CN110317246A - People MOG epitope peptide, antigen, antibody, application and chemical luminescence reagent kit - Google Patents
People MOG epitope peptide, antigen, antibody, application and chemical luminescence reagent kit Download PDFInfo
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- CN110317246A CN110317246A CN201810265084.XA CN201810265084A CN110317246A CN 110317246 A CN110317246 A CN 110317246A CN 201810265084 A CN201810265084 A CN 201810265084A CN 110317246 A CN110317246 A CN 110317246A
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- C—CHEMISTRY; METALLURGY
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
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Abstract
The present invention relates to people MOG epitope peptide, antigen, antibody, application, external diagnosis reagent case and the chemical luminescence reagent kits for detecting people MOG-Ab.The amino acid sequence of the people MOG epitope peptide is one of sequence shown in SEQ ID NO.1 and SEQ ID NO.2.The chemical luminescence reagent kit of the detection people MOG-Ab detects people MOG-Ab using dual-antigen sandwich method, and includes chemiluminescent substrate.People's MOG epitope peptide of the invention has good antigenicity, the monoclonal antibody and polyclonal antibody of high degree of specificity can be generated, the chemoluminescence method kit has many advantages, such as that high sensitivity, high specificity, cheap, stable and validity period is long, method is stable quickly, detection range is wide, high degree of automation easy to operate.
Description
Technical field
The invention belongs to chemiluminescent polypeptides and field of immunology, and in particular to the anti-myelin oligodendroglia glycoprotein of people
(MOG) epitope peptide, the MOG specific antigen and corresponding monoclonal antibody or Anti-TNF-α that are prepared with the epitope peptide
Body, the antigen are preparing the application on people MOG-Ab vitro detection kit, people MOG-Ab external diagnosis reagent case, Yi Jiyi
Chemical luminescence reagent kit and preparation method thereof of the kind for people MOG-Ab in quantitative detection sample.
Background technique
Unclear about the definite pathogenic mechanism of MOG-Ab disease at present, most of research is thought, identifies the spy of MOG antigen
Specific B cells are likely to be present in peripheral blood, but due to lacking MOG antigen presentation in marrow, make MOG specificity immature B cells
In state of anergy, simultaneously because lacking corresponding T helper cell (Th cell) booster action, identify that the specific b of MOG is thin
Born of the same parents cannot activate, and only be proliferated in peripheral blood.When neurotropic virus infects body, blood brain barrier is destroyed, MOG antigen
Periphery is leak into, CD4+T cell is activated, MOG specific b cells are raised and activated with increase, generates a large amount of MOG-IgG；Promote simultaneously
Scorching T cell enters maincenter, raises MOG specific b cells and flows into maincenter, generates corresponding antibodies.It has been demonstrate,proved in some experiment in vitro
Real MOG-IgG can pass through complement and antibody approach mediated cell lethal effect.And CD4+T cell is in some cytokine inductions
Under be divided into Th1, Th17, Th9 cell, the factors such as secretion TFN- γ, interleukins (IL) -12, IL-23, IL-17A pass through
Chemotactic factor (CF) attracts variety classes immunocyte, such as myeloid cell, macrophage, induces inflammatory cascade reaction, mediating central
Myelinoclasis.There are also researcher it can be observed that spontaneous in the transgenic mouse of the specific t-cell receptor of expression identification MOG
Optic neuritis, support T cell MOG-Ab disease in play an important role.
The amount of MOG-Ab (anti-MOG antibody) can be increased in the blood of MOG-Ab patient, using Enzyme-linked Immunosorbent Assay
Method (ELISA) or Western blotting, cell transfection technique (cell-basedassay, CBA), tetramer radioimmunology
(tetramer radioimmunoassay) can detect MOG-Ab.
The present invention uses chemiluminescence, prepares the kit of quantitative detection MOG-Ab.Therefore, finding suitably has
The specific MOG antigen of the MOG epitope peptide of immunogenicity, preparation and the kit for developing quantitative detection MOG-Ab are
At emphasis.
Summary of the invention
For solve it is above-mentioned the problems of in the prior art, the present invention provides people MOG epitope peptides, antigen, anti-
Body, application and chemical luminescence reagent kit and preparation method thereof.
Specifically, the present invention provides:
A kind of people MOG epitope peptide, wherein the amino acid sequence of the MOG epitope peptide is one of both:
(1)Tyr-Arg-Ile-Ser-Pro-Gly-Lys-Asn-Ala-Thr-Gly-Met-Glu；
(2)Tyr-Gln-Glu-Glu-Ala-Ala-Met-Glu-Leu-Lys-Val-Glu-Asp-Pro-Phe。
The present invention also provides a kind of MOG antigen, be by make people MOG epitope peptide (1) of the present invention with
What carrier protein couplet was prepared.
The present invention also provides a kind of MOG antigen, be by make people MOG epitope peptide (2) of the present invention with
What carrier protein couplet was prepared.
It is the monoclonal being prepared by MOG antigen of the present invention the present invention also provides a kind of people MOG antibody
Antibody or polyclonal antibody, wherein the MOG antigen is by keeping the people MOG epitope peptide (1) and carrier protein even
What connection was prepared.
It is the monoclonal being prepared by MOG antigen of the present invention the present invention also provides a kind of people MOG antibody
Antibody or polyclonal antibody, wherein the MOG antigen is by keeping the people MOG epitope peptide (2) and carrier protein even
What connection was prepared.
The present invention also provides MOG antigens according to the present invention on preparation people MOG-Ab vitro detection kit
Using.
The present invention also provides a kind of chemical luminescence reagent kit for people MOG-Ab in quantitative detection sample, the reagents
Box detects the people MOG-Ab using dual-antigen sandwich method, and the kit includes chemiluminescent substrate, in which:
The dual-antigen sandwich method is using the first MOG antigen as capture antigen, and the first MOG antigenic source is in people
One of MOG epitope peptide (1) and (2)；And
The dual-antigen sandwich method is using the 2nd MOG antigen as detection antigen, and the 2nd MOG antigenic source is in people
The other of MOG epitope peptide (1) and (2)；
The people MOG epitope peptide (1) and (2) are respectively as follows:
(1)Tyr-Arg-Ile-Ser-Pro-Gly-Lys-Asn-Ala-Thr-Gly-Met-Glu；
(2)Tyr-Gln-Glu-Glu-Ala-Ala-Met-Glu-Leu-Lys-Val-Glu-Asp-Pro-Phe。
Preferably, the first MOG antigen is by making one of the people MOG epitope peptide (1) and (2) and load
Body protein coupling is prepared.
Preferably, the 2nd MOG antigen be by make the other of the people MOG epitope peptide (1) and (2) with
What carrier protein couplet was prepared.
Preferably, the capture antigen is coupled with magnetic bead.Preferably, the dosage of the capture antigen and the magnetic bead
Than for (1-4ng): (1-3mg).
Preferably, the concentration of the capture antigen is 2ng/ml to 4ng/ml.
Preferably, the concentration of the combination antigen is 6ng/ml to 8ng/ml.
Preferably, the chemical luminescence reagent kit includes luminol salt system as the chemiluminescent substrate, the Shandong
Minot salt system includes urea peroxide, luminol, 4- iodophenol and Tween-20.
Preferably, in the luminol salt system, the concentration of the luminol is 0.6g/L-0.9g/L；The 4- iodine
The concentration of phenol is 0.1g/L-0.3g/L；The concentration of Tween-20 is 0.3ml/L-0.6ml/L；The concentration of urea peroxide are as follows:
0.3g/L-0.5g/L。
Preferably, the chemical luminescence reagent kit is for people's MOG-Ab albumen in quantitative detection serum and blood plasma.
The present invention also provides a kind of methods for preparing chemical luminescence reagent kit according to the present invention comprising following
Step:
1) the first MOG antigen is provided as capture antigen；
2) the 2nd MOG antigen is provided as detection antigen；And
3) chemiluminescent substrate is provided.
Preferably, in step 1), the concentration of the capture antigen is 2ng/ml to 4ng/ml.
Preferably, in step 1), the first MOG antigen and magnetic bead are coupled, and the first MOG antigen is dense
Spending is 2ng/ml to 4ng/ml, and the amount ratio of the first MOG antigen and the magnetic bead is (1-4ng): (1-3mg).
Preferably, in step 2), the concentration of the detection antigen is 6ng/ml to 8ng/ml.
Preferably, in step 3), the chemiluminescent substrate is luminol salt system, and the luminol salt system includes
Urea peroxide, luminol, 4- iodophenol and Tween-20.
Preferably, in the luminol salt system, the concentration of the luminol is 0.6g/L-0.9g/L；The 4- iodine
The concentration of phenol is 0.1g/L-0.3g/L；The concentration of Tween-20 is 0.3ml/L-0.6ml/L；The concentration of urea peroxide is
0.3g/L-0.5g/L。
The present invention also provides a kind of people MOG-Ab external diagnosis reagent cases, and it includes MOG antigen of the present invention works
For envelope antigen.
Preferably, the kit also includes in conjunction with antigen, and the combination antigen is MOG antigen of the present invention, and
And when the combination antigenic source is when one of the people MOG epitope peptide (1) and (2), the envelope antigen comes
Derived from the other of the people MOG epitope peptide (1) and (2).
Compared with the prior art, the present invention has the following advantages and good effect:
1. people's MOG epitope peptide of the invention has good antigenicity, exempted from antigen prepared therefrom (immunogene)
Epidemic disease animal can generate the monoclonal antibody and polyclonal antibody of high degree of specificity.
2. with MOG antigen prepared by the present invention can high special with blood sample (especially serum and plasma sample)
In MOG-Ab combine.
3. blood sample (especially serum sample can be effectively detected in people MOG-Ab chemical luminescence reagent kit of the invention
Originally the level of the MOG-Ab in).
4. the present invention combines chemiluminescence with dual-antigen sandwich method, provide a kind of for quantitative detection sample
The chemical luminescence reagent kit of middle people MOG-Ab, it is easy to operate, quick with the people MOG-Ab in kit detection sample, and
Accuracy in detection and precision it is good, it is specific it is high, sensitivity is good, stability is good, can the assisted diagnosis state of an illness in time rapidly, prison
Survey prognosis.
5. the present invention is touched during preparing the chemical luminescence reagent kit of the people MOG-Ab by largely testing
Rope optimizes the preparation condition of various aspects, when so that being detected with chemical luminescence reagent kit of the invention, detection sensitivity and
Result credibility is improved；In addition, kit of the present invention joined reinforcing agent and stabilization on the basis of single luminous substrate
Agent enhances luminous intensity and time compared with this is with a kind of traditional only luminescent substrate component (such as luminol) the case where,
Overcome that original luminescence reagent is unstable, disadvantage not easy to maintain.Kit of the invention can be stored for more than one year.
Specific embodiment
The following describes the present invention further through the description of specific embodiments, but it is to limit of the invention that this, which is not,
System, those skilled in the art's basic thought according to the present invention can make various modifications or improvements, but without departing from this
The basic thought of invention, is all within the scope of the present invention.
One, people MOG epitope peptide
People's MOG albumen described herein be it is known in the art, amino acid sequence is known in the art, Ke Yi
It is found in the specialized databases such as NCBI.
The present invention provides people MOG epitope peptide (1) and (2), and amino acid sequence is respectively such as sequence table SEQ ID
Shown in No.1 and SEQ ID No.2, are as follows:
(1) Y-R-I-S-P-G-K-N-A-T-G-M-E and
(2)Y-Q-E-E-A-A-M-E-L-K-V-E-D-P-F。
The present inventor gropes by a large amount of theoretical research and experiment, finally screens to obtain two kinds with good
Antigenic epitope peptide.
MOG epitope peptide (1) includes people MOG albumen (NCBI accession number CAA52617.1's) N-terminal the 54th to the 65th
Peptide fragment, and Y is added in the N-terminal of the peptide fragment, to constitute the MOG epitope peptide (1) for containing 13 amino acid.
MOG epitope peptide (2) includes people MOG albumen (NCBI accession number CAA52617.1) N-terminal the 134th to the 148th
The peptide fragment of position constitutes the MOG epitope peptide (2) for containing 15 amino acid.
Compared with the sequence of MOG albumen other positions, the selected peptide fragment section of the present invention and length are in hydrophily, antigen
Property and synthetic aspect are more preferable.Selected and addition additional amino acid has advanced optimized hydrophily, antigenicity and conjunction
It becomes second nature.Therefore, two epitope peptides of the invention have the characteristics that excellent hydrophilic, antigenicity is strong and is readily synthesized.
Currently, the research of the invention finds that, MOG epitope peptide of the invention has following function:
1. having antigenicity；2. the antibody of specificity is generated after connecting with carrier protein as immune primary stimuli animal；3.
It can be specifically in conjunction with people MOG-Ab with antigen prepared by epitope peptide.
The preparation method of MOG epitope peptide of the invention can use chemical synthesis: certainly using American AB I431A type polypeptide
Dynamic synthesizer, passes through Solid phase synthesis epitope peptide.The molecular weight of epitope peptide (1) and (2) of the invention is respectively
1639.78,2051.18, it can be determined with mass spectrum, and the epitope peptide sequence synthesized by polypeptide sequence measurement identification
Column.The purity of peptide fragment can be evaluated with thin-layer chromatography and high performance liquid chromatography, and measure the concentration of epitope peptide.
Two, MOG envelope antigen, MOG combination antigen
The present invention also provides MBP antigen, it can be used as the envelope antigen in dual-antigen sandwich method and combine antigen, thus
It is used to prepare people's MOG-Ab external diagnosis reagent case.The MBP antigen by make people MOG epitope peptide (1) of the invention and
One of (2) it is prepared with carrier protein couplet.Specifically, the present invention provides MOG antigen (1) and (2), it is described
MOG antigen (1) is by being prepared people MOG epitope peptide (1) of the invention with carrier protein couplet；The MOG antigen
(2) by the way that people MOG epitope peptide (2) of the invention is prepared with carrier protein couplet.When MOG antigen (1) is selected
When for envelope antigen, MOG antigen (2) is then selected as combining antigen；Vice versa.
MOG antigen of the invention has immunogenicity and specificity, is a kind of immunogene, can be with MOG-Ab specificity knot
It closes, to detect the MOG-Ab in sample, it is also possible to animal is immunized to prepare the MBP antibody of specificity.In the present invention,
The example of available carrier protein includes KLH (keyhole limpet hemocyanin), bovine serum albumin(BSA) (BSA), ovalbumin OVA etc..By
Strong in KLH (keyhole limpet hemocyanin) immunogenicity, binding site is more, and immune effect is preferable, and with immune animal affiliation
Farther out, it uses it to be not easy to cause cross reaction as carrier protein, is therefore preferred.
Three, MOG monoclonal antibody, MOG polyclonal antibody and people's MOG external diagnosis reagent case
The present invention also provides people MOG monoclonal antibody and people's MOG polyclonal antibody, the antibody can be each with this
The MOG antigen (1) or (2) (immunogene) of invention are immunized animal and are prepared.The ordinary skill in the art can be used in preparation method,
For details, reference can be made to embodiments 2.
Four, MOG envelope antigen, MOG combination antigen and people's MOG-Ab external diagnosis reagent case
MOG antigen of the invention can be used as the envelope antigen (MOG envelope antigen) in dual-antigen sandwich method and combine antigen
(MOG combination antigen), for preparing people MOG-Ab external diagnosis reagent case, which can be based on immunization method to people
MOG-Ab in body tissue, cell or body fluid is detected, preferably to the MOG-Ab in blood preparation, especially serum and blood plasma
It is detected.
Therefore, the present invention provides a kind of people MOG-Ab external diagnosis reagent cases, and it includes MOG envelope antigens of the invention
And/or MOG combination antigen.
It is currently known that can be used for the immunization experiment method of clinical examination mainly include following several: ELISA method, chemiluminescence
Method, fluorescent chromatographic method, colloid gold immune measuring method etc..
And ELISA method includes following several types: double antibody sandwich method detects antigen, dual-antigen sandwich method detects antibody,
Indirect method surveys antibody, competition law surveys antibody, competition law surveys antigen, capture coating method surveys antibody etc..
People MOG-Ab external diagnosis reagent case of the invention uses ELISA dual-antigen sandwich method preferably to detect MOG-Ab egg
It is white.The kit may include the combination antigen and/or necessary tool and reagent etc. of envelope antigen, enzyme label.The enzyme mark
Note can be horseradish peroxidase, alkaline phosphatase etc..The combination antigen itself can also be marked without enzyme, but
First addition adds enzyme additionally again after combining antigen to realize detection in reaction.The example of enzyme is known in the art.
Here, term " envelope antigen " refers to the antigen being coated on the ELISA Plate of solid phase, it can be with test antibodies spy
The opposite sex combines.Preferably, the people MOG-Ab external diagnosis reagent case includes MOG envelope antigen and MOG combination antigen,
In, when the envelope antigen is from one of people MOG epitope peptide (1) of the invention and (2), the combination antigen
From the other of the epitope peptide (1) and (2).Here, term " in conjunction with antigen " refers to another kind in kit
The antigen that can be specifically bound with test antibodies.Test antibodies distinguish specific recognition envelope antigen and combine the difference on antigen
Epitope.
People MOG-Ab external diagnosis reagent case of the invention preferably detects the MOG-Ab in serum.
, can also be comprising any reagent or tool needed for detection in kit of the invention, such as pre-coated plate, wash
Wash liquid, color developing agent, terminate liquid etc..
Five, the chemical luminescence reagent kit for quantitative detection people MOG-Ab
The present invention also provides a kind of chemical luminescence reagent kit for people MOG-Ab in quantitative detection sample, the reagents
Box detects the people MOG-Ab using chemiluminescence dual-antigen sandwich method, and the kit includes chemiluminescent substrate,
In:
Using the first MOG antigen as capture antigen, the first MOG antigen comes the chemiluminescence dual-antigen sandwich method
Derived from one of people MOG epitope peptide (1) and (2)；And
Using the 2nd MOG antigen as detection antigen, the 2nd MOG antigen comes the chemiluminescence dual-antigen sandwich method
Derived from the other of people MOG epitope peptide (1) and (2)；
The people MOG epitope peptide (1) and (2) are respectively as follows:
(1)Tyr-Arg-Ile-Ser-Pro-Gly–Lys-Asn-Ala-Thr-Gly-Met-Glu；
(2)Tyr-Gln-Glu-Glu-Ala-Ala-Met-Glu-Leu-Lys-Val-Glu-Asp-Pro-Phe。
In chemical luminescence reagent kit of the present invention, term " capture antigen " is referred to by test antibodies specificity in sample
The antigen for identifying and combining.
In the present invention, term " detection antigen " refers to that another kind is referred to by test antibodies specific recognition in sample simultaneously
In conjunction with antigen, test antibodies can distinguish specific recognition capture antigen and detect antigen on different epitopes.Detecting antigen can
In conjunction with detectable substance or it itself is marked with detectable substance (such as enzyme label), to realize the detection of test antibodies.
In some embodiments, the chemical luminescence reagent kit of the detection people MOG-Ab includes that the detection of enzyme label is anti-
Original, the enzyme label can be horseradish peroxidase (HRP), alkaline phosphatase etc., wherein preferably horseradish peroxidase.
In other embodiments, the detection antigen itself can also be marked without enzyme, but first addition detection in the reaction
Additionally addition enzyme is reacted and realizes detection again after antigen.The example of enzyme is known in the art.
In the present invention, the first MOG antigen can be by making one in the people MOG epitope peptide (1) and (2)
Person is prepared with carrier protein couplet；And the 2nd MOG antigen can be by making the people MOG epitope peptide (1)
The other of (2) it is prepared with carrier protein couplet.
In the present invention, the example of available carrier protein includes KLH (keyhole limpet hemocyanin), bovine serum albumin(BSA)
(BSA), ovalbumin OVA etc..Since KLH (keyhole limpet hemocyanin) immunogenicity is strong, binding site is more, and immune effect is preferable,
And it farther out with immune animal affiliation, uses it to be not easy to cause cross reaction as carrier protein, is therefore preferred.
The present inventor gropes through a large number of experiments, to each ingredient in chemical luminescence reagent kit of the invention
It is optimized, so that the result for meeting clinical detection requirement can be obtained for people MOG-Ab, that is, accuracy and precision
It spends, specificity is high, sensitivity is good, stability is good.
In the present invention, the capture antigen is preferably coupled with magnetic bead.It is preferred that will be caught using methods described below
It obtains antigen and magnetic bead is coupled.
Preferably, in chemical luminescence reagent kit of the invention, the concentration of the capture antigen is 2ng/ml to 4ng/ml,
More preferably 3ng/ml to 4ng/ml, most preferably 3ng/ml.
Preferably, it is described capture antigen and the magnetic bead amount ratio be (1ng to 4ng): (and 1mg to 3mg), more preferably
(2ng to 4ng): (1mg to 2.5mg), most preferably 3ng:2mg.
Preferably, in chemical luminescence reagent kit of the invention, the concentration of the detection antigen is 6ng/ml to 8ng/ml,
More preferably 6.5ng/ml to 7.5ng/ml, most preferably 7ng/ml.
Preferably, chemiluminescent substrate used in chemical luminescence reagent kit of the invention is luminol salt system.Rumi
The chemical name of promise is 5- amino -2,3- dihydro-Isosorbide-5-Nitrae phthalazine diketone, shines and shines for oxidation reaction, it is under alkaline condition
It is catalyzed by HRP, is aoxidized and generated the excitation state intermediate of 3- amino phthalic acid, the hair when it returns to ground state by urea peroxide
Photon out gives off the chemiluminescence that maximum emission wavelength is 425nm.
The composition of luminol salt system and the usage ratio of each component is optimized in the present invention.Preferably, the Shandong
Minot salt system includes urea peroxide, luminol, 4- iodophenol (4-Iodophenol, linear formula: IC6H4) and tween-OH
20.Wherein urea peroxide is as oxidant, and luminol is as luminous agent, and 4- iodophenol is as reinforcing agent, and Tween-20 is as stabilization
Agent.Inventors of the present invention have surprisingly found that this combination not only enhances luminous intensity and time, but also overcome conventional lightening agents
Unstable, not easy to maintain disadvantage, so that chemiluminescent substrate of the invention can be stored for more than one year.
Preferably, in luminol salt system of the invention, the concentration of luminol is 0.6g/L to 0.9g/L, more preferably
0.7g/L to 0.9g/L, most preferably 0.8g/L.Preferably, in luminol salt system of the invention, the concentration of 4- iodophenol
For 0.1g/L to 0.3g/L, more preferably 0.2g/L.Preferably, in luminol salt system of the invention, urea peroxide it is dense
Degree is 0.3g/L to 0.5g/L, more preferably 0.4g/L.Preferably, in luminol salt system of the invention, Tween-20 it is dense
Degree is 0.3ml/L to 0.6ml/L, more preferably 0.5ml/L.
Preferably, chemical luminescence reagent kit of the invention also includes calibration object, and the concentration gradient of calibration object is preferably
150ng/ml, 100ng/ml, 50ng/ml, 25ng/ml, 12.5ng/ml, calibration object, can be from examples from the MOG-Ab of purifying
It is obtained as Abcan company buys.
Chemical luminescence reagent kit of the invention preferably detects the MOG-Ab in serum and blood plasma.
In a specific embodiment, chemical luminescence reagent kit of the invention when in use, (preferably by blood to be measured
For serum or blood plasma) sample and coupling have the magnetic bead of purifying MOG antigen (i.e. capture antigen) to mix, the test antibodies in sample
(MOG-Ab) capture antigen-test antibodies compound is combined into capture antigentic specificity.Be washed out removal be not associated with it is to be measured
The ingredient of antibody, thus separating trap antigen-test antibodies compound.Then horseradish peroxidase (HRP) (following letter is added
Claim enzyme) label detection antigen of the invention, the enzyme label detection antigen and capture antigen-determined antigen compound in
It surveys antibody specificity to combine, forms capture antigen-test antibodies-enzyme label detection antigenic compound.It is washed out removal not
In conjunction with ingredient, thus separating trap antigen-test antibodies-enzyme label detection antigenic compound.Then institute of the present invention is added
The luminol salt system stated excites intermediate using the free energy of chemical reaction release, returns to excitation state from ground state, energy is with light
The form release of son.Later, with the luminous value of Chemiluminescence Apparatus detection photon release, the concentration of MOG-Ab is calculated according to luminous value
Value.
On the other hand, the present invention provides it is a kind of prepare it is of the present invention for people MOG- in quantitative detection sample
The method of the chemical luminescence reagent kit of Ab comprising following steps:
1) the first MOG antigen is provided as capture antigen；
2) the 2nd MOG antigen is provided as detection antigen；And
3) chemiluminescent substrate is provided.
The present inventor is groped by largely test, is optimized prepare and of the invention is used to detect people MOG-Ab's
The condition of each step of the method for chemical luminescence reagent kit, including antigen and magnetic bead will be captured and be coupled, determine capture antigen and
The concentration for detecting antigen, selects suitable chemiluminescent substrate etc., so that chemical luminescence reagent kit of the invention being capable of needle
The result for meeting clinical detection requirement is obtained to people MOG-Ab, that is, accuracy and precision it is good, it is specific it is high, sensitivity is good, steady
It is qualitative good.
Concentration preferably as the first MOG antigen of the capture antigen is 2ng/ml to 4ng/ml, more preferably
3ng/ml to 4ng/ml, most preferably 3ng/ml.
It is further preferred that be coupled the first MOG antigen and magnetic bead in step 1), and the first MOG antigen
Concentration is 2ng/ml to 4ng/ml, and the amount ratio of the first MOG antigen and the magnetic bead is that (1ng to 4ng): (1mg is extremely
3mg), more preferably (2ng to 4ng): (1mg to 2.5mg), most preferably 3ng:2mg.
Concentration preferably as the 2nd MOG antigen of the detection antigen is 6ng/ml to 8ng/ml, more preferably
6.5ng/ml to 7.5ng/ml, most preferably 7ng/ml.
Preferably, in step 3), use luminol salt system as the chemiluminescent substrate, luminol salt system
System includes urea peroxide, luminol, 4- iodophenol and Tween-20.
It is further preferred that the concentration of luminol is 0.6g/L-0.9g/L, more preferably in the luminol salt system
0.8g/L.Preferably, in the luminol salt system, the concentration of 4- iodophenol is 0.1g/L to 0.3g/L, more preferably
0.2g/L.Preferably, in luminol salt system of the invention, the concentration of urea peroxide is 0.3g/L to 0.5g/L, more preferably
For 0.4g/L.Preferably, in luminol salt system of the invention, the concentration of Tween-20 is 0.3ml/L to 0.6ml/L, more excellent
It is selected as 0.5ml/L.
In one embodiment of the invention, the step 1) includes: to take out magnetic bead from 4 DEG C of refrigerators, and ultrasound is washed
It washs 2 minutes；It takes 200 μ l magnetic beads to be placed on magnet stand, removes supernatant, remaining about 20 μ l magnetic beads；2.5 times of volumes are added into magnetic bead
First washing lotion (9.76g MES (a water morpholino b acid), is dissolved in 500ml water, adjust pH to 6.0), supersound washing 1 divides
Clock, 3 times repeatedly；300 μ l just washing lotion is added；Prepare 25mg/ml EDC (1- (3- dimethylamino-propyl) -3- ethyl carbon respectively again
Diimmonium salt hydrochlorate) solution and NHS (n-hydroxysuccinimide) solution, EDC and NHS solution respectively adds in the ratio of 1:1 volume
Enter 100 μ l；Room temperature reaction 30 minutes, removes supernatant, washs；Coupling buffer (H is added3BO31.24g is added to 100ml's
In water, wiring solution-forming 1., Na2B4O7·10H2O 1.91g is added in 100ml water, wiring solution-forming 2., take 50ml solution 1. and
2., 100ml water is added in mixing to 14.5ml solution, the use of 2N sodium hydroxide adjustment pH is 8.5, constant volume to 200ml), 3 times of magnetic beads
Volume removes supernatant after washing 3 times；The MOG antigen of 3ng/ml is added, reacts 2 hours, removes supernatant；It is added the 1% of 4 times of volumes
(w/v) BSA confining liquid reacts at room temperature 1 hour, removes supernatant；The Tris end washing lotion of 0.1M is added, 2.5 times of volumes wash 3 times, most
3 times of volume constant volumes afterwards.
In one embodiment of the invention, to determine suitable capture antigen concentration and detection antigen concentration,
Capture antigen concentration is set as 3ng/ml, MOG-Ab calibration object concentration is set as 150,100,50,25,12.5ng/ml, detection is anti-
Original takes 6ng/ml, 6.5ng/ml, 7ng/ml, 7.5ng/ml, 8ng/ml respectively in the range of 6ng/ml-8ng/ml, and these are dense
Degree is tested, final to determine detection antigen optium concentration.By comparison result it is found that detection antigen concentration is preferably 6.5ng/ml
To 7.5ng/ml, most preferably 7ng/ml.Similarly, it will test antigen concentration and be set as 7ng/ml, by capture antigen concentration difference
It is set as 2ng/ml, 2.5ng/ml, 3ng/ml, 3.5ng/ml and 4ng/ml, is compared, it is final to determine capture antigen concentration.By
Comparison result is it is found that capture antigen concentration is preferably 3ng/ml to 4ng/ml, most preferably 3ng/ml.
In one embodiment of the invention, the oxidation reaction of luminol carries out in alkaline buffer, therefore
It is preferred that with the 0.1mol/L pH8.6 borate buffer solution (Na of 0.05mol/L2B4O7With the H of 0.2mol/L3BO3According to 5.5:4.5
Volume ratio be formulated, pH=8.6) make substrate solution.Luminol and urea peroxide also can slow spontaneous hairs when no HRP is catalyzed
Light, to cause blank to interfere in the measurement of last luminous intensity, therefore preferably that luminous substrate is separately formulated at two parts, it may be assumed that A
Liquid and B liquid, A liquid include urea peroxide, buffer；B liquid includes 4- iodophenol, luminol, Tween-20, buffer (A liquid and B liquid
In buffer all can be above-mentioned 0.1mol/L pH8.6 borate buffer solution), A liquid and B liquid are being mixed at once using preceding.
In addition, for example certain phenol reagents (such as adjacent iodine phenol) of HRP luminescence enhancer or firefly luciferase can enhance HRP catalysis luminol oxygen
The reaction of change and extension fluorescent lifetime, improve the susceptibility that shines.Using 4- iodophenol as reinforcing agent in this system.
The contents of the present invention are further explained and described below by way of the mode of example, but these examples are understood not to
Limitation to protection scope of the present invention.
Embodiment
Unless otherwise indicated, solution as described below is aqueous solution, and the percentage in solution is percentage by volume.
The preparation of embodiment 1:MOG epitope peptide (1) and (2).
Preparation method chemical synthesis: American AB I431A type polypeptide automatic synthesizer is utilized, is closed respectively by solid phase method
At MOG epitope peptide (1) and (2).The purity of epitope peptide is evaluated with high performance liquid chromatography, and measures the dense of peptide fragment
Degree.The molecular weight of epitope peptide (1) and (2) of the invention is respectively 1639.78,2051.18, is determined using mass spectrum,
Pass through the synthesized polypeptide sequence of polypeptide sequence measurement identification.
One, the synthesis of MOG epitope peptide (1) and (2)
Above-mentioned peptide fragment uses Solid phase synthesis.The main thought of Solid phase peptide synthesis is: first by the carboxyl for the peptide chain of being synthesized
The carboxyl of end amino acid same insoluble high-molecular compound (resin) in the form of covalent bond is connected, and is then tied with this
Amino acid on solid phase carrier is closed as moiety, through sloughing amino protecting group and with excessive activated carboxyl component it is anti-
It answers, spreading peptide chain.Such step repeatedly can repeatedly go on, the length of the peptide chain synthesized required for finally reaching.
This synthesis process is as follows.
Specific preparation process is as follows for MOG epitope peptide (1) and (2) of the invention respective:
1. raw materials used:
HMP resin (P- hydroxymethyl phenoxy methyl poly vinyl, be purchased from sigma company)
Fmoc-AA (amino acid of 9- fluorenylmethoxycarbonyl carbonyl acyl group protection, be purchased from Merck company)
NMP (N-methyl pyrrolidones is purchased from sigma company)
DCM (methylene chloride is purchased from Central Plains chemical company)
MeoH (methanol is purchased from Central Plains chemical company)
Piperidines (Piperidine is purchased from sigma company)
DMAP (dimethyl aminopyridine is purchased from sigma company)
HOBT (hydroxybenzotriazole is purchased from sigma company)
DCC (dicyclohexylcarbodiimide is purchased from sigma company)
TFA (trifluoroacetic acid is purchased from sigma company)
EDT (1,2- dithioglycol is purchased from sigma company)
Thioanisole is purchased from Guangzhou Wei Bai Chemical Co., Ltd.
Crystalline phenol is purchased from Sinopharm Chemical Reagent Co., Ltd.
Acetonitrile is purchased from Sinopharm Chemical Reagent Co., Ltd.
2. using instrument:
Polypeptide automatic synthesizer, model 431A are purchased from ABI company
Rotary Evaporators, model R-201 are purchased from Shanghai Shen Shun company
High performance liquid chromatograph, Waters 600, is purchased from Waters, US
Freeze drier, model VFD-2000 are purchased from the rich doctor Kanggong department in Beijing
3. synthetic method and process:
HMP resin 100mg is weighed, replacing equivalent is 1.0meq, i.e., 0.1mmol is placed in American AB I431A type polypeptide certainly
In the reaction chamber of dynamic synthesizer, specific amino acid is connected in a different order automatically by synthesizer, Conjugate ratio reaches
99%.It reacts as follows:
(1) activation (HOBt/DCC method) of amino acid
Fmoc-protected amino acid
(2) it connects on amino acid to resin
(3) the Fmoc protecting group of amino acid is sloughed
(4) activation (HOBt/DCC method) of another amino acid
(5) it is coupled
Peptide-resin of new coupling
(6) step (3) to (5) are repeated until synthesis terminates.
Respectively obtain the peptide resin 126mg of peptide resin 145mg and the MOG peptide fragment (2) of MOG peptide fragment (1).
(7) peptide resin:
Peptide chain is cut with TFA (trifluoroacetic acid), is removed with EDT (2.5 volume %), thioanisole (2.5 volume %)
Agent is reacted 3.0 hours at room temperature, is removed cutting reagent, then is extracted with ether, and the thick of MOG peptide fragment (1) and (2) is respectively obtained
Product.
Two, the purifying of MOG epitope peptide (1) and (2) crude product:
It is purified using high performance liquid chromatography separation:
Condition: chromatographic column: 10 × 100mm of C8 is purchased from Waters, US
Chromatograph: Waters 600, Waters, US
Mobile phase: A:0.1%TFA (trifluoroacetic acid) aqueous solution
B:0.1%TFA (trifluoroacetic acid) is in 60% acetonitrile
Detection wavelength: 214nm
Flow velocity: 4ml/ minutes
Gradient: 20-60%B, 30 minutes
HPLC (high performance liquid chromatography) analysis
Chromatographic column: 4.6 × 150mm of C18 is purchased from Waters, US's mobile phase: A:0.1%TFA (trifluoroacetic acid)
Aqueous solution
B:0.1%TFA (trifluoroacetic acid) is in acetonitrile
Detection wavelength: 214nm
Flow velocity: 1ml/ minutes
Gradient: 0-60%B, 30 minutes
The purity of peptide piecewise analysis MOG epitope peptide (1) and (2) of the invention as the result is shown is 95% or more.
Three, the identification of MOG epitope peptide (1) and (2)
1. measuring the molecular weight for purifying resulting MOG epitope peptide (1) and (2) respectively using mass spectrum.
(1) reagent raw material
TFA (trifluoroacetic acid is purchased from sigma company)
HCCA (alpha-cyano -4- hydroxycinnamic acid, be purchased from sigma company)
Acetonitrile (is purchased from Sinopharm Chemical Reagent Co., Ltd.)
(2) instrument
Matrix-Assisted Laser Desorption Ionization Time of Flight instrument MALDI-TOF-MS (model: REFLEX III, Germany
Bruker company)；
(3) matrix liquid: α-CCA being dissolved in the 50%ACN solution containing 0.1%TFA, saturated solution is made, and centrifugation takes
Clearly；
(4) instrument testing conditions: reflection detection mode；Flight pipe range 3m；Nitrogen laser: wavelength 337nm, acceleration voltage
20KV；Reflected voltage 23KV.
(5) operating procedure: taking the sample of the above-mentioned purified polypeptide of 1 μ L (1) and (2) respectively, respectively and in the saturation matrix of 1 μ L
The isometric mixing of clear liquid mixing, takes 1 μ L point on sample target respectively, is sent into ion source and is detected.
As a result, the molecular weight for measuring gained MOG epitope peptide (1) is the molecule of 1639.63, MOG epitope peptide (2)
Amount is 2051.32, consistent with theoretical molecular weight 1639.78,2051.18, it was demonstrated that synthesis polypeptide is purpose product.
2. the sequence of MOG epitope peptide (1) and (2) as obtained by polypeptide sequence measurement identification respectively.
(1) principle: the basic principle of polypeptid acid sequence analysis is Edman degradation, is that a circulating chemistry is anti-
Answer process.Including three main chemical steps: (1) be coupled: the end the N- residue of phenyl isothiocyanate and proteins and peptides is anti-
It answers, forms phenylamino formyl sulfide (PTC) derivative, i.e. PTC- peptide.(2) cyclisation cracking: PTC- peptide cyclisation cracking.(3) it converts: thiophene
Azoles purine ketone phenylamino (ATZ) is converted into the different sulphur urine amino acid of benzene (PTH- amino acid).Stay in the solution reduce an amino acid
The peptide of residue, which repeats, carries out above-mentioned reaction process, and entire sequencing procedure is carried out automatically by sequenator now.
(2) instrument: 491 type protein/polypeptide -terminal amino acid sequenator of American AB I company
(3) reagent raw material
Phenyl isothiocyanate PITC is purchased from sigma company
Normal heptane is purchased from Sinopharm Chemical Reagent Co., Ltd.
Trimethylamine TMA aqueous solution, is purchased from Sinopharm Chemical Reagent Co., Ltd.
TFA (trifluoroacetic acid is purchased from sigma company)
Ethyl acetate is purchased from Sinopharm Chemical Reagent Co., Ltd.
Chlorobutane is purchased from sigma company
Acetonitrile is purchased from Sinopharm Chemical Reagent Co., Ltd.
(4) it measures
It is carried out by instrument specification.
As a result: identified, the sequence of gained MOG epitope peptide (1) and (2) is respectively as follows:
(1) Y-R-I-S-P-G-K-N-A-T-G-M-E and
(2)Y-Q-E-E-A-A-M-E-L-K-V-E-D-P-F。
The result is consistent with target section of synthesized peptide.
Embodiment 2: the resulting MOG epitope peptide (1) of embodiment 1 and (2) are connect with carrier protein to prepare respectively
Animal is immunized using gained antigen (1) and (2) in MOG antigen (1) and (2) respectively, to prepare specificity using antigen (1)
Monoclonal antibody and polyclonal antibody, and specific monoclonal antibody and polyclonal antibody are prepared using antigen (2).
1. the preparation of antigen: using BDB (Bis-diazotizedbenzidine dichloride) method by MOG peptide fragment (1)
(2) it is connect respectively with carrier protein KLH (keyhole limpet hemocyanin) (being purchased from sigma company) and is prepared into MOG antigen (1) and (2).
MOG peptide fragment (1) or (2) 8.0mg are taken, is dissolved with 1ml 0.1M PBS buffer solution (pH 7.4)；KLH 10mg is used
0.2M borate buffer solution (pH 8.6) 20ml dissolution；Then the two is mixed, is cooled to 0 DEG C, takes BDBCl2110 μ L, room temperature
Lower reaction 1.5h is dispensed after dialysed overnight, -20 DEG C of preservations.
In embodiments, the formula of PBS buffer solution (if use) are as follows: the Na of 0.2mol/L2HPO481ml adds
The NaH of 0.2mol/L2PO419ml is mixed.
The formula of borate buffer solution are as follows: 0.05mol/L borax 80ml adds 0.2mol/L boric acid 20ml to mix.
2. immune animal prepares monoclonal antibody:
2.1. the MOG antigen (1) and (2) (immunogene) for taking above-mentioned preparation (are purchased with isometric Freund's complete adjuvant respectively
From sigma company) be sufficiently mixed after, be immunized Balb/c mouse, 50 μ g antigens/only, subcutaneous multi-point injection.Serum effect is surveyed after 4 weeks
Valence selects the good mouse of immunoreactivity booster immunization again: taking antigen and isometric incomplete Freund's adjuvant (purchased from sigma
Company) be sufficiently mixed after, only, subcutaneous multi-point injection, the number of booster immunization is 6 times to 25 μ g/ of antigen dose, every minor tick 2-3
In week, in addition twice, every minor tick 1-2 weeks, extracting spleen cell and Sp2/0 myeloma cell are by normal later for continuous booster immunization before merging
Rule method is mediated with 50%PEG (MW4000) (being purchased from Bao Yu biotech firm) and is merged, and (is purchased from HAT conditioned medium
Sigma company) selection culture.CO is put into after fusion237 DEG C after culture 9~11 days in incubator, appearance biggish cell hole in
Clone.Start to be screened with indirect ELISA method within 11 days.4 time clonings are carried out using limiting dilution assay to the hole of the primary dcreening operation positive
Change culture (even if screening after a large amount of schizogamies of cell), later amplifying cells, freeze, prepare ascites.
2.2. Balb/c mouse is only handled with norphytane (being purchased from sigma company) 0.5ml/, intraperitoneal inoculation is miscellaneous after a week
Friendship oncocyte 2 × 106/only, ascites is collected after 10 days.
2.3. antibody titer is measured: the monoclonal antibody (1) with indirect ELISA method measurement using MOG antigen (1) preparation
Potency, the potency of monoclonal antibody reaches 1:32000 or more as the result is shown.
It is also measured, is imitated using identical method using the potency of the monoclonal antibody (2) of MOG antigen (2) preparation
Valence also reaches 1:32000 or more.
3. immune animal prepares polyclonal antibody:
3.1. select the New Zealand White Rabbit that three monthly ages, weight are about 2.5kg or so as immune animal.Fundamental immunity
In, the MOG antigen (1) of the above-mentioned preparation of 1-2mg and (2) (immunogene) (are purchased from isometric complete Freund's adjuvant respectively
Sigma biotech firm) mixing-it is fully emulsified after rabbit back carry out multiple spot subcutaneous injection, it is primary every 4 weeks booster immunizations,
Booster immunization 6 times altogether.After antigen and incomplete Freund's adjuvant are fully emulsified later, only it is subcutaneously injected in back multiple spot with 100 μ g/
Carry out final boost.Arteria carotis bloodletting in 10th day after final boost separates serum.
3.2. it measures antibody titer: utilizing the polyclonal antibody (1) of MOG antigen (1) preparation with indirect elisa method measurement
Potency, antibody titer reaches 1:32000 or more as the result is shown.
It is also measured, is imitated using identical method using the potency of the polyclonal antibody (2) of MOG antigen (2) preparation
Valence also reaches 1:32000 or more.
3.3. take blood and separation serum: arteria carotis intubation takes blood, separates serum.
4. isolating and purifying antibody: after ammonium sulfate precipitation, then through Protein G (being purchased from sigma company) affinity purification.
5. being lyophilized after antibody packing, cryo-conservation.
Embodiment 3: the specificity identification of people MOG monoclonal antibody (1) and (2)
It is detected with ELISA.Respectively with people MOG albumen, myelin associated glucoprotein (MAG), people's myelin alkaline protein
(MBP) (being purchased from Shanghai Lian Shuo company) is that envelope antigen is coated with elisa plate, and it is mono- to detect prepared MOG respectively by ELISA
The specific reaction of clonal antibody (1) and (2) and people MOG albumen makees negative control, PBS liquid with normal BALB/c mouse serum
Make blank control.
As a result: MOG monoclonal antibody (1) and (2) only reacts respectively with MOG for the positive (P/N > 2.1), and related to myelin
Glycoprotein (MAG), people's myelin alkaline protein (MBP) reaction are feminine gender, illustrate MOG monoclonal antibody (1) and (2) of the invention
It is respectively provided with specificity.
Embodiment 4: the specificity identification of people MOG polyclonal antibody (1) and (2)
It is identified using method identical with above-mentioned identification monoclonal antibody specificity.
As the result is shown: MOG polyclonal antibody (1) and (2) are reacted respectively with MOG for positive (P/N > 2.1), and with myelin phase
Close glycoprotein (MAG), people's myelin alkaline protein (MBP) reaction is feminine gender, illustrate MOG polyclonal antibody (1) of the invention and
(2) it is respectively provided with specificity.
Embodiment 5: the specific reaction identification of MOG antigen and MOG-Ab prepared by the present invention
It is detected with ELISA.Respectively with people MOG-Ab, people's myelin alkaline protein antibody (MBP-Ab) (on being purchased from
Hai Lianshuo company) it is that coated antibody is coated with elisa plate, detect the spy of prepared MOG antigen and MOG-Ab respectively by ELISA
Opposite sex reaction, makees negative control with normal BALB/c mouse serum, PBS liquid makees blank control.
As a result: MOG antigen prepared by the present invention only react with people MOG-Ab for the positive (P/N > 2.1), and with people's myelin
Basic protein antibody (MBP-Ab) reaction is feminine gender, illustrates MOG antigen and MOG-Ab specific reaction of the invention.
Embodiment 6: MOG-Ab external diagnosis reagent case is prepared using MOG antigen (1) and MOG antigen (2).
In the present embodiment, by the MOG antigen (1) prepared in embodiment 2 using MOG epitope peptide (1) as this examination
Envelope antigen in agent box；By the MOG antigen (2) prepared in embodiment 2 using MOG epitope peptide (2) as in conjunction with antigen.
The preparation and operation of MOG-Ab external diagnosis reagent case are as follows:
1. the preparation of various buffers and reagent:
A, it is coated with buffer: the CB (carbonate buffer solution) of 0.050M, pH9.6
Na2CO3: 16.0 grams
NaHCO3: 29.0 grams
It distills water-soluble to 1000ml
B, sample/washing buffer: 10 × PBS-Tween 20 of pH7.2
Na2HPO4·12H2O:58 grams
KH2PO4: 4 grams
NaCl:100 grams
KCl:4 grams
It distills water-soluble to 1000ml
Add Tween 20:20ml
C, color developing agent A:
Citric acid: 35.5 grams
Urea peroxide: 10 grams
It distills water-soluble to 1000ml
Tween 20:10ml
D, color developing agent B:
Citric acid: 120 grams
EDTA-2Na:1 grams
TMB2HCl:2 grams
It distills water-soluble to 1000ml
E, terminate liquid: 2M H2SO4
The concentrated sulfuric acid (95-98%): 22.2ml
Distilled water: 177.3ml
The concentrated sulfuric acid is slowly dropped into distilled water by timing, is shaken well while adding.
2. the preparation of pre-coated plate:
In the carbonate buffer solution for the 0.05M that MOG antigen (1) is dissolved in pH=9.6, pre- packet liquid is made, and (envelope antigen is dense
Spend 3ng/ml), 100 μ l are added in every hole on ELISA Plate (being purchased from Shenzhen Jin Canhua company), set 4 DEG C of placements 18-24 hours, take
Out, coating buffer is got rid of, is washed with sample/washing buffer, 16 hours is closed through 1 (w/v) %BSA-0.05M ethanol amine, stays overnight
It is fitted into after drying in aluminide-coating bag and vacuumizes sealing, be placed in 4 DEG C of preservations.
3. combining antigen (MOG antigen (2)) and enzyme-linked object (goat anti-rabbit igg antibody of horseradish peroxidase-labeled) (purchase
From Beijing company, Zhong Shan Golden Bridge) dilution ratio by square matrix titration experiments determine, made with horseradish peroxidase-labeled antigen
It is carried out with the conventional method of this field and condition.
4. the composition of kit:
Pre-coated plate: 48/96 hole
MOG-Ab calibration object (raw material be purchased from Abcan company): 5: 5 × 1.0ml (concentration be respectively 150ng/ml,
100ng/ml、50ng/ml、25ng/ml、12.5ng/ml)
The MOG combination antigen of enzyme label: 1 × 10ml (concentration 7ng/ml)
Concentrated cleaning solution (25 × PBS-Tween 20): 1 × 20ml
Color developing agent A:1 × 6.0ml
Color developing agent B:1 × 6.0ml
Terminate liquid: 1 × 6.0ml
5. the operating procedure of kit:
It is separately added into 100 hole μ l/ of serum and calibration object to be checked in each hole of pre-coated plate, is diplopore, 37 DEG C incubate
It educates 60 minutes, is washed 5 times, patted dry with 1 × washing buffer.Addition enzyme label 00 hole μ l/ of MOG combination antigen 1 in each hole, 37
DEG C be incubated for 30 minutes, washed 5 times, patted dry with 1 × washing buffer.Color developing agent A, B liquid is added, every each 50 μ l in hole is mixed, 37 DEG C
It is incubated for 15 minutes.Add 50 hole μ l/ of terminate liquid to terminate reaction, is used with enzyme detector (model RT-6000 is purchased from Lei Du company)
Dual wavelength (450nm, 620nm) detects absorbance.
6. result judgement:
Table 1: calibration object concentration and corresponding mean light absorbency (OD) value
| Concentration ng/ml | 12.5 | 25 | 50 | 100 | 150 |
| Mean OD value | 0.085 | 1.653 | 3.680 | 6.881 | 10.156 |
Standard curve, the R of standard curve are drawn with the logarithm of calibration object concentration and corresponding absorbance2=0.976.
The MOG-Ab concentration results in sample detected are calculated according to standard curve.
Serum MOG-Ab detection is carried out in a manner described to 68 patients and 151 healthy persons, the MOG- in patients serum
Ab content is apparently higher than healthy control group, and difference is statistically significant (P < 0.01), is shown in Table 2.
2: two groups of sample MOG-Ab concentration of table compare
From the above data, kit of the invention can effectively and specifically detect the MOG-Ab content in serum,
To detect the MOG-Ab content difference between patient and normal person.
Embodiment 7: for detecting the preparation of the chemical luminescence reagent kit of people MOG-Ab in sample.
In the present embodiment, by the MOG antigen (1) prepared in embodiment 2 using MOG epitope peptide (1) as this examination
Capture antigen in agent box；By the MOG antigen (2) prepared in embodiment 2 using MOG epitope peptide (2) as detection antigen.
The preparation and operation of MOG-Ab chemical luminescence reagent kit are as follows:
1. buffer
A, first wash buffer
9.76 grams of MES (a water morpholino b acid) is weighed, is dissolved in 500 milliliters of water, PH to 6.0 is adjusted.
B, coupling buffer
Weigh boric acid (H3BO3) 1.24 grams, it is dissolved in 100 milliliters of water, is configured to solution 1；
1.91 grams of sodium tetraborate (borax) is weighed, is dissolved in 100 milliliters of water, is configured to solution 2；
50 milliliters of solution 1 and 14.5 milliliter solution 2 are taken, mixes, 100 milliliters of water is added；
PH to 8.5 is adjusted using 2N sodium hydroxide, is settled to 200 milliliters.
C, Block buffer
Ethanol amine is taken, 0.08M solution is configured to；
BSA is added into the solution, and its final solid content is made to reach 1% (w/v).
D, whole wash buffer
Tween-20 is added in purified water, until solution concentration 0.2% (v/v)；
BSA is added into solution, and its final solid content is made to reach 1%.
2. the coupling of magnetic bead and capture antigen:
It is taken out magnetic bead (being purchased from Tianjin Bei Sile company, product article No. 3412) from 4 DEG C of refrigerators, supersound washing 2 minutes；It takes
200 μ l magnetic beads are placed on magnet stand, remove supernatant, remaining about 20 μ l magnetic beads；The previous cleaning buffering of 2.5 times of volumes is added into magnetic bead
Liquid, supersound washing 1 minute, repeatedly 3 times；300 μ l just wash buffer is added；Prepare 25mg/ml EDC (1- (3- diformazan respectively again
Aminopropyl) -3- ethyl-carbodiimide hydrochloride) solution and NHS (n-hydroxysuccinimide) solution, EDC and NHS solution presses
100 μ l are respectively added in the ratio of 1:1 volume；Room temperature reaction 30 minutes, removes supernatant, washs；Coupling buffer, 3 times of magnetic beads are added
Volume removes supernatant after washing 3 times；The 80 μ l of MOG antigen of 3ng/ml is added, reacts at room temperature 2 hours, removes supernatant；4 times of bodies are added
Long-pending Block buffer reacts at room temperature 1 hour, removes supernatant；The whole wash buffer of 0.1M is added, 2.5 times of volumes wash 3 times, most
3 times of volume constant volumes afterwards.
The detection antigen (MOG antigen (2)) of enzyme 3. (horseradish peroxidase is purchased from company, Beijing Zhong Shan Golden Bridge) label
Concentration and dilution ratio are determined by square matrix titration experiments.
4. the composition of kit:
The magnetic bead being coupled with capture antigen: 1 × 2ml
MOG-Ab calibration object (raw material be purchased from Abcan company): 5,5 × 1.0ng/ml (concentration be respectively 150ng/ml,
100ng/ml、50ng/ml、25ng/ml、12.5ng/ml)
The MOG of enzyme label detects antigen (MOG antigen (2)): 1 × 10ml (concentration 7ng/ml)
Concentrated cleaning solution (25 × PBS-Tween 20): 1 × 20ml
(A liquid includes 0.4g/L urea peroxide, 0.1mol/L pH8.6 borate buffer solution to luminous substrate；B liquid includes
0.2g/L 4- iodophenol, 0.8g/L luminol, 0.5ml/L Tween-20,0.1mol/L pH8.6 borate buffer solution): 2 ×
6.0ml
5. the operating procedure of kit:
Reagents are taken out from 4 DEG C of refrigerators, equilibrium at room temperature 15 minutes；Concentrated cleaning solution 1:25 is diluted to working solution.It will be to be measured
Serum, plasma sample and calibration object are respectively positioned in the reaction cup of the magnetic bead containing 20ul, and each concentration of calibration object respectively sets one glass, are separately set
One glass is made blank control.Sample or calibration object add 100 μ l in respective reaction cup respectively, set 37 DEG C of isothermal reactions 60 minutes, wash.
The reaction solution in reaction cup is absorbed, about 400 μ l of cleaning solution is added in each reaction cup, stands 20 seconds or so, removes wherein liquid, washes
It washs.It so washes altogether three times, liquid in reaction cup is blotted in last time.In addition to blank reaction cup, each enzyme label of reaction cup
(2) 100 μ l of MOG antigen, sets 37 DEG C of isothermal reactions 60 minutes.The reaction solution in reaction cup is absorbed, cleaning solution is added in each reaction cup
About 400 μ l stand 20 seconds or so, remove wherein liquid, washing.It so washes altogether three times, liquid in reaction cup is inhaled in last time
It is dry.Each 50 μ l of chemiluminescent substrate A, B liquid is added in every reaction cup.The luminous value for measuring each hole on the 10th minute after adding substrate
RLU (detecting instrument: the chemiluminescent analyzer that Jiangsu pool is produced at company, model C IA600).
6. result judgement:
Table 3: calibration object concentration and corresponding average irradiance RLU
| Concentration ng/ml | 12.5 | 25 | 50 | 100 | 150 |
| Luminous value RLU | 8125 | 16201 | 32620 | 64003 | 96524 |
Standard curve, the R of standard curve are drawn with the logarithm of calibration object concentration and corresponding luminous value2=0.978.According to
Standard curve calculates the MOG-Ab concentration results in sample detected.
Due to the R of standard curve2=0.978, therefore the calibration detected with MOG-Ab chemical luminescence reagent kit of the invention
Product concentration and the actual concentrations fitting degree of calibration object are higher, this illustrates the detection accuracy of chemical luminescence reagent kit of the invention
It is excellent with confidence level.
Luminol salt luminescent system parameter constant, the capture antigen of other concentration and the testing result such as table 4 for detecting antigen
It is shown.
Table 4
The concentration for capturing antigen and detection antigen is constant, the luminol salt luminescent system (carbamide peroxide concentration of different content
Testing result 0.4g/L) is as shown in table 5.
Table 5
7.MOG-Ab chemical luminescence reagent kit performance evaluation
1) sensitivity: 20 replications are carried out to 12.5ng/ml calibration object, luminous value RLU value is calculated, uses standard curve
Inverse goes out the corresponding concentration value of its x+2s, the result is that 12.45ng/ml, shows MOG-Ab chemical luminescence reagent kit spirit of the invention
Sensitivity is good.
2) accuracy: being measured with recovery test, is added 10 in 1 part of volume calibration product that MOG-Ab concentration is 150ng/ml
Calibration object and the mixing that part volume MOG-Ab concentration is 12.5ng/ml measure rate of recovery range 85~115%, show this hair
Bright MOG-Ab chemical luminescence reagent kit accuracy is good.
3) precision: detecting MOG-Ab concentration respectively with MOG-Ab chemical luminescence reagent kit of the invention is respectively 25ng/
The calibration object serum of ml and 150ng/ml repeats detection 10 times, carries out withinrun precision measurement.
The calibration object serum of three different batches of above-mentioned 2 concentration is measured daily, 1 day 1 time, continuously surveys 20
It, carries out betweenrun precision measurement.As a result criticizing interior CV (coefficient of variation) is 4.5% and 5.7% respectively, and CV is respectively between batch
9.4% and 9.0%.CV is respectively less than 10% between criticizing interior CV and criticizing, and illustrates MOG-Ab chemical luminescence reagent kit accuracy of the invention
Well.
4) stability: placing 3 days, 5 days, 7 days with MOG-Ab chemical luminescence reagent kit of the invention in 37 DEG C of incubators,
Test result standard curve is done with these kits, observe placement 3 days, 5 days and places 0 day without significant difference, is placed 7 days
Afterwards, maximum A value is lower, and linear poor, learns that MOG-Ab chemiluminescence of the invention tries using Arrhenius equation conversion
Agent box 12 months internal stabilities after factory are good.
5) specific: to use MOG-Ab chemical luminescence reagent kit of the invention, detect containing MBP-Ab and MOG-Ab feminine gender is faced
Bed serum and plasma sample, with calibration object dilution (PBS, 10% (w/v) calf serum and 0.03% (w/v) biological preservative
It is formulated) dilution, luminous value RLU is measured, the cross reaction situation with MBP-Ab is calculated.Test result shows and MBP-Ab
Cross reacting rate be 0.23%, MOG-Ab chemical luminescence reagent kit specificity of the invention is good.
SEQUENCE LISTING
<110>Anqun Bioengineering Co., Ltd., Shenzhen
<120>people MOG epitope peptide, antigen, antibody, application and chemical luminescence reagent kit
<130> FI-180871-59:52/C
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 13
<212> PRT
<213>artificial sequence (Artificial sequence)
<400> 1
Tyr Arg Ile Ser Pro Gly Lys Asn Ala Thr Gly Met Glu
1 5 10
<210> 2
<211> 15
<212> PRT
<213>artificial sequence (Artificial sequence)
<400> 2
Tyr Gln Glu Glu Ala Ala Met Glu Leu Lys Val Glu Asp Pro Phe
1 5 10 15
Claims (13)
1. a kind of people MOG epitope peptide, wherein the amino acid sequence of the MOG epitope peptide is one of both:
(1)Tyr-Arg-Ile-Ser-Pro-Gly-Lys-Asn-Ala-Thr-Gly-Met-Glu；
(2)Tyr-Gln-Glu-Glu-Ala-Ala-Met-Glu-Leu-Lys-Val-Glu-Asp-Pro-Phe。
2. a kind of MOG antigen is by making people MOG epitope peptide (1) described in claim 1 and carrier protein couplet system
Made of standby.
3. a kind of MOG antigen is by making people MOG epitope peptide (2) described in claim 1 and carrier protein couplet system
Made of standby.
4. a kind of people MOG antibody is the monoclonal antibody being prepared by MOG antigen as claimed in claim 2 or polyclonal
Antibody.
5. a kind of people MOG antibody is the monoclonal antibody being prepared by MOG antigen as claimed in claim 3 or polyclonal
Antibody.
6. application of the MOG antigen according to claim 2 or 3 on preparation people MOG-Ab vitro detection kit.
7. a kind of chemical luminescence reagent kit for people MOG-Ab in quantitative detection sample, the kit uses double antigens sandwich
Method detects the people MOG-Ab, and the kit includes chemiluminescent substrate, in which:
The dual-antigen sandwich method is using the first MOG antigen as capture antigen, and the first MOG antigenic source is in claim
One of epitope peptide described in 1 (1) and (2)；And
The dual-antigen sandwich method is using the 2nd MOG antigen as detection antigen, and the 2nd MOG antigenic source is in claim
The other of epitope peptide described in 1 (1) and (2).
8. chemical luminescence reagent kit according to claim 7, wherein the capture antigen is coupled with magnetic bead, and institute
The amount ratio for stating capture antigen and the magnetic bead is (1-4ng): (1-3mg).
9. chemical luminescence reagent kit according to claim 7, wherein the concentration of the capture antigen is 2ng/ml to 4ng/
The concentration of ml, the detection antigen are 6ng/ml to 8ng/ml.
10. chemical luminescence reagent kit according to claim 7, wherein the chemiluminescent substrate is luminol salt system,
The luminol salt system includes urea peroxide, luminol, 4- iodophenol and Tween-20.
11. chemical luminescence reagent kit according to claim 10, wherein in the luminol salt system, the luminol
Concentration be 0.6g/L-0.9g/L；The concentration of the 4- iodophenol is 0.1g/L-0.3g/L；The concentration of Tween-20 is 0.3ml/
L-0.6ml/L；The concentration of urea peroxide is 0.3g/L-0.5g/L.
12. a kind of people MOG-Ab external diagnosis reagent case, it is anti-as coating that it includes MOG antigens described in claim 2 or 3
It is former.
13. people MOG-Ab external diagnosis reagent case according to claim 12, wherein the kit also includes in conjunction with anti-
Original, the combination antigen are MOG antigen described in claim 2 or 3, and when the combination antigenic source is in the people
When one of MOG epitope peptide (1) and (2), the envelope antigen from the people MOG epitope peptide (1) and
The other of (2).
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