassay methods can be used to measure the biomarkers of the invention. In general, a biomarker immunoassay method may be competitive or noncompetitive. Competitive methods typically employ an immobilized or immobilizable antibody to the biomarker (anti-biomarker such as anti-Serpinel) and a labeled form of the biomarker (such as labeled Serpinel). Sample biomarker and labeled biomarker compete for binding to anti-biomarker. After separation of the resulting labeled biomarker that has become bound to anti-biomarker (bound fraction) from that which has remained unbound (unbound fraction), the amount of the label in either bound or unbound fraction is measured and may be correlated with the amount of biomarker in the biological sample in any conventional manner, e.g. , by comparison to a standard curve.

Preferably, a noncompetitive method is used for the determination of one or more radiation biomarkers of the invention, with the most common method being the "sandwich" method. In this assay, two anti-biomarker antibodies, such as two anti-serpinel antibodies, are employed. One of the anti-biomarker antibodies is directly or indirectly labeled (also referred to as the "detection antibody") and the other is immobilized or immobilizable (also referred to as the "capture antibody"). The capture and detection antibodies can be contacted simultaneously or sequentially with the biological sample. Sequential methods can be accomplished by incubating the capture antibody with the sample, and adding the detection antibody at a predetermined time thereafter (sometimes referred to as the "forward" method); or the detection antibody can be incubated with the sample first and then the capture antibody added (sometimes referred to as the "reverse" method). After the necessary incubation(s) have occurred, to complete the assay, the capture antibody is separated from the liquid test mixture, and the label is measured in at least a portion of the separated capture antibody phase or the remainder of the liquid test mixture. Generally, it is measured in the capture antibody phase since it comprises the biomarker bound by ("sandwiched" between) the capture and detection antibodies.

In a typical two-site immunometric assay for a biomarker, one or both of the capture and detection antibodies are polyclonal antibodies. The label used in the detection antibody can be selected from any of those known conventionally in the art. As with other embodiments of the protein detection assay, the label can be an enzyme or a chemiluminescent moiety, for example, or a radioactive isotope, a fluorophore, a detectable ligand (e.g., detectable by a secondary binding by a labeled binding partner for the ligand), and the like. Preferably, the antibody is labeled with an enzyme that is detected by adding a substrate that is selected so that a reaction product of the enzyme and substrate forms fluorescent complexes. The capture antibody is selected so that it provides a mode for being separated from the remainder of the test mixture. Accordingly, the capture antibody can be introduced to the assay in an already immobilized or insoluble form, or can be in an immobilizable form, that is, a form which enables immobilization to be accomplished subsequent to introduction of the capture antibody to the assay. An immobilized capture antibody can comprise an antibody covalently or noncovalently attached to a solid phase such as a magnetic particle, a latex particle, a microtiter multi-well plate, a bead, a cuvette, or other reaction vessel. An example of an immobilizable capture antibody is an antibody that has been chemically modified with a ligand moiety, e.g., a hapten, biotin, or the like, and that can be subsequently immobilized by contact with an immobilized form of a binding partner for the ligand, e.g., m antibody, avidin, or the like. In an embodiment, the capture antibody can be immobilized using a species specific antibody for the capture antibody that is bound to the solid phase.

A particular sandwich immunoassay method of the invention employs two antibodies reactive against a biomarker of the invention, a second antibody having specificity against an antibody reactive against the biomarker labeled with an enzymatic label, and a fluorogenic substrate for the enzyme. In an embodiment, the enzyme is alkaline phosphatase (ALP) and the substrate is 5-fluorosalicyl phosphate. ALP cleaves phosphate out of the fluorogenic substrate, 5-fluorosalicyl phosphate, to produce 5-fluorosalicylic acid (FSA). 5- Fluorosalicylic acid can then form a highly fluorescent ternary complex of the form FSA- Tb(3+)-EDTA, which can be quantified by measuring the Tb³⁺ fluorescence in a time- resolved mode. Fluorescence intensity is typically measured using a time-resolved fluorometry as described herein.

The above-described immunoassay methods and formats are intended to be exemplary and are not limiting since, in general, it will be understood that any immunoassay method or format can be used in the present invention.

The protein detection methods, devices, and kits of the invention can utilize nanowire sensor technology (Zhen et al, Nature Biotechnology, 2005, 23(10):1294-1301 ; Lieber et al, Anal. Chem., 2006, 78(13):4260-4269, which are incorporated herein by reference) or microcantilever technology (Lee et al, Biosens. Bioelectron, 2005, 20(10):2157-2162; Wee et al, Biosens. Bioelectron., 2005, 20(10):1932-1938; Campbell and Mutharasan, Biosens. Bioelectron., 2005, 21(3):462-473; Campbell and Mutharasan, Biosens. Bioelectron., 2005, 21(4): 597-607; Hwang et al, Lab Chip, 2004, 4(6):547-552; Mukhopadhyay et al, Nano. Lett., 2005, 5(12):2835-2388, which are incorporated herein by reference) for detection of one or more biomarkers of the invention in samples. In addition, Huang et al. describe a prostate specific antigen immunoassay on a commercially available surface plasmon resonance biosensor {Biosens. Bioelectron., 2005, 21(3):483-490, which is incorporated herein by reference) which may be adapted for detection of one or more biomarkers of the invention. High-sensitivity miniaturized immunoassays may also be utilized for detection of the biomarkers (Cesaro-Tadic et al, Lab Chip, 2004, 4(6):563-569; Zimmerman et al, Biomed. Microdevices, 2005, 7(2):99-l 10, which are incorporated herein by reference).

Nucleic Acids

Nucleic acids including naturally occurring nucleic acids, oligonucleotides, antisense oligonucleotides, and synthetic oligonucleotides that hybridize to target nucleic acids within biomarker genes or transcripts of the invention (e.g., encoding biomarker polypeptides of the invention), are useful as agents to detect the presence of radiation biomarkers of the invention in blood samples or blood derived samples of subjects, preferably in blood samples or blood derived samples from those exposed to radiation or at risk for radiation exposure. The present invention contemplates the use of nucleic acid sequences corresponding to the coding sequence of radiation biomarkers of the invention and to the complementary sequence thereof, as well as sequences complementary to the biomarker transcript sequences occurring further upstream or downstream from the coding sequence (e.g., sequences contained in, or extending into, the 5' and 3' untranslated regions) for use as agents for detecting the expression of radiation biomarkers of the invention in samples of subjects.

The preferred oligonucleotides for detecting the presence of radiation biomarkers of the invention in samples are those that are complementary to at least part of the cDNA sequence encoding the biomarker. These complementary sequences are also known in the art as "antisense" sequences. These oligonucleotides may be oligoribonucleotides or oligodeoxyribonucleotides. In addition, oligonucleotides may be natural oligomers composed of the biologically significant nucleotides, . e. , A (adenine), dA (deoxyadenine), G (guanine), dG (deoxyguanine), C (cytosine), dC (deoxycytosine), T (thymine) and U (uracil), or modified oligonucleotide species, substituting, for example, a methyl group or a sulfur atom for a phosphate oxygen in the inter-nucleotide phosphodiester linkage. Additionally, these nucleotides themselves, and/or the ribose moieties may be modified.

The oligonucleotides may be synthesized chemically, using any of the known chemical oligonucleotide synthesis methods well described in the art. For example, the oligonucleotides can be prepared by using any of the commercially available, automated nucleic acid synthesizers. Alternatively, the oligonucleotides may be created by standard recombinant DNA techniques, for example, inducing transcription of the noncoding strand. The DNA sequence encoding the biomarker may be inverted in a recombinant DNA system, e.g., inserted in reverse orientation downstream of a suitable promoter, such that the noncoding strand now is transcribed.

Although any length oligonucleotide may be utilized to hybridize to a target nucleic acid within biomarker genes or transcripts of the invention (e.g. , to a nucleic acid encoding a biomarker polypeptide), oligonucleotides typically within the range of 8-100 nucleotides are preferred. Most preferable oligonucleotides for use in detecting biomarkers in blood samples or blood derived samples are those within the range of 1 -50 nucleotides.

In some embodiments, the substrate (e.g. , solid support) of the array of the invention has no more than 500 oligonucleotides attached to it. In some embodiments, the substrate has no more than 100 oligonucleotides attached to it. In some embodiments, the substrate has no more than 50 oligonucleotides attached to it. In some embodiments, the substrate has no more than 20 oligonucleotides attached to it. In some embodiments, the substrate has no more than 10 oligonucleotides attached to it. In some embodiments, the substrate has no more than 5 oligonucleotides attached to it. In some embodiments, the substrate has no more than 4 oligonucleotides attached to it. In some embodiments, the substrate has no more than 3 oligonucleotides attached to it. In some embodiments, the substrate has no more than 2 oligonucleotides attached to it. In some embodiments, the substrate has no more than 1 oligonucleotide attached to it.

When referring to hybridization of one nucleic to another, "low stringency conditions" means in 10% formamide, 5X Denhart's solution, 6X SSPE, 0.2% SDS at 42 °C, followed by washing in IX SSPE, 0.2% SDS, at 50 °C; "moderate stringency conditions" means in 50% formamide, 5X Denhart's solution, 5X SSPE, 0.2% SDS at 42 °C, followed by washing in 0.2X SSPE, 0.2% SDS, at 65 °C; and "high stringency conditions" means in 50% formamide, 5X Denhart's solution, 5X SSPE, 0.2% SDS at 42 °C, followed by washing in 0.1X SSPE, and 0.1% SDS at 65 °C. The phrase "stringent hybridization conditions" means low, moderate, or high stringency conditions.

The oligonucleotide selected for hybridizing to the biomarker nucleic acid molecule, whether synthesized chemically or by recombinant DNA technology, can be isolated and purified using standard techniques and then preferably labeled {e.g., with S or P) using standard labeling protocols. Oligonucleotides can be attached or immobilized to a suitable solid support using methods known in the art.

The present invention also contemplates the use of oligonucleotide pairs (e.g., primers) in polymerize chain reactions (PCR) to detect the expression of the biomarker in biological samples. The oligonucleotide pairs include a forward primer and a reverse primer.

The presence of biomarkers in a sample from a subject may be determined by nucleic acid hybridization, such as but not limited to Northern blot analysis, dot blotting, Southern blot analysis, fluorescence in situ hybridization (FISH), and PCR. Chromatography, preferably HPLC, and other known assays may also be used to determine messenger RNA levels of biomarkers in a sample.

Nucleic acid molecules encoding a biomarker of the present invention can be found in the biological fluids inside a biomarker-positive cell that is present in a biological sample under investigation, e.g., blood. Nucleic acids encoding biomarkers can also be found directly (i.e., cell-free) in the fluid or biological sample, e.g., blood.

In one aspect, the present invention contemplates the use of nucleic acids as agents for detecting radiation biomarkers of the invention in blood samples or blood derived samples of subjects, wherein the nucleic acids are labeled. The nucleic agents may be labeled with a radioactive label, a fluorescent label, an enzyme, a chemiluminescent tag, a colorimetric tag or other labels or tags that are discussed above or that are known in the art.

In another aspect, the present invention contemplates the use of Northern blot analysis to detect the presence of biomarker mRNA in a blood sample or blood derived sample. The first step of the analysis involves separating a sample containing biomarker nucleic acid by gel electrophoresis. The dispersed nucleic acids are then transferred to a nitrocellulose filter or another filter. Subsequently, the labeled oligonucleotide is exposed to the filter under suitable hybridizing conditions, e.g., 50% formamide, 5 x SSPE, 2 x Denhardt's solution, 0.1% SDS at 42° C, as described in Molecular Cloning: A Laboratory Manual, Maniatis et al. (1982, CSH Laboratory). Other useful procedures known in the art include solution hybridization, dot and slot RNA hybridization, and probe based microarrays. Measuring the radioactivity of hybridized fragments, using standard procedures known in the art quantitates the amount of biomarker nucleic acid present in the sample of a subject.

Dot blotting involves applying samples containing the nucleic acid of interest to a membrane. The nucleic acid can be denatured before or after application to the membrane. The membrane is incubated with a labeled probe. Dot blot procedures are well known to the skilled artisan and are described more fully in U.S. Patent Nos. 4,582,789 and 4,617,261, the disclosures of which are incorporated herein by reference.

Polymerase chain reaction (PCR) is a process for amplifying one or more target nucleic acid sequences present in a nucleic acid sample using primers and agents for polymerization and then detecting the amplified sequence. The extension product of one primer when hybridized to the other becomes a template for the production of the desired specific nucleic acid sequence, and vice versa, and the process is repeated as often as is necessary to produce the desired amount of the sequence. The skilled artisan to detect the presence of desired sequence (U.S. Patent No. 4,683,195) routinely uses polymerase chain reaction.

A specific example of PCR that is routinely performed by the skilled artisan to detect desired sequences is reverse transcript PCR (RT-PCR; Saiki et al, Science, 1985, 230:1350; Scharf et al, Science, 1986, 233:1076). RT-PCR involves isolating total RNA from biological fluid, denaturing the RNA in the presence of primers that recognize the desired nucleic acid sequence, using the primers to generate a cDNA copy of the RNA by reverse transcription, amplifying the cDNA by PCR using specific primers, and detecting the amplified cDNA by electrophoresis or other methods known to the skilled artisan.

In a preferred embodiment, the methods of detecting biomarker nucleic acid in blood samples or blood derived samples of subjects include Northern blot analysis, dot blotting, Southern blot analysis, FISH, and PCR. Devices

The methods of the invention can be carried out on a solid support. The solid supports used may be those which are conventional for the purpose of assaying an analyte in a biological sample, and are typically constructed of materials such as cellulose, polysaccharide such as Sephadex, and the like, and may be partially surrounded by a housing for protection and/or handling of the solid support. The solid support can be rigid, semi-rigid, flexible, elastic (having shape-memory), etc., depending upon the desired application. Radiation biomarkers of the invention can be detected in a sample in vivo or in vitro {ex vivo). When, according to an embodiment of the invention, the amount of biomarker in a sample is to be determined without removing the sample from the body {i.e., in vivo, such as with an indwelling catheter or probe), the support should be one which is harmless to the subject and may be in any form convenient for insertion into an appropriate part of the body. For example, the support may be a probe made of polytetrafluoroethylene, polystyrene or other rigid non-harmful plastic material and having a size and shape to enable it to be introduced into a subject. The selection of an appropriate inert support is within the competence of those skilled in the art, as are its dimensions for the intended purpose.

A contacting step in an assay (method) of the invention can involve contacting, combining, or mixing the biological sample and the solid support, such as a reaction vessel, microvessel, tube, microtube, well, multi-well plate, or other solid support. In an embodiment of the invention, the solid support to be contacted with the biological sample {e.g., blood) has an absorbent pad or membrane for lateral flow of the liquid medium to be assayed, such as those available from Millipore Corp. (Bedford, MA), including but not limited to Hi-Flow Plus™ membranes and membrane cards, and SureWick™ pad materials.

The diagnostic device useful in carrying out the methods of the invention can be constructed in any form adapted for the intended use. Thus, in one embodiment, the device of the invention can be constructed as a disposable or reusable test strip or stick to be contacted with a blood sample or blood derived sample for which the presence of the biomarker or biomarker level is to be determined. In another embodiment, the device can be constructed using art recognized micro-scale manufacturing techniques to produce needle- like embodiments capable of being implanted or injected into an anatomical site, such as a vein or artery, for indwelling diagnostic applications. In other embodiments, devices intended for repeated laboratory use can be constructed in the form of an elongated probe or catheter, for sampling of blood. In preferred embodiments, the devices of the invention comprise a solid support (such as a strip or dipstick), with a surface that functions as a lateral flow matrix defining a flow path for a biological sample such as blood.

Immunochromatographic assays, also known as lateral flow test strips or simply strip tests, for detecting various analytes of interest, have been known for some time, and may be used for detection of radiation biomarkers of the invention. The benefits of lateral flow tests include a user-friendly format, rapid results, long-term stability over a wide range of climates, and relatively low cost to manufacture. These features make lateral flow tests ideal for applications involving home testing, rapid point of care testing, and testing in the field for various analytes. The principle behind the test is straightforward. Essentially, any ligand that can be bound to a visually detectable solid support, such as dyed microspheres, can be tested for, qualitatively, and in many cases even semi-quantitatively. For example, a one-step lateral flow immunostrip for the detection of free and total prostate specific antigen in serum is described in Fernandez-Sanchez et al. (J. Immuno. Methods, 2005, 307(1 -2):1-12, which is incorporated herein by reference) and may be adapted for detection of biomarkers of the invention in a biological sample such as blood.

Some of the more common immunochromatographic assays currently on the market are tests for pregnancy (as an over-the-counter (OTC) test kit), Strep throat, and Chlamydia. Many new tests for well-known antigens have been recently developed using the immunochromatographic assay method. For instance, the antigen for the most common cause of community acquired pneumonia has been known since 1917, but a simple assay was developed only recently, and this was done using this simple test strip method (Murdoch, D.R. et al. J Clin Microbiol, 2001, 39:3495-3498). Human immunodeficiency virus (HIV) has been detected rapidly in pooled blood using a similar assay (Soroka, S.D. et al. J Clin Virol, 2003, 27:90-96). A nitrocellulose membrane card has also been used to diagnose schistosomiasis by detecting the movement and binding of nanoparticles of carbon (van Dam, G.J. et al. J Clin Microbiol, 2004, 42:5458-5461).

The two common approaches to the immunochromatographic assay are the noncompetitive (or direct) and competitive (or competitive inhibition) reaction schemes (TechNote #303, Rev. #001, 1999, Bangs Laboratories, Inc., Fishers, IN). The direct (double antibody sandwich) format is typically used when testing for larger analytes with multiple antigenic sites such as luteinizing hormone (LH), human chorionic gonadotropin (hCG), and HIV. In this instance, less than an excess of sample analyte is desired, so that some of the microspheres will not be captured at the capture line, and will continue to flow toward the second line of immobilized antibodies, the control zone. This control line uses species- specific anti-immunoglobulin antibodies, specific for the conjugate antibodies on the microspheres. Free antigen, if present, is introduced onto the device by adding sample (blood, etc.) onto a sample addition pad. Free antigen then binds to antibody-microsphere complexes. Antibody 1, specific for epitope 1 of sample antigen, is coupled to dye microspheres and dried onto the device. When sample is added, microsphere-antibody complex is rehydrated and carried to a capture zone and control lines by liquid. Antibody 2, specific for a second antigenic site (epitope 2) of sample antigen, is dried onto a membrane at the capture line. Antibody 3, a species-specific, anti-immunoglobulin antibody that will react with antibody 1, is dried onto the membrane at the control line. If antigen is present in the sample (i.e., a positive test), it will bind by its two antigenic sites, to both antibody 1 (conjugated to microspheres) and antibody 2 (dried onto membrane at the capture line). Antibody 1 -coated microspheres are bound by antibody 3 at the control line, whether antigen is present or not. If antigen is not present in the sample (a negative test), microspheres pass the capture line without being trapped, but are caught by the control line.

The competitive reaction scheme is typically used when testing for small molecules with single antigenic determinants, which cannot bond to two antibodies simultaneously. As with double antibody sandwich assay, free antigen, if present is introduced onto the device by adding sample onto a sample pad. Free antigen present in the sample binds to an antibody- microsphere complex. Antibody 1 is specific for sample antigen and couple to dyed microspheres. An antigen-carrier molecule (typically BSA) conjugate is dried onto a membrane at the capture line. Antibody 2 (Ab2) is dried onto the membrane at the control line, and is a species-specific anti-immunoglobulin that will capture the reagent particles and confirm that the test is complete. If antigen is present in the sample (a positive test), antibody on microspheres (Abl) is already saturated with antigen from sample and, therefore, antigen conjugate bound at the capture line does not bind to it. Any microspheres not caught by the antigen carrier molecule can be caught by Ab2 on the control line. If antigen is not present in the sample (a negative test), antibody-coated dyed microspheres are allowed to be captured by antigen conjugate bound at the capture line.

Normally, the membranes used to hold the antibodies in place on these devices are made of primary hydrophobic materials, such as nitrocellulose. Both the microspheres used as the solid phase supports and the conjugate antibodies are hydrophobic, and their interaction with the membrane allows them to be effectively dried onto the membrane. Samples and/or biomarker-specific binding agents may be arrayed on the solid support, or multiple supports can be utilized, for multiplex detection or analysis. "Arraying" refers to the act of organizing or arranging members of a library (e.g., an array of different samples or an array of devices that target the same target molecules or different target molecules), or other collection, into a logical or physical array. Thus, an "array" refers to a physical or logical arrangement of, e.g., biological samples. A physical array can be any "spatial format" or physically gridded format" in which physical manifestations of corresponding library members are arranged in an ordered manner, lending itself to combinatorial screening. For example, samples corresponding to individual or pooled members of a sample library can be arranged in a series of numbered rows and columns, e.g., on a multi-well plate. Similarly, binding agents can be plated or otherwise deposited in microtitered, e.g., 96-well, 384-well, or-1536 well, plates (or trays). Optionally, biomarker- specific binding agents may be immobilized on the solid support.

Detection of biomarkers of the invention and other radiation biomarkers, and other assays that are to be carried out on samples, can be carried out simultaneously or sequentially with the detection of other target molecules, and may be carried out in an automated fashion, in a high-throughput format.

The biomarker-specific binding agents can be deposited but "free" (non-immobilized) in the conjugate zone, and be immobilized in the capture zone of a solid support. The biomarker-specific binding agents may be immobilized by non-specific adsorption onto the support or by covalent bonding to the support, for example. Techniques for immobilizing binding agents on supports are known in the art and are described for example in U.S. Patent Nos. 4,399,217, 4,381,291, 4,357,311, 4,343,312 and 4,260,678, which are incorporated herein by reference. Such techniques can be used to immobilize the binding agents in the invention. When the solid support is polytetrafluoroethylene, it is possible to couple hormone antibodies onto the support by activating the support using sodium and ammonia to aminate it and covalently bonding the antibody to the activated support by means of a carbodiimide reaction (yon Klitzing, Schultek, Strasburger, Fricke and Wood in "Radioimmunoassay and Related Procedures in Medicine 1982", International Atomic Energy Agency, Vienna (1982), pages 57-62.).

The diagnostic device of the invention can utilize lateral flow strip (LFS) technology, which has been applied to a number of other rapid strip assay systems, such as over-the- counter early pregnancy test strips based on antibodies to human chorionic gonadotropin (hCG). As with many other diagnostic devices, the device utilizes a binding agent to bind the target molecule (biomarker of the invention). The device has an application zone for receiving a biological sample such as blood, a labeling zone containing label which binds to biomarker in the sample, and a detection zone where biomarker label is retained.

Binding agent retained in the detection zone gives a signal, and the signal differs depending on whether biomarker levels in the biological sample are lower than, equal to, or greater than a given threshold concentration.

A sample from a subject having a biomarker level equal to or greater than the given reference biomarker concentration can be referred to as a "threshold level", "threshold amount", or "threshold sample". The application zone in the device is suitable for receiving the biological sample to be assayed. It is typically formed from absorbent material such as blotting paper. The labeling zone contains binding agent that binds to any biomarker in the sample. In one embodiment, the binding agent is an antibody (e.g., monoclonal antibody, polyclonal antibody, antibody fragment). For ease of detection, the binding agent is preferably in association with a label that provides a signal that is visible to the naked eye, e.g., it is tagged with a fluorescent tag or a colored tag such as conjugated colloidal gold, which is visible as a pink color.

The detection zone retains biomarker to which the binding agent has bound. This will typically be achieved using an immobilized binding agent such as an immobilized antibody. Where the binding agent in the labeling zone and the detection zone are both antibodies, they will typically recognize different epitopes on the target molecule (biomarker protein). This allows the formation of a "sandwich" comprising antibody-biomarker-antibody.

The detection zone is downstream of the application zone, with the labelling zone typically located between the two. A sample will thus migrate from the application zone into the labeling zone, where any biomarker in the sample binds to the label. Biomarker-binding agent complexes continue to migrate into the detection zone together with excess binding agent. When the biomarker-binding agent complex encounters the capture reagent, the complex is retained whilst the sample and excess binding agent continue to migrate. As biomarker levels in the sample increase, the amount of binding agent (in the form of biomarker-binding agent complex) retained in the detection zone increases proportionally.

In preferred embodiments, the device of the invention has the ability to distinguish between samples according to the threshold concentration. This can be achieved in various ways.

One type of device includes a reference zone that includes a signal of fixed intensity against which the amount of binding agent retained in the detection zone can be compared— when the signal in the detection zone equals the signal in the reference zone, the sample is a threshold sample; when the signal in the detection zone is less intense than the reference zone, the sample contains less biomarker than a threshold sample; when the signal in the detection zone is more intense than the reference zone, the sample contains more biomarker than a threshold sample.

A suitable reference zone can be prepared and calibrated without difficulty. For this type of device, the binding agent will generally be present in excess to biomarker in the sample, and the reference zone may be upstream or, preferably, downstream of the detection zone. The signal in the reference zone will be of the same type as the signal in the detection zone, i.e., they will typically both be visible to the naked eye, e.g., they will use the same tag. A preferred reference zone in a device of this type comprises immobilized protein (e.g., bovine serum albumin) which is tagged with colloidal gold.

In another device of the invention, the reference zone is downstream of the detection zone and includes a reagent which captures binding agent (e.g., an immobilized anti-binding agent antibody). Binding agent that flows through the device is not present in excess, but is at a concentration such that 50% of it is bound by a sample having biomarker at the threshold concentration. In a threshold sample, therefore, 50% of the binding agent will be retained in the detection zone and 50% in the reference zone. If the biomarker level in the sample is greater than in a threshold sample, less than 50% of the binding agent will reach the reference zone and the detection zone will give a more intense signal than the reference zone; conversely, if the biomarker level in the sample is less than in a threshold sample, less than 50% of the binding agent will be retained in the detection zone and the reference zone will give a more intense signal than the detection zone.

In another device of the invention which operates according to similar principles, the reference zone is downstream of the detection zone and includes a limiting amount of a reagent which captures binding agent (e.g., an immobilized anti-binding agent antibody). The reagent is present at a level such that it retains the same amount of label which would bind to the detection zone for a threshold sample, with excess label continuing to migrate beyond the reference zone.

In these three types of device, therefore, a comparison between the detection zone and the reference zone is used to compare the sample with the threshold concentration. The detectionireference binding ratio can preferably be determined by eye. Close juxtaposition of the detection and reference zones is preferred in order to facilitate visual comparison of the signal intensities in the two zones. In a fourth type of device, no reference zone is needed, but the detection zone is configured such that it gives an essentially on/off response, e.g., no signal is given below the threshold concentration but, at or above the threshold, signal is given.

In a fifth type of device, no reference zone is needed, but an external reference is used which corresponds to the threshold concentration. This can take various forms, e.g., a printed card against which the signal in the detection zone can be compared, or a machine reader which compares an absolute value measured in the detection zone (e.g., a calorimetric signal) against a reference value stored in the machine.

In some embodiments of the invention, the device includes a control zone downstream of the detection zone. This will generally be used to capture excess binding agent that passes through the detection and/or reference zones (e.g., using immobilized anti- binding agent antibody). When binding agent is retained at the control zone, this confirms that mobilization of the binding agent and migration through the device have both occurred. It will be appreciated that this function may be achieved by the reference zone.

In a preferred embodiment, the detection, reference and control zones are preferably formed on a nitrocellulose support.

Migration from the application zone to the detection zone will generally be assisted by a wick downstream of the detection zone to aid capillary movement. This wick is typically formed from absorbent material such as blotting or chromatography paper.

The device of the invention can be produced simply and cheaply, conveniently in the form of a dipstick. Furthermore, it can be used very easily, for instance by the home user. The invention thus provides a device which can be used as a screen for radiation exposure.

Kits for detecting ionizing radiation exposure, determination of absorbed dose, and time of exposure

The present invention also concerns kits comprising the required elements for detecting radiation exposure in a subject and, optionally, determining the absorbed dose of radiation, and the time of exposure. In one embodiment, the kits comprise one or more containers for collecting a blood sample or blood derived sample from a subject and an agent for detecting the presence of radiation biomarker polypeptides or nucleic acids encoding the polypeptides in the fluid. The radiation biomarker components of the kits can be packaged either in aqueous medium or in lyophilized form.

The methods of the invention can be carried out using a diagnostic kit for qualitatively or quantitatively detecting one or more radiation biomarkers of the invention in a sample such as blood. By way of example, the kit can contain binding agents (e.g., antibodies) specific for biomarkers of the invention, antibodies labeled with a detectable label or substance that can bind to the binding agents. If the detectable substance is an enzyme, then the kit can comprise a substrate for the enzyme. The kit can also contain a solid support (such as microtiter multi-well plates, nitrocellulose, etc.), standards, assay diluent, wash buffer, adhesive plate covers, and/or instructions for carrying out a method of the invention using the kit. In one embodiment, the kit includes one or more protease inhibitors (e.g., a protease inhibitor cocktail) and/or nuclease inhibitors to be applied to the biological sample to be assayed (such as blood).

Kits for detecting radiation exposure determining absorbed dose containing one or more agents that detect the biomarker polypeptides, such as but not limited to biomarker antibodies, fragments thereof, or biomarker binding partners, can be prepared. The agent(s) can be packaged with a container for collecting the biological sample from a patient. When the antibodies or binding partner are used in the kits in the form of conjugates in which a label is attached, such as a radioactive metal ion or a moiety, the components of such conjugates can be supplied either in fully conjugated form, in the form of intermediates, or as separate moieties to be conjugated by the user of the kit.

Kits containing one or more agents that detect biomarker nucleic acids, such as but not limited to the full length biomarker nucleic acid, biomarker oligonucleotides, and pairs of biomarker primers can also be prepared. The agent(s) can be packaged with a container for collecting biological samples from a patient. The nucleic acid can be in the labeled form or unlabeled form.

Other components of the kit may include, but are not limited to, means for collecting biological samples (e.g., lancets, needles), means for labeling the detecting agent (binding agent), means for immobilizing the biomarker protein or biomarker nucleic acid in the biological sample (e.g., support membranes, nitrocellulose, etc.), means for applying the biological sample to an immobilizing support means for binding the agent to the biomarker in the biological sample of a subject, a second antibody, a means for isolating total RNA or mRNA from a biological fluid of a subject, means for performing gel electrophoresis, means for generating cDNA from isolated total RNA or mRNA, means for performing hybridization assays, and means for performing PCR, etc.

Gene specific primers and methods of using same are described in U.S. Patent No. 5,994,076. In addition to the specific primers designed to selectively amplify the biomarker nucleic acids, the kits of the invention can also include reagents such as dNTPs (labeled or unlabeled) and/or rNTPs, buffers, enzymes, etc.

As used herein, the term "ELIS A" includes an enzyme-linked immunoabsorbent assay that employs an antibody or antigen bound to a solid phase and an enzyme-antigen or enzyme-antibody conjugate to detect and quantify the amount of an antigen {e.g., biomarker of the invention) or antibody present in a sample. A description of the ELISA technique is found in Chapter 22 of the 4^th Edition of Basic and Clinical Immunology by D.P. Sites et al, 1982, published by Lange Medical Publications of Los Altos, Calif, and in U.S. Patent Nos. 3,654,090; 3,850,752; and 4,016,043, the disclosures of which are herein incorporated by reference. ELISA is an assay that can be used to quantitate the amount of antigen, proteins, or other molecules of interest in a sample. In particular, ELISA can be carried out by attaching on a solid support (e.g., polyvinylchloride) an antibody specific for an antigen or protein of interest. Cell extract or other biological sample of interest such as blood can be added for formation of an antibody-antigen complex, and the extra, unbound sample is washed away. An enzyme-linked antibody, specific for a different site on the antigen is added. The support is washed to remove the unbound enzyme-linked second antibody. The enzyme-linked antibody can include, but is not limited to, alkaline phosphatase. The enzyme on the second antibody can convert an added colorless substrate into a colored product or can convert a non-fluorescent substrate into a fluorescent product. The ELISA-based assay method provided herein can be conducted in a single chamber or on an array of chambers and can be adapted for automated processes.

In these exemplary embodiments, the antibodies can be labeled with pairs of FRET dyes, bioluminescence resonance energy transfer (BRET) protein, fluorescent dye-quencher dye combinations, beta gal complementation assays protein fragments. The antibodies may participate in FRET, BRET, fluorescence quenching or beta-gal complementation to generate fluorescence, colorimetric or enhanced chemiluminescence (ECL) signals, for example.

These methods are routinely employed in the detection of antigen-specific antibody responses, and are well described in general immunology text books such as Immunology by Ivan Roitt, Jonathan Brostoff and David Male (London: Mosby, cl998. 5th ed. and Immunobiology: Immune System in Health and Disease/Charles A. Janeway and Paul Travers. Oxford: Blackwell Sci. Pub., 1994), the contents of which are herein incorporated by reference. Definitions

A "sample" (biological sample) can be any composition of matter of interest from a human or non-human subject, in any physical state {e.g., solid, liquid, semi-solid, vapor) and of any complexity. The sample can be any composition reasonably suspecting of containing one or more biomarkers of the invention that can be analyzed by the methods, devices, and kits of the invention. Preferably, the sample is a fluid (biological fluid such as blood). Samples can include human or animal samples. The sample may be contained within a test tube, culture vessel, multi-well plate, or any other container or supporting substrate. The sample can be, for example, a cell culture, human or animal tissue. Fluid homogenates of cellular tissues are biological fluids that may contain biomarkers for detection by the invention. Preferably, the sample is a blood sample or blood derived sample.

The "complexity" of a sample refers to the relative number of different molecular species that are present in the sample.

The terms "body fluid" and "bodily fluid", as used herein, refer to a composition obtained from a human or animal subject. Bodily fluids include, but are not limited to, whole blood, blood plasma, serum, urine, bladder wash, bladder barbotage specimen, tears, semen, saliva, sputum, exhaled breath, nasal secretions, pharyngeal exudates, bronchoalveolar lavage, tracheal aspirations, interstitial fluid, lymph fluid, meningal fluid, amniotic fluid, glandular fluid, feces, perspiration, mucous, vaginal or urethral secretion, cerebrospinal fluid, and transdermal exudate. Bodily fluid also includes experimentally separated fractions of all of the preceding solutions or mixtures containing homogenized solid material, such as feces, tissues, and biopsy samples.

The term "ex vivo," as used herein, refers to an environment outside of a subject. Accordingly, a sample of bodily fluid collected from a subject is an ex vivo sample of bodily fluid as contemplated by the subject invention. In-dwelling embodiments of the method and device of the invention obtain samples in vivo.

As used herein, the term "conjugate" refers to a compound comprising two or more molecules bound together, optionally through a linking group, to form a single structure. The binding can be made by a direct connection {e.g., a chemical bond) between the molecules or by use of a linking group.

As used herein, the terms solid "support", "substrate", and "surface" refer to a solid phase which is a porous or non-porous water insoluble material that can have any of a number of shapes, such as strip, rod, particle, beads, or multi-welled plate. In some embodiments, the support has a fixed organizational support matrix that preferably functions as an organization matrix, such as a microtiter tray. Solid support materials include, but are not limited to, cellulose, polysaccharide such as Sephadex, glass, polyacryloylmorpholide, silica, controlled pore glass (CPG), polystyrene, polystyrene/latex, polyethylene such as ultra high molecular weight polyethylene (UPE), polyamide, polyvinylidine fluoride (PVDF), polytetrafluoroethylene (PTFE; TEFLON), carboxyl modified Teflon, nylon, nitrocellulose, and metals and alloys such as gold, platinum and palladium. The solid support can be biological, non-biological, organic, inorganic, or a combination of any of these, existing as particles, strands, precipitates, gels, sheets, pads, cards, strips, dipsticks, test strips, tubing, spheres, containers, capillaries, pads, slices, films, plates, slides, etc., depending upon the particular application. Preferably, the solid support is planar in shape, to facilitate contact with a biological sample such as whole blood, plasma, serum, urine, peritoneal fluid, or ascites fluid. Other suitable solid support materials will be readily apparent to those of skill in the art. The solid support can be a membrane, with or without a backing (e.g., polystyrene or polyester card backing), such as those available from Millipore Corp. (Bedford, MA), e.g., Hi-Flow™ Plus membrane cards. The surface of the solid support may contain reactive groups, such as carboxyl, amino, hydroxyl, thiol, or the like for the attachment of nucleic acids, proteins, etc. Surfaces on the solid support will sometimes, though not always, be composed of the same material as the support. Thus, the surface can be composed of any of a wide variety of materials, such as polymers, plastics, resins, polysaccharides, silica or silica- based materials, carbon, metals, inorganic glasses, membranes, or any of the aforementioned support materials {e.g., as a layer or coating).

As used herein, the terms "label" and "tag" refer to substances that may confer a detectable signal, and include, but are not limited to, enzymes such as alkaline phosphatase, glucose-6-phosphate dehydrogenase, and horseradish peroxidase, ribozyme, a substrate for a replicase such as QB replicase, promoters, dyes, fluorescers, such as fluorescein, isothiocynate, rhodamine compounds, phycoerythrin, phycocyanin, allophycocyanin, o- phthaldehyde, and fluorescamine, chemiluminescers such as isoluminol, sensitizers, coenzymes, enzyme substrates, radiolabels, particles such as latex or carbon particles, liposomes, cells, etc., which may be further labeled with a dye, catalyst or other detectable group.

As used herein, the term "receptor" and "receptor protein" are used herein to indicate a biologically active proteinaceous molecule that specifically binds to (or with) other molecules such as biomarkers of the invention. As used herein, the term "ligand" refers to a molecule that contains a structural portion that is bound by specific interaction with a particular receptor protein.

As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions (fragments) of immunoglobulin molecules, i.e., molecules that contain an antibody combining site or paratope. The term is inclusive of monoclonal antibodies and polyclonal antibodies.

As used here, the terms "monoclonal antibody" or "monoclonal antibody composition" refer to an antibody molecule that contains only one species of antibody combining site capable of immunoreacting with a particular antigen. A monoclonal antibody composition thus typically displays a single binding affinity for any antigen with which it immunoreacts. A monoclonal antibody composition is typically composed of antibodies produced by clones of a single cell called a hybridoma that secretes (produces) only one type of antibody molecule. The hybridoma cell is formed by fusing an antibody-producing cell and a myeloma or other self-perpetuating cell line. Such antibodies were first described by Kohler and Milstein, Nature, 1975, 256:495-497, the disclosure of which is herein incorporated by reference. An exemplary hybridoma technology is described by Niman et al, Proc. Natl. Acad. Sci. U.S.A., 1983, 80:4949-4953. Other methods of producing monoclonal antibodies, a hybridoma cell, or a hybridoma cell culture are also well known. See e.g., Antibodies: A Laboratory Manual, Harlow et al, Cold Spring Harbor Laboratory, 1988; or the method of isolating monoclonal antibodies from an immunological repertoise as described by Sasatry, et al, Proc. Natl. Acad. Sci. USA, 1989, 86:5728-5732; and Huse et al, Science, 1981, 246:1275-1281. The references cited are hereby incorporated herein by reference.

As used herein, a semi-permeable membrane refers to a bio-compatible material which is impermeable to liquids and capable of allowing the transfer of gases through it. Such gases include, but are not limited to, oxygen, water vapor, and carbon dioxide. Semipermeable membranes are an example of a material that can be used to form a least a portion of an enclosure defining a flow chamber cavity. The semi-permeable membrane may be capable of excluding microbial contamination {e.g., the pore size is characteristically small enough to exclude the passage of microbes that can contaminate the analyte, such as cells). In a particular aspect, a semi-permeable membrane can have an optical transparency and clarity sufficient for permitting observation of an analyte, such as cells, for color, growth, size, morphology, imaging, and other purposes well known in the art. As used herein, the term "bind" refers to any physical attachment or close association, which may be permanent or temporary. The binding can result from hydrogen bonding, hydrophobic forces, van der Waals forces, covalent, or ionic bonding, for example.

As used herein, the term "particle" includes insoluble materials of any configuration, including, but not limited to, spherical, thread-like, brush-like, and irregular shapes. Particles can be porous with regular or random channels inside. Particles can be magnetic. Examples of particles include, but are not limited to, silica, cellulose, Sepharose beads, polystyrene (solid, porous, derivatized) beads, controlled-pore glass, gel beads, magnetic beads, sols, biological cells, subcellular particles, microorganisms (protozoans, bacteria, yeast, viruses, and other infectious agents), micelles, liposomes, cyclodextrins, and other insoluble materials.

A "coding sequence" or "coding region" is a polynucleotide sequence that is transcribed into mR A and/or translated into a polypeptide. For example, a coding sequence may encode a polypeptide of interest. The boundaries of the coding sequence are determined by a translation start codon at the 5'-terminus and a translation stop codon at the 3'-terminus. A coding sequence can include, but is not limited to, mRNA, cDNA, and recombinant polynucleotide sequences.

As used herein, the term "polypeptide" refers to any polymer comprising any number of two or more amino acids, and is used interchangeably herein with the terms "protein", "gene product", and "peptide".

As used herein, the term "nucleoside" refers to a molecule having a purine or pyrimidine base covalently linked to a ribose or deoxyribose sugar. Exemplary nucleosides include adenosine, guanosine, cytidine, uridine and thymidine.

The term "nucleotide" refers to a nucleoside having one or more phosphate groups joined in ester linkages to the sugar moiety. Exemplary nucleotides include nucleoside monophosphates, diphosphates and triphosphates.

The terms "polynucleotide", "nucleic acid molecule", and "nucleotide molecule" are used interchangeably herein and refer to a polymer of nucleotides joined together by a phosphodiester linkage between 5' and 3' carbon atoms. Polynucleotides can encode a polypeptide such as biomarker polypeptide (whether expressed or non-expressed), or may be short interfering RNA (siRNA), antisense nucleic acids (antisense oligonucleotides), aptamers, ribozymes (catalytic RNA), or triplex-forming oligonucleotides (i.e., antigene), for example. As used herein, the term "RNA" or "RNA molecule" or "ribonucleic acid molecule" refers generally to a polymer of ribonucleotides. The term "DNA" or "DNA molecule" or deoxyribonucleic acid molecule" refers generally to a polymer of deoxyribonucleotides. DNA and RNA molecules can be synthesized naturally (e.g., by DNA replication or transcription of DNA, respectively). RNA molecules can be post-transcriptionally modified. DNA and RNA molecules can also be chemically synthesized. DNA and RNA molecules can be single-stranded (i.e., ssRNA and ssDNA, respectively) or multi-stranded (e.g., double stranded, i.e., dsRNA and dsDNA, respectively). Based on the nature of the invention, however, the term "RNA" or "RNA molecule" or "ribonucleic acid molecule" can also refer to a polymer comprising primarily (i.e., greater than 80% or, preferably greater than 90%) ribonucleotides but optionally including at least one non-ribonucleotide molecule, for example, at least one deoxyribonucleotide and/or at least one nucleotide analog.

As used herein, the term "nucleotide analog" or "nucleic acid analog", also referred to herein as an altered nucleotide/nucleic acid or modified nucleotide/nucleic acid refers to a non-standard nucleotide, including non-naturally occurring ribonucleotides or deoxyribonucleotides. Preferred nucleotide analogs are modified at any position so as to alter certain chemical properties of the nucleotide yet retain the ability of the nucleotide analog to perform its intended function. For example, locked nucleic acids (LNA) are a class of nucleotide analogs possessing very high affinity and excellent specificity toward complementary DNA and RNA. LNA oligonucleotides have been applied as antisense molecules both in vitro and in vivo (Jepsen J.S. et ah, Oligonucleotides, 2004, 14(2): 130- 146).

As used herein, the term "RNA analog" refers to a polynucleotide (e.g., a chemically synthesized polynucleotide) having at least one altered or modified nucleotide as compared to a corresponding unaltered or unmodified RNA but retaining the same or similar nature or function as the corresponding unaltered or unmodified RNA. As discussed above, the oligonucleotides may be linked with linkages which result in a lower rate of hydrolysis of the RNA analog as compared to an RNA molecule with phosphodiester linkages. Exemplary RNA analogues include sugar- and/or backbone-modified ribonucleotides and/or deoxyribonucleotides. Such alterations or modifications can further include addition of non- nucleotide material, such as to the end(s) of the RNA or internally (at one or more nucleotides of the RNA).

As used herein, the terms "radiation", "ionizing radiation", and "IR" refer to electromagnetic radiation (e.g., x-rays and gamma rays) and/or particulate radiation (e.g., alpha, beta+, and beta-). Gamma-rays have the highest energy of any of these and are emitted from certain radio-isotopes that may be released from a dirty bomb, nuclear power plant accident, or nuclear bomb. While these radio-isotopes may release other forms of IR, gamma-rays are able to penetrate skin and travel long distances, therefore affecting more people and causing more DNA damage than other types of radiation. It is considered the primary hazard to humans during a radiological emergency.

The terms "comprising", "consisting of and "consisting essentially of are defined according to their standard meaning. The terms may be substituted for one another throughout the instant application in order to attach the specific meaning associated with each term.

The terms "isolated" or "biologically pure" refer to material that is substantially or essentially free from components which normally accompany the material as it is found in its native state.

As used in this specification, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a biomarker" includes more than one such biomarker. A reference to "an antibody" includes more than one such antibody. A reference to "a molecule" includes more than one such molecule, and so forth.

The practice of the present invention can employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA technology, electrophysiology, and pharmacology that are within the skill of the art. Such techniques are explained fully in the literature (see, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989); DNA Cloning, Vols. I and II (D. N. Glover Ed. 1985); Perbal, B., A Practical Guide to Molecular Cloning (1984); the series, Methods In Enzymology (S. Colowick and N. Kaplan Eds., Academic Press, Inc.); Transcription and Translation (Hames et al. Eds. 1984); Gene Transfer Vectors For Mammalian Cells (J. H. Miller et al. Eds. (1987) Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.); Scopes, Protein Purification: Principles and Practice (2nd ed., Springer- Verlag); and PCR: A Practical Approach (McPherson et al. Eds. (1991) IRL Press)), each of which are incorporated herein by reference in their entirety.

Following are examples that illustrate materials, methods, and procedures for practicing the invention. The examples are illustrative and should not be construed as limiting. MATERIALS AND METHODS

Patients and Volunteers. Informed consent for study participation was obtained from four pediatric cancer patients at the Children's Hospital of Birmingham and nine healthy volunteers according to guidelines set forth by the Institutional Review Board (IRB) at the University of Alabama at Birmingham (UAB). All studies were approved by the IRB at UAB Cancer patients used in this study were diagnosed with Acute Lymphocytic Leukemia (ALL) and were undergoing myeloablative therapy with total body irradiation (TBI). None of these patients were being treated with chemotherapy during the course of TBI. All patients received 2 Gy twice a day for three consecutive days (total dose of 12 Gy). Whole blood was collected prior to irradiation (baseline) and at approximately 5, 23 and 48 hours after the first fraction of TBI. Whole blood collected from nine healthy volunteers served as control samples.

Irradiation of C57BL6 Mouse. C57BL6 mice were obtained from NCI-Frederick (Frederick, MD) and were allowed to acclimate for one to two weeks prior to treatment. 8- week old mice were anesthetized with a ketamine/xylazine mixture (dose of 85 mg/kg ketamine; 10 mg/kg xylazine) followed by sham or total body irradiation using a ⁶⁰Co Teletherapy unit (Picker, Cleveland OH). Mice were euthanized via exsanguination and cervical dislocation at 0, 5, 12, 23, or 48 hours following irradiation. All experiments were approved and conducted under guidelines set forth by the UAB Institutional Animal Care and Use Committee.

A total of 166 mice were included in a training set that was used for development of all statistical models. The training set was comprised of 70 controls (non-irradiated) as well as 8 mice per treatment group (1, 2, or 6 Gy) per time point (5, 12, 23, and 48 hours post- TBI). A total of 45 mice were utilized as an independent validation set for testing statistical models developed in the training set. The validation set contained 9 control (non-irradiated) mice and 3-4 mice per treatment group (1, 2, 4, 6, or 8 Gy) at two time points (12 or 48 hours).

Blood Collection and RNA Isolation. Whole blood samples were collected from four patients and nine healthy volunteers via venipuncture. Mouse blood was collected through intra-cardiac puncture. All blood samples were drawn directly into PAXgene Blood RNA tubes (PreAnalytiX by BD/Qiagen, Franklin Lakes NJ) and total RNA was isolated with the PAXgene Blood RNA kit (Qiagen, Valencia CA). All samples underwent on-column DNA digestion using the Qiagen RNase-free Dnase set (Qiagen, Valencia CA) according to manufacturer's instructions. RNA was reverse transcribed to cDNA with the High Capacity cDNA Archive kit (Applied Biosystems, Foster City CA) following manufacturer's instructions.

Real-time quantitative Low Density Array (RTQ-LDA) design. To identify potential biodosimetry genes in humans exposed to known amounts of radiation, a rationally-designed RTQ-LDA was utilized to examine differences in expression of 46 independent genes in blood samples collected from healthy volunteers and from ALL patients at baseline (prior to irradiation) and at 5, 23, and 48 hours following the first fraction of TBI. Selection of these genes ensued after an earlier study conducted by the inventors' laboratory which identified seventeen potential biodosimetry genes in blood samples collected from a separate set of six pediatric ALL patients undergoing treatment with TBI (unpublished results). The remaining twenty-nine genes were included because they met one of the following criteria: 1) reported as radiation responsive by at least two independent investigators; 2) demonstrated radiation response in multiple models (in vivo, in vitro or ex vivo); 3) identified in array studies of TBI cancer patients. The complete RTQ-LDA also included two housekeeping genes, RPLPO and 18S, for a total of 48 independent genes (Table IB). These 48 genes were formatted onto a RTQ-LDA (Applied Biosystems, Foster City CA) as previously described by the inventors' laboratory (33, 34).

Real-time Quantitative PCR (RTQ). Genes identified as potential biodosimetry genes from RTQ-LDA analysis in patients were analyzed via single gene RTQ in blood samples from mice. Individual RTQ primer probe sets for mouse Acta2, Bbc3, Ccngl, Cdknla, Gadd45a, Mdk, Serpinel, and Tnfrsf10b were obtained from Applied Biosystems (Foster City CA). PCR amplification for both RTQ-LDA and single gene RTQ analysis was performed on an ABI 7900HT Sequence Detection System. Cycling conditions for RTQ- LDA analysis were as follows: 50°C for 2 minutes and 94.5°C for 10 minutes followed by 40 cycles at 97°C for 15 seconds and 59.7°C for 1 minute. Cycling conditions for single-gene RTQ were as follows: 50°C for 2 minutes and 95°C for 10 minutes followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. All single-gene RTQ reactions, including no template controls, were performed in triplicate and samples were normalized to the mouse Rps9 housekeeping gene. Expression values for all analyses were calculated as previously described (33-36).

Statistical Analysis. The overall approach in the current study was to identify potential biodosimetry genes in patients exposed to TBI and further evaluate these genes in a murine model in which dose of exposure and time of sample collection could be varied. Due to the limited number of patients included in this study (n=4), p-values resulting from statistical testing of patient gene expression were not considered definitive and the following filtering criteria were applied. Genes with no detectable expression (C_t=40) or with only marginal changes in expression following exposure to radiation (<3 standard deviations from average baseline expression) were not examined in the mouse model. In addition, genes whose expression varied (>3 standard deviations) between healthy volunteers and average expression in baseline patient samples were not further examined due to a potential association with cancer diagnosis and/or treatment. Those genes remaining after application of these filtering criteria were further analyzed in mice.

Prior to single-gene RTQ analysis in irradiated mice, a power analysis was performed to ensure the ability to detect statistically significant changes in gene expression. Using a two-sided two-group t-test of equal mean and significance level of 0.05, it was determined that 8 mice per experimental group was sufficient to detect an effect size of 1.5 to 2 standard deviations. Significant differences in mouse gene expression were determined at each time point with One-way ANOVA analysis followed by Tukey's Multiple Comparison Test. Dose-dependent changes in mouse gene expression were assessed with linear regression analysis. All statistical analyses of mouse gene expression values were performed in GraphPad Prism Software with p-values <0.05 considered significant. Only genes whose expression changed significantly following irradiation in mice were included in class prediction and multiple linear regression analyses.

Using expression data from the mouse training set, a weighted voting algorithm (37,

38) was utilized to develop a class prediction model at each time point capable of distinguishing irradiated from non-irradiated mice. Non-irradiated and irradiated mice were defined as classes 0 and 1, respectively. Genes whose expression best distinguished these two classes were identified based on the signal-to-noise statistic defined as: S_x= (μ_class0- c_lass1/s _class0+s _class1), where μ=mean expression and s=standard deviation. Thereafter, the weighted voting classification algorithm was applied. Briefly, each gene in the class prediction model casts a vote for either class 0 or 1 depending on whether the expression level in the sample is closer to the mean of class 0 or 1. The weight of the vote (V) reflects the deviation of the sample expression level (X) from the mean of each class: V= |X-( μ_class0+ μ_class1)/2|. The votes are summed to obtain the total number of votes for class 0 and class 1 with the class with the most votes deemed as the winning class. The confidence of the prediction of the winning class was calculated as (V_win-V_lose)/(V_win+V_lose). Using leave-one- out cross-validation, the number of class prediction errors in the training set was determined using different combinations of genes to identify relevant marker genes capable of distinguishing the two classes with the highest accuracy. These molecular class prediction profiles were then applied to expression data obtained from an independent validation set of 45 mice. Prior to analyses, gene expression data in the validation set was corrected based on the medians of each set. Weighted voting analyses were performed using GenePattern software available at http://www.broadinstitute.org.

Development of a continuous model capable of predicting dose of radiation exposure was performed using multiple linear regression analysis (39). Modeling was performed using all possible combinations of independent variables and the r-squared statistic and their associated p-values were used to determine the global fit of all models. Optimal models developed using data obtained from the training set were utilized to determine the predicted dose of exposure based on gene expression data within the validation set of mice.

EXAMPLE 1— PATIENT TREATMENT DEMOGRAPHICS Initial studies were conducted on blood samples obtained from four pediatric patients diagnosed with Acute Lymphocytic Leukemia (ALL) undergoing fractionated therapy with TBI prior to bone marrow transplantation (treatment schema illustrated in Figure 1). Blood collection times corresponded with previously scheduled time points used to monitor white blood cell counts. Sufficient RNA for expression analysis could not be isolated from blood samples collected after the 48-hour time point. As shown in Table 1A, white blood cell counts varied prior to TBI and decreased dramatically following completion of myeloablative therapy.

EXAMPLE 2— RTQ-LDA ANALYSIS IN PATIENTS UNDERGOING TOTAL BODY

IRRADIATION

To identify potential biodosimetry genes, RTQ-LDA analysis was performed in blood samples collected from four ALL patients prior to and following TBI. Nine healthy volunteers were also analyzed to examine variation in gene expression in a disease free population. Of the forty-eight genes represented on the RTQ-LDA (forty-six target and two housekeeping genes), the expression of nineteen target genes fell below the limits of detection (Footnote³ in Table IB). Seven genes demonstrated variability (greater than 3 standard deviations) between baseline patient samples and healthy volunteers (Footnote^b in Table IB) suggesting that the expression of these genes may be altered by disease state or prior treatment for cancer. Eight genes (bolded in Table IB) demonstrated increased expression in irradiated patient samples compared to baseline patient samples (Figure 2). Linear regression analysis demonstrated a significant correlation (p<0.05) between time (post-TBI) and mRNA levels of CDKN1A, MDK, SERPINE1, and TNFRSF10B (Table 2). Due to the fractionated nature of TBI therapy, it could not be determined if the observed changes in gene expression resulted from the accumulating dose of radiation or from increasing time after exposure. To elucidate the effects of radiation dose and time from exposure, these eight genes were further evaluated in an irradiated murine model.

Table 1C lists the 8 genes of Table 2, in humans and mice, including National Center for Biotechnology Information (NCBI) accession numbers (25 sequences, corresponding to SEQ ID Os:l-25).

EXAMPLE 3— GENE EXPRESSION ANALYSIS FOLLOWING TOTAL BODY

IRRADIATION OF C57BL6 MICE

Expression analysis of the mouse homologs of the eight genes identified in patient samples was performed in C57BL6 mice using single-gene RTQ. One gene (Mdk) demonstrated no detectable expression. Two genes (Acta2 and Gadd45a) showed no significant increase in expression between non-irradiated and irradiated mice at any time point (data not shown). Five genes (Bbc3, Ccngl, Cdknla, Serpinel, and Tnfrsf10b) demonstrated increased expression in irradiated mice compared to non-irradiated controls (Figure 3). Stable baseline expression (no significant variation) was observed for all five genes in non-irradiated mice over the 48 hours evaluated.

Four genes (Bbc3, Ccngl, Cdknla, and Tnfrsf10b) demonstrated significant differences in expression between non-irradiated mice and at least one irradiated group (1, 2, or 6 Gy) at every time point. Expression of Serpinel in mice was significantly increased 12 hours after exposure to 6 Gy and at 48 hours in both the2 Gy and 6 Gy exposure groups. While significant increases in expression were observed for most genes at early time points, dose-dependence was more pronounced with increasing time post-exposure. As shown in Table 3, linear regression analysis demonstrated a significant correlation (p<0.05) between dose and expression levels of Bbc3, Ccngl, Cdknl a, and Serpinel at 48 hours post- irradiation. Collectively, these data suggests that these five genes may be useful for determining exposure status (non-irradiated vs. irradiated) at earlier time points and level of exposure (doses) at 48 hours. EXAMPLE — DETERMINING EXPOSURE STATUS (NON-IRRADIATED vs.

IRRADIATED) IN C57BL6 MICE

Using the gene expression data obtained from mice in the training set (Figure 3), we employed a weighted voting algorithm to develop class prediction models at each time point that could distinguish non-irradiated from irradiated mice. Mice were grouped into either class 0 (non-irradiated) or class 1 (irradiated) based on their exposure status. Because there was no significant difference in expression between the non-irradiated groups, all seventy non-irradiated mice (n=14 per time point) were included in class 0 regardless of time of sample collection. Mice exposed to 1 , 2, or 6 Gy TBI were grouped into class 1 for a total of twenty-four irradiated mice (n=8 per dose) at each independent time point. All five candidate predictor genes (Bbc3, Ccngl, Cdknla, Serpinel, and Tnfrsfl0b) were evaluated independently and in combination to identify the optimal profile that could discriminate non- irradiated from irradiated mice at each time point. Highest accuracy was obtained using unique class prediction models comprised of one or two genes per profile. While all three profiles (Ccngl at 5 and 12 hours; Bbc3 and Ccngl at 23 hours; and Ccngl and Cdknla at 48 hours) contained Ccngl, none of the four class prediction signatures included Serpinel. Internal examination of this training set via leave-one-out cross validation demonstrated 94.7, 93.6, 95.7, and 92.6% accuracy in correctly classifying mice as non-irradiated or irradiated at 5, 12, 23, or 48 hours, respectively (Table 5). Specificity for all models was =97.1% with no more than two false positives at any time point. One mouse was misclassified as irradiated using the prediction models developed at all four time points indicating abnormally high expression levels. As expected, the sensitivity of each class prediction model increased with increasing dose. There were no more than five false negatives at any time point. Further, all mice in the 6 Gy treatment group were correctly classified as irradiated at 12, 23, and 48 hours post-irradiation. Overall, these data demonstrate that increased expression of these genes is indicative of radiation exposure and mice exposed to higher doses of irradiation can be correctly classified with higher accuracy. Results are summarized in the upper panel of Table 4.

Class prediction models developed at 12 and 48 hours using gene expression data from the mouse training set were independently tested in a separate validation set of mice. This validation set included mice irradiated with 1, 2, 4, 6, or 8 Gy (n=3 or 4 per time point) and nine non-irradiated mice. These mice were classified into corresponding exposure groups with 92.6 and 96.3% accuracy at 12 and 48 hours post-TBI, respectively (Table 5). There were no false positives using either class prediction model. Importantly, the two mice misclassified as non-irradiated at 12 hours were in the 1 or 2 Gy exposure groups. At 48 hours, one mouse in the 1 Gy exposure group was incorrectly classified but overall sensitivity remained at 94.4%. This data again demonstrates that the sensitivity to predict exposure status increases with dose. Results are summarized in the lower panel of Table 4.

EXAMPLE 5— DETERMINING DOSE IN C57BL6 MICE Expression of all five genes demonstrated a significant linear correlation with dose at 48 hours post-irradiation (Table 3) suggesting the potential to develop a continuous model capable of determining dose of exposure at this time point. Multiple linear regression analysis was performed using expression data obtained from mice in the training set. All possible combinations of genes were used to determine the model that was most capable of estimating dose of exposure. The model with the highest adjusted r-squared value was chosen for validation.

Multiple linear regression analysis of gene expression data from the mouse training set revealed that the model defined by expression of Ccngl and Cdknla had the highest adjusted r-squared value. The regression model for this data is predicted dose= - 0.7674 + 0.6343 * [Ccngl] + 0.0804 * [Cdknl a] (adjusted r²=0.8400, p<0.0001). This model was initially validated within the training set to determine predicted dose based on expression of Ccngl and Cdknla. The relationship of actual dose versus predicted dose is visualized in Figure 4A. There was a significant linear correlation between these two parameters ( r²=.8487, p<0.0001). The average predicted doses for the 0, 1, 2, and 6 Gy treatment groups in the training set were 0.1±0.5, 1.2±0.3, 2.3±0.5, 5.3±1.6 Gy, respectively (upper panel of Table 5).

This regression model was independently tested for its ability to predict dose using the gene expression data obtained from the validation set. Importantly, this set contained two additional treatment groups (4 and 8 Gy) to test the boundaries of this continuous model. As shown in Figure 4B, there was a significant linear correlation between actual dose and predicted dose (1^=0.7113, p<0.0001). The average predicted doses for the 0, 1 , 2, 4, 6, and 8 Gy treatment groups were 0.0±0.2, 1.6±1.0, 2.9±1.4, 5.1±2.0, 5.3±0.7, and 10.5±5.6 Gy, respectively (lower panel of Table 5). Variability in the predicted dose was greatest in the 8 Gy treatment group. Overall, the multiple linear regression model slightly overestimated dose.

Currently, chromosome aberration cytogenetics remains the only validated method capable of retrospective radiation dose assessment determined directly from biological samples. While advances have been made to automate this method (40), it remains both time- and labor-intensive and would not be amenable to mass casualty situations. Recent microarray studies examining the transcriptional response to ionizing radiation have identified potential molecular markers of radiation exposure. With this in mind, we sought to develop statistical models of gene expression with the ability to not only distinguish non- irradiated from irradiated individuals, but also estimate the actual dose of exposure.

Unique to this study, a "reverse-translational" approach was utilized whereby we initially identified potential biodosimetry genes in human samples prior to assessment of dose-dependent radiation responses in other experimental models. Further, gene expression analysis was performed utilizing a rationally-designed RTQ-LDA comprised of genes previously characterized as responsive to ionizing radiation by other investigators in combination with genes we identified in our preliminary studies (Table 1). In the current study, eight potential biodosimetry genes were identified from the whole blood of four cancer patients undergoing treatment with TBI (Figure 2). Interestingly, this is the first report to our knowledge demonstrating radiation response of four genes (CCNG1, MDK, SERPINE1, and TNFRSF10B) in human blood.

While analysis of transcriptional response to radiation in human subjects is imperative for validation of biodosimetry genes, the population of individuals exposed to known amounts of radiation is very limited. Further, the effects of single acute doses of radiation and dose-dependent responses cannot be assessed in patients undergoing stringent treatment protocols, which include fractionated radiotherapy. Although in vitro cell lines and ex vivo whole blood models could be used to examine mRNA profiles after irradiation, these models lack the endogenous cellular microenvironment which may influence the overall transcriptional response. Therefore, we elected to assess dose-dependent changes in gene expression in C57BL6 mice, a strain of mice with intermediate radiation sensitivity compared to radiosensitive BALB/cJ and radioresistant 128/J mice (41). Mice were irradiated with doses between 1 Gy and 8 Gy, representing exposures that would initiate varying severities of acute radiation syndrome³. Of the eight radiation responsive genes identified in patients, expression of Bbc3, Ccngl, Cdknla, Serpinel, and Tnfrsf10b (Figure 3) increased significantly in irradiated mice (p<0.05). Most of these genes are involved in apoptosis (Bbc3 and Tnfrsf10b) or cell cycle regulation (Ccngl and Cdknla) and have very well characterized radiation responses. However, while Serpinel mRNA and protein levels have been shown to increase in numerous tissue types after ionizing radiation (42-45), its response in whole blood has never been characterized. It has been suggested that increased SERPINE1 levels may contribute to more efficient thrombus formation following vascular injury (46), which may be important following exposure to ionizing radiation.

To determine if changes in gene expression are capable of qualitative (non- irradiated/irradiated) and/or quantitative (dose) assessment of radiation exposure, class prediction (weighted voting) and multiple linear regression models were developed at each time point. Using data obtained from a training set of mice, we developed class prediction models (using the expression of Ccngl and Cdknla) capable of distinguishing non-irradiated from irradiated mice in a separate validation set with an accuracy, specificity and sensitivity of 96.3, 100.0 and 94.4%, respectively, at 48 hours (Table 5). Applying this model back to the original set of human samples (non-irradiated controls and irradiated cancer patients) demonstrates an impressive accuracy, specificity and sensitivity of 93.7, 92.3 and 100%, respectively at 48 hours. Collectively, these data suggest that gene expression analysis may facilitate rapid identification of exposed individuals in need of further evaluation through other dosimetry methods, such as monitoring of clinical symptoms or cytogenetic analysis.

The significant linear dose-dependent increase in the expression of all five genes

(Bbc3, Ccngl, Cdknla, Serpinel, and Tnfrsfl Ob) at 48 hours post-irradiation (Table 3) suggested that a continuous model could be developed to predict radiation dose. In the training set of mice, a multiple linear regression model capable of predicting dose of exposure was developed using the expression of Ccngl and Cdknl a (predicted dose= - 0.7674 + 0.6343 * [Ccngl] + 0.0804 * [Cdknla]). Examination of this model in an independent validation set of mice predicted doses for the 0, 1, 2, 4, 6, and 8 Gy treatment groups as 0.0±0.2, 1.6±1.0, 2.9±1.4, 5.1±2.0, 5.3±0.7, and 10.5±5.6 Gy, respectively. This model could not be directly applied to predict dose in the original set of human samples (non- irradiated controls and irradiated cancer patients) due to the fractionated nature of the radiation therapy administered. However, these studies provide a "proof of principle" suggesting that gene expression may be used for determining dose of radiation exposure.

In conclusion, the current study: 1.) identified biodosimetry genes in cancer patients whose expression changed with exposure to radiation; 2.) utilized these genes in an in vivo mouse model to develop classification models capable of distinguishing non-irradiated from irradiated individuals and 3.) utilized a continuous regression model to demonstrate that estimation of dose of exposure was possible. These studies show that incorporating gene expression analysis into current dosimetry protocols may facilitate qualitative (non- irradiated/irradiated) and quantitative (dose) assessment of radiation exposure. Even more, incorporating the analysis of protein levels of these gen es may be used in protein assays that could potentially be self-administered.

All patents, patent applications, provisional applications, and publications referred to or cited herein, supra or infra, are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application.

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REFERENCES

1. N. Dainiak, P. Berger, and J. Albanese, Relevance and feasibility of multi-parameter assessment for management of mass casualties from a radiological event Exp Hematol, 35, 17-23 (2007).

2. I. Turai, K. Veress, B. Gunalp, and G. Souchkevitch, Medical response to radiation incidents and radionuclear threats. Bmj, 328, 568-572 (2004).

3. J.K. Waselenko, T.J. MacVittie, W.F. Blakely, N. Pesik, A.L. Wiley, W.E. Dickerson, H. Tsu, D.L. Confer, C.N. Coleman, and N. Dainiak, Medical management of the acute radiation syndrome: recommendations of the Strategic National Stockpile Radiation Working Group. Ann Intern Med, 140, 1037-1051 (2004).

4. E. Hall and A. Giaccia, DNA Strand Breaks and Chromosomal Aberrations, in Radiobiology for the Radiologist. 2006, Lippincott Williams and Wilkins: Philadelphia, PA. p. 16-29. 5. M. Shrivastav, L.P. De Haro, and J. A. Nickoloff, Regulation of DNA double-strand break repair pathway choice. Cell Res, 18, 134-147 (2008).

6. M.A. Bender and P.C. Gooch, Somatic chromosome aberrations induced by human whole-body irradiation: the "Recuplex" criticality accident Radiat Res, 29, 568-582 (1966).

7. B.N. Ford, D. Wilkinson, E.M. Thorleifson, and B.L. Tracy, Gene expression responses in lymphoblastoid cells after radiation exposure. Radiat Res, 156, 668-671 (2001).

8. S.A. Amundson, M. Bittner, Y. Chen, J. Trent, P. Meltzer. and A J. Fornace, Jr., Fluorescent cDNA microarray hybridization reveals complexity and heterogeneity of cellular genotoxic stress responses. Oncogene, 18, 3666-3672 (1999).

9. H.P. Wang, X.H. Long, Z.Z. Sun, O. Rigaud, Q.Z. Xu, Y.C. Huang, J.L. Sui, B. Bai, and P.K. Zhou, Identification of differentially transcribed genes in human lymphoblastoid cells irradiated with 0.5 Gy of gamma-ray and the involvement of low dose radiation inducible CHD6 gene in cell proliferation and radiosensitivity. Int J Radiat Biol, 82, 181-190 (2006).

10. G.S. Akerman, B.A. Rosenzweig, O.E. Domon, C.A. Tsai, M.E. Bishop, L.J.

McGarrity, J.T. Macgregor, F.D. Sistare, J.J. Chen, and S.M. Morris, Alterations in gene expression profiles and the DNA-damage response in ionizing radiation-exposed TK6 cells. Environ Mol Mutagen, 45, 188-205 (2005).

1 1. . Y. Jen and V.G. Cheung, Identification of novel p53 target genes in ionizing radiation response. Cancer Res, 65, 7666-7673 (2005).

12. O. Zschenker, K. Borgmann, T. Streichert, I. Meier, A. Wrona, and E. Dikomey, Lymphoblastoid cell lines differing in p53 status show clear differences in basal gene expression with minor changes after irradiation. Radiother Oncol, 80, 236-249 (2006).

13. K.Y. Jen and V.G. Cheung, Transcriptional response of lymphoblastoid cells to ionizing radiation. Genome Res, 13, 2092-2100 (2003).

14. S.A. Amundson, R.A. Lee, C.A. Koch-Paiz, M.L. Bittner, P. Meltzer, J.M. Trent, and A.J. Fornace, Jr., Differential responses of stress genes to low dose-rate gamma irradiation. Mol Cancer Res, 1, 445-452 (2003).

15. S.A. Amundson, M. Bittner, and A.J. Fornace, Jr., Functional genomics as a window on radiation stress signaling. Oncogene, 22, 5828-5833 (2003).

16. V.G. Tusher, R. Tibshirani, and G. Chu, Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci U SA, 98, 5116-5121 (2001).

17. CM. Kang, K.P. Park, J.E. Song, D.I. Jeoung, C.K. Cho, T.H. Kim, S. Bae, S.J. Lee, and Y.S. Lee, Possible biomarkers for ionizing radiation exposure in human peripheral blood lymphocytes. Radiat Res, 159, 312-319 (2003).

18. R. Lin, Y. Sun, C. Li, C. Xie, and S. Wang, Identification of differentially expressed genes in human lymphoblastoid cells exposed to irradiation and suppression of radiation- induced apoptosis with antisense oligonucleotides against caspase-4. Oligonucleotides, 17, 314-326 (2007).

19. X.H. Long, Z.Q. Zhao, X.P. He, H.P. Wang, Q.Z. Xu, J. An, B. Bai, J.L. Sui, and P.K. Zhou, Dose-dependent expression changes of early response genes to ionizing radiation in human lymphoblastoid cells. M J Mol Med, 19, 607-615 (2007).

20. S.A. Amundson, K.T. Do, L.C. Vinikoor, R.A. Lee, C.A. Koch-Paiz, J. Ahn, M.

Reimers, Y. Chen, D.A. Scudiero, and A.J. Fornace, Jr., Integrating global gene expression and radiation survival parameters across the 60 cell lines of the National Cancer Institute Anticancer Drug Screen. Cancer Res, 68, 415-424 (2008). 21. G. Gruel, C. Lucchesi, A. Pawlik, V. Frouin, O. Alibert, T. Kortulewski, A. Zarour, B. Jacquelin, X. Gidrol, and D. Tronik-Le Roux, Novel microarray-based method for estimating exposure to ionizing radiation. Radiat Res, 166, 746-756 (2006).

22. H.K. Dressman, G.G. Muramoto, N.J. Chao, S. Meadows, D. Marshall, G.S.

Ginsburg, J.R. Nevins, and J.P. Chute, Gene expression signatures that predict radiation exposure in mice and humans. PLoS Med, 4, el 06 (2007).

23. W.Y. Park, C.I. Hwang, C.N. Im, M.J. Kang, J.H. Woo, J.H. Kim, Y.S. Kim, J.H. Kim, H. Kim, and J.S. Seo, Identification of radiation-specific responses from gene expression profile. Oncogene, 21, 8521-8528 (2002).

24. E. Kis, T. Szatmari, M. Keszei, R. Farkas, O. Esik, K. Lumniczky, A. Falus, and G.

Safrany, Microarray analysis of radiation response genes in primary human fibroblasts. Int J Radiat Oncol Biol Phys, 66, 1506-1514 (2006).

25. M. Mori, M.A. Benotmane, I. Tirone, E.L. Hooghe-Peters, and C. Desaintes, Transcriptional response to ionizing radiation in lymphocyte subsets. Cell Mol Life Sci, 62, 1489-1501 (2005).

26. S.A. Amundson, K.T. Do, S. Shahab, M. Bittner, P. Meltzer, J. Trent, and A.J.

Fornace, Jr., Identification of potential mRNA biomarkers in peripheral blood lymphocytes for human exposure to ionizing radiation. Radiat Res, 154, 342-346 (2000).

27. A. Turtoi, I. Brown, D. Oskamp, and F.H. Schneeweiss, Early gene expression in human lymphocytes after gamma-irradiation-a genetic pattern with potential for

biodosimetry. Int J Radiat Biol, 84, 375-387 (2008).

28. S. Paul and S.A. Amundson, Development of gene expression signatures for practical radiation biodosimetry. Int J Radiat Oncol Biol Phys, 71, 1236-1244 (2008).

29. S.A. Amundson, M.B. Grace, C.B. McLeland, M.W. Epperly, A. Yeager, Q. Zhan, J.S. Greenberger, and A.J. Fornace, Jr., Human in vivo radiation-induced biomarkers: gene expression changes in radiotherapy patients. Cancer Res, 64, 6368-6371 (2004).

30. M.B. Grace, C.A. Salter, J.R. Bullard, P.G. Prasanna, G.L. Manglapus, and W.F. Blakely. Gene-Expression Biomarkers for Application to High-Throughput Radiation Biodosimetry. in NATO Human Factors and Medicine Panel Research Task Group 099 "Radiation Bioe fects and Countermeasures". 2005. Bethesda, Maryland. 31. M.B. Grace, C.B. McLeland, and W.F. Blakely, Real-time quantitative RT-PCR assay of GADD45 gene expression changes as a biomarker for radiation biodosimetry. IntJ Radiat Biol, 78, 1011-1021 (2002).

32. K. Bishay, K. Ory, M.F. Olivier, J. Lebeau, C. Levalois, and S. Chevillard, DNA damage-related RNA expression to assess individual sensitivity to ionizing radiation.

Carcinogenesis, 22, 1179-1183 (2001).

33. A. Steg, S.M. Vickers, M. Eloubeidi, W. Wang, LA. Eltoum, W.E. Grizzle, M.W. Saif, A.F. Lobuglio, A.R. Frost, and M.R. Johnson, Hedgehog pathway expression in heterogeneous pancreatic adenocarcinoma: implications for the molecular analysis of clinically available biopsies. Diagn Mol Pathol, 16, 229-237 (2007).

34. A. Steg, W. Wang, C. Blanquicett, J.M. Grunda, I.A. Eltoum, K. Wang, D.J.

Buchsbaum, S.M. Vickers, S. Russo, and M.R. Johnson, Multiple gene expression analyses in paraffin-embedded tissues by TaqMan low-density array: Application to hedgehog and Wnt pathway analysis in ovarian endometrioid adenocarcinoma. J Mol Diagn, 8, 76-83 (2006). 35. C. Blanquicett, M.R. Johnson, M. Heslin, and R.B. Diasio, Housekeeping gene variability in normal and carcinomatous colorectal and liver tissues: applications in pharmacogenomic gene expression studies. Anal Biochem, 303, 209-214 (2002).

36. M.R. Johnson, K. Wang, J.B. Smith, M.J. Heslin, and R.B. Diasio, Quantitation of dihydropyrimidine dehydrogenase expression by real-time reverse transcription polymerase chain reaction. Anal Biochem, 278, 175-184 (2000).

37. T.R. Golub, D.K. Slonim, P. Tamayo, C. Huard, M. Gaasenbeek, J.P. Mesirov, H. Coller, M.L. Loh, J.R. Downing, and E.S. Lander, Molecular classification of cancer: class discovery and class prediction by gene expression monitoring. Science, 286, 531-537 (1999).

38. S. Ramaswamy, K.N. Ross, E.S. Lander, and T.R. Golub, A molecular signature of metastasis in primary solid tumors. Nat Genet, 33, 49-54 (2003).

39. L.E. Eberly, Multiple linear regression. Methods Mol Biol, 404, 165-187 (2007).

40. P.R. Martin, R.E. Berdychevski, U. Subramanian, W.F. Blakely, and P.G. Prasanna, Sample Tracking in an Automated Cytogenetic Biodosimetry Laboratory for Radiation Mass Casualties. Radiat Meas, 42, 1119-1124 (2007).

41. E.L. Green, ed. Biology of the Laboratory Mouse. 2 ed. 1966, Dover Publications, Inc.: New York City. 42. J. Hageman, B J. Eggen, T. Rozema, K. Damman, H.H. Kampinga, and R.P. Coppes, Radiation and transforming growth factor-beta cooperate in transcriptional activation of the profibrotic plasminogen activator inhibitor-1 gene. Clin Cancer Res, 11, 5956-5964 (2005).

43. W. Zhao, D.R. Spitz, L.W. Oberley, and M.E. Robbins, Redox modulation of the pro- fibrogenic mediator plasminogen activator inhibitor-1 following ionizing radiation. Cancer

Res, 61, 5537-5543 (2001).

44. W. Zhao, Y. O'Malley, S. Wei, and M.E. Robbins, Irradiation of rat tubule epithelial cells alters the expression of gene products associated with the synthesis and degradation of extracellular matrix Int J Radiat Biol, 76, 391-402 (2000).

45. W. Zhao, Y. O'Malley, and M.E. Robbins, Irradiation of rat mesangial cells alters the expression of gene products associated with the development of renal fibrosis. Radiat Res, 152, 160-169 (1999).

46. C.F. Reilly and R.C. McFall, Platelet-derived growth factor and transforming growth factor-beta regulate plasminogen activator inhibitor-1 synthesis in vascular smooth muscle cells. J Biol Chem, 266, 9419-9427 (1991).

47. A. Turtoi and F.H. Schneeweiss, Effect of (21 l)At alpha-particle irradiation on expression of selected radiation responsive genes in human lymphocytes. Int J Radiat Biol, 85, 403-412 (2009).

48. Ofir R, Zhang LC, Kyne AP, Houtzager V, O'Connor L, Adams JM., Gamma- radiation-induced growth arrest and apoptosis in p53-null lymphoma cells is accompanied by modest transcriptional changes in many genes. DNA Cell Biol. 2000 Jan;19(l):29-37.

49. Papathanasiou MA, Fomace AJ Jr. DNA-damage inducible genes. Cancer Treat Res. 1991; 57:13-36.

50. Sakamoto-Hojo ET, Mello SS, Pereira E, Fachin AL, Cardoso RS, Junta CM, Sandrin-Garcia P, Donadi EA, Passos GA. Gene expression profiles in human cells submitted to genotoxic stress. Mutat Res. 2003 Nov;544(2-3):403-13.

51. Long XH, Zhao ZQ, He XP, Wang HP, Xu QZ, An J, Bai B, Sui JL, Zhou PK. Dose- dependent expression changes of early response genes to ionizing radiation in human lymphoblastoid cells. Int JMol Med. 2007 Apr; 19(4):607-15.

Claims

CLAIMS We claim:

1. A method of identifying a mammalian subject exposed to ionizing radiation, comprising: detecting the presence of, and/or quantifying the level of, one or more biomarker nucleic acids or proteins in a blood sample or blood derived sample from the subject, wherein the presence of the biomarker, or a level or concentration of the biomarker above a predetermined threshold is indicative of ionizing radiation exposure in the subject, and wherein the one or more biomarkers comprise or consist of one or m ore among Bbc3, Ccngl, Cdknla, Serpinel, and Tnfrsf10b.

2. The method according to claim 1, wherein the one or more biomarkers comprise or consist of one or two from among Bbc3, Ccngl, and Cdknla.

3. The method according to claim 1, wherein the one or more biomarkers comprise or consist of Bbc3 and Ccngl.

4. The method according to claim 1, wherein the one or more biomarkers comprise or consist of Ccngl.

5. The method according to claim 1, wherein the one or more biomarkers comprise or consist of Ccngl and Cdknl .

6. The method according to claim 1, wherein the one or more biomarkers comprise or consist of one or two from among Bbc3, Ccngl, and Cdknla.

7. The method according to any preceding claim, wherein the presence of the biomarker, or a level or concentration of the biomarker above a pre-determined threshold is further indicative of ionizing radiation exposure in the subject at a point in time.

8. The method according to any preceding claim, wherein the presence of the biomarker, or a level or concentration of the biomarker above a pre-determined threshold is further indicative of a quantitative dose of ionizing radiation exposure in the subject.

9. The method according to claim 8, wherein the one or more biomarkers comprise or consist of Ccngl and Cdknla.

10. The method according to claim 1, wherein a level or concentration of the biomarker(s) above a pre-determined threshold is indicative of ionizing radiation exposure of equal to or greater than 2 grays (Gy) in the subject, and wherein a level or concentration of the biomarker(s) below a pre-determined threshold is indicative of ionizing radiation exposure of less than 2 Gy in the subject.

11. The method according to claim 10, wherein the one or more biomarkers comprise or consist of one or both biomarkers from among Ccngl and Tnfrsf10b.

12. The method according to claim 1, wherein a level or concentration of the biomarker above a pre-determined threshold is indicative of ionizing radiation exposure in the subject within 5 to 48 hours.

13. The method according to any preceding claim, wherein the subject is human.

14. The method according to any preceding claim, wherein the sample is a blood sample.

15. The method according to claim 14, wherein the blood sample is a peripheral blood sample.

16. The method according to claim 1, wherein the sample comprises whole blood, serum, or plasma.

17. The method according to claim 1, wherein the sample is obtained within 48 hours of exposure of the individual to radiation.

18. The method according to claim 1, wherein the sample is obtained within 5 hours of exposure of the individual to radiation.

19. The method according to claim 1, wherein the sample is obtained within 5 hours to 48 hours of exposure of the individual to radiation.

20. The method according to any preceding claim, wherein said method comprises detecting the presence of, and/or quantifying the level of, one or more biomarker proteins.

21. The method according to any preceding claim, wherein said method comprises detecting the presence of, and/or quantifying the level of, one or more biomarker nucleic acids.

22. The method according to claim 21, wherein the one or more biomarker nucleic acids is RNA.

23. The method according to claim 22, further comprising extracting the target RNA from blood cells of the subject, and analyzing the RNA for expression of one or more genes comprising or consisting of Bbc3, Ccngl, Cdknla, Serpinel, and Tnfrsf10b.

24. The method according to claim 23, wherein the extracted target RNA is amplified.

25. The method according to claim 1, wherein the detecting comprises: (a) contacting the sample with a binding agent that binds a biomarker protein to form a complex; and (b) detecting and/or quantifying the complex; and correlating the detected complex to the amount of biomarker protein in the sample, wherein the presence of one or more biomarkers, or the presence of elevated levels of the biomarker protein, is indicative of ioizing radiation exposure.

26. The method according to claim 25, wherein the detecting of step (b) further comprises linking or incorporating a label onto the agent, or using ELISA-based immunoenzymatic detection.

27. The method according to claim 1, wherein said method further comprises comparing the level of one or more biomarkers in the sample with the level of biomarker(s) present in a normal control sample, wherein a higher level of biomarker in the sample as compared to the level in the normal control sample is indicative of exposure to ionizing radiation.

28. The method according to claim 1, wherein the nucleic acid is detected using an oligonucleotide that is complementary with at least a portion of the nucleic acid.

29. The method according to claim 1, wherein the nucleic acid is detected using a nucleic acid hybridization method.

30. The method according to claim 29, wherein the nucleic acid hybridization method is Northern blot analysis, dot blotting, Southern blot analysis, fluorescence in situ hybridization (FISH), or polymerase chain reaction (PCR) analysis.

31. The method according to claim 30, wherein the PCR is reverse transcription PCR (RT-PCR).

32. The method according to claim 1, wherein the protein is detected using an aptamer, peptide, or antibody that binds to the protein.

33. The method according to claim 1, wherein the protein is detected using an electrophoretic, chromatographic, or spectrometric method.

34. The method according to claim 1, wherein the protein is detected using mass spectrometry.

35. The method according to claim 1, wherein the presence of the biomarker, or a level or concentration of the biomarker is indicative of ionizing radiation exposure in the subject, and wherein said method further comprises treating the subject for radiation exposure.

36. The method according to claim 1, wherein the presence of the biomarker, or a level or concentration of the biomarker is indicative of ionizing radiation exposure in the subject, and wherein said method further comprises verifying that the mammalian subject is suffering from radiation exposure.

37. The method according to claim 1, wherein the subject is human, wherein the presence of the biomarker, or a level or concentration of the biomarker is indicative of ionizing radiation exposure in the subject, and wherein said method further comprises informing the subject of the exposure or potential exposure to ionizing radiation.

38. A kit comprising an array of nucleic acids attached to a solid substrate, wherein said nucleic acids comprise or consist of one or more oligonucleotides that will hybridize to one or more target nucleic acid molecules from among one or more of the biomarkers Bbc3, Ccngl, Cdknla, Serpinel, and Tnfrsf10b.

39. The kit according to claim 38, wherein the one or more biomarkers comprise or consist of one or two from among Bbc3, Ccngl, and Cdknla.

40. The kit according to claim 38, wherein the one or more biomarkers comprise or consist of Bbc3 and Ccngl .

41. The kit according to claim 38, wherein the one or more biomarkers comprise or consist of Ccngl.

42. The kit according to claim 38, wherein the one or more biomarkers comprise or consist of Ccngl and Cdknl.

43. The kit according to claim 38, wherein the one or more biomarkers comprise or consist of one or both biomarkers from among Ccngl and Tnfrsf10b.

44. The kit according to claim 38, further comprising one or more oligonucleotides that hybridize to nucleic acid molecules of other radiation-responsive genes.

45. The kit according to claim 44, wherein said other radiation-responsive genes are dose-dependent radiation responsive genes.

46. The kit according to claim 38, wherein said array further comprises one or more oligonucleotides that hybridize to nucleic acids from among those in Table IB.

47. The kit according to claim 38, further comprising printed instructions for detecting hybridization of said one or more oligonucleotides to said one or more target nucleic acid molecules.

48. A kit comprising primers for amplifying target nucleic acid sequences within one or more biomarkers comprising or consisting of Bbc3, Ccngl, Cdknla, Serpinel, and Tnfrsf10b.

49. The kit according to claim 48, wherein the one or more biomarkers comprise or consist of one or two from among Bbc3, Ccngl, and Cdknla.

50. The kit according to claim 48, wherein the one or more biomarkers comprise or consist of Bbc3 and Ccngl.

51. The kit according to claim 48, wherein the one or more biomarkers comprise or consist of Ccngl.

52. The kit according to claim 48, wherein the one or more biomarkers comprise or consist of Ccngl and Cdknl.

53. The kit according to claim 48, wherein the one or more biomarkers comprise or consist of one or both biomarkers from among Ccngl and Tnfrsf10b.

54. The kit according to claim 48, further comprising primers for one or more other radiation-responsive genes.

55. The kit according to claim 54, wherein said other radiation-responsive genes are dose-dependent radiation responsive genes.

56. The kit according to claim 48, wherein said kit further comprises one or more primers for one or more genes from among those listed in Table IB.

57. The kit according to claim 48, further comprising printed instructions for detecting amplicons produced from amplification of the target nucleic acids.

58. A device comprising an application zone for receiving a sample of blood; a labeling zone containing a binding agent that binds to one or more biomarkers in the sample; and a detection zone where a biomarker-bound binding agent is retained to give a signal, wherein the signal given for a sample from a subject with a biomarker level lower than a threshold concentration is different from the signal given for a sample from a subject with a biomarker level equal to or greater than a threshold concentration, and wherein the one or more biomarkers comprise or consist of one or more from among Bbc3, Ccngl, Cdknl a, Serpinel, and Tnfrsf10b.

59. The device according to claim 58, wherein the one or more biomarkers comprise or consist of one or two from among Bbc3, Ccngl, and Cdknl a.

60. The device according to claim 58, wherein the one or more biomarkers comprise or consist of Bbc3 and Ccngl.

61. The device according to claim 58, wherein the one or more biomarkers comprise or consist of Ccngl .

62. The device according to claim 58, wherein the one or more biomarkers comprise or consist of Ccngl and Cdknl.

63. The device according to claim 58, wherein the one or more biomarkers comprise or consist of one or both biomarkers from among Ccngl and Tnfrsf10b.

64. A method of identifying an expression profile in a sample from a mammalian subject that is indicative of the subject's exposure or non-exposure to ionizing radiation, comprising: providing a subject expression profile from the mammalian subject; providing one or more reference profiles, wherein each reference profile is associated with exposure or non- exposure to ionizing radiation, wherein the subject expression profile and each reference profile has a value, wherien each value represents the expression level of one or more biomarkers comprising or consisting of Bbc3, Ccngl, Cdknla, Serpinel, and Tnfrsf10b; and identifying the reference profile most similar to the subject expression profile, to thereby identify an expression profile that is indicative of exposure or non-exposure to ionizing radiation.

65. The method according to claim 64, wherein the most similar reference profile is identified by weighting a comparison value for each value of each reference profile using a weight value associated with the corresponding biomarker.

66. The method according to claim 64, wherein the identified reference profile is associated with exposure to ionizing radiation, and wherein said method further comprises treating the mammalian subject for radiation exposure.

67. The method according to claim 64, wherein the identified reference profile is associated with exposure to ionizing radiation, and wherein said method further comprises verifying that the mammalian subject is suffering from radiation exposure.

68. The method according to claim 64, wherein the mammalian subject is human, wherein the identified reference profile is associated with exposure to ionizing radiation, and wherein said method further comprises informing the subject of the exposure or potential exposure to ionizing radiation.

69. The method according to claim 64, wherein the one or more reference profiles comprises a plurality of reference profiles.

70. The method according to claim 69, wherein each reference profile of the plurality of reference profiles is associated with exposure to a different dose of ionizing radiation.

71. The method according to claim 64, wherein the one or more biomarkers comprise or consist of one or two from among Bbc3, Ccngl, and Cdknla.

72. The method according to claim 64, wherein the one or more biomarkers comprise or consist of Bbc3 and Ccngl.

73. The method according to claim 64, wherein the one or more biomarkers comprise or consist of Ccngl.

74. The method according to claim 64, wherein the one or more biomarkers comprise or consist of Ccngl and Cdknl.

75. The method according to claim 64, wherein the one or more biomarkers comprise or consist of one or both biomarkers from among Ccngl and Tnfrsf10b.