JP5499405B2 - Test method for relapsing polychondritis and test kit used therefor - Google Patents

Description

  The present invention relates to a biomarker for relapsing polychondritis. More specifically, the present invention relates to a test method for relapsing polychondritis using the biomarker and a test kit used therefor.

  Relapsing polychondritis (abbreviated as “RP”) is a chronic disease of unknown cause that causes inflammation in cartilage tissue throughout the body. Symptoms include pain, redness and swelling in the cartilage tissue. In particular, lesions in the nasal root and auricle are characteristic, and many cases are associated with polyarthritis. In addition, examples of airway obstruction due to cartilage lesions of the pharynx, trachea and bronchi have been reported. Although there are no accurate statistics on relapsing polychondritis in Japan, it is estimated that there are 400-500 patients in Japan. In the 1986 report, the 10-year survival rate was 55%, but in the 1998 report, the 8-year survival rate was reported to be 94%.

  The findings of blood tests for relapsing polychondritis include 1) increased erythrocyte sedimentation rate, 2) increased serum C-reactive protein (hereinafter referred to as “CRP”), and 3) sphere 4) Eosinophilia (about 10%), 5) Antinuclear antibody positive (about 22-66%), 6) Anti-type II collagen antibody positive (about 50% or less), 7 ) Rheumatoid factor positive (about 16%), 8) Anti-neutrophil cytoplasmic antibody positive (partial), etc. have been reported. However, no useful test method for relapsing polychondritis has been established so far, such as cases in which CRP values are within the normal range even when cartilage destruction has progressed. Has been made.

  On the other hand, by measuring the amount of Triggering Receptor Expressed on Myeloid cells-1 (hereinafter abbreviated as “TREM-1”) protein in blood cells in blood, the inflammatory response due to autoimmune diseases (eg, rheumatoid arthritis) is measured. A diagnosis method has been proposed (see Patent Document 1). Patent Document 1 proposes a method for diagnosing an inflammatory response due to an autoimmune disease by measuring the amount of TREM-1 protein in blood cells, but actually diagnosing an autoimmune disease by measuring the amount of TREM-1 protein. The results are not shown.

JP-T-2004-522742

  For various human diseases, testing methods using biomarkers specific to the diseases as an index have become widespread. Since this method does not require large-scale equipment and places little burden on the subject, it can be widely applied to many subjects who have no subjective symptoms. However, no specific biomarker for relapsing polychondritis has been reported so far, and no specific testing method has been established. Therefore, the examination and treatment of relapsing polychondritis have been delayed, and there have been many cases in which severe sequelae and significant QOL decrease have occurred. Under such circumstances, there is an increasing social demand for a test method for relapsing polychondritis.

  This invention is made | formed in view of this point, and makes it the 1st objective to provide the biomarker of relapsing polychondritis.

  The second object of the present invention is to provide a test method for relapsing polychondritis and a test kit for relapsing polychondritis.

  The present inventor has comprehensively analyzed cytokines and chemokines in the serum of patients with relapsing polychondritis and found that TREM-1 can be a specific biomarker for relapsing polychondritis. Further studies were made to complete the present invention.

That is, the present invention relates to the following method for examining relapsing polychondritis.
[1] A method for examining relapsing polychondritis, comprising measuring the amount of TREM-1 protein in a biological sample collected from a subject.
[2] The test according to [1], further comprising the step of determining that the subject is likely to have relapsing polychondritis when the amount of the TREM-1 protein is greater than a predetermined value. Method.
[3] The examination method according to [1] or [2], wherein the biological sample is serum.
[4] The test method according to any one of [1] to [3], wherein the amount of the TREM-1 protein is measured by an immunoassay.

The present invention also relates to the following test kit for relapsing polychondritis.
[5] A test kit for relapsing polychondritis having an anti-TREM-1 antibody or a fragment thereof.

  According to the present invention, examination (diagnosis) of relapsing polychondritis, evaluation of disease activity, evaluation of therapeutic effect, and the like can be performed quickly and easily. Therefore, according to the present invention, it becomes possible to test and treat relapsing polychondritis at an early stage and improve the patient's QOL.

ROC curve showing sensitivity and specificity of TREM-1 for relapsing polychondritis Graph showing serum TREM-1 concentration in healthy subjects and patients with relapsing polychondritis A graph showing the concentrations of six inflammation-related factors in serum in healthy subjects and patients with relapsing polychondritis ROC curve showing sensitivity and specificity of six inflammation-related factors for relapsing polychondritis Graph showing serum TREM-1 concentration in healthy subjects, patients with relapsing polychondritis, patients with systemic scleroderma, and patients with HTLV-I related myelopathy

1. Test Method for Recurrent Polychondritis of the Present Invention The test method of the present invention includes a step of measuring the amount of TREM-1 protein in a biological sample collected from a subject.

  TREM-1 (Triggering Receptor Expressed on Myeloid cells-1) is a transmembrane receptor belonging to the immunoglobulin superfamily, and is expressed in myeloid cells such as monocytes / macrophages and neutrophils. TREM-1 transmits a signal into a cell via DAP12 having ITAM (immunoreceptor tyrosine-based activation motief). Stimulation of TREM-1 using an agonist antibody induces the expression of inflammatory cytokines such as TNFα and IL-1, chemokines such as MCP-1, and surface molecules such as cell adhesion molecules. TREM-1 also has the effect of dramatically enhancing the inflammatory reaction in cooperation with TLR and NLR. From these facts, TREM-1 is considered to play a role in determining the intensity of the inflammatory response. There is also a soluble type of TREM-1. This soluble TREM-1 is a transmembrane TREM-1 cleaved by a metalloproteinase.

  The present inventor has exhaustively searched for biomarkers specific to relapsing polychondritis, and soluble TREM-1 protein in a biological sample (particularly plasma) becomes a biomarker of relapsing polychondritis. I found out. According to the study of the present inventor, the amount of soluble TREM-1 protein specifically increases in the biological sample (especially plasma) of patients with relapsing polychondritis. As shown in the Examples, the test method of the present invention for measuring soluble TREM-1 protein can be a highly sensitive and highly specific test method by setting an appropriate cut-off value.

  Although the kind of biological sample used as a measuring object is not specifically limited, Blood, plasma, serum, these dilution liquids, etc. can be used. When the biological sample is serum, it is easy to collect the biological sample, and the test can be performed in a short time without invasion compared to biopsy or the like.

  The method for measuring the amount of TREM-1 protein in a biological sample is not particularly limited, and is known to those skilled in the art such as immunoassay, mass spectrometry, electrophoresis, liquid chromatography, and gas chromatography. It can be selected appropriately from the law.

  Examples of immunoassays include ELISA, radioimmunoassay (RIA), enzyme immunoassay (EIA), chemiluminescence immunoassay (CLIA), chemiluminescence enzyme immunoassay (CLEIA), electrochemiluminescence Examples include immunoassay (ECLIA), fluorescent immunoassay (FIA), western blotting, latex agglutination, immunochromatography, and the like. For example, after capturing TREM-1 protein in a biological sample using a solid phase carrier (for example, immunoplate or latex particles) to which an antibody is bound by physical adsorption or chemical bonding, it is immobilized on the solid phase carrier. What is necessary is just to quantify the TREM-1 protein captured using a labeled antibody (an antibody labeled with an enzyme or a fluorescent substance) having a different antigen recognition site from the antibody.

When the amount of TREM-1 protein is measured by immunoassay, the antibody used is not particularly limited as long as it is an antibody or a fragment thereof that can specifically bind to soluble TREM-1 protein. Examples of antibodies include monoclonal antibodies and polyclonal antibodies. Examples of antibody fragments include Fab fragments, F (ab) ′ ₂ fragments, single chain antibodies (scFv), and the like. In addition, these antigens may be labeled with enzymes, radioisotopes, fluorescent dyes, avidin, biotin and the like.

  For example, as a result of measuring the amount of TREM-1 protein in a biological sample, when the amount of TREM-1 protein is greater than a predetermined cut-off value, the subject has recurrent polychondritis. When it is determined that the possibility is high and the amount of the TREM-1 protein is smaller than a predetermined cut-off value, it is determined that the subject is highly likely not to have TREM-1 protein. The cut-off value of TREM-1 protein can be set using, for example, an ROC curve (see Examples).

  The test method of the present invention can rapidly and easily test relapsing polychondritis by using TREM-1 protein as a biomarker for relapsing polychondritis. The test method of the present invention is useful as an auxiliary test when a medical worker diagnoses whether relapsing polychondritis or not. The test method of the present invention can also be applied in the evaluation of disease activity of relapsing polychondritis, confirmation of therapeutic effects, monitoring of clinical course, and the like.

2. Test Kit for Recurrent Polychondritis of the Present Invention The test kit of the present invention is a kit for measuring the amount of TREM-1 protein in a biological sample by an immunoassay, and can be used for the test method of the present invention. it can.

  The test kit of the present invention has an antibody that can specifically bind to soluble TREM-1 protein. The test kit of the present invention detects the antigen-antibody reaction between this antibody and soluble TREM-1 protein, and measures the amount of TREM-1 protein in the biological sample.

The antibody is not particularly limited as long as it is an antibody or a fragment thereof that can specifically bind to soluble TREM-1 protein, and may be a commercially available antibody. Examples of antibodies include monoclonal antibodies and polyclonal antibodies. Examples of antibody fragments include Fab fragments, F (ab) ′ ₂ fragments, single chain antibodies (scFv), and the like. These antibodies may be immobilized on a carrier or may not be immobilized. In addition, these antigens may be labeled with enzymes, radioisotopes, fluorescent dyes, avidin, biotin and the like.

  Various test kits for measuring the amount of protein in a biological sample are commercially available depending on the type of protein. The test kit of the present invention can also be constituted by each element used in a test kit known to those skilled in the art, except that an antibody or fragment thereof that can specifically bind to soluble TREM-1 protein is used. it can.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail with reference to an Example, this invention is not limited by these Examples.

[Example 1]
Example 1 shows that serum TREM-1 concentration can be used as a biomarker for relapsing polychondritis.

  After completing the procedure for ensuring ethics, blood was collected from 16 patients with relapsing polychondritis and 16 healthy individuals to obtain serum (sample). Diagnosis of relapsing polychondritis was based on Damiani's diagnostic criteria (Damiani, JM and Levine, HL, "Relapsing polychondritis-report of ten cases", Laryngoscope, Vol.89, pp.929-946.) .

  The concentration of TREM-1 contained in each sample (serum) was measured using a commercially available ELISA kit (Human TREM-1 Quantikine ELISA kit; R & D Systems). The measurement procedure was performed as follows according to the protocol attached to the kit.

  First, 100 μL of a diluted solution (Assay Diluent RD1-52) was added to each well of a 96-well microplate (TREM-1 Microplate) on which a mouse anti-TREM-1 monoclonal antibody was immobilized. Next, 100 μL of sample (serum) or standard (TREM-1 Standard) was added to each well and allowed to react at room temperature for 2 hours. After washing 4 times, 200 μL of HRP-labeled anti-TREM-1 antibody solution (TREM-1 Conjugate) was added to each well and allowed to react at room temperature for 2 hours. After washing 4 times again, 200 μL of a substrate solution (Substrate Solution) was added to each well and allowed to react at room temperature for 30 minutes. After the reaction, 50 μL of Stop Solution was added to each well, and the absorbance of each well was measured at a wavelength of 450 nm. A standard curve was drawn using spreadsheet software (Excel; Microsoft), and the concentration of TREM-1 in each sample (serum) was calculated.

  The ROC curve shown in FIG. 1 was prepared using the data of serum TREM-1 concentration of each subject (recurrent polychondritis patient and healthy subject). The area under the ROC curve (AUC) was about 0.9. From this, it can be said that the serum TREM-1 concentration is excellent in sensitivity and specificity as a biomarker of relapsing polychondritis.

  FIG. 2 is a graph showing serum TREM-1 concentrations in healthy subjects and patients with relapsing polychondritis. The broken line in the figure indicates the cutoff value (155 pg / mL) obtained from the ROC curve in FIG. Here, the TREM-1 concentration in serum corresponding to the point at which the distance from the upper left corner in the graph of FIG. Thus, when the cutoff value is 155 pg / mL, the test can be performed with a sensitivity of 81.25% and a specificity of 87.5%.

[Example 2]
In Example 2, the serum concentration of patients with relapsing polychondritis and healthy subjects was measured for 25 types of inflammation-related factors (including TREM-1) such as cytokines (including chemokines) and matrix metalloproteinases. Indicates.

  The same serum (16 patients with relapsing polychondritis, 16 healthy subjects) as in Example 1 was prepared, and the concentration of inflammation-related factors contained in each sample (serum) was measured using a commercially available kit. .

  The concentration of TREM-1 was measured according to the protocol attached to the kit using the Human TREM-1 Quantikine ELISA kit (R & D Systems) in the same procedure as in Example 1.

  The concentration of IL-17 was measured using a Human IL-17A ELISA kit (Tepnel Life Sciences) according to the protocol attached to the kit.

  The concentration of MMP-1 and the concentration of MMP2 were measured using a Human Pro-MMP-1 Quantikine ELISA kit and a Human MMP-2 Quantikine ELISA kit (both R & D Systems) according to the protocol attached to the kit.

  About the density | concentration of MMP-3, it measured according to the protocol attached to a kit using Human MMP-3 ELISA kit (1st fine chemical).

  About the density | concentration of MMP-13, it measured according to the protocol attached to a kit using Human MMP-13 Assay (GE Healthcare).

  IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, TNF, IFN-γ, GM-CSF, MIP-1α, Concentrations of MIP-1β, MCP-1, IP-10, RANTES, VEGF, and fractalkine were measured by the following procedure using a Cytometric Bead Array Flex set system (BD Biosciences).

  First, the cytokine standards to be measured simultaneously are dissolved in a diluting solution (Assay Diluent) and combined into one, and then diluted series (0, 5, 10, 20, 40, 80, 156, 312.5, 625, 1250). 2500 pg / mL). On the other hand, each sample (serum) was diluted 4 times (RANTES only 40 times) with a diluted solution (Assay Diluent). Next, 50 μL of a mixture containing beads corresponding to cytokines to be measured simultaneously (Capture Beads) was placed in all FACS tubes. Sample dilutions and standards were placed in 50 μL aliquots sequentially into a FACS tube and incubated at room temperature for 1 hour. Subsequently, 50 μL of a mixture in which PE detection reagent for cytokine to be measured simultaneously was mixed was placed in all FACS tubes and incubated at room temperature for 2 hours under light-shielded conditions. After washing, data was measured using a flow cytometer (FACSCalibur; BD Biosciences). The obtained data was analyzed using analysis software (FCAP Array Software; BD Biosciences), and the concentration of cytokine in each sample was measured.

  As a result of measuring the concentration of 25 types of inflammation-related factors (including TREM-1) in each sample, 5 types of inflammation-related factors including TREM-1, TREM-1, MMP-3, VEGF, MIP-1β, IP-10) was significantly elevated in the group of patients with relapsing polychondritis (p <0.05). On the other hand, no statistically significant difference was observed for the remaining 20 inflammation-related factors (such as TNF-α and IL-6).

  3A to E show healthy subjects of five types of inflammation-related factors (TREM-1, MMP-3, VEGF, MIP-1β, IP-10) that were significantly elevated in the group of patients with relapsing polychondritis FIG. 3F is a graph showing serum concentrations in patients with relapsing polychondritis, and FIG. 3F shows healthy subjects and relapsing polycartilage of inflammation-related factor (MCP-1) for which no statistically significant difference was observed. It is a graph which shows the density | concentration in serum in the patient of a flame. FIG. 3A is a graph showing serum TREM-1 concentration (p <0.0001), FIG. 3B is a graph showing serum MMP-3 concentration (p = 0.0083), and FIG. FIG. 3D is a graph showing serum VEGF concentration (p = 0.123), FIG. 3D is a graph showing serum MIP-1β concentration (p = 0.0041), and FIG. 3E is serum IP-10 concentration. (P = 0.413) and FIG. 3F is a graph showing serum MCP-1 concentration (p = 0.6652).

  FIG. 4 is an ROC curve for the six types of inflammation-related factors (TREM-1, MMP-3, VEGF, MIP-1β, IP-10, MCP-1). From this graph, among the five types of inflammation-related factors (TREM-1, MMP-3, VEGF, MIP-1β, IP-10) that were significantly elevated in the group of patients with relapsing polychondritis, TREM- 1 is the most excellent biomarker for relapsing polychondritis both in sensitivity and specificity.

[Example 3]
Example 3 shows the results of measuring serum TREM-1 concentration in patients with other autoimmune diseases (systemic scleroderma and HTLV-I related myelopathy) as well as patients with relapsing polychondritis .

  About the serum of patients with relapsing polychondritis and healthy subjects, the same sera as in Example 1 (16 patients with relapsing polychondritis, 16 healthy subjects) were prepared. Moreover, about the serum of the patient with systemic scleroderma and the patient with HTLV-I related myelopathy, after completing the procedure for ensuring ethics, 10 patients with systemic scleroderma and HTLV-I related Blood was collected from 14 patients with myelopathy and serum (sample) was obtained. Diagnosis of systemic scleroderma and HTLV-I related myelopathy was made based on the results of clinical diagnosis.

  The concentration of TREM-1 contained in each sample (serum) was measured in the same procedure as in Example 1 using a commercially available ELISA kit (Human TREM-1 Quantikine ELISA kit; R & D Systems).

  FIG. 5 is a graph showing serum TREM-1 levels in healthy subjects, patients with relapsing polychondritis, patients with systemic scleroderma, and patients with HTLV-I-related myelopathy. As shown in this graph, in patients with relapsing polychondritis, the serum TREM-1 concentration is not only for healthy subjects but also for patients with systemic scleroderma and HTLV-I-related myelopathy. There was a significant increase (p <0.05).

  From the above results, it can be seen that the serum TREM-1 concentration hardly increases in patients with other autoimmune diseases and can be used as a specific marker for relapsing polychondritis.

The method for examining relapsing polychondritis according to the present invention does not require large-scale equipment and has little burden on the subject, and is thus useful, for example, as a diagnostic auxiliary blood test for relapsing polychondritis.

Claims (4)

To test the relapsing polychondritis, how you measure the amount TREM-1-proteins in a biological sample collected from a subject. The biological sample is serum, methods who claim 1. The amount of the TREM-1 protein is determined by immunoassay, method who claim 1.   A test kit for relapsing polychondritis, comprising an anti-TREM-1 antibody or a fragment thereof.