JP5252339B2 - Method for measuring PAD4 and anti-PAD4 antibody and method for detecting rheumatoid arthritis - Google Patents

Description

  The present invention relates to a method for measuring PAD4 using an anti-PAD4 monoclonal antibody, a method for measuring anti-PAD4 antibody by a sandwich method using the monoclonal antibody, and a method for detecting rheumatoid arthritis.

  The serological diagnostic method for rheumatoid arthritis was the most reliable method for measuring rheumatoid factor, but in recent years, the measurement of autoantibodies against cyclized citrullinated peptides (anti-CCP antibody) exceeds that of rheumatoid factor. It is said that it is an effective method (nonpatent literature 1). For rheumatoid arthritis, useful treatments have been developed and it is important to diagnose them at an earlier stage. Although measurement of CCP antibodies is the measurement method that can be diagnosed earliest at present, it is not yet a sufficient method from the viewpoint of early diagnosis.

  Protein citrullination is a reaction in which the constituent amino acid arginine is converted to citrulline by an enzyme called peptidylarginine deiminase (PAD). There are 5 types of isoforms in PAD (type 1, type 2, type 3, type 4/5 type and type 6), and the tissue distribution and substrate specificity in the living body are different. CCP autoantibodies detected in patients with rheumatoid arthritis are thought to be produced in the patient's body against citrullinated proteins (responsible proteins are not known), but protein citrullination involved in rheumatoid arthritis is type 4 PAD (PAD4 ). Considering earlier diagnosis, it is considered that blood PAD4 or anti-PAD4 autoantibody, which is likely to appear in blood earlier than CCP autoantibodies, enables early diagnosis of rheumatoid arthritis.

  However, since the amount of PAD4 protein present in the blood is small, it cannot be purified and it is difficult to establish an anti-PAD4 antibody and a method for measuring blood PAD4 that can be preferably used for rheumatoid arthritis diagnosis. is there.

  As a method for diagnosing rheumatoid arthritis by measuring anti-PAD4 autoantibodies, the method described in Non-Patent Document 2 is known. However, in the method of Non-Patent Document 2, immunoassay is performed by directly binding PAD4 antigen to a solid phase. In such a method, there is a possibility that the three-dimensional structure of the immobilized PAD4 may change, and a non-specific reaction is likely to occur unless the purity of the antigen is increased, so that highly accurate measurement is difficult.

Critical Reviews in Clinical Laboratory Science, 44 (4): 339-363 (2007) Scand J Rheumatol 2005; 34: 212-215

  An object of the present invention is to provide means useful for detecting rheumatoid arthritis with high accuracy and capable of accurately detecting even PAD4 in blood.

  As a result of earnest research, the inventors of the present application established a monoclonal antibody capable of detecting PAD4, and when immunoassay of PAD4 in a test sample using the monoclonal antibody was performed, the calcium ion chelating agent was added to the sample. It was found that PAD4 can be accurately measured even in samples such as serum containing a lot of calcium ions by removing calcium ions. Also, when measuring anti-PAD4 autoantibodies, the monoclonal antibodies are bound to a solid phase and immunoassay is performed by a sandwich method to measure the autoantibodies with higher accuracy than directly immobilizing the antigen PAD4 itself. I found that I can do it. And it discovered that rheumatoid arthritis could be detected with high precision by combining these measuring methods as appropriate, and completed the present invention.

That is, the present invention performs immunoassay on a test sample brought into contact with a calcium ion chelating agent using a monoclonal antibody or antigen-binding fragment thereof that reacts with PAD4 in an antigen-antibody, and is contained in the test sample. Provided is a method for measuring PAD4 in a test sample, comprising measuring the obtained PAD4. The present invention also provides rheumatoid arthritis using as an index the amount of PAD4 present in the test sample, which is measured by performing the PAD4 measurement method of the present invention on a test sample obtained from a living body. Provide a detection method.

  The present invention provides a measurement method capable of measuring PAD4 and anti-PAD4 autoantibodies with high accuracy. According to the PAD4 measurement method of the present invention, PAD4 can be measured with high accuracy, and in particular, PAD4 in serum and plasma samples can also be measured with high accuracy. Further, according to the anti-PAD4 autoantibody measurement method of the present invention, since the PAD4 antigen is used by binding to the solid phase via a specific antibody, the PAD4 antigen on the solid phase is used rather than the method of directly immobilizing the PAD4 antigen. Thus, the three-dimensional structure can be preferably maintained, and further, adverse effects due to contaminants mixed in the PAD4 antigen can be avoided, so that anti-PAD4 autoantibodies can be measured with high accuracy. Furthermore, by appropriately combining these measurement methods, it has become possible to diagnose rheumatoid arthritis with high accuracy. Conventionally, the method of measuring autoantibodies against cyclized citrullinated peptides (anti-CCP antibodies) is said to be the earliest method for diagnosing rheumatoid arthritis, but is involved in the production of cyclized citrullinated peptides. Since the enzyme is PAD4, it is considered that rheumatoid arthritis can be diagnosed earlier than the diagnosis by measurement of anti-CCP antibody by measuring PAD4 or anti-PAD4 autoantibodies. That is, according to the detection method of the present invention in which measurement of PAD4 and anti-PAD4 autoantibodies is appropriately combined, rheumatoid arthritis can be detected earlier than the conventional method.

  The method for measuring PAD4 of the present invention is performed by immunoassay using an anti-PAD4 monoclonal antibody or an antigen-binding fragment thereof that reacts with PAD4 as an antigen antibody. The anti-PAD4 monoclonal antibody has high specificity for PAD4 and does not substantially react with other proteins for antigen-antibody reaction. “Substantially no antigen-antibody reaction” means that no antigen-antibody reaction occurs at a detectable level, or even if an antigen-antibody reaction occurs, the degree of the reaction is clearly weaker than the antigen-antibody reaction with PAD4, The absence of antigen-antibody reaction with PAD4 means that it reacts only to a degree that is obvious to those skilled in the art.

In the PAD4 measurement method of the present invention, a test sample is brought into contact with a calcium ion chelating agent. For example, immunoassay can be performed after adding a chelating agent to the test sample. When immunoassay is performed using an antibody-immobilized solid phase, a test sample and a chelating agent may be added separately to the solid phase, but the chelating agent may be added to the test sample in advance. It is preferable to add and use. As described in the Examples below, when an immunoassay is performed without adding EDTA (ethylenediaminetetraacetic acid) to a serum or plasma sample, a normal measurement value cannot be obtained. The following can be considered as the cause. That is, in the screening process, the use of PAD4 protein in Ca ²⁺ absence recognizes the conformation of PAD4 protein Ca ²⁺ absence antibody is screened. On the other hand, PAD4 present in serum or plasma samples is in the presence of high concentrations of Ca ²⁺ . Therefore, it is considered that the anti-PAD4 monoclonal antibody prepared by the above-mentioned method cannot properly recognize the three-dimensional structure taken by PAD4 in the presence of a high concentration of Ca ²⁺ . Therefore, by adding a calcium chelating agent such as EDTA to a serum or plasma sample and removing Ca ²⁺ , PAD4 can be returned to the structure in the absence of Ca ²⁺ , thereby allowing the anti-PAD4 monoclonal antibody to It will be able to recognize properly. The chelating agent is not particularly limited as long as it can chelate calcium ions, and may be capable of chelating other metal ions. Specific examples include EDTA (ethylenediaminetetraacetic acid), EGTA, and the like. When the concentration of the chelating agent used is about 1 to 10 mM, preferably about 3 to 7 mM, PAD4 in the sample can be measured with preferable accuracy.

  The anti-PAD4 monoclonal antibody used in the method of the present invention can be prepared, for example, by a known hybridoma method using a PAD4 protein prepared by a known genetic engineering technique or synthesized by a known peptide synthesizer as an immunogen. it can. That is, a PAD4 recombinant protein is prepared, and this is used as an immunogen to immunize animals (excluding humans), and antibody-producing cells are obtained from the animals and fused with immortalized cells such as myeloma cells. An anti-PAD4 monoclonal antibody can be obtained by preparing a hybridoma that produces an antibody having a desired reactivity (specific reactivity to PAD4) from the hybridoma and recovering the antibody from the screened hybridoma it can.

  As a method for producing PAD4 by genetic engineering techniques, for example, cDNA of PAD4 gene is prepared by RT-PCR from total RNA extracted from tissues or cells expressing PAD4, and the cDNA is incorporated into an expression vector. And a method of introducing PAD4 into the host cell and expressing PAD4 in the host cell to obtain a PAD4 recombinant protein. The gene sequence and amino acid sequence of PAD4 are known as registered under accession number AB017919 in NCBI GenBank, and are described in SEQ ID NOS: 1 and 2, respectively. A PAD4 recombinant protein can be easily prepared based on these sequence information. In this field, PAD4 is sometimes called PAD5. RNA extraction, RT-PCR, cDNA integration into a vector, and introduction of a vector into a host cell can be performed by well-known methods. Further, vectors and host cells to be used are well known, and various types are commercially available.

  The prepared PAD4 protein may optionally be checked for enzyme activity prior to use as an immunogen. A PAD4 protein that can exhibit enzymatic activity can have a three-dimensional structure equivalent to that of a PAD4 protein produced in vivo, and thus is considered to be highly effective as an immunogen for producing an anti-PAD4 antibody. The enzyme activity of the prepared PAD4 recombinant protein is, for example, in the presence of about 10 mM to 30 mM calcium ion using benzoyl-L-arginine ethyl ester (BAEE) as a substrate, as detailed in the Examples below. It can be confirmed by performing an enzyme reaction at a temperature of about 37 ° C. to 55 ° C. for a predetermined time (for example, about 1 hour) and measuring the absorbance using a known colorimetric reagent. Although it is not particularly limited, it can be evaluated that it is PAD4 having enzyme activity if activity of about 0.01 U / ml or more can be confirmed.

  Methods for fusing antibody-producing cells and immortalized cells obtained from immunized animals, methods for screening hybridomas, and methods for recovering antibodies from hybridomas can be performed by conventional methods well known in the art.

It is well known in the art that an antibody fragment (referred to as “antigen-binding fragment” in the present invention) having binding ability to PAD4, which is a corresponding antigen, can be used for immunoassay as well as an anti-PAD4 monoclonal antibody. It is as follows. Therefore, not only the anti-PAD4 monoclonal antibody but also its antigen-binding fragment can be used in the PAD4 measurement method of the present invention. Examples of antigen-binding fragments include, for example, immunoglobulin Fab fragments and F (ab ′) ₂ fragments. As is well known, these fragments can be obtained by treating a monoclonal antibody with a proteolytic enzyme such as papain or pepsin. The antigen-binding fragment is not limited to the Fab fragment or the F (ab ′) ₂ fragment, and may be any fragment that maintains the binding property with the corresponding antigen. It may be prepared. In addition, for example, an antibody in which a single chain fragment of variable region (scFv) is expressed in E. coli can be used by genetic engineering techniques. The method for producing scFv is also well known. The hybridoma mRNA produced as described above is extracted, single-stranded cDNA is prepared, and PCR is performed using primers specific to immunoglobulin H chain and L chain. ScFv by amplifying globulin H chain gene and L chain gene, ligating them with a linker, adding an appropriate restriction enzyme site and introducing it into a plasmid vector, transforming E. coli, and recovering scFv from E. coli Can be produced. Such scFv is also included in the “antigen-binding fragment” referred to in the present invention.

  The immunoassay method itself is well known in this field, and any known immunoassay method can be adopted in the PAD4 measurement method of the present invention. That is, when classified based on the reaction format, there are a sandwich method, a competitive method, an agglutination method, and the like, and when classified based on a label, there are an enzyme immunoassay, a radioimmunoassay, a fluorescence immunoassay, etc. And can be employed in the measurement method of the present invention.

  In the PAD4 measurement method of the present invention, when PAD4 is immunoassay by the sandwich method, a monoclonal antibody is used as one of the anti-PAD4 antibody to be immobilized and the anti-PAD4 antibody used as a labeled antibody, and the other is used as a polyclonal antibody. Also good. However, from the viewpoint of improving measurement accuracy, it is preferable to use both antibodies as monoclonal antibodies. As is well known, the solid-phased antibody and the labeled antibody must recognize different epitopes from each other, which is particularly problematic when both are used as monoclonal antibodies. It can be easily examined by actually performing an immunoassay. If desired, the recognition site of the antibody may be identified to see if it recognizes a different epitope. Identification of the recognition site of the antibody can be performed by a conventional method well known in this field. Briefly, for example, the corresponding antigen PAD4 protein is partially digested with a proteolytic enzyme such as trypsin, and the digest is bound to the affinity column to which the antibody to be examined for binding is bound through the partially digested solution. Then, the bound digest is eluted and subjected to mass spectrometry in the usual manner, whereby the recognition site of the antibody can be identified.

  The test sample used in the PAD4 measurement method of the present invention is a biological component collected from a mammal such as a human, preferably serum or plasma.

  The present invention also provides a method for measuring anti-PAD4 autoantibodies in a test sample by sandwich immunoassay using anti-PAD4 monoclonal antibodies. As used herein, “autoantibody” refers to an antibody produced against PAD4 protein expressed in vivo, and includes not only antibodies induced against PAD4 in a healthy organism, but also rheumatoid arthritis, etc. An antibody induced against the highly expressed PAD4 protein in a living body in which the expression level of the PAD4 protein is increased due to a disease is also included. As described above, since the antigen-binding fragment of an anti-PAD4 monoclonal antibody can be used for immunoassay in the same manner as the antibody, the antigen-binding fragment should also be used in the method for measuring an anti-PAD4 autoantibody of the present invention. Can do. Hereinafter, in the description of the present invention, reference to “antigen-binding fragment” may be omitted, but it is not intended to exclude the antigen-binding fragment from the scope of the present invention, and is used in immunoassays. Monoclonal antibodies also include antigen-binding fragments thereof.

  The method for measuring PAD4 autoantibodies of the present invention is performed by immunoassay by a sandwich method using a solid phase on which PAD4 antigen is immobilized. Here, the PAD4 antigen used for capturing the PAD4 autoantibody is not directly immobilized on the solid phase, but is immobilized on the solid phase via an anti-PAD4 monoclonal antibody or an antigen-binding fragment thereof. That is, in this measurement method, a PAD4 antigen is bound to a solid-phased anti-PAD4 monoclonal antibody or the like to prepare a solid phase, and PAD4 autoantibody is measured by an immunoassay by a sandwich method using the solid phase.

  As used herein, “PAD4 antigen” refers to a PAD4 protein or fragment thereof that can bind to an anti-PAD4 autoantibody in a sample by an antigen-antibody reaction, or a polypeptide having a certain degree of homology thereto (hereinafter referred to as the polypeptide for convenience). (Referred to as "modified polypeptide"). As will be described later, even a fragment of PAD4 can be used as an antigen for immunoassay. In general, a protein antigen has almost the same antigenicity as the original protein even when a small number of amino acid residues in the amino acid sequence of the protein are substituted, deleted, inserted, or added. It is well known to those skilled in the art that The modified polypeptide has a homology of 80% or more, preferably 90% or more, more preferably 95% or more, and further preferably 98% or more with the amino acid sequence of the PAD4 protein (SEQ ID NO: 2) or a fragment thereof. Alternatively, the modified polypeptide is also preferably one in which one to several amino acids are substituted, deleted, inserted and / or added in the amino acid sequence of the PAD4 protein (SEQ ID NO: 2) or a fragment thereof. .

  The PAD4 antigen may be a commercially available PAD4 protein purified product, or may be a PAD4 recombinant protein produced by a known genetic engineering technique (described above). PAD4 produced by genetic engineering techniques usually contains some host cell components (for example, E. coli components), but humans have antibodies against E. coli components, so contamination of these E. coli components is not human. It becomes a cause which raises background when measuring a derived sample. As described above, by using a solid phase in which PAD4 antigen is indirectly immobilized via a monoclonal antibody, an increase in background due to contamination with E. coli components can be suppressed, and the measurement accuracy of PAD4 autoantibodies can be improved. In addition, if the PAD4 antigen is directly bound to the solid phase, the three-dimensional structure may change, which may make it difficult to measure autoantibodies that recognize the three-dimensional structure of PAD4. When immobilized on a solid phase, there is a high possibility that the three-dimensional structure of PAD4 can be maintained, so that the measurement accuracy of PAD4 autoantibodies can be increased. Actually, as described in Examples below, the measurement accuracy of PAD4 autoantibodies is improved by immobilizing PAD4 antigen on a solid phase via a monoclonal antibody, rather than directly immobilizing PAD4 antigen.

  The PAD4 antigen is not limited to one consisting of the full length of the PAD4 protein (SEQ ID NO: 2), and may be a fragment thereof. Since the PAD4 autoantibody to be measured is a collection of antibodies that recognize a plurality of sites on the PAD4 protein, that is, a polyclonal antibody, even if it is a PAD4 protein fragment, an antibody that can recognize the fragment in the polyclonal antibody Can be included. Therefore, PAD4 fragments can also be used to measure PAD4 autoantibodies in test samples, as with PAD4 full-length proteins, as long as they have binding properties to anti-PAD4 monoclonal antibodies that link the solid phase and PAD4 antigen. Are included within the scope of the present invention. The PAD4 fragment comprises a sequence of 7 amino acid residues or more, preferably 10 amino acid residues or more in SEQ ID NO: 2, and is a fragment that undergoes an antigen-antibody reaction with the anti-PAD4 monoclonal antibody and the PAD4 autoantibody in the test sample. It is. It is known in this field that a protein fragment having about 7 amino acid residues or more exhibits antigenicity. However, if the number of amino acid residues is too small, there is a high possibility of cross-reacting with a non-measurement antibody present in the sample. Therefore, from the viewpoint of improving measurement accuracy, the number of amino acid residues of the PAD4 antigen is preferably 30 or more, more preferably 100 or more, further preferably 300 or more, 500 or more, and the like. PAD4 antigen is most preferred.

  The solid phase used in the anti-PAD4 autoantibody measurement method of the present invention can be any solid phase used in immunoassay by the usual sandwich method, and is not particularly limited. For example, a commercially available ELISA plate can be preferably used.

  The test sample used in the anti-PAD4 autoantibody measurement method of the present invention is a biological component collected from a mammal such as a human, preferably serum or plasma.

  The present invention further provides a method for detecting rheumatoid arthritis using at least one of the above-described PAD4 measurement method and anti-PAD4 autoantibody measurement method of the present invention. In the detection method, at least one of PAD4 measurement and anti-PAD4 autoantibody measurement is performed on a test sample obtained from a living body, and the amount of PAD4 and / or anti-PAD4 autoantibody present in the sample is used as an index. To determine whether or not rheumatoid arthritis. In patients with rheumatoid arthritis, as described in the Examples below, PAD4 and / or anti-PAD4 autoantibodies are significantly more present than in healthy subjects, so measurement of PAD4 and / or anti-PAD4 autoantibodies can detect rheumatoid arthritis. Is possible. From the viewpoint of improving detection accuracy, PAD4 and / or anti-PAD4 autoantibodies are measured for one or a plurality of healthy subject samples to obtain healthy subject reference values, and the measured values of the target organism are compared with the normal subject reference values. It is preferable to do. In order to further improve the detection accuracy, RAD is determined for patients with rheumatoid arthritis by examining the amount of PAD4 and / or anti-PAD4 autoantibodies for samples obtained from a large number of patients known to have rheumatoid arthritis. The measured value of the target living body may be compared with both the healthy subject reference value and the rheumatoid arthritis patient reference value. The reference value can be determined, for example, by quantifying the amount of PAD4 and / or anti-PAD4 autoantibody in each sample and calculating the average value. In addition, the healthy subject reference value and the rheumatoid arthritis patient reference value can be determined in advance by examining the amount of PAD4 and / or anti-PAD4 autoantibodies for a large number of healthy subjects and rheumatoid arthritis patients. Therefore, a predetermined reference value may be used when comparing with a reference value by the rheumatoid arthritis detection method of the present invention.

  In the rheumatoid arthritis detection method of the present invention, it is not always necessary to perform both PAD4 measurement and anti-PAD4 autoantibody measurement for one test sample, and rheumatoid arthritis can be detected by only one of them. Specifically, as described in the Examples below, rheumatoid arthritis patients can be divided into two types: those with PAD4 autoantibodies and those without PAD4 autoantibodies. Indicates a high price. Therefore, if either one of PAD4 measurement or anti-PAD4 autoantibody measurement is performed on the test sample, and the measurement value is significantly higher than the reference value for healthy subjects, the other measurement must be performed. However, it can be determined that the living body has rheumatoid arthritis. As a result of either one of the measurements, if a measurement value significantly higher than the healthy person reference value is not obtained, the other measurement is performed to determine whether or not the living body has rheumatoid arthritis. Can do. For example, first, PAD4 autoantibody is measured, and if the autoantibody measurement value is higher than the normal standard value, it can be determined that the living body has rheumatoid arthritis at that time. When the autoantibody measurement value is not significantly higher than the healthy subject reference value, PAD4 measurement is performed on the sample. If the PAD4 measurement value is significantly higher than the healthy subject reference value, the living body is judged to have rheumatoid arthritis. When the measured value of PAD4 is not higher than the normal value, it can be determined that the living body does not suffer from rheumatoid arthritis. On the contrary, PAD4 may be measured first, and PAD4 autoantibody may be measured according to the result. Further, for one test sample, both PAD4 measurement and anti-PAD4 autoantibody measurement may be performed simultaneously or sequentially. When there are multiple test samples, one of the measurements may be performed, and the other measurement may be performed for a sample that cannot be determined to be rheumatoid arthritis, or for all the test samples Two measurements may be performed simultaneously or sequentially.

  The test sample used in the method for detecting rheumatoid arthritis is also a biological component collected from mammals such as humans, and preferably serum or plasma, as in the two measurement methods of the present invention.

  The present invention also provides a kit for carrying out the above-described PAD4 measurement method of the present invention. The PAD4 measurement kit includes the above-described anti-PAD4 monoclonal antibody or an antigen-binding fragment thereof. The antibody or antigen-binding fragment thereof may be in a form bound to a solid phase such as a known immunoassay plate. In addition, the kit may further contain known reagents necessary for various immunoassays.

  Similarly, the present invention provides a kit for carrying out the above-described method for measuring an anti-PAD4 antibody of the present invention. The anti-PAD4 autoantibody measurement kit includes the above-described anti-PAD4 monoclonal antibody or an antigen-binding fragment thereof and the PAD4 antigen. The monoclonal antibody or antigen-binding fragment thereof may be in a form bound to a solid phase such as a known immunoassay plate, and further, the PAD4 antigen is bound to the immobilized antibody or the like. The form may be sufficient. In addition, the kit may further contain known reagents necessary for various immunoassays.

  These kits can also be used to carry out the rheumatoid arthritis detection method of the present invention. That is, the present invention also provides a rheumatoid arthritis detection kit comprising the PAD4 measurement kit and / or the anti-PAD4 autoantibody measurement kit. In the rheumatoid arthritis detection method of the present invention, when both PAD4 measurement and anti-PAD4 autoantibody measurement are performed, the rheumatoid arthritis measurement kit comprises the PAD4 measurement kit and the anti-PAD4 autoantibody measurement kit. In the rheumatoid arthritis measurement kit, the PAD4 measurement kit and the anti-PAD4 autoantibody measurement kit may not be included in a form that can be clearly distinguished. Specifically, for example, the anti-PAD4 monoclonal antibody or antigen-binding fragment thereof contained in the two measurement kits described above is divided into an antibody for measuring PAD4 and the like and an antibody for measuring anti-PAD4 autoantibody and the like. It may be included in one container.

  Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.

1. Preparation of human type 4 peptidylarginine deiminase (PAD4) recombinant protein
The PAD4 gene (SEQ ID NO: 1) was isolated by RT-PCR using total RNA extracted from retinoic acid-treated human leukemia cells HL60. That is, cDNA was synthesized from the above total RNA using an oligo-dT primer, and pENTR-hPAD4-F 5′-CACCATGGCCCAGGGGACATTGA-3 ′ (23 mer) (SEQ ID NO: 3) and pENTR-hPAD4-R 5 using the cDNA as a template. PCR was performed using '-CCGAATTCGCGGCCGCGAGCTCTTGCTTGCCACAC-3' (35 mer) (SEQ ID NO: 4) as a primer (94 ° C-1 min, 55 ° C-1 min, 72 ° C-2 min 30 cycles, Taq polymerase (Invitrogen) ) And the attached buffer), PAD4 cDNA was amplified. The obtained PAD4 cDNA was inserted into the pENRT / D-TOPO vector (Invitrogen) using Gateway Technology (Invitrogen). Next, in order to prepare a PAD4 recombinant protein, the PAD4 gene was recombined into a pDEST17 vector (Invitrogen) having 6 histidine (6 × His) sequences at the N-terminus, and 6 × His− using E. coli BL21-AI. A recombinant protein was prepared as PAD4. The 6 × His-PAD4 recombinant protein was purified to a level at which a single band (molecular weight of about 74 kDa) was obtained by electrophoresis using Ni-NTA agarose beads (Qiagen) (FIG. 1). In addition, the enzyme activity of the PAD4 recombinant protein was measured using benzoyl-L-arginine ethyl ester (BAEE) as a substrate. That is, 25 μl each of PAD4 recombinant protein, 200 mM Tris-HCl (pH 7.6), 20 mM CaCl ₂ , 10 mM DTT, and 20 mM BAEE were mixed and reacted at 50 ° C. for 1 hour. After 1 hour, 12.5 μl of 5 M perchloric acid was added to stop the reaction and left on ice for 20 minutes. Add 50 μl of the reaction solution to the colorimetric reagent (0.0416% FeCl ₃ · 6H ₂ O: H ₂ SO ₄ : H ₂ PO ₄ (50:25:20), 0.5% diacetyl monooxime, 0.01% thiosemilubazide in 2: 1 Mixing) 950 μl was added, and after 5 minutes of reaction in a boiling water bath, the absorbance was measured at 530 nm. PAD4 activity was approximately 10 U / ml.

2. Establishment of anti-PAD4 monoclonal antibody Anti-PAD4 monoclonal antibody is prepared by immunizing the footpad of BALB / C mouse with the PAD4 recombinant protein shown in 1 above and fusing the lymph node lymphocytes with myeloma cells. did. Namely, BALB / C mice were first immunized with 25-50 μg / mouse of PAD4 recombinant protein emulsified with Freund's complete adjuvant, and after 3 weeks, only recombinant PAD4 (25-50 μg / mouse) Boosted. Three days later, lymphocytes were removed from the mouse hind limb lymph nodes, mixed with mouse myeloma cells (P3U1) and spleen cells previously cultured in RPMI-1640 medium at a ratio of 1: 3, and PEG (Boehringer) was added. Cell fusion was performed. The fused cells were suspended in a HAT medium, dispensed into a 96-well culture plate, and cultured in a 37 ° C. CO ₂ incubator.

  Screening for PAD4-specific antibody-producing cells was performed by antigen solid-phase ELISA. That is, 50 μl / well of the PAD4 recombinant protein shown in 1. above was dispensed to a 96-well ELISA plate (Falcon) at a concentration of 1 μg / ml and allowed to stand overnight at 4 ° C. After blocking with 1% skim milk (Difco), wash 3 times with Wash Buffer (PBS containing 0.05% Tween 20), add 50 μl of the culture supernatant of the cell-fused plate, and react at 37 ° C for 1 hour. It was. Similarly, after washing 3 times with Wash Buffer, POD-labeled anti-mouse immunoglobulin antibody (DACO) was added, and further reacted at 37 ° C. for 1 hour. After washing 4 times with Wash Buffer, substrate ABTS (Wako Pure Chemical Industries, Ltd.) was added and wells showing color development were selected. In this way, wells of anti-PAD4 antibody-producing cells were selected. Finally, PAD4-541 antibody-producing cells and PAD4-543 antibody-producing cells were established, and the antibodies produced from these cells were used for examination of the ELISA system.

3. Establishment of PAD4 measurement sandwich ELISA system
A sandwich ELISA system was established using PAD4 recombinant protein as an antigen. Specifically, PAD4-541 antibody was diluted with PBS 7.4 to a concentration of 10 μg / ml on an ELISA plate (POLYSORB) manufactured by NUNC, and 100 μl / well was added and allowed to stand overnight at 4 ° C. for adsorption. Next, 200 μl / well of 5% skim milk-containing Tris buffer (pH 7.4) was added and left at 37 ° C. for 5 hours for masking. After washing 3 times with a washing buffer solution (PBS 7.4 containing 0.05% Tween20), the PAD4 recombinant protein was adjusted to 10 ng / ml with 1% BSA-PBS, added at 100 μg / well, and reacted at 37 ° C. for 1 hour. In order to confirm the specificity, a recombinant protein was prepared for PAD1, 2 and 3 and adjusted to 20 ng / ml and reacted in the same manner. After washing 3 times with a washing buffer, 100 μl / well of alkaline phosphatase (ALP) -labeled PAD4-543 antibody was added and reacted at 37 ° C. for 1 hour. After thoroughly washing with a washing buffer, 100 μl / well of the substrate PNPP was added and allowed to stand at room temperature for 2 hours at 37 ° C., and then the wavelength of 405 nm was measured. As a result, as shown in FIG. 2, the absorbance is high only when PAD4, which is an antigen of the solid phase antibody and the labeled antibody, is reacted (hPAD-4), while it has about 60% amino acid homology with PAD4. It was confirmed that the other PAD families, PAD1, PAD2, and PAD3, hardly bind with the combination of the solid phase antibody and the labeled antibody (hPAD-1, hPAD-2, and hPAD-3). Thus, it was confirmed that the PAD4 recombinant protein can be specifically measured by the combination of the solid phase antibody and the labeled antibody.

4. Measurement of PAD4 in blood and addition of EDTA PAD4 in serum / plasma was measured by the sandwich ELISA measurement system established in 3 above.

  First, instead of the PAD4 recombinant protein of 3. above, ELISA was performed using a sample obtained by diluting healthy subject serum or plasma three times with PBS containing bovine serum and an irrelevant monoclonal antibody (pH 7.4). As a result, a clear value was not obtained. Therefore, the same measurement was performed using the above samples (Hu. Serum-1 and 2) to which a certain amount (5 ng / ml) of PAD4 recombinant protein was added. As a control sample, PBS alone or a PBS containing a monoclonal antibody unrelated to bovine serum albumin (BSA) to which a fixed amount (5 ng / ml) of PAD4 recombinant protein was added was prepared and measured in the same manner.

As a result, as shown in FIG. 3 (a), in the sample in which the PAD4 recombinant protein was added to the serum / plasma, the reaction was inhibited and an appropriate measurement value could not be obtained. On the other hand, when EDTA was added to the serum / plasma sample at a final concentration of 5 mM, the reaction inhibition was eliminated, and an appropriate measurement value similar to that of the control was obtained (FIG. 3 (b)). This is probably because PAD4 is a Ca ²⁺ -requiring enzyme. The PAD4 recombinant protein used for immunogens and screening is used in the absence of Ca ^2+, but blood PAD4 is thought to have a different steric structure because it is in the presence of a high concentration of Ca ²⁺ . Therefore, it is considered necessary to remove Ca ²⁺ by EDTA and return to the structure in the absence of Ca ²⁺ .

5. Measurement of PAD4 in serum / plasma of patients with rheumatoid arthritis Measurement of PAD4 in serum / plasma of 23 patients with rheumatoid arthritis and 10 healthy subjects was performed using the sandwich ELISA measurement system established in 3 and 4 above. . That is, instead of the PAD4 recombinant protein of 3. above, the serum or plasma of rheumatoid arthritis patients and healthy subjects is diluted 3-fold with PBS (pH 7.4) containing bovine serum and an irrelevant monoclonal antibody, and ELISA was performed using a sample to which EDTA having a final concentration of 5 mM was added.

  As a result, as shown in FIG. 4, 13 out of 23 patients with rheumatoid arthritis showed a high value of M + 2SD (0.20 ng / ml) or higher in healthy subjects. Nine of 23 patients with rheumatoid arthritis had a PAD4 value of zero. On the other hand, 0 values were not observed in healthy subjects. CRP (C-reactive protein), ESR30 (30 minute erythrocyte sedimentation rate), ESR60 (60 minute erythrocyte sedimentation rate), RF (rheumatic factor), MMP-3, anti-GAL antinuclear antibody, anti-CCP antibody, C3 (complement) ), C4 (complement), WBC (leukocyte), Hb (hemoglobin), Plt (platelet) and other clinical laboratory items were examined, but none showed significant correlation with PAD4. In addition, data other than anti-CCP antibodies that significantly determined these rheumatoid arthritis patients could not be obtained. Therefore, it was shown that the PAD4 ELISA system shown in the above 3 and 4 is so sensitive that it cannot be detected by existing clinical test items, and that it can specifically diagnose rheumatoid arthritis.

6. Measurement of PAD4 autoantibodies in rheumatoid arthritis patients serum / plasma In order to clarify the cause of PAD4 value being zero in 9 of 23 rheumatoid arthritis patients, PAD4 self in serum / plasma of rheumatoid arthritis patients The presence or absence of antibodies was examined.

  In order to examine the presence of PAD4 autoantibodies, a sandwich ELISA system was established using PAD4 recombinant protein as an antigen. That is, PAD4 was diluted with PBS 7.4 to a concentration of 2.5 μg / ml on an ELISA plate (POLYSORB) manufactured by NUNC, and 100 μl / well was added and allowed to stand overnight at 4 ° C. for adsorption. Next, 200 μl / well of 5% skim milk (Difco) -containing Tris buffer (pH 7.4) was added and left at 37 ° C. for 5 hours for masking. After washing 3 times with a washing buffer (PBS 7.4 containing 0.05% Tween20), serum / plasma of patients with rheumatoid arthritis was diluted 50-fold with bovine serum albumin-containing PBS and added at 100 μl / well and reacted at 37 ° C. for 1 hour. . After washing 3 times with a washing buffer, 100 μl / well of horseradish peroxidase (POD) labeled human IgG antibody (manufactured by DAKO) was added and reacted at 37 ° C. for 1 hour. After thoroughly washing with a washing buffer, 100 μl / well of substrate ABTS was added and allowed to stand at 37 ° C. for 30 minutes, and then a wavelength of 405 nm was measured. FIG. 5 shows the measurement results of 7 patients with rheumatoid arthritis and the 32 healthy subjects whose PAD4 values shown in 5 above were 0. The presence of PAD4 autoantibodies was suggested in 6 out of 7 patients with rheumatoid arthritis. Therefore, when ELISA reaction was performed, PAD4 recombinant protein was added in advance to the plasma of patients with rheumatoid arthritis and incubated. In 5 of 6 cases, inhibition of color development was confirmed, and the presence of autoantibodies was confirmed. Therefore, it was also confirmed that the PAD4 autoantibody measurement system was specific.

  PAD4 autoantibodies were also confirmed by Western blotting. That is, the PAD4 recombinant protein was electrophoresed and transferred to a PVDF membrane (Millipore). Serum / plasma of 5 patients with rheumatoid arthritis and 5 healthy subjects who showed positive PAD4 autoantibodies by ELISA was diluted 200-fold with PBS and sprinkled on PVDF membrane to cause antigen-antibody reaction. PAD4-541 antibody was used as an antibody positive control. The HRP-labeled human IgG antibody was used as the secondary antibody, and the band was detected by ECL chemiluminescence (Amersham Pharmacia). Thereafter, the PVDF membrane was stained with CBB (Coomassie Brilliant Blue). As a result, a band was confirmed in all 5 patients with rheumatoid arthritis who showed positive PAD4 autoantibodies by ELISA, and it was confirmed that all 5 rheumatoid patients had autoantibodies. On the other hand, no autoantibodies were present in 5 healthy subjects who were PAD4 negative by ELISA. Therefore, it was shown that patients with rheumatoid arthritis can be divided into two types: those who have PAD4 autoantibodies and those who do not. Rheumatoid arthritis patients who did not have PAD4 autoantibodies showed high levels of PAD4 in serum / plasma as shown in Section 5 above.

  As described above, joints have a probability of 87% or more by combining two types of ELISA measurement systems: (1) measurement of presence / absence of PAD4 autoantibodies in serum / plasma and (2) measurement of PAD4 levels in serum / plasma. Diagnosis of rheumatism is possible.

7. Detection method of PAD4 autoantibodies using PAD4-541 solid phase plate Next, the method for detecting PAD4 autoantibodies described in 6. above was improved in order to reduce the background. In the above 6., the antigen is directly immobilized, whereas the improved method is characterized in that the antigen is indirectly captured by Mab. That is, PAD4-541, an anti-PAD4Mab, was adsorbed by dispensing 100 μl / well at a concentration of 10 μg / ml in a 96-well ELISA plate (poly sorp manufactured by NUNC) and leaving it overnight at 4 ° C. After blocking with 1% skim milk, the plate was washed 3 times with Wash Buffer (PBS containing 0.05% Tween 20), 100 μl / well of recombinant PAD4 (0.2 μg / ml) was added, and reacted at 37 ° C. for 1 hour. Similarly, after washing with Wash Buffer three times, 34 patients' rheumatism patient plasma including newly added specimens were diluted 100-fold with 1% BSA-containing PBS 7.4 and added at 100 μl / well and reacted at 37 ° C. for 1 hour. Next, after washing 3 times with Wash Buffer, alkaline phosphatase-labeled anti-human IgG antibody (manufactured by Zymet) was added, and further reacted at 37 ° C. for 1 hour. After washing 4 times with Wash Buffer, the substrate pNPP was added and allowed to stand at 37 ° C for 1 hour, and the absorbance at 405 nm was measured. As a result, as shown in Table 1 below, it was confirmed that this autoantibody measurement method (PAD541 solid phase sandwich ELISA method) showed a higher positive rate than the direct antigen solid phase method described in 6. above.

It is an electrophoretic image of the human PAD4 recombinant protein produced and purified in the examples. It is a graph showing the result of having measured the PAD recombinant protein dilution sample by the PAD4 measurement sandwich ELISA system established in the Example. hPAD-1 is human PAD1 recombinant protein 20ng / ml, hPAD-2 is human PAD2 recombinant protein 20ng / ml, hPAD-3 is human PAD3 recombinant protein 20ng / ml, hPAD-4 is human PAD4 recombinant protein 10ng / ml ml. It is a graph showing the result of having measured the sample prepared by adding PAD4 recombinant protein by the PAD4 measurement system established in the Example. (a) is the result of measuring each sample without addition of EDTA, and (b) is the result of measurement with addition of EDTA. PBS; PBS sample with PAD4 added, BSA / PBS; PBS sample with BAD4 added and BSA and unrelated monoclonal antibody, Hu.Serum-1 and 2; PBS with PAD4 added human serum containing BSA and unrelated monoclonal antibody Dilute sample. It is a figure showing the result of having measured PAD4 in the serum / plasma sample of a rheumatoid arthritis patient and a healthy subject by the PAD4 measuring system established in the Example. It is a figure showing the result of having measured the anti-PAD4 autoantibody in the serum / plasma sample of a rheumatoid arthritis patient and a healthy subject by ELISA using the solid phase which couple | bonded the PAD4 antigen directly.

Claims (6)

  A test sample brought into contact with a calcium ion chelating agent is subjected to immunoassay using a monoclonal antibody or antigen-binding fragment thereof that reacts with PAD4 for antigen-antibody, and PAD4 that can be contained in the test sample is measured. A method for measuring PAD4 in a test sample.   The method according to claim 1, wherein the calcium ion chelating agent is ethylenediaminetetraacetic acid.   The method according to claim 1 or 2, wherein the test sample is serum or plasma.   The abundance of PAD4 in the test sample, which is measured by performing the PAD4 measurement method according to any one of claims 1 to 3 on a test sample obtained from a living body, is used as an index. , Detection method of rheumatoid arthritis. The PAD4 measurement method is performed on the test sample, and then at least a test sample in which PAD4 is not detected is bound to a solid phase with a monoclonal antibody or an antigen-binding fragment thereof that reacts with PAD4 and an antigen antibody, In vivo, PAD4 can be contained in a test sample by a sandwich method using an antigen-immobilized solid phase prepared by binding PAD4 antigen to the monoclonal antibody or antigen-binding fragment thereof on the solid phase. The method according to claim 4 , wherein the measurement is performed by performing a method for measuring an anti-PAD4 autoantibody in a test sample, which comprises immunoassay of the anti-PAD4 autoantibody produced in this manner. The amount of PAD4 present in the test sample and the presence of anti-PAD4 autoantibodies are measured by performing both the PAD4 measurement method and the anti-PAD4 autoantibody measurement method for all of the test samples. The method according to claim 5 , wherein the quantity is used as an index.