CN108267576A - Modified CCP antigens and application thereof and antiCCP antibody detection kit and its manufacturing method - Google Patents
Modified CCP antigens and application thereof and antiCCP antibody detection kit and its manufacturing method Download PDFInfo
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- CN108267576A CN108267576A CN201710006139.0A CN201710006139A CN108267576A CN 108267576 A CN108267576 A CN 108267576A CN 201710006139 A CN201710006139 A CN 201710006139A CN 108267576 A CN108267576 A CN 108267576A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—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
Abstract
The invention belongs to vitro diagnostic techniques fields, and in particular to a kind of modified CCP antigens, the CCP antigens have the sequence shown in following formula I：Wherein, the carboxylic hydroxyl of the glutamic acid E in Formulas I is replaced by R group, and the R group is selected from：C1‑10Alkyl oxy, C1‑10Alkyl CO NH bases and C1‑10Alkyl CO NH NH bases；The C1‑10Alkyl is optionally selected from C by one or more1‑4Alkyl, C1‑4The substituent group substitution of alkyl oxy, hydroxyl, amino, nitro and phenyl.It is used to detect the purposes of antiCCP antibody, antiCCP antibody detection kit and its manufacturing method including the antigen the invention further relates to the antigen.The advantage of the present invention includes the antigen active higher of the derivative after modified CCP antigens connection protein carrier；The precision higher of antiCCP antibody detection；The accuracy higher of antiCCP antibody detection.
Description
Technical field
The invention belongs to vitro diagnostic techniques fields, and in particular to a kind of modified CCP antigens, the antigen are used to detect
The purposes of antiCCP antibody, antiCCP antibody detection kit and its manufacturing method including the antigen.
Background technology
Rheumatoid arthritis (Rheumatoid Arthritis, RA) is a kind of polyphyly involved based on periarticular
System, inflammatory, autoimmune, teratogenesis disease.The disease American-European countries incidence for 1%, China for 0.32%~
0.36%.The disease may occur in which that irreversible Bones and joints destroy in onset 2 years, be the main original for causing crowd's disability
One of because.
The researchs such as Guo great Wen find that cyclic citrullinated peptid has predictive value (Guo to the morbidity of rheumatoid arthritis
Big text, Wang Ye, Gao Honghua wait predictive value [J] China that cyclic citrullinated peptids fall ill to rheumatoid arthritis real
Test diagnostics, 2005,9 (1)：13-14).
Anti- cyclic citrullinated peptide (Cyclic Citrullinated Peptide, CCP) antibody is anti-based on IgG types
Body.AntiCCP antibody is the bone-marrow-derived lymphocyte Autocrine by RA patient, and other diseases patient and normal population bone-marrow-derived lymphocyte
Not Autocrine antiCCP antibody.Therefore, antiCCP antibody has higher specificity to RA.The detection of antiCCP antibody is existing more
Using solid-phase synthesis come the artificial synthesized peptide fragment containing citrulling.The initially use linear citrulling peptide of straight chain for containing 19 amino acid
Section, amino acid sequence are：SHQESTXGRSRGRSGRSGS.However research discovery is substrate with the linear citrulling peptide fragment of straight chain,
It can often be led to the failure with ELISA method to detect cyclic citrullinated peptid, the reason is that since this peptide fragment cannot be adsorbed in polyphenyl second
On alkene.Later, in order to improve the antigen active of citrulling peptide chain, overcome the shortcomings of the linear citrulling peptide fragment of straight chain, Dutch scholar
Schellekens GA (Schellekens GA, Visser H, de Jong BA, van den Hoogen FH, Hazes JM,
Breedveld FC et al.The diagnostic properties of rheumatoid arthritis
antibodies recognizing a cyclic citrullinated peptide.Arthritis Rheum 2000；
43：Two serines in an above-mentioned citrulling peptide chain being made of 19 amino acid residues 155-163) are substituted for half Guang
Propylhomoserin, and cysteine is made to be cyclized to form the similar disulfide bond of β-corner structure, and as cyclic citrullinated peptide.
Specificity of the diagnosis of RA with height can be used in the detection of Anti-CCP antibody (specificity is more than 90%)
In the early diagnosis of RA.About 70% RA patient may occur in which Anti-CCP antibody, and antibody in morbidity early stage, serum
Positive patient easily develops into the Bone mineral density that can be detected by radiometric method than the patient of negative antibody.Tiercy
JM (Rheumatology, 2002,41 (7)：809) research Anti-CCP antibody, AKA and IgM-RF are in difference RA and other rheumatism
It is found during meaning in property disease：The sensibility that IgM-RF diagnoses RA is high (75%), secondly Anti-CCP antibody (68%)；
And the specific highest (96%) that Anti-CCP antibody diagnoses RA, secondly it is AKA (94%)；And the spy that IgM-RF diagnoses RA
The opposite sex only 74%.So when diagnosing RA, Anti-CCP antibody has higher specificity than IgM-RF.
Current Anti-CCP diagnostic techniques mainly includes enzyme linked immune assay (ELISA), chemiluminescence (CLIA), is immunized
Chromatography (colloidal gold or latex particle method), time-resolved fluoroimmunoassay (TRFIA), immunodot test, these sides
Method has respective advantage and shortcoming.
At present, the detection of antiCCP antibody is mainly studied and applied chemistry luminescent immunoassay.CLIA is detecting anti-CCP
In antibody, detection sensitivity is high, and preferably, detection range is wide, and stable reagent nonhazardous is pollution-free for specificity and repeatability, operation letter
Single, detection process takes short and easy to automate and single sample detection, is the antiCCP antibody of current mainstream
Detection method.
In the scheme of existing chemiluminescence immunoassay detection antiCCP antibody, generally carried using CCP antigens and albumen
Body connects the derivative to be formed coating solid phase carrier, and uses luminescent label anti-human igg secondary antibody.In detection serum sample
When, one side Anti-CCP antibody is combined by CCP antigens with solid phase carrier, another aspect Anti-CCP antibody and anti-human igg
Two anti-bindings finally using solid phase carrier isolation technics, achieve the purpose that accurately to detect Anti-CCP antibody in serum sample.
CCP antigens are small molecule ring type polypeptides, and sequence is：HQCHQEST-cit-GRSRGRCGRSGS (can with three characters
It is expressed as NH2-His-Gln-Cys-His-Gln-Glu-Ser-Thr-Cit-Gly-Arg-Ser-Arg- Gly-Arg-Cys-
Gly-Arg-Ser-Gly-Ser-COOH), disulfide bond：C3=C16.Citrulling (Citrulline, Cit) is wherein important exempts from
Epidemic disease antigenic sites.CCP antigens are coupled by carboxyl and protein carrier.
The prior art still connects CCP antigens the antigen active of the derivative after protein carrier there are requirements at the higher level, and right
There are requirements at the higher level for the preci-sion and accuracy of antiCCP antibody detection.
Invention content
In order to overcome one or more technical problems in the prior art, inventor has carried out lasting research.Pass through
Constantly experiment has been surprisingly found that a kind of modified CCP antigens can realize the technical purpose of the present invention.
The CCP antigens have the sequence shown in following formula I：
The carboxylic hydroxyl (- OH) of glutamic acid E (i.e. the 6th amino acid) wherein in Formulas I is replaced by R group, obtains-COR bases
Group, the R group are selected from one or more of：C1-10Alkyl oxy, C1-10Alkyl-CO-NH- bases and C1-10Alkyl-CO-NH-
NH- bases；The C1-10Alkyl represents the alkyl containing 1-10 carbon atom, such as methyl, ethyl, isopropyl, tertiary butyl, new penta
Base, n-nonyl etc.；Oxygroup expression-the O-；- the CO- represents ketone group；
The C1-10Alkyl is optionally replaced by one or more conventional substituent groups.The substituent group may be selected from C1-4Alkyl (ratio
Such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl group), C1-4Alkyl oxy (i.e. C1-4Alkyl oxy), hydroxyl (HO-), ammonia
Base (NH2-), nitro (NO2-) and phenyl (C6H5-).
Preferably, the R group is selected from one or more of：C1-5Alkyl oxy, C1-5Alkyl-CO-NH- bases and C1-5
Alkyl-CO-NH-NH- bases.
Preferably, the R group is selected from one or more of：Methyl oxygroup, ethyl oxygroup, tertiary butyl oxygroup, benzene first
Base oxygroup, benzhydryl oxygroup ((C6H5) 2CHO-), acetamido (CH3CHO-NH-) and acetyl hydrazine (CH3CHO-NH-
NH-)。
In another embodiment, the present invention relates to above-mentioned modified CCP antigens to be used to prepare antiCCP antibody detection
The purposes of reagent.
In another embodiment, the present invention relates to the antiCCP antibodies including above-mentioned modified CCP antigens to detect examination
Agent.
In another embodiment, the present invention relates to the drugs for including above-mentioned modified CCP antigens.
In another embodiment, the present invention relates to a kind of antiCCP antibody detection kit, the kit includes group
Divide A and component B, wherein the component A includes magnetic microsphere, the magnetic microsphere is coupled above-mentioned through modification with protein carrier
CCP antigen coats, the component B includes the luminous marker solution that is marked by mouse anti-human igg secondary antibody.
Preferably, the protein carrier is selected from：Bovine serum albumin(BSA) (BSA), cationic bovine serum albumin(BSA) (cBSA), blood
Azurin (KLH), ovalbumin (OVA), seralbumin HSA, ox gamma Globulin and people's gamma Globulin etc. be animal derived or people
Endogenous binding protein.
Preferably, the magnetic microsphere, i.e. magnetic ball can be any one commercially available magnetic ball, including but not limited to：Table
Face with epoxy group, sulfonyl, aldehyde radical, amide groups, amino, carboxyl, sulfydryl, hydroxyl modified magnetic ball.
Preferably, the luminous marker is selected from：Luminol, different luminol and its derivative, horseradish peroxidase
(Horseradish Peroxidase, HRP) and alkaline phosphatase etc..
Preferably, the working concentration of the magnetic microsphere is 1-10mg/mL (such as 1,2,3,4,5,6,7,8,9 or 10mg/
ML) and/or the working concentration of the modified CCP antigens for 50-100ng/mL (such as 50,55,60,65,70,75,80,
85th, 90,96 or 100ng/mL) and/or the protein carrier working concentration for 10-40ng/mL (such as 10,15,20,25,
30th, 35 or 40ng/mL) and/or the mouse anti-human igg secondary antibody working concentration for 100-300ng/mL (such as 100,150,
200th, 250 or 300ng/mL) and/or the luminous marker working concentration for 10-30ng/mL (such as 10,15,20,25 or
30ng/mL)。
Preferably, the kit further include low concentration calibration product solution (such as 2,3,4,5 or 6U/mL, specifically such as
3.91U/mL) and high concentration calibration object solution (such as 50,100,150,200,250,300,350,400,450 or 500U/mL).
Preferably, each component A and B in the kit contains BSA and preservative, and BSA mass volume basis is dense
It spends for 0.1%-0.5%, preservative is potassium sorbate, any in sodium benzoate, Sodium azide, sodium nitrite, Proclin series
Kind or their mixture.
In another embodiment, the present invention relates to a kind of manufacturing method of antiCCP antibody detection kit, the sides
Method includes：
1) makes above-mentioned modified CCP antigens be coupled with protein carrier, it is preferable that the modified CCP antigens and egg
The molar ratio of Bai Zaiti is 20: 1-80: 1, such as 30: 1,40: 1,50: 1,60: 1 or 70: 1；
2) makes the conjugate coating magnetic microsphere that step 1) obtains, and obtains component A, the magnetic microsphere and conjugate
Weight ratio is g-1mg: 40 μ g of 1mg: 10 μ, for example uses 15,20,25,30 or 35 μ g conjugates per 1mg magnetic microspheres；
3) reacts mouse anti-human igg secondary antibody and luminous marker, obtains component B；
4) component A and component B are assembled into kit by.
Preferably, the manufacturing method further includes the above-mentioned low concentration calibration product solution of manufacture and high concentration calibration object solution,
And use that they are placed among kit or is arranged in pairs or groups.
Specifically, one embodiment of the invention is related to a kind of detection kit for being used to detect antiCCP antibody
Preparation method, core preparation process are as follows：
Prepare 1：The modified CCP antigens (hereinafter referred to as novel C CP antigens) of the present invention are formed into idol with protein carrier
Join object
1) novel C CP antigens with crosslinking agent are reacted in the presence of a catalyst, obtains novel C CP antigen-reactive liquid；
Wherein, the crosslinking agent includes but not limited to glutaraldehyde, BS (PEG) 5, BS (PEG) 8, PEG400, PEG800, fourth
Dicarboxylic anhydride etc., dosage are 1eq to 4eq；
The catalyst includes but not limited to triethylamine, pyridine, potassium hydroxide, potassium carbonate, and dosage is 1eq to 4eq；
2) protein carrier is added in into the novel C CP antigen-reactive liquid of step 1) to be reacted, it is purified, it obtains novel
The conjugate that CCP antigens are formed with protein carrier；
Wherein, the molar ratio of novel C CP antigens and protein carrier is 20: 1-80: 1.
Prepare 2：The preparation of the coated magnetic ball suspension of novel C CP antigen conjugates
Magnetic microsphere (the limited public affairs of Shenzhen's NPD projects biomedical engineering share are coated with using 1 conjugate obtained is prepared
Department's production, 80% particle diameter distribution are 1-5 μm, the intensity of magnetization 4000), a concentration of 100mg/mL of magnetic ball, hydroxyl value 95.Specifically
Step is as follows：
1) magnetic ball is placed in be stirred by ultrasonic in acetate buffer liquor and is cleaned, supernatant liquor is removed by Magneto separate, is repeated
This step three times obtains acetate buffer solution suspension magnetic ball；
2) using magnetic ball connection CDC methods (magnetic ball-CDC- antigen/antibodies), CDC is added in into acetate buffer solution suspension magnetic ball
It with novel C CP antigens-protein carrier conjugate, is placed in isothermal vibration water bath and reacts, obtain novel C CP antigen conjugates packets
The magnetic ball reaction solution of quilt；
Wherein the ratio of magnetic ball and novel C CP antigens-protein carrier conjugate is 1mg: 10 μ g to 1mg: 40 μ g；
3) reaction solution that step 2) obtains is removed into supernatant liquor by Magneto separate, and it is clear to add in the stirring of magnetic ball cleaning solution
It washes, repeats this step and clean four times；
Wherein magnetic ball cleaning solution is：The PBS buffer solution of 0.1M, pH7.4 add in the BSA (Roche productions) of 0.5% (w/v),
Mixing；
4) it after cleaning, adds in magnetic ball suspension and suspends, obtain the coated magnetic ball of novel C CP antigen conjugates and hang
Supernatant liquid；
Wherein magnetic ball suspension be 0.1M, the PBS buffer solution of pH7.4.
Prepare 3：The preparation of the luminous marker of mouse anti-human igg secondary antibody label
1) mouse anti-human igg secondary antibody carbonic acid buffer is fitted into the bag filter of suitable interception to be placed in dialyzate and is carried out thoroughly
Analysis, wherein, the dialyzate is carbonic acid buffer；
2) luminous marker is added in into the solution that step 1) has been dialysed to be reacted；Wherein, the temperature of reaction is 25-37
DEG C, reaction time 8-12h；
The luminous marker may be selected from：Luminol and its derivative, different luminol and its derivative, horseradish peroxidase
Enzyme, alkaline phosphatase etc.；
3) reaction solution that step 2) obtains by G-25 gel columns is purified, collects the solution for peak value occur, obtain
The luminous marker solution of mouse anti-human igg secondary antibody label；
4) the luminous marker solution that the mouse anti-human igg secondary antibody for obtaining step 3) marks adds in BSA protection liquid, for use.
One embodiment of the invention is related to a kind of chemiluminescence immunoassay method for being used to detect antiCCP antibody,
Include the following steps：
1) sample to be tested solution, high concentration calibration object solution and low concentration calibration product solution are added separately to differential responses
In cup aperture；
2) respectively to step 1-) in add in the special magnetic microsphere of novel C CP antigen coats for preparing of the present invention, make and treat
Substance is surveyed to combine；
3) luminous marker for adding in mouse anti-human igg secondary antibody label into step 2) respectively is incubated, and resists the mouse
Human IgG secondary antibody is combined with the magnetic microsphere and test substance of novel C CP antigen coats；
Wherein, incubation temperature is 25-37 DEG C, incubative time 10-20min；
The luminous marker may be selected from：Luminol and its derivative, different luminol and its derivative, horseradish peroxidase
Enzyme, alkaline phosphatase etc.；
4) Magneto separate obtains the combination of mouse anti-human igg secondary antibody and the magnetic microsphere and test substance of novel C CP antigen coats
Object；
5) conjugate obtained to step 4) adds in the excitation substrate that shines, and detects light signal strength；
When luminous marker is different luminol, the excitation substrate that shines can be NaOH and H2O2；When luminous marker uses it
During its substance, shine excitation substrate can respective change be other common substances；
6) it by the revised working curve of calibration object, is calculated automatically according to pattern detection luminous intensity, obtains sample to be tested
AntiCCP antibody concentration.
The invention further relates to the kit manufactured by the above-mentioned manufacture method, the application method of kit uses the present invention
The detection method of kit detects the method for antiCCP antibody using the modified CCP antigens of the present invention and implements this method
Equipment and instrument.
Compared to the prior art, advantages of the present invention includes：
The antigen active higher of derivative after the modified CCP antigens connection protein carrier of the present invention；AntiCCP antibody is examined
The precision higher of survey；The accuracy higher of antiCCP antibody detection.
Specific embodiment
With reference to specific embodiment, the invention will be further described.The scope of the present invention is not only restricted to this.
Embodiment is related to following term,
Marker：The term " marker " used in the present invention refers to the substance that can be detected in immune detection.It is described
Marker may be selected from any nano particle class marker, also can refer to include " labeled complex " of the marker.
Labelled antigen：The term " labelled antigen " used in the present invention is what is combined in immune detection with purpose antibody to be measured
Antigen can also be labelled protein.
Ligand：The term " ligand " used in the present invention is can be with the molecule of label specific recognition, such as antibody, parent
With element etc..
Label：The term " label " used in the present invention includes but not limited to more peptide or proteins, biotin or is them
With the composition of peptide or protein, more peptide or proteins mentioned here are more in addition in the kit envelope antigen any one section
Peptide or protein.
Alkoxylate：The reaction of alkoxy is introduced in organic compound molecule.
Following embodiment uses the Maglumi of Shenzhen New Industries Biomedical Engineering Co., Ltd.'s production
2000plus chemical illumination immunity analysis instruments are detected.
The raw material sources being related to are as follows：
Novel C CP antigenic sources：Shenzhen New Industries Biomedical Engineering Co., Ltd. provides；Novel C CP resists
The carboxylic hydroxyl of glutamic acid E is substituted by R bases by original on the basis of traditional CCP antigens (HQCHQEST-cit-GRSRGRCGRSGS)
Group；
ABEI (N- (4- ammonia butyl)-N- ethyls different luminol) source：Shenzhen's NPD projects biomedical engineering share has
Limit company provides；
Mouse anti-human igg secondary antibody source：Sigma companies；
Magnetic microsphere：Shenzhen New Industries Biomedical Engineering Co., Ltd. produces, and 80% particle diameter distribution is 1-5 μ
M, the sedimentation time is 10-15 seconds when the intensity of magnetization is 4000 Gauss, a concentration of 0.8mg-1.2mg of protein adsorption when BSA is 30mg.
Prepare 1：Novel C CP antigens form the preparation of derivative with protein carrier
23.4mg novel C CP polypeptide antigens are dissolved in dimethyl sulfoxide and are configured to 100mg/mL reaction solutions, 21.3mg is added in and hands over
Join agent BS (PEG) 5 (4eq, thermo), 5.6 μ L catalyst of triethylamine (4eq), react 2.5h by 25 DEG C, 250rpm.
In novel C CP antigens and the ratio of ox IgG molar ratios 20: 1,40 μ L reaction solutions is taken to be added to 1mL ox IgG solution
(10mg/mL, 0.1M NaHCO3) in, room temperature, 250rpm reacts 2.5h.G25 gel-purifieds, buffer solution are 0.01M pH 7.4
PBS.
Prepare 2：The preparation of the coated magnetic ball suspension of novel C CP antigenic derivants
Magnetic microsphere (Shenzhen's NPD projects biomedical engineering is coated with using the 1 novel C CP antigenic derivants obtained are prepared
Limited company produces, and 80% particle diameter distribution is 1-5 μm, the intensity of magnetization 4000), a concentration of 100mg/mL, hydroxyl value is
95 nano-magnetic microsphere.It is as follows：
The processing procedure of magnetic ball：
1) configuration of 3.6 acetate buffer solutions of 0.05mol/L pH：
It weighs after 2.55g sodium acetate trihydrates are purified with 4500mL and add 14mL acetic acid mixings after water dissolution, is settled to
5000mL obtains the acetate buffer solution of 0.05mol/L pH 3.6.
2) magnetic ball connection CDC methods (magnetic ball-CDC- antigen/antibodies)
The 0.05mol/L and pH that 5 times of coating volumes are added in little Bai bottles are 3.6 acetate buffer liquor, are put into
Stirring and washing 2-3 minutes on one side while ultrasound in ultrasonic instrument, be subsequently placed on magnet, after supernatant is limpid, pour out
Clear liquid.The step is in triplicate.
3) antigen/antibody sample-adding is with reacting
The pH3.6 acetate buffer solution suspension magnetic ball concentration 20mg/mL of coating volume equivalent are added in, are added a concentration of
The CDC of 10mg/mL adds in novel C CP antigens-ox IgG derivatives of purifying in the ratio of 1mg: 12 μ g, is put into isothermal vibration water
It is reacted 24 hours for 37 DEG C in bath cabinet
4) cleaning of magnetic ball：
1. the configuration of magnetic ball cleaning solution
The BSA (Roche productions) of 0.5% (w/v) of addition in the PBS buffer solution of 0.1M, pH7.4, mixing, for use.
2. the good magnetic ball of warm bath is poured into beaker, it is subsequently placed in after being precipitated on magnet, outwells supernatant, adds in 5 times of bodies
Long-pending magnetic ball cleaning solution stirring and washing, is then placed on magnet, and supernatant is outwelled after supernatant is limpid, repeats cleaning step
Rapid four times.
5) suspension of magnetic ball：
After cleaning, the magnetic ball suspension of coating volume, suspended concentration 20mg/mL are added in.
Prepare 3：The preparation of the ABEI of mouse anti-human igg secondary antibody label
1) configuration of dialyzate (F solution)：
Na is added in 5000mL beakers2CO314.31g NaHCO326.46g adds water to be settled to 4500mL.It prepares
F solution be placed in it is spare on magnetic stirring apparatus.
2) bag filter of suitable interception (common 14000) is selected, measurement is suitably sized, and one end is tightened after wetting, pure
Change water leak test 3 times (need to be without leakage).
3) 1mg mouse anti-human igg secondary antibodies is taken to be adjusted to 1mL with the carbonic acid buffer (F solution) of 0.1mol/L pH9.5.It tightens
The other end is put into dialyzate, is dialysed 2 hours (mixing speed 400).
4) solution dialysed is fitted into little Bai bottles (every bottle of 1mL), adds in 300 μ g ABEI Acibenzolars, and 37 DEG C of reactions 2 are small
When.
5) G-25 gel columns are installed, with purifying water elution it is clean after, then be balanced with the PBS buffer solution of pH value 7.4
Elution.
6) after the elution of gel column equilibration, the ABEI marked is crossed into column purification, then collects the solution for peak value occur.
7) protein solution that will be gathered adds in isometric BSA protection liquid containing 50mg/mL.
Embodiment 1：
The peptide sequence of novel C CP antigens that the present embodiment uses for：HQCHQE (R) ST-cit-GRSRGRCGRSGS, two
Sulfide linkage：C3=C16, wherein R bases are methoxyl group CH3O-。
Kit includes following components：
1) component A：The novel C CP antigenic derivant solution of magnetic ball is coated with, novel C CP antigenic derivants are resisted by novel C CP
Original is coupled with ox IgG protein carriers, wherein：The working concentration of magnetic ball：1mg/mL, the working concentration of novel C CP antigens are
50ng/mL, a concentration of 10ng/mL of ox IgG protein carriers；
2) component B：The ABEI solution of mouse anti-human igg secondary antibody label, wherein, the working concentration of ABEI is 30ng/mL, and mouse resists
The working concentration of human IgG secondary antibody is 300ng/mL.
3) calibration object solution：The low concentration calibration product solution of concentration 3.91U/mL and the high concentration school of concentration 250.0U/mL
Quasi- product solution.
Above-mentioned each component is containing BSA and preservative, and BSA mass concentration expressed in percentage by volume is 0.1%, preservative main component
For Sodium azide (NaN3), quality concentration expressed in percentage by volume is 0.2%.
The preparation method of the kit each component of the present embodiment is：
The novel C CP antigens-ox IgG formed according to above-mentioned preparation 1 preparation novel C CP antigens with ox IgG protein carriers spreads out
Biology；
The magnetic ball suspension for being coated with novel C CP antigens-ox IgG conjugates is prepared according to above-mentioned preparation 2；
The ABEI solution of mouse anti-human igg secondary antibody label is prepared according to above-mentioned preparation 3.
Using antiCCP antibody standard items, using cow's serum as solvent, the standard solution of 10 parts of various concentrations is prepared, is used
Reagent constituents manufactured in the present embodiment are detected 10 parts of solution, and detecting step is as follows：
A) 10 μ L samples to be tested solution, high concentration calibration object solution and low concentration calibration product solution are added separately to difference
It reacts in cup aperture；
B) it is separately added into the special magnetic microsphere of the novel C CP antigen coats of 20 μ L；
C) it is separately added into the ABEI of the mouse anti-human igg secondary antibody label of 100 μ L；
D) 37 DEG C of warm bath 15min are placed under magnetic environment and clean 3 times；
E) 200 μ Lindenmayer system washing lotions are added in；
F) it is separately added into 2 (H of luminous substrate 1 (NaOH) and luminous substrate2O2), detect light signal strength；
G) it by the revised working curve of calibration object, is calculated according to pattern detection luminous intensity, is obtained by working curve automatically
Go out the antiCCP antibody concentration of sample to be tested.
Embodiment 2：
The R bases of novel C CP antigens that the present embodiment uses is amoxy CH3(CH2)4O-, remaining and embodiment 1 are identical.
Embodiment 3：
The R bases of novel C CP antigens that the present embodiment uses is acetamido (CH3CHO-NH-), remaining and 1 phase of embodiment
Together.
Embodiment 4：
The R bases of novel C CP antigens that the present embodiment uses is acetyl hydrazine (CH3CHO-NH-NH-), remaining and embodiment 1
It is identical.
Comparative example：
This comparative example using tradition CCP antigens (HQCHQEST-cit-GRSRGRCGRSGS), i.e., R bases be hydroxyl, remaining with
Embodiment 1 is identical.
Performance Evaluation
The performance of the kit of embodiment 1,2 and comparative example is assessed as follows：
1 blank limits
Use the antiCCP antibody assay kit replication zero-dose calibration object 20 prepared in embodiment and comparative example
It is secondary, record the relative luminous intensity (RLU) of 20 tests.
1.1 data processing
It calculates 20 times and measures the average value (M) of RLU and standard deviation (SD), obtain M+2SD, the value of M+2SD is substituted into reagent
Box working curve (can be used directly the mating user software of instrument and calculate corresponding concentration when RLU values are M+2SD), corresponding concentration
Value is blank limit.
2 precision
2.1 withinrun precision
Basic, normal, high 3 concentration samples (being selected as far as possible close to medical science decision level concentration) prepared using zero serum
With kit quality-control product as sample is measured, repeated using the antiCCP antibody assay kit prepared in embodiment and comparative example
It measures each sample 20 times, records each 20 measurement results of sample.
2.2 betweenrun precision
Basic, normal, high 3 concentration samples (being selected as far as possible close to medical science decision level concentration) prepared using zero serum
With kit quality-control product as sample is measured, each sample of measure is continuously repeated respectively using three batches of reagents each 20 times, record is every
A 60 measurement results of sample.
2.3 data processing
The average value (M) of measurement result and standard deviation (SD) in calculating batch and between criticizing respectively, CV is calculated according to formula (1)
Value.
CV=SD/M × 100%..................................... (1)
3 accuracy
3.1 experiment acceptance criterias
Relative deviation should be in the range of ± 10%.
3.2 test method
Compound concentration is about the cyclic citrullinated peptide company standard product of 100U/mL (tolerance is ± 10%), as
Sample after replication 3 times, records its result mean value, its measurement deviation is calculated according to formula (2).
3.3 data processing
Deviation is calculated according to formula (2)：
Measured deviation=(measuring average value-theoretical value)/theoretical value × 100%.................. (2)
The assessment of the blank limit of kit, testing result are as shown in table 1；
The assessment of the withinrun precision of kit, as a result as shown in table 2,3,4,5；
The assessment of the betweenrun precision of kit, the results are shown in Table 6；
The assessment of the accuracy of measurement of kit, the results are shown in Table 7.
Table 1：Blank limits
Conclusion：By embodiment 1, embodiment 2, from the point of view of the blank limit testing result of embodiment 3, embodiment 4 and comparative example,
After novel C CP antigens have been used in embodiment 1, embodiment 2, embodiment 3 and embodiment 4, RLU is limited relative to the blank of comparative example
Value reduces 40%, is obviously improved the sex-limited energy of blank of antibody CCP antibody assay kits.
Table 2：Criticize internal difference (embodiment 1vs. comparative examples)
Table 3：Criticize internal difference (embodiment 2vs. comparative examples)
Table 4：Criticize internal difference (embodiment 3vs. comparative examples)
Table 5：Criticize internal difference (embodiment 4vs. comparative examples)
Conclusion：From the point of view of embodiment 1, embodiment 2, embodiment 3, batch internal difference testing result of embodiment 4 and comparative example,
After novel C CP antigens have been used in embodiment, low value sample, intermediate value sample, the CV values of high level sample are respectively less than comparative example, so
Novel C CP antigens can be obviously improved the withinrun precision of antiCCP antibody detection kit.
Table 6-1：Difference between batch (embodiment 1)
Table 6-2：Difference between batch (embodiment 2)
Table 6-3：Difference between batch (embodiment 3)
Table 6-4：Difference between batch (embodiment 4)
Table 6-5：Difference between batch (comparative example)
Conclusion：By embodiment 1, embodiment 2, embodiment 3, embodiment 4 with from the point of view of the difference between batch testing result of comparative example,
After novel C CP antigens have been used in embodiment, three lot numbers (Lot1, Lot2, Lot3) detect low value sample, and intermediate value sample is high
The CV values of value sample are respectively less than comparative example, thus novel C CP antigens can be obviously improved antiCCP antibody detection kit batch between
Precision.
Table 7：Accuracy
Conclusion：By embodiment 1, embodiment 2, embodiment 3, embodiment 4 with from the point of view of the accuracy testing result of comparative example,
After novel C CP antigens have been used in embodiment, the value of detection error is respectively 0.12%, 0.73%, 0.38% and 4.44%,
Respectively less than the 8.41% of comparative example, this illustrates that the accuracy of embodiment is better than the accuracy of comparative example, therefore the present invention's is new
Type CCP antigens can promote the accuracy of antiCCP antibody detection kit.
Inventor has found that novel C CP antigens of the invention can surprisingly improve antiCCP antibody detection by above-described embodiment
The sex-limited energy of blank of kit, preci-sion and accuracy.
Under the premise of not limited by principle, inventor attempts to make description below to the principle of the present invention：The present invention
Novel anti-CCP antigens by using modified glutamic acid replace tradition CCP antigens in natural glutamate, make the new of synthesis
Glutamic acid in type CCP antigens no longer contains carboxyl.This novel C CP antigens pass through C-terminal well when connecting protein carrier
Carboxyl be coupled, avoid protein carrier and be connected to influence after the carboxyl of glutamic acid to CCP antigen actives, therefore obtain
Superior technique effect, realize present invention contemplates that technical purpose.
In traditional unmodified CCP antigens, the carboxyl contained by glutamic acid is apart from immunocompetence critical sites melon ammonia
Acid is close, and the two is only separated by 2 amino acid.When carrier protein is connected by the carboxyl of glutamic acid, certainly will cause in CCP antigens
Citrulling too close to protein carrier, so as to be influenced by space steric effect, after reducing CCP antigens connection protein carrier
Derivative antigen active, and then reduce detection antiCCP antibody preci-sion and accuracy.
On the other hand, the derivative coating magnetic ball that the present invention is formed using the novel C CP antigens with protein carrier, with right
Small molecule antigens carry out derivative amplification, improve the preci-sion and accuracy of the anti-Anti-CCP antibody of subsequent detection.This is also contributed to
The realization of the technology of the present invention purpose.
The above content is a further detailed description of the present invention in conjunction with specific preferred embodiments, it is impossible to assert
The specific implementation of the present invention is confined to these explanations.For those of ordinary skill in the art to which the present invention belongs, exist
Under the premise of not departing from present inventive concept, several simple deduction or replace can also be made, should all be considered as belonging to the present invention's
Protection domain.
Claims (12)
1. a kind of modified CCP antigens, the CCP antigens have the sequence shown in following formula I：
It is characterized in that, the carboxylic hydroxyl of the glutamic acid E in Formulas I is replaced by R group,
The R group is selected from one or more of：C1-10Alkyl oxy, C1-10Alkyl-CO-NH- bases and C1-10Alkyl-CO-
NH-NH- bases；The C1-10Alkyl is optionally selected from C by one or more1-4Alkyl, C1-4Alkyl oxy, hydroxyl, amino, nitro and
The substituent group substitution of phenyl.
2. modified CCP antigens as described in claim 1, which is characterized in that the R group is selected from following a kind of or more
Kind：C1-5Alkyl oxy, C1-5Alkyl-CO-NH- bases and C1-5Alkyl-CO-NH-NH- bases.
3. modified CCP antigens as described in claim 1, which is characterized in that the R group is selected from following a kind of or more
Kind：Methyl oxygroup, ethyl oxygroup, tertiary butyl oxygroup, benzyl oxygroup, benzhydryl oxygroup, acetamido and acetyl hydrazine.
4. as the modified CCP antigens of claim 1-3 any one of them are used to prepare the use of antiCCP antibody detection reagent
On the way.
5. a kind of antiCCP antibody detection kit, the kit includes component A and component B, which is characterized in that
The component A is including magnetic microsphere, described in the claim any one of 1-3 that the magnetic microsphere is coupled with protein carrier
Modified CCP antigen coats,
The component B includes the luminous marker solution marked by mouse anti-human igg secondary antibody.
6. kit as claimed in claim 5, which is characterized in that the luminous marker is selected from：Luminol, different luminol and
Its derivative, horseradish peroxidase and alkaline phosphatase.
7. kit as claimed in claim 5, which is characterized in that the protein carrier is selected from：Bovine serum albumin(BSA) (BSA),
Cationic bovine serum albumin(BSA) (cBSA), hemocyanin (KLH), ovalbumin (OVA), seralbumin HSA, ox gamma Globulin
With people's gamma Globulin.
8. kit as claimed in claim 5, which is characterized in that the kit meets following one or more：
The working concentration of magnetic microsphere is 1-10mg/mL；
The working concentration of the modified CCP antigens is 50-100ng/mL；
The working concentration of the protein carrier is 10-40ng/mL；
The working concentration of the mouse anti-human igg secondary antibody is 100-300ng/mL；
The working concentration of the luminous marker is 10-30ng/mL.
9. a kind of preparation method of antiCCP antibody detection kit, which is characterized in that including：
1) makes the modified CCP antigens of claim 1-3 any one of them be coupled with protein carrier；
2) makes the conjugate coating magnetic microsphere that step 1) obtains, and obtains component A；
3) reacts mouse anti-human igg secondary antibody and luminous marker, obtains component B；
4) component A and component B are assembled into kit by.
10. preparation method as claimed in claim 9, which is characterized in that
The molar ratio of the modified CCP antigens and protein carrier is 20: 1-80: 1；And/or
The weight ratio of the magnetic microsphere and conjugate is g-1mg: 40 μ g of 1mg: 10 μ.
11. preparation method as claimed in claim 9, which is characterized in that the step 1) is including making the CCP antigens and crosslinking
Agent is reacted in the presence of a catalyst, then adds in the protein carrier.
12. preparation method as claimed in claim 9, which is characterized in that
The crosslinking agent is selected from：Glutaraldehyde, BS (PEG) 5, BS (PEG) 8, PEG400, PEG800 and succinic anhydride；And/or
The catalyst is selected from：Triethylamine, pyridine, potassium hydroxide and potassium carbonate.
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