JP2008249618A - External secretion disorder diagnostic reagent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a diagnostic reagent, a diagnostic method and a diagnostic kit for symptoms brought on by an external secretion disorder, a dry mouth, a dry eye, a dry skin, a dry vagina and a gland tissue destruction, which cause a subject to feel no pain in sample extraction. <P>SOLUTION: The diagnostic reagent, the kit containing the diagnostic reagent, the diagnostic method using the diagnostic reagent or the kit, and the like are characterized by detecting a midkine family protein in a body fluid of the subject such as a lacrimal fluid, saliva, sweat, a vaginal secretion fluid or the like. The diagnostic reagent for the external secretion disorder, the dry mouth, the dry eye, the dry skin or the dry vagina comprises an antibody or its fragment against the midkine family protein such as midkine, pleiotrophin or the like. Especially, the diagnostic reagent is used for diagnosing whether or not the external secretion disorder, the dry mouth, the dry eye, the dry skin or the dry vagina is the gland tissue destruction or Sjogren's syndrome. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for diagnosing an exocrine disorder including detecting midkine in a body fluid, and a diagnostic agent and a diagnostic kit used in the method.

  Exocrine secretion means that secretory fluid is secreted from the glandular tissue into the body surface and digestive tract via a conduit. For example, tear fluid, saliva, sweat, vaginal fluid, gastric fluid, etc. are secreted by exocrine secretion. Exocrine disorders include cystic fibrosis, exocrine gland tumors (salivary gland cancer, gastric adenocarcinoma, pancreatic ductal adenocarcinoma, cystadenocarcinoma), acute pancreatitis, Sjogren's syndrome, benign lymphoepithelial lesions, irradiation, diuretics / anticholinergic drugs, etc. Drug administration, salivary gland disease, dehydration, mouth breathing, Eaton-Lambert syndrome, diabetes, scar pemphigoid, Stevens-Johnson syndrome, trachoma, vitamin A deficiency, malnutrition due to protein or caloric deficiency, hypothyroidism, chronic Caused by hypocalcemia, viral gastroenteritis, etc., dry eye, dry mouse, dry skin, driver jain, etc., dry tears, keratoconjunctivitis deficient in water, corneal softening, blepharitis, oral infection, bad breath Secretion of saliva, pancreatic juice, tears, digestive enzymes, etc., such as decreased taste, gingivitis, chronic obstructive pulmonary disease, pancreatic exocrine dysfunction It has been known to cause a variety of symptoms and disease states by all.

  Treatment methods for patients with symptoms of exocrine disorders should be selected according to their causes and diseases. Currently, it is not always possible to diagnose the causes and diseases of patients who exhibit such symptoms specific to exocrine disorders. In many cases, it is not easy to make a comprehensive judgment based on the experience of the doctor based on the results of diagnosis such as medical examination, blood test, and other systemic symptoms.

  In particular, in the treatment of gland tissue destruction, which is one of the causes of exocrine disorders, local humidification with artificial saliva, artificial tears, skin cream, and the like is performed. However, for example, in the case of Sjögren's syndrome, which is considered a kind of autoimmune disease that causes glandular tissue destruction due to attack of the secretory gland and causes exocrine disorders, the respiratory tract such as aspiration pneumonia and anaerobic pneumonia Abnormal function of mucosal glands in the lung, systemic symptoms such as liver disease, kidney damage, lymphoma, fibromyalgia, etc., and immunity with nonsteroidal anti-inflammatory drugs, corticosteroids, immunomodulators, etc. Treatment for symptoms may be necessary.

  The destruction of glandular tissue is caused by damage to the secretory glands that make tears, saliva, etc., resulting in dry symptoms such as eyes and mouth seen in exocrine disorders. Have difficulty. In particular, patients with Sjogren's syndrome have dry symptoms of the eyes, mouth, skin, and vagina, which are symptoms of exocrine disorders. When the symptoms worsen, subjective symptoms such as foggy vision, foreign body sensation, changes in taste, and difficulty swallowing may appear. Therefore, it is not possible to determine Sjogren's syndrome based on these symptoms alone, and more types of tests are required.

  Currently, diagnostic methods for Sjogren's syndrome have been proposed in various countries, but no accurate diagnostic criteria have been established, and the establishment of diagnostic criteria that are universally accepted is required. For example, in Japan, when lymphocyte infiltration into the salivary gland, decreased salivary secretion, decreased tear secretion, autoantibody (SS-A antibody, SS-B antibody) positive in two or more items are positive Patients with Sjogren's syndrome diagnosed in the US-Europe joint study group who have no complications of related diseases, including ocular symptoms, oral symptoms, visual objective findings, histopathological findings, salivary gland disorders, autoantibodies If the histopathological findings or serum antibodies are positive and 4 out of 6 items are satisfied, or 3 out of 4 items of objective findings are satisfied, primary Sjoegren's syndrome is assumed. However, the sensitivity and specificity in these tests are not fully satisfactory as a diagnostic method, and since multiple types of tests must be performed, there are time and money burdens, and self-organization and blood samples are required. Therefore, there are problems such as the pain of the subject in collecting the sample for diagnosis and the fact that these tests can be diagnosed only in a limited facility.

  Midkine (hereinafter referred to as “MK”) is a growth / differentiation factor discovered as a product of a gene that is transiently expressed during the process of induction of differentiation of embryonic cancer cells by retinoic acid. It is a cysteine-rich polypeptide with a molecular weight of 13 kDa (see, for example, Non-Patent Document 1 and Non-Patent Document 2). The amino acid sequence of MK shows 50% homology with pleiotrophin (hereinafter referred to as “PTN”), and these are considered to be heparin-binding family proteins.

  MK is known to be one of the molecules that occupy the core in the process of inflammation imaging. For example, it is known that the formation of neointima when a blood vessel is injured and the development of nephritis at the time of ischemic injury are reduced in knockout mice lacking the MK gene. It is also known that rheumatism and post-surgical adhesions are greatly reduced in knockout mice (see, for example, Patent Document 1, Patent Document 2, and Patent Document 3). MK is also known to promote the migration (migration) of inflammatory cells such as macrophages and neutrophils. Since this movement is necessary for the establishment of an inflammatory image, it is thought that if MK is deleted, a disease based on inflammation is unlikely to occur. (For example, refer to Patent Document 4).

  As a diagnostic agent using MK, the amount of MK in synovial fluid and serum increases in patients with rheumatoid arthritis, and macrophage-like cells and fibroblast-like cells in the synovial tissue of patients with rheumatoid arthritis Since cells express MK, a method for diagnosing arthritis by measuring MK in blood or synovial tissue has been disclosed (see Patent Document 3).

In addition, although a method for diagnosing cancer has been disclosed as a diagnostic method using MK, the relationship between MK and exocrine disorders has not been reported so far.
International Publication No. 2000/10608 Pamphlet International Publication No. 2004/078210 Pamphlet International Publication No. 2004/085642 Pamphlet International Publication No. 1999/03493 Pamphlet Kadomatsu, K. et al .: (1988) Biochem. Biophys. Res. Commun., 151: p.1312-1318 Tomokura, M. et al .: (1999) J. Biol. Chem, 265: p.10765-10770

  The present invention is a diagnostic agent, diagnostic method or diagnosis for symptoms based on exocrine disorders, dry mouth, dry eye, dry skin, driver gyna, gland tissue destruction, which can be diagnosed easily and without limited facilities. It is an object to provide a kit. The present invention also provides a diagnostic agent, diagnostic method, or diagnostic kit for symptoms based on exocrine disorders, dry mice, dry eyes, dry skin, driver gyna, gland tissue destruction, which does not cause the subject's pain in sample collection. Is an issue. The present invention further provides a diagnostic agent, diagnostic method or diagnostic kit for symptoms based on exocrine disorder, dry mouth, dry eye, dry skin, driver gyna, gland tissue destruction, which is more accurate and less time-consuming. The issue is to provide. In particular, an object of the present invention is to provide a diagnostic agent, a diagnostic method, or a diagnostic kit for Sjogren's syndrome.

  The present inventors have determined whether or not a subject has Sjogren's syndrome by detecting an MK family protein in a body fluid of a patient presenting with exocrine disorder, dry mouth, dry eye, dry skin, driver gina, or glandular tissue destruction. It was found that can be diagnosed. In particular, we are in the secretions of patients with exocrine disorders, dry mice, dry eyes, dry skin, driver gina, gland tissue destruction symptoms (especially tears, saliva, sweat, or vaginal secretions). By detecting the MK family protein, it was found that the subject can diagnose whether or not the subject has Sjogren's syndrome, and the present invention has been completed.

  The present invention relates to a diagnostic agent, a diagnostic kit and a diagnostic method for symptoms based on exocrine disorders, dry mice, dry eyes, dry skin, driver gyna, gland tissue destruction, which comprise a reagent for detecting MK family proteins. In particular, the present invention relates to a diagnostic agent, a diagnostic kit, and a diagnostic method for Sjogren's syndrome comprising a reagent for detecting an MK family protein.

More specifically, the present invention relates to the following diagnostic agents, diagnostic methods or diagnostic kits.
(1) A diagnostic agent for an exocrine disorder, comprising a reagent for detecting an MK family protein.
(2) The diagnostic agent according to (1), wherein the exocrine disorder is dry mouth, dry eye, dry skin, or driver jainer.
(3) The diagnostic agent according to (1) or (2), for diagnosing whether or not the exocrine disorder is based on glandular tissue destruction.
(4) The diagnostic agent according to (1) or (2), for diagnosing whether or not the exocrine disorder is based on Sjogren's syndrome.
(5) A diagnostic agent for glandular tissue destruction comprising a reagent for detecting an MK family protein.
(6) The diagnostic agent according to (5) for diagnosing whether glandular tissue destruction is based on Sjogren's syndrome.
(7) A diagnostic agent for Sjogren's syndrome comprising a reagent for detecting an MK family protein.
(8) The diagnostic agent according to any one of (1) to (7), for detecting an MK family protein in a body fluid of a subject.
(9) The diagnostic agent according to (8), wherein the body fluid is a secretory fluid.
(10) The diagnostic agent according to (8), wherein the body fluid is tear fluid, saliva, sweat, or vaginal secretion.
(11) The diagnostic agent according to (8), wherein the body fluid is saliva.
(12) The diagnostic agent according to any one of (1) to (11), wherein the reagent for detecting the MK family protein is an antibody against the MK family protein or a fragment thereof.
(13) The diagnostic agent according to any one of (1) to (12), wherein the MK family protein is MK or PTN.
(14) A kit for diagnosing an exocrine disorder, comprising the diagnostic agent according to any one of (1) to (13).
(15) A method for diagnosing an exocrine disorder, comprising a step of preparing a sample and a step of detecting an MK family protein.
(16) The diagnostic method according to (15), wherein the exocrine disorder is dry mouth, dry eye, dry skin, or driver jainer.
(17) The diagnostic method according to (15) or (16), comprising a step of diagnosing whether or not the exocrine disorder is based on glandular tissue destruction.
(18) The diagnostic method according to (15) or (16), comprising a step of diagnosing whether or not the exocrine disorder is based on Sjogren's syndrome.
(19) A diagnostic agent for glandular tissue destruction comprising a step of detecting an MK family protein.
(20) The diagnostic method according to (19), comprising a step of diagnosing whether or not glandular tissue destruction is based on Sjogren's syndrome.
(21) A method for diagnosing Sjogren's syndrome, comprising a step of detecting an MK family protein.
(22) The diagnostic method according to any one of (15) to (21), wherein the step of adjusting the sample includes adjusting the sample from a body fluid obtained from the subject.
(23) The diagnostic method according to (22), wherein the body fluid is a secretory fluid.
(24) The diagnostic method according to (22), wherein the body fluid is tear fluid, saliva, sweat, or vaginal secretion.
(25) The diagnostic method according to (22), wherein the body fluid is saliva.
(26) The process according to any one of (15) to (25), wherein the step of detecting the MK family protein includes a step of detecting binding between an antibody against the MK family protein or a fragment thereof and the MK family protein. Diagnosis method.
(27) The method according to any one of (15) to (26), wherein the MK family protein is MK or PTN.

  In the present invention, the “midkine family protein” refers to a protein that has an approximate sequence of MK or a site that expresses the function of MK and has the same or similar function as the function of MK. . Even if the protein has low sequence homology, a protein having the same or similar function as that of MK is included in the MK family protein. Examples of such MK family proteins include MK, MK-like protein (see International Publication No. 2004/052928), abbreviated MK (see US Patent Publication No. 2004/0219614), and PTN. In the above, the function of MK is not particularly limited as long as it is a function possessed by MK, and examples thereof include a cell proliferation function, an apoptosis inhibitory function, a heparin binding ability, a cell migration ability, or a differentiation inducing ability. Note that the MK family protein of the present invention does not have to have all these functions, and substances exhibiting at least one of these functions are also included in the MK family protein of the present invention.

  In the present invention, “detecting a midkine family protein” means confirming the presence of the MK family protein, for example, detection of binding to the MK family protein, and physical and chemical properties of the MK family protein. Alternatively, detection using a biological property can be exemplified, and detection of binding to an MK family protein is preferable.

  In the present invention, detection of MK family proteins includes both quantitative and qualitative measurement methods. In the case of quantitative measurement, for example, the concentration or amount of MK family protein exceeding the cut-off value can be made positive. The cut-off value can be determined as appropriate using a receiver operating characteristic curve (ROC curve) or the like from the sensitivity and specificity according to the distribution of measured values.

  In the present invention, when detection of MK family protein is detected by binding to a substance that binds to MK family protein, the detection method used is a method that can detect the substance by measuring the binding to the substance. If there is no particular limitation, for example, enzyme immunoassay method (EIA method), simplified EIA method, enzyme-linked immunosorbent assay method (ELISA method), radioimmunoassay method (RIA method), fluorescent immunoassay method (FIA method), etc. Labeled immunoassay; immunoblotting method such as Western blotting method; immunochromatography method such as colloidal gold aggregation method; chromatography method such as ion exchange chromatography method and affinity chromatography method; turbidimetric method (TIA method); NIA method); colorimetric method; latex agglutination method (LIA method); Child counting method (CIA method); chemiluminescence measuring method (CLIA method, CLEIA method); precipitation reaction method; surface plasmon resonance method (SPR method); resonant mirror detector method (RMD method); comparative interference method it can.

  In the present invention, the “reagent for detecting a midkine family protein” is not particularly limited as long as it is a reagent capable of detecting an MK family protein. Examples of the reagent for detecting the MK family protein include a substance that binds to the MK family protein, a reagent for measuring physical, chemical, or biological properties of the MK family protein, and preferably MK. A substance that binds to a family protein, more preferably a substance that specifically binds to an MK family protein, still more preferably a substance that specifically binds to MK or PTN, and most preferably specifically to MK It is a substance that binds.

  Examples of the substance that binds to the MK family protein include nucleic acids such as aptamers; antibodies, antibody fragments, proteins and peptides such as receptors and heparins; and low molecular weight compounds. An antibody or a fragment thereof that binds, more preferably an antibody or a fragment thereof that specifically binds to an MK family protein, more preferably an antibody that specifically binds to MK or PTN, most preferably an MK. Is an antibody that specifically binds to.

  In addition, when an antibody that binds to an MK family protein or a fragment thereof is used as a reagent for detecting a midkine family protein, the immunoglobulin class of the antibody is not particularly limited, and IgG, IgM, IgA, IgE, IgD Any immunoglobulin class of IgY may be used. In addition, antibodies that bind to MK family proteins include antibodies of any isotype.

In the present invention, an antibody fragment refers to a part of an antibody (partial fragment) or a peptide containing a part of an antibody, which retains the action of the antibody on the antigen. Examples of such antibody fragments include F (ab ′) ₂ , Fab ′, Fab, single chain Fv (hereinafter referred to as “scFv”), disulfide bond Fv (hereinafter referred to as “dsFv”), Examples thereof include a polymer, a dimerized V region (hereinafter referred to as “Diabody”), or a peptide containing CDR.

Substances that bind to MK family proteins include radioactive labels such as ³² P, ³ H, ¹²⁵ I, ¹⁴ C; β-galactosidase, peroxidase, alkaline phosphatase, glucose oxidase, lactate oxidase, alcohol oxidase, monoamine oxidase, horseradish peroxidase, etc. Coenzymes or prosthetic groups such as FAD, FMN, ATP, biotin, and heme; fluorescein derivatives (fluorescein isothiocyanate (FITC), fluorescein ovalbamyl, etc.), rhodamine derivatives (tetramethylrhodamine, trimethylrhodamine (RITC) , Texas Red, Rhodamine 110, etc.), Cy dyes (Cy3, Cy5, Cy5.5, Cy7), Cy-chrome, Spectrum Green, Spectrum Fluorescent labels such as orange, propidium iodide, allophycocyanin (APC), R-phycoerythrin (R-PE); bioluminescent labels such as luciferase; or luminol, isoluminol, N- (4-aminobutyl)- Chemiluminescence of luminol derivatives such as N-ethylisoluminose ester, acridinium derivatives such as N-methylacridinium ester, N-methylacridinium acylsulfonamide ester, lucigenin, adamantyl dioxetane, indoxyl derivatives, ruthenium complexes Label: A detectable label such as a metal such as colloidal gold may be bound.

  In the present invention, the “exocrine disorder” is a symptom that causes some disorder in the exocrine function, either primary or secondary. Symptoms of exocrine disorders include, for example, dry symptoms such as dry eyes, dry mice, dry skin, driver jain; aqueous tear deficiency keratoconjunctivitis, corneal softening, blepharitis, oral infection, bad breath, decreased taste, Examples include gingivitis, chronic obstructive pulmonary disease, pancreatic exocrine dysfunction, saliva, pancreatic juice, tears, digestive enzymes and other symptoms and pathological conditions, preferably dry eye, dry mouse, dry skin or A driver gina is more preferable, and dry eye or dry mouth is more preferable.

  In the present invention, “gland tissue destruction” refers to a disease that exhibits dry symptoms such as eyes and mouth, which are seen in exocrine disorders, due to damage to the secretory glands that secrete tears and saliva.

  In the present invention, “Sjogren's syndrome” is a chronic autoimmune disease characterized by dry symptoms such as a decrease in tears and saliva. Features of Sjogren's syndrome include, for example, dry mouth, cracker sign, burning sensation of the oral mucosa, hardening or swelling of the parotid gland, dental caries, redness of the oral mucosa, dry sticky oral mucosal surface, irritation and absence Oral symptoms or signs such as decreased salivary flow during stimulation, inflammation of salivary glands on small salivary gland biopsy, frequent occurrence of chronic fungal infection; foreign body sensation, inability to cry, intolerable to see light, anesthesia Low signs of shama test, no tear film destruction time, twisted mucus thread on corneal surface, tears reduction, eye symptoms or signs such as observation of dry spots by rose bengal test; and chronic joints Rheumatoid or other connective tissue diseases, fatigue, fever, lymphocyte infiltration into the lungs, kidney / muscle / nerve and liver disorders, blood abnormal globulins, excess γ-globulin, rheumatoid factor (RF), antinuclear antibodies ANA), Ro / SS-A autoantibodies, La / SS-B autoantibodies, etc., and patients with Sjogren's syndrome of the present invention are among these systemic or extraglandular symptoms or signs Present with one or more symptoms or signs.

Current standards include clear dry keratoconjunctivitis, positive lip biopsy to confirm the presence of immune cells or lymphocytes as the cause of xerostomia, extraglandular connective tissue disease (joint, skin or muscle) or chronic joint If there are two or more signs of complications such as rheumatism or systemic lupus erythematosus, you are diagnosed with Sjögren's syndrome. More specifically, satisfy at least two of the following (1) to (4) Sjogren's syndrome is diagnosed: (1) biopsy histopathological findings (a) oral glandular tissue 4 mm ² per focus (more than 50 lymphocyte infiltrates around the conduit) or higher, (b) lacrimal gland tissue 4 mm per ² 1Focus (conduit around 50 or more lymphocyte infiltration) to admit or more of any of the positive findings, (2) in the oral test, (a) in the salivary gland contrast Stage1 (1m diameter (B) (small dot-like shading) or more abnormal findings, (b) positive findings of salivary gland secretion (gum test 10 mL or less / 10 minutes, saxon test 2 g or less / 2 minutes) and salivary gland scintigraphy (3) Ophthalmological findings: (a) Shamma test 5 mm or less / 5 minutes, Rose Bengal test score 3 or more, (b) Shamma test 5 mm or less / 5 minutes, positive fluorescent dye test (4) Serum test shows positive findings of either (a) anti-SS-A antibody positive or (b) anti-SS-B antibody positive. In the development of a new diagnostic method, patients that cannot be detected by the conventional diagnostic method may be detected. Therefore, Sjogren's syndrome in the present invention is not limited to a disease that is determined to be positive by the above-described diagnostic method.

  In the present invention, "diagnosis of exocrine disorder" or "diagnosis of dry eye, dry mouse, dry skin or driver jain" means that a subject who exhibits an exocrine disorder or dry eye, dry mouse, dry skin or driver jainer symptoms, Diagnosing whether or not glandular tissue destruction or Sjogren's syndrome, preferably diagnosing whether a subject exhibiting exocrine disorder or dry eye, dry mouse, dry skin or driver Jain symptoms has Sjogren's syndrome That is.

  In the present invention, “diagnosis of glandular tissue destruction” or “diagnosis of Sjogren's syndrome” is to diagnose whether the subject has glandular tissue destruction or whether the subject has Sjögren's syndrome. .

  In the present invention, “body fluid” refers to a liquid that a human or animal has in the body, a liquid that fills between tissues, in a body cavity, a tube, or the circulatory system, and a liquid that is discharged from the body by a human or animal. Say that. Examples of the body fluid include blood; lymph fluid; tissue fluid; body cavity fluid such as joint fluid and aqueous humor; and secretory fluid such as saliva, gastric fluid, pancreatic juice, bile, intestinal fluid, sweat, tears, nasal discharge, urine, vaginal secretion, and milk. Can be mentioned. The body fluid of the present invention is preferably a secretory fluid, more preferably an exocrine fluid, still more preferably tears, saliva, sweat, and vaginal secretion, and most preferably saliva.

  The kit of the present invention is not particularly limited as long as it contains a reagent for detecting an MK family protein. For example, the reagent contained in the kit for detecting an MK family protein may be solid, gel, or liquid. In addition, the reagent for detecting the MK family protein may be contained or fixed in a resin, a membrane, a film, a container, or the like, or may be dissolved in a solvent.

  The kit of the present invention may contain a coloring reagent, a reaction stopping reagent, a standard antigen reagent, a sample pretreatment reagent, a blocking reagent, and the like, if necessary. The kit of the present invention is, for example, nitrocellulose, sepharose, nylon, vinylon, polyester, acrylic, polyolefin, polyurethane, rayon, polynosic, cupra, lyocell, acetate, polyvinylidene difluoride, silicone rubber, latex, polystyrene, polypropylene. , Polyethylene, polyvinyl chloride, polyvinylidene chloride, polystyrene, polyvinyl acetate, fluorinated resin, ABS resin, AS resin, acrylic resin, polymer alloy, glass fiber, carbon fiber, glass, gelatin, polyamino acid and / or magnetic response A plate, a tube, a chip (for example, a protein chip, a laboratory chip, etc.), a bead, a membrane, an absorber, and / or a particle containing a functional material or the like may be provided.

  Examples of the kit of the present invention include a kit comprising an anti-MK mouse monoclonal antibody-immobilized plate, biotin-labeled anti-MK rabbit polyclonal antibody solution, streptavidin POD solution, washing solution, TMB reagent, 2M HCl, and standard MK. it can.

  Examples of the kit of the present invention include a kit comprising an anti-MK family protein mouse monoclonal antibody, an anti-MK family protein mouse monoclonal antibody-binding gold colloid, a rabbit immunoglobulin-binding gold colloid, and a test plate. In the kit, the test plate is a specimen collection part for inserting a specimen, a sensitized gold colloid application part containing an anti-MK family protein mouse monoclonal antibody-binding gold colloid, and a judgment part (test line) containing an anti-MK family protein mouse monoclonal antibody. And a determination part (reference line) containing an anti-rabbit immunoglobulin polyclonal antibody, an absorbent, and a membrane filter.

  The diagnostic agent, diagnostic method or diagnostic kit of the present invention can be carried out even if the examination is easy and the facility is not limited. Moreover, in another aspect, the diagnostic agent, diagnostic method, or diagnostic kit of the present invention does not accompany the suffering of the subject during sample collection. Furthermore, in another aspect, the diagnostic agent, diagnostic method, or diagnostic kit of the present invention has less time and money burden. In particular, the diagnostic agent, diagnostic method or diagnostic kit of the present invention is suitable for diagnosing Sjogren's syndrome, which has been difficult to diagnose.

1. Reagent for Detecting Binding with MK Family Protein The reagent for detecting the MK family protein used in the diagnostic agent or kit of the present invention can be obtained by a method well known to those skilled in the art as a method for obtaining a substance that binds to a protein. it can.

(1) Antibody When an antibody is used as a substance that binds to an MK family protein, for example, MK is optionally used as an immunostimulant (for example, mineral oil or aluminum precipitate and heat-killed bacteria or lipopolysaccharide, Freund's complete adjuvant. Or a Freund's incomplete adjuvant or the like) and a non-human mammal or bird. An antibody that binds to MK can be preferably prepared by immunizing MK knockout mice (Japanese Patent Laid-Open No. 2002-85058, Nakamura, E. et al .: Genes Cells 3, p. 811-). 822.).

  The MK family protein used as an immunogen can be obtained by introducing an expression vector containing cDNA encoding the MK family protein into E. coli, yeast, insect cells, animal cells, etc. and expressing them. Here, the MK family protein is not particularly limited as long as it is a mammalian MK family protein, but is preferably human MK or human PTN. For example, mouse, rabbit, and human MKs have already been cloned, and the sequence of human MKs has been reported (Japanese Patent Laid-open No. 5-91880, Tsutui, J. et al .: Biochem. Biophys. Res. Commun. 176, pages 792-797). Therefore, a recombinant MK can be prepared by incorporating a DNA encoding MK into an expression vector, introducing the vector into Pichia yeast for expression, and collecting it from the culture supernatant (Japanese Patent Laid-Open No. 9-95454). (See the publication).

  MK family proteins can also be produced by chemical synthesis using the Fmoc method or the Boc method. For example, the C-terminal amino acid of a peptide having an MK family protein is immobilized on a polystyrene carrier, and an amino acid protected with a 9-fluorenylmethyloxycarbonyl group (Fmoc group) or a tert-butoxycarbonyl group (Boc group) is converted to diisopropyl A peptide having a desired amino acid sequence can be obtained by binding by reacting with a condensing agent such as carbodiimide (DIC) and repeating the steps of washing and deprotection.

  MK family proteins can also be synthesized using an automatic peptide synthesizer. Examples of such peptide synthesizers include PSSM-8 (Shimadzu Corporation); model 433A peptide synthesizer (Applied Biosystems, Inc.); ACT396 Apex (Advanced ChemTech Inc.) Etc. MK can also be synthesized according to a reported method (Inui, T. et al .: J. Peptide Sci., 2: p. 28-39, 1996).

  The immunized animal is not particularly limited as long as it can produce hybridomas such as mice, rats, hamsters, rabbits, chickens and ducks, but is preferably a mouse or rat, more preferably a mouse. is there. When MK is used as the immunogen, MK knockout mice are most preferred.

  Administration of the immunogen to the animal can be performed by, for example, subcutaneous injection, intraperitoneal injection, intravenous injection, intradermal injection, intramuscular injection, or footpad injection, preferably by subcutaneous injection or intraperitoneal injection. is there. The amount of the immunogen to be used is not particularly limited as long as it is an amount capable of producing an antibody, but is preferably 0.1 to 1000 μg, more preferably 1 to 500 μg, still more preferably 10 to 10 μg. 100 μg. Immunization can be performed once or several times at appropriate intervals. Preferably, immunization is performed once every 1 to 5 weeks 2 to 5 times in total, and more preferably, immunization once every 3 weeks is performed 3 times in total. One to two weeks after the last immunization, blood is collected from the orbit or tail vein of the immunized animal, and the antibody titer is measured using the serum. Antibody titer can be measured by methods well known to those skilled in the art. For example, a radioisotope immunoassay (RIA method), a solid-phase enzyme immunoassay (ELISA method), a fluorescent antibody method, and a passive hemagglutination method can be exemplified, and the ELISA method is preferable. The antibody of the present invention can be obtained by purification from the serum of an animal exhibiting a sufficient antibody titer.

  When a monoclonal antibody is used as an antibody, it can be obtained by culturing a hybridoma obtained by fusing an antibody-producing cell obtained from an immunized animal immunized by the above-described method and a myeloma cell (myeloma cell). it can. Examples of the fusion method include the method of Milstein et al. (Galfre, G. & Milstein, C., Methods Enzymol. 73: 3-46, 1981).

  The antibody-producing cells to be used can be collected from the spleen, pancreas, lymph nodes, and peripheral blood of mice or rats that have been immunized by the above-described method and have a sufficient antibody titer, and are preferably collected from the spleen.

  The myeloma cells to be used are not particularly limited as long as they are cells derived from mammals such as mice, rats, guinea pigs, hamsters, rabbits or humans, and can proliferate in vitro. Examples of such cells include P3-X63Ag8 (X63) (Nature, 256, 495, 1975), P3 / NS1 / 1-Ag4-1 (NS1) (Eur. J. Immunol., 6, 292, 1976). ), P3X63Ag8U1 (P3U1) (Curr. Top. Microbiol. Immunol., 81, 1, 1978), P3X63Ag8.653 (653) (J. Immunol., 123, 1548, 1979), Sp2 / 0-Ag14 (Sp2 / S) O) (Nature, 276, 269, 1978), Sp2 / O / FO-2 (FO-2) (J. Immunol. Methods, 35, 1, 1980) and the like, preferably P3U1 .

  The antibody-producing cells and myeloma cells obtained according to the above method are washed with a medium, PBS (Phosphate Buffered Saline), etc., and then added with a cell-aggregating medium such as polyethylene glycol (hereinafter referred to as “PEG”), Merge (Elsevier Publishing, 1988). The ratio of antibody-producing cells and myeloma cells at the time of fusion is, for example, 2: 1 to 1: 2. After cell fusion, the hybridoma is selectively proliferated by culturing in a medium such as HAT (Hypoxanthine-aminopterin-thymidine) medium. After culturing, the culture supernatant is collected, and a sample that binds to the antigen protein and does not bind to the non-antigen protein is selected by ELISA or the like. The sample is made into a single cell by the limiting dilution method, and cells that stably show a high antibody titer are selected.

  The monoclonal antibody can be obtained by culturing the hybridoma obtained by the above method in vitro and purifying the culture solution. Monoclonal antibodies can also be obtained by transplanting hybridomas into syngeneic animals or immunodeficient animals in which pristane has been administered intraperitoneally in advance, then ascites, and purifying the collected ascites.

  Purification of the monoclonal antibody can be obtained by collecting the IgG fraction using a protein A column, protein G column or the like after centrifugation. When the antibody class is IgY or IgM, the antibody can be purified by a column using mercaptopyridine as a ligand. Further, it can be purified using an MK solid phase column, ion exchange chromatography, hydrophobic interaction chromatography or the like, regardless of the antibody class.

(2) Aptamer When an aptamer is used as a substance that binds to an MK family protein, for example, RNA having a sequence that binds to the MK family protein is selected from an RNA pool having a random sequence, and after reverse transcription, PCR is performed. By repeating amplification by (SELEX method), aptamers that bind to MK family proteins can be obtained.

(3) Others The substance that binds to the MK family protein binds to the MK family protein by, for example, subjecting a natural or artificial compound, nucleic acid, protein, or peptide library to affinity chromatography with the MK family protein immobilized. A powerful protein or peptide can be obtained.

(4) Label The label can be bound to a substance that binds to the MK family protein by a method common in the art. For example, when fluorescently labeling a protein or peptide, after washing the protein or peptide with a phosphate buffer, a dye adjusted with DMSO, buffer, etc. is added, mixed, and then allowed to bind at room temperature for 10 minutes. be able to. In addition, as a commercially available labeling kit, a biotin labeling kit (Biotin Labeling Kit-NH2, Biotin Labeling Kit-SH: Dojin Chemical Laboratory Co., Ltd.), an alkaline phosphatase labeling kit (Alkaline Phosphatase Labeling Kit-NH2, Alkaline Phosphatase Kit) SH: Dojindo Laboratories Co., Ltd.), peroxidase labeling kit (Peroxidase Labeling Kit-NH2, Peroxidase Labeling Kit-NH2: Dojindo Laboratories Co., Ltd.), phycobiliprotein labeling kit (Allophycocyanin Labeling Kit-NH2, Allophylabin) ing Kit-SH, B-Phycoerythrin Labeling Kit-NH2, B-Phycoerythrin Labeling Kit-SH, R-Phycothyrin Labeling Kit-NH2, R-Phycoerythrin Labeling Kit Fluorescent Kit Labeling Kit-NH2, HiLyte Fluor (registered trademark) 555 Labeling Kit-NH2, HiLyte Fluor (registered trademark) 647 Labeling Kit-NH2: Dojin Chemical Laboratory Co., Ltd., DyLight 547, DyLight 647 (Technochemical Co., Ltd.) Trademark) Alexa Fluor (registered trademark) Antibody labeling kit, Qdot (TM) antibody labeling kit (Invitrogen), can be labeled using EZ-Label Protein Labeling Kit (Funakoshi Corporation), and the like.

2. Method for measuring MK family protein When measuring MK family protein by detecting the binding between the MK family protein in the sample of the subject and the substance that binds to the MK family protein, the binding of the substance can be detected. Any method known to those skilled in the art can be used without any particular limitation. Examples of such methods include enzyme immunoassay (EIA method), simplified EIA method, enzyme-linked immunosorbent assay (ELISA method), radioimmunoassay method (RIA method), fluorescence immunoassay method (FIA method), etc. Labeled immunoassay; immunoblotting method such as Western blotting method; immunochromatography method such as colloidal gold aggregation method; chromatography method such as ion exchange chromatography method and affinity chromatography method; turbidimetric method (TIA method); Colorimetric method; Latex aggregation method (LIA method); Particle counting method (CIA method); Chemiluminescence measurement method (CLIA method, CLEIA method); Precipitation reaction method; Surface plasmon resonance method (SPR method); Resonant mirror Detector method (RMD method); comparative interference method and the like.

  When the detectable label is bound to a substance that binds to the MK family protein, the MK family protein is detected by measuring the label such as radioactivity and fluorescence intensity by a method suitable for the label. Can be detected. In addition, when a detectable label is not bound to a substance that binds to the MK family protein, for example, measurement is performed using a labeled secondary substance that binds to a substance that binds to the MK family protein, or binding to the MK family protein. The binding to the MK family protein can be detected by measuring a physical or chemical change caused by the binding of the substance to be bound to the MK family protein.

  The sample used in the detection of the MK family protein is not particularly limited as long as it is a body fluid obtained from a subject, preferably a secretory fluid, more preferably tears or saliva. The body fluid obtained from the subject may be used directly, but may be diluted, purified, chemically modified and / or concentrated as necessary.

  When measuring by the EIA method, after reacting an antibody that recognizes the MK family protein with the MK family protein in the sample of the subject, the antibody that recognizes the labeled MK family protein is bound, and the unbound antibody is removed. The formation of the complex can be measured by measuring by a method suitable for the labeling substance.

  For example, when detecting an MK family protein using a biotin-labeled antibody as the EIA method, a sample or a recombinant MK family protein prepared by diluting a body fluid of a subject and a biotin-labeled antibody solution are immobilized on an anti-MK antibody. After adding to each well of the phased 96-well plate and reacting at room temperature, each well was washed with a washing solution, added with a substrate solution (TMB) and reacted at room temperature, then added with a reaction stop solution (2M HCl), It can be measured by measuring the absorbance at 450 nm with a plate reader. The concentration of MK in the sample can be calculated using a calibration curve prepared with a standard solution.

  In the case of measurement by immunochromatography, the sample can be detected by binding the sample to a labeled antibody, followed by chromatography using the capillary phenomenon of a nitrocellulose membrane and binding to a solid-phased antigen-specific antibody. In the immunochromatography method, by using gold colloid as a label, the binding between the immobilized antibody and the sample / labeled antibody complex can be visually confirmed.

  For example, a kit including an anti-MK family protein mouse monoclonal antibody, an anti-MK family protein mouse monoclonal antibody-binding gold colloid, a rabbit immunoglobulin-binding gold colloid, and a test plate, wherein the test plate inserts a sample. , Sensitized gold colloid coating part containing anti-MK family protein mouse monoclonal antibody-conjugated gold colloid, judgment part containing anti-MK family protein mouse monoclonal antibody (test line), judgment part containing anti-rabbit immunoglobulin polyclonal antibody (reference line) MK family proteins can be detected using a kit comprising an absorption part and a membrane filter. MK detection using this kit involves dropping a sufficient amount of sample onto the specimen collection part, and the MK family protein in the sample that has moved through the membrane filter contacts and binds to the antibody-bound gold colloid at the sensitized gold colloid coating part. It can be detected by moving with these gold colloids and binding to the monoclonal antibody at the test line. In addition, when this kit is used, the measurement is completed accurately by detecting the binding between the rabbit immunoglobulin-binding gold colloid contained in the sensitized gold colloid coating part and the anti-rabbit immunoglobulin polyclonal antibody contained in the reference line. You can confirm that.

  For example, when the CLIA method is used as a chemiluminescence measurement method, an antibody that recognizes an MK family protein labeled with a luminol derivative or an acridinium derivative is reacted with an MK family protein in a sample of a subject, and then an unbound antibody is used. After the separation, detection can be performed by emitting a chemiluminescent label adsorbed on the solid phase.

3. Diagnosis method By measuring MK in the sample by the above method, whether or not the exocrine disorder such as dry mouth, dry eye, dry skin, or driver jain is due to glandular tissue destruction, especially Sjogren's syndrome Can be diagnosed. That is, a patient or sample in which MK is detected in the sample or a patient with a large amount of MK in the sample (eg, exceeding the cutoff value) is likely to have Sjogren's syndrome and MK is not detected in the sample. Patients with low levels of MK (eg, not exceeding the cut-off value) can be diagnosed as having a low probability of having Sjogren's syndrome.

Example 1. Measurement of MK in human saliva with dry mouth symptoms Healthy subjects, Sjogren's syndrome patients with dry mouse symptoms, and non-Sjogren's syndrome patients with dry mouth symptoms, saliva collected from these subjects, saliva The MK in the medium was measured.

  Saliva collected from the subject was diluted with 1% BSA-PBS to prepare a sample. Recombinant MK was diluted with 1% BSA-PBS to prepare a standard solution. A diluted saliva sample or standard solution (20 μL) and a biotin-labeled antibody solution (100 μL) were added to each well of a 96-well plate on which an anti-MK antibody was immobilized, and reacted at room temperature for 4 hours. After the reaction, each well was washed with a washing solution, and a substrate solution (TMB) was added at 100 μL / well and allowed to react at room temperature for 10 minutes. Thereafter, 50 μL / well of a reaction stop solution (2M HCl) was added to stop the reaction. After stopping the reaction, the absorbance at 450 nm was measured with a plate reader, and the amount of MK was calculated using a calibration curve prepared from the absorbance of the standard solution.

  The results are shown in Table 1. The amount of MK in saliva was significantly increased (p = 0.0072) in patients with Sjogren's syndrome dry mice compared to non-Sjogren's syndrome dry mice.

  The diagnostic agent, kit and diagnostic method of the present invention can be used for diagnosing patients with exocrine disorders such as dry mouth, dry eye, dry skin, or driver jainer. In particular, the diagnostic agent, kit and diagnostic method of the present invention can be used for diagnosis of glandular tissue destruction, particularly Sjogren's syndrome.

Claims (19)

  A diagnostic agent for an exocrine disorder comprising a reagent for detecting a midkine family protein.   The diagnostic agent according to claim 1, wherein the exocrine disorder is dry mouth, dry eye, dry skin, or driver jainer.   A diagnostic agent for glandular tissue destruction comprising a reagent for detecting a midkine family protein.   A diagnostic agent for Sjogren's syndrome, comprising a reagent for detecting a midkine family protein.   The diagnostic agent of any one of Claims 1-4 for detecting the midkine family protein in a test subject's bodily fluid.   The diagnostic agent according to claim 5, wherein the body fluid is a secretory fluid.   6. The diagnostic agent according to claim 5, wherein the body fluid is tear fluid, saliva, sweat, or vaginal secretion.   The diagnostic agent of any one of Claims 1-7 whose reagent which detects a midkine family protein is the antibody with respect to a midkine family protein, or its fragment | piece.   The diagnostic agent according to any one of claims 1 to 8, wherein the midkine family protein is midkine or pleiotrophin.   A kit comprising the diagnostic agent according to any one of claims 1 to 9.   A method for diagnosing an exocrine disorder comprising the steps of preparing a sample and detecting a midkine family protein.   The diagnostic method according to claim 11, wherein the exocrine disorder is dry mouth, dry eye, dry skin, or driver jainer.   A method for diagnosing glandular tissue destruction, comprising a step of detecting a midkine family protein.   A diagnostic agent for Sjogren's syndrome comprising a step of detecting a midkine family protein.   The diagnostic method according to any one of claims 11 to 14, wherein the step of adjusting the sample includes adjusting the sample from a body fluid obtained from a subject.   The diagnostic method according to claim 15, wherein the body fluid is a secretory fluid.   The diagnostic method according to claim 15, wherein the body fluid is tear fluid, saliva, sweat, or vaginal secretion.   18. The method according to claim 11, wherein the step of detecting a midkine family protein includes a step of detecting binding of an antibody against the midkine family protein or a fragment thereof and the midkine family protein. Diagnostic method.   The method according to any one of claims 11 to 18, wherein the midkine family protein is midkine or pleiotrophin.