KR102573606B1 - Composition and kit for distinguishing between rheumatic disease and ankylsoing spondylitis using serum - Google Patents

Abstract

The present invention relates to a composition capable of distinguishing a patient suffering from ankylosing spondylitis among rheumatoid disease patients by confirming the expression of caspase-1 in a serum sample. By confirming the expression level of -1, it is possible to distinguish ankylosing spondylitis among rheumatic diseases. Since only ankylosing spondylitis can be significantly diagnosed, the present invention is expected to help establish a treatment strategy for ankylosing spondylitis at an early stage.

Description

Composition and kit for distinguishing between rheumatic disease and ankylosing spondylitis using serum}

The present invention relates to a composition capable of distinguishing a patient suffering from ankylosing spondylitis among rheumatoid disease patients by confirming the expression of caspase-1 in a serum sample, and more specifically, caspase-1 in the separated serum sample. It relates to a composition for diagnosing rheumatoid disease and ankylosing spondylitis, including an agent for measuring the expression level of caspase-1, and a kit containing the same.

Ankylosing spondylitis (AS) is a chronic autoimmune disease affecting the axial skeleton and peripheral joints. Several etiologies have been proposed for ankylosing spondylitis, but no definitive cause has been identified so far. Activated caspase-1 in the nucleotide-binding oligomerization domain-like receptor pyrin domain-containing-3 (NLRP3) inflammasome -Converts interleukin ((IL)-1β) and pro-IL-18 to their biologically active forms, IL-1β and IL-18, respectively. Assuming that ankylosing spondylitis is an inflammatory disease, some inflammasomes activated during the immune response, particularly the NLRP3 inflammasome, appear to contribute to the development and progression of ankylosing spondylitis. However, there is a lack of research on a technique capable of significantly diagnosing only ankylosing spondylitis using the inflammasome-related marker when a rheumatoid disease occurs.

Under the above background, the present inventors have made efforts to study a method for specifically diagnosing ankylosing spondylitis among various rheumatoid diseases, and as a result, the expression level of caspase-1 in serum is very high in ankylosing spondylitis, which The present invention was completed by confirming that only ankylosing spondylitis among rheumatoid diseases can be specifically diagnosed through

Accordingly, the present invention is a method for diagnosing and distinguishing between rheumatic disease and ankylosing spondylitis, including an agent for measuring the expression level of caspase-1 in a separated serum sample. It aims to provide a composition.

In addition, another object of the present invention is to provide a kit for diagnosing a rheumatoid disease (rheumatic disease) and ankylosing spondylitis (ankylsoing spondylitis) comprising the above composition.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention as described above, the present invention, including a preparation for measuring the expression level of caspase-1 (caspase-1) in the separated serum sample, rheumatoid disease (rheumatic disease) and spasticity A composition for distinguishing and diagnosing spondylitis (ankylsoing spondylitis) is provided.

In one embodiment of the present invention, the rheumatoid disease may be gout or rheumatoid arthritis.

In addition, the present invention provides a kit for diagnosing rheumatoid disease (rheumatic disease) and ankylosing spondylitis (ankylsoing spondylitis) containing the composition.

The composition of the present invention can distinguish ankylosing spondylitis among rheumatoid diseases by checking the expression level of caspase-1 in serum. Since only ankylosing spondylitis can be significantly diagnosed, the composition of the present invention is expected to help establish a treatment strategy for ankylosing spondylitis at an early stage.

However, the effects of the present invention are not limited to the above effects, and should be understood to include all effects that can be inferred from the detailed description of the present invention or the configuration of the invention described in the claims.

The present inventors confirmed that the expression level of caspase-1 in serum was very high in ankylosing spondylitis, and through this, it was confirmed that only ankylosing spondylitis among rheumatoid diseases could be specifically diagnosed, thereby completing the present invention.

Accordingly, the present invention is a method for diagnosing and distinguishing between rheumatic disease and ankylosing spondylitis, including an agent for measuring the expression level of caspase-1 in a separated serum sample. composition is provided.

The rheumatoid disease is not limited thereto, but is not limited to, psoriatic arthritis, tenosynovitis, ankylosing spondylitis, systemic lupus erythematosus, gout or It may be rheumatoid arthritis, preferably ankylosing spondylitis, gout or rheumatoid arthritis.

In the present invention, the composition of the present invention can diagnose rheumatic diseases and ankylosing spondylitis, except for the ankylosing spondylitis.

The present inventors confirmed through specific examples that rheumatoid disease and ankylosing spondylitis can be differentiated and diagnosed through a method for confirming the expression level of caspase-1 in serum.

In one embodiment of the present invention, it was confirmed that the serum caspase-1 expression level was significantly higher in patients with ankylosing spondylitis than patients suffering from rheumatic diseases (gout and rheumatoid arthritis). In addition, these results are based on the high expression of caspase-1 in patients with high CRP levels (group suffering from rheumatoid diseases) through a separate sample, in the case of patients suffering from rheumatoid diseases, caspase Although the expression of -1 increases, the expression level of serum caspase-1 in patients with ankylosing spondylitis increases significantly, so it was confirmed that rheumatic diseases excluding ankylosing spondylitis and ankylosing spondylitis can be diagnosed separately (Example 3 reference).

Through the above results, the present inventors can specifically diagnose only ankylosing spondylitis among rheumatoid diseases with similar symptoms even by checking the caspase-1 expression level of serum, which is most commonly collected and tested in daily practice. confirmed that there is Therefore, the composition of the present invention and a kit containing the same can be usefully utilized for the diagnosis of ankylosing spondylitis, and it is expected that an ankylosing spondylitis treatment strategy can be established early through this.

As another aspect of the present invention, the present invention provides a kit for diagnosing rheumatoid disease (rheumatic disease) and ankylosing spondylitis (ankylsoing spondylitis) comprising the above composition.

The kit of the present invention may include not only an agent capable of measuring the expression level of caspase-1 in a serum sample, but also one or more types of compositions, solutions, or devices suitable for analyzing the expression level. For example, the kit may include a substrate, an appropriate buffer solution, a secondary antibody labeled with a detection label, and a chromogenic substrate for immunological detection of the antibody.

In addition, it may further include a secondary antibody capable of detecting the antibody or aptamer. The secondary antibody is not particularly limited thereto, but can bind to the caspase-1 protein capable of binding to the protein chip. It can be used by being labeled with a fluorescent substance, a radioactive isotope, etc.

Specifically, the kit may be a kit characterized in that it includes essential elements required to perform ELISA in order to implement various ELISA methods such as an ELISA kit and a sandwich ELISA. The ELISA kit may include antibodies or aptamers specific to the proteins. The antibody has high specificity and affinity for the caspase-1 protein and little cross-reactivity to other proteins, and may be a monoclonal antibody, a polyclonal antibody, or a recombinant antibody. Also, the ELISA kit may include an antibody or aptamer specific for a control protein. Other ELISA kits may include reagents capable of detecting bound antibodies, such as labeled secondary antibodies, chromophores, enzymes and their substrates, or other substances capable of binding to antibodies.

In addition, the kit may include Western blot, enzyme linked immunosorbent assay (ELISA), immunoprecipitation assay, complement fixation assay, flow cytometry (Fluorecence Activated Cell Sorter, FACS), or It may be a kit for implementing a protein chip, etc., and may further include additional components suitable for each analysis method. Through these analysis methods, it is possible to accurately diagnose only ankylosing spondylitis disease among rheumatoid diseases by comparing the amount of antigen-antibody complex formation, and accordingly, it is possible to provide a customized treatment method for ankylosing spondylitis patients.

Hereinafter, a preferred embodiment is presented to aid understanding of the present invention. However, the following examples are provided to more easily understand the present invention, and the content of the present invention is not limited by the following examples.

[Example]

Example 1. Experiment preparation and method

For the experiment, between June 2017 and August 2018 at Keimyung University Dongsan Hospital in Daegu, 126 study participants (20 patients with ankylosing spondylitis, 23 patients with gout, 62 patients with rheumatoid arthritis) (rheumatoid arthritis, RA) patients and 21 healthy controls) were enrolled and the study was conducted. The study was approved by the institutional review board (IRB; 2017-06-021) and was conducted in accordance with the Declaration of Helsinki. Consent related to the experiment was obtained from all participants. A blood sample was collected from each patient prior to initiation of medical treatment. Patients with rheumatic disease consisted of patients who met the 1984 New York criteria for ankylosing spondylitis (Arthritis Rheum 1984; 27:361-8). In the case of rheumatoid arthritis patients, patients who met the 2010 rheumatoid arthritis classification criteria (Arthritis Rheum 2010; 62:2569-81) were included. Gout patients were composed of patients suffering from acute gout according to classification (Arthritis Rheum 1977; 20:895-900). The control group consisted of healthy health care workers. We collected age, sex, disease duration, and blood chemistry data from all participants.

A second cohort of 22 ankylosing spondylitis patients was provided by an outpatient rheumatology clinic for rheumatoid disease at Korea University Hospital, and their serum caspase-1 levels were compared with indicators of ankylosing spondylitis disease activity. compared. The study was approved by the IRB of Korea University Hospital (2008-09-001). Written informed consent was obtained from all patients. Blood samples were collected between September 2017 and March 2019 from patients with ankylosing spondylitis.

Example 2. Confirmation of serum caspase-1 expression level in rheumatoid disease

The present inventors measured serum caspase-1 level, serum erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), and the Bath Ankylosing Spondylitis Disease Activity Index. and Ankylosing Spondylitis Disease Activity Score. Caspase-1, IL-1β, IL-18, and NLRP3 levels were measured using ELISA kits (Quantikine, R&D Systems, Minneapolis, Minn., USA; Aviva Systems Biology, San Diego, Calif., USB). The results obtained through this method were performed using statistical analysis.

Analysis results were expressed as median (interquartile range) or frequency (%). For continuous variables, the Mann-Whitney U test was used to compare the high CRP group (high expression of CRP in rheumatoid disease) to the normal CRP group, and the Kruskal-Wallis test was used to compare ankylosing spondylitis, gout, rheumatoid arthritis and healthy A control group was used for comparison between groups. For categorical variables, Fisher's exact test was used to compare the frequency of high levels of caspase-1 (>125 pg/mL) between groups. P < 0.05 was considered statistically significant.

Example 3. Diagnosis of ankylosing spondylitis according to serum caspase-1 expression level

The results obtained through Example 2 are shown in Table 1. P values showing statistically significant values are shown in bold, and ψP values were determined using the Kruskal-Wallis test.

Feature Ankylosing spondylitis (N=20) Gout
(N=23) Rheumatoid arthritis (N=62) Healthy controls
(N=21) P Age (yr) 35.5 (21.0) 56.0 (25.0) 61.5 (9.0) 41.0 (23.0) <0.001 Sex (M/F) 18/2 20/3 24/38 1/20 Sex (M/F) ESR (mm/hr) 20.0 (41.5) 43.5 (30.0) (N=10) 40.5 (56.0) NA 0.316 CRP (mg/dL) 0.2 (1.6) 2.41 (8.14) (N=10) 0.32 (1.75) NA 0.020 WBC (×109/L) 7.1 (2.6) 8.2 (6.0) (N=11) 7.6 (3.8) NA 0.460 Caspase-1 (pg/mL) 201.1 (109.8) 137.3 (91.7) 144.6 (150.8) 71.5 (72.9) <0.001 IL-1 (pg/mL) 2.39 (3.95) 4.1 (1.9) 2.1 (3.5) 6.1 (2.5) <0.001 IL-18 (pg/mL) 1,585 (127) 1,520 (176) 1,572 (118) 1,695 (199) 0.014 NLRP3 (pg/mL) 0.2 (0.3) 0.5 (1.0) 0.6 (1.3) 0.0 (0.1) <0.001 Caspase-1 >125 pg/mL‡ 16 (80.0%) 15 (65.2%) 39 (62.9%) 3 (14.3%) <0.001

It was confirmed that the mean serum caspase-1 level was significantly higher in the ankylosing spondylitis patient group than in the other groups (P<0.001). The frequency of caspase-1 levels > 125 pg/mL was also higher in the AS group than in gout, rheumatoid arthritis and healthy controls.

The present inventors compared serum caspase-1 levels according to high and normal CRP levels in independent sample sets, and the results are shown in Table 2 below.

Feature* High CRP (≥0.8 mg/L) (N=12) Normal CRP (<0.8 mg/L) (N=10) P Age (yr) 29.5 (4.0) 30.0 (9.0) 0.205 Sex (M/F) 11/1 10/0 Sex (M/F) Disease duration (months) 21.0 (85.5) 33.5 (86.0) 0.865 ESR (mm/hr) 42.5 (34.5) 4.5 (7.0) <0.001 Caspase-1 (pg/mL) 86.7 (130.1) 32.1 (56.2) 0.041 BASDAI 5.1 (2.1) 1.9 (3.4) 0.021 ASDAS-CRP 3.8 (1.0) 1.8 (1.8) 0.002 Peripheral arthritis 4 (33%) 5 (50%)

Serum caspase-1 level was significantly higher in the group showing high CRP than in the group showing normal CRP level (P = 0.041).

Through the above results, the present inventors confirmed that a higher caspase-1 expression level was shown in the group showing higher CRP levels due to inflammation than in patients with other diseases and healthy controls, and among several diseases related to this, spasticity The highest expression level of caspase-1 was confirmed in the serum of patients with spondylitis. Through this, it was confirmed that ankylosing spondylitis and other rheumatic diseases can be distinguished by measuring the level of caspase-1 in serum.

The description of the present invention described above is for illustrative purposes, and those skilled in the art can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. There will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not limiting.

Claims (3)

Measuring the expression level of caspase-1 in a serum sample isolated from the patient; and
If the expression level of caspase-1 is 180 pg/mL or more, judging it as ankylosing spondylitis rather than a rheumatic disease; A method for providing information to differentiate between ankylosing spondylitis and ankylosing spondylitis.
According to claim 1,
The method of providing information, characterized in that the rheumatoid disease is gout or rheumatoid arthritis.
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