CN102175873A - Combined parallel detection method of cardiovascular disease protein marker and diagnosis kit of cardiovascular disease protein marker - Google Patents
Combined parallel detection method of cardiovascular disease protein marker and diagnosis kit of cardiovascular disease protein marker Download PDFInfo
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- CN102175873A CN102175873A CN2011100043836A CN201110004383A CN102175873A CN 102175873 A CN102175873 A CN 102175873A CN 2011100043836 A CN2011100043836 A CN 2011100043836A CN 201110004383 A CN201110004383 A CN 201110004383A CN 102175873 A CN102175873 A CN 102175873A
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Abstract
The invention discloses a combined parallel detection method of a cardiovascular disease protein marker and a diagnosis kit of the cardiovascular disease protein marker. The combined parallel detection method is characterized in capability of detecting multiple protein markers in the same sample at one time. The method comprises the following steps of: forming a quadruplet complex of 'signal-labeled detection body-protein markers of cardiovascular diseases-capture antibody-microsphere' through preparation of a liquid phase chip; and determining existence and content of different protein markers of cardiovascular diseases in samples to be detected by detecting marking signals of different microspheres in the quadruplet complex. The invention also discloses compositions of the diagnosis kit and application of the diagnosis kit. The method and the kit have the advantages of high throughput, high sensibility and specificity, and fast and rapid detection, and can be used for qualitative and quantitative detection of multiple protein markers of cardiovascular diseases at the same time.
Description
Technical field
The present invention relates to a kind of in-vitro diagnosis detection method and diagnostic kit thereof, particularly relate to the liquid-phase chip combined parallel detecting method and the diagnostic kit thereof of cardiovascular and cerebrovascular disease protein marker.
Background technology
Cardiovascular and cerebrovascular disease is one of the principal disease of Chinese city population and western developed country and major causes of death, comprises that mainly with coronary heart disease, hypertension etc. be the angiocardiopathy of representative, and ischemic cerebrovascular disease and hemorrhagic cerebrovascular disease.Ischemic cerebrovascular disease claims acute ischemic cerebrovascular disease syndrome (AICS) again, comprises that cerebral infarction (also claims cerebral infarction, CI) with TIA outbreak (TIA); Hemorrhagic cerebrovascular disease comprises cerebral hemorrhage and subarachnoid hemorrhage.The joint-detection of cardiovascular and cerebrovascular disease protein marker has become the hazards examination of cardiovascular and cerebrovascular disease or the very important index that prediction, early diagnosis, disease process, curative effect monitoring and prognosis are judged, and the rapid property of the high flux of many indexs associating parallel detection, accuracy and stability are just most important.
The relevant protein marker of Recent study generation development that find and cardiovascular and cerebrovascular disease comprises vascular endothelial growth factor (VEGF), monocyte chemoattractant protein-1 (MCP-1), metal matrix proteinase-9 (MMP-9), people's matrix metalloproteinase inhibiting factor-1 (TIMP-1), interferon-(IFN-γ).
At present, for the existing multiple detection method of the mensuration of cardiovascular and cerebrovascular disease protein marker, comprise immunoturbidimetry analysis, enzyme-linked immuno assay (ELISA), radiommunoassay (RIA), the separation enzyme-linked immunoassay of magnetic (EIMA) etc.But these technology all exist the limitation that detects flux, and complex operation, sensitivity are relatively poor, can not really satisfy the needs that clinical diagnosis detects.Repeatability is poor, insufficient sensitivity good and the shortcoming of complex operation and the solid phase biological chip technology exists.
Liquid-phase chip (xMAP) claim liquid chip again, be a kind of biochip technology of new generation that is widely used in multiple biological respinses such as protein, gene, receptor/ligand, mainly comprise color-coded microballoon (Color-coded Beads), probe molecule, detected material and four kinds of compositions of reporter molecules.In the middle of the manufacture process of microballoon, mixed two kinds of different redness classification fluorescence,, sphere matrix has been divided into 100 kinds according to the ratio difference of these two kinds of fluorescence, 100 kinds of different probe molecules on can mark, can be simultaneously in the sample nearly 100 kinds of different target molecules detect.In the course of reaction, probe and reporter molecules all combine with the target molecule specificity respectively.After reaction finishes, make single microballoon, use red, green two-color laser simultaneously the green report fluorescence on classification fluorescence of the redness on the microballoon and the reporter molecules to be detected, can determine the kind and the quantity of the detection thing of institute's combination by sense channel.
The present invention is based on the high flux, high sensitivity, high specific, good reproducibility of liquid-phase chip technology, easy and simple to handle, outstanding advantage such as the range of linearity is wide, detection is rapid, the multiple protein mark relevant to cardiovascular and cerebrovascular disease carries out parallel detection, can obtain better application on clinical detection.
Summary of the invention
One of technical matters to be solved by this invention provides a kind of liquid-phase chip combined parallel detecting method that is used for the cardiovascular and cerebrovascular disease protein marker;
Two of technical matters to be solved by this invention provides a kind of diagnostic kit that detects the cardiovascular and cerebrovascular disease protein marker;
Three of technical matters to be solved by this invention provides the application of above-mentioned diagnostic kit.
For solving the problems of the technologies described above, the liquid-phase chip combined parallel detecting method of a kind of cardiovascular and cerebrovascular disease protein marker of the present invention may further comprise the steps:
(1) with microballoon (Beads) coupling of capture antibody with different numberings, form " capture antibody-microballoon " bigeminy complex, the anti-respectively a kind of cardiovascular and cerebrovascular disease protein marker of described each capture antibody, thus make cardiovascular and cerebrovascular disease protein marker and capture antibody in the testing sample form " cardiovascular and cerebrovascular disease protein marker-capture antibody-microballoon " three complexs; Described cardiovascular and cerebrovascular disease protein marker comprise following mark any one or arbitrarily multiple or add the combination of other mark: VEGF, MCP-1, MMP-9, TIMP-1, IFN-γ; Be described multiple protein mark can be comprise among VEGF, MCP-1, MMP-9, TIMP-1, the IFN-γ any one, any two kinds, any three kinds, any four kinds, any five kinds or add the combination of other mark.
(2) different detection antibody is carried out the signal mark, wherein said each detect the anti-respectively a kind of cardiovascular and cerebrovascular disease protein marker of antibody and corresponding to capture antibody, and be incorporated into the different epitopes of this protein marker respectively with capture antibody;
(3) the detection antibody that has the signal mark in three complexs that step (1) is formed and the step (2) mixes, thus formation " the detection antibody-cardiovascular and cerebrovascular disease protein marker-capture antibody-microballoon of signal mark " tetrad complex;
(4) detect the marking signal of different microballoons in the tetrad complex with liquid-phase chip method (xMAP), thereby determine the existence and the content of various cardiovascular and cerebrovascular disease protein markers in the detected sample.
In the step (4), the content of various cardiovascular and cerebrovascular disease protein markers in described definite detected sample, its concrete grammar is: detectable label signal and the typical curve measured in the step (4) compared, thus the content of various cardiovascular and cerebrovascular disease protein markers in definite detected sample.
The mean diameter of described microballoon can be 5.6 μ m, and this microballoon is the polyphenyl alkene microballoon that combines different fluorescent dyes, i.e. color coding microballoon (Color-coded Beads).
Described signal mark refers to adopt biotin (the Biotin)-mark of Streptavidin-phycoerythrin (Streptavidin-PE) or the immune labeled method of other this area routine such as fluorescein, enzyme, chemiluminescence agent etc.
Described in the step (1) and the microballoon capture antibody coupling can use activated microballoon (Activated Beads), can be directly and capture antibody covalent coupling (as the free thoil group by the antibody surface); If (COOH) microballoon must activate microballoon earlier, normally adopts chemical substance such as EDC/S-NHS to activate, and microballoon just can be contained molecule (as the antibody etc.) covalent bond of free amino group group (Amino Group) like this to use carboxyl.The microballoon activation generally can be adopted following method: microballoon activates in 3 mixed liquors of being made up of 1-ethyl-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDC) solution, N-hydroxy thiosuccinimide (S-NHS) solution and activation damping fluid, wherein, the amount ratio of 3 mixed liquors is: when microballoon quantity is 2.5 * 10
6When individual, need 50mg/mlEDC solution 10 μ l: 50mg/mlS-NHS solution 10 μ l: 100mM NaH
2PO
4, pH 6.3 activation damping fluid 80 μ l, the consumption of mixed liquor can be adjusted accordingly according to the quantity of microballoon.
Described capture antibody and detect antibody be at following 5 kinds can independent assortment the cardiovascular and cerebrovascular disease protein marker capture antibody and detect antibody:
VEGF、MCP-1、MMP-9、TIMP-1、IFN-γ。
Detect with liquid-phase chip method (xMAP) in the step described in the detection method (4), described detectable label signal is a fluorescence signal.
In another aspect of this invention, provide a kind of diagnostic kit that detects the cardiovascular and cerebrovascular disease protein marker, mainly comprise following component:
(1) microballoon of coupling antibody: the microballoon of different capture antibodies that contained respectively coupling comprises following any one or any multiple microballoon: coupling the microballoon of vascular endothelial growth factor VEGF capture antibody, coupling the microballoon of monocyte chemoattractant protein-1MCP-1 capture antibody, coupling the microballoon of metal matrix proteinase-9MMP-9 capture antibody, coupling the microballoon of people's matrix metalloproteinase inhibiting factor-1TIMP-1 capture antibody, coupling the microballoon of interferon-IFN-γ capture antibody; Every kind of capture antibody resists a kind of cardiovascular and cerebrovascular disease protein marker respectively and is coupled to different microballoons of numbering, and forms the bigeminy complex of " antibody-microballoon ";
(2) the detection antibody of signal mark: comprise following any one or any multiple detection antibody that has the signal mark: the IFN-γ that the TIMP-1 that the MMP-9 that the MCP-1 that the VEGF of mark detects antibody, mark detects antibody, mark detects antibody, mark detects antibody, mark detects antibody; Wherein said each detect the anti-respectively a kind of corresponding cardiovascular and cerebrovascular disease protein marker of antibody and corresponding to capture antibody, and be incorporated into the different epitopes of this protein marker respectively with capture antibody;
(3) standard items: the standard items that comprise various cardiovascular and cerebrovascular disease protein markers (antigen);
(4) quality-control product: comprise positive control and negative control.
The detection antibody of described signal mark can be the detection antibody that adopts biotin (Biotin) mark; As adopt biotin labeling to detect antibody, this diagnostic kit also comprises Streptavidin-phycoerythrin (Streptavidin-PE), wherein Streptavidin can combine with the biotin specificity, form the fluorescein-labeled detection antibody of band phycoerythrin (PE), the green laser by the liquid-phase chip instrument excites phycoerythrin to carry out fluoroscopic examination.
Wherein said capture antibody and detect antibody be at following 5 kinds can independent assortment the cardiovascular and cerebrovascular disease protein marker capture antibody and detect antibody:
VEGF、MCP-1、MMP-9、TIMP-1、IFN-γ。
The application of above-mentioned diagnostic kit also is provided in another aspect of this invention.A kind of diagnostic kit that detects the cardiovascular and cerebrovascular disease protein marker of the present invention can be used for detecting the existence and the content of cardiovascular and cerebrovascular disease protein marker in the vitro samples, and be used for the hazards examination of cardiovascular and cerebrovascular disease or prediction, diagnostic detection, curative effect monitoring and prognosis are judged, also can be used for the diagnostic detection or the prediction of other disease except that cardiovascular and cerebrovascular disease.
Because the present invention has utilized liquid-phase chip technology, multiple protein mark to cardiovascular and cerebrovascular disease carries out the associating parallel detection, the feature of combined parallel detecting method is the multiple biomarker in can the same sample of disposable detection, make detection method and kit have outstanding advantages such as high sensitivity, high specific, high flux, good stability, detection be rapid, accurate, can carry out qualitative and detection by quantitative simultaneously to multiple relevant protein marker, can on clinical detection, obtain better application.
Description of drawings
The present invention is further detailed explanation below in conjunction with accompanying drawing and embodiment:
Fig. 1 is the concentration profile that detects VEGF in cardio-cerebral vascular disease patient and the normal control group in the embodiment of the invention 3;
Fig. 2 is the concentration profile that detects MCP-1 in cardio-cerebral vascular disease patient and the normal control group in the embodiment of the invention 3;
Fig. 3 is the concentration profile that detects MMP-9 in cardio-cerebral vascular disease patient and the normal control group in the embodiment of the invention 3;
Fig. 4 is the concentration profile that detects TIMP-1 in cardio-cerebral vascular disease patient and the normal control group in the embodiment of the invention 3;
Fig. 5 is the concentration profile that detects IFN-γ in cardio-cerebral vascular disease patient and the normal control group in the embodiment of the invention 3.
Embodiment
Experiment material:
Used 5 kinds of cardiovascular and cerebrovascular disease protein marker VEGF, MCP-1, MMP-9, TIMP-1, IFN-γ (antigen) and the corresponding antibodies of the present invention derives from Abcam, Santa Cruz Biotechnology, Fitzgerald, Biodesign and R ﹠amp; DSystems company;
Carboxyl microballoon (surperficial carboxyl modified), the Streptavidin-phycoerythrin of different numberings are all purchased the company in QIAGEN;
1-ethyl-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDC), N-hydroxy thiosuccinimide (S-NHS) and N-hydroxy thiosuccinimide biotin (S-NHS-Biotin) are purchased the company in Pierce.
The damping fluid preparation:
Activation damping fluid (Activation buffer): 100mM NaH
2PO
4, pH 6.3;
Coupling buffer (Coupling buffer): 50mM HEPES, pH 7.4;
Phosphate buffer (PBS): 10mM NaH
2PO
4, 150mM NaCl, pH 7.4;
Analysis buffer (Assay Buffer): PBS with 1% BSA, 0.05% Sodium Azide, pH 7.4;
Cleansing solution (Wash Buffer): PBS with 0.05% Tween 20,0.05% Proclin, pH 7.4.
The liquid-phase chip associated detecting method of embodiment 1:2 kind cardiovascular and cerebrovascular disease protein marker
Concrete detection method comprises the steps:
Selection is numbered 2 kinds of microballoons of 11, No. 15
1. the activation of required microballoon
1.1 full speed vortex microballoon storage liquid is 3min at least, forms the microballoon suspension of homogeneous;
1.2 take by weighing respectively among the EDC and S-NHS to two centrifuge tube of 10mg;
1.3 making its final concentration with deionized water dissolving is 50mg/ml;
1.4 get the centrifugal 3min of microballoon suspension 10000g of 1ml, carefully remove supernatant;
1.5 it is resuspended with microballoon to add the activation damping fluid of 80 μ l;
1.6 add the EDC solution (50mg/ml) of 10 μ l and the S-NHS solution (50mg/ml) of 10 μ l respectively, mix, room temperature (15-25 ℃), lucifuge, 20min is hatched in vibration.
2. the microballoon coupling of corresponding capture antibody and activation
2.1 with coupling buffer capture antibody being diluted to volume is 500 μ l, concentration is the solution of 0.1mg/ml; (can not contain foreign protein in the antibody-solutions, azide, aminoacetic acid, Tris or other any reagent that contains amino.If contain these reagent, remove by dialysis or gel permeation chromatography.)
2.2 microballoon at the centrifugal 3min of 10000g, is carefully removed supernatant;
2.3 add and diluted good antibody-solutions (500 μ l) in the step 2.1;
2.4 with the microballoon and the antibody-solutions of activation, under room temperature (15-25 ℃), lucifuge, 2h is hatched in vibration; (centrifuge tube must wrap up lucifuge with tinfoil)
2.5 microballoon at the centrifugal 3min of 10000g, is carefully removed supernatant;
2.6 it is resuspended with microballoon to add 500 μ l PBS, the centrifugal 3min of 10000g carefully removes supernatant;
2.7 it is resuspended with microballoon to add the 1ml analysis buffer;
2.8 microballoon is counted by thrombocytometer;
3. biotin (Biotin) marker detection antibody
3.1 (S-NHS-Biotin) takes out from the reefer of refrigerator with biotin reagent, at room temperature balance makes it return to room temperature;
3.2, antibody dilution is arrived 1mg/ml with PBS according to the concentration that detects antibody;
If being dissolved in, antibody contains in the amino solution, should be earlier except that deaminizing, and being replaced as does not have amino damping fluid; 3.3 biotin solution with ultrapure water configuration 10mM;
3.4 1: 20 in molar ratio (antibody: biotin), calculate the needed amount of biotin, join in the antibody-solutions that concentration is 1mg/ml;
Computing formula:
3.5 reactant liquor is hatched 2h on ice, or at incubated at room 30min;
3.6 reactant liquor is transferred to dialysis cassette, removes wherein unreacted S-NHS-Biotin.
4. the configuration of antigen standard items
VEGF and MCP-1 by 31250,6250,1250,250,50,10, the concentration of 0pg/ml prepares, the mark mixed liquor is labeled as STD6, STD5, STD4, STD3, STD2, STD1, STD0 respectively.
5. the preparation of the microballoon mixed liquor of coupling capture antibody (I mixed liquor)
The microballoon of capture antibody of 2 kinds of cardiovascular and cerebrovascular disease protein markers of having got coupling respectively, as following: VEGF capture antibody microballoon 11, MCP-1 capture antibody microballoon 15, equal proportion is mixed, and makes the final concentration of every kind of microballoon be respectively 200/μ l, and 4 ℃ keep in Dark Place.
6. contain the preparation of biotin labeled detection antibody mixed liquor (II mixed liquor)
Get respectively and carried out that biotin labeled VEGF detects antibody, MCP-1 detects antibody, add analysis buffer, make the final concentration of every kind of detection antibody be respectively 10 μ g/ml.
7. quality-control product
Quality-control product comprises positive control and negative control.
8. the content detection of 2 kinds of cardiovascular and cerebrovascular disease biomarkers in the blood serum sample
Blood serum sample comprises 12 parts in normal human serum sample (normal control group 1-12 sample), 12 parts in cardiovascular and cerebrovascular disease patients serum sample (cardiovascular and cerebrovascular disease group be for 1-3 number patients with coronary heart disease, 4-6 number number be the sample of Patients with Hemorrhagic Cerebrovascular Disease for ischemic cerebrovascular disease patient, 10-12 for hyperpietic, 7-9 number).
Prewet 8.1 add the cleansing solution in 200 μ l/ holes in the miillpore filter plate (96 hole) respectively, 5min is hatched in concussion at room temperature, removes liquid with the vacuum pump suction filtration;
8.2 add analysis buffer respectively in the miillpore filter plate, 25 μ l/ holes;
8.3 the microballoon mixed liquor (I mixed liquor) that adds the coupling capture antibody respectively is in the miillpore filter plate, 25 μ l/ holes;
8.4 the normal human serum sample that add standard items (STD0, STD1, STD2, STD3, STD4, STD5, STD6), quality-control product (positive control and negative control), patients serum's sample 1-12 number that diluted respectively, diluted 1-12 number, 25 μ l/ holes;
8.5 with the potpourri of mixing up and down of volley of rifle fire tenderness, 60min is hatched in lucifuge concussion at room temperature;
8.6 the vacuum pump suction filtration is removed liquid in the hole, with cleansing solution 200 μ l/ holes, washs 2 times; Vacuum filtration is removed cleansing solution;
8.7 add respectively and contain biotin labeled detection antibody mixed liquor (II mixed liquor), 25 μ l/ holes, 30min is hatched in lucifuge concussion at room temperature;
8.8 Streptavidin-phycoerythrin (Streptavidin-PE) is diluted to the solution that concentration is 200 μ g/ml with analysis buffer, every hole adds the good Streptavidin phycoerythrin 25 μ l of dilution, with the potpourri of mixing up and down of volley of rifle fire tenderness, 20min is hatched in lucifuge concussion at room temperature;
8.9 the vacuum pump suction filtration is removed liquid in the hole, with cleansing solution 200 μ l/ holes, washs 2 times; Vacuum filtration is removed cleansing solution;
8.10 every hole adds the resuspended microballoon of 100 μ l analysis buffer respectively;
8.11 on liquid-phase chip instrument (LiquiChip 200 or Luminex 200), the potpourri of reaction is analyzed, drawing standard curve automatically, and calculate the content of 2 kinds of cardiovascular and cerebrovascular disease marks in the test sample according to typical curve.
8.12 testing result and analysis
Specifically referring to table 1-3.
The concentration value of table 1 cardiovascular and cerebrovascular disease histone mark
The concentration value of table 2 normal control histone mark
The protein marker mean value contrast of table 3 cardiovascular and cerebrovascular disease patient and normal group
Above result shows, can carry out the associating parallel detection to 2 kinds of cardiovascular and cerebrovascular disease protein markers simultaneously with the inventive method, the concentration mean value of 2 kinds of marks as can be seen from table 1-3,2 kinds of protein markers of this of cardiovascular and cerebrovascular disease patient, the VEGF group exceeds 4.7 times of normal control groups, the MCP-1 group exceeds 3.3 times of normal control groups, has significant difference, can be used as the important indicator that the cardiovascular and cerebrovascular disease clinical diagnosis detects or predicts.
The liquid-phase chip associated detecting method of embodiment 2:3 kind cardiovascular and cerebrovascular disease protein marker
Concrete detection method comprises the steps:
Selection is numbered 3 kinds of microballoons of 21,33, No. 37
The 1-3 step is with embodiment 1.
4. the configuration of antigen standard items
MMP-9 and TIMP-1 by 31250,6250,1250,250,50,10, the concentration of 0ng/ml prepares, IFN-γ by 31.25,6.25,1.25,0.25,0.05,0.01, the concentration of 0pg/ml prepares, the mark mixed liquor is labeled as STD6, STD5, STD4, STD3, STD2, STD1, STD0 respectively.
5. the preparation of the microballoon mixed liquor of coupling capture antibody (I mixed liquor).
Get bag respectively by the microballoon of capture antibody of 3 kinds of cardiovascular and cerebrovascular protein markers, as following: MMP-9 capture antibody microballoon 21, TIMP-1 capture antibody microballoon 33, IFN-γ capture antibody microballoon 37, equal proportion is mixed, make the final concentration of every kind of microballoon be respectively 200/μ l, 4 ℃ keep in Dark Place.
6. contain the preparation of biotin labeled detection antibody mixed liquor (II mixed liquor)
Get respectively and carried out biotin labeled MMP-9 and detect that antibody, TIMP-1 detect antibody, IFN-γ detects antibody, adds analysis buffer, makes the final concentration of every kind of detection antibody be respectively 10 μ g/ml.
7. quality-control product
Quality-control product comprises positive control and negative control.
8. the content detection of 3 kinds of cardiovascular and cerebrovascular disease protein markers in the blood serum sample
Blood serum sample comprises 12 parts in normal human serum sample (normal control group 1-12 sample), 12 parts in cardiovascular and cerebrovascular disease patients serum sample (cardiovascular and cerebrovascular disease group be for 1-3 number patients with coronary heart disease, 4-6 number number be the sample of Patients with Hemorrhagic Cerebrovascular Disease for ischemic cerebrovascular disease patient, 10-12 for hyperpietic, 7-9 number).
Prewet 8.1 add the cleansing solution in 200 μ l/ holes in the miillpore filter plate (96 hole) respectively, 5min is hatched in concussion at room temperature, removes liquid with the vacuum pump suction filtration;
8.2 add analysis buffer respectively in the miillpore filter plate, 25 μ l/ holes;
8.3 the microballoon mixed liquor (I mixed liquor) that adds the coupling capture antibody respectively is in the miillpore filter plate, 25 μ l/ holes;
8.4 the normal human serum sample that add standard items (STD0, STD1, STD2, STD3, STD4, STD5, STD6), quality-control product (positive control and negative control), patients serum's sample 1-12 number that diluted respectively, diluted 1-12 number, 25 μ l/ holes;
8.5 with the potpourri of mixing up and down of volley of rifle fire tenderness, 60min is hatched in lucifuge concussion at room temperature;
8.6 the vacuum pump suction filtration is removed liquid in the hole, with cleansing solution 200 μ l/ holes, washs 2 times; Vacuum filtration is removed cleansing solution;
8.7 add respectively and contain biotin labeled detection antibody mixed liquor (II mixed liquor), 25 μ l/ holes, 30min is hatched in lucifuge concussion at room temperature;
8.8 Streptavidin-phycoerythrin (Streptavidin-PE) is diluted to the solution that concentration is 200 μ g/ml with analysis buffer, every hole adds the good Streptavidin phycoerythrin 25 μ l of dilution, with the potpourri of mixing up and down of volley of rifle fire tenderness, 20min is hatched in lucifuge concussion at room temperature;
8.9 the vacuum pump suction filtration is removed liquid in the hole, with cleansing solution 200 μ l/ holes, washs 2 times; Vacuum filtration is removed cleansing solution;
8.10 every hole adds the resuspended microballoon of 100 μ l analysis buffer respectively;
8.11 on liquid-phase chip instrument (LiquiChip 200 or Luminex 200), the potpourri of reaction is analyzed, drawing standard curve automatically, and calculate the content of 3 kinds of cardiovascular and cerebrovascular disease protein markers in the test sample according to typical curve.
8.12 testing result and analysis
Referring to table 4-6.
Table 4 cardiovascular and cerebrovascular disease histone mark concentration value
Table 5 normal control histone mark concentration value
The cardiovascular and cerebrovascular disease protein marker mean value contrast of table 6 cardiovascular and cerebrovascular disease patient and normal group
Above result shows, can carry out the associating parallel detection to 3 kinds of cardiovascular and cerebrovascular disease protein markers simultaneously with the inventive method, but also can carry out the joint-detection of various combination according to the actual requirements to 3 kinds of cardiovascular and cerebrovascular disease protein markers.The concentration mean value of 3 kinds of marks as can be seen from table 4-6,3 kinds of protein markers of this of cardiovascular and cerebrovascular disease patient, wherein the MMP-9 group exceeds 5.1 times of normal control groups, the TIMP-1 group exceeds 2.6 times of normal control group, IFN-γ group exceeds 3.2 times of normal control group, have significant difference, can be used as the important indicator that the cardiovascular and cerebrovascular disease clinical diagnosis detects or predicts.
The liquid-phase chip associated detecting method of embodiment 3:5 kind cardiovascular and cerebrovascular disease protein marker
Concrete detection method comprises the steps:
Selection is numbered 5 kinds of microballoons of 11,15,21,33, No. 37
The 1-3 step is with embodiment 1.
4. the configuration of antigen standard items
VEGF and MCP-1 by 31250,6250,1250,250,50,10, the concentration of 0pg/ml prepares, MMP-9 and TIMP-1 by 31250,6250,1250,250,50,10, the concentration of 0ng/ml is configured, IFN-γ by 31.25,6.25,1.25,0.25,0.05,0.01, the concentration of 0pg/ml prepares, the mark mixed liquor is labeled as STD6, STD5, STD4, STD3, STD2, STD1, STD0 respectively.
5. the preparation of the microballoon mixed liquor of coupling capture antibody (I mixed liquor)
Get bag respectively by the microballoon of capture antibody of 5 kinds of cardiovascular and cerebrovascular diseases mark albumen, as following: VEGF capture antibody microballoon 11, MCP-1 capture antibody microballoon 15, MMP-9 capture antibody microballoon 21, TIMP-1 capture antibody microballoon 33, IFN-γ capture antibody microballoon 37, equal proportion is mixed, make the final concentration of every kind of microballoon be respectively 200/μ l, 4 ℃ keep in Dark Place.
6. contain the preparation of biotin labeled detection antibody mixed liquor (II mixed liquor)
Get respectively and carried out biotin labeled VEGF and detect antibody, MCP-1 and detect antibody, MMP-9 and detect that antibody, TIMP-1 detect antibody, IFN-γ detects antibody, adds analysis buffer, makes the final concentration of every kind of detection antibody be respectively 10 μ g/ml.
7. quality-control product
Quality-control product comprises positive control and negative control.
8. the content detection of 5 kinds of cardiovascular and cerebrovascular disease protein markers in the blood serum sample
Blood serum sample comprises 20 parts in normal human serum sample (normal control group 1-20 sample), 20 parts in cardiovascular and cerebrovascular disease patients serum sample (cardiovascular and cerebrovascular disease group be for 1-5 number patients with coronary heart disease, 6-10 number number be the sample of Patients with Hemorrhagic Cerebrovascular Disease for ischemic cerebrovascular disease patient, 16-20 for hyperpietic, 11-15 number).
Prewet 8.1 add the cleansing solution in 200 μ l/ holes in the miillpore filter plate (96 hole) respectively, 5min is hatched in concussion at room temperature, removes liquid with the vacuum pump suction filtration;
8.2 add analysis buffer respectively in the miillpore filter plate, 25 μ l/ holes;
8.3 the microballoon mixed liquor (I mixed liquor) that adds the coupling capture antibody respectively is in the miillpore filter plate, 25 μ l/ holes;
8.4 the normal human serum sample that add standard items (STD0, STD1, STD2, STD3, STD4, STD5, STD6), quality-control product (positive control and negative control), patients serum's sample 1-20 number that diluted respectively, diluted 1-20 number, 25 μ l/ holes;
8.5 with the potpourri of mixing up and down of volley of rifle fire tenderness, 60min is hatched in lucifuge concussion at room temperature;
8.6 the vacuum pump suction filtration is removed liquid in the hole, with cleansing solution 200 μ l/ holes, washs 2 times; Vacuum filtration is removed cleansing solution;
8.7 add respectively and contain biotin labeled detection antibody mixed liquor (II mixed liquor), 25 μ l/ holes, 30min is hatched in lucifuge concussion at room temperature;
8.8 Streptavidin-phycoerythrin (Streptavidin-PE) is diluted to the solution that concentration is 200 μ g/ml with analysis buffer, every hole adds the good Streptavidin phycoerythrin 25 μ l of dilution, with the potpourri of mixing up and down of volley of rifle fire tenderness, 20min is hatched in lucifuge concussion at room temperature;
8.9 the vacuum pump suction filtration is removed liquid in the hole, with cleansing solution 200 μ l/ holes, washs 2 times; Vacuum filtration is removed cleansing solution;
8.10 every hole adds the resuspended microballoon of 100 μ l analysis buffer respectively;
8.11 on liquid-phase chip instrument (LiquiChip 200 or Luminex 200), the potpourri of reaction is analyzed, drawing standard curve automatically, and calculate the content of 5 kinds of cardiovascular and cerebrovascular disease protein markers in the test sample according to typical curve.
8.12 testing result and analysis
Referring to table 7-9 and Fig. 1-5.
Table 7 cardiovascular and cerebrovascular disease histone mark concentration value
Table 8 normal control histone mark concentration value
The protein marker mean value contrast of table 9 cardiovascular and cerebrovascular disease patient and normal group
Show from the above result of table 7-9 and Fig. 1-Fig. 5 demonstration, can carry out the associating parallel detection to 5 kinds of cardiovascular and cerebrovascular disease protein markers simultaneously with the inventive method, but also can carry out the joint-detection of various combination according to the actual requirements to 5 kinds of cardiovascular and cerebrovascular disease protein markers.The concentration mean value of 5 kinds of marks as can be seen from table 7-9,5 kinds of protein markers of this of cardiovascular and cerebrovascular disease patient, the VEGF group exceeds 4.1 times of normal control groups, the MCP-1 group exceeds 3.6 times of normal control groups, the MMP-9 group exceeds 4.5 times of normal control groups, and the TIMP-1 group exceeds 2.8 times of normal control group, and IFN-γ group exceeds 3.9 times of normal control group, have significant difference, can be used as the important indicator that the cardiovascular and cerebrovascular disease clinical diagnosis detects or predicts.
About after the those set forth of the present invention, those skilled in the art can do various modifications or variation to the present invention more than having read, and these equivalent form of values belong to the scope defined in the application's appended claims equally.
Claims (9)
1. the combined parallel detecting method of a cardiovascular and cerebrovascular disease protein marker is characterized in that, comprises following key step:
(1) with the microballoon Beads coupling of capture antibody with different numberings, form " capture antibody-microballoon " bigeminy complex, the anti-respectively a kind of cardiovascular and cerebrovascular disease protein marker of described each capture antibody, thus make cardiovascular and cerebrovascular disease protein marker and capture antibody in the testing sample form " cardiovascular and cerebrovascular disease protein marker-capture antibody-microballoon " three complexs; Described cardiovascular and cerebrovascular disease protein marker comprise following mark any one or arbitrarily multiple or add the combination of other mark: vascular endothelial growth factor VEGF, monocyte chemoattractant protein-1MCP-1, metal matrix proteinase-9MMP-9, people's matrix metalloproteinase inhibiting factor-1TIMP-1, interferon-IFN-γ;
(2) different detection antibody is carried out the signal mark, wherein said each detect the anti-respectively a kind of cardiovascular and cerebrovascular disease protein marker of antibody and corresponding to capture antibody, and be incorporated into the different epitopes of this protein marker respectively with capture antibody;
(3) three complexs that step (1) is formed mix with the detection antibody that has the signal mark in the step (2), thus formation " the detection antibody-cardiovascular and cerebrovascular disease protein marker-capture antibody-microballoon of signal mark " tetrad complex;
(4) detect the marking signal of different microballoons in the tetrad complex with liquid-phase chip method xMAP, thereby determine the existence and the content of various cardiovascular and cerebrovascular disease protein markers in the detected sample.
2. the combined parallel detecting method of cardiovascular and cerebrovascular disease protein marker as claimed in claim 1, it is characterized in that, in the step (4), the content of various cardiovascular and cerebrovascular disease protein markers in described definite detected sample, its concrete grammar is: marking signal and the typical curve measured in the step (4) compared, thus the content of various cardiovascular and cerebrovascular disease protein markers in definite detected sample.
3. the combined parallel detecting method of cardiovascular and cerebrovascular disease protein marker as claimed in claim 1, it is characterized in that, in the step (1), described microballoon is a kind of color coding microballoon Color-coded Beads, and combines the polyphenyl alkene microballoon of different fluorescent dyes.
4. the combined parallel detecting method of cardiovascular and cerebrovascular disease protein marker as claimed in claim 1, it is characterized in that described capture antibody and detection antibody are at the capture antibody of the cardiovascular and cerebrovascular disease protein marker of following 5 kinds of mark independent assortments and detect antibody:
Vascular endothelial growth factor VEGF, monocyte chemoattractant protein-1MCP-1, metal matrix proteinase-9MMP-9, people's matrix metalloproteinase inhibiting factor-1TIMP-1, interferon-IFN-γ.
5. the combined parallel detecting method of cardiovascular and cerebrovascular disease protein marker as claimed in claim 1 is characterized in that, detects with liquid-phase chip method xMAP in the described step (4), and detectable marking signal is a fluorescence signal.
6. a diagnostic kit that detects the cardiovascular and cerebrovascular disease protein marker is characterized in that, comprises following main constituent:
(1) microballoon of coupling antibody: the microballoon of different capture antibodies that contained respectively coupling comprises following any one or any multiple microballoon: coupling the microballoon of vascular endothelial growth factor VEGF capture antibody, coupling the microballoon of monocyte chemoattractant protein-1MCP-1 capture antibody, coupling the microballoon of metal matrix proteinase-9MMP-9 capture antibody, coupling the microballoon of people's matrix metalloproteinase inhibiting factor-1TIMP-1 capture antibody, coupling the microballoon of interferon-IFN-γ capture antibody; Every kind of capture antibody resists a kind of cardiovascular and cerebrovascular disease protein marker respectively and is coupled to different microballoons of numbering, and forms the bigeminy complex of " antibody-microballoon ";
(2) the detection antibody of signal mark: comprise following any one or any multiple detection antibody that has the signal mark: the interferon-IFN-γ that people's matrix metalloproteinase inhibiting factor-1TIMP-1 that metal matrix proteinase-9MMP-9 that monocyte chemoattractant protein-1MCP-1 that the vascular endothelial growth factor VEGF of mark detects antibody, mark detects antibody, mark detects antibody, mark detects antibody, mark detects antibody; Wherein said each detect the anti-respectively a kind of corresponding cardiovascular and cerebrovascular disease protein marker of antibody and corresponding to capture antibody, and be incorporated into the different epitopes of this protein marker respectively with capture antibody;
(3) standard items: the standard items that comprise various cardiovascular and cerebrovascular disease protein markers;
(4) quality-control product: comprise positive control and negative control.
7. the diagnostic kit of detection cardiovascular and cerebrovascular disease protein marker as claimed in claim 6, it is characterized in that described capture antibody and detection antibody are capture antibody and the detection antibody that comprises at the cardiovascular and cerebrovascular disease protein marker of following 5 kinds of independent assortments:
Vascular endothelial growth factor VEGF, monocyte chemoattractant protein-1MCP-1, metal matrix proteinase-9MMP-9, people's matrix metalloproteinase inhibiting factor-1TIMP-1, interferon-IFN-γ.
8. one kind as the existence of claim 6 or 7 described diagnostic kits cardiovascular and cerebrovascular disease protein marker in detecting vitro samples and the application in the content, and the application in the hazards examination of cardiovascular and cerebrovascular disease or prediction, diagnostic detection, curative effect monitoring and prognosis are judged.
9. one kind as the application in the diagnostic detection of the other diseases except that cardiovascular and cerebrovascular disease or prediction, curative effect monitoring and prognosis are judged of claim 6 or 7 described diagnostic kits.
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