JP6944700B2 - How to test for autoimmune disorders - Google Patents

Description

The present invention relates to a method for testing an autoimmune abnormality of a test animal, a reagent kit for testing, a program for testing, and a device for testing.

Autoimmune disease is a disease in which self-cells, which should be exempted from immune attack targets, become attack targets and are destroyed for some reason. In a healthy immune system, "self-tolerance", which clearly distinguishes between self and non-self, works, but in autoimmune diseases, this self-tolerance system has collapsed. There are systemic and organ-specific autoimmune diseases, all of which target and gradually destroy nerves, digestive organs, respiratory organs, skeletal tissues, skin, and all other tissues. The pain of the patients is unbearable, and the treatment is long-term and extremely difficult. However, in recent years, many treatment methods have been developed, and the earlier the treatment method is, the higher the therapeutic effect is and the higher the possibility of remission. Early detection significantly reduces the burden on not only the patient but also the supporting family members, leading to an improvement in the QOL of the people involved.

Autoimmune diseases are not diagnosed until they become aware of the abnormality, but if early diagnosis is missed, the therapeutic effect will be significantly reduced. Diagnosis of autoimmune diseases includes the Coombs test, erythrocyte sedimentation rate (ESR) measurement, and blood autoantibody measurement, which is a disease marker. However, this is a diagnostic item when an autoimmune disease develops and is suspected, and sprouting cannot be detected.

It is important to capture the sprouting of onset before the patient becomes aware of the symptoms. If we can capture the "sprouting" of autoimmune diseases, we can greatly increase the possibility of remission and complete cure. What is needed is to keep up with the timing of the emergence of very early autoimmune diseases, but such tools do not yet exist.

It has been reported that patients with siliceous pneumonia frequently have autoimmune diseases such as rheumatism, scleroderma, and ANCA-related vasculitis in addition to respiratory symptoms (Non-Patent Document 1).

Lee S, et al., Allergy and Immunotoxicology in Occupational Health, p 15-26, 2016

An object of the present invention is to capture the germination of humans and non-human animals before they develop an autoimmune disease or before they become aware of the symptoms of an autoimmune disease. Another object of the present invention is to provide a test method, a test program, a test device, and a test reagent kit for autoimmune abnormalities in the nascent stage of the onset of autoimmune diseases.

In order to solve the above problems, the present inventors have repeatedly studied inflammatory cytokines, soluble cytokine receptors, etc., which are considered to be related to the inflammatory reaction, and various immunoglobulins which are the main body of the antibody. As a result, they have found that by measuring the amount of at least three factors in a sample collected from a test animal, it is possible to test for autoimmunity abnormalities before the onset of autoimmune disease with simple and high measurement accuracy. Was completed.

That is, the present invention comprises the following.
1. 1. A method for testing an autoimmunity abnormality of a test animal using an autoimmunity prediction score as an index, which comprises the following steps (1) and (2);
(1) A step of measuring the amount of at least three factors selected from the group consisting of the factors contained in the following (a) to (d) in the sample collected from the test animal;
(A) Inflammatory Cytokines (b) Soluble Cytokine Receptors (c) TNF / TNF Receptor Superfamily (d) Immunoglobulins (2) Measured values obtained in the steps of (1) are subjected to multiple regressions prepared in advance. The process of calculating the autoimmunity prediction score by applying it to the formula.
2. The test method according to item 1 above, wherein IgG4 is contained in the three factors.
3. 3. The test method according to item 1 or 2 above, wherein the three factors include soluble FAS.
4. The inspection method according to any one of the above items 1 to 3, wherein IL-6 is included in the three factors.
5. The test method according to item 1 above, wherein the three factors include IgG4, soluble FAS and IL-6.
6. The inspection method according to any one of items 1 to 5 above, wherein the multiple regression equation prepared in advance is the following mathematical expression 1.
(Formula 1) Autoimmunity prediction score = d + a × [IL-6 amount] + b × [soluble FAS amount] + c × [IgG4 amount]
[However, a, b, c, d in Equation 1 are non-zero real numbers]
7. The test method according to any one of items 1 to 6 above, further comprising a step of determining an immune abnormality when the autoimmunity prediction score is equal to or higher than a cutoff value.
8. The test method according to any one of the above items 1 to 7, wherein the test animal is a human.
9. The test method according to any one of items 1 to 8 above, wherein the sample is blood, serum or plasma.
10. It is a program for testing autoimmunity abnormality that functions a computer as a means for calculating an autoimmunity prediction score, and the autoimmunity prediction score is included in the following (a) to (d) in a sample collected from a test animal. A program for testing autoimmunity abnormalities calculated from measurements of the amount of at least three factors selected from the group consisting of factors and a pre-prepared multiple regression equation;
(A) Inflammatory Cytokines (b) Soluble Cytokine Receptors (c) TNF / TNF Receptor Superfamily (d) Immunoglobulins.
11. An device for testing autoimmunity abnormalities provided with a means for calculating an autoimmunity prediction score, and the autoimmunity prediction score is obtained from the following factors (a) to (d) in a sample collected from a test animal. A device for testing autoimmunity abnormalities calculated from a measured value of the amount of at least three factors selected from the group and a pre-prepared multiple regression equation;
(A) Inflammatory Cytokines (b) Soluble Cytokine Receptors (c) TNF / TNF Receptor Superfamily (d) Immunoglobulins.
12. Self, comprising at least 3 or more reagents for measuring the amount of at least 3 factors selected from the group consisting of the factors contained in the following (a)-(d) in a sample collected from a test animal. Reagent kit for testing autoimmunity abnormalities using the immune prediction score as an index;
(A) Inflammatory Cytokines (b) Soluble Cytokine Receptors (c) TNF / TNF Receptor Superfamily (d) Immunoglobulins.

INDUSTRIAL APPLICABILITY According to the present invention, the immune status of an autoimmune disease preparatory animal (including an autoimmune disease preparatory person) and a healthy animal (including a healthy person) who do not develop the symptoms of an autoimmune disease can be clearly distinguished for the first time. Became. Capturing preparatory animals for the onset of autoimmune disease can greatly increase the chances of remission or complete cure.

In the present invention, autoimmune abnormalities, which are the germs of autoimmune diseases, can be captured easily and with high accuracy. In the present invention, autoimmune abnormalities can be detected from changes in three or more factors that are not autoantibodies in a sample. When the sample is blood (serum, plasma), the sprouting of autoimmune disease can be detected by the fluctuation of 3 or more blood factors that are not autoantibodies, so the test is simple and the burden on the test animal is small, and it is general. Easy to incorporate into inspection.

In addition, by adding the test of the present invention to the general health examination items, it becomes possible to save people who are considered to be unrelated to autoimmune diseases and lose the opportunity for early diagnosis. In particular, save these people by adding them to the health check items of pneumoconiosis patients, people exposed to various chemical substances, such as silicosis, and young women with frequent autoimmune diseases. be able to.

(A) is a graph showing the ROC curve from the autoimmunity prediction score. (B) It is a table which shows the score (coordinates of ROC curve) for drawing an optimum ROC curve (Example 2). It is a graph which shows the distribution of the autoimmunity prediction score in a healthy person (HV) and the autoimmune disease onset preliminary group (autoimmune disease undeveloped silicosis case (SIL)) (Example 2).

The test method, test program, test device, and test reagent kit of the present invention test for autoimmune abnormalities. The state of the animal's immune system before and after the onset of symptoms of autoimmune disease is different from that of healthy animals. As used herein, the term autoimmune disorder means an abnormal state of the immune system before and after the onset of such symptoms of an autoimmune disease. The test target of the present invention may be an animal before the onset of symptoms of autoimmune disease or an animal after onset, but since there is no other test means, an animal before the onset of symptoms of autoimmune disease is preferable. Be inspected.

As used herein, the test animal may be any animal. Preferred are mammals, including humans. Examples of non-human animals include pet animals, zoo-reared animals, laboratory animals, livestock, and the like, and specifically include mice, rats, rabbits, sheep, pigs, cows, horses, cats, dogs, and dogs. Examples include monkeys and chimpanzees. A more preferred test animal is a human or pet animal, and a more preferred test animal is a human.

When the test animal is a human in the present specification, the test target may be any human being. For example, the subject may be a subject such as a general health examination or a human dock. It may be a pneumoconiosis patient represented by silicosis or a person exposed to various chemical substances. It may be a young woman or the like who frequently has an autoimmune disease.

In the present specification, the sample may be a sample obtained from a test animal. For example, blood, urine, cerebrospinal fluid, tissue obtained for examination, excised tissue, and the like can be mentioned. Further, a sample obtained by treating or fractionating blood or tissue is also included in the sample in the present invention. Blood and the treated plasma or serum are preferable as the sample because the burden on the test animal and the simplification of the test.

The test method, test program, test device, and test reagent kit of the present invention use the autoimmunity prediction score as an index of autoimmunity abnormality. The autoimmunity prediction score was obtained by measuring the amount of at least three factors selected from the group consisting of the factors contained in the following (a) to (d) in the sample collected from the test animal. The value is calculated by applying it to a pre-created multiple regression equation.
(A) Inflammatory Cytokines (b) Soluble Cytokine Receptors (c) TNF / TNF Receptor Superfamily (d) Immunoglobulins

In the present specification, the factor includes not only the factor itself, but also a factor (molecule) composed of a part of the factor (molecule), mRNA encoding the factor, and the like.

Inflammatory cytokines are cytokines associated with inflammation and include IL-6, IL-10, IL-1α, TGF-β1, G-CSF, IL-8 and the like. IL-6 is an example of an inflammatory cytokine suitable as a measurement target.

Soluble cytokine receptors consist of a part of cytokine receptors and are present in blood and the like. Examples of soluble cytokine receptors include IL-2Rα, IL-6sR, IL-12R and the like.

TNF / TNF receptor superfamily means TNF superfamily and TNF receptor superfamily. As used herein, the TNF / TNF receptor superfamily also includes soluble factors (molecules) that are part of the factors (molecules) of the TNF / TNF receptor superfamily. Factors included in the TNF / TNF receptor superfamily include, for example, FAS, soluble FAS, DcR3, BAFF and the like. Soluble FAS is a factor (molecule) in which FAS is cleaved and solubilized. Since blood and plasma or serum treated therein are preferable as the sample, a soluble factor (molecule) is preferable as the factor contained in the TNF / TNF receptor superfamily. Soluble factors include soluble FAS, BAFF, DcR3 and the like. Soluble FAS (hereinafter, also referred to as sFAS) is mentioned as a soluble factor suitable as a measurement target.

Immunoglobulins are classified into five classes of IgG, IgA, IgE, IgM and IgD, and further classified into subclasses IgG1, IgG2, IgG3 and IgG4. Any immunoglobulin to be measured may be used. IgG4 is an example of an immunoglobulin suitable as a measurement target.

In order to calculate the autoimmunity prediction score, the amount of at least three factors selected from the group consisting of the factors contained in the following (a) to (d) in the sample collected from the test animal is measured.
(A) Inflammatory Cytokines (b) Soluble Cytokine Receptors (c) TNF / TNF Receptor Superfamily (d) Factors contained in immunoglobulins (a) to (d) are (a) (b) (c) ) And (d). At least three factors may belong to (a) to (d) in any way. It may belong to each group dispersedly, or all factors may belong to one group. For example, when the measurement target is three factors, the three factors are (a) one factor selected from inflammatory cytokines, (c) one factor selected from the TNF / TNF receptor superfamily, and (d) immunity. It can be a three factor consisting of one factor selected from globulin, and can be (a) three factors selected from an inflammatory cytokine.

The at least three factors preferably include IgG4 and sFAS. More preferably, IgG4, sFAS and IL-6 are included. By using IgG4, sFAS and IL-6 as measurement targets, it is possible to discriminate between immunocompromised persons and healthy persons before the onset of autoimmune disease with high accuracy.

The number of factors to be measured to calculate the autoimmunity prediction score is 3 or more in order to increase the sensitivity and specificity, preferably 10 or less, preferably 8 from the viewpoint of the complexity of the test and the sensitivity and specificity. The following is more preferable, 6 or less is further preferable, 5 or less is further preferable, and 4 or less is further preferable. The number is most preferably three to simplify the inspection. By targeting the three factors of IgG4, sFAS and IL-6 as measurement targets, it is possible to carry out a simple and highly accurate test.

The amount of these factors in the sample can be measured by any method well known to those of skill in the art. For example, it can be measured by the ELISA method and the EIA method. A preferred measuring method is the ELISA method. The sample can be measured as it is or after pretreatment. If the sample is blood, plasma, or the like, it can be measured as it is, and if the sample is a tissue, each factor may be extracted before measurement.

In the present specification, the amount of a factor is represented by the weight, concentration, activity, relative amount, etc. of the factor. Since blood and plasma or serum treated therein are preferable as the sample, the amount of the factor is preferably expressed in concentration.

The amount of each measured factor is applied to a pre-prepared multiple regression equation to calculate the autoimmunity prediction score. The multiple regression equation is obtained by multiple regression analysis of the amounts of factors in a sample of a plurality of animals that are in the preparatory group for the onset of autoimmune disease. The factor is the same as at least three factors to be measured in the test animal. That is, the multiple regression equation is the amount of at least three factors selected from the group consisting of the factors included in the following (a) to (d) in the sample collected from the animal which is the preparatory group for the onset of autoimmune disease. It is a mathematical formula obtained by measuring and performing multiple regression analysis using the measured value as an independent variable.
(A) Inflammatory Cytokines (b) Soluble Cytokine Receptors (c) TNF / TNF Receptor Superfamily (d) Immunoglobulins

The preferred formula for the multiple regression equation is the IL-6 amount, the sFAS amount, and the IgG4 amount as independent variables. For example, the following formula 1 is preferable.
(Formula 1) Autoimmunity prediction score = d + a × [IL-6 amount] + b × [sFAS amount] + c × [IgG4 amount]
[However, a, b, c, d in Equation 1 are non-zero real numbers]

The specific values of a, b, c, and d in Equation 1 may be those set by multiple regression analysis. For example, the following formula 2 is exemplified.
(Formula 2) Autoimmunity prediction score = 0.063 + 0.059 x [IL-6 amount] +0.119 x [sFAS amount] +1.063 x [IgG4 amount]
([IL-6 amount] is the plasma concentration of IL-6 (pg / ml), [sFAS amount] is the plasma concentration of sFAS (ng / ml), and [IgG4 amount] is the plasma concentration of IgG4 (μg). / Ml))

The test method of the present invention may further include a step of determining an immune abnormality when the autoimmunity prediction score is equal to or higher than the cutoff value. The value having the largest value of {sensitivity − (1-unique)} in the coordinates of the ROC curve (FIG. 1b) can be set as the cutoff value.

The multiple regression equation can be updated by adding the test animal test measurements to the independent variables of the multiple regression analysis. The updated multiple regression equation is used to calculate the autoimmunity prediction score using the measurements obtained thereafter. By performing the multiple regression analysis including the acquired measured values, the setting of the coefficient, the determination term, and the cutoff value in the multiple regression equation is updated from time to time.

The reagent kit for testing autoimmune disorders of the present invention measures the amount of at least three factors selected from the group consisting of the factors contained in the following (a) to (d) in a sample collected from a test animal. Contains at least 3 or more reagents for this purpose.
(A) Inflammatory Cytokines (b) Soluble Cytokine Receptors (c) TNF / TNF Receptor Superfamily (d) Immunoglobulins

The type and number of reagents included in the test reagent kit of the present invention may increase or decrease depending on the number of factors to be measured. Preferably, a reagent for measuring the amount of IgG4 is included. Further, preferably, a reagent for measuring the amount of sFAS is included. Also preferably, a reagent for measuring the amount of IL-6 is included. More preferably, a reagent for measuring the amount of IgG4 and a reagent for measuring the amount of sFAS are included. More preferably, a reagent for measuring the amount of IgG4, a reagent for measuring the amount of sFAS, and a reagent for measuring the amount of IL-6 are included.

Reagents that measure the amount of each factor can include antibodies that can bind to each factor. The antibody may be a known antibody, or an antibody produced by an existing general production method or a commercially available antibody can be used. The antibody may be a polyclonal antibody or a monoclonal antibody. The antibody may be labeled with a labeling substance. Specifically, as the labeling substance, an enzyme, a radioactive isotope, a fluorescent dye, a biotin, a dye sol, and an insoluble carrier such as colloidal gold or latex particles can be used. Further, although labeling can be performed by a known method, the labeling substance may be bound to an antibody. The above antibody is included in the detection reagent kit of the present invention as a reagent (reagent composition). Examples of the reagent containing the antibody include reagents that can be stored for a long period of time, such as in a form diluted with physiological saline or a buffer solution, or in a lyophilized form. The antibody may be dissolved in a solution containing an additive (for example, a carrier, an excipient, a diluent, etc.), a stabilizer, or the like, and then freeze-dried to prepare a reagent. Stabilizers include monosaccharides such as glucose, disaccharides such as saccharose and maltose, sugar alcohols such as mannitol and sorbitol, neutral salts such as sodium chloride, amino acids such as glycine, polyethylene glycol and polyoxyethylene-polyoxy. Nonionic surfactants such as propylene copolymer (Pluronic) and polyoxyethylene sorbitan fatty acid ester (Tween), human glucose and the like are exemplified, and it is preferable that about 1 to 10 w / v% is added. Further, the detection reagent kit of the present invention may appropriately contain a blocking solution, a reaction solution, a reaction terminator solution and the like.

Reagents for measuring the amount of each factor may include reagents for detecting mRNA of each factor. The reagent for detecting mRNA of each factor contains a polynucleotide capable of detecting the expression of each factor. In the present specification, a polynucleotide refers to a biopolymer in which two or more nucleotides composed of a base, a sugar, and a phosphoric acid are conjugated with a phosphate ester, and includes nucleic acids such as DNA and RNA. The polynucleotide has a length of 8 nucleotides (base) or more. The polynucleotide functions as a probe capable of specifically hybridizing to a specific mRNA, a primer capable of specifically annealing to a specific mRNA, and a primer capable of amplifying a nucleic acid fragment in combination with the primer. The base sequence of the polynucleotide does not have to be a sequence that is 100% complementary to the base sequence of the gene whose expression is detected, and is about 1 to 5 bases (preferably about 1 to 4 bases, more preferably 1 to 2). It may be deleted (as much as a base), or it may be replaced, inserted or added with a non-complementary base. When the polynucleotide is a primer, it is preferably about 17 to 25 nucleotides, and when it is a probe, it is preferably about 8 to 40 nucleotides, and more preferably about 10 to 30 nucleotides. The primers may be included in the detection reagent kit of the present invention as a pair of primer sets.

The autoimmunity abnormality test program of the present invention makes a computer function as a means for calculating an autoimmunity prediction score. The autoimmunity prediction score is a measured value of the amount of at least three factors selected from the group consisting of the factors contained in the following (a) to (d) in the sample collected from the test animal, and prepared in advance. It is calculated from the calculated multiple regression equation.
(A) Inflammatory Cytokines (b) Soluble Cytokine Receptors (c) TNF / TNF Receptor Superfamily (d) Immunoglobulins

The program of the present invention may have means for multiple regression analysis using the measured values of the amounts of various factors measured for a plurality of samples as independent variables. Further, the inspection program of the present invention may have a storage means for storing the measured value. In addition, it may have a selection means for selecting additional factors to be used as independent variables in the multiple regression equation. Further, it may have an updating means for updating the multiple regression equation by performing multiple regression analysis on the measured values obtained from the sample collected from the test animal in addition to the stored measured values. Further, it may have a means for determining the cutoff value, and may have a determining means for determining the presence or absence of an autoimmune abnormality based on the cutoff value.

The device for testing autoimmunity abnormality of the present invention includes means for calculating an autoimmunity prediction score. The autoimmunity prediction score calculation means includes means for inputting measured values of the amounts of various factors in a sample collected from a test animal, and means for applying the input values to a pre-prepared multiple regression equation. The inspection apparatus of the present invention may include means for multiple regression analysis of measured values of various factors. Further, the inspection device of the present invention may include measuring means for measuring various factors in a sample collected from a test animal. Further, a storage means for storing the measured value may be provided. In addition, it may have a selection means for selecting additional factors to be used as independent variables in the multiple regression equation. Further, the measured value obtained from the sample collected from the test animal may be subjected to multiple regression analysis in addition to the stored measured value, and may have an updating means for updating the multiple regression equation. Further, it may have a means for determining the cutoff value, and may have a determining means for determining the presence or absence of an autoimmune abnormality based on the cutoff value. Further, a display means for showing a determination result or a measured value based on the determination means may be provided.

In order to help the understanding of the present invention, the present invention will be specifically described with reference to Examples below, but the present invention is not limited to the present examples.

(Example 1) Acquisition of measured values of various factors Silicosis patients frequently develop autoimmune diseases. From this, patients with silicosis who had not developed an autoimmune disease were regarded as a preliminary group for the onset of autoimmune disease, and peripheral blood factors involved in the inflammatory reaction were measured.

With the approval of the Institutional Review Board of Kawasaki Medical School Hospital, healthy subjects (19 males, 11 females, average age 44.84 ± 8.58 years), and preparatory group for the onset of autoimmune disease (preliminary group for autoimmune disease onset) Peripheral blood was collected from (20 cases of silicosis without autoimmune disease) (20 males, 19 males, 1 female, average age 74.85 ± 5.35 years), and the concentration of the plasma factor thereof was measured. As the factors to be measured, inflammatory cytokines, soluble cytokine receptors, etc., which are considered to be related to the inflammatory response, and various immunoglobulins which are the main body of the antibody were selected (Table 1).

An Enzyme-Linked Immuno Solvent Assay (ELISA method) was used for the measurement. Reagents for measuring each factor are shown below.
IgG4: Human IgG Subclass Profile ELISA Kit (Invitrogen)
sFAS: Human Fas / TNFRSF6 Quantikine® ELISA (R & D)
IL-6: Human IL-6 Quantikine® ELISA (R & D)
DcR3: LEGEND MAX ^TM Human Soluble DcR3 / TNFRSF6B ELISA kit (BioLegend®)
IL-2Rα: Human IL-2Rα Quantikine® ELISA (R & D)
IL-10: Human IL-10 Quantikine® ELISA (R & D)
IL-1α: RayBio® Human IL-1α ELISA Kit (RayBiotech, Inc.)
IL-6sR: Human IL-6sR Quantikine® ELISA (R & D)
TGF-β1: Human TGF-β1 Quantikine® ELISA (R & D)
G-CSF: Human G-CSF Quantikine® ELISA (R & D)
IgG: Human IgG ELISA Kit (Bethyl Laboratories, Inc.)
IgA: Human IgA ELISA Kit (Bethyl Laboratories, Inc.)
IgE: Human IgE ELISA Kit (Bethyl Laboratories, Inc.)
IgM: Human IgM ELISA Kit (Bethyl Laboratories, Inc.)
IgG1: Human IgG Subclass Profile ELISA Kit (Invitrogen)
IgG2: Human IgG Subclass Profile ELISA Kit (Invitrogen)
IgG3: Human IgG Subclass Profile ELISA Kit (Invitrogen)
BAFF: Human BAFF / BLys / TNFSF13B Quantikine® ELISA (R & D)
LAG3: Human lymphocyte activation gene 3 protein (LAG3) ELISA kit (CUSABIO)

(Example 2) Preparation of multiple regression equation The measured values of each factor obtained in Example 1 were subjected to multiple regression analysis by automatic linear modeling using statistical software (IBM SPSS Statistics Ver.22). Plasma concentrations of all the factors shown in Table 1 were introduced as independent variables, and the dependent variable was analyzed with an autoimmunity prediction score (healthy subjects: 1, autoimmune disease onset preliminary group: 2).

As a result, the following formula for obtaining the autoimmunity prediction score was obtained (Table 2).
Autoimmunity prediction score = 0.063 + 0.059 x [IL-6 amount] +0.119 x [sFAS amount] +1.063 x [IgG4 amount]
([IL-6 amount] is the plasma concentration of IL-6 (pg / ml), [sFAS amount] is the plasma concentration of sFAS (ng / ml), and [IgG4 amount] is the plasma concentration of IgG4 (μg). / Ml))

ROC (Receiver Operating Characteristic) analysis was performed from the autoimmunity prediction score, and the results were obtained with a sensitivity of 94.4% and a cutoff value of 1.3935 (Fig. 1). From the autoimmunity prediction score, when the healthy subjects and the silicosis cases were divided by the cutoff value, the results were quite credible, with no false positives in the healthy subjects and one false negative in the silicosis cases (Fig. 2).

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to clearly distinguish the immune status between the autoimmune disease preparatory group and the healthy animal group in which the symptoms of autoimmune disease have not developed. Capturing autoimmune disease preparatory individuals and autoimmune disease preparatory animals can greatly increase the likelihood of remission or complete cure. Since the test method of the present invention is simple, it can be added to general health examination items, and it becomes possible to save people and animals who are considered to be unrelated to autoimmune diseases and lose the opportunity for early diagnosis. ..

Claims (7)

A method for testing an autoimmunity abnormality of a test animal using an autoimmunity prediction score as an index, which comprises the following steps (1) and (2);
(1) A step of measuring the amount of at least three factors selected from the group consisting of the factors contained in the following (a) to (d) in the sample collected from the test animal;
(A) Inflammatory Cytokines (b) Soluble Cytokine Receptors (c) TNF / TNF Receptor Superfamily (d) Immunoglobulins (2) Measured values obtained in the steps of (1) are subjected to multiple regressions prepared in advance. The process of calculating the autoimmunity prediction score by applying it to the formula ,
Here, the three factors include IgG4, soluble FAS and IL-6, and the pre-prepared multiple regression equation is Equation 1 below.
(Formula 1) Autoimmunity prediction score = d + a x [IL-6 amount] + b x [soluble FAS amount] + c x [IgG4 amount]
[However, a, b, c, and d in Equation 1 are non-zero real numbers]. The test method according to claim 1 , further comprising a step of detecting a sample having an immunological abnormality when the autoimmunity prediction score is equal to or higher than a cutoff value. The test method according to claim 1 or 2 , wherein the test animal is a human. The test method according to any one of claims 1 to 3 , wherein the sample is either blood, serum or plasma. It is a program for testing autoimmunity abnormality that functions a computer as a means for calculating an autoimmunity prediction score, and the autoimmunity prediction score is included in the following (a) to (d) in a sample collected from a test animal. A program for testing autoimmunity abnormalities calculated from measurements of the amount of at least three factors selected from the group consisting of factors and a pre-prepared multiple regression equation;
(A) Inflammatory Cytokines (b) Soluble Cytokine Receptors (c) TNF / TNF Receptor Superfamily (d) Immunoglobulins ,
Here, the three factors include IgG4, soluble FAS and IL-6, and the pre-prepared multiple regression equation is Equation 1 below.
(Formula 1) Autoimmunity prediction score = d + a x [IL-6 amount] + b x [soluble FAS amount] + c x [IgG4 amount]
[However, a, b, c, and d in Equation 1 are non-zero real numbers]. An device for testing autoimmunity abnormalities provided with a means for calculating an autoimmunity prediction score, and the autoimmunity prediction score is obtained from the following factors (a) to (d) in a sample collected from a test animal. A device for testing autoimmunity abnormalities calculated from a measured value of the amount of at least three factors selected from the group and a pre-prepared multiple regression equation;
(A) Inflammatory Cytokines (b) Soluble Cytokine Receptors (c) TNF / TNF Receptor Superfamily (d) Immunoglobulins,
Here, the three factors include IgG4, soluble FAS and IL-6, and the pre-prepared multiple regression equation is Equation 1 below.
(Formula 1) Autoimmunity prediction score = d + a x [IL-6 amount] + b x [soluble FAS amount] + c x [IgG4 amount]
[However, a, b, c, and d in Equation 1 are non-zero real numbers]. Self, comprising at least 3 or more reagents for measuring the amount of at least 3 factors selected from the group consisting of the factors contained in the following (a)-(d) in a sample collected from a test animal. A reagent kit for testing autoimmunity abnormalities for use in the test method according to any one of claims 1 to 3, using the immune prediction score as an index;
(A) Inflammatory Cytokines (b) Soluble Cytokine Receptors (c) TNF / TNF Receptor Superfamily (d) Immunoglobulins.

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