CN109580955A - For detecting the magnetic microparticle separating chemiluminescence immunoassay of Tau albumen (TAU) - Google Patents
For detecting the magnetic microparticle separating chemiluminescence immunoassay of Tau albumen (TAU) Download PDFInfo
- Publication number
- CN109580955A CN109580955A CN201811392665.6A CN201811392665A CN109580955A CN 109580955 A CN109580955 A CN 109580955A CN 201811392665 A CN201811392665 A CN 201811392665A CN 109580955 A CN109580955 A CN 109580955A
- Authority
- CN
- China
- Prior art keywords
- tau
- albumen
- reagent
- antibody
- magnetic
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
-
- 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
- G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
-
- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/28—Neurological disorders
- G01N2800/2814—Dementia; Cognitive disorders
- G01N2800/2821—Alzheimer
Abstract
The invention discloses a kind of human body Tau albumen (TAU) magnetic microparticle separating chemiluminescence immunologic detection methods, kit forms include: calibration object, quality-control product reagent A, reagent B, cleaning concentrated liquor, luminous substrate liquid, wherein, calibration object is a series of Tau albumen (TAU) antigen containing concentration, for establishing standard curve；Quality-control product is that the buffer of the albumen of Tau containing a certain concentration (TAU) antigen forms；Reagent A is Tau albumen (TAU) antibody-solutions of the label of magnetic particle containing a certain concentration；Reagent B is Tau albumen (TAU) antibody-solutions of the alkali phosphatase enzyme mark containing a certain concentration；Cleaning concentrated liquor is for preparing cleaning solution；Luminous substrate liquid is the luminous substrate solution of alkaline phosphatase (ALP) catalysis.Present invention greatly enhances the signal strength of immune response and sensitivity, make low content substance that can also generate very strong chemiluminescence signal when carrying out immune combine, provides a kind of more acurrate, accurate, convenient, fast and simple method for the detection of human body Tau albumen (TAU).
Description
Technical field
The invention belongs to technical field of immune assay, the Magneto separate chemistry for detecting Tau albumen (TAU) is provided
Luminescence immunoassay is suitable for human serum Tau albumen (TAU) quantitative detection.
Background technique
Tau albumen is also known as microtubule bindin (Microtubulue Association Protein, MAP), is present in
In normal cerebral tissue's neuron axon, have the function of synthesizing and stablizing neuronal cell bone.Tau albumen is thin in normal brain activity
Born of the same parents' function is to promote it to polymerize to form micro-pipe with tubulin binding；In conjunction with the micro-pipe of formation, microtubule stability is maintained, is reduced
The dissociation of tubulin molecule, and induce micro-pipe bunchy.Tau protein molecular contains 2-3 phosphate, and alzheimer ' in normal brain activity
Tau albumen in silent disease (AD) patient's brain then abnormal hyperphosphorylation, per molecule Tau albumen can contain 5-9 phosphate, and lose
Normal bio function.
In the cell, Tau albumen can be modified, most important of which is that phosphorylation, and the function of Tau albumen depends on
Its phosphorylation level.It data show, insoluble, the Tau egg of Hyperphosphorylationof is switched to after soluble T au protein hyperphosphorylation
It is white, it can finally form NFT.
The main reason for some researches show that AD causes a disease simultaneously is since Tau protein hyperphosphorylation leads to neural fibre in brain
Amyloid beta (β amyloid, A β) largely deposits in dimension entanglement (neuro fibrillary tangles, NFT) and brain
Senile plaque is constituted, and then causes neuronal function abnormal.
Have been reported that display, Patients with Mild Cognitive Impairment cerebrospinal fluid Tau protein level obviously increases.The report such as Hampel, brain
Phosphorylated Tau protein is the optimum detection marker of alzheimer's disease in spinal fluid.There is zoopery discovery, continuing Low perfusion makes
Hyperphosphorylationof occurs for rat hippocampus Tau albumen, and assembles it in nerve cell, causes the cognition dysfunction of rat.
These are studies have shown that Tau albumen and cognitive function are closely related.It pretends as a kind of nervous specific albumen, Tau albumen exists
Cerebrospinal fluid can be discharged into after cerebral injury from nerve cell, peripheral blood is further leaked by the blood-brain barrier being destroyed.Therefore,
Cerebrospinal fluid, peripheral blood Tau protein concentration can react the severity of brain injury to a certain extent.
Also some researches show that Tau protein hyperphosphorylation is the important initial step of AD brain neuron degradation, Tau albumen
The increase of the product P-Tau-181 albumen of Hyperphosphorylationof, can start the generation of the P-Tau-181 albumen outside blood-brain barrier, most
Lead to the increase of P-Tau-181 content in blood eventually.Therefore, the content for detecting P-Tau-181 albumen in serum has diagnosis AD
Higher clinical value.
Tau albumen (TAU) detection method being currently known mainly has RIA (radioimmunoassay radiommunoassay
Method) method, homogeneous enzyme immunoassay detection method, enzyme-linked immunization (ELISA), high performance liquid chromatography, gas chromatography and gas-chromatography
With Mass Spectrometry (GC-MS).But these methods all have the defects that certain, the pollution of RIA method is big, ELISA method automates journey
Spend low and complicated for operation cumbersome, high performance liquid chromatography, gas chromatography and gas-chromatography and Mass Spectrometry are not suitable for extensive
For clinical detection.
Therefore, it is still necessary to a kind of pollutions at present small, high sensitivity, easy to operate, high degree of automation, specific good, cost
The fast quantitative measurement method for detecting of low Tau albumen (TAU).
Summary of the invention
The present invention is directed to develop a kind of high sensitivity, pollution-free, easy to operate, specific good and low-cost Tau
The fast quantitative measurement method for detecting of albumen (TAU).
Based on above-mentioned purpose, the present invention provides a kind of human body Tau albumen (TAU) magnetic microparticle separating chemiluminescence immune detection
Method, kit forms include: calibration object, quality-control product reagent A, reagent B, cleaning concentrated liquor, luminous substrate liquid, wherein school
Quasi- product are a series of Tau albumen (TAU) antigen containing concentration, for establishing standard curve；Quality-control product is the egg of Tau containing a certain concentration
The buffer of white (TAU) antigen forms；Reagent A is that Tau albumen (TAU) antibody of the label of magnetic particle containing a certain concentration is molten
Liquid；Reagent B is Tau albumen (TAU) antibody-solutions of the alkali phosphatase enzyme mark containing a certain concentration；Cleaning concentrated liquor is for preparing
Cleaning solution；Luminous substrate liquid is the luminous substrate solution of alkaline phosphatase (ALP) catalysis；Detection method includes:
(1) it is immunoreacted: sample to be tested and quality-control product, 50 μ l reagent As, 50 μ l reagent B being sequentially added in reaction tube, 37
Mixing incubates 15min under the conditions of DEG C；
(2) Magneto separate: settling magnetic particle in magnetic field, removes supernatant, and 200-500 μ l cleaning solution is added, and removes magnetic field, so
It settles magnetic particle in magnetic field again afterwards, removes supernatant；It so repeats 2-4 times, to remove unbonded antibody and impurity；
(3) readings: being added 150 μ l of luminous substrate liquid, and alkaline phosphatase (ALP) catalysis substrate uses Li Deman certainly after shining
Grind chemiluminescence detector measurement relative luminous intensity (RLU)；
(4) it calculates content: the standard curve of the luminous intensity of sample to be tested and calibration object being compared, four parameters are used
Logistic equation model can calculate the content of Tau albumen (TAU) in sample to be tested.
Another program, human body Tau albumen (TAU) the magnetic microparticle separating chemiluminescence immunologic detection method, feature exist
In the configuration method of the reagent A includes: that the magnetic particle that carboxyl (COOH-) active group is contained on surface carries out Magneto separate, so
It is cleaned 3 times with activation buffer afterwards；Activator is added after suspension, the magnetic particle solution after activation is pressed with Tau albumen (TAU) antibody
Ratio mixing, so that Tau albumen (TAU) antibody is by covalent coupling on magnetic particle surface.
Another program, human body Tau albumen (TAU) magnetic microparticle separating chemiluminescence immunologic detection method, which is characterized in that institute
(2- (N- morpholine) ethanesulfonic acid) MES, pH6.0 that activation buffer is molar concentration 0.05M is stated, the activator is 1-
ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride(EDC)。
Another program, human body Tau albumen (TAU) magnetic microparticle separating chemiluminescence immunologic detection method, which is characterized in that institute
Stating ratio is 20:1.
Another program, human body Tau albumen (TAU) magnetic microparticle separating chemiluminescence immunologic detection method, which is characterized in that institute
The configuration method for stating reagent B includes: that Tau albumen (TAU) antibody, alkaline phosphatase (ALP) are separately added into activator, desalination；It is living
Tau albumen (TAU) antibody, alkaline phosphatase (ALP) after change mix in proportion；It isolates and purifies, removes not connected Tau albumen
(TAU) antibody and alkaline phosphatase (ALP), are stored in 4 DEG C for attachment.
Another program, human body Tau albumen (TAU) magnetic microparticle separating chemiluminescence immunologic detection method, which is characterized in that
The activator that Tau albumen (TAU) antibody is added is 2-Iminothiolane hydrochloride (2-IT), alkaline phosphatase
(ALP) activator being added is Succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-
carboxylate(SMCC)。
Another program, human body Tau albumen (TAU) magnetic microparticle separating chemiluminescence immunologic detection method, which is characterized in that institute
Stating ratio is 1:1.
Another program, human body Tau albumen (TAU) magnetic microparticle separating chemiluminescence immunologic detection method, which is characterized in that institute
State desalination be using Sephadex G25 gel columns desalination, it is described to isolate and purify to use Supperdex200 gel chromatography column
It isolates and purifies.
Another program, human body Tau albumen (TAU) magnetic microparticle separating chemiluminescence immunologic detection method, which is characterized in that build
The method of day-mark directrix curve includes:
(1) be immunoreacted: by the calibration object of 50 μ l of various concentration respectively with quality-control product, 50 μ l reagent As, 50 μ l reagent B
It sequentially adds in reaction tube, mixing incubates 15min under the conditions of 37 DEG C；
(2) Magneto separate: settling magnetic particle in magnetic field, removes supernatant, and 200-500 μ l cleaning solution is added, and removes magnetic field, so
It settles magnetic particle in magnetic field again afterwards, removes supernatant；It so repeats 2-4 times, to remove unbonded antibody and impurity；
(3) readings: being added 150 μ l of luminous substrate liquid, and alkaline phosphatase (ALP) catalysis substrate uses Li Deman certainly after shining
Grind chemiluminescence detector measurement relative luminous intensity (RLU)；
(4) four parameter Logistic equation models are used according to the numerical value of detection, obtains Tau albumen (TAU) concentration-and shines
It is worth standard curve.
Another program, human body Tau albumen (TAU) magnetic microparticle separating chemiluminescence immunologic detection method, which is characterized in that school
The series of concentrations of quasi- product is respectively 0ng/ml, 0.8ng/ml, 2.5ng/ml, 9.5ng/ml, 25ng/ml, 50ng/ml.
Above-mentioned steps show the reaction pattern for the competition law that the present invention uses, micro- with magnetic using chemiluminescence detection technology
The principle that the immune isolation technics of grain combines, Tau albumen (TAU) content in quantitative detection human serum or plasma sample, it is ensured that
The sensitivity of detection, since HOOK effect is not present using competition law in experiment, pollution-free, high specificity, it is easy to operate and
To the pre-treatment of sample require it is low, can fast high-flux detect high-volume sample, be convenient for clinical reagent application.The present invention is clinic
Tau albumen (TAU) in detection human serum provides a kind of more acurrate, accurate, convenient, fast and simple method.
Detailed description of the invention
Fig. 1 is human body Tau albumen (TAU) magnetic microparticle separating chemiluminescence immunologic detection method flow chart of the present invention.
Specific embodiment
The purpose of the present invention is to provide one kind to have wide detection range, high sensitivity, easily to operate detection human body Tau
Albumen (TAU) tubular type magnetic microparticle separating chemiluminescence immunologic detection method.
Above-mentioned purpose of the invention is achieved through the following technical solutions:
Human body Tau albumen (TAU) magnetic microparticle separating chemiluminescence immunologic detection method principle is as follows:
The present invention is double antibody sandwich method, combines and determines with chemiluminescence immunoassay system using magnetic particle isolation technics
The content of Tau albumen (TAU) in amount detection serum sample.As shown in Figure 1, the technical principle of reaction are as follows: the Tau of magnetic particle connection
Tau albumen (TAU) antibody of albumen (TAU) antibody and alkaline phosphatase (ALP) (AP) label with sample to be tested or calibration object or
Tau albumen (TAU) in quality-control product is immunoreacted, in conjunction with the compound of formation " sandwich " structure.Then adding magnetic outside
Direct precipitation in, the compound that immune response is formed and unbonded other materials Magneto separate.Precipitating is cleaned after removing supernatant
Compound, be added enzyme-catalyzed chemical luminescence substrate.Substrate, by catalytic pyrolysis, is formed among unstable excitation state under enzyme effect
Body just issues photon when excitation state intermediate returns to ground state, forms luminescence-producing reaction, that is, the hair of light-emitting appearance detection reaction can be used
Luminous intensity.Luminous intensity is directly proportional to the concentration of Tau albumen (TAU) in sample, can using four parameter Logistic equation models
Calculate the content of Tau albumen (TAU) in sample.
Measure the composition of the magnetic microparticle chemiluminescence detection kit of human body Tau albumen (TAU) content
The kit forms packet of human body Tau albumen (TAU) magnetic microparticle separating chemiluminescence immunoassays that the present invention generates
It includes: 1, calibration object (a series of Tau albumen (TAU) antigen containing concentration, for establishing standard curve)；2, quality-control product is (containing certain
The buffer of concentration Tau albumen (TAU) antigen forms)；3, reagent A (the Tau albumen of the label of magnetic particle containing a certain concentration
(TAU) antibody-solutions)；4, reagent B (Tau albumen (TAU) antibody-solutions of alkaline phosphatase containing a certain concentration (ALP) label)；
5, clean concentrated liquor (for preparing cleaning solution)；6, substrate solution (luminous substrate of enzymatic).
Below by specific embodiment, the present invention is described in detail, but does not limit the present invention.
Reagent
All components in detection kit of the present invention can pass through commercial sources from biological reagent or chemical reagents corporation
It is commercially available.Label alkaline phosphatase (ALP) antibody used is the production of Fitzgerald company, article No. 70R- in the present invention
34649；Mark magnetic particle antibody for the production of Fitzgerald company, article No. 70R-32554；Antigen is raw for Shanghai Ling Chao company
It produces, article No. are as follows: L2B006；Alkaline phosphatase (ALP) is purchased from Britain BBI company, model are as follows: ALPI12G；SMCC is purchased from thermo
Fisher scientific company, article No. are as follows: 22360；2-IT is purchased from thermo fisher scientific company, CAS:
4781-83-3；Article No. are as follows: 26101；Carboxyl modified particle is purchased from thermo fisher scientific company；EDC is purchased from
SIMGA company, CAS:25952-53-8, article No.: E7750.Luminous substrate, cleaning concentrate are all from Beijing Li Deman biochemistry stock
Part Co., Ltd finished product kit.
Embodiment 1: magnetic particle marks Tau albumen (TAU) antibody
20mg carboxyl modified Nanoparticle Solution is taken, will have superparamagnetism, uniform particle sizes, it is living that carboxyl (COOH-) is contained on surface
The magnetic particle of property group settles (Magneto separate) 10 minutes under magnetic fields, removes supernatant, the magnetic particle activation buffer of sedimentation
(2- (N- morpholine) ethanesulfonic acid) MES of molar concentration 0.05M, pH6.0 buffer solution for cleaning 3 times, each dosage 2ml.
By magnetic particle after washing with (2- (N- morpholine) ethanesulfonic acid) of 1.0ml activation buffer molar concentration 0.05M
Activator 1-ethyl-3- [3-dimethylaminopropyl] is added after being sufficiently suspended in the buffer of MES, pH6.0
Carbodiimide hydrochloride (EDC), room temperature, which is suspended, reacts 30 minutes, and it is 7.5mM that EDC, which reacts molar concentration,.
Tau albumen (TAU) antibody for taking 1.0mg, is concentrated into 2.5mg/mL.
20mg is added by Tau albumen (TAU) antibody of 1.0mg in Tau albumen (TAU) antibody for being concentrated into 2.5mg/mL to live
Magnetic particle solution mixing after change, jog are mixed, are reacted 6.5 hours under the conditions of 4 DEG C of suspensions, so that Tau albumen (TAU) antibody
By covalent coupling on magnetic particle surface, reagent A is made.
Embodiment 2: alkaline phosphatase (ALP) marks Tau albumen (TAU) antibody
Tau albumen (TAU) antibody for taking 1.0mg is concentrated into 2.5mg/mL, and the activator that concentration is 13.76mg/mL is added
5 μ L of 2-Iminothiolane hydrochloride (2-IT) solution is reacted at room temperature 15 minutes.It is solidifying using Sephadex G25
The sub- desalination of rubber column gel column, the antibody after collecting activation.
1.2mg alkaline phosphatase (ALP) is taken, 2.5mg/mL is concentrated into, the activator that concentration is 6.69mg/mL is added
12 μ L of Succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) solution, room
Temperature reaction 15 minutes.Alkaline phosphatase (ALP) using Sephadex G25 gel columns desalination, after collecting activation.
Tau albumen (TAU) antibody after activation is added with alkaline phosphatase (ALP) by Tau albumen (TAU) antibody of 1.0mg
The ratio mixing for entering 1.0mg alkaline phosphatase (ALP), places and reacts 18 hours at 4 DEG C.
Using Supperdex200 gel chromatography column separating purification, not connected Tau albumen (TAU) antibody and alkalinity are removed
Attachment is stored in 4 DEG C by phosphatase (ALP), and reagent B is made.
Embodiment 3: human body Tau albumen (TAU) magnetic microparticle separating chemiluminescence immunologic detection method
(1) be immunoreacted: by the calibration object of 50 μ l of embodiment 1 series (concentration is respectively 0,0.8,2.5,9.5,25,
50ng/ml) the 50 μ l reagent B respectively with quality-control product, 50 μ l reagent As of embodiment 1, embodiment 2 are sequentially added in reaction tube, and 37
Mixing incubates 15min under the conditions of DEG C；
(2) Magneto separate: settling magnetic particle in magnetic field, removes supernatant, and 200-500 μ l cleaning solution is added, and removes magnetic field, so
It settles magnetic particle in magnetic field again afterwards, removes supernatant；It so repeats 2-4 times, to remove unbonded antibody and impurity；
(3) readings: being added 150 μ l of luminous substrate liquid, and alkaline phosphatase (ALP) catalysis substrate uses Li Deman certainly after shining
Grind chemiluminescence detector measurement relative luminous intensity (RLU)；
(4) four parameter Logistic equation models are used according to the numerical value of detection, obtains Tau albumen (TAU) concentration-and shines
It is worth standard curve.
(5) 50 μ l reagent B of 50 μ l reagent As of the sample to be tested of 50 μ l and embodiment 1, embodiment 2 are sequentially added instead
Ying Guanzhong, mixing incubates 15min under the conditions of 37 DEG C；
(6) Magneto separate: settling magnetic particle in magnetic field, removes supernatant, and 200-500 μ l cleaning solution is added, and removes magnetic field, so
It settles magnetic particle in magnetic field again afterwards, removes supernatant；It so repeats 2-4 times, to remove unbonded antibody and impurity；
(7) it readings: is added after 150 μ l, ALP catalysis substrate of luminous substrate liquid shines and is examined using Li Deman from chemiluminescence is ground
Survey instrument measurement relative luminous intensity (RLU)；
(8) standard curve of the luminous intensity of sample to be tested and step (4) is compared, uses four parameter Logistic equations
Fitting can calculate the content of Tau albumen (TAU) in sample to be tested.
Embodiment 4: kit performance test
Detect sensitivity, the range of linearity, accuracy, precision of this kit.Specific steps are as follows:
One, reagent prepares:
Before experiment, reagent A (Tau albumen (TAU) antibody-solutions of magnetic particle label), reagent B (alkaline phosphatase are first taken out
(ALP) mark Tau albumen (TAU) antibody-solutions), calibration object, luminous substrate.
Two, instrument prepares:
This kit is suitable for Beijing Leaderman Biochemistry Co., Ltd CI1000, CI2000 Full-automatic chemiluminescence and exempts from
Epidemic disease analyzer, Britain IDS type chemical illumination immunity analysis instrument.
Three, operating procedure:
1, dose-response curve is linear: using kit maximum concentration point calibration object (F point) with the project calibration object dilution
Doubling dilution forms several concentration point samples to close to the project analysis sensitivity.Gradient sample is tested after kit calibration, often
A diluted concentration is tested 3 times, finds out the mean value (yi) of measurement result respectively.With diluted concentration (xi) for independent variable, to measure knot
Fruit mean value (yi) is that dependent variable finds out equation of linear regression.The related coefficient (r) of linear regression is calculated by formula (1).
…………(1)
Criterion of acceptability: in 1.0-200ng/ml, r > 0.9900.
2, accuracy: newly preparing and qualified reagent (including STD, R1, R2, M) is examined to be put into 37 DEG C of incubators places 7 days altogether
(can be more than 7 days).Several days taking-up reagents, after calibration (with the qualified calibration objects of 4 DEG C of preservations) detection reagent accuracy, Quality Control deviation
Less than 20%.
Criterion of acceptability: the calibration object range of decrease is less than 10%, and less than 50%, accuracy meets the requirements the kit range of decrease.
3, minimum detection limit: using zero-dose calibration object to be detected as sample, replication 20 times, obtains 20 measurements
As a result relative luminous intensity (RLU) value, calculates its average value (M) and standard deviation (SD), M+2SD is obtained, according to zero-dose school
Concentration-RLU between quasi- product and adjacent calibration object carries out two o'clock regression fit and obtains linear function, by M+2SD (positive reaction) or
The RLU value of M-2SD (negative reaction) is brought into above-mentioned equation, and corresponding concentration value, as minimum detection limit are found out.
4, it precision: analysis method (in analysis): is examined with the quality controlled serum of high and low two level concentrations or fresh human serum
Test agent retest 10 times, calculates the mean value of measurement result as followsWith the coefficient of variation (CV).
In formula: Xi -- the measurement result of test serum sample；X -- the mean value of test serum sample；N -- measurement time
Number, is herein 10.
Analysis method (between analysis): testing in three times, with the quality controlled serum or fresh human serum of high and low two level concentrations
Detection reagent, retest 10 times, each sample standard deviation obtains 30 as a result, calculating the mean value of measurement result as follows
With the coefficient of variation (CV).
In formula: Xi -- the measurement result of test serum sample；-- the mean value of test serum sample；N -- measurement
Number is herein 30.
Criterion of acceptability: in analysis: being not more than 10%；Between analysis: being not more than 15%.
5, specific: to press reagent kit product standard requirements, prepare certain density cross reaction object as sample reagent
Box is detected, and the ratio (%) of testing result and sample compound concentration is the cross reacting rate to the sample.
Criterion of acceptability: < 1.0%.
Performance indicator testing result:
1, dose-response curve is linear: four parameter Logistic equation models are used, within the scope of 0.5-500ng/mL,
Dose-response curve correlation coefficient r >=0.99.
2, accuracy: being recycled by dilution and evaluate its accuracy, and acquired results dilute the rate of recovery between 85%-115%.
3, minimum detection limit: < 0.05ng/mL.
4, precision: in analysis≤8%, between analysis≤10%.
5, specific: crossing-over rate is respectively less than 0.1%.
Above-mentioned steps show the reaction pattern for the competition law that the present invention uses, micro- with magnetic using chemiluminescence detection technology
The principle that the immune isolation technics of grain combines, Tau albumen (TAU) content in quantitative detection human serum or plasma sample, it is ensured that
The sensitivity of detection, since HOOK effect is not present using competition law in experiment, pollution-free, high specificity, it is easy to operate and
To the pre-treatment of sample require it is low, can fast high-flux detect high-volume sample, be convenient for clinical reagent application.The present invention is clinic
Tau albumen (TAU) in detection human serum provides a kind of more acurrate, accurate, convenient, fast and simple method.
The magnetic microparticle chemiluminescence detection kit of human body Tau albumen (TAU) content provided by the invention can with it is complete from
Dynamic chemiluminescent analyzer combination, operating procedure greatly simplify, and increase detection speed and detection flux, improve detection effect
Rate, while avoiding error caused by manual operation.
It should be understood by those ordinary skilled in the art that: the discussion of any of the above embodiment is exemplary only, not
It is intended to imply that the scope of the present invention (including claim) is limited to these examples；Under thinking of the invention, above embodiments
Or it can also be combined between the technical characteristic in different embodiments, and there are different aspects present invention as described above
Many other variations, in order to it is concise they do not provided in details.Therefore, all within the spirits and principles of the present invention,
Any omission, modification, equivalent replacement, improvement for being made etc., should all be included in the protection scope of the present invention.
Claims (10)
- Human body Tau albumen 1. (TAU) magnetic microparticle separating chemiluminescence immunologic detection method, which is characterized in that kit forms packet It includes: calibration object, quality-control product reagent A, reagent B, cleaning concentrated liquor, luminous substrate liquid, wherein calibration object is containing a series of concentration Tau albumen (TAU) antigen, for establishing standard curve；Quality-control product is the buffering of the albumen of Tau containing a certain concentration (TAU) antigen Liquid is formulated；Reagent A is Tau albumen (TAU) antibody-solutions of the label of magnetic particle containing a certain concentration；Reagent B is containing certain dense Spend Tau albumen (TAU) antibody-solutions of alkali phosphatase enzyme mark；Cleaning concentrated liquor is for preparing cleaning solution；Luminous substrate liquid For the luminous substrate solution of alkaline phosphatase (ALP) catalysis；Detection method includes:(1) it is immunoreacted: sample to be tested and quality-control product, 50 μ l reagent As, 50 μ l reagent B being sequentially added in reaction tube, 37 DEG C of items Mixing incubates 15min under part；(2) Magneto separate: settling magnetic particle in magnetic field, removes supernatant, and 200-500 μ l cleaning solution is added, and removes magnetic field, then again It is secondary to settle magnetic particle in magnetic field, remove supernatant；It so repeats 2-4 times, to remove unbonded antibody and impurity；(3) readings: being added 150 μ l of luminous substrate liquid, and alkaline phosphatase (ALP) catalysis substrate uses Li Deman from grinding after shining Learn luminometer measurement relative luminous intensity (RLU)；(4) it calculates content: the standard curve of the luminous intensity of sample to be tested and calibration object being compared, four parameter Logistic are used Equation model can calculate the content of Tau albumen (TAU) in sample to be tested.
- 2. human body Tau albumen (TAU) magnetic microparticle separating chemiluminescence immunologic detection method according to claim 1, feature It is, the configuration method of the reagent A includes: that the magnetic particle that carboxyl (COOH-) active group is contained on surface carries out Magneto separate, Then it is cleaned 3 times with activation buffer；Activator is added after suspension, the magnetic particle solution after activation and Tau albumen (TAU) antibody It mixes in proportion, so that Tau albumen (TAU) antibody is by covalent coupling on magnetic particle surface.
- 3. according to claim 2 human body Tau albumen (TAU) magnetic microparticle separating chemiluminescence immunologic detection method, which is characterized in that The activation buffer is (2- (N- morpholine) ethanesulfonic acid) MES, pH6.0 of molar concentration 0.05M, and the activator is 1- ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride(EDC)。
- 4. according to claim 2 human body Tau albumen (TAU) magnetic microparticle separating chemiluminescence immunologic detection method, which is characterized in that The ratio is 20:1.
- 5. human body Tau albumen (TAU) magnetic microparticle separating chemiluminescence immunologic detection method according to claim 1, which is characterized in that The configuration method of the reagent B includes: that Tau albumen (TAU) antibody, alkaline phosphatase (ALP) are separately added into activator, desalination； Tau albumen (TAU) antibody, alkaline phosphatase (ALP) after activation mix in proportion；It isolates and purifies, removes not connected Tau egg White (TAU) antibody and alkaline phosphatase (ALP), are stored in 4 DEG C for attachment.
- 6. according to claim 5 human body Tau albumen (TAU) magnetic microparticle separating chemiluminescence immunologic detection method, which is characterized in that The activator that Tau albumen (TAU) antibody is added is 2-Iminothiolane hydrochloride (2-IT), alkaline phosphatase (ALP) activator being added is Succinimidyl 4- (N-maleimidomethyl) cyclohexane-1- carboxylate(SMCC)。
- 7. according to claim 5 human body Tau albumen (TAU) magnetic microparticle separating chemiluminescence immunologic detection method, which is characterized in that The ratio is 1:1.
- 8. according to claim 5 human body Tau albumen (TAU) magnetic microparticle separating chemiluminescence immunologic detection method, which is characterized in that The desalination be using Sephadex G25 gel columns desalination, it is described to isolate and purify to use Supperdex200 gel chromatography Column separating purification.
- 9. human body Tau albumen (TAU) magnetic microparticle separating chemiluminescence immunologic detection method according to claim 1, which is characterized in that The method for establishing standard curve includes:(1) be immunoreacted: by the calibration object of 50 μ l of various concentration respectively with quality-control product, 50 μ l reagent As, 50 μ l reagent B successively It is added in reaction tube, mixing incubates 15min under the conditions of 37 DEG C；(2) Magneto separate: settling magnetic particle in magnetic field, removes supernatant, and 200-500 μ l cleaning solution is added, and removes magnetic field, then again It is secondary to settle magnetic particle in magnetic field, remove supernatant；It so repeats 2-4 times, to remove unbonded antibody and impurity；(3) readings: being added 150 μ l of luminous substrate liquid, and alkaline phosphatase (ALP) catalysis substrate uses Li Deman from grinding after shining Learn luminometer measurement relative luminous intensity (RLU)；(4) four parameter Logistic equation models are used according to the numerical value of detection, obtains Tau albumen (TAU) concentration-luminous value mark Directrix curve.
- 10. according to claim 9 human body Tau albumen (TAU) magnetic microparticle separating chemiluminescence immunologic detection method, feature exists In the series of concentrations of calibration object is respectively 0ng/ml, 0.8ng/ml, 2.5ng/ml, 9.5ng/ml, 25ng/ml, 50ng/ml.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811392665.6A CN109580955A (en) | 2018-11-21 | 2018-11-21 | For detecting the magnetic microparticle separating chemiluminescence immunoassay of Tau albumen (TAU) |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811392665.6A CN109580955A (en) | 2018-11-21 | 2018-11-21 | For detecting the magnetic microparticle separating chemiluminescence immunoassay of Tau albumen (TAU) |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109580955A true CN109580955A (en) | 2019-04-05 |
Family
ID=65923574
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811392665.6A Withdrawn CN109580955A (en) | 2018-11-21 | 2018-11-21 | For detecting the magnetic microparticle separating chemiluminescence immunoassay of Tau albumen (TAU) |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109580955A (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112162101A (en) * | 2020-10-13 | 2021-01-01 | 南京立顶医疗科技有限公司 | Kit for detecting biomarkers of Alzheimer's disease and detection method thereof |
| CN112684166A (en) * | 2020-12-02 | 2021-04-20 | 北京利德曼生化股份有限公司 | Magnetic particle chemiluminescence detection kit for determining content of human gastrin G17 |
| CN112730839A (en) * | 2021-01-20 | 2021-04-30 | 宁波海壹生物科技有限公司 | Kit for measuring content of cytokeratin 19 fragments by magnetic particle chemiluminescence method |
| CN113533746A (en) * | 2021-07-22 | 2021-10-22 | 深圳市天大生物医疗器械有限公司 | P-Tau protein chemiluminescence detection kit and preparation method thereof |
| CN114002435A (en) * | 2021-10-13 | 2022-02-01 | 华中科技大学 | Three kits for detecting human phosphorylated Tau protein and preparation method thereof |
| CN114034872A (en) * | 2022-01-12 | 2022-02-11 | 山东大学齐鲁医院 | Kit for early diagnosis of Alzheimer's disease and application thereof |
| CN114778856A (en) * | 2022-05-30 | 2022-07-22 | 苏州宇测生物科技有限公司 | Phosphorylated tau protein detection kit |
| CN115047183A (en) * | 2022-06-08 | 2022-09-13 | 广州康盛生物科技股份有限公司 | Detection reagent and preparation method and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994018560A1 (en) * | 1993-02-12 | 1994-08-18 | Teijin Limited | Method of assaying human tau protein, kit therefor, and diagnostic method |
| US20110065197A1 (en) * | 2006-09-06 | 2011-03-17 | National University Corporation Tottori University | Diagnostic kit for alzheimer's disease, diagnostic marker, and detection method for indicator of pathological state thereof |
| US20130217147A1 (en) * | 2009-02-24 | 2013-08-22 | Nanosphere, Inc. | Detection of complexes of tau and amyloid |
| CN103901215A (en) * | 2014-04-11 | 2014-07-02 | 苏州浩欧博生物医药有限公司 | Chemiluminescence quantitative detection kit for food allergens, and preparation method and detection method thereof |
| CN105388302A (en) * | 2015-12-22 | 2016-03-09 | 中国医学科学院输血研究所 | Detection method for content of Tau protein antibodies in human immune globulin product |
-
2018
- 2018-11-21 CN CN201811392665.6A patent/CN109580955A/en not_active Withdrawn
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994018560A1 (en) * | 1993-02-12 | 1994-08-18 | Teijin Limited | Method of assaying human tau protein, kit therefor, and diagnostic method |
| US20110065197A1 (en) * | 2006-09-06 | 2011-03-17 | National University Corporation Tottori University | Diagnostic kit for alzheimer's disease, diagnostic marker, and detection method for indicator of pathological state thereof |
| US20130217147A1 (en) * | 2009-02-24 | 2013-08-22 | Nanosphere, Inc. | Detection of complexes of tau and amyloid |
| CN103901215A (en) * | 2014-04-11 | 2014-07-02 | 苏州浩欧博生物医药有限公司 | Chemiluminescence quantitative detection kit for food allergens, and preparation method and detection method thereof |
| CN105388302A (en) * | 2015-12-22 | 2016-03-09 | 中国医学科学院输血研究所 | Detection method for content of Tau protein antibodies in human immune globulin product |
Non-Patent Citations (1)
| Title |
|---|
| 胡元元等: "建立超敏ELISA-双酶底物循环法检测tau蛋白", 《生物化学与生物物理进展》 * |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112162101A (en) * | 2020-10-13 | 2021-01-01 | 南京立顶医疗科技有限公司 | Kit for detecting biomarkers of Alzheimer's disease and detection method thereof |
| CN112684166A (en) * | 2020-12-02 | 2021-04-20 | 北京利德曼生化股份有限公司 | Magnetic particle chemiluminescence detection kit for determining content of human gastrin G17 |
| CN112730839A (en) * | 2021-01-20 | 2021-04-30 | 宁波海壹生物科技有限公司 | Kit for measuring content of cytokeratin 19 fragments by magnetic particle chemiluminescence method |
| CN112730839B (en) * | 2021-01-20 | 2024-01-30 | 宁波海尔施智造有限公司 | Kit for measuring content of cytokeratin 19 fragments by magnetic particle chemiluminescence method |
| CN113533746A (en) * | 2021-07-22 | 2021-10-22 | 深圳市天大生物医疗器械有限公司 | P-Tau protein chemiluminescence detection kit and preparation method thereof |
| CN114002435A (en) * | 2021-10-13 | 2022-02-01 | 华中科技大学 | Three kits for detecting human phosphorylated Tau protein and preparation method thereof |
| CN114034872A (en) * | 2022-01-12 | 2022-02-11 | 山东大学齐鲁医院 | Kit for early diagnosis of Alzheimer's disease and application thereof |
| CN114778856A (en) * | 2022-05-30 | 2022-07-22 | 苏州宇测生物科技有限公司 | Phosphorylated tau protein detection kit |
| CN115047183A (en) * | 2022-06-08 | 2022-09-13 | 广州康盛生物科技股份有限公司 | Detection reagent and preparation method and application thereof |
| CN115047183B (en) * | 2022-06-08 | 2023-04-11 | 广州康盛生物科技股份有限公司 | Detection reagent and preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109580955A (en) | For detecting the magnetic microparticle separating chemiluminescence immunoassay of Tau albumen (TAU) | |
| CN109521004A (en) | For detecting the magnetic microparticle separating chemiluminescence immunoassay of glial fibrillary acid protein (GFAP) | |
| McCaman et al. | Fluorimetric method for the determination of phenylalanine in serum | |
| CN104634980B (en) | The super quick detection kit of cardiac muscle troponin I and super quick detection method | |
| CN102735833B (en) | Thyroperoxidase antibody homogeneous-phase luminescent immunoassay kit and detection method thereof | |
| CN106353506A (en) | Kit for detecting osteocalcin content and testing method thereof | |
| CN106018388B (en) | A kind of pepsinogen Cgene or II assay kit and preparation method thereof | |
| CN109580956A (en) | For detecting the magnetic microparticle separating chemiluminescence immunoassay of amyloid beta (A β) | |
| JP5554247B2 (en) | Method for measuring cystatin C in human body fluid | |
| CN107422129A (en) | A kind of super quick cardiac muscle troponin I magnetic microsphere immunoturbidimetry detection method and detection kit | |
| CN112485445B (en) | Kit for quantitatively detecting GFAP and application thereof | |
| CN107817232A (en) | For the automation immunoassay system for the diagnostic assay for carrying out allergy and autoimmune disease | |
| CN111735965A (en) | Cardiac troponin I detection reagent, preparation method thereof and cardiac troponin I detection kit | |
| CN113092786B (en) | Buffer solution and application thereof in central nervous system specific protein detection kit | |
| CN112630430B (en) | Kit for quantitatively detecting UCHL-1 and application thereof | |
| CN109100516A (en) | A kind of detection cytomegalovirus IgG antibody chemical luminescence immune assay determination reagent kit and preparation method thereof | |
| CN108152507A (en) | A kind of I type precollagen amino terminal peptides detection kit and its detection method | |
| CN108169486A (en) | A kind of kit and its test method for measuring squamous cell carcinoma-related antigen content | |
| CN112162101A (en) | Kit for detecting biomarkers of Alzheimer's disease and detection method thereof | |
| CN114034872A (en) | Kit for early diagnosis of Alzheimer's disease and application thereof | |
| CN113804896A (en) | Magnetic particle chemiluminescence kit for measuring interleukin-8 content in human serum | |
| CN108931652A (en) | A kind of kit with Magnetism particulate immuno chemistry luminescence method detection myoglobin content | |
| CN113125696A (en) | Estradiol homogeneous phase chemiluminescence detection kit and application thereof | |
| CN110531085A (en) | A kind of magnetic microparticle chemiluminescence detection kit and preparation method thereof measuring human nerve silk light chain protein content | |
| CN114624448B (en) | Kit for detecting acidic protein in glial fibers |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WW01 | Invention patent application withdrawn after publication | ||
| WW01 | Invention patent application withdrawn after publication |
Application publication date: 20190405 |