CN108948174A - A kind of citrulling modified peptides and its application - Google Patents
A kind of citrulling modified peptides and its application Download PDFInfo
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- CN108948174A CN108948174A CN201810496268.7A CN201810496268A CN108948174A CN 108948174 A CN108948174 A CN 108948174A CN 201810496268 A CN201810496268 A CN 201810496268A CN 108948174 A CN108948174 A CN 108948174A
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- citrulling
- modified peptides
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Classifications
-
- 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 present invention provides a kind of citrulling modified peptides antigen and the composition including citrulling modified peptides antigen.The present invention also provides citrulling modified peptides antigen or the composition including citrulling modified peptides antigen is preparing the purposes in the diagnostic tool for detecting anti-citrulling modified peptides IgG antibody and/or IgA antibody.Citrulling modified peptides antigen of the present invention solves the problems, such as that the RA patient of clinic 30% lacks clear marker for detecting IgG antibody and/or IgA antibody marker.Antigen corresponding to autoantibody of the diagnostic tool provided by the invention using novel rheumatoid arthritis associated biomarkers, detect autoantibody in human serum, have the advantages that specificity is good, high sensitivity, accuracy is good, the range of linearity is wide, safe and reliable, easy-operating, the screening and diagnosis of the undetectable negative rheumatoid arthritis patients of especially suitable Present clinical CCP2.
Description
Technical field
The present invention relates to field of biomedicine technology, and in particular to anti-citrulling modified peptides IgG antibody and/or IgA antibody
Serologic detection technology, more particularly to citrulling modified peptides antigen, diagnostic tool or preparation method and in CCP feminine gender class wind
Application in wet arthritic's autoantibody detection.
Background technique
Rheumatoid arthritis (RA, Rheumatoid Arthritis) be the most common systemic autoimmune disease it
One, about 0.5~1% crowd of world wide suffers from this disease.RA is systemic autoimmune diseases, and patient is often with joint chronic inflammation
Disease reaction, by disease chronic process, can lead to progressive destruction of joint, bone erosion, deformity etc..Rheumatoid arthritis RA is most heavy
The serology discovery wanted: the autoantibody containing specific recognition citrulling modification albumen in patients serum.About 70% RA patient
Albumen autoantibody (ACPA, Anti-citrullinated protein is modified containing identification citrulling in serum
Antibody), these citrulling modification albumen is referred to as autoantigen.ACPAs antibody test is the RA diagnosis important inspection of goldstandard
Survey index.
Research shows that anti-citrulling modifies the generation of albumen autoantibody with RA patient disease process, in clinical diagnosis side
Face, ACPAs have very high specificity and sensitivity to RA patient, also have higher sensitivity to RA early stage patient.For super
Early stage RA patient, show clinical symptoms before in addition the more than ten years before, can detect citrulling autoantibody exist.Simultaneously
Anti- citrulling autoantibody plays an important role to RA patient's prognosis, and disease state of development can be predicted.75% rheumatoid arthritis is suffered from
Person made a definite diagnosis in 1~2 year, it may occur that irreversible Bones and joints corrode, and ACPAs antibody positive is invaded compared with ACPAs negative antibody RA patient bone
Degree of corrosion is more serious, and ACPAs antibody level is higher, and RA morbidity's time is shorter.
The examination of clinical use commercialization cyclic citrullinated peptide (CCP, cyclic citrullinated peptide) detection at present
For agent box as RA patient's auxiliary diagnosis detection method, CCP detection kit can detecte anti-cyclic citrulline modification in patients serum
Peptide autoantibody.First generation citrulling detection kit CCP1, antigen come from profilaggrin (Profilaggrin), use
Elisa detection method, coating profilaggrin citrulling modified peptides detect citrulling in patients serum and modify egg as antigen
White autoantibody.Based on CCP1 detection kit, CCP2 detection kit is developed, CCP1 citrulling modified peptides are changed
It makes, two serines in polypeptide sequence is replaced with into cysteine, synthesize cyclic citrulline modified peptides, that is, CCP (cyclic
Citrullinated peptide), the stability of antigen is not only increased, while also improving sensitivity and the spy of CCP detection
It is anisotropic.CCP2 is commercialized detection kit and listed in 2002, and is widely used in clinical detection rapidly, is currently still RA
The clinical the most frequently used goldstandard of detection.Current at least six kinds of common agents boxes, respectively from Axis-Shield (US), Euro-
Diagnostica(The Netherlands)、Euroimmun(Germany)、Inova(US)、Phadia(Sweden/
Germany),Abbott(US).CCP2 detection kit is based on same CCP2 Antigenic Peptide, has slightly difference in context of detection,
Detection sensitivity is 66.7%~77.7%, and specificity is 86.1%~98.8%.
CCP antibody shows preferable potential applicability in clinical practice as RA medical diagnosis on disease and tick mark object.It is examined with CCP2
There is the patient-specific citrulling modification protein antibodies of many RA, such as Anti-citrullinated in the research for surveying target spot
Fibrinogen antibodies, Anti-citrullinated fibronection, Anti-citrullinated
Vimentin/Anti-Sa antibody, Anti-mutated and citrullinated vimentin (MCV) etc., but this
A little markers are not yet widely used in clinical detection.With the development of molecular diagnostic techniques, RA molecular diagnosis further updates, closely
There is within several years CCP third generation detection reagent CCP3.1 (Inova, US) and obtain extensive concern, CCP3.1 antibody can detect patient's blood
Clear IgG antibody and IgA are horizontal, and CCP3 peptide sequence is undisclosed, constrain CCP3 laboratory research, and some researches show that answer in RA diagnosis
It is lower than CCP2 with aspect CCP3.1 specificity.Anti-CarP (Anti-Carbamylated protein) antibody is to reflect in recent years
Novel RA specific detection antibody is made, can recognize modified types after different protein translations, 43% RA compared with CCP antibody
There are anti-carbamylation antibody by patients serum.
CCP2 detection kit has very high specificity and sensitivity to RA patient, however silk polymeric protein is in joint
It does not express, thus CCP2 cannot function as joint specific antigen.It is generally acknowledged that expressing in RA disease process in joint
Certain antigen causes antibody response, and CCP2 simulates its epitope, thus can be identified by autoantibody, clinical detection
Sensitivity is up to 70%.
All the time, the emphasis of people's research is mostly CCP antibody positive RA patient, and patent CN1602426A discloses one
The method that kind detects autoantibody from the patient with rheumatoid arthritis, including the autoantibody and includes XG motif
Peptide unit, and comprising XnonG motif peptide unit contact.Patent CN101918432A discloses a kind of selected synthetic peptide mould
The three dimensional matrix of quasi-ordering column, what the synthetic peptide simulated series were preferentially detected from the patient with rheumatic arthritis
Autoimmune antibody is identified that the synthetic peptide simulated series can be improved to presymptomatic patient, show rheumatic
The patient of arthritic symptom and these autoantibodies being diagnosed as in rheumatic arthritis positive patient are detected special
Property and sensibility.Patent CN101957365A discloses a kind of kit for detecting CCP and IgG bispecific antibody, the reagent
Box, which can detecte, is present in the natural bispecific antibody of one of rheumatoid arthritis patients serum, easy to use, simple easy
Row.Patent CN102323402A discloses a kind of kit and preparation method thereof of CCP antibody in vitro detection, which can answer
Auxiliary diagnosis for rheumatoid arthritis.Patent CN102796173A discloses a kind of epitope of rheumatoid arthritis
And its application, the epitope only in conjunction with the IgG in patients serum, without reacting with Healthy Human Serum, can be used for preparing and examine
The drug of disconnected rheumatoid arthritis.
But clinic still has about 30% rheumatoid arthritis patients there has been no clear Testing index at present, and this kind of patient is known as
Negative (CCP-) rheumatoid arthritis patients of anti-citrulling.Citrulling detection reagent generally targets single protein molecular, to RA
Subgroups have good specificity.It is a series of research shows that positive (CCP+) patient of anti-citrulling and anti-citrulling are negative (CCP-)
RA patient has dramatically different clinical manifestation, for the missing of CCP- rheumatoid arthritis patients clinical diagnosis information, part
It is limited to current detection method system to fail to detect the specific indicators that CCP- patient carries, CCP- patient may be containing spy
RA patient's hypotype of anisotropic citrulling marker.The Multiple detection for merging multiple target spots is significantly to attempt, and can be segmented
RA patient's autoantibody type, to preferably provide foundation for clinical diagnosis and treatment.
Patent CN106950365A disclose a kind of ACPA feminine gender RA diagnosis marker and its application, specially deoxidation it is auxiliary
Albumen dioxygenase, that is, DOHH or its segment are preparing the rheumatoid arthritis for diagnosing anti-citrulline polypeptide negative antibody
Purposes in the reagent of disease.Patent CN106950366A discloses RA diagnosis marker and its application of a kind of ACPA feminine gender,
Specially Pentaxin GAP-associated protein GAP 3 is that PTX3 or its segment are preparing the class wind for diagnosing anti-citrulline polypeptide negative antibody
Purposes in the reagent of wet arthritis disease.But the specificity of these negative rheumatoid arthritis is only 90%.
Summary of the invention
To solve the problems, such as that the RA patient of clinic 30% lacks clear marker, the present inventor identifies to obtain citrulling modification
Peptide, and applying it in the detection of CCP feminine gender rheumatoid arthritis patients, while providing a kind of sensitivity, specificity good melon
Propylhomoserin antibody assay kit.
The first aspect of the present invention is related to a kind of citrulling modified peptides antigen, the sequence of the citrulling modified peptides antigen
Selected from one of following amino acid sequences:
1) all or part of the amino acid sequence of citrulling modified peptides antigen sequence as shown in SEQ ID NO:1；
2) amino acid sequence of the citrulling modified peptides antigen be with SEQ ID NO:1 have at least 90%, 91%,
92%, the sequence of 93%, 94%, 95%, 96%, 97%, 98% or 99% identity degree；
3) difference of sequence shown in the amino acid sequence of the citrulling modified peptides antigen and SEQ ID NO:1 be no more than 5,
4,3,2 or 1 amino acid；
4) amino acid sequence of the citrulling modified peptides antigen be SEQ ID NO:1 variant, wherein the variant with
The difference of SEQ ID NO:1 include replace, missing and/or be inserted into one or more amino acid residues sequence or at least one
The end N-/C- extends.
Preferably, the amino acid sequence of the citrulling modified peptides antigen be with SEQ ID NO:1 have at least 90%,
91%, the sequence of 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity degree, and the citrulling
7th to the 9th amino acid of the amino acid sequence of modified peptides antigen is L- (Cit)-G；
Preferably, the difference of sequence shown in the amino acid sequence of the citrulling modified peptides antigen and SEQ ID NO:1 is not
More than 5,4,3,2 or 1 amino acid, and the 7th to the 9th ammonia of the amino acid sequence of the citrulling modified peptides antigen
Base acid is L- (Cit)-G；
Preferably, the amino acid sequence of the citrulling modified peptides antigen is the variant of SEQ ID NO:1, wherein the change
The difference of body and SEQ ID NO:1 include replacing, lack and/or being inserted into the sequence or at least one of one or more amino acid residues
A end N-/C- extends, and the 7th to the 9th amino acid of the amino acid sequence of the citrulling modified peptides antigen is
L-(Cit)-G。
It is further preferred that the amino acid sequence of the citrulling modified peptides antigen sequence is SEQ ID NO:1:
NLNGGLL-(Cit)-GIEILNQ
Preferably, the citrulling modified peptides antigen sequence is prepared by chemical synthesis process.
Preferably, the sequence of the citrulling modified peptides antigen can combine rheumatoid arthritis patients autoantibody.Into
One step is preferred, and the autoantibody is selected from IgG or IgA.
Preferably, a kind of citrulling modified peptides detecting CCP feminine gender arthritic's autoantibody markers, including melon ammonia
The all or part of sequence shown in sour modified peptides antigen SEQ ID NO:1 or with SEQ ID NO:1 have at least 90%, 91%,
92%, the sequence of 93%, 94%, 95%, 96%, 97%, 98% or 99% identity degree or with shown in SEQ ID NO:1
The difference of sequence is no more than 5,4,3,2 or 1 amino acid or the variant of SEQ ID NO:1, wherein the variant and SEQ ID
The difference of NO:1 includes replacing, lack and/or being inserted into sequence or at least one end N-/C- of one or more amino acid residues
Extend.
The second aspect of the present invention is related to a kind of nucleotides sequence for encoding citrulling modified peptides antigen described in first aspect
Column.
The third aspect of the present invention is related to the genophore comprising nucleotide sequence described in second aspect.
The fourth aspect of the present invention is related to the cell comprising genophore described in the third aspect.
The fifth aspect of the present invention, is related to a kind of composition, and the composition includes that citrulling described in first aspect is repaired
Adorn peptide antigen and carrier.
Preferably, the carrier is solid phase carrier.
Solid phase carrier of the present invention be selected from one or both of ELISA Plate, 96 hole microwell plates, colloidal gold, microballoon with
On combination.Preferably, it is poly- to be selected from silicon dioxide microsphere, polystyrene microsphere, magnetic microsphere or large biological molecule for the microballoon
Close the combination of one or more of object microballoon.Most preferably, the microballoon is polystyrene microsphere.
In a specific embodiment of the invention, the carrier is the polystyrene microsphere for being combined with fluorescent dye.
Preferably, the amount of the citrulling modified peptides antigen coat to solid phase carrier is 1-25 μ g/1, and 000,000 micro-
Ball.It is further preferred that the amount of the citrulling modified peptides antigen coat to solid phase carrier is 5 μ g/1,000,000 microballoon.
Preferably, the preparation method of the composition includes:
1) support-activated: carrier being added in activation buffer, and sequentially adds Sulfo-NHS solution, EDC (1-
Ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride) solution；
2) it citrulling modified peptides antigen coat or is coupled at carrier surface: being added into the carrier solution after step 1) activation
Citrulling modified peptides antigen is coated with or is coupled.
Preferably, the composition can be used as is examined based on liquid-phase chip CCP feminine gender rheumathritis patient's autoantibody
Test agent box.
Preferably, Sulfo-NHS solution in the step 1), EDC solution working concentration be 50mg/mL.
It is further preferred that the preparation method of the composition includes:
A) microballoon pre-processes: after microballoon ultrasound, abandoning supernatant；Microballoon is resuspended in deionized water, abandons supernatant；
B) microballoon activates: microballoon is resuspended with activation buffer, sequentially adds Sulfo-NHS solution, EDC solution, concussion is mixed
Even, room temperature is protected from light incubation 5-30 minutes, abandons supernatant；
C) citrulling modified peptides antigen and microballoon are coupled: microballoon is resuspended with coupling buffer, abandons supernatant；It is even that 500 μ L are added
Joining buffer, and citrulling modified peptides antigen is added and carries out coupling reaction to microballoon is resuspended, room temperature is protected from light incubation 0.5-3 hours,
Supernatant is abandoned, microballoon is cleaned with cleaning buffer solution and coupling microballoon is resuspended afterwards twice to get composition.
Still more preferably, it is 20 minutes that room temperature, which is protected from light incubation time, in the step b).
Still more preferably, it is 2 hours that room temperature, which is protected from light incubation time, in the step c)；
The sixth aspect of the present invention is related to citrulling modified peptides antigen or the present invention described in a kind of first aspect present invention
Composition described in 5th aspect is in the diagnostic tool that preparation detects anti-citrulling modified peptides IgG antibody and/or IgA antibody
Purposes, the diagnostic tool include at least combination described in citrulling modified peptides antigen described in first aspect or the 5th aspect
Object.
Preferably, combination described in citrulling modified peptides antigen or fifth aspect present invention described in first aspect present invention
Purposes of the object in the diagnostic tool for preparing rheumatoid arthritis, wherein the rheumatoid arthritis is that negative rheumatoid is closed
Section is scorching.
It preferably, further include preservative in the diagnostic tool for the ease of saving.
The seventh aspect of the present invention is related to a kind of diagnosis for detecting anti-citrulling modified peptides IgG antibody and/or IgA antibody
Tool, the diagnostic tool include at least combination described in citrulling modified peptides antigen described in first aspect or the 5th aspect
Object, the diagnostic tool in kit, diagnostic reagent, genetic chip, protein chip or immunity test strip one
Kind.
The eighth aspect of the present invention is related to a kind of reagent for detecting anti-citrulling modified peptides IgG antibody and/or IgA antibody
Box, the kit include described in citrulling modified peptides antigen or fifth aspect present invention described in first aspect present invention
Composition.
Preferably, the kit further includes the secondary antibody, Block buffer, cleaning buffer solution for being marked with marker.
Kit of the present invention can be diagnostic purpose, or non-diagnostic purpose.
Preferably, the method for the anti-citrulling modified peptides IgG antibody of detection and/or IgA antibody is indirectly legal
Detection.
Preferably, described to be detected as indirect method qualitative detection.
Preferably, the use step of the kit includes:
(1) composition described in fifth aspect present invention is prepared；Preferably, citrulling modified peptides antigen in the composition
IgG antibody and/or IgA antibody can be combined.It is further preferred that the IgG antibody and/or IgA antibody are autoantibody.
(2) sample to be detected is contacted with the composition that step (1) prepares；
(3) secondary antibody for being marked with marker is added, forms citrulling modified peptides antigen-IgG antibody and/or IgA antibody-mark
Remember secondary antibody compound；
(4) compound that washing step (3) obtains, and detected for marker.
Preferably, sample to be detected described in step (2) is selected from serum, blood plasma or antibody.It is further preferred that described
Sample to be detected is serum.
It preferably, further include Sample Dilution to be detected before sample to be detected is contacted with composition in the step (2).Into one
Step is preferred, and the Sample Dilution multiple to be detected is 1:20-1:200.Still more preferably, the detection Sample Dilution times
Number is 1:50.
Preferably, the secondary antibody having is marked to be selected from the egg for being marked with enzyme or fluorescein described in the step (3)
White or antibody and its conjugate.Wherein, the enzyme is alkaline phosphatase or peroxidase or their combination.Described is glimmering
Light element is selected from one of anthocyanidin Cy line fluorescent element, Alexa Fluor line fluorescent element or fluorescein isothiocynate or two
Kind or more combination.The albumen or antibody and its conjugate are selected from Avidin, Streptavidin, DigiTAb, histidine
The combination of one or more of antibody, affinity molecule containing Ni or fluorescent molecule antibody.
It is further preferred that described mark the secondary antibody having anti-for the anti-human igg of Alexa555 label or anti-human IgA
Body.
Preferably, the addition concentration for the secondary antibody that the label has is 1-10 μ g/mL.It is further preferred that institute
The addition concentration for the secondary antibody that the label stated has is 4 μ g/mL.
Preferably, signal detection is detected as described in the step (4).It is further preferred that the signal detection choosing
One of the detection of autofluorescence method, development process detection, Electrochemical Detection, mechanics detection, DNA encoding hybridization or sequencing detection or two
Kind or more combination.
In the specific embodiment of the present invention, the signal detection is Fluorometric assay；The signal inspection
It surveys to carry out fluorescence detection in wavelength 532nm and 635nm.
In the specific embodiment of the present invention, anti-citrulling modified peptides IgG antibody in the serum and/or
IgA antibody detection method includes: to take 96 hole microwell plates, and 100 μ L Block buffers are added, and 37 DEG C are closed 1 hour；Citrulling is added
Modified peptides antigen is coupled microballoon and detection sample, 4 DEG C of overnight incubations；Cleaning buffer solution washs three times；Fluorescent marker secondary antibody is added；
Cleaning buffer solution washs three times, and microballoon is resuspended in detection buffer, is detected using instrument.
Citrulling modified peptides antigen of the present invention is for detecting IgG antibody and/or IgA antibody marker, for inspection
It is high (98%) to survey rheumatoid arthritis patients specificity, the diagnosis of especially suitable CCP feminine gender rheumatoid arthritis, and realize primary
The index of reaction detection three kinds or more provides foundation for clinical diagnosis and treatment.Diagnostic tool provided by the invention has simultaneously
Wide, safe and reliable, the easy-operating advantage of the range of linearity.
"and/or" of the present invention includes selecting the project and any amount of projects combo that one lists.
" comprising " of the present invention is open description, containing described specified ingredient or step, and will not
Other the specified ingredients or step substantially influenced.
When " diagnosis " of the present invention refers to find out that patient goes over, diagnoses or whether future suffers from disease or illness,
Either find out the progress or possible progress, or the reaction of assessment patient for treatment in the future of disease.
Detailed description of the invention
Hereinafter, carrying out the embodiment that the present invention will be described in detail in conjunction with attached drawing, in which:
Fig. 1: rheumatoid arthritis autoantibody detection kit detection schematic diagram；
Fig. 2: SEQ ID NO:1 peptide antigen synthesizes Mass Spectrometer Method result；
Fig. 3: rheumatoid arthritis detection kit screening Serum experiments group inspection of the application containing SEQ ID NO:1 peptide antigen
Survey result, wherein HC: healthy group, α-CCP-_RA: citrulling negative antibody rheumatoid arthritis patients group, MFI (Median
Fluorescent intensity): fluorescence signal value median；
Fig. 4: rheumatoid arthritis detection kit screening serum validation group inspection of the application containing SEQ ID NO:1 peptide antigen
Survey result, wherein HC: healthy group, α-CCP-_RA: citrulling negative antibody rheumatoid arthritis patients group, MFI (Median
Fluorescent intensity): fluorescence signal value median；
Fig. 5: rheumatoid arthritis detection kit screening Serum experiments group of the application containing SEQ ID NO:1 peptide antigen
ROC curve；
Fig. 6: rheumatoid arthritis detection kit screening serum validation group of the application containing SEQ ID NO:1 peptide antigen
ROC curve.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiment is only section Example of the invention, rather than all.Based in the present invention
Embodiment, every other embodiment obtained by those of ordinary skill in the art without making creative efforts, all
Belong to the scope of protection of the invention.
The synthesis of 1 citrulling modified peptides of embodiment
1, the sequence of citrulling modified peptides is as follows:
NLNGGLL- (Cit)-GIEILNQ (SEQ ID NO:1)
2, synthesis sequence: from sequence C end to N-terminal, steps are as follows:
1) it weighs 0.3g resin to be put into glass reactor, DCM (methylene chloride) is added and is swollen 30 minutes；
2) DCM is taken out, first amino acid 0.6g in amino acid sequence is added, 0.6g DIEA (diisopropyl second is added
Amine), DMF (dimethylformamide), DCM, nitrogen blistering reaction 60 minutes, take out reaction solution, DMF, MEOH washing be added three times；
3) second amino acid 0.6g in amino acid sequence is added in reactor, HBTH (1- hydroxyl, benzo, trichlorine is added
Azoles tetramethyl hexafluorophosphate) and DIEA, nitrogen blistering reaction 30 minutes, wash off liquid, ninhydrin detection, then with pyridine and
Acetic anhydride sealing end.It finally cleans, liquid removal Fmoc (9-fluorenylmethyloxycarbonyl) protecting group of raising one's hat in right amount is added, cleans, ninhydrin inspection
It surveys；
4) according to step 3), the amino acid in sequence is sequentially added；
5) it after resin is dried with nitrogen, is removed in reaction column.Move to 10mL centrifuge tube, be added 6mL cutting liquid (95%TFA,
2% dithioglycol, 2% tri isopropyl silane, 1% water), concussion filters resin；
6) a large amount of ether are added in filtrate, crude product is precipitated, is then centrifuged for, up to crude product after cleaning；
7) crude product is purified to 90% by peptide purification, high performance liquid chromatography；
8) polypeptide is lyophilized, and liquid is put into freeze dryer and is concentrated after purification, is lyophilized into white powder.
3, peptide synthesis Mass Spectrometer Method result
The SEQ ID NO:1 peptide antigen mass spectral results of synthesis are as shown in Figure 2.Sequent synthesis is correct, and its molecular weight is
1979.38Dalton。
Embodiment 2 is based on microballoon detection technique citrulling modified peptides in the detection of CCP feminine gender rheumatoid arthritis patients
Using
1, experimental material and reagent
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-dimethylaminopropyl] 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 buffer PBS:2mM KH2PO4, 10mM Na2HPO4, 137mM NaCl, 2.7mM KCl, pH7.4
(Sigma-Aldrich,St.Louis,MO,USA)；
6) detection/cleaning buffer solution: 0.05% (v/v) Tween 20 is dissolved in 0.1MPBS buffer；
7) Block buffer: PBS-TBN, 1%BSA are dissolved in 0.1MPBS buffer；
8) antibody: anti-human IgG or anti-human IgA antibody (4 μ of Alexa555 label is detected
g/mL)(Jackson ImmunoResearch,PA,USA)。
2, experimental method:
(1) preparation of kit
Kit includes composition and preservative.
The preparation of composition (coupling microballoon): 1,000,000 three kinds of microballoon is taken to be placed in 1.5mL centrifuge tube, microballoon ultrasound
Afterwards, centrifuge tube is placed in magnetic frame 1 minute, abandons supernatant；Microballoon is resuspended in 100 μ L deionized waters, abandons supernatant；80 μ L activation buffering is added
Microballoon is resuspended in liquid, sequentially adds 10 μ L Sulfo-NHS solution, EDC solution, and concussion mixes, and room temperature is protected from light incubation 20 minutes；It incubates
After educating, supernatant is abandoned；250 μ L coupling buffers are added, microballoon is resuspended, abandons supernatant；500 μ L coupling buffers are added, and 5 μ g are added
Citrulling modified peptides antigen carries out coupling reaction to microballoon is resuspended, and room temperature is protected from light incubation and is incubated for 2 hours；Supernatant is abandoned, it is slow with cleaning
Fliud flushing washs microballoon twice, and coupling microballoon is resuspended in Block buffer；Incubation at room temperature 30 minutes；Supernatant is abandoned, is washed with cleaning buffer solution
Wash microballoon three times, the coupling microballoon of acquisition is composition.Flow cytomery couples microballoon quantity, and detection buffer will be micro-
Ball concentration is adjusted to 2000/100 μ L, stores for future use.
(2) detecting step
96 hole microwell plates are taken, 100 μ L Block buffers are added, 37 DEG C are closed 1 hour；150 μ L cleaning buffer solutions washing 1
It is secondary；Every hole is added 100 μ L and couples microspheres solution, and 2 μ L serum are added, 4 DEG C of overnight incubations；Cleaning buffer solution washs three times；Add
Enter anti-human IgG or anti-human the IgA antibody of Alexa555 label, forms citrulling modified peptides antigen-
Autoantibody-label secondary antibody compound；Cleaning buffer solution washs three times, and microballoon is resuspended in detection buffer, is detected using instrument,
Middle 532nm reads fluorescent marker secondary antibody value detection signal, and 635nm reads microballoon ID number.
3, Data Processing in Experiment
It reads value detection signal MFI (Median fluorescent intensity), and maps.
4, sample information
110, Healthy People sample are collected altogether, and CCP feminine gender rheumatoid arthritis patients 114, data are analyzed the blood of screening
Final proof is originally randomly divided into experimental group (Training) and validation group (Validation), and experimental group Healthy People 52, CCP feminine gender class
Rheumathritis patient 53, validation group Healthy People 58, CCP feminine gender rheumatoid arthritis patients 61.Clinical sample information
As shown in Table 1 and Table 2, wherein HC: healthy group, α-CCP-_RA: citrulling negative antibody rheumatoid arthritis patients.Rheumatoid
Arthritis autoantibody detection kit detection schematic diagram is shown in Fig. 1.
1 clinical sample information of table
2 clinical sample information of table
5, testing result
The testing result of kit screening Serum experiments group and validation group is shown in Fig. 3 and Fig. 4, wherein IgA:IgA autoantibody
Detection；The detection of IgG:IgG autoantibody.Kit screening Serum experiments group and the testing result of validation group result ROC curve are shown in
Fig. 5 and Fig. 6, wherein experimental group sensitivity and specificity are respectively 11.32%, 98%, validation group sensitivity and specificity difference
It is 24.59%, 98%.
The preferred embodiment of the present invention has been described above in detail, still, during present invention is not limited to the embodiments described above
Detail within the scope of the technical concept of the present invention can be with various simple variants of the technical solution of the present invention are made, this
A little simple variants all belong to the scope of protection of the present invention.
It is further to note that specific technical features described in the above specific embodiments, in not lance
In the case where shield, can be combined in any appropriate way, in order to avoid unnecessary repetition, the present invention to it is various can
No further explanation will be given for the combination of energy.
Claims (10)
1. a kind of citrulling modified peptides antigen, which is characterized in that the sequence of the citrulling modified peptides antigen is selected from following amino
One of acid sequence:
1) all or part of the amino acid sequence of citrulling modified peptides antigen sequence as shown in SEQ ID NO:1；
2) amino acid sequence of the citrulling modified peptides antigen be with SEQ ID NO:1 have at least 90%, 91%, 92%,
93%, the sequence of 94%, 95%, 96%, 97%, 98% or 99% identity degree；
3) difference of sequence shown in the amino acid sequence of the citrulling modified peptides antigen and SEQ ID NO:1 be no more than 5,4,3,
2 or 1 amino acid；
4) amino acid sequence of the citrulling modified peptides antigen is the variant of SEQ ID NO:1, wherein the variant and SEQ
The difference of ID NO:1 includes replacing, lack and/or being inserted into the sequence or at least one N-/C- of one or more amino acid residues
End extends.
2. a kind of nucleotide sequence of citrulling modified peptides antigen described in coding claim 1.
3. a kind of genophore comprising nucleotide sequence as claimed in claim 2.
4. a kind of cell comprising genophore as claimed in claim 3.
5. a kind of composition, which is characterized in that the composition includes citrulling modified peptides antigen described in claim 1 and load
Body.
6. composition according to claim 5, which is characterized in that the carrier is solid phase carrier, and the solid phase carries
Body is selected from the combination of one or more of ELISA Plate, 96 hole microwell plates, colloidal gold, microballoon；Wherein, the microballoon is selected from
One or more of silicon dioxide microsphere, polystyrene microsphere, magnetic microsphere or large biological molecule polymer microballoon
Combination.
7. a kind of preparation method of composition described in claim 5 or 6, which is characterized in that the preparation side of the composition
Method includes:
1) support-activated: carrier being added in activation buffer, and sequentially adds Sulfo-NHS solution, EDC solution；
2) it citrulling modified peptides antigen coat or is coupled at carrier surface: melon ammonia is added into the carrier solution after step 1) activation
Sour modified peptides antigen is coated with or is coupled.
8. prepared by a kind of citrulling modified peptides antigen described in claim 1 or any composition of claim 5-6
Detect the purposes in the diagnostic tool of anti-citrulling modified peptides IgG antibody and/or IgA antibody；Preferably, the citrulling is repaired
Adorn the purposes of peptide antigen or composition in the diagnostic tool for preparing negative rheumatoid arthritis.
9. a kind of diagnostic tool for detecting anti-citrulling modified peptides IgG antibody and/or IgA antibody, which is characterized in that described examines
Disconnected tool includes citrulling modified peptides antigen described in claim 1 or composition described in claim 5 or 6, wherein described
Diagnostic tool be selected from one of kit, diagnostic reagent, genetic chip, protein chip or immunity test strip.
10. diagnostic tool according to claim 9, which is characterized in that the diagnostic tool is kit, the examination
Agent box further includes the secondary antibody, Block buffer and/or cleaning buffer solution for being marked with marker.
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| CN116162134A (en) * | 2022-12-29 | 2023-05-26 | 旦生(北京)医学科技有限责任公司 | Polypeptide or polypeptide composition |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116162134A (en) * | 2022-12-29 | 2023-05-26 | 旦生(北京)医学科技有限责任公司 | Polypeptide or polypeptide composition |
| CN116162134B (en) * | 2022-12-29 | 2023-12-01 | 旦生(北京)医学科技有限责任公司 | Polypeptide or polypeptide composition |
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