Active il-18 detection kit and method for running test

Abstract

To provide an easy method and a kit for detecting active IL-18 alone.SOLUTION: There are constructed a method and a kit for immunologically measuring active IL-18 alone by using an antibody which recognizes a neoepitope generated in association with activation of IL-18. Examinations were made by using a plurality of bodies which recognizes activation cross edges of IL-18 and a plurality of antibodies which specifically recognizes IL-18 and the inventors have found that the combination of a monoclonal antibody 9-10.2 which recognizes activation cross edges of IL-18 and a rabbit-derived polyclonal antibody which specifically recognizes IL-18 detects active IL-18 the most sensitively. The immunoassay and the measurement kit by that combination can detect active IL-18 in concentration as low as a pg level.SELECTED DRAWING: Figure 7

Description

Technical Field

[0001] The present invention relates to a kit that specifically recognizes active IL-18 and a detection method. In particular, it relates to an ELISA kit widely used clinically.

Background Art

[0002] IL-18 has been suggested to be involved in various diseases. IL-18 is a cytokine of the IL-1β family and is mainly expressed in macrophages, but is also expressed in various cells such as dendritic cells, epithelial cells, and keratinocytes. In addition, the IL-18 receptor is expressed in various cells such as NK cells, NKT cells, CD4 T cells, as well as B cells, neutrophils, macrophages, vascular endothelial cells, and smooth muscle cells. The diversity of IL-18-expressing cells and receptor-expressing cells indicates the diversity of IL-18 functions and is known to be involved in various immune systems.

[0003] Due to its extensive involvement with the immune system, IL-18-related diseases that are caused or exacerbated by overexpression of IL-18 include a variety of diseases. For example, adult-onset Still's disease (AOSD), systemic juvenile idiopathic arthritis (sJIA), pancreatic cancer, lung cancer, colorectal cancer and other malignant tumors, cryopyrin-associated periodic syndrome, systemic lupus erythematosus (SLE), multiple sclerosis, juvenile idiopathic arthritis (JIA), bronchial asthma, bronchiectasis, chronic obstructive pulmonary disease (COPD), transfusion-related acute lung injury, bronchopulmonary dysplasia (BPD), acute respiratory distress syndrome (ARDS), interstitial lung disease (ILD), idiopathic pulmonary fibrosis, cystic fibrosis, rheumatoid arthritis, metabolic bone disease, severe organ damage in the liver and intestine, heart failure, amyotrophic lateral sclerosis (ALS), dry eye disease (DED), keratitis, corneal neovascularization, pathologic intraocular neovascularization, iritis, glaucoma, macular degeneration, Sjögren's syndrome, autoimmune uveitis, Behçet's disease, conjunctivitis, allergic conjunctivitis, eyelid dermatitis, allergic rhinitis, type 2 diabetes, non-alcoholic fatty liver disease (NAFLD), steatohepatitis, ischemia-reperfusion injury, familial Mediterranean fever, TNF receptor-associated periodic syndrome, hyper IgD syndrome, gout, Schnitzler's syndrome, microscopic polyangiitis, granulomatosis with polyangiitis, eosinophilic granulomatosis with polyangiitis, ANCA-associated vasculitis, Hashimoto's disease, Crohn's disease, ulcerative colitis, immunoglobulin G4 (IgG4)-related disease, pulmonary hypertension, atopic dermatitis, NLRC4-related autoinflammatory disease (or NLRC4 inflammasome disease), etc. are said to be included.

[0004] Unlike cytokines such as TNF, IL-18 is not regulated at the mRNA level in many cells and is abundantly present intracellularly as an inactive precursor (pro-IL-18). Pro-IL-18 becomes active and is released extracellularly by cleavage of the precursor by caspase 1 or caspase 4. Therefore, even if the mRNA expression level or the intracellular precursor protein is measured, the activity of IL-18 cannot be measured. To analyze the function of IL-18, it is essential to analyze the active form of IL-18 cleaved by caspase.

[0005] As described above, there are many diseases in which the involvement of IL-18 is suggested. However, not only active IL-18 has been measured, but many cases have only detected precursor IL-18, and the details of the degree of its involvement have not been clarified. Conventionally, attempts have been made to distinguish active and precursor IL-18 by molecular weight using Western blotting and detect active IL-18. However, with Western blotting, it is impossible to detect IL-18 contained at only pg levels in 1 ml, and even in diseases where the involvement of IL-18 is suspected, the presence of active IL-18 has not been examined in the clinical setting.

[0006] The present inventors have prepared an antibody that recognizes only active IL-18 and disclosed its application to the treatment of inflammatory bowel disease (Patent Documents 1 and 2). Analyzing the increase in active IL-18 in IL-18-related diseases leads to predicting the therapeutic effect of antibody drugs capable of neutralizing active IL-18, so a method for accurately and simply measuring active IL-18 in patient serum is required. Therefore, it has been required to detect only active IL-18 by a method widely used clinically, such as ELISA, by using an antibody that recognizes only active IL-18. However, in reality, a kit for sensitively detecting active IL-18 could not be constructed.

[0007] Despite using an antibody that recognizes active IL-18, the inability to accurately detect active IL-18 is thought to be due not only to the low concentration of active IL-18 contained in the specimen but also to the large amount of precursor IL-18 contained. Therefore, even when using an antibody that recognizes only active IL-18, the detection sensitivity was low and it could not be put into practical use as a clinical test.

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Patent Document 2

[0009] As described above, there has been no simple method for measuring only active IL-18 until now. An object of the present invention is to provide a simple method and kit capable of detecting only active IL-18. [Means for Solving the Problems]

[0010] Active IL-18 can be detected with high sensitivity by the following detection kit and detection method. (1) A test kit for specifically detecting only active IL-18, which is detected by an immunological measurement method using a monoclonal antibody that recognizes the active fragment of IL-18 and an antibody that recognizes a region other than the active fragment of active IL-18. Specifically detecting only active IL-18 has been required for analyzing the involvement of active IL-18 in IL-18-related diseases and for future treatment using active IL-18 antibodies. However, since the concentration of active IL-18 in serum is very low and a large amount of precursor is contained in cells, no kit has been put into practical use. The present inventors prepared a monoclonal antibody that recognizes the active fragment of IL-18, and thus succeeded in developing a kit.

[0011] (2) The test kit according to (1), wherein the monoclonal antibody that recognizes the active fragment of IL-18 is the 9-10.2 antibody, and the antibody that recognizes a region other than the active fragment of active IL-18 is a polyclonal antibody. There has been no simple kit that can specifically detect only active IL-18 until now. The inventors of the present invention examined combinations of multiple antibodies that recognize the active fragment of IL-18 and multiple antibodies that recognize regions other than the fragment, and studied combinations that can detect active IL-18 with high sensitivity. As a result, it was revealed that by combining the 9-10.2 antibody, which is an antibody that recognizes the active fragment, and a polyclonal antibody, active IL-18 can be detected with high sensitivity, and a kit was constructed.

[0012] (3) The test kit according to (2), wherein the test kit is an ELISA kit, and when the 9-10.2 antibody is used as the immobilized antibody, the polyclonal antibody is used as the detection antibody, and when the polyclonal antibody is used as the immobilized antibody, the 9-10.2 antibody is used as the detection antibody. With the combination of the above two antibodies, active IL-18 can be detected with high sensitivity and specificity. By using it as a widely used ELISA kit, it is also useful as a clinical test kit for tests for IL-18-related diseases and the like.

[0013] (4) A method for specifically detecting only active IL-18, characterized by detecting by an immunoassay using a monoclonal antibody that recognizes the active fragment of IL-18 and an antibody that recognizes a region other than the active fragment of active IL-18. There is no simple method for specifically detecting only active IL-18, and there has been a demand for a method that can be easily examined clinically, such as for the examination of IL-18-related diseases. In addition, when an anti-active IL-18 antibody is used as a pharmaceutical composition in the future, it is necessary to measure the concentration of active IL-18 in the patient's serum. By combining an antibody that recognizes the active fragment and an antibody that recognizes other regions, only active IL-18 can be detected.

[0014] (5) The detection method according to (4), wherein the monoclonal antibody that recognizes the active fragment of IL-18 is the 9-10.2 antibody, and the antibody that recognizes a region other than the active fragment of active IL-18 is a polyclonal antibody. By combining the 9-10.2 antibody that recognizes the active fragment and a polyclonal antibody, active IL-18 can be detected with high sensitivity and simply.

[0015] (6) The detection method is ELISA. When the 9-10.2 antibody is used as the immobilized antibody, the polyclonal antibody is used as the detection antibody. When the polyclonal antibody is used as the immobilized antibody, the 9-10.2 antibody is used as the detection antibody. The detection method according to (5), characterized in that. Since ELISA is widely used both clinically and in research settings, this method that can be detected by ELISA is a very useful method.

Brief Description of the Drawings

[0016]

Figure 1

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Figure 7

Modes for Carrying Out the Invention

[0017] Active IL-18 refers to the IL-18 molecule (SEQ ID NO: 2) in which the precursor pro-IL-18 (uniprot: Q14116, SEQ ID NO: 1) is cleaved by caspase 1 / 4 into a peptide from position 37 to position 193. An antibody that recognizes only active IL-18 refers to an antibody that recognizes the cleavage end sequence generated by cleavage by caspase 1 / 4, i.e., a neoepitope. In this specification, an antibody that recognizes only active IL-18 is referred to as an antibody that recognizes an active cleavage end, or an antibody that recognizes a neoepitope, or simply an activated cleavage end antibody.

[0018] In addition, although the construction of the ELISA kit is specifically disclosed in the specification, any method may be used as long as it is a method of testing by combining the following two types of antibodies. As the solid phase, beads may be used instead of plates, and as the detection system, antibodies labeled with radioisotopes or fluorescent dyes may also be used in addition to antibodies labeled with enzymes. Needless to say, known methods used in this field, such as color development using chromogenic substrates and luminescent substances, can be utilized.

[0019] In addition, as shown below, a rabbit polyclonal antibody that specifically binds to IL-18 is used in the construction of the sandwich ELISA kit. However, any polyclonal antibody may be used, and polyclonal antibodies obtained by immunizing animals other than rabbits, such as goats and sheep, can also be used.

[0020] [Construction of Human Active IL-18 ELISA Kit] 1. Selection of Activated Cleavage End Antibody To construct a human active IL-18 measurement ELISA kit, the selection of activated cleavage end antibodies was carried out. A competition experiment against the activated cleavage end peptide (YFGKLESKC, SEQ ID NO: 3) was performed using several types of monoclonal antibodies that recognize the human IL-18 active cleavage end. Here, as an example, the results obtained using antibodies 9-10.2 and 8-4.1 are shown (Figure 1). The activated cleavage end peptide and BSA were cross-linked, and 50 mM NaHCO 3It was adjusted to 5 ng / 100 μl using [relevant reagent] and coated on an ELISA plate at 4°C overnight. The ELISA plate was blocked and washed by conventional methods, and 9-10.2 or 8-4.1 antibody solutions adjusted to 0, 100, 300, 1000 ng / 100 μl were added, and the reaction was carried out at 25°C for 1 hour. Only the washing solution was added to the wells without antibody.

[0021] Next, each HRP-labeled antibody (0.5 μg / μl) was diluted 500-fold with the washing solution. 100 μl of HRP-labeled 9-10.2 antibody was added to the wells where the 8-4.1 antibody was bound to the peptide as the primary antibody, and 100 μl of HRP-labeled 8-4.1 antibody was added to the wells where the 9-10.2 antibody was bound to the peptide, and incubated at 25°C for 1 hour. Then, washing was performed, color development was carried out, and the absorbance was measured (upper part of Figure 1). It was shown that both antibodies bound to the peptide and competed with each other, but it was shown that the 9-10.2 antibody bound more strongly to the peptide. In addition, when a competitive experiment was conducted between a monoclonal antibody against the G196 tag (DLVPR, SEQ ID NO: 4, Patent Document 3) (mAbProtein Co., Ltd.) and the 9-10.2 antibody, no competitive inhibition was observed for the antibody against the G196 tag, which is a sequence unrelated to IL-18 (lower part of Figure 1).

[0022] 2. Examination of antibody combinations From the above results, as an antibody that recognizes active IL-18, the more sensitive 9-10.2 antibody was used as the detection antibody or the immobilized antibody, and the examination of the antibody to be combined was carried out.

[0023] (1) Examination of the combination with the 9-10.2 antibody as the detection antibody First, the highly sensitive 9-10.2 antibody was used as the detection antibody, and the 11-4.1, 7-6.3, 7-2.5, 9-4.2, 4-18.1, and 7-12.3 antibodies that recognize the amino acid sequence from position 58 to position 72 of IL-18, and the 6-2.6 and 12-7.6 antibodies that recognize the amino acid sequence from position 128 to position 142 were used as the immobilized antibodies to examine the detection sensitivity (Figure 2). The ELISA plate was coated overnight at 4°C with the immobilized antibody adjusted to 0.5 μg / 100 μl or 1 μg / 100 μl. Next, it was reacted with the active IL-18 protein adjusted to 2 ng / 100 μl. After reacting with the HRP-labeled 9-10.2 (0.5 μg / μl diluted 500-fold) antibody solution at 25°C for 1 hour, an enzyme substrate was added and measurement was performed in the same manner as above. The active IL-18 protein was obtained by mixing the precursor IL-18 protein expressed and purified in E. coli with the active caspase-4 also expressed and purified in E. coli. 105-377 Also, the ELISA plate was blocked by a conventional method, and washing was performed with a washing solution between each step. As shown in Figure 2, when the 9-10.2 antibody was used as the detection antibody, the highest detection sensitivity was obtained when the 7-12.3 antibody was used as the immobilized antibody.

[0024] (2) Examination of the combination with the 9-10.2 antibody as the immobilized antibody Next, the immobilized antibody was fixed to the 9-10.2 antibody, and the 11-4.1, 9-4.2, 4-18.1, and 7-12.3 antibodies that recognize the amino acid sequence from position 58 to position 72 of IL-18 and the 6-2.6 antibody that recognizes the amino acid sequence from position 128 to position 142 as the detection antibody were HRP-labeled and examined in the same manner as above using them as the detection antibody (Figure 3). Also when the 9-10.2 antibody was used as the immobilized antibody, the highest detection sensitivity was obtained when combined with the 7-12.3 antibody.

[0025] (3) Examination of the combination of the rabbit polyclonal antibody and the 9-10.2 antibody Since polyclonal antibodies are a mixture of antibodies that detect multiple epitopes, their detection power is often higher than that of monoclonal antibodies. Therefore, the detection sensitivity was examined using a combination of polyclonal antibodies and 9-10.2 antibodies. The polyclonal antibody was obtained by immunizing rabbits with IL-18 protein from the activated fragment to the C-terminus and purifying it with Protein A (mAbProtein Co., Ltd.). As a comparison, monoclonal antibody 7-12.3, which had the highest detection sensitivity in the above examination results, was used.

[0026] The detection antibody was 9-10.2 antibody, and the combination with the highest detection sensitivity in the above experimental results, 7-12.3 antibody, and the IL-18 polyclonal antibody were used as the immobilized antibodies, and the measurement was performed in the same manner as above (Figure 4). As a result, when the polyclonal antibody was used as the immobilized antibody, the detection sensitivity was higher than when the 7-12.3 antibody was used. The combination of the IL-18 specific rabbit polyclonal antibody as the immobilized antibody and the 9-10.2 antibody as the detection antibody was the best combination. The amino acid sequence of the H chain (SEQ ID NO: 5) and the amino acid sequence of the L chain (SEQ ID NO: 6) of the 9-10.2 antibody are as already determined and disclosed.

[0027] Active IL-18 serially diluted from 5000 pg / ml or 1000 pg / ml was measured using the IL-18 specific rabbit polyclonal antibody as the immobilized antibody and the 9-10.2 antibody as the detection antibody. The absorbance was analyzed using the analysis software Prism (GraphPad Software) to analyze the correlation between the absorbance and the concentration (Figure 5). Active IL-18 could be detected at a concentration of 50 pg / ml or less. By creating a calibration curve using active IL-18 as a standard substance, the concentration of active IL-18 in the sample can be quantified. This ELISA can also detect changes in the pg concentration of active IL-18. Therefore, it becomes possible to measure and analyze active IL-18 in patient serum clinically.

[0028] Next, it was measured whether the ELISA kit constructed above could detect active IL-18 in patient samples.

[0029] [Example 1] Measurement of Active IL-18 in the Serum of Patients with Polymyositis / Dermatomyositis (PM / DM) Using the ELISA kit constructed above, the concentrations of active IL-18 in the sera of 152 patients with polymyositis / dermatomyositis and 34 healthy subjects were measured (Figure 6). The serum IL-18 concentrations of each group were compared between the two groups by the Mann-Whitney U test. As a result, a significant difference in the active IL-18 concentration was observed between polymyositis / dermatomyositis (IIM in Figure 6) and healthy subjects (HC in Figure 6) (P < 0.0001). Although not shown here, although no statistical significance was found, the concentration of active IL-18 in the sera of patients with interstitial lung disease (ILD) complicated cases (125 cases) in polymyositis / dermatomyositis tended to be higher than that in the sera of patients without ILD complication (27 cases).

[0030] [Example 2] Measurement of Active IL-18 in the Serum of Crohn's Disease Patients Resistant to Anti-TNF-α Antibody Preparations Sera of 31 active Crohn's disease patients resistant to anti-TNF-α antibody preparations and receiving anti-IL12 / 23 antibody preparation (ustekinumab) were used. They were classified into responder (17 cases) and non-responder (14 cases) according to the therapeutic responsiveness to anti-IL12 / 23 antibody. Responders were defined as patients whose Clinical Disease Activity Index (CDAI) decreased by 100 or more compared with that before treatment initiation at 24 weeks after treatment initiation, or patients with a CDAI of less than 150. The patient background is as shown in Table 1. Before treatment initiation, non-responders had a significantly lower CDAI score and a higher serum hematocrit compared with responders.

[0031]

Table 1

[0032] The active IL-18 concentrations of responders and non-responders were measured using this ELISA kit (Figure 7). The concentration of active IL-18 in the sera of non-responders was significantly higher (P<0.01) compared to that of responders. This result suggests that treatment with anti-active IL-18 antibody is effective for patients (non-responders) who are resistant to anti-TNF-α antibody formulations and show no therapeutic effect against anti-IL12 / 23 antibody.

[0033] As shown above, active IL-18 can be detected with high sensitivity using a combination of rabbit polyclonal antibody and 9-10.2 antibody. So far, there has been no simple method for detecting active IL-18, and this method will be a very useful tool for patient selection in future treatment with anti-active IL-18 antibody. In addition, although there are many diseases conventionally referred to as IL-18-related diseases, there are still many unclear points regarding the involvement of IL-18. It is expected that the use of this kit will also advance research on the onset of IL-18-related diseases. Furthermore, since the sequence of the IL-18 activation cleavage fragment is the same in monkeys, it can also be applied to experimental animals, monkeys.

Claims

1. An assay kit for specifically detecting only active IL-18, comprising: a monoclonal antibody that recognizes the active fragment of IL-18, and an antibody that recognizes a region other than the active fragment of active IL-18, and detecting by an immunoassay method.

2. The monoclonal antibody that recognizes the active fragment of IL-18 is the 9-10.2 antibody, and The antibody according to claim 1, wherein the antibody that recognizes a region other than the active fragment of active IL-18 is a polyclonal antibody.

3. The above assay kit is an ELISA kit, When the 9-10.2 antibody is used as the solid-phase antibody, the polyclonal antibody is used as the detection antibody, and The assay kit according to claim 2, wherein when the polyclonal antibody is used as the solid-phase antibody, the 9-10.2 antibody is used as the detection antibody.

4. A method for specifically detecting only active IL-18, comprising: a monoclonal antibody that recognizes the active fragment of IL-18, and detecting by an immunoassay method using an antibody that recognizes a region other than the active fragment of active IL-18.

5. The monoclonal antibody that recognizes the active fragment of IL-18 is the 9-10.2 antibody, and The detection method according to claim 4, wherein the antibody that recognizes a region other than the active fragment of active IL-18 is a polyclonal antibody.

6. The above detection method is ELISA, When the 9-10.2 antibody is used as the solid-phase antibody, the polyclonal antibody is used as the detection antibody, and The detection method according to claim 5, wherein when the polyclonal antibody is used as the solid-phase antibody, the 9-10.2 antibody is used as the detection antibody.

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