WO2019111797A1 - Biomarker for diagnosing muscle disease - Google Patents

Abstract

The present invention aims to provide a biomarker for early diagnosis of muscle disease. In addition, the present invention aims to provide a measurement technology that facilitates early diagnosis of muscle disease using said biomarker. The biomarker for muscle disease and comprising anti-DDB1 antibodies is useful as a biomarker for early diagnosis of muscle disease. A muscle disease detection method including a step in which anti-DDB1 antibodies in a blood sample derived from a test subject are detected is useful for the early detection of muscle disease. Ideally said muscle disease is polymyositis.

Description

Biomarkers for the diagnosis of muscle disease

The present invention relates to a biomarker for early diagnosis of muscle disease.

Inflammatory myopathy (IM) is a systemic autoimmune disease that includes not only muscle but also skin and organs such as lung, heart and joints. The IM includes polymyositis and dermatomyositis classified according to Bohan and Peter's diagnostic criteria (Non-Patent Document 1) proposed in 1975.

In general, diagnosis of myositis includes clinical symptoms including muscle symptoms, and laboratory findings (specifically, blood tests (myogenic enzyme levels such as creatine kinase and aldolase levels) and physiological tests (needle electromyography) Etc), imaging (MRI), histology (muscle biopsy)). Muscle biopsy plays an important role especially in the definitive diagnosis of IM.

Recently, many myositis-specific autoantibodies (MSA) and myositis-associated autoantibodies (MAA) have been reported. These MSAs and MAAs are noteworthy in that they are closely related to the clinical features. So far, detection of MSA and MAA in the serum of patients has facilitated disease diagnosis, clinical course prediction, and treatment decisions in the early stages of the disease. Such autoantibodies are useful in that they make it easy to determine the cause of symptoms such as muscle weakness or abnormalities in laboratory findings, and serve as a basis for determining the implementation of aggressive examinations such as MRI and muscle biopsy at an early stage. .

On the other hand, DNA damage-binding protein 1 (DDB1) has been found as a protein involved in the process of DNA repair. DDB1 has also been reported to be associated with ultraviolet light (Non-patent Documents 3 and 4) and with viral infection (Non-patent Documents 5 to 11).

MSA and MAA can be used as useful biomarkers for patients who are positive for them. However, there are only a few types of MSA and MAA that can actually be measured at present clinical sites, and the positive rate is about 50% of the cases of myo-inflammatory cases. If these antibodies are judged to be negative and myositis is not positively suspected, it is often the case that early diagnosis is not achieved and treatment is delayed. Especially in the case of polymyositis, unlike dermatomyositis, there are cases where typical skin findings are not shown and cases where interstitial pneumonia is not observed, this problem is made more serious.

For example, even if there are some abnormalities in a blood test or a physiological test, the abnormalities may depend on individual circumstances such as the patient's lifestyle, living environment, muscle movement condition at the time of consultation, etc. It is difficult to make a positive diagnosis as it is a clear anomaly that originates. Even if the indication of diagnostic imaging is to be determined for further examination, the hurdle is high for the convenience of the equipment. In addition, even if a slight abnormal image is found when the imaging diagnosis is indicated, it is also invasive and therefore the patient's psychology to determine the indication of the muscle biopsy for the further examination. Above all the hurdles are significantly higher. For this reason, follow-up measures are often taken in cases where blood tests, physiological tests or imaging tests do not show clear abnormalities. If symptoms progress slowly, follow-up measures are further promoted. Under these circumstances, early diagnosis is extremely difficult.

In addition, in recent years where internal medicine has been subdivided, patients are often referred to a department other than internal medicine for treating polymyositis (especially general internal medicine) as described above. In such families, cases that do not show clear abnormalities in blood tests, physiological tests or imaging tests are more likely to be followed up without scrutiny and diagnosis. It is thought that the cases that caused muscle weakness and / or muscle atrophy during the follow-up are overwhelmingly more frequent than the patients who visited the department of internal diseases of collagen diseases.

In addition, in the case of inclusion body myositis treated mainly by neurology, the presence of myofibers with bordered vacuoles (inclusions) is a major clue for diagnosis, but in cases where such a finding is not found, it is clear It is not diagnosed with various inclusion body myositis, and there are not a few cases treated according to inclusion body myositis with the course showing chronic muscle weakness and / or muscle atrophy. Alternatively, in the group of diseases that show muscle weakness that are not diagnosed as clear collagen disease and are treated in other departments such as neurology, those that progress chronically and slowly progress to effective diagnosis In some cases, no active treatment has been introduced because no treatment has been established. Even in cases such as these, cases where recovery of muscle strength can be expected by appropriate diagnosis and measures, that is, cases in which recovery of muscle strength can be expected if appropriate diagnosis and therapeutic intervention have been made are potential. It is thought that there are many.

Muscle weakness and / or muscle atrophy caused by the diagnosis of muscle disease and delayed action can not be returned to the original state because they are irreversible symptoms. Muscle weakness and / or muscle atrophy greatly affect the patient's QOL. Thus, the loss of lifelong health due to delayed diagnosis and action is very serious.

Therefore, the present invention is broadly directed to muscle diseases which can not be detected by conventional MSA or MMA, or muscle diseases which have been missed not only in internal diseases of collagen diseases but also in general medicine or neurology. The purpose is to provide a biomarker for early diagnosis. Furthermore, another object of the present invention is to provide a measurement technique suitable for early diagnosis of muscle disease using a biomarker as described above.

The inventor conducted intensive studies, and based on serum samples and clinical information collected from the patients and healthy controls, there were only minor symptoms such as no or missed skin symptoms, blood tests, physiological tests, Or, in cases where there is no obvious abnormality derived from muscle disease on imaging examination and DDB1 protein is common in the cases in which a definitive diagnosis of muscle disease has been made by intentionally taking a muscle biopsy, DDB1 protein Were found to have antibodies against DDB1 protein is a protein that has not been known to be associated with muscle disease. The present invention has been completed by further studies based on this finding.

That is, the present invention provides the invention of the aspects listed below.
Item 1. A biomarker for muscle disease consisting of anti-DDB1 antibodies.
Item 2. The biomarker according to item 1, wherein the muscle disease is polymyositis.
Item 3. A method for detecting muscle disease, comprising the step of detecting an anti-DDB1 antibody in a blood sample derived from a subject.
Item 4. Item 4. The detection method according to Item 3, wherein the blood sample is serum.
Item 5. Item 5. The detection method according to Item 3 or 4, wherein the clinical finding of the subject does not substantially exhibit skin symptoms.
Item 6. The detection method according to any one of Items 3 to 5, wherein the myopathy is polymyositis.
Item 7. 7. The detection method according to any one of items 3 to 6, wherein the blood test findings of the test subject have normal values for myogenic enzymes.
Item 8. 7. The detection method according to any one of Items 3 to 6, wherein the blood test findings of the subject are creatine kinase 300 U / L or more.
Item 9. Item 9. The detection method according to any one of Items 3 to 6 and 8, wherein the blood test findings of the subject are aldolase 6.2 IU / L or more.
Item 10. The detection method according to any one of Items 3 to 9, wherein the detection is performed using an antigen consisting of DDB1 protein or a partial peptide thereof.
Item 11. Item 11. The detection method according to Item 10, wherein the detection is performed by ELISA.
Item 12. A reagent for testing a muscle disease, which comprises an antigen consisting of DDB1 protein or a partial peptide thereof.
Item 13. A diagnostic kit for muscle disease, comprising the test reagent according to Item 12.
Item 14. A method for diagnosing muscle disease, comprising: detecting an anti-DDB1 antibody of a blood sample collected from a subject; and diagnosing muscle disease based on the detection result obtained in the step.
Item 15. Use of an antigen consisting of DDB1 protein or a partial peptide thereof for the production of a reagent for testing a muscle disease.

According to the present invention, since biomarkers for early diagnosis of muscle diseases are provided, muscle diseases which can not be detected by conventional MSA or MMA, or collagen diseases as well as general medicine or neurology, etc. It is useful for early diagnosis of muscle disease, since screening for muscle diseases that have been missed in other families can be widely targeted. Furthermore, according to the present invention, a test reagent capable of easily detecting a biomarker as described above and a diagnostic kit comprising the test reagent are provided, so that screening for a wide range of subjects can be performed for early diagnosis of muscle disease. It is useful.

The electrophoresis result of the protein immunoprecipitated from the serum of the inflammatory myopathy patient is shown. Common to the patients (lanes 1 to 6), an anti-120 kDa antibody indicated by an arrow was detected. Immunoaffinity chromatography from the serum of patients inflammatory myopathies by (immunoaffinity chromatography), 1M NaCl, 1M MgCl 2, and 3M antigens stepwise purified using MgCl ₂ (antigen anti 120kDa antibody) as elution liquid The results of SDS-PAGE electrophoresis gel silver staining (FIG. 2 (A)) and the results of western blot analysis (FIG. 2 (B)) were carried out using the same patient serum as the primary antibody to confirm the corresponding protein. The lane 1 shows the 1 M NaCl elution fraction, the lane 2 shows the 1 M MgCl ₂ elution fraction, and the lane 3 shows the 3 M MgCl ₂ elution fraction. A band of about 120 kDa in lane 3 was identified as DDB1 by PMF. The results of Western blot analysis for human DDB1 human recombinant protein are shown, using serum and normal control serum of inflammatory myopathic patients and commercially available anti-DDB1 antibody as primary antibody. It was confirmed that the target autoantigen of the anti-120 kDa antibody in the patient's serum is indeed DDB1 and the anti-120 kDa antibody is an anti-DDB1 antibody.

1. Biomarker The biomarker for muscle disease of the present invention is characterized by comprising an anti-DDB1 antibody. Hereinafter, the biomarker of the present invention will be described in detail.

In the present invention, “muscle disease” is a disease to be diagnosed when the biomarker of the present invention is positive, and specifically refers to a disease exhibiting muscle weakness and / or muscle atrophy as one of the symptoms. . As far as this is done, the myopathy in the present invention is not particularly limited, and examples thereof include both inflammatory myopathy and non-inflammatory myopathy. Inflammatory myopathies include idiopathic inflammatory myopathy (IIM). Idiopathic inflammatory myopathy includes polymyositis (PM), dermatomyositis (DM), and inclusion body myositis (Sporadic Inclusion Body Myositis; sIBM). Among these, polymyositis is particularly effective because the biomarker of the present invention is effective since there is no skin symptom. In addition, when the dermatomyositis is overlooked because the degree of skin symptoms is extremely slight, the biomarker of the present invention is effective. The biomarker of the present invention is effective when inclusion body myositis is overlooked because there is no inclusion body pathologically. The biomarkers of the present invention enable early diagnosis of these myopathies.

Anti-DDB1 antibody is an autoantibody that specifically recognizes DDB1 protein as an antigen. DDB1 (DNA damage-binding protein 1) is involved in the process of DNA repair, and is further known to be associated with ultraviolet light and viral infection. The specific amino acid sequence of DDB1 and the nucleotide sequence encoding it can be referred to the accession number Q16531 of UniProt (the universal protein resource) (http://www.uniprot.org/uniprot/Q16531 ).

In the present invention, the class of the antibody is not particularly limited as long as it can be detected, and may be any of IgG (including IgG1, IgG2, IgG3, and IgG4), IgD, IgE, IgA, sIgA, IgM and the like. As a preferred class of antibodies in the present invention, IgG is exemplified. Furthermore, the biomarkers of the present invention are also used for binding fragments of antibodies (eg, F (ab ') ₂ , Fab', Fab, Fv, sFv, dsFv, sdAb) etc. as long as they specifically bind to the antigen It can be used as

2. Method for detecting muscle disease The present invention provides a method for detecting muscle disease. The detection method of the present invention is characterized by comprising the step of detecting an anti-DDB1 antibody in a blood sample collected from a subject.

2-1. Subject The detection target sample of the detection method of the present invention is a blood sample, and the detection target substance is the aforementioned biomarker (ie, anti-DDB1 antibody).

2-2. Test subject The muscle disease to be analyzed by the test method of the present invention corresponds to a muscle disease which affects a test subject from which a blood sample to be analyzed is derived. The subject may be any animal, and specifically, experimental animals such as rodents such as mice, rats, hamsters and guinea pigs and rabbits; domestic animals such as pigs, cows, goats, horses and sheep; Examples include pets such as dogs and cats; and primates such as humans, monkeys, orangutans and chimpanzees. The subject in the present invention is preferably a primate, more preferably a human. Furthermore, as the test subjects, internal diseases such as internal diseases of collagen diseases, neurology and general internal medicine, and any other departments, and all patients who receive medical examinations and the like correspond.

As a pathological condition which a test subject has, a subjective symptom and an objective symptom, and those presence or absence do not ask. In particular, the present invention is a pathological condition which does not show a clear abnormality in clinical findings and laboratory findings (blood test, physiological test and imaging test) as objective symptoms even in light of conventional diagnostic criteria, It is effective for a pathological condition that can be definitively diagnosed by muscle biopsy, that is, a pathological condition that can not be definitively diagnosed without muscle biopsy.

Specifically, the clinical findings include an embodiment in which skin symptoms are not substantially exhibited. The skin symptoms referred to here include skin inflammation such as Heliotrope rash, Gottron papule, Gottron symptoms, nail erythema, polymorphic skin atrophy, Mechanics hand and the like. The absence of substantial skin symptoms refers to the absence of skin symptoms (especially in the case of polymyositis and inclusion body myositis) and the case where skin symptoms are so slight as to be overlooked under normal diagnostic criteria ( In particular, dermatomyositis is applicable.

Among the laboratory findings, in blood tests, for example, those with normal values of myogenic enzymes such as creatine kinase (CK) and aldolase (ALD); those with myogenic enzymes are higher than normal values Although the degree of increase is slight, it is difficult to make a clear abnormal judgment; and even if the value of the myogenic enzyme shows an abnormal value beyond the normal value Good.

Even if the value of myogenic enzyme is normal, muscle biopsy may lead to a definitive diagnosis of muscle disease. Therefore, even when the myogenic enzyme value is normal, the detection method using the biomarker of the present invention is effective.

In addition, because the value of myogenic enzymes depends not only on muscle disease but also on individual circumstances such as the patient's lifestyle, living environment, and exercise conditions of muscles at the time of medical examination, the value of blood test itself is normal. Even if it is not (that is, a value slightly above the normal value or showing an abnormal value beyond the normal value), it is difficult to make a judgment as a clear abnormality attributable to a muscle disease. Therefore, the biomarker of the present invention is effective even when the myogenic enzyme value is not normal. More specifically, creatine kinase is above the normal range (43 to 157 U / L for human) and / or the normal range of aldolase (for human, 2.1 to 6.1 IU / L). L), preferably when the test subject is a human, when the creatine kinase is 300 U / L or more, and / or the aldolase is 6.2 IU / L or more, the biomarker of the present invention is used It is also preferable to carry out the same detection method.

Among the laboratory findings, as a physiological examination, needle electromyography etc. may be mentioned. Physiological examination findings may be either in the absence of abnormal findings or in the case of findings of myogenic pattern. In addition, in the case where findings of the myogenic pattern are observed, either the myogenic pattern alone or a mixture of the myogenic and neurogenic patterns may be used.

Among the examination findings, imaging examinations include nuclear magnetic resonance imaging (MRI), computed tomography (CT) and the like. As the findings of the imaging examination, any mode may be used in the case where there is no abnormal finding and in the case where a finding of a muscle tissue inflammation image is found.

2-3. Blood sample In the present invention, a blood sample collected from a subject whose muscle disease is to be diagnosed is used. The type of blood sample is not particularly limited as long as the biomarker can be detected, and examples include whole blood, serum, plasma and the like. Serum can be mentioned as a suitable example from a viewpoint of being able to prepare simply and detecting the said biomarker more correctly.

Collection and preparation of various blood samples can be performed according to known methods. For example, in the case of serum, it can be prepared by removing blood cells and collected blood coagulation factors such as fibrinogen (factor I), prothrombin (factor II), factor V, factor VIII and the like from collected blood (whole blood) Or the supernatant obtained after leaving the blood standing, or the supernatant obtained by subjecting the blood to centrifugation. The blood sample is used after dilution to an appropriate concentration, as necessary.

2-4. Antigen In the detection method of the present invention, detection of anti-DDB1 antibody is carried out by using DDB1 protein or its partial peptide as an antigen, and specifically binding to the aforementioned biomarker (anti-DDB1 antibody) in a blood sample.

The antigen used to detect the biomarker in the blood sample in the detection method of the present invention is specifically recognized by the biomarker (anti-DDB1 antibody) present in the blood sample collected from the subject. Although it is not particularly limited as long as it can be obtained, an antigen derived from an animal belonging to the same species as the subject is preferred. For example, when the test subject is a human, it is preferable that the antigen used for detection of the biomarker is of human origin.

Also, the antigen used to detect the biomarker in the blood sample may be a full-length protein or a partial peptide thereof as long as it can be specifically recognized by an antibody. In the present invention, the partial peptide used as an antigen is not particularly limited in its length as long as it contains an antigenic determinant that can be recognized by an anti-DDB1 antibody derived from a test subject, and is detected as the biomarker Can be appropriately set according to the type of antibody. In general, since the number of amino acid residues constituting an antigenic determinant is about 5 to 20, the partial peptide includes, for example, those composed of 5 or more amino acid residues.

Antigens can be obtained by known techniques. For example, an antigen can be collected from an animal such as a human exemplified above as a test subject. Harvesting can be performed according to a conventionally known method of isolating and purifying a protein or peptide from tissue or cultured cells, for example, cell lysate after disrupting the tissue or cells expressing the antigen with a homogenizer And the method of isolating and purifying the antigen in the extract by chromatography.

Alternatively, an antigen can be obtained by culturing a transformant into which an expression vector containing a nucleic acid encoding the antigen has been introduced, and isolating and purifying the antigen from the culture. Alternatively, based on the amino acid sequence of the antigen, it can be prepared as a polypeptide by a conventionally known peptide synthesis method. Examples of peptide synthesis methods include solid phase synthesis and liquid phase synthesis, and any method may be used in the present invention.

Furthermore, partial peptides to be used as antigens may be prepared by cleaving each polypeptide produced by the above method with a peptidase.

In addition, the antigen may be modified by phosphate, sugar or sugar chain, phospholipid, lipid, nucleotide or the like. In addition, the antigen may be one to which a known tag is linked in order to facilitate purification treatment and the like. Such tags include, for example, glutathione-S-transferase (GST), FLAG tag, His tag and the like.

2-5. Detection of Biomarker The detection method of the present invention may be carried out by detecting the presence or absence of the biomarker in a blood sample collected from a subject. In the detection method of the present invention, the detection of the biomarker may be performed by measuring the amount of the biomarker in a blood sample. A more accurate analysis can be performed by measuring the amount of the biomarker and comparing the measured value to the value of the biomarker in a normal control group and / or a subject suffering from muscle disease. Thus, the detection method of the present invention is preferably carried out by a method capable of quantifying said biomarker in a blood sample.

Specifically, as a method of detecting the biomarker present in a blood sample using the antigen, specifically, the antigen is brought into contact with the blood sample to specifically measure the antigen and the biomarker (antibody). Methods include direct or indirect detection of binding. Specific examples of such detection methods include immunoassays such as ELISA, Western blotting, immunoprecipitation, radioimmunoassay (RIA) and fluorescence immunoassay. When the biomarker is detected by these immunoassays, an antibody that specifically binds to the biomarker (antibody) is bound to a label such as an enzyme label, a chromogenic label, a radio label or a luminescent label, and this label is detected Or it can carry out by measuring.

In the present invention, although any of the detection methods can be used to analyze muscle diseases, the ELISA method is mentioned as a preferable detection method from the viewpoint of simplicity.

The conditions for carrying out the immunoassay are not particularly limited as long as specific binding between the biomarker and the antigen in the blood sample can be detected, and may be set based on conventionally known conditions. For example, when the biomarker of the present invention is detected by ELISA, a blood sample collected from a subject is added to each well of the multiwell plate on which the antigen or antigen is immobilized, and the antigen in the well and the bio in the blood sample are added. React with a marker (anti-DDB1 antibody). Then, after adding a labeled antibody that specifically binds to the antibody derived from the test subject and reacting each well, a blood sample is obtained by detecting and / or quantifying the reaction product obtained by adding the enzyme substrate. Detection and / or quantification of the biomarkers in can be performed.

Here, the labeled antibody that specifically binds to the antibody derived from the test subject may be appropriately selected based on the animal of the test subject from which a blood sample is collected, but for example, when the test subject is a human, Non-human labeled antibodies (for example, rabbit-derived anti-human IgG antibodies) that specifically bind human antibodies can be mentioned.

In addition, an enzyme used for labeling a labeled antibody that specifically binds to an antibody derived from a test subject can also be appropriately selected from those commonly used and used, for example, peroxidase, alkaline phosphatase, luciferase, Esterase, glucose oxidase, β-D-galactosidase, β-D-glucuronidase and the like can be mentioned. The enzyme substrate may be appropriately selected from known substrates depending on the type of enzyme. For example, when the enzyme is peroxidase, 3,3 ', 5,5'-tetramethylbenzidine (TMB) Can be used as a substrate.

The detection and / or quantification of the reaction product generated by the reaction of the enzyme and the substrate can be carried out by measuring the absorbance of the reaction product, for example, 3,3 ', 5,5'-tetramethylbenzidine ( When TMB) is used as the enzyme substrate, it can be performed by measuring the absorbance at 450 nm.

In the case of radioimmunoassay (RIA), the antigen is labeled with a radioactive isotope, reacted with the biomarker in a blood sample to form an immune complex, and detected based on the radioactivity released from the radioactive isotope be able to.

In the case of a fluorescent immunoassay, the antigen is immobilized on a plate or the like, a blood sample is added thereto and reacted, and then an antibody which specifically binds to an antibody present in a blood sample to be tested is further reacted. Can be performed by detecting fluorescent coloring. As the antibody that specifically binds to the antibody derived from the test subject, one that is as described in the above-mentioned ELISA method and labeled with a fluorescent dye is used. Examples of fluorescent dyes include FITC, PE, APC, Cy-3, Cy-5 and the like.

In the case of immunoprecipitation, it can be detected by reacting the antigen with a blood sample to form an immune complex, and using an active adsorbent such as protein A or protein G to precipitate as an insoluble. Furthermore, immunoprecipitation and Western blotting can be combined and detected. More specifically, the antigen to which a tag such as FLAG is linked is reacted with a blood sample, and if the biomarker is present in the sample, an immune complex is formed, so that it is precipitated by the above-mentioned active adsorbent . The resulting precipitate is then subjected to Western blotting. That is, the precipitate is separated and developed by SDS-PAGE, transferred to a nitrocellulose membrane, PVDF membrane or the like, and then the antibody against the tag and the antigen-antibody reaction on the transfer membrane cause the biomarker to be present in the blood sample. If it does, it can be detected as a band.

2-6. Determination of morbidity of muscle disease According to the detection method of the present invention, when the biomarker is detected in a blood sample collected from the subject, it can be judged that the subject is suffering from muscle disease .

In subjects suffering from muscle disease, the amount of the biomarker of the present invention in the blood sample is significantly elevated as compared to the normal control group. Therefore, when quantitatively detecting the biomarker of the present invention, muscle disease is detected by comparing the amount of the biomarker in the blood sample of the normal control group with the amount of the biomarker in the blood sample from the subject. You can make a diagnosis.

In the present invention, when the amount of biomarker contained in the blood sample is larger than that in the normal control group, specifically, the 95th percentile value of the normal control group is set as the cutoff value for the amount of biomarker in the test subject. And the case where it is more than the said cutoff value. If it is more than the said cutoff value, it can be judged that a subject is likely to suffer from muscle disease. In addition, if it is less than the cut-off value, it may be judged that the subject is unlikely to suffer from muscle disease. Also, the more the amount of the biomarker of the present invention in the blood sample, the more severe the symptoms of muscle disease can be predicted.

If a biomarker of the invention is detected, the application of muscle biopsy can be determined. The objective fact that the biomarkers of the present invention are positive can make it easier to make an open muscle biopsy, both clinically and psychologically. If a definitive diagnosis of muscle disease is made by performing a muscle biopsy, treatment of muscle disease and an appropriate dosing schedule and follow-up measures to suppress or ameliorate the progression of symptoms (patients with hospital admission without medication Can plan various measures, such as the period of time), including isolation from daily life. Alternatively, if the biomarker of the present invention is detected, the muscle biopsy should be performed because the site to be subjected to the muscle biopsy is a site that is difficult to remove or for other reasons, or for muscle biopsy. If it is judged that the treatment needs to be started sooner than the operation, etc., the possibility of muscle disease is judged to be extremely high without muscle biopsy, and treatment of muscle disease and symptoms It is also possible to design various measures such as appropriate dosing schedule and transitional measures (including isolation of patients from daily life by hospitalization and rest without medication) to control or improve the progression of

The prognosis of a subject can also be predicted by detecting the biomarkers of the present invention. For example, the treatment effect can be monitored by performing the detection method of the present invention after treatment. In this case, the treatment can be terminated when the biomarker of the present invention is not detected or when the detected amount is less than the cut-off value. In addition, by performing the detection method of the present invention during follow-up, it is also possible to monitor the timing of start or resumption of treatment. In this case, treatment can be started or resumed when the biomarker of the present invention is detected, or when the detected amount reaches or exceeds the cut-off value.

3. Reagent for examination of muscle disease The present invention provides a reagent for examination of muscle disease, which comprises an antigen consisting of DDB1 protein or a partial peptide thereof. The test reagent of the present invention can be used to carry out the above-described muscle disease detection method.

About the said antigen used for the test reagent of this invention, it is as above-mentioned.

In the test reagent of the present invention, the antigen may be provided in an immobilized state on an insolubilizing carrier. The material of the insolubilized carrier is not particularly limited as long as it does not interfere with the detection of the biomarker, and for example, polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, polyvinyl chloride, fluorocarbon resin, crosslinked dextran, paper, silicon, glass, metal, Agarose etc. can be illustrated. In addition, two or more of these materials may be used in combination. The shape of the insolubilized carrier may be any shape such as, for example, microplate, tray, sphere, fiber, rod, disc, container, cell, test tube and the like.

Immobilization of the antigen on the insolubilized carrier can be performed according to a conventionally known method.

The amount of the antigen immobilized on the insolubilizing carrier is not particularly limited as long as it is a sufficient amount to specifically bind to the antibody against the antigen, and for example, in the solution used when immobilized on the insolubilizing carrier Of 1 to 10 μg / mL.

When the biomarker detected by the test reagent of the present invention is an antibody capable of recognizing a plurality of antigenic determinants, an antibody specifically recognizing a plurality of antigenic determinants present in each antigen (the above-mentioned biomarkers Each antigen is preferably a full-length protein, from the viewpoint that detection sensitivity can be improved by comprehensively detecting

The test reagent of the present invention may contain, in addition to the above-mentioned antigen, a buffer, a stabilizer, a preservative, etc., and may be formulated according to a conventionally known method.

4. Diagnostic kit for muscle disease The present invention provides a kit for diagnosing a muscle disease, which comprises the above-described reagent for testing for muscle disease.

The diagnostic kit of the present invention may further comprise a labeled antibody (eg, a human antibody) which specifically binds to an antibody derived from a subject animal, which may be required to carry out detection of the antibody, in addition to the above-mentioned reagents. It may contain a non-human labeled antibody to be bound, a detection agent for a labeling substance, a solubilizer, a detergent, a reaction stopping solution, a control sample, a test protocol and the like. The test protocol includes information such as operations and procedures for performing the muscle disease detection method described above.

5. Method of diagnosing muscle disease The present invention provides a method of diagnosing muscle disease. The diagnostic method comprises the steps of detecting an anti-DDB1 antibody in a blood sample collected from a subject, and diagnosing a muscle disease based on the result obtained in the step.

In the diagnostic method of the present invention, the method for detecting an antibody, the criteria for determining that the subject is afflicted with muscle disease, etc. are as described above.

EXAMPLES The present invention will be more specifically described below by way of Examples, but the present invention is not limited thereto.

[1. Patient background]
Serum samples and clinical information were collected from 370 patients with autoimmune disease and 20 healthy controls (HC). These patients may have polymyositis (n = 100), dermatomyositis (n = 60), inclusion body myositis (n = 5), systemic lupus erythematosus (n = 88), idiopathic pulmonary fibrosis (n = 21) ), IgG4-related disease (n = 20), rheumatoid arthritis (n = 20), scleroderma (n = 20), Sjögren's syndrome (n = 10), anti-neutrophil cytoplasmic antibody related vasculitis syndrome (n = 3) And diseases such as psoriatic arthritis (n = 3) and ankylosing spondylitis (n = 2). All connective tissue diseases (CTD) are most commonly accepted criteria (Ann Neurol. 1995; 38: 705-13, Arthritis Rheum. 1997 40: 1725, Am J Respir Crit Care Med. 2011; 183: 788). 2010: 62: 2569-81 / Ann Rheum Dis. 2010; 69: 1580-8, Ann Rheum Dis. 2013; 72: 1747-55, Ann-824, Mod Rheumatol. 2012; 22: 1-14, Arthritis Rheum. Rheum Dis. 2002; 61: 554-8, Ann Rheum Dis. 2007; 66: 222-227, Arthritis Rheum. 54: 2665-73, 2006, and Arthritis Rheum. 1984; 27: 361-8). It was done. Other patients were not diagnosed with suspected CTD. Intervention of Interstitial Lung Disease (ILD), Official Clinical Practice Guidelines of the American Thoracic Society: Classification, Evaluation, and Management of Infant Periodontologic Lung Disease (Am J Respir Crit Care Med. 2013; 188: 376 The diagnosis was made based on the multidisciplinary approach proposed in -94). Clinical data were collected retrospectively from the clinical chart review of all patients. Before taking serum samples, informed consent was obtained from all patients and healthy controls according to the Helsinki Declaration. This study was approved by the Medical Ethics Committee of Kyoto University Graduate School of Medicine.

[2. Method]
[2-1. Detection of autoantibodies-immunoprecipitation and autoradiography]
Screening for autoantibodies was performed by immunoprecipitation (IPP) with radiolabeled HeLa cell extract as follows. The following method is based on the method described in J Biol Chem. 1986; 261: 2274-8.

1 × 10 ⁷ HeLa cells were labeled with 18.5 MBq of [ ³⁵ S] methionine (Perkin Elmer, Waltham, Mass., USA) in 30 ml of methionine-free minimal essential medium and incubated at 37 ° C. for 18 hours. After washing four times with IPP buffer (10 mM Tris-HCl, 500 mM NaCl, 0.1% Nonidet P-40, pH 8.0), [ ³⁵ S] methionine-labeled HeLa cells are Misonix Microson (Misonix, Farmingdale, NY, USA) Sonicated with. The soluble supernatant in IPP buffer was collected by centrifugation (10,000 × g for 10 minutes) to obtain [ ³⁵ S] methionine labeled HeLa cell extract.

15 microliters of serum is contacted with 2 mg of Protein A CL-4 B sepharose beads (GE Healthcare, Uppsala, Sweden) in IPP buffer with stirring for 2 hours at room temperature to bind serum sample IgG to Sepharose beads The The IgG conjugated Sepharose beads were washed four times in IPP buffer and mixed with [ ³⁵ S] methionine labeled HeLa cell extract for 2 hours at 4 ° C. The beads were washed 4 times with 500 μl of IPP buffer and once with 500 μl of distilled water. The beads were then resuspended in sodium dodecyl sulfate (SDS) sample buffer and heated to 100 ° C. for 5 minutes. The supernatant was fractionated by 10% SDS-polyacrylamide gel electrophoresis (PAGE). After drying the gel, radiolabeled polypeptide components were analyzed by autoradiography using Fuji Bio-Imaging Analyzer System-5000 (Fuji Photo-Film, Tokyo, Japan).

[2-2. Identification of autoantibodies]
[2-2-1. Immunoaffinity chromatography and electrophoresis]
IgG was purified from 5 ml of patient serum using an IgG purification kit (ImmunoPure (G) IgG Purification Kit, Pierce, Rockford, IL, USA). The purified IgG was dialyzed and coupled to cyanogen bromide (CNBr) activated Sepharose 4B beads (GE Healthcare, Uppsala, Sweden) (procedure was according to the product's protocol). The IgG bound Sepharose 4B beads are poured onto a glass column (Bio Rad, Hercules, CA, USA) to make an immunoaffinity column, and the extracted 6 × 10 ⁸ HeLa cell extract is added to the column at 4 ° C. After filtering over time, IgG was bound to HeLa cell protein (antigen). After washing the column with 200 ml of Tris-buffered saline (TBS, 10 mM Tris-HCl, 150 mM NaCl, pH 7.5) containing 0.05% Tween-20, the antigen bound to the IgG was 1 M NaCl, 1 M MgCl ₂ and Elution was carried out with 3 M MgCl ₂ (adjusted to pH 7.0 with Tris) using a step-wise gradient of ionic strength. The eluate is dialyzed against TBS containing 0.05% Tween-20 to purify the protein, concentrated with an Amicon Centriprep concentrator (Millipore, Billerica, Mass., USA), and subjected to SDS-PAGE electrophoresis for analysis of antigen identification. went.

2-2-2. Peptide mass fingerprinting (PMF)]
The proteins eluted from the immunoaffinity column in 2-2-1 above were subjected to SDS-PAGE electrophoresis, and then the protein bands visualized by silver staining were excised from the gel. The excised band is extracted by digestion with trypsin (Promega, Madison, WI, USA) and treated with α-cyano-4-hydroxycinnamic acid in 50% acetonitrile / 0.1% TFA, Electrophoresis. 1998; 19: 1036 MALDI-TOF analysis (Microflex LRF 20, Flex Analysis 3.0 software, Bruker Daltonics) based on the general procedure described in -45 (Method for protein identification from SDS-PAGE gel or PVDF membrane using MALDI-TOF-MS) (Billrica, Mass., USA) to identify proteins by PMF.

Spectra were collected from 300 shots per spectrum in the m / z range 600-3000 and calibrated by two-point internal calibration with tryptic autodigestion peaks (m / z 842.5099, 2211.1046). Peak lists were created using Flex Analysis 3.0 software. The threshold used for peak picking was 500 with a minimum resolution of monoisotopic mass and 5 with a S / N ratio. The search program MASCOT developed by Matrixscience (http://www.matrixscience.com/) was used for protein identification by PMF. The following parameters were used for database search: Trypsin as Enzyme, maximum of one missed cleavage; iodoacetate (cys) as complete modification, oxidation (Mrt) as partial modificaiton, monoisotopic masses; ± 0.1 Da as mass tolerance.

2-2-3. Immunoblotting (Western blotting)]
The proteins extracted in 2-2-2 above were electrophoresed and analyzed by western blotting. Western blotting is described in Towbin H, Staehelin T, Gordon J. Proc Natl Acad Sci US A. 1979; 76: 4350-4. (Electrical transfer of proteins from acrylamide gel to nitrocellulose membrane: method and application) This is done by modifying the general method. Specifically, the extracted proteins ^{10% Mini-PROTEAN (R)} TGXTM gel (BIO RAD, California, USA) was electrophoretically separated on SDS-PAGE by, proteins were transferred to nitrocellulose membranes. After blocking and washing 3 times with Bullet Blocking One for Western Blotting (13779-01, Nakalai Tesque, Kyoto, Japan), the membrane is used as a primary antibody in 6 patient sera, healthy control sera and commercial Incubated with available anti-DDB1 antibody (GTX100129, GENETEX, CA, USA). The membrane was washed 5 times with Bullet Blocking One for Western Blotting for Western blotting and incubated with a secondary antibody against human IgG (H + L) (anti-human IgG (H + L) AP Conjugate, Promega, WI, USA) . After washing 5 times with Bullet Blocking One for Western Blotting for Western blotting, the blots were visualized using BCIP / NBT chromogenic substrate (S3771, Promega, WI, USA).

[3. result]
3-1. Screening of anti-120 kDa antibody (detection of autoantibody)]
Analysis of the electrophoresis gel of a sample in which [ ³⁵ S] -labeled HeLa cell extract was bound to immunoprecipitates of serum collected from 370 patients and 20 healthy control proteins was analyzed by autoradiography. The 120 kDa protein binding was commonly observed in IIM patients, confirming the presence of anti-120 kDa antibodies. Thus, these six IIM patients are commonly considered to be anti-120 kDa antibody positive. Again, autoradiography was similarly performed on the six IIM patients, and a photo of the visualized membrane (electrophoretic gel) is shown in FIG. In FIG. 1, a band indicated by an arrow indicates a 120 kDa common immunoprecipitated protein, lanes 1-6 have 6 patients (anti-120 kDa antibody-positive patients), lane 7 has a healthy control (HC), lane MM Indicates a molecular marker.

Thus, while the sera of six IIM patients commonly immunoprecipitated the 120 kDa (FIG. 1) protein (that is, they were anti 120 kDa antibody positive), although not shown in the data, they are other autoantibodies. Neither MSA nor MAA had (ie other autoantibodies were negative). Also, although not shown in the data, other patients positive for other autoantibodies (MSA or MAA) and sera from HC did not immunoprecipitate the protein (ie, anti-120 kDa antibody negative) ). Therefore, the anti-120 kDa antibody is a new autoantibody for muscle disease since the six patients specifically have the anti-120 kDa antibody and no other auto-antibodies (MSA or MAA). It confirmed that it was.

[3-2. Clinical and laboratory findings and course of patients with anti-120 kDa antibody]
The clinical and laboratory profiles of 6 (4 women) patients who were anti-120 kDa antibody positive were evaluated (Table 1). All patients had muscle soreness, and 83% (5/6 cases) had muscle weakness. Muscle weakness was observed in 67% (4/6 cases), muscle atrophy in 17% (1/6 cases), arthritis in 50% (3/6 cases), and chronic ILD in 17% (1/6 cases) . None of the patients had cancer-related myositis. Only one patient was low titer and ANA positive. Myogenic enzymes were elevated in all patients. Three out of five patients who received electromyography showed idiopathic myopathy. All patients who received MRI or muscle biopsy showed edema and inflammation respectively. Also, none of the patients exhibited substantially skin symptoms. Below, individual clinical and laboratory findings and courses are shown.

Patient No. 1 had gradually progressed in muscle weakness for 20 years. The patient had been examined at several other hospitals but had not been unambiguously diagnosed. Thereafter, when the inventor examined the patient, no skin symptoms were observed in the patient, but an increase in serum myogenic enzyme was observed, showing a finding of muscle weakness accompanied by atrophy. Magnetic resonance imaging (MRI) T2-weighted imaging shows findings of muscle inflammation and electromyography (EMG) shows a mixture of myogenic and neurogenic patterns consistent with the diagnosis of myositis. The examination confirmed pathological findings consistent with the diagnosis of myositis. Therefore, this patient was diagnosed with polymyositis (PM). Although treatment with an immunosuppressant drug was recommended, the patient refused treatment and observed the clinical course without medication, although the elevated myogenic enzyme levels diminished over time, but muscle weakness and atrophy progressed I kept doing it.

Patient No. 2 had a subjective symptom of muscle weakness for one year with a marked rise in myogenic enzymes. Myositis was strongly suspected but further testing was done. Although no skin symptoms were observed in the patient, T2-weighted MRI images show muscle inflammation and EMG show myogenic patterns, so when a muscle biopsy was performed based on MRI findings, myositis was noted. Supporting pathological findings were shown. Therefore, this patient was diagnosed with polymyositis (PM). Treatment with high doses of glucocorticoid rapidly improved clinical findings and symptoms.

Patient No. 3 showed an acute onset of myalgia, muscle weakness and fatigue and a marked increase in myogenic enzymes. Interestingly, despite the marked rise in myogenic enzymes, no skin symptoms were noted and MRI showed only slight muscle edema. When a muscle biopsy was performed, pathological findings showed muscle disease. Therefore, this patient was diagnosed with polymyositis (PM). Thereafter, without medication, the patient's symptoms gradually improved, and the CK level decreased to less than 1,000 IU / l within one month. After discharge, there was no apparent recurrence of symptoms and CK levels spontaneously improved to the normal range.

Patient No. 4 showed elevated CK and had muscle pain and fatigue for 10 years, but was considered to be a secondary symptom of fibromyalgia or chronic fatigue syndrome. The patient was referred for myalgia and muscle weakness with elevated CK, mild fever (> 37.0 degrees), dyspnea, and tracheal aspiration. Although no skin symptoms were noted, MRI findings showed slight inflammation at a localized site of the right inguinal muscle, but it was a very difficult site to perform a muscle biopsy. Therefore, the patient was diagnosed with suspected polymyositis (PM). No further examinations or medications were performed as resting at the hospital spontaneously improved the symptoms and myogenic enzymes elevation.

Patient No. 5 has been coughing for over 1 year and muscle weakness and myogenic enzymes have been rising for 3 months. The patient became treatment-introduced (inhaled bronchodilator, leukotriene receptor antagonist, carbocystin, macrolide antibiotic) based on the diagnosis of asthma due to respiratory symptoms, but none showed any effect. Exacerbation of urinary symptoms was noted. For this reason, it was thought that close examination for myositis was required, but prior to that, treatment hospitalization with glucocorticoid started. The patient had no skin symptoms, but he was suspected of having polymyositis (PM) because of exacerbation of respiratory symptoms and myalgia. Treatment with glucocorticoids improved muscle weakness, respiratory symptoms, and elevation of myogenic enzymes within one week and ended treatment two weeks later without further inflammation. EMG was performed three months after admission and no apparent abnormalities were noted.

Patient No. 6 had continued upper arm fatigue and severe arthritis for one month. Dermatologic symptom in Table 1 is daringly described as "+" because there was a skin symptom that may be slightly Gottron papules, but the skin symptom is extremely slight, and usually it is usually taken as an internal medicine of collagen disease. Even in dermatology, it was not a level at which it was diagnosed that there was a skin symptom, but it was "-" in fact, and it was not enough to doubt the disease (that is, it corresponds to the embodiment that does not show skin symptoms substantially in the present invention). In blood tests, CK values were in the normal range, but only a little higher in aldolase. CT showed ILD with increasing ferritin concentration. As a result of the muscle biopsy, the patient was diagnosed with dermatomyositis (DM). All symptoms of the patient improved within one week without treatment and were discharged without further examination.

When Bohan & Peter's diagnostic criteria were applied to these anti-120 kDa antibody-positive patients (the skin symptoms of patient No. 6 were daringly applied as being a standard skin symptom), only 33% of positive cases were applied. Staying in (2/6 cases), 67% (4/6 cases) did not lead to sure cases. It should be noted that, at the normal diagnostic level, the skin symptoms of the patient No. 6 are not usually diagnosed as falling under the standard skin symptoms, so when applying Bohan & Peter's diagnostic criteria at the normal diagnostic level, certain cases The number is further reduced to 17% (1/6 cases).

[3-3. Identification of Autoantigen Recognized by Anti-120 kDa Antibody]
Figure 2 (A) shows the results of SDS-PAGE electrophoresis of antigens purified stepwise by elution with a buffer of different ionic strength by immunoaffinity chromatography to determine the antigen required for autoantigen analysis. Show. Specifically, HeLa cell extract (protein) is bound to immunoaffinity chromatography prepared by binding IgG of 6 patient sera (anti-120 kDa antibody positive sera) to CNBr-activated Sepharose 4B beads, Proteins eluted with a buffer (1 M NaCl, 1 M MgCl ₂ , and 3 M MgCl ₂ ) of increasing ionic strength on a dynamic gradient are electrophoresed on an 8% SDS-polyacrylamide gel (lane 1: 1 M NaCl elution) Fraction, lane 2: 1 M MgCl ₂ elution fraction, lane 3: 3 M MgCl ₂ elution fraction), results of visualization with silver staining are shown. The band corresponding to the autoantigen band detected in the above-described immunoblot analysis (indicated by the arrow in lane 3) was cleaved and used for PMF analysis.

In order to confirm that the 120 kDa protein obtained by this immunoaffinity chromatography is a protein captured by the antibody contained in patient serum, immunoblot analysis was carried out using the same serum used for immunoaffinity chromatography. The result is shown in FIG. 2 (B). Specifically, polypeptides of six patient sera (anti-120 kDa antibody positive sera) eluted with buffer (1 M NaCl, 1 M MgCl ₂ , and 3 M MgCl ₂ ) respectively using the above-mentioned immunoaffinity column, After subjecting to electrophoresis in the same manner as described above (Lane 1: 1 M NaCl eluted fraction, Lane 2: 1 M MgCl ₂ eluted fraction, Lane 3: 3 M MgCl ₂ eluted fraction), the immunoaffinity chromatography was carried out by Western blotting. The results of the reaction with the same patient serum as used in It was confirmed that a band of about 120 kDa reacted with patient serum in the 3 M MgCl ₂ eluate (lane 3). That is, it was confirmed that a band of about 120 kDa was indeed an antigen protein that reacts with an antibody contained in patient serum.

The antigen protein was extracted from the approximately 120 kDa silver-stained gel band cleaved in FIG. 2 (A) and subjected to PMF, and as a result, it was identified as DDB1 (coverage: 21%). Therefore, it was identified that the antigen protein that reacts with anti-120 kDa antibody positive serum is DDB1.

[3-4. Confirmation that anti-120 kDa antibody is anti-DDB1 antibody]
DDB1 human recombinant protein (H00001642-P01, Abnova, Taipei, Taiwan) was electrophoresed, and 6 patients' anti-120 kDa antibody positive serum samples and normal control sera as primary antibodies, and commercially available anti-DDB1 antibody (GTX 100 129) The immunoblotting for DDB1 protein was carried out using GENETEX, CA, USA), and the results of further examination for specific reactivity with DDB1 human recombinant protein are shown in FIG. In FIG. 3, lane 1 shows the results of using the anti-DDB1 antibody, lanes 2 to 7 show the anti-120 kDa antibody positive sera of 6 patients, and lane 8 shows the results of using a healthy subject (HC) as the primary antibody. DDB1 human recombinant protein was recognized by anti-DDB1 antibody and all anti-120 kDa antibody positive sera but not by HC sera. Therefore, it was confirmed that the antigen protein (target autoantigen of anti-120 kDa antibody) that reacts with the patient's anti-120 kDa antibody-positive serum is indeed DDB1, and the anti-120 kDa antibody in patient serum is an anti-DDB1 antibody.

From the above, it was shown that anti-DDB1 antibody is useful as a biomarker for detecting muscle disease. In particular, muscle diseases that can not be detected by conventional MSA and MMA, and muscle diseases that do not substantially exhibit skin symptoms can be effectively detected, which makes it possible to diagnose early and take appropriate measures early. Can be It has also been shown that the biomarker is easily detectable by DDB1 protein.

Claims (13)

1. A biomarker for muscle disease consisting of anti-DDB1 antibodies.
2. The biomarker according to claim 1, wherein the myopathy is polymyositis.
3. A method for detecting muscle disease, comprising the step of detecting an anti-DDB1 antibody in a blood sample derived from a subject.
4. The detection method according to claim 3, wherein the blood sample is serum.
5. The detection method according to claim 3 or 4, wherein the clinical finding of the subject does not substantially exhibit skin symptoms.
6. The detection method according to any one of claims 3 to 5, wherein the myopathy is polymyositis.
7. The detection method according to any one of claims 3 to 6, wherein the blood test findings of the test subject have normal values for myogenic enzymes.
8. The detection method according to any one of claims 3 to 6, wherein the blood test findings of the subject are creatine kinase 300 U / L or more.
9. The detection method according to any one of claims 3 to 6 and 8, wherein the blood test findings of the subject are aldolase 6.2 IU / L or more.
10. The detection method according to any one of claims 3 to 9, wherein the detection is performed using an antigen consisting of DDB1 protein or a partial peptide thereof.
11. The detection method according to claim 10, wherein the detection is performed by ELISA.
12. A reagent for testing a muscle disease, which comprises an antigen consisting of DDB1 protein or a partial peptide thereof.
13. A diagnostic kit for muscle disease, comprising the test reagent according to claim 12.

Images