Testing method and test reagents

Abstract

This invention provides a COVID-19 severe disease risk assessment method that can accurately determine the risk of progressing from mild to moderate to severe, and the risk of progressing from mild to moderate to severe, in relation to the risk of severe COVID-19. [Solution] A method for testing the risk of severe illness from SARS-CoV-2 infection (COVID-19), wherein the risk of severe illness is the second-degree severe illness risk, which is the transition from stage (2) to stage (3) or (4) in the classification of severity, and the method includes a quantitative step of quantifying calprotectin in a sample, and further includes an evaluation step of comparing the quantitative value of calprotectin with a cutoff value that serves as an indicator for the second-degree severe illness risk, and evaluating the magnitude of the quantitative value in the sample, wherein the cutoff value is one point within the range of 3.5 to 8.1 μg / mL.

Description

[Technical Field]

[0001] This invention relates to a testing method and a testing reagent. [Background technology]

[0002] The options for detecting infection with the novel coronavirus (hereinafter also referred to as "SARS-CoV-2") are increasing, including PCR, antibody tests, and antigen tests. On the other hand, the rapid deterioration of symptoms during observation of positive patients makes elective treatment difficult, and there is a need for a testing method that does not require complicated procedures and can contribute to the early prediction of the risk of severe illness.

[0003] Patent Document 1 describes a method for testing the risk of severe SARS-CoV-2 infection (hereinafter also simply referred to as "COVID-19") using liver-type fatty acid-binding proteins in urine. Specifically, Patent Document 1 discloses a method for testing the risk of severe COVID-19, which includes a step of quantifying liver-type fatty acid-binding proteins in urine collected from a subject, and the results of the quantification are used for testing the risk of severe SARS-CoV-2 infection (COVID-19). [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Patent No. 6933834 [Overview of the project] [Problems that the invention aims to solve]

[0005] The risk factors for severe COVID-19 include the risk of progressing from mild to moderate to severe, and the risk of progressing from mild to moderate to severe. There was a need for testing methods that could accurately assess these risks.

[0006] This invention was made to solve the above problems, and the object of this invention is to provide a method for testing the risk of severe COVID-19, which can accurately determine the risk of progressing from mild and moderate to severe cases, and the risk of progressing from mild to moderate to severe cases. [Means for solving the problem]

[0007] As a result of diligent research to solve the above problems, the inventors of this invention have found that calprotectin, IFN-λ3, IL-6, CRP, white blood cell count, platelet count, neutrophil count to total white blood cell count, lymphocyte count to total white blood cell count, total bilirubin, urea nitrogen, creatinine, and TARC are useful indicators (biomarkers, etc.) for predicting the risk of severe COVID-19, and have completed the present invention. In other words, the present invention is a method for testing the risk of severe illness from SARS-CoV-2 infection (COVID-19), wherein the risk of severe illness is the first severe illness risk, which is the transition from stage (2) or stage (3) to stage (4) in the classification of severity below, and the method for testing the risk of severe illness from COVID-19 includes a quantitative step of quantifying at least one indicator selected from the group consisting of calprotectin, IFN-λ3, IL-6, CRP, white blood cell count, platelet count, ratio of neutrophils to total white blood cells, ratio of lymphocytes to total white blood cells, total bilirubin, urea nitrogen, creatinine, and TARC in the sample. <Classification of severity> (1) Oxygen administration is not required. (2) Oxygen administration is required. (3) Requires a high-flow nasal cannula, a non-invasive ventilator, or both. (4) Requires an invasive ventilator, extracorporeal membrane oxygenation (ECMO), or both.

[0008] Another aspect of the present invention is a method for testing the risk of severe illness from SARS-CoV-2 infection (COVID-19), wherein the risk of severe illness is a second-degree risk of progression from stage (2) to stage (3) or (4) in the classification of severity below, and the method for testing the risk of severe illness from COVID-19 includes a quantitative step of quantifying at least one indicator selected from the group consisting of calprotectin, IFN-λ3, IL-6, CRP, white blood cell count, platelet count, ratio of neutrophils to total white blood cells, ratio of lymphocytes to total white blood cells, total bilirubin, urea nitrogen, creatinine, and TARC in a sample. <Classification of severity> (1) Oxygen administration is not required. (2) Oxygen administration is required. (3) Requires a high-flow nasal cannula, a non-invasive ventilator, or both. (4) Requires an invasive ventilator, extracorporeal membrane oxygenation (ECMO), or both.

[0009] Another aspect of the present invention is a test reagent for predicting the risk of severe COVID-19, comprising at least one antibody selected from the group consisting of anti-calprotectin antibody, anti-IFN-λ3 antibody, anti-IL-6 antibody, anti-CRP antibody, anti-creatinine antibody, and anti-TARC antibody, which is used in the test method of the present invention described above. The diagnostic reagent of the present invention is used in the above quantitative step to quantify at least one biomarker selected from the group consisting of calprotectin, IFN-λ3, IL-6, CRP, creatinine, and TARC in a sample. [Effects of the Invention]

[0010] According to the present invention, it is possible to provide a method for testing the risk of severe COVID-19, which can accurately determine the risk of progressing from mild and moderate cases to severe cases, and the risk of progressing from mild to moderate and severe cases. [Brief explanation of the drawing]

[0011] [Figure 1]Figure 1 is a plot showing the calprotectin concentrations in the sera of Group 1 and Group 2 measured by the enzyme immunoassay (ELISA). [Figure 2] Figure 2 is a plot showing the calprotectin concentrations in the sera of Group 1 and Group 2 measured by the immunochromatography method. [Figure 3] Figure 3 is a plot showing the IFN-λ3 concentrations in the sera of Group 1 and Group 2 measured by the chemiluminescent enzyme immunoassay (CLEIA). [Figure 4] Figure 4 is a plot showing the IL-6 concentrations in the sera of Group 1 and Group 2 measured by the electrochemiluminescent immunoassay (ECLIA). [Figure 5] Figure 5 is a plot showing the CRP concentrations in the sera of Group 1 and Group 2 measured by the latex agglutination nephelometry method. [Figure 6] Figure 6 is a plot showing the white blood cell counts in the whole blood of Group 1 and Group 2 measured by a multi-item automated hematology analyzer. [Figure 7] Figure 7 is a plot showing the platelet counts in the whole blood of Group 1 and Group 2 measured by a multi-item automated hematology analyzer. [Figure 8] Figure 8 is a plot showing the ratio of neutrophil count to total white blood cell count in the whole blood of Group 1 and Group 2 measured by a multi-item automated hematology analyzer. [Figure 9] Figure 9 is a plot showing the ratio of lymphocyte count to total white blood cell count in the whole blood of Group 1 and Group 2 measured by a multi-item automated hematology analyzer. [Figure 10] Figure 10 is a plot showing the total bilirubin concentrations in the sera of Group 1 and Group 2 measured by the chemical oxidation method. [Figure 11] Figure 11 is a plot showing the urea nitrogen concentrations in the sera of Group 1 and Group 2 measured by the urease method. [Figure 12] Figure 12 is a plot showing the creatinine concentrations in the sera of Group 1 and Group 2 measured by the enzymatic method. [Figure 13]Figure 13 is a plot showing the TARC concentrations in the serum of Group 1 and Group 2, measured by chemiluminescent enzyme immunoassay (CLEIA). [Figure 14] Figure 14 shows the ROC curves for calprotectin concentrations in the serum of Group 1 and Group 2, measured by enzyme immunosorbent assay (ELISA). [Figure 15] Figure 15 shows the ROC curves for serum calprotectin concentrations in groups 1 and 2, measured by immunochromatography. [Figure 16] Figure 16 shows the ROC curves for IFN-λ3 concentrations in serum from groups 1 and 2, measured by chemiluminescent enzyme immunoassay (CLEIA). [Figure 17] Figure 17 shows the ROC curves for serum IL-6 concentrations in Group 1 and Group 2, measured by electrochemiluminescence immunoassay (ECLIA). [Figure 18] Figure 18 shows the ROC curves for CRP concentrations in serum from groups 1 and 2, measured by latex agglutination turbidimetry. [Figure 19] Figure 19 shows the ROC curves for the white blood cell count in whole blood of Group 1 and Group 2, measured using a multi-parameter automated hematology analyzer. [Figure 20] Figure 20 shows the ROC curves for platelet counts in whole blood of Group 1 and Group 2, measured using a multi-parameter automated hematology analyzer. [Figure 21] Figure 21 shows the ROC curves for the ratio of neutrophils to total white blood cells in the whole blood of Group 1 and Group 2, measured using a multi-parameter automated hematology analyzer. [Figure 22] Figure 22 shows the ROC curves for the ratio of lymphocytes to total white blood cells in the whole blood of Group 1 and Group 2, measured using a multi-parameter automated hematology analyzer. [Figure 23] Figure 23 shows the ROC curves for total bilirubin concentrations in serum of Group 1 and Group 2, measured by chemical oxidation. [Figure 24] Figure 24 shows the ROC curves for serum urea nitrogen concentrations in groups 1 and 2, measured by the urease method. [Figure 25] Figure 25 shows the ROC curves for serum creatinine concentrations in groups 1 and 2, measured by the enzymatic method. [Figure 26] Figure 26 shows the ROC curves for TARC concentrations in serum of Group 1 and Group 2, measured by chemiluminescent enzyme immunoassay (CLEIA). [Figure 27] Figure 27 is a plot showing the calprotectin concentrations in the serum of groups 3 and 4, as measured by enzyme immunosorbent assay (ELISA). [Figure 28] Figure 28 is a plot showing the calprotectin concentrations in the serum of groups 3 and 4, as measured by immunochromatography. [Figure 29] Figure 29 is a plot showing the IFN-λ3 concentrations in the serum of groups 3 and 4, as measured by chemiluminescent enzyme immunoassay (CLEIA). [Figure 30] Figure 30 is a plot showing the IL-6 concentrations in the serum of groups 3 and 4, as measured by electrochemiluminescence immunoassay (ECLIA). [Figure 31] Figure 31 is a plot showing the CRP concentrations in the serum of groups 3 and 4, measured by latex agglutination turbidimetry. [Figure 32] Figure 32 is a plot showing the white blood cell counts in the whole blood of groups 3 and 4, as measured by a multi-parameter automated blood cell analyzer. [Figure 33] Figure 33 is a plot showing the platelet counts in whole blood of groups 3 and 4, as measured by a multi-parameter automated hematology analyzer. [Figure 34] Figure 34 is a plot showing the ratio of neutrophils to total white blood cells in the whole blood of groups 3 and 4, as measured by a multi-parameter automated hematology analyzer. [Figure 35] Figure 35 is a plot showing the ratio of lymphocytes to total white blood cells in the whole blood of groups 3 and 4, as measured by a multi-parameter automated hematology analyzer. [Figure 36] Figure 36 is a plot showing the total bilirubin concentrations in the serum of groups 3 and 4, as measured by chemical oxidation. [Figure 37] Figure 37 is a plot showing the urea nitrogen concentrations in the serum of groups 3 and 4, as measured by the urease method. [Figure 38] Figure 38 is a plot showing the serum creatinine concentrations of groups 3 and 4, measured by the enzymatic method. [Figure 39] Figure 39 is a plot showing the TARC concentrations in the serum of groups 3 and 4, as measured by chemiluminescent enzyme immunoassay (CLEIA). [Figure 40] Figure 40 shows the ROC curves for serum calprotectin concentrations in groups 3 and 4, measured by enzyme immunosorbent assay (ELISA). [Figure 41] Figure 41 shows the ROC curves for serum calprotectin concentrations in groups 3 and 4, measured by immunochromatography. [Figure 42] Figure 42 shows the ROC curves for IFN-λ3 concentrations in serum from groups 3 and 4, measured by chemiluminescent enzyme immunoassay (CLEIA). [Figure 43] Figure 43 shows the ROC curves for serum IL-6 concentrations in groups 3 and 4, measured by electrochemiluminescence immunoassay (ECLIA). [Figure 44] Figure 44 shows the ROC curves for CRP concentrations in serum from groups 3 and 4, measured by latex agglutination turbidimetry. [Figure 45] Figure 45 shows the ROC curves for the white blood cell count in whole blood of groups 3 and 4, measured using a multi-parameter automated hematology analyzer. [Figure 46] Figure 46 shows the ROC curves for platelet counts in whole blood of groups 3 and 4, measured using a multi-parameter automated hematology analyzer. [Figure 47] Figure 47 shows the ROC curves for the ratio of neutrophils to total white blood cells in the whole blood of groups 3 and 4, measured using a multi-parameter automated hematology analyzer. [Figure 48]Figure 48 shows the ROC curves for the ratio of lymphocytes to total white blood cells in whole blood of groups 3 and 4, measured using a multi-parameter automated hematology analyzer. [Figure 49] Figure 49 shows the ROC curves for total bilirubin concentrations in serum of groups 3 and 4, measured by chemical oxidation. [Figure 50] Figure 50 shows the ROC curves for serum urea nitrogen concentrations in groups 3 and 4, measured by the urease method. [Figure 51] Figure 51 shows the ROC curves for serum creatinine concentrations in groups 3 and 4, measured by the enzymatic method. [Figure 52] Figure 52 shows the ROC curves for TARC concentrations in serum from groups 3 and 4, measured by chemiluminescent enzyme immunoassay (CLEIA). [Modes for carrying out the invention]

[0012] (First Embodiment) The inspection method according to the first embodiment of the present invention will be described in detail below. A testing method according to the first embodiment of the present invention is a testing method for the risk of severe illness of SARS-CoV-2 infection (COVID-19), wherein the risk of severe illness is the first severe illness risk, which is the transition from stage (2) or stage (3) to stage (4) in the classification of severity below, and includes a quantitative step of quantifying at least one indicator selected from the group consisting of calprotectin, IFN-λ3, IL-6, CRP, white blood cell count, platelet count, ratio of neutrophils to total white blood cells, ratio of lymphocytes to total white blood cells, total bilirubin, urea nitrogen, creatinine, and TARC in the sample. <Classification of severity> (1) Oxygen administration is not required. (2) Oxygen administration is required. (3) Requires a high-flow nasal cannula, a non-invasive ventilator, or both. (4) Requires an invasive ventilator, extracorporeal membrane oxygenation (ECMO), or both.

[0013] Calprotectin is a component found in neutrophils. It is known that when inflammation occurs in tissue, neutrophils gather in that area and release calprotectin.

[0014] IFN-λ3 is a type of cytokine and is known as one of the inhibitors of viral infection.

[0015] IL-6 is a type of inflammatory cytokine known to be elevated in infections, trauma, and autoimmune diseases. It is also known to activate the immune response.

[0016] CRP is known as one of the acute-phase response proteins secreted by liver and spleen cells when inflammation or tissue destruction occurs.

[0017] White blood cells are known as cells that play a role in eliminating foreign substances such as bacteria and viruses that have entered the body from outside, as well as tumor cells.

[0018] Platelets are a type of cellular component found in blood, and they are known to gather together when the blood vessel wall is damaged, sealing the wound and stopping bleeding.

[0019] Neutrophils are a type of white blood cell that exhibits migratory behavior towards inflammatory cytokines and components of bacteria and fungi. They are known to gather at the site of inflammation and protect the body by phagocytosing, killing, and breaking down foreign substances such as bacteria and fungi.

[0020] Lymphocytes are a type of white blood cell and are known to possess various immune responses, such as antibody production.

[0021] Bilirubin is one of the main components of red blood cells, and abnormally elevated levels are known to indicate some kind of disease. Total bilirubin refers to the total amount of direct and indirect bilirubin in the blood.

[0022] Urea nitrogen is a unit that indicates the amount of nitrogen derived from urea, and an increase in its measured value is known to be related to decreased renal function.

[0023] Creatinine is known as the final metabolite of creatine, which is synthesized in the liver and kidneys and used in muscle contraction.

[0024] TARC is a CC chemokine (CCL17) and is known as a molecule that causes lymphocytes (Th2 cells expressing CCR4) to migrate to the site of inflammation.

[0025] When humans are infected with SARS-CoV-2, the expression levels of calprotectin, IFN-λ3, IL-6, and CRP increase, while white blood cell count, neutrophil ratio to total white blood cell count, total bilirubin, urea nitrogen, and creatinine increase, and platelet count, lymphocyte ratio to total white blood cell count, and TARC expression decrease. In particular, patients at high risk of developing severe illness (I-1) show even greater increases in these expression levels. Therefore, these indicators can be used to assess the risk of developing severe illness (I-1). For example, when using calprotectin, IFN-λ3, IL-6, or CRP as indicators, the risk of developing severe illness (I-1) can be assessed by quantitative values ​​obtained from these biomarkers. In the following explanation, if it is not necessary to distinguish between calprotectin, IFN-λ3, IL-6, CRP, white blood cell count, platelet count, neutrophil ratio to total white blood cell count, lymphocyte ratio to total white blood cell count, total bilirubin, urea nitrogen, creatinine, and TARC, then "any one of the following indicators" will also be referred to simply as "the above indicators."

[0026] We will now explain more specific methods for testing for the risk of developing severe illness (level 1). When a person is infected with SARS-CoV-2, if the above indicators are above or below a certain value (i.e., a cutoff value), the risk of developing severe illness (first-degree severe illness) increases.

[0027] Therefore, the testing method according to the first embodiment of the present invention may include an evaluation step in which the quantitative value of at least one indicator selected from the group consisting of calprotectin, IFN-λ3, IL-6, CRP, white blood cell count, platelet count, ratio of neutrophils to total white blood cells, ratio of lymphocytes to total white blood cells, total bilirubin, urea nitrogen, creatinine, and TARC is compared with a predetermined numerical value (i.e., a cutoff value) that serves as an indicator for the risk of first-degree severe illness, and the magnitude of the quantitative value of at least one indicator selected from the group consisting of calprotectin, IFN-λ3, IL-6, CRP, white blood cell count, platelet count, ratio of neutrophils to total white blood cells, ratio of lymphocytes to total white blood cells, total bilirubin, urea nitrogen, creatinine, and TARC in the sample is evaluated.

[0028] In the testing method according to the first embodiment of the present invention, the specimen for measuring at least one indicator from the group consisting of calprotectin, IFN-λ3, IL-6, CRP, white blood cell count, platelet count, neutrophil count to total white blood cell count, lymphocyte count to total white blood cell count, total bilirubin, urea nitrogen, creatinine, and TARC is not particularly limited, but at least one selected from the group consisting of body fluids, cells, and tissues can be used. Furthermore, the body fluid is preferably at least one selected from the group consisting of blood (whole blood, etc.), urine, cerebrospinal fluid, saliva, lymph, pleural fluid, ascites, and bile. Among these, blood is more preferred, and it is even more preferred to be one of whole blood, serum, or plasma.

[0029] In performing the testing method according to the first embodiment of the present invention, the timing of sample collection is preferably within 3 days after the start of oxygen inhalation, and more preferably within 1 day.

[0030] For determining the risk of severe illness, the cutoff value for calprotectin is preferably one point within the range of 8.2 to 19.0 μg / mL, and more preferably one point within the range of 10.9 to 16.3 μg / mL, when using serum as the sample. If the calprotectin concentration in a sample is measured and found to be higher than the cutoff value, the patient from whom the sample was collected can be judged to be at high risk of developing severe illness (level 1).

[0031] For determining the risk of developing severe illness (level 1), the cutoff value for IFN-λ3 is preferably one point within the range of 18.8 to 43.8 pg / mL, and more preferably one point within the range of 25.0 to 37.6 pg / mL, when using serum as the sample. If the concentration of IFN-λ3 in a sample is measured and the result is higher than the cutoff value, the patient from whom the sample was collected can be judged to be at high risk of developing severe illness (level 1).

[0032] For determining the risk of severe illness, the IL-6 cutoff value is preferably one point within the range of 25.4 to 59.4 pg / mL, and more preferably one point within the range of 33.9 to 50.9 pg / mL, when using serum as the sample. If the concentration of IL-6 in a sample is measured and the result is higher than the cutoff value, the patient from whom the sample was collected can be judged to be at high risk of developing severe illness (level 1).

[0033] For determining the risk of developing a first-degree severe illness, the cutoff value for CRP is preferably one point within the range of 1.80 to 4.20 mg / dL, and more preferably one point within the range of 2.40 to 3.60 mg / dL, when using serum as the sample. If the CRP concentration in a sample is measured and the result is higher than the cutoff value, the patient from whom the sample was collected can be judged to be at high risk of developing severe illness (level 1).

[0034] For determining the risk of developing a first-degree severe illness, the cutoff value for white blood cell count is preferably one point within the range of 4.80 to 11.20 cells / nL, and more preferably one point within the range of 6.40 to 9.60 cells / nL, when whole blood is used as the sample. If the white blood cell count in a sample is measured and the result is higher than the cutoff value, the patient from whom the sample was collected can be judged to be at high risk of developing severe illness (level 1).

[0035] The platelet count cutoff value for determining the risk of severe illness (level 1) is, for example, 12.9 to 30.1 × 10⁻¹⁴ when using whole blood as the sample. 4 It is preferable that the point be within the range of individual units / μL units / , and is 17.2~25.8×10 4 It is more preferable that the point be within the range of particles / μL. If the platelet count in a sample is measured and found to be lower than the cutoff value, the patient from whom the sample was collected can be judged to be at high risk of developing severe illness (level 1).

[0036] For determining the risk of first-degree severe illness, the cutoff value for the ratio of neutrophils to total white blood cells is preferably one point within the range of 54.0-100.0%, and more preferably one point within the range of 72.0-100.0%, when whole blood is used as the sample. If the percentage of neutrophils in a sample is measured and the result is higher than the cutoff value, the patient from whom the sample was collected can be judged to be at high risk of developing severe illness (level 1).

[0037] For determining the risk of developing a first-degree severe illness, the cutoff value for the ratio of lymphocytes to total white blood cells is preferably one point within the range of 4.8-11.2%, and more preferably one point within the range of 6.4-9.6%, when whole blood is used as the sample. If the percentage of lymphocytes in a sample is measured and the result is lower than the cutoff value, the patient from whom the sample was collected can be judged to be at high risk of developing severe illness (level 1).

[0038] For determining the risk of developing a primary severe illness, the cutoff value for total bilirubin is preferably one point within the range of 0.3 to 0.6 mg / dL, and more preferably one point within the range of 0.4 to 0.5 mg / dL, when using serum as the sample. If the total bilirubin concentration in a sample is measured and found to be higher than the cutoff value, the patient from whom the sample was collected can be judged to be at high risk of developing severe illness (level 1).

[0039] For determining the risk of severe illness, the urea nitrogen cutoff value is preferably one point within the range of 13.2 to 30.8 mg / dL, and more preferably one point within the range of 17.6 to 26.4 mg / dL, when using serum as the sample. If the urea nitrogen concentration in a sample is measured and found to be higher than the cutoff value, the patient from whom the sample was collected can be judged to be at high risk of developing severe illness (level 1).

[0040] For determining the risk of developing a first-degree severe illness, the creatinine cutoff value is preferably one point within the range of 0.6 to 1.4 mg / dL, and more preferably one point within the range of 0.8 to 1.2 mg / dL, when using serum as the sample. If the creatinine concentration in a sample is measured and found to be higher than the cutoff value, the patient from whom the sample was collected can be judged to be at high risk of developing severe illness (level 1).

[0041] For determining the risk of severe illness (level 1), the TARC cutoff value is preferably one point within the range of 60.0 to 140.0 pg / mL, and more preferably one point within the range of 80.0 to 120.0 pg / mL, when using serum as the sample. If the TARC concentration in a sample is measured and found to be lower than the cutoff value, the patient from whom the sample was collected can be judged to be at high risk of developing severe illness (level 1).

[0042] The above cutoff value can be set, for example, by creating an ROC curve (Receiver Operating Characteristic Curve) from the quantitative values ​​of the above indicator in samples from patient groups that did not progress from stage (2) or stage (3) to stage (4) in the classification of severity above, and from the quantitative values ​​of the above indicator in samples from patient groups that progressed from stage (2) or stage (3) to stage (4). Furthermore, the area under the ROC curve is preferably 0.65 or greater, more preferably 0.75 or greater, and even more preferably 0.80 or greater.

[0043] Furthermore, since the cutoff value can vary depending on the testing method, it is preferable to set it appropriately for each testing method.

[0044] In the testing method according to the first embodiment of the present invention, the quantification of calprotectin, IFN-λ3, IL-6, CRP, creatinine, and TARC among the above indicators (i.e., when the quantification step is a quantification step in which at least one biomarker selected from the group consisting of calprotectin, IFN-λ3, IL-6, CRP, creatinine, and TARC in the sample) is preferably performed by immunological measurement. Furthermore, it is more preferable that the quantification by immunological measurement uses an antibody against at least one biomarker selected from the group consisting of calprotectin, IFN-λ3, IL-6, CRP, creatinine, and TARC (i.e., an anti-calprotectin antibody, an anti-IFN-λ3 antibody, an anti-IL-6 antibody, an anti-CRP antibody, an anti-creatinine antibody, and an anti-TARC antibody). By measuring the above biomarkers using immunological assays, the biomarkers can be quantified with high sensitivity, accuracy, speed, and convenience. In the testing method according to the first embodiment of the present invention, among the above indicators, the quantitative determination of white blood cell count, platelet count, neutrophil count as the total white blood cell count, lymphocyte count as the total white blood cell count, total bilirubin, and urea nitrogen can be performed using known methods or the like. In addition to the immunological quantification method described above, creatinine can also be quantified using an enzymatic method.

[0045] In the testing method according to the first embodiment of the present invention, monoclonal antibodies and / or polyclonal antibodies against the biomarker can be used for quantitative analysis by immunoassay. Furthermore, monoclonal antibodies are used because they exhibit good specificity for the above biomarkers.

[0046] In the testing method according to the first embodiment of the present invention, examples of immunological measurement methods include radioimmunoassay (RIA), fluorescence immunoassay (FIA), enzyme immunoassay (EIA), luminescence immunoassay (CLIA), electrochemiluminescence immunoassay (ECLIA), and immunochromatography. Of these, fluorescence immunoassay (FIA), enzyme immunoassay (EIA), and luminescence immunoassay (CLIA) are more preferred from the viewpoint of measurement sensitivity, and enzyme immunoassay (EIA) is even more preferred. As for enzyme immunoassay (EIA), chemiluminescent enzyme immunoassay (CLEIA) and ELISA are preferred, using a chemiluminescent substance as the enzyme substrate.

[0047] In the testing method according to the first embodiment of the present invention, when quantifying the above biomarker in a sample by enzyme immunoassay (EIA), the biomarker and / or its analog introduced from an external source, the antibody against the biomarker, or the secondary antibody is labeled for chemical color development and luminescence. The label is not particularly limited, but is preferably alkaline phosphatase, β-galactosidase, peroxidase (POD), microperoxidase, glucose oxidase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, luciferase, tyrosinase, or acid phosphatase. Furthermore, from the viewpoint of detection sensitivity and other factors, it is more preferable to use alkaline phosphatase, peroxidase, or glucose oxidase as the label used for chemical color development and luminescence, and it is even more preferable to use peroxidase.

[0048] In the testing method according to the first embodiment of the present invention, when quantifying the biomarker by immunoassay, an antibody against the biomarker, or the biomarker and / or its analog, etc., is supported on a solid support, and the biomarker is quantified using the solid support. The solid-phase support used in the inspection method according to the first embodiment of the present invention is not particularly limited, but examples include glass beads, polystyrene beads, magnetic particles such as magnetic silica particles, microplates, latex, etc. Of these, magnetic particles are preferred from the viewpoint of detection sensitivity and other factors.

[0049] In the testing method according to the first embodiment of the present invention, the immunological measurement can be, for example, the measurement described in Enzyme Immunoassay (published May 15, 1987, 3rd edition, Igaku-Shoin Co., Ltd.), and may be a non-competitive immunological measurement or a competitive immunological measurement. Of these, a non-competitive immunological measurement is preferred from the viewpoint of detection sensitivity, etc.

[0050] In this specification, non-competitive immunological measurement means a measurement that includes the step of conjugating the biomarker in a sample with an antibody against the biomarker without adding the biomarker and / or its analog from an external source and without competition. In this specification, competitive immunoassay means a measurement that includes the step of adding the biomarker and / or its analog from an external source, causing the biomarker in the sample to compete with the biomarker and / or its analog introduced from an external source to bind to an antibody against the biomarker.

[0051] The following describes a first non-competitive immunoassay, which is an example of a testing method for quantifying the above biomarker in a sample by non-competitive immunoassay according to the first embodiment of the present invention.

[0052] (1) Preparation process for inspection materials In this process, the materials to be used for the test are prepared: a solid support carrying antibodies against the biomarker, an immunoassay buffer, and labeled antibodies against the biomarker.

[0053] We will now describe the preparation of a solid support carrying antibodies against the above biomarkers. The method for loading antibodies against the above biomarker onto a solid support is not particularly limited, and conventional methods can be used. The solid phase support is not particularly limited, but examples include glass beads, polystyrene beads, magnetic particles such as magnetic silica particles, microplates, and latex. Of these, glass beads and / or magnetic silica particles are preferred from the viewpoint of detection sensitivity and other factors. Furthermore, antibodies against the above biomarkers supported on a solid phase carrier will also be referred to simply as "antibodies supported on a solid phase" below.

[0054] Next, we will explain the buffer solutions used for immunoassays. The buffer solution for the immunoassay is preferably one that has a buffering effect at pH 5.0 to 10.0, and more preferably one that has a buffering effect at pH 6.0 to 9.0. Examples of such buffer solutions for immunoassays include phosphate buffer, Tris buffer, Good's buffer, glycine buffer, and borate buffer.

[0055] Next, we will describe the preparation of antibodies against the labeled biomarkers mentioned above. The label attached to the antibody against the above biomarker may be a radioisotope label or a chemically color-developing or luminescent label. Of these, a chemically color-developing or luminescent label is preferred from a safety standpoint. These labels can be attached to antibodies against the above biomarkers by conventional methods. Furthermore, antibodies against the labeled biomarkers mentioned above will also be referred to simply as "labeled antibodies" below.

[0056] (2) Calibration curve creation process (2-1) Steps for preparing biomarker standard solutions The above biomarker is added to the above immunoassay buffer to prepare several biomarker standard solutions at different concentrations.

[0057] (2-2) Step of forming antibody-biomarker complex supported on a solid support First, the biomarkers in the biomarker standard solutions of each concentration are reacted with the antibodies supported on the solid support. The immunoassay buffer is used as the buffer for this reaction. This reaction allows for the formation of antibody-biomarker complexes supported on a solid support. Subsequently, the unreacted biomarkers are removed from the reaction system.

[0058] (2-3) Step of forming an antibody-biomarker-labeled antibody complex supported on a solid phase carrier Next, the labeled antibody is added to the reaction system, and the antibody-biomarker complex supported on the solid support is reacted with the antibody against the labeled biomarker. This reaction can form an antibody-biomarker-labeled antibody complex supported on a solid support. Subsequently, the unreacted target antibody is removed from the reaction system.

[0059] (2-4) Marker counting step Next, the labels on the antibody-biomarker-labeled antibody complexes supported on the solid support remaining in the reaction system are counted. The number of signs can be counted using conventional methods, depending on the type of sign.

[0060] (2-5) Calibration curve creation step Based on the concentrations of each standard solution for the biomarkers mentioned above, and the resulting count of labels, a calibration curve is created showing the relationship between the biomarker concentration and the count of labels.

[0061] (3) Steps for forming antibody-biomarker complexes supported on a solid support The above-mentioned immunoassay buffer is used as the buffer for the reaction system, and the biomarker in the sample is reacted with the antibody supported on the solid support. This reaction allows for the formation of antibody-biomarker complexes supported on a solid support. Subsequently, the unreacted biomarkers are removed from the reaction system.

[0062] (4) Process for forming antibody-biomarker-labeled antibody complexes supported on a solid support Next, the labeled antibody is added to the reaction system, and the antibody-biomarker complex supported on the solid support reacts with the labeled antibody. This reaction can form an antibody-biomarker-labeled antibody complex supported on a solid support. Subsequently, the unreacted labeled antibody is removed from the reaction system.

[0063] (5) Label counting process Next, the labels on the antibody-biomarker-labeled antibody complexes supported on the solid support remaining in the reaction system are counted. The number of signs can be counted using conventional methods, depending on the type of sign.

[0064] (6) Quantification process Next, the concentration of the biomarker in the sample is calculated based on the obtained count and the created calibration curve. Through the above process, the above biomarkers in the sample can be quantified.

[0065] In addition, in the testing method according to the first embodiment of the present invention, non-competitive immunological measurement may be performed using a labeled secondary antibody instead of a labeled antibody.

[0066] Next, a second non-competitive immunoassay, which is an example of a testing method for quantifying the above biomarker in a sample by a non-competitive immunoassay relating to the first embodiment of the present invention, will be described.

[0067] (1) Preparation process for inspection materials In this process, the materials to be used for testing are prepared: a solid support, an immunoassay buffer, an antibody against the first biomarker to which the secondary antibody cannot bind (hereinafter also simply referred to as the "first antibody"), an antibody against the second biomarker to which the secondary antibody can bind (hereinafter also simply referred to as the "second antibody"), and a labeled secondary antibody.

[0068] First, the first antibody is loaded onto a solid support. The same type of solid support described in the first non-competitive immunoassay can be used.

[0069] Furthermore, a label is attached to the secondary antibody to produce a labeled secondary antibody. The label attached to the secondary antibody may be a radioactive isotope label, or a chemically color-developing or luminescent label. Of these, a chemically color-developing or luminescent label is preferred from a safety standpoint. These labels can be attached to secondary antibodies using conventional methods.

[0070] As the immunoassay buffer, the same immunoassay buffer described in the first non-competitive immunoassay above can be used.

[0071] (2) Calibration curve creation process (2-1) Steps for preparing biomarker standard solutions The above biomarker is added to the above immunoassay buffer to prepare several biomarker standard solutions at different concentrations.

[0072] (2-2) First antibody-biomarker complex formation step The biomarkers in the biomarker standard solutions of each concentration are reacted with the first antibody supported on a solid support. In this process, the immunoassay buffer is used as the buffer for the reaction system. This reaction allows for the formation of the first antibody-biomarker complex. Subsequently, the unreacted biomarkers are removed from the reaction system.

[0073] (2-3) Step of forming a first antibody-biomarker-second antibody complex Next, the second antibody is added to the reaction system, and the first antibody-biomarker complex reacts with the second antibody. This reaction can form a first antibody-biomarker-second antibody complex. Next, the unreacted second antibody is removed from the reaction system.

[0074] (2-4) Labeled secondary antibody binding step Next, the labeled secondary antibody is added to the reaction system, and the first antibody-biomarker-second antibody complex is reacted with the labeled secondary antibody. This allows for the formation of a first antibody-biomarker-second antibody-labeled secondary antibody complex. Next, the unreacted labeled secondary antibody is removed from the reaction system.

[0075] (2-5) Marker counting step Next, the labels of the first antibody-biomarker-second antibody-labeled secondary antibody complex remaining in the reaction system are counted. The number of signs can be counted using conventional methods, depending on the type of sign.

[0076] (2-6) Calibration curve creation step Based on the concentrations of each of the above biomarker standard solutions and the number of labels obtained, a calibration curve is created showing the relationship between the biomarker concentration and the number of labels.

[0077] (3) First antibody-biomarker complex formation step The above-mentioned immunoassay buffer is used as the buffer for the reaction system, and the biomarker in the sample is reacted with the first antibody supported on a solid support. This reaction allows for the formation of the first antibody-biomarker complex. Subsequently, the unreacted biomarkers are removed from the reaction system.

[0078] (4) First antibody-biomarker-second antibody complex formation process Next, the second antibody is added to the reaction system, and the first antibody-biomarker complex reacts with the second antibody. This reaction can form a first antibody-biomarker-second antibody complex. Subsequently, the unreacted second antibody is removed from the reaction system.

[0079] (5) Labeled secondary antibody binding step Next, the labeled secondary antibody is added to the reaction system, and the first antibody-biomarker-second antibody complex is reacted with the labeled secondary antibody. This allows for the formation of a first antibody-biomarker-second antibody-labeled secondary antibody complex. Subsequently, the unreacted labeled secondary antibody is removed from the reaction system.

[0080] (6) Label counting process Next, the labels of the first antibody-biomarker-second antibody-labeled secondary antibody complex remaining in the reaction system are counted. The number of signs can be counted using conventional methods, depending on the type of sign.

[0081] (7) Quantification process Next, the concentration of the biomarker in the sample is calculated based on the obtained count and the created calibration curve. Through the above process, the above biomarkers in the sample can be quantified.

[0082] An example of a testing method for quantifying the above biomarker in a sample by competitive immunoassay according to the first embodiment of the present invention is described below. For convenience, in the following explanation of competitive immunological measurements, the above biomarkers that are not labeled will be referred to as "unlabeled biomarkers," and the above biomarkers that are labeled will be referred to as "labeled biomarkers."

[0083] (1) Preparation process for inspection materials In this process, the materials to be used for the test are prepared: a solid support carrying an antibody against the biomarker, an immunoassay buffer, and a labeled biomarker. The solid support carrying the antibody against the above biomarker and the immunoassay buffer can be those described in the explanation of the first non-competitive immunoassay above. Furthermore, antibodies against the above biomarkers supported on a solid support will hereafter be simply referred to as "antibodies supported on a solid support."

[0084] This section explains labeled biomarkers. Labeled biomarkers can be prepared by labeling unlabeled biomarkers. The label may be a radioactive isotope label, or it may be a chemically colored or luminescent label. Of these, a chemically colored or luminescent label is preferred from a safety standpoint. These labels can be attached to unlabeled biomarkers using conventional methods.

[0085] (2) Calibration curve creation process (2-1) Steps for preparing unlabeled biomarker standard solutions Unlabeled biomarkers are added to the above-mentioned immunoassay buffer to prepare several unlabeled biomarker standard solutions of different concentrations.

[0086] (2-2) Preparation step of unlabeled biomarker-labeled biomarker mixture First, a certain amount of labeled biomarker is added to each unlabeled biomarker standard solution and mixed to prepare an unlabeled biomarker-labeled biomarker mixture.

[0087] (2-3) Steps for forming antibody-biomarker complexes supported on a solid support Next, the unlabeled biomarker and labeled biomarker in the unlabeled biomarker-labeled biomarker mixture are reacted with the antibody supported on the solid support. During this reaction, the unlabeled biomarker and the labeled biomarker compete to bind to the antibody supported on the solid support. This reaction can form antibody-unlabeled biomarker complexes supported on a solid support and antibody-labeled biomarker complexes supported on a solid support. In this reaction, if the amount of unlabeled biomarker in the unlabeled biomarker-labeled biomarker mixture is large, the amount of antibody-labeled biomarker complex supported on the solid support will be small. Furthermore, if the amount of unlabeled biomarker in the unlabeled biomarker-labeled biomarker mixture is small, the amount of antibody-labeled biomarker complex supported on the solid support will increase. Subsequently, unreacted unlabeled and labeled biomarkers are removed.

[0088] (2-4) Marker counting step Next, the labels of the antibody-labeled biomarker complexes supported on the solid support remaining in the reaction system are counted. The number of signs can be counted using conventional methods, depending on the type of sign.

[0089] (2-5) Calibration curve creation step Based on the concentrations of each unlabeled biomarker standard solution and the resulting number of labeled biomarkers, a calibration curve is created showing the relationship between the unlabeled biomarker concentration and the number of labeled biomarkers.

[0090] (3) Preparation of unlabeled biomarker-labeled biomarker mixture Next, the sample and a certain amount of labeled biomarker are added to the immunoassay buffer and mixed to prepare a sample-labeled biomarker mixture. Since the above biomarkers in the sample are not labeled, they will be referred to as "unlabeled biomarkers" in the following explanation.

[0091] (4) Steps for forming antibody-biomarker complexes supported on a solid support Next, the unlabeled and labeled biomarkers in the sample-labeled biomarker mixture are reacted with the antibody supported on the solid support. During this reaction, the unlabeled and labeled biomarkers compete to bind to the antibody supported on the solid support. This reaction can form antibody-unlabeled biomarker complexes supported on a solid support and antibody-labeled biomarker complexes supported on a solid support. Subsequently, unreacted unlabeled and labeled biomarkers are removed.

[0092] (5) Label counting process Next, the labels of the antibody-labeled biomarker complexes supported on the solid support remaining in the reaction system are counted. The number of signs can be counted using conventional methods, depending on the type of sign.

[0093] (6) Quantification process Next, the concentration of the biomarker (unlabeled biomarker) in the sample is calculated based on the obtained count and the created calibration curve. Through the above process, the above biomarkers in the sample can be quantified.

[0094] (Second Embodiment) A testing method according to a second embodiment of the present invention is a testing method for the risk of severe illness of SARS-CoV-2 infection (COVID-19), wherein the risk of severe illness is a second-degree risk of progression from stage (2) to stage (3) or (4) in the classification of severity below, and includes a quantitative step of quantifying at least one indicator selected from the group consisting of calprotectin, IFN-λ3, IL-6, CRP, white blood cell count, platelet count, ratio of neutrophils to total white blood cells, ratio of lymphocytes to total white blood cells, total bilirubin, urea nitrogen, creatinine, and TARC in the sample. <Classification of severity> (1) Oxygen administration is not required. (2) Oxygen administration is required. (3) Requires a high-flow nasal cannula, a non-invasive ventilator, or both. (4) Requires an invasive ventilator, extracorporeal membrane oxygenation (ECMO), or both.

[0095] The classification of severity in the examination method according to the second embodiment of the present invention is the same as the classification of severity in the examination method according to the first embodiment of the present invention.

[0096] In the examination method according to the second embodiment of the present invention, the risk of progression to severe illness from stage (2) to stage (3) or stage (4) is referred to as the "second severe illness risk."

[0097] When humans are infected with SARS-CoV-2, the expression levels of calprotectin, IFN-λ3, IL-6, and CRP increase, while white blood cell count, the ratio of neutrophils to total white blood cells, total bilirubin, urea nitrogen, and creatinine increase, and platelet count, the ratio of lymphocytes to total white blood cells, and TARC expression decrease. In particular, patients at high risk of developing 2 severe illness tend to have more increased or decreased levels of these factors. Therefore, these indicators can be used to assess the risk of developing 2 severe illness. For example, when using calprotectin, IFN-λ3, IL-6, or CRP as indicators, the risk of developing 2 severe illness can be assessed by measuring the quantitative values ​​of these biomarkers.

[0098] We will now explain more specific methods for testing for the risk of developing a second severe illness. When a person is infected with SARS-CoV-2, if the above indicators are above or below a certain value (i.e., a cutoff value), the risk of developing a second-degree severe illness, progressing from mild to moderate or severe, increases.

[0099] Therefore, the testing method according to the second embodiment of the present invention may include an evaluation step in which the quantitative value of at least one indicator selected from the group consisting of calprotectin, IFN-λ3, IL-6, CRP, white blood cell count, platelet count, ratio of neutrophils to total white blood cells, ratio of lymphocytes to total white blood cells, total bilirubin, urea nitrogen, creatinine, and TARC is compared with a predetermined numerical value (i.e., a cutoff value) that serves as an indicator for the risk of second-degree severe illness, and the magnitude of the quantitative value of at least one indicator selected from the group consisting of calprotectin, IFN-λ3, IL-6, CRP, white blood cell count, platelet count, ratio of neutrophils to total white blood cells, ratio of lymphocytes to total white blood cells, total bilirubin, urea nitrogen, creatinine, and TARC in the sample is evaluated.

[0100] In the testing method according to the second embodiment of the present invention, the preferred sample for measuring at least one indicator from the group consisting of calprotectin, IFN-λ3, IL-6, CRP, white blood cell count, platelet count, neutrophil count to total white blood cell count, lymphocyte count to total white blood cell count, total bilirubin, urea nitrogen, creatinine, and TARC is the same as the preferred sample in the testing method according to the first embodiment of the present invention. In performing the inspection method according to the second embodiment of the present invention, the preferred time for collecting the sample is the same as the preferred time for collecting the sample according to the first embodiment of the present invention.

[0101] For determining the risk of developing a second severe illness, the cutoff value for calprotectin is preferably one point within the range of 3.5 to 8.1 μg / mL, and more preferably one point within the range of 4.6 to 7.0 μg / mL, when using serum as the sample. If the calprotectin concentration in a sample is measured and found to be higher than the cutoff value, the patient from whom the sample was collected can be judged to be at high risk of developing a second-degree severe illness.

[0102] For determining the risk of developing a second severe illness, the cutoff value for IFN-λ3 is preferably one point within the range of 10.0 to 23.2 pg / mL, and more preferably one point within the range of 13.3 to 19.9 pg / mL, when using serum as the sample. If the concentration of IFN-λ3 in a sample is measured and the result is higher than the cutoff value, the patient from whom the sample was collected can be judged to be at high risk of developing a second-degree severe illness.

[0103] For determining the risk of developing a second severe illness, the IL-6 cutoff value is preferably one point within the range of 25.4 to 59.4 pg / mL, and more preferably one point within the range of 33.9 to 50.9 pg / mL, when using serum as the sample. If the concentration of IL-6 in a sample is measured and the result is higher than the cutoff value, the patient from whom the sample was collected can be judged to be at high risk of developing a second-degree severe illness.

[0104] For determining the risk of developing a second severe illness, the cutoff value for CRP is preferably one point within the range of 1.50 to 3.50 mg / dL, and more preferably one point within the range of 2.00 to 3.00 mg / dL, when using serum as the sample. If the CRP concentration in a sample is measured and the result is higher than the cutoff value, the patient from whom the sample was collected can be judged to be at high risk of developing a second-degree severe illness.

[0105] For determining the risk of developing a second severe disease, the cutoff value for white blood cell count is preferably one point within the range of 5.16 to 12.04 cells / nL, and more preferably one point within the range of 6.88 to 10.32 cells / nL, when whole blood is used as the sample. If the white blood cell count in a sample is measured and the result is higher than the cutoff value, the patient from whom the sample was collected can be judged to be at high risk of developing a second-degree severe illness.

[0106] The cutoff platelet count for determining the risk of developing a second severe illness is, for example, 13.2 to 30.8 × 10⁻⁶ when using whole blood as the sample.4 It is preferable that the point be within the range of particles / μL, specifically 17.6 to 26.4 × 10⁻⁶. 4 It is more preferable that the point be within the range of particles / μL. If the platelet count in a sample is measured and found to be lower than the cutoff value, the patient from whom the sample was collected can be judged to be at high risk of developing a second-degree severe illness.

[0107] When determining the risk of developing a second severe disease, the cutoff value for the ratio of neutrophils to total white blood cells is preferably one point within the range of 48.0-100.0%, and more preferably one point within the range of 64.0-96.0%, if whole blood is used as the sample. If the percentage of neutrophils in a sample is measured and the result is higher than the cutoff value, the patient from whom the sample was collected can be judged to be at high risk of developing a second-degree severe illness.

[0108] When determining the risk of developing a second severe disease, the cutoff value for the ratio of lymphocytes to total white blood cells is preferably one point within the range of 6.6 to 15.4%, and more preferably one point within the range of 8.8 to 13.2%, if whole blood is used as the sample. If the percentage of lymphocytes in a sample is measured and the result is lower than the cutoff value, the patient from whom the sample was collected can be judged to be at high risk of developing a second-degree severe illness.

[0109] For determining the risk of developing a second severe illness, the cutoff value for total bilirubin is preferably one point within the range of 0.3 to 0.6 mg / dL, and more preferably one point within the range of 0.4 to 0.5 mg / dL, when using serum as the sample. If the total bilirubin concentration in a sample is measured and found to be higher than the cutoff value, the patient from whom the sample was collected can be judged to be at high risk of developing a second-degree severe illness.

[0110] For determining the risk of developing a second severe illness, the urea nitrogen cutoff value is preferably one point within the range of 13.2 to 30.8 mg / dL, and more preferably one point within the range of 17.6 to 26.4 mg / dL, when using serum as the sample. If the urea nitrogen concentration in a sample is measured and found to be higher than the cutoff value, the patient from whom the sample was collected can be judged to be at high risk of developing a second-degree severe illness.

[0111] For determining the risk of developing a second severe illness, the creatinine cutoff value is preferably one point within the range of 0.36 to 0.84 mg / dL, and more preferably one point within the range of 0.48 to 0.72 mg / dL, when using serum as the sample. If the creatinine concentration in a sample is measured and the result is higher than the cutoff value, the patient from whom the sample was collected can be judged to be at high risk of developing a second-degree severe illness.

[0112] When determining the risk of developing a second severe illness, the cutoff value for TARC is preferably one point within the range of 60.0 to 140.0 pg / mL, and more preferably one point within the range of 80.0 to 120.0 pg / mL, for example, when using serum as the sample. If the TARC concentration in a sample is measured and found to be lower than the cutoff value, the patient from whom the sample was collected can be judged to be at high risk of developing a second-degree severe illness.

[0113] The above cutoff value can be set, for example, by creating an ROC curve (Receiver Operating Characteristic Curve) from the quantitative values ​​of the above indicator in samples from patient groups that did not progress from stage (2) to stage (3) or from stage (4) in the classification of severity above, and from the quantitative values ​​of the above indicator in samples from patient groups that progressed from stage (2) to stage (3) or stage (4). Furthermore, the area under the ROC curve is preferably 0.65 or greater, more preferably 0.75 or greater, and even more preferably 0.80 or greater.

[0114] The quantitative determination of the indicator in the inspection method according to the second embodiment of the present invention can be performed in the same manner as the quantitative determination of the indicator in the inspection method according to the first embodiment of the present invention.

[0115] (Third embodiment) The test reagent for predicting the risk of severe COVID-19 disease according to the third embodiment of the present invention comprises at least one antibody selected from the group consisting of anti-calprotectin antibody, anti-IFN-λ3 antibody, anti-IL-6 antibody, anti-CRP antibody, anti-creatinine antibody, and anti-TARC antibody, which are used in the test method according to the first embodiment and the test method according to the second embodiment of the present invention. (The above-mentioned test reagent is used in a test method in which the quantitative step is to quantify at least one biomarker selected from the group consisting of calprotectin, IFN-λ3, IL-6, CRP, creatinine, and TARC in the sample.)

[0116] The test reagent according to the third embodiment of the present invention may include, in addition to the antibody against the biomarker, a solid support on which the antibody is carried, an immunoassay buffer, the labeled biomarker and / or its analog, a labeled secondary antibody, and the like. [Examples]

[0117] The inspection methods of the present invention will be further described below with reference to examples, but the inspection methods of the present invention are not limited to these.

[0118] <Preparation of test reagents> The following reagents were prepared as test reagents for measuring calprotectin, IFN-λ3, IL-6, CRP, total bilirubin, urea nitrogen, creatinine, and TARC. Calprotectin assay reagent (enzyme-linked immunosorbent assay (ELISA)): Manufactured by BUHLMANN, product name "BUHLMANN sCAL® ELISA" Reagent for calprotectin measurement (immunochromatography): Manufactured by BUHLMANN, product name "Quantum Blue® sCAL" IFN-λ3 measurement reagent (chemiluminescent enzyme immunoassay (CLEIA method)): Manufactured by Sysmex Corporation, product name "HISCL® IFN-λ3 Reagent" IL-6 measurement reagent (electrochemiluminescence immunoassay (ECLIA)): Manufactured by Roche Diagnostics, product name "ECLIA® Reagent IL-6" CRP measurement reagent (latex agglutination turbidimetry method): Manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., product name "LT Auto Wako CRP-HSII" Total bilirubin measurement reagent (chemical oxidation method): Manufactured by Fujifilm Wako Pure Chemical Corporation, product name "Total Bilirubin E-HA Test Wako" Urea nitrogen measurement reagent (urease method): Manufactured by Sinotest Co., Ltd., product name "Cygnus Auto UN" Creatinine measurement reagent (enzymatic method): Manufactured by Sinotest Co., Ltd., product name "Cygnus Auto CRE" TARC assay reagent (chemiluminescent enzyme immunoassay (CLEIA method)): Manufactured by Shionogi & Co., Ltd., product name "HISCL® TARC Reagent" White blood cell count, platelet count, neutrophil count (percentage of neutrophils to total white blood cells), and lymphocyte count (percentage of lymphocytes to total white blood cells) were measured using the Sysmex Corporation XN-2000 / XN-9000 multi-parameter automated hematology analyzer.

[0119] <Measurement of each indicator> For 14 SARS-CoV-2 infected individuals who required oxygen therapy, whole blood was collected after the start of oxygen administration and used as a sample for measuring white blood cell count, platelet count, neutrophil count (percentage of neutrophils to total white blood cells), and lymphocyte count (percentage of lymphocytes to total white blood cells). Separately collected whole blood was centrifuged after blood coagulation, and serum was collected and used as a sample for measuring calprotectin, IFN-λ3, IL-6, CRP, total bilirubin, urea nitrogen, creatinine, and TARC. It should be noted that only patient No. 9, listed in Table 1 below, had a <Patient Severity Classification> of (3) at the time of whole blood collection. All other patients had a <Patient Severity Classification> of (2) at the time of whole blood collection. Therefore, the data for patient No. 9 was excluded from the examination of the second severe disease risk described below.

[0120] (Measurement of calprotectin, IFN-λ3, IL-6, CRP, white blood cell count, platelet count, neutrophil ratio to total white blood cell count, lymphocyte ratio to total white blood cell count, total bilirubin, urea nitrogen, creatinine, and TARC) The concentrations of calprotectin, IFN-λ3, IL-6, CRP, total bilirubin, urea nitrogen, creatinine, and TARC in the obtained samples, as well as the white blood cell count, platelet count, neutrophil count (calculated as the ratio of neutrophils to total white blood cells), and lymphocyte count (calculated as the ratio of lymphocytes to total white blood cells), were measured according to the methods described in the accompanying documentation for each reagent. The results are shown in Table 1. Furthermore, data on CRP, white blood cell count, platelet count, neutrophil count (percentage of total white blood cells), and lymphocyte count (percentage of total white blood cells) could not be obtained from patient No. 6, and data on total bilirubin could not be obtained from patient No. 14. Therefore, the analysis described below was performed using only the data that could be obtained.

[0121] [Table 1]

[0122] <Classification of patient severity> SARS-CoV-2 infected individuals from whom samples were collected were monitored and classified as follows (2) to (4). The results are shown in Table 1. (2) Although oxygen inhalation was required during the recovery period, high-flow nasal cannula, non-invasive ventilator, invasive ventilator, and extracorporeal membrane oxygenation (ECMO) were not required. (3) The patient required high-flow nasal cannula, non-invasive ventilator, or both during recovery, but did not require invasive ventilator or extracorporeal membrane oxygenation (ECMO). (4) The patient required invasive mechanical ventilation, extracorporeal membrane oxygenation (ECMO), or both during recovery or death.

[0123] The number of SARS-CoV-2 infected individuals classified as stage (2) was 8. The number of SARS-CoV-2 infected individuals classified as stage (3) was 2. The number of SARS-CoV-2 infected individuals classified as stage (4) was 4.

[0124] <Risk Level 1 for Severe Illness> The patient groups classified into stages (2) and (3) above were designated as Group 1, and the patient groups classified into stage (4) above were designated as Group 2. Next, a comparison was made between Group 1 and Group 2 based on the obtained concentrations of calprotectin, IFN-λ3, IL-6, CRP, total bilirubin, urea nitrogen, creatinine, and TARC, as well as white blood cell count, platelet count, neutrophil count to total white blood cell count, and lymphocyte count to total white blood cell count. Figure 1 is a plot showing the calprotectin concentrations in the serum of Group 1 and Group 2, as measured by enzyme immunosorbent assay (ELISA). Figure 2 is a plot showing the calprotectin concentrations in the serum of Group 1 and Group 2, as measured by immunochromatography. Figure 3 is a plot showing the IFN-λ3 concentrations in the serum of Group 1 and Group 2, as measured by chemiluminescent enzyme immunoassay (CLEIA). Figure 4 is a plot showing the serum IL-6 concentrations of Group 1 and Group 2 as measured by electrochemiluminescent enzyme immunoassay (ECLIA). Figure 5 is a plot showing the CRP concentrations in the serum of Group 1 and Group 2, as measured by latex agglutination turbidimetry. Figure 6 is a plot showing the white blood cell counts in the whole blood of Group 1 and Group 2, as measured by a multi-parameter automated blood cell analyzer. Figure 7 is a plot showing the platelet counts in whole blood of Group 1 and Group 2, measured using a multi-parameter automated hematology analyzer. Figure 8 is a plot showing the ratio of neutrophils to total white blood cells in the whole blood of Group 1 and Group 2, as measured by a multi-parameter automated hematology analyzer. Figure 9 is a plot showing the ratio of lymphocytes to total white blood cells in the whole blood of Group 1 and Group 2, as measured by a multi-parameter automated hematology analyzer. Figure 10 is a plot showing the total bilirubin concentrations in the serum of Group 1 and Group 2, measured by chemical oxidation. Figure 11 is a plot showing the urea nitrogen concentrations in the serum of Group 1 and Group 2, measured by the urease method. Figure 12 is a plot showing the creatinine concentrations in the serum of Group 1 and Group 2, measured by the enzymatic method. Figure 13 is a plot showing the TARC concentrations in the serum of Group 1 and Group 2, measured by chemiluminescent enzyme immunoassay (CLEIA).

[0125] The median calprotectin concentration in the serum of group 1, as measured by ELISA, was 5.1 μg / mL. The median calprotectin concentration in the serum of group 2, as measured by ELISA, was 14.0 μg / mL.

[0126] The median calprotectin concentration in the serum of group 1, as measured by immunochromatography, was 4.9 μg / mL. The median calprotectin concentration in the serum of group 2, as measured by immunochromatography, was 19.4 μg / mL.

[0127] The median IFN-λ3 concentration in the serum of Group 1, as measured by chemiluminescent enzyme immunoassay (CLEIA), was 3.7 pg / mL. The median IFN-λ3 concentration in the serum of Group 2, as measured by chemiluminescent enzyme immunoassay (CLEIA), was 45.1 pg / mL.

[0128] The median serum IL-6 concentration in Group 1, measured by electrochemiluminescence immunoassay (ECLIA), was 9.5 pg / mL. The median serum IL-6 concentration in Group 2, measured by electrochemiluminescence immunoassay (ECLIA), was 115.0 pg / mL.

[0129] The median CRP concentration in the serum of Group 1, as measured by latex agglutination turbidimetry, was 1.69 mg / dL. The median CRP concentration in the serum of Group 2, as measured by latex agglutination turbidimetry, was 4.50 mg / dL.

[0130] The median white blood cell count in whole blood of Group 1, as measured by a multi-parameter automated hematology analyzer, was 6.00 cells / nL. The median white blood cell count in whole blood of group 2, as measured by a multi-parameter automated hematology analyzer, was 8.46 cells / nL.

[0131] The median platelet count in whole blood of Group 1, measured using a multi-parameter automated hematology analyzer, was 23.1 × 10⁴. 4 The concentration was individual particles / μL. The median platelet count in whole blood of group 2, measured using a multi-parameter automated hematology analyzer, was 14.1 × 10⁴. 4 The concentration was individual particles / μL.

[0132] The median ratio of neutrophils to total white blood cells in the whole blood of Group 1, as measured by a multi-parameter automated hematology analyzer, was 72.9%. The median ratio of neutrophils to total white blood cells in the second group, as measured by a multi-parameter automated hematology analyzer, was 90.5%.

[0133] The median ratio of lymphocytes to total white blood cells in the whole blood of Group 1, as measured by a multi-parameter automated hematology analyzer, was 20.3%. The median ratio of lymphocytes to total white blood cells in the whole blood of group 2, as measured by a multi-parameter automated hematology analyzer, was 6.3%.

[0134] The median total bilirubin level in the serum of Group 1, measured by chemical oxidation, was 0.3 mg / dL. The median total bilirubin level in the serum of Group 2, measured by chemical oxidation, was 0.5 mg / dL.

[0135] The median serum urea nitrogen level in Group 1, measured by the urease method, was 16.0 mg / dL. The median serum urea nitrogen level in Group 2, as measured by the urease method, was 24.6 mg / dL.

[0136] The median serum creatinine level in Group 1, measured by enzymatic method, was 0.60 mg / dL. The median serum creatinine level in Group 2, measured by enzymatic method, was 1.27 mg / dL.

[0137] The median TARC concentration in the serum of Group 1, as measured by chemiluminescent enzyme immunoassay (CLEIA), was 139.5 pg / mL. The median TARC concentration in the serum of group 2, as measured by chemiluminescent enzyme immunoassay (CLEIA), was 70.5 pg / mL.

[0138] (ROC analysis of each indicator in the sample) Based on the measurement results of each indicator (biomarker concentration, etc.) in the obtained samples (whole blood or serum), ROC analysis was performed on each indicator (biomarker concentration, etc.) in the samples of Group 1 vs. Group 2. The ROC curves are shown in Figures 14 to 26. Figure 14 shows the ROC curves for calprotectin concentrations in the serum of Group 1 and Group 2, measured by enzyme immunosorbent assay (ELISA). Figure 15 shows the ROC curves for serum calprotectin concentrations in groups 1 and 2, measured by immunochromatography. Figure 16 shows the ROC curves for IFN-λ3 concentrations in serum from groups 1 and 2, measured by chemiluminescent enzyme immunoassay (CLEIA). Figure 17 shows the ROC curves for serum IL-6 concentrations in Group 1 and Group 2, measured by electrochemiluminescence immunoassay (ECLIA). Figure 18 shows the ROC curves for CRP concentrations in serum from groups 1 and 2, measured by latex agglutination turbidimetry. Figure 19 shows the ROC curves for the white blood cell count in whole blood of Group 1 and Group 2, measured using a multi-parameter automated hematology analyzer. Figure 20 shows the ROC curves for platelet counts in whole blood of Group 1 and Group 2, measured using a multi-parameter automated hematology analyzer. Figure 21 shows the ROC curves for the ratio of neutrophils to total white blood cells in the whole blood of Group 1 and Group 2, measured using a multi-parameter automated hematology analyzer. Figure 22 shows the ROC curves for the ratio of lymphocytes to total white blood cells in the whole blood of Group 1 and Group 2, measured using a multi-parameter automated hematology analyzer. Figure 23 shows the ROC curves for total bilirubin concentrations in serum of Group 1 and Group 2, measured by chemical oxidation. Figure 24 shows the ROC curves for serum urea nitrogen concentrations in groups 1 and 2, measured by the urease method. Figure 25 shows the ROC curves for serum creatinine concentrations in groups 1 and 2, measured by the enzymatic method. Figure 26 shows the ROC curves for TARC concentrations in serum of Group 1 and Group 2, measured by chemiluminescent enzyme immunoassay (CLEIA).

[0139] The p-value for the calprotectin concentration between group 1 and group 2, as measured by ELISA, was 0.024. In this specification, the p-value refers to the value calculated using the Mann-Whitney U test. In the ROC curves for calprotectin concentrations in serum from groups 1 and 2, measured by ELISA, the area under the ROC curve was 0.90. The cutoff value calculated from Youden's Index was 13.6 μg / mL. Furthermore, the accuracy of the test when the cutoff value was set at 13.6 μg / mL was as follows. The overall agreement rate was 92.9%. The sensitivity was 100.0%. The specificity was 100.0%. The positive predictive value was 100.0%. The negative predictive value was 90.9%.

[0140] The p-value for the calprotectin concentration between group 1 and group 2, as measured by immunochromatography, was 0.14. In the ROC curves for calprotectin concentrations in serum from groups 1 and 2, measured by immunochromatography, the area under the ROC curve was 0.78. The cutoff value calculated from Youden's Index was 13.4 μg / mL. Furthermore, the accuracy of the test when the cutoff value was set at 13.4 μg / mL was as follows. The overall agreement rate was 85.7%. The sensitivity was 75.0%. The specificity was 90.0%. The positive predictive value was 75.0%. The negative predictive value was 90.0%.

[0141] The p-value for IFN-λ3 concentration between group 1 and group 2, as measured by chemiluminescent enzyme immunoassay (CLEIA), was 0.11. In the ROC curves for IFN-λ3 concentrations in serum from groups 1 and 2, measured by chemiluminescent enzyme immunoassay (CLEIA), the area under the ROC curve was 0.80. Furthermore, the cutoff value calculated from Youden's Index was 31.3 pg / mL. Furthermore, the accuracy of the test when the cutoff value was set at 31.3 pg / mL was as follows: The overall agreement rate was 92.9%. The sensitivity was 75.0%. The specificity was 100.0%. The positive predictive value was 100.0%. The negative predictive value was 90.9%.

[0142] The p-value for IL-6 concentration between group 1 and group 2, as measured by electrochemiluminescent enzyme immunoassay (ECLIA), was 0.004. In the ROC curves for IL-6 concentrations in serum from groups 1 and 2, measured by electrochemiluminescent enzyme immunoassay (ECLIA), the area under the ROC curve was 0.98. Furthermore, the cutoff value calculated from Youden's Index was 42.4 pg / mL. Furthermore, the accuracy of the test when the cutoff value was set at 42.4 pg / mL was as follows: The overall agreement rate was 92.9%. The sensitivity was 100.0%. The specificity was 90.0%. The positive predictive value was 80.0%. The negative predictive value was 100.0%.

[0143] The p-value for CRP concentration between Group 1 and Group 2, measured by latex agglutination turbidimetry, was 0.414. In the ROC curves for CRP concentrations in serum from groups 1 and 2, measured by latex agglutination turbidimetry, the area under the ROC curve was 0.67. Furthermore, the cutoff value calculated from Youden's Index was 3.00 mg / dL. Furthermore, the accuracy of the test when the cutoff value was set to 3.00 mg / dL was as follows: The overall agreement rate was 69.2%. The sensitivity was 100.0%. The specificity was 55.6%. The positive predictive value was 50.0%. The negative predictive value was 100.0%.

[0144] The p-value for white blood cell counts between Group 1 and Group 2, as measured by a multi-parameter automated blood cell analyzer, was 0.414. In the ROC curves for white blood cell counts in whole blood of groups 1 and 2, measured using a multi-parameter automated hematology analyzer, the area under the ROC curve was 0.67. Furthermore, the cutoff value calculated from Youden's Index was 8.00 units / nL. Furthermore, the inspection accuracy when the cutoff value was set to 8.00 pieces / nL was as follows: The overall agreement rate was 69.2%. The sensitivity was 75.0%. The specificity was 66.7%. The positive predictive value was 50.0%. The negative predictive value was 85.7%.

[0145] The p-value for platelet counts between Group 1 and Group 2, as measured by a multi-parameter automated hematology analyzer, was 0.148. In the ROC curves for platelet counts in whole blood of groups 1 and 2, measured using a multi-parameter automated hematology analyzer, the area under the ROC curve was 0.78. Furthermore, the cutoff value calculated from Youden's Index is 21.5 × 10 4 The concentration was individual particles / μL. Also, the cutoff value is 21.5 × 10 4 The inspection accuracy when expressed as particles / μL was as follows: The overall agreement rate was 69.2%. The sensitivity was 100.0%. The specificity was 55.6%. The positive predictive value was 50.0%. The negative predictive value was 100.0%.

[0146] The p-value for the ratio of neutrophils to total white blood cells between Group 1 and Group 2, as measured by a multi-parameter automated hematology analyzer, was 0.106. In the ROC curves for the ratio of neutrophils to total white blood cells in the whole blood of groups 1 and 2, measured using a multi-parameter automated hematology analyzer, the area under the ROC curve was 0.81. Furthermore, the cutoff value calculated from Youden's Index was 90.0%. Furthermore, the test accuracy when the cutoff value was set to 90.0% was as follows: The overall agreement rate was 92.3%. The sensitivity was 75.0%. The specificity was 100.0%. The positive predictive value was 100.0%. The negative predictive value was 90.0%.

[0147] The p-value for the ratio of lymphocytes to total white blood cells between Group 1 and Group 2, as measured by a multi-parameter automated hematology analyzer, was 0.148. In the ROC curves for the ratio of lymphocytes to total white blood cells in whole blood of groups 1 and 2, measured using a multi-parameter automated hematology analyzer, the area under the ROC curve was 0.78. Furthermore, the cutoff value calculated from Youden's Index was 8.0%. Furthermore, the accuracy of the test when the cutoff value was set to 8.0% was as follows: The overall agreement rate was 84.6%. The sensitivity was 75.0%. The specificity was 88.9%. The positive predictive value was 75.0%. The negative predictive value was 88.9%.

[0148] The p-value for total bilirubin between group 1 and group 2, measured by chemical oxidation, was 0.371. In the ROC curves for total bilirubin in serum from groups 1 and 2, measured by chemical oxidation, the area under the ROC curve was 0.70. Furthermore, the cutoff value calculated from Youden's Index was 0.45 mg / dL. Also, when the cut-off value was set at 0.45 mg / dL, the test accuracy was as follows. The overall agreement rate was 76.9%. The sensitivity was 66.7%. The specificity was 80.0%. The positive predictive value was 50.0%. The negative predictive value was 88.9%.

[0149] The p-value of urea nitrogen between the first group and the second group measured by the urease method was 0.036. In the ROC curve for serum urea nitrogen in the first group and the second group measured by the urease method, the area under the ROC curve was 0.88. Also, the cut-off value calculated from Youden's Index was 22.0 mg / dL. Also, when the cut-off value was set at 22.0 mg / dL, the test accuracy was as follows. The overall agreement rate was 85.7%. The sensitivity was 100.0%. The specificity was 80.0%. The positive predictive value was 66.7%. The negative predictive value was 100.0%.

[0150] The p-value of creatinine between the first group and the second group measured by the enzymatic method was 0.027. In the ROC curve for serum creatinine in the first group and the second group measured by the enzymatic method, the area under the ROC curve was 0.89. Also, the cut-off value calculated from Youden's Index was 1.00 mg / dL. Also, when the cut-off value was set at 1.00 mg / dL, the test accuracy was as follows. The overall agreement rate was 92.9%. The sensitivity was 75.0%. The specificity was 100.0%. The positive predictive value was 100.0%. The negative predictive value was 90.9%.

[0151] The p-value for TARC concentration between group 1 and group 2, as measured by chemiluminescent enzyme immunoassay (CLEIA), was 0.014. In the ROC curves for TARC concentrations in serum from groups 1 and 2, measured by chemiluminescent enzyme immunoassay (CLEIA), the area under the ROC curve was 0.93. Furthermore, the cutoff value calculated from Youden's Index was 100.0 pg / mL. Furthermore, the accuracy of the test when the cutoff value was set to 100.0 pg / mL was as follows: The overall agreement rate was 85.7%. The sensitivity was 100.0%. The specificity was 80.0%. The positive predictive value was 66.7%. The negative predictive value was 100.0%.

[0152] <Risk level 2 for severe illness> The group of patients classified into stage (2) above was designated as Group 3, and the group of patients classified into stages (3) and (4) above was designated as Group 4. Next, a comparison was made between Group 3 and Group 4 based on the obtained concentrations of calprotectin, IFN-λ3, IL-6, CRP, total bilirubin, urea nitrogen, creatinine, and TARC, as well as white blood cell count, platelet count, neutrophil count to total white blood cell count, and lymphocyte count to total white blood cell count. Figure 27 is a plot showing the calprotectin concentrations in the serum of groups 3 and 4, as measured by enzyme immunosorbent assay (ELISA). Figure 28 is a plot showing the calprotectin concentrations in the serum of groups 3 and 4, as measured by immunochromatography. Figure 29 is a plot showing the IFN-λ3 concentrations in the serum of groups 3 and 4, as measured by chemiluminescent enzyme immunoassay (CLEIA). Figure 30 is a plot showing the IL-6 concentrations in the serum of groups 3 and 4, as measured by electrochemiluminescence immunoassay (ECLIA). Figure 31 is a plot showing the CRP concentrations in the serum of groups 3 and 4, measured by latex agglutination turbidimetry. Figure 32 is a plot showing the white blood cell counts in the whole blood of groups 3 and 4, as measured by a multi-parameter automated blood cell analyzer. Figure 33 is a plot showing the platelet counts in whole blood of groups 3 and 4, as measured by a multi-parameter automated hematology analyzer. Figure 34 is a plot showing the ratio of neutrophils to total white blood cells in the whole blood of groups 3 and 4, as measured by a multi-parameter automated hematology analyzer. Figure 35 is a plot showing the ratio of lymphocytes to total white blood cells in the whole blood of groups 3 and 4, as measured by a multi-parameter automated hematology analyzer. Figure 36 is a plot showing the total bilirubin concentrations in the serum of groups 3 and 4, as measured by chemical oxidation. Figure 37 is a plot showing the urea nitrogen concentrations in the serum of groups 3 and 4, as measured by the urease method. Figure 38 is a plot showing the serum creatinine concentrations of groups 3 and 4, measured by the enzymatic method. Figure 39 is a plot showing the TARC concentrations in the serum of groups 3 and 4, as measured by chemiluminescent enzyme immunoassay (CLEIA).

[0153] The median calprotectin concentration in the serum of group 3, as measured by ELISA, was 4.7 μg / mL. The median calprotectin concentration in the serum of group 4, as measured by ELISA, was 13.8 μg / mL.

[0154] The median calprotectin concentration in the serum of group 3, as measured by immunochromatography, was 4.7 μg / mL. The median calprotectin concentration in the serum of group 4, as measured by immunochromatography, was 18.9 μg / mL.

[0155] The median IFN-λ3 concentration in the serum of Group 3 measured by chemiluminescent enzyme immunoassay (CLEIA) was 3.2 pg / mL. The median IFN-λ3 concentration in the serum of Group 4 measured by ELISA was 40.4 pg / mL.

[0156] The median IL-6 concentration in the serum of Group 3 measured by electrochemiluminescence immunoassay (ECLIA) was 9.2 pg / mL. The median IL-6 concentration in the serum of Group 4 measured by electrochemiluminescence immunoassay (ECLIA) was 69.0 pg / mL.

[0157] The median CRP concentration in the serum of Group 3 measured by latex agglutination nephelometry was 1.69 mg / dL. The median CRP concentration in the serum of Group 4 measured by latex agglutination nephelometry was 4.60 mg / dL.

[0158] The median white blood cell count in the whole blood of Group 3 measured by a multi-item automatic blood cell analyzer was 6.00 cells / nL. The median white blood cell count in the whole blood of Group 4 measured by a multi-item automatic blood cell analyzer was 8.22 cells / nL.

[0159] The median platelet count in the whole blood of Group 3 measured by the multi-item automatic blood cell analyzer was 29.9×10 4 cells / μL. The median platelet count in the whole blood of Group 4 measured by the multi-item automatic blood cell analyzer was 12.7×10 4 cells / μL.

[0160] The median ratio of the number of neutrophils to the total white blood cell count in the whole blood of Group 3 measured by the multi-item automatic blood cell analyzer was 72.0%. The median ratio of the number of neutrophils to the total white blood cell count in the whole blood of Group 4 measured by the multi-item automatic blood cell analyzer was 90.1%.

[0161] The median ratio of lymphocytes to total white blood cells in the whole blood of Group 3, as measured by a multi-parameter automated hematology analyzer, was 21.0%. The median ratio of lymphocytes to total white blood cells in whole blood of Group 4, as measured by a multi-parameter automated hematology analyzer, was 6.9%.

[0162] The median total bilirubin level in the serum of Group 3, measured by chemical oxidation, was 0.3 mg / dL. The median total bilirubin level in the serum of Group 4, measured by chemical oxidation, was 0.6 mg / dL.

[0163] The median serum urea nitrogen level in Group 3, as measured by the urease method, was 15.9 mg / dL. The median serum urea nitrogen level in Group 4, measured by the urease method, was 23.6 mg / dL.

[0164] The median serum creatinine level in Group 3, measured by enzymatic method, was 0.55 mg / dL. The median serum creatinine level in Group 4, measured by enzymatic method, was 1.19 mg / dL.

[0165] The median serum IL-6 concentration in group 3, as measured by chemiluminescent enzyme immunoassay (CLEIA), was 139.5 pg / mL. The median serum IL-6 concentration in Group 4, as measured by chemiluminescent enzyme immunoassay (CLEIA), was 81.3 pg / mL.

[0166] (ROC analysis of each indicator in the sample) Based on the measurement results of each indicator (biomarker concentration, etc.) in the obtained samples (whole blood or serum), ROC analysis was performed on each indicator (biomarker concentration, etc.) in the samples of Group 3 vs. Group 4. The ROC curves are shown in Figures 40 to 52. Figure 40 shows the ROC curves for serum calprotectin concentrations in groups 3 and 4, measured by enzyme immunosorbent assay (ELISA). Figure 41 shows the ROC curves for serum calprotectin concentrations in groups 3 and 4, measured by immunochromatography. Figure 42 shows the ROC curves for IFN-λ3 concentrations in serum from groups 3 and 4, measured by chemiluminescent enzyme immunoassay (CLEIA). Figure 43 shows the ROC curves for serum IL-6 concentrations in groups 3 and 4, measured by electrochemiluminescence immunoassay (ECLIA). Figure 44 shows the ROC curves for CRP concentrations in serum from groups 3 and 4, measured by latex agglutination turbidimetry. Figure 45 shows the ROC curves for the white blood cell count in whole blood of groups 3 and 4, measured using a multi-parameter automated hematology analyzer. Figure 46 shows the ROC curves for platelet counts in whole blood of groups 3 and 4, measured using a multi-parameter automated hematology analyzer. Figure 47 shows the ROC curves for the ratio of neutrophils to total white blood cells in the whole blood of groups 3 and 4, measured using a multi-parameter automated hematology analyzer. Figure 48 shows the ROC curves for the ratio of lymphocytes to total white blood cells in whole blood of groups 3 and 4, measured using a multi-parameter automated hematology analyzer. Figure 49 shows the ROC curves for total bilirubin concentrations in serum of groups 3 and 4, measured by chemical oxidation. Figure 50 shows the ROC curves for serum urea nitrogen concentrations in groups 3 and 4, measured by the urease method. Figure 51 shows the ROC curves for serum creatinine concentrations in groups 3 and 4, measured by the enzymatic method. Figure 52 shows the ROC curves for TARC concentrations in serum from groups 3 and 4, measured by chemiluminescent enzyme immunoassay (CLEIA).

[0167] The p-value for the calprotectin concentration between group 3 and group 4, as measured by ELISA, was 0.011. In the ROC curves for calprotectin concentrations in serum from groups 3 and 4, measured by ELISA, the area under the ROC curve was 0.93. Furthermore, the cutoff value calculated from Youden's Index was 5.8 μg / mL. Furthermore, the accuracy of the test when the cutoff value was set at 5.8 μg / mL was as follows. The overall agreement rate was 84.6%. The sensitivity was 100.0%. The specificity was 75.0%. The positive predictive value was 71.4%. The negative predictive value was 100.0%.

[0168] The p-value for the calprotectin concentration between group 3 and group 4, as measured by immunochromatography, was 0.045. In the ROC curves for calprotectin concentrations in serum from groups 3 and 4, measured by immunochromatography, the area under the ROC curve was 0.85. The cutoff value calculated from Youden's Index was 5.8 μg / mL. Furthermore, the accuracy of the test when the cutoff value was set at 5.8 μg / mL was as follows. The overall agreement rate was 84.6%. The sensitivity was 80.0%. The specificity was 87.5%. The positive predictive value was 80.0%. The negative predictive value was 87.5%.

[0169] The p-value for IFN-λ3 concentration between group 3 and group 4, as measured by chemiluminescent enzyme immunoassay (CLEIA), was 0.065. The area under the curve of the ROC curve for IFN-λ3 concentrations in serum from groups 3 and 4, measured by chemiluminescent enzyme immunoassay (CLEIA), was 0.83. Furthermore, the cutoff value calculated from Youden's Index was 16.6 pg / mL. Furthermore, the accuracy of the test when the cutoff value was set at 16.6 pg / mL was as follows: The overall agreement rate was 85.6%. The sensitivity was 80.0%. The specificity was 87.5%. The positive predictive value was 80.0%. The negative predictive value was 87.5%.

[0170] The p-value for IL-6 concentration between group 3 and group 4, as measured by electrochemiluminescent enzyme immunoassay (ECLIA), was 0.019. In the ROC curves for IL-6 concentrations in serum from groups 3 and 4, measured by electrochemiluminescent enzyme immunoassay (ECLIA), the area under the ROC curve was 0.90. Furthermore, the cutoff value calculated from Youden's Index was 42.4 pg / mL. Furthermore, the accuracy of the test when the cutoff value was set at 42.4 pg / mL was as follows: The overall agreement rate was 84.6%. The sensitivity was 80.0%. The specificity was 87.5%. The positive predictive value was 80.0%. The negative predictive value was 87.5%.

[0171] The p-value for CRP concentration between group 3 and group 4, measured by latex agglutination turbidimetry, was 0.343. In the ROC curves for CRP concentrations in serum from groups 3 and 4, measured by latex agglutination turbidimetry, the area under the ROC curve was 0.69. Furthermore, the cutoff value calculated from Youden's Index was 2.50 mg / dL. Furthermore, the accuracy of the test when the cutoff value was set to 2.50 mg / dL was as follows. The overall agreement rate was 75.5%. The sensitivity was 100.0%. The specificity was 57.1%. The positive predictive value was 62.5%. The negative predictive value was 100.0%.

[0172] The p-value for white blood cell counts between group 3 and group 4, as measured by a multi-parameter automated hematology analyzer, was 0.639. In the ROC curves for white blood cell counts in whole blood of groups 3 and 4, measured using a multi-parameter automated hematology analyzer, the area under the ROC curve was 0.60. Furthermore, the cutoff value calculated from Youden's Index was 8.60 units / nL. Furthermore, the inspection accuracy when the cutoff value was set to 8.60 pieces / nL was as follows. The overall agreement rate was 75.0%. The sensitivity was 40.0%. The specificity was 100.0%. The positive predictive value was 100.0%. The negative predictive value was 70.0%.

[0173] The p-value for platelet counts between group 3 and group 4, as measured by a multi-parameter automated hematology analyzer, was 0.048. In the ROC curves for platelet counts in whole blood of groups 3 and 4, measured using a multi-parameter automated hematology analyzer, the area under the ROC curve was 0.86. Furthermore, the cutoff value calculated from Youden's Index is 22.0 × 10 4 The concentration was individual particles / μL. Also, the cutoff value is 22.0 × 10 4 The inspection accuracy when expressed as particles / μL was as follows: The overall agreement rate was 83.3%. The sensitivity was 100.0%. The specificity was 71.4%. The positive predictive value was 71.4%. The negative predictive value was 100.0%.

[0174] The p-value for the ratio of neutrophils to total white blood cells between Group 3 and Group 4, as measured by a multi-parameter automated hematology analyzer, was 0.073. In the ROC curves for the ratio of neutrophils to total white blood cells in whole blood of groups 3 and 4, measured using a multi-parameter automated hematology analyzer, the area under the ROC curve was 0.83. Furthermore, the cutoff value calculated from Youden's Index was 80.0%. Furthermore, the accuracy of the test when the cutoff value was set to 80.0% was as follows: The overall agreement rate was 83.3%. The sensitivity was 80.0%. The specificity was 85.7%. The positive predictive value was 80.0%. The negative predictive value was 85.7%.

[0175] The p-value for the ratio of lymphocytes to total white blood cells between Group 3 and Group 4, as measured by a multi-parameter automated hematology analyzer, was 0.106. In the ROC curves for the ratio of lymphocytes in whole blood to total white blood cells in groups 3 and 4, measured using a multi-parameter automated hematology analyzer, the area under the ROC curve was 0.80. Furthermore, the cutoff value calculated from Youden's Index was 11.0%. Furthermore, the accuracy of the test when the cutoff value was set at 11.0% was as follows: The overall agreement rate was 83.3%. The sensitivity was 80.0%. The specificity was 85.7%. The positive predictive value was 80.0%. The negative predictive value was 85.7%.

[0176] The p-value for total bilirubin between group 3 and group 4, measured by chemical oxidation, was 0.119. In the ROC curves for total bilirubin in serum from groups 3 and 4, measured by chemical oxidation, the area under the ROC curve was 0.80. Furthermore, the cutoff value calculated from Youden's Index was 0.45 mg / dL. Furthermore, the accuracy of the test when the cutoff value was set to 0.45 mg / dL was as follows: The overall agreement rate was 83.9%. The sensitivity was 75.0%. The specificity was 87.5%. The positive predictive value was 75.0%. The negative predictive value was 87.5%.

[0177] The p-value for urea nitrogen between group 3 and group 4, as measured by the urease method, was 0.045. In the ROC curves for urea nitrogen in serum from groups 3 and 4, measured by the urease method, the area under the ROC curve was 0.85. Furthermore, the cutoff value calculated from Youden's Index was 22.0 mg / dL. Furthermore, the accuracy of the test when the cutoff value was set at 22.0 mg / dL was as follows: The overall agreement rate was 84.6%. The sensitivity was 80.0%. The specificity was 87.5%. The positive predictive value was 80.0%. The negative predictive value was 87.5%.

[0178] The p-value for creatinine between group 3 and group 4, as measured by enzymatic method, was 0.021. In the ROC curves for serum creatinine in groups 3 and 4, measured by enzymatic methods, the area under the ROC curve was 0.89. Furthermore, the cutoff value calculated from Youden's Index was 0.60 mg / dL. Furthermore, the accuracy of the test when the cutoff value was set to 0.60 mg / dL was as follows. The overall agreement rate was 76.9%. The sensitivity was 100.0%. The specificity was 62.5%. The positive predictive value was 62.5%. The negative predictive value was 100.0%.

[0179] The p-value for TARC concentration between group 3 and group 4, as measured by chemiluminescent enzyme immunoassay (CLEIA), was 0.065. The area under the curve of the ROC curve for TARC concentration in serum from groups 3 and 4, measured by chemiluminescent enzyme immunoassay (CLEIA), was 0.83. Furthermore, the cutoff value calculated from Youden's Index was 100.0 pg / mL. Furthermore, the accuracy of the test when the cutoff value was set to 100.0 pg / mL was as follows: The overall agreement rate was 84.6%. The sensitivity was 80.0%. The specificity was 87.5%. The positive predictive value was 80.0%. The negative predictive value was 87.5%.

[0180] These results demonstrate that calprotectin, IFN-λ3, IL-6, CRP, white blood cell count, platelet count, neutrophil ratio to total white blood cell count, lymphocyte ratio to total white blood cell count, total bilirubin, urea nitrogen, creatinine, and TARC are highly sensitive and specific indicators for assessing the risk of first- and second-degree severe illness. [Industrial applicability]

[0181] The present invention is useful for clinical tests, particularly for testing the risk of progression from mild and moderate to severe cases of COVID-19, and the risk of progression from mild to moderate and severe cases.

Claims

1. A method for testing the risk of developing severe SARS-CoV-2 infection (COVID-19), The aforementioned risk of severe illness is the second-degree risk of progressing from stage (2) to stage (3) or (4) in the classification of severity below. The process includes a quantitative step for quantifying calprotectin in the sample, The process further includes an evaluation step of comparing the quantitative value of calprotectin with a cutoff value that serves as an indicator for the second severe disease risk, and evaluating the magnitude of the quantitative value in the sample. The aforementioned cutoff value is one point within the range of 3.5 to 8.1 μg / mL, which is a method for testing the risk of severe COVID-19. <Classification of severity> (1) Oxygen administration is not required. (2) The patient requires oxygen administration but does not require a high-flow nasal cannula, non-invasive ventilator, invasive ventilator, or extracorporeal membrane oxygenation (ECMO). (3) Requiring a high-flow nasal cannula, a non-invasive ventilator, or both, but not an invasive ventilator or extracorporeal membrane oxygenation (ECMO). (4) Requires an invasive ventilator, extracorporeal membrane oxygenation (ECMO), or both.

2. The testing method according to claim 1, wherein the quantification is performed by immunological measurement.

3. The testing method according to claim 2, wherein the quantification by immunological measurement is quantification using an antibody against calprotectin.

4. The testing method according to claim 2, wherein the quantification by immunoassay is performed by an enzyme immunoassay method.

5. The testing method according to claim 1, wherein the specimen is at least one selected from the group consisting of body fluids, cells, and tissues.

6. The examination method according to claim 5, wherein the body fluid is at least one selected from the group consisting of blood, urine, cerebrospinal fluid, saliva, lymph, pleural fluid, ascites, and bile.

7. The testing method according to claim 6, wherein the body fluid is serum or plasma.

8. A test reagent for predicting the risk of severe COVID-19, used in the test method according to any one of claims 1 to 7, wherein the quantitative step is a quantitative step of quantifying calprotectin in a sample, and the test reagent for predicting the risk of severe COVID-19 contains an anti-calprotectin antibody.

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