CN106932571A - Cardiovascular disease detection method and kit based on RCN2 marker detections - Google Patents
Cardiovascular disease detection method and kit based on RCN2 marker detections Download PDFInfo
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Abstract
The present invention provides a kind of cardiovascular disease detection method based on RCN2 marker detections and the kit using the method, belong to cardiovascular disease vitro detection technical field, its utilize Avidin biotin combination ELISA method detect human blood sample in RCN2 concentration levels so as to detect cardiovascular disease, the concentration of RCN2 is drawn by using ELIASA mensuration absorbance, when RCN2 concentration levels are less than 20ng/ml, the affiliated human body of sample is healthy individuals, when RCN2 concentration levels are that, more than 20ng/ml, the affiliated human body of sample is individual risk high.The detection method that the present invention is provided has the advantages that high specificity, Detection accuracy are high.
Description
Technical field
The invention belongs to cardiovascular disease vitro detection technical field, more particularly to one kind is based on RCN2 in blood of human body sample
Application of the cardiovascular disease detection method and RCN2 marks of the concentration level of mark in cardiovascular disease detection.
Background technology
Cardiovascular disease is the general designation of cardiovascular disease, refer to due to hyperlipidemia, sticky blood, atherosclerosis,
Ischemic or hemorrhagic disease that the caused heart such as hypertension, brain and body tissue occur.Cardiovascular disease is a kind of tight
The mankind, particularly more than the 50 years old common disease of the elderly's health, with illness rate high, disability rate high and high mortality are threatened again
The characteristics of, even if using treatment means most advanced, perfect at present, the cerebrovas-cularaccident survivor that can still have more than 50% lives
Can not completely take care of oneself, the number that cardiovascular disease is died from the whole world every year is up to 15,000,000 people, occupy the various causes of the death the first.
Atherosclerosis is the main cause that coronary heart disease, ishemic stroke and peripheral arterial disease occur.Although dynamic
The death rate of pulse atherosclerosis coming drastically to decline due to effective prevention and the treatment of stanin fat-reducing medicament over the past thirty years,
But this disease still deprives more people's life than any other disease.OxLDL ELISA is attached to subendothelial layer and draws
The arterial inflammation reaction for rising is the critical process that atherosclerotic plaque occurs, develops even rupture.Current most worthy
Atherosclerosis mark be C-reactive protein (CRP), CRP is mainly produced in liver, is not to move
Pulse atherosclerosis are specific.High-caliber CRP shows to there occurs the acute phase response of inflammation-induced, such as infection, wound,
Allergy etc..It is personal because the elevated CRP of gene is usually displayed without further and the foundation level of CRP is determined by gene
The risk of angiocardiopathy is suffered from increase.Therefore, a vascular wall that can exactly predict OxLDL ELISA induction is set up
The label of inflammation will be an important process to anti-cardiovascular disease.
RCN2（Net calcium albumen 2）The one kind in CALU is belonging to, is a kind of low affine in mammal body
Power calbindin, has expression in endoplasmic reticulum/sarcoplasmic reticulum and golgiosome.
Platelet activation is one of atherosclerosis generation and thrombotic important pathogenesis.Research is found through solidifying
After the activation such as hemase, used as secreted protein, RCN2 can be by autocrine and other point for the RCN2 in releasable its kytoplasm of blood platelet
The mode of secreting participates in AP and thrombotic pathomechanism.Immunohistochemical analysis show have artery athero- in the mankind
RCN2 is can detect on the vascular wall of plaque to exist, and normal blood vessels Duan Zewei has found the expression of RCN2, further grinds
Study carefully and find that RCN2 is the essential condition factor that Intravascular Thrombus are formed, while RCN2 can influence the performance of fibrocyte, promote its turn
Myofibroblast is turned to, the formation of atherosclerotic lesion is participated in.
There is above-mentioned situation, the present invention provides a kind of cardiovascular disease detection method based on RCN2 marker detections and uses
The kit of the method, it utilizes the method for Avidin-Biotin combination ELISA to detect RCN2 concentration levels so as to detect painstaking effort
Pipe disease, the concentration of RCN2 is drawn by using ELIASA mensuration absorbance, when RCN2 concentration levels are less than 20ng/ml, sample
Affiliated human body is healthy individuals, when RCN2 concentration levels are that, more than 20ng/ml, the affiliated human body of sample is individual risk high.
The detection method that the present invention is provided has the advantages that high specificity, Detection accuracy are high.
A kind of cardiovascular disease detection method based on RCN2 marker detections, it passes through to detect RCN2 concentration levels so as to examine
Thought-read angiosis.
The cardiovascular disease detection method of RCN2 marker detections is preferably based on using Avidin-Biotin combination ELISA
Method detect RCN2 concentration levels so as to detect cardiovascular disease.
The cardiovascular disease detection method for being preferably based on RCN2 marker detections comprises the following steps：
Step S111 coated antibodies：Coating buffer solution writes on ELISA microwell plates the dilution of RCN2 specific antibodies admittedly；
Step S112 adds sample：To be added to after testing sample and standard protein dilution in ELISA ELISA Plates hole, 25 ~ 40 DEG C incubate
Educate 1-2 hours；
Step S113 adds detection antibody：The RCN2 specific antibodies of biotin labeling are separately added into, 25 ~ 40 DEG C are incubated 1-2 hours
Afterwards, cleaning solution is washed 1 ~ 5 time, 1 ~ 5min every time；
Step S114 introduces enzyme mark：Avidin crosslinking horseradish peroxidase conjugate is separately added into, 25 ~ 40 DEG C of incubation 1-2 are small
Shi Hou, cleaning solution is washed 1 ~ 5 time, 1 ~ 5min every time；
Step S115 substrate reactions：Chromogenic substrate 15 ~ 30min of colour developing is separately added into, terminate liquid stopped reaction is added；
Step S116 is detected：Using ELIASA at 450nm determination sample absorbance, calculate RCN2 concentration；
Step S117 results judge：When RCN2 concentration levels are that, less than 20ng/ml, the affiliated human body of sample is healthy individuals, when
RCN2 concentration levels are that, more than 20ng/ml, the affiliated human body of sample is individual risk high.
A kind of cardiovascular disease detection kit based on RCN2 marker detections, the kit is ELISA detection kit,
Including：It is coated with the ELISA ELISA Plates of RCN2 antibody, the detection antibody of RCN2 antigens, enzyme conjugates, standard protein, sample dilute
Release liquid, coating buffer solution, confining liquid, ELISA Plate cleaning solution, nitrite ion and terminate liquid.
Preferably, in described ELISA detection kit：
Described ELISA Plate coated antibody is the anti-human RCN2 monoclonal antibodies of mouse；
Described detection antibody is rabbit-anti people's RCN2 antibody of biotin labeling；
Described enzyme conjugates is Avidin crosslinking horseradish peroxidase conjugate；
Described standard protein is recombined human RCN2 albumen；
Described coating buffer solution is 1 × PBS, pH：7.4；
Described confining liquid is the confining liquid of 5% bovine serum albumin(BSA)+PBS solution；
Described ELISA Plate cleaning solution is 1 × PBS solution containing 0.25%Tween-20；
Described sample diluting liquid is 1 × PBS；
Described nitrite ion is 3,3', 5,5'- tetramethyl benzidines；
Described terminate liquid is the sulfuric acid solution of 2mol/L.
Further, the detection method that described detection kit is used is comprised the following steps：
Step S111 coated antibodies：Coating buffer solution dilutes RCN2 specific antibodies, is added in every hole of ELISA ELISA Plates
100ul, is placed in 4 DEG C of overnight incubations after shrouding, standby；
Step S112 adds sample：Sample sample diluting liquid to be checked is diluted with certain proportion, is added with the volume in 100ul/ holes
Sample；Standard protein sample diluting liquid is diluted to various concentrations gradient, is loaded with the volume in 100ul/ holes, and 37 DEG C are incubated 2 hours；
Step S113 adds detection antibody：Sample in hole is discarded, the RCN2 specific antibodies of biotin labeling are added, per hole 100ul,
After 37 DEG C are incubated 1 hour, hole interior antibody is discarded, add cleaning solution to wash 3 times, per hole 300ul, each 2min, dried；
Step S114 introduces enzyme mark：Avidin crosslinking horseradish peroxidase conjugate is added, after 37 DEG C are incubated 1 hour, is discarded
Hole interior antibody, adds cleaning solution to wash 5 times, per hole 300ul, each 2min, dries；
Step S115 substrate reactions：TMB chromogenic substrates are added, per hole 90ul, after colour developing 20min, 50ul terminate liquids is added per hole
Stopped reaction；
Step S116 is detected：Using ELIASA at 450nm determination sample absorbance, tuning wavelength be 540nm or 570nm,
Calculate RCN2 concentration.
Further, the above-mentioned cardiovascular disease detection kit based on RCN2 marker detections is applied in cardiovascular diagnosis.
Wherein, described cardiovascular disease includes coronary heart disease（CHD）, peripheral arterial disease（PAD）.
Wherein, when risk is judged, Healthy People RCN2 concentration levels are less than 20ng/ml, when more than 20ng/ml
When, then representing has risk higher.
Brief description of the drawings
Fig. 1, cardiovascular disease detection kit detection normal population and patients with coronary heart disease based on RCN2 marker detections
RCN2 concentration results comparison diagrams.
Fig. 2, cardiovascular disease detection kit detection normal population and peripheral arterial disease based on RCN2 marker detections
The RCN2 concentration results comparison diagrams of patient.
Fig. 3, the statistics dependency relation of RCN2 concentration and high-density lipoprotein concentration in 92 CHD blood samples of patients samples
Figure.
Fig. 4, the statistics dependency relation figure of RCN2 concentration and diastolic pressure in 92 CHD blood samples of patients samples.
Specific embodiment
With reference to specific embodiment, the invention will be further described.
Embodiment one：A kind of cardiovascular disease detection kit based on RCN2 marker detections.
The kit is ELISA detection kit, including：The ELISA ELISA Plates of RCN2 antibody, detection RCN2 is coated with to resist
Former antibody, enzyme conjugates, standard protein, sample diluting liquid, coating buffer solution, confining liquid, ELISA Plate cleaning solution, nitrite ion and
Terminate liquid, wherein：
Described ELISA Plate coated antibody is the anti-human RCN2 monoclonal antibodies of mouse；
Described detection antibody is rabbit-anti people's RCN2 antibody of biotin labeling；
Described enzyme conjugates is Avidin crosslinking horseradish peroxidase conjugate；
Described standard protein is recombined human RCN2 albumen；
Described coating buffer solution is 1 × PBS, pH：7.4；
Described confining liquid is the confining liquid of 5% bovine serum albumin(BSA)+PBS solution；
Described ELISA Plate cleaning solution is 1 × PBS solution containing 0.25%Tween-20；
Described sample diluting liquid is 1 × PBS；
Described nitrite ion is 3,3', 5,5'- tetramethyl benzidines；
Described terminate liquid is the sulfuric acid solution of 2mol/L.
The detection method for using is matched with the kit to comprise the following steps：
Step S111 coated antibodies：Coating buffer solution dilutes RCN2 specific antibodies, is added in every hole of ELISA ELISA Plates
100ul, is placed in 4 DEG C of overnight incubations after shrouding, standby；
Step S112 adds sample：Sample sample diluting liquid to be checked is diluted with certain proportion, is added with the volume in 100ul/ holes
Sample；Standard protein sample diluting liquid is diluted to various concentrations gradient, is loaded with the volume in 100ul/ holes, and 37 DEG C are incubated 2 hours；
Step S113 adds detection antibody：Sample in hole is discarded, the RCN2 specific antibodies of biotin labeling are added, per hole 100ul,
After 37 DEG C are incubated 1 hour, hole interior antibody is discarded, add cleaning solution to wash 3 times, per hole 300ul, each 2min, dried；
Step S114 introduces enzyme mark：Avidin crosslinking horseradish peroxidase conjugate is added, after 37 DEG C are incubated 1 hour, is discarded
Hole interior antibody, adds cleaning solution to wash 5 times, per hole 300ul, each 2min, dries；
Step S115 substrate reactions：TMB chromogenic substrates are added, per hole 90ul, after colour developing 20min, 50ul terminate liquids is added per hole
Stopped reaction；
Step S116 is detected：Using ELIASA at 450nm determination sample absorbance, tuning wavelength be 540nm or 570nm,
Calculate RCN2 concentration.
Embodiment two：A kind of cardiovascular disease detection method based on RCN2 marker detections.
According to the following steps using the cardiovascular disease detection kit based on RCN2 marker detections to healthy normal person, coronary disease
The blood sample of patient and peripheral arterial disease patient carries out RCN2 concentration level detections：
Step S111 coated antibodies：Coating buffer solution writes on ELISA microwell plates the dilution of RCN2 specific antibodies admittedly；
Step S112 adds sample：In adding to ELISA ELISA Plates hole after the dilution of sample to be checked and standard protein, 35 DEG C of incubations
1.52 hours；
Step S113 adds detection antibody：The RCN2 specific antibodies of biotin labeling are separately added into, after 35 DEG C are incubated 1.5 hours,
Cleaning solution is washed 3 times, each 3min；
Step S114 introduces enzyme mark：Avidin crosslinking horseradish peroxidase conjugate is separately added into, 35 DEG C are incubated 1.5 hours
Afterwards, cleaning solution is washed 3 times, each 3min；
Step S115 substrate reactions：Chromogenic substrate colour developing 20min is separately added into, terminate liquid stopped reaction is added；
Step S116 is detected：Using ELIASA at 450nm determination sample absorbance, calculate RCN2 concentration；
Step S117 results judge：When RCN2 concentration levels are that, less than 20ng/ml, the affiliated human body of sample is healthy individuals, when
RCN2 concentration levels are that, more than 20ng/ml, the affiliated human body of sample is individual risk high.
As shown in Figures 1 and 2, the RCN2 concentration of healthy normal person is 18 ng/ml to result, patients with coronary heart disease
RCN2 concentration is 55ng/ml, and the RCN2 concentration of peripheral arterial disease patient is 92ng/ml.
Embodiment three：RCN2 concentration is detected with the dependency relation of correlated variables in patient's body.
Measurement RCN2 concentration and human body high-density lipoprotein concentration level, the related of vasodilation pressure are closed according to the following steps
System：
Step S111 measures RCN2 concentration：92 blood samples of cardiovascular patient are extracted as experiment sample, implementation is used
The detection method that the kit and kit that example one is provided are used detects 92 RCN2 concentration of sample；
Step S112 measures high-density lipoprotein concentration：The height of patient is detected using conventional high density lipoprotein concentration detection method
Density lipoprotein concentration, and statistical analysis is carried out to RCN2 concentration and high-density lipoprotein concentration using statistical method；
Step S113 measures diastolic pressure：Patients' blood's diastolic pressure is detected using conventional blood pressure detecting method, and uses statistics side
Method carries out statistical analysis to RCN2 concentration and diastolic pressure.
As shown in Figure 3, RCN2 concentration and high-density lipoprotein concentration show statistic analysis result in significantly negatively correlated
RCN2 concentration is bigger, and high-density lipoprotein concentration is smaller.Cholesterol in HDL delivery surrounding tissue, is then converted to
Bile acid directly passes through bile from enteron aisle discharge, and angiography proves that HDL-C content is narrow with arterial lumen
Narrow degree is in significant negatively correlated.So HDL is a kind of plasma lipoprotein of antiatherosclerosis, it is coronary disease
The protective factors of disease, are commonly called as " blood vessel scavenger ".There is certain relation in the concentration and cardiovascular disease illness risk for proving RCN2,
RCN2 can be applied as detection mark in cardiovascular disease detection.
In addition it can also be seen that RCN2 concentration and diastolic pressure show that RCN2 concentration is got in notable positive correlation from accompanying drawing 4
Greatly, the pressure of diastolic pressure is bigger, and cardiovascular disease illness risk is bigger.The concentration and cardiovascular disease illness risk for proving RCN2 are present
Certain relation, RCN2 can be applied as detection mark in cardiovascular disease detection.
Embodiment described above only expresses several embodiments of the invention, and its description is more specific and detailed, but simultaneously
Therefore the limitation to the scope of the claims of the present invention can not be interpreted as.It should be pointed out that for one of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to guarantor of the invention
Shield scope.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Claims (9)
1. a kind of cardiovascular disease detection method based on RCN2 marker detections, it is characterised in that it passes through to detect RCN2 concentration
Level is so as to detect cardiovascular disease.
2. the cardiovascular disease detection method based on RCN2 marker detections according to claim 1, it is characterised in that its profit
Detect RCN2 concentration levels so as to detect cardiovascular disease with the method for Avidin-Biotin combination ELISA.
3. the cardiovascular disease detection method based on RCN2 marker detections according to claim 2, it is characterised in that its bag
Include following steps：
Step S111 coated antibodies：Coating buffer solution writes on ELISA microwell plates the dilution of RCN2 specific antibodies admittedly；
Step S112 adds sample：To be added to after testing sample and standard protein dilution in ELISA ELISA Plates hole, 25 ~ 40 DEG C incubate
Educate 1-2 hours；
Step S113 adds detection antibody：The RCN2 specific antibodies of biotin labeling are separately added into, 25 ~ 40 DEG C are incubated 1-2 hours
Afterwards, cleaning solution is washed 1 ~ 5 time, 1 ~ 5min every time；
Step S114 introduces enzyme mark：Avidin crosslinking horseradish peroxidase conjugate is separately added into, 25 ~ 40 DEG C of incubation 1-2 are small
Shi Hou, cleaning solution is washed 1 ~ 5 time, 1 ~ 5min every time；
Step S115 substrate reactions：Chromogenic substrate 15 ~ 30min of colour developing is separately added into, terminate liquid stopped reaction is added；
Step S116 is detected：Using ELIASA at 450nm determination sample absorbance, calculate RCN2 concentration；
Step S117 results judge：When RCN2 concentration levels are that, less than 20ng/ml, the affiliated human body of sample is healthy individuals, when
RCN2 concentration levels are that, more than 20ng/ml, the affiliated human body of sample is individual risk high.
4. a kind of cardiovascular disease detection kit based on RCN2 marker detections, it is characterised in that the kit is ELISA inspections
Test agent box, including：It is coated with ELISA ELISA Plates, antibody, enzyme conjugates, the standard egg of detection RCN2 antigens of RCN2 antibody
In vain, sample diluting liquid, coating buffer solution, confining liquid, ELISA Plate cleaning solution, nitrite ion and terminate liquid.
5. the cardiovascular disease detection kit based on RCN2 marker detections according to claim 4, it is characterised in that institute
In the ELISA detection kit stated：
Described ELISA Plate coated antibody is the anti-human RCN2 monoclonal antibodies of mouse；
Described detection antibody is rabbit-anti people's RCN2 antibody of biotin labeling；
Described enzyme conjugates is Avidin crosslinking horseradish peroxidase conjugate；
Described standard protein is recombined human RCN2 albumen；
Described coating buffer solution is 1 × PBS, pH：7.4；
Described confining liquid is the confining liquid of 5% bovine serum albumin(BSA)+PBS solution；
Described ELISA Plate cleaning solution is 1 × PBS solution containing 0.25%Tween-20；
Described sample diluting liquid is 1 × PBS；
Described nitrite ion is 3,3', 5,5'- tetramethyl benzidines；
Described terminate liquid is the sulfuric acid solution of 2mol/L.
6. the cardiovascular disease detection kit based on RCN2 marker detections according to claim 4, it is characterised in that its
The detection method for being used is comprised the following steps：
Step S111 coated antibodies：Coating buffer solution dilutes RCN2 specific antibodies, is added in every hole of ELISA ELISA Plates
100ul, is placed in 4 DEG C of overnight incubations after shrouding, standby；
Step S112 adds sample：Sample sample diluting liquid to be checked is diluted with certain proportion, is added with the volume in 100ul/ holes
Sample；Standard protein sample diluting liquid is diluted to various concentrations gradient, is loaded with the volume in 100ul/ holes, and 37 DEG C are incubated 2 hours；
Step S113 adds detection antibody：Sample in hole is discarded, the RCN2 specific antibodies of biotin labeling are added, per hole 100ul,
After 37 DEG C are incubated 1 hour, hole interior antibody is discarded, add cleaning solution to wash 3 times, per hole 300ul, each 2min, dried；
Step S114 introduces enzyme mark：Avidin crosslinking horseradish peroxidase conjugate is added, after 37 DEG C are incubated 1 hour, is discarded
Hole interior antibody, adds cleaning solution to wash 5 times, per hole 300ul, each 2min, dries；
Step S115 substrate reactions：TMB chromogenic substrates are added, per hole 90ul, after colour developing 20min, 50ul terminate liquids is added per hole
Stopped reaction；
Step S116 is detected：Using ELIASA at 450nm determination sample absorbance, tuning wavelength be 540nm or 570nm,
Calculate RCN2 concentration.
7. a kind of cardiovascular disease detection kit based on RCN2 marker detections as described in claim 4 to 6 is any is in the heart
Application in vascular diagnostic.
8. the cardiovascular disease detection kit based on RCN2 marker detections according to claim 7 is in cardiovascular diagnosis
Application, it is characterised in that described cardiovascular disease include coronary heart disease（CHD）, peripheral arterial disease（PAD）.
9. the cardiovascular disease detection kit based on RCN2 marker detections according to claim 8 is in cardiovascular diagnosis
Application, Healthy People RCN2 concentration levels be less than 20ng/ml, when more than 20ng/ml, then represent have ill wind higher
Danger.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115040637A (en) * | 2022-05-24 | 2022-09-13 | 中南大学湘雅医院 | Application of RCN2 recombinant protein in preparation of drugs for preventing and/or treating osteoporosis and improving immunity |
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| US8529888B2 (en) * | 2007-09-19 | 2013-09-10 | Pluristem Ltd. | Adherent cells from adipose or placenta tissues and use thereof in therapy |
| CN104198727A (en) * | 2014-08-22 | 2014-12-10 | 广西南宁隆吉维特生物科技有限公司 | Human serum CCL2 enzyme-linked immunosorbent assay kit as well as preparation and use methods thereof |
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| CN115040637A (en) * | 2022-05-24 | 2022-09-13 | 中南大学湘雅医院 | Application of RCN2 recombinant protein in preparation of drugs for preventing and/or treating osteoporosis and improving immunity |
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