Compounds bound to free interleukin-18, methods for their production, and kits for the quantitative determination of free interleukin-18.

Abstract

This method accurately quantifies free interleukin-18 in serum. [Solution] This invention provides a compound (NanoLuc-IL-18BP) that combines interleukin-18 binding protein (IL-18BP) with NanoLuc®, enabling the quantification of free interleukin-18. It also provides a method for producing NanoLuc-IL-18BP, characterized by its production in insect cells. Furthermore, it provides a method for quantifying free interleukin-18 using NanoLuc-IL-18BP.

Description

Technical Field

[0001] The present invention relates to a binding compound (NanoLuc-IL-18BP) with human free interleukin-18 and a method for producing the same. The present invention also relates to a method for quantifying free interleukin-18 and a kit for quantification.

Background Art

[0002] Interleukin-18 (hereinafter also referred to as IL-18) is a kind of cytokine and has a function of regulating the immune response. Abnormal expression of interleukin-18 is said to cause immune disorders such as autoimmune diseases. For example, when systemic juvenile idiopathic arthritis, adult-onset Still's disease, and some hereditary autoinflammatory diseases transform into hemophagocytic lymphohistiocytosis, it is known that interleukin-18 explosively increases. Therefore, interleukin-18 has attracted attention as a marker that can reflect the disease state.

[0003] In serum, in addition to free interleukin-18 existing alone, there is also bound interleukin-18 that binds to an antagonistic inhibitory molecule, IL-18 binding protein (hereinafter also referred to as IL-18BP), to form a complex. Since it is free interleukin-18 that exhibits more activity in lesions, a technique capable of accurately measuring the content of free interleukin-18 in serum is required.

[0004] One method for detecting and quantifying interleukin-18 is the ELISA method (enzyme-linked immunosorbent assay). Non-Patent Document 1 discloses the results of measuring free interleukin-18 by the ELISA method. However, the disclosed measurement method has a limit in the measurable range.

[0005] If recombinant IL-18BP that can label free interleukin-18 can be created, it will be possible to quantify free interleukin-18 with high accuracy. Non-patent document 2 discloses a technology for producing interleukin-18, receptor proteins, and recombinant IL-18BP proteins using E. coli and insect cell expression systems. However, the yield of IL-18BP in insect cell expression systems is extremely low, making it difficult to provide a practical measurement system. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Girard C, et al. "Elevated serum levels of free interleukin-18 in adult - onset Still's disease" Rheumatology, Volume 55, Issue 12, pp. 2237-2247, 2016 [Non-Patent Document 2] T. Kimura, et al., “Expression, Purification and Structural Analysis of Human IL-18 Binding Protein: A Potent Therapeutic Molecule for Allergy,” Allegology International, Volume 57, Issue 4, pp. 367-376, 2008. [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] There is a need for a technology to accurately quantify free interleukin-18. To that end, there is a need for a technology to produce recombinant IL-18BP protein that labels free interleukin-18.

[0008] This invention has been made in view of the current situation, and aims to provide a recombinant protein that labels free interleukin-18 and a technology for quantifying free interleukin-18. [Means for solving the problem]

[0009] This invention relates to a compound capable of labeling free interleukin-18. The compound of this invention is a conjugated compound (NanoLuc-IL-18BP) of interleukin-18 binding protein (IL-18BP) and NanoLuc®, capable of quantifying free interleukin-18.

[0010] In the present invention, the conjugated compound (NanoLuc-IL-18BP) is preferably a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 13, which is the interleukin-18 binding protein.

[0011] The present invention also provides a polynucleotide comprising a nucleotide sequence encoding an amino acid sequence contained in a conjugated compound of an interleukin-18 binding protein and NanoLuc®, which is capable of labeling free interleukin-18.

[0012] The polynucleotide of the present invention preferably comprises a nucleotide sequence represented by SEQ ID NO: 4, which encodes NanoLuc®, and a nucleotide sequence represented by SEQ ID NO: 6, which encodes an interleukin-18 binding protein.

[0013] The present invention also provides an expression vector comprising a nucleotide encoding an interleukin-18 binding protein.

[0014] The present invention provides a method for producing a compound (NanoLuc-IL-18BP) by combining interleukin-18 binding protein (IL-18BP) and NanoLuc®, which allows for the quantification of free interleukin-18. The production method of the present invention is characterized by producing the compound (NanoLuc-IL-18BP) in insect cells or silkworms.

[0015] The present invention further provides an ELISA kit for free interleukin-18. The ELISA kit for free interleukin-18 of the present invention comprises an interleukin-18 binding protein, which is a fusion of a protein that is a specific binding agent for interleukin-18 and luciferase, characterized in that the interleukin-18 binding protein is a protein produced by insect cells or silkworms.

[0016] The present invention further provides a method for quantifying free interleukin-18 using a conjugated compound (NanoLuc-IL-18BP). The quantification method of the present invention is characterized by comprising the steps of: binding the conjugated compound (NanoLuc-IL-18BP) to free interleukin-18; and causing the conjugated compound (NanoLuc-IL-18BP) bound to the free interleukin-18 to emit light and measuring the emission intensity. [Effects of the Invention]

[0017] The present invention provides a conjugated compound (NanoLuc-IL-18BP) capable of labeling free interleukin-18.

[0018] The NanoLuc-IL-18BP of the present invention can be mass-produced using an insect cell culture system or a silkworm protein expression system.

[0019] By using the conjugated compound of the present invention (NanoLuc-IL-18BP), it becomes possible to quantify free interleukin-18 with high accuracy. [Brief explanation of the drawing]

[0020] [Figure 1] Figure 1 is a design diagram of the expression vector used for the preparation of the binding compound (NanoLuc-IL-18BP) of the present invention. [Figure 2] Figure 2 shows the result of confirming the molecular weight of the binding compound (NanoLuc-IL-18BP) of the present invention produced from silkworms by electrophoresis. [Figure 3] Figure 3 is a diagram schematically showing the main measurement steps of free interleukin-18 of the present invention. [Figure 4] Figure 4 is a diagram showing the three-dimensional structure of the trimer formed by the 125-2H antibody, interleukin-18, and the binding compound (NanoLuc-IL-18BP) of the present invention. [Figure 5] Figure 5 is a diagram showing the result of the binding test between the antibody and interleukin-18 by BIACORE. [Figure 6] Figure 6 is a diagram showing the result of the binding test between the antibody, interleukin-18, and the binding compound (NanoLuc-IL-18BP) of the present invention by BIACORE. [Figure 7] Figure 7 is a diagram showing the result of measuring the activity of free interleukin-18 using the interleukin-18 receptor expressed in HEK293T cells. [Figure 8] Figure 8 is a graph showing an example of the measurement results of the serum concentration of total interleukin-18 by the conventional method for measuring interleukin-18 and the serum concentration of free interleukin-18 measured using the ELISA kit for free interleukin-18 of the present invention. [Figure 9] Figure 9 is a graph showing another example of the measurement results of the serum concentration of total interleukin-18 by the conventional method for measuring interleukin-18 and the serum concentration of free interleukin-18 measured using the ELISA kit for free interleukin-18 of the present invention.

Mode for Carrying Out the Invention

[0021] ]The following describes compounds capable of labeling free interleukin-18 according to the present invention. The compounds of the present invention are conjugated compounds of interleukin-18 binding protein (IL-18BP) and NanoLuc® (hereinafter also referred to as NanoLuc-IL-18BP).

[0022] Nanoluc-IL-18BP in this embodiment is a polypeptide containing the amino acid sequences shown in (a) to (d) below. (a) A peptide that acts as a secretion signal, represented by Sequence ID No. 10. (b) The polypeptide corresponding to Nanoluc®, represented by Sequence ID No. 11. (c) The linker peptide represented by Sequence ID No. 12. (d) The polypeptide corresponding to IL-18BP, represented by sequence number 13.

[0023] The amino acid sequences for sequence numbers 10 to 13 are as follows: Sequence ID 10: MPMLSAIVLYVLLAAAAHSAFA Sequence ID 11:MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA Sequence ID 12: GGSGGGS Sequence ID 13:TPVSQTTTAATASVRSTKDPCPSQPPVFPAAKQCPALEVTWPEVEVPLNGTLSLSCVACSRFPNFSILYWLGNGSFIEHLPGRLWEGSTSRERGSTGTQLCKALVLEQLTPALHSTNFSCVLVDPEQVVQRHVVLAQLWAGLRATLPPTQEALPSSHSSPQQQG

[0024] The Nanoluc-IL-18BP of the present invention binds to free interleukin-18. Furthermore, because it incorporates Nanoluc®, it emits light through an enzymatic reaction. By binding Nanoluc-IL-18BP to free interleukin-18 and then measuring the emission intensity, the amount of free interleukin-18 can be quantified.

[0025] Next, we will describe the method for producing NanoLuc-IL-18BP. Nanoluc-IL-18BP can be produced in insect cells or silkworm body fluid. Figure 1 shows the design of an expression vector for expressing Nanoluc-IL-18BP in insect cells.

[0026] In this embodiment, a baturovirus expression vector is used as the vector to be incorporated. Examples of nucleotide sequences encoding the amino acid sequence contained in Nanoluc-IL-18BP are shown from SEQ ID NOs: 1 to 9.

[0027] The nucleotide sequence of SEQ ID NO: 1 encodes the restriction enzyme EcoR1. The nucleotide sequence of SEQ ID NO: 2 is the Kozak sequence. The nucleotide sequence of SEQ ID NO: 3 encodes the secretory signal peptide. The nucleotide sequence of SEQ ID NO: 4 encodes NanoLuc®. The nucleotide sequence of SEQ ID NO: 5 encodes the linker. The nucleotide sequence of SEQ ID NO: 6 encodes the interleukin-18 binding protein. The nucleotide sequence of SEQ ID NO: 7 encodes the His tag. The nucleotide sequence of SEQ ID NO: 8 encodes the stop codon. The nucleotide sequence of SEQ ID NO: 9 encodes the endonuclease Not1.

[0028] The nucleotide sequences of sequence numbers 1 through 9 are as follows: Sequence ID 1: GAATTC Sequence ID 2: GCCACC அக்க்குத்துக்கு3:ATGCCCATGTTAAGCGCTATTGTTTTATATGTGCTTTTGGCGGCGGCGGCGCATTCTGCCTTTGCG Sequence number 4: ATGGTCTTCACACTCGAAGAATTTCGTTGGGGACTGGCGACAGACAGCCGGCTACAACCTGGAACCAAGTCCTTGAACAGGGAGGTGTGCCAGTTTGTTCCAGAATCTCGGGGTGTCCGTAACTCCGATCCAAAGGATTGTTCCTGAGCGGTGGAAAATGGGCCAGATCGACATCCATGTCATCATCCGTGATGGCACACTGGTAATCGCACGGGGTTACGCCGAACATGATCGACTATTTCGGACGGCCGTATGAAGGCATCGCGTGTTTCGACGGCAAAAGATCACTGTAACAGGGACCCTGTGGGAACGGCAAAATTATCGACGAGCCGCGGTGTTCCGAGTAACCATCAACGGAGTGACCGGCTGGCGGCTGTGTGCGAACGCATCTTGCG அக்குக்க்குக்கு5:GGTGGTAGCGGTGGTAGCGGTGGTAGC SEQ ID NO: 6:ACACCTGTCTCGCAGACCACCACAGCTGCCACTGCCTCAGTTAGAAGCACAAAGGACCCCTGCCCCTCCCAGCCCCCAGTGTTCCCAGCAGCTAAGCAGTGTCCAGCATTGGAAGTGA CCTGGCCAGAGGTGGAAGTGCCACTGAATGGAACGCTGAGCTTATCCTGTGTGGCCTGCAGCCGCTTCCCCAACTTCAGCATCCTCTACTGGCTGGGCAATGGTTCCTTCATTGAGCACCTCCCA GGCCGACTGTGGGAGGGGAGCACCAGCCGGGAACGTGGGAGCACAGGTACGCAGCTGTGCAAGGCCTTGGTGCTGGAGCAGCTGACCCCTGCCCTGCACAGCACCAACTTCTCCTGTGTGCTCG TGGACCCTGAACAGGTTGTCCAGCGTCACGTCGTCCTGGCCCAGCTCTGGGCTGGGCTGAGGGCAACCTTGCCCCCACCCAAGAAGCCCTGCCCTCCAGCCACAGCAGTCCACAGCAGCAGGGT Sequence ID 7: CATCATCACCATCACCAT Sequence ID 8: TGA Sequence ID 9: GCGGCCGC

[0029] The nucleotide sequence of Sequence ID No. 6, which encodes the interleukin-18 binding protein, has been confirmed not to be cleaved by the restriction enzyme EcoR1 and the endonuclease Not1.

[0030] A preferred embodiment of a method for expressing Nanoluc-IL-18BP in insect cells is shown below. In this embodiment, silkworm larvae were used as the insect. The expression method includes the following steps 1 to 12. (Step 1) Insert the nucleotides shown in SEQ ID NOs. 1 to SEQ ID NOs. 9 into pFastBac1 to create a plasmid. (Step 2) Add plasmid to E. coli (BmDH10Bac) (Step 3) After standing on ice for 30 minutes, perform a heat shock at 42°C for 40 seconds, stand on ice for 2 minutes, add 1.4 ml of SOC medium and incubate at 37°C for 3 hours. (Step 4) Add 5 ml of LB medium containing tetracycline and incubate overnight at 37°C. (Step 5) Add gentamicin and incubate at 37°C for 4 hours. (Step 6) Spread the stock solution of the culture medium obtained in Step 5 and a 10-fold diluted version of the culture medium obtained in Step 5 onto LB agar medium and incubate overnight at 37°C. (Step 7) Select white colonies, perform colony PCR and electrophoresis, and confirm that a band appears around 3500 bp to confirm that the target gene has been inserted into the bacmid DNA. (Step 8) Colonies in which the target gene has been confirmed to be inserted by gene sequence analysis are placed in LB medium containing kanamycin and gentamicin and cultured overnight at 37°C. (Step 9) Purify bacmid DNA from the proliferated E. coli. (Step 10) Prepare the inoculum DNA by mixing bacmid DNA with insect cell culture medium and transfection reagent so that the amount of bacmid DNA per silkworm is 1000 μg. (Step 11) Inoculate the silkworm larvae with inoculation DNA. (Step 12) Observe the silkworms for about a week. Once the silkworms' body color has changed to black and infection has been established, collect the body fluid from the silkworm larvae.

[0031] The body fluid containing Nanoluc-IL-18BP recovered from silkworm larvae can be purified by liquid chromatography. Specifically, Nanoluc-IL-18BP can be purified by His-tag affinity purification and gel filtration.

[0032] Figure 2 shows the results of confirming the molecular weight of NanoLuc-IL-18BP purified by liquid chromatography using electrophoresis. The molecular weight of NanoLuc-IL-18BP is approximately 40 kDa, and it was confirmed that NanoLuc-IL-18BP can be isolated and purified by His-tag affinity purification and gel filtration.

[0033] Using the expression method of this embodiment, 703.3 μg of NanoLuc-IL-18BP was extracted from 29 silkworms. This corresponds to a production amount of 24.3 μg per silkworm, which is significantly higher than the amount obtained by conventional IL-18BP production methods using E. coli or insect cell expression systems.

[0034] Next, we will describe a measurement method for quantifying free interleukin-18 using NanoLuc-IL-18BP.

[0035] Free interleukin-18 can be quantified by causing NanoLuc-IL-18BP bound to free interleukin-18 to emit light and measuring the emission intensity. ELISA is a preferred method for this measurement.

[0036] The measurement method preferably includes the following measurement steps. (Measurement step 1) An antibody that binds to interleukin-18 is immobilized onto a sample plate. (Measurement process 2) Blocking process using a blocking buffer. (Measurement step 3) Add the sample containing free interleukin-18. (Measurement step 4) Add NanoLuc-IL-18BP. (Measurement step 5) Add a substrate that reacts with Nanoluc (2-Furanylmethyldeoxy-coelenterazine (Furimazine)). (Measurement step 6) Measurement of luminescence intensity.

[0037] Figure 3 schematically shows the state of interleukin-18 and its reactants in the main measurement step of this embodiment. In this embodiment, 125-2H antibody is used as the antibody that is immobilized and binds to interleukin-18. The binding site of the 125-2H antibody to interleukin-18 is designed to be different from the binding site of interleukin-18 to NanoLuc-IL-18BP. Figure 4 shows the three-dimensional structure of the trimer formed by the 125-2H antibody, interleukin-18, and NanoLuc-IL-18BP.

[0038] The reaction generated by the measurement method of this embodiment was confirmed by a reactivity test using BIACORE. The results are shown in Figures 5 and 6.

[0039] Figure 5 shows the results of the binding affinity test between the 125-2H antibody and interleukin-18. Figure 6 shows the results of the binding affinity test between the 125-2H antibody, interleukin-18, and NanoLuc-IL-18BP. In the BIACORE test of the 125-2H antibody, interleukin-18, and NanoLuc-IL-18BP, the decrease in RU (response units) over time was small, confirming high binding affinity. Furthermore, the maximum response value Rmax for the 125-2H antibody and interleukin-18 was 20.93 RU, while the maximum response value Rmax for the 125-2H antibody, interleukin-18, and NanoLuc-IL-18BP was 84.68 RU. The actual molecular weights are approximately 18 kDa for interleukin-18 and approximately 40 kDa for NanoLuc-IL-18BP, but the maximum response values ​​in this study were slightly higher than expected. One possible reason for this is that some NanoLuc-IL-18BP molecules may be dimerized.

[0040] If both free interleukin-18 and conjugated interleukin-18 already bound to IL-18BP and forming a complex are present in the sample, the 125-2H antibody will bind to both free interleukin-18 and conjugated interleukin-18. However, Nanoluc-IL-18BP does not bind to conjugated interleukin-18. The substrate added in measurement step 5 reacts with Nanoluc, so it will emit light in proportion to the amount of free interleukin bound to Nanoluc-IL-18BP. As a result, the emission intensity measured in measurement step 6 will reflect only the amount of free interleukin-18 in the sample.

[0041] This document describes an ELISA kit for quantifying free interleukin-18 using NanoLuc-IL-18BP. The ELISA kit includes a plate with an antibody against interleukin-18 immobilized on the plate and NanoLuc-IL-18BP. A 125-2H antibody can be used as the antibody.

[0042] This report presents the results of confirming the binding state of free interleukin-18 and Nanoluc-IL-18BP using HEK293T cells. The HEK293T cells used have been transfected with a plasmid incorporating the luciferase gene and the IL-18Rβ (IL-18 receptor) gene, causing them to express the IL-18 receptor on their cell surface. By adding a sample containing interleukin-18 and Nanoluc-IL-18BP to HEK293T cells and measuring the activity of NF-κB luciferase, which is downstream of the interleukin-18 signaling pathway, it is possible to quantify the suppression of the physiological activity of free interleukin-18 caused by the binding of free interleukin-18 to Nanoluc-IL-18BP.

[0043] We prepared HEK293T cells without interleukin-18 (described as unstimulated), and HEK293T cells with the following added: free interleukin-18 (2 ng / ml) alone (described as control), free interleukin-18 + NanoLuc-IL-18BP (10 ng / ml), free interleukin-18 + NanoLuc-IL-18BP (100 ng / ml), free interleukin-18 + NanoLuc-IL-18BP (5000 ng / ml), and free IL-18 + 125-2H antibody (Ab 500 ng / ml).

[0044] Figure 7 shows the relative activity units of luciferase luminescence measured in each HEK293T cell to evaluate the transcriptional activity of NF-κB. If interleukin-18 is active, signal transduction occurs when it binds to the IL-18 receptor, and NF-κB downstream is activated, enhancing luminescence. On the other hand, if the added Nanoluc-IL-18BP binds to free interleukin-18 in the same binding state as normal IL-18BP, the activity of interleukin-18 is inhibited, signal transduction does not occur, and NF-κB is not activated, so luminescence is not enhanced. As shown in Figure 7, the amount of luminescence indicating NF-κB activation decreases as the amount of added Nanoluc-IL-18BP increases, confirming that Nanoluc-IL-18BP binds to free interleukin-18 in the same binding state as IL-18BP.

[0045] Instead of using Nanoluc-IL-18BP in the ELISA method, free interleukin-18 can also be quantified using absorbance measurement with HRP Anti-6X His tag antibody in an ELISA method. However, this measurement method requires a long time for quantification. Furthermore, it has been confirmed that measuring luminescence using NanoLuc provides a wider dynamic range.

[0046] Figure 8 shows the results of measuring the serum concentration of total interleukin-18 using a conventional measurement method and the serum concentration of free interleukin-18 using the measurement method of the present invention for three patients who progressed from systemic juvenile idiopathic arthritis (sJIA) to macrophage activation syndrome (MAS).

[0047] It is known that the amount of free interleukin-18 in the serum of patients with systemic juvenile idiopathic arthritis (sJIA) is trace compared to conjugated interleukin-18, and in all measurements, the amount of free interleukin-18 detected was less than the amount of total interleukin-18 detected.

[0048] In Case 1, free interleukin-18 was detected when macrophage activation syndrome developed after hospitalization and again when the patient relapsed after remission. In Case 2, free interleukin-18 was detected when macrophage activation syndrome developed, and although the patient subsequently experienced two relapses, free interleukin-18 was not detected during the relapses. In Case 3, free interleukin-18 was detected at the time of onset, during relapse, when macrophage activation syndrome developed, and during relapse.

[0049] The difference in serum concentrations of free interleukin-18 relative to total interleukin-18 during relapses among cases is thought to be influenced by factors such as the disease state, the expression of other inflammatory cytokines, and the medications used.

[0050] Figure 9 shows the results of measuring total interleukin-18 serum concentration using a conventional measurement method and the results of measuring free interleukin-18 serum concentration using the measurement method of the present invention for patients different from cases 1-3. These patients experienced a relapse of systemic juvenile idiopathic arthritis, but did not develop macrophage activation syndrome. Furthermore, free interleukin-18 was not detected at the time of symptom relapse.

[0051] As described above, by using NanoLuc-IL-18BP, a compound formed by combining the interleukin-18 binding protein of the present invention with NanoLuc®, it becomes possible to accurately quantify free interleukin-18 in serum, which was previously difficult to quantify. NanoLuc-IL-18BP of the present invention can be produced using an insect cell culture system or a silkworm protein expression system. [Industrial applicability]

[0052] The NanoLuc-IL-18BP of the present invention can be used for the quantification of free interleukin-18. Furthermore, the NanoLuc-IL-18BP of the present invention can be produced in large quantities using an insect cell culture system or a silkworm protein expression system, and IL-18BP can be isolated by cleaving NanoLuc and IL-18BP using a protease in the next step. The isolated IL-18BP can be used as a reagent or pharmaceutical as an interleukin-18 inhibitor.

Claims

1. A compound (NanoLuc-IL-18BP) is a combination of interleukin-18 binding protein (IL-18BP) and NanoLuc®, which allows for the quantification of free interleukin-18.

2. The binding compound according to claim 1 (NanoLuc-IL-18BP), characterized in that the interleukin-18 binding protein is a polypeptide consisting of the amino acid sequence represented by Sequence ID No.

13.

3. A polynucleotide containing a nucleotide sequence that encodes the amino acid sequence contained in a compound of interleukin-18 binding protein and NanoLuc®.

4. It consists of a nucleotide sequence represented by Sequence ID No. 4, and a nucleotide sequence that codes for NanoLuc (registered trademark), It consists of a nucleotide sequence represented by Sequence ID No. 6, which encodes an interleukin-18 binding protein, The polynucleotide according to claim 3, characterized by containing the following:

5. An expression vector comprising the polynucleotide described in claim 3.

6. A method for producing the conjugated compound (NanoLuc-IL-18BP) according to claim 1, characterized by producing the conjugated compound (NanoLuc-IL-18BP) in insect cells.

7. A method for producing the conjugated compound (NanoLuc-IL-18BP) according to claim 1, characterized by producing the conjugated compound (NanoLuc-IL-18BP) in silkworms.

8. It contains an interleukin-18 binding protein, which is formed by the fusion of a specific binding agent for interleukin-18 with luciferase. An ELISA kit for free interleukin-18, characterized in that the interleukin-18 binding protein is a silkworm-produced protein.

9. A method for quantifying free interleukin-18 using the conjugated compound (NanoLuc-IL-18BP) described in claim 1, The process involves binding the aforementioned conjugation compound (NanoLuc-IL-18BP) to free interleukin-18, A step of causing the binding compound (NanoLuc-IL-18BP) bound to free interleukin-18 to emit light and measuring the emission intensity, A method for quantifying free interleukin-18, characterized by comprising the following:

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