KR20220106542A - Radiodosimetrical biomarker and detecting method thereof - Google Patents

Abstract

The present invention relates to a composition for diagnosing radiation exposure and a diagnostic method using the same. Provided are: a composition capable of diagnosing radiation exposure rapidly and accurately by measuring expression levels of ATM, p53, H2AX and MCP-1 proteins, which have high sensitivity to radiation, in samples such as blood, based on the fact that the expression levels thereof are significantly changed depending on the radiation dose and exposure time; and a diagnostic method using the same. According to the present invention, biological dosimetry can be predicted, and it is possible to provide useful information for predicting or diagnosing a degree of radiation exposure from a person handling radiation and human exposure to background radiation.

Description

Radiation exposure diagnostic composition and diagnostic method using the same

The present invention relates to a composition for diagnosing radiation exposure for diagnosing radiation exposure and a diagnostic method using the same

Ionizing radiation (IR) was first used for cancer treatment by Helsper in 1967. Currently, radiation and radioactive isotopes are used in various fields such as diagnosis and treatment, aerospace, semiconductor, biotechnology and food production as well as various industrial uses, and are closely located in our daily life. In particular, in the case of Korea, not only relying on nuclear power for a significant portion of electricity production, but also small-sized radiation irradiation devices are used in industrial sites for non-destructive structural diagnosis of buildings, and accordingly, the number of radiation-related workers increases. are doing The increase in the use of radiation, which is closely located in our daily life, is also increasing the exposure of the human body.

The general public is also constantly exposed to low-dose radiation of less than 0.1 Gy from cosmic radiation or radioactive materials on Earth. Research on the biological effects of low-dose radiation has attracted attention for nearly 20 years, and the results suggest that there are quantitative and qualitative differences in cellular responses to low-dose and high-dose radiation (Ding et al., 2005). ). Although much effort and time have been invested worldwide, the biological effects of low-dose radiation have not been clearly elucidated, and opinions are divided on the correlation between radiation dose and risk. In addition, the number of cases of exposure to radiation is increasing, such as the heavy water leakage accident at the Wolsong nuclear power plant in 1999 or the accident at the Tokaimura uranium plant in Japan. Radiation exposure causes apoptosis by causing DNA repair, cell circulation abnormality, and cell death, and causes chromosomal abnormalities in peripheral blood lymphocytes (PBL), tissue and organ damage, birth defects, and cancer. is known However, no method has yet been developed to monitor the effects of radiation exposure on the human body or to rapidly predict radiation damage in the event of an unexpected accident such as a nuclear accident.

Biological side effects that occur during radiation exposure include physical side effects and genetic side effects. Physical side effects originate from damage to somatic cells constituting the human body, and the effects of the side effects are limited to the exposed person. Acute side effects occur within about one month after exposure to radiation and cause hematopoietic tissue damage, digestive system damage, central nervous system damage and skin damage, and in severe cases, even death. Chronic side effects occur about 3 months after exposure to radiation and cause symptoms such as cataracts, tumor development, and decreased fertility. Genetic side effects originate from germ cell damage caused by chromosomal abnormalities, and the effects of side effects can occur even after several generations due to gene mutations or chromosomal mutations.

When normal organs of the human body are exposed to radiation exceeding the limiting dose, their intrinsic function is partially or permanently lost, resulting in chronic side effects. Radiation fibrosis that occurs during radiation therapy not only aggravates the pain of patients by inducing edema or pain, but also significantly reduces the function of normal organs, leading to death in severe cases. Therefore, when a patient exposed to radiation occurs, it is essential to determine the patient's treatment policy and to determine the prognosis to accurately determine the radiation dose received by the patient as soon as possible. In addition, confirming the radiation dose will play an important role in predicting the chronic effects of radiation that may occur in the future and identifying the causes of diseases that may be caused by radiation exposure.

The dose range can be estimated clinically by using the symptoms and signs of the patient after radiation exposure, but this is a possible method when exposed to relatively high-dose radiation. method suitable for As a more objective and accurate method, it can be confirmed using a worn dosimeter, but if it is not possible to confirm through the dosimeter, a physical method that reconstructs the exposure situation and calculates the radiation dose can be used. However, this method can be difficult to calculate accurately depending on the environment and circumstances exposed to radiation, and since there may also be individual differences in the biological response exposed to radiation, the amount of radiation exposed from the sample that can be obtained from the patient can be accurately calculated. It is necessary to develop a biological method that can measure it. The biological measurement method mainly used so far can mainly cope with the relatively high-dose radiation exposure of 0.2 Gy - 5 Gy, and it takes several days to a week to read the exposure dose. Therefore, it is necessary to develop damage control factors or exposure biomarkers that can respond to low-dose radiation from biological samples and can be identified within the same day.

Methods for measuring radiation exposure include isolation of lymphocytes from blood to confirm chromosomal abnormalities, measurement of micronucleus formation, measurement of glycoporin A mutation in red blood cells, DNA cleavage degree, and tooth enamel. Although it relies on methods such as EST (electron spin resonance), there are disadvantages in that the reactivity is different for each individual, it is almost impossible to measure for low-dose radiation, and it takes a long time to measure. In addition, in order to measure the exposure to biological radiation, it is necessary to develop a method to increase the accuracy because measuring the degree of exposure by a single method is risky.

Against this background, the study was further expanded to search for biomarkers of exposure that can be found in normal tissues exposed to radiation, and these exposure biomarkers were used to determine the effects and prognosis of the human body due to exposure to nuclear power plant accidents, terrorism, or radiation therapy, etc. It is expected to be of great use.

Japanese Patent Laid-Open No. 2007-093341 (published on April 12, 2007) Republic of Korea Patent Registration No. 10-1885596 (Registered on July 31, 2018)

In order to solve the above-mentioned problems, the present inventors performed a study on a biomarker capable of accurately and rapidly diagnosing radiation exposure by quantitatively responding to radiation exposure dose in a liquid sample that is easy to collect.

As a result, it was confirmed that diagnostic information on radiation exposure can be provided using ATM, p53, H2AX and MCP-1 proteins or phosphorylated proteins thereof, thereby completing the present invention.

It is an object of the present invention to provide a composition for diagnosis of radiation exposure comprising an agent for measuring any one or more proteins selected from the group consisting of ATM, p53, H2AX, and MCP-1, phosphorylated proteins or fragments thereof. More preferably, it is to provide a composition for diagnosis of radiation exposure comprising an agent for measuring ATM, p53, H2AX and MCP-1 proteins, phosphorylated proteins or fragments thereof.

Another object of the present invention is to provide a kit for diagnosis of radiation exposure comprising an agent for measuring at least one protein selected from the group consisting of ATM, p53, H2AX, and MCP-1, a phosphorylated protein thereof, or a fragment thereof. More preferably, it is to provide a kit for diagnosis of radiation exposure comprising an agent for measuring ATM, p53, H2AX and MCP-1 proteins, phosphorylated proteins or fragments thereof.

Another object of the present invention is to provide a method for diagnosing radiation exposure comprising measuring the expression level of one or more proteins selected from the group consisting of ATM, p53, H2AX, and MCP-1, fragments thereof, or phosphorylation thereof in an isolated sample is to do More preferably, there is provided a method for diagnosing radiation exposure comprising measuring the level of ATM, p53, H2AX and MCP-1 proteins, fragments thereof, or phosphorylation expression levels thereof in the isolated sample.

The present invention provides a composition for diagnosis of radiation exposure comprising an agent for measuring any one or more proteins selected from the group consisting of ATM, p53, H2AX, and MCP-1, phosphorylated proteins or fragments thereof. More preferably, there is provided a radiation exposure diagnostic composition comprising an agent for measuring ATM, p53, H2AX and MCP-1 proteins, phosphorylated proteins or fragments thereof.

The present invention provides a kit for diagnosing radiation exposure comprising an agent for measuring one or more proteins selected from the group consisting of ATM, p53, H2AX, and MCP-1, phosphorylated proteins or fragments thereof. More preferably, there is provided a radiation exposure diagnostic kit comprising an agent for measuring ATM, p53, H2AX and MCP-1 proteins, phosphorylated proteins or fragments thereof.

The present invention provides a method for diagnosing radiation exposure comprising measuring the expression level of one or more proteins selected from the group consisting of ATM, p53, H2AX and MCP-1, fragments thereof, or phosphorylation thereof in an isolated sample. More preferably, there is provided a method for diagnosing radiation exposure, comprising measuring the expression level of ATM, p53, H2AX and MCP-1 proteins, fragments thereof, or phosphorylation thereof in the isolated sample.

According to the present invention, by using the proteins of ATM, p53, H2AX, and MCP-1 or their phosphorylated proteins having high sensitivity to radiation for diagnosis of radiation exposure, radiation exposure can be diagnosed. In particular, since the present invention can use a sample as a liquid sample, such as blood, it is possible to quickly and accurately diagnose whether or not to be exposed to radiation. , it can be used to easily predict or diagnose the degree of radiation exposure from radiation-related workers and radiation exposure around nuclear power plants.

1 is a result of observing the phosphorylation change of ATM protein according to radiation dose and time in human-derived lymphocytes.
2 is a result of observing changes in phosphorylation of p53 protein according to radiation dose and time in human-derived lymphocytes.
3 is a result of observing the phosphorylation change of H2AX protein according to radiation dose and time in human-derived lymphocytes.
4 is a result of observing the change of MCP-1 protein according to radiation dose and time in human-derived lymphocytes.
5 is a result of observing changes in MIF protein according to radiation dose and time in human-derived lymphocytes.
6 is a result of observing changes in GDF15 protein according to radiation dose and time in human-derived lymphocytes.
7 is a result of observing the phosphorylation change of ATM protein from the hematocyte lysate isolated from human peripheral blood.
8 is a result of observing the phosphorylation change of the p53 protein from the hematocyte lysate isolated from human peripheral blood.
9 is a result of observing the change in phosphorylation of H2AX protein from hematocyte lysate isolated from human peripheral blood.
10 is a result of observing the change of MCP-1 protein from the hematocyte lysate isolated from human peripheral blood.

Hereinafter, to help the understanding of the present invention, examples will be described in detail. However, the following examples are merely illustrative of the content of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

The terms of the present invention will be described below.

As used herein, the term “irradiation” refers to exposure to electromagnet radiation, including, for example, X-rays, ultraviolet rays, alpha particles and gamma rays, or the action of alpha or beta particles. Radiation can affect both cancer cells and normal cells, but various methods and techniques are used to treat cancer by destroying a lot of cancer cells with less effect on normal cells. This does not immediately kill cancer cells, but destroys the ability of cancer cells to divide and proliferate, preventing new cancer cells from dividing and creating, and cancer cells that do not divide anymore die at the end of their lifespan. With each treatment, more cells die, and the dead cells are broken down and transported into the bloodstream to be excreted from the body, reducing the size of the tumor. Most of the normal cells are recovered, but some normal cells are not, resulting in side effects of radiation therapy. Changes in cells and tissues that may be caused by side effects include changes in cell division timing, inhibition of apoptosis and necrosis, tissue damage, and decreased immune function.

As used herein, the term “gray (Gy)” refers to a radiation dose capable of emitting 1 Joule from 1 kg of biological tissue.

As used herein, the term “measurement” refers to determining the degree of radiation exposure or irradiation in cells. In one embodiment, the present invention can determine the degree of radiation exposure or irradiation by measuring the mRNA expression level of the gene or the expression level of its protein from a biological sample obtained from an individual, and comparing it with the expression level of a normal control sample.

As used herein, the term “diagnosing” refers to ascertaining the presence or characteristics of pathophysiology. Diagnosis in the present invention is to confirm whether and/or the extent of radiation exposure.

During radiation exposure, somatic cells are damaged, and acute side effects such as hematopoietic tissue damage, digestive system damage, central nervous system damage, or skin damage, or chronic side effects such as cataracts, tumors, and decreased fertility may occur. In addition, it can cause germ cell damage caused by chromosomal abnormalities. Therefore, there is a need for a marker that can quantitatively diagnose radiation exposure.

In addition, although radiation therapy plays a very important role in anticancer therapy, damage to normal tissues around the tumor occurs irresistibly due to radiation exposure during radiation therapy. Damage to the normal tissue around the tumor is an obstacle to improving radiation therapy. In addition, when radiation above the limit dose is irradiated to a normal organ due to radiation therapy, the intrinsic function of the organ is partially lost or the function is permanently damaged, resulting in chronic side effects. Therefore, there is a need for a marker capable of monitoring the damage of normal and cancerous tissues in response to radiation exposure, and through these markers, patient-specific treatment is possible.

As used herein, the term "marker" refers to a substance that can be diagnosed by distinguishing between a normal group individual and a radiation-exposed individual, and a polypeptide, protein or nucleic acid showing an increase or decrease in the radiation-exposed group compared to the normal group individual. , including organic biomolecules such as genes, lipids, glycolipids, glycoproteins or sugars.

As used herein, the term "expression level measurement" refers to a process of confirming the expression level of a protein expressed in a radiation exposure diagnostic gene in a biological sample for diagnosing radiation exposure, and according to an embodiment of the present invention, Proteins can be identified using an antibody or antigen-binding fragment that binds selectively. Analysis methods for this include western blotting, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony. ) Immune diffusion method, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS (Fluorescence Activated Cell Sorter), protein chip chip), but is not limited thereto.

As used herein, the term “antibody” is a term known in the art and refers to a specific immunoglobulin directed against an antigenic site. The antibody in the present invention refers to an antibody that specifically binds to the radiation exposure marker of the present invention, the gene is cloned into an expression vector to obtain a protein encoded by the gene, and from the obtained protein, a conventional method in the art Antibodies can be prepared according to The form of the antibody includes a polyclonal antibody or a monoclonal antibody, and all immunoglobulin antibodies are included. The antibody refers to a complete form having two full-length light chains and two full-length heavy chains. Moreover, the said antibody also includes special antibodies, such as a humanized antibody.

As used herein, the term "biological sample" refers to a sample obtained from an organism. The biological sample may be blood, plasma, platelets, serum, ascites fluid, bone marrow fluid, lymph fluid, saliva, tear fluid, mucosal fluid, amniotic fluid, or a combination thereof. When the biological sample is blood or plasma, since blood or plasma, which is easy to collect, is used as a sample, organ tissues of the individual are not extracted, so that analysis can be performed conveniently without burdening the individual. In addition, since blood circulates through the human body at a high speed, it can react sensitively to radiation exposure, and thus the accuracy and sensitivity of diagnosis are high, and thus it can be usefully used for radiation exposure diagnosis. The biological sample may include an intact protein. The intact protein may be a protein isolated from a biological sample without further modification of the protein. An intact protein may be a protein that has been isolated from a biological sample, for example, without proteolysis by proteolytic enzymes.

The present invention provides a composition for diagnosing radiation exposure comprising at least one protein selected from the group consisting of ATM, p53, H2AX, and MCP-1, a phosphorylated protein thereof, or a fragment thereof.

Specifically, there is provided a composition for diagnosis of radiation exposure comprising the proteins of ATM, p53, H2AX, and MCP-1, phosphorylated proteins thereof, or fragments thereof.

Ataxia telangiectasia mutated (ATM) protein is known as an activator of DNA damage response by DNA double-strand breaks (DSBs). After the development of DSBs, ATM is known to be recruited into the most extensive signaling network by responding to specific stimuli and directly or indirectly modulating various targets. For example, GenBank: AAI37170.1 (Gene ID: 472, UniProtKB: Q13315) and the like.

p53 is a cancer suppressor protein, and in humans it is encoded by the TP53 gene. P53 is known to be important for cancer prevention as a cancer suppressor in the cell cycle of multicellular organisms. For example, GenBank: BAC16799.1 (Gene ID: 7157, UniProtKB: P04637) can be referred to as a protein.

H2AX (H2A histone family member X) H2AX (H2A histone family member X) is a type of histone protein of the H2A family. have. For example, Gene ID: 472, UniProtKB: Q13315, and the like.

Monocyte Chemoattractant Protein-1 (MCP-1) is a small cytokine belonging to the CC chemokine family and is known to play a role in recruiting monocytes, memory T cells, and dendritic cells to sites of inflammation generated by tissue damage or infection. For example, GenBank: AAB29926.1 (Gene ID: 6347, UniProtKB: P13500) and the like.

The composition for diagnosis of radiation exposure according to the present invention may further include an agent for measuring the expression of MIF, GDF15 or both proteins, phosphorylated proteins or fragments thereof.

The expression of MIF, GDF15, or both of them may increase according to radiation exposure.

MIF (macrophage migration inhibitory factor) is known as a protein related to the regulation of macrophage function in the process of host defense by suppressing the anti-inflammatory effect of glucocorticoids. For example, Gene ID: 4282, UniProtKB: P14174, and the like.

GDF15 (Growth/differentiation factor 15) acts as a pleiotropic cytokine and is known to be involved in the cellular stress response after cell damage. Elevated protein levels are associated with disease states such as hypoxia, inflammation, acute injury and oxidative stress. For example, GeneID: 9518, UniProtKB: Q99988, and the like.

ATM, p53 and H2AX according to the present invention may be provided in the composition for diagnosis of radiation exposure to provide information on the initial stage before 1 day after exposure. According to the present invention, since the expression levels of ATM, p53 and H2AX increase rapidly immediately after exposure, direct information on whether or not exposure is immediately after exposure can be provided.

MCP-1 according to the present invention may be to provide information of the late stage after 1 day of exposure. In addition, MCP-1 can also provide additional information about exposure to high doses or long-term exposure to low doses.

That is, according to the present invention, not only can a quick diagnostic test be provided when exposure is directly suspected, but also diagnostic information on whether or not exposure has passed even when a certain time or more has passed since exposure can be provided. Through the combination, it is possible to provide specific and accurate information about the timing of exposure and the level of exposure.

The composition may further include a sample required for diagnosis of radiation exposure.

The composition for diagnosing radiation exposure according to the present invention can be used to measure protein expression levels in blood cells or samples obtained from blood, which are particularly easy to obtain among individuals suspected of radiation exposure. That is, as described above, by using a liquid sample such as blood, diagnostic information can be provided quickly and accurately, and thus has more improved characteristics than the existing marker system.

The present invention provides a kit for diagnosing radiation exposure comprising an agent for measuring one or more proteins selected from the group consisting of ATM, p53, H2AX, and MCP-1, phosphorylated proteins or fragments thereof. More preferably, there is provided a radiation exposure diagnostic kit comprising an agent for measuring ATM, p53, H2AX and MCP-1 proteins, phosphorylated proteins or fragments thereof.

The kit may further include a sample necessary for diagnosing radiation exposure. The kit may include an antibody, a substrate for immunological detection of the antibody, a suitable buffer, a secondary antibody labeled with a chromogenic enzyme or a fluorescent material, or a chromogenic substrate.

In another embodiment, the kit may be an ELISA kit, a protein chip kit, a rapid kit, or a multiple reaction monitoring (MRM) kit.

The kit for diagnosis of radiation exposure according to the present invention may further include an agent for measuring the expression of MIF, GDF15 or both proteins, phosphorylated proteins or fragments thereof.

The present invention provides a method for diagnosing radiation exposure comprising measuring the expression level of one or more proteins selected from the group consisting of ATM, p53, H2AX and MCP-1, fragments thereof, or phosphorylation thereof in an isolated sample. More preferably, there is provided a method for diagnosing radiation exposure, comprising measuring the expression level of ATM, p53, H2AX and MCP-1 proteins, fragments thereof, or phosphorylation thereof in the isolated sample.

The isolated sample may be a sample isolated from a subject. The subject may be a subject suspected of being exposed to radiation, or may be a mammal including a human.

The measuring may include incubating the biological sample with an antibody that specifically binds to the above-mentioned protein or fragment thereof. Incubation can be performed in vitro . Measurement methods include, for example, Western blotting, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion method, rocket immunization. Electrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, protein chip, and the like may be included.

The measuring may include performing mass spectrometry of the biological sample. The mass spectrometry of the biological sample may include measuring a peptide derived from a corresponding protein.

The method comprises comparing the measured expression level with the above-mentioned protein expression level of a normal control. The normal control may be an individual not exposed to radiation, or a biological sample isolated from the same individual prior to exposure to radiation.

Since the measured expression level of the protein increases relative to the normal control in proportion to the time and/or dose of exposure to radiation, the expression level of the protein is compared with that of the normal control. Depending on the degree to which the level increases, whether and/or the extent of radiation exposure, for example, the radiation exposure dose, predicts the effect on radiation therapy, or radiation therapy, The degree of damage can be diagnosed and predicted.

The method includes a method of providing information for diagnosing radiation exposure. For example, if there is a statistically significant increase compared to the control group, it can be judged to have been exposed.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example 1. Irradiation

In order to check the expression level of radiation exposure marker factors that change according to the intensity of radiation, human lymphocyte cell lines and human peripheral blood mononuclear cells were irradiated with various doses of 1 mGy to 2 Gy, 30 minutes to 72 hours. Samples were obtained at various times until the protein was extracted, and Western blot assay or enzyme-linked immunosorbent assay (ELISA) was performed.

Example 1-1: Irradiation of human lymphocyte cell lines

IM-9 cells, a human B lymphocyte cell line, and HuT 78 cells, a human T lymphocyte cell line, were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-spreptomycin (10,000 U/ml) for 5 % CO ₂ and maintenance culture at 37°C.

Each cell line was aliquoted into a T75 culture vessel with an appropriate number of cells, and after 24 hours had elapsed, transferred to a 15 ml tube, and irradiated with radiation from 1 mGy to 2 Gy using an irradiator (0). , 1, 5, 10, 25, 50, 75, 100, 200, 500, 1000, 2000 mGy), transferred to a T25 culture vessel and continued incubation. Samples were obtained at 1, 3, 8, 24, 30, 48, and 72 hours from 30 minutes after irradiation.

The obtained sample was washed with phosphate buffered saline (PBS), and the supernatant was removed and the pellet was stored at -80°C and used for Western blot analysis.

Example 1-2: Irradiation of Human Peripheral Blood Mononuclear Cells (PBMC)

Primary human mononuclear cells (MNCs) isolated from human peripheral blood were purchased from STEMCELL Technologies, and the frozen human peripheral blood mononuclear cells (PBMC) were prepared using 10% fetal bovine serum (FBS) and 1 % Penicillin-Spreptomycin (10,000 U/ml) is thawed in RPMI-1640 medium, 5% CO ₂ and 37 ℃ conditions, the appropriate number of cells in a T75 culture vessel, 24 hours have elapsed, and then irradiated was carried out.

PBMC cells were transferred to a 15 ml tube and irradiated with radiation from 1 mGy to 2 Gy using an irradiator (0, 1, 5, 25, 100, 500, 1000, 2000 mGy), T25 After transferring to the culture vessel, the culture was continued. Samples were obtained from 1 hour to 3, 8, and 24 hours after irradiation.

The obtained sample was washed with a phosphate buffer solution, and the pellet from which the supernatant was removed was stored at -80°C, and then used for enzyme-linked immunosorbent assay (ELISA).

Example 2. Analysis of protein changes according to radiation exposure in human lymphocyte cells

Phosphorylated protein or phosphorylated protein targeting ATM, CHK1, CHK2, p53, H2AX, BRCA1, EGFR, NBS, IL-1α, MCP-1, IL-7, MIF, GDF15, MIP, etc. Protein expression was confirmed by Western analysis.

Example 2-1: Western blot assay

The cell line was treated with TNN containing proteolysis inhibitor (1 mM phenylmethylsulfonyl fluoride (PMSF), 1 μg/ml aprotinin, 1 μg/ml leupeptin and 1 mM Na ₃ VO ₄ ). Cell lysates were obtained using a cell lysis buffer (cell lysis buffer, 1 M Tris-HCl, 5M NaCl and 10% NP-40) The amount of protein was quantified using the Bradford method, and the same amount of Protein was separated by SDS-polyacrylamide gel and transferred to a nitrocellulose membrane.The membrane to which the protein was transferred was TBS-T (Tris-buffered saline/TBS-T) containing 5% non-fat milk to reduce non-specific binding. After blocking with a 0.05% Tween-20) solution at room temperature for 30 minutes, the primary antibody (1:1000 dilution) was reacted overnight at 4° C. The next day, the secondary antibody (1:5000) was incubated for 1 hour. During the reaction at room temperature, the immunoreactive band was detected using an enhanced chemiluminescent reagent.

Example 2-2: Protein expression quantification and curve fitting analysis (Curve fitting)

The protein bands obtained through the above Western blot analysis were analyzed using ImageJ (v. 1.52)

Quantification was performed using a gram, and the obtained quantitative value was normalized, and then expressed as a graph using the GraphPad Prism 8 program. A curve fitting analysis was performed from the graph to evaluate the change in protein expression according to the radiation dose. Table 1 shows the results of significant changes in time or dose for each candidate factor for the IM-9 cell line in a table.

IR dose (cGy)

Time 0 One 5 10 25 50 75 100 200 500 1000 2000 IL-1a 30 h 100.0 255.2 291.8 253.0 258.3 224.5 278.8 224.9 197.7 86.3 142.6 47.3 72 h 100.0 215.8 166.7 205.5 193.9 211.3 179.5 210.6 229.5 192.3 220.2 169.9 MIF 24 h 100.0 102.9 205.0 175.0 233.3 218.6 201.5 286.7 252.7 404.3 371.7 454.0 48 h 100.0 197.2 137.1 132.6 156.2 105.4 193.8 132.1 1998.8 186.7 183.6 315.2 MCP-1 24 h 100.0 97.2 68.1 96.5 124.7 128.3 127.6 222.0 215.0 252.0 322.4 254.7 72 h 100.0 237.6 140.3 182.1 197.1 230.7 295.7 285.2 329.1 281.6 235.6 217.3 GDF15 8 h 100.0 99.3 164.5 112.7 181.5 190.4 171.7 209.4 229.7 203.4 281.5 274.4 30 h 100.0 168.7 206.6 206.3 192.9 162.8 192.5 168.8 182.5 74.4 144.5 35.6 72 h 100.0 177.9 55.0 164.6 224.0 225.8 233.0 201.2 186.0 205.6 167.0 171.8 IL-7 30 h 100.0 140.7 172.2 143.5 142.0 115.1 150.5 138.0 158.7 86.3 117.6 54.5 72 h 100.0 157.5 93.7 134.3 119.6 125.4 133.7 150.4 159.5 131.0 133.7 125.8 MIP 72 h 100.0 145.2 61.9 67.8 98.1 124.0 116.8 103.3 126.4 113.0 110.4 99.5 pATM 3 h 100.0 118.8 116.6 117.4 140.1 124.0 124.9 140.6 155.4 172.5 190.6 263.8 pH2AX 30 min 100.0 181.3 113.2 137.9 114.5 105.0 156.7 99.8 129.8 161.4 175.4 231.8 24 h 100.0 133.4 195.1 172.6 195.5 242.6 217.9 267.4 231.5 315.5 299.7 415.0 pBRCA1 30 min 100.0 181.2 197.8 94.2 209.4 49.7 225.2 111.4 99.5 161.7 61.4 72.9 8 h 100.0 102.9 119.2 84.9 109.6 103.3 112.6 69.4 82.4 98.3 113.3 77.5 pEGFR 30 min 100.0 105.4 138.8 81.1 152.2 114.5 133.5 112.5 86.3 156.0 94.6 285.7 24 h 100.0 78.7 79.0 81.9 80.3 88.1 105.1 117.0 106.0 130.3 100.5 154.5 pCHK1 8 h 100.0 104.4 87.4 83.0 85.4 88.0 111.7 106.2 125.9 137.7 95.5 97.0 24 h 100.0 64.2 49.4 64.2 71.8 116.6 119.6 131.2 127.2 138.7 142.5 156.9 pCHK2 30 min 100.0 131.7 117.1 108.6 118.2 116.5 147.0 168.1 112.5 179.7 249.0 334.4 p-TP53 30 min 100.0 103.7 160.1 183.6 181.2 250.6 192.3 238.5 261.1 594.5 808.6 1036.9 3 h 100.0 119.8 131.6 95.0 93.6 96.0 114.6 91.3 97.7 153.4 220.9 309.8 NBS1 3 h 100.0 149.5 166.1 145.1 169.0 145.6 165.9 169.3 177.9 158.7 149.4 120.2 24 h 100.0 112.6 122.0 135.2 136.6 152.7 124.2 142.4 141.9 130.5 86.4 77.0

According to the results in Table 1, ATM, p53, H2AX, MCP-1, MIF, and GDF15 showed significant expression change patterns.

Specifically, the phosphorylation of ATM increased by 1.2 to 2.6 times in a dose-dependent manner at 3 hours, and gH2AX showed an increase from 30 to 24 hours. It was confirmed that pp53 also increased up to 10-fold in a dose-dependent manner from the 30th minute, and maintained a 3-fold increase even after 3 hours. MCP-1 showed a dose-dependent 1.2-3.2-fold increase in 24 hours over 25 cGy, followed by a 1.4-3.3-fold increase in expression at a low dose at 72 hours.

MIF and GDF15 also showed a generally increased expression pattern.

That is, from the expression changes, it was confirmed that the expression of ATM, p53, and H2AX greatly increased after radiation exposure. In addition, it was confirmed that the expression of MCP-1 was increased by high radiation exposure or long-term low-dose exposure. In addition, MIF and GDF15 also provided information on the availability of auxiliary diagnostic factors.

The results of significant changes in time or dose for each candidate factor for the HuT 78 cell line are summarized in a table and presented in Table 2.

IR dose (cGy)

Time 0 One 5 10 25 50 75 100 200 500 1000 2000 IL-1a 30 h 100.0 175.9 188.4 178.6 185.0 257.5 75.0 166.5 198.8 61.1 226.6 199.1 72 h 100.0 124.3 147.7 192.6 209.7 204.3 166.7 154.6 145.6 128.9 140.0 93.1 MIF 8 h 100.0 203.8 167.6 221.4 199.3 191.3 193.4 199.9 230.2 207.5 222.5 179.1 24 h 100.0 102.9 205.0 175.0 233.3 218.6 201.5 286.7 252.7 404.3 371.7 454.0 48 h 100.0 197.2 137.1 132.6 156.2 105.4 193.8 132.1 1998.8 186.7 183.6 315.2 MCP-1 24 h 100.0 164.8 209.8 232.8 228.8 226.0 260.4 256.6 268.9 260.8 426.1 289.8 72 h 100.0 191.8 209.3 258.3 343.2 215.7 281.7 240.4 241.9 171.6 212.1 191.7 GDF15 24 h 100.0 56.3 76.0 76.8 122.6 95.7 176.8 291.5 371.9 247.8 172.8 406.3 30 h 100.0 174.0 221.0 154.9 262.2 233.9 116.3 196.1 208.4 44.6 149.1 191.1 IL-7 30 h 100.0 136.0 121.5 120.8 153.6 144.6 141.9 112.0 138.8 29.6 114.5 111.2 MIP 72 h 100.0 96.8 88.0 95.4 104.8 111.1 88.8 98.4 110.5 97.5 109.2 90.9 pATM 1 h 100.0 171.9 384.6 396.4 501.6 406.2 457.1 413.3 424.1 595.1 503.1 547.3 3 h 100.0 162.1 193.2 209.6 110.6 128.1 108.7 93.6 93.1 173.6 251.0 274.7 pH2AX 30 min 100.0 131.6 192.6 284.6 201.3 144.9 264.0 47.3 125.4 261.9 170.0 222.0 3 h 100.0 228.8 273.7 284.4 247.4 226.1 245.4 215.8 164.4 252.2 228.5 238.1 pBRCA1 30 min 100.0 115.5 130.7 132.8 107.6 78.1 129.1 41.2 99.6 120.7 126.6 112.6 8 h 100.0 95.8 69.9 80.8 40.3 62.1 44.5 29.3 25.0 32.5 49.6 54.3 pEGFR 30 min 100.0 99.1 121.5 96.1 157.5 110.0 167.7 138.0 140.3 222.1 154.6 270.2 24 h 100.0 102.1 87.8 82.7 90.6 49.9 57.8 61.6 63.1 36.9 17.4 38.0 pCHK1 8 h 100.0 100.9 76.9 94.1 91.3 109.5 104.5 76.9 55.1 50.0 47.1 43.0 24 h 100.0 112.5 97.5 77.8 105.8 95.7 113.9 112.5 125.8 105.8 68.1 88.6 pCHK2 30 min 100.0 88.1 115.3 126.7 153.2 136.8 163.7 156.6 156.5 184.8 177.4 196.6 p-TP53 30 min 100.0 99.2 134.6 143.2 135.8 102.8 116.8 62.7 91.9 98.6 167.5 127.6 3 h 100.0 103.0 139.3 144.9 145.2 122.7 137.0 143.5 123.7 126.0 115.2 114.9 NBS1 3 h 100.0 133.0 136.3 123.0 123.6 130.2 139.7 107.9 136.8 137.3 121.8 127.5 24 h 100.0 123.6 172.3 157.1 185.2 185.2 166.2 160.6 160.7 154.6 116.1 62.7

ATM phosphorylation showed a faster and stronger response than in IM-9 cells, increasing 1.7 -6.0 times at 1 hour, and gH2AX increased 1.3 - 2.8 times from 30 minutes to 3 hours. The phosphorylation expression of p53 showed an increase of approximately 20-30% from 30 min without a dose dependence. It was confirmed that MCP-1 also increased 1.6 - 4.3 times after 24 hours, and 1.7 - 3.4 times at 72 hours.

MIF was confirmed to increase 1.7 - 2.3 times from 8 hours, 1.8 - 4.6 times at 24 hours, and 1.3 - 3.2 times at 48 hours, showing a stronger response than in IM-9 cells. The expression of GDF15 was increased 1.2-3.7-fold at 24 hours and 1.2-2.6-fold at 30 hours.

That is, similar to the previous results, it was confirmed that ATM, p53, H2AX, MCP-1, MIF, and GDF15 showed significant expression change patterns to provide diagnostic information on radiation exposure.

In particular, this expression change shows excellent diagnostic efficacy in that it provides the overall analysis result for the treated Gy and exposure time, unlike the literature that provides information on simple expression changes in the past.

The expression of the protein according to the radiation dose was analyzed using a five-parameter asymmetry sigmoidal with 5PL curve, and the curve joining result was evaluated through the coefficient of determination (R ² ).

Referring to FIG. 1 , ATM protein phosphorylation increased in a dose-dependent manner from 200 mGy or more at 30 minutes after irradiation, and the coefficient of determination was 0.9973 as a result of verifying the significance by formulating the response curve slope. 3 hours after irradiation, it increased significantly from 50 mGy and showed a coefficient of determination of 0.9915.

The phosphorylation of TP53 protein of FIG. 2 showed an increasing change from 5 mGy at 30 minutes after irradiation, and the coefficient of determination was 0.9921. At 3 hours after irradiation, the slope also decreased, but showed a coefficient of determination of 0.9912 with an increasing trend.

H2AX protein phosphorylation of FIG. 3 showed an increasing change from the 200 mGy dose at 30 minutes after irradiation, and the coefficient of determination was 0.9855. At 24 hours after irradiation, it increased in a dose-dependent manner from a lower 1 mGy, and the coefficient of determination was 0.9892.

The expression of MCP-1 protein of FIG. 4 showed an increasing change from the 25 mGy radiation dose at 24 hours after irradiation, and the coefficient of determination was 0.9989. It showed an increasing response curve even at 48 hours after irradiation, and the coefficient of determination was 0.9960.

Referring to FIG. 5 , the expression of MIF protein showed an increasing change from a low dose of 5 mGy at 24 hours after irradiation, and the coefficient of determination was 0.9885 as a result of verifying the significance by formulating the response curve slope. At 48 hours after irradiation, it also increased from 500 mGy to 2000 mGy, and the coefficient of determination was 0.9329.

The expression of GDF15 protein of FIG. 6 showed a dose-dependent increase from 25 mGy at 8 hours after irradiation, and the coefficient of determination was 0.9933.

Example 3. Analysis of protein changes according to radiation exposure in human peripheral blood

In order to derive a diagnostic factor for exposure to radiation, the expression level of protein or phosphorylated protein was confirmed by enzyme-linked immunosorbent test for candidate factors derived through Western blot. For the test, RayBiotech's MCP-1, p-ATM (S1981), p-H2AX (S139), and p-TP53 (S15) ELISA Kits were used.

Example 3-1: Enzyme-linked immunosorbent assay (ELISA)

Cell lysates were obtained from human peripheral blood mononuclear cells stored after irradiation according to the method attached to the kit, and a 5-fold diluted sample was used for antibody reaction attached to a 96-well plate, overnight at 4°C. reacted. The subsequent process was also performed according to the manufacturer's instructions, and finally, the protein expression was analyzed from the result of measuring the absorbance at 450 nm.

Example 3-2: Protein Expression Normalization and Curve Fitting Analysis

The results obtained through the above enzyme-linked immunosorbent test were analyzed in Microsoft's Excel Pro.

After normalization using a gram, it was expressed as a graph using the GraphPad Prism 8 program. From the graph, curve fitting analysis was performed to evaluate the change in phosphorylated protein expression according to the radiation dose. The expression of the protein according to the radiation dose was analyzed using a five-parameter asymmetry sigmoidal with 5PL curve, and the curve joining result was evaluated through the coefficient of determination (R ² ).

Referring to FIG. 7 , ATM protein phosphorylation showed a change that increased from 5 mGy irradiation dose to 2000 mGy at 1 hour after irradiation, and the coefficient of determination was 0.8799 as a result of verifying significance by formulating the reaction curve slope.

8, TP53 protein phosphorylation showed a dose-dependently increasing change in all time intervals after irradiation (1, 3, 8, 24 hours), and the coefficient of determination at 1 hour was 0.9585, 3 At the hour, it was 0.9113, at the eighth hour, 0.9777, and at the 24 hour, 0.6548.

The H2AX protein phosphorylation of FIG. 9 confirmed a dose-dependently significant increase at 24 hours after irradiation, and the coefficient of determination was 0.9493.

Referring to FIG. 10 , the expression of MCP1 protein showed a change that increased from 1 mGy to 100 mGy at 24 hours after irradiation, and the coefficient of determination was 0.96 as a result of verifying the significance by formulating the response curve slope.

As can be seen from the above results, the combination of MCP-1, ATM, H2AX, and p53 as four markers according to the present invention provides accurate and fast information on radiation exposure. In particular, since it can provide not only a quick diagnostic test when exposure is directly suspected, but also diagnostic information on whether or not exposure has occurred even after a certain period of time has elapsed after exposure, the combination of the above It can provide specific and accurate information about the timing and level of exposure.

From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the claims described below and their equivalents.

Claims (11)

A composition for diagnosis of radiation exposure, comprising an agent for measuring ATM, p53, H2AX and MCP-1 proteins, phosphorylated proteins or fragments thereof. The composition for diagnosis of radiation exposure according to claim 1, further comprising an agent for measuring the expression of MIF, GDF15 or a protein thereof, a phosphorylated protein thereof, or a fragment thereof. The composition for diagnosis of radiation exposure according to claim 1, wherein the agent to be measured is an antibody or antigen-binding fragment thereof that specifically binds to a protein, a phosphorylated protein thereof, or a fragment thereof. The composition for diagnosis of radiation exposure according to claim 3, wherein the antibody is a polyclonal antibody or a monoclonal antibody. A kit for diagnosing radiation exposure comprising the composition according to any one of claims 1 to 4. The kit for diagnosing radiation exposure according to claim 5, wherein the kit is an ELISA kit, a protein chip kit, a rapid kit, or a multiple reaction monitoring (MRM) kit. A method for diagnosing radiation exposure, comprising measuring the level of ATM, p53, H2AX and MCP-1 proteins, fragments thereof, or phosphorylation expression levels thereof in the isolated sample. The method of claim 7, wherein the diagnostic method further comprises measuring the expression level of MIF, GDF15 or both proteins, fragments thereof, or phosphorylation thereof. According to claim 7, Western blotting, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA: Radioimmunoassay), radioimmunodiffusion (radioimmunodiffusion), Ouchterlony (Ouchterlony) immunodiffusion method, rocket immunoelectrophoresis Electrophoresis, tissue immunostaining, immunoprecipitation assay (Immunoprecipitation Assay), Complement Fixation Assay (Complement Fixation Assay), FACS and protein chip (protein chip), including measurement by any one method selected from the group consisting of, Radiation exposure diagnosis method. The method for diagnosing radiation exposure according to claim 7, wherein ATM, p53 and H2AX provide information on the initial stage before 1 day after exposure. The method according to claim 7, wherein MCP-1 provides information on the late stage 1 day after exposure.

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