JP5924659B2 - Methods for comprehensive analysis of immune complexes and novel rheumatoid arthritis biomarkers - Google Patents

Description

  The present invention provides a method for discovering novel biomarkers of various diseases by comprehensively analyzing immune complexes collected from a subject and comparatively analyzing it with that of a healthy donor, and a joint found by the method The present invention relates to a test kit and test for rheumatoid arthritis targeting a novel rheumatoid biomarker, and a method for evaluating drug efficacy.

An antibody is a type of protein produced from B lymphocytes. It recognizes non-self (bacteria, viruses, etc.) that invade from outside the body and binds to them to activate leukocytes and macrophages for ecological protection. Plays a big role. Thus, the antibody has a function of recognizing and eliminating non-self xenobiotics. However, when an antibody that recognizes its own normal cells and tissues is produced at some opportunity, an immune complex (immunocomplex) is formed, which may be deposited on tissues such as organs and blood vessel walls and cause damage. Such diseases are called autoimmune diseases, and a large number of diseases including rheumatoid arthritis, systemic lupus erythematosus, and Graves' disease are known. In addition, immune complexes are known to show high levels in viruses, bacterial infections, and tumors. For this reason, in many infectious diseases and autoimmune diseases involving humoral immunity, pathological condition elucidation, diagnosis, and severity determination are generally performed based on the presence and amount of antibodies produced. However, there are many reports of antibody-negative cases and cases in which the amount of antibody does not match the severity, and it cannot be said that it is sufficiently effectively used in clinical settings. In addition, even if the antigen or immune complex is expected to be the etiology or marker, there are many diseases in which the detailed specification of foreign / self antigens has not been achieved.
On the other hand, in recent years, proteome analysis has begun to be used in many bioanalytical science research fields, and has been regarded as promising as a means for elucidating the cause and onset mechanism of diseases. In particular, shotgun proteomics has been developed, in which all proteins are fragmented by enzymatic degradation and analyzed by high performance liquid chromatography / tandem mass spectrometry (LC-MS / MS) (Non-patent Document 1). Etc. can be performed quickly, efficiently and with good reproducibility. In addition, for the purpose of elucidating specific diseases, an example in which an antigen is purified using an antibody and the identification is performed by LC-MS / MS has been reported (Non-patent Document 2).
However, from the viewpoint of means for clinical diagnosis and pathological elucidation, attention has been paid to the antigen itself forming the immune complex, and a method for comprehensive analysis of this has not been developed so far.

D. Figeys, Anal. Chem. , 75, 2891-2905, 2003 H. Tjalsma et al. , Proteomics Clin. Appl. , 2, 167-180, 2008

  An object of the present invention is to provide a novel biomarker search method for clinical diagnosis of disease, a diagnosis method of disease, a test for diagnosis of rheumatoid arthritis using the new biomarker identified by the search method, a method for evaluating drug efficacy, and a test It is to provide kits and the like.

The present inventors trypsinize immune complexes exhaustively collected from the blood of healthy donors and autoimmune disease (rheumatoid arthritis) patients, and subject them to high-performance liquid chromatography / tandem mass spectrometry. Analysis was performed, and thereby, an antigen molecule different from the conventionally known rheumatoid arthritis marker was found.
As a result of further studies based on these findings, the present inventors have completed the present invention.

That is, the present invention
[1] A comprehensive search method for disease biomarkers, including the following steps;
(1) a step of recovering an immune complex from each sample derived from a disease patient and a healthy person,
(2) a step of decomposing each immune complex and separating a degradation product;
(3) a step of comprehensively identifying antigens contained in each sample by mass spectrometry of the separated degradation products;
(4) A step of selecting an antigen specifically identified in a diseased patient as compared with a healthy person as the disease biomarker [2] The disease is an infection, autoimmune disease, malignant tumor, arteriosclerosis, diabetes The method according to [1] above, which is selected;
[3] The method according to [1] above, wherein the sample is blood, serum or tissue;
[4] The method according to [1] above, wherein in step (1), the immune complex is recovered using protein G or protein A;
[5] The method according to [1] above, wherein in step (2), the degradation product is separated by liquid chromatography;
[6] The method according to [1] above, wherein in step (3), mass spectrometry is performed by tandem mass spectrometry;
[7] A method for examining whether or not to be affected by a disease or predicting whether or not to be affected in the future, including the following steps:
(1) a step of recovering immune complexes from each sample derived from a subject and a disease patient,
(2) a step of decomposing each immune complex and separating a degradation product;
(3) a step of comprehensively identifying and quantifying antigens contained in each sample by mass spectrometry of the separated degradation products;
(4) A step of testing whether or not the subject is affected by the disease or predicting whether or not the subject will be affected in the future by comparing the identified antigens between the subject and the diseased patient [8] The method according to [7] above, wherein the disease is selected from infectious diseases, autoimmune diseases, malignant tumors, arteriosclerosis, and diabetes;
[9] The method according to [7] above, wherein the sample is blood, serum or tissue;
[10] The method according to [7] above, wherein in step (1), the immune complex is recovered using protein G or protein A;
[11] The method according to [7] above, wherein in step (2), the degradation product is separated by liquid chromatography;
[12] The method according to [7] above, wherein in step (3), mass spectrometry is performed by tandem mass spectrometry;
[13] Rheumatoid arthritis test method for detecting the presence or absence of thrombospondin-1 and / or platelet factor 4 in an immune complex recovered from a sample derived from a subject;
[14] The method according to [13] above, wherein the immune complex is recovered using protein G or protein A;
[15] detecting the presence or absence of thrombospondin-1 and / or platelet factor 4 in an immune complex collected from a sample collected over time from a patient being treated or treated with rheumatoid arthritis, A method for evaluating the therapeutic effect on patients with rheumatoid arthritis;
[16] The method according to [15] above, wherein the immune complex is recovered using protein G or protein A;
[17] A test kit for rheumatoid arthritis comprising an antibody that specifically recognizes thrombospondin-1 and / or an antibody that specifically recognizes platelet factor 4 is provided.

  It is possible to exhaustively search for disease-specific biomarkers by collecting immune complexes from patients clinically determined to have a specific disease and analyzing the antigen. In addition, for subjects who are suspected of suffering from some kind of disease but have not made a specific clinical judgment, the immune complex is collected and the analyzed antigen and a known antigen marker of the specific disease Can be tested to determine which disease the subject suffers from or predict whether it will suffer in the future. Furthermore, in the present invention, by examining the presence or absence of a novel biomarker found in a rheumatoid arthritis patient as an immune complex, it is possible to quickly and easily examine and evaluate rheumatoid arthritis.

The present invention is described in detail below.
The present invention relates to a method for searching for disease biomarkers by comprehensively examining immune complexes derived from patients suffering from any disease.

  The disease to which the disease biomarker search method of the present invention is applied is not particularly limited, and examples thereof include infectious diseases, autoimmune diseases, malignant tumors, arteriosclerosis, diabetes and the like. Examples of infectious diseases include parasitic protozoal infections (malaria, amoeba dysentery, toxoplasmosis, leishmaniasis, cryptosporidium, etc.), viral infections (dengue fever, West Nile fever, influenza, acquired immune deficiency syndrome, rabies, Marburg hemorrhagic fever, severe acute respiratory syndrome, chickenpox, shingles, hand-foot-and-mouth disease, Ebola hemorrhagic fever, pharyngeal conjunctival fever, infectious erythema, infectious mononucleosis, smallpox, rubella, acute gray leukitis, measles, etc.), Bacterial infections (Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus, Listeria, Neisseria meningitidis, Neisseria gonorrhoeae, Pathogenic Escherichia coli, Neisseria pneumoniae, Proteus, Pertussis, Pseudomonas aeruginosa, Serratia, Citrobacter, Acinetobacter, Entero Various infections caused by Bacter, Mycoplasma, Chlamydia, Clostridium, etc., tuberculosis, cholera, di Terrier, dysentery, scarlet fever, anthrax, trachoma, syphilis, tetanus, leprosy, Legionella, Leptospira, Lyme disease, tularemia, etc. Q fever), and the like. Examples of autoimmune diseases include rheumatoid arthritis, systemic lupus erythematosus, primary biliary cirrhosis, polymyositis, mixed connective tissue disease, nodular periarteritis, Graves' disease, myasthenia gravis, Sjogren's syndrome, anti-phosphorus Examples include lipid antibody syndrome, multiple sclerosis, idiopathic thrombocytopenic purpura, pemphigus, relapsing polychondritis, and multigland autoimmune syndrome. Examples of malignant tumors include ovarian cancer, cervical cancer, endometrial cancer, stomach cancer, liver cancer, colon cancer, pancreatic cancer, gallbladder cancer, kidney cancer, lung cancer and the like. Examples of diabetes include type 1 diabetes and type 2 diabetes.

The comprehensive search method for disease biomarkers of the present invention includes the following steps.
(1) a step of recovering an immune complex from each sample derived from a disease patient and a healthy person,
(2) a step of decomposing each immune complex and separating a degradation product;
(3) a step of comprehensively identifying antigens contained in each sample by mass spectrometry of the separated degradation products;
(4) A step of selecting, as the disease biomarker, an antigen that is specifically identified in a disease patient as compared with a healthy subject

  The disease patient to which the search method of the present invention can be applied is not particularly limited, and examples thereof include disease patients clinically determined to suffer from the above diseases, such as humans, monkeys, cows, horses, pigs, Mammals such as mice, rats, guinea pigs, hamsters, dogs, cats, rabbits, sheep, goats and the like can be mentioned. Preferably it is a human. In addition, a healthy person includes, for example, a person who is determined not to suffer from at least any of the above diseases.

  Samples used in the search method of the present invention are those collected from patients with the above diseases and healthy individuals, and those containing an immune complex comprising an antigen to be detected and an antibody that binds to the antigen. There is no particular limitation. Examples of such samples include whole blood, plasma, serum, lymph, saliva, cerebrospinal fluid, body fluid such as joint fluid, or fractions thereof, mucous membrane, tissue specimen obtained by biopsy, and the like. Blood, plasma, serum, lymph, saliva, etc. are particularly preferred because they can be collected quickly and easily and are less invasive to diseased patients and healthy subjects. However, when the disease is a malignant tumor, etc. It may be a tissue containing the malignant tumor. The antibody concentration in the blood is expected to be about 10 mg / ml, and even if it is assumed that 0.001% of the antibody forms an immune complex, the antigen amount of the immune complex obtained using 10 μl of blood (average) The molecular weight of 10,000) is expected to exceed the detection limit of a general LC-MS / MS apparatus. Therefore, when blood, plasma, serum, or lymph is used as a sample, it is preferable to use at least 10 μl.

  The immune complex in the sample can be separated and purified using a method known per se. For example, immunoglobulin separation and purification methods [Example: salting-out method, alcohol precipitation method, isoelectric precipitation method, electrophoresis method, adsorption / desorption method by ion exchanger (eg DEAE), ultracentrifugation method, gel filtration method, A specific purification method in which only an immune complex and an antibody are collected by an active adsorbent such as protein A or protein G, and the binding to the active adsorbent is dissociated to obtain an immune complex can be separated and purified. Specifically, as described in the following examples, only immune complexes and antibodies are directly obtained from blood and serum by a carrier (sepharose column, magnetic beads, etc.) bound with an active adsorbent such as protein A or protein G. Can be collected. Even when the sample is a tissue, a protein fraction can be prepared based on a known method, and the immune complex contained in the fraction can be separated and purified according to the above-described method. Further, when the immune complex is derived from a human, the antibody constituting the immune complex may be any subclass, including IgG, IgM, IgA, IgD or IgE. IgG1, IgG2, IgG3, IgG4 may be included. Therefore, in place of protein A and protein G, for example, by fixing an anti-IgE antibody or the like on a carrier, it is possible to analyze an early stage of infection or allergic diseases.

  The immune complex collected from the sample may be separated as it is, or may be reduced alkylated in advance in order to reduce the disulfide bond of the antibody. Examples of the reducing agent include dithiothreitol (DTT). Furthermore, it is preferable to decompose the antibody and antigen that form an immune complex with a protease or peptidase into peptide fragments and separate the degradation products. Examples of peptidase (protease) include trypsin and pepsin.

  The decomposition product can be separated according to a method known per se. Such methods mainly utilize differences in molecular weight such as methods utilizing solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis. Method using charge difference such as ion exchange chromatography, method utilizing specific affinity such as affinity chromatography, method utilizing hydrophobic difference such as liquid chromatography, isoelectric focusing A method using the difference in isoelectric point, such as a method, is used. These methods can be combined as appropriate. In order to separate the degradation product of the present invention, it is preferable to use liquid chromatography because the degradation product is a peptide, and it is easy to perform the subsequent mass spectrometry.

  The carrier for the packing material for liquid chromatography used in the separation is preferably silica gel or a spherical or crushed synthetic resin such as polyvinyl alcohol. The average particle size is preferably from about 0.5 μm to about 100 μm, more preferably from about 1 μm to about 50 μm, still more preferably from about 3 μm to about 5 μm. A person skilled in the art can select a suitable particle size in consideration of the fact that the smaller the average particle size, the larger the theoretical number of columns per unit length, but the greater the pressure loss of the column. In addition, the carrier of the packing material for liquid chromatography used in the present invention is one in which a substituted or unsubstituted hydrocarbon group is bonded to the carrier, and in particular, one having a chain length of 8 or more carbon atoms. Is preferred. Examples of such a hydrocarbon group are not particularly limited, and include an octyl group, an octadecyl group, and a functional group obtained by modifying these ends, and an octadecyl group is preferably used. In addition, since the holding force depends on the chain length of the hydrocarbon group, those skilled in the art can adjust the holding force in the stationary phase by adjusting the number of carbon atoms in the chain length of the hydrocarbon group. Moreover, the polarity of a stationary phase can also be adjusted using a phenyl group, a diol group, a nitrile group, an amino group, etc. as a functional group.

  Examples of the substituted or unsubstituted hydrocarbon group having a chain length of 8 or less carbon atoms include a trimethyl group, a butyl group, a phenyl group, a cyanopropyl group, and an aminopropyl group.

  Suitable as the packing material for liquid chromatography of the present invention is, for example, a packing material for liquid chromatography in which a commercially available octyl group is chemically bonded to silica gel [Acclaim (R) PepMap100 C8], and an octadecyl group on silica gel. Examples include chemically bonded packing materials for liquid chromatography [Acclaim (R) PepMap300 C18] and [Acclaim (R) PepMap100 C18]. These fillers are suitable in terms of stable quality and easy availability, but are not limited thereto.

  As a solvent to be brought into contact with the packing material for liquid chromatography, a salt is contained in a mixed solvent system such as water and an organic solvent such as methanol, ethanol, propanol, isopropanol, acetonitrile, ethyl acetate, dioxane, tetrahydrofuran (THF) or DMSO. Use things. In the case of the present invention, for example, in the examples described later, a solvent in which formic acid is mixed in acetonitrile is brought into contact with the packing material for liquid chromatography, but is not limited thereto. In addition, a concentration gradient (gradient) is given to the solvent, and molecules with high polarity are eluted with a solvent with low acetonitrile concentration, for example, and the molecules with low polarity are eluted successively by increasing the acetonitrile concentration. Can be made. Moreover, those skilled in the art can also set suitably about the elution time at the time of elution.

  In addition, if necessary, using a salt such as ammonium sulfate, sodium citrate, potassium citrate, potassium phosphate, sodium phosphate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium chloride, potassium chloride, etc. The pH of the solvent can be adjusted, but is not limited thereto.

  The ionization of the solute can be controlled by adjusting the pH of the solvent. However, in the present invention, the positive ion mode for detecting a cation is selected as the ion mode. Is preferable, and 2-3 is more preferable. Moreover, although stable results can be obtained by keeping the temperature constant, the temperature in this embodiment is preferably 0 to 40 ° C, more preferably 10 to 30 ° C.

  Mass spectrometry is a method of measuring an ionized analyte in the gas phase. The analyzer is roughly classified into an ion source and a mass spectrometer that measures a mass-to-charge ratio (m / z) of the ionized analyte. And an ion detector that records the number of ions at each m / z value. The most commonly used techniques for ionizing samples such as proteins and peptides include, for example, electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and matrix-assisted laser desorption / ionization (MALDI). Is mentioned. ESI is a method in which a solution-like sample is introduced into a capillary and sprayed or ionized by applying a high voltage, and can be analyzed in combination with a separation means such as chromatography or electrophoresis. APCI is a method for forcibly evaporating a sample solution by heating at a high temperature of 400 to 500 ° C., and then generating ions using the discharge of a corona needle. MALDI is a method in which a sample obtained by mixing a sample in a matrix (such as an aromatic organic compound) is sublimated and ionized by laser pulses. In the case of the present invention, since the protein to be the antigen is the subject of analysis, it is preferable that the applicable molecular weight range is large. Therefore, ESI or MALDI is preferably used for ionization of the sample of the present invention, but ESI is more preferable because expensive ions are easily generated.

  The sample ionized by the ion source is applied and pulled into the mass spectrometer at a high voltage. Such a voltage can be appropriately set by those skilled in the art as long as the sample can be analyzed, but if the voltage is too low, the ionized sample is not sufficiently transferred to the mass analyzer, and if the voltage is too high, Fragmentation occurs in the ionized sample. In carrying out the present invention, for example, a voltage of 1.0 kV to 2.5 kV, preferably 1.2 kV to 2.0 kV can be applied.

  The mass analyzer separates ions within a selected mass to charge ratio (m / z). It plays an important role in the sensitivity and resolution of analytical data, the accuracy of mass and the abundance of information obtained from mass spectral data. Ion separation methods can be classified into six basic types: magnetic field type, electric field type, ion trap type, time-of-flight (TOF) type, quadrupole type, and Fourier transform cyclotron. It is a type. Each of these has advantages and disadvantages, and can be used alone or connected to each other.

  A tandem mass (MS / MS) analysis method is used as a mass analysis method for fragmenting a sample in the middle of mass analysis and analyzing fragmented ions. Any of the mass spectrometry methods described above may be used to carry out the method of the present invention, and those skilled in the art can implement the method of the present invention by substantially the same or similar methods. In the present invention, tandem mass spectrometry is preferably used. For example, ion trap tandem mass spectrometry is used in the examples described later.

  A spectrum obtained by mass spectrometry (for example, an MS / MS spectrum obtained by tandem mass spectrometry) can be analyzed by a known method, but an antigen can also be determined by comparison with a public protein / peptide database. Such databases include, for example, the protein database of the International Protein Index provided by The Europe Bioinformatics Institute, the protein database published in the National Center Biotechnology Information (NCBI), and the protein database published in Swiss-Prot. Etc.

  The search method of the present invention uses the above-described method to identify an antigen that specifically forms an immune complex in a diseased patient as compared with a healthy person, whereby the immune complex containing the antigen is identified as a disease-specific biomolecule. Can be selected as a marker. Here, “specific” means that the frequency of detection of an antigen forming an immune complex is higher in a diseased patient than in a healthy subject. For example, as shown in the examples described below, thrombospondin-1 and platelet factor 4 are detected more frequently in patients with rheumatoid arthritis than in healthy subjects (based on the criteria of the American College of Rheumatology (ACR) in the examples below). In patients with rheumatoid arthritis, thrombospondin-1 is detected at a frequency of 17/21 and platelet factor 4 is detected at a frequency of 10/21, whereas none is detected in healthy subjects. Therefore, it can be judged that an immune complex containing thrombospondin-1 and an immune complex containing platelet factor 4 are disease markers for rheumatoid arthritis. In this case, “high detection frequency” means that the detection frequency of a specific antigen in a disease patient is 45% or more, preferably 80% or more, and most preferably 100% with respect to the total number of disease patients from which the sample is derived. And 5% or less, preferably 2% or less, and most preferably 0% of the healthy individuals from which the sample was derived. . Further, even when the specificity of the antigen cannot be confirmed between the healthy subject and the diseased patient, the antigen whose detection amount varies in the diseased patient compared to the healthy subject is a disease-specific marker. Judgment can be made. The comparison of the detection amount level is preferably performed based on the presence or absence of a significant difference.

In addition, the search method of the present invention can be provided as a method for examining whether or not a specific disease is affected or predicting whether or not the disease will be affected in the future.
That is, the present invention provides a method for examining whether or not to be affected by a disease or predicting whether or not to be affected in the future, including the following steps.
(1) a step of recovering immune complexes from each sample derived from a subject and a disease patient,
(2) a step of decomposing each immune complex and separating a degradation product;
(3) a step of comprehensively identifying antigens contained in each sample by mass spectrometry of the separated degradation products;
(4) A step of examining whether or not the subject is affected by the disease or predicting whether or not the subject will be affected in the future by comparing the identified antigens between the subject and the diseased patient.

  The subject to which the test or prediction method of the present invention can be applied is not particularly limited, and examples thereof include subjects who are likely to suffer from or suspected of suffering from the disease. Is mentioned. Preferably it is a human. The disease patient includes, for example, a person who is clinically determined to suffer from the disease. In addition, the sample used in the test or prediction method of the present invention, the method for collecting immune complexes, the separation and purification, the mass spectrometry method and the like are as described above.

  The test or prediction method of the present invention examines whether or not the subject suffers from the disease by comparing the antigens identified between the subject and the disease patient by the above-described method, or in the future. Whether or not it can be predicted. As described above, as shown in the Examples described later, the detection frequency of thrombospondin-1 and platelet factor 4 forming immune complexes is higher in patients with rheumatoid arthritis than in healthy individuals. Thus, for example, if thrombospondin-1 and / or platelet factor 4 forming an immune complex is detected in a subject, the subject may suffer from rheumatoid arthritis or in the future. It can be judged that there is. In addition, the specificity of the antigen cannot be confirmed between the healthy subject and the disease patient, but in the case of a disease having an antigen whose detection amount varies in the disease patient compared to the healthy subject as a biomarker, the subject and the disease patient By comparing the detected amounts of the antigens identified between each other, it is possible to test whether or not the subject suffers from the disease or to predict whether or not the subject will suffer from the future.

  Alternatively, an immune complex is obtained from an individual confirmed to have no specific disease (negative control) and an individual clinically determined to have the specific disease (positive control). It can be collected, analyzed and tested or predicted compared to that collected from the subject. Alternatively, a correlation diagram between the antigen level for a specific disease and the rate at which it is clinically determined to be affected by the disease is prepared in advance, and the detection level of the immune complex recovered from the subject is correlated. May be compared. The comparison of detection levels is preferably performed based on the presence or absence of a significant difference.

  The present inventors tried to identify a novel rheumatoid arthritis marker from a rheumatoid arthritis patient using the above-described disease marker search method. In patients with rheumatoid arthritis who met the criteria of the American College of Rheumatology (ACR) in Example 1 described later, thrombospondin-1 was detected at a frequency of 17/21 and platelet factor 4 was detected at a frequency of 10/21. On the other hand, none is detected in healthy persons. Moreover, in the early rheumatic patients of Example 2 described later, thrombospondin-1 was detected at a frequency of 9/16 from patients who were negative for rheumatoid factors and anti-CCP antibodies, which are known rheumatic markers. As a result, the present inventors have found that an immune complex containing thrombospondin-1 and an immune complex containing platelet factor 4 are reliable disease markers for rheumatoid arthritis. Therefore, the present invention also provides a method for examining rheumatoid arthritis.

  The present invention relates to whether or not the subject suffers from rheumatoid arthritis, comprising detecting the presence or absence of thrombospondin-1 and / or platelet factor 4 in an immune complex recovered from a sample derived from the subject. It relates to the inspection method. The human nucleotide sequence (amino acid sequence) of thrombospondin-1 is Genbank as accession number NM_003246.2 (NP_003237.2), and the human nucleotide sequence of platelet factor 4 (amino acid sequence) is accession number NM_002619.2. It is registered as (NP_002616.1).

  The subject to which the test method of the present invention can be applied is not particularly limited. Individuals such as humans, monkeys, cows, horses, pigs, mice, rats, guinea pigs, hamsters, dogs, cats, rabbits, sheep, goats and the like can be mentioned. Preferably, it is a human.

  The sample used in the test method of the present invention is not particularly limited as long as it is collected from a subject and contains an immune complex composed of an antigen to be detected and an antibody that binds to the antigen. Examples of such samples include whole blood, plasma, serum, saliva, lymph, cerebrospinal fluid, body fluid such as joint fluid, or fractions thereof, mucous membrane, tissue specimen obtained by biopsy, and the like. Blood, plasma, serum, saliva, lymph, etc. are particularly preferred because they can be collected quickly and easily and have less invasion to the subject. However, since the disease to be examined is rheumatoid arthritis, A tissue sample such as a synovium can also be preferably used.

  As described above, the immune complex in the sample can be separated and purified using a method known per se. That is, immunoglobulin separation and purification methods [Example: salting out method, alcohol precipitation method, isoelectric precipitation method, electrophoresis method, adsorption / desorption method by ion exchanger (eg DEAE), ultracentrifugation method, gel filtration method, A specific purification method in which only an immune complex and an antibody are collected by an active adsorbent such as protein A or protein G, and the binding to the active adsorbent is dissociated to obtain an immune complex can be separated and purified. Preferably, as described in the examples below, only immune complexes are collected directly from blood or serum using a carrier (sepharose column, magnetic beads, etc.) bound with an active adsorbent such as protein A or protein G. Can do. In addition, even when the sample is a joint or synovial tissue, a protein fraction is prepared based on a known method, and the immune complex contained in the fraction is separated and purified according to the above method. Can do.

  Detection of the presence or absence of thrombospondin-1 and / or platelet factor 4 in the immune complex recovered from the subject-derived sample is specific to the antibody specifically recognizing thrombospondin-1 and platelet factor 4 respectively. Can be performed by immunoassay (eg, ELISA, FIA, RIA, Western blot, etc.), and thrombospondin-1 and / or platelet factor 4 can be detected by MALDI. It can also be performed using mass spectrometry such as -TOFMS.

When the test subject is a human, the antibody specifically recognizing thrombospondin-1 and the antibody specifically recognizing platelet factor 4 include thrombospondin-1 and platelet factor 4 polypeptide and their antigenicity. A partial peptide having a portion corresponding to the whole or an epitope of the peptide sequence shown in SEQ ID NO: 2 for thrombospondin-1 and SEQ ID NO: 4 for platelet factor 4 as an immunogen. It can be manufactured by a general manufacturing method. In the present specification, the antibody includes a polyclonal antibody, a natural antibody such as a monoclonal antibody (mAb), a chimeric antibody that can be produced using a gene recombination technique, a humanized antibody or a single chain antibody, and binding properties thereof. Fragments are included, but are not limited to these. Preferably, the antibody is a polyclonal antibody, a monoclonal antibody or a binding fragment thereof. The binding fragment means a partial region of the aforementioned antibody having specific binding activity, and specifically includes, for example, F (ab ′) ₂ , Fab ′, Fab, Fv, sFv, dsFv, sdAb and the like. (Exp. Opin. Ther. Patents, Vol. 6, No. 5, p. 441-456, 1996). The class of the antibody is not particularly limited, and includes antibodies having any isotype such as IgG, IgM, IgA, IgD, or IgE. IgG or IgM is preferable, and IgG is more preferable in consideration of ease of purification.

  In applying each immunological measurement method to the test method of the present invention, special conditions, operations and the like are not required to be set. A measurement system for thrombospondin-1 and platelet factor 4 may be constructed by adding ordinary technical considerations to those skilled in the art to the usual conditions and procedures in each method. For details of these general technical means, it is possible to refer to reviews, books and the like. For example, Hiroshi Irie “Radioimmunoassay” (Kodansha, published in 1974), Hiroshi Irie “Continue Radioimmunoassay” (published in Kodansha, 1974), “Enzyme Immunoassay” edited by Eiji Ishikawa et al. 53)), edited by Eiji Ishikawa et al. “Enzyme Immunoassay” (2nd edition) (Medical Shoin, published in 1982), edited by Eiji Ishikawa et al. 62)), “Methods in ENZYMOLOGY” Vol. 70 (Immunochemical Technologies (Part A)), ibid., Vol. 73 (Immunochemical Technologies (Part B)), ibid., Vol. 74 (Immunochemical Technologies (Part C)), ibid., Vol. 84 (Immunochemical Technologies (Part D: Selected Immunoassays)), ibid., Vol. 92 (Immunochemical Technologies (Part E: Monoclonal Antibodies and General Immunoassay Methods)), ibid., Vol. 121 (Immunochemical Technologies (Part I: Hybridoma Technologies and Monoclonal Antibodies)) (published by Academic Press).

  According to the test method of the present invention, the subject suffers from rheumatoid arthritis by detecting the presence or absence of thrombospondin-1 and / or platelet factor 4 in the immune complex recovered from the sample derived from the subject by the above method. Can be inspected. For example, if a significant difference in the detected amount can be found in the subject compared to the detected amount of thrombospondin-1 and / or platelet factor 4 in the immune complex recovered from the sample derived from a healthy subject, Can be determined to have rheumatoid arthritis. Rheumatoid arthritis that can be judged here is rheumatoid arthritis that can meet the criteria of the American College of Rheumatology (ACR), or rheumatoid arthritis that does not meet the criteria of the American College of Rheumatology (ACR), but that a rheumatologist can judge to require treatment Is also included. Alternatively, it may include rheumatoid arthritis that may be determined by a rheumatologist to start DMARDs (Disease Modifying Antitherapeutic Drugs).

  For example, an immune complex is obtained from a sample derived from an individual confirmed to have no rheumatoid arthritis (negative control) and an individual clinically determined to have rheumatoid arthritis (positive control). The level of detection of thrombospondin-1 and / or platelet factor 4 in the immune complex collected and recovered from the sample from the subject is compared to that of the positive and negative controls.

  If the detection level of thrombospondin-1 and / or platelet factor 4 to be measured is relatively higher than the comparison result of the detection level of thrombospondin-1 and / or platelet factor 4, It can be determined that the probability of being rheumatic is relatively high. Conversely, when the detection level of thrombospondin-1 and / or platelet factor 4 to be measured is relatively low, it can be determined that the probability of rheumatoid arthritis is relatively low.

  By applying the above-mentioned rheumatoid arthritis test method to patients who have been treated for rheumatoid arthritis or experimental animals (eg, monkeys, dogs, rats, mice, etc.) that have been administered a therapeutic drug for rheumatoid arthritis. Can be evaluated. That is, thrombospondin-1 and / or platelet factor 4 in the immune complex collected from a sample collected over time from a rheumatoid arthritis patient who is being treated or treated is measured and compared, and compared with that before treatment. Thus, it can be determined that a therapeutic effect is observed if the level of immune complex containing thrombospondin-1 and / or platelet factor 4 after treatment is suppressed.

  The present invention also provides a test kit for testing rheumatoid arthritis that can be preferably used in the test method of the present invention. The test kit contains an antibody that specifically recognizes thrombospondin-1 and / or an antibody that specifically recognizes platelet factor 4. When the agent to be tested contains two or more of the above-mentioned antibodies, each antibody can specifically recognize different epitopes of thrombospondin-1 and platelet factor 4 with respect to each other. In order to exclude free antigens that do not form immune complexes from detection targets, antibodies that specifically recognize thrombospondin-1 and / or platelet factor 4 are collected as immune complexes and then detected. It is preferable to carry out. Examples of the antibody specifically recognizing thrombospondin-1 and the antibody specifically recognizing platelet factor 4 include the antibodies described above in the test method of the present invention.

  In addition to an antibody specifically recognizing thrombospondin-1 and / or an antibody capable of specifically detecting platelet factor 4 as a reagent constituting the test kit of the present invention, thrombospondin-1 and / or It may further contain other substances necessary for the reaction for detecting platelet factor 4 that do not adversely affect the reaction when stored in the coexisting state. Alternatively, the reagent may be provided with a separate reagent containing thrombospondin-1 and / or other substances necessary in the reaction to detect platelet factor 4. For example, when the reaction for detecting thrombospondin-1 and / or platelet factor 4 is an antigen-antibody reaction, examples of the other substance include a reaction buffer solution, a competent antibody, a labeled secondary antibody ( For example, when the primary antibody is rabbit anti-human thrombospondin-1 and / or rabbit anti-human platelet factor 4 antibody, mouse anti-rabbit IgG labeled with peroxidase, alkaline phosphatase, etc.), blocking solution, etc. may be mentioned. In addition, the test kit may contain protein A or protein G for collecting immune complexes as a reagent, and these may be immobilized on a carrier (sepharose column, magnetic beads, etc.). Good.

  Examples of the present invention are shown below.

Example 1
Sample Collection Serum samples were collected from 21 patients with rheumatoid arthritis (AR) at Sasebo Central Hospital, which meet the criteria of the American College of Rheumatology (ACR). Serum was also collected from healthy donors (13 persons) and used as a control. All experiments were approved by the Organizational Ethics Committee of Sasebo Central Hospital and Graduate School of Biomedical Sciences, Nagasaki University, and were carried out in accordance with the Declaration of Helsinki. Informed consent was obtained from patients and healthy donors.

Collection and digestion of immune complex (IC) The immune complex was purified using magnetic beads (PureProteome, Millipore) on which protein G was immobilized. 40 μl of the beads were washed with 500 μl of phosphate buffered saline (PBS, Wako Chemicals) and incubated with 100 μl of serum diluted with PBS (serum: PBS = 1: 9, v / v) for 30 minutes with gentle agitation. The beads to which the immune complex was bound were collected with a magnet and washed three times with 500 μl of PBS. The beads were resuspended in 100 μl of 10 mM dithiothreitol and incubated at 56 ° C. for 45 minutes, after which 100 μl of 55 mM iodoacetamide was added and incubated in the dark at room temperature for 30 minutes. Subsequently, trypsin solution (25 mM ammonium bicarbonate) was added at a final concentration of 0.5 mg / ml and the mixture was incubated overnight at 37 ° C. TFA (10%, v / v) was added to stop the digestion, and then the supernatant containing the antigen and antibody-derived peptide digest was collected using a magnet. Finally, the mixture was concentrated to about 80 μl under reduced pressure.

Protein identification by nano-HPLC-MS / MS LC pump with a LC flow splitter (Accurate, Dionex, Sunnyvale, CA, USA) (Surveyor MS pump, Thermo Fiscientific, Waltham, MA, USA, USA, USA) A peptide mixture (1 μl) was tested on LC-electrospray ionization-tandem MS (LCQ Fleet, Thermo Fisher Scientific, Waltham, Mass., USA) equipped with a custom nano LC system consisting of Analysics, Zwingen, Switzerland. Samples were injected into a nano-precolumn (300 μm id × 5.0 mm, LC-18, Chemicals and Evaluation and Research, Tokyo, Japan) in the injection loop, using 1% TFA in 2% acetonitrile. And washed. Peptides were separated on a nano HPLC column (75 μm, id, Acclaim PepMap100C18, 3 μm, Dionex, Sunnyvale, CA, USA) and ion sprayed onto the MS at a spray voltage of 1.2 to 2.0 kV. Separation was carried out by setting the mobile phase gradient conditions as follows: the mobile phase B content in the mobile phase was increased from 5 to 23% in 10 minutes (0.1% formic acid in 90% acetonitrile [Kanto Kagaku , Tokyo, Japan]) (mobile phase A: 0.1% formic acid); mobile phase B content increased from 24 to 34% in 70 minutes; mobile phase B content increased from 34 to 50% in 16 minutes; mobile phase Increase B content from 50 to 100% in 0.1 minutes; maintain mobile phase B content of 100% for 9 minutes (total separation time: 115 minutes). Processed from full sample analysis to three tandem MS scans of the three most intense precursor masses (determined in real time by Xcaliber® software [Thermo Fisher Scientific, Waltham, MA, USA]) The mass spectrometer was set up to optimize the duty cycle length with data quality. The collision energy was 35%. The entire spectrum was measured with a total mass / charge ratio range of 400-1500. The transfer capillary temperature was set to 200 ° C. MS / MS data was extracted using Bioworks V3.3 (Thermo Fisher Scientific, Waltham, MA, USA). From the non-redundant protein database published by the International Protein Index version 3.67 (download date; December 10, 2009) provided by the European Institute of Bioinformatics (EBI, Cambridge, UK) The spectra were searched with the following search parameters: mass, monoisotopic precursors and fragments; enzyme, trypsin (KR); enzyme limit, total enzymatic cleavage allowing up to 2 mis-cleavages; 0 atomic mass units; fragment ion tolerance, 1.0 atomic mass units; number of score results, 250; measured ions and ion series, B and Y ions; static correction, C (carboxymethylation); specific modification, M (oxidation); N and Q Amide decomposition). Empirically determined protein false discovery rate (FDR) 0, ie, filter criteria (single, double, triple charge peptides with associated factors [XCorr] and protein probabilities [P]) so that random hits are zero. It was adjusted. The FDR was calculated by dividing the number of unique peptides in the reverse database by the number of key unique peptides in the forward database. Proteins were identified for peptides longer than 6 amino acids. If an amino acid sequence having a peptide in which a plurality of proteins were found was shared, the protein with the lowest probability was judged to be the best match, and keratin and trypsin were excluded as potential matches.

Results Proteins identified by extensive proteome analysis for rheumatoid arthritis patients and healthy donors are abbreviated in Table 1-1 and Table 1-2.

Peptides derived from IgG, trypsin and keratin were excluded from the analysis. Peptide assignment, corrected and determined based on FDR = 0, was confirmed by manual examination of the fragmentation spectrum. 36 known human proteins such as apolipoprotein, complement, coagulation protein and adhesion protein were identified from two or more independent samples. Deposits of immune complexes containing several proteins identified in this analysis (clusterin, apolipoprotein E, fibronectin and vitronectin) have been found in arthritic joints (PA Monach et al., Proceeding of The National Academy of Science, 106, 15867-15872, 2009), the inventors' results suggest that circulating immune complexes accumulate in joint lesions.
Of the identified antigenic proteins, thrombospondin-1 (TSP-1) was detected most frequently in patients with rheumatoid arthritis and was very specific. TSP-1 is produced in multiple cells including neutrophils, endothelial cells and fibroblasts. TSP-1 has been thought to be involved in the physiological functions of rheumatoid arthritis patients and is present in the synovial tissue of rheumatoid arthritis patients (I. Gotis-Graham et al., Arthritis & Rheumatism, 40, 1780-). 1787, 1997), and its plasma levels have been reported to be high (M. Rico et al., Translation Research, 152, 95-98, 2008). In recent studies, the TSP-1 / transforming growth factor / connective tissue growth factor (CTGF) pathway plays a role in the progression of rheumatoid arthritis, in which TSP-1 expression is triggered. Up-regulation of downstream CTGF has been suggested to be associated with angiogenesis and erosive arthritic lesions.
The most famous diagnostic marker is rheumatoid factor (RF), but rheumatoid arthritis has low specificity (80.8%) (L. De Ryche et al., Ann. Rheum. Dis., 63, 1587-1593). 2004), also observed in other diseases (Y. Renaudineau et al., Autoimmunity, 38, 11-16, 2005; T. Dorner et al., Current Opinion in Rumology, 16, 246-253, 2004). On the other hand, protein / peptide antibodies (anti-CCP) containing citrulline have a much higher specificity for rheumatoid arthritis than rheumatoid factor. The majority of rheumatoid arthritis patients have one or both of these antibodies, but 20% do not. TSP-1 immune complexes are highly specific for rheumatoid arthritis (100%), have high detection sensitivity (81.0%) and are promising disease biomarkers.
To find non-specific binding of the two proteins, fibronectin and TSP-1, to the beads with protein G, a standard protein solution at a concentration of 10 times higher than the standard value in blood was used instead of the serum sample. The absence of fibronectin or TSP-1 suggests that their detection in the plasma of rheumatoid arthritis patients or controls is not due to non-specific binding to the beads.
Anti-CCP antibodies are now widely recognized as specific markers of rheumatoid arthritis, and citrullinated fibrinogen is present in the immune complex of sera from rheumatoid arthritis patients (X. Zhao et al., Arthritis Res. Ther. , 10, R94, 2008), the inventors also searched MS spectra for peptides in which citrulline was replaced with arginine. Citrullinated thymidine kinase 2, GRAM domain-containing protein 2 and prothrombin were found in 2 of 21 patients with rheumatoid arthritis, while only 4 healthy individuals found citrullinated prothrombin. It was done.
On the other hand, the rheumatoid factor that binds to the Fc domain of IgG and has been used in diagnostic tests for rheumatoid arthritis was detected by a proteomic approach in seven rheumatoid arthritis patients. For the anti-CCP antibody, the amino acid sequence was not entered in the database and was not detected in this example.
Although the detection sensitivity is lower than that of TSP-1 (52.4%), platelet factor 4 (PF4) was also specifically detected in patients with rheumatoid arthritis (Tables 2-1 and 2-2). Several studies have reported that platelet factor 4 in synovial fluid is increased, suggesting that platelet factor 4 may be involved in chronic rheumatoid arthritis.

Example 2
Sample collection Next, serum samples were collected from 26 patients with early rheumatoid arthritis (AR) at Nagasaki University Hospital (a group that did not meet the ACR criteria, but was recognized as needing treatment by a rheumatologist and was not treated). . Of the 26 patients, 16 were rheumatoid factor (RF) positive seropositive populations. Furthermore, 11 out of 16 seropositive populations were also positive for ACCP antibodies. The remaining 10 patients were negative for RF and ACCP antibodies (serum negative population). Serum was also collected from healthy donors (11 persons) and used as a control.

  The immune complex (IC) was collected and digested, and protein identification by nano-HPLC-MS / MS was performed in the same manner as in Example 1. The identified proteins are abbreviated in Table 3-1 and Table 3-2.

The detection frequency of TSP-1 was detected with a frequency of 56.3% in seropositive-positive early rheumatic patients and 50.0% in seronegative early rheumatic patients, and as a whole about 54% as early-stage rheumatic patients. Detection frequency. TSP-1 can be expected as a promising disease biomarker even in patients with early rheumatoid arthritis because detection frequency is 50% even in patients who are negative for RF and anti-CCP antibodies, which are known rheumatoid biomarkers.
For PF4, 18.8% of seropositive positive rheumatic patients (3 positive patients in which PF4 and PF4 variant were detected. Table 3-2 includes 1 patient who was both positive) However, it was not confirmed in patients with seronegative early rheumatism.

According to the present invention, it is possible to comprehensively search for disease-specific markers by collecting immune complexes from patients clinically determined to be suffering from a specific disease and analyzing the antigen. Is possible. In addition, for a subject who has not been clinically judged, the immune complex is collected and compared with the analyzed antigen and a known disease marker to examine which disease the subject is suffering from or You can predict whether you will be affected in the future. Furthermore, in the present invention, a test for rheumatoid arthritis can be performed quickly and easily by examining the detection level of an immune complex of a novel biomarker found in rheumatoid arthritis.
This application is based on Japanese Patent Application No. 2010-231935 (filing date: October 14, 2010) filed in Japan, the contents of which are incorporated in full herein.

Claims (15)

A comprehensive search method for disease biomarkers, comprising the following steps.
(1) a step of recovering an immune complex from each blood or serum derived from a disease patient and a healthy person,
(2) a step of decomposing each immune complex and separating a degradation product;
(3) a step of comprehensively identifying antigens contained in each sample by mass spectrometry of the separated degradation products;
(4) A step of selecting, as the disease biomarker, an antigen that is specifically identified in a disease patient as compared with a healthy subject   The method according to claim 1, wherein the disease is selected from infectious diseases, autoimmune diseases, malignant tumors, arteriosclerosis, diabetes.   The method according to claim 1, wherein in step (1), the immune complex is recovered using protein G or protein A.   The method according to claim 1, wherein in step (2), the degradation products are separated by liquid chromatography.   The method according to claim 1, wherein in step (3), mass spectrometry is performed by tandem mass spectrometry. A method for examining whether or not to be affected by a disease or predicting whether or not to be affected in the future, comprising the following steps.
(1) recovering immune complexes from each blood or serum derived from a subject and a disease patient,
(2) a step of decomposing each immune complex and separating a degradation product;
(3) a step of comprehensively identifying and quantifying antigens contained in each sample by mass spectrometry of the separated degradation products;
(4) A step of examining whether or not the subject is affected by the disease or predicting whether or not the subject will be affected in the future by comparing the identified antigens between the subject and the diseased patient.   7. The method according to claim 6, wherein the disease is selected from infectious diseases, autoimmune diseases, malignant tumors, arteriosclerosis, diabetes.   The method according to claim 6, wherein in the step (1), the immune complex is recovered using protein G or protein A.   The method according to claim 6, wherein in step (2), the degradation product is separated by liquid chromatography.   The method according to claim 6, wherein in step (3), mass spectrometry is performed by tandem mass spectrometry.   A method for examining rheumatoid arthritis, which detects the presence or absence of thrombospondin-1 and / or platelet factor 4 in an immune complex recovered from a sample derived from a subject.   The method according to claim 11, wherein the immune complex is recovered using protein G or protein A. Rheumatoid arthritis patient comprising detecting the presence or absence of thrombospondin-1 and / or platelet factor 4 in an immune complex collected from a sample collected over time from a patient undergoing treatment or undergoing treatment Measurement method for evaluating the therapeutic effect on a patient.   14. The method according to claim 13, wherein the immune complex is recovered using protein G or protein A.   A test kit for rheumatoid arthritis, comprising an antibody specifically recognizing thrombospondin-1 and / or an antibody specifically recognizing platelet factor 4.