JP4363767B2 - Test method for multiple sclerosis - Google Patents

Description

以上の結果より、血液中のヒトメダラシン値による多発性硬化症の診断は非常に信頼性の高い診断方法であることが認められた。
【００３６】
多発性硬化症患者のメダラシン値の水準を男女別に分類したところ次の結果を得た（図３参照。）。

また、多発性硬化症患者のメダラシン値の水準を年齢別に分類した結果は次の通りである（図４参照。）。

これらの結果によると、男女による差及び年齢による差は認められないことが判った。
【００３７】
【発明の効果】
本発明の多発性硬化症の診断方法により、特別の装置を必要とせずに患者等の被検者から採取した血液を分析対象とし、被検者に大きな苦痛を与えることなく、安価かつ容易に多発性硬化症の発症及び／又は病勢を診断することが可能になる。
【図面の簡単な説明】
【図１】 実施例１記載の酵素免疫測定方法を用いてヒトメダラシン（標準物質）を測定し、その吸光度を抗原濃度の関数としてプロットして作成したヒトメダラシン測定用の検量線である。
【図２】 血液試料中のヒトメダラシン値（μg/10⁸ 顆粒球）を健常人、多発性硬化症及び非炎症性神経性疾患患者別にプロットしたものである。
【図３】 血液試料中のヒトメダラシン値（μg/10⁸ 顆粒球）を多発性硬化症患者の男女別にプロットしたものである。
【図４】 血液試料中のヒトメダラシン値（μg/10⁸ 顆粒球）を多発性硬化症患者の年齢別にプロットしたものである。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for diagnosing multiple sclerosis, and more particularly to a method for easily diagnosing multiple sclerosis from the amount of human medalacin in blood.
[0002]
[Prior art]
Medalacin, a kind of serine protease, is present in granulocytes and the like, and is considered to play an important role in the defense mechanism of the body widely including inflammation, especially the expression of chronic inflammation. Granulocyte medalacin increases in the exacerbations of many chronic inflammatory diseases and normalizes in remission, but it increases markedly several days before exacerbations in patients with multiple sclerosis and may normalize prior to remission It recognized. Multiple sclerosis is characterized by the appearance of localized demyelinating lesions and gliosis in the white matter of the central nervous system, progressing in remission and progression, many of which are chronic inflammations that die over the course of 10-15 years The cause of the disease is not yet clearly understood, but it is thought to be a kind of autoimmune disease in which viruses and bacteria stimulate the immune system and antibodies attack their nerve tissue Yes. In addition, the diagnostic method is quite difficult, and it is currently performed by nuclear magnetic resonance imaging (MRI), bone marrow fluid examination, etc., but MRI uses a very large apparatus and has a skilled measurement operation. It is a costly and expensive method, and the method of examining bone marrow has problems such as causing great pain to the patient, and it is possible to diagnose disease, grasp disease state, estimate prognosis, etc. with a simple test Have been explored, and the possibility of diagnosis by the amount of granulocyte medalacin in the blood has been proposed.
[0003]
[Problems to be solved by the invention]
However, whether or not multiple sclerosis can be diagnosed with the amount of medalacin in the blood needs to be determined by examining a considerable amount of clinical data, but such data has not existed until now. In addition, the measurement value of granulocyte medalacin in blood varies widely and it is difficult to obtain an accurate measurement value, so it is difficult to make a correct diagnosis, and whether or not multiple sclerosis can be diagnosed with the amount of medalacin in blood The fact is that there is no confirmation. The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a simple method for accurately measuring the amount of medalacin in blood and diagnosing multiple sclerosis based on the magnitude and / or increase / decrease of the measured value. And
[0004]
[Means for Solving the Problems]
As a result of intensive studies to achieve the above object, the present inventors measured the amount of human medalacin in a human blood sample, and the magnitude and increase / decrease of the measured values were the onset of multiple sclerosis and its disease state, etc. It has been found that they are closely related, and based on these findings, the present invention has been completed.
[0005]
Therefore, the first of the present invention is to accurately measure the amount of human medalacin in a human blood sample using an immunological method, and to determine the onset and / or disease state of multiple sclerosis by the magnitude and / or increase / decrease of the measured value. The present invention relates to a method for diagnosing multiple sclerosis characterized by diagnosis.
[0006]
In the second aspect of the present invention, a human blood sample has an osmotic pressure of 250 mOsm / kg · H. ₂ Aqueous liquid lower than O or 310mOsm / kg ・ H ₂ O After treatment with an aqueous liquid higher than leukocytes to completely destroy leukocytes, human medalacin is measured using an anti-human medalacin antibody, and the onset and / or disease state of multiple sclerosis is determined by the magnitude and / or increase / decrease of the measured value. The present invention relates to a method for diagnosing multiple sclerosis characterized by diagnosis.
[0007]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in more detail.
Most of the human medalacin in the human blood sample used in the method for diagnosing multiple sclerosis according to the present invention is present inside granulocytes, which are one of the leukocyte components present in blood. It is an essential requirement to obtain an accurate measurement value after completely destroying and releasing all human medalacin out of the cell. Therefore, when this essential requirement is not completely satisfied, the measured values vary widely and only data with poor reproducibility can be obtained.
[0008]
As a method of completely destroying granulocytes in a blood sample, a method of treating with an aqueous liquid having an osmotic pressure different from that of blood is the simplest and practical. The osmotic pressure of human blood is about 280-290mOsm / kg ・ H ₂ Since it is in the range of O, 250-310mOsm / kg ・ H ₂ It is difficult to completely destroy granulocytes present in blood with aqueous liquids in the O range. Therefore, complete destruction of granulocytes in human blood has an osmotic pressure of 250 mOsm / kg · H. ₂ Aqueous liquid lower than O, or 310mOsm / kg ・ H ₂ This can be achieved by diluting the blood with an aqueous liquid higher than O. Such aqueous liquids include purified water that may contain a water-miscible organic solvent, inorganic acid salts, organic acid salts, sugars, sugar alcohols, amino acids, and solutes composed of water-soluble substances such as proteins. An aqueous solution such as an aqueous solution having a very high concentration or an aqueous solution having a very low concentration of these solutes and having an osmotic pressure capable of completely destroying granulocytes can be used. Moreover, the usage-amount of this aqueous liquid is 50-100,000 times by volume unit with respect to a blood sample, Preferably it is 100-10,000 times, Most preferably, it is 500-2,000 times.
[0009]
The method for immunoassay of human medalacin using an aqueous dilution of a human blood sample obtained by completely destroying the granulocytes thus obtained is as follows. The measurement sample is prepared in the presence of a labeled antigen or antibody. It comprises an immune reaction stage in which it is brought into contact with an antibody and captured as a labeled immune complex by an antigen-antibody reaction, and a detection stage in which the generated immune complex is measured using a labeling substance present in the molecule. Although the method of antigen-antibody reaction in the immune reaction stage is arbitrary, for example, the following methods can be used without limitation.
[0010]
(1) Sandwich method in which an antibody bound to an insoluble carrier is captured with an antigen to be measured and then reacted with a labeled antibody;
(2) A two-antibody method in which an antibody derived from a different animal species than an antibody bound to an insoluble carrier is used in the sandwich method, and the resulting sandwich complex is further reacted with a labeled second antibody against the antibody,
(3) A competitive method in which an antibody to be measured is reacted with an antibody bound to an insoluble carrier in the presence of a peroxidase enzyme-labeled antigen.
(4) An aggregation precipitation method for detecting a labeled substance in an immune complex separated by centrifugation after allowing a labeled antibody that reacts specifically with the sample to act on a sample containing the antigen to be measured, as well as
(5) Biotin-avidin method in which labeled avidin is reacted with a biotin-labeled antibody.
[0011]
When an insoluble carrier is used in the immunoassay of medalacin useful for the method for diagnosing multiple sclerosis of the present invention, examples of the insoluble carrier include polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluororesin, cross-linking Examples thereof include polymer compounds such as dextran and polysaccharide, glass, metal, magnetic particles, and combinations thereof. The insoluble carrier can be used in various shapes such as a tray shape, a spherical shape, a fiber shape, a rod shape, a disk shape, a container shape, a cell, a microplate, and a test tube. Furthermore, the immobilization method of the antigen or antibody to these insoluble carriers is arbitrary, but a physical adsorption method, a covalent bond method, an ionic bond method or the like can be used.
[0012]
The immunoglobulin class of antibodies used for the immunological measurement of medalacin in the present invention is arbitrary, but an IgG class antibody is preferably used. The antibody can be either a monoclonal antibody or a polyclonal antibody, but a monoclonal antibody is preferred. These forms include whole antibodies or F (ab ') ₂ Fragments such as Fab and the like can be used. The origin of the antibody is arbitrary and is not particularly limited, but an antibody derived from mouse, rat, rabbit, sheep, goat, chicken or the like is preferably used.
[0013]
Next, it is preferable to use an enzyme, a fluorescent substance, a luminescent substance, a radioactive substance or the like as a labeling substance for measuring the labeled immune complex of human medalacin captured in this manner at the detection stage. Peroxidase, alkaline phosphatase, β-D-galactosidase, etc. as enzymes, fluorescein isocyanate, phycobiliprotein, etc. as fluorescent substances, luminols, luminols, dioxetanes, acridinium salts, etc., radioactive substances as ¹²⁵ I, ¹³¹ I, ¹¹¹ In, ^99m Tc etc. can be mentioned without limitation. When the labeling substance is an enzyme, a substrate, and if necessary, a color former, a fluorescent agent, and a luminescent agent can be used for measuring the activity. When peroxidase is used as an enzyme, hydrogen peroxide or the like is used as a substrate, and 2,2′-azinodi [3-ethylbenzthiazolinesulfonic acid] ammonium salt (ABTS), 5-aminosalicylic acid, o-phenylenediamine as a color former. , 4-aminoantipyrine, 3,3 ′, 5,5′-tetramethylbenzidine, etc., fluorescent agents such as 4-hydroxyphenylacetic acid, 3- (4-hydroxyphenyl) propionic acid, etc., luminescent agents such as luminols, When using alkaline phosphatase as an enzyme, such as a lucigenin charge transfer complex, when using 4-nitrophenyl phosphate, 4-methylumbelliferyl phosphate, cortisol-21-phosphate as a substrate, or β-D-galactosidase as an enzyme Is 2-nitrophenyl-β-D-galactoside, 4-methyl Rumbelliferyl-β-D-galactoside, 3- (2′-spiroadamantane) -4-methoxy-4 (3 ″ -β-D-galactosyloxyphenyl) -1,2-dioxetane (AMPGD), etc. are used. be able to.
[0014]
The polyclonal antibody that can be used for the immunological measurement of human medalacin in the method for diagnosing multiple sclerosis of the present invention is an antibody component of an anti-human medalacin antiserum obtained by immunizing an animal using human medalacin as an antigen by a conventionally known method. The product separated and purified as is preferable. Among them, for example, goat anti-human medalacin-polyclonal antibody and rabbit anti-human medalacin-polyclonal antibody are preferably used. A monoclonal antibody that can be used in the present invention and a method for producing the same are described in detail in Japanese Patent Application Laid-Open No. 11-151085 filed earlier.
[0015]
That is, an anti-human medalacin monoclonal antibody that can be used for immunological measurement of human medalacin useful for the diagnosis of multiple sclerosis is an antibody collected from an animal immunized with human medalacin extracted from granulocytes isolated from healthy human blood A hybridoma prepared by cell fusion between a production cell and a myeloma cell is cultured on a medium, or is administered into an abdominal cavity of an animal and proliferated in ascites, and then is collected from the culture or ascites. be able to.
[0016]
Hybridomas that produce anti-human medalacin monoclonal antibodies can be obtained by selectively proliferating hybridomas obtained by fusing antibody-producing cells collected from animals immunized with medalacin as an antigen with myeloma cells, and searching and cloning from the hybridomas Can be manufactured.
[0017]
Examples of the antibody-producing cells include spleen cells, lymph node cells, B-lymphocytes and the like obtained from animals immunized with human medalacin or a composition or cells containing the same. Examples of animals to be immunized include mice, rats, rabbits, goats, sheep and horses. Immunization is carried out, for example, by administering human medalacin as it is or with an appropriate adjuvant, subcutaneously, intramuscularly or intraperitoneally at about 1 μg to 1 mg / time 1-2 times / month for 1-6 months. Separation of antibody-producing cells is performed by collecting from the immunized animal 2 to 4 days after the final immunization.
As myeloma cells, those derived from mice and rats can be used. The antibody-producing cells and the myeloma cells are preferably derived from the same species.
[0018]
The method of cell fusion is arbitrary, but for example, it can be performed by mixing antibody-producing cells and myeloma cells in the presence of a fusion promoter such as polyethylene glycol in a medium such as Darcope modified Eagle medium (DMEM).
After cell fusion is completed, the hybridoma is selected by appropriately diluting with DMEM or the like, centrifuging, and suspending and culturing the precipitate in a selective medium such as HAT medium. Subsequently, antibody-producing hybridomas are searched for by the enzyme antibody method using the culture supernatant, and cloning is performed by the limiting dilution method or the like, so that a hybridoma producing an anti-human medalacin monoclonal antibody can be obtained.
[0019]
The antibody-producing hybridoma thus obtained is cultured in a medium or in vivo, and a monoclonal antibody is collected from the culture. In order to produce a large amount of monoclonal antibody, a method may be employed in which the hybridoma is administered into the abdominal cavity of an animal of the same species as the myeloma cell-derived cells, the monoclonal antibody is accumulated in the ascites, and collected from the ascites.
[0020]
Separation of the monoclonal antibody from the culture or ascites can be performed by an ammonium sulfate fractionation method commonly used for IgG purification, chromatography using an anion exchanger, or a column such as protein A or G.
There are four types of anti-human medalacin monoclonal antibodies obtained in this way, 3F03, 3G03, 2E04, and 1G12, depending on the type of hybridoma that produces them. All of these monoclonal antibodies are IgG in the globulin class and IgG in the subclass. ₁ Each antibody specifically reacts with the antigen human medalacin, and is useful for immunoassay of human medalacin for the diagnosis of multiple sclerosis.
[0021]
The method for diagnosing multiple sclerosis according to the present invention is based on the measured human medalacin concentration in a blood sample and the number of granulocytes in the blood sample measured using the same blood sample. ⁸ The amount of human medalacin in each granulocyte was calculated, and its value and cut-off value (the amount of medalacin in healthy persons (μg / 10 ⁸ Granulocytes) Average + 2SD (standard deviation)) The method of judging the onset of multiple sclerosis by comparing the magnitude with the above, or determining the change with time of the disease by comparing the increase / decrease in the value of time-measurement is preferably used.
[0022]
Of 112 patients with multiple sclerosis measured by this method, 85 medalacin levels were positive more than the cut-off value, the positive rate was as high as 75.8%, and various non-inflammatory neurological diseases as controls Of the 80 patients, 13 were positive with a cut-off value or higher, with a low positive rate of 16.3%. Diagnosis of multiple sclerosis using human medalacin levels in the blood is a highly reliable diagnostic method. It is recognized that In addition, the positive rate of medalacin levels in healthy individuals was 0% (see Table 1 and FIG. 2). In addition, it was found that there was no difference between men and women in the level of medalasin levels in multiple sclerosis patients (see FIG. 3) and age (see FIG. 4).
[0023]
【Example】
Hereinafter, the present invention will be specifically described with reference to examples. However, the present invention is not limited to the following examples.
[0024]
[Reference Example 1]
Preparation of purified human medalacin
A 6% physiological saline solution of dextran (molecular weight 200,000 to 300,000) is mixed in a ratio of blood: dextran aqueous solution = 2: 1 to 400 ml of healthy human blood, lightly stirred with a glass rod, etc., and then at a temperature of 4 ° C. to 8 ° C. The precipitate was separated from the supernatant by centrifugation at 15,000 rpm, and the precipitate was collected to obtain leukocytes. Next, an extraction solution consisting of 1M potassium phosphate buffer (PKB) of pH 7.0 containing 1 mM ethylenediaminetetraacetic acid disodium salt (EDTA) and 1 mM p-chloromercurybenzoic acid (PCMB) is added to the leukocytes. Incubate for 20 minutes at 37 ° C under agitation, then completely disrupt cells by sonication for 15 seconds, and further incubate for 20 minutes at 37 ° C, then 12,000 rpm at 4 ° C. The supernatant was collected by centrifuging for 10 minutes, and the supernatant was dialyzed against distilled water. The precipitate was extracted several times by repeating the same operation as described above. The extract was then passed through a CM-Sepharose gel column equilibrated with 50 mM PKB (pH 6.0), washed with the same buffer, and the adsorbate was eluted with 1 M PKB (pH 6.0). The eluate was dialyzed against distilled water overnight and desalted, and then concentrated with a collodion membrane to obtain 1.5 mg of purified human medalacin.
[0025]
[Reference Example 2]
Preparation of anti-human medalacin monoclonal antibody
(1) Preparation of hybridoma by cell fusion of antibody-producing cells and myeloma cells
Human medalacin extracted and purified from human granulocytes in Reference Example 1 was emulsified with Freund's complete adjuvant and administered subcutaneously to 7-week-old BALB / C mice in an amount of 50 μg / mouse. After 4 weeks, this mouse was boosted in the same manner as the first time, and after 7 days, it was confirmed that the amount of antibody had increased in the blood. After 7 days, 50 μg of antigen was injected into the peritoneal cavity as the final immunization. The dose was administered per animal. On the other hand, mouse myeloma cells P3-X63-Ag8-U1 (P3U1) were maintained and cultured in Dulbecco's modified Eagle (DMEM) medium supplemented with 20% fetal bovine serum. Three days after the final immunization, Spleen cells were collected and fused with P3U1 using polyethylene glycol 4000 and plated on 96-well microplates. After cell fusion, the medium is replaced with DMEM (HAT medium) supplemented with 100 μM hypoxanthine, 0.4 μM aminopterin, and 16 μM thymidine, and is a fusion of spleen cells and myeloma cells by selective culture for 2-3 weeks. A hybridoma was obtained.
[0026]
(2) Screening for hybridomas producing anti-human medalacin antibodies
Next, the antibody activity in the culture solution of this hybridoma was screened by ELISA (Enzyme-Linked Immunosorbent Assay). Specifically, human medalacin was adsorbed on a microplate for ELISA, subjected to blocking treatment with a solution of 2% bovine serum albumin (BSA) added to 10 mM phosphate buffered saline (PBS) at pH 7.4, and then hybridoma. Add 50 μl of the culture solution to this microplate and leave it for 1 hour, then remove and wash the hybridoma culture solution, add 100 μl of a 2 μg / ml PBS solution of peroxidase-labeled goat anti-mouse IgG-Fc specific antibody, The reaction was carried out at 37 ° C for 1 hour. Next, this enzyme-labeled antibody solution was removed and washed, and then 200 μl of 0.1 M phosphate citrate buffer (pH 4.6) containing 0.05% ABTS and 0.0034% hydrogen peroxide was added to cause color development, thereby causing anti-human medalacin antibody Productive hybridomas were selected.
[0027]
(3) Cloning of antibody-producing strains and preparation of monoclonal antibodies
This anti-human medalacin antibody-producing hybridoma culture solution was collected and cloned by limiting dilution to finally obtain four types of single clone hybridomas. Each of these hybridomas was administered to the peritoneal cavity of pristane-administered BALB / C mice to proliferate, and ascites containing monoclonal antibodies was obtained. Next, 50% saturated ammonium sulfate is added to the obtained ascites to precipitate the antibody, the precipitate is separated and dissolved in PBS, and dialyzed against 50 mM Tris-HCl buffer (pH 7.8) containing 3 M NaCl. 3F03, 3GO3, 2E04, 3E03, 3GO3, 2E04, by purifying by applying to a protein A-Sepharose CL4B column (Pharmacia), and eluting the adsorbed antibody with 0.1 M glycine-hydrochloric acid buffer (pH 5.0) and neutralizing it. And 4 types of monoclonal antibodies consisting of 1G12 were obtained.
[0028]
(4) Properties of monoclonal antibodies
[Western blotting method]
Antigen specific for the monoclonal antibody was immobilized using Western blotting.
First, human granulocyte-derived medalacin was subjected to SDS-polyacrylamide gel electrophoresis, then a solution containing 25 mM glycine and 20% methanol in an electrolyte buffer, and a protein from a slab gel under conditions of a voltage gradient of 7 V / cm for 2 hours. Was transferred to a nitrocellulose sheet. Next, each lane of the nitrocellulose sheet was separated, one sheet was protein-stained with amide black, and the other was subjected to the following enzyme immunoassay. Specifically, after blocking with 2% BSA / PBS, a mouse anti-human medalacin monoclonal antibody is added as a primary antibody, and a peroxidase-labeled goat anti-mouse IgG-Fc specific antibody is added as a secondary antibody to react and washed. By adding a substrate solution composed of PBS containing 0.04% 3,3′-diaminobenzidine and 0.0034% hydrogen peroxide, the four mouse anti-human medalacin monoclonal antibodies recognize medalacin derived from human granulocytes. It was confirmed.
[0029]
[Inhibition assay]
Human medalacin immobilized on an ELISA microplate was reacted with a biotinylated first antibody and an unlabeled second antibody in the presence of avidin, and then reacted with avidinized peroxidase. Inhibition assay, in which the amount of biotinylated antibody reaction is measured by adding color, the biotinylated antibody reaction amount does not change in any two combinations. All of the monoclonal antibodies were confirmed to recognize different epitopes (antigen sites).
[0030]
[Example 1]
Creating a calibration curve for measuring human medalacin
(1) Preparation of monoclonal antibody-immobilized beads
After thoroughly washing the polystyrene beads (diameter 6 mm), leave it in PBS (pH 7.4) solution containing 10 μg / ml of mouse anti-human medalacin monoclonal antibody (3F03) at a temperature of 4 ° C. for one day and then with PBS. The antibody was washed and allowed to stand for 1 day at night at a temperature of 4 ° C. in a 1% BSA aqueous solution to give a blocking treatment to obtain monoclonal antibody-immobilized beads.
[0031]
(2) Preparation of peroxidase-labeled monoclonal antibody
To a PBS solution containing 1.0 mg / ml of mouse anti-human medalacin monoclonal antibody (2E04), 0.1 ml of a dimethylformamide solution of N- (m-maleimidobenzoic acid) -N-succinimide ester (MBS) at a concentration of 10 mg / ml was added. Add and react for 30 minutes at a temperature of 25 ° C. Next, this reaction mixture was subjected to gel filtration with a 0.1M phosphate buffer (pH 6.O) using a column packed with Sephadex G-25 to separate the maleimidated monoclonal antibody and unreacted MBS.
On the other hand, an ethanol solution of N-succinimidyl-3- (2-pyridylthio) propionate (SPDP) at a concentration of 10 mg / ml was added to a 1.0 mg / ml PBS solution of horseradish peroxidase (HRP) as a peroxidase enzyme. React for 30 minutes at a temperature of 25 ° C. Next, the reaction mixture was purified by gel filtration with 10 mM acetate buffer (pH 4.5) using a column packed with Sephadex G-25, and a fraction containing pyridyl disulfide HRP was collected. Concentrate about 10 times under ice-cooling in collodion bag. Next, 1 ml of 0.1 M acetic acid buffered saline (pH 4.5) containing 0.1 M dithiothreitol was added thereto, and the pyridyl disulfide group introduced into the HRP molecule was stirred at a temperature of 25 ° C. for 30 minutes. After the reduction, this reaction mixture was subjected to gel filtration using a column packed with Sephadex G-25, and a fraction containing thiolated HRP was obtained.
Next, maleimidated monoclonal antibody and thiolated HRP were mixed, concentrated to a protein concentration of 4 mg / ml under ice-cooling using collodion bag, and allowed to stand at 4 ° C. for 24 hours, and then Ultrogel AcA44 (SEPRACOR) was used. Gel filtration was performed using a packed column to obtain a peroxidase enzyme-labeled monoclonal antibody.
[0032]
(3) Human medalacin sandwich enzyme immunoassay method
50 μl of 2% BSA-containing PBS solution containing 2 μl of BSA containing 2% BSA, each containing 1 mouse anti-human medalacin monoclonal antibody (3F03) immobilized, and purified human medalacin (standard) at concentrations of 0,1,10,100,200 ng / ml After adding 350 μl of PBS solution and incubating at 37 ° C. for 30 minutes, the solution in the test tube was removed by suction, washed with physiological saline, and 0.2 μg of HRP-labeled mouse anti-human medalasin monoclonal antibody (2E04) was added. A test tube was filled with 400 μl of 2% BSA-containing PBS solution at a concentration of / ml and incubated at a temperature of 37 ° C. for 30 minutes. Next, the solution in the test tube is removed by suction, washed with physiological saline, and 400 μl each of 0.1 M phosphate citrate buffer (pH 4.6) containing 0.05% ABTS and 0.0034% hydrogen peroxide. After adding to each test tube and incubating for 30 minutes at a temperature of 37 ° C., 1 ml of 0.1N oxalic acid aqueous solution was added as a reaction terminator to stop the enzyme reaction. Next, the absorbance of the solution at a wavelength of 420 nm is measured using a spectrophotometer, and this is plotted against the standard substance concentration, whereby a calibration curve having a good concentration dependency as shown in FIG. 1 is obtained. It was.
[0033]
[Example 2]
Calculation of human medalacin levels in blood samples and diagnosis of diseases
Samples of healthy individuals, multiple sclerosis patients, and non-inflammatory neurological disease patients were collected and cryopreserved and returned to room temperature. Thaw 10 μl and purified water (osmotic pressure = 0 mOsm / kg).・ H ₂ O) The sample solution was added to 2 ml and mixed well using a vortex mixer, and 10 μl thereof was added to a test tube, and 390 μl of 2% BSA-containing PBS (pH 7.4) was added thereto for dilution. Next, one bead each immobilized with a mouse anti-human medalacin monoclonal antibody (3F03) was added to the test tube, incubated at 37 ° C. for 30 minutes, and then the solution in the test tube was removed by suction, followed by physiological saline. After washing with HRP, 400 μl of 2% BSA-containing PBS solution containing HRP-labeled mouse anti-human medalacin monoclonal antibody (2E04) at a concentration of 0.2 μg / ml was filled into a test tube and incubated at a temperature of 37 ° C. for 30 minutes. Next, after washing, enzymatic reaction, and stopping the reaction by exactly the same operations as those for creating the calibration curve described above, the absorbance at a wavelength of 420 nm was measured using a spectrophotometer, and the human medalacin concentration was determined from the calibration curve. Asked.
From the obtained human medalacin concentration and the number of granulocytes measured using each blood sample, 10 ⁸ Human medalacin level (μg / 10) indicating the amount of medalacin in individual granulocytes ⁸ Granulocytes) are calculated and shown in FIG.
[0034]
FIG. 2 shows a comparison of the respective medalacin values of healthy individuals, multiple sclerosis patients and non-inflammatory neurological disease patients. From the figure, the following results were obtained.
Patients with multiple sclerosis: 355 ± 117 (n = 112)
Non-inflammatory neurological disease patients: 233 ± 66 (n = 80)
Healthy people: 213 ± 34 (n = 25)
[0035]
The above result is cut-off value (281 μg / 10 ⁸ Table 1 shows the number and positive rate by classifying positive and negative in comparison with granulocytes.
[Table 1]

From the above results, it was confirmed that the diagnosis of multiple sclerosis by the human medalacin level in the blood is a very reliable diagnostic method.
[0036]
The following results were obtained when the levels of medalacin levels in patients with multiple sclerosis were classified by gender (see FIG. 3).

Moreover, the result of classifying the level of medalacin value in patients with multiple sclerosis according to age is as follows (see FIG. 4).

According to these results, it was found that there was no difference between gender and age.
[0037]
【The invention's effect】
According to the multiple sclerosis diagnosis method of the present invention, blood collected from a subject such as a patient without the need for a special device is used as an analysis target, and it is inexpensive and easy without causing great pain to the subject. It becomes possible to diagnose the onset and / or disease state of multiple sclerosis.
[Brief description of the drawings]
FIG. 1 is a calibration curve for measuring human medalacin prepared by measuring human medalacin (standard substance) using the enzyme immunoassay method described in Example 1 and plotting the absorbance as a function of antigen concentration.
[Figure 2] Human medalacin levels in blood samples (μg / 10 ⁸ (Granulocytes) are plotted according to healthy subjects, multiple sclerosis and non-inflammatory neurological disease patients.
[Figure 3] Human medalacin level in blood sample (μg / 10 ⁸ (Granulocytes) are plotted by gender in patients with multiple sclerosis.
[Fig. 4] Human medalacin level in blood sample (μg / 10 ⁸ (Granulocytes) are plotted by age of patients with multiple sclerosis.

Claims (6)

A human blood sample is treated with an aqueous liquid having an osmotic pressure lower than 250 mOsm / kg · H ₂ O or an aqueous liquid higher than 310 mOsm / kg · H ₂ O to completely destroy leukocytes, and then the human blood sample is obtained using an anti-human medalacin antibody. The amount of human medalacin (μg / 10 ⁸ granulocytes) released into the blood sample was measured by an immunological method, and the measured value was the cut-off value (average of the amount of medalacin in healthy persons (μg / 10 ⁸ granulocytes)) A test method for multiple sclerosis, characterized by examining the onset of multiple sclerosis according to the magnitude of the value + 2SD (standard deviation)). A human blood sample is treated with an aqueous liquid having an osmotic pressure lower than 250 mOsm / kg · H ₂ O or an aqueous liquid higher than 310 mOsm / kg · H ₂ O to completely destroy leukocytes, and then the human blood sample is obtained using an anti-human medalacin antibody. The amount of human medalacin (μg / 10 ⁸ granulocytes) released into the blood sample was measured by an immunological method, and the measured value was the cut-off value (average of the amount of medalacin in healthy persons (μg / 10 ⁸ granulocytes)) A method for examining multiple sclerosis, characterized by examining time-dependent changes in the disease state after the onset of multiple sclerosis based on the magnitude of the value + 2SD (standard deviation)) and the increase / decrease of the value by measurement over time. The method for examining multiple sclerosis according to claim 1 or 2, wherein the aqueous liquid is purified water or a buffer solution that may contain a water-miscible organic solvent. The method for examining multiple sclerosis according to any one of claims 1 to 3, wherein the amount of the aqueous liquid used is 50 to 100,000 times by volume of the human blood sample. The immunoassay method comprises sandwiching an antigen-antibody reaction by contacting human medalacin released into a human blood sample after the destruction of leukocytes with an anti-human medalacin antibody and a labeled anti-human medalacin antibody immobilized on an insoluble carrier. The method for measuring the amount of the human medalacin in the human blood sample, comprising forming a complex and capturing the human medalacin on an insoluble carrier, and then quantifying the label in the complex. Test method for multiple sclerosis. The method for examining multiple sclerosis according to claim 1, 2, or 5, wherein the anti-human medalacin antibody is an anti-human medalacin monoclonal antibody.

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