CN114573663B - Citrulline peptide and application thereof, and detection reagent and kit for rheumatoid arthritis - Google Patents

Citrulline peptide and application thereof, and detection reagent and kit for rheumatoid arthritis Download PDF

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CN114573663B
CN114573663B CN202210190265.7A CN202210190265A CN114573663B CN 114573663 B CN114573663 B CN 114573663B CN 202210190265 A CN202210190265 A CN 202210190265A CN 114573663 B CN114573663 B CN 114573663B
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citrullinated peptide
seq
detection reagent
peptide
rheumatoid arthritis
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CN114573663A (en
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代双
夏良雨
吕伟华
柯拓
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Guangdong Fapon Biotech Co Ltd
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Guangdong Fapon Biotech Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • 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
    • G01N2410/00—Assays, e.g. immunoassays or enzyme assays, involving peptides of less than 20 animo acids
    • 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

Abstract

The invention discloses citrullinated peptide and application thereof, and a detection reagent and a kit for rheumatoid arthritis, and relates to the technical field of detection of rheumatoid arthritis. The amino acid sequence of the citrullinated peptide disclosed by the invention is shown as any one of SEQ ID NO. 1-9. The citrullinated peptide provided by the invention can be used as an antigen of an anti-CCP antibody for detecting the anti-CCP antibody of a rheumatoid arthritis patient, and has the characteristics of high sensitivity and strong specificity.

Description

Citrulline peptide and application thereof, and detection reagent and kit for rheumatoid arthritis
Technical Field
The invention relates to the technical field of detection of rheumatoid arthritis, in particular to citrullinated peptide, application thereof, and detection reagent and kit for the rheumatoid arthritis.
Background
Rheumatoid arthritis (Rheumatoid Arthritis, RA) is one of the most common autoimmune diseases, and is also the most common chronic inflammatory joint disease, with a global incidence of about 1% and a chinese incidence of 0.32% -0.36%. RA is frequent in women, with a ratio of about 1:2 to 1:4 for men and women. It is a systemic disease characterized by inflammation of the synovium of the joint, the symmetry of inflammation developing from facet joints to large joints, eventually leading to joint damage at the later stages of the disease, with soft tissue damage.
The Cyclic Citrullinated Peptide (CCP) antigen originated in 1998 and developed to now have a total of three generations, the first generation of antigens was citrulline-containing linear polypeptides of 19 amino acid residues synthesized by humans, i.e. citrullinated peptides, the detection sensitivity of the first generation of CCP antigens was 49% and the specificity 96%. The two serines in the 19 peptide were later replaced with cysteines, which would form disulfide bonds between them, thereby forming a cyclic citrullinated peptide, yielding a second generation CCP antigen (CCP 2). The second generation CCP antigen not only maintains higher detection specificity (98%), but also has higher sensitivity (69%). The third generation CCP antigen remains cyclic citrullinated peptide but has increased therein an epitope that is not recognized by Anti-CCP2 (Anti-CCP 2 antibody) with slightly increased detection sensitivity (73%).
At present, detection of RA by anti-CCP antibodies has been commonly used in clinic, and has been gradually used in clinic since 2001 in China. The anti-cyclic citrullinated peptide antibody detection kit in the markets at home and abroad mainly uses an enzyme-linked immunosorbent assay (ELISA), an immunoturbidimetry and a chemiluminescence method.
Although the ELISA method has low cost, manual operation is generally adopted, the operation is tedious and the efficiency is low, the human influence factors are large, and the result can only be qualitatively or inaccurately quantified. Therefore, in the high-efficiency automation age, the full-automatic detection method with high specificity and simple operation has good market prospect.
At present, an in-vitro diagnostic reagent company develops an anti-cyclic citrullinated peptide antibody kit detected by a latex enhanced turbidimetry method and is put into clinical application. The level of anti-CCP antibodies in serum of RA patients was examined by Bizzaro N et al, and as a result, it was revealed that the average level of anti-CCP antibodies in serum of RA patients was 1100AU/ml (range: 57 to 3419 AU/ml), whereas the average level of anti-CCP antibodies in control group was only 6.8AU/ml (range: 1 to 39 AU/ml). The detection sensitivity of the existing latex-enhanced immunoturbidimetry, such as the kits disclosed in the patent CN 102507918B and the patent CN 104198725B, is more than or equal to 1.25AU/ml, the linear measurement range is below 200AU/ml, and the clinical detection requirements are difficult to meet.
The chemiluminescent immunoassay is a new immunoassay technology developed after fluorescence, radioisotope and enzyme immunoassay, and according to a large amount of experimental results and clinical application data, the chemiluminescent immunoassay in the non-radioactive labeling analysis technology is in the leading position in terms of practicality, stability, accuracy and development prospect, represents the development direction and trend of the current world, has the specificity of immune reaction and has chemiluminescent reactionHigh sensitivity (detection limit can reach 10 -15 ～10 -18 mol/L). The chemiluminescent immunoassay technology has the advantages of high sensitivity, rapidness, accuracy, good repeatability, long effective period, safety, no toxicity, no pollution and the like, and becomes the first choice for replacing the radioimmunoassay and enzyme immunoassay technology.
However, in general, there is a need for improved sensitivity and specificity in the detection of anti-CCP antibodies using existing cyclic citrullinated peptide kits.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide a novel citrullinated peptide, application thereof, and a detection reagent and a kit for rheumatoid arthritis. The citrullinated peptide provided by the invention can be used as an antigen of an anti-CCP antibody for detecting the anti-CCP antibody of a rheumatoid arthritis patient, and has the characteristics of high sensitivity and strong specificity.
The invention is realized in the following way:
in a first aspect, embodiments of the present invention provide a citrullinated peptide having an amino acid sequence as shown in any one of SEQ ID NO. 1-9.
The citrullinated peptide shown in SEQ ID NO.1-9 provided by the invention has the advantages that through reasonable optimization and modification of the sequence, 2 amino acid modifications such as lysine (Lys, K) containing amino side chains are used at the N end of the citrullinated peptide so as to enlarge a binding site, and simultaneously, corner amino acid modifications such as glycine (Gly, G) are added so as to increase the flexibility of the citrullinated peptide serving as an antigen, and through reasonable optimization and modification, the sensitivity and the specificity of the citrullinated peptide when the citrullinated peptide is used as the antigen to detect an anti-CCP antibody can be obviously improved, so that a more reliable detection result is provided for detecting rheumatoid arthritis, and a new selection and detection strategy is provided for detecting rheumatoid arthritis.
In an alternative embodiment, the above citrullinated peptide has a cyclic structure formed by disulfide bonds between the cysteine residues at positions 4 and 17.
Compared with the linear structure, the cyclic citrullinated peptide has higher sensitivity and specificity when combined with the anti-CCP antibody, and is more beneficial to improving the reliability of detection results.
However, it should be noted that the citrullinated peptide provided by the present invention may have a linear structure or a cyclic structure, and the other structure may be for detecting the anti-CCP antibody, which falls within the scope of the present invention.
In a second aspect, embodiments of the present invention provide the use of a citrullinated peptide selected from at least one of SEQ ID nos. 1 to 9 as an antigen of rheumatoid arthritis in the manufacture of a rheumatoid arthritis test agent.
Experimental results in the embodiment of the invention show that any one citrullinated peptide of SEQ ID NO.1-9 or any combination thereof can be used as an antigen of an anti-CCP antibody, and the anti-CCP antibody has higher sensitivity and specificity when being detected.
In an alternative embodiment, the citrullinated peptide is selected from any one of the following combinations: a combination of SEQ ID NO.1 and 8, a combination of SEQ ID NO.2 and 8, a combination of SEQ ID NO.1, 2 and 8, and a combination of SEQ ID NO.1, 2, 4 and 8.
Detection of an anti-CCP antibody in a combination has higher sensitivity and specificity than detection of an anti-CCP antibody with a single citrullinated peptide, for example, detection of an anti-CCP antibody with a combination of SEQ ID nos. 1 and 8, a combination of SEQ ID nos. 2 and 8, a combination of SEQ ID nos. 1, 2 and 8, or a combination of SEQ ID nos. 1, 2, 4 and 8 as an antigen.
In an alternative embodiment, the cysteine residue at position 4 and the cysteine residue at position 17 of the citrullinated peptide shown in any of SEQ ID nos. 1-9 form a cyclic structure via disulfide bonds.
In a third aspect, embodiments of the present invention provide an antigen for use in the detection of rheumatoid arthritis comprising a citrullinated peptide as shown in any one or more of SEQ ID NO. 1-9.
The antigen provided by the invention can realize detection of the anti-CCP antibody, and has higher sensitivity and specificity.
In alternative embodiments, the antigen is selected from any one of the following combinations: a combination of SEQ ID NO.1 and 8, a combination of SEQ ID NO.2 and 8, a combination of SEQ ID NO.1, 2 and 8, and a combination of SEQ ID NO.1, 2, 4 and 8.
In an alternative embodiment, the cysteine residue at position 4 and the cysteine residue at position 17 of the citrullinated peptide shown in any of SEQ ID nos. 1-9 form a cyclic structure via disulfide bonds.
In a fourth aspect, embodiments of the present invention provide a detection reagent for rheumatoid arthritis, comprising citrullinated peptides having amino acid sequences as shown in any one or more of SEQ ID NO. 1-9.
The detection reagent provided by the invention can detect the anti-CCP antibody in the sample, so as to detect the rheumatoid arthritis, and has the characteristics of high sensitivity and strong specificity.
In an alternative embodiment, the citrullinated peptide described above is present in a form coupled to a solid phase particle.
In an alternative embodiment, the solid phase particles are magnetic beads or latex particles.
In an alternative embodiment, the surface of the solid phase particle carries a modifying group, and the modifying group is at least one selected from the group consisting of: amino, carboxyl, tosyl and epoxy.
In an alternative embodiment, the surface of the magnetic bead is modified with amino or carboxyl groups, and the citrullinated peptide is coupled to the surface of the magnetic bead by a coupling agent.
In an alternative embodiment, when the surface of the magnetic bead is modified with carboxyl groups, the coupling agent is selected from at least one of EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide); when the surface of the magnetic bead is modified with an amino group, the coupling agent is at least one selected from the group consisting of SMCC (Succinimidoyl 4- (N-maleimidomethyl) cyclohexane-1-caxboxylate), sulfo-SMCC (Sulfosumidinyl 4- (N-maleimidomethyl) cyclohexane-1-caxboxylate), SMPEG2, SMPEG4, SMPEG 6, SMPEG8, SMPEG12, SMPEG24, LC-SMCC (Succinimidyl- [4- (N-maleimidomethyl) ] -cyclohexane-1-carboxylic acid- (6-aminocaproate)), 2-IT2-Iminothiolane HCl), STAT (N-Succinimidyl) acetate, and P (N-Succinimidyl-3-acetylthiopropionate).
Wherein SMPEG2/4/6/8/12/24 refers to SMCC-derived bifunctional cross-linking agents with varying amounts of polyethylene glycol.
The coupling agents such as SMPEG2/4/6/8/12/24, LC-SMCC and the like have longer coupling arms, can better retain the activity of citrullinated peptide and are favorable for the combination with anti-CCP antibodies.
In a fifth aspect, an embodiment of the present invention provides a method for preparing a detection reagent according to the foregoing embodiment, including: the citrullinated peptide shown in any one or more of SEQ ID NO.1-9 is obtained.
In an alternative embodiment, the above preparation method comprises: mixing the obtained citrullinated peptide with solid phase particles to couple the citrullinated peptide to the solid phase particles.
The cyclic citrulline polypeptide adopted in the current market is relatively short and contains-COOH and-NH 2 The number of amino acids of the side chain group such as-OH is small, and thus, the conventional method of use is for ELISA, for example, european diagnosis and the like. Although some products have added sequences such as HQC at one end of the polypeptide, the coupling efficiency per se is not high.
The outer loop citrulline polypeptide is very small, latex microspheres or magnetic beads are coupled on a biochemical or chemiluminescent platform, the binding capacity of antigen and antibody is optimized through cyclization treatment, but the binding with the antibody is greatly influenced due to the influence of steric hindrance, and a plurality of products can be coupled with BSA or other hydrophobic proteins in an indirect coating manner to improve the binding efficiency, but the process is complex, the batch-to-batch difference is difficult to control, and in addition, animal-derived proteins such as BSA and the like are introduced, so that adverse effects can be brought in the aspect of specificity.
The citrullinated peptide has more side chain groups such as amino, hydroxyl and carboxyl which are favorable for coupling, and the side chain genes are favorable for coupling the citrullinated peptide with solid-phase particles, so that the coupling efficiency is improved, and the citrullinated peptide can be suitable for detection by various detection methods.
In an alternative embodiment, the solid phase particles are magnetic beads or latex particles.
In an alternative embodiment, the surface of the solid phase particle carries a modifying group, and the modifying group is at least one selected from the group consisting of: amino, carboxyl, hydroxyl and epoxy groups.
In an alternative embodiment, the solid phase particles are magnetic beads, and the magnetic beads are modified with carboxyl groups or amino groups.
In an alternative embodiment, 5-100. Mu.g of the citrullinated peptide is added per mg of the magnetic beads for mixing, more preferably 20-50. Mu.g of citrullinated peptide is added for mixing.
In an alternative embodiment, after addition of the citrullinated peptide described above, the resulting mixture is allowed to react at 15-45℃for 15-300min, more preferably 30-120min.
The coupling efficiency of the citrullinated peptide and the magnetic beads is improved by adopting the proper reaction conditions, and the citrullinated peptide coupled with the magnetic beads can keep higher antigen activity.
In an alternative embodiment, the above preparation method further comprises, prior to mixing the above citrullinated peptide with the above magnetic beads: an activation step;
the activation step comprises the following steps: the magnetic beads are resuspended by using a buffer solution, and a coupling agent is added into the resuspension for activation.
Preferably, when the surface of the magnetic bead is modified with carboxyl groups, the coupling agent is selected from at least one of EDC and NHS; when the surface of the magnetic bead is modified with amino groups, the coupling agent is at least one selected from SMCC, sulfo-SMCC, SMPEG2, SMPEG4, SMPEG 6, SMPEG8, SMPEG12, SMPEG24, LC-SMCC, 2-IT, STAT and STAP.
In an alternative embodiment, the buffer is Mes or PBS.
Mes buffers are commonly used for carboxyl-modified bead activation and PBS for amino-modified bead activation.
In a sixth aspect, the present embodiment provides a kit for detecting rheumatoid arthritis, which comprises the citrullinated peptide according to the previous embodiment, the antigen according to the previous embodiment or the detection reagent according to the previous embodiment.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram showing the reaction process of carboxyl magnetic bead coupling CCP in example 15 of the present invention.
FIG. 2 is a schematic diagram showing the reaction process of amino magnetic bead coupled CCP in example 16 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Example 1
The example provides a cyclic citrullinated peptide, the amino acid sequence of which is shown in SEQ ID NO.1, the specific sequence of which is shown in Table 1, and the cysteine residue at position 4 and the cysteine residue at position 17 form a cyclic structure through disulfide bonds.
Examples 2 to 9
The cyclic citrullinated peptides provided in examples 2-9 have the amino acid sequences shown in Table 1, with the cysteine residue at position 4 and the cysteine residue at position 17 forming a cyclic structure via disulfide bonds.
TABLE 1 sequences of cyclic citrullinated peptides provided in examples 1-9
In the table: cit represents citrulline in the sequences in the table, xaa represents citrulline in the sequence table.
Example 10
The antigen for detecting rheumatoid arthritis provided in this example is a cyclic citrullinated peptide as shown in any one of SEQ ID NO. 1-9.
Example 11
The antigen for detecting rheumatoid arthritis provided in this example is a combination of cyclic citrullinated peptides shown in SEQ ID No.1 and SEQ ID No. 8.
Example 12
The antigen for detecting rheumatoid arthritis provided in this example is a combination of cyclic citrullinated peptide shown as SEQ ID NO.2 and SEQ ID NO. 8.
Example 13
The antigen for detecting rheumatoid arthritis provided in this example is a combination of cyclic citrullinated peptides shown in SEQ ID No.1, 2 and 8.
Example 14
The antigen for detecting rheumatoid arthritis provided in this example is a combination of cyclic citrullinated peptides shown in SEQ ID No.1, 2, 4 and 8.
Example 15
This example provides a detection reagent for detecting rheumatoid arthritis comprising cyclic citrullinated peptides as shown in SEQ ID NO.1-9, wherein each cyclic citrullinated peptide is coupled to carboxyl-modified magnetic beads via a coupling agent EDC.
The embodiment also provides a preparation method of the detection reagent, which comprises the following steps:
(1) Dialyzing any one of the cyclic citrullinated peptides of SEQ ID No.1-9 with 1 XPBS;
(2) Suspending the carboxyl modified magnetic beads with 0.1M MES pH5.5, adding EDC (10 mg/mg solid phase particle) and NHS (10 mg/mg solid phase particle) for activation, and reacting at 25 ℃ for 1h to obtain activated carboxyl magnetic beads;
(3) The activated carboxyl magnetic beads are separated by a magnetic separator, and the dialyzed cyclic citrullinated peptide is mixed with the activated carboxyl magnetic beads according to the proportion of 20ug/mg (20 ug of cyclic citrullinated peptide is corresponding to each mg of carboxyl magnetic beads) and is placed at 25 ℃ for reaction for 2 hours. The coupling reaction is described with reference to FIG. 1.
(4) And separating the reactants by a magnetic separator to obtain the detection reagent for detecting the rheumatoid arthritis. When in use, the magnetic beads are diluted by the magnetic bead reagent buffer solution to be used as working solution.
Example 16
This example provides a detection reagent for detecting rheumatoid arthritis comprising cyclic citrullinated peptides as shown in SEQ ID NO.1-9, wherein each cyclic citrullinated peptide is coupled to amino modified magnetic beads via coupling agents SMPEG8 and 2-IT.
The embodiment also provides a preparation method of the detection reagent, which comprises the following steps:
(1) Dialyzing any one of the cyclic citrullinated peptides of SEQ ID No.1-9 with 1 XPBS;
(2) Suspending amino modified magnetic beads by using 1 XPBS, adding a coupling agent SMPEG2 with a crosslinking arm to activate the magnetic beads, then adding the magnetic beads and 2-IT to activate the magnetic beads, and reacting for 30min at 25 ℃ to obtain activated amino magnetic beads;
(3) Separating the activated magnetic beads by a magnetic separator, adding one of the cyclic citrullinated peptides selected in the step (1), mixing with the activated amino magnetic beads in a ratio of 20ug/mg, and reacting for 2 hours at 25 ℃; the coupling reaction is described with reference to FIG. 2.
(4) Separating the reactant by a magnetic separator to obtain the detection reagent for detecting the rheumatoid arthritis of the embodiment; when in use, the magnetic beads are diluted by the magnetic bead reagent buffer solution to be used as working solution.
Experimental example 1
Detecting the activity of the cyclic citrullinated peptide in the detection reagents provided in example 15 and example 16, and providing a control cyclic citrullinated peptide having a sequence which does not have the KKG sequence at the N-terminus compared to SEQ ID nos. 1-9; the control cyclic citrullinated peptide was coupled to carboxyl magnetic beads using a preparation similar to that of example 15.
The detection method comprises the following steps:
the immune solid phase magnetic bead particles in the embodiment are detected by adopting an indirect method, and the specific principle and the process are as follows: the solid-phase magnetic beads coated with CCP antigen capture the corresponding antibody in the sample, after magnetic separation, the corresponding antibody is repeatedly washed for 4 times, then anti-human IgG secondary antibody marked with alkaline phosphatase is added, finally, the magnetic bead-marked antigen-antibody anti-human IgG secondary antibody-ALP compound is formed, after magnetic separation, the corresponding antibody is repeatedly washed for 4 times, after the substrate AMPPD is added, the catalytic reaction is carried out for 5-6min, and an instrument photomultiplier collects RLU and converts the RLU into a digital signal RLU. The results are shown in Table 2 below. The numerical values in the table refer to relative luminescence values, the higher the value, the higher the activity.
Table 2 activity of different cyclic citrullinated peptides coupled with different coupling agents in examples 15 and 16
As can be seen from table 2, compared with the citrullinated peptide sequence without KKG modification, the cyclic citrullinated peptide provided in the examples of the present invention has higher activity after KKG modification, and in addition, the cyclic citrullinated peptide still has higher activity after coupling to the magnetic beads by different coupling agents, especially coupling by using the SMPEG24 coupling agent, and the activity of the obtained cyclic citrullinated peptide coupled to the magnetic beads is higher than that of the cyclic citrullinated peptide coupled by EDC.
Experimental example 2
Detection of sensitivity and specificity of different cyclic citrullinated peptides or combinations thereof
Sample: 104 RA specimens collected in hospitals and 342 healthy human specimens;
the detection method comprises the following steps:
the test reagent of example 16 was selected, the above samples were tested by indirect method, 2.1 times of the average relative light intensity of the negative samples (342 healthy human samples) was used as the Cutoff value, and the values of ≡cutoff were judged positive, the values of lower than Cutoff were judged negative, and the results of statistical sensitivity and specificity were shown in Table 3 below.
TABLE 3 Table 3
Cyclocitrullinated peptides Sensitivity to Specificity (specificity)
SEQ ID NO：1 83.7％ 98.8％
SEQ ID NO：2 84.6％ 99.4％
SEQ ID NO：3 76.0％ 99.7％
SEQ ID NO：4 82.7％ 99.4％
SEQ ID NO：5 70.2％ 98.2％
SEQ ID NO：6 78.8％ 99.4％
SEQ ID NO：7 72.1％ 99.1％
SEQ ID NO：8 84.6％ 99.7％
SEQ ID NO：9 69.2％ 99.4％
SEQ ID NO：1+8 86.5％ 98.8％
SEQ ID NO：2+8 85.6％ 99.1％
SEQ ID NO：1+2+8 92.1％ 98.8％
SEQ ID NO：1+2+4+8 94.6％ 99.8％
Sensitivity: sample ratio of detection signal higher than the Cutoff value in 104 RA samples. Specificity: sample proportion of detection signals lower than the Cutoff value in 342 healthy human samples.
As can be seen from the results in table 3, the cyclic citrullinated peptides provided by the examples of the present invention, either alone or in combination, are more sensitive and specific for detection of anti-CCP antibodies than the cyclic citrullinated peptides without KKG modification, in particular as defined in SEQ ID NO: when the combination of 1+2+4+8 is detected, the sensitivity reaches 94.6%, and the specificity reaches 99.8%. The sensitivity of the detection is 67% and the specificity is 95% by adopting the detection of the prior art. Compared with the prior art, the cyclic citrullinated peptide provided by the invention has the advantage that the sensitivity and the specificity of anti-CCP antibody detection are obviously improved.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
SEQUENCE LISTING
<110> Guangdong Fit biological Co., ltd
<120> citrullinated peptide and use thereof, detection reagent and kit for rheumatoid arthritis
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<211> 17
<212> PRT
<213> artificial sequence
<400> 9
Lys Lys Gly Cys Ala Arg Phe Gln Met Arg His Xaa Arg Leu Ile Arg
1 5 10 15
Cys

Claims (20)

1. The citrulline peptide is characterized in that the amino acid sequence of the citrulline peptide is shown as any one of SEQ ID NO.2-9, xaa in the sequence table represents citrulline.
2. The citrullinated peptide of claim 1, wherein the cysteine residue at position 4 and the cysteine residue at position 17 of the citrullinated peptide form a cyclic structure via disulfide bonds.
3. The application of citrullinated peptide as antigen of rheumatoid arthritis in preparing rheumatoid arthritis detecting reagent is characterized in that the amino acid sequence of citrullinated peptide is selected from at least one of SEQ ID NO.2-9, xaa in the sequence table represents citrulline.
4. Use according to claim 3, characterized in that the citrullinated peptide is selected from the group of SEQ ID nos. 2 and 8.
5. The use according to claim 3, wherein the cysteine residue at position 4 and the cysteine residue at position 17 of the citrullinated peptide represented by any of SEQ ID nos. 2-9 form a cyclic structure via disulfide bonds.
6. An antigen for detecting rheumatoid arthritis, which comprises citrullinated peptide shown as any one or more of SEQ ID NO.2-9, xaa in the sequence Listing represents citrulline.
7. The antigen of claim 6, wherein the antigen is selected from the group consisting of SEQ ID nos. 2 and 8.
8. The antigen as claimed in claim 6, wherein the cysteine residue at position 4 and the cysteine residue at position 17 of the citrullinated peptide of any one of SEQ ID NO.2-9 form a cyclic structure via disulfide bonds.
9. A detection reagent for rheumatoid arthritis is characterized by comprising citrullinated peptide with an amino acid sequence shown as any one or more of SEQ ID NO.2-9, wherein Xaa in a sequence table represents citrulline.
10. The detection reagent of claim 9, wherein the citrullinated peptide is present in a form coupled to a solid phase particle.
11. The detection reagent according to claim 10, wherein the solid phase particles are magnetic beads or latex microparticles.
12. The detection reagent according to claim 10, wherein the surface of the solid phase particle has a modifying group selected from at least one of the following groups: amino, carboxyl, tosyl and epoxy.
13. The detection reagent according to claim 11, wherein the surface of the magnetic bead is modified with an amino group or a carboxyl group, and the citrullinated peptide is coupled to the surface of the magnetic bead by a coupling agent.
14. The detection reagent according to claim 13, wherein the surface of the magnetic bead is modified with an amino group or a carboxyl group, the citrullinated peptide is coupled by a coupling agent, and when the surface of the magnetic bead is modified with a carboxyl group, the coupling agent is at least one selected from EDC and NHS; when the surface of the magnetic bead is modified with amino groups, the coupling agent is at least one selected from SMCC, sulfo-SMCC, SMPEG2, SMPEG4, SMPEG 6, SMPEG8, SMPEG12, SMPEG24, LC-SMCC, 2-IT, STAT and STAP.
15. The method for producing a detection reagent according to any one of claims 9 to 14, comprising: the citrulline peptide shown in any one or more of SEQ ID NO.2-9 is obtained, xaa in the sequence table represents citrulline.
16. The method for producing a detection reagent according to claim 15,
comprising the following steps: mixing the citrullinated peptide obtained with solid phase particles to couple the citrullinated peptide to the solid phase particles;
the solid phase particles are magnetic beads or latex particles;
the surface of the solid phase particle is provided with a modification group, and the modification group is selected from at least one of the following: amino, carboxyl, tosyl and epoxy.
17. The method for producing a detection reagent according to claim 16, wherein
The solid-phase particles are magnetic beads, and carboxyl or amino groups are modified by the magnetic beads;
adding 5-100 mug of citrullinated peptide into each mg of magnetic beads for mixing;
after the citrullinated peptide is added, the obtained mixed system is placed at 15-45 ℃ for reaction for 15-300min.
18. The method for producing a detection reagent according to claim 17, wherein
Adding 10-50 mug of citrullinated peptide into each mg of magnetic beads for mixing;
after the citrullinated peptide is added, the obtained mixed system is placed at 15-45 ℃ for reaction for 30-120min.
19. The method of preparing according to claim 16, further comprising, prior to mixing the citrullinated peptide with the magnetic beads: an activation step;
the activating step comprises the following steps: suspending the magnetic beads by using a buffer solution, and adding a coupling agent into the suspension for activation;
when the surface of the magnetic bead is modified with carboxyl, the coupling agent is at least one of EDC and NHS; when the surface of the magnetic bead is modified with amino groups, the coupling agent is at least one selected from SMCC, sulfo-SMCC, SMPEG2, SMPEG4, SMPEG 6, SMPEG8, SMPEG12, SMPEG24, LC-SMCC, 2-IT, STAT and STAP;
the buffer is Mes or PBS.
20. A kit for detecting rheumatoid arthritis, comprising the citrullinated peptide of claim 1 or 2, the antigen of claim 6, 7 or 8, or the detection reagent of claim 9, 10, 11, 12, 13, or 14.
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