JP3735676B1 - Method for detecting autoantibodies present in sera of patients with aplastic anemia - Google Patents

Abstract

A test method and test reagent for aplastic anemia are provided.
A method for testing aplastic anemia, comprising detecting an autoantibody that specifically recognizes moesin. Also provided is a reagent for testing aplastic anemia comprising a substance that detects an antibody that specifically recognizes moesin. Since autoimmune aplastic anemia and hematopoietic stem cell abnormal aplastic anemia can be discriminated, it is an extremely useful test method for detecting the presence of an immunological condition, and has high utility value.
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Description

  The present invention relates to a method for diagnosing an immunopathological condition of aplastic anemia and a reagent for detecting an autoantibody. Specifically, the present invention relates to a method for examining aplastic anemia, which comprises detecting an antibody that specifically recognizes moesin present in the serum of aplastic anemia patients, and a reagent therefor.

  Aplastic anemia is a disease characterized by bone marrow hypoplasia (reduction in bone marrow cell density) and pancytopenia (reduction of all red blood cells, white blood cells, and platelets in the blood). Aplastic anemia, once an intractable disease, is now a disease that is likely to be cured with appropriate treatment. Therefore, early accurate diagnosis and appropriate treatment are necessary. Diagnosis of aplastic anemia is carried out based on the diagnostic standards of the Ministry of Health, Labor and Welfare (Ministry of Health, Labor and Welfare specific disease idiopathic hematopoietic disorder research group 1989). Aplastic anemia is a syndrome that is diagnosed only after excluding other diseases that cause bone marrow hypoplasia and pancytopenia, and no specific marker that reflects the disease state is known. Aplastic anemia is known to be autoimmune and hematopoietic stem cell abnormal, but a method for discriminating both is not known. Moreover, since about 70% of aplastic anemia is improved by immunosuppressive therapy, it is important to prove the existence of an immunological condition. However, there is no established immunopathological marker. In general, in autoimmune diseases, it is known that many types of antibodies (autoantibodies) against autologous cell components are detected, and the antibodies are closely related to clinical symptoms. In recent years, it has been revealed that the corresponding antigens (autoantigens) of many autoantibodies are intracellular macromolecules and proteins that are conserved across species. However, little analysis has been made on autoantibodies and their corresponding antigens in autoimmune aplastic anemia patients.

Moesin has been isolated from bovine uterus as a membrane-organizing extension spike protein and has been suggested to be a receptor protein for heparin sulfate (Non-patent Document 1). Moreover, it has been shown by cDNA cloning that it usually consists of 577 amino acids in humans (Non-patent Document 2). Recently, moesin was found to be localized with actin in the apical villi of endothelial cells and certain epithelial cells, but its function has not been elucidated. In 1996, it was shown that autoantibodies against moesin are present in the serum of about 30% of patients with rheumatoid arthritis (Patent Document 1). However, it has never been known that such anti-moesin antibodies are found in patients with blood diseases in which the function of bone marrow is reduced, such as aplastic anemia.
Japanese Patent Application No. 8-509375 Rankes, WT et al., Biochem. J., (UK), Vol. 251, p. 831-842, 1988 Rankes, WT et al., Proceedings of the National Academy of Sciences of the United States of America (Proc. Natl. Acad. Sci. U.S.A.), (USA), Vol. 88, p. 8297-8301, 1991

  The present invention aims to detect an antibody that reacts with a specific self-antigen from the viewpoint of an aplastic anemia from the viewpoint of an autoimmune disease, focusing on autoantibodies present in the serum of an aplastic anemia patient.

The present invention was completed by finding that the antigen derived from hematopoietic stem cells that reacts with autoantibodies in the serum of aplastic anemia patients is moesin. Accordingly, the present invention relates to:
(1) A method for testing aplastic anemia, comprising detecting an antibody specifically recognizing moesin in a biological sample,
(2) The method according to (1), wherein the aplastic anemia is autoimmune,
(3) The method according to (1), which is a method for discriminating between autoimmune aplastic anemia and hematopoietic stem cell abnormal aplastic anemia,
(4) The method according to (1), which is a method for determining the suitability of immunosuppressive therapy for aplastic anemia,
(5) A reagent for testing aplastic anemia comprising a substance for detecting an antibody that specifically recognizes moesin in a biological sample,
(6) The reagent according to (5), wherein the substance is moesin or a partial peptide thereof.

  By using the method of the present invention, the conventional diagnosis of aplastic anemia was based on the exclusion diagnosis, but a simple method such as serum separation, antibody titer measurement, etc. enables aplastic anemia, particularly autoimmunity. It is possible to objectively diagnose sexual aplastic anemia. Therefore, the present invention enables diagnosis of an immunopathological condition of aplastic anemia, and is simple and highly reliable as a serum diagnostic method based on a specific antigen-antibody reaction.

  The subject to which the inspection method of the present invention can be applied is not particularly limited as long as it is an animal, and examples thereof include mammals. Mammals include, for example, primates, laboratory animals, livestock, pets and the like, and are not particularly limited. Specifically, for example, humans, monkeys, rats, mice, rabbits, horses, cows , Goats, sheep, dogs, cats and the like. Preferably, the target animal is a human.

  Although it does not specifically limit as a biological sample which can be used for the method of this invention, For example, the tissue derived from the animal which is a test object, a cell, a cell extract component, a body fluid, etc. are mentioned. Examples of tissues include spleen and lymph nodes, examples of cells include spleen cells, lymphocytes, antibody-producing cells, and examples of body fluids include blood, serum, plasma, urine, and sweat. In view of ease of detection and the like, the biological sample is preferably a body fluid, particularly serum / plasma.

  Moesin recognized by an antibody (hereinafter sometimes referred to as “anti-moesin antibody”) detected by the test method of the present invention is a protein belonging to a protein family (Ezrin Radixin Moesin: ERM family) generally bound to the cytoskeleton. In humans, it usually consists of 577 amino acids. The moesin is not particularly limited as long as it is derived from the above-mentioned mammals, but is preferably human-derived moesin. Examples of human moesin include a protein comprising the amino acid sequence shown in SEQ ID NO: 2 (GenBank accession number NM002444), or a natural allelic variant thereof.

The class of the antibody is not particularly limited, and may be any of IgG, IgD, IgE, IgA, sIgA, IgM and the like. Antibody binding fragments (Fab, Fab ′, F (ab ′) ₂ etc.) and the like are also included in the “antibody” as long as they specifically bind to moesin.

  The antibody detected in the present invention is an autoantibody that specifically recognizes its own moesin. For example, if the biological sample is derived from a human, a human antibody that specifically recognizes human moesin is preferably detected.

  As a method for detecting an antibody specifically recognizing moesin in a biological sample, a method known per se can be used, and is not particularly limited. However, a reaction that occurs in a liquid phase or a solid phase (for example, an antigen-antibody reaction) is directly performed. A measurement method, a method of measuring inhibition of an immune reaction by adding an inhibitor, and the like can be used.

  Examples of the method include a method in which moesin or a partial peptide thereof is brought into contact with a biological sample, and specific binding of an antibody in the biological sample to moesin or a partial peptide thereof is detected directly or indirectly.

  The moesin used in the above method is not particularly limited as long as it can be specifically recognized by the anti-moesin antibody that is an autoantibody of the test animal among the above-described moesin proteins, but preferably derived from the test animal. Moesin. For example, if the test object is a human, it is preferable to use human moesin (for example, a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or a natural allelic variant thereof).

  In the present invention, the partial peptide of moesin is not particularly limited as long as it is a partial peptide containing an antigenic determinant recognized by the anti-moesin antibody detected in the present invention. In general, since an antigenic determinant of a protein antigen is composed of at least 5 to 6 amino acid residues, moesin containing at least 5 or more, preferably 8 or more, more preferably 10 or more amino acid residues. These partial peptides can be used in the present invention.

  Moesin or a partial peptide thereof may be modified. Examples of the modification include modification with phosphoric acid, sugar or sugar chain, phospholipid, lipid, nucleotide and the like.

  Moesin or a partial peptide thereof used in the present invention can be obtained from the aforementioned humans and other animals by a known method. For example, moesin can be purified using a moesin-expressing tissue such as spleen, uterus, kidney or the like, or a cultured cell thereof, or a moesin-expressing cell line such as UT-7. Specifically, after homogenizing the animal tissue or cells, extraction with an acid or the like is performed, and the extract is purified and isolated by combining chromatography such as reverse phase chromatography or ion exchange chromatography. it can.

  The moesin of the present invention or a partial peptide thereof is obtained by culturing a transformant introduced with an expression vector containing a nucleic acid encoding moesin or a partial peptide thereof to produce moesin or a partial peptide thereof. It can also be produced by separating and purifying the partial peptide.

  Moesin or a partial peptide thereof used in the present invention can also be produced by a known peptide synthesis method. The peptide synthesis method may be, for example, either a solid phase synthesis method or a liquid phase synthesis method. Moesin or a partial peptide thereof can be produced by condensing a partial peptide or amino acid capable of constituting moesin with the remaining portion and removing the protective group when the product has a protective group.

  The partial peptide of moesin used in the present invention can also be produced by cleaving moesin obtained by any of the methods described above or below with an appropriate peptidase.

  Moesin or a partial peptide thereof may be one in which an appropriate tag is linked for the purpose of facilitating purification work and the like. Examples of the tag include an immunoglobulin Fc region, maltose binding protein (MBP), glutathione-S-transferase (GST), c-Myc tag, FLAG tag, HA tag, His tag and the like.

Although it does not specifically limit as this detection method, More specifically, the following method is mentioned.
(1) An agglutination reaction in which moesin or a partial peptide (antigen) thereof is coated on the surface of blood cells or gelatin particles, and an antigen-antibody reaction is caused by adding a biological sample to form an aggregate.
(2) A double immunodiffusion method (DID: double immunodiffusion) in which an extract containing moesin or a partial peptide thereof and a biological sample are diffused in an agar gel to cause a precipitation reaction.
(3) After immobilizing purified moesin or its partial peptide on a plate and adding a biological sample to react,
i) ELISA method in which a secondary antibody conjugated with an enzyme is further reacted to detect color development of the substrate with a spectrophotometer,
ii) Fluorescence immunoassay (FIA) in which a secondary antibody conjugated with a fluorescent dye is further reacted to measure fluorescence development, or
iii) a chemiluminescence immunoassay (CLIA) in which a secondary antibody bound to a chemiluminescent substance is further reacted to measure chemiluminescence.
(4) An immunoturbidimetric method in which the surface of latex particles or glass beads is coated with moesin or a partial peptide thereof, and light is applied to the agglutination reaction solution that occurs when the particles encounter an antibody, and the transmitted light is measured. Immuno-comparison method (nephrometry method) to measure the scattered light,
(5) Radioimmunoassay in which moesin or a partial peptide thereof is labeled with a radioisotope and reacted with a biological sample to detect an antigen-antibody reaction,
(6) A frozen thin section or cell of a tissue containing moesin or a partial peptide thereof is attached to a glass slide, reacted by dropping a biological sample on the section, and further reacted with a secondary antibody bound to a fluorescent dye. A fluorescent antibody method for detecting fluorescence under a microscope,
(7) A surface plasmon resonance analysis method for checking affinity by immobilizing moesin or its partial peptide on a chip and flowing a biological sample.
(8) Western blotting method in which moesin or a partial peptide thereof in a gel separated and developed by electrophoresis is transferred to a nitrocellulose membrane and reacted with a biological sample to detect an antigen-antibody reaction.

  For example, when the detection means is an ELISA method, specifically, detection and / or quantification can be performed as follows. That is, according to a conventional ELISA method, for example, a biological sample is provided to each well of a multi-well plate coated with moesin or a partial peptide thereof, and an enzyme-labeled secondary antibody is added to each well for reaction, and the enzyme substrate is allowed to react. After the addition, the antigen-antibody reaction can be detected and / or quantified by detecting and / or quantifying the product produced by the enzyme.

  In the case of the ELISA method, the enzyme used for labeling may be a conventional enzyme usually used for the ELISA method. For example, peroxidase, alkaline phosphatase, β-D-galactosidase, glucose oxidase, luciferase, esterase, β -D-glucuronidase etc. are mentioned. Peroxidase or alkaline phosphatase is desirable from the viewpoint that it is possible to achieve more sensitive and stable detection. The enzyme substrate can be appropriately selected depending on the enzyme used. For example, 3,3 ′, 5,5′-tetramethylbenzidine or the like is used in the case of peroxidase, and paranitrophenyl phosphate in the case of alkaline phosphatase. Sodium or the like is used.

  Detection and / or quantification of the product produced by the enzyme can be performed by measuring the absorbance of the product. For example, when 3,3 ′, 5,5′-tetramethylbenzidine is used as the enzyme substrate, the absorbance at 450 nm may be measured.

  For example, when the detection means is a fluorescence immunoassay (FIA), examples of the fluorescent dye include FITC (fluorescein isothiocyanate), PE (phycoerythrin), APC (Allophycocyanin), Cy-3, and Cy-5.

  For example, when the detection means is a chemiluminescence immunoassay (CLIA), examples of chemiluminescence include acridinium ester.

  When an antibody that specifically recognizes moesin is detected in a biological sample, it can be determined that the subject from which the biological sample is derived has a high possibility of developing / being aplastic anemia. In this case, the higher the antibody titer that specifically recognizes moesin in the biological sample, the higher the possibility of developing aplastic anemia. Conversely, if an antibody that specifically recognizes moesin is not detected in a biological sample, it can be determined that the subject from which the biological sample is derived is less likely to develop / be developing aplastic anemia. .

  When judging the possibility of the onset, the judgment criteria are not limited to detection / non-detection of antibodies. For example, an average value ± 3SD of the amount of antibody specifically recognizing moesin in a biological sample derived from a healthy subject is set as a cut-off value, and if the cut-off value or higher, the subject develops aplastic anemia / On the other hand, if the target is less than or equal to the cutoff value, it may be determined that the subject is less likely to develop / behave aplastic anemia.

  In addition, moesin is specifically recognized by the test method of the present invention in patients who have been found to develop aplastic anemia using the reticulocyte count, platelet count, white blood cell count, etc. in the blood as an index. If the antibody to be detected is detected, it can be determined that the patient is likely to develop autoimmune aplastic anemia. In this case, it can also be determined that the patient is suitable for immunosuppressive therapy. If no antibody specifically recognizing moesin is detected, it can be determined that the patient is likely to develop hematopoietic stem cell abnormal aplastic anemia or anemia due to other causes. In this case, it can also be determined that the patient is not suitable for immunosuppressive therapy. The test method of the present invention is particularly excellent for determining whether aplastic anemia is autoimmune aplastic anemia or hematopoietic stem cell abnormal aplastic anemia.

  In the present specification, autoimmune aplastic anemia means aplastic anemia caused by hematopoietic stem cells being attacked and reduced by their own lymphocytes, antibodies, etc., and hematopoietic stem cell abnormal regeneration Aplastic anemia means aplastic anemia caused by hematopoietic stem cells not proliferating / differentiating due to acquired genetic abnormalities.

  The present invention also provides a reagent for testing aplastic anemia comprising a substance that detects an antibody that specifically recognizes moesin in a biological sample.

  The substance is not particularly limited as long as it can achieve detection of an antibody that specifically recognizes moesin in the above-described method, but is preferably moesin or a partial peptide thereof. In addition, when the anti-moesin antibody detected in the present invention is an antibody group that recognizes a plurality of antigenic determinants, the antibodies specifically recognizing each of the many antigenic determinants present in moesin are exhausted. From the viewpoint of improving detection sensitivity by detection, the substance is preferably moesin (full length of protein).

  Moesin or a partial peptide thereof may be provided in the form of powder, solution, etc., or alternatively, blood cells, gelatin particles, plates, latex particles, glass beads, glass slides, chips, microtiter plates, centrifuge tubes, microbeads, You may provide with the form carry | supported by insoluble support | carriers, such as a membrane and a paper disk. In the carrier on the container, moesin or a partial peptide thereof is carried at the site where the solution held on the carrier comes into contact, for example, in the case of a microtiter plate. It should be noted that moesin or a partial peptide thereof can be supported on an insoluble carrier by a known method.

  If the test reagent of the present invention is used, aplastic anemia can be easily tested by the above-described method.

  The test reagent of the present invention can also be a regenerative anemia test kit further including a reagent used in the detection method described above. Specific examples of the reagent include a buffer solution for diluting the reagent and biological sample, a fluorescent dye, a reaction vessel, a positive control, a negative control, and an instruction document describing a test protocol. These elements can be mixed in advance if necessary. By using this kit, the test for aplastic anemia of the present invention is simplified, and it is very useful for early treatment policy determination.

  Hereinafter, the present invention will be described in detail using examples, but the present invention is not limited to the following examples.

(Example 1) Antigen-antibody reaction by Western blotting using lysate of UT-7 cells derived from human acute myeloid leukemia About 1 × 10 ⁷ UT-7 cells were treated with Dulbecco's PBS (137 mM NaCl, 1.4 mM phosphate). After washing twice with potassium, 4.3 mM sodium phosphate, 2.7 mM KCl), a loading solution for electrophoresis (62.5 mM Tris.HCl pH 6.8, 10% glycerol, 5% β-mercaptoethanol, 0.8%). It was dissolved in 500 μl of 001% bromophenol blue (BPB), 2% sodium dodecyl sulfate (SDS), and sonicated. The lysate was loaded at 10 μl per well, and electrophoresed on a 10% polyacrylamide gel with a 1 × electrophoresis solution containing 0.1% SDS (25 mM Tris · HCl, 192 mM glycine pH 8.4). A part of the gel was stained with Coomassie Brilliant Blue (CBB) to confirm the size of the band. The results are shown in lane 1 of FIG. In the figure, lane m is a molecular weight marker, and lane 1 is Coomassie brilliant blue staining.
Proteins in the unstained gel were electrically transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, Mass.) Over 1.5 hours at a constant current of 190 mA in a methanol-containing transfer buffer. The PVDF membrane was blocked overnight at 4 ° C. in 5% skim milk solution. Thereafter, normal human serum, aplastic anemia patient serum, or anti-moesin antibody diluted 200-fold with 1% BSA-TBS (25 mM tris (hydroxymethyl) aminomethane (Tris) -buffered solution containing 150 mM NaCl, 2.5 mM KCl) (Clone 38/87, Neomarkers) was reacted at room temperature for 1 to 2 hours, and then washed 3 times with TBS-T (TBS and 0.05% Tween 20 (TBS-T, pH 7.5)). Washed once more with TBS and diluted 2000 times with 1% BSA-TBS with alkaline phosphatase-labeled sheep anti-human IgG Fcγ fraction (Jackson ImmunoResearch) or alkaline phosphatase-labeled rabbit anti-mouse IgG (Chemicocom) for 1 hour at room temperature Reacted. After washing 3 times with TBS-T and once with TBS, color was developed using BCIP / NBT phosphate substrate system (KPL), and it was confirmed that the band reacting with the serum of aplastic anemia patient was 77 kD . The 77 kD band reacted with 4 of 9 sera from aplastic anemia patients. On the other hand, normal human serum was found to react with 2 of 10 samples. The typical pattern is shown in FIG. Lanes 2 to 5 are normal human sera, lanes 6 to 9 are aplastic anemia patient sera, and lane 10 is an anti-moesin antibody.

Example 2 Analysis of 77 kD Band by Mass Spectrometry About 1 × 10 ⁷ UT-7 cells were incubated in 0.5 ml PBS at 37 ° C. for 1 hour, and then centrifuged at 500 rpm for 3 minutes in a Kendro Biofuge Freso. The obtained supernatant was dissolved in the above-described electrophoresis loading solution, followed by electrophoresis, and CBB staining. After cutting out the 77 kD band in the CBB-stained gel, 100 mM Na ₂ S ₂ O ₃ , 30 mM [Fe (CN) ₆ ] K ₃ was prepared in advance, prepared 1: 1 for use, and decolorized in a few minutes. . The gel which had become decolored and became yellow was washed with water until it became transparent visually. After changing water several times, it was confirmed that the gel became transparent in about 10 to 15 minutes. Next, the water used for washing was drained, and the gel was subdivided into about 1 mm ³ and dried. The protein of interest was fragmented by treatment with 50 mM ammonia bicarbonate pH 8.0 2% acetonitrile containing the required amount of Trypsin (Promega) overnight at 37 ° C. The fragmented peptide was simultaneously extracted, desalted and concentrated using ZipTip. Finally, the amino acid sequence was determined by mass spectrometry (MALDI-TOF / MS (peptide mass fingerprinting method)) and searched from a database. As a result, the 77 kD protein was found to be moesin.

(Example 3) Production of antigenic protein
1. Construction of moesin expression vector
Moesin cDNA was obtained from the Department of Cell Information Pharmacology, Nagoya University Graduate School of Medicine, and the region containing the coding site was cut out using Kpn I and incorporated into pET-44c (Novagen, Madison, WI).
2. Expression of recombinant moesin by E. coli
Escherichia coli BL21 (Novagen) was transformed with the expression plasmid pET-44c-moesin obtained in 1 of Example 3, and the resulting single colony was used as an inoculum for expression experiments. Four independent clones were spread on one surface of an LB (Luria Betani) plate containing 100 μg / ml of ampicillin, and allowed to stand at 37 ° C. overnight. The cells on the plate were scraped with a platinum loop, planted in 3 ml of ampicillin-containing LB medium, and cultured with shaking at 37 ° C. for about 1.5 hours. Thereafter, the absorbance at 550 nm (OD550) was measured, the culture solution was added to 20 ml of the same medium so that OD550 = 0.2, and the shaking culture was continued. After culturing, when OD550 = 0.8 was reached in about 2 hours, IPTG (isopropyl-1-thio-β-D-galactoside) was added to a final concentration of 1 mM. At 0, 4, and 18 hours after the addition of IPTG, OD550 = 1.0 equivalent of the bacteria was collected after centrifugation at 13000 rpm for 2 minutes. The sample solution was dissolved in the electrophoresis loading solution, 10 μl of the lysate was loaded into the well, and 10% polyacrylamide gel electrophoresis was performed to confirm the expression of recombinant moesin. Expression of the cells for purification was performed on a 100 mL scale, and the culture was continued for 18 to 20 hours, and the cells were collected by centrifugation at 3000 rpm for 30 minutes with RL-131 manufactured by Tommy Seiko.
3. Purification of transgenic moesin
The cells were suspended in 40 ml of 1 × Binding Buffer (His BindKits, Novagen), then dispensed 1 ml at a time into Eppendorf tubes, repeated approximately 1 minute of ultrasonic disruption 3 times, and then centrifuged at 3000 rpm for 30 minutes. And a sediment was obtained. The precipitate was washed with 20 ml of 1 × Binding Buffer, placed on ice with 5 ml of 1 × Binding Buffer containing 6M urea for 60 minutes, and the eluted supernatant was collected by centrifugation. Recombinant moesin with a His tag contained in the supernatant was chromatographed according to the protocol under the denaturing conditions of His · BindKits (Novagen) to obtain an antigenic protein.

Example 4 Detection of anti-moesin antibody in patient serum using genetically modified moesin Western blotting
The antigen protein obtained in Example 3 was electrophoresed at 200 ng per well. The antigenic protein in the gel was electrically transferred onto a PVDF membrane in a methanol-containing transfer buffer at a constant current of 190 mA over 1.5 hours. The PVDF membrane was blocked overnight at 4 ° C. in 5% skim milk solution. Thereafter, the mixture was reacted with normal human serum, patient serum or anti-moesin antibody diluted 200-fold with 1% BSA-PBS for 1 to 2 hours at room temperature, and then washed 3 times with TBS-T. Further, it was washed once with TBS and diluted 2000-fold with 1% BSA-PBS and diluted with alkaline phosphatase-labeled sheep anti-human IgG Fcγ (Jackson ImmunoResearch) or alkaline phosphatase-labeled rabbit anti-mouse IgG (Chemicocom) for 1 hour at room temperature. Reacted. After washing three times with TBS-T and further once with TBS, color development was caused using BCIP / NBT phosphate substrate system (KPL). FIG. 2 shows a typical pattern. Lane m is a molecular weight marker, lanes 1 to 2 are normal human sera, lanes 3 to 5 are sera from aplastic anemia patients, and lane 6 is a result of anti-moesin antibody.
2. ELISA
250-500 ng of the recombinant protein obtained in Example 3 was coated on 96-well Nunc-Immuno Plate (Nalge-Nunc International, Roskilde, Denmark) at 4 ° C. overnight. Phosphate-buffered saline (PBS) containing 10% fetal calf serum (PBS-FCS) was washed three times, PBS-FCS was added, and the plate blocked overnight at 4 ° C. was used as an ELISA plate. 50 μl of 1000-fold diluted serum was added and incubated at room temperature for 60 minutes. After washing 3 times with PBS-FCS, peroxidase-labeled anti-human IgG (1: 5000, Jackson ImmunoResearch) was added, incubated at room temperature for 60 minutes, and washed in the same manner. Thereafter, color development operation was performed with 3,3 ′, 5,5′-tetramethylbenzidine (TMB) substrate (Pierce, Rockford, IL), and the absorbance of OD450 was measured. The result is shown in FIG. When the mean value ± 3SD of normal human serum (B) was taken as the cut-off value for each antigen (OD450 = 0.284), 47% of patients with aplastic anemia showed a significantly high antibody titer. That is, by using this ELISA system, aplastic anemia having an immune pathological condition can be diagnosed before treatment.

  As is clear from the results described above, using the test method of the present invention, it is possible to determine whether aplastic anemia is either autoimmune aplastic anemia and hematopoietic stem cell abnormal aplastic anemia, An appropriate treatment strategy can be quickly determined. As described above, the immunological detection method using the self-antigen protein is a serum diagnostic method based on the autoantigen of aplastic anemia and is extremely useful because it is simple and reliable.

It is the pattern which electrophoresed the antigen protein roughly purified from the human acute myeloid leukemia cell line UT-7. It is a figure which shows the result (lane 2-9) which carried out the Western blotting analysis using the coomassie brilliant blue dyeing | staining result (lane 1), and normal human serum or an aplastic patient's serum. It is a figure which shows what electrophoresed the recombinant type | mold moesin obtained from colon_bacillus | E._coli, and carried out the western blotting analysis using the serum of an aplastic anemia patient or a healthy subject's serum. The band is detected at about 150 kD as a fusion protein. It is a figure which shows the difference in the reactivity by ELISA of aplastic anemia patient serum (A) and normal human serum (B) when recombinant moesin is used as an antigen.

Claims (2)

  A test method for aplastic anemia, comprising detecting an antibody that specifically recognizes moesin in a biological sample. A reagent for testing aplastic anemia comprising moesin or a partial peptide thereof.