WO2019174104A1 - 一种测定抗环瓜氨酸肽抗体的试剂盒及其应用、检测方法 - Google Patents

一种测定抗环瓜氨酸肽抗体的试剂盒及其应用、检测方法 Download PDF

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WO2019174104A1
WO2019174104A1 PCT/CN2018/083989 CN2018083989W WO2019174104A1 WO 2019174104 A1 WO2019174104 A1 WO 2019174104A1 CN 2018083989 W CN2018083989 W CN 2018083989W WO 2019174104 A1 WO2019174104 A1 WO 2019174104A1
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peptide
kit
citrulline
branched
branched peptide
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PCT/CN2018/083989
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English (en)
French (fr)
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崔利歌
柳乐
杨苏清
赵婷
郅晓乐
黎静雯
徐乐
李裕明
李庆春
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江苏浩欧博生物医药股份有限公司
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Priority to US16/980,561 priority Critical patent/US20210172946A1/en
Publication of WO2019174104A1 publication Critical patent/WO2019174104A1/zh

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    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • 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
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2440/00—Post-translational modifications [PTMs] in chemical analysis of biological material
    • G01N2440/18—Post-translational modifications [PTMs] in chemical analysis of biological material citrullination
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders
    • G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
    • G01N2800/102—Arthritis; Rheumatoid arthritis, i.e. inflammation of peripheral joints

Definitions

  • the invention belongs to the technical field of magnetic particle chemiluminescence immunodiagnosis, and particularly relates to a kit for determining an anti-cyclic citrullinated peptide antibody, an application thereof and a detection method.
  • RA Rheumatoid arthritis
  • the global incidence rate is about 1%, and the incidence rate in China is 0.32-0.36%.
  • RA is more common in women, and the ratio of male to female is about 1:2-1:4. It is a systemic disease characterized by inflammation of the joint synovium, symmetry of inflammation from small joints to large joints, and eventually joint damage in the later stages of the disease, accompanied by soft tissue damage.
  • Autoantibodies with higher specificity to RA include: rheumatoid factor (RF), anti-keratin antibody (AKA), anti-peripheral factor (APF), RA33 and anti-cyclic citrullinated peptide antibody (CCP).
  • RF rheumatoid factor
  • AKA anti-keratin antibody
  • APF anti-peripheral factor
  • RA33 anti-cyclic citrullinated peptide antibody
  • Rheumatoid factor is an antibody produced by an infectious agent (bacteria, virus, etc.) with denatured IgG (an antibody) as an antigen.
  • IgM type is an infectious agent
  • IgG type is an antibody
  • IgA type is an antibody
  • IgE type is an antibody that is a non-specific antibody that may be present in healthy elderly, autoimmune and infectious diseases, with poor specificity.
  • anti-siltin monoclonal antibody including anti-peripheral factor APF, anti-keratin protein AKA, APF is an anti-human buccal mucosal cytoplasmic keratin protein granule
  • APF is an anti-human buccal mucosal cytoplasmic keratin protein granule
  • the antibody, the pro-serin is its main target antigen
  • AKA is an antibody in the serum of RA that can react with the stratum corneum of the rat esophagus
  • the silk fibroin is its main target antigen
  • pre-silver protein is highly phosphorylated from 10 to Twelve repetitive silk fibroin fragments are ligated by a short peptide of 7 amino acids, which are dephosphorylated and cleavage from the ligated short peptide to form a functional texis polypeptide.
  • the specificity of these two antibodies is higher. However, the sensitivity is very low, APF or AKA negative results can not rule out RA
  • Citrulline is a non-standard amino acid that is not involved in protein translation in vivo. It is produced by arginine residues modified by arginine deiminase. In the human mucosal keratinocytes, the silk protein precursor is in the cell. During the differentiation process, the enzyme is decomposed into a silk protein subunit. During this process, the protein is dephosphorylated and the arginine residue is converted to a citrulline residue by enzymatic catalysis. Through comparative studies, it was found that the use of cyclic citrullinated peptide instead of linear citrullinated peptide as the antigenic substrate of ELISA can increase the sensitivity from 49% to 68%. Anti-CCP is a new high specificity index of RA. .
  • Anti-CCP has the following three aspects in the clinical application of RA.
  • Anti-CCP has great clinical significance for RA, this detection technology has attracted much attention, and the development of commercial kits is very rapid.
  • the CCP antigen originated in 1998 and has been developed for three generations.
  • the first generation of antigen is a linear peptide containing citrulline, which is a 19 amino acid residue, which is a citrullin peptide.
  • Detection of the first generation CCP antigen Sensitivity is 49% and specificity is 96%.
  • the two serines in the 19 peptide were replaced with cysteine, and a disulfide bond was formed between the cysteine to form a cyclic citrulline peptide, resulting in a second generation CCP antigen (CCP2).
  • the second-generation CCP antigen not only maintained a high detection specificity (98%), but also a significant increase in sensitivity (69%).
  • the third-generation CCP antigen is still a cyclic citrullinated peptide, but in which an antigenic determinant that cannot be recognized by Anti-CCP2 is added, the detection sensitivity is slightly increased (73%).
  • citrullinated autoantigens there are many kinds of citrullinated autoantigens in synovial fluid, and there are a large number of corresponding heterogeneous antibodies in the body fluid.
  • the citrullinated antigen or peptide alone can not improve the specificity and sensitivity of clinical tests.
  • the CCP antigen currently used on the market is a single citrullin peptide/cyclic citrullinated peptide, or a mixture of various citrullinated peptides/cyclic citrullinated peptides, and both are used in an ELISA detection platform, which is qualitative detection and requires labor. Manual operation takes a long time (about 2 hours).
  • the combination of multiple antigens ensures high detection sensitivity and specificity, but makes the preparation and debugging process of the reagents very complicated and difficult to control, resulting in a large difference between reagent batches.
  • the use of a single antigen reduces the sensitivity of the assay.
  • CN1796997A discloses a detection kit for diagnosing RA, a method of preparing and completing a quality inspection standard, the detection kit comprising a cartridge, an ELISA plate provided in the cartridge, a control and a liquid reagent, and in the ELISA plate Each well has a branched antigen peptide structure of a specific antigen CCP coated with a liquid and blocked by an unrelated protein or serum.
  • the branched antigen peptide structure has a polylysine as a core matrix and has 2 to 8
  • the CCP branch structure preferably has four branch structures.
  • the kit has a longer detection time, takes 2 hours, and has a lower negative compliance rate.
  • the technical problem to be solved by the present invention is to provide a kit for measuring anti-cyclic citrullinated peptide antibody with good stability, high sensitivity, good repeatability and short detection time, and application and detection method thereof.
  • the present invention adopts the following technical solutions:
  • An object of the present invention is to provide a kit for assaying an anti-cyclic citrullinated peptide antibody, the kit comprising a biotinylated antigen, the biotinylated antigen being a plurality of branched peptide units having a branched peptide chain It is formed by lysine coupling, and the branched peptide unit is formed by coupling lysine with a plurality of citrulline peptides.
  • the citrulline peptide is one or more selected from the following amino acid sequences:
  • SEQ ID NO: 1 SHQESTRGRSRGXSGRSGS;
  • SEQ ID NO: 2 SHQESTRGRSRGRSGXSGS
  • SEQ ID NO: 3 SHQESTXGXSRGRSGRSGS;
  • SEQ ID NO: 4 SHQESTXGRSXGRSGRSGS.
  • a plurality of citrulline peptides as shown in SEQ ID NO: 1 form a first branched peptide unit
  • a plurality of citrulline peptides as shown in SEQ ID NO: 2 form a second branched peptide unit
  • a plurality of The citrulline peptide as shown in SEQ ID NO: 3 forms a third branched peptide unit
  • a plurality of citrulline peptides as shown in SEQ ID NO: 4 form a fourth branched peptide unit, the biotinylated antigen comprising The first branched peptide unit, the second branched peptide unit, the third branched peptide unit, and the fourth branched peptide unit.
  • the lysine of the first branched peptide unit and the lysine of the second branched peptide unit are respectively coupled to a lysine
  • the third branched peptide unit is Lysine and the lysine of the fourth branched peptide unit are coupled to another lysine, respectively.
  • each of said branched peptide units has 4 to 6 said citrulline peptides, and said biotinylated antigen has 4 to 6 of said branched peptide units.
  • each of said branched peptide units has 4 of said citrulline peptides, and said biotinylated antigen has 4 of said branched peptide units.
  • biotin is coupled to each of the citrulline peptides.
  • the preparation method of the biotinylated antigen comprises the following steps:
  • Step 1 coupling the citrulline peptide with lysine to form a citrulline branched peptide
  • Step 2 mixing 2 to 5 mg of the synthesized citrulline branched peptide with 0.5 to 0.8 mg of N-hydroxysuccinimide activated biotin, and mixing at 22 to 25 ° C for 25 to 40 minutes;
  • Step 3 Add 15 to 20 uL of the substance in a concentration of 0.04 to 0.06 mol/L of tris buffer, mix at 28 to 32 ° C for 15 to 30 minutes, and then add 550 to 650 uL of glycerol. Biotinylated CCP branched peptide antigen, stored at -20 ° C for use;
  • Step 4 Dilute the biotinylated citrulline branched peptide antigen into a mixed solution having a concentration of 1 to 5 ug/ml with a phosphate buffer solution having a pH of 7 to 7.5 and a substance concentration of 0.01 mol/L. Biotinylated antigen working fluid.
  • the kit further comprises a magnetic particle separation reagent, an alkaline phosphatase-labeled anti-human IgG antibody, a chemiluminescent substrate, a calibrator, and a control.
  • the magnetic fine particles in the magnetic particle separating agent have a diameter of 0.1 to 0.5 ⁇ m, and the magnetic fine particles are superparamagnetic and have a streptavidin group on the surface.
  • the chemiluminescent substrate employed in the present invention is an enzymatic chemiluminescent substrate of alkaline phosphatase disclosed in Application No. CN201510359183.0.
  • the chemiluminescent substrate of the invention has the advantages of high strength, high sensitivity, long duration, good stability and the like. Since AMPPD can act as a co-surfactant, the chemiluminescent substrate can be better incorporated into the chemiluminescent buffer system, thereby greatly increasing the chemiluminescence efficiency and releasing photons under the catalysis of alkaline phosphatase.
  • Another object of the present invention is to provide an application of the kit for detecting the content of an anti-cyclic citrullinated peptide antibody.
  • a third object of the present invention is to provide a method for detecting the content of an anti-cyclic citrullinated peptide antibody using the kit, comprising the steps of: separating a sample to be examined with a magnetic particle separation reagent and a biotinylated antigen The reaction is carried out at 36 to 38 ° C for 10 to 25 minutes to obtain a first complex. After washing, an alkaline phosphatase-labeled anti-human IgG antibody is added, and the reaction is carried out at 36 to 38 ° C for 10 to 25 minutes to obtain a second complex. After washing, chemiluminescence is added. The substrate was reacted at 36 to 38 ° C for 5 to 10 minutes for detection.
  • the present invention has the following advantages compared with the prior art:
  • the detection sensitivity of the invention is high, the specificity is good, and the product is superior to the products of well-known foreign manufacturers.
  • the present invention is capable of performing quantitative detection, and the time for completing all the processes is 45 minutes, which greatly shortens the detection time compared to ELISA.
  • the present invention employs an alkaline phosphatase (AP)-adamantane (AMPPD) system, which is 10 times more sensitive than the ELISA detection system.
  • AP alkaline phosphatase
  • AMPPD adamantane
  • the invention is a fully automatic closed operating system with high reliability, good stability and good repeatability of test results.
  • the invention realizes full automation from the steps of dilution, sample loading, incubation, cleaning and detection, avoids the deviation of results caused by human operation, and effectively improves the detection efficiency and saves labor cost.
  • Figure 1 is a schematic view showing the structure of each branched peptide unit and biotinylated antigen
  • Figure 2 is a schematic diagram of the detection
  • Fig. 3 is a linear regression diagram of the actual detection concentration and the theoretical concentration detected by the detection method of Example 6 for the detection kit of the anti-cyclic citrullinated peptide antibody IgG of Example 5.
  • the present invention will be further described in detail below with reference to specific embodiments, but the invention is not limited to the following examples.
  • the implementation conditions employed in the examples can be further adjusted according to the different requirements of the specific use.
  • the unspecified implementation conditions are conventional conditions in the industry, and the reagents in the present invention are commercially available.
  • citrulline and arginine According to the structural characteristics of citrulline and arginine, bioinformatics related software is used to predict the spatial structure changes.
  • the known citrullinated antigens are analyzed, classified, and the citrulline-containing peptide library is selected, which will contain cucurbitamide.
  • the acid peptide library and the rheumatoid arthritis serum were screened, and a group of citrulline peptides were obtained, and the four citrulline peptides with the highest detection sensitivity were screened, and the lysine had the characteristics of two amino groups, and each melon was used.
  • the peptide chain is coupled into one molecule with four identical branched peptide chains, and finally four citrulline four-branched peptides are obtained, and the four different branched peptide molecules are coupled into one molecule by lysine.
  • a citrulline 16-branched peptide in which a method of coupling a branched peptide with lysine is a conventional method.
  • amino acid sequence of the screened citrulline is:
  • SEQ ID NO: 1 SHQESTRGRSRGXSGRSGS, ie Ser His Gln Glu Ser Thr Arg Gly Arg Ser Arg Gly Xaa Ser Gly Arg Ser Gly Ser, wherein X, Xaa respectively represent citrulline, as shown in SEQ ID NO: 1 in the Sequence Listing;
  • SEQ ID NO: 2 SHQESTRGRSRGRSGXSGS, ie Ser His Gln Glu Ser Thr Arg Gly Arg Ser Arg Gly Arg Ser Gly Xaa Ser Gly Ser, wherein X, Xaa represent citrulline, respectively, as shown in SEQ ID NO: 2 in the Sequence Listing;
  • SEQ ID NO: 3 SHQESTXGXSRGRSGRSGS, ie Ser His Gln Glu Ser Thr Xaa Gly Xaa Ser Arg Gly Arg Ser Gly Arg Ser Gly Ser, wherein X and Xaa represent citrulline, respectively, as shown in SEQ ID NO: 3 in the Sequence Listing;
  • SEQ ID NO: 4 SHQESTXGRSXGRSGRSGS, ie Ser His Gln Glu Ser Thr Xaa Gly Arg Ser Xaa Gly Arg Ser Gly Arg Ser, wherein X and Xaa represent citrulline, respectively, as shown in SEQ ID NO: 4 in the Sequence Listing.
  • Step 1 Mix 2 mg of the synthesized citrulline 16-branched peptide with 0.5 mg of N-hydroxysuccinimide-activated biotin, and mix and react at 25 ° C for 30 min;
  • Step 2 Add 20 uL of the substance to a concentration of 0.05 mol/L of tris buffer, mix and react at 30 ° C for 30 min, and then add 600 uL of glycerol to obtain a biotinylated CCP branch peptide antigen. Store at -20 °C for use;
  • Step 3 Diluting the biotinylated citrulline branched peptide antigen into a mixed solution having a concentration of 2 ug/ml with a phosphate buffer having a pH of 7.5 and a substance concentration of 0.01 mol/L, thereby obtaining a biotinylated antigen.
  • Working fluid Diluting the biotinylated citrulline branched peptide antigen into a mixed solution having a concentration of 2 ug/ml with a phosphate buffer having a pH of 7.5 and a substance concentration of 0.01 mol/L
  • the structure of the biotinylated antigen is shown in Fig. 1, wherein the citrulline peptide 1 is the citrulline peptide represented by SEQ ID NO: 1, and the citrullin peptide 2 is the citrullin peptide represented by SEQ ID NO: 2, and the cucurbitamide Acid peptide 3 is a citrulline peptide represented by SEQ ID NO: 3, and citrulline peptide 4 is a citrulline peptide represented by SEQ ID NO: 4.
  • Instrument G-25 gel column, reagent cryopreservation tank, Supperdex200 gel purification column, analytical balance, biochemical incubator;
  • Step 1 3 mg of anti-human IgG antibody was added to 40 mL of a concentration of 10 mg / mL of 2-iminothiolane hydrochloride coupling agent, and allowed to stand at 20 ° C for 20 min;
  • Step 2 Add 2 mL of 0.08 mol/L glycine solution, let stand at 20 ° C for 4 min, remove the salt with G-25 gel column, collect the activated anti-human IgG antibody, and store at 5 ° C for use;
  • Step 3 Add 3 mg of alkaline phosphatase solution to 4 mg/mL of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimidyl ester solution at 25 ° C After standing for 30 min, the salt was removed by G-25 gel column, and the activated alkaline phosphatase was collected and stored at 5 ° C for use;
  • Step 4 The activated anti-human IgG antibody and the activated alkaline phosphatase were mixed, allowed to stand at 5 ° C for 20 h, and purified by a Supperdex 200 gel purification column to obtain a concentrated solution of the conjugate, which was stored at 5 ° C for use;
  • Step 5 Dilute the connate solution in step 4 with a mass ratio of 1% bovine serum albumin, a pH of 7.8-8.0, and a substance concentration of 0.05 mol/L of tris buffer.
  • the alkaline phosphatase-labeled anti-human IgG antibody was obtained at a concentration of 1 ⁇ g/mL to an alkaline phosphatase-labeled anti-human IgG antibody.
  • the anti-citrullin peptide antibody was selected and diluted with a phosphate buffer solution having a pH of 7.5 and a concentration of 0.01 mol/L, and the standards were adjusted to a concentration of 20 RU/ml and 200 RU/ml, respectively. Calibrator.
  • the anti-citrullin peptide antibody was selected and diluted with a phosphate buffer solution having a pH of 7.5 and a substance concentration of 0.01 mol/L, and the concentration was 10 RU/ml and 80 RU/ml, respectively, according to the standard product. Quality control products.
  • Example 5 Detection kit for an anti-cyclic citrullinated peptide antibody IgG
  • a kit for detecting an anti-cyclic citrullinated peptide antibody IgG in the present embodiment comprising:
  • Biotinylated antigen prepared according to the method of Example 1 (concentration of 2 ⁇ g / mL), 5 mL;
  • Alkaline phosphatase-labeled anti-human IgG antibody (concentration 1 ⁇ g / mL) prepared according to the method of Example 2, 30 mL;
  • the magnetic particle separation reagent was purchased from Thermo Fisher, 5 mL;
  • control product prepared according to the method of Example 4, 1 mL;
  • the chemiluminescent substrate is shown in Formulation 2 of Table 1.2 APSH-1 in an enzymatic chemiluminescent substrate of alkaline phosphatase disclosed in Application No. CN201510359183.0, ie
  • the kit of Example 6 was tested using a fully automated chemiluminescence meter, and specifically includes the following steps:
  • Step 1 The detection kit of the embodiment 5 is matched with the matched automatic chemiluminescence measuring instrument, and the reagent box is placed in the corresponding position of the reagent chamber of the fully automatic chemiluminescence measuring instrument, and the information of the kit is input into the instrument system through the barcode scanner. Or set by the instrument supporting software;
  • Step 2 Place the calibrator in the instrument sample bin, identify the calibrator information through the barcode scanner, and assign the calibrator position in the instrument system;
  • Step 3 Place the quality control product/test sample in the instrument sample bin, and edit the corresponding test information through the instrument supporting software;
  • Step 4 Start the running program, and all the calibrators/controls/samples to be tested will be automatically executed.
  • the samples to be tested are reacted with the magnetic particle separation reagent and the biotinylated antigen at 37 ° C for 15 min to obtain the first complex.
  • alkaline phosphatase-labeled anti-human IgG antibody was added and reacted at 37 ° C for 15 min to obtain a second complex.
  • a chemiluminescent substrate was added and reacted at 37 ° C for 5 min to carry out detection.
  • test kit When the test kit is used with the fully automatic chemiluminescence analyzer, it is fully automated from dilution, sample loading, incubation, cleaning and detection steps, and can be operated unattended. Fully automatic closed operating system, not only easy to operate, high reliability, good stability, good repeatability of test results, avoiding the deviation of results caused by human operation, and effectively improving the detection efficiency and saving labor costs.
  • Yin and positive coincidence rate The content of CCP was measured in 250 clinical serum samples using the kit of Example 5 of the present invention, and compared with the CCP antibody ELISA test kit of EURO DIAGNOSTICA (Table 1). The results showed that compared with the ELISA test kit, the negative compliance rate of the CCP kit of the present invention was 99.0% (198/200), and the positive coincidence rate was 94.0% (48/50), indicating that the kit and the existing anti-CCP antibody on the market. The detection reagents are highly consistent. The data is shown in Table 1.
  • the lowest detection quantity reference products (L1, L2, and L3) of the detection enterprise are respectively detected, and each repeated detection is performed three times.
  • L1 reference product concentration concentration is 60RU/ml, concentration error is not more than 10%;
  • L2 reference product concentration concentration is 40RU/ml, concentration error is not more than 10%;
  • L3 reference concentration concentration is 10RU/ml, concentration error is not more than 10%.
  • Sample recovery rate measured concentration of added sample / (0.1 * sample A measured concentration + 0.9 * sample B measured concentration) * 100%
  • Example 5 of the present invention was placed at 4 ° C for 7 days and 37 ° C for 7 days, respectively, and the measurement was high (measured concentration was about 200 RU/ml), medium (measured concentration was about 80 RU/ml). , low (measured concentration is about 10RU / ml) three kinds of concentration control signal retention rate. The results were all >90%, and the reagents showed that the kit was stable and met the clinical requirements. The data is shown in Table 4.

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Abstract

一种测定抗环瓜氨酸肽抗体的试剂盒及其应用、检测方法，该试剂盒包括多个具有分支肽链的分支肽单元用赖氨酸偶联形成的生物素化抗原，其分支肽单元为多条瓜氨酸肽用赖氨酸偶联形成。该试剂盒和方法检测灵敏度高，特异性好，瓜氨酸偶联后作为单组份抗原，相比混合抗原降低了抗原组分的复杂程度和试剂的质控难度，提高了批间一致性，能够进行定量检测，且完成所有流程出结果时间为45min。

Description

一种测定抗环瓜氨酸肽抗体的试剂盒及其应用、检测方法
相关申请的交叉引用
本申请要求2018年3月13日提交的申请号为CN201810202734.6的中国专利申请的优先权，其全部内容通过引用的方式并入本发明中。
技术领域
本发明属于磁微粒化学发光免疫诊断技术领域，具体涉及一种测定抗环瓜氨酸肽抗体的试剂盒及其应用、检测方法。
背景技术
类风湿关节炎(Rheumatoid Arthritis，RA)是一种最常见的自身免疫性疾病，也是最多发的慢性炎症性关节疾病，全球发病率1％左右，中国发病率为0.32-0.36％。RA多发于女性，男女比例约为1∶2-1∶4。它是一种全身性疾病，其特征为关节滑膜出现炎症，炎症对称性的从小关节发展到大关节，最终在疾病后期导致关节损坏，并伴有软组织损伤。
与RA具有较高特异性的自身抗体包括：类风湿因子(RF)，抗角蛋白抗体(AKA)，抗核周因子(APF)，RA33和抗环瓜氨酸肽抗体(CCP)。
类风湿因子是由于感染因子(细菌、病毒等)引起体内产生的以变性IgG(一种抗体)为抗原的一种抗体，常见的类风湿因子有IgM型、IgG型、IgA型和IgE型。在RA中的阳性率为60-80％，但是RF是一种非特异性抗体，在健康的老年人、自身免疫性疾病和感染性疾病患者体内都可能存在，特异性较差。
还有一类在RA患者血清中可检测出来的特异性抗体，即抗丝集蛋白自身抗体，包括抗核周因子APF，抗角质蛋白AKA，APF是一种抗人颊黏膜细胞浆内角质蛋白颗粒的抗体，前丝集蛋白是其主要靶抗原，AKA是RA血清中一种能与鼠食管角质层反应的抗体，丝集蛋白是其主要靶抗原(前丝集蛋白是高度磷酸化由10至12个重复的丝集蛋白片段经7个氨基酸的短肽连接而成，经过去磷酸化，从连接短肽处断裂，形成有功能的丝集蛋白多肽)，这两种抗体的特异性较高，但灵敏度很低，APF或者AKA阴性结果不能排除RA，研究发现丝集蛋白中稀有的氨基酸瓜氨酸是其主要的抗原表位。
瓜氨酸是一个非标准的氨基酸，不参与体内蛋白质翻译，它是由精氨酸残 基通过精氨酸脱亚氨酶修饰产生，在人黏膜细胞角质颗粒中的丝集蛋白前体在细胞分化过程中被酶分解成丝集蛋白亚基，在此过程中，蛋白被去磷酸化，精氨酸残基在酶催化下转化成瓜氨酸残基。通过比较研究发现，用环状的瓜氨酸肽代替线性的瓜氨酸肽作为ELISA的抗原基质，可将灵敏度从49％提高到68％，Anti-CCP是RA的一个高特异性的新指标。
Anti-CCP在RA的临床应用中有以下三个方面的意义，
RA早期诊断的重要指标：10％的RA在首个临床症状出现前10年左右即可检测阳性；40％的RA发作和70％的RA首次就诊前1年出现阳性；可早期预测未分化关节炎向类风湿关节炎的发展：Anti-CCP阳性患者93％进展为类风湿关节炎；Anti-CCP阴性患者25％进展为类风湿关节炎。减少RF阴性患者漏诊，20％-57％RF诊断为阴性的RA，Anti-CCP为阳性；Anti-CCP与RF具有相同的灵敏度，但Anti-CCP的特异性更高(Anti-CCP：96-100％，RF：63％)。Anti-CCP还可预测关节损害的独立指标，Anti-CCP阳性的RA患者出现严重关节损坏明显多于阴性患者。
因为Anti-CCP对于RA有很重大的临床意义，该检测技术备受关注，商品化试剂盒的发展非常迅速。
1998年Schellkens等按照前丝集蛋白cDNA序列合成了31条可能含有抗原表位的9至19个氨基酸短肽，并将分子中不同部位的精氨酸替换为瓜氨酸，以抗丝集蛋白阳性的RA患者血清采用竞争ELISA进行筛选。在同一段肽链中，不同部位瓜氨酸化，以及瓜氨酸的数量均影响抗原与抗体的亲和力，说明瓜氨酸是形成抗原决定簇的必要成分。
CCP抗原起源于1998年，发展到现在共有三代，第一代抗原是由人工合成19个氨基酸残基的含有瓜氨酸的直链型多肽，即瓜氨酸肽，第一代CCP抗原的检测灵敏度49％，特异性96％。后来将19肽中的两个丝氨酸替换为半胱氨酸，半胱氨酸之间会形成二硫键，从而形成环瓜氨酸肽，得到第二代CCP抗原(CCP2)。第二代CCP抗原不仅保持了较高的检测特异性(98％)，灵敏度也有较大提高(69％)。第三代CCP抗原依然是环瓜氨酸肽，但是在其中增加了不能被Anti-CCP2识别的抗原决定簇，检测灵敏度略微提高(73％)。
关节滑膜液中瓜氨酸化自身抗原多种多样，患者体液中也存在着大量的相应的异质性抗体，单独的瓜氨酸化抗原或者多肽并不能本质提高临床检验的特异性和敏感度，目前市场上使用的CCP抗原为单个瓜氨酸肽/环瓜氨酸肽，或 多种瓜氨酸肽/环瓜氨酸肽混合，且均用于ELISA检测平台，属于定性检测，且需要人工手动操作，得出结果所需时间较长(约2小时)。多种抗原混合使用虽然保证了较高的检测灵敏度和特异性，但会使试剂的制备和调试工艺变得十分复杂和难以控制，从而导致试剂批间差大。而采用单个抗原则会降低检测灵敏度。
公开号CN1796997A公开了诊断RA的检测试剂盒、制备及完成质量检测标准的方法，该检测试剂盒包括盒体、设在盒体内的酶标板、对照物和液体试剂，并且在酶标板的每个孔内有包被液包被、且被无关蛋白或血清封闭的特异性抗原CCP的分支抗原肽结构，该分支抗原肽结构以多聚赖氨酸为核心基质，且具有2～8个CCP分支结构，优选具有4个分支结构。但该试剂盒的检测时间较长，需要2个小时，且阴性符合率较低。
发明内容
本发明所要解决的技术问题是提供一种稳定性好、灵敏度高、重复性好且检测时间短的测定抗环瓜氨酸肽抗体的试剂盒及其应用、检测方法。
为解决以上技术问题，本发明采取如下技术方案：
本发明的一个目的是提供一种测定抗环瓜氨酸肽抗体的试剂盒，所述的试剂盒包括生物素化抗原，所述的生物素化抗原为多个具有分支肽链的分支肽单元用赖氨酸偶联形成，所述的分支肽单元为多条瓜氨酸肽用赖氨酸偶联形成。
优选地，所述的瓜氨酸肽为选自如下氨基酸序列中的一种或几种：
SEQ ID NO：1：SHQESTRGRSRGXSGRSGS；
SEQ ID NO：2：SHQESTRGRSRGRSGXSGS；
SEQ ID NO：3：SHQESTXGXSRGRSGRSGS；
SEQ ID NO：4：SHQESTXGRSXGRSGRSGS。
进一步优选地，多条如SEQ ID NO：1所示的瓜氨酸肽形成第一分支肽单元，多条如SEQ ID NO：2所示的瓜氨酸肽形成第二分支肽单元，多条如SEQ ID NO：3所示的瓜氨酸肽形成第三分支肽单元，多条如SEQ ID NO：4所示的瓜氨酸肽形成第四分支肽单元，所述的生物素化抗原包括所述的第一分支肽单元、所述的第二分支肽单元、所述的第三分支肽单元和所述的第四分支肽单元。
更为优选地，所述的第一分支肽单元的赖氨酸和所述的第二分支肽单元的赖氨酸分别与一个赖氨酸相偶联，所述的第三分支肽单元的赖氨酸和所述的第四分支肽单元的赖氨酸分别与另一个赖氨酸相偶联。
优选地，每个所述的分支肽单元分别有4～6条所述的瓜氨酸肽，所述的生物素化抗原有4～6个所述的分支肽单元。
进一步优选地，每个所述的分支肽单元分别有4条所述的瓜氨酸肽，所述的生物素化抗原有4个所述的分支肽单元。
优选地，所述的生物素化抗原中，每条所述的瓜氨酸肽上偶联有生物素。
所述的生物素化抗原的制备方法包括如下步骤：
步骤1：用赖氨酸将所述的瓜氨酸肽偶联形成瓜氨酸分支肽；
步骤2：将2～5mg合成的瓜氨酸分支肽与0.5～0.8mg N-羟基琥珀酰亚胺活化的生物素混合，在22～25℃下混匀反应25～40min；
步骤3：加入15～20uL的物质的量浓度为0.04～0.06mol/L的三羟甲基氨基甲烷缓冲液，在28～32℃下混匀反应15～30min，再加入550～650uL甘油，获得生物素化的CCP分支肽抗原，在-20℃保存备用；
步骤4：用pH为7-7.5、物质的量浓度为0.01mol/L的磷酸盐缓冲液将生物素化的瓜氨酸分支肽抗原稀释成浓度为1～5ug/ml的混合溶液，即得生物素化抗原工作液。
优选地，所述的试剂盒还包括磁微粒分离试剂、碱性磷酸酶标记的抗人IgG抗体、化学发光底物、校准品、质控品。
进一步优选地，所述的磁微粒分离试剂中磁微粒的直径为0.1～0.5μm，所述的磁微粒具有超顺磁性且表面含有链霉亲和素基团。
本发明中采用的化学发光底物为申请号为CN201510359183.0公开的一种碱性磷酸酶的酶促化学发光底物。本发明的化学发光底物具有强度高、灵敏度高、持续时间长、稳定性好等优点。由于AMPPD可以起到共表面活性剂的作用，能使该化学发光底物更好的结合到化学发光缓冲体系中，从而大幅度提高化学发光效率，在碱性磷酸酶的催化下释放光子。
本发明的另一个目的是提供一种所述的试剂盒在检测抗环瓜氨酸肽抗体的含量中的应用。
本发明的第三个目的是提供一种采用所述的试剂盒进行抗环瓜氨酸肽抗体的含量的检测方法，包括如下步骤：将待检样本与磁微粒分离试剂以及生物素化抗原在36～38℃反应10～25min，得到第一复合物，清洗后加入碱性磷酸酶标记的抗人IgG抗体，在36～38℃反应10～25min，得到第二复合物，清洗后加入化学发光底物，在36～38℃反应5～10min，进行检测。
由于以上技术方案的实施，本发明与现有技术相比具有如下优点：
1.本发明的检测灵敏度高，特异性好，与国外知名厂家产品相比更优。
2.瓜氨酸偶联后作为单组份抗原，相比混合抗原，降低了抗原组分的复杂程度，大幅度降低了试剂的质控难度，提高了批间一致性。
3.本发明能够进行定量检测，且完成所有流程出结果时间为45min，相比ELISA大大缩短了检测时间。
4.本发明采用碱性磷酸酶(AP)-金刚烷(AMPPD)系统，相比ELISA检测系统，灵敏度提高10倍。
5.本发明为全自动封闭操作系统，可靠性高，稳定性好，检测结果重复性好。
6.本发明从稀释、加样、孵育、清洗以及检测步骤实现了全自动化，避免了人为操作带来的结果偏差，同时有效的提高了检测效率及节省人力成本。
附图说明
附图1为各分支肽单元和生物素化抗原的结构示意图；
附图2为检测原理图；
附图3为实施例5的抗环瓜氨酸肽抗体IgG的检测试剂盒按照实施例6的检测方法检测的实际检测浓度与理论浓度的线性回归图。
具体实施方式
下面结合具体实施例对本发明做进一步详细的说明，但本发明并不限于以下实施例。实施例中采用的实施条件可以根据具体使用的不同要求做进一步调整，未注明的实施条件为本行业中的常规条件，本发明中的试剂均可市购获得。
实施例1：生物素化抗原的制备
瓜氨酸16分支肽的合成：
根据瓜氨酸与精氨酸的结构特征，应用生物信息学相关软件预测其空间结构变化，对已知的瓜氨酸化抗原分析、分类、筛选出含有瓜氨酸的多肽文库，将含有瓜氨酸的多肽库与类风湿关节炎血清筛选，获得了一组瓜氨酸肽，筛选其中组合检测灵敏度最高的四种瓜氨酸肽，利用赖氨酸具备两个氨基的特性，将每种瓜氨酸肽链偶联成为具有四条相同分支肽链的一个分子，最终得到四种瓜氨酸四分支肽，再将这四种不同的分支肽分子用赖氨酸偶联成一个分子，即为瓜氨酸16分支肽，其中，用赖氨酸将分支肽进行偶联的方法为常规方法。
筛选出的瓜氨酸的氨基酸序列为：
SEQ ID NO：1：SHQESTRGRSRGXSGRSGS，即Ser His Gln Glu Ser Thr Arg Gly Arg Ser Arg Gly Xaa Ser Gly Arg Ser Gly Ser，其中，X、Xaa分别代表瓜氨酸，如序列表中序列1所示；
SEQ ID NO：2：SHQESTRGRSRGRSGXSGS，即Ser His Gln Glu Ser Thr Arg Gly Arg Ser Arg Gly Arg Ser Gly Xaa Ser Gly Ser，其中，X、Xaa分别代表瓜氨酸，如序列表中序列2所示；
SEQ ID NO：3：SHQESTXGXSRGRSGRSGS，即Ser His Gln Glu Ser Thr Xaa Gly Xaa Ser Arg Gly Arg Ser Gly Arg Ser Gly Ser，其中，X、Xaa分别代表瓜氨酸，如序列表中序列3所示；
SEQ ID NO：4：SHQESTXGRSXGRSGRSGS，即Ser His Gln Glu Ser Thr Xaa Gly Arg Ser Xaa Gly Arg Ser Gly Arg Ser Gly Ser，其中，X、Xaa分别代表瓜氨酸，如序列表中序列4所示。
生物素化抗原的具体制备方法：
1、材料与仪器：
材料：合成的瓜氨酸16分支肽、N-羟基琥珀酰亚胺活化的生物素、三羟甲基氨基甲烷缓冲液、甘油、磷酸盐缓冲液；
仪器：试剂低温保存箱、生化培养箱。
2、制备步骤：
步骤1：将2mg合成的瓜氨酸16分支肽与0.5mg N-羟基琥珀酰亚胺活化的生物素混合，在25℃下混匀反应30min；
步骤2：加入20uL的物质的量浓度为0.05mol/L的三羟甲基氨基甲烷缓冲液，在30℃下混匀反应30min，再加入600uL甘油，获得生物素化的CCP分支肽抗原，在-20℃保存备用；
步骤3：用pH为7.5、物质的量浓度为0.01mol/L的磷酸盐缓冲液将生物素化的瓜氨酸分支肽抗原稀释成浓度为2ug/ml的混合溶液，即得生物素化抗原工作液。
生物素化抗原的结构参见图1，其中，瓜氨酸肽1为SEQ ID NO：1所示瓜氨酸肽，瓜氨酸肽2为SEQ ID NO：2所示瓜氨酸肽，瓜氨酸肽3为SEQ ID NO：3所示瓜氨酸肽，瓜氨酸肽4为SEQ ID NO：4所示瓜氨酸肽。
实施例2：碱性磷酸酶标记的抗人IgG抗体的制备：
材料与仪器：
材料：抗人IgG抗体、碱性磷酸酶、偶联剂2-亚胺基硫烷盐酸盐、甘氨酸、三羟甲基氨基甲烷缓冲液。
仪器：G-25凝胶柱、试剂低温保存箱、Supperdex200凝胶纯化柱、分析天平、生化培养箱；
操作步骤：
步骤1：将3mg抗人IgG抗体加入到40mL浓度为10mg/mL的2-亚胺基硫烷盐酸盐偶联剂中，在20℃下静置20min；
步骤2：加入2mL的0.08mol/L甘氨酸溶液，在20℃下静置4min，用G-25凝胶柱除盐，收集活化后抗人IgG抗体，5℃保存备用；
步骤3：将3mg碱性磷酸酶溶液加入到4mg/mL的4-(N-马来酰亚胺基甲基)环己烷-1-羧酸琥珀酰亚胺酯溶液中，在25℃下静置30min，用G-25凝胶柱除盐，收集活化后碱性磷酸酶，5℃保存备用；
步骤4：将活化后的抗人IgG抗体和活化后的碱性磷酸酶混合，在5℃下静置20h，用Supperdex200凝胶纯化柱纯化，获得连接物浓溶液，在5℃保存备用；
步骤5：将步骤4中的连接物浓溶液用含质量比为1％的牛血清白蛋白、pH为7.8-8.0、物质的量浓度为0.05mol/L的三羟甲基氨基甲烷缓冲液稀释到含碱性磷酸酶标记的抗人IgG抗体的浓度为1μg/mL，即得碱性磷酸酶标记的抗人IgG抗体。
实施例3：校准品的制备：用于校准曲线。
1、材料与仪器：
材料：抗瓜氨酸肽抗体、磷酸盐缓冲液、标准品；
2、制备步骤：
选取抗瓜氨酸肽抗体，用pH为7.5、物质的量浓度为0.01mol/L的磷酸盐缓冲液按一定的比例稀释，参照标准品，配制成浓度分别为20RU/ml与200RU/ml的校准品。
实施例4：质控品的制备：
1、材料与仪器：
材料：抗瓜氨酸肽抗体、磷酸盐缓冲液、标准品；
2、制备步骤：
选取抗瓜氨酸肽抗体，用pH为7.5、物质的量浓度为0.01mol/L的磷酸盐缓冲液按一定的比例稀释，参照标准品，配制成浓度分别为10RU/ml与80RU/ml的质控品。
实施例5：一种抗环瓜氨酸肽抗体IgG的检测试剂盒
本实施例中的一种抗环瓜氨酸肽抗体IgG的检测试剂盒，包括：
按照实施例1方法制备的生物素化抗原(浓度为2μg/mL)，5mL；
按照实施例2方法制备的碱性磷酸酶标记的抗人IgG抗体(浓度为1μg/mL)，30mL；
磁微粒分离试剂外购自Thermo Fisher，5mL；
按照实施例3方法制备的校准品，1mL；
按照实施例4方法制备的质控品，1mL；
化学发光底物为申请号为CN201510359183.0公开的一种碱性磷酸酶的酶促化学发光底物中的表1.2APSH-1的配方二所示，即
Figure PCTCN2018083989-appb-000001
实施例6：试剂盒的测试
实施例6的试剂盒采用全自动化学发光测定仪进行测试，具体包括以下步骤：
步骤1：实施例5的检测试剂盒与相适配的全自动化学发光测定仪配套使用，将试剂盒放入全自动化学发光测定仪试剂仓相应位置，试剂盒信息通过条形码扫描仪输入仪器系统或通过仪器配套软件设定；
步骤2：将校准品置于仪器样本仓，通过条形码扫描仪识别校准品信息，并在仪器系统中分配校准品位置；
步骤3：将质控品/待测样本置于仪器样本仓，通过仪器配套软件编辑相应检测信息；
步骤4：启动运行程序，所有校准品/质控品/待检样本处理步骤将自动执行，其中，待检样本与磁微粒分离试剂以及生物素化抗原在37℃反应15min，得到第一复合物，清洗后加入碱性磷酸酶标记的抗人IgG抗体，在37℃反应15min，得到第二复合物，清洗后加入化学发光底物，在37℃反应5min，进行检测。
当检测试剂盒配合全自动化学发光测定仪配套使用时，从稀释、加样、孵育、清洗以及检测步骤均实现了全自动化，可以无人值守流水操作。全自动封闭操作系统，不仅操作简易方便、可靠性高、稳定性好、检测结果重复性好，避免了人为操作带来的结果偏差，同时有效的提高了检测效率及节省人力成本。
实施例7：检测的实施和检测效果的评价
(1)样本比对
阴、阳性符合率：采用本发明实施例5的试剂盒对250例临床血清检测CCP的含量，并与EURO DIAGNOSTICA公司的CCP抗体ELISA检测试剂盒进行比对(表1)。结果表明，和ELISA检测试剂盒相比，本发明CCP试剂盒阴性符合率99.0％(198/200)，阳性符合率94.0％(48/50)，说明本试剂盒和市场上现有抗CCP抗体检测试剂一致程度很高。数据见表1。
表1：样本比对
Figure PCTCN2018083989-appb-000002
(2)灵敏度：本发明实施例5的检测试剂盒的LOD为0.039RU/ml，而EURO DIAGNOSTICA公司的CCP抗体ELISA检测试剂盒的灵敏度为2RU/ml。
采用本发明实施例5的试剂盒对检测企业最低检出量参考品(L1、L2、L3)分别进行检测，各重复检测3次。按试剂盒说明书操作，评价检测结果。
L1参考品浓度：浓度为60RU/ml，浓度误差不大于10％；
L2参考品浓度：浓度为40RU/ml，浓度误差不大于10％；
L3参考品浓度：浓度为10RU/ml，浓度误差不大于10％。
(3)线性：将一份高值血清(理论浓度400RU/ml，实测浓度449.4RU/ml)按照1/1、1/2、1/8、1/20、1/80、1/200，用本发明实施例5的试剂盒检测稀释样本，以稀释比例和检测浓度做回归曲线。求出相关系数R的平方值。结果见图3，表明CCP试剂盒的线性相关系数为0.9998，结果大于0.9900，斜率为1.0924。
(4)准确度：通过加样回收评估本发明实施例5的试剂盒的准确度。以一份高值血清H(实测浓度约为300RU/ml)、一份中值血清M(实测浓度约为100RU/ml)、一份低值血清L(实测浓度约为50RU/ml)按照1∶9添加到对应3份基础血清(实测浓度＜10RU/ml)中，计算其浓度。结果血清加样回收率在85％-115％之间。数据见表2。
注：加样回收率＝添加后样本实测浓度/(0.1*样本A实测浓度+0.9*样本B实测浓度)*100％
表2 准确度
Figure PCTCN2018083989-appb-000003
(5)精密度：采用本发明实施例5的试剂盒对三种浓度的质控品进行检测， 每天两次，分上下午检测，每次进行4个重复，共检测10天，每种浓度共测定80次，计算变异系数(变异系数CV＝浓度均值/标准差*100％)，结果表明变异系数在10％以内。
表3：精密度
Figure PCTCN2018083989-appb-000004
(6)稳定性：本发明实施例5的试剂盒分别在4℃放置7天和37℃放置7天，测定高(实测浓度约为200RU/ml)、中(实测浓度约为80RU/ml)、低(实测浓度约为10RU/ml)3种浓度质控的信号保留率。结果均＞90％，将试剂表明试剂盒稳定好，符合临床要求。数据见表4。
表4：稳定性
Figure PCTCN2018083989-appb-000005
(7)特异性：对高(300RU/ml)、中(100RU/ml)、低(20RU/ml)不同浓度值的血清添加不同浓度的胆红素、血红蛋白、类风湿因子、甘油三酯、人抗小鼠抗体，检测结果显示：添加物质对本发明实施例5的试剂盒检测结果没有影响。数据见表5。
表5：特异性结果
干扰物 添加浓度 交叉反应率(％)
胆红素 20mg/dL 0.89
血红蛋白 1000mg/dL 1.52
甘油三酯 2000mg/dL 3.01
人抗小鼠抗体 2000ng/mL 2.64
类风湿因子 1000IU/mL 1.86
表5的结果表明，以上各添加物质对实施例5的抗环瓜氨酸肽抗体检测试剂盒的检测结果没有影响。
(8)批间差：用3个不同批号的本发明实施例5的试剂盒分别测定浩欧博内部精密度参考品(RP1：10RU/ml、RP2：20RU/ml、RP3：100RU/ml)，各重复10次，计算30次测定结果的平均值(M)、标准差(SD)和批间变异系数(CV)，结果应符合要求CV＜15％。计算公式为：
Figure PCTCN2018083989-appb-000006
式中：
CV——变异系数；
SD——30次测定结果的标准差；
M——30次测定结果的平均值。
表6 批间差
Figure PCTCN2018083989-appb-000007
Figure PCTCN2018083989-appb-000008
从表6可以看出，批间差＜10％，批间差较小，符合行业要求！
以上对本发明做了详尽的描述，其目的在于让熟悉此领域技术的人士能够了解本发明的内容并加以实施，并不能以此限制本发明的保护范围，凡根据本发明所作的等效变化或修饰，都应涵盖在本发明的保护范围内。

Claims (11)

  1. 一种测定抗环瓜氨酸肽抗体的试剂盒，其特征在于：所述的试剂盒包括生物素化抗原，所述的生物素化抗原为多个具有分支肽链的分支肽单元用赖氨酸偶联形成，所述的分支肽单元为多条瓜氨酸肽用赖氨酸偶联形成。
  2. 根据权利要求1所述的测定抗环瓜氨酸肽抗体的试剂盒，其特征在于：所述的瓜氨酸肽为选自如下氨基酸序列中的一种或几种：
    SEQ ID NO：1：SHQESTRGRSRGXSGRSGS；
    SEQ ID NO：2：SHQESTRGRSRGRSGXSGS；
    SEQ ID NO：3：SHQESTXGXSRGRSGRSGS；
    SEQ ID NO：4：SHQESTXGRSXGRSGRSGS。
  3. 根据权利要求2所述的测定抗环瓜氨酸肽抗体的试剂盒，其特征在于：多条如SEQ ID NO：1所示的瓜氨酸肽形成第一分支肽单元，多条如SEQ ID NO：2所示的瓜氨酸肽形成第二分支肽单元，多条如SEQ ID NO：3所示的瓜氨酸肽形成第三分支肽单元，多条如SEQ ID NO：4所示的瓜氨酸肽形成第四分支肽单元，所述的生物素化抗原包括所述的第一分支肽单元、所述的第二分支肽单元、所述的第三分支肽单元和所述的第四分支肽单元。
  4. 根据权利要求3所述的测定抗环瓜氨酸肽抗体的试剂盒，其特征在于：所述的第一分支肽单元的赖氨酸和所述的第二分支肽单元的赖氨酸分别与一个赖氨酸相偶联，所述的第三分支肽单元的赖氨酸和所述的第四分支肽单元的赖氨酸分别与另一个赖氨酸相偶联。
  5. 根据权利要求1所述的测定抗环瓜氨酸肽抗体的试剂盒，其特征在于：每个所述的分支肽单元分别有4～6条所述的瓜氨酸肽，所述的生物素化抗原有4～6个所述的分支肽单元。
  6. 根据权利要求1所述的测定抗环瓜氨酸肽抗体的试剂盒，其特征在于：所述的生物素化抗原中，每条所述的瓜氨酸肽上偶联有生物素。
  7. 根据权利要求1所述的测定抗环瓜氨酸肽抗体的试剂盒，其特征在于： 所述的试剂盒还包括磁微粒分离试剂、碱性磷酸酶标记的抗人IgG抗体、化学发光底物。
  8. 根据权利要求7所述的测定抗环瓜氨酸肽抗体的试剂盒，其特征在于：所述的磁微粒分离试剂中磁微粒的直径为0.1～0.5μm，所述的磁微粒具有超顺磁性且表面含有链霉亲和素基团。
  9. 根据权利要求1所述的测定抗环瓜氨酸肽抗体的试剂盒，其特征在于，所述的生物素化抗原通过如下步骤制得：
    步骤1：用赖氨酸将所述的瓜氨酸肽偶联形成瓜氨酸分支肽；
    步骤2：将2～5mg合成的瓜氨酸分支肽与0.5～0.8mg N-羟基琥珀酰亚胺活化的生物素混合，在22～25℃下混匀反应25～40min；
    步骤3：加入15～20uL的物质的量浓度为0.04～0.06mol/L的三羟甲基氨基甲烷缓冲液，在28～32℃下混匀反应15～30min，再加入550～650uL甘油，获得生物素化的CCP分支肽抗原，在-20℃保存备用；
    步骤4：用pH为7-7.5、物质的量浓度为0.01mol/L的磷酸盐缓冲液将生物素化的瓜氨酸分支肽抗原稀释成浓度为1～5ug/ml的混合溶液，即得生物素化抗原工作液。
  10. 一种如权利要求1至9中任一项所述的试剂盒在检测抗环瓜氨酸肽抗体的含量中的应用。
  11. 一种采用权利要求1至9中任一项所述的试剂盒进行抗环瓜氨酸肽抗体的含量的检测方法，包括如下步骤：将待检样本与磁微粒分离试剂以及生物素化抗原在36～38℃反应10～25min，得到第一复合物，清洗后加入碱性磷酸酶标记的抗人IgG抗体，在36～38℃反应10～25min，得到第二复合物，清洗后加入化学发光底物，在36～38℃反应5～10min，进行检测。
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CN116284440A (zh) * 2022-12-30 2023-06-23 安必进(南京)生物技术有限公司 Ccp肽段及其应用以及ccp抗原和试剂盒
CN116284440B (zh) * 2022-12-30 2023-11-14 安必进(南京)生物技术有限公司 Ccp肽段及其应用以及ccp抗原和试剂盒

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