CN106053791A - Anti-mullerian hormone chemiluminescence immunoassay kit and preparation method and application thereof - Google Patents
Anti-mullerian hormone chemiluminescence immunoassay kit and preparation method and application thereof Download PDFInfo
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- CN106053791A CN106053791A CN201610512550.0A CN201610512550A CN106053791A CN 106053791 A CN106053791 A CN 106053791A CN 201610512550 A CN201610512550 A CN 201610512550A CN 106053791 A CN106053791 A CN 106053791A
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- G—PHYSICS
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- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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Abstract
The invention discloses an anti-mullerian hormone chemiluminescence immunoassay kit and a preparation method and application thereof. The anti-mullerian hormone chemiluminescence immunoassay kit comprises a solid-phase carrier enveloped by an anti-mullerian hormone monoclonal antibody, and an anti-mullerian hormone monoclonal antibody marked by a chemiluminescence marker. The anti-mullerian hormone chemiluminescence immunoassay kit can complete the assay of anti-mullerian hormone by taking a full-automatic chemiluminescence immunoassay analyzer as an assay tool. Through experiments, the assay sensitivity of the anti-mullerian hormone chemiluminescence immunoassay kit can reach 0.01ng/mL, and is improved by at last ten times compared with the traditional anti-mullerian hormone assay method, and the assay precision of the anti-mullerian hormone chemiluminescence immunoassay kit is higher.
Description
Technical field
The present invention relates to vitro detection field, particularly relate to a kind of anti-Miao's Le Shi pipe hormone chemiluminescence immunoassay detectable
Box and its preparation method and application.
Background technology
Miao Le Shi pipe hormone (MIS), the most anti-Miao's Le Shi pipe hormone (Anti-M ü llerian hormone, AMH), be
The glycoprotein dimer of the 140kU that two 72kDa monomers form is connected by disulphide bridges.MIS/AMH belongs to β-conversion growth
The factor (TGF-β) family Superfamily.First found in nineteen forty-seven by Profess Alfred Jost.AMH is immature by testis
After Sertoli cell and birth, the granular cell secretion of ovarian growth follicle, has regulation cell development and differentiation, makes male embryo
The effect that tire gyneduct is degenerated.Male, initially express in 8 weeks fetal sertoli cells, start from embry ogenesis, and pass through
Wear life all the time.During fetal development, AMH can cause Miao's Le Shi pipe to degenerate, and forms normal male genetic pipeline.For female
For property, AMH generates at 36 weeks prenatal follicular cells, until Women’s climacteric, AMH gradually decreases down cannot
The level of detection.
Can be used for true and false androgynous diagnosis, the specific marker of tumor, the labelling etc. of spermiogenesis tail clinically.
AMH is applied to be mainly used in monitoring ovarian reserve power, the ovary response of prediction external fertilization associating embryo transfer technology (IVF)
Property, prevention ovarian hyperstimulation syndrome (OHSS), the assessment etc. of male reproductive function.At polycystic ovarian syndrome (PCOS)
Diagnosis also replaces Antral follicles (AFC) index with this.
Women's ovary function and age, FSH (FSH), basis inhibin B (INHB), basis estradiol
(E2), clomifene provocative test (CCCT), GuRH-A (GnRH-a) provocative test, Antral follicles,
Ovarian Volume is relevant with many factors such as Ovarian Blood Flow abundance and AMH (AMH) levels.AMH is the most most sensitive
Index.
The detection method of traditional AMH mainly uses enzyme linked immunosorbent assay, but, it is limited to the feature of enzyme linked immunological,
The detection method of traditional AMH is in open space during detection, easily causes the cross-contamination between various reagent,
And using manual operations owing to detection process, the dosage of reagent or sample is not bery accurate, and operating process is the most loaded down with trivial details and multiple
Miscellaneous, it is susceptible to bust, accuracy of detection is poor.
Summary of the invention
Based on this, it is necessary to provide anti-Miao's Le Shi pipe hormone chemiluminescence immunoassay detectable that a kind of accuracy of detection is higher
Box and its preparation method and application.
A kind of anti-Miao's Le Shi pipe hormone chemiluminescence immune detection reagent kit, including: anti-Miao's Le Shi pipe hormone monoclonal anti
The coated solid phase carrier of body and anti-Miao's Le Shi pipe hormone monoclonal antibody of chemiluminescent labels labelling.
In one embodiment, in described anti-Miao's Le Shi pipe coated solid phase carrier of hormone monoclonal antibody, described solid phase
Carrier is that surface is with the magnetic particle for protein chemistry reaction forming group.
In one embodiment, described surface is surface band with the magnetic particle for protein chemistry reaction forming group
There is the magnetic particle of amino, carboxyl, tosyl or Oxyranyle.
In one embodiment, described surface with the particle diameter of the magnetic particle for protein chemistry reaction forming group is
0.05 μm～3 μm.
In one embodiment, in described anti-Miao's Le Shi pipe coated solid phase carrier of hormone monoclonal antibody, described solid phase
On carrier, coupling has Streptavidin, Avidin or neutral Avidin, coupling in described anti-Miao's Le Shi pipe hormone monoclonal antibody
There is biotin.
In one embodiment, in anti-Miao's Le Shi pipe hormone monoclonal antibody of described chemiluminescent labels labelling, institute
Stating chemiluminescent labels is different luminol, tris (bipyridine) ruthenium, acridinium ester, alkali phosphatase or horseradish peroxidase.
In one embodiment, in anti-Miao's Le Shi pipe hormone monoclonal antibody of described chemiluminescent labels labelling, institute
State coupling on chemiluminescent labels and have Streptavidin, Avidin or neutral Avidin, described anti-Miao Le Shi pipe hormone Dan Ke
On grand antibody, coupling has biotin.
In one embodiment, in anti-Miao's Le Shi pipe hormone monoclonal antibody of described chemiluminescent labels labelling, institute
State chemiluminescent labels be directly connected to by chemical reaction key with described anti-Miao's Le Shi pipe hormone monoclonal antibody or pass through egg
White cross-linking agent connects.
In one embodiment, at least during described protein-crosslinking agent is selected from phosphinylidyne diimmonium salt and butanimide
Kind.
In one embodiment, described protein-crosslinking agent selected from 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine,
1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate, N-hydroxy-succinamide and sulfonated N-hydroxysuccinimidyl
At least one in acid imide.
In one embodiment, also include that anti-Miao's Le Shi pipe hormone calibrates product.
The preparation method of a kind of above-mentioned anti-Miao's Le Shi pipe hormone chemiluminescence immune detection reagent kit, including walking as follows
Rapid:
Anti-Miao's Le Shi pipe hormone monoclonal antibody and solid phase carrier are mixed and fully react, obtains anti-Miao's Le Shi pipe hormone
The coated solid phase carrier of monoclonal antibody；And
Chemiluminescent labels and anti-Miao's Le Shi pipe hormone monoclonal antibody are mixed and fully react, obtains chemiluminescence
Anti-Miao's Le Shi pipe hormone monoclonal antibody of label labelling.
In one embodiment, in described anti-Miao's Le Shi pipe coated solid phase carrier of hormone monoclonal antibody, described solid phase
Carrier is that surface is with the magnetic particle for protein chemistry reaction forming group；
Described anti-Miao's Le Shi pipe hormone monoclonal antibody and solid phase carrier mixed and fully reacts, obtaining anti-Miao Le Shi and manage
The operation of the coated solid phase carrier of hormone monoclonal antibody is: take surface with the magnetic for protein chemistry reaction forming group
The suspension of microgranule, Magneto separate is resuspended with buffer after removing supernatant, and then activator activates described surface with for protein
The Surface attachment groups of the magnetic particle of chemical reaction linking group, is subsequently added into anti-Miao's Le Shi pipe hormone monoclonal antibody, room temperature
Lower fully reaction, Magneto separate is resuspended after removing supernatant, obtains anti-Miao's Le Shi pipe hormone monoclonal antibody coated surface band useful
Magnetic particle in protein chemistry reaction forming group.
In one embodiment, in described anti-Miao's Le Shi pipe coated solid phase carrier of hormone monoclonal antibody, described solid phase
On carrier, coupling has Streptavidin, Avidin or neutral Avidin, coupling in described anti-Miao's Le Shi pipe hormone monoclonal antibody
There is biotin；
Described anti-Miao's Le Shi pipe hormone monoclonal antibody and solid phase carrier mixed and fully reacts, obtaining anti-Miao Le Shi and manage
The operation of the coated solid phase carrier of hormone monoclonal antibody is: coupling has Streptavidin, Avidin or neutral Avidin
Solid phase carrier and coupling have anti-Miao's Le Shi pipe hormone monoclonal antibody of biotin to mix in buffer, fully obtain after reaction
Anti-Miao's Le Shi pipe coated solid phase carrier of hormone monoclonal antibody.
In one embodiment, in anti-Miao's Le Shi pipe hormone monoclonal antibody of described chemiluminescent labels labelling, institute
State coupling on chemiluminescent labels and have Streptavidin, Avidin or neutral Avidin, described anti-Miao Le Shi pipe hormone Dan Ke
On grand antibody, coupling has biotin；
Described chemiluminescent labels and anti-Miao's Le Shi pipe hormone monoclonal antibody mixed and fully reacts, obtaining chemistry
The operation of anti-Miao's Le Shi pipe hormone monoclonal antibody of luminescent label is: coupling is had Streptavidin, Avidin or
Chemiluminescent labels and the coupling of neutral Avidin have anti-Miao's Le Shi pipe hormone monoclonal antibody of biotin in buffer
Anti-Miao's Le Shi pipe hormone monoclonal antibody of chemiluminescent labels labelling is obtained after mixing, fully reaction.
In one embodiment, in anti-Miao's Le Shi pipe hormone monoclonal antibody of described chemiluminescent labels labelling, institute
State chemiluminescent labels to be directly connected to by chemical reaction key with described anti-Miao's Le Shi pipe hormone monoclonal antibody；
Described chemiluminescent labels and anti-Miao's Le Shi pipe hormone monoclonal antibody mixed and fully reacts, obtaining chemistry
The operation of anti-Miao's Le Shi pipe hormone monoclonal antibody of luminescent label is: by anti-Miao's Le Shi pipe hormone monoclonal antibody and
Chemiluminescent labels mixes in buffer, fully obtains anti-Miao's Le Shi pipe hormone of chemiluminescent labels labelling after reaction
Monoclonal antibody.
In one embodiment, in anti-Miao's Le Shi pipe hormone monoclonal antibody of described chemiluminescent labels labelling, institute
State chemiluminescent labels to be connected by protein-crosslinking agent with described anti-Miao's Le Shi pipe hormone monoclonal antibody；
Described chemiluminescent labels and anti-Miao's Le Shi pipe hormone monoclonal antibody mixed and fully reacts, obtaining chemistry
The operation of anti-Miao's Le Shi pipe hormone monoclonal antibody of luminescent label is: by anti-Miao's Le Shi pipe hormone monoclonal antibody,
Chemiluminescent labels and protein-crosslinking agent mix in buffer, fully obtain the anti-of chemiluminescent labels labelling after reaction
Miao's Le Shi pipe hormone monoclonal antibody.
The detection method of the concentration of a kind of anti-Miao's Le Shi pipe hormone, uses above-mentioned anti-Miao's Le Shi pipe hormone chemiluminescence immunoassay
Detection kit, the detection method of the concentration of described anti-Miao's Le Shi pipe hormone comprises the steps:
Use anti-Miao's Le Shi pipe coated solid phase carrier of hormone monoclonal antibody and anti-Miao of chemiluminescent labels labelling
Le Shi pipe hormone monoclonal antibody carries out sandwich immunoassay test to sample to be tested, the anti-Miao's Le Shi pipe hormone in described sample to be tested
With anti-Miao's Le Shi pipe coated solid phase carrier of hormone monoclonal antibody and anti-Miao's Le Shi pipe hormone of chemiluminescent labels labelling
Monoclonal antibody reactive, forms sandwich complex, after over cleaning separates, records the luminous intensity of sample to be tested；
Use anti-Miao's Le Shi pipe coated solid phase carrier of hormone monoclonal antibody and anti-Miao of chemiluminescent labels labelling
Le Shi pipe hormone monoclonal antibody antagonism Miao's Le Shi pipe hormone calibration product carry out sandwich immunoassay test, described anti-Miao's Le Shi pipe hormone
Anti-Miao's Le Shi pipe hormone in calibration product and anti-Miao's Le Shi pipe coated solid phase carrier of hormone monoclonal antibody and chemiluminescence mark
Anti-Miao's Le Shi pipe hormone monoclonal antibody reactive of note substance markers, forms sandwich complex, after over cleaning separates, records anti-Miao
The luminous intensity of Le Shi pipe hormone calibration product, according to luminous intensity and the described anti-Miao Le of described anti-Miao's Le Shi pipe hormone calibration product
The standard that the concentration of family name's pipe hormone calibration product builds anti-Miao's Le Shi pipe hormone that transverse and longitudinal coordinate is respectively concentration and luminous intensity is bent
Line；And
The luminous signal of described sample to be tested is brought in the standard curve of described anti-Miao's Le Shi pipe hormone, be calculated institute
State the concentration of anti-Miao's Le Shi pipe hormone in sample to be tested.
This anti-Miao's Le Shi pipe hormone chemiluminescence immune detection reagent kit can be with automatic chemiluminescence immunoassay
Instrument is detection instrument, completes the detection of anti-Miao's Le Shi pipe hormone.This anti-Miao's Le Shi pipe hormone chemiluminescence immunoassay detectable
Box, through experiment, its detection sensitivity reaches 0.01ng/mL, relative to the detection method spirit of traditional anti-Miao's Le Shi pipe hormone
Sensitivity at least improves 10 times, and the accuracy of detection of this anti-Miao's Le Shi pipe hormone chemiluminescence immune detection reagent kit is higher.
Accompanying drawing explanation
Fig. 1 is the canonical plotting of anti-Miao's Le Shi pipe hormone that embodiment 3 obtains；
Fig. 2 is the canonical plotting of anti-Miao's Le Shi pipe hormone that embodiment 7 obtains.
Detailed description of the invention
Understandable for enabling the above-mentioned purpose of the present invention, feature and advantage to become apparent from, real with concrete below in conjunction with the accompanying drawings
Execute example the detailed description of the invention of the present invention is described in detail.Elaborate a lot of detail in the following description so that
Fully understand the present invention.But the present invention can implement to be much different from alternate manner described here, art technology
Personnel can do similar improvement in the case of intension of the present invention, and therefore the present invention is not embodied as by following public
Restriction.
Anti-Miao's Le Shi pipe hormone chemiluminescence immune detection reagent kit of one embodiment, including: anti-Miao's Le Shi pipe hormone
The coated solid phase carrier of monoclonal antibody and anti-Miao's Le Shi pipe hormone monoclonal antibody of chemiluminescent labels labelling.
In anti-Miao's Le Shi pipe coated solid phase carrier of hormone monoclonal antibody, anti-Miao's Le Shi pipe hormone monoclonal antibody is permissible
Behaviour source, gene engineering expression or animal sources.
In anti-Miao's Le Shi pipe hormone monoclonal antibody of chemiluminescent labels labelling, anti-Miao's Le Shi pipe hormone monoclonal anti
Body can be people source, gene engineering expression or animal sources.
Anti-Miao's Le Shi pipe coated solid phase carrier of hormone monoclonal antibody and anti-Miao Le Shi of chemiluminescent labels labelling
In pipe hormone monoclonal antibody, the anti-Miao's Le Shi pipe hormone monoclonal antibody each used can be identical, it is also possible to differs.
In one embodiment, in anti-Miao's Le Shi pipe coated solid phase carrier of hormone monoclonal antibody, solid phase carrier is
Surface is with the silica-base material that can carry out protein physical absorption for the magnetic particle of protein chemistry reaction forming group, surface
Or the magnetic particle through macromolecule surface process.
Solid phase carrier is that surface is with the magnetic particle for protein chemistry reaction forming group or through macromolecule surface
During the magnetic particle processed, it is preferred that anti-Miao's Le Shi pipe hormone monoclonal antibody is 1:20～100 with the mass ratio of magnetic particle.
Surface can be that surface is with amino, carboxyl, first with the magnetic particle for protein chemistry reaction forming group
Benzenesulfonyl or the magnetic particle of Oxyranyle.
Surface is preferably 0.05 μm～3 μm with the particle diameter of the magnetic particle for protein chemistry reaction forming group.
By solid phase carrier for surface with as a example by the magnetic particle of carboxyl, anti-Miao's Le Shi pipe hormone monoclonal antibody is coated
The preparation process of solid phase carrier can be: taking the suspension of carboxylated magnetic particle, Magneto separate uses MES buffer after removing supernatant
Resuspended, it is subsequently added into EDC aqueous solution, the surface carboxyl groups of the magnetic particle of activated carboxyl, it is subsequently added into anti-Miao's Le Shi pipe hormone list
Clonal antibody, suspendible 2h～10h under room temperature, Magneto separate is resuspended with Tris buffer after removing supernatant, obtains anti-Miao Le Shi pipe and swashs
The element coated carboxylated magnetic particle of monoclonal antibody.Wherein, the concentration of MES (2-(N-morpholine) ethyl sulfonic acid) buffer is
0.02M, pH are 5.5；The concentration of Tris buffer is 0.1M and is 8.0 containing 2%BSA, pH；EDC (1-ethyl-3-(3-bis-
Methyl aminopropyl)-carbodiimides) concentration of aqueous solution is 10mg/mL～20mg/mL, the ratio of EDC and carboxylated magnetic particle
Example is 0.05:0.1～1.
In another embodiment, in anti-Miao's Le Shi pipe coated solid phase carrier of hormone monoclonal antibody, solid phase carrier
Upper coupling has Streptavidin, Avidin or neutral Avidin, and in anti-Miao's Le Shi pipe hormone monoclonal antibody, coupling has biotin.
Anti-Miao's Le Shi pipe coated solid phase carrier of hormone monoclonal antibody can be that the commercialization Avidin directly bought is solid
Phase carrier, it is also possible to be the Avidin solid phase carrier prepared by Avidin chemical bond and other solid phase carriers of coupling.
In anti-Miao's Le Shi pipe hormone monoclonal antibody of chemiluminescent labels labelling, chemiluminescent labels is different Rumi
Promise, tris (bipyridine) ruthenium, acridinium ester, alkali phosphatase or horseradish peroxidase.
Preferably, in the chemiluminescent labels of inhibin labeling of monoclonal antibody, anti-Miao's Le Shi pipe hormone monoclonal anti
Body is 50:1～10 with the ratio of chemiluminescent labels.
In one embodiment, in anti-Miao's Le Shi pipe hormone monoclonal antibody of chemiluminescent labels labelling, chemistry is sent out
On signal thing, coupling has Streptavidin, Avidin or neutral Avidin, coupling in anti-Miao's Le Shi pipe hormone monoclonal antibody
There is biotin.
Having Streptavidin with coupling on chemiluminescent labels, in anti-Miao's Le Shi pipe hormone monoclonal antibody, coupling has life
As a example by thing element, as a example by chemiluminescent labels is acridinium ester, anti-Miao's Le Shi pipe hormone monoclonal of chemiluminescent labels labelling
The preparation process of antibody is as follows: takes anti-Miao's Le Shi pipe hormone monoclonal antibody solution, adds the phosphate-buffered of 500 μ L pH8.0
Liquid, adds 0.1mg biotin succinimide acid imide (Biotin-NHS) mixing, and lucifuge reaction under room temperature is taken out after 1h～2h, centrifugal
Desalting column desalting processing, processes with pure water and TBS buffer in desalination processes the most respectively, collects the liquid in desalination pipe
Body preserves, and obtains biotin labeled anti-Miao's Le Shi pipe hormone monoclonal antibody；Take solution of streptavidin, add 0.1M～
The carbonate buffer solution of 0.2M pH9.0～9.5, mixing, it is subsequently adding acridinium ester mixing, lucifuge reaction under room temperature, after 1～2h
Take out, be centrifuged desalting column desalting processing with zeba, desalination processes processes with pure water and TBS buffer the most respectively,
It is eventually adding the Streptavidin of the acridinium ester label obtained；By biotin labeled anti-Miao's Le Shi pipe hormone monoclonal antibody and
The Streptavidin mixing of acridinium ester label, obtains anti-Miao's Le Shi pipe hormone monoclonal antibody of acridinium ester label.
In another embodiment, in anti-Miao's Le Shi pipe hormone monoclonal antibody of chemiluminescent labels labelling, chemistry is sent out
Signal thing is directly connected to by chemical reaction key with anti-Miao's Le Shi pipe hormone monoclonal antibody or is connected by protein-crosslinking agent.
Protein-crosslinking agent can be at least one in phosphinylidyne diimmonium salt and butanimide.
Preferably, protein-crosslinking agent is selected from 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine, 1-ethyl-(3-bis-
Dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate, in N-hydroxy-succinamide and sulfonated N-hydroxy-succinamide extremely
Few one.
As a example by chemiluminescent labels is as acridinium ester, chemiluminescent labels and anti-Miao's Le Shi pipe hormone monoclonal antibody
It is directly connected to prepare the process of anti-Miao's Le Shi pipe hormone monoclonal antibody of chemiluminescent labels labelling by chemical reaction key
As follows: to take anti-Miao's Le Shi pipe hormone monoclonal antibody, add carbonate buffer solution, mixing, be subsequently adding acridinium ester mixing, room temperature
Lower lucifuge is reacted, and takes out after 1h～2h, centrifugal desalting column desalting processing, delays with pure water and TBS the most respectively in desalination processes
Rush liquid to process, obtain anti-Miao's Le Shi pipe hormone monoclonal antibody of acridinium ester label.
With chemiluminescent labels as alkali phosphatase, protein-crosslinking agent is 1-ethyl-(3-dimethylaminopropyl) carbon
As a example by acyl diimmonium salt hydrochlorate and N-hydroxy-succinamide, chemiluminescent labels and anti-Miao's Le Shi pipe hormone monoclonal antibody
The process being connected the anti-Miao's Le Shi pipe hormone monoclonal antibody preparing chemiluminescent labels labelling by protein-crosslinking agent is as follows:
Take 1mg anti-Miao Le Shi pipe hormone monoclonal antibody solution, 1mg alkali phosphatase, add the 2-(N-morpholine) of 500 μ L pH5.5
Ethyl sulfonic acid (MES) buffer, adds 1mg1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate and 1mg N-hydroxyl
Base butanimide mixes, lucifuge reaction under room temperature, takes out after 1h～2h, and centrifugal desalting column desalting processing is first in desalination processes
Process with pure water and TBS buffer the most respectively, collect the fluid preservation in desalination pipe, obtain alkali phosphatase enzyme mark
Anti-Miao's Le Shi pipe hormone monoclonal antibody.
In other examples, above-mentioned anti-Miao's Le Shi pipe hormone chemiluminescence immune detection reagent kit also includes that chemistry is sent out
Light substrate solution.
Chemoluminescent substrate includes A liquid and B liquid.A liquid can be H2O2Solution, B liquid can be NaOH solution.
In the present embodiment, A liquid be concentration be the H of 0.1mol/L2O2Solution, B liquid be concentration be that the NaOH of 0.25mol/L is molten
Liquid.
In other examples, above-mentioned anti-Miao's Le Shi pipe hormone chemiluminescence immune detection reagent kit also includes anti-Miao Le
Family name's pipe hormone calibration product.
The preparation process of anti-Miao's Le Shi pipe hormone calibration product is as follows: uses calibration to savor buffer and is joined by anti-Miao's Le Shi pipe hormone
Make the solution that concentration is respectively anti-Miao's Le Shi pipe hormone of 0～30ng/mL.
Concrete, anti-Miao's Le Shi pipe hormone calibration product be concentration be respectively 0ng/mL, 0.7ng/mL, 2.5ng/mL,
The solution of anti-Miao's Le Shi pipe hormone of 4.65ng/mL, 13.58ng/mL and 28.56ng/mL.
This anti-Miao's Le Shi pipe hormone chemiluminescence immune detection reagent kit can be with automatic chemiluminescence immunoassay
Instrument is detection instrument, completes the detection of anti-Miao's Le Shi pipe hormone.This anti-Miao's Le Shi pipe hormone chemiluminescence immunoassay detectable
Box, through experiment, its detection sensitivity reaches 0.01ng/mL, relative to the detection method spirit of traditional anti-Miao's Le Shi pipe hormone
Sensitivity at least improves 10 times, and the accuracy of detection of this anti-Miao's Le Shi pipe hormone chemiluminescence immune detection reagent kit is higher.
This anti-Miao's Le Shi pipe hormone chemiluminescence immune detection reagent kit is exempted from first with Full-automatic chemiluminescence when using
Epidemic disease analyser antagonism Miao's Le Shi pipe hormone calibration product detect, and draw standard curve, are built in computer software, test actual sample
This, calculate concentration of specimens, finally antagonism Miao's Le Shi pipe hormone automatic chemiluminescence immunoassay system according to sample luminous value
Carry out the evaluation of performance (sensitivity, linear, precision, interference).
This anti-Miao's Le Shi pipe hormone chemiluminescence immune detection reagent kit, has the advantage that
1, selection acridinium ester is as marker material, and is applied to chemiluminescence immunoassay system, and this luminescence system is straight
Connecing chemiluminescence, compared with traditional enzyme-catalyzed chemical luminescence, this reaction need not the participation of enzyme, more cost-effective；
2, the acridinium ester selected or alkali phosphatase chemiluminescence immunoassay system detection sensitivity are high, it is possible to reach
0.01ng/mL, the detection method sensitivity comparing other at least improves 10 times；
3, select the chemiluminescence immunoassay system range of linearity width of acridinium ester, more than 25ng/mL can be reached；
4, acridinium ester chemiluminescent immunoassay system repeatability is high, and in batch and difference between batch is all within 5%, this is other
Chemiluminescence immunoassay system is unapproachable；
5, chemiluminescence immunoassay system has realized the quantitative of sample, by built-in standard curve to test software, only
Need test sample just can directly obtain the concentration value of sample；
6, chemiluminescence immunoassay system can realize the interpolation of full-automation, reagent and sample has instrument to complete entirely,
Operate easier, decrease artificial error.
In sum, this anti-Miao's Le Shi pipe hormone chemiluminescence immune detection reagent kit testing cost low, highly sensitive,
Detection the range of linearity is wide, repeatability is high, can be quantitative, simple to operate.
Additionally, use this anti-Miao's Le Shi pipe hormone chemiluminescence immune detection reagent kit, by measuring specific crowd
AMH concentration level or distribution, experimenter's AMH concentration compares therewith, if the AMH concentration level of deviation demarcation crowd, then prompting is subject to
Examination person be non-health or function weak.Same experimenter can also be carried out continuous AMH level monitoring, be correlated with merit assessing reproduction
Energy.Can be used for auxiliary diagnosis or predictive disease, monitor and predict health status: diagnosis GCT, diagnostic abnormal development,
Diagnosis of polycystic ovary syndrome, monitoring ovarian reserve power, the ovary response of prediction external fertilization associating embryo transfer technology (IVF)
Property, prevention ovarian hyperstimulation syndrome (OHSS), assessment male reproductive function etc..
The invention also discloses the preparation side of a kind of above-mentioned anti-Miao's Le Shi pipe hormone chemiluminescence immune detection reagent kit
Method, comprises the steps:
S110, anti-Miao's Le Shi pipe hormone monoclonal antibody and solid phase carrier mixed and fully reacts, obtaining anti-Miao Le Shi
The coated solid phase carrier of pipe hormone monoclonal antibody.
In anti-Miao's Le Shi pipe coated solid phase carrier of hormone monoclonal antibody, anti-Miao's Le Shi pipe hormone monoclonal antibody is permissible
Behaviour source, gene engineering expression or animal sources.
In one embodiment, in anti-Miao's Le Shi pipe coated solid phase carrier of hormone monoclonal antibody, solid phase carrier is
Surface is with the silica-base material that can carry out protein physical absorption for the magnetic particle of protein chemistry reaction forming group, surface
Or the magnetic particle through macromolecule surface process.
Solid phase carrier is that surface is with the magnetic particle for protein chemistry reaction forming group or through macromolecule surface
During the magnetic particle processed, it is preferred that anti-Miao's Le Shi pipe hormone monoclonal antibody is 1:20～100 with the mass ratio of magnetic particle.
Surface can be that surface is with amino, carboxyl, first with the magnetic particle for protein chemistry reaction forming group
Benzenesulfonyl or the magnetic particle of Oxyranyle.
Surface is preferably 0.05 μm～3 μm with the particle diameter of the magnetic particle for protein chemistry reaction forming group.
When, in anti-Miao's Le Shi pipe coated solid phase carrier of hormone monoclonal antibody, solid phase carrier is that surface is with for albumen
During the magnetic particle of matter chemical reaction linking group, S110 is: take surface with the magnetic for protein chemistry reaction forming group
The suspension of microgranule, Magneto separate is resuspended with buffer after removing supernatant, and then activator activating surface is with for protein chemistry
The Surface attachment groups of the magnetic particle of reaction forming group, is subsequently added into anti-Miao's Le Shi pipe hormone monoclonal antibody, fills under room temperature
Dividing reaction, Magneto separate is resuspended after removing supernatant, obtains the coated surface of anti-Miao's Le Shi pipe hormone monoclonal antibody with for egg
The magnetic particle of white matter chemical reaction linking group.
By solid phase carrier for surface with as a example by the magnetic particle of carboxyl, anti-Miao's Le Shi pipe hormone monoclonal antibody is coated
The preparation process of solid phase carrier can be: taking the suspension of carboxylated magnetic particle, Magneto separate uses MES buffer after removing supernatant
Resuspended, it is subsequently added into EDC aqueous solution, the surface carboxyl groups of the magnetic particle of activated carboxyl, it is subsequently added into anti-Miao's Le Shi pipe hormone list
Clonal antibody, suspendible 2h～10h under room temperature, Magneto separate is resuspended with Tris buffer after removing supernatant, obtains anti-Miao Le Shi pipe and swashs
The element coated carboxylated magnetic particle of monoclonal antibody.Wherein, the concentration of MES (2-(N-morpholine) ethyl sulfonic acid) buffer is
0.02M, pH are 5.5；The concentration of Tris buffer is 0.1M and is 8.0 containing 2%BSA, pH；EDC (1-ethyl-3-(3-bis-
Methyl aminopropyl)-carbodiimides) concentration of aqueous solution is 10mg/mL～20mg/mL, the ratio of EDC and carboxylated magnetic particle
Example is 0.05:0.1～1.
In another embodiment, in anti-Miao's Le Shi pipe coated solid phase carrier of hormone monoclonal antibody, solid phase carrier
Upper coupling has Streptavidin, Avidin or neutral Avidin, and in anti-Miao's Le Shi pipe hormone monoclonal antibody, coupling has biotin.
Anti-Miao's Le Shi pipe coated solid phase carrier of hormone monoclonal antibody can be that the commercialization Avidin directly bought is solid
Phase carrier, it is also possible to be the Avidin solid phase carrier prepared by Avidin chemical bond and other solid phase carriers of coupling.
In anti-Miao's Le Shi pipe coated solid phase carrier of hormone monoclonal antibody, on solid phase carrier coupling have Streptavidin,
Avidin or neutral Avidin, when in anti-Miao's Le Shi pipe hormone monoclonal antibody, coupling has biotin, S110 is: coupling is had chain
Mould Avidin, Avidin or the solid phase carrier of neutral Avidin and coupling have anti-Miao's Le Shi pipe hormone monoclonal antibody of biotin
Buffer mixes, fully obtains anti-Miao's Le Shi pipe coated solid phase carrier of hormone monoclonal antibody after reaction.
S120, chemiluminescent labels and anti-Miao's Le Shi pipe hormone monoclonal antibody mixed and fully reacts, being changed
Learn anti-Miao's Le Shi pipe hormone monoclonal antibody of luminescent label.
The order of S110 and S120 can be replaced, and does not affect this method.
In anti-Miao's Le Shi pipe hormone monoclonal antibody of chemiluminescent labels labelling, anti-Miao's Le Shi pipe hormone monoclonal anti
Body can be people source, gene engineering expression or animal sources.
Anti-Miao's Le Shi pipe coated solid phase carrier of hormone monoclonal antibody and anti-Miao Le Shi of chemiluminescent labels labelling
In pipe hormone monoclonal antibody, the anti-Miao's Le Shi pipe hormone monoclonal antibody each used can be identical, it is also possible to differs.
In anti-Miao's Le Shi pipe hormone monoclonal antibody of chemiluminescent labels labelling, chemiluminescent labels is different Rumi
Promise, tris (bipyridine) ruthenium, acridinium ester, alkali phosphatase or horseradish peroxidase.
Preferably, in the chemiluminescent labels of inhibin labeling of monoclonal antibody, anti-Miao's Le Shi pipe hormone monoclonal anti
Body is 50:1～10 with the ratio of chemiluminescent labels.
In one embodiment, in anti-Miao's Le Shi pipe hormone monoclonal antibody of chemiluminescent labels labelling, chemistry is sent out
On signal thing, coupling has Streptavidin, Avidin or neutral Avidin, coupling in anti-Miao's Le Shi pipe hormone monoclonal antibody
There is biotin.
In anti-Miao's Le Shi pipe hormone monoclonal antibody of chemiluminescent labels labelling, on chemiluminescent labels, coupling has
Streptavidin, Avidin or neutral Avidin, when in anti-Miao's Le Shi pipe hormone monoclonal antibody, coupling has biotin, S120
For: coupling have Streptavidin, Avidin or the chemiluminescent labels of neutral Avidin and coupling have anti-Miao of biotin
Le Shi pipe hormone monoclonal antibody mixes in buffer, fully obtains anti-Miao Le Shi of chemiluminescent labels labelling after reaction
Pipe hormone monoclonal antibody.
Concrete, there is Streptavidin with coupling on chemiluminescent labels, in anti-Miao's Le Shi pipe hormone monoclonal antibody
As a example by coupling has biotin, as a example by chemiluminescent labels is acridinium ester, anti-Miao Le Shi pipe of chemiluminescent labels labelling swashs
The preparation process of element monoclonal antibody is as follows: takes anti-Miao's Le Shi pipe hormone monoclonal antibody solution, adds the phosphorus of 500 μ L pH8.0
Phthalate buffer, adds 0.1mg biotin succinimide acid imide (Biotin-NHS) mixing, and lucifuge reaction under room temperature, after 1h～2h
Take out, centrifugal desalting column desalting processing, desalination processes processes with pure water and TBS buffer the most respectively, collect de-
Fluid preservation in salt pipe, obtains biotin labeled anti-Miao's Le Shi pipe hormone monoclonal antibody；Take solution of streptavidin, add
Enter the carbonate buffer solution of 0.1M～0.2M pH 9.0～9.5, mixing, it is subsequently adding acridinium ester mixing, under room temperature, lucifuge is anti-
Should, take out after 1～2h, be centrifuged desalting column desalting processing with zeba, the most respectively with pure water and TBS buffer in desalination processes
Process, be eventually adding the Streptavidin of the acridinium ester label obtained；By biotin labeled anti-Miao's Le Shi pipe hormone list
The Streptavidin mixing of clonal antibody and acridinium ester label, obtains anti-Miao's Le Shi pipe hormone monoclonal anti of acridinium ester label
Body.
In another embodiment, in anti-Miao's Le Shi pipe hormone monoclonal antibody of chemiluminescent labels labelling, chemistry is sent out
Signal thing is directly connected to by chemical reaction key with anti-Miao's Le Shi pipe hormone monoclonal antibody or is connected by protein-crosslinking agent.
Protein-crosslinking agent can be at least one in phosphinylidyne diimmonium salt and butanimide.
Preferably, protein-crosslinking agent is selected from 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine, 1-ethyl-(3-bis-
Dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate, in N-hydroxy-succinamide and sulfonated N-hydroxy-succinamide extremely
Few one.
In anti-Miao's Le Shi pipe hormone monoclonal antibody of chemiluminescent labels labelling, chemiluminescent labels and anti-Miao Le
When family name's pipe hormone monoclonal antibody is directly connected to by chemical reaction key, S120 is: by anti-Miao's Le Shi pipe hormone monoclonal antibody
Mixing in buffer with chemiluminescent labels, the anti-Miao Le Shi pipe fully obtaining chemiluminescent labels labelling after reaction swashs
Element monoclonal antibody.
Concrete, as a example by chemiluminescent labels is as acridinium ester, chemiluminescent labels and anti-Miao's Le Shi pipe hormone list
Clonal antibody is directly connected to prepare anti-Miao's Le Shi pipe hormone monoclonal anti of chemiluminescent labels labelling by chemical reaction key
The process of body is as follows: take anti-Miao's Le Shi pipe hormone monoclonal antibody, adds carbonate buffer solution, mixing, is subsequently adding acridinium ester
Mixing, lucifuge reaction under room temperature, to take out after 1h～2h, centrifugal desalting column desalting processing, the most respectively with pure in desalination processes
Water and TBS buffer process, and obtain anti-Miao's Le Shi pipe hormone monoclonal antibody of acridinium ester label.
In anti-Miao's Le Shi pipe hormone monoclonal antibody of chemiluminescent labels labelling, chemiluminescent labels and anti-Miao Le
When family name's pipe hormone monoclonal antibody is connected by protein-crosslinking agent, S120 is: by anti-Miao's Le Shi pipe hormone monoclonal antibody, chemistry
Luminous marker and protein-crosslinking agent mix in buffer, fully obtain the anti-Miao Le of chemiluminescent labels labelling after reaction
Family name's pipe hormone monoclonal antibody.
Concrete, with chemiluminescent labels as alkali phosphatase, protein-crosslinking agent is 1-ethyl-(3-dimethylamino
Propyl group) as a example by phosphinylidyne diimmonium salt hydrochlorate and N-hydroxy-succinamide, chemiluminescent labels and anti-Miao's Le Shi pipe hormone list
Clonal antibody connects anti-Miao's Le Shi pipe hormone monoclonal antibody of prepared chemiluminescent labels labelling by protein-crosslinking agent
Process is as follows: takes 1mg anti-Miao Le Shi pipe hormone monoclonal antibody solution, 1mg alkali phosphatase, adds the 2-of 500 μ L pH5.5
(N-morpholine) ethyl sulfonic acid (MES) buffer, adds 1mg1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate
Mix with 1mg N-hydroxy-succinamide, lucifuge reaction under room temperature, take out after 1h～2h, centrifugal desalting column desalting processing, de-
Process with pure water and TBS buffer the most respectively during salt, collect the fluid preservation in desalination pipe, obtain alkalescence phosphorus
Anti-Miao's Le Shi pipe hormone monoclonal antibody of acid enzyme labelling.
In other examples, anti-Miao's Le Shi pipe hormone chemiluminescence immune detection reagent kit also includes at the bottom of chemiluminescence
Thing liquid.
Chemoluminescent substrate includes A liquid and B liquid.A liquid can be H2O2Solution, B liquid can be NaOH solution.
In the present embodiment, A liquid be concentration be the H of 0.1mol/L2O2Solution, B liquid be concentration be that the NaOH of 0.25mol/L is molten
Liquid.
In other examples, anti-Miao's Le Shi pipe hormone chemiluminescence immune detection reagent kit also includes that anti-Miao Le Shi manages
Hormone calibration product.
The preparation process of anti-Miao's Le Shi pipe hormone calibration product is as follows: uses calibration to savor buffer and is joined by anti-Miao's Le Shi pipe hormone
Make the solution that concentration is respectively anti-Miao's Le Shi pipe hormone of 0～30ng/mL.
Concrete, anti-Miao's Le Shi pipe hormone calibration product be concentration be respectively 0ng/mL, 0.7ng/mL, 2.5ng/mL,
The solution of anti-Miao's Le Shi pipe hormone of 4.65ng/mL, 13.58ng/mL and 28.56ng/mL.
The invention also discloses and manage with anti-Miao Le Shi of above-mentioned anti-Miao's Le Shi pipe hormone chemiluminescence immune detection reagent kit
The detection method of the concentration of hormone, comprises the steps:
S210, use anti-Miao's Le Shi pipe coated solid phase carrier of hormone monoclonal antibody and chemiluminescent labels labelling
Anti-Miao's Le Shi pipe hormone monoclonal antibody carries out sandwich immunoassay test to sample to be tested, the anti-Miao's Le Shi pipe hormone in sample to be tested
With anti-Miao's Le Shi pipe coated solid phase carrier of hormone monoclonal antibody and anti-Miao's Le Shi pipe hormone of chemiluminescent labels labelling
Monoclonal antibody reactive, forms sandwich complex, after over cleaning separates, records the luminous signal of sample to be tested.
S220, use anti-Miao's Le Shi pipe coated solid phase carrier of hormone monoclonal antibody and chemiluminescent labels labelling
Anti-Miao's Le Shi pipe hormone monoclonal antibody antagonism Miao's Le Shi pipe hormone calibration product carry out sandwich immunoassay test, anti-Miao's Le Shi pipe hormone
Anti-Miao's Le Shi pipe hormone in calibration product and anti-Miao's Le Shi pipe coated solid phase carrier of hormone monoclonal antibody and chemiluminescence mark
Anti-Miao's Le Shi pipe hormone monoclonal antibody reactive of note substance markers, forms sandwich complex, after over cleaning separates, records anti-Miao
The luminous intensity of Le Shi pipe hormone calibration product, according to luminous intensity and anti-Miao's Le Shi pipe hormone of anti-Miao's Le Shi pipe hormone calibration product
The concentration of calibration product builds the standard curve that transverse and longitudinal coordinate is respectively anti-Miao's Le Shi pipe hormone of concentration and luminous intensity.
The order of S210 and S220 can be replaced, and does not affect this method.
The preparation process of anti-Miao's Le Shi pipe hormone calibration product is as follows: uses calibration to savor buffer and is joined by anti-Miao's Le Shi pipe hormone
Make the solution that concentration is respectively anti-Miao's Le Shi pipe hormone of 0～30ng/mL.
Concrete, anti-Miao's Le Shi pipe hormone calibration product be concentration be respectively 0ng/mL, 0.7ng/mL, 2.5ng/mL,
The solution of anti-Miao's Le Shi pipe hormone of 4.65ng/mL, 13.58ng/mL and 28.56ng/mL.
S230, the luminous signal of the sample to be tested obtained by S210 bring the standard of anti-Miao's Le Shi pipe hormone that S220 obtains into
In curve, the concentration of the anti-Miao's Le Shi pipe hormone being calculated in sample to be tested.
It it is below specific embodiment.
Embodiment 1: the preparation of anti-Miao's Le Shi pipe hormone chemiluminescence immune detection reagent kit
(1) preparation of AMH monoclonal antibody coated magnetic granule:
Take carboxylated magnetic particle (MagnaBindTM, the article No. 21353) suspension that 50mg particle diameter is 0.05 μm～3 μm,
Magneto separate removes supernatant, resuspended with the MES buffer that 0.02M, pH are 5.5, adds 10mg/mL's newly configured for 0.5mL～2mL
EDC aqueous solution, activated magnetic beads surface carboxyl groups, add the AMH monoclonal antibody (Anshlabs, AA011) of 1mg～3mg, under room temperature
Suspendible 2h～10h, Magneto separate, remove supernatant, be resuspended to 1mg/mL with the Tris buffer that 0.1M pH is 8.0 containing 2%BSA,
Obtain AMH monoclonal antibody coated magnetic granule, every bottle of 5mL subpackage be stored in 4 DEG C standby.
(2) preparation of the AMH monoclonal antibody of acridinium ester label:
Take AMH monoclonal traget antibody (Anshlabs, the AA012) solution of 500 μ L 1.0mg/mL, add 500 μ L
The carbonate buffer solution of 0.1-0.2M pH 9.0-9.5, mixing, it is subsequently adding the acridinium ester mixing of 10～20 μ L 5mg/mL, room
The lower lucifuge reaction of temperature, takes out after 1～2h, is centrifuged desalting column desalting processing with the zeba of 2mL, the most respectively with pure in desalination processes
Water purification and TBS buffer process, and are eventually adding the AMH monoclonal antibody solution of the acridinium ester label obtained, and collect centrifugal
Liquid in pipe is in control the AMH monoclonal antibody of acridinium ester label to preserving, every bottle of 1mL subpackage be stored in 4 DEG C standby.
(3) preparation of AMH calibration product:
With standard substance buffer (40mM Tris-HCl, 0.5%BSA, 1%NaCl, pH 8.0) by humanization AMH albumen
Being configured to concentration is 0ng/mL, 0.7ng/mL, 2.5ng/mL, 4.65ng/mL, 13.58ng/mL, 28.56ng/mL, every bottle
0.5mL subpackage, after lyophilised process, 4 DEG C save backup.
Embodiment 2:AMH acridinium ester chemiluminescent immunologic detection method
Anti-Miao's Le Shi pipe hormone chemiluminescence that the present invention prepares with Full-automatic chemiluminescence immunoassay analysis meter and embodiment 1
Immunity detection reagent is detection instrument, the methodology pattern of the present invention be sandwich assay, i.e. instrument be sequentially added into 50 μ L sample,
The AMH monoclonal antibody coated magnetic granule of 50 μ L and the AMH monoclonal antibody of the acridinium ester label of 50 μ L, react 10min
After, carrying out Magneto separate, reactant mixture is sent into darkroom by instrument, is sequentially added into luminous substrate A liquid (containing 0.1M HNO3With 0.5%
H2O2) and B liquid (containing 0.25M NaOH) carry out luminescence-producing reaction, finally record luminous intensity, calculate sample from standard curve
AMH content.
Embodiment 3: anti-Miao's Le Shi pipe hormone chemiluminescence immune detection reagent kit performance evaluation that embodiment 1 prepares
Use the antagonism Miao's Le Shi pipe hormone calibration product of the method in embodiment 2 to detect, obtain drawing standard curve such as
Shown in Fig. 1.
The detection of sensitivity:
With reference to CLSI EP17-A file recommendation experimental program, calculate the sensitivity of AMH chemiluminescence immunoassay kit, ask
The sensitivity obtained is 0.006ng/mL.
Linear detection:
It is that 0.01ng/mL, 0.5ng/mL, 2ng/mL, 10ng/mL, 25ng/mL standard substance do linear analysis to concentration, meter
Calculating linearly dependent coefficient, r=0.9991, it addition, this test kit is 0.01-25ng/mL to the range of linearity of AMH sample detection.
Precision measures:
Taking concentration is 0.1ng/mL and two AMH samples of 10ng/mL, each concentration of each sample respectively do 3 parallel, with three
Batch test kit detects, and calculates test kit and criticizes interior and difference between batch, and result shows that this test kit is criticized interior and difference between batch and is respectively less than
5%.
Interference is tested:
Take pooled serum to add chaff interference respectively and include: conjugated bilirubin, unconjugated bilirubin, hemoglobin, ascorbic acid,
Glyceride, adds mass ratio and carries out according to 1:20, pooled serum after measuring pooled serum respectively and with the addition of various chaff interference
Measured value, calculates deviation therebetween, with ± 10% as tolerance interval.Result shows, interference all reaches the file of NCCLS
Standard, can be used for the accurate evaluation of clinical laboratory's AMH situation.
Embodiment 4, the contrast experiment of anti-Miao's Le Shi pipe hormone chemiluminescence immune detection reagent kit
Respectively with the chemical luminescence detection method in embodiment 2 and traditional enzyme linked immunosorbent assay be 0 to concentration,
The AMH sample of 50pg/mL detects, and zero-dose sample carries out 20 retests, calculate zero-dose sample average (M) and
SD, brings in calibration equation by the RLU value of M+2SD, obtains the concentration value of correspondence, is detection sensitivity.Two kinds of method detections
Sensitivity is compared, and data are as shown in the table:
As can be seen from the above table, the sensitivity of chemical luminescence detection method relatively enzyme linked immunosorbent assay improves about 50 times.
Embodiment 5: the preparation of anti-Miao's Le Shi pipe hormone chemiluminescence immune detection reagent kit
(1) preparation of AMH monoclonal antibody coated magnetic granule:
Take magnetic particle (Dynal, the 30110D) suspension of the tosylation that 50mg particle diameter is 0.05 μm～2 μm, magnetic
Supernatant is removed in separation, and the borate buffer solution with pH9.0～11.0 is resuspended, adds the AMH monoclonal antibody of 1mg～3mg
(Anshlabs, AA011), adds 0.5mL～2mL saturated ammonium sulfate solution, rotates hybrid reaction 20h～30h at 37 DEG C, and magnetic divides
From, remove supernatant, be resuspended to 1mg/mL with the Tris buffer that 0.1M pH is 8.0 containing 2%BSA, obtain AMH monoclonal anti
Body coated magnetic granule, every bottle of 5mL subpackage be stored in 4 DEG C standby.
(2) preparation of the AMH monoclonal antibody of alkali phosphatase enzyme mark:
Take AMH monoclonal traget antibody (Anshlabs, AA012) of 500 μ L 2.0mg/mL, add 500 μ L pH5.0's
MES acidic buffer, adds the mixing of 1mg alkali phosphatase, is subsequently adding 1mg 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne
Diimmonium salt hydrochlorate (EDC.HCL) cross-linking agent or addition 1mg 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine hydrochloric acid
Salt (EDC.HCL) and 1mg N-hydroxy-succinamide (NHS) mixed cross-linker, lucifuge reaction under room temperature, take out after 1h～2h,
Centrifugal desalting column desalting processing, processes with pure water and TBS buffer in desalination processes the most respectively, collects in desalination pipe
Fluid preservation.AKTA protein purification system on desalinization liquor is purified, collects the AMH Dan Ke obtaining alkali phosphatase enzyme mark
Grand antibody, every bottle of 1mL subpackage be stored in 4 DEG C standby.
(3) preparation of AMH calibration product:
With standard substance buffer (40mM Tris-HCl, 0.5%BSA, 1%NaCl, pH 8.0) by humanization AMH albumen
Being configured to concentration is 0ng/mL, 0.7ng/mL, 2.5ng/mL, 4.65ng/mL, 13.58ng/mL, 28.56ng/mL, every bottle
0.5mL subpackage, after lyophilised process, 4 DEG C save backup.
Embodiment 6:AMH alkali phosphatase chemical luminous immune detection method
Anti-Miao's Le Shi pipe hormone chemiluminescence that the present invention prepares with Full-automatic chemiluminescence immunoassay analysis meter and embodiment 5
Immunity detection reagent is detection instrument, the methodology pattern of the present invention be sandwich assay, i.e. instrument be sequentially added into 20 μ L sample,
The AMH monoclonal antibody coated magnetic granule of 50 μ L and the AMH antibody of the alkali phosphatase enzyme mark of 50 μ L, after reaction 10min,
Carrying out Magneto separate, darkroom sent into by reactant mixture by instrument, adds AMPPD luminous substrate and carries out luminescence-producing reaction, and finally record is sent out
Light intensity, calculates the AMH content of sample from standard curve.
Embodiment 7: anti-Miao's Le Shi pipe hormone chemiluminescence immune detection reagent kit performance evaluation that embodiment 5 prepares
Use the antagonism Miao's Le Shi pipe hormone calibration product of the method in embodiment 6 to detect, obtain drawing standard curve such as
Shown in Fig. 2.
The detection of sensitivity:
With reference to CLSI EP17-A file recommendation experimental program, calculate the sensitivity of AMH chemiluminescence immunoassay kit, ask
The sensitivity obtained is 0.06ng/mL.
Embodiment 8: the preparation of anti-Miao's Le Shi pipe hormone chemiluminescence immune detection reagent kit
(1) preparation of AMH monoclonal antibody coated magnetic granule.
Consistent with (1) in embodiment 5.
(2) preparation of biotin labeling AMH monoclonal antibody:
Take the AMH monoclonal traget antibody of 500 μ L 2.0mg/mL, add the phosphate buffer of 500 μ L pH8.0, add
Enter 0.1mg biotin succinimide acid imide (Biotin-NHS) mixing, lucifuge reaction under room temperature, take out after 1-2h, centrifugal desalting column
Desalting processing, processes with pure water and TBS buffer in desalination processes the most respectively, collects the liquid in desalination pipe and protects
Deposit.Obtain biotin labeled AMH monoclonal antibody, every bottle of 1mL subpackage be stored in 4 DEG C standby.
(3) preparation of the Streptavidin of acridinium ester label:
Taking the Streptavidin of 500 μ L 1.0mg/mL, the carbonate adding 500 μ L 0.1-0.2M pH 9.0-9.5 delays
Rush liquid, mixing, it is subsequently adding the acridinium ester mixing of 10-20 μ L 5mg/mL, lucifuge reaction under room temperature, takes out after 1-2h, use 2mL
Zeba be centrifuged desalting column desalting processing, desalination processes processes with pure water and TBS buffer the most respectively, finally adds
Enter the Streptavidin of the acridinium ester label obtained, collect the liquid in centrifuge tube to preserving the strepto-being in control acridinium ester label
Avidin, every bottle of 1mL subpackage be stored in 4 DEG C standby.
(4) preparation of the AMH monoclonal antibody of acridinium ester label: by biotin labeling AMH monoclonal antibody and acridinium ester
The Streptavidin of labelling mixes according to 4:1 ratio, obtains the AMH monoclonal antibody of acridinium ester label.
(5) preparation of AMH calibration product:
With standard substance buffer (40mM Tris-HCl, 0.5%BSA, 1%NaCl, pH 8.0) by humanization AMH albumen
Being configured to concentration is 0ng/mL, 0.7ng/mL, 2.5ng/mL, 4.65ng/mL, 13.58ng/mL, 28.56ng/mL, every bottle
0.5mL subpackage, after lyophilised process, 4 DEG C save backup.
Embodiment 9: the method for Avidin-Biotin systemic immunity detection AMH
The anti-Miao's Le Shi pipe hormone chemiluminescence immunoassay inspection prepared with Full-automatic chemiluminescence immunoassay analysis meter and embodiment 8
Test agent box be detection instrument, i.e. instrument be sequentially added into the sample of 10 μ L, 50 μ L AMH monoclonal antibody coated magnetic granule with
And 50 biotin labeled AMH antibody of μ L, the Streptavidin of 50 μ L acridinium ester label, after reaction 20min, carry out magnetic and divide
From, reactant mixture is sent into darkroom by instrument, is sequentially added into luminous substrate A liquid (containing 0.1M HNO3And 0.5%H2O2) and B liquid
(containing 0.25M NaOH) carries out luminescence-producing reaction, finally records luminous intensity, and the AMH calculating sample from standard curve contains
Amount.
Embodiment 10: anti-Miao's Le Shi pipe hormone chemiluminescence immune detection reagent kit performance evaluation that embodiment 8 prepares
The detection of sensitivity:
With reference to CLSI EP17-A file recommendation experimental program, calculate the sensitivity of AMH chemiluminescence immunoassay kit, ask
The sensitivity obtained is 0.001ng/mL.
Embodiment described above only have expressed the several embodiments of the present invention, and it describes more concrete and detailed, but also
Therefore the restriction to the scope of the claims of the present invention can not be interpreted as.It should be pointed out that, for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, it is also possible to make some deformation and improvement, these broadly fall into the guarantor of the present invention
Protect scope.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (18)
1. anti-Miao's Le Shi pipe hormone chemiluminescence immune detection reagent kit, it is characterised in that including: anti-Miao's Le Shi pipe hormone
The coated solid phase carrier of monoclonal antibody and anti-Miao's Le Shi pipe hormone monoclonal antibody of chemiluminescent labels labelling.
Anti-Miao's Le Shi pipe hormone chemiluminescence immune detection reagent kit the most according to claim 1, it is characterised in that described
In anti-Miao's Le Shi pipe coated solid phase carrier of hormone monoclonal antibody, described solid phase carrier is that surface is with for protein chemistry
The magnetic particle of reaction forming group.
Anti-Miao's Le Shi pipe hormone chemiluminescence immune detection reagent kit the most according to claim 2, it is characterised in that described
Surface is that surface is with amino, carboxyl, tosyl or ring with the magnetic particle for protein chemistry reaction forming group
The magnetic particle of oxirane group.
4. according to the anti-Miao's Le Shi pipe hormone chemiluminescence immune detection reagent kit described in Claims 2 or 3, it is characterised in that
Described surface is 0.05 μm～3 μm with the particle diameter of the magnetic particle for protein chemistry reaction forming group.
Anti-Miao's Le Shi pipe hormone chemiluminescence immune detection reagent kit the most according to claim 1, it is characterised in that described
In anti-Miao's Le Shi pipe coated solid phase carrier of hormone monoclonal antibody, on described solid phase carrier, coupling has Streptavidin, affine
Plain or neutral Avidin, in described anti-Miao's Le Shi pipe hormone monoclonal antibody, coupling has biotin.
Anti-Miao's Le Shi pipe hormone chemiluminescence immune detection reagent kit the most according to claim 1, it is characterised in that described
In anti-Miao's Le Shi pipe hormone monoclonal antibody of chemiluminescent labels labelling, described chemiluminescent labels be different luminol,
Tris (bipyridine) ruthenium, acridinium ester, alkali phosphatase or horseradish peroxidase.
7. according to the anti-Miao's Le Shi pipe hormone chemiluminescence immune detection reagent kit described in claim 1 or 6, it is characterised in that
In anti-Miao's Le Shi pipe hormone monoclonal antibody of described chemiluminescent labels labelling, on described chemiluminescent labels, coupling has
Streptavidin, Avidin or neutral Avidin, in described anti-Miao's Le Shi pipe hormone monoclonal antibody, coupling has biotin.
8. according to the anti-Miao's Le Shi pipe hormone chemiluminescence immune detection reagent kit described in claim 1 or 6, it is characterised in that
In anti-Miao's Le Shi pipe hormone monoclonal antibody of described chemiluminescent labels labelling, described chemiluminescent labels is anti-with described
Miao's Le Shi pipe hormone monoclonal antibody is directly connected to by chemical reaction key or is connected by protein-crosslinking agent.
Anti-Miao's Le Shi pipe hormone chemiluminescence immune detection reagent kit the most according to claim 8, it is characterised in that described
At least one in phosphinylidyne diimmonium salt and butanimide of protein-crosslinking agent.
Anti-Miao's Le Shi pipe hormone chemiluminescence immune detection reagent kit the most according to claim 9, it is characterised in that institute
State protein-crosslinking agent selected from 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine, 1-ethyl-(3-dimethylaminopropyl)
At least one in phosphinylidyne diimmonium salt hydrochlorate, N-hydroxy-succinamide and sulfonated N-hydroxy-succinamide.
11. anti-Miao's Le Shi pipe hormone chemiluminescence immune detection reagent kits according to claim 1, it is characterised in that also
Product are calibrated including anti-Miao's Le Shi pipe hormone.
Anti-Miao's Le Shi pipe hormone chemiluminescence immune detection reagent kit according to any one of 12. 1 kinds of claim 1～11
Preparation method, it is characterised in that comprise the steps:
Anti-Miao's Le Shi pipe hormone monoclonal antibody and solid phase carrier are mixed and fully react, obtains anti-Miao Le Shi pipe hormone Dan Ke
The coated solid phase carrier of grand antibody；And
Chemiluminescent labels and anti-Miao's Le Shi pipe hormone monoclonal antibody are mixed and fully react, obtains chemiluminescent labeling
Anti-Miao's Le Shi pipe hormone monoclonal antibody of substance markers.
13. anti-Miao's Le Shi pipe hormone chemiluminescence immune detection reagent kits according to claim 12, it is characterised in that institute
Stating in anti-Miao's Le Shi pipe coated solid phase carrier of hormone monoclonal antibody, described solid phase carrier is that surface is with for protein
Learn the magnetic particle of reaction forming group；
Described anti-Miao's Le Shi pipe hormone monoclonal antibody and solid phase carrier mixed and fully reacts, obtaining anti-Miao's Le Shi pipe hormone
The operation of the coated solid phase carrier of monoclonal antibody is: take surface with the magnetic particle for protein chemistry reaction forming group
Suspension, Magneto separate goes after supernatant resuspended with buffer, and then activator activates described surface with for protein chemistry
The Surface attachment groups of the magnetic particle of reaction forming group, is subsequently added into anti-Miao's Le Shi pipe hormone monoclonal antibody, fills under room temperature
Dividing reaction, Magneto separate is resuspended after removing supernatant, obtains the coated surface of anti-Miao's Le Shi pipe hormone monoclonal antibody with for egg
The magnetic particle of white matter chemical reaction linking group.
14. anti-Miao's Le Shi pipe hormone chemiluminescence immune detection reagent kits according to claim 12, it is characterised in that institute
Stating in anti-Miao's Le Shi pipe coated solid phase carrier of hormone monoclonal antibody, on described solid phase carrier, coupling has Streptavidin, parent
With plain or neutral Avidin, in described anti-Miao's Le Shi pipe hormone monoclonal antibody, coupling has biotin；
Described anti-Miao's Le Shi pipe hormone monoclonal antibody and solid phase carrier mixed and fully reacts, obtaining anti-Miao's Le Shi pipe hormone
The operation of the coated solid phase carrier of monoclonal antibody is: coupling has Streptavidin, Avidin or the solid phase of neutral Avidin
Carrier and coupling have anti-Miao's Le Shi pipe hormone monoclonal antibody of biotin to mix in buffer, fully obtain anti-Miao after reaction
The Le Shi pipe coated solid phase carrier of hormone monoclonal antibody.
15. anti-Miao's Le Shi pipe hormone chemiluminescence immune detection reagent kits according to claim 12, it is characterised in that institute
Stating in anti-Miao's Le Shi pipe hormone monoclonal antibody of chemiluminescent labels labelling, on described chemiluminescent labels, coupling has chain
Mould Avidin, Avidin or neutral Avidin, in described anti-Miao's Le Shi pipe hormone monoclonal antibody, coupling has biotin；
Described chemiluminescent labels and anti-Miao's Le Shi pipe hormone monoclonal antibody mixed and fully reacts, obtaining chemiluminescence
The operation of anti-Miao's Le Shi pipe hormone monoclonal antibody of label labelling is: coupling has Streptavidin, Avidin or neutrality
The chemiluminescent labels of Avidin and coupling have anti-Miao's Le Shi pipe hormone monoclonal antibody of biotin to mix in buffer,
Fully obtain anti-Miao's Le Shi pipe hormone monoclonal antibody of chemiluminescent labels labelling after reaction.
16. anti-Miao's Le Shi pipe hormone chemiluminescence immune detection reagent kits according to claim 12, it is characterised in that institute
State in anti-Miao's Le Shi pipe hormone monoclonal antibody of chemiluminescent labels labelling, described chemiluminescent labels and described anti-Miao
Le Shi pipe hormone monoclonal antibody is directly connected to by chemical reaction key；
Described chemiluminescent labels and anti-Miao's Le Shi pipe hormone monoclonal antibody mixed and fully reacts, obtaining chemiluminescence
The operation of anti-Miao's Le Shi pipe hormone monoclonal antibody of label labelling is: by anti-Miao's Le Shi pipe hormone monoclonal antibody and chemistry
Luminous marker mixes in buffer, fully obtains anti-Miao Le Shi pipe hormone Dan Ke of chemiluminescent labels labelling after reaction
Grand antibody.
17. anti-Miao's Le Shi pipe hormone chemiluminescence immune detection reagent kits according to claim 12, it is characterised in that institute
State in anti-Miao's Le Shi pipe hormone monoclonal antibody of chemiluminescent labels labelling, described chemiluminescent labels and described anti-Miao
Le Shi pipe hormone monoclonal antibody is connected by protein-crosslinking agent；
Described chemiluminescent labels and anti-Miao's Le Shi pipe hormone monoclonal antibody mixed and fully reacts, obtaining chemiluminescence
The operation of anti-Miao's Le Shi pipe hormone monoclonal antibody of label labelling is: by anti-Miao's Le Shi pipe hormone monoclonal antibody, chemistry
Luminous marker and protein-crosslinking agent mix in buffer, fully obtain the anti-Miao Le of chemiluminescent labels labelling after reaction
Family name's pipe hormone monoclonal antibody.
The detection method of the concentration of 18. 1 kinds of anti-Miao's Le Shi pipe hormones, uses anti-Miao as according to any one of claim 1～11
Le Shi pipe hormone chemiluminescence immune detection reagent kit, it is characterised in that the detection side of the concentration of described anti-Miao's Le Shi pipe hormone
Method comprises the steps:
Use anti-Miao's Le Shi pipe coated solid phase carrier of hormone monoclonal antibody and anti-Miao Le Shi of chemiluminescent labels labelling
Pipe hormone monoclonal antibody carries out sandwich immunoassay test to sample to be tested, and the anti-Miao's Le Shi pipe hormone in described sample to be tested is with anti-
Miao's Le Shi pipe coated solid phase carrier of hormone monoclonal antibody and anti-Miao Le Shi pipe hormone Dan Ke of chemiluminescent labels labelling
Grand antibody response, forms sandwich complex, after over cleaning separates, records the luminous intensity of sample to be tested；
Use anti-Miao's Le Shi pipe coated solid phase carrier of hormone monoclonal antibody and anti-Miao Le Shi of chemiluminescent labels labelling
Pipe hormone monoclonal antibody antagonism Miao's Le Shi pipe hormone calibration product carry out sandwich immunoassay test, and described anti-Miao's Le Shi pipe hormone is calibrated
Anti-Miao's Le Shi pipe hormone in product and anti-Miao's Le Shi pipe coated solid phase carrier of hormone monoclonal antibody and chemiluminescent labels
Anti-Miao's Le Shi pipe hormone monoclonal antibody reactive of labelling, forms sandwich complex, after over cleaning separates, records anti-Miao Le Shi
The luminous intensity of pipe hormone calibration product, according to luminous intensity and described anti-Miao Le Shi pipe of described anti-Miao's Le Shi pipe hormone calibration product
The concentration of hormone calibration product builds the standard curve that transverse and longitudinal coordinate is respectively anti-Miao's Le Shi pipe hormone of concentration and luminous intensity；With
And
The luminous signal of described sample to be tested is brought in the standard curve of described anti-Miao's Le Shi pipe hormone, be calculated described in treat
The concentration of the anti-Miao's Le Shi pipe hormone in test sample basis.
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