CN108948153B - Citrulline modified peptide antigen combination and application thereof - Google Patents

Citrulline modified peptide antigen combination and application thereof Download PDF

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CN108948153B
CN108948153B CN201810495338.7A CN201810495338A CN108948153B CN 108948153 B CN108948153 B CN 108948153B CN 201810495338 A CN201810495338 A CN 201810495338A CN 108948153 B CN108948153 B CN 108948153B
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peptide antigen
seq
citrullinated peptide
detection
microspheres
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CN108948153A (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
    • 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
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    • 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
    • G01N33/68—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
    • 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

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Abstract

The invention provides a citrullinated peptide antigen combination and a composition comprising the same. The invention also provides the use of a citrullinated peptide antigen combination or a composition comprising a citrullinated peptide antigen combination for the manufacture of a diagnostic tool for the detection of anti-citrullinated peptide IgG and/or IgA antibodies. The citrullinated peptide antigen combination 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 combination 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 easy operation, and is particularly suitable for screening and diagnosing negative rheumatoid arthritis patients who cannot be detected by the current clinical CCP 2.

Description

Citrulline modified peptide antigen combination 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) that recognize citrullinated proteins, which are called autoantigens. 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-citrullinated protein antibodies, Anti-citrullinated protein antibody, Anti-citrullinated protein/Anti-Sa antibody, Anti-mutated and citrullinated protein (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 three citrullinated peptides, applies the three citrullinated peptides to the detection of CCP negative rheumatoid arthritis patients, and provides a kit comprising any combination of the three citrullinated peptides, wherein the kit has good sensitivity and specificity.
In a first aspect of the present invention, a citrullinated peptide antigen combination is provided, wherein the amino acid sequence of the citrullinated peptide antigen combination is selected from (1) SEQ ID NO: 1 or SEQ ID NO: 1, or (2) all or part of the variant sequence of SEQ ID NO: 2 or SEQ ID NO: 2, or (3) all or part of the variant sequence of SEQ ID NO: 3 or SEQ ID NO: 3 in whole or in part, or a combination of two or more thereof.
Preferably, the citrullinated peptide antigen combination is selected from the group consisting of:
(1) SEQ ID NO: 1 or SEQ ID NO: 1, and (2) all or part of the variant sequence of SEQ ID NO: 2 or SEQ ID NO: 2, all or part of the variant sequence of 2.
Or
(2) SEQ ID NO: 2 or SEQ ID NO: 2, and (3) all or part of the variant sequence of SEQ ID NO: 3 or SEQ ID NO: 3, all or part of a variant sequence of 3.
Or
(1) SEQ ID NO: 1 or SEQ ID NO: 1, and (3) all or part of the variant sequence of SEQ ID NO: 3 or SEQ ID NO: 3, all or part of a variant sequence of 3.
Or
(1) SEQ ID NO: 1 or SEQ ID NO: 1, and (2) all or part of the variant sequence of SEQ ID NO: 2 or SEQ ID NO: 2, and (3) all or part of the variant sequence of SEQ ID NO: 3 or SEQ ID NO: 3, all or part of a variant sequence of 3.
In one embodiment of the present invention, the citrullinated peptide antigen combination is (1) SEQ id no: 1 or SEQ ID NO: 1, and (2) all or part of the variant sequence of SEQ ID NO: 2 or SEQ ID NO: 2, and (3) all or part of the variant sequence of SEQ ID NO: 3 or SEQ ID NO: 3, all or part of a variant sequence of 3.
Preferably, the citrullinated peptide antigen combination can be a dimer, a trimer or a multimer.
The variants according to the invention are selected from sequences having a degree of identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% to the sequence, or which differ from the sequence by no more than 5, 4, 3, 2 or 1 amino acid, or which differ from the sequence by substitution, deletion and/or insertion of one or more amino acid residues or at least one N-/C-terminal extension.
Preferably, the sequence of SEQ ID NO: 1, is a variant of SEQ ID NO: 1, and the sequence of SEQ ID NO: 1 is P- (Cit) -S at amino acids from position seven to ninth of the amino acid sequence of the variant;
preferably, the sequence of SEQ ID NO: 1 and SEQ ID NO: 1 by NO more than 5, 4, 3, 2 or 1 amino acids, and said SEQ ID NO: 1 is P- (Cit) -S at amino acids from position seven to ninth of the amino acid sequence of the variant;
preferably, the sequence of SEQ ID NO: 1, wherein the variant hybridizes to SEQ ID NO: 1, comprises a sequence or at least one N-/C-terminal extension in which one or more amino acid residues are substituted, deleted and/or inserted, and said sequence of seq id no: 1 is P- (Cit) -S at amino acids from position seven to ninth of the amino acid sequence.
Preferably, the sequence of SEQ ID NO: 2, is a variant of SEQ ID NO: 2, and the sequence of SEQ ID NO: 2 is F- (Cit) -L at amino acids from position seven to ninth of the amino acid sequence;
preferably, the sequence of SEQ ID NO: 2 and SEQ ID NO: 2 by NO more than 5, 4, 3, 2 or 1 amino acids, and the sequence of SEQ ID NO: 2 is F- (Cit) -L at amino acids from position seven to ninth of the amino acid sequence;
preferably, the sequence of SEQ ID NO: 2, wherein the variant hybridizes to SEQ ID NO: 2, comprises a sequence of substitutions, deletions and/or insertions of one or more amino acid residues or at least one N-/C-terminal extension, and said SEQ id no: 2 is F- (Cit) -L at amino acids from position seven to ninth of the amino acid sequence.
Preferably, the sequence of SEQ ID NO: 3, is a variant of SEQ ID NO: 3, and the sequence of SEQ ID NO: 3 is L- (Cit) -G at amino acids from position seven to ninth of the amino acid sequence;
preferably, the sequence of SEQ ID NO: 3 and SEQ ID NO: 3 by NO more than 5, 4, 3, 2 or 1 amino acids, and the sequence of SEQ ID NO: 3 is L- (Cit) -G at amino acids from position seven to ninth of the amino acid sequence;
preferably, the sequence of SEQ ID NO: 3, wherein the variant hybridizes to SEQ ID NO: 3, comprises a sequence of substitutions, deletions and/or insertions of one or more amino acid residues or at least one N-/C-terminal extension, and said SEQ id no: 3 is L- (Cit) -G at amino acids from position seven to ninth of the amino acid sequence.
Preferably, the citrullinated peptide antigen sequence is selected from one or more of the following amino acid sequences:
GSGMSNP-(Cit)-SLEEEKY(SEQ ID NO：1)
RLAADDF-(Cit)-LKYENEV(SEQ ID NO：2)
NLNGGLL-(Cit)-GIEILNQ(SEQ ID NO：3)
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, a citrullinated peptide antigen combination for detecting autoantibody markers of CCP negative arthritis patients, comprising the citrullinated peptide antigen combination of the first aspect.
In a second aspect, the present invention relates to a nucleotide sequence encoding each amino acid sequence of the citrullinated modified peptide antigen combination of the first aspect.
In a third aspect, the present invention relates to a gene 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 combination of the first aspect and a carrier.
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 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 the citrullinated peptide antigen combination of the first aspect or the composition of the fifth aspect 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 the citrullinated peptide antigen combination of the first aspect or the composition of the fifth aspect.
Preferably, the citrullinated peptide antigen combination 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 combination 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 invention relates to a kit for detecting an anti-citrullinated peptide IgG antibody and/or an anti-IgA antibody, wherein the kit comprises the citrullinated peptide antigen combination of the first aspect or the composition of the fifth aspect.
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 preparation method of the kit 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 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 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 kit comprises:
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.
Further preferably, the incubation time in step b) is 20 minutes at room temperature in the absence of light.
Further preferably, the incubation time in step c) is 2 hours at room temperature in the dark;
preferably, the kit is used in steps comprising:
(a) preparing a composition according to the fifth aspect; 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.
(b) Contacting a sample to be tested with the composition prepared in step (a);
(c) adding a secondary antibody marked with a marker to form a citrullinated peptide antigen-IgG antibody and/or IgA antibody-marked secondary antibody compound;
(d) washing the complex obtained in step (c) and detecting for the label.
Preferably, the sample to be detected in step (b) is selected from serum, plasma or antibodies. Further preferably, the sample to be detected is serum.
Preferably, the step (b) further comprises diluting the sample to be tested before contacting the sample to be tested 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 step (c) is selected from a protein or an antibody labeled with an enzyme or fluorescein, and a conjugate 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 step (d) 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 combination 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.
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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: SEQ ID NO: 2, synthesizing peptide antigen into mass spectrum detection result;
FIG. 4: SEQ ID NO: 3, detecting the result of peptide antigen synthesis mass spectrum;
FIG. 5: screening the detection result of the serum experimental group by applying the citrullinated peptide antigen combination joint detection kit, wherein the ratio of HC: healthy group, α -CCP- _ RA: citrulline antibody negative rheumatoid arthritis patients, p < 0.05;
FIG. 6: screening the detection result of the serum verification group by applying the citrullinated peptide antigen combination joint detection kit, wherein the ratio of HC: healthy group, α -CCP- _ RA: citrulline antibody negative rheumatoid arthritis patients, p < 0.05;
FIG. 7: screening a ROC curve of a serum experimental group by using a citrullinated peptide antigen combination joint detection kit;
FIG. 8: and screening a serum verification group ROC curve by using a citrullinated peptide antigen combination joint detection kit.
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:
GSGMSNP-(Cit)-SLEEEKY(SEQ ID NO：1)
RLAADDF-(Cit)-LKYENEV(SEQ ID NO：2)
NLNGGLL-(Cit)-GIEILNQ(SEQ ID NO：3)
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: 1. SEQ ID NO: 2. SEQ ID NO: the results of mass spectrometry of the 3 peptide antigens are shown in FIG. 2, FIG. 3 and FIG. 4, respectively. The sequence synthesis is correct, and the molecular weights are 2039.22Dalton, 2194.55Dalton and 1979.38Dalton respectively.
Example 2 application of citrullinated peptide in CCP-negative rheumatoid arthritis patient detection based on microsphere detection technology 1, experimental 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 ] carbodiimidate 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 each microsphere 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 experimental data were obtained using logistic regression and Bayesian Information Criterion (BIC) with the following data fitting formula:
Figure BDA0001668821310000131
asn ═ 9.45e-4 xmi (CP001) -1.65e-4 xmi (CP002) +5.6e-5 xmi (CP003) -8.2e-1MFI (media fluorescence intensity): median fluorescence signal value.
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 BDA0001668821310000141
TABLE 2 clinical sample information
Figure BDA0001668821310000142
5. The result of the detection
The detection results of the serum screening experimental group and the verification group are shown in fig. 5 and fig. 6. The results of ROC curve detection in serum screening test and validation test are shown in fig. 7 and 8. The sensitivity of the citrulline modified peptide antigen combined detection experimental group is 22.64 percent, and the specificity is 98 percent; the sensitivity of the verification group is 19.67%, and the specificity is 98%.
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 (5)

1. The application of the citrullinated peptide antigen combination or the composition comprising the citrullinated peptide antigen combination and a carrier in the preparation of a diagnostic tool for negative rheumatoid arthritis is characterized in that the amino acid sequence of the citrullinated peptide antigen combination is (1) SEQ ID NO: 1, and (2) SEQ ID NO: 2, and (3) SEQ ID NO: 3, in combination.
2. The use of claim 1, wherein the carrier is a solid phase carrier selected from one or a combination of more than two 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.
3. Use according to claim 1 or 2, characterized in that the composition is prepared by a process comprising 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 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.
4. The use of claim 1, wherein the diagnostic means is selected from one of a kit, a diagnostic reagent, a protein chip, or an immunoassay strip.
5. The use according to claim 4, wherein the diagnostic means is a kit further comprising a secondary antibody labeled with a label, a blocking buffer and/or a wash buffer.
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