CN109696552A - Luminous ELISA external diagnosis reagent case and vitro detection equipment for headstroke - Google Patents
Luminous ELISA external diagnosis reagent case and vitro detection equipment for headstroke Download PDFInfo
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Abstract
The present invention provides a kind of external diagnosis reagent case for the ELISA that shines, it include: humoral sample stabilisation aqueous solution, contain the significant protein ingredient of neurotrosis in the humoral sample, in the humoral sample stabilisation aqueous solution, solute includes human serum, animal serum albumin, inorganic alkaline metal salt, Tris alkali, protein denaturant and nonionic surface active agent, and the pH value of the aqueous solution is 6.7~7.6；And the antibody (for example, anti-glial fibrillary acidic albumen (glial fibrillary acidic protein, GFAP) antibody) for resisting the significant protein ingredient of the neurotrosis optionally included.A kind of vitro detection equipment is also provided.Using shine ELISA method from humoral sample (such as, peripheral blood) in detection neural cell injury marker protein ingredient GFAP when, the accuracy and repeatability of external diagnosis reagent case and vitro detection equipment of the invention are high, the GFAP stabilization of pg rank in body fluid can accurately be detected.
Description
Technical field
The present invention relates to field of biological medicine, specifically, being related to a kind of for cerebral apoplexy (headstroke) patient's
Humoral sample, which stabilizes, uses aqueous solution, vitro detection kit and detection system.
Background technique
Cerebral apoplexy is commonly called as apoplexy, and by cerebrovascular occlusion or cerebral injury caused by rupturing causes, and endangers extremely serious.Epidemic disease
It learns the study found that the disability rate of current China apoplexy ranks the first, case fatality rate is number two, and is only second to cancer.Without which kind of disease
Disease can be as apoplexy, and moment makes us facial paralysis, numb limb, is paralyzed in bed, and people is allowed to lose life dignity.Currently, I
It is more than 3,700,000 that state increases apoplexy case newly every year, nearly 20,000,000 people of the patients with cerebral apoplexy of survival, and patient populations are in and rapidly rise
Phase.Hypertension, diabetes, heart disease are all the risk factors of headstroke, and the middle-aged and the old is apoplexy people at highest risk.Therefore, it is necessary to
Establish the method for quickly accurately diagnosing cerebral apoplexy.
For the diagnostic method of current headstroke based on iconography, accuracy is low, expense is high, brings to social hygiene's cause
Significant burden.In addition paralytic's change of illness state is rapid, individual difference is obvious, brings great difficulty to Clinics and Practices.
In addition to imaging diagnosis, in the method for diagnosis headstroke, nerve in ELISA detection cerebrospinal fluid is utilized there are also a kind of
The method of glial fibrillary acidic protein (GFAP) content.GFAP is one of highest albumen of central nervous system content.
Acute ischemic cerebral apoplexy directly contributes a large amount of nerve cell death, and dead nerve cell then decomposes and it is a large amount of to release
Structural proteins, including GFAP.The reason of because of blood-brain barrier, GFAP is mostly trapped in cerebrospinal fluid.It detects in cerebrospinal fluid
GFAP, the method for generalling use thecal puncture.But this method again results in serious body and mind pain to patient, to society and
Family brings tremendous economic to bear.
As a kind of method of GFAP in detection cerebrospinal fluid, more generally MBP enzyme linked immuno-adsorbent assay (ELISA) method,
The basic principle is that using antigen, the specific reaction of antibody, with known antigens or antibody test unknown antigen or antibody.Due to
ELISA test method have the characteristics that it is sensitive, special, economical, easy, safe, at present the diagnosis of disease and observation of curative effect with
And preventive medicine etc. application is especially extensive.
As a kind of measurement trace antigen, the classical way of antibody, ELISA method is widely used in clinical examination diagnosis,
Technology maturation, widely used, sensitivity for analysis is high, analysis specificity is good, easy to operate.Basic operation process are as follows: the first step, it will
Antigen (or antibody) is coated on solid phase carrier (such as polystyrene plastics ELISA Plate), puts 4 DEG C of refrigerator overnights.Second step, with slow
Fliud flushing wash bags three times, put 37 DEG C of incubators (or room temperature) reactions 1~2.5 hour after adding sample to be measured by plate.Third step uses buffer
Board-washing three times, react 1~2.5 hour by 37 DEG C of incubators (or room temperature) of enzyme labeling antibody postposition.4th step, with buffer board-washing three
It is secondary, add substrate solution to develop the color, sulfuric acid stopped reaction, upper machine judging result.
As the state-of-the-art technology of ELISA development, luminophore is coupled to detection antibody, directly measures it by the ELISA that shines
Institute's luminous quantity quantifies antigen, has not only reduced enzyme/substrate reactions bring additional interference, but also largely improve
Detectivity.
Either detected by imaging diagnosis or ELISA cerebrospinal fluid, currently, the Major Difficulties of stroke diagnosis have:
Asymptomatic in early days or mild, it is difficult to detect；Real-time diagnosis is difficult to differentiate between hemorrhagic stroke or ishemic stroke；Long-term treatment
It can not judge damaged cell type and/or therapeutic effect, cause stroke diagnosis horizontal universal low.Therefore, it early diagnoses, quickly
Diagnosis, Precise Diagnosis, such diagnosis can be completed by obtaining cerebrospinal fluid particularly without thecal puncture, be suffered from for reducing headstroke
The disability rate and the death rate of person has especially important effect.
Summary of the invention
The present inventor has carried out for a long time and in-depth study the humoral sample especially peripheral blood of Patients with Stroke, as a result
It was found that also there is the GFAP of denier (< 100pg/mL) to enter peripheral blood system.Based on the discovery, the present invention provides one kind
Technological means carries out the ELISA detection that shines to by the body fluid of representative of peripheral blood, measures the significant egg in the peripheral blood serum
The concentration of Bai Chengfen (such as GFAP) realizes the diagnosis to cerebral apoplexy with this, while being also avoided that the thecal puncture of high risk.
Shine ELISA the characteristics of be with identification small-signal ability, sensitivity be it is traditional, using enzymatic as base
10~1,000 times of the ELISA of plinth.This detection sensitivity greatly improves, the positive true and false to detection system resoluting signal
Ability proposes the effect of high requirement, especially detection reagent.
It is of the invention in order to which the ELISA that makes to shine can reduce noise while enhance signal to significantly reducing false positive
First aspect provides a kind of humoral sample stabilisation aqueous solution for luminous ELISA, contains nerve damage in the humoral sample
Hurt significant protein ingredient, in the humoral sample stabilisation aqueous solution, solute include human serum, animal serum albumin,
Inorganic alkaline metal salt, Tris alkali, protein denaturant and nonionic surface active agent, the pH value of the aqueous solution is 6.7~
7.6。
It is 7.0~7.4 that the preferably described humoral sample, which is stabilized with the pH value of aqueous solution,.
The animal serum albumin comes from ox, sheep, horse, rabbit, chicken, and preferably bovine serum albumin(BSA).
Preferably, in the humoral sample stabilisation aqueous solution described in every 100ml, the concentration of human serum is 10 volume %
~50 volume %；The concentration of bovine serum albumin(BSA) is 0.1 mass/volume of mass/volume %~10 %；Inorganic alkaline metal salt it is dense
Degree is 110mM~200mM；The concentration of Tris alkali is 10mM~25mM；The concentration of protein denaturant is 0.01mM~0.5mM, with
And the concentration of nonionic surface active agent is 0.05 volume of volume %~5.0 %.
Inorganic alkaline metal salt is preferably selected from alkali metal chloride；Protein denaturant is selected from urea and lauryl sodium sulfate；
The nonionic surface active agent is selected from Tween-20, Tween-40, Tween-60, Tween-80 and Triton X-100.
Preferably, the inorganic alkaline metal salt is sodium chloride and potassium chloride, and the protein denaturant is urea, described non-
Ionic surface active agent is Tween-20.
It is further preferred that the concentration of the human serum is 10 in the humoral sample stabilisation aqueous solution described in every 100ml
The volume of volume %~35 %；The concentration of the bovine serum albumin(BSA) is 0.5 mass/volume of mass/volume %~5 %；Tween-20
Concentration be 0.1 volume of volume %~3.0 %；The concentration of urea is 0.05mM~0.25mM；The concentration of sodium chloride be 110mM~
150mM；The concentration of potassium chloride is 2.0mM~3mM；And the concentration of Tris alkali is 15mM~25mM.
At least part of the sodium chloride, potassium chloride and the Tris alkali is provided in the form of Tris buffered saline.
The significant protein ingredient of neurotrosis is selected from glial fibrillary acidic albumen (GFAP), neurofilament
Albumen (NFP), neurofilament protein heavy chain (NF-H), chain (NF-M) in neurofilament protein, neurofilament protein light chain (NF-L) and
Neural enolase.
The body fluid is blood, serum, cerebrospinal fluid, tissue fluid, urine, saliva or sweat, preferably human peripheral blood
Clearly.It is further preferred that containing GFAP in the human peripheral serum as the significant protein ingredient of the neurotrosis；Further
It is further preferred that GFAP contained in every milliliter of human peripheral serum is pg grades horizontal.
The human peripheral serum is suffered from from asymptomatic volunteer, the Patients with Stroke of light symptoms, headstroke acute stage
Person, headstroke reconvalescent or cerebral apoplexy sequela phase patient.
The luminous ELISA is Double antibody sandwich ELISA.
Second aspect of the present invention provides a kind of external diagnosis reagent case for the ELISA that shines, including above-mentioned first aspect
The humoral sample stabilisation aqueous solution, and what is optionally included resist the anti-of the significant protein ingredient of the neurotrosis
Body.
The antibody for resisting the significant protein ingredient of the neurotrosis is preferably anti-GFAP protein antibodies.
The anti-GFAP protein antibodies include: to capture antibody and detection antibody；Wherein the capture antibody and the detection
Antibody is the anti-GFAP antibody for being directed to GFAP albumen difference epitope respectively.
Preferably, luminophore has been coupled on the detection antibody.
The illumination mode of the luminophore includes chemical mode or physics mode, and the chemical mode preferably includes enzyme
Catalysis or chemiluminescence, the physics mode preferably include appointing in electroluminescent, fluorescence and fluorescence resonance energy transfer
Meaning one.
Preferably, the capture antibody is monoclonal antibody or polyclonal antibody, and the detection antibody is Dan Ke
Grand antibody or polyclonal antibody.
The polyclonal antibody from mouse, rabbit, chicken, sheep, cavy, donkey, Malaysia and China any one.
External diagnosis reagent case of the invention can be used for headstroke, hemorrhagic cerebral disorders, traumatic cerebral disorders, nerve and move back
(Alzheimer disease, cerebrovascular dementia, Parkinson's disease, Heng Yandunshi chorea, muscular dystrophy aixs cylinder are hard for row disease
Change disease etc.) in-vitro diagnosis.Preferably, the external diagnosis reagent case is used for the diagnosis of headstroke.
Third aspect present invention provides a kind of vitro detection equipment for stroke diagnosis or prognostic evaluation, including shines
ELISA analyzer and the external diagnosis reagent case according to above-mentioned second aspect for the ELISA that shines.
Preferably, the luminous ELISA analyzer is highly sensitive analyzer, and detectivity is lower than 10 skins
Gram.
Advantageous effects of the invention:
1, humoral sample stabilisation of the invention can be effectively reduced the interference of matrix effect with aqueous solution, improve noise
Than promoting the accuracy and repeatability of testing result, being particularly suitable for the ELISA that shines.
2, external diagnosis reagent case of the invention can accurately detect GFAP albumen in the peripheral blood of Patients with Stroke
Concentration can be used for clinical diagnosis, the early screening of headstroke, it may also be used for the monitoring for the treatment of curative effect and prognosis are commented with risk of recurrence
Estimate.
3, the present invention can effectively avoid the thecal puncture of high risk.By technological innovation, based in detection peripheral blood
Micro GFAP and diagnose cerebral apoplexy, and testing cost can be greatly reduced.
Detailed description of the invention
Fig. 1 shows in test example 2 GFAP content difference in patient with cerebral apoplexy and the serum sample of normal healthy controls crowd.
Fig. 2 shows GFAP contents in patient with cerebral apoplexy serum sample to extend and raised situation with disease time.
Fig. 3 is MSD GFAP ELISA standard curve.
Specific embodiment
The following describes the present invention further through the description of specific embodiments, but it is to limit of the invention that this, which is not,
System.Those skilled in the art's basic thought according to the present invention, can make various modifications or improvements, but without departing from this
The basic thought of invention, is all within the scope of the present invention.
One, humoral sample stabilisation uses aqueous solution
Although the inventors discovered that also with the presence of micro GFAP albumen in the body fluid of Patients with Stroke, due to the mark
Content of the will albumen in body fluid extremely pettiness, generally pg rank, at the same exist again in body fluid a large amount of non-antigen at
Point, so detection can not be completed using conventional ELISA method.
In view of the above-mentioned problems, the inventors discovered that, if using specific humoral sample stabilisation aqueous solution to body fluid
Sample carries out dilution appropriate, then can mask the interference component in humoral sample to be measured very significantly, and letter/hot-tempered is made to compare pole
The earth improves, so as to stablize and reliably detected using the luminous ELISA that humoral sample carries out headstroke.
First aspect present invention provides one kind and contains for diluting Patients with Stroke body fluid to detect GFAP albumen therein
The humoral sample stabilisation of amount uses aqueous solution.In the present invention, the humoral sample may include blood, urine, saliva, sweat
Liquid, tissue fluid, cerebrospinal fluid etc..Consider from the content of GFAP, the complexity of acquisition and processing of sample etc., it is preferably described
Humoral sample be blood.The blood can be new blood, be also possible to desiccation blood sample.It is particularly preferred that the blood sample
Product are centrifuged the peripheral blood serum of acquisition after standing for the fresh whole blood of Patients with Stroke or suspected patient.
In humoral sample stabilisation aqueous solution for the ELISA that shines of the invention, solute includes human serum, animal blood
Pure albumen, inorganic alkaline metal salt, Tris alkali, protein denaturant and nonionic surface active agent, the pH of the aqueous solution are
6.7~7.6.
The animal serum albumin can come from other mammals other than people, more preferably from ox, sheep,
Horse, rabbit, chicken etc., and be even more preferably bovine serum albumin(BSA).
The inorganic alkaline metal salt can be alkali metal chloride, alkali metal sulfates and alkali metal phosphate.Every
Humoral sample described in 100ml is stabilized in aqueous solution, and the concentration of inorganic alkaline metal salt can be within the scope of 110~200mM, more
It is preferred that within the scope of 120~150mM.
The protein denaturant can be urea or lauryl sodium sulfate.The humoral sample described in every 100ml is stablized
Change in aqueous solution, the concentration of protein denaturant can be within the scope of 0.01~0.5mM, more preferably in 0.05~0.25mM range
It is interior.
The nonionic surface active agent can be Tween-20, Tween-40, Tween-60, Tween-80 and Triton
X-100.In the humoral sample stabilisation aqueous solution described in every 100ml, the concentration of nonionic surface active agent can be
Within the scope of 0.05~5.0 volume %, more preferably within the scope of 0.1~3.0 volume %.
Preferably, the humoral sample stabilisation is blood serum sample stabilisation aqueous solution with aqueous solution.
In a particularly preferred embodiment, humoral sample stabilisation aqueous solution described in 100ml is by 10 volume %
The human serum of~50 volume %, the bovine serum albumin(BSA) of 0.1~5.0 mass/volume %, 0.05~5.0 volume % tween-
20, the urea of 0.05~0.25mM, the sodium chloride of 110~150mM, the potassium chloride of 2.0~3.0mM, 15~25mM Tris alkali,
And sterile distilled water composition.The stabilisation is preferred for the dilution of blood sample to be checked with aqueous solution.Use the blood sample
Stabilisation aqueous solution can make blood sample processing, dilution are primary to complete, easy to operate, and effectively reduce other in serum
The interference of ingredient.
For example, the humoral sample stabilisation can be prepared as follows with aqueous solution:
Prepare a small amount of sterile distilled water, normal human serum is added in 10~50% ratio of volume
Bovine serum albumin(BSA) (Sigma, USA) is added in 0.1~5.0 mass/volume % ratio in (BioreclamationIVT, USA),
Polysorbas20 (Sigma-Aldrich, USA) is added in 0.05~5.0% ratio of volume, the ratio of 0.05~0.25mM by weight is added
Urea (Sigma-Aldrich, USA), the ratio of 110~150mM by weight are added sodium chloride (Sigma-Aldrich, USA), press
Potassium chloride (Sigma-Aldrich, USA) is added in weight 2.0~3.0mM ratio, and Tris alkali is added in the ratio of 15~25mM by weight
(Sigma-Aldrich, USA) is eventually adding sterile distilled water and is uniformly mixed so that total volume is 100ml.
Two, external diagnosis reagent cases
Second aspect of the present invention provides a kind of external diagnosis reagent case for the ELISA that shines, it is preferred to use ELISA is dual anti-
Body sandwich method detects GFAP albumen.
ELISA method is a kind of common antibody or antigen measuring method.ELISA method mainly includes the separation of antigen and net
Change, three links are reacted in sero-fast preparation and antiserum.It is immune to Ag-Ab that ELISA is mainly based upon associated enzyme
Compound occurs enzymic catalytic reaction and is measured.Detection for antigen, there are commonly Direct Determination antigens, competition by ELISA
Method measures the methods of antigen, double antibody sandwich method measurement antigen.
Double antibody sandwich method is the current measurement most common solution of antigen.It, will be known anti-in double antibody sandwich method
Body is adsorbed in the aperture on microtiter plate (plastic plate), washed once；Add determined antigen (for example, containing GFAP albumen
Test serum), specific antigen-antibody occurs and combines；Then unbonded Excess antibody is removed；It is added with determined antigen and is in
The detection antibody that the preparatory and enzyme or luminophore of idiosyncrasy have coupled, makes to form " sandwich "；The substrate of enzyme is added, or necessary
Luminous inducer.Substrate can generate coloring matter or luminophore after being degraded by enzymes can generate visible light or fluorescence, by
Corresponding ELISA analyzer can carry out quantitative detection to color or light, may thereby determine that the presence or absence of antigen and quantity.
The inventors discovered that the content of the significant albumen of neurotrosis (such as GFAP) is extremely in the body fluid of Patients with Stroke
Pettiness, generally pg rank, in order to accurately detect its content, the present invention provides a kind of luminous ELISA diagnostic kit.
Specifically, luminous ELISA diagnostic kit of the invention includes that the sample according to above-mentioned first aspect is steady
Fixedization aqueous solution, also comprising capturing antibody, detection antibody, corresponding zymolyte as needed and operation instructions etc..
In luminous ELISA diagnostic kit of the invention, luminophore has been coupled on detection antibody.With tradition
Unlike ELISA, the reaction carriers for the ELISA that shines are opaque plate (hereinafter, sometimes referred to simply as " ELISA Plate "), are shone
System is photonic absorption, and what detecting instrument utilized is the photonic absorption of photomultiplier tube, and the sensitivity for the ELISA that shines is higher.
The illumination mode of the luminophore may include chemical mode or physics mode.The chemical mode includes enzyme
Catalysis or chemiluminescence, the physics mode include electroluminescent, fluorescence or fluorescence resonance energy transfer.
Chemical luminous immune detection method have high specificity, stablize quickly, detection range is wide, simple operation and other advantages.
The principle of chemiluminescence system is enzyme effect in luminous substrate.Under the action of enzyme, it is anti-that chemistry occurs luminous substrate for substrate
A large amount of energy should and be released, the intermediate of excitation state is generated.This excitation state intermediate, when it returns to stable ground state
When, photon can be launched simultaneously.It can measure quantum yield of luminscence, the quantum yield of luminscence and sample using luminous signal measuring instrument
In test substance amount it is directly proportional.It is possible thereby to establish standard curve and calculate the content of test substance in sample.Specifically
Come, luminol (Luminol) substrate luminescent system can be catalyzed using horseradish peroxidase (HRP).
As an example of chemiluminescent solution, for example, preparing three (methylol) ammonia containing luminol, p-cresol
Methylmethane solution is another to prepare to contain citric acid, anhydrous Na as A liquid2HPO4, carbamide peroxide solution as B liquid, using
It is preceding to mix A liquid and B liquid by 1: 1.
Electrogenerated chemiluminescence (electrogenerated chemiluminescence, ECL) is luminescent substance in electrode
Surface forms the excitation state of high energy after electrochemistry and chemical reaction, then the process of light is generated through relaxation.Common ECL examination
Agent has 9,10- diphenylanthrancene (DPA), ruthenium bipyridyl (Ru (byp)3 2+), peroxyoxalate, luminol and quantum dot etc..
Fluorescence and fluorescence resonance energy transfer (fluorescence resonance energy transfer, FRET)
It in interaction of biomacromolecules, can be immunized in the tool of biological living and vitro detection nanoscale distance change as a kind of
Analysis, detection of nucleic acids etc. are widely used.FRET has many advantages, such as that high sensitivity, widely applicable, analysis speed is fast.Its
In, unique optical properties possessed by quantum dot (QDs) (width absorption, narrow transmitting, anti-light bleaching and fluorescence are adjustable) make it very
It is studied suitable for FRET.
External diagnosis reagent case for the ELISA that shines of the invention preferably uses mouse GFAP monoclonal antibody to be used as and catches
Catch antibody.Here, term " capturing antibody " refers to the antibody being coated on the ELISA Plate (for example, microtiter plate) of solid phase.This
Outside, the external diagnosis reagent case further preferably includes chicken source GFAP polyclonal antibody as detection antibody.Here, term " detection
Antibody " refers to the specific antibody that can not be coated in conjunction with determined antigen and on solid phase ELISA Plate in kit.It " captures anti-
Body " and " detection antibody " can respectively use commercial product, or utilize the ordinary skill in the art, using hybridoma skill
Art or immune animal are prepared.
External diagnosis reagent case for the ELISA that shines of the invention can be used for traumatic brain injury or primary brain
The quick diagnosis of damage.For example, external diagnosis reagent case of the invention can be used for headstroke, hemorrhagic cerebral disorders, wound
Wound property cerebral disorders, neurodegenerative disease (Alzheimer disease, cerebrovascular dementia, Parkinson's disease, Heng Yandunshi dancing
Disease, muscular dystrophy aixs cylinder sclerosis etc.) etc. diseases quick diagnosis.
Preferably, the external diagnosis reagent case for the ELISA that shines of the invention can be used for Patients with Stroke
GFAP albumen in serum is detected.
, can also be comprising any reagent or tool needed for detection in kit of the invention, such as pre-coated plate, wash
Wash liquid, color developing agent, terminate liquid etc..If in kit including the ELISA Plate (for example, microtiter plate) of solid phase, the preferably enzyme
Target is the pre-coated plate that micropore was coated with by the capture antibody.
Three, vitro detection systems
The present invention also provides a kind of vitro detection equipment for stroke diagnosis or prognostic evaluation comprising shines
ELISA analyzer and the external diagnosis reagent case according to above-mentioned second aspect for the ELISA that shines.
For the ELISA analyzer that shines used in the present invention, it may for example comprise but it is not limited to U.S. Meso Scale
The MSD ELISA Imagers of Discovery, the AlphaLISA of PerkinElmer company, the U.S., Quanterix company, the U.S.
Simoa HD1.
Specifically, if using the blood of patient with cerebral apoplexy for humoral sample to be checked, using the ELISA analyzer into
Row detection when basic operational steps include:
The preparation of test serum
Venous puncture patient with cerebral apoplexy blood；1~2h is placed under room temperature state, makes haemocyte and serum natural layering point
From；Serum is uniformly mixed in 2: 1~1: 10 ratios with the blood serum sample stabilisation with aqueous solution, it is spare.
Enzyme-linked Immunosorbent Assay
Prepare 48 or 96 hole microtiter plates (ELISA Plate).GFAP monoclonal antibody is adsorbed on small on microtiter plate
Kong Li washed once with PBS；Patient with cerebral apoplexy serum after dilution produced above is added into microtiter plate well；So
Unbonded Excess antibody is removed afterwards；Then the detection antibody coupled in advance with luminophore, the coupling are added into aperture
Connection detection antibody is preferably the antiserum (that is, polyclonal antibody) of animal origin.It is washed out aperture 3-4 times of the ELISA Plate.
Antigenic content measurement
After washing, it is (such as chemiluminescent that the luminophore inducer is added into the aperture of ELISA Plate if needed
Zymolyte etc.), which can lead to luminophore and generates color or shine.After substrate is added, ELISA Plate is put into phase immediately
The highly sensitive ELISA detector answered reads signal data.
By the way that highly sensitive luminous ELISA detector is diluted and used to blood serum sample, the present invention uses ELISA
Method confrontation GFAP antibody is detected, and can detect the GFAP protein content of the pg rank in Patients with Stroke serum.
The present inventor's experimental studies have found that, after headstroke serum GFAP protein content caused by brain damage extremely increase
Add.Serum GFAP protein level directly reflect brain damage degree, damaging cells type as caused by apoplexy (be ischemic also
It is that hemorrhagic brain cell is impaired), it can be used as paralytic's therapeutic evaluation and formulate the important reference indicator of rehabilitation scheme.According to this
Protein content, the present invention can be used in diagnosing the impaired type of Patients with Stroke brain cell and relative populations, can diagnose asymptomatic
Or headstroke, headstroke acute stage, headstroke convalescence, cerebral apoplexy sequela phase of light symptoms etc..The present invention can also be enough
It is monitored in headstroke early warning and curative effect and the course of disease, stroke prevention and physical examination is instructed to detect.
In addition to blood sample, the vitro detection equipment for stroke diagnosis or prognostic evaluation of the invention is also applied for examining
Survey other humoral samples, such as cerebrospinal fluid, tissue fluid, urine, saliva, sweat etc..
The contents of the present invention are further explained and described below by way of the mode of example, but these examples are understood not to
Limitation to protection scope of the present invention.
Embodiment
Preparation example 1
Humoral sample stabilizes the preparation for using aqueous solution 1
Prepare 50mL sterile distilled water, is added 10mL normal human serum (BioreclamationIVT, USA), 1g cow's serum
Albumin (Sigma, USA), 2mL polysorbas20 (Sigma-Aldrich, USA), ultimate density are the urea (Sigma- of 0.05mM
Aldrich, USA), ultimate density is the sodium chloride (Sigma-Aldrich, USA) of 110mM, and ultimate density is the chlorination of 2.0mM
The Tris alkali (Sigma-Aldrich, USA) that potassium (Sigma-Aldrich, USA) and ultimate density are 10mM, is eventually adding
Surplus sterile distilled water is uniformly mixed and is settled to 100mL.
Preparation example 2
Humoral sample stabilizes the preparation for using aqueous solution 2
Prepare 50mL sterile distilled water, is added 20mL normal human serum (BioreclamationIVT, USA), 2g cow's serum
Albumin (Sigma, USA), 2mL polysorbas20 (Sigma-Aldrich, USA), ultimate density are the urea (Sigma- of 0.08mM
Aldrich, USA), ultimate density is the sodium chloride (Sigma-Aldrich, USA) of 125mM, and ultimate density is the chlorination of 2.3mM
Potassium (Sigma-Aldrich, USA), ultimate density are the Tris alkali (Sigma-Aldrich, USA) of 15mM, are eventually adding surplus
Sterile distilled water is uniformly mixed and is settled to 100mL.
Preparation example 3
Humoral sample stabilizes the preparation for using aqueous solution 3
Prepare 50mL sterile distilled water, is added 30mL normal human serum (BioreclamationIVT, USA), 2g cow's serum
Albumin (Sigma, USA), 2mL polysorbas20 (Sigma-Aldrich, USA), ultimate density are the urea (Sigma- of 0.1mM
Aldrich, USA), ultimate density is the sodium chloride (Sigma-Aldrich, USA) of 137mM, and ultimate density is the chlorination of 2.7mM
Potassium (Sigma-Aldrich, USA), ultimate density are the Tris alkali (Sigma-Aldrich, USA) of 19mM, are eventually adding sterile
Distilled water is uniformly mixed and is settled to 100mL.
Preparation example 4
Humoral sample stabilizes the preparation for using aqueous solution 4
Prepare 20mL sterile distilled water, is added 50mL normal human serum (BioreclamationIVT, USA), 1g Sheep Blood
Pure albumen (Sigma, USA), 5mL polysorbas20 (Sigma-Aldrich, USA), ultimate density are the urea of 0.25mM
(Sigma-Aldrich, USA), ultimate density are the sodium chloride (Sigma-Aldrich, USA) of 150mM, ultimate density 3mM
Potassium chloride (Sigma-Aldrich, USA), ultimate density be 25mM Tris alkali (Sigma-Aldrich, USA), finally plus
Enter sterile distilled water to be uniformly mixed and be settled to 100mL.
Preparation example 5
The preparation of GFAP capture antibody (mouse GFAP monoclonal antibody) solution
Mouse GFAP monoclonal antibody (Abcam, anti-GFAP mouse monoclonal antibody 2A5) is used into phosphate buffer
(PBS) it is diluted to 1 μ g/mL, 4 DEG C save backup.
Preparation example 6
Detect the preparation of antibody (rabbit polyclonal antibody)
Rabbit source polyclonal antibody (Abcam, anti-GFAP, ab7260) is illustrated by producer to the sulfo group label NHS ester with MSD
(SULFO-Tag-NHS-Ester) it is coupled (MSD, R91AO-1), 4 DEG C save backup.
The effect of 1 present invention detection serum GFAP albumen of test example, tests
1, experimental method
(1) MSD ELISA GFAP kit detecting step:
Prepare a 96 hole MSD ELISA ELISA Plates, captures antibody for being coated with GFAP.Specific procedure is: 30 μ L are resisted
Liquid solution (1 μ g/mL, mouse GFAP monoclonal antibody are made according to the preparation method of preparation example 5) is added in ELISA Plate hole, gently
Micro- shake makes antibody uniformly be laid on hole bottom.4 DEG C are placed in overnight, so that antibody is fixed on bottom hole.With 120 μ L PBS by enzyme
Target hole is washed 2 times, and the ingredients such as loose antibody are removed.Each ELISA Plate is added in 120 μ L confining liquids (3%BSA/PBS)
ELISA Plate is placed in microwell plate oscillator and is mixed at room temperature 1 hour with 200~500RPM speed by Kong Zhong.
Preparation standard ox GFAP protein sample (USBiological Life Sciences, Bovine GFAP).By standard
Humoral sample stabilisation aqueous solution 1 obtained in ox GFAP protein sample preparation example 1 is diluted to 8 set by step (2)
A concentration gradient.Two adjacent ELISA Plate holes (as repeated sample) are added in sample after two part of 25 μ L is diluted, by enzyme mark
Plate is placed on microwell plate oscillator to be mixed 1 hour at room temperature with 200~500RPM speed.
Detection antibody (rabbit source GFAP polyclonal antibody) is coupled into (Sulfo-tag, according to MSD Products in advance
Handbook；4C is saved).
Detection antibody will be coupled before experiment, and with solution, (solution is by being dissolved with TRIS buffer
Obtained from 2% bovine serum albumin(BSA) and 0.2% polysorbas20) 2 μ g/mL are diluted to, enzyme mark is added in 25 μ L antibody diluents
ELISA Plate is placed in microwell plate oscillator and is mixed at room temperature 1 hour with 200~500RPM speed by plate hole.
By ELISA Plate, with TBST cleaning solution, (cleaning solution is by that will be spat with TRIS buffer (TBS)
Temperature 20 is diluted to prepared by 0.2% by volume) it washes 4 times.By 150 μ L MSD luminescent solutions (1X), (it is by the way that 4xMSD shines
Obtained from liquid (MSD Read Buffer (4x) Cat#R92TC) is diluted with sterile distilled water) ELISA Plate hole is added, immediately will
ELISA disk is put into the reading signal data of MSD Imager 2400 and referring to MSD Benchwork pre-set programs, calculates automatically dense
Degree.
(2) production and sensitivity (the Lower Limit of Detection of MSD ELISA GFAP standard curve；
LLOD measurement): standard ox GFAP albumen is carried out according to following gradient concentration with humoral sample stabilisation with aqueous solution 1 dilute
It releases: 0,2.5,9.8,39.1,156.3,625,2500 and 10,000 (pg/mL).Relevant concentration is calculated according to above-mentioned steps (1)
With minimum measurement concentration, standard curve is made.
(3) stability and precision measure of MSD GFAP ELISA kit:
3 experiments are carried out according to experimental procedure (1) and (2) to obtain 3 independent standard curve measured values.Each experiment
Between be separated by 8 months.At least 2 years detection stability and measurement essence of kit have been obtained by comparing the result of 3 experiments
Degree.
2, experimental result
(1) MSDGFAP ELISA standard curve and sensitivity:
3 experiments are carried out according to experimental procedure (1) and (2).
3 times experimental result shows that the range of linearity is wide, high sensitivity.3 experiments are sensitive to the detection of blood GFAP albumen
Spend (minimal detectable concentration；LLOD) it is respectively 17.9,21.1 and 20.8pg/mL, is below the concentration of 22pg/mL, has very big
Diagnostic value.Experimental result is shown in table 1 and attached drawing 3.
Table 1:MSD GFAP ELISA standard curve and sensitivity
Experiment 1
Experiment 2
Experiment 3
(2) stability and precision of MSDGFAP ELISA kit:
It is shown according to 6 independent experimental results for being separated by 4 months, GFAP kit of the invention has highly stable
Testing result and detection accuracy.Relative error (the Relative Error that 6 independent experimental results calculate；RE) and become
Different coefficient (Coefficient of variation；CV) 10% is below in the concentration range of 156pg/mL or more.Experiment
As a result it is shown in table 2.
The stability and precision of table 2:MSD GFAP ELISA kit
The statistical result of 6 experiments:
2 MSD ELISA method detection Patients with Stroke serum GFAP content of test example
1, experimental method
(1) stroke patient collects condition:
1. being included in standard: patient age 40~85 years old, admission time was in morbidity 48h；It is diagnosed using classical ways such as CT
For ischemic or hemorrhagic acute apoplexy.
2. exclusion criteria: with mental disease or the serious disturbance of consciousness or communication obstacle, cannot complete to check；Previously
There are severe head wound or stroke morbidity history in 3 months；Non- compressing position arteriopuncture in the past 14d, or receive capital operation；
Intracranial arteriovenous malformation or aneurysm.
3. sample collection method: each case collects 3~5 serum samples, specifically includes:
Acute stage: after stroke generation in 7 days: acquiring serum sample 1 time, amount to 2~3 times every other day；
Convalescence: 8~30 days after stroke generation: acquisition serum sample 1 time；Sequela stage: acquisition serum sample 1 time.
The normal person that 34 patient with cerebral apoplexies and 25 ages, genders match is included in experiment.
(2) blood sample acquisition and processing:
It is 0h with admission time, 0,8 hour, 24 hours, 72 hours (the 3rd day), 120 hours (the 5th day), 192 hours
(the 8th day) and 240 hours (the 10th day), patient 1mL blood sample was collected respectively.Normal human blood sample is collected in 8 points of morning.It is stored at room temperature
After 1~2h, separation serum saves backup after dry powder is made using frozen drying instrument.
(3) blood sample GFAP protein content detects:
By desiccation blood sample 1mL dissolved in purified water.By sample stabilisation aqueous solution prepared in sample and preparation example 1
1 is mixed in 1: 1 ratio, 25 μ L dilute samples (as repeated sample) is separately added into 2 ELISA Plate holes, according to test example 1
In experimental procedure (1) test sample in GFAP content.
2, experimental result
To 67 blood samples in total, (wherein, 42 samples belong to stroke patient to the present invention and 25 samples belong to health
People) testing result discovery, the average value of the blood GFAP content of patient with cerebral apoplexy group is 79.81pg/mL, hence it is evident that is higher than health
The average value 2.72pg/mL of people's control group.Testing result is shown in table 3 and attached drawing 1.And it was found that as shown in Fig. 2, in brain
The lengthening of time after wind occurs, the content of GFAP is on a declining curve in blood.
Table 3: the blood GFAP content of patient with cerebral apoplexy and Healthy People control group
The present invention obtains high sensitivity using the ELISA method detection Patients with Stroke serum GFAP protein content that shines.
The present invention can accurately measure the GFAP protein content change level as caused by apoplexy in patient with cerebral apoplexy peripheral blood, compensate for
The defect of traditional image method or cerebrospinal fluid analytic approach, and asymptomatic or light symptoms stroke diagnosis can be carried out.Of the invention
It promotes and applies, it is expected to reduce incidence rate of stroke, curing apoplexy scheme is formulated in guidance, is lost after assessment apoplexy curative effect, mitigation apoplexy
Disease.Therefore, there is important and extensive clinical diagnostics value.
Claims (10)
1. external diagnosis reagent case of the one kind for Enzyme-linked Immunosorbent Assay (ELISA) that shine, comprising:
Humoral sample, which stabilizes, uses aqueous solution, contains the significant protein ingredient of neurotrosis in the humoral sample, in the body fluid
Sample is stabilized in aqueous solution, and solute includes human serum, animal serum albumin, inorganic alkaline metal salt, Tris alkali, albumen change
Property agent and nonionic surface active agent, the pH value of the aqueous solution is 6.7~7.6；And
What is optionally included resists the antibody of the significant protein ingredient of the neurotrosis.
2. the external diagnosis reagent case according to claim 1 for the ELISA that shines, wherein
The antibody for resisting the significant protein ingredient of the neurotrosis is anti-glial fibrillary acidic albumen
(GFAP) antibody, the anti-GFAP antibody include:
Antibody is captured, and
Detect antibody；
Wherein antibody and the detection antibody of capturing is the anti-GFAP antibody for being directed to GFAP albumen difference epitope respectively,
The animal serum albumin comes from ox, sheep, horse, rabbit, chicken, and preferably bovine serum albumin(BSA).
3. the external diagnosis reagent case according to claim 2 for the ELISA that shines, wherein
Luminophore has been coupled on the detection antibody,
The illumination mode of the luminophore includes chemical mode or physics mode,
The chemical mode preferably includes enzymatic or chemiluminescence,
The physics mode preferably includes any one in electroluminescent, fluorescence and fluorescence resonance energy transfer (FRET).
4. the external diagnosis reagent case according to claim 2 for the ELISA that shines, wherein
The capture antibody is monoclonal antibody or polyclonal antibody, and the detection antibody is monoclonal antibody or polyclonal
Antibody.
5. the external diagnosis reagent case according to claim 4 for the ELISA that shines, wherein
The polyclonal antibody from mouse, rabbit, chicken, sheep, cavy, donkey, Malaysia and China any one.
6. the external diagnosis reagent case according to claim 2 for the ELISA that shines, wherein
The concentration of the human serum is 10 volume of volume %~50 %；
The concentration of the bovine serum albumin(BSA) is 0.1 mass/volume of mass/volume %~10 %；
The concentration of the inorganic alkaline metal salt is 110mM~200mM；
The concentration of the Tris alkali is 10mM~25mM；
The concentration of the protein denaturant is 0.01mM~0.5mM, and
The concentration of the nonionic surface active agent is 0.05 volume of volume %~5.0 %.
7. the external diagnosis reagent case according to claim 1 for the ELISA that shines, wherein
The inorganic alkaline metal salt is selected from alkali metal chloride；
The protein denaturant is selected from least one of urea and lauryl sodium sulfate；And
The nonionic surface active agent is selected from Tween-20, Tween-40, Tween-60, in Tween-80 and Triton X-100
At least one.
8. the external diagnosis reagent case according to any one of claims 1 to 7 for the ELISA that shines, wherein
The body fluid is blood, serum, cerebrospinal fluid, tissue fluid, urine, saliva or sweat, preferably human peripheral serum；More
It is preferred that containing GFAP in the human peripheral serum as the significant protein ingredient of the neurotrosis；It is even more preferably every
GFAP contained in the milliliter human peripheral serum is pg grades horizontal；
The external diagnosis reagent case is preferred for headstroke, hemorrhagic cerebral disorders, traumatic cerebral disorders, neurodegenerative disease
(Alzheimer disease, cerebrovascular dementia, Parkinson's disease, Heng Yandunshi chorea, muscular dystrophy aixs cylinder sclerosis)
In-vitro diagnosis.
9. a kind of vitro detection equipment for stroke diagnosis or prognostic evaluation, including shine ELISA analyzer and according to
External diagnosis reagent case according to any one of claims 1 to 8 for the ELISA that shines.
10. the vitro detection equipment according to claim 9 for stroke diagnosis or prognostic evaluation, wherein described
The detectivity of ELISA analyzer is less than 10pg.
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| CN117268877A (en) * | 2023-11-21 | 2023-12-22 | 军科正源（北京）药物研究有限责任公司 | Method for detecting nerve growth factor in human tear and method for treating tear |
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