Citrullinated SETDB1 polypeptide and application thereof in diagnosis of rheumatoid arthritis

Abstract

The present invention relates to the field of immunological detection, in particular immunological diagnosis. In particular, the present invention provides a method for diagnosing rheumatoid arthritis based on the level of an anti-citrullinated SETDB1 antibody, and a kit for use in the method. The invention further provides a polypeptide of citrullinated SETDB1 for diagnosis and application of the polypeptide to diagnosis of rheumatoid arthritis.

Description

Technical Field

[0001] The present invention relates to immunological detection, in particular to the field of immunological diagnosis. Specifically, the present invention provides a method for diagnosing rheumatoid arthritis based on the level of anti-citrullinated SETDB1 antibodies, and a kit for the method. The present invention also provides a polypeptide of citrullinated SETDB1 used for the above diagnosis and its use in diagnosing rheumatoid arthritis. Background Art

[0002] Citrullination is one of the post-translational modifications of proteins. It is a post-translational modification that occurs under the catalysis of Ca+-dependent peptidylarginine deiminase (PAD) (Wang, R., et al., Gut stem cell necroptosis by genome instability triggers bowel inflammation. Nature, 2020. 580(7803): p. 386-390.). Since citrullination fundamentally changes the structure and function of proteins, when abnormal citrullination increases in the body, the level of autoantibodies against citrullinated residue epitopes will increase abnormally, leading to the occurrence of diseases. Diseases such as rheumatoid arthritis, systemic lupus erythematosus, and tumors have all been reported to be related to citrullination.

[0003] Among them, citrullination is most commonly studied in rheumatoid arthritis. Rheumatoid arthritis is a systemic autoimmune disease of unknown etiology, affecting 0.5-1.0% of adults worldwide. It generally involves the synovium of joints and is often manifested as joint pain and joint swelling. In severe cases, joint deformity and even disability may occur (MCINNES IB, SCHETT G. The pathogenesis of rheumatoid arthritis [J]. N Engl J Med, 2011, 365 (23): 2205-2219.). A study found that 2 of the 33 medical causes of motor function loss in adults in the United States are caused by arthritis (HOOTMAN JM, HELMICK CG, BRADY T JA public health approach to addressing arthritis in older adults: the most common cause of disability [J]. Am J Public Health, 2012, 102 (3): 426-433.). Therefore, rheumatoid arthritis can no longer be simply regarded as an ordinary medical disease, but a public health issue that needs to be taken seriously. In addition, some health economists have conducted relevant research on the economic burden caused by rheumatoid arthritis. The results show that by reducing risk factors or intervening in the disease as early as possible, the cost is much lower than the cost of hospitalization and surgery in the later stage (LANES SF, LANZA LL, RADENSKY PW, et al. Resource utilization and cost of care for rheumatoid arthritis and osteoarthritis in a managed care setting: the importance of drug and surgery costs [J]. Arthritis Rheum, 1997, 40 (8): 1475-1481.). In summary, to control the occurrence and development of rheumatoid arthritis, it is necessary to achieve "early detection, early diagnosis, and early treatment".

[0004] Studies have shown that the formation of autoantibodies is one of the characteristics of rheumatoid arthritis that is different from other inflammatory arthritis, such as psoriatic arthritis, reactive arthritis and osteoarthritis (KOURILOVITCH M, GALARZA-MALDONADO C, ORTIZ-PRADO E. Diagnosis and classification of rheumatoid arthritis [J]. J Autoimmun, 2014, 48-49 (26-30).). Currently, the diagnosis of rheumatoid arthritis in laboratory tests mainly relies on rheumatoid factors (RFs) and anti-citrullinated protein antibodies (ACPA). The production of rheumatoid factors is due to the stimulation of the body by persistent infection with foreign pathogens such as viruses and mycoplasmas. Therefore, positive results can also be detected in patients with other immune diseases, chronic infections, and 5% of healthy people. It is generally used as an indicator for early screening of diseases. Among anti-citrullinated protein antibodies, anti-cyclic citrullinated polypeptide (CCP) antibodies have become the most commonly used clinical diagnostic markers in clinical diagnosis due to their high specificity. Studies have shown that the specificity of this marker can reach 90%, but the sensitivity is only about 70% (Wegner, N., Lundberg, K., Kinloch, A., Fisher, B., V.,Feldmann,M.,&Venables,PJ(2010).Autoimmunity to specific citrullinated proteins gives the first clues to theetiology of rheumatoid arthritis.Immunological reviews,233(1),34-54.https: / / doi.org / 10.1111 / j.0105-2896.2009.00850.x). Due to the low sensitivity, patients are often missed, so it is urgent to screen new RA diagnostic markers. In addition, CCP is an artificially designed cyclic citrullinated peptide, which can only be used for the diagnosis of clinical RA and has little significance for the exploration of disease mechanisms.

[0005] In summary, citrullination is involved in the occurrence and development of RA. Therefore, the citrulline markers naturally present in RA patients are expected to become high-performance markers for its diagnosis and mechanism exploration. Screening for high levels of citrulline markers naturally present in patients can not only solve the problem of the current single clinical marker, but also is expected to find the key factors in the pathogenesis of RA. Summary of the invention

[0006] Histone methyltransferase SETDB1 (SETDB1) belongs to the SUV39 protein family and is a member of the SET domain-containing protein lysine methyltransferases (PKMTs) involved in epigenetic regulation. It can trimethylate histone H3 at the ninth lysine (H3K9me3). Existing studies mainly believe that SETDB1 is closely related to cancer. In many cancers, the expression level of SETDB1 tends to increase and affects the occurrence and development of cancer, such as colorectal cancer, liver cancer, gastric cancer, lung cancer, etc.; SETDB1 can be used as one of the joint targets for tumor treatment. For example, inhibiting SETDB1 can enhance the sensitivity of cetuximab in colorectal cancer and overcome its drug resistance. In melanoma cells with high expression of SETDB1, treatment with SETDB1 inhibitors combined with BAKR and MEK inhibitors can effectively kill cells resistant to targeted therapy.

[0007] The inventors of the present application innovatively screened the serum of patients with rheumatoid arthritis for citrullinated markers by constructing chemical derivatives, and discovered for the first time a citrullinated SETDB1 epitope polypeptide, which has a high level of autoantibodies in RA patients, showing diagnostic value in rheumatoid arthritis. Thus, the inventors provided a method for RA diagnosis based on the detection of anti-citrullinated SETDB1 antibody levels, and a citrullinated SETDB1 polypeptide used in the method, which can effectively reduce the missed diagnosis rate of early RA patients, and is even expected to be better than current clinical diagnostic markers.

[0008] Isolated polypeptide or variant thereof

[0009] In a first aspect, the present invention provides an isolated polypeptide or a variant thereof, wherein the polypeptide consists of at least 11 consecutive amino acid residues of the MBD domain of the SETDB1 protein;

[0010] wherein at least one of the consecutive amino acid residues is an arginine residue, and at least one of the arginine residues is citrullinated;

[0011] The variant differs from the polypeptide from which it is derived only in the substitution (e.g., conservative substitution or non-conservative substitution; e.g., conservative substitution) of one or several (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acid residues, and retains the biological function of the polypeptide from which it is derived (e.g., the activity of being recognized and bound by an anti-citrullinated SETDB1 antibody).

[0012] Herein, the biological function of the polypeptide or variant thereof of the present invention includes, but is not limited to, the activity of being recognized and bound by an anti-citrullinated antibody (eg, an anti-citrullinated SETDB1 antibody) as an epitope peptide.

[0013] In this document, when referring to the amino acid position of the MBD domain of the SETDB1 protein, the description is made with reference to the sequence shown in SEQ ID NO: 1. For example, the expression "amino acid residue at position 2 of the MBD domain of the SETDB1 protein" refers to the amino acid residue at position 2 of the sequence shown in SEQ ID NO: 1 and its corresponding position. The corresponding position refers to the position in the sequence to be compared that is equivalent to the specific amino acid position in SEQ ID NO: 1 when the sequence to be compared is optimally aligned with SEQ ID NO: 1 (i.e., to obtain the highest percentage identity).

[0014] In certain embodiments, all of the arginine residues in the consecutive amino acid residues are citrullinated (ie, are citrullinated).

[0015] In certain embodiments, the isolated polypeptide has an arginine residue at at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or all 9) of the amino acid positions corresponding to the following positions of the MBD domain of the SETDB1 protein: 2, 16, 17, 21, 22, 23, 26, 43, and / or 50; and at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or all 9) of the arginine residues is citrullinated.

[0016] In certain embodiments, the isolated polypeptide comprises at least amino acid residues 16-26 of the MBD domain of the SETDB1 protein, wherein at least one of the consecutive amino acid residues 16-26 is an arginine residue, and at least one of the arginine residues is citrullinated. In certain embodiments, all of the arginine residues in the consecutive amino acid residues 16-26 are citrullinated.

[0017] In certain embodiments, the isolated polypeptide has an arginine residue at at least one (e.g., 1, 2, 3, 4, 5, or all 6) of the amino acid positions corresponding to the following positions of the MBD domain of the SETDB1 protein: 16, 17, 21, 22, 23, and / or 26; and at least one (e.g., 1, 2, 3, 4, 5, or all 6) of the arginine residues is citrullinated. In certain embodiments, all of the arginine residues are citrullinated.

[0018] In certain embodiments, the isolated polypeptide comprises the sequence shown in SEQ ID NO: 8 at positions corresponding to positions 16-26 of the MBD domain of the SETDB1 protein.

[0019] In certain embodiments, the isolated polypeptide further comprises an arginine residue at at least one (e.g., 1, 2, or all 3) of the amino acid positions corresponding to the following positions of the MBD domain of the SETDB1 protein: 2, 43, and / or 50; and at least one (e.g., 1, 2, or all 3) of the arginine residues is citrullinated.

[0020] In certain embodiments, the isolated polypeptide consists of consecutive amino acid residues selected from the group consisting of amino acid residues 5-27, 8-27, 12-27, 14-27, 16-27, 16-26, 1-27, or 5-32 of the MBD domain of the SETDB1 protein; wherein at least one of the consecutive amino acid residues is an arginine residue, and at least one of the arginine residues is citrullinated. In certain embodiments, all of the arginine residues in the consecutive amino acid residues are citrullinated.

[0021] In certain embodiments, the isolated polypeptide has an arginine residue at at least one (e.g., 1, 2, 3, 4, 5, or all 6) of the amino acid positions corresponding to the following positions of the MBD domain of the SETDB1 protein: 16, 17, 21, 22, 23, and / or 26; and at least one (e.g., 1, 2, 3, 4, 5, or all 6) of the arginine residues is citrullinated.

[0022] In certain embodiments, the isolated polypeptide further comprises an arginine residue at the amino acid position corresponding to position 2 of the MBD domain of the SETDB1 protein, which is optionally citrullinated.

[0023] In certain embodiments, the variants described herein have exactly the same citrulline characteristics as the polypeptide from which they are derived, ie, have the same number and position of citrulline.

[0024] In certain embodiments, the variant does not include an amino acid substitution in the amino acid positions corresponding to the following positions of the MBD domain of the SETDB1 protein compared to the polypeptide from which it is derived: 16, 17, 21, 22, 23 and 26. That is, the variant is identical to the polypeptide from which it is derived at the amino acid positions corresponding to 16, 17, 21, 22, 23 and 26 of the MBD domain of the SETDB1 protein. In certain embodiments, the variant further does not include an amino acid substitution in the amino acid positions corresponding to positions 2, 43 and / or 50 of the MBD domain of the SETDB1 protein. That is, the variant is identical to the polypeptide from which it is derived at the amino acid positions corresponding to positions 2, 43 and / or 50 of the MBD domain of the SETDB1 protein.

[0025] In certain embodiments, the variants of the present invention differ from the polypeptide from which they are derived only in the substitution of 1, 2, 3 or 4 amino acid residues (e.g., conservative substitutions or non-conservative substitutions; e.g., conservative substitutions), and retain the biological function of the polypeptide from which they are derived (e.g., the activity of being recognized and bound by an anti-citrullinated SETDB1 antibody).

[0026] In certain embodiments, the polypeptides of the present invention and variants thereof are arginine at amino acid positions corresponding to positions 16, 17, 21, 22, 23 and 26 of the MBD domain of the SETDB1 protein, and at least one (e.g., 1, 2, 3, 4, 5 or all 6) of the arginine residues are citrullinated.

[0027] In certain embodiments, the polypeptides and variants thereof of the present invention are all citrullinated arginine (ie, citrulline) at the amino acid positions corresponding to positions 16, 17, 21, 22, 23, and 26 of the MBD domain of the SETDB1 protein.

[0028] In certain embodiments, the above polypeptides and variants thereof further contain an arginine residue at the amino acid position corresponding to position 2 of the MBD domain of the SETDB1 protein, which is optionally citrullinated.

[0029] In certain embodiments, the above polypeptides and variants thereof further comprise an arginine residue at the amino acid position corresponding to position 43 of the MBD domain of the SETDB1 protein, which is optionally citrullinated.

[0030] In certain embodiments, the above polypeptides and variants thereof further comprise an arginine residue at the amino acid position corresponding to position 50 of the MBD domain of the SETDB1 protein, which is optionally citrullinated.

[0031] In certain embodiments, the isolated polypeptide consists of no more than 72 (e.g., no more than 70, no more than 69, no more than 68, no more than 67, no more than 66, no more than 65, no more than 64, no more than 63, no more than 62, no more than 61, no more than 60, no more than 59, no more than 58, no more than 57, no more than 56, no more than 55, no more than 54, no more than 53, no more than 52, no more than 51, no more than 50, no more than 52, no more than 51, no more than 50, no more than 53, no more than 52, no more than 51, no more than 50, no more than 51, no more than 50, no more than 52 ... No more than 49, no more than 48, no more than 47, no more than 46, no more than 45, no more than 44, no more than 43, no more than 42, no more than 41, no more than 40, no more than 39, no more than 38, no more than 37, no more than 36, no more than 35, no more than 34, no more than 33, no more than 32, no more than 31, no more than 30, no more than 29, or no more than 28 consecutive amino acid residues.

[0032] In certain embodiments, the isolated polypeptide consists of 11 to 30 (e.g., 11-28) consecutive amino acid residues of the MBD domain of the SETDB1 protein. In certain embodiments, the isolated polypeptide consists of 11, 12, 14, 16, 20, 23, 27 or 28 consecutive amino acid residues of the SETDB1 MBD domain.

[0033] In certain embodiments, the polypeptides and variants thereof of the present invention comprise at least amino acid residues 2 to 26 of the MBD domain of the SETDB1 protein.

[0034] In certain embodiments, the isolated polypeptide has arginine residues at amino acid positions corresponding to positions 2, 16, 17, 21, 22, 23 and 26 of the MBD domain of the SETDB1 protein, and at least one (e.g., 1, 2, 3, 4, 5, 6 or all 7) of the arginine residues is citrullinated.

[0035] In certain embodiments, the isolated polypeptide is citrullinated at arginine residues corresponding to positions 2, 16, 17, 21, 22, 23, and 26 of the MBD domain of the SETDB1 protein.

[0036] In certain embodiments, the variant does not include amino acid substitutions in the following amino acid positions corresponding to the MBD domain of the SETDB1 protein compared to the polypeptide from which it is derived: 2, 16, 17, 21, 22, 23 and 26. That is, the variant is identical to the polypeptide from which it is derived at amino acid positions corresponding to 2, 16, 17, 21, 22, 23 and 26 of the MBD domain of the SETDB1 protein.

[0037] In certain embodiments, the isolated polypeptide and variants thereof consist of no more than 50 (e.g., no more than 49, no more than 48, no more than 47, no more than 46, no more than 45, no more than 44, no more than 43, no more than 42, no more than 41, no more than 40, no more than 39, no more than 38, no more than 37, no more than 36, no more than 35, no more than 34, no more than 33, no more than 32, no more than 31, or no more than 30) consecutive amino acid residues of the SETDB1 MBD domain.

[0038] In certain embodiments, the isolated polypeptide and variants thereof consist of 25 to 30 consecutive amino acid residues of the MBD domain of the SETDB1 protein.

[0039] In certain embodiments, the SETDB1 protein MBD domain has a sequence as shown in SEQ ID NO: 1, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0040] In certain embodiments, the isolated polypeptide has a sequence as shown in any one of SEQ ID NOs: 3-10. In certain embodiments, the variant has a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity compared to the sequence shown in any one of SEQ ID NOs: 3-10, and has exactly the same citrulline position and number.

[0041] Derivatized polypeptide or variant thereof

[0042] The polypeptide of the present invention or its variant can be derivatized, for example, by linking other functional units, such as detectable markers, protein tags, solid phase carriers, etc.

[0043] In one embodiment, the isolated polypeptide or variant thereof is connected to the surface of a solid support, or carries a modifying group that can be connected to a solid support. In certain embodiments, the N-terminus of the isolated polypeptide or variant thereof is connected to the surface of a solid support, or carries a modifying group that can be connected to a solid support. In certain embodiments, the modifying group is biotin or avidin. In certain embodiments, the solid support is selected from magnetic beads or microtiter plates (e.g., microtiter plates or ELISA plates).

[0044] In another embodiment, the isolated polypeptide or variant thereof carries a detectable label. In certain embodiments, the detectable label is selected from an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridinium ester compound), a fluorescent dye, or biotin.

[0045] Preparation of peptides

[0046] The polypeptide or variant thereof of the present invention is not limited by the production method thereof, for example, it can be produced by genetic engineering methods (recombinant technology) or by chemical synthesis methods.

[0047] In another aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide of the present invention or a variant thereof.

[0048] In another aspect, the present invention also provides a vector comprising the isolated nucleic acid molecule as described above. The vector of the present invention can be a cloning vector or an expression vector. In a preferred embodiment, the vector of the present invention is, for example, a plasmid, a cosmid, a phage, a cosmid, or the like.

[0049] In another aspect, the present invention also provides host cells comprising isolated nucleic acid molecules of the present invention or vectors. Such host cells include, but are not limited to, prokaryotic cells such as Escherichia coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.).

[0050] In another aspect, the present invention also provides a method for preparing the polypeptide of the present invention or its variant, which comprises culturing the host cell of the present invention under conditions allowing expression of the polypeptide or its variant, and recovering the polypeptide or its variant from the culture of the cultured host cell.

[0051] Detection Kits

[0052] In a second aspect, the present invention provides a kit comprising an isolated polypeptide or a variant thereof selected from the first aspect.

[0053] In certain embodiments, the kit of the present invention comprises a capture reagent, and the capture reagent is selected from the isolated polypeptide or variant thereof described in the first aspect. In certain embodiments, the kit further comprises instructions for using the isolated polypeptide or variant thereof as a capture reagent to detect antibodies specific to citrullinated SETDB1 protein in a sample, or instructions for using the isolated polypeptide or variant thereof as a capture reagent to determine the level of antibodies specific to citrullinated SETDB1 protein in a sample from a subject, thereby determining whether the subject suffers from rheumatoid arthritis.

[0054] In certain embodiments, the subject is a mammal, such as a human.

[0055] In some embodiments, the capture reagent is connected to the surface of the solid phase carrier. In certain embodiments, the N-terminus of the capture reagent is connected to the solid phase carrier. In this article, the solid phase carrier includes a concave plate, a test tube, beads (e.g., latex particles) or a film (e.g., a nitrocellulose membrane) made or coated by a polymer material (e.g., polyvinyl chloride, polystyrene, polyacrylamide or cellulose), or a magnetic bead pre-coated by a functional group (e.g., amino, carboxyl, biotin or avidin). In certain embodiments, the solid phase carrier is selected from magnetic beads or microtiter plates (e.g., microtiter plates or ELISA plates).

[0056] In other embodiments, the capture reagent carries a modifying group that can be connected to a solid phase carrier. In certain embodiments, the N-terminus of the capture reagent carries a modifying group that can be connected to a solid phase carrier. In such embodiments, the kit may further comprise a coating reagent for coating the capture reagent on the solid phase carrier, such as a coating buffer (e.g., a carbonate buffer, a phosphate buffer, a Tris-HCL buffer, or a borate buffer). Methods for coating proteins or polypeptides on solid phase carriers are well known in the art, such as physical adsorption, covalent coupling achieved by amino or carboxylation of the surface, or mediated binding achieved by an avidin-biotin system, a polylysine pre-coated surface, a protein A or a protein G pre-coated surface. In certain embodiments, the modifying group is biotin or avidin, and the solid phase carrier surface carries a corresponding linking group.

[0057] In certain embodiments, the kit comprises at least a solid support coated with avidin or streptavidin, and the above-mentioned capture reagent in separate containers or in separate compartments of a single container unit.

[0058] In some embodiments, the method of determining the level of anti-citrullinated SETDB1 antibodies in a sample from a subject to determine whether the subject suffers from rheumatoid arthritis is performed in an indirect method. Determining the level of antibodies in a sample by indirect methods is well known to those skilled in the art. In such determinations, a "capture antigen" first forms an immune complex with an antibody in the sample, and then the captured antibody is detected by a secondary antibody (e.g., an anti-immunoglobulin antibody).

[0059] Therefore, in certain embodiments, the kit further comprises a detection reagent selected from a secondary antibody with a detectable label. In certain embodiments, the detectable label is selected from an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridinium ester compound), a fluorescent dye, or biotin. In certain exemplary embodiments, the detectable label is selected from an enzyme (e.g., horseradish peroxidase or alkaline phosphatase).

[0060] In certain embodiments, the secondary antibody is specific for an antibody from the species from which the test antibody is derived (eg, human).

[0061] In certain embodiments, the secondary antibody is an anti-immunoglobulin antibody.

[0062] In certain exemplary embodiments, the kit is used to detect anti-citrullinated SETDB1 protein IgG antibodies. In such embodiments, the anti-immunoglobulin antibody is selected from anti-IgG antibodies, such as anti-human IgG antibodies.

[0063] In certain exemplary embodiments, the kit is used to detect IgM antibodies against citrullinated SETDB1 protein. In such embodiments, the anti-immunoglobulin antibody is selected from anti-IgM antibodies, such as anti-human IgM antibodies.

[0064] In certain exemplary embodiments, the kit is used to detect anti-citrullinated SETDB1 protein IgA antibodies. In such embodiments, the anti-immunoglobulin antibody is selected from anti-IgA antibodies, such as anti-human IgA antibodies.

[0065] In other embodiments, the method of determining the level of anti-citrullinated SETDB1 antibodies in a sample from a subject to determine whether the subject suffers from rheumatoid arthritis is performed in a double antigen sandwich format. Determining the level of antibodies in a sample by a double antigen sandwich method is well known to those skilled in the art. In such determinations, the "capture antigen" and "detection antigen" allow the sample antibody to form a bridge between two specific antigens, so that the two antigens are usually the same, or have the same core epitope, or have immunological cross-reactivity, so that one antibody can bind to both antigens.

[0066] Therefore, in certain embodiments, the kit further comprises a detection reagent, which is selected from the isolated polypeptide or variant thereof described in the first aspect.

[0067] In certain embodiments, the detection reagent carries a detectable label. In certain embodiments, the detectable label is selected from an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridinium ester compound), a fluorescent dye, or biotin. In certain exemplary embodiments, the detectable label is selected from an enzyme (e.g., horseradish peroxidase or alkaline phosphatase).

[0068] In certain embodiments, the polypeptide sequences contained in the detection reagent and the capture reagent are identical or substantially identical. In certain embodiments, the substantially identical means that the two polypeptide sequences contained in the detection reagent and the capture reagent contain the same core epitope, and the core epitope includes at least the 16th-26th consecutive amino acid residues of the MBD domain of the SETDB1 protein, at least one of the consecutive amino acid residues is an arginine residue, and at least one of the arginine residues is citrullinated (preferably all citrullinated). In certain embodiments, the core epitope includes at least the sequence shown in SEQ ID NO:8.

[0069] In certain embodiments, the kit of the present invention may also include one or more reagents or devices selected from the following: (i) a device for collecting or storing a sample from a subject (e.g., a blood collection device); (ii) other reagents required for performing the assay (e.g., buffers, diluents, blocking solutions, and / or standards).

[0070] Detection purposes and methods

[0071] In a third aspect, the present invention provides a method for detecting antibodies specific to citrullinated SETDB1 protein in a sample, comprising the following steps:

[0072] (1) contacting the sample with a capture reagent to obtain an antigen-antibody immune complex; the capture reagent is selected from the isolated polypeptide or a variant thereof described in the first aspect;

[0073] (2) Determine the amount of the antigen-antibody immune complex obtained in step (1).

[0074] In certain embodiments, the methods are performed in vitro.

[0075] In certain embodiments, the methods are used for non-diagnostic purposes.

[0076] In certain embodiments, in step (2), the amount of the immune complex is determined by immunological detection.

[0077] In certain embodiments, the immunological detection is selected from an enzyme immunoassay (eg, ELISA), a chemiluminescent immunoassay, a fluorescent immunoassay, or a radioimmunoassay.

[0078] In some embodiments, the capture reagent is attached to the surface of a solid support.

[0079] In other embodiments, the capture agent has a modification group that can be connected to a solid phase carrier. In such embodiments, before step (1), the method further comprises the step of coating the capture agent on the surface of a solid phase carrier.

[0080] In certain embodiments, the N-terminus of the capture agent is connected to the surface of a solid support, or carries a modification group that can be connected to a solid support.

[0081] In some embodiments, the assay is performed in an indirect method. Determining the antibody level in a sample by an indirect method is well known to those skilled in the art. In such assays, the "capture antigen" first forms an immune complex with the antibody in the sample, and then the captured antibody is detected by a secondary antibody (e.g., an anti-immunoglobulin antibody).

[0082] Therefore, in certain embodiments, in step (2), a detection reagent is used to detect the amount of the immune complex, and the detection reagent is selected from a secondary antibody with a detectable label.

[0083] In certain embodiments, the detectable label is selected from an enzyme (eg, horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (eg, an acridinium ester compound), a fluorescent dye, or biotin.

[0084] In certain embodiments, the secondary antibody is specific for an antibody from the species from which the test antibody is derived (eg, human).

[0085] In certain embodiments, the secondary antibody is an anti-immunoglobulin antibody.

[0086] In certain exemplary embodiments, the antibody being detected is an IgG antibody. In such embodiments, the anti-immunoglobulin antibody is selected from an anti-IgG antibody, such as an anti-human IgG antibody.

[0087] In certain exemplary embodiments, the antibody being detected is an IgM antibody. In such embodiments, the anti-immunoglobulin antibody is selected from an anti-IgM antibody, such as an anti-human IgM antibody.

[0088] In certain exemplary embodiments, the antibody being detected is an IgA antibody. In such embodiments, the anti-immunoglobulin antibody is selected from an anti-IgA antibody, such as an anti-human IgA antibody.

[0089] In other embodiments, the assay is performed in a double antigen sandwich format. Determination of antibody levels in a sample by a double antigen sandwich method is well known to those skilled in the art. In such assays, the "capture antigen" and "detection antigen" allow the sample antibody to form a bridge between two specific antigens, so that the two antigens are usually the same, or have the same core epitope, or have immunological cross-reactivity, so that one antibody can bind to both antigens.

[0090] Therefore, in certain embodiments, in step (2), a detection reagent is used to detect the amount of the immune complex, and the detection reagent is selected from the isolated polypeptide or a variant thereof described in the first aspect.

[0091] In certain embodiments, the detection reagent is selected from the isolated polypeptide or its variant described in the first aspect, and the isolated polypeptide or its variant carries a detectable label. In certain embodiments, the detectable label is selected from an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridinium ester compound), a fluorescent dye, or biotin. In certain exemplary embodiments, the detectable label is selected from an enzyme (e.g., horseradish peroxidase or alkaline phosphatase).

[0092] In certain embodiments, the polypeptide sequences contained in the detection reagent and the capture reagent are identical or substantially identical. In certain embodiments, the substantially identical means that the two polypeptide sequences contained in the detection reagent and the capture reagent contain the same core epitope, and the core epitope includes at least the 16th-26th consecutive amino acid residues of the MBD domain of the SETDB1 protein, at least one of the consecutive amino acid residues is an arginine residue, and at least one of the arginine residues is citrullinated (preferably all citrullinated). In certain embodiments, the core epitope includes at least the sequence shown in SEQ ID NO:8.

[0093] In another aspect, the present invention also relates to the use of the isolated polypeptide or variant thereof described in the first aspect in preparing a kit for detecting antibodies specific to citrullinated SETDB1 protein in a sample. In certain embodiments, the kit detects antibodies specific to citrullinated SETDB1 protein in a sample by the method described in the third aspect.

[0094] Diagnostic Uses and Methods

[0095] In a fourth aspect, the present invention provides a method for determining whether a subject suffers from rheumatoid arthritis, comprising:

[0096] (1) determining the level of antibodies specific for citrullinated SETDB1 protein in a sample from the subject; and,

[0097] (2) Comparing the level with a reference value.

[0098] In certain embodiments, if the level is higher than a reference value, the subject is diagnosed with rheumatoid arthritis.

[0099] In certain embodiments, the methods are performed in vitro.

[0100] Herein, the reference value is a value derived from a subject not suffering from rheumatoid arthritis or a healthy person (e.g., a subject with no detectable disease and no history of rheumatoid arthritis), or indicates the level of antibodies specific to citrullinated SETDB1 protein in a corresponding sample of a subject not suffering from rheumatoid arthritis or a healthy person.

[0101] In certain embodiments, the sample is a blood sample (eg, whole blood, plasma, or serum) or a tissue fluid sample (eg, synovial fluid).

[0102] In certain embodiments, the subject is a mammal, such as a human.

[0103] In certain embodiments, the level of antibodies specific for citrullinated SETDB1 protein in the sample is determined by an immunological assay. In certain embodiments, the immunological assay is selected from an enzyme immunoassay (eg, ELISA), a chemiluminescent immunoassay, a fluorescent immunoassay, or a radioimmunoassay.

[0104] In certain embodiments, the assay comprises using the isolated polypeptide or variant thereof according to the first aspect as a capture reagent.

[0105] In certain embodiments, step (1) comprises the following steps:

[0106] (1a) contacting a sample from the subject with a capture reagent to obtain an antigen-antibody immune complex; the capture reagent is selected from the isolated polypeptide or a variant thereof described in the first aspect;

[0107] (1b) determining the amount of the antigen-antibody immune complex obtained in step (1a).

[0108] In certain embodiments, in step (1b), the amount of the immune complex is determined by immunological detection. In certain embodiments, the immunological detection is selected from enzyme immunoassay (eg, ELISA), chemiluminescent immunoassay, fluorescent immunoassay, or radioimmunoassay.

[0109] In some embodiments, the capture reagent is attached to the surface of a solid support.

[0110] In other embodiments, the capture agent has a modification group that can be connected to a solid phase carrier. In such embodiments, before step (1), the method further comprises the step of coating the capture agent on the surface of a solid phase carrier.

[0111] In certain embodiments, the N-terminus of the capture agent is connected to the surface of a solid support, or carries a modification group that can be connected to a solid support.

[0112] In some embodiments, the assay is performed in an indirect method. Determining the antibody level in a sample by an indirect method is well known to those skilled in the art. In such assays, the "capture antigen" first forms an immune complex with the antibody in the sample, and then the captured antibody is detected by a secondary antibody (e.g., an anti-immunoglobulin antibody).

[0113] Therefore, in certain embodiments, in step (1b), the amount of the immune complex is detected using a detection reagent, wherein the detection reagent is selected from a secondary antibody with a detectable label.

[0114] In certain embodiments, the detectable label is selected from an enzyme (eg, horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (eg, an acridinium ester compound), a fluorescent dye, or biotin.

[0115] In certain embodiments, the secondary antibody is specific for an antibody from the species from which the test antibody is derived (eg, human).

[0116] In certain embodiments, the secondary antibody is an anti-immunoglobulin antibody.

[0117] In certain exemplary embodiments, the antibody being detected is an IgG antibody. In such embodiments, the anti-immunoglobulin antibody is selected from an anti-IgG antibody, such as an anti-human IgG antibody.

[0118] In certain exemplary embodiments, the antibody being detected is an IgM antibody. In such embodiments, the anti-immunoglobulin antibody is selected from an anti-IgM antibody, such as an anti-human IgM antibody.

[0119] In certain exemplary embodiments, the antibody being detected is an IgA antibody. In such embodiments, the anti-immunoglobulin antibody is selected from an anti-IgA antibody, such as an anti-human IgA antibody.

[0120] In other embodiments, the assay is performed in a double antigen sandwich format. Determination of antibody levels in a sample by a double antigen sandwich method is well known to those skilled in the art. In such assays, the "capture antigen" and "detection antigen" allow the sample antibody to form a bridge between two specific antigens, so that the two antigens are usually the same, or have the same core epitope, or have immunological cross-reactivity, so that one antibody can bind to both antigens.

[0121] Therefore, in certain embodiments, in step (1b), a detection reagent is used to detect the amount of the immune complex, and the detection reagent is selected from the isolated polypeptide or a variant thereof described in the first aspect.

[0122] In certain embodiments, the detection reagent is selected from the isolated polypeptide or its variant described in the first aspect, and the isolated polypeptide or its variant carries a detectable label. In certain embodiments, the detectable label is selected from an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridinium ester compound), a fluorescent dye, or biotin. In certain exemplary embodiments, the detectable label is selected from an enzyme (e.g., horseradish peroxidase or alkaline phosphatase).

[0123] In certain embodiments, the polypeptide sequences contained in the detection reagent and the capture reagent are identical or substantially identical. In certain embodiments, the substantially identical means that the two polypeptide sequences contained in the detection reagent and the capture reagent contain the same core epitope, and the core epitope includes at least the 16th-26th consecutive amino acid residues of the MBD domain of the SETDB1 protein, at least one of the consecutive amino acid residues is an arginine residue, and at least one of the arginine residues is citrullinated (preferably all citrullinated). In certain embodiments, the core epitope includes at least the sequence shown in SEQ ID NO:8.

[0124] In certain embodiments, the method in any of the above embodiments further comprises: providing a sample from the subject before step (1).

[0125] In certain embodiments, the method described in any of the above embodiments further comprises: after step (2), administering a therapeutically effective amount of a therapy that can treat rheumatoid arthritis (e.g., chemotherapy, radiotherapy and / or immunotherapy) to the subject diagnosed as having rheumatoid arthritis.

[0126] In certain embodiments, the therapy for treating rheumatoid arthritis is selected from surgery, chemotherapy (such as hydroxychloroquine sulfate, sulfasalazine, leflunomide, methotrexate, prednisone, celecoxib, ibuprofen, diclofenac sodium, loxoprofen sodium), targeted therapy (such as tumor necrosis factor antagonists), immunotherapy (such as tocilizumab) and combination therapy (such as chemotherapy + surgery).

[0127] In another aspect, the present invention also relates to the use of a reagent capable of detecting antibodies specific to citrullinated SETDB1 protein in preparing a kit for determining whether a subject suffers from rheumatoid arthritis.

[0128] In certain embodiments, the reagent is capable of determining the level of antibodies specific to citrullinated SETDB1 protein by immunological assay. In certain embodiments, the immunological assay is selected from enzyme immunoassay (eg, ELISA), chemiluminescent immunoassay, fluorescent immunoassay, or radioimmunoassay.

[0129] In certain embodiments, the subject is a mammal, such as a human.

[0130] In certain embodiments, the sample is a blood sample (eg, whole blood, plasma, or serum) or a tissue fluid sample (eg, synovial fluid).

[0131] In certain embodiments, the kit determines whether a subject has rheumatoid arthritis by:

[0132] (1) determining the level of antibodies specific for citrullinated SETDB1 protein in a sample from the subject; and,

[0133] (2) Comparing the level with a reference value.

[0134] In certain embodiments, if the level is higher than a reference value, the subject is diagnosed with rheumatoid arthritis.

[0135] In certain embodiments, the reagent capable of detecting antibodies specific to citrullinated SETDB1 protein comprises a capture reagent, and the capture reagent is selected from the isolated polypeptide or a variant thereof described in the first aspect.

[0136] In certain embodiments, the above step (1) comprises the following steps:

[0137] (1a) contacting a sample from the subject with a capture reagent to obtain an antigen-antibody immune complex; the capture reagent is selected from the isolated polypeptide or a variant thereof described in the first aspect;

[0138] (1b) determining the amount of the antigen-antibody immune complex obtained in step (1a).

[0139] In certain embodiments, in step (1b), the amount of the immune complex is determined by immunological detection. In certain embodiments, the immunological detection is selected from enzyme immunoassay (eg, ELISA), chemiluminescent immunoassay, fluorescent immunoassay, or radioimmunoassay.

[0140] In some embodiments, the capture reagent is attached to the surface of a solid support.

[0141] In other embodiments, the capture agent has a modification group that can be connected to a solid phase carrier. In such embodiments, before the above step (1), the method further comprises the step of coating the capture agent on the surface of a solid phase carrier.

[0142] In certain embodiments, the N-terminus of the capture agent is connected to the surface of a solid support, or carries a modification group that can be connected to a solid support.

[0143] In some embodiments, the reagent capable of detecting antibodies specific to citrullinated SETDB1 protein further comprises a detection reagent, wherein the detection reagent is selected from a secondary antibody with a detectable label.

[0144] In certain embodiments, in the above step (1b), the detection reagent is used to detect the amount of the immune complex.

[0145] In certain embodiments, the detectable label is selected from an enzyme (eg, horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (eg, an acridinium ester compound), a fluorescent dye, or biotin.

[0146] In certain embodiments, the secondary antibody is specific for an antibody from the species from which the test antibody is derived (eg, human).

[0147] In certain embodiments, the secondary antibody is an anti-immunoglobulin antibody.

[0148] In certain exemplary embodiments, the antibody being detected is an IgG antibody. In such embodiments, the anti-immunoglobulin antibody is selected from an anti-IgG antibody, such as an anti-human IgG antibody.

[0149] In certain exemplary embodiments, the antibody being detected is an IgM antibody. In such embodiments, the anti-immunoglobulin antibody is selected from an anti-IgM antibody, such as an anti-human IgM antibody.

[0150] In certain exemplary embodiments, the antibody being detected is an IgA antibody. In such embodiments, the anti-immunoglobulin antibody is selected from an anti-IgA antibody, such as an anti-human IgA antibody.

[0151] In other embodiments, the reagent capable of detecting antibodies specific to citrullinated SETDB1 protein further comprises a detection reagent, and the detection reagent is selected from the isolated polypeptide or variant thereof described in the first aspect.

[0152] In certain embodiments, in the above step (1b), the detection reagent is used to detect the amount of the immune complex.

[0153] In certain embodiments, the detection reagent is selected from the isolated polypeptide or its variant described in the first aspect, and the isolated polypeptide or its variant carries a detectable label. In certain embodiments, the detectable label is selected from an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridinium ester compound), a fluorescent dye, or biotin. In certain exemplary embodiments, the detectable label is selected from an enzyme (e.g., horseradish peroxidase or alkaline phosphatase).

[0154] In certain embodiments, the polypeptide sequences contained in the detection reagent and the capture reagent are identical or substantially identical. In certain embodiments, the substantially identical means that the two polypeptide sequences contained in the detection reagent and the capture reagent contain the same core epitope, and the core epitope includes at least the 16th-26th consecutive amino acid residues of the MBD domain of the SETDB1 protein, at least one of the consecutive amino acid residues is an arginine residue, and at least one of the arginine residues is citrullinated (preferably all citrullinated). In certain embodiments, the core epitope includes at least the sequence shown in SEQ ID NO:8.

[0155] Definition of terms

[0156] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Meanwhile, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0157] As used herein, the term "citrullination" refers to a form of post-translational modification (PTM), specifically, a chemical change in which the amino acid arginine (R) is deiminated to citrulline (C) by peptidyl arginine deiminase (PAD), resulting in the release of a nitrogen moiety. In a citrullinated sequence, at least one arginine residue is replaced with a citrulline residue. Therefore, the expressions "arginine residues are citrullinated" and "arginine residues are replaced with citrulline residues" have the same meaning.

[0158] As used herein, the terms "citrulline" and "cit" are used interchangeably and refer to 2-amino-5-(carbamoylamino)pentanoic acid, an α-amino acid having the formula: H2NC(O)NH(CH2)3CH(NH2)CO2H.

[0159] As used herein, the term "anti-cyclic citrullinated peptide (CCP) antibody" refers to an autoantibody with a synthetic cyclic citrullinated peptide (CCP) as an antigen, mainly of the IgG type. Anti-CCP antibodies are spontaneously secreted by B lymphocytes of patients with rheumatoid arthritis, while B lymphocytes of patients with other diseases and normal people do not spontaneously secrete anti-CCP antibodies. Therefore, anti-CCP antibodies have a high specificity for rheumatoid arthritis.

[0160] As used herein, the term "SETDB1" refers to a histone methyltransferase that methylates lysine at position 9 of histone H3 (H3K9) (Loyola A, et al., EMBO Reports. 2009; 10(7): 769-775; Gurard-Levin ZA, et al., Annu Rev Biochem. 2014; 83: 487-517). The human SETDB1 gene is located on human chromosome 1q21. The sequence of SETDB1 is well known to those skilled in the art, for example, the sequence of the human SETDB1 gene can be found in the Ensembl database accession number: ENSG00000143379. In this article, "citrullinated SETDB1 protein" refers to a SETDB1 protein in which at least one arginine residue is replaced by a citrulline residue. Preferably, at least one of the arginine residues at positions 16-26 of the MBD domain of the citrullinated SETDB1 protein is citrullinated (preferably all are citrullinated). Preferably, the citrullinated SETDB1 protein has the sequence shown in SEQ ID NO: 8 at positions 16-26 of its MBD domain.

[0161] As used herein, the term "SETDB1 protein MBD (methyl-CpG-binding domain) domain" refers to the methylated CpG binding domain contained in the N-terminus of the SETDB1 protein, which is located at positions 594-665 of the SETDB1 protein. The sequence of the SETDB1 protein MBD domain is well known to those skilled in the art, and an exemplary sequence thereof is provided in SEQ ID NO: 1.

[0162] As used herein, when referring to the amino acid sequence of the MBD domain of the SETDB1 protein, it is described using the sequence shown in SEQ ID NO: 1. For example, the expression "amino acid residues 16-26 of the MBD domain of the SETDB1 protein" refers to the amino acid residues 16-26 of the polypeptide shown in SEQ ID NO: 1. However, it is understood by those skilled in the art that mutations or variations may occur naturally or be artificially introduced into the amino acid sequence of the MBD domain of the SETDB1 protein without affecting its biological function. Therefore, in the present invention, the term "MBD domain of the SETDB1 protein" and similar expressions shall include all such sequences, including, for example, the sequence shown in SEQ ID NO: 1 and its natural or artificial variants (e.g., variants having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity). Furthermore, when describing a sequence fragment of the MBD domain of the SETDB1 protein, it includes not only a sequence fragment of SEQ ID NO: 1, but also a corresponding sequence fragment in a natural or artificial variant thereof. For example, the expression "amino acid residues 16-26 of the MBD domain of SETDB1 protein" includes amino acid residues 16-26 of SEQ ID NO: 1, and corresponding fragments in variants thereof (natural or artificial). According to the present invention, the expression "corresponding sequence fragment" or "corresponding fragment" refers to a fragment located at an equivalent position in the compared sequences when the sequences are optimally aligned, i.e., when the sequences are aligned to obtain the highest percentage identity.

[0163] As used herein, the term "specific binding" or "specific for..." refers to a non-random binding reaction between two molecules (i.e., a binding molecule and a target molecule), such as the reaction between an antibody and the antigen to which it is directed. The binding affinity between two molecules can be described by a KD value. The KD value refers to the dissociation constant obtained by the ratio of kd (the dissociation rate of a specific binding molecule-target molecule interaction; also known as koff) to ka (the association rate of a specific binding molecule-target molecule interaction; also known as kon), or refers to kd / ka expressed as a molar concentration (M). The smaller the KD value, the tighter the binding of the two molecules and the higher the affinity. In certain embodiments, an antibody that specifically binds to an antigen (or an antibody that is specific to an antigen) refers to an antibody that binds to an antigen with a specificity of less than about 10 -5 M, for example, less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 The KD value can be determined by methods well known in the art, such as using surface plasmon resonance (SPR) in a BIACORE instrument.

[0164] As used herein, the term "immunological assay" refers to an assay performed using the specific interaction / binding affinity between an antigen and an antibody, which is generally useful for detecting the presence or level of a specific antigen or antibody in a sample. Such immunological assays are well known to those skilled in the art, and include, but are not limited to, enzyme immunoassay (EIA), chemiluminescent immunoassay (CLIA), radioimmunoassay (RIA), fluorescent immunoassay (FIA), Western blotting, immunoturbidimetry, surface plasmon resonance, and the like. In certain embodiments, the immunological assay is an enzyme immunoassay (EIA), such as an ELISA assay, an Elispot assay, or a CLEIA assay. For a detailed description of immunological assays, see, for example, Fundamental Immunology, Ch.7 Paul, W., ed., 2nd edition, Raven Press, NY (1989).

[0165] As used herein, the term "epitope" refers to a site on an antigen that is specifically bound by an immunoglobulin or antibody. "Epitope" is also referred to as an "antigenic determinant" in the art. For example, an epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 continuous or non-continuous amino acids in a unique spatial conformation, which can be "linear" or "conformational". In a linear epitope, all points of interaction between a protein and an interacting molecule (e.g., an antibody) exist linearly along the primary amino acid sequence of the protein. In a conformational epitope, the points of interaction exist across separate protein amino acid residues.

[0166] As used herein, the term "detectable label" can be any substance detectable by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means. It is particularly preferred that such labels are suitable for use in immunological assays (e.g., enzyme-linked immunosorbent assays, radioimmunoassays, fluorescent immunoassays, chemiluminescent immunoassays, etc.). Such labels are well known in the art and include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3 H. 125 I. 35 S. 14 C or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g., Cy7, Alexa 750)), luminescent substances (e.g., chemiluminescent substances such as acridinium ester compounds), magnetic beads (e.g., ), calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to avidin (e.g., streptavidin) modified with the above labels. Patents teaching the use of such labels include, but are not limited to, U.S. Patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241 (all of which are incorporated herein by reference). The labels encompassed in the present invention can be detected by methods known in the art. For example, radioactive labels can be detected using photographic film or a scintillation counter, and fluorescent labels can be detected using a photodetector to detect emitted light. Enzyme labels are generally detected by providing a substrate to the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate, and calorimetric labels are detected by simple visualization of a colored label. In certain embodiments, detectable labels as described above can be attached to detection antibodies or antigens via linkers of varying lengths to reduce potential steric hindrance.

[0167] As used herein, the term "antibody" refers to an immunoglobulin molecule that is usually composed of two pairs of polypeptide chains, each pair having a light chain (LC) and a heavy chain (HC). Antibody light chains can be classified as κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the isotype of the antibody is defined as IgM, IgD, IgG, IgA and IgE, respectively. Within the light and heavy chains, the variable region and the constant region are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2 and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain CL. The constant domain is not directly involved in the binding of antibodies to antigens, but exhibits a variety of effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can also be subdivided into regions with high variability (called complementary determining regions (CDRs)), interspersed with more conservative regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain / light chain pair form antigen binding sites, respectively.

[0168] As used herein, the term "isolated" or "isolated" refers to that obtained from the natural state by artificial means. If a certain "isolated" substance or component appears in nature, it may be that the natural environment in which it is located has changed, or the substance has been separated from the natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity separated from this natural state is called isolated. The term "isolated" or "isolated" does not exclude the presence of artificial or synthetic substances, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.

[0169] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art, and include but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses, etc. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site.

[0170] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0171] As used herein, the term "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match). Typically, the comparison is made when the two sequences are aligned to produce maximum identity. Such an alignment can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol. Biol. 48: 444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0172] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or change the expected properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace a corresponding amino acid residue with another amino acid residue from the same side chain family. In addition, amino acid residues can also be divided into categories defined by optional physical and functional properties. For example, alcohol-containing residues (S and T), aliphatic residues (I, L, V and M), cycloalkenyl-related residues (F, H, W and Y), hydrophobic residues (A, C, F, G, H, I, L, M, R, T, V, W and Y), negatively charged residues (D and E), polar residues (C, D, E, H, K, N, Q, R, S and T), positively charged residues (H, K and R), small residues (A, C, D, G, N, P, S, T and V), very small residues (A, G and S), residues involved in turn formation (A, C, D, E, G, H, K, N, Q, R, S, P and T), and flexible residues (Q, T, K, S, G, P, D, E and R). Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10): 879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94: 412-417 (1997), which are incorporated herein by reference).

[0173] The compilation of the twenty conventional amino acids involved in this article follows conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, ES Golub and DR Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. And in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0174] As used herein, the term "subject" includes, but is not limited to, various animals, particularly mammals, such as humans.

[0175] Beneficial Effects

[0176] The present invention provides a citrullinated SETDB1 polypeptide and a detection method based on the level of anti-citrullinated SETDB1 antibodies. The technical solution of the present invention has high specificity and high sensitivity, and can realize the early diagnosis of rheumatoid arthritis. The technical solution of the present invention is easy to operate. Compared with the disease diagnosis of patients through imaging, the patient's disease can be preliminarily diagnosed by detecting the level of anti-citrullinated SETDB1 antibodies in the patient's serum. The detection positive rate of the technical solution of the present invention in the anti-cyclic citrullinated polypeptide antibody negative population can reach 50%, which greatly improves the detection rate of patients and solves the problem of low sensitivity of diagnostic markers in the prior art, which leads to missed diagnosis of early RA patients. Compared with artificially designed anti-CCP antibodies, the technical solution of the present invention is a protein naturally present in the patient's body, which is more beneficial to discover the pathogenesis of rheumatoid arthritis.

[0177] In summary, the citrullinated SETDB1 polypeptide and the detection method based on the anti-citrullinated SETDB1 antibody level provided by the present invention provide an effective solution to the problem that the RA diagnostic marker in the prior art is single and prone to missed diagnosis, and are also of great significance for the study of the pathogenesis of RA. BRIEF DESCRIPTION OF THE DRAWINGS

[0178] Figure 1A-1B :Evaluation of antibody levels and diagnostic value of different citrullinated peptides in rheumatoid arthritis. Figure 1A is the antibody level of different citrullinated peptides in rheumatoid arthritis; Figure 1B Evaluation of the diagnostic value of different citrullinated peptides in rheumatoid arthritis.

[0179] Figure 2:Comparison of the diagnostic value of citrullinated SETDB1 short peptide (cit-4) and anti-citrullinated peptide antibody (CCP2).

[0180] Figure 3 :Study on the expression level of citrullinated short peptide (cit-4) in anti-citrullinated peptide antibody (CCP2) negative population.

[0181] Figure 4A-4B :Study on the antibody level and diagnostic value of citrullinated SETDB1 short peptide (cit-4) in patients with different autoimmune diseases. Figure 4A It is the antibody level of cit-4 in patients with different autoimmune diseases; Figure 4B It is the evaluation of the diagnostic value of CIT-4 in patients with different autoimmune diseases.

[0182] Figure 5A-5B :The detection results of SETDB1 citrullinated peptides of different lengths in rheumatoid arthritis. Among them, Figure 5A is the antibody level of SETDB1 citrullinated peptides of different lengths in rheumatoid arthritis; Figure 5B The diagnostic value of SETDB1 citrullinated peptides of different lengths in rheumatoid arthritis was evaluated.

[0183] Sequence information

[0184] Table 1: Information on the sequences involved in the present invention is provided in the table below.

[0185]

[0186]

[0187] X stands for citrulline DETAILED DESCRIPTION

[0188] The invention is now described in the following non-limiting examples.

[0189] Those skilled in the art will appreciate that the embodiments describe the present invention by way of example and are not intended to limit the scope of protection claimed in the present application. The experimental methods in the embodiments are conventional methods unless otherwise specified. If specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

[0190] The ELISA test reagents and consumables used in the following examples were donated by Beijing Wantai Biopharmaceutical Co., Ltd.; the detection antigens used were synthesized by Nanjing GenScript Biotechnology Co., Ltd.

[0191] Example 1: Study on the antibody level of citrullinated SETDB1 short peptide (cit-4) in rheumatoid arthritis

[0192] First, the serum of patients with rheumatoid arthritis was screened for citrulline markers by mass spectrometry, and the initial citrulline short peptides obtained included: cit-2 (SEQ ID NO: 11), cit-3 (SEQ ID NO: 12), cit-4 (SEQ ID NO: 3), cit-5 (SEQ ID NO: 13), cit-8 (SEQ ID NO: 14), and cit-9 (SEQ ID NO: 15). Subsequently, these short peptides were coupled with biotin and chemically synthesized to provide biotin-labeled short peptides for further ELISA screening.

[0193] The expression level of autoantibodies to citrulline markers was detected by indirect ELISA. Briefly, a short peptide with biotin coupled to the N-terminus was first coated on the PABC plate at a concentration of 50 ng / well. After coating, sera from 24 patients with rheumatoid arthritis (RA) and 24 healthy subjects (HC) were diluted 10 times as primary antibodies and incubated for 30 minutes. HRP-labeled goat anti-human IgG (GAH-HRP) was used as the secondary antibody, incubated for 30 minutes, and finally the color was developed and read. The results are shown in Figure 1. Figure 1A The results showed that the level of autoantibodies of cit-4 in rheumatoid arthritis was significantly increased, with statistical significance. At the same time, the test results were analyzed by ROC curve. The results are as follows Figure 1B As shown, cit-4 has a high diagnostic value in rheumatoid arthritis, and its AUC value can reach 0.9462, which is significantly better than other short peptides.

[0194] Example 2: Study on the diagnostic value of citrullinated SETDB1 short peptide (cit-4) for rheumatoid arthritis

[0195] The expression level of citrulline marker autoantibodies in patients with rheumatoid arthritis was detected by indirect ELISA (see Example 1 for specific methods). In addition, anti-citrullinated polypeptide antibody (CCP2) was used as a control. The anti-citrullinated polypeptide antibody kit was purchased from Beijing Bell Biotechnology Co., Ltd. and the relevant detection was performed according to the kit instructions. The results are shown in Figure 2 middle. Figure 2 The results showed that the AUC value of cit-4 was 0.9887, which was greater than that of CCP2 (AUC value was 0.9667), and had a higher diagnostic effect compared with CCP2.

[0196] Example 3: Study on the expression level of citrullinated short peptide (cit-4) in anti-citrullinated peptide antibody (CCP2) negative population

[0197] The cit-4 antibody level was detected in patients with negative CCP2 test using indirect ELISA method (see Example 1 for details). The results are shown in Figure 3 middle. Figure 3 The results showed that in the CCP2-negative patient population, the positive rate of cit-4 could reach 50%, indicating that the sensitivity of cit-4 was better than that of CCP2.

[0198] Example 4: Study on the diagnostic value of citrullinated SETDB1 short peptide (cit-4) in patients with different autoimmune diseases

[0199] The levels of citrullinated SETDB1 autoantibodies in the serum of patients with different autoimmune diseases (RA rheumatoid arthritis, SLE systemic lupus erythematosus, PSS primary Sjögren's syndrome, SSc scleroderma) were detected by indirect ELISA method (see Example 1 for details). The results are shown in FIG4 . Figure 4A The results showed that among autoimmune diseases, citrullinated SETDB1 had the highest autoantibody level in rheumatoid arthritis. At the same time, the ROC curve analysis of the test results was performed, and the results were as follows Figure 4B As shown, among autoimmune diseases, citrullinated SETDB1 has the best diagnostic value for patients with rheumatoid arthritis.

[0200] Example 5: Expression levels of citrullinated SETDB1 truncated variants of different lengths in serum of patients with rheumatoid arthritis

[0201] Based on the citrullinated short peptide cit-4 (SEQ ID NO: 3), citrullinated SETDB1 truncated variants of different lengths (SEQ ID NOs: 4-10) were further designed and synthesized, all of which contained SEQ ID NO: 8 (i.e., amino acid residues 16-26 of the MBD domain). These citrullinated SETDB1 truncated variants (SEQ ID NOs: 3-10) were used to detect the expression level of citrullinated marker autoantibodies in patients with rheumatoid arthritis according to the indirect ELISA method (see Example 1 for specific methods). The results are shown in Figure 5. Figure 5A The results showed that the expression level of autoantibodies of citrullinated SETDB1 truncated peptides of different lengths in the serum of patients with rheumatoid arthritis was significantly increased. At the same time, the ROC curve analysis of the test results was performed. The results are as follows Figure 5B As shown, citrullinated SETDB1 truncated peptides of different lengths can be used for the diagnosis of rheumatoid arthritis.

[0202] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The full scope of the present invention is given by the attached claims and any equivalents thereof.

Claims

1. An isolated polypeptide or a variant thereof, wherein: The polypeptide consists of at least 11 consecutive amino acid residues of the MBD domain of the SETDB1 protein; wherein at least one of the consecutive amino acid residues is an arginine residue, and at least one of the arginine residues is citrullinated; The variant differs from the polypeptide from which it is derived only in the substitution (e.g., conservative substitution or non-conservative substitution) of 1 or several (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acid residues, and retains the biological function of the polypeptide from which it is derived (e.g., the activity of being recognized and bound by an anti-citrullinated SETDB1 antibody).

2. The isolated polypeptide or variant thereof according to claim 1, wherein The isolated polypeptide has an arginine residue at at least one of the amino acid positions corresponding to the following positions of the MBD domain of the SETDB1 protein: 2, 16, 17, 21, 22, 23, 26, 43 and / or 50; and at least one of the arginine residues is citrullinated; Preferably, the isolated polypeptide has an arginine residue at at least one of the amino acid positions corresponding to the following positions of the MBD domain of the SETDB1 protein: 16, 17, 21, 22, 23 and / or 26.

3. The isolated polypeptide or variant thereof according to claim 1 or 2, wherein The isolated polypeptide comprises at least amino acid residues 16 to 26 of the MBD domain of the SETDB1 protein; wherein at least one of the consecutive amino acid residues 16 to 26 is an arginine residue, and at least one of the arginine residues is citrullinated; Preferably, the isolated polypeptide has an arginine residue at at least one (e.g., 1, 2, 3, 4, 5 or all 6) of the amino acid positions corresponding to the following positions of the MBD domain of the SETDB1 protein: 16, 17, 21, 22, 23 and / or 26; and at least one (e.g., 1, 2, 3, 4, 5 or all 6) of the arginine residues is citrullinated; Preferably, the isolated polypeptide comprises at least the sequence shown in SEQ ID NO: 8; Preferably, the isolated polypeptide further comprises an arginine residue at at least one (e.g., 1, 2 or all 3) of the amino acid positions corresponding to the following positions of the MBD domain of the SETDB1 protein: 2, 43 and / or 50; and at least one (e.g., 1, 2 or all 3) of the arginine residues is citrullinated.

4. The isolated polypeptide or variant thereof according to any one of claims 1 to 3, wherein The isolated polypeptide consists of consecutive amino acid residues selected from the group consisting of amino acid residues 5-27, 8-27, 12-27, 14-27, 16-27, 16-26, 1-27, or 5-32 of the MBD domain of the SETDB1 protein; wherein at least one of the consecutive amino acid residues is an arginine residue, and at least one of the arginine residues is citrullinated; Preferably, the isolated polypeptide has an arginine residue at at least one (e.g., 1, 2, 3, 4, 5 or all 6) of the amino acid positions corresponding to the following positions of the MBD domain of the SETDB1 protein: 16, 17, 21, 22, 23 and / or 26; and at least one (e.g., 1, 2, 3, 4, 5 or all 6) of the arginine residues is citrullinated; Preferably, the isolated polypeptide further comprises an arginine residue at the amino acid position corresponding to position 2 of the MBD domain of the SETDB1 protein, which is optionally citrullinated.

5. The isolated polypeptide or variant thereof according to any one of claims 1 to 4, wherein The isolated polypeptide consists of no more than 72 (e.g., no more than 70, no more than 65, no more than 60, no more than 55, no more than 50, no more than 45, no more than 40, no more than 35 or no more than 30) consecutive amino acid residues of the MBD domain of the SETDB1 protein.

6. The isolated polypeptide or variant thereof according to any one of claims 1 to 5, wherein The variant does not include amino acid substitutions in the amino acid positions corresponding to the following positions of the MBD domain of the SETDB1 protein: 16, 17, 21, 22, 23, and 26; Preferably, the variant further does not comprise an amino acid substitution in the amino acid position corresponding to position 2, 43 and / or 50 of the MBD domain of the SETDB1 protein.

7. The isolated polypeptide or variant thereof according to any one of claims 1 to 6, wherein The isolated polypeptide comprises at least amino acid residues 2-26 of the MBD domain of the SETDB1 protein; Preferably, the isolated polypeptide has arginine residues at amino acid positions corresponding to positions 2, 16, 17, 21, 22, 23 and 26 of the MBD domain of the SETDB1 protein, and at least one (e.g., 1, 2, 3, 4, 5, 6 or all 7) of the arginine residues is citrullinated; Preferably, the isolated polypeptide has arginine residues at amino acid positions corresponding to positions 2, 16, 17, 21, 22, 23 and 26 of the MBD domain of the SETDB1 protein all citrullinated.

8. The isolated polypeptide or variant thereof according to any one of claims 1 to 7, wherein The MBD domain of the SETDB1 protein has the sequence shown in SEQ ID NO:

1.

9. The isolated polypeptide or variant thereof according to any one of claims 1 to 8, wherein The isolated polypeptide has a sequence as shown in any one of SEQ ID NOs: 3-10; Preferably, the variant has a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity compared to the sequence shown in any one of SEQ ID NOs: 3-10, and has exactly the same citrulline position and number.

10. The isolated polypeptide or variant thereof according to any one of claims 1 to 9, wherein The isolated polypeptide or variant thereof is connected to the surface of a solid support, or carries a modification group that can be connected to a solid support; Preferably, the modifying group is biotin or avidin; Preferably, the solid phase carrier is selected from magnetic beads or microtiter plates (eg, microtiter plates or ELISA plates).

11. The isolated polypeptide or variant thereof according to any one of claims 1 to 9, wherein The isolated polypeptide or variant thereof is detectably labeled; Preferably, the detectable label is selected from an enzyme (such as horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (such as an acridinium ester compound), a fluorescent dye or biotin.

12. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the isolated polypeptide or variant thereof according to any one of claims 1 to 9.

13. A vector comprising the isolated nucleic acid molecule of claim 12.

14. A host cell comprising the isolated nucleic acid molecule of claim 12 or the vector of claim 13.

15. A kit comprising the isolated polypeptide or variant thereof according to any one of claims 1 to 9.

16. The kit of claim 15, comprising a capture agent selected from the isolated polypeptide or variant thereof according to any one of claims 1 to 9; Preferably, the kit further comprises instructions for using the isolated polypeptide or variant thereof as a capture reagent to determine the level of antibodies specific for citrullinated SETDB1 protein in a sample from a subject, and optionally to determine whether the subject suffers from rheumatoid arthritis; Preferably, the capture agent is selected from the isolated polypeptide or variant thereof according to claim 10; Preferably, the subject is a mammal, such as a human.

17. The kit according to claim 16, further comprising a detection reagent, wherein The detection reagent is selected from a secondary antibody with a detectable label; Preferably, the detectable label is selected from an enzyme (such as horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (such as an acridinium ester compound), a fluorescent dye or biotin; Preferably, the secondary antibody is specific for an antibody of the species (e.g., human) from which the antibody to be tested originates; Preferably, the secondary antibody is an anti-immunoglobulin antibody; Preferably, the anti-immunoglobulin antibody is selected from anti-IgG antibody, anti-IgM antibody, anti-IgA antibody.

18. The kit according to claim 16, further comprising a detection reagent, wherein The detection agent is selected from the isolated polypeptide or variant thereof according to claim 11.

19. Use of a reagent capable of detecting antibodies specific to citrullinated SETDB1 protein in the preparation of a kit for determining whether a subject suffers from rheumatoid arthritis; Preferably, the reagent is capable of detecting antibodies specific for citrullinated SETDB1 protein by immunological assay; preferably, the immunological assay is selected from enzyme immunoassay (eg ELISA), chemiluminescent immunoassay, fluorescent immunoassay or radioimmunoassay; Preferably, the subject is a human; Preferably, the reagent is used to determine the level of antibodies specific to citrullinated SETDB1 protein in a sample from the subject; Preferably, the sample is a blood sample (eg, whole blood, plasma or serum) or a tissue fluid sample (eg, synovial fluid).

20. The use according to claim 19, wherein The reagent capable of determining antibodies specific to citrullinated SETDB1 protein comprises a capture reagent, wherein the capture reagent is selected from the isolated polypeptide or variant thereof according to any one of claims 1 to 7; Preferably, the capture agent is selected from the isolated polypeptide or a variant thereof according to claim 10.

21. The use according to claim 20, wherein The reagent capable of determining antibodies specific to citrullinated SETDB1 protein further comprises a detection reagent, wherein the detection reagent is selected from a secondary antibody with a detectable label; Preferably, the detectable label is selected from an enzyme (such as horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (such as an acridinium ester compound), a fluorescent dye or biotin; Preferably, the secondary antibody is specific for an antibody of the species (e.g., human) from which the antibody to be tested originates; Preferably, the secondary antibody is an anti-immunoglobulin antibody; Preferably, the anti-immunoglobulin antibody is selected from anti-IgG antibody, anti-IgM antibody or anti-IgA antibody; preferably, the anti-immunoglobulin antibody is anti-IgG antibody.

22. The use according to claim 20, wherein The reagent capable of detecting antibodies specific to citrullinated SETDB1 protein further comprises a detection reagent, and the detection reagent is selected from the isolated polypeptide or variant thereof according to claim 11.

23. Use of the isolated polypeptide or variant thereof according to any one of claims 1 to 11 in the preparation of a kit for detecting antibodies specific to citrullinated SETDB1 protein in a sample.

24. A method for detecting antibodies specific for citrullinated SETDB1 protein in a sample, comprising the following steps: (1) contacting the sample with a capture reagent to obtain an antigen-antibody immune complex; the capture reagent is selected from the isolated polypeptide or a variant thereof according to any one of claims 1 to 9; (2) determining the amount of the antigen-antibody immune complex obtained in step (1); Preferably, in step (2), the amount of the immune complex is determined by immunological detection; Preferably, the immunological detection is selected from enzyme immunoassay (e.g. ELISA), chemiluminescent immunoassay, fluorescent immunoassay or radioimmunoassay; Preferably, the capture agent is selected from the isolated polypeptide or variant thereof according to claim 10; Preferably, the method is performed in vitro; Preferably, the method is used for non-diagnostic purposes.

25. The method of claim 24, wherein: In step (2), the amount of the immune complex is detected using a detection reagent, wherein the detection reagent is selected from a secondary antibody with a detectable label; Preferably, the detectable label is selected from an enzyme (such as horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (such as an acridinium ester compound), a fluorescent dye or biotin; Preferably, the secondary antibody is specific for an antibody of the species (e.g., human) from which the antibody to be tested originates; Preferably, the secondary antibody is an anti-immunoglobulin antibody; Preferably, the anti-immunoglobulin antibody is selected from anti-IgG antibody, anti-IgM antibody or anti-IgA antibody; preferably, the anti-immunoglobulin antibody is anti-IgG antibody.

26. The method of claim 24, wherein: In step (2), the amount of the immune complex is detected using a detection reagent, wherein the detection reagent is selected from the isolated polypeptide or a variant thereof according to claim 11.

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