WO2020239090A1 - 一种类风湿关节炎相关的特异性多肽及其应用 - Google Patents

一种类风湿关节炎相关的特异性多肽及其应用 Download PDF

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WO2020239090A1
WO2020239090A1 PCT/CN2020/093402 CN2020093402W WO2020239090A1 WO 2020239090 A1 WO2020239090 A1 WO 2020239090A1 CN 2020093402 W CN2020093402 W CN 2020093402W WO 2020239090 A1 WO2020239090 A1 WO 2020239090A1
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cit
citrulline
rheumatoid arthritis
specific polypeptide
specific
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French (fr)
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杨翔
楼建荣
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广州市雷德生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/64—Cyclic peptides containing only normal peptide links
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

Definitions

  • the present invention relates to related technologies for the detection of rheumatoid arthritis, in particular to a polypeptide design of Multi-Cirtrullin Similar Motif (MCSM) and its application in the development of detection kits related to rheumatoid arthritis Applications.
  • MCSM Multi-Cirtrullin Similar Motif
  • Rheumatoid arthritis is a chronic systemic autoimmune disease. my country adopts the RA classification standard revised by the American College of Rheumatology (ACR) in 1987, with a specificity of 89% and a sensitivity of 91% to 94 %, but this standard is easy to miss some early or atypical patients.
  • Rheumatoid factor (rheumatoid factor, RF) is an antibody against the epitopes of the Fc fragment of human or animal IgG molecules, and is an autoantibody with denatured IgG as the target antigen. RF was first found in the serum of patients with rheumatoid arthritis (RA) by Rose et al. (1984).
  • anti-CCP antibodies can appear in the early stage of arthritis and are related to the destruction of bones and joints, but their sensitivity is 60% and specificity is 90%.
  • anti-cyclic citrulline antibody anti-CCP antibody
  • RF rheumatoid factor
  • AKA anti-keratin antibody
  • AFA anti-filaggin autoantibody
  • APF anti-pernuclear factor
  • anti-vimentin autoantibody anti-vimentin autoantibody
  • the CCP commercial test kit was launched in 2002 and is quickly and widely used in clinical testing. It is still the most commonly used clinical gold standard for RA testing. At present, there are at least six common kits from Axis-Shield (US), Euro-Diagnostica (The Netherlands), Euroimmun (Germany), Inova (US), Phadia (Sweden/Germany), Abbott (US).
  • the CCP2 detection kit is a multi-peptide optimized after a large number of screenings on the basis of CCP1. The sequence has not been published, and there is a slight difference in detection. The detection sensitivity is 66.7% to 77.7%, and the specificity is 86.1% to 98.8%.
  • CCP detection reagent CCP3.1 In recent years, the third generation of CCP detection reagent CCP3.1 (Inova, US) has appeared to detect the serum antibody IgG and IgA levels of patients. The peptide sequence of CCP3 is not disclosed. Studies have shown that the specificity of CCP3.1 is lower than CCP2 in the application of RA .
  • Anti-CarP (Anti-Carbamylated protein) antibody is a new type of RA specific detection antibody identified in recent years. Compared with CCP antibody, it can recognize different types of protein post-translational modification. 43% of RA patients have anti-carbamylation in the serum antibody.
  • CN1602426A discloses a method for detecting autoantibodies from patients with rheumatoid arthritis, including the autoantibody and peptide units containing XG motifs. , And contact the peptide unit containing the XnonG motif.
  • CN101918432A discloses a three-dimensional matrix of selected synthetic peptide mimic sequences, the synthetic peptide mimic sequences being preferentially recognized by autoimmune antibodies detected from patients suffering from rheumatoid arthritis, the synthetic peptide mimic sequences It can improve the specificity and sensitivity of detecting these autoantibodies in patients before the onset of symptoms, patients with rheumatoid arthritis symptoms, and patients diagnosed with rheumatoid arthritis positive.
  • CN101957365A discloses a kit for detecting CCP and IgG bispecific antibodies. The kit can detect a natural bispecific antibody in the serum of patients with rheumatoid arthritis, and is convenient to use, simple and easy to implement.
  • CN102323402A discloses a kit for in vitro detection of CCP antibodies and a preparation method thereof. The kit can be applied to the auxiliary diagnosis of rheumatoid arthritis.
  • CN102796173A discloses a kind of epitope of rheumatoid arthritis and its application. The epitope only binds to IgG in patient's serum and does not react with healthy human serum, and can be used to prepare drugs for diagnosing rheumatoid arthritis.
  • CCP- anti-citrulline-negative rheumatoid arthritis patients.
  • Citrulline detection reagents generally target a single protein molecule and have good specificity for patients with a certain subtype of RA.
  • a series of studies have shown that patients with anti-citrulline-positive (CCP+) and anti-citrulline-negative (CCP-) RA patients have significantly different clinical manifestations.
  • the lack of clinical diagnostic information in patients with CCP-rheumatoid arthritis is partially limited by The current detection method system fails to detect the specific indications carried by CCP-patients, and CCP-patients may be a subtype of RA patients with specific citrulline markers. Combining multiple detections with multiple targets is a very meaningful attempt to subdivide the types of autoantibodies in RA patients, so as to provide a better basis for clinical diagnosis and treatment.
  • CN106950365A discloses an ACPA-negative RA diagnostic marker and its application, specifically deoxycoprotein dioxygenase, DOHH or fragments thereof, in preparation for diagnosing rheumatoid arthritis disease with negative anti-citrulline polypeptide antibody Use in reagents.
  • CN106950366A discloses an ACPA-negative RA diagnostic marker and its application, specifically Pentaxin-related protein 3, PTX3 or fragments thereof, in the preparation of a reagent for diagnosing rheumatoid arthritis disease with negative anti-citrulline polypeptide antibody use.
  • the diagnostic specificity of these negative rheumatoid arthritis is only 90%.
  • RA autoantigens are the first to appear in the body of RA patients, and the accumulation of autoantigens reaches a certain degree of reaction. Autoantigens stimulate the T cells and memory cells in the patient's body to produce corresponding autoantibodies. In rheumatoid arthritis patients, antibodies against citrullinated autoantigens are considered to have high specificity (1) .
  • Citrulline is a non-protein amino acid. In nature, there is no tRNA that corresponds to citrulline. The citrullination of proteins in the body is catalyzed by the PADI enzyme. There are five types of PADI enzymes, among which PADI2 and PADI4 are found in the joint fluid of patients with rheumatoid arthritis.
  • Citrullination has some functions in the body’s normal physiology.
  • citrullinated filaggrin is related to the formation of the stratum corneum of the skin
  • citrullination of histones in the nucleus is related to the regulation of gene expression; in inflammation, trauma, cell
  • citrullinated protein will also increase in old age (4,5,6,7) .
  • arginine on the protein some are easy to be citrullinated, some are not easy, some are citrullinated faster, and some are slower (2) , and the structure of the protein, arginine in the protein 3
  • the position in the dimensional folding structure is related to the amino acid sequence around arginine.
  • Citrullination changes the polarity of amino acid side chains and therefore also changes the antigenicity (3) .
  • the change from arginine to citrulline has little effect on the molecular weight change of the protein, due to the change in charge, there is still a huge difference in the recognition of antibody antigens.
  • citrullinated peptides In people with HLA-DR4 genes, the affinity between citrullinated peptides and HLA-DR4 MHC molecules is much higher than that of the corresponding argininated peptides.
  • Research on CCP antibodies found that most of CCP autoantibodies are related to the high-affinity peptides of HLA-DR4.
  • the corresponding citrullinated antigen in vivo has not yet been discovered.
  • proteins were found to be citrullinated, such as structural protein filaggrin, collagen; gene regulatory protein histones; intracellular protein wave type protein, enolase; extracellular Protein fibrinogen and so on.
  • citrullinated antigens Although there are citrullinated antigens in RA serum, the autoantigens that specifically cause the disease have not yet been determined. The presence of citrullinated antigen is not unique to rheumatoid arthritis. There are also reports of elevated citrullinated antigen in other diseases, such as ankylosing spondylitis, psoriatic arthritis, undifferentiated spondyloarthritis, and joint damage. Multiple myeloma, osteoarthritis, gout, pseudogout, chondrocalcinosis, etc.
  • citrullinated protein in animal models, the deposition of citrullinated protein in joint tissues and the severity of arthritis Related, but the concentration of citrullinated protein in the joints of patients with rheumatoid arthritis has little to do with the severity of the disease; on the contrary, in some other diseases, citrullinated protein is sometimes found, such as scleroderma In the block (11) ; the hippocampus of Alzheimer's disease; in the renal tubules of obstructive nephropathy (12, 13) ; therefore, the identification of rheumatoid arthritis-specific citrullinated proteins is directly related to the detection Specificity.
  • rheumatoid arthritis-related antigens include human cartilage glycoprotein (HCgp-39), which is produced by chondrocytes, synovial fibroblasts, macrophages and neutrophils, and participates in tissue reconstruction and extracellular matrix degradation.
  • HCgp-39 is highly expressed in articular cartilage and synovium of RA patients.
  • Studies have shown that HCgp-39 polypeptide can be recognized by the peripheral blood mononuclear cells (PBMC) of RA patients, causing PBMC proliferation; and can induce chronic invasive arthritis in BALB/c mice.
  • PBMC peripheral blood mononuclear cells
  • Type II collagen can stimulate the recollection of T cells in RA patients and cause proliferation. It is now known that CII and some bacteria or bacteria (such as Mycobacterium tuberculosis, Epstein-Barr virus, etc.) have a common epitope QK/RRAA. Identify pathogenicity through molecular simulation or fuzzy identification. Many studies in recent years have shown that the amino acid sequence at positions 261-273 of CII is the main epitope of CII.
  • Aggrecan is the main macromolecular proteoglycan component of the cartilage matrix.
  • the degradation of polymerin in RA patients is accelerated, which may cause the destruction of articular cartilage.
  • Tests have confirmed that polymerin can activate T cells in the peripheral blood of RA patients to induce autoimmune responses. It is generally believed that the main T cell epitope of polymerin-induced arthritis exists in the G1 globular domain of the antigen.
  • the 280-292 amino acids of the main peptide epitope can not only induce T cell expansion and interferon (IFN)- ⁇ secretion, but also induce IgG2 antibody response.
  • IFN interferon
  • Glycoprotein P68 a molecular chaperone protein
  • BiP immunoglobulin binding protein
  • P205 cartilage connexin (LP), CH65, osteopontin (osteopontin), etc. may be used as autoantigens in the joints to participate in the pathological process of RA.
  • Calpastatin an antibody (ACAST-C27) against the amino acid epitope at the C-terminal 27 of calpastatin can be found in seronegative early RA, which may be of significance for the early diagnosis of RA.
  • glucose 6-phosphate isomerase is an important enzyme in glycolysis and gluconeogenesis.
  • the citrullination of arginine (R) residues in the amino acids 306-324 of filaggrin (SHQESTRCRSRGRSGRSGS) provides the main epitope recognized by AFA in patients with RA.
  • the process of protein recognition is crucial. It is believed that these peptides may also be one of the sources of citrullinated antigens and may have the characteristics of citrullinated sites.
  • the current RA antigen design is mainly based on selecting specific antigen regions from natural proteins, which is relatively inefficient and results in unsatisfactory results.
  • the purpose of the present invention is to overcome the limitations of the prior art, and provide a new artificially designed specific polypeptide related to rheumatoid arthritis and its application.
  • the first aspect of the present invention provides:
  • a specific polypeptide related to rheumatoid arthritis or a cyclic peptide and aggregate thereof, the specific polypeptide has the following characteristics:
  • citrulline or between citrulline and the citrulline-like amino acid, there are independently 0 to 2 spacer amino acids;
  • citrulline-like amino acids are independently selected from arginine, glutamine, or asparagine.
  • the specific polypeptide includes a citrulline core (Cit)-X-(Cit), where X is a spacer amino acid, such as (Cit)-G-(Cit), (Cit)-S- (Cit), (Cit)-T-(Cit).
  • X is a spacer amino acid
  • citrulline-like amino acids are distributed on the left and right sides of the citrulline core in the specific polypeptide; further, citrulline-like amino acids are distributed at intervals, in particular, two adjacent citrulline There is only one amino acid between similar amino acids.
  • the total number of citrulline in the specific polypeptide is 1-2. If there are 3 citrullines, there are 4 to 5 spacer amino acids between the third citrulline and the first two citrullines.
  • Specific polypeptides and cyclized peptides can be further multimerized in the form of monomers, dimers, trimers, and multimers.
  • the aggregates include aggregates of specific polypeptides and aggregates of cyclized peptides of the specific polypeptides.
  • the type of spacer amino acid is not particularly limited, and it may be various natural amino acids other than citrulline-like amino acids.
  • the total length of the specific polypeptide is 10-40 amino acids, preferably 16-30 amino acids. This is conducive to synthesis, while having strong antigenicity.
  • the spacer amino acid directly connected to the C-terminus of citrulline is selected from the group consisting of glycine, serine, threonine, alanine and proline.
  • the side chains of these spacer amino acids are small, small in size or easy to fold, and have less influence on the display of citrulline side chains, and are more conducive to obtaining specific peptides with better effects.
  • Proline has a turning result, and can further provide the effect of part of the cyclic peptide.
  • the specific polypeptide is at least one of the following polypeptides, wherein Cit refers to citrulline:
  • the second aspect of the present invention provides:
  • the specific polypeptide or its cyclic peptide and aggregates are used in preparing antigens for distinguishing citrullinated autoantibodies, and the sequence of the specific polypeptide is as described in the first aspect of the present invention.
  • the citrullinated autoantibody is derived from rheumatoid arthritis serum, joint fluid, or body fluid.
  • the third aspect of the present invention provides:
  • a method for inducing citrullinated autoantibodies includes the operation of immunizing animals with specific polypeptides or cyclic peptides and aggregates thereof, and the sequence of the specific polypeptides is as described in the first aspect of the present invention.
  • the fourth aspect of the present invention provides:
  • the application of the specific polypeptide or its cyclic peptide and aggregates according to the first aspect of the present invention includes:
  • the antibody is an anti-citrullinated autoantibody specific for detecting rheumatoid arthritis.
  • the fifth aspect of the present invention provides:
  • kits containing at least one specific polypeptide or a cyclic peptide and aggregate thereof, the sequence of the specific polypeptide is as described in the first aspect of the present invention.
  • the antibody is induced by the specific polypeptide or at least one of the cyclic peptide and aggregate thereof; and/or
  • At least one detection antibody which is anti-fibrinogen, collagen, wave type protein, enolase, human IgG, human IgM or human IgA.
  • the diagnostic accuracy of the kit can be further improved.
  • the kit is used to detect citrullinated autoantibodies and assess the condition of rheumatoid arthritis.
  • the specific polypeptides related to rheumatoid arthritis of the present invention have high sensitivity and specificity in identifying autoimmune antibodies of patients with rheumatoid arthritis, and can be directly used for the detection of RA autoimmune antibodies, and can also be used In immunized animals, it produces polyclonal antibodies or monoclonal antibodies that can be used to detect citrullinated proteins, and is used in the production of identification kits for citrullinated proteins in rheumatoid arthritis serum, joint fluid and body fluids.
  • the specific polypeptides related to rheumatoid arthritis of the present invention can also be cyclized. These specific polypeptides and their cyclized peptides can be further multimerized in the form of monomers, dimers, trimers, and multimers.
  • Figure 1 is a comparison of the side chain groups of citrulline and its similar amino acids
  • Fig. 2 is a schematic diagram of a specific polypeptide related to rheumatoid arthritis of the present invention
  • Figure 3 is a different peptide coating, testing CCP calibrator, and drawing a standard curve
  • Figure 4 shows the results of detecting RA-related specific antibodies in the serum of 20 RA patients coated with different polypeptides
  • Figure 5 shows the test results of 20 RA patients with different MCSM monoclonal antibodies combined with different secondary antibodies.
  • the invention found that around 1 to 3 citrulline, there are 3 to 5 amino acids similar to citrulline side chain on the side of citrulline, or the special structure formed around citrulline, named citrulline-like Acid side chain repeat structure; there are spacer amino acids between citrulline and similar citrulline side chain amino acids, consisting of 0 to 2 amino acids.
  • the pattern of the repeating structure is shown in Figure 2. This repeated structure has high sensitivity and specificity in the recognition of autoimmune antibodies in patients with rheumatoid arthritis.
  • Example 1 Design and synthesis of Multi-Cirtrullin Similar Motif (MCSM) polypeptide:
  • the designed peptide sequence is as follows:
  • polypeptides designed above were commissioned to be synthesized by a peptide chemical synthesis company.
  • biotinylation can be added to the synthesis;
  • cysteine at both ends can be added to form a cyclic polypeptide.
  • Example 2 Comparison of different citrullinated and MSCM polypeptides to detect differences in anti-citrullinated antibodies in human serum
  • Coating buffer PBS, pH7.4
  • Washing solution PBS+Triton X-100, pH7.4
  • Blocking solution PBS+1% BSA, pH7.4
  • Enzyme-labeled antibody HRP-labeled mouse anti-human IgG (for 1:15000)
  • Enzyme-labeled antibody diluent PBS+1%BSA+0.1%Triton X-100, pH7.4
  • Blocking 200ul/well add blocking solution, incubate at room temperature, 200rpm for 3 hours;
  • the SA coated ELISA plate can be used directly within a week and stored at 2 ⁇ 8°C; long-term storage needs to be dried: inverted and placed in a 37°C blast drying oven for 2 hours; aluminum foil bag and desiccant should be used to package the ELISA plate. Store at 2 ⁇ 8°C.
  • Reading value of microplate reader OD450, 630nm.
  • Calibrator 2 0.050 0.055 0.274 0.069 0.092 Calibrator 3 0.051 0.052 0.740 0.085 0.197 Calibrator 4 0.050 0.055 2.121 0.209 0.762 Calibrator 5 0.052 0.059 2.677 0.466 1.514 BLANK 0.051 0.052 0.055 0.055 0.052 BLANK 0.051 0.051 0.053 0.051 0.054 BLANK 0.049 0.052 0.053 0.050 0.051 To To To To To To To To To To To To To To To To To To To To To To To To To RA01 0.058 0.447 1.914 1.175 0.338 RA02 0.058 0.187 1.178 2.153 0.461 RA03 0.057 0.071 2.202 2.203 0.069 RA04 0.057 0.104 2.824 1.308 2.431 RA05 0.060 0.107 2.873 1.590 1.385 RA06 0.068 0.120 2.848 2.298 1.0
  • Figure 3 is the standard curve obtained by using peptide coating to detect CCP calibrator (in the figure, the data of P04 and P05 are overlapped);
  • Figure 4 is the different peptide coating, detecting RA in the serum of 20 RA patients and normal people Results of related specific antibodies.
  • the P04 polypeptide without citrulline can not distinguish the serum of healthy people from RA patients, and the detection rate is 0;
  • Example 3 Cross-link the synthetic polypeptide with BSA, and after purification, immunize rabbits to produce polyclonal antibodies
  • the synthetic peptide is coupled to the carrier protein BSA or OVA by EDC method or glutaraldehyde cross-linking method;
  • the antibody in the serum is purified using ProteinA/G affinity filler.
  • the purified polyclonal antibody combined with one or more antibodies against vimentin, fibrinogen, type II collagen, and enolase can constitute a kit for detecting specific antigens of rheumatoid arthritis.
  • Synthetic peptides are coupled to the carrier protein BSA or OVA by EDC method or glutaraldehyde cross-linking method;
  • the adjuvant can be Freund's complete adjuvant or a new type of immune adjuvant;
  • the purified monoclonal antibody is matched with one or more antibodies against vimentin, fibrinogen, type II collagen, and enolase, and can be used to prepare a kit for detecting specific antigen of rheumatoid arthritis.
  • Example 5 Assembly of a kit for detecting autoantigens
  • Coating buffer 0.1M ⁇ 0.5M carbonate buffer, pH 9.0 ⁇ 9.6;
  • Blocking solution 1 ⁇ 5% BSA (or 5% skimmed milk powder), 0.05M ⁇ 0.5M PBS buffer, 0.01% ⁇ 0.5% thimerosal, pH 6.0 ⁇ 8.5;
  • Diluent of negative and positive reference substance 1 ⁇ 5% BSA, 0.05M ⁇ 0.5M PBS buffer, 0.01% ⁇ 0.5% thimerosal, pH 6.0 ⁇ 8.5;
  • Enzyme-labeled antibody dilution solution 1 ⁇ 5% BSA, 0.05M ⁇ 0.5M PBS buffer, 0.01% ⁇ 0.5% thimerosal, pH 6.0 ⁇ 8.5;
  • Chromogenic buffer 0.01M ⁇ 1M phosphate-citrate buffer, pH 3.0 ⁇ 5.0;
  • Enzyme-labeled streptavidin can be obtained through ordinary purchase and screening.
  • the serum of patients with rheumatoid arthritis diagnosed with rheumatoid arthritis and normal human serum are collected from the hospital, and after processing, they are diluted with a negative and positive reference substance dilution in an appropriate ratio (1:3 to 1:100, preferably 1:20).
  • the kit includes the following aspects: 96-well ELISA plate, negative control, positive control, critical control, sample diluent, washing solution, enzyme-labeled antibody, color reagent, stop solution, sealing film and instructions.
  • Washing Wash the incubated ELISA plate, discard the solution and pat dry, and wash 4-6 times with the washing solution;
  • the antibody detection kit goes directly to the next step, and the antigen detection kit is incubated with enzyme-labeled streptavidin, and then goes to the next step after washing;
  • Measured value add 100 ⁇ l/well of the stop solution to the microtiter plate, tap and mix well, set the microplate reader wavelength to 450nm and 630nm for dual-wavelength measurement of the OD value of each well, and compare and calculate with the standard or reference result.
  • Example 7 Comparison of different anti-MSCM monoclonal antibodies in detecting RA specific antigens in human serum
  • Coating antibodies several strains of mouse monoclonal antibodies G4, G6, G9, G11 against the MSCM polypeptide (obtained after a series of steps including immunization of mice, cell fusion, and antibody screening in Example 4).
  • Coating buffer CBS, pH9.0 ⁇ 9.6
  • Washing solution PBS+0.05% Triton X-100, pH7.4
  • Blocking solution PBS+3% BSA, pH7.4
  • Enzyme-labeled antibody diluent PBS+1%BSA+0.1%Triton X-100, pH7.4
  • Serum of healthy controls 20 cases (N01 ⁇ N20)
  • Serum of clinically diagnosed RA patients 20 cases (RA01 ⁇ RA20)
  • Blocking 200ul/well add blocking solution, incubate at room temperature, 200rpm for 3 hours;
  • Drying is required for long-term storage: Place it upside down in a blast drying oven at 37°C for 1 to 3 hours; encapsulate the ELISA plate with an aluminum foil bag and desiccant, and store at 2-8°C.
  • Reading value of microplate reader OD450, 630nm.
  • Figure 5 is the test results of 20 RA patients with different MCSM monoclonal antibodies combined with different secondary antibodies.
  • the detection rate of anti-fibrinogen secondary antibody for G4 was 16/20 (80%); the detection rate of anti-vimentin secondary antibody for G4 was 15/20 (75%); Cutoff set OD450-630 ⁇ 0.2.
  • the detection rate of anti-fibrinogen secondary antibody for G6 is 18/20 (90%); the detection rate of anti-vimentin secondary antibody for G6 is 17/20 (85%);
  • the detection rate of anti-fibrinogen secondary antibody for G11 is 18/20 (90%); the detection rate of anti-vimentin secondary antibody for G11 is 18/20 (90%).
  • Example 8 MCSM polypeptide autoantibody rheumatoid arthritis diagnostic kit expands sample detection volume
  • sample serum is diluted 1:100 and added 100ul per well to the sample air of the reaction plate. Select the remaining wells to add standard or reference products, react at room temperature for 1 hour, after washing, add HRP-labeled anti-human IgG antibody, react at room temperature for 30 minutes, and wash Then add TMB substrate, react at room temperature for 15 minutes, add stop solution to terminate the reaction, use a spectrophotometer to detect the light absorption value at 450nm and 630nm, and compare the calculated results with the standard or reference.
  • Sample size and type A total of 350 clinical sera, including 214 normal people and 136 RA patients. The experimental results are shown in Table 1.
  • Table 1 Statistical Table of Clinical Diagnosis Results of MCSM Autoantibody Test Kit Vs Rheumatoid Arthritis
  • the kit that uses citrulline-like multimeric structural peptides (MSCM peptides) to detect autoantibodies has a positive coincidence rate of 81.6% and a negative coincidence rate of 97.7 compared with the results of clinical judgments. %; the overall agreement is 91.4%.
  • This kit has a sensitivity of 81% and a specificity of 91%, which can be used to assist doctors in making judgments.
  • the combination of different peptides can improve the sensitivity and specificity of detection.
  • the sample serum is diluted by 10 ⁇ 50 times and then 100ul per well is added to the sample air of the reaction plate, and the remaining wells are added Standard or reference product, react at room temperature for 1 hour, add biotin-labeled composite primary antibody (anti-fibrinogen antibody and anti-vimentin antibody, 1:1 ratio) after washing, react at room temperature for 1 hour, add dilution after washing A good enzyme-labeled streptavidin, react at room temperature for 30 minutes, add TMB substrate after washing, react at room temperature for 15 minutes, add stop solution to terminate the reaction, use a spectrophotometer to detect the light absorption value at 450nm and 630nm, and the standard or Comparing calculation results with reference products.
  • Sample size and type A total of 350 clinical sera, including 214 normal people and 136 RA patients. The experimental results are shown in Table 2.
  • the sensitivity of the rheumatoid arthritis autoantigen diagnostic kit prepared by immunizing animals with citrulline-like multimeric structural peptides (MSCM polypeptides) to produce antibodies is 86%, specificity is 96%, both sensitivity and specificity are superior to other commonly used detection kits, and can also be used to assist doctors in making judgments.
  • the combination of different coating antibodies and the combination of different recognition antibodies can further improve the sensitivity and specificity of detection.
  • the present invention uses the above embodiments to illustrate the detailed methods of the present invention, but the present invention is not limited to the above detailed methods, which does not mean that the present invention must rely on the above detailed methods to be implemented.
  • Those skilled in the art should understand that any improvement to the present invention, the equivalent replacement of each raw material of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc. fall within the scope of protection and disclosure of the present invention.

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Abstract

提供一种类风湿关节炎相关的特异性多肽及其应用。特异性多肽包括瓜氨酸，瓜氨酸的至少一侧连接有瓜氨酸类似氨基酸，瓜氨酸和所述瓜氨酸类似氨基酸之间还设有间隔氨基酸。提供的类风湿关节炎相关的特异性多肽，在识别类风湿关节炎患者的自身免疫性抗体中具有较高的灵敏度与特异性，可以直接用于RA自身免疫性抗体的检测，也可以用于免疫动物，生产可用于检测瓜氨酸化蛋白的多克隆抗体或单克隆抗体，用于类风湿关节炎血清，关节液及体液中瓜氨酸化蛋白的识别试剂盒的生产。

Description

一种类风湿关节炎相关的特异性多肽及其应用 技术领域
本发明涉及类风湿关节炎检测的相关技术，特别涉及一种多瓜氨酸类似氨基酸重复结构(Multi-Cirtrullin Similar Motif，MCSM)的多肽设计及其在类风湿关节炎相关的检测试剂盒开发中的应用。
背景技术
类风湿关节炎(rheumatoid factor,RA)是一种慢性系统性自身免疫疾病，我国采用1987年美国风湿病学会(ACR)修订的RA分类标准，其特异性为89％，灵敏度为91％～94％，但该标准容易漏诊一些早期或不典型的患者。类风湿因子(rheumatoid factor，RF)是一种抗人或动物IgG分子Fc片段抗原决定簇的抗体，是以变性IgG为靶抗原的自身抗体。RF最初由Rose等(1984年)在类风湿性关节炎(RA)患者血清中发现，可在疾病早期出现，检测方便、敏感性高(80％左右)，但是特异性较低，在正常人中RF阳性率也可达到5％，其他一些自身免疫性疾病中也大量出现。此外抗CCP抗体可在关节炎早期出现，并与骨关节的破坏相关，但其灵敏度60％，特异性90％。临床实验目前检测RA疾病最常用检测指标除了抗环瓜氨酸抗体(抗-CCP抗体)和类风湿因子(RF)，还有抗角蛋白抗体(AKA)、抗聚角蛋白微丝蛋白抗体(anti-filaggin autoantibody,AFA)、抗细胞核周围因子(anti-pernuclear factor,APF)、抗波形蛋白(anti-vimentin autoantibody)抗体等的发现为临床早期诊断RA、指导治疗、预测预后提供重要依据。
1998年Schellekens和Girbal Neuhause等根据filaggrin的cDNA序列合成的多肽证实瓜氨酸残基是类风湿关节炎特异的抗中间丝相关蛋白(filaggrin)抗体识别表位的必需组成。通过对基因文库中各个序列号的filggrin氨基酸序列进行分析，合成了一条以瓜氨酸代替精氨酸的20个左右氨基酸残基的肽链。并通过一定的试验显示了瓜氨酸是RA患者血清中抗filaggrin相关抗体识别的主要组成性抗原决定簇成分。在2000年，Schellekens将一条由19个氨基酸残基组成的瓜氨酸肽链中的两个丝氨酸替换为半胱氨酸，形成与β-转角具有相似结构的二硫键，合成环瓜氨酸肽(cyclic citrullinated peptide,CCP)。采用环瓜氨酸肽为抗原基质用ELISA法检测RA患者血清中的抗环瓜氨酸肽抗体，敏感性和特异性均有明显提高。这是二代CCP产品。合成的环瓜氨酸修饰肽不仅增加了抗原的稳定性，同时也提高了CCP检测的灵敏度和特异性。CCP商品化检测试剂盒于2002年上市，并迅速广泛的应用于临床检测，现在仍然是RA检测临床最常用金标准。目前至少有六种常见试剂盒，分别来自Axis-Shield(US)、Euro-Diagnostica(The Netherlands)、Euroimmun(Germany)、Inova(US)、Phadia(Sweden/Germany)、Abbott(US)。CCP2检测试剂盒是在CCP1的基础上通过大量筛选后优化的多个多肽，序列未公布，在检测方面有稍许差别，检测灵敏度为66.7％～77.7％，特异性为86.1％～98.8％。近几年出现CCP第三代检测试剂CCP3.1(Inova，US)可检测患者血清抗体IgG及IgA水平，CCP3肽序列未公开，有研究表明在RA诊断应用方面CCP3.1特异性低于CCP2。Anti-CarP(Anti-Carbamylated protein)抗体是近几年鉴定出新型RA特异性检测抗体，与CCP抗体相比可识别不同的蛋白翻译后修饰类型，43％的RA患者血清存在抗氨甲酰化抗体。
一直以来，人们研究的重点大多为CCP抗体阳性RA患者，CN1602426A公开了一种从患有类风湿关节炎的患者中检测自身抗体的方法，包括所述的自身抗体与包含XG基序的肽单元，和包含XnonG基序的肽单元接触。CN101918432A公开了一种选定的合成肽模拟序列的三维基质，所述的合成肽模拟序列优先被从患有风湿性关节炎的患者中检测到的自身免疫抗体所识别，所述合成肽模拟序 列能够提高对症状发生前的患者、表现出风湿性关节炎症状的患者以及确诊为风湿性关节炎阳性患者中的这些自身抗体进行检测的特异性和敏感性。CN101957365A公开了一种检测CCP和IgG双特异性抗体的试剂盒，该试剂盒可以检测存在于类风湿关节炎患者血清中的一种天然双特异性抗体，使用方便，简单易行。CN102323402A公开了一种CCP抗体体外检测的试剂盒及其制备方法，该试剂盒可应用于类风湿性关节炎的辅助诊断。CN102796173A公开了一种类风湿关节炎的抗原表位及其应用，该抗原表位只与患者血清中的IgG结合，而不与健康人血清反应，可用于制备诊断类风湿关节炎的药物。
但目前临床仍然有约30％类风湿关节炎患者尚未有明确检测指标，这类患者称为抗瓜氨酸阴性(CCP-)类风湿关节炎患者。瓜氨酸检测试剂一般靶向单个蛋白分子，对RA某亚型患者有良好特异性。一系列研究表明抗瓜氨酸阳性(CCP+)患者与抗瓜氨酸阴性(CCP-)RA患者有着显著不同的临床表现，对于CCP-类风湿关节炎患者临床诊断信息的缺失，部分受限于目前检测方法体系未能检测出CCP-患者携带的特异性指征，CCP-患者可能是含有特异性瓜氨酸标志物的RA患者亚型。合并多个靶点的多重检测是非常有意义的尝试，可细分RA患者自身抗体类型，从而更好地为临床诊治提供依据。
CN106950365A公开了一种ACPA阴性的RA诊断标志物及其应用，具体为脱氧辅蛋白双加氧酶即DOHH或其片段在制备用于诊断抗瓜氨酸多肽抗体阴性的类风湿性关节炎疾病的试剂中的用途。CN106950366A公开了一种ACPA阴性的RA诊断标志物及其应用，具体为Pentaxin相关蛋白3即PTX3或其片段在制备用于诊断抗瓜氨酸多肽抗体阴性的类风湿性关节炎疾病的试剂中的用途。但这些阴性类风湿关节炎的诊断特异性仅为90％。
早期诊断早期治疗是保护关节功能阻止关节破坏发生的关键。目前仍然有一部分的早期患者不能被确诊。国际公认的CCP法检测RA，灵敏度最多才能到70％，而且无法诊断早期患者。土耳其伊斯坦布尔大学kasapcopur等报告，与成人类风湿性关节炎(RA)不同，幼年型特发性关节炎(JIA)患者血清抗环瓜氨酸肽(CCP)抗体检出率很低。研究人员对122例JIA患儿和27例成人RA患者进行了抗CCP抗体检测。结果显示，仅3例多关节JIA患儿为抗CCP抗体阳性，且这些患儿都为女孩，都有腐蚀性关节病变，类风湿因子(RF)也都为阳性；而成人RA患者的抗CCP抗体阳性率则高达70％。
RA患者的体内最先出现RA自身抗原，经过自身抗原的积累到达一定的反应程度，自身抗原经过刺激患者机体内的T细胞及记忆细胞产生相应的自身抗体。在类风湿关节炎患者体内，针对瓜氨酸化自身抗原的抗体被认为具有很高的特异性 (1)。瓜氨酸是非蛋白质氨基酸，在自然界中，并没有一种tRNA是对应瓜氨酸的，体内蛋白上的瓜氨酸化是由PADI酶催化产生的。PADI酶分为5种，其中PADI2型和PADI4型在类风湿关节炎患者的关节液中有被发现。瓜氨酸化在机体的正常生理中有一些功能，如瓜氨酸化的丝聚蛋白与皮肤的角质层形成有关；细胞核内的组蛋白的瓜氨酸化与基因表达调控有关；在炎症，创伤，细胞凋亡，年老的情况下，瓜氨酸化蛋白也会升高 (4,5,6,7)。
在蛋白上所有的精氨酸中，有的容易被瓜氨酸化，有的不容易，有的瓜氨酸化较快，有的较慢 (2)，与蛋白的结构，精氨酸在蛋白3维折叠结构中所处位置有关，也和精氨酸周围的氨基酸序列有关。瓜氨酸化改变了氨基酸侧链的极性，因此也带来了抗原性的改变 (3)。尽管从精氨酸到瓜氨酸的变化对蛋白的分子量变化影响很小，但由于电荷的变化，在抗体抗原的识别中，还是产生了巨大的不同。在拥有HLA-DR4基因人群中，瓜氨酸化多肽与HLA-DR4的MHC分子之间的亲和力比对应的精氨酸化多肽大大提高了。对CCP抗体的研究发现，CCP自身抗体大部分与HLA-DR4的高亲和性多肽有关。但体内与之相应的瓜氨酸化抗原尚未发现。在瓜氨酸化抗原的 检测中，很多种蛋白都被发现有瓜氨酸化，如结构蛋白丝聚蛋白，胶原蛋白；基因调节类蛋白组蛋白；胞内蛋白波型蛋白，烯醇化酶；胞外蛋白纤维蛋白原等。
尽管RA血清中存在瓜氨酸化的抗原，但具体引发疾病的自身抗原至今尚未被确定。瓜氨酸化抗原的存在并非类风湿关节炎特有，其他一些疾病中也有瓜氨酸化抗原升高的报道，如强直性脊柱炎，银屑性关节炎，非分化性脊椎关节病，关节受损的多发性骨髓瘤，骨关节炎，痛风，假性痛风,软骨钙质沉着病等 (8，9，10)，在动物模型中，瓜氨酸化蛋白在关节组织中的沉积与关节炎的严重程度相关，但在类风湿关节炎病人的关节中的瓜氨酸化蛋白的浓度与疾病的严重程度关系不大；相反，在一些其他疾病中，瓜氨酸化蛋白时有发现，如硬皮病的斑块中 (11)；阿尔兹海默症的海马区；梗阻性肾病的肾小管中 (12，13)；因此，对类风湿关节炎特异性的瓜氨酸化蛋白的鉴别，直接关系到检测的特异性。
其他类风湿关节炎相关的抗原还有人软骨糖蛋白(HCgp-39)，由软骨细胞，滑膜成纤维细胞、巨噬细胞及中性粒细胞产生，参与组织重建和细胞外基质的降解。HCgp-39在RA患者的关节软骨及滑膜高表达。研究显示，HCgp-39多肽可被RA患者的外周血单个核细胞(PBMC)识别，引起PBMC增生；并可诱导BALB/c鼠发生慢性侵袭性关节炎。
II型胶原(CII)可刺激RA患者T细胞的回忆反应引起增生，现已知CII与某些细菌或病菌(如结核分支杆菌、EB病毒等)的蛋白均存在共同表位QK/RRAA，可能通过分子模拟或模糊识别致病。近年来的不少研究显示，CII261-273位氨基酸序列是CII的主要抗原表位。
聚合素(aggrecan)是构成软骨基质的主要大分子蛋白多糖成分。RA患者聚合素降解加速，可能导致关节软骨的破坏。试验证实，聚合素可激活RA患者外周血T细胞诱导自身免疫反应。一般认为，聚合素诱导关节炎的主要T细胞表位存在于该抗原的G1球状结构域。其中主要肽表位280-292位氨基酸除诱导T细胞扩增及干扰素(IFN)-γ分泌外，尚可诱导IgG2型抗体反应。
糖蛋白P68，一种分子伴侣蛋白，也称免疫球蛋白结合蛋白(BiP)。实验证实P68可刺激RA患者外周血及滑液T细胞增生。P68抗体对RA诊断敏感性为64％，特异性为99％。
P205、软骨连接蛋白(LP)、CH65、骨桥蛋白(osteopontin)等均可能做为关节局部的自身抗原参与RA免疫病理过程。
钙蛋白酶抑素(calpastatin)，针对钙蛋白酶抑素C端27位氨基酸表位的抗体(ACAST-C27)可见于血清阴性的早期RA，可能对于RA的早期诊断有一定意义。
文献报导，葡萄糖6-磷酸异构酶(GPI)是糖酵解和糖异生过程的重要酶类，RA患者滑液及血浆存在高滴度GPI抗体及可溶性GPI分子。聚丝蛋白第306-324位氨基酸(SHQESTRCRSRGRSGRSGS)中精氨酸(R)残基的瓜氨酸化提供了RA患者AFA识别的主要表位，其中心位置的瓜氨酸残基在RA对聚丝蛋白的识别过程至关重要。认为根据这些肽也可能是瓜氨酸化的抗原的来源之一，可能具有瓜氨酸化位点的特点。
现有的RA抗原设计，主要还是通过在天然蛋白中选取特异性抗原区，效率较为低下，结果也不尽如人意。
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发明内容
本发明的目的在于克服现有技术的局限，提供一种全新的人工设计的类风湿关节炎相关的特异性多肽及其应用。
本发明所采取的技术方案是：
本发明的第一个方面，提供：
一种类风湿关节炎相关的特异性多肽或其环肽和聚集体，所述特异性多肽具有如下特征：
包括1～3个瓜氨酸，2～5个瓜氨酸类似氨基酸；
瓜氨酸之间，或瓜氨酸和所述瓜氨酸类似氨基酸之间独立设有0～2个间隔氨基酸；
所述瓜氨酸类似氨基酸独立选自精氨酸、谷氨酰胺或天门冬酰胺。
在一些实例中，所述特异性多肽包括一个瓜氨酸核心(Cit)-X-(Cit)，X为一个间隔氨基酸，如：(Cit)-G-(Cit)、(Cit)-S-(Cit)、(Cit)-T-(Cit)。实验数据显示这样可以带来更好的效果。
在一些实例中，所述特异性多肽中瓜氨酸核心的左右两侧均有瓜氨酸类似氨基酸分布；进一步的，瓜氨酸类似氨基酸间隔分布，特别的，相邻的两个瓜氨酸类似氨基酸之间仅有一个氨基酸。
在一些实例中，所述特异性多肽中瓜氨酸的总数量为1～2个。如果存在3个瓜氨酸，则第3个瓜氨酸与前两个瓜氨酸之间存在4～5个间隔氨基酸。
特异性多肽及其环化肽还可以进一步以单体、二聚体、三聚体以及多聚体的形式被多聚化。所述聚集体包括特异性多肽的聚集体，以及所述特异性多肽的环化肽的聚集体。间隔氨基酸的种类没有特殊限制，可以是瓜氨酸类似氨基酸外的各种天然氨基酸。
在一些实例中，所述特异性多肽的总长度为10～40个氨基酸，优选为16～30个氨基酸。这样有利于合成，同时具有较强的抗原性。
在一些实例中，与瓜氨酸C端直接相连的间隔氨基酸选自甘氨酸，丝氨酸，苏氨酸，丙氨酸和脯氨酸。这些间隔氨基酸的侧链较小，体积较小或易折叠，对瓜氨酸侧链的展示影响较小，更有利于得到效果更好的特异性多肽。脯氨酸具有转折的结果，可以进一步提供部分环肽的效果。
在一些实例中，所述特异性多肽为以下多肽中的至少一种，其中Cit指瓜氨酸：
P01：HQDQG(Cit)S(Cit)NRAA
P02：DCDGQW(Cit)GP(Cit)SVE(Cit)HQSAKCK
P03：KCKQFRN(Cit)G(Cit)SPRAADCD
P05：SHQEST(Cit)G(Cit)SRG(Cit)SG(Cit)SGS
P06：HQDNQ(Cit)S(Cit)QNAA
P07：HQGQG(Cit)G(Cit)SQGQGRA
P08：HNGNG(Cit)G(Cit)SNGRGNA
P10：HANN(Cit)T(Cit)SNNGA
P11：RSQFNW(Cit)S(Cit)SRPR
P12：HQFRN(Cit)Q(Cit)RSRNHA
P13：HQYNFQW(Cit)S(Cit)GRPR
P14：GRFQM(Cit)H(Cit)RLIRH
P15：RSQFNF(Cit)P(Cit)SRPR
P16：KAAN(Cit)NNDAL(Cit)QAKQ
P17：HQYNFQW(Cit)S(Cit)GRP(Cit)NQAA
P18：DCDGQV(Cit)S(Cit)GRP(Cit)NQAAKCK
P19：HQMREL(Cit)S(Cit)GQVDQL(Cit)NDKAR。
本发明的第二个方面，提供：
特异性多肽或其环肽和聚集体在制备区分瓜氨酸化自身抗体的抗原中应用，所述特异性多肽的序列如上本发明的第一个方面所述。
在一些实例中，所述瓜氨酸化自身抗体来自类风湿关节炎血清、关节液或体液中。
本发明的第三个方面，提供：
一种诱导瓜氨酸化自身抗体的方法，包括使用特异性多肽或其环肽和聚集体免疫动物的操作，所述特异性多肽的序列如本发明的第一个方面所述。
本发明的第四个方面，提供：
本发明的第一个方面所述特异性多肽或其环肽和聚集体的应用，所述应用包括：
诱导产生单克隆抗体；
用于评价抗体效价；
所述抗体为检测类风湿关节炎特异的抗瓜氨酸化自身抗体。
本发明的第五个方面，提供：
一种试剂盒，其含有至少一种特异性多肽或其环肽和聚集体，所述特异性多肽的序列如本发明的第一个方面所述。
在一些试剂盒的实例中，所述特异性多肽或其环肽和聚集体中的至少一种诱导得到的抗体；和/或
至少一种检测抗体，所述检测抗体抗纤维蛋白原、胶原蛋白、波型蛋白、烯醇化酶、人IgG、人IgM或人IgA。
通过与其他检测抗体联合使用，可以进一步提高试剂盒的诊断准确性。
在一些试剂盒的实例中，所述试剂盒用于：检测瓜氨酸化自身抗体，评估类风湿关节炎病情。
本发明的有益效果是：
本发明的类风湿关节炎相关的特异性多肽，在识别类风湿关节炎患者的自身免疫性抗体中具有较高的灵敏度与特异性，可以直接用于RA自身免疫性抗体的检测，也可以用于免疫动物，生产可用于检测瓜氨酸化蛋白的多克隆抗体或单克隆抗体，用于类风湿关节炎血清，关节液及体液中瓜氨酸化蛋白的识别试剂盒的生产。
本发明的类风湿关节炎相关的特异性多肽，还可被环化。这些特异性多肽及其环化肽还可以进一步以单体、二聚体、三聚体以及多聚体的形式被多聚化。
附图说明
图1是瓜氨酸及其类似氨基酸的侧链基团比较情况；
图2是本发明的类风湿关节炎相关的特异性多肽的模式示意图；
图3是不同多肽包被，检测CCP校准品，绘制标准曲线；
图4是不同多肽包被，在20例RA患者血清中检测RA相关特异性抗体的结果；
图5是不同MCSM单抗配合不同二抗对20例RA患者血清的检测结果。
具体实施方式
发明人研究发现，如图1所示，从瓜氨酸的侧链结构上看，谷氨酰胺和天门冬酰胺与瓜氨酸有一定的相似度。发明发现，在1～3 个瓜氨酸的周围，由3到5个类似瓜氨酸侧链的氨基酸在瓜氨酸的一侧，或围绕瓜氨酸形成的特殊结构，命名为类瓜氨酸侧链重复结构；瓜氨酸和类似瓜氨酸侧链氨基酸之间还有间隔氨基酸，由0到2个氨基酸组成。其重复结构的模式如图2所示。此重复结构在类风湿关节炎患者中对自身免疫性抗体的识别中有较高的灵敏度与特异性。
为更进一步阐述本发明所采取的技术手段及其效果，以下结合本发明的优选实施例来进一步说明本发明的技术方案，但本发明并非局限在实施例范围内。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购获得的常规产品。
实施例1：多瓜氨酸类似氨基酸重复结构(Multi-Cirtrullin Similar Motif，MCSM)多肽的设计和合成：
设计的多肽序列如下：
P01：HQDQG(Cit)S(Cit)NRAA(SEQ ID NO.：1)
P02：DCDGQW(Cit)GP(Cit)SVE(Cit)HQSAKCK(SEQ ID NO.：2)
P03：KCKQFRN(Cit)G(Cit)SPRAADCD(SEQ ID NO.：3)
P04：HQCHQESTRGRSRGRCGRSGS(SEQ ID NO.：4)
P05：SHQEST(Cit)G(Cit)SRG(Cit)SG(Cit)SGS(SEQ ID NO.：5)
P06：HQDNQ(Cit)S(Cit)QNAA(SEQ ID NO.：6)
P07：HQGQG(Cit)G(Cit)SQGQGRA(SEQ ID NO.：7)
P08：HNGNG(Cit)G(Cit)SNGRGNA(SEQ ID NO.：8)
P10：HANN(Cit)T(Cit)SNNGA(SEQ ID NO.：9)
P11：RSQFNW(Cit)S(CIT)SRPR(SEQ ID NO.：10)
P12：HQFRN(Cit)Q(Cit)RSRNHA(SEQ ID NO.：11)
P13：HQYNFQW(Cit)S(Cit)GRPR(SEQ ID NO.：12)
P14：GRFQM(Cit)H(Cit)RLIRH(SEQ ID NO.：13)
P15：RSQFNF(Cit)P(Cit)SRPR(SEQ ID NO.：14)
P16：KAAN(Cit)NNDAL(Cit)QAKQ(SEQ ID NO.：15)
P17：HQYNFQW(Cit)S(Cit)GRP(Cit)NQAA(SEQ ID NO.：16)
P18：DCDGQV(Cit)S(Cit)GRP(Cit)NQAAKCK(SEQ ID NO.：17)
P19：HQMREL(Cit)S(Cit)GQVDQL(Cit)NDKAR(SEQ ID NO.：18)。
上述设计得到的多肽委托多肽化学合成公司合成。为了后续检测的目的，合成中可加上生物素化；为了肽链的稳定性和检测的灵敏度提高还可加上两端的半胱氨酸，形成环状多肽。
实施例2:比较不同瓜氨酸化和MSCM多肽检测人血清中抗瓜氨酸化抗体的差异
实验材料：
酶标板：NUNC446469
链霉亲和素：杭州纽龙rc-SA
包被缓冲液：PBS，pH7.4
洗涤液：PBS+Triton X-100，pH7.4
封闭液：PBS+1％BSA，pH7.4
样本稀释液：PBS+0.1％Triton X-100+1％BSA
酶标抗体：HRP标记的鼠抗人IgG(1：15000用)
酶标抗体稀释液：PBS+1％BSA+0.1％Triton X-100，pH7.4
不同生物化多肽：
P04(无瓜氨酸化)
P05(有瓜氨酸化，无MSCM特征构象)
P11(有瓜氨酸化，有MSCM特征构象)
P18(有瓜氨酸化，有MSCM特征构象)
欧蒙CCP
样本：
健康对照：20例
RA，欧蒙CCP+：20例
校准品：欧蒙CCP试剂盒校准品。
实验方法：
SA包被酶标板的制备
1)用PBS将SA稀释到10ug/ml，100ul/孔加入空白酶标板底部，2～8℃，200rpm孵育过夜；
2)弃液，拍干；
3)包被不同生物素化多肽；
4)将不同生物素化多肽分别稀释到5ug/ml，100ul/孔加入空白酶标板底部，室温，200rpm孵育1小时；
5)弃液，拍干；
6)洗涤1次：200ul/孔加入洗涤液，静置2min，弃液，拍干；
7)封闭：200ul/孔加入封闭液，室温，200rpm孵育3小时；
8)弃液，拍干；得到SA包被酶标板。
SA包被酶标板一周内可直接用，置于2～8℃保存；长期保存需干燥：倒置放在37℃鼓风干燥箱干燥2小时；需用铝箔袋加干燥 剂封装酶标板，置于2～8℃保存。
检测样本
1)将样本用样本稀释液稀释100倍，100ul/孔加入包被酶标板中；
2)室温，200rpm孵育1小时；
3)洗涤3次；
4)100ul/孔加入酶标抗体(HRP标记的鼠抗人IgG，1:15000)，室温，200rpm孵育30分钟；
5)洗涤3次；
6)显色15min，终止；
7)酶标仪读值：OD450，630nm。
布板：
  1 2 3 4 5 6
A 校准品1 RA01 RA09 RA17 N05 N13
B 校准品2 RA02 RA10 RA18 N06 N14
C 校准品3 RA03 RA11 RA19 N07 N15
D 校准品4 RA04 RA12 RA20 N08 N16
E 校准品5 RA05 RA13 N01 N09 N17
F BLANK RA06 RA14 N02 N10 N18
G BLANK RA07 RA15 N03 N11 N19
H BLANK RA08 RA16 N04 N12 N20
实验结果：
板1：包被P04酶标板
  1 2 3 4 5 6
A 0.049 0.058 0.060 0.058 0.057 0.055
B 0.050 0.058 0.057 0.060 0.055 0.055
C 0.051 0.057 0.059 0.057 0.057 0.059
D 0.050 0.057 0.064 0.057 0.060 0.053
E 0.052 0.060 0.062 0.058 0.054 0.071
F 0.051 0.068 0.062 0.057 0.089 0.056
G 0.051 0.059 0.056 0.080 0.058 0.062
H 0.049 0.068 0.058 0.057 0.057 0.061
板2：包被P05酶标板
  1 2 3 4 5 6
A 0.051 0.447 0.140 0.196 0.064 0.059
B 0.055 0.187 1.341 0.679 0.057 0.059
C 0.052 0.071 0.090 0.066 0.063 0.068
D 0.055 0.104 0.433 0.091 0.067 0.058
E 0.059 0.107 0.168 0.075 0.057 0.068
F 0.052 0.120 1.786 0.076 0.063 0.066
G 0.051 0.193 0.083 0.085 0.064 0.059
H 0.052 0.436 0.064 0.069 0.060 0.065
板3：欧蒙CCP
  1 2 3 4 5 6
A 0.104 1.914 2.565 2.639 0.062 0.059
B 0.274 1.178 2.626 2.111 0.062 0.063
C 0.740 2.202 2.742 1.854 0.063 0.068
D 2.121 2.824 2.668 2.792 0.066 0.057
E 2.677 2.873 2.860 0.072 0.058 0.078
F 0.055 2.848 2.340 0.093 0.061 0.059
G 0.053 2.820 1.084 0.093 0.059 0.062
H 0.053 2.987 0.995 0.066 0.063 0.064
板4：包被p11酶标板
  1 2 3 4 5 6
A 0.052 1.175 0.731 2.231 0.067 0.059
B 0.069 2.153 2.498 0.653 0.058 0.089
C 0.085 2.203 1.677 1.464 0.058 0.079
D 0.209 1.308 2.738 1.939 0.067 0.057
E 0.466 1.590 2.293 0.080 0.059 0.080
F 0.055 2.298 2.305 0.085 0.082 0.080
G 0.051 1.248 2.714 0.136 0.063 0.098
H 0.050 2.622 1.045 0.070 0.061 0.067
板5：包被p18酶标板
  1 2 3 4 5 6
A 0.065 0.338 1.779 1.468 0.062 0.057
B 0.092 0.461 0.507 0.377 0.055 0.056
C 0.197 0.069 0.293 0.187 0.056 0.061
D 0.762 2.431 1.760 1.813 0.062 0.062
E 1.514 1.385 0.763 0.059 0.056 0.066
F 0.052 1.089 0.362 0.061 0.058 0.055
G 0.054 2.439 0.228 0.082 0.058 0.063
H 0.051 2.939 0.061 0.066 0.058 0.058
板1～5结果总结如下表：
样本号 P04 P05 欧蒙CCP P11 P18
校准品1 0.049 0.051 0.104 0.052 0.065
校准品2 0.050 0.055 0.274 0.069 0.092
校准品3 0.051 0.052 0.740 0.085 0.197
校准品4 0.050 0.055 2.121 0.209 0.762
校准品5 0.052 0.059 2.677 0.466 1.514
BLANK 0.051 0.052 0.055 0.055 0.052
BLANK 0.051 0.051 0.053 0.051 0.054
BLANK 0.049 0.052 0.053 0.050 0.051
           
RA01 0.058 0.447 1.914 1.175 0.338
RA02 0.058 0.187 1.178 2.153 0.461
RA03 0.057 0.071 2.202 2.203 0.069
RA04 0.057 0.104 2.824 1.308 2.431
RA05 0.060 0.107 2.873 1.590 1.385
RA06 0.068 0.120 2.848 2.298 1.089
RA07 0.059 0.193 2.820 1.248 2.439
RA08 0.068 0.436 2.987 2.622 2.939
RA09 0.060 0.140 2.565 0.731 1.779
RA10 0.057 1.341 2.626 2.498 0.507
RA11 0.059 0.090 2.742 1.677 0.293
RA12 0.064 0.433 2.668 2.738 1.760
RA13 0.062 0.168 2.860 2.293 0.763
RA14 0.062 1.786 2.340 2.305 0.362
RA15 0.056 0.083 1.084 2.714 0.228
RA16 0.058 0.064 0.995 1.045 0.061
RA17 0.058 0.196 2.639 2.231 1.468
RA18 0.060 0.679 2.111 0.653 0.377
RA19 0.057 0.066 1.854 1.464 0.187
RA20 0.057 0.091 2.792 1.939 1.813
Cutoff＝0.2 0阳性 6阳性 20阳性 20阳性 17阳性
           
N01 0.058 0.075 0.072 0.080 0.059
N02 0.057 0.076 0.093 0.085 0.061
N03 0.080 0.085 0.093 0.136 0.082
N04 0.057 0.069 0.066 0.070 0.066
N05 0.057 0.064 0.062 0.067 0.062
N06 0.055 0.057 0.062 0.058 0.055
N07 0.057 0.063 0.063 0.058 0.056
N08 0.060 0.067 0.066 0.067 0.062
N09 0.054 0.057 0.058 0.059 0.056
N10 0.089 0.063 0.061 0.082 0.058
N11 0.058 0.064 0.059 0.063 0.058
N12 0.057 0.060 0.063 0.061 0.058
N13 0.055 0.059 0.059 0.059 0.057
N14 0.055 0.059 0.063 0.089 0.056
N15 0.059 0.068 0.068 0.079 0.061
N16 0.053 0.058 0.057 0.057 0.062
N17 0.071 0.068 0.078 0.080 0.066
N18 0.056 0.066 0.059 0.080 0.055
N19 0.062 0.059 0.062 0.098 0.063
N20 0.061 0.065 0.064 0.067 0.058
N均值 0.061 0.065 0.066 0.075 0.060
SD 0.009 0.007 0.010 0.019 0.006
N均值+3*SD 0.088 0.086 0.097 0.131 0.079
图3是使用多肽包被检测CCP校准品，绘制得到的标准曲线(图中，P04和P05的数据重叠了)；图4是不同多肽包被，在20例RA患者和正常人血清中检测RA相关特异性抗体的结果。
实验结果表明：
1)无瓜氨酸的P04多肽完全无法区分健康人与RA患者血清，检出率为0；
2)有瓜氨酸化，无MSCM特征构象的多肽P05，即使拥有4个瓜氨酸化位点，仍无法很好地区分健康人与RA患者血清，在20个RA血清中检出率仅为6个；
3)有瓜氨酸化，也有MSCM特征构象的多肽P11可以很好地区分健康人与RA患者血清，20个健康人均测为阴性，20个RA均测为阳性，与欧蒙CCP试剂盒检出率完全一致；
4)有瓜氨酸化，有部分MSCM特征构象的多肽P18，20个健康人均测为阴性，20个RA测出17个阳性，略低与欧蒙CCP试剂盒。
实施例3：合成多肽与BSA交联，纯化后，免疫兔子，生产多克隆抗体
1)合成多肽利用EDC法或戊二醛交联法偶联到载体蛋白BSA或OVA上；
2)挑选健康的6周大小的新西兰大白兔两只(约2Kg)；
3)将1ml的弗氏完全佐剂与准备好的1ml抗原充分混匀，呈乳白色；
4)初次免疫400ug抗原，后续免疫每次为100ug；背部皮下注射；
5)两周免疫一次，总共4～5次，验证血清中抗体浓度达标后最终采血；
6)血清中的抗体利用ProteinA/G亲和填料纯化。
纯化的多克隆抗体配合针对波形蛋白，纤维蛋白原，II型胶原蛋白，烯醇化酶的一种或多种抗体，可构成检测类风湿关节炎的特异性抗原的试剂盒。
实施例4：合成多肽与BSA交联纯化后，免疫小鼠，筛选单克隆抗体
1)合成多肽利用EDC法或戊二醛交联法偶联到载体蛋白BSA或OVA上；佐剂可采用弗氏完全佐剂，也可采用新型免疫佐剂；
2)选用6～8周龄BALB/C小鼠，背部免疫4～5次，每次间隔2～3周；最后一次免疫后10天，取血验证效价达标后，取脾融合 前三天加强免疫；第一次免疫用50ug每只小鼠，以后25ug每只每次；
3)选取血清效价最高的小鼠的取脾细胞与小鼠骨髓瘤细胞SP20融合；可采用电融合法，化学融合法，病毒融合法；
4)有限稀释融合细胞，96孔板克隆化；
5)HAT筛选杂交瘤细胞；检测出抗原检测阳性细胞，扩大培养；
6)液氮冻存细胞；
7)腹水法制备或杂交瘤细胞培养法制备抗体；
8)利用ProteinA/G亲和填料纯化抗体。
纯化的单克隆抗体配合针对波形蛋白，纤维蛋白原，II型胶原蛋白，烯醇化酶的一种或多种抗体，可用于制备检测类风湿关节炎的特异性抗原的试剂盒。
实施例5：检测自身抗原试剂盒的组装
1)、主要试剂：
(1)多克隆或单克隆抗MCSM抗体；
(2)包被缓冲液：0.1M～0.5M碳酸缓冲液，pH 9.0～9.6；
(3)封闭液：1～5％BSA(或5％脱脂奶粉)，0.05M～0.5M PBS缓冲液，0.01％～0.5％硫柳汞，pH 6.0～8.5；
(4)样本稀释液：1～5％BSA，0.05M～0.5M Tri-HCL缓冲液，0.01％～0.5％硫柳汞，pH 6.0～8.5；
(5)阴阳性对照品稀释液：1～5％BSA，0.05M～0.5M PBS缓冲液，0.01％～0.5％硫柳汞，pH 6.0～8.5；
(6)洗涤液：0.1％～0.5％Tween-20，0.05M～0.5M PBS缓冲液，0.01％～0.5％硫柳汞，pH 6.0～8.5；
(7)酶标抗体稀释液：1～5％BSA，0.05M～0.5M PBS缓冲液，0.01％～0.5％硫柳汞，pH 6.0～8.5；
(8)显色缓冲液：0.01M～1M磷酸盐-柠檬酸缓冲液，pH 3.0～5.0；
(9)显色液：TMB；
(10)终止液：硫酸。
2)、反应板工作液配制
采用生产的抗特殊结构瓜氨酸肽的多抗或单抗以筛选后的最佳浓度包被，过夜(16～20h)后弃废液，拍干，加入封闭液封闭；拍干后放入37℃温箱干燥1～3h后将反应板放入带有干燥剂的用铝箔袋密封包装。
生物素化抗波型蛋白，抗纤维蛋白原抗体，抗胶原蛋白抗体，抗烯醇化酶抗体或针对其他与类风湿关节炎相关的抗原的抗体单独或采用一定比例混合，按照最佳工作浓度配制成一抗工作液。
酶标记链霉亲和素可以普通采购筛选获得。
3)、阴阳性对照配制
采用从医院收集确诊为类风湿性关节炎患者的血清和正常人血清，经过处理后用阴阳性对照品稀释液按照适当比例进行稀释(1：3到1：100，优选为1：20)。
组建的试剂盒包括以下几个方面：96孔酶标板、阴性对照、阳性对照、临界对照，样本稀释液、洗涤液、酶标抗体、显色液、终止液、封板膜及说明书。
实施例6：试剂盒的使用方法
1)预处理：将试剂盒各组分置于室温平衡0.5～1h，用样本稀释液将样本进行(1：3到1：100)稀释，可根据具体实验确定；
2)加样：在酶标板相对应的孔中加入已稀释好的待测样品、阴阳性对照各100μl/孔，用封板膜封板；
3)孵育：将已加样的酶标板置200～280rpm振荡器室温孵育30～60min；
4)洗涤：将孵育好的酶标板，弃液拍干，用洗涤液洗涤4～6次；
5)加一抗或酶标抗体按照100μl/孔加入酶标板中，用封板膜封板；
6)孵育：将已加样的酶标板置200～280rpm振荡器室温孵育30～60min；
7)洗涤：将孵育好的酶标板，弃液拍干，用洗涤液洗涤4～6次；
8)抗体检测试剂盒直接进入下一步，抗原检测试剂盒则添加酶标链霉亲和素后孵育，洗涤后进入下一步；
9)显色：底物A与底物B按照1:10进行稀释混合均匀，按照100μl/孔加入酶标板中，置400rpm振荡器室温孵育10～15min；
10)测值：将终止液按照100μl/孔加入酶标板中，轻拍混匀，设定酶标仪波长450nm和630nm进行双波长测定各孔的OD值，与标准品或参考品比较计算结果。
实施例7：比较不同抗MSCM单抗检测人血清中RA特异性抗原的差异
实验材料：
酶标板：NUNC446469
包被抗体：几株抗MSCM多肽的鼠单抗G4、G6、G9、G11(由实施例4免疫小鼠，细胞融合，抗体筛选等一系列步骤后得到)。
包被缓冲液：CBS，pH9.0～9.6
洗涤液：PBS+0.05％Triton X-100，pH7.4
封闭液：PBS+3％BSA，pH7.4
样本稀释液：PBS+0.1％Triton X-100+1％BSA
一抗：
羊抗人纤维蛋白原多抗
羊抗人波形蛋白多抗
酶标二体：
HRP标记的兔抗羊IgG
酶标抗体稀释液：PBS+1％BSA+0.1％Triton X-100，pH7.4
样本：
健康对照者血清：20例(N01～N20)
临床确诊RA患者血清：20例(RA01～RA20)
实验方法：
不同抗MSCM单抗包被酶标板的制备
1)用CBS将包被抗体稀释到1到5ug/ml，100ul/孔加入空白酶标板底部，2～8℃，200rpm孵育过夜；
2)弃液，拍干；
3)洗涤1次：200ul/孔加入洗涤液，静置2min，弃液，拍干；
4)封闭：200ul/孔加入封闭液，室温，200rpm孵育3小时；
5)弃液，拍干；
6)一周内可直接用，置于2～8℃保存；
7)长期保存需干燥：倒置放在37℃鼓风干燥箱干燥1～3小时；需用铝箔袋加干燥剂封装酶标板，置于2～8℃保存。
检测样本
1)将样本用样本稀释液稀释，100ul/孔加入包被酶标板中；
2)室温，200rpm孵育1小时；
3)洗涤3次；
4)一抗(羊抗人纤维蛋白原按1：1000稀释；羊抗人波形蛋白按1：2500稀释)和酶标二抗(同RACP酶标二抗稀释度1：2500)按1：1比例混合，100ul/孔加入酶标板,室温，200rpm孵育30分钟；
5)洗涤5次；
6)显色15min，终止；
7)酶标仪读值：OD450，630nm。
布板：
Figure PCTCN2020093402-appb-000002
Figure PCTCN2020093402-appb-000003
实验结果
包被：G4
Figure PCTCN2020093402-appb-000004
包被：G6
Figure PCTCN2020093402-appb-000005
包被：G9
Figure PCTCN2020093402-appb-000006
包被：G11
Figure PCTCN2020093402-appb-000007
结果分析：
Figure PCTCN2020093402-appb-000008
Figure PCTCN2020093402-appb-000009
图5是不同MCSM单抗配合不同二抗对20例RA患者血清的检测结果。
结论：
1)不同MSCM单抗对RA患者的检出率不同，
2)不同二抗对检出的结果也有影响
3)G4用抗纤维蛋白原二抗的检出率为16/20(80％)；G4用抗波形蛋白二抗的检出率为15/20(75％)；Cutoff设为OD450-630≥0.2.
4)G6用抗纤维蛋白原二抗的检出率为18/20(90％)；G6用抗波形蛋白二抗的检出率为17/20(85％)；
5)G9用抗纤维蛋白原二抗的检出率为17/20(85％)；G9用抗波形蛋白二抗的检出率为17/20(85％)；
6)G11用抗纤维蛋白原二抗的检出率为18/20(90％)；G11用抗波形蛋白二抗的检出率为18/20(90％)。
实施例8：MCSM多肽自身抗体类风湿关节炎诊断试剂盒扩大样本检测量
以链霉亲和素包板，结合人工合成的生物素化的MSCM混合多肽(环化的P11，P07，P18)后，封闭，烘干。样品血清经1：100稀释后按每孔100ul加入反应板样品空中，选取其余孔加入标准品或参考品，室温反应1h，经洗涤后加入HRP标记的抗人IgG抗体，室温反应30min，经洗涤后加入TMB底物，室温反应15min，加入终止液终止反应，用分光光度计在450nm和630nm下检测光吸收值，与标准品或参考品比较计算结果。
样本数量及类型：共计350例临床血清，其中正常人214例，RA患者136例，实验结果如表1所示。
表1：MCSM自身抗体检测试剂盒Vs类风湿关节炎临床诊断结果统计表
Figure PCTCN2020093402-appb-000010
Figure PCTCN2020093402-appb-000011
从表1的结果可以看出，采用类瓜氨酸多聚结构肽(MSCM多肽)来检测自身抗体的试剂盒，与临床判断的结果相比的阳性符合率为81.6％；阴性符合率为97.7％；总一致性为91.4％。本试剂盒灵敏度为81％，特异性为91％，可以用来辅助医生做判断。
不同多肽的组合可以提高检出的灵敏度和特异性。
实施例9：类风湿关节炎自身抗原类风湿关节炎诊断试剂盒扩大样本检测量
以1～5ug/ml抗MSCM抗体(G11，G6的组合，1：1比例)包被酶标板，样品血清经10～50倍稀释后按每孔100ul加入反应板样品空中，选取其余孔加入标准品或参考品，室温反应1h，经洗涤后加入生物素标记的复合一抗(抗纤维蛋白原抗体与抗波形蛋白抗体的组合，1：1比例)，室温反应1h，经洗涤后加入稀释好的酶标链酶亲和素，室温反应30min，经洗涤后加入TMB底物，室温反应15min，加入终止液终止反应，用分光光度计在450nm和630nm下检测光吸收值，与标准品或参考品比较计算结果。
样本数量及类型：共计350例临床血清，其中正常人214例，RA患者136例。实验结果如表2所示。
表2 MCSM特异性抗原检测试剂盒Vs临床诊断结果统计表
Figure PCTCN2020093402-appb-000012
从表2的结果可以看出，采用类瓜氨酸多聚结构肽(MSCM多肽)免疫动物，生产抗体，制备的类风湿关节炎自身抗原诊断试剂盒，与临床判断的结果相比，灵敏度为86％，特异性为96％，无论是灵敏度还是特异性均优于其他常用检测试剂盒，也可以用来辅助医生做判断。
不同包被抗体的组合，不同识别抗体的组合可以进一步提高检出的灵敏度和特异性。
本发明通过上述实施例来说明本发明的详细方法，但本发明并不局限于上述详细方法，即不意味着本发明必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。

Claims (10)

  1. 一种类风湿关节炎相关的特异性多肽或其环肽和聚集体，所述特异性多肽具有如下特征：
    包括1～3个瓜氨酸，2～5个瓜氨酸类似氨基酸；
    瓜氨酸之间，或瓜氨酸和所述瓜氨酸类似氨基酸之间独立设有0～2个间隔氨基酸；
    所述瓜氨酸类似氨基酸独立选自精氨酸、谷氨酰胺或天门冬酰胺；
    进一步的，所述特异性多肽包括一个瓜氨酸核心(Cit)-X-(Cit)，X为一个间隔氨基酸；
    进一步的，所述瓜氨酸核心的左右两侧均有瓜氨酸类似氨基酸分布；
    进一步的，瓜氨酸类似氨基酸间隔分布，特别的，相邻的两个瓜氨酸类似氨基酸之间仅有一个氨基酸。
  2. 根据权利要求1所述的特异性多肽或其环肽和聚集体，其特征在于：所述特异性多肽的总长度为10～40个氨基酸，优选为16～30个氨基酸。
  3. 根据权利要求1或2所述的特异性多肽或其环肽和聚集体，其特征在于：与瓜氨酸C端直接相连的间隔氨基酸选自甘氨酸，丝氨酸，苏氨酸，丙氨酸和脯氨酸。
  4. 根据权利要求1所述的特异性多肽或其环肽和聚集体，其特征在于：所述特异性多肽为以下多肽中的至少一种，其中Cit指瓜氨酸：
    P01：HQDQG(Cit)S(Cit)NRAA
    P02：DCDGQW(Cit)GP(Cit)SVE(Cit)HQSAKCK
    P03：KCKQFRN(Cit)G(Cit)SPRAADCD
    P05：SHQEST(Cit)G(Cit)SRG(Cit)SG(Cit)SGS
    P06：HQDNQ(Cit)S(Cit)QNAA
    P07：HQGQG(Cit)G(Cit)SQGQGRA
    P08：HNGNG(Cit)G(Cit)SNGRGNA
    P10：HANN(Cit)T(Cit)SNNGA
    P11：RSQFNW(Cit)S(Cit)SRPR
    P12：HQFRN(Cit)Q(Cit)RSRNHA
    P13：HQYNFQW(Cit)S(Cit)GRPR
    P14：GRFQM(Cit)H(Cit)RLIRH
    P15：RSQFNF(Cit)P(Cit)SRPR
    P16：KAAN(Cit)NNDAL(Cit)QAKQ
    P17：HQYNFQW(Cit)S(Cit)GRP(Cit)NQAA
    P18：DCDGQV(Cit)S(Cit)GRP(Cit)NQAAKCK
    P19：HQMREL(Cit)S(Cit)GQVDQL(Cit)NDKAR。
  5. 特异性多肽或其环肽和聚集体在制备区分瓜氨酸化自身抗体的抗原中应用，所述特异性多肽的序列如权利要求1～4任一项所述。
  6. 根据权利要求5所述的应用，其特征在于：所述瓜氨酸化自身抗体来自类风湿关节炎血清、关节液或体液中。
  7. 一种诱导瓜氨酸化抗体的方法，包括使用特异性多肽或其环肽和聚集体免疫动物的操作，其特征在于：所述特异性多肽的序列如权利要求1～4任一项所述。
  8. 特异性多肽或其环肽和聚集体的应用，所述特异性多肽的序列如权利要求1～4任一项所述，所述应用包括：
    诱导产生单克隆抗体；
    用于评价抗体效价；
    所述抗体为检测类风湿关节炎特异的抗瓜氨酸化自身抗体。
  9. 一种试剂盒，其含有至少一种特异性多肽或其环肽和聚集体，其特征在于：所述特异性多肽的序列如权利要求1～4任一项所述；
    所述试剂盒用于：检测类风湿关节炎特异的抗瓜氨酸化自身抗体，评估类风湿关节炎病情。
  10. 根据权利要求9所述的试剂盒，还包括：
    所述特异性多肽或其环肽和聚集体中的至少一种诱导得到的抗体；和/或
    至少一种检测抗体，所述检测抗体抗纤维蛋白原、胶原蛋白、波型蛋白、烯醇化酶、人IgG、人IgM或人IgA。
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