CCP peptide segment, CCP antigen, rheumatoid arthritis detection reagent and its kit

Abstract

The present invention provides a CCP peptide segment, a CCP antigen, a rheumatoid arthritis detection reagent and a kit thereof. The CCP peptide segment has the structure shown in Formula I: NH2-S1-S2-X-COOH......Formula 1; wherein, S1 is a reaction site region having at least 1 lysine; S2 is a linker region; and X is a cyclized citrulline epitope region. The present invention solves the technical problem of low detection sensitivity of CCP antigens in the prior art during detection, and achieves the technical effect of improving the clinical detection sensitivity of anti-cyclic citrullinated peptide antibodies in the diagnosis of RA diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of medical detection, and in particular, to a CCP peptide segment, a CCP antigen, a rheumatoid arthritis detection reagent and a kit thereof. Background Art

[0002] Rheumatoid Arthritis (RA) is a common autoimmune disease characterized by chronic inflammatory responses in synovial joints and joint damage, which can ultimately lead to joint deformity and various system complications in the body. The prevalence of RA accounts for about 0.2 - 1% of the world's population, and the incidence rate in China is 0.28% - 0.41%, with women being more susceptible to the disease. Early identification and treatment of rheumatoid arthritis are important means to prevent irreversible joint damage. In the RA diagnostic criteria proposed by the American College of Rheumatology (ACR) and the European League Against Rheumatism (EULAR) in 2010, rheumatoid factor (RF) and anti-cyclic citrullinated peptide antibody (Anti-CCP antibody) were listed as serological detection markers. Compared with RF, the Anti-CCP antibody has extremely high detection specificity (up to over 95%) and good sensitivity, especially having high diagnostic value and prognostic potential for the early diagnosis of RA. The Anti-CCP antibody usually appears 2 - 10 years before the clinical manifestations of RA and is suitable for physical examination screening of healthy people.

[0003] Multiple autoantibodies that recognize citrullinated epitopes can be detected in the bodies of RA patients, including anti-perinuclear factor antibody, anti-keratin antibody, anti-citrullinated filaggrin antibody, anti-cyclic citrullinated peptide antibody, anti-α-vimentin antibody, etc. Citrulline is a non-coding amino acid in the body. Arginine in the protein is catalyzed by peptidylarginine deiminase (PAD) to produce citrulline, and the formed citrulline antigen causes an immune response in the body during cell damage or uncontrolled apoptosis, thereby leading to the production of autoantibodies against citrulline peptides or protein epitopes. Therefore, simulating citrulline epitope antigens in vitro is an important basis for serum antibody detection.

[0004] In 1998, Schellekens et al. screened out a linear peptide segment of about 20 amino acids from the sequence of filaggrin, which could detect antibodies in the sera of early-stage patients. In 2000, based on the previous work, Schellekens et al. replaced serine in the linear filaggrin peptide segment with cystine and introduced disulfide bonds to form a cyclic conformation, thus obtaining CCP with a 21-amino acid sequence, namely the first-generation CCP (CCP1). Compared with linear citrullinated peptides, the sensitivity of CCP1 in diagnosing RA increased (from 49% to 68%), and the specificity remained almost unchanged. However, the sensitivity of CCP1 was slightly lower than that of RF. In 2002, scientists constructed multiple polypeptide libraries, screened out different series of antigen peptides with good reactivity according to the reaction results of the libraries with the sera of RA patients, and found that the combined use of multiple CCP antigens could significantly improve the detection sensitivity. The sensitivity of CCP2 in diagnosing RA is 64.4% - 96%, and it has become the most commonly used antigen form in the current Anti-CCP antibody detection market.

[0005] The existing problems are as follows: The CCP2 antigen is usually a cyclic polypeptide of 12 - 19 amino acids, with a short structure. The shorter antigen peptide has fewer active sites and a low coupling rate with the carrier. After binding to the solid phase, it has a small active space, which to a certain extent limits the binding process with antibodies, thereby affecting the sensitivity of reagent detection and resulting in the problem of missed detection. Summary of the Invention

[0006] The present invention solves the technical problem of low detection sensitivity of CCP antigens in detection in the prior art, and achieves the technical effect of improving the clinical detection sensitivity of anti-cyclic citrullinated peptide antibodies in the diagnosis of RA diseases.

[0007] To solve the above problems, the present invention provides a CCP peptide segment, which has the structure shown in Formula I: NH2 - S1 - S2 - X - COOH... Formula 1; wherein, S1 is a reaction site region with at least 1 lysine; S2 is a linker region; X is a cyclized citrulline epitope region.

[0008] Compared with the prior art, the technical effects achieved by adopting this technical solution are as follows: In the above peptide segment, the antigenic epitope region containing citrulline is the key region that directly recognizes and binds to the antibodies in the test sample. The linker in the side chain can provide a larger active space for the antigenic epitope region, increase the reaction probability with antibodies, improve the signal-to-noise ratio and sensitivity of the reagent, and further improve the clinical detection specificity of anti-cyclic citrullinated peptide antibodies in the diagnosis of RA diseases. The introduction of terminal lysine can increase the active sites of the polypeptide, improve the coupling efficiency, and further optimize the performance of the detection reagent.

[0009] In an example of the present invention, S1 has 1 to 4 amino acids, and at least one of them is lysine.

[0010] Compared with the prior art, the technical effects achieved by adopting this technical solution: S1 is the amino acid residue region used to form a ring to form the CCP peptide segment. Further, to improve the coupling efficiency, S1 has at least one lysine to increase the polypeptide active site.

[0011] In one example of the present invention, S2 is any one or more of Mini-PEG, PEG4, PEG8, PEG12, and 6-aminohexanoic acid.

[0012] Compared with the prior art, the technical effects achieved by adopting this technical solution: The linker arm is used to improve the stability of polypeptide connection. The above reagent is a hydrophilic linker arm, which can improve solubility. The polyethylene glycol polymer chain is covalently linked to the polypeptide. The polypeptide modified with polyethylene glycol, and the increase in the molecular mass of the peptide can also protect the polypeptide from being cleaved by proteases and improve the half-life of biological activity. In addition, polyethylene glycolylation can also make the polypeptide not produce an immune response and improve pharmacokinetics. The incorporation of 6-aminohexanoic acid provides a straight-chain space of six carbons, reduces the steric hindrance of the reaction, improves the reaction efficiency, and reduces the reaction difficulty.

[0013] In one example of the present invention, X has 10-16 amino acids, wherein at least one citrulline is contained in the amino acid segment at positions 4-9, and the amino acids at the head and tail of the X region form a ring.

[0014] Compared with the prior art, the technical effects achieved by adopting this technical solution: The polypeptide sequence is too short to form a cyclic structure, and if the sequence is too long, it is easy to lose the conformational advantage of the cyclic structure, and at the same time, it is easy to introduce other non-essential binding sites. Therefore, it is reasonably set according to needs in practical applications. At least one citrulline is contained in the amino acid segment at positions 4-9. Preferably, the citrulline located at the most central position has better detection sensitivity and improves the signal-to-noise ratio at the same time.

[0015] In one example of the present invention, the CCP peptide segment exists in the form of a CCP-carrier protein conjugate, CCP-biotin, or CCP-fluorescein isothiocyanate.

[0016] Compared with the prior art, the technical effects achieved by adopting this technical solution are as follows: After coupling the CCP peptide segment with the carrier protein, the presence of the carrier protein will to a certain extent reduce the steric hindrance of antigen-antibody recognition, and the CCP polypeptide with antigenic epitopes is more easily recognized by antibody molecules, thus ensuring the high sensitivity of the reagent. By biotin-modifying the CCP peptide segment, the formed cyclic citrullinated peptide structure is more stable. Fluorescein isothiocyanate has relatively high activity. Generally speaking, introducing this kind of fluorescent group during the solid-phase synthesis process is easier than other fluoresceins, and no activating reagent needs to be added during the reaction. Linking the side chain of the CCP peptide segment to fluorescein isothiocyanate directly plays a role in reducing the steric hindrance, thereby improving the reaction efficiency.

[0017] In one example of the present invention, the carrier protein in the CCP-carrier protein conjugate is an animal-derived carrier protein or a human-derived carrier protein.

[0018] Compared with the prior art, the technical effects achieved by adopting this technical solution are as follows: Using an animal-derived carrier protein or a human-derived carrier protein as the carrier protein can not only increase the size of the antigen but also enhance the immunogenicity.

[0019] In one example of the present invention, the carrier protein is any one of the following: bovine serum albumin, cationic bovine serum albumin, hemocyanin, ovalbumin, human serum albumin, bovine γ-globulin, and human γ-globulin.

[0020] Compared with the prior art, the technical effects achieved by adopting this technical solution are as follows: For the selection of the carrier protein, factors such as its solubility, size, coupling group, immunogenicity, and cost price are mainly considered. The above-mentioned carrier proteins are easily obtained and can be selected according to actual needs.

[0021] The present invention also provides a CCP antigen, and the CCP antigen includes one or more CCP peptide segments of any one of the above examples.

[0022] Compared with the prior art, the technical effects achieved by adopting this technical solution are as follows: The CCP antigen provided by the present invention has all the beneficial effects of the CCP peptide segments of any one of the above examples. Therefore, the CCP antigen formed by combining one or several of them has not only high detection sensitivity but also high detection specificity when used for detecting the serum of RA patients.

[0023] The present invention also provides a rheumatoid arthritis detection reagent, and the reagent includes a CCP antigen, and the CCP antigen is the antigen of the above example.

[0024] Compared with the prior art, the technical effects achieved by adopting this technical solution: The rheumatoid arthritis detection reagent provided by the present invention has all the beneficial effects of the CCP antigen in the above examples. Therefore, on the premise of maintaining the existing detection specificity of the CCP antigen, it has higher clinical detection sensitivity and reduces the risk of false negatives and missed detections.

[0025] The present invention also provides a rheumatoid arthritis detection kit, and the kit includes the detection reagent in the above example.

[0026] Compared with the prior art, the technical effects achieved by adopting this technical solution: The rheumatoid arthritis detection kit provided by the present invention has all the beneficial effects of the detection reagent in the above examples. By reacting the detection reagent in the above rheumatoid arthritis detection kit with the serum of a patient, it can be detected by a semi-automatic immunoassay analyzer or a fully automatic immunoassay analyzer, which is convenient for detection and has a high degree of automation. Detailed implementation manners

[0027] To make the above objects, features, and advantages of the present invention more obvious and understandable, the specific embodiments of the present invention will be described in detail below.

[0028] Example 1:

[0029] This example provides a CCP peptide segment, and the CCP peptide segment has the structure shown in Formula I: NH2-S1-S2-X-COOH …………… Formula 1; wherein, S1 is a reaction site region having at least 1 lysine; S2 is a linker region; X is a cyclized citrulline epitope region.

[0030] Specifically, in the above peptide segment, the antigenic epitope region containing citrulline is the key region that directly recognizes and binds to the antibody in the test sample. The linker in the side chain can provide a larger activity space for the antigenic epitope region, increase the reaction probability with the antibody, improve the signal-to-noise ratio and sensitivity of the reagent, and further improve the clinical detection sensitivity of anti-cyclic citrullinated peptide antibody in the diagnosis of RA disease. The introduction of terminal lysine can increase the polypeptide active site, improve the coupling efficiency, and further optimize the performance of the detection reagent.

[0031] Further, S1 has 1 to 4 amino acids, and at least one of them is lysine.

[0032] Specifically, S1 is the amino acid residue region for connecting to form a ring to form a CCP peptide segment. Further, to improve the coupling efficiency, S1 has at least one lysine to increase the polypeptide active site.

[0033] Preferably, S1 is a combination of glycine and lysine.

[0034] Further, S2 is any one or more of Mini-PEG, PEG4, PEG8, PEG12, and 6-aminohexanoic acid.

[0035] Specifically, the linker is used to improve the stability of polypeptide ligation. The above reagents are hydrophilic linkers, which can improve solubility. The polyethylene glycol polymer chain is covalently linked to the polypeptide. The polypeptide modified with polyethylene glycol, and the increase in the molecular mass of the peptide can also protect the polypeptide from being cleaved by proteases and improve the half-life of biological activity. In addition, polyethylene glycolylation can also make the polypeptide not produce an immune response and improve pharmacokinetics. The incorporation of 6-aminohexanoic acid provides a straight-chain space of six carbons, reduces the steric hindrance of the reaction, improves the reaction efficiency, and reduces the reaction difficulty.

[0036] Preferably, the linker length is 2 - 12 PEG lengths.

[0037] More preferably, the linker length is 4 - 8 PEG lengths.

[0038] Further, X has 10 - 16 amino acids, wherein at least one citrulline is contained in the amino acid segment at positions 4 - 9, and the amino acids at the head and tail of the X region form a ring.

[0039] Specifically, if the polypeptide sequence is too short, it is difficult to form a cyclic structure. If the sequence is too long, it is easy to lose the conformational advantage of the cyclic structure and at the same time easy to introduce other non-essential binding sites. Therefore, it is reasonably set according to needs in practical applications. Containing at least one citrulline in the amino acid segment at positions 4 - 9, preferably, the citrulline located at the most central position has more excellent detection sensitivity and at the same time improves the signal-to-noise ratio.

[0040] Preferably, X has 12 - 14 amino acids.

[0041] Specifically, the amino acids at the head and tail of the X region form a ring. Preferably, the first and last amino acids form a ring; more preferably, the first and last are cysteines and form a ring with a disulfide bond.

[0042] Further, the CCP peptide segment exists in the form of a CCP-carrier protein conjugate, CCP-biotin, or CCP-fluorescein isothiocyanate.

[0043] Specifically, after coupling the CCP peptide segment with a carrier protein, the presence of the carrier protein will to a certain extent reduce the steric hindrance of antigen-antibody recognition, and the CCP polypeptide with antigenic epitopes is more easily recognized by antibody molecules, thus ensuring the high sensitivity of the reagent. By modifying the CCP peptide segment with biotin, the formed cyclic citrullinated peptide structure is made more stable. Fluorescein isothiocyanate has relatively high activity. Generally speaking, introducing this kind of fluorescent group during the solid-phase synthesis process is easier than other fluoresceins, and no activating reagent needs to be added during the reaction. Linking the side chain of the CCP peptide segment to fluorescein isothiocyanate directly reduces the steric hindrance, thereby improving the reaction efficiency.

[0044] Further, the carrier protein in the CCP-carrier protein conjugate is an animal-derived carrier protein or a human-derived carrier protein.

[0045] Specifically, using an animal-derived carrier protein or a human-derived carrier protein as the carrier protein can not only increase the size of the antigen but also enhance the immunogenicity.

[0046] Further, the carrier protein is any one of the following: bovine serum albumin, cationic bovine serum albumin, hemocyanin, ovalbumin, human serum albumin, bovine γ-globulin, and human γ-globulin.

[0047] Specifically, for the selection of the carrier protein, factors such as its solubility, size, coupling groups, immunogenicity, and cost price are mainly considered. The above carrier proteins are easily obtained and can be selected according to actual needs.

[0048] In a specific embodiment, the present application provides three polypeptides, specifically:

[0049] The sequence of the polypeptide of Comparative Example S0:

[0050] biotin-C-HQFRF cit G cit SRAA-C

[0051] The sequence of the polypeptide of Example S1:

[0052] biotin-KG-PEG-PEG-PEG-PEG-C-HQFRF cit G cit SRAA-C

[0053] The sequence of the polypeptide of Example S2:

[0054] biotin-KG-PEG-PEG-PEG-PEG-PEG-PEG-PEG-PEG-C-HQFRF cit G cit SRAA-C

[0055] Among them, two cysteines in the polypeptide form an intramolecular disulfide bond to achieve polypeptide cyclization.

[0056] It should be noted that the polypeptides of Comparative Example S0 and Examples S1 and S2 were synthesized by Gil Biochemical (Shanghai) Co., Ltd. using the FMOC solid-phase synthesis method, analyzed and purified by HPLC, and the antigen purity reached over 95%.

[0057] Example Two:

[0058] This example provides a CCP antigen, which includes one or more CCP peptide segments of any of the above examples.

[0059] Preferably, the CCP antigen provided in this example has all the beneficial effects of the CCP peptide segments of any of the above examples. Therefore, the CCP antigen formed by combining one or several of them not only has high detection sensitivity but also high detection specificity when used to detect the serum of RA patients.

[0060] Specifically, based on Example One, this example provides three CCP antigens, specifically:

[0061] CCP Antigen One, which includes Polypeptide S0;

[0062] CCP Antigen Two, which includes Polypeptide S1;

[0063] CCP Antigen Three, which includes Polypeptide S2.

[0064] Furthermore, the chemiluminescence immunoassay method and the indirect method principle are used to detect the content of Anti-CCP antibody in the sample. The three CCP antigens are respectively coated on streptavidin magnetic microspheres according to the ratio of 5 μg polypeptide: 1 mg magnetic beads to prepare Solid Phase Working Solution One, Solid Phase Working Solution Two, and Solid Phase Working Solution Three.

[0065] Example Three:

[0066] This example provides a rheumatoid arthritis detection reagent, which includes a CCP antigen, and the CCP antigen is the antigen of the above example.

[0067] Preferably, the rheumatoid arthritis detection reagent provided in this example has all the beneficial effects of the CCP antigen of the above example. Therefore, on the premise of maintaining the existing detection specificity of the CCP antigen, it has higher clinical detection sensitivity and reduces the risk of missed detection.

[0068] Example Four:

[0069] This example provides a rheumatoid arthritis detection kit, which includes the detection reagent of the above example.

[0070] Preferably, the rheumatoid arthritis detection kit provided in this embodiment has all the beneficial effects of the detection reagents in the above examples. The detection reagent in the above rheumatoid arthritis detection kit is used to react with the patient's serum, and can be detected by a semi-automatic immunoassay analyzer or a fully automatic immunoassay analyzer, which is convenient and highly automated.

[0071] Specifically, the kit also includes: a luminescent working solution containing acridinium ester-labeled mouse anti-human IgG antibody and a sample diluent.

[0072] In a specific embodiment, the test kit is used to detect sera from RA patients, and 30 positive and 20 negative sera from clinical diagnosis and other commercial reagent screening and verification are collected as test samples.

[0073] 1. Test negative test samples. Add 10ul of diluted negative test sample to three reaction cups, then add 30ul of solid phase working solution 1, solid phase working solution 2, and solid phase working solution 3 respectively. Incubate at 25-37°C for 15-20 minutes. After magnetic separation and cleaning, add 50ul of luminescent working solution to each of the three reaction cups. Incubate at 25-37°C for 15-20 minutes. After magnetic separation and cleaning, add luminescent excitation substrate to each reaction cup and measure the light signal intensity. The test results are shown in Table 1.

[0074] 2. The detection method for positive samples is the same as above, so it will not be repeated here. The test results are shown in Table 2.

[0075] It is worth noting that, in the kits used in the following examples and comparative examples, except for the CCP antigen in the solid phase working solution, the other components are the same.

[0076] Table 1 Negative sample detection results corresponding to each embodiment and comparative example

[0077]

[0078] Table 2 Positive sample detection results corresponding to each embodiment and comparative example

[0079]

[0080]

[0081] From the test results of negative and positive samples, it can be seen that compared with Comparative Example S0, in Examples S1 and S2, without obvious change in the cut-off value, the signal values of positive samples are significantly improved. Especially for some weakly positive samples, such as the signal values of positive samples 13# and 14# can increase up to 33 times. Therefore, the rheumatoid arthritis detection kit provided in this example can improve the sensitivity of the Anti-CCP antibody diagnostic reagent and reduce the possibility of sample missed detection.

[0082] Although the present invention is disclosed as above, the present invention is not limited thereto. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be subject to the scope defined by the claims.

Claims

1. A CCP peptide segment, characterized in that, The CCP peptide segment has the structure shown in Formula I: NH2-S1-S2-X-COOH …………… Formula 1; wherein, S1 is a reaction site region having at least 1 lysine; S2 is a linker arm region; X is a cyclized citrulline epitope region; S1 has 1 to 4 amino acids, wherein at least one is lysine, the lysine is a terminal lysine, and S1 is a combination of glycine and lysine; S2 is any one or more of Mini-PEG, PEG4, PEG8, PEG12.

2. The CCP peptide segment according to claim 1, wherein X has 10 - 16 amino acids, wherein at least one citrulline is contained in the amino acid segment at positions 4 - 9, and the amino acids at the head and tail of the X region form a ring.

3. The CCP peptide segment according to claim 1, characterized in that, The CCP peptide segment exists in the form of a CCP-carrier protein conjugate, CCP-biotin, or CCP-fluorescein isothiocyanate.

4. The CCP peptide segment according to claim 3, wherein The carrier protein in the CCP-carrier protein conjugate is an animal-derived carrier protein or a human-derived carrier protein.

5. The CCP peptide segment according to claim 4, wherein The carrier protein is any one of the following: bovine serum albumin, cationic bovine serum albumin, hemocyanin, ovalbumin, human serum albumin, bovine γ-globulin, and human γ-globulin.

6. A CCP antigen, characterized in that, The CCP antigen includes one or more CCP peptide segments described in any one of claims 1 to 5.

7. A rheumatoid arthritis detection reagent, characterized in that, The reagent includes a CCP antigen, and the CCP antigen is the antigen described in claim 6.

8. A rheumatoid arthritis detection kit, characterized in that The kit includes the detection reagent described in claim 7.

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