CN108948174B - Citrulline modified peptide and application thereof - Google Patents

Citrulline modified peptide and application thereof Download PDF

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CN108948174B
CN108948174B CN201810496268.7A CN201810496268A CN108948174B CN 108948174 B CN108948174 B CN 108948174B CN 201810496268 A CN201810496268 A CN 201810496268A CN 108948174 B CN108948174 B CN 108948174B
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peptide antigen
citrullinated peptide
detection
composition
citrullinated
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CN108948174A (en
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于晓波
王红叶
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Academy of Military Medical Sciences AMMS of PLA
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BEIJING PROTEOME RESEARCH CENTER
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4713—Autoimmune diseases, e.g. Insulin-dependent diabetes mellitus, multiple sclerosis, rheumathoid arthritis, systemic lupus erythematosus; Autoantigens
    • 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
    • G01N33/531—Production of immunochemical test materials
    • 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
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • 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 provides a citrullinated peptide antigen and a composition comprising the same. The invention also provides the use of a citrullinated peptide antigen or a composition comprising a citrullinated peptide antigen for the manufacture of a diagnostic tool for the detection of anti-citrullinated peptide IgG and/or IgA antibodies. The citrullinated peptide antigen is used for detecting IgG antibody and/or IgA antibody markers, and solves the problem that 30% of RA patients lack clear markers clinically. The diagnosis tool provided by the invention adopts the antigen corresponding to the autoantibody of the novel rheumatoid arthritis related biomarker to detect the autoantibody in human serum, has the advantages of good specificity, high sensitivity, good accuracy, wide linear range, safety, reliability and easiness in operation, and is particularly suitable for screening and diagnosing negative rheumatoid arthritis patients who cannot be detected by current clinical CCP 2.

Description

Citrulline modified peptide and application thereof
Technical Field
The invention relates to the technical field of biomedicine, in particular to an anti-citrullinated peptide IgG antibody and/or IgA antibody serological detection technology, and particularly relates to a citrullinated peptide antigen, a diagnostic tool or a preparation method thereof and application thereof in detection of autoantibodies of CCP negative rheumatoid arthritis patients.
Background
Rheumatoid Arthritis (RA) is one of the most common systemic autoimmune diseases, and is encountered in about 0.5-1% of the world's population. RA is a systemic autoimmune disease, and patients often suffer from chronic processes of disease with chronic inflammatory reactions of the joints, which can lead to progressive joint destruction, bone erosion, deformity, and the like. The most important serological findings of rheumatoid arthritis RA: the serum of the patient contains autoantibodies which specifically recognize citrulline-modified proteins. About 70% of RA patients contain antibodies (ACPA) recognizing citrullinated proteins, which are called autoantigens, in their sera. ACPAs antibody detection is an important detection index of RA diagnosis gold standard.
Research shows that the generation of the anti-citrullinated protein autoantibody is accompanied with the disease process of RA patients, and in the aspect of clinical diagnosis, ACPAs have high specificity and sensitivity to RA patients and high sensitivity to RA early patients. In patients with very early RA, the presence of citrulline autoantibodies can be detected before they show clinical symptoms, even more than a decade ago. Meanwhile, the anti-citrulline autoantibody plays an important role in the prognosis of RA patients and can predict the disease development state. Within 1-2 years of accurate diagnosis of 75% of rheumatoid arthritis patients, irreversible bone and joint erosion can occur, the bone erosion degree of ACPAs antibody positive RA patients is more serious than that of ACPAs antibody negative RA patients, the ACPAs antibody level is higher, and the disease attack time of RA patients is shorter.
At present, a commercial Cyclic Citrullinated Peptide (CCP) detection kit is clinically used as an auxiliary diagnosis detection method for RA patients, and the CCP detection kit can detect an anti-cyclic citrullinated peptide autoantibody in serum of the patients. The first generation citrulline detection kit CCP1, antigen derived from Profilaggrin (Profilaggrin), coated with the serine Profilaggrin-modified peptide as antigen, detects citrulline-modified protein autoantibodies in patient serum using the Elisa method. Based on a CCP1 detection kit, a CCP2 detection kit is developed, CCP1 citrulline modified peptide is modified, two serines in a polypeptide sequence are replaced by cysteine, and cyclic citrulline modified peptide (CCP) is synthesized, so that the stability of the antigen is increased, and the sensitivity and specificity of CCP detection are improved. CCP2 commercial test kit is marketed in 2002 and is rapidly and widely applied to clinical tests, and is still the most common gold standard in RA tests. There are at least six common kits available today, respectively from Axis-Shield (US), Euro-Diagnostica (the Netherlands), Euroimmun (Germany), Inova (US), Phadia (Sweden/Germany), Abbott (US). The CCP2 detection kit is based on the same CCP2 antigen peptide, and has slight difference in detection, the detection sensitivity is 66.7-77.7%, and the specificity is 86.1-98.8%.
CCP antibody as RA disease diagnosis and progress marker shows good clinical application prospect. With the research of CCP2 for detecting target, many antibodies against the citrullinated protein specific for RA patients, such as Anti-citrated fibrous antibodies, Anti-citrated fibrous binding, Anti-citrated viral/Anti-Sa antibody, Anti-Mutated and Citrated Viral (MCV), have appeared, but these markers have not been widely used in clinical tests. With the development of molecular diagnostic technology, RA molecular diagnosis is further updated, in recent years, CCP third generation detection reagent CCP3.1(Inova, US) is widely concerned, CCP3.1 can detect serum antibody IgG and IgA levels of patients, CCP3 peptide sequence is not disclosed, CCP3 laboratory research is restricted, and some researches show that CCP3.1 is lower in specificity than CCP2 in the aspect of RA diagnostic application. Anti-CarP (Anti-Carbamylated protein) antibodies are novel RA-specific detection antibodies identified in recent years, recognizing a different type of post-translational modification of proteins compared to CCP antibodies, and Anti-carbamoylation antibodies were present in the serum of 43% of RA patients.
The CCP2 detection kit has high specificity and sensitivity to RA patients, however, silk polymeric protein is not expressed in joints, so CCP2 cannot be used as joint-specific antigen. It is believed that during the course of RA disease, certain antigens expressed in the joints elicit an antibody response, and CCP2 mimics its epitope and is therefore recognized by autoantibodies with a clinical detection sensitivity of up to 70%.
Since the focus of research has been on CCP-antibody positive RA patients, patent CN1602426A discloses a method for detecting autoantibodies from patients suffering from rheumatoid arthritis, comprising contacting said autoantibodies with a peptide unit comprising an XG motif and a peptide unit comprising an XnonG motif. Patent CN101918432A discloses a three-dimensional matrix of selected synthetic peptide mimic sequences preferentially recognized by autoimmune antibodies detected in patients with rheumatoid arthritis, which are capable of improving the specificity and sensitivity of detection of these autoantibodies in patients before symptoms occur, patients showing symptoms of rheumatoid arthritis and patients diagnosed as positive for rheumatoid arthritis. Patent CN101957365A discloses a kit for detecting a CCP and IgG bispecific antibody, which can detect a natural bispecific antibody existing in the serum of a rheumatoid arthritis patient, and is convenient to use, simple and easy to implement. Patent CN102323402A discloses a CCP antibody in-vitro detection kit and a preparation method thereof, and the kit can be applied to the auxiliary diagnosis of rheumatoid arthritis. The patent CN102796173A discloses an epitope of rheumatoid arthritis and application thereof, the epitope is only combined with IgG in serum of a patient, but not reacted with serum of healthy people, and can be used for preparing a medicament for diagnosing rheumatoid arthritis.
However, about 30 percent of patients with rheumatoid arthritis still have no clear detection index in clinic, and the patients are called patients with anti-citrulline negative (CCP-) rheumatoid arthritis. The citrulline detection reagent generally targets a single protein molecule and has good specificity on a certain subtype of RA. A series of researches show that a patient with positive resistance to citrulline (CCP +) has obviously different clinical manifestations from a patient with negative resistance to citrulline (CCP-) RA, and the lack of clinical diagnosis information of the patient with CCP-rheumatoid arthritis is partially limited by the fact that the existing detection method system cannot detect specific indications carried by the CCP-patient, and the CCP-patient can be an RA patient subtype containing a specific citrulline marker. Combining multiple detection of multiple targets is a very meaningful attempt, and the autoantibody types of RA patients can be subdivided, so that basis is better provided for clinical diagnosis and treatment.
Patent CN106950365A discloses an ACPA negative RA diagnostic marker and application thereof, in particular to application of deoxyco-protein dioxygenase, namely DOHH or fragments thereof in preparing a reagent for diagnosing anti-citrullinated polypeptide antibody negative rheumatoid arthritis diseases. Patent CN106950366A discloses an ACPA negative RA diagnostic marker and application thereof, in particular to application of Pentaxin related protein 3, namely PTX3 or fragments thereof in preparation of a reagent for diagnosing anti-citrullinated polypeptide antibody negative rheumatoid arthritis diseases. However, the diagnostic specificity of these negative rheumatoid arthritis was only 90%.
Disclosure of Invention
In order to solve the problem that 30% of RA patients lack clear markers clinically, the inventor identifies and obtains citrulline modified peptide, applies the citrulline modified peptide to the detection of CCP negative rheumatoid arthritis patients, and provides a citrulline antibody detection kit with good sensitivity and specificity.
In a first aspect, the present invention relates to a citrullinated modified peptide antigen, wherein the sequence of the citrullinated modified peptide antigen is selected from one of the following amino acid sequences:
1) the amino acid sequence of the citrulline modified peptide antigen is shown as SEQ ID NO: 1, or a portion or all of a sequence set forth in 1;
2) the amino acid sequence of the citrulline modified peptide antigen is similar to that of SEQ ID NO: 1 with a degree of identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%;
3) the amino acid sequence of the citrulline modified peptide antigen is similar to the amino acid sequence of SEQ ID NO: 1 differ by no more than 5, 4, 3, 2, or 1 amino acid;
4) the amino acid sequence of the citrullinated peptide antigen is SEQ ID NO: 1, wherein the variant hybridizes to SEQ ID NO: 1 includes a sequence in which one or more amino acid residues are substituted, deleted and/or inserted or at least one N-/C-terminal extension.
Preferably, the amino acid sequence of the citrullinated peptide antigen is identical to SEQ ID NO: 1, and the amino acids from position seventh to ninth of the amino acid sequence of the citrullinated peptide antigen are L- (Cit) -G;
preferably, the amino acid sequence of the citrullinated modified peptide antigen is identical to SEQ ID NO: 1 by no more than 5, 4, 3, 2 or 1 amino acid difference, and the amino acids from the seventh position to the ninth position of the amino acid sequence of the citrullinated peptide antigen are L- (Cit) -G;
preferably, the amino acid sequence of the citrullinated peptide antigen is SEQ ID NO: 1, wherein the variant hybridizes to SEQ ID NO: 1 or at least one N-/C-terminal extension, and the amino acids in positions seventh to ninth of the amino acid sequence of the citrullinated peptide antigen are L- (Cit) -G.
Further preferably, the amino acid sequence of the citrullinated peptide antigen sequence is SEQ ID NO: 1:
NLNGGLL-(Cit)-GIEILNQ
preferably, the citrullinated peptide antigen sequence is prepared by a chemical synthesis method.
Preferably, the sequence of the citrullinated peptide antigen is capable of binding to an autoantibody in a rheumatoid arthritis patient. Further preferably, the autoantibody is selected from IgG or IgA.
Preferably, the citrullinated peptide for detecting the autoantibody marker of the CCP negative arthritis patient comprises the citrullinated peptide antigen SEQ ID NO: 1 or a sequence identical to all or part of the sequence shown in SEQ ID NO: 1 or a sequence having a degree of identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% to SEQ ID NO: 1 by NO more than 5, 4, 3, 2 or 1 amino acids or SEQ ID NO: 1, wherein the variant hybridizes to SEQ ID NO: 1 includes a sequence in which one or more amino acid residues are substituted, deleted and/or inserted or at least one N-/C-terminal extension.
In a second aspect, the present invention relates to a nucleotide sequence encoding the citrullinated modified peptide antigen of the first aspect.
In a third aspect, the present invention relates to a genetic vector comprising the nucleotide sequence of the second aspect.
In a fourth aspect, the present invention relates to a cell comprising the gene vector of the third aspect.
In a fifth aspect, the present invention relates to a composition comprising the citrullinated peptide antigen of the first aspect and a carrier.
Preferably, the carrier is a solid phase carrier.
The solid phase carrier is selected from one or the combination of more than two of an enzyme label plate, a 96-hole microporous plate, colloidal gold and microspheres. Preferably, the microspheres are selected from one or a combination of more than two of silica microspheres, polystyrene microspheres, magnetic microspheres or biomacromolecule polymer microspheres. Most preferably, the microspheres are polystyrene microspheres.
In a specific embodiment of the present invention, the carrier is a polystyrene microsphere combined with a fluorescent dye.
Preferably, the citrullinated peptide antigen is coated onto the solid support in an amount of 1-25 μ g per 1,000,000 microspheres. Further preferably, the citrullinated peptide antigen is coated onto the solid support in an amount of 5 μ g per 1,000,000 microspheres.
Preferably, the preparation method of the composition comprises the following steps:
1) activating a carrier: adding the carrier into an activation buffer solution, and sequentially adding a Sulfo-NHS solution and an EDC (1-ethyl-3- [3-dimethylamino propyl ] carbodiimide hydrochloride) solution;
2) coating or coupling citrullinated peptide antigen on the surface of a carrier: adding citrulline modified peptide antigen into the activated carrier solution obtained in the step 1) for coating or coupling.
Preferably, the composition can be used as a detection kit for the self-antibody of a liquid-phase chip CCP-based patient with rheumatoid arthritis.
Preferably, the working concentration of the Sulfo-NHS solution and the EDC solution in the step 1) is 50 mg/mL.
Further preferably, the preparation method of the composition comprises the following steps:
a) pretreatment of microspheres: after the microspheres are subjected to ultrasonic treatment, discarding the supernatant; resuspending the microspheres with deionized water, and discarding the supernatant;
b) activation of microspheres: resuspending the microspheres by using an activation buffer solution, sequentially adding a Sulfo-NHS solution and an EDC solution, shaking and uniformly mixing, incubating for 5-30 minutes at room temperature in a dark place, and discarding the supernatant;
c) coupling citrullinated peptide antigen with microspheres: resuspending the microspheres with a coupling buffer solution, and discarding the supernatant; adding 500 mu L of coupling buffer solution, adding citrulline modified peptide antigen into the heavy suspension microspheres for coupling reaction, incubating for 0.5-3 hours at room temperature in a dark place, discarding the supernatant, washing the microspheres twice with washing buffer solution, and then re-suspending the coupling microspheres to obtain the composition.
Still more preferably, the incubation time in step b) is 20 minutes at room temperature in the absence of light.
Still more preferably, the incubation time in step c) is 2 hours at room temperature in the dark;
in a sixth aspect, the present invention relates to the use of a citrullinated peptide antigen according to the first aspect of the invention or a composition according to the fifth aspect of the invention for the manufacture of a diagnostic tool for the detection of anti-citrullinated peptide IgG and/or IgA antibodies, said diagnostic tool comprising at least a citrullinated peptide antigen according to the first aspect or a composition according to the fifth aspect.
Preferably, the citrullinated peptide antigen according to the first aspect of the invention or the composition according to the fifth aspect of the invention is used for preparing a diagnostic tool for rheumatoid arthritis, wherein the rheumatoid arthritis is negative rheumatoid arthritis.
Preferably, for ease of preservation, the diagnostic tool further comprises a preservative.
The seventh aspect of the present invention relates to a diagnostic tool for detecting an anti-citrullinated peptide IgG antibody and/or an anti-IgA antibody, wherein the diagnostic tool comprises at least the citrullinated peptide antigen of the first aspect or the composition of the fifth aspect, and the diagnostic tool is one selected from a kit, a diagnostic reagent, a gene chip, a protein chip, and an immunoassay test paper.
In an eighth aspect, the present invention relates to a kit for detecting an anti-citrullinated peptide IgG antibody and/or an anti-IgA antibody, said kit comprising the citrullinated peptide antigen of the first aspect of the invention or the composition of the fifth aspect of the invention.
Preferably, the kit further comprises a secondary antibody marked with a marker, a blocking buffer and a washing buffer.
The kit of the invention may be for diagnostic or non-diagnostic purposes.
Preferably, the method for detecting the anti-citrullinated peptide IgG antibody and/or IgA antibody is indirect qualitative detection.
Preferably, the detection is indirect qualitative detection.
Preferably, the kit is used in steps comprising:
(1) preparing a composition according to the fifth aspect of the invention; preferably, the citrullinated peptide antigen in the composition is capable of binding to an IgG antibody and/or an IgA antibody. More preferably, the IgG antibody and/or the IgA antibody are autoantibodies.
(2) Contacting a sample to be detected with the composition prepared in the step (1);
(3) adding a secondary antibody marked with a marker to form a citrullinated peptide antigen-IgG antibody and/or IgA antibody-marked secondary antibody compound;
(4) washing the complex obtained in step (3) and detecting for the label.
Preferably, the sample to be detected in step (2) is selected from serum, plasma or antibodies. Further preferably, the sample to be detected is serum.
Preferably, the step (2) further comprises diluting the sample to be detected before contacting the sample to be detected with the composition. Further preferably, the dilution factor of the sample to be detected is 1:20-1: 200. Still more preferably, the test sample is diluted by a factor of 1: 50.
Preferably, the secondary antibody labeled with a label in the step (3) is selected from proteins or antibodies labeled with enzymes or fluorescein and conjugates thereof. Wherein the enzyme is alkaline phosphatase or peroxidase or a combination thereof. The fluorescein is one or the combination of more than two of anthocyanin Cy series fluorescein, Alexa Fluor series fluorescein or fluorescein isothiocyanate. The protein or antibody and the conjugate thereof are selected from one or the combination of more than two of avidin, streptavidin, digoxin antibody, histidine antibody, Ni-containing affinity molecule or fluorescent molecule antibody.
Further preferably, the secondary antibody labeled with a label is an Alexa 555-labeled anti-human IgG or anti-human IgA antibody.
Preferably, the secondary antibody labeled with the marker is added at a concentration of 1 to 10. mu.g/mL. Further preferably, the secondary antibody labeled with the marker is added at a concentration of 4. mu.g/mL.
Preferably, the detection in the step (4) is signal detection. Further preferably, the signal detection is selected from one or a combination of two or more of fluorescence detection, chromogenic detection, electrochemical detection, mechanical detection, DNA-encoding hybridization, and sequencing detection.
In one embodiment of the invention, the signal detection is a fluorescence detection; the signal detection is fluorescence detection at the wavelength of 532nm and 635 nm.
In one embodiment of the present invention, the method for detecting an anti-citrullinated peptide IgG antibody and/or IgA antibody in serum comprises: adding 100 mu L of sealing buffer solution into a 96-hole microporous plate, and sealing for 1 hour at 37 ℃; adding citrulline modified peptide antigen coupled microspheres and a detection sample, and incubating overnight at 4 ℃; washing with a washing buffer solution for three times; adding a fluorescence labeled secondary antibody; washing with a washing buffer solution for three times, resuspending the microspheres with a detection buffer solution, and detecting with an instrument.
The citrullinated peptide antigen is used for detecting IgG antibody and/or IgA antibody markers, has high specificity (98%) for detecting rheumatoid arthritis patients, is particularly suitable for diagnosing CCP negative rheumatoid arthritis, realizes three or more indexes of one-time reaction detection, and provides basis for clinical diagnosis and treatment. Meanwhile, the diagnostic tool provided by the invention has the advantages of wide linear range, safety, reliability and easiness in operation.
The term "and/or" as used herein includes a list of items in the alternative as well as any number of combinations of items.
The terms "comprises" and "comprising" as used herein are intended to be open-ended terms that specify the presence of the stated elements or steps, and not substantially affect the presence of other stated elements or steps.
"diagnosis" as used herein refers to the determination of whether a patient has suffered from a disease or condition in the past, at the time of diagnosis, or in the future, or the determination of the progression or likely progression of a disease in the future, or the assessment of a patient's response to a therapy.
Drawings
Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1: a schematic detection diagram of a rheumatoid arthritis autoantibody detection kit;
FIG. 2: SEQ ID NO: 1, synthesizing peptide antigen and detecting the mass spectrum;
FIG. 3: use of a polypeptide comprising SEQ ID NO: 1 peptide antigen, wherein the peptide antigen has a structure shown in the specification, and the peptide antigen has a structure shown in the specification, wherein the peptide antigen has a structure shown in the specification: healthy group, α -CCP- _ RA: citrulline antibody negative rheumatoid arthritis patient group, mfi (media fluorescence intensity): median fluorescence signal value;
FIG. 4: use of a polypeptide comprising SEQ ID NO: 1 peptide antigen, wherein the peptide antigen has a detection result of a serum verification group, and the detection result of the peptide antigen comprises the following components: healthy group, α -CCP- _ RA: citrulline antibody negative rheumatoid arthritis patient group, mfi (media fluorescence intensity): median fluorescence signal value;
FIG. 5: use of a polypeptide comprising SEQ ID NO: 1 screening ROC curves of a serum experimental group by using a peptide antigen rheumatoid arthritis detection kit;
FIG. 6: use of a polypeptide comprising SEQ ID NO: 1 peptide antigen for rheumatoid arthritis test kit screening serum verification group ROC curve.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 Synthesis of citrullinated peptides
1. The sequence of the citrullinated peptide is as follows:
NLNGGLL-(Cit)-GIEILNQ(SEQ ID NO：1)
2. the synthesis sequence is as follows: from the C end to the N end of the sequence, the steps are as follows:
1) weighing 0.3g of resin, putting the resin into a glass reactor, adding DCM (dichloromethane) and swelling for 30 minutes;
2) removing DCM, adding 0.6g of the first amino acid in the amino acid sequence, adding 0.6g of DIEA (diisopropylethylamine), DMF (dimethylformamide) and DCM, carrying out nitrogen bubbling reaction for 60 minutes, removing the reaction solution, adding DMF and MEOH, and washing for three times;
3) the second amino acid 0.6g in the amino acid sequence was added to the reactor, HBTH (1-hydroxy, benzo, trichloroazoltetramethylhexafluorophosphate) and DIEA were added, the reaction was bubbled with nitrogen for 30 minutes, the liquid was washed off, ninhydrin detection was performed, and then end capping with pyridine and acetic anhydride was performed. Finally, cleaning, adding a proper amount of decapping liquid to remove the Fmoc (9-fluorenylmethyloxycarbonyl) protecting group, cleaning, and detecting ninhydrin;
4) adding amino acids in the sequence in sequence according to step 3);
5) and taking down the reaction column after the resin is dried by nitrogen. Transferring to a 10mL centrifuge tube, adding 6mL cutting fluid (95% TFA, 2% ethanedithiol, 2% triisopropylsilane and 1% water), shaking and filtering off the resin;
6) adding a large amount of diethyl ether into the filtrate to separate out a crude product, centrifuging, and cleaning to obtain the crude product;
7) purifying the polypeptide, namely purifying the crude product to 90% by high performance liquid chromatography;
8) freeze-drying the polypeptide, concentrating the purified liquid in a freeze dryer, and freeze-drying to obtain white powder.
3. Peptide synthesis mass spectrum detection result
Synthetic SEQ ID NO: the results of mass spectrometry for peptide antigens 1 are shown in FIG. 2. The sequence was correctly synthesized and its molecular weight was 1979.38 Dalton.
Example 2 application of citrullinated peptide in CCP-negative rheumatoid arthritis patient detection based on microsphere detection technology
1. Test materials and reagents
1) Activation buffer: 0.1M NaH2PO4(Sigma-Aldrich,St.Louis,MO,USA)，pH 6.2；
2) EDC solution: 50mg/mL EDC (1-ethyl-3- [ 3-methylenepropyl ] carbodiimide hydrochloride, Thermo Fisher Scientific, IL, USA);
3) Sulfo-NHS solution: 50mg/ml sulfo-NHS (Thermo Fisher Scientific, IL, USA);
4) coupling buffer: 50mM MES, pH5.0(Sigma-Aldrich, St. Louis, MO, USA);
5) phosphate buffered saline PBS: 2mM KH2PO4，10mM Na2HPO4，137mM NaCl，2.7mM KCl，pH7.4(Sigma-Aldrich,St.Louis,MO,USA)；
6) Detection/wash buffer: 0.05% (v/v) Tween 20 in 0.1MPBS buffer;
7) blocking buffer: PBS-TBN, 1% BSA in 0.1MPBS buffer;
8) detecting an antibody: alexa 555-labeled anti-human IgG or anti-human IgA antibody (4. mu.g/mL) (Jackson ImmunoResearch, Pa., USA).
2. The experimental method comprises the following steps:
(1) preparation of the kit
The kit includes a composition and a preservative.
Preparation of the composition (coupled microspheres): putting 1,000,000 three microspheres into a 1.5mL centrifuge tube, carrying out ultrasonic treatment on the microspheres, putting the centrifuge tube into a magnetic frame for 1 minute, and discarding the supernatant; resuspending the microspheres in 100. mu.L of deionized water, and discarding the supernatant; adding 80 mu L of activation buffer solution for resuspending microspheres, sequentially adding 10 mu L of Sulfo-NHS solution and EDC solution, shaking and uniformly mixing, and incubating for 20 minutes at room temperature in a dark place; after incubation, the supernatant was discarded; adding 250 mu L of coupling buffer solution for resuspending the microspheres, and discarding the supernatant; adding 500 mu L of coupling buffer solution, adding 5 mu g of citrulline modified peptide antigen into the heavy suspension microspheres for coupling reaction, and incubating for 2 hours at room temperature in a dark place; discarding the supernatant, washing the microspheres twice with a washing buffer, and resuspending the coupled microspheres with a blocking buffer; incubation for 30 minutes at room temperature; discarding the supernatant, washing the microspheres with a washing buffer solution for three times to obtain the coupled microspheres, namely the composition. Detecting the number of coupled microspheres by a flow cytometer, adjusting the concentration of the microspheres to 2000/100 mu L by using a detection buffer solution, and storing for later use.
(2) Detection step
Adding 100 mu L of sealing buffer solution into a 96-hole microporous plate, and sealing for 1 hour at 37 ℃; washing 1 time with 150. mu.L of washing buffer; adding 100 mu L of coupling microsphere solution into each hole, adding 2 mu L of serum, and incubating overnight at 4 ℃; washing with a washing buffer solution for three times; adding Alexa 555-labeled anti-human IgG or anti-human IgA antibody to form a citrulline modified peptide antigen-autoantibody-labeled secondary antibody complex; washing the microspheres with a washing buffer solution for three times, detecting the microspheres with a detection buffer solution, and detecting with an instrument, wherein the detection value of a fluorescence-labeled secondary antibody signal is read at 532nm, and the ID number of the microspheres is read at 635 nm.
3. Experimental data processing
The signal detection value mfi (media fluorescent intensity) is read and plotted.
4. Sample information
In total, 110 healthy human samples and 114 CCP-negative rheumatoid arthritis patients were collected, and the selected serum samples were randomly divided into an experimental group (Training) and a verification group (Validation) in the data analysis, wherein the experimental group comprises 52 healthy human samples, 53 CCP-negative rheumatoid arthritis patients, 58 verification group healthy human samples and 61 CCP-negative rheumatoid arthritis patients. Clinical sample information is shown in tables 1 and 2, where HC: healthy group, α -CCP- _ RA: citrulline antibody negative rheumatoid arthritis patients. The detection schematic diagram of the rheumatoid arthritis autoantibody detection kit is shown in figure 1.
TABLE 1 clinical sample information
Figure BDA0001669008510000111
TABLE 2 clinical sample information
Figure BDA0001669008510000121
5. The result of the detection
The detection results of the serum screening experimental group and the verification group are shown in fig. 3 and fig. 4, wherein the ratio of IgA: detecting IgA autoantibodies; IgG: and detecting IgG autoantibodies. The detection results of ROC curves of the results of the serum screening experiment group and the verification group of the kit are shown in fig. 5 and fig. 6, wherein the sensitivity and the specificity of the experiment group are respectively 11.32 percent and 98 percent, and the sensitivity and the specificity of the verification group are respectively 24.59 percent and 98 percent.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.

Claims (11)

1. A citrullinated peptide antigen, wherein the amino acid sequence of the citrullinated peptide antigen is as shown in SEQ ID NO: 1 is shown.
2. A nucleic acid encoding the citrullinated peptide antigen of claim 1.
3. A genetic vector comprising the nucleic acid of claim 2.
4. A cell comprising the gene vector of claim 3.
5. A composition comprising the citrullinated peptide antigen of claim 1 and a carrier.
6. The composition of claim 5, wherein the carrier is a solid phase carrier selected from one or a combination of two or more of an elisa plate, a 96-well microplate, colloidal gold, and microspheres; wherein, the microsphere is selected from one or the combination of more than two of silica microsphere, polystyrene microsphere, magnetic microsphere or biological macromolecular polymer microsphere.
7. A method of preparing the composition of claim 5 or 6, comprising:
1) activating a carrier: adding the carrier into an activation buffer solution, and sequentially adding a Sulfo-NHS solution and an EDC solution;
2) coating or coupling citrullinated peptide antigen on the surface of a carrier: adding citrulline modified peptide antigen into the activated carrier solution obtained in the step 1) for coating or coupling.
8. Use of a citrullinated peptide antigen according to claim 1 or a composition according to any of claims 5 to 6 for the manufacture of a diagnostic tool for the detection of anti-citrullinated peptide IgG and/or IgA antibodies.
9. Use of the citrullinated peptide antigen of claim 1 or the composition of any of claims 5 to 6 for the preparation of a diagnostic tool for negative rheumatoid arthritis.
10. A diagnostic tool for detecting IgG and/or IgA antibodies, wherein the diagnostic tool comprises the citrullinated peptide antigen of claim 1 or the composition of claim 5 or 6, wherein the diagnostic tool is selected from one of a kit, a diagnostic reagent, a protein chip, and an immunoassay strip.
11. The diagnostic tool of claim 10, wherein the diagnostic tool is a kit further comprising a secondary antibody labeled with a label, a blocking buffer and/or a wash buffer.
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Citations (3)

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Publication number Priority date Publication date Assignee Title
WO2001046222A2 (en) * 1999-12-21 2001-06-28 Innogenetics N.V. Peptides designed for the diagnosis and treatment of rheumatoid arthritis
CN102796173A (en) * 2012-06-27 2012-11-28 中国人民解放军军事医学科学院基础医学研究所 Epitope of rheumatoid arthritis and application thereof
CN104262489A (en) * 2014-09-30 2015-01-07 陈仁奋 Cyclic chimeric citrullinated peptide antigen and application thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001046222A2 (en) * 1999-12-21 2001-06-28 Innogenetics N.V. Peptides designed for the diagnosis and treatment of rheumatoid arthritis
CN102796173A (en) * 2012-06-27 2012-11-28 中国人民解放军军事医学科学院基础医学研究所 Epitope of rheumatoid arthritis and application thereof
CN104262489A (en) * 2014-09-30 2015-01-07 陈仁奋 Cyclic chimeric citrullinated peptide antigen and application thereof

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