WO2019111797A1 - Biomarqueur pour diagnostiquer une maladie musculaire - Google Patents

Biomarqueur pour diagnostiquer une maladie musculaire Download PDF

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WO2019111797A1
WO2019111797A1 PCT/JP2018/043998 JP2018043998W WO2019111797A1 WO 2019111797 A1 WO2019111797 A1 WO 2019111797A1 JP 2018043998 W JP2018043998 W JP 2018043998W WO 2019111797 A1 WO2019111797 A1 WO 2019111797A1
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biomarker
muscle
antibody
muscle disease
ddb1
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PCT/JP2018/043998
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English (en)
Japanese (ja)
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祐司 細野
経世 三森
蘭 笹井
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国立大学法人京都大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

Definitions

  • the present invention relates to a biomarker for early diagnosis of muscle disease.
  • IM Inflammatory myopathy
  • Bohan Bohan and Peter's diagnostic criteria
  • 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.
  • MSA myositis-specific autoantibodies
  • MAA myositis-associated autoantibodies
  • DDB1 DNA damage-binding protein 1
  • Non-patent Documents 3 and 4 DNA damage-binding protein 1
  • Non-patent Documents 5 to 11 DNA damage-binding protein 1
  • MSA and MAA can be used as useful biomarkers for patients who are positive for them.
  • MSA and MAA 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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. The detection method according to Item 3, wherein the blood sample is serum.
  • 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.
  • 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.
  • 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.
  • the lane 1 shows the 1 M NaCl elution fraction
  • the lane 2 shows the 1 M MgCl 2 elution fraction
  • 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.
  • Biomarker The biomarker for muscle disease of the present invention is characterized by comprising an anti-DDB1 antibody.
  • an anti-DDB1 antibody the biomarker of the present invention will be described in detail.
  • 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. .
  • 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).
  • polymyositis is particularly effective because the biomarker of the present invention is effective since there is no skin symptom.
  • the biomarker of the present invention is effective when the dermatomyositis is overlooked because the degree of skin symptoms is extremely slight.
  • 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
  • DDB1 DNA damage-binding protein 1
  • 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 ).
  • 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.
  • IgG is exemplified.
  • 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
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • the test subject is a human
  • 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.
  • Physiological examination findings may be either in the absence of abnormal findings or in the case of findings of myogenic pattern.
  • findings of the myogenic pattern either the myogenic pattern alone or a mixture of the myogenic and neurogenic patterns may be used.
  • imaging examinations include nuclear magnetic resonance imaging (MRI), computed tomography (CT) and the like.
  • MRI nuclear magnetic resonance imaging
  • CT computed tomography
  • 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.
  • 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.
  • serum 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.
  • anti-DDB1 antibody 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.
  • the biomarker anti-DDB1 antibody
  • an antigen derived from an animal belonging to the same species as the subject is preferred.
  • the antigen used for detection of the biomarker is of human origin.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • partial peptides to be used as antigens may be prepared by cleaving each polypeptide produced by the above method with a peptidase.
  • the antigen may be modified by phosphate, sugar or sugar chain, phospholipid, lipid, nucleotide or the like.
  • the antigen may be one to which a known tag is linked in order to facilitate purification treatment and the like.
  • tags include, for example, glutathione-S-transferase (GST), FLAG tag, His tag and the like.
  • 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.
  • 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.
  • the detection method of the present invention is preferably carried out by a method capable of quantifying said biomarker in a blood sample.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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, ⁇ -D-galactosidase, ⁇ -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.
  • TMB 3,3 ', 5,5'-tetramethylbenzidine
  • 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.
  • RIA radioimmunoassay
  • 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.
  • 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.
  • 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.
  • an active adsorbent such as protein A or protein G
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the prognosis of a subject can also be predicted by detecting the biomarkers of the present invention.
  • 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.
  • 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.
  • Reagent for examination of muscle disease 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.
  • 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 ⁇ g / mL.
  • 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
  • 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.
  • 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.
  • 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.
  • the method for detecting an antibody, the criteria for determining that the subject is afflicted with muscle disease, etc. are as described above.
  • 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.
  • 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
  • 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).
  • IgG immunoaffinity chromatography and electrophoresis
  • 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).
  • CBr cyanogen bromide
  • 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).
  • 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.
  • PMF Peptide mass fingerprinting
  • 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 membrane 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).
  • 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.
  • 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.
  • EMG electromyography
  • 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.
  • PM polymyositis
  • 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.
  • PM polymyositis
  • 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.
  • 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.
  • PM polymyositis
  • 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.
  • Figure 2 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.
  • 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.
  • polypeptides of six patient sera eluted with buffer (1 M NaCl, 1 M MgCl 2 , and 3 M MgCl 2 ) respectively using the above-mentioned immunoaffinity column
  • buffer 1 M NaCl, 1 M MgCl 2 , and 3 M MgCl 2
  • the immunoaffinity chromatography was carried out by Western blotting.
  • 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.
  • DDB1 human recombinant protein H00001642-P01, Abnova, Taipei, Taiwan
  • GTX 100 129 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.
  • 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
  • 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.
  • anti-DDB1 antibody is useful as a biomarker for detecting muscle disease.
  • 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.
  • the biomarker is easily detectable by DDB1 protein.

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Abstract

La présente invention a pour objectif de fournir un biomarqueur pour le diagnostic précoce d'une maladie musculaire. De plus, la présente invention vise à fournir une technologie de mesure qui facilite le diagnostic précoce d'une maladie musculaire au moyen dudit biomarqueur. Le biomarqueur pour une maladie musculaire et comprenant des anticorps anti-DDB1 est utile en tant que biomarqueur pour le diagnostic précoce d'une maladie musculaire. Un procédé de détection de maladie musculaire comprenant une étape dans laquelle des anticorps anti-DDB1 dans un échantillon de sang dérivé d'un sujet d'essai sont détectés est utile pour la détection précoce d'une maladie musculaire. Idéalement, ladite maladie musculaire est la polymyosite.
PCT/JP2018/043998 2017-12-05 2018-11-29 Biomarqueur pour diagnostiquer une maladie musculaire WO2019111797A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013008930A1 (fr) * 2011-07-13 2013-01-17 国立大学法人 金沢大学 Méthode d'inspection pour le diagnostic de la dermatomyosite
JP2016153736A (ja) * 2015-02-20 2016-08-25 一三 西野 炎症性筋疾患鑑別マーカー及びそれを用いた炎症性筋疾患と非炎症性筋疾患の鑑別方法
CN107643405A (zh) * 2017-09-19 2018-01-30 上海市肺科医院 一种检测抗tsc22d3等血液自身抗体的肺癌诊断试剂盒和检测方法及其应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013008930A1 (fr) * 2011-07-13 2013-01-17 国立大学法人 金沢大学 Méthode d'inspection pour le diagnostic de la dermatomyosite
JP2016153736A (ja) * 2015-02-20 2016-08-25 一三 西野 炎症性筋疾患鑑別マーカー及びそれを用いた炎症性筋疾患と非炎症性筋疾患の鑑別方法
CN107643405A (zh) * 2017-09-19 2018-01-30 上海市肺科医院 一种检测抗tsc22d3等血液自身抗体的肺癌诊断试剂盒和检测方法及其应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MIMORI ET AL: "Polymyositis , Dermatomyositis, Inclusion Body Myositis, Necrotising Myopathy: Autoantibodies in myositis", NIPPON RINSHO, vol. 73, no. Suppl. 7, pages 554 - 560 *

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