CN103376313A - Liquid-phase chip kit for detection of acute coronary syndrome based on serum exosomes and preparation method - Google Patents
Liquid-phase chip kit for detection of acute coronary syndrome based on serum exosomes and preparation method Download PDFInfo
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Abstract
The invention relates to a kit used for auxiliary judgment of risk stratification of patients with acute coronary syndrome based on detection of serum exosomes and a preparation method. The kit mainly comprises exosomes extraction reagents, relevant microballoons capturing antibodies (the above antibodies are one or some selected from CD11c, CD81, MHCII, CD86, CD106, CD54 and CD3), biotin labeled detection antibodies and streptavidin phycoerythrin. After being optimized, the diagnostic kit has advantages of high detection efficiency, less amount of required samples, high specificity, high sensitivity, fast and accurate detection and the like, so the risk stratification conditions of patients can be evaluated comprehensively, and relevant references are provided for clinical intervention.
Description
Technical field
The present invention relates to the technical field of external kit, a kind of external kit to the Acute Coronary Syndrome Patients risk stratification specifically, particularly relate to based on the serum allochthon, i.e. the liquid-phase chip combined parallel detecting method of exosomes and kit and preparation method thereof.
Background technology
Atherosclerotic (atherosclerosis, AS) the property vascular diseases have become " the No.1 killer " who threatens compatriots' health, along with China's expanding economy, the incidence of disease is year by year ascendant trend, the annual medical expense that is used for such disease of China is up to hundred billion yuans, and the control of strengthening AS vascular diseases and complication thereof has become the main task of China's major disease control.
The acute coronary syndrome (Acute coronary syndrome, ACS) that the AS vascular diseases cause is the most common and the most serious clinical performance, and the secondary thrombus formation of breaking of coronary artery vulnerable plaque is the main pathologic basis of its generation.Can not well identify vulnerable plaque as the coronarography of diagnosis of coronary heart disease " goldstandard " at present, the identification of vulnerable plaque and diagnosis mainly depend on the interventional imaging sections of learning to do such as intravascular ultrasound and angioscope, the pathology that can show vascular wall, accurately distinguish the character of pathology and identify vulnerable plaque, but the inflammatory activity in can not detection of plaque, more since its for the inspection of wound property is arranged, technical conditions require costliness of height and expense, have limited applying of it.Therefore, searching can reflect the responsive and specific serologic marker thing of AS vulnerable plaque, and by effectively intervening, and before vulnerable plaque breaks, in early days with its identification and to make it trend stable, is expected to reach and reduces the purpose that ACS occurs.
Exosomes is allochthon, it is a class size has the double-layer of lipoid membrane structure between 10-100nm biological imitated vesicle structure, it mainly is cell a large amount of secretions after being stimulated, arrive the extracellular by many vesicas corpusculum exocytosis, find that the earliest it is mainly derived from blood platelet, have short solidifying effect, studies confirm that in a large number afterwards: except blood platelet, endothelial cell, vascular smooth muscle cell, leucocyte, lymphocyte, red blood cell etc. all can discharge exosomes.The exosomes diacrisis among the various disease patients serum that studies confirm that in recent years increases, and its specificity that is used for diagnosis and prediction can be used as a kind of index of disease risk stratification apparently higher than blood serum designated object.
But existing diagnostic kit focuses mostly in the diagnostic sensitivity and the specificity aspect that improve by the Peripheral Blood mark ACS, in related kit or a blank of improving aspect patient's the risk stratification, simultaneously the specificity of related kit is not high, and this respect exactly at present clinical patients administrative institute be badly in need of.
At present, mensuration for the exosomes mark mainly relies on flow cytometry, but flow cytometry once can only detect for a mark, and complex operation, susceptibility are different, can not really satisfy clinical needs, in addition because above-mentioned exosomes mark has different expression in the course of disease of ACS, the problems referred to above are so that each index is different in ACS patient's risk stratification and the value in the prognosis judgement, if can detect simultaneously, then can increase substantially the accuracy that ACS patient's risk stratification and prognosis are judged.And the solid phase biological chip technology exists the shortcoming of the not high and complex operation of poor repeatability, susceptibility.
Summary of the invention
The object of the present invention is to provide a kind of improved liquid phase chip reagent box that detects acute coronary syndrome based on the serum allochthon and preparation method thereof, it can overcome in the prior art in related kit or a blank of improving aspect patient's the risk stratification, the simultaneously not high some shortcomings of specificity of related kit.
To achieve these goals, technical scheme of the present invention is: a kind of liquid phase chip reagent box based on serum allochthon detection acute coronary syndrome, and it mainly comprises, it is characterized in that:
Described liquid phase chip reagent box includes following component:
1) coated microballoon: microballoon mainly comprises two classes, a kind of diameter is the polystyrene microsphere of 5.6 μ m, another kind is the magnetic microsphere of 6.5 μ m for diameter, above-mentioned two class microballoons be coated with respectively the CD11c capture antibody microballoon, be coated with the CD81 capture antibody microballoon, be coated with MHC II capture antibody microballoon, be coated with the CD86 capture antibody microballoon, be coated with the CD106 capture antibody microballoon, be coated with the microballoon of CD54 capture antibody and be coated with the microballoon of CD3 capture antibody, above-mentioned microballoon has respectively the different colours coding;
2) biotin labeling detects antibody: contain respectively and detect antibody with biotin labeled CD11c, CD81, MHC II, CD86, CD106, CD54 and CD3;
3) streptavidin phycoerythrin.
A kind ofly detect the preparation method of the liquid phase chip reagent box of acute coronary syndrome based on the serum allochthon, it is characterized in that: described preparation method comprises the steps:
1) according to the composition of kit, Dispersal risk is coated with microballoon, and the preparation method of every kind of coated microballoon of capture antibody is identical:
After the activation of 50ul microballoon, through the high speed centrifugation precipitation, centrifugal condition is 〉=7500g 5 minutes;
Supernatant is abandoned in suction, microballoon is resuspended in the MES solution of 50mM of 230-280 μ l of pH5.0, vortex vibration 25-30 second, ultrasonic 25-30 second, after microballoon is centrifugal, centrifugal condition is 〉=8000g centrifugal 1-2 minute; Repeat above-mentioned steps;
Supernatant is abandoned in suction, microballoon is resuspended in the solution of MES of 100 μ l 50mM pH5.0 vortex vibration 25-30 second, ultrasonic 25-30 second;
In the microballoon that suspends, add corresponding 0.8-1.2 μ g antibody, with the MES solution of the pH5.0 of 50mM cumulative volume is mended the l to 450-550 μ;
With vortex oscillator mixing, room temperature lucifuge vibration 2-2.5 hour;
With the microballoon centrifugation of the good antibody of coupling, centrifugal condition is 〉=8000g centrifugal 1-2 minute;
Supernatant is abandoned in suction, microballoon is resuspended in the 500 μ l PBS-TBN solution vortex vibration 25-30 second, sonic oscillation 25-30 second;
Room temperature lucifuge vibration 30 minutes; Centrifugal collection microballoon again, centrifugal condition is 〉=8000g centrifugal 1-2 minute;
Supernatant is abandoned in suction, microballoon is resuspended in the PBS-TBN solution of 1ml vortex vibration 25-30 second, sonic oscillation 25-30 second; Centrifugal collection microballoon, centrifugal condition are 〉=8000g centrifugal 1-2 minute; Repeat this step once;
Supernatant is abandoned in suction, microballoon is resuspended in the PBS-TBN solution of 500-1000 μ l;
Every kind of coated good microballoon is placed on 2-8 degree lucifuge by Luminex instrument counting and preserves separately, during use, needs equal proportion to mix according to monitoring, and the concentration of every kind of capture antibody coupling microballoon is 125/μ l in the mixed liquor;
2) according to the composition of kit, carry out every kind of biotin labeling that detects antibody:
Calculate the volume that the detection antibody-solutions is diluted to 1mg/ml according to detecting antibody concentration, this volume is as target volume;
Use dimethyl sulfoxide solution, the N-hydroxy thiosuccinimide biotin reaction liquid of configuration 10mg/ml;
1:100 adds respectively detection antibody and N-hydroxy thiosuccinimide biotin reaction liquid in reaction tube in molar ratio;
Adding volume is the sodium bicarbonate solution of the pH8.9 of target volume 1/10, and the PBS that adds subsequently pH7.4 mends to target volume;
Wrap aluminium foil or put into the lucifuge magazine, reaction tube or lucifuge magazine are placed on the circumference vibration shaking table, lucifuge was hatched 4-5 hour in 25 degree constant temperature ovens, and rotating speed is 900rpm-1000rpm;
Reactant liquor is gone in the dialysis cassette, in PBS damping fluid dialysed overnight, to remove unreacted N-hydroxy thiosuccinimide biotin reaction liquid;
During use as required that mark is good every kind detect the antibody equal proportion and mix.
3) have the extracting of the biological imitated vesicle structure of double-layer of lipoid membrane structure:
Can adopt ultracentrifugation, ultrafiltration or commercial special-purpose extraction agent extracting film vesicles from fixed amount serum (at least 1ml), and carry out the standardization that protein quantification is finished sample, be prepared into testing sample.
During use, the present invention is according to before a large amount of documents and materials combinations Research of predicting markers being screened, therefrom determine these seven kinds biomarkers that help ACS patient's risk stratification that come from exosomes of CD11c, CD81, MHC II, CD86, CD106, CD54 and CD3, utilized the liquid-phase chip platform to carry out the detection of running simultaneously of These parameters.
The inventive method detects 7 kinds of exosomes marks synchronously in conjunction with characteristic and the highly sensitive characteristic of double antibody sandwich method of the parallel detection of many indexs of liquid-phase chip platform high channel, significantly improves the accuracy rate that the Acute Coronary Syndrome Patients risk stratification is judged.
Liquid-phase chip disclosed in this invention can this react the quantitative detection of finishing simultaneously multiple exosomes mark, thereby reach the judgement to the comprehensive patient's of acute coronary risk stratification, also have the detection efficiency height, required sample size is few, high specificity, the advantages such as sensitivity height.
In addition, preparation method of the present invention is simple, good stability, and the various technological parameters in its technical scheme such as the amount of microballoon and antibody, course of reaction etc. all draw on the great many of experiments basis, are the parameter value of preparation process the best.
Embodiment:
The invention will be further described below in conjunction with embodiment.
The present invention comprises that mainly described liquid phase chip reagent box includes following component:
1) coated microballoon: microballoon mainly comprises two classes, a kind of diameter is the polystyrene microsphere of 5.6 μ m, another kind is the magnetic microsphere of 6.5 μ m for diameter, above-mentioned two class microballoons be coated with respectively the CD11c capture antibody microballoon, be coated with the CD81 capture antibody microballoon, be coated with MHC II capture antibody microballoon, be coated with the CD86 capture antibody microballoon, be coated with the CD106 capture antibody microballoon, be coated with the microballoon of CD54 capture antibody and be coated with the microballoon of CD3 capture antibody, above-mentioned microballoon has respectively the different colours coding;
2) biotin labeling detects antibody: contain respectively and detect antibody with biotin labeled CD11c, CD81, MHC II, CD86, CD106, CD54 and CD3;
3) streptavidin phycoerythrin.
The working concentration of the microballoon of every kind of coated capture antibody is 120-130/μ l; The working concentration of every kind of biotin labeling detection antibody is 1-4 μ g/ml.
Liquid-phase chip technology also is called the streaming fluorescent technique, this technology by a kind of microballoon as reaction carriers, the surface can be for chemical coupling after processing by pendant carboxylic group, the carboxyl that the biomacromolecule such as antigen, antibody passes through amino and microsphere surface is again finished the coated of microballoon through the chemical reaction covalent bond.Existing microballoon adds respectively infrared according to different proportion in manufacture process and two kinds of fluorescent dyes of far infrared are encoded, and can distinguish the microballoon of hundreds of different coding by the identification of instrument.During use, the capture antibody with CD11c, CD81, MHC II, CD86, CD106, CD54 and CD3 is coated in respectively on the microballoon of different colours coding first, uses respectively simultaneously the above-mentioned detection antibody of biotin labeling.Coated five kinds of good microballoons are mixed, be suspended in liquid phase, add again sample, the corresponding some epi-position opposite sex ground combination that detects thing in the capture antibody of mark and the sample on the microballoon in suspension, adding afterwards biotin labeled detection antibody another epitope specificity of corresponding detection thing in sample is combined, rear adding fluorescent material---the streptavidin of phycoerythrin mark reacts completely, because streptavidin phycoerythrin (SA-PE) can be combined with the biotin high degree of specificity, therefore form at last the detection compound of " microballoon-capture antibody+thing to be detected+biotin labeled detection antibody+SA-PE " in the reaction system, take microballoon as carrier, detect by Luminex series liquid-phase chip analytical instrument or flow cytometer, read the color numbers of microballoon and the fluorescent value of SA-PE.By realizing finishing the microballoon of number, realize the corresponding one by one of test item, simultaneously because SA-PE fluorescent value and each detection substrate concentration are proportionate, by detecting CD11c, CD81, MHC II, CD86, CD106, CD54 and the fluorescent value of CD3 standard items under variable concentrations, can obtain each and detect index standard items concentration-fluorescent value typical curve and typical curve equation.Sample to be tested is detected the content that gained fluorescent value substitution typical curve equation can be tried to achieve respectively the above-mentioned blood serum designated object in the sample to be tested.
Because institute responds and all is in liquid phase environment, more is conducive to keep the natural conception of protein, makes the reaction of probe and detected material faster more complete, so detection sensitivity and the range of linearity all are greatly improved.
Another technical issues that need to address of the present invention provide the preparation method of liquid-phase chip.
Described preparation method comprises the steps:
1) according to the composition of kit, Dispersal risk is coated with microballoon, and the preparation method of every kind of coated microballoon of capture antibody is identical:
After the activation of 50ul microballoon, through the high speed centrifugation precipitation, centrifugal condition is 〉=7500g 5 minutes;
Supernatant is abandoned in suction, microballoon is resuspended in the MES solution of 50mM of 230-280 μ l of pH5.0, vortex vibration 25-30 second, ultrasonic 25-30 second, after microballoon is centrifugal, centrifugal condition is 〉=8000g centrifugal 1-2 minute; Repeat above-mentioned steps;
Supernatant is abandoned in suction, microballoon is resuspended in the solution of MES of 100 μ l 50mM pH5.0 vortex vibration 25-30 second, ultrasonic 25-30 second;
In the microballoon that suspends, add corresponding 0.8-1.2 μ g antibody, with the MES solution of the pH5.0 of 50mM cumulative volume is mended the l to 450-550 μ;
With vortex oscillator mixing, room temperature lucifuge vibration 2-2.5 hour;
With the microballoon centrifugation of the good antibody of coupling, centrifugal condition is 〉=8000g centrifugal 1-2 minute;
Supernatant is abandoned in suction, microballoon is resuspended in the 500 μ l PBS-TBN solution vortex vibration 25-30 second, sonic oscillation 25-30 second;
Room temperature lucifuge vibration 30 minutes; Centrifugal collection microballoon again, centrifugal condition is 〉=8000g centrifugal 1-2 minute;
Supernatant is abandoned in suction, microballoon is resuspended in the PBS-TBN solution of 1ml vortex vibration 25-30 second, sonic oscillation 25-30 second; Centrifugal collection microballoon, centrifugal condition are 〉=8000g centrifugal 1-2 minute; Repeat this step once;
Supernatant is abandoned in suction, microballoon is resuspended in the PBS-TBN solution of 500-1000 μ l;
Every kind of coated good microballoon is placed on 2-8 degree lucifuge by Luminex instrument counting and preserves separately, during use, needs equal proportion to mix according to monitoring, and the concentration of every kind of capture antibody coupling microballoon is 125/μ l in the mixed liquor;
2) according to the composition of kit, carry out every kind of biotin labeling that detects antibody:
Calculate the volume that the detection antibody-solutions is diluted to 1mg/ml according to detecting antibody concentration, this volume is as target volume;
Use dimethyl sulfoxide solution, the N-hydroxy thiosuccinimide biotin reaction liquid of configuration 10mg/ml;
1:100 adds respectively detection antibody and N-hydroxy thiosuccinimide biotin reaction liquid in reaction tube in molar ratio;
Adding volume is the sodium bicarbonate solution of the pH8.9 of target volume 1/10, and the PBS that adds subsequently pH7.4 mends to target volume;
Wrap aluminium foil or put into the lucifuge magazine, reaction tube or lucifuge magazine are placed on the circumference vibration shaking table, lucifuge was hatched 4-5 hour in 25 degree constant temperature ovens, and rotating speed is 900rpm-1000rpm;
Reactant liquor is gone in the dialysis cassette, in PBS damping fluid dialysed overnight, to remove unreacted N-hydroxy thiosuccinimide biotin reaction liquid;
During use as required that mark is good every kind detect the antibody equal proportion and mix.
3) have the extracting of the biological imitated vesicle structure of double-layer of lipoid membrane structure:
Can adopt ultracentrifugation, ultrafiltration or commercial special-purpose extraction agent extracting film vesicles from fixed amount serum, and carry out the standardization that protein quantification is finished sample, be prepared into testing sample, described fixed amount serum volume is more than or equal to 1ml.
The step of described activation microballoon is as follows:
With vortex oscillator or supersonic wave suspended microballoon;
Get 50 μ l microballoon centrifugal collecting precipitations, centrifugal condition is 〉=8000g centrifugal 1-2 minute;
Remove supernatant, microballoon is suspended from the distilled water of 100 μ l, with vortex oscillator or supersonic wave suspended microballoon 20-25 second, centrifugal collecting precipitation, centrifugal condition are 〉=8000g centrifugal 1-2 minute;
Supernatant is abandoned in suction, adds the phosphate buffer of 80 μ l, pH6.2, vortex vibration 20-25 second, supersonic wave suspended microballoon 20-25 second;
The N-hydroxy thiosuccinimide (S-NHS) that adds 10 μ l 50mg/ml is used lightly mixing of vortex oscillator;
Add the 1-ethyl of 10 μ l 50mg/ml-(3-dimethylamino third class)-3-ethyl-carbodiimide hydrochloride (EDC), with vortex oscillator mixing lightly;
The room temperature lucifuge leaves standstill 20min, uses lightly mixing of vortex oscillator in per 10 minutes.
The prescription of described various solution is as follows:
1, the prescription of the MES damping fluid of 50mM: will be dissolved in available from the MES 4.88g of Sigma company in the 450ml distilled water, the sodium hydroxide solution that adds the good 0.1M of configured in advance is regulated pH value to 5.0, and after 0.22 μ m sterile filters was filtered, 4 degree refrigerators were for subsequent use.
2, PBS prescription: will be dissolved in the 1000ml DDW available from 10 in the PBS tablet of Takara company, wherein contain NaCl 8,000 mg, KCl 200 mg, Na2HPO4 1150 mg, KH2PO4 200 mg, normal temperature is for subsequent use behind the autoclaving.
3, PBS-TBN prescription: add the BSA 1g available from Amresco company in the PBS solution that 1L is realized preparing, 0.2ml is available from Tween-20 and the Sodium azide 500mg of Sigma company, and stirring is mixed even.
According to the composition of kit, Dispersal risk is coated with microballoon, has referred to be coated with the different microballoons of CD11c, CD81, MHC II, CD86, CD106, CD54 and CD3 capture antibody; According to the composition of kit, carry out every kind of biotin labeling that detects antibody and refer to: contain the detection antibody of using respectively biotin labeled CD11c, CD81, MHC II, CD86, CD106, CD54 and CD3.
In the enforcement, capture antibody of the present invention is the monoclonal antibody of difference energy correspondence with CD11c, CD81, MHC II, CD86, CD106, CD54 and CD3 specific binding; Described detection antibody for respectively can with monoclonal or the polyclonal antibody of CD11c, CD81, MHC II, CD86, CD106, CD54 and CD3 specific binding.
The capture antibody of used " CD11c, CD81, MHC II, CD86, CD106, CD54 and CD3 " and detection antibody are respectively available from Biolegend, Abcam, Santa cruz, Invitrogen and Millipore company in the present embodiment.
Microballoon (surperficial carboxyl modified), the SA-PE of different numberings are all purchased the Luminex company in the U.S..
EDC, S-NHS and S-NHS-Biotin all purchase the company in Pierce.
In the present embodiment, the prescription of described various solution is as follows:
1, the MES damping fluid (pH5.0) of 50mM prescription (500ml): will be M5287 available from the MES(article No. of Sigma company) 4.88g is dissolved in the 450ml distilled water, the sodium hydroxide solution that adds the good 0.1M of configured in advance is regulated pH value to 5.0, after 0.22 μ m sterile filters was filtered, 4 degree refrigerators were for subsequent use.
2, PBS prescription: will be dissolved in (NaCl 8,000 mg, KCl 200 mg, Na2HPO4 1150 mg, KH2PO4 200 mg) in the 1000ml DDW available from 10 in the PBS tablet (article No. T900) of Takara company, normal temperature is for subsequent use behind the autoclaving.
3, the PBS-TBN prescription (is to contain 0.1% BSA among the PBS, 0.02% Tween-20,0.05% Sodium azide, pH is 7.4), to be 0903 available from the BSA(article No. of Amresco company) 1g, Tween-20(article No. available from Sigma company is P2287) 0.2ml and Sodium azide 500mg(article No. be S8032), be dissolved in 1L and realize that the PBS solution (available from takara company, article No. T900) for preparing stirs and evenly mixs.
4, the liquid phase chip reagent box of Acute Coronary Syndrome Patients risk stratification includes: 1) coated microballoon: contain the different microballoons that have been coated with respectively CD11c, CD81, MHC II, CD86, CD106, CD54 and CD3 capture antibody; 2) biotin labeling detects antibody: contain the detection antibody of using respectively biotin labeled CD11c, CD81, MHC II, CD86, CD106, CD54 and CD3; 3) streptavidin phycoerythrin (SA-PE, 10 μ g/ml); 4) exsomes extraction agent; 5) supporting analysis buffer; 6) microballoon standard items; 7) control liquid one; 8) control liquid two; 9) serum matrix liquid; 10) sealed membrane; 11) filter plate.
5, prepare above-mentioned liquid phase chip reagent box, include following steps:
1) according to the composition of mentioned reagent box, Dispersal risk is coated with microballoon, (preparation method of every kind of coated microballoon of capture antibody is identical):
Choose respectively different microballoon (purchasing the Luminex company in the U.S.), through vortex or ultrasound suspending 20-25 second;
Get 50 μ l microballoons in the 1.5ml centrifuge tube, centrifugal condition is 〉=8000g centrifugal 1-2 minute;
Suck supernatant, microballoon is resuspended in the distilled water of 100 μ l, with vortex or ultrasound suspending 20-25 second, centrifugal collecting precipitation, centrifugal condition are 〉=8000g centrifugal 1-2 minute;
The S-NHS and the EDC that successively add each 10 μ l 50mg/ml are through the vortex mixing;
The room temperature lucifuge left standstill 25-30 minute, used the vortex mixing once in per 10 minutes;
Microballoon after the centrifugal collection activation, centrifugal condition is 〉=8000g centrifugal 1-2 minute;
Suck supernatant, microballoon be resuspended in the MES(pH5.0 of 250 μ l 50mM) solution in, vortex 25-30 second, ultrasonic 25-30 second, the microballoon after the centrifugal collection activation, centrifugal condition is 〉=8000g centrifugal 1-2 minute; Repeat this step once;
Suck supernatant, microballoon be resuspended in the MES(pH5.0 of 100 μ l 50mM) solution in, vortex 25-30 second, ultrasonic 25-30 second, add the antibody of 1 μ g, with the MES(pH5.0 of 50mM) solution cumulative volume is mended to 500 μ l; Through vortex concussion mixing, room temperature lucifuge vibration 3 hours;
The microballoon of centrifugal collection coupling antibody, centrifugal condition are 〉=8000g centrifugal 2-3 minute; Suck supernatant, microballoon is resuspended in the PBS-TBN solution of 500 μ l, vortex 25-30 second, ultrasonic 25-30 second, room temperature lucifuge concussion 30 minutes, centrifugal collection microballoon, centrifugal condition are 〉=8000g centrifugal 1-2 minute;
Suck supernatant, microballoon is resuspended in the PBS-TBN solution of 1ml, vortex 25-30 second, ultrasonic 25-30 second, centrifugal collection microballoon, centrifugal condition are 〉=8000g that centrifugal 1-2 minute repeats this step once;
Suck supernatant, microballoon is resuspended in the PBS-TBN solution of 600 μ l, coated good microballoon is sent to Luminex instrument or Flow cytometry;
The microballoon of coated good antibody places the 2-8 degree to keep in Dark Place, and the microballoon of every kind of antibody coupling is preserved separately, during use, selects equal proportion to mix according to test item.
2) according to the composition of mentioned reagent box, carry out every kind of biotin labeling that detects antibody:
Calculate reaction system according to protein concentration: calculate the volume that antibody-solutions is diluted to 1mg/ml, be target volume (V), preparation NHS-Biotin reactant liquor (with the DMSO dissolving, final concentration is 10mg/ml); The ratio of 1:100 adds respectively detection antibody and N-Biotin reactant liquor in reaction tube in molar ratio; The sodium bicarbonate solution (pH8.9) that adds 1/10V; Adding PBS solution mends to target volume;
Wrap aluminium foil or put into the lucifuge magazine, reaction tube or lucifuge magazine are placed on the circumference oscillator, lucifuge was hatched 5 hours in 25 degree constant temperature ovens, and rotating speed is 1000rpm;
To the dialysis card, dialysed overnight twice in the PBS of 1000 times of volumes damping fluid to remove unreacted NHS-Biotin, is measured protein concentration with the liquid rotating in the reaction tube.
3) use ultracentrifugation, ultrafiltration or commercial exosomes extraction agent from serum extracting exosomes, and carry out the standardization that protein quantification is finished sample, be prepared into testing sample.
6, detect, may further comprise the steps:
1) uses front all reagent that take out, place balance to room temperature 15-30 minute;
2) dilution of standard items: each standard items arranges 6 dilutabilitys, then equal proportion is mixed, so that final concentration separately is required concentration, the standard items of each concentration must with the dilution that just can be used for next concentration behind the thorough mixing of vortex three times, can use high speed centrifugation to reduce issuable foam to the impact of follow-up dilution after having diluted in the blending process.The dilution process of each pipe and theoretical concentration:
3) according to the settle the standard position of product, quality-control product, testing sample and blank well of the order of the layout of different orifice plates (96 or 384 hole filter opening plate) and instrument readings; In the orifice plate layout that sets, every hole adds the analysis buffer of 25 μ l, and standard items, quality-control product and the testing sample that adds respectively again 25 μ l is in hole separately, and blank well adds the analysis buffer of 25 μ l;
4) take out respectively six kinds of capture antibody coupling microballoons of above-mentioned preparation, through vortex and ultrasonic process respectively 30 seconds after equal proportion mix so that the concentration of microballoon is 125/μ l in the mixed liquor.The mixing suspension 25 μ l of six kinds of capture antibody coupling microballoons that add in every hole.Microballoon should be at mixing before use, and should at once use behind the mixing, avoids the microballoon precipitation;
5) seal all wells with sealed membrane, and encase orifice plate or put into the lucifuge magazine with lucifuge with tinfoil paper, 25 degree are placed on the microwell plate oscillator and shake with 800 rotary speeds, hatched 1 hour, then suck damping fluid by the vacuum filtration device, use the lavation buffer solution washed twice, add again damping fluid;
6) every hole adds the mixed liquor of the biotin labeled detection antibody of the above-mentioned preparation of 25 μ l subsequently, seal entire plate with sealed membrane, and wrap up lucifuge with tinfoil paper, 25 degree are placed on the microwell plate oscillator and shake with 800 rotary speeds, hatched again 1 hour, hatch and suck damping fluid by the vacuum filtration device after finishing, use the lavation buffer solution washed twice, add again damping fluid;
7) every hole adds 25 μ l streptavidin phycoerythrin, seal entire plate with sealed membrane, and wrap up with tinfoil paper, 25 degree are placed on the microwell plate oscillator and shake with 800 rotary speeds, hatch half an hour again, hatch and suck damping fluid by the vacuum filtration device after finishing, use the lavation buffer solution washed twice, add again damping fluid;
8) reading result on the liquid-phase chip analyser, the drawing standard curve also calculates the numerical value of testing sample.
9) aforesaid operations is for cooperating polystyrene microsphere, use filter opening plate and the quick washing operation of vacuum suction apparatus realization entire plate manually or automatically, when cooperate be magnetic microsphere the time, then both can use the aforesaid operations flow process, and also can use magnetic attached plate and automatic or manual instead and wash panel assembly and finish quick washing operation.
7, interpretation: will directly react the Gensini integration of coronary artery changes in patients of coronary heart disease and several indexs of exosomes and all be remarkable positive correlation (p＜0.05), relevant clinical data is carried out stepwise regression analysis.The result shows that These parameters is the independentpredictor of Gensini integration.
Above result shows, can carry out the associating parallel detection to above-mentioned 7 kinds of exosomes marks simultaneously with the inventive method, but also can carry out the joint-detection of various combination to above-mentioned mark according to the reagent demand, 7 kinds of biomarkers of exosomes can help clinical ACS patient to be carried out better risk stratification assessment simultaneously.
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 (6)
1. one kind is detected the liquid phase chip reagent box of acute coronary artery syndrome based on the serum allochthon, and it mainly comprises, it is characterized in that: described liquid phase chip reagent box includes following component:
1) coated microballoon: microballoon mainly comprises two classes, a kind of diameter is the polystyrene microsphere of 5.6 μ m, another kind is the magnetic microsphere of 6.5 μ m for diameter, above-mentioned two class microballoons be coated with respectively the CD11c capture antibody microballoon, be coated with the CD81 capture antibody microballoon, be coated with MHC II capture antibody microballoon, be coated with the CD86 capture antibody microballoon, be coated with the CD106 capture antibody microballoon, be coated with the microballoon of CD54 capture antibody and be coated with the microballoon of CD3 capture antibody, above-mentioned microballoon has respectively the different colours coding;
2) biotin labeling detects antibody: contain respectively and detect antibody with biotin labeled CD11c, CD81, MHC II, CD86, CD106, CD54 and CD3;
3) streptavidin phycoerythrin.
2. according to claim 1ly a kind ofly detect the liquid phase chip reagent box of acute coronary artery syndrome based on the serum allochthon, it is characterized in that: the working concentration of the microballoon of every kind of coated capture antibody is 120-130/μ l; The working concentration of every kind of biotin labeling detection antibody is 1-4 μ g/ml.
3. one kind is detected the preparation method of the liquid phase chip reagent box of acute coronary artery syndrome based on the serum allochthon, and it is characterized in that: described preparation method comprises the steps:
1) according to the composition of kit, Dispersal risk is coated with microballoon, and the preparation method of every kind of coated microballoon of capture antibody is identical:
After the activation of 50ul microballoon, through the high speed centrifugation precipitation, centrifugal condition is 〉=7500g 5 minutes;
Supernatant is abandoned in suction, microballoon is resuspended in the MES solution of 50mM of 230-280 μ l of pH5.0, vortex vibration 25-30 second, ultrasonic 25-30 second, after microballoon is centrifugal, centrifugal condition is 〉=8000g centrifugal 1-2 minute; Repeat above-mentioned steps;
Supernatant is abandoned in suction, microballoon is resuspended in the solution of MES of 100 μ l 50mM pH5.0 vortex vibration 25-30 second, ultrasonic 25-30 second;
In the microballoon that suspends, add corresponding 0.8-1.2 μ g antibody, with the MES solution of the pH5.0 of 50mM cumulative volume is mended the l to 450-550 μ;
With vortex oscillator mixing, room temperature lucifuge vibration 2-2.5 hour;
With the microballoon centrifugation of the good antibody of coupling, centrifugal condition is 〉=8000g centrifugal 1-2 minute;
Supernatant is abandoned in suction, microballoon is resuspended in the 500 μ l PBS-TBN solution vortex vibration 25-30 second, sonic oscillation 25-30 second;
Room temperature lucifuge vibration 30 minutes; Centrifugal collection microballoon again, centrifugal condition is 〉=8000g centrifugal 1-2 minute;
Supernatant is abandoned in suction, microballoon is resuspended in the PBS-TBN solution of 1ml vortex vibration 25-30 second, sonic oscillation 25-30 second; Centrifugal collection microballoon, centrifugal condition are 〉=8000g centrifugal 1-2 minute; Repeat this step once;
Supernatant is abandoned in suction, microballoon is resuspended in the PBS-TBN solution of 500-1000 μ l;
Every kind of coated good microballoon is placed on 2-8 degree lucifuge by Luminex instrument counting and preserves separately, during use, needs equal proportion to mix according to monitoring, and the concentration of every kind of capture antibody coupling microballoon is 125/μ l in the mixed liquor;
2) according to the composition of kit, carry out every kind of biotin labeling that detects antibody:
Calculate the volume that the detection antibody-solutions is diluted to 1mg/ml according to detecting antibody concentration, this volume is as target volume;
Use dimethyl sulfoxide solution, the N-hydroxy thiosuccinimide biotin reaction liquid of configuration 10mg/ml;
1:100 adds respectively detection antibody and N-hydroxy thiosuccinimide biotin reaction liquid in reaction tube in molar ratio;
Adding volume is the sodium bicarbonate solution of the pH8.9 of target volume 1/10, and the PBS that adds subsequently pH7.4 mends to target volume;
Wrap aluminium foil or put into the lucifuge magazine, reaction tube or lucifuge magazine are placed on the circumference vibration shaking table, lucifuge was hatched 4-5 hour in 25 degree constant temperature ovens, and rotating speed is 900rpm-1000rpm;
Reactant liquor is gone in the dialysis cassette, in PBS damping fluid dialysed overnight, to remove unreacted N-hydroxy thiosuccinimide biotin reaction liquid;
During use as required that mark is good every kind detect the antibody equal proportion and mix;
3) have the extracting of the biological imitated vesicle structure of double-layer of lipoid membrane structure:
Can adopt ultracentrifugation, ultrafiltration or commercial special-purpose extraction agent extracting film vesicles from fixed amount serum, and carry out the standardization that protein quantification is finished sample, be prepared into testing sample, described fixed amount serum volume is more than or equal to 1ml.
4. according to claim 3ly a kind ofly detect the liquid phase chip reagent box of acute coronary artery syndrome based on the serum allochthon, it is characterized in that: the step of described activation microballoon is as follows:
With vortex oscillator or supersonic wave suspended microballoon;
Get 50 μ l microballoon centrifugal collecting precipitations, centrifugal condition is 〉=8000g centrifugal 1-2 minute;
Remove supernatant, microballoon is suspended from the distilled water of 100 μ l, with vortex oscillator or supersonic wave suspended microballoon 20-25 second, centrifugal collecting precipitation, centrifugal condition are 〉=8000g centrifugal 1-2 minute;
Supernatant is abandoned in suction, adds the phosphate buffer of 80 μ l, pH6.2, vortex vibration 20-25 second, supersonic wave suspended microballoon 20-25 second;
The N-hydroxy thiosuccinimide (S-NHS) that adds 10 μ l 50mg/ml is used lightly mixing of vortex oscillator;
Add the 1-ethyl of 10 μ l 50mg/ml-(3-dimethylamino third class)-3-ethyl-carbodiimide hydrochloride (EDC), with vortex oscillator mixing lightly;
The room temperature lucifuge leaves standstill 20min, uses lightly mixing of vortex oscillator in per 10 minutes.
5. the preparation method of a kind of liquid phase chip reagent box based on serum allochthon acute coronary syndrome according to claim 3, it is characterized in that: the prescription of described various solution is as follows:
1, the prescription of the MES damping fluid of 50mM: will be dissolved in available from the MES 4.88g of Sigma company in the 450ml distilled water, the sodium hydroxide solution that adds the good 0.1M of configured in advance is regulated pH value to 5.0, and after 0.22 μ m sterile filters was filtered, 4 degree refrigerators were for subsequent use;
2, PBS prescription: will be dissolved in the 1000ml DDW available from 10 in the PBS tablet of Takara company, wherein contain NaCl 8,000 mg, KCl 200 mg, Na2HPO4 1150 mg, KH2PO4 200 mg, normal temperature is for subsequent use behind the autoclaving;
3, PBS-TBN prescription: add the BSA 1g available from Amresco company in the PBS solution that 1L is realized preparing, 0.2ml is available from Tween-20 and the Sodium azide 500mg of Sigma company, and stirring is mixed even.
6. the preparation method of a kind of liquid phase chip reagent box based on serum allochthon acute coronary syndrome according to claim 3, it is characterized in that: according to the composition of kit, Dispersal risk is coated with microballoon, has referred to be coated with the different microballoons of CD11c, CD81, MHC II, CD86, CD106, CD54 and CD3 capture antibody; According to the composition of kit, carry out every kind of biotin labeling that detects antibody and refer to: contain the detection antibody of using respectively biotin labeled CD11c, CD81, MHC II, CD86, CD106, CD54 and CD3.
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