CN110470848A - A kind of MBP ELISA antibody assay kit and preparation method thereof - Google Patents
A kind of MBP ELISA antibody assay kit and preparation method thereof Download PDFInfo
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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
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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Abstract
The present invention relates to chemiluminescent polypeptides and immunological technique field, specifically disclose a kind of MBP ELISA antibody assay kit, and the kit detects the content of the people MBP antibody using dual-antigen sandwich method；It include coating plate, calibration object, MBP combination antigen, enzyme conjugates, quality-control product, color developing agent A, color developing agent B, terminate liquid and concentrated cleaning solution in the kit；The coating plate is coated with MBP envelope antigen, the MBP envelope antigen is by the way that the X segment in the people MBP epitope peptide and carrier protein couplet is prepared, the MBP combination antigen is by the way that the Y segment in the people MBP epitope peptide and carrier protein couplet is prepared, and the MBP combination antigenic mark has biotin.The present invention overcomes the deficiencies in the prior art, easy to operate, quick using the content of dual-antigen sandwich method quantitative detection people's MBP antibody, and accuracy in detection and precision it is good, it is specific it is high, sensitivity is good, stability is good.
Description
Technical field
The present invention relates to chemiluminescent polypeptides and immunological technique field, particularly belong to a kind of MBP ELISA antibody test examination
Agent box.
Background technique
Unclear about the definite pathogenic mechanism of MBP-Ab disease at present, most of research is thought, identifies the spy of MBP antigen
Specific B cells are likely to be present in peripheral blood, but due to lacking MBP antigen presentation in marrow, make MBP 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 MBP 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, MBP antigen
Periphery is leak into, CD4+T cell is activated, MBP specific b cells are raised and activated with increase, generates a large amount of MBP-IgG；Promote simultaneously
Scorching T cell enters maincenter, raises MBP specific b cells and flows into maincenter, generates corresponding antibodies.It has been demonstrate,proved in some experiment in vitro
Real MBP-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 MBP
Optic neuritis, support T cell MBP-Ab disease in play an important role.
The amount of MBP-Ab (anti-MBP antibody) can be increased in the blood of MBP-Ab patient, how quantitative, efficient
The content for detecting MBP antibody become urgent problem to be solved in the industry.
Summary of the invention
The object of the present invention is to provide a kind of MBP ELISA antibody assay kits, overcome the prior art not
Foot, it is easy to operate, quick using the content of dual-antigen sandwich method quantitative detection people's MBP antibody, and accuracy in detection and precision
It spends, specificity is high, sensitivity is good, stability is good.
To solve the above problems, the technical solution used in the present invention is as follows:
A kind of MBP ELISA antibody assay kit, the kit detect the people using dual-antigen sandwich method
The content of MBP antibody；It include coating plate, calibration object, MBP combination antigen, enzyme conjugates, quality-control product, colour developing in the kit
Agent A, color developing agent B, terminate liquid and concentrated cleaning solution；
The coating plate is coated with MBP envelope antigen, and the MBP envelope antigen is by making the people MBP epitope
What X segment and carrier protein couplet in peptide were prepared, the MBP combination antigen is by making the people MBP epitope
What Y segment and carrier protein couplet in peptide were prepared, the MBP combination antigenic mark has biotin, the people MBP antigen
The NCBI accession number of epitope peptide is CAA35179.1.
Further, the concentration of the MBP envelope antigen is 22 μ g/L, and the concentration of the MBP combination antigen is 24 μ g/L, institute
The horseradish peroxidase that enzyme conjugates is streptomysin label is stated, concentration is 3 μ g/L.
Further, the color developing agent A is the urea peroxide element solution of 10g/L, and the color developing agent B is the TMB of 2g/L
The volume ratio of 2HCl solution, the color developing agent A and color developing agent B are 1:1.
Further, from human serum, the concentration of the quality-control product is 7ng/L, described for the quality-control product and calibration object
The concentration gradient of calibration object is 10 μ g/L, 5 μ g/L, 2 μ g/L, 1 μ g/L, 0.5 μ g/L, 0 μ g/L.
Further, the concentrated cleaning solution is the phosphate buffer of 25 times of concentrations, and the terminate liquid is 2M H2SO4Solution.
A kind of preparation method based on above-mentioned MBP ELISA antibody assay kit kit, the method includes
Following steps:
S1, preparation MBP envelope antigen and preparation MBP combination antigen；
MBP combination is prepared in the coupling of S2, the horseradish peroxidase marked by MBP combination antigen and streptomysin
Antigen-HRP marker；
The preparation of S3, various buffers and reagent；
S4, coating buffer is used to dilute MBP envelope antigen with certain proportion, 100 holes μ L/ are added to ELISA Plate, envelope
Loaded in the aluminum foil bag added with desiccant, coating is finished；Specifically:
MBP envelope antigen is dissolved in the coating buffer of the 0.05M of pH=9.6, pre-coated liquid is made, on ELISA Plate
100 μ l are added by 0.1 hole μ g/ in every hole, set 4 DEG C of placements 18-24 hours, take out, coating buffer are got rid of, with sample/washing buffer
Washing is fitted into aluminide-coating bag after 1 (w/v) %BSA-0.05M ethanol amine is closed 16 hours, is dried overnight and vacuumizes sealing, and
It is placed in 4 DEG C of preservations.
Compared with prior art, implementation result of the invention is as follows by the present invention:
1. the present invention uses dual-antigen sandwich method, blood sample can be effectively detected (especially using the detection kit
Serum sample) in MBP antibody level, it is easy to operate, quick, and accuracy in detection and precision are high, can be rapid
The assisted diagnosis state of an illness in time monitors prognosis.
2. stability of the present invention is good, strong antijamming capability is as a result stable, reproducible, and kit of the invention can be reserved for
1 year or more.
Specific embodiment
The present invention will be further described with reference to the examples below, but the present invention is not limited to these instances, and is being de-
Under the premise of from present inventive concept, carried out by it is any improvement be within the scope of the present invention.
Embodiment 1
A kind of composition of MBP ELISA antibody assay kit:
1) MBP is coated with plate: being coated with the microwell plate of MBP.48 holes/96 orifice plates
2) human serum, concentration 0,0.5,1,2,5,10g/L 1 set of A-F of MBP calibration object: are derived from.
1×0.5ml
3) MBP combination antigen: the human myelin basic protein (MBP) of biotin labeling.
1×2.5ml/1×5.0ml
4) MBP-Ab enzyme conjugates: the horseradish peroxidase of streptomysin label.
1×5.0ml/2×5.0ml
5) MBP quality-control product: deriving from human serum, and Quality Control range is ± 1.05 μ g/L of 7 μ g/L.1×0.5ml
6) color developing agent A: main component is urea peroxide element.1×5.0ml
7) color developing agent B: main ingredient 3,3,5,5 ,-tetramethyl biphenyl amine hydrochlorate (TMB2HCl).1×5.0ml
8) concentrated cleaning solution (25 ×): the phosphate buffer of concentration is diluted using preceding by 1:24.
1×20.0ml
9) terminate liquid: 2mol/L sulfuric acid solution.1×5.0ml
10) sealing plate film 2 is opened
11) valve bag 1
Embodiment 2
A kind of preparation method of MBP ELISA antibody assay kit:
1) MBP envelope antigen and preparation MBP combination antigen are prepared:
X segment and Y segment are intercepted respectively in people's MBP epitope peptide, respectively convert X segment and Y segment to large intestine
It is cultivated in bacillus, collects the albumen for obtaining recombination precipitating after thallus is centrifuged, recombinant protein is purified respectively, is obtained
To destination protein, it is respectively designated as X protein and Y albumen, X protein is MBP envelope antigen, and Y albumen is MBP combination antigen.
2) MBP combination antigen-HRP marker is prepared:
The coupling of MBP labelled antigen and HRP specifically use NaIO4 oxidizing process, comprise the following steps that
2.1) by MBP labelled antigen obtained in step S1 in PBS solution 4 DEG C of dialysed overnights；
2.2) ultraviolet specrophotometer measures MBP labelled antigen concentration；
2.3) using assay balance weigh 10mgHRP be dissolved in prepared in 2mL ultrapure water final concentration of 5mg/mL HRP it is molten
Liquid, assay balance weigh NaIO4, are dissolved in the NaIO4 solution for standby that final concentration of 20mg/mL is prepared in ultrapure water；
2.4) 225 μ LNaIO4 solution are slowly added dropwise in 2mLHRP solution, are protected from light standing at room temperature 20 minutes；
2.5) 20 μ L ethylene glycol are added in HRP mixed solution, stands 30 minutes at room temperature later, obtains activated
HRP solution；
2.6) it takes the MBP labelled antigen of 1mL2.1mg/mL to be slowly added dropwise in activated HRP solution, obtains MBP
Labelled antigen-HRP conjugate；
2.7) by MBP labelled antigen-HRP conjugate in the solution of 50mMCBpH9.6 4 DEG C be protected from light dialysis 2.5 hours；
2.8) assay balance weighing NaBH4 is soluble in water, prepares the NaBH4 solution of final concentration of 4mg/mL；
2.9) the NaBH4 solution of 162 μ L is added into the bag filter of step 2.7), gently shakes 2 on shaking table at room temperature
A hour；
2.10) 4 DEG C of dialysed overnights in PBS solution；
2.11) glycerol is added in 1:1 ratio after dialysing, and -20 degree save, as MBP labelled antigen-HRP marker.
3) 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
Distilled water dissolution, is settled to 1000ml；
B, sample/washing buffer: 10 × PBS-Tween 20 of pH7.2
Na2HPO412H2O:58 grams
KH2PO4:4 grams
NaCl:100 grams
KCl:4 grams
Distilled water dissolution, is settled to 1000ml
Add Tween 20:20ml；
C, enzyme marker dilution
10 × PBS-Tween 20:10ml
FCS (calf serum): 20ml
Distilled water dissolution, is settled to 1000ml
Enzyme stabilizers (are purchased from Shanghai Xi Bao company): 1 gram
Biological preservative (is purchased from Shanghai Xi Bao company): 1ml；
D, color developing agent A:
Citric acid: 35.5 grams
Urea peroxide: 10 grams
Distilled water dissolution, is settled to 1000ml
Tween 20:10ml；
E, color developing agent B:
Citric acid: 120 grams
EDTA-2Na:1 grams
TMB2HCl:2 grams
Distilled water dissolution, is settled to 1000ml；
F, 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) preparation of pre-coated plate:
MBP envelope antigen is dissolved in the carbonate buffer solution of the 0.05M of pH=9.6, pre-coated liquid is made, in ELISA Plate
100 μ l are added by 0.1 hole μ g/ in every hole on (being purchased from Shenzhen Jin Canhua company), set 4 DEG C of placements 18-24 hours, take out, get rid of
Coating buffer is washed with sample/washing buffer, is filled after 1 (w/v) %BSA-0.05M ethanol amine is closed 16 hours, is dried overnight
Enter and vacuumize sealing in aluminide-coating bag, is placed in 4 DEG C of preservations.
3) combine the dilution ratio of antigen and enzyme conjugates (being purchased from company, Beijing Zhong Shan Golden Bridge) by square matrix titration experiments
It determines.The horseradish peroxidase of streptomysin label uses enzyme marker diluted.
Embodiment 3
A kind of detection method of MBP ELISA antibody assay kit, comprising the following steps:
1) preparation of cleaning solution: the 25 × concentrated cleaning solution stored at 2-8 DEG C may be formed in bottom of bottle and be crystallized, and be used
Whole bottle concentrated cleaning solution is completely dissolved in 480ml distilled water or deionized water by 500ml container.
2) kit is balanced to after room temperature (20 DEG C -30 DEG C), required capillary strip taking-up is fixed on grillage, is compiled
Good sequence；
3) it is loaded: 50ul sample to be tested/MBP calibration object/MBP quality-control product is first added, adds 50ul MBP combination antigen
In corresponding aperture, mixing is patted, if blank well；
4) it incubates: batten being sealed and (prevents from polluting) with sealed membrane, 37 DEG C incubate 30 minutes；
5) board-washing: reaction plate is taken out, gets rid of liquid in most plate, every hole fills cleaning solution board-washing 5 times, pats dry, in washing process
In generation bubble should be avoided；
6) enzyme: two drops are added in every hole or the MBP-Ab enzyme conjugates of 100ul, blank well are not added；
7) it incubates: being sealed plate with sealed membrane or plastic glue strip, 37 DEG C incubate 30 minutes；
8) board-washing: reaction plate is taken out, gets rid of liquid in most plate, every hole fills cleaning solution board-washing 5 times, pats dry, in washing process
In generation bubble should be avoided；
9) develop the color: each drop of color developing agent A, B solution or each 50ul is added in every hole, mixes well, and sets 37 DEG C and incubates 15 minutes；
10) terminate: the drop of terminate liquid 1 (50l) is added as early as possible and pats mixing for every hole；
11) it measures: being returned to zero with microplate reader (450nm wavelength) with blank control and (or blank is not set, with 450/630nm double wave
Long measurement), measure each hole OD. value.
12) quality controls: if MBP-Ab maximum concentration calibration object OD. >=0.8, test result is effective.
As a result it calculates: abscissa is made with calibration object concentration, OD. value makees ordinate, is fitted with double-log and draws calibration curve.
Its corresponding concentration value can be found on calibration curve by the OD. value of sample to be tested.
Table 1: calibration object concentration and corresponding mean light absorbency (OD) value
| Concentration μ g/L | 0 | 0.5 | 1 | 2 | 5 | 10 |
| Mean OD value | 0.036 | 0.181 | 0.223 | 0.437 | 0.667 | 1.132 |
Embodiment 4
60 patients and 112 healthy persons are carried out with the detection of serum MBP-Ab in a manner described, in patients serum
MBP-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 MBP-Ab concentration of table compare
| Group | Number | MBP-Ab concentration mean value (μ g/L) |
| Patient | 60 | 5.63 |
| Control group | 112 | 2.18 |
From the above data, kit of the invention can effectively and specifically detect the MBP-Ab content in serum,
To detect the MBP-Ab content difference between patient and normal person, its sensitivity of this kit is 96.2%, and specificity is
98.6%, accuracy rate 97.3%.
Above content is only to present inventive concept example and explanation, affiliated those skilled in the art couple
Described specific embodiment does various modifications or additions or is substituted in a similar manner, without departing from invention
Conceive or beyond the scope defined by this claim, is within the scope of protection of the invention.
Claims (6)
1. a kind of MBP ELISA antibody assay kit, it is characterised in that: the kit is examined using dual-antigen sandwich method
Survey the content of the people MBP antibody；It include coating plate, calibration object, MBP combination antigen, enzyme conjugates, Quality Control in the kit
Product, color developing agent A, color developing agent B, terminate liquid and concentrated cleaning solution；
The coating plate is coated with MBP envelope antigen, and the MBP envelope antigen is by making in the people MBP epitope peptide
X segment and carrier protein couplet be prepared, the MBP combination antigen is by making in the people MBP epitope peptide
Y segment and carrier protein couplet be prepared, the MBP combination antigenic mark has biotin, the people MBP epitope
The NCBI accession number of peptide is CAA35179.1.
2. a kind of MBP ELISA antibody assay kit according to claim 1, it is characterised in that: the MBP packet
It is 22 μ g/L by the concentration of antigen, the concentration of the MBP combination antigen is 24 μ g/L, and the enzyme conjugates is streptomysin label
Horseradish peroxidase, concentration are 3 μ g/L.
3. a kind of MBP ELISA antibody assay kit according to claim 1, it is characterised in that: the color developing agent
A is the urea peroxide element solution of 10g/L, and the color developing agent B is the TMB2HCl solution of 2g/L, the color developing agent A and color developing agent
The volume ratio of B is 1:1.
4. a kind of MBP ELISA antibody assay kit according to claim 1, it is characterised in that: the quality-control product
With calibration object from human serum, the concentration of the quality-control product is 7ng/L, and the concentration gradient of the calibration object is 10 μ g/L, 5
μg/L、2μg/L、1μg/L、0.5μg/L、0μg/L。
5. a kind of MBP ELISA antibody assay kit according to claim 1, it is characterised in that: the concentration is washed
The phosphate buffer that liquid is 25 times of concentrations is washed, the terminate liquid is 2M H2SO4Solution.
6. a kind of preparation side based on MBP ELISA antibody assay kit kit described in claim 1-5 any one
Method, it is characterised in that: described method includes following steps:
S1, preparation MBP envelope antigen and preparation MBP combination antigen；
The coupling of S2, the horseradish peroxidase marked by MBP combination antigen and streptomysin, are prepared MBP combination antigen-
HRP marker；
The preparation of S3, various buffers and reagent；
S4, coating buffer is used to dilute MBP envelope antigen with certain proportion, 100 holes μ L/ are added to ELISA Plate, are packaged in
In aluminum foil bag added with desiccant, coating is finished；Specifically:
MBP envelope antigen is dissolved in the coating buffer of the 0.05M of pH=9.6, pre-coated liquid is made, every hole on ELISA Plate
100 μ l are added by 0.1 hole μ g/, set 4 DEG C of placements 18-24 hours, takes out, gets rid of coating buffer, washed with sample/washing buffer,
It is fitted into aluminide-coating bag after 1 (w/v) %BSA-0.05M ethanol amine is closed 16 hours, is dried overnight and vacuumizes sealing, be placed in 4
DEG C save.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111089972A (en) * | 2019-12-18 | 2020-05-01 | 天津天海新域生物科技有限公司 | Kit for detecting anti-human myelin basic protein antibody and application thereof |
| CN112557672A (en) * | 2020-12-21 | 2021-03-26 | 安徽恩禾生物技术有限公司 | Myelin basic protein antibody detection kit and preparation method thereof |
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| WO2012112013A2 (en) * | 2011-02-18 | 2012-08-23 | Korea Research Institute Of Bioscience And Biotechnology | A marker comprising anti-ck8/18 complex autoantibody and its use for diagnosing cancer |
| CN105541992A (en) * | 2015-11-04 | 2016-05-04 | 武汉云克隆诊断试剂研究所有限公司 | Human MBP 18.5kD variant antigen epitope peptide and specific detection kits |
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| US20030092089A1 (en) * | 2001-11-14 | 2003-05-15 | Moscarello Mario Anthony | Method for diagnosing multiple sclerosis and an assay therefore |
| WO2012112013A2 (en) * | 2011-02-18 | 2012-08-23 | Korea Research Institute Of Bioscience And Biotechnology | A marker comprising anti-ck8/18 complex autoantibody and its use for diagnosing cancer |
| CN105541992A (en) * | 2015-11-04 | 2016-05-04 | 武汉云克隆诊断试剂研究所有限公司 | Human MBP 18.5kD variant antigen epitope peptide and specific detection kits |
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| CN111089972A (en) * | 2019-12-18 | 2020-05-01 | 天津天海新域生物科技有限公司 | Kit for detecting anti-human myelin basic protein antibody and application thereof |
| CN111089972B (en) * | 2019-12-18 | 2021-05-14 | 天津天海新域生物科技有限公司 | Kit for detecting anti-human myelin basic protein antibody and application thereof |
| CN112557672A (en) * | 2020-12-21 | 2021-03-26 | 安徽恩禾生物技术有限公司 | Myelin basic protein antibody detection kit and preparation method thereof |
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