JP4512828B2 - Detection marker and detection kit for pulmonary sarcoidosis and ocular sarcoidosis - Google Patents

Description

The present invention relates to detection markers and detection kits for pulmonary sarcoidosis and ocular sarcoidosis.

Pulmonary sarcoidosis is a disease of unknown cause that claims systemic non-buty granuloma lesion formation, but as a disease-specific detection marker, serum angiotensin
Converting enzyme: ACE and lysozyme have been reported below, but the ACE positive rate in serum is about 50%, and it is said that serum lysozyme has poor disease specificity.

  Sarcoidosis is a systemic disease and is often found from ocular lesions (ocular sarcoidosis). Ocular sarcoidosis has distinctive ocular lesions and ocular sarcoidosis is strongly suspected, but there are few positive findings on systemic examination, and it is not uncommon to remain suspected without meeting diagnostic criteria.

  For diagnosis of pulmonary sarcoidosis, a bronchoalveolar lavage test (BAL) is performed. In pulmonary sarcoidosis, it has been reported that an increase in many inflammatory cells, especially CD4 positive lymphocytes, has been confirmed in the BAL fluid. There is a suggestion that this may be achieved (see, for example, Non-Patent Document 1 below).

  By the way, the SEREX (Serologic analysis of recombinant cDNA expression libraries) method can detect even a few self-antigen-expressing cDNAs out of about 10,000 cDNAs, and detect a small amount of self-antigens. This is a suitable method. Various tumor-related antigens including lung cancer have been identified by this SEREX method. The identified antigens include novel genes in addition to autoantibodies against transcription factors, cell differentiation antigens, cell constituent proteins, and the like. In other words, the SEREX method is a powerful and useful technique in the search for self-antigen, and as a report using this method, there is only one document regarding the search for sarcoidosis-related autoantibodies (see Non-Patent Document 2 below).

  In the search for T cell Vα / β chain gene rearrangement in BAL fluid by PCR (Polymerase Chain Reaction) method, antigen-specific T cell proliferation was observed in pulmonary sarcoidosis, and was contained in the antigen-containing groove of HLA class II antigen This means that a T cell repertoire having a T cell antigen receptor having a structure that appropriately binds to a complex of an antigen peptide and an HLA antigen has proliferated by an antigen-specific immune reaction, suggesting the presence of a disease-related antigen. (See, for example, Non-Patent Document 3 below).

Gernot Z et al., “TCR Vβ Family in T cell clones from sarcoid lun parenchyma, BAL, and blood”, Am J Respir Crit Care Med, 1997, pp. 159-153. Ebe et al., "Proliferative response of peripheral blood mononuclear cells and levels of antibody to recombinant protein from Propionibacterium acnes DNA expression library in Japanese patients with sarcoidosis", Sarcoidosis Vasc Diffuse Lung Dis 2000 years, Vol. 17, pp. 256-265 Dohi M et al., “Accumulation of multiple T cell clonotypes in lungs of health individuals and patients with pulmonary sarcoidosis, 198, 152, J Immun.

  Certainly, the above Non-Patent Document 1 suggests that CD4-positive T cells are involved in some pathological condition, but most of the mechanisms by which CD4-positive T cells increase and retinitis are combined. There are many cases in which it is difficult to diagnose pulmonary sarcoidosis only by increasing peripheral blood ACE. Since the size of the ocular lesion detected by an ophthalmologist is several tens of microns to several millimeters or less, if the lesion is confined in the eye, even if ACE or the like flows from the intraocular lesion into the bloodstream, The activity in the whole body unit is extremely low, and it is below the detection limit in a normal test. Since there is currently no disease marker specific to ocular sarcoidosis, it is very difficult to diagnose ocular sarcoidosis even if uveitis is the initial symptom.

  Moreover, most of the SEREX methods are used for identification of tumor-associated antigens, and there are very few examples where they are applied to other than tumor-associated antigens. There is a report of sarcoidosis using this in only one document of Non-Patent Document 2 described above. This report is a SEREX analysis using a phage incorporating Propionibacterium acnes DNA, and includes humans including alveolar epithelium. SEREX analysis using a phage into which cDNA expressed by the cells of FIG.

  Furthermore, in the report described in Non-Patent Document 3 above, antigen-specific T cell proliferation is recognized, but the HLA antigen differs between cases only by the PCR method, and the recognized antigen depends on the antigen-presenting cell. Since it is fragmented, it is extremely difficult to determine the antigen recognized by the sarcoidosis-related autoantibody.

  In view of the above problems, an object of the present invention is to provide a detection marker and a detection kit for detecting pulmonary sarcoidosis and ocular sarcoidosis.

  T cells that infiltrate into the BAL fluid or lung interstitium have Vα / Vβ chains in their immunoglobulin variable region, and have disease-specific antigens in the alveoli, which are disease-specific in the lungs. If an antigen / antibody reaction occurs, the assistance of CD4 positive T cells is indispensable for antibody production by B cells. First, we also analyzed the Vβ chain of CD4 positive T cells in BAL fluid of pulmonary sarcoidosis cases, and in all cases, specific proliferation of specific CD4 positive T cells was observed. This finding supported the previous reports of pulmonary sarcoidosis in the BAL fluid, and resulted in the proliferation of CD4-positive T cells against a specific antigen in the alveolar region, suggesting the existence of an active antigen-antibody reaction. It is not uncommon for mediastinal lymphadenopathy and intrapulmonary shadow to accompany ocular sarcoidosis, and it is possible that an immune response to a common self-antigen occurs in the alveoli and the eye. If such autoantigens are present, there is almost no periocular lymphadenopathy, and if pulmonary sarcoidosis is complicated, mediastinal lymphadenopathy often accompanies. It is suggested that antigen stimulation accompanying sarcoidosis is strongly present in the lung.

  Then, when the present inventors further examined the above, the alveolar lumen includes type II alveolar epithelium and alveolar macrophages as its constituent cells. If a sarcoidosis-specific antigen-antibody reaction occurred in the alveolar space, it was inferred that the proteins of alveolar epithelium and macrophages, which occupy most of the constituent cells, acted as antigens. It was found that autoantibodies present in sera of patients with pulmonary sarcoidosis can be detected by analyzing proteins expressed by alveolar epithelium by a very sensitive method (SEREX method). As specifically shown below, the nine types of autoantibodies we discovered are present in the serum of pulmonary sarcoidosis cases with relatively high frequency, one of which is retinal short-chain dehydrogenase / reductase (ret SDR2). And trabecular meshwork constituent protein (accession number: CD676906, CD677025, CD677068), ocular pericite constituent protein (CD066293), retinal constituent protein (BQ637781), and lens constituent protein (CD674,39) are common to alveolar epithelium and retina Because it is a protein, self-antigen protein that flows out of the alveolar epithelium and macrophages in the alveolar cavity is used as an antigen to produce antibodies, and this protein is also present in the retina. For that represents protein, these abnormal autoantibodies are contributing to the pathogenesis of ocular lesions of the eye sarcoidosis found that measurement of these autoantibodies is useful in the diagnosis of eye sarcoidosis. By combining these, an accurate serological diagnosis of pulmonary sarcoidosis and ocular sarcoidosis becomes possible.

  That is, the detection marker of pulmonary sarcoidosis according to the present invention includes anti-poly binding protein 1 antibody, anti-1-phosphatidylinositol-4-phosphate 5 kinase isoformc antibody, anti-retinshot-chain dehydrogenase antibody, protein synthesis initiation factor 4a-II antibody, anti-S-adenosylhomocysteine synthetic antibody, anti-macrophage migration inhibitory factor antibody, anti-cytoreductive antibody It comprises at least one of a ccase protein antibody and an anti-transforming growth factor beta 2 antibody. In addition, detection markers for ocular sarcoidosis are anti-retinal short-chain dehydrogenase / reductase (ret SDR2) antibody, anti-retinal short-chain dehydrase / bacterial protein 70 (reverse SDR2) antibody, anti-retinal short-chain dehydrogenase It comprises at least one of a CD677068) antibody, an anti-occlusive peritite constituent protein (CD066293) antibody, an anti-retinal constituent protein (BQ637781) antibody, and an anti-lens constituent protein (CD674,39) antibody.

  In addition, the detection kit for pulmonary sarcoidosis according to the present invention includes poly a binding protein 1, 1-phosphotylated-lintositol 4-phosphonate 5 kinase isoform, c, retinshort re-in 2 , S-adenosyl homosteinine hydrolase, macromigration migration factor, cytochrome product core protein, oxidoreductase protein protein, It is characterized by adsorbing at least one antigen protein of forming growth factor beta 2 to a substrate. In addition, detection kits for ocular sarcoidosis include retinal short-chain dehydrogenase / reductase (ret SDR2), trabecular meshwork protein (accession number: CD6760p, CD6767025, CD677025, CD667025, CD667025, CD677025, Further, it is characterized in that at least one antigen protein of the lens constituent protein (CD674, 39) is adsorbed on a base material. Here, the substrate is not limited as long as the antigen protein can be adsorbed and the antibody reaction can be confirmed, but includes a membrane and a plate scattered in the ELISA method. In this case, it is also desirable that the antigen protein is labeled, and the label is labeled with a His-tag label.

  In addition, as a method according to the present invention, poly a binding protein 1, 1-phosphatidylinositol-4-phosphate 5 kinase isoform c, retinal short-chain dehydrogenase / reducetitereduction adenosyl homosteinine hydrolase, macromigration migration factor, cytochrome reductase core protein, oxidoreductase protein complex low factor beta 2, trabecular meshwork constituent protein (accession number: CD676906, CD677025, CD677068), ocular pericite constituent protein (CD066633), retinal constituent protein (BQ637781), lens constituent protein CD3967 It is characterized in that blood or BAL solution is applied to a plate adsorbed with the antigen protein.

  As described above, a detection marker and a detection kit for detecting pulmonary sarcoidosis and ocular sarcoidosis can be provided. In particular, the 15 types of autoantibodies we have discovered have not been reported in the past, and most autoantibodies are not detected in cases other than sarcoidosis, which is highly useful as a diagnostic test.

  By using the present invention, it is possible to continuously analyze disease-specific autoantibodies using BAL fluid before and after treatment or serum collected over time. In particular, measurement of autoantibodies using His-tag labeled autoantigen protein is an optimal test method for disease follow-up and determination of therapeutic effects such as steroids.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings.

  As an embodiment of the present invention, a detection marker and detection kit of pulmonary sarcoidosis and ocular sarcoidosis can be employed. Examples of pulmonary sarcoidosis detection markers include anti-poly a binding protein 1 antibody, anti-1-phosphophatidylinositol-4-phosphate 5-kinase isoform antibody, anti-retinshot / retin anti-DR2 -II antibody, anti-s-adenosyl homocysteine hydrochloride antibody, anti-macrophage migration inhibitory factor antibody, anti-cytochrome reductase core protein antibody, anti-oxidorducuc It is supposed to comprise at least one of a case protein antibody and an anti-transforming growth factor beta 2 antibody. Anti-retinal short-chain dehydrogenase / reductase (ret SDR2) antibody, anti-retin short-chain 70 gene, anti-retinsh-chain 70-antibody ) Antibody, anti-occlusive peritite constituent protein (CD066293) antibody, anti-retinal constituent protein (BQ637781) antibody, and anti-lens constituent protein (CD674,39) antibody. That is, blood or BAL fluid is extracted from a patient suspected of having pulmonary sarcoidosis or ocular sarcoidosis, and the presence or absence of this antibody is examined. If present, it is determined that pulmonary sarcoidosis or ocular sarcoidosis is suspected. can do. This makes it possible to investigate with high accuracy.

  In addition, as an embodiment of the present invention, poly a binding protein 1, 1-phosphotylino sito 4- phosphatate 5 kinase isoform form c hydrolase, macromigration migration factor, cytochrome reductase core protein, oxidoreductase protein, transforming gr wth factor beta 2, trabecular meshwork constituent protein (accession number: CD676906, CD677025, CD677068), ocular pericite constituent protein (CD066633), retinal constituent protein (BQ637781), lens constituent protein CD (67) A kit for detecting pulmonary sarcoidosis and ocular sarcoidosis, wherein a protein is adsorbed on a substrate, can also be used. Whether blood or BAL fluid is extracted from a patient suspected of having pulmonary sarcoidosis or ocular sarcoidosis, and whether or not this antigenic protein reacts with an antibody to determine whether suspicion of pulmonary sarcoidosis or ocular sarcoidosis is present. Can be determined. Specifically, these antigen proteins are adsorbed on a substrate, reacted with an antigen protein by applying a BAL solution or serum, etc., and further reacted with a secondary antibody having a luminescence or color development site, thereby causing luminescence or the like. It is possible to determine whether or not the antibody is present based on the amount of the pulmonary sarcoidosis and ocular sarcoidosis. In this case, it is desirable that the antigen protein is labeled from the viewpoint of producing the antigen protein. In particular, according to the present embodiment, by using a membrane or plate adsorbed with a self-antigen protein prepared in advance, rapid examination is possible, and a result can be obtained in about one hour. Therefore, it is possible to promptly respond to severe sarcoidosis cases whose medical condition changes every moment. When pulmonary sarcoidosis worsens, it is necessary to treat it as soon as possible, but it often takes time to exclude other complications (infections, malignant tumors) and is difficult to diagnose. The present invention can provide very important information in distinguishing whether the pulmonary sarcoidosis itself has deteriorated or other complications, and can lead to a prompt response.

  In the following, studies were conducted to verify the effects of the invention according to this embodiment. This will be described below.

  Since the T cell Vβ chain is composed of 25 types of subfamilies, first, primers specific to each subfamily were prepared. The prepared primer is shown in FIG. In the figure, Vβ1, Vβ2,..., Vβ25 are used as forwards, and Cβ is commonly used as reverse.

  BAL was performed on 20 cases of pulmonary sarcoidosis, and the BAL was extracted. Then, CD4 positive T cells are separated from this BAL solution using Dyna beads, total RNA is extracted, the Vβ chain variable region of T cells is amplified by RT-PCR, and this PCR product is electrophoresed with 2% agarose. By staining with ethidium bromide, only a small part of the Vβ chain subgroup was expressed (see FIG. 2).

  Next, an outline of the SEREX method is shown in FIG. MRNA is extracted from a type II alveolar epithelial cancer culture, and a cDNA library is prepared. The resulting cDNA library is incorporated into a protein expression vector (ZAP expression vector) and infected with Escherichia coli. The protein encoded by was expressed in E. coli. Then, plaques that bind to immunoglobulin in pulmonary sarcoidosis serum and BAL fluid were selected by colony hybridization, and positive phages were converted into phagemid vectors, and then the base sequences of specific proteins were determined by sequencing. The structure of the protein was determined by homology search, and autoantibodies recognized in the serum and BAL fluid of pulmonary sarcoidosis / ocular sarcoidosis were identified. As a result of the above, FIGS. 4 and 5 show the antigens recognized by the cases and the autoantibodies detected for the cases.

  According to FIG. 4 and FIG. 5, anti-poly binding protein 1 antibody was present in the serum and BAL fluid of 5 cases (50%) out of 10 cases analyzed by SEREX method among 15 types of antibodies. Anti-1-phosphatidylinoside-4-phosphate 5 kinase isoform c antibody and anti-retinal short-chain dehydrogenase / reductase (ret SDR2) antibodies were present in 4 cases (40%) in 10 cases of pulmonary sarcoidosis and BAL (40%). . Anti-protein synthesis initiation factor 4a-II antibody was present in the serum and BAL fluid of 3 (30%) of 10 pulmonary sarcoidosis cases. Anti s-adenosylhomocysteine hydrolase antibody, anti-macrophage migration inhibitory factor antibodies, anti-cytochrome c reductase core protein antibody, anti-oxidoreductase protein antibody, anti-transforming growth factor beta 2 antibodies, anti-trabecular meshwork structure protein (accession number: CD676906, CD677025, CD677068) Antibody, anti-occlusive peritite constituent protein (CD066293) antibody, anti-retinal constituent protein (BQ637781) antibody, anti-lens constituent protein (CD674,39) antibody is 1 in 10 cases It was present in 10%). Thus, it was confirmed that the presence or absence of pulmonary sarcoidosis and ocular sarcoidose can be determined by detecting autoantibodies.

In 4 cases (Fig. 6) of the 10 cases analyzed by the SEREX method, an increase in ACE was observed, but among the antibodies found this time, four autoantibodies (anti-poly) with a high positive rate were observed.
a binding protein 1 antibody, anti-1-phosphatidylinositol-4-phosphate
5 kinase isoform c antibody, anti-retinal short-chain dehydrogenase / reductase
(ret SDR2) antibody, anti-protein synthesis initiation factor 4a-II antibody), 9 out of 10 cases (90%) were positive for new autoantibodies and 6 cases were negative for ACE All were autoantibodies positive. Of the common antigens that are expressed in both the alveoli and the retina, anti-retinal short-chain dehydrogenase / reductase (ret SDR2) antibodies, which were detected frequently this time, were present in 3 (43%) of 7 ocular sarcoidosis cases. It was positive. In the remaining 1 patient who is positive for anti-retinal short-chain dehydrogenase / reductase (ret SDR2) antibody and currently has no symptoms of ocular sarcoidosis, accumulation in the eye was observed by gallium scintigraphy, and sarcoidosis lesions were also observed in the eye. It is suggested that there is a potential. Autoantibodies against alveolar and ocular common antigens, especially anti-retinal short-chain dehydrogenase / reductase (ret SDR2) antibodies, are useful as diagnostic markers for ocular sarcoidosis.

  In addition, among the 15 types of autoantigen proteins detected this time, three types of proteins (poly a binding protein a 1, 1-phosphatidylinositol-4-phosphate 5-kinase isoform c, retinal short-chain dehydrogenase / For SDR2)), primers for the whole autoantibody-recognizing antigen gene were prepared and analyzed by RT-PCR to confirm the expression of each gene. FIG. 7 shows the primers designed here. Two types of autoantigen proteins (poly a binding protein a 1, retin short-chain dehydrogenase / reductase (ret SDR2)) are classified into type II alveolar epithelial cancer culture (A549) and monocyte culture ( Expression could be confirmed for both THP-1). Expression of the remaining one type of autoantigen protein (1-phosphatidylinositol-4-phosphate 5 kinase isoform c) was observed only in the type II alveolar epithelial cancer culture (A549). Pulmonary sarcoidosis is thought to have a lesion site in the alveolar space, and autoantibody-recognizing antigen protein is expressed from both type II alveolar epithelium and alveolar macrophages that occupy most of the constituent cells on the alveolar space side. The possibility of being produced was confirmed. The result is shown in FIG. Further, the discovered autoantibody-recognizing antigen protein was artificially prepared in Escherichia coli as a His-tag labeled protein. The outline of the protein production method performed below is shown.

  The PCR product obtained by the above RT-PCR method was ligated to a His-tag labeled protein expression vector (Pqe-30UA) and transformed into competent M15 cells. The obtained His-tag labeled protein-expressing colony protein was transferred to a nitrocellulose filter, and then the filter was transferred to a plate containing IPTG to express a 6 × his tag labeled protein. The obtained filter was immunostained using Penta-HisHRP Conjugate to select autoantibody recognition antigen protein expression colonies. The obtained colonies were picked up and grown in a culture solution, and 6 × his tag-labeled protein was extracted. After confirming protein expression by Western blot method, autoantibody recognition antigen protein (His-tag labeled protein) was purified from E. coli mass culture.

  The 15 types of pulmonary sarcoidosis-specific His-tag labeled autoantigen proteins thus obtained were adsorbed on a nylon membrane as 6 mm dots using The Convertible (Biometer). Thus, the obtained protein-adsorbing membrane was hybridized with the serum and BAL fluid of the case, and anti-human IgG antibody was bound as a secondary antibody, and eleven types of autoantibody expression were detected simultaneously by chemiluminescence. In the search by the dot blot method and the western blot method, a positive band for His-tag labeled protein can be detected only when lung sarcoidosis serum and bronchoalveolar lavage fluid are used. There was a tendency to be strongly detected when the disease worsened.

  Furthermore, the 15 types of pulmonary sarcoidosis-specific His-tag labeled autoantigen proteins obtained as described above were detected by the Ni-NT (FIG. 4) SEREX method, and the self-antigen protein recognized by the pulmonary sarcoidosis-specific autoantibodies and their expression frequencies. I investigated. It was added to an A-labeled ELISA plate and incubated overnight at 4 degrees for fixation. As a result, the His-tag labeled protein could be bound to Ni-NTA, and the concentration of autoantibodies bound to the self-antigen protein was detected using the case serum and the BAL fluid using the ELISA plate thus obtained. In the ELISA analysis using serum and BAL fluid with a plate adsorbed with a His-tag labeled autoantigen protein, a tendency was found that the antibody titer was detected higher when the disease state worsened. The ELISA analysis of the present invention was a useful index for follow-up of pulmonary sarcoidosis and ocular sarcoidosis.

The figure which shows the primer used for PCR method with respect to T cell V (beta) chain | strand subfamily. Example of analysis of T cell Vβ chain subfamily in pulmonary sarcoidosis. It is a figure which shows the schematic of a SEREX method. The figure which shows the self-antigen protein which the lung sarcoidosis specific autoantibody recognized by the SEREX method recognizes, and its expression frequency. The figure which shows the alveoli recognized by the pulmonary sarcoidosis specific autoantibody detected by the SEREX method, an ocular common autoantigen protein, and its expression frequency. The figure which shows the expression condition of three types of autoantibodies with high expression frequency. The figure which shows the PCR primer used in order to amplify the pulmonary sarcoidosis autoantibody recognition antigen gene. The figure which analyzed the expression in the type II alveolar epithelial cancer culture strain (A549) and monocyte culture strain (THP-1) of three types of autoantigen proteins by RT-PCR method. 1: retinal short-chain dehydrogenase / reductase (ret SDR2), 2: 1-phosphatidylinosideol-4-phosphate 5 kinase isoform cell, TH: poly a binding clot

Claims (4)

A marker for detecting pulmonary sarcoidosis comprising an anti-poly a binding protein 1 antibody . poly a
A detection kit for pulmonary sarcoidosis comprising binding protein 1 antigen protein adsorbed on a base material. The pulmonary sarcoidosis detection kit according to claim 2, wherein the antigen protein is labeled. The kit for detecting pulmonary sarcoidosis according to claim 3, wherein a His-tag label is attached as the label.