CN103163295B - A kind of liquid phase chip reagent box for acute coronary syndrome is with its preparation method - Google Patents
A kind of liquid phase chip reagent box for acute coronary syndrome is with its preparation method Download PDFInfo
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Abstract
The present invention relates to a kind of liquid phase chip reagent box for acute coronary syndrome and preparation method thereof.Does liquid-phase chip mainly include: bag is by oxidative low density lipoprotein (oxidized respectively low density lipoprotein, ox-LDL), soluble CD 40 ligand (soluble CD40 ligand, sCD40L), GELB (matrix metallopeptidase 9, MMP-9), tissue factor (tissue factor, TF), nethike embrane element (omentin) and endogenous solubility RAGE(endogenous secretory receptor of advanced glycation end products, esRAGE) microballoon of capture antibody, biotin labeled detection antibody and streptavidin phycoerythrin.Liquid-phase chip provided by the present invention has that high, the required sample size of detection efficiency is few, high specificity, highly sensitive, detect the advantage such as quick and precisely.Meanwhile, this chip reflects the height of machine vivo protective and damaging factor level simultaneously, so that the risk stratification of comprehensive assessment patient and prognosis situation more comprehensively.Preparation method of the present invention is simple simultaneously, and good stability, the amount, course of reaction etc. of the various technological parameters in its technical scheme as microballoon and antibody all draw on great many of experiments basis, are the parameter value of preparation process the best.
Description
Technical field:
The external kit technical field that the present invention relates to, specifically a kind of liquid phase chip reagent box for acute coronary syndrome is with its preparation method.
Background technology:
Atherosclerotic (atherosclerosis, AS) property vascular diseases have become " number one killer " that threaten compatriots' health, along with China's expanding economy, the incidence of disease is in ascendant trend year by year, China every year for the medical expense of such disease up to hundred billion yuans, the control strengthening AS vascular diseases and complication thereof has become the main task of China's major disease control.
AS vascular diseases cause acute coronary syndrome (Acutecoronarysyndrome, ACS) be the most common and the most serious clinical performance, and coronary artery vulnerable plaque break secondary thrombus formed be its generation main pathologic basis.Coronarography at present as diagnosis of coronary heart disease " goldstandard " well can not identify vulnerable plaque, the identification of vulnerable plaque and diagnosis depend on the interventional imaging such as intravascular ultrasound and the angioscope section of learning to do, the pathology of vascular wall can be shown, accurately distinguish the character of pathology to identify vulnerable plaque, but can not inflammatory activity in detection of plaque, more because it is for there being the inspection of wound property, technical conditions require high and expense costly, limit applying of it.Therefore, searching can reflect the responsive and specific serologic marker thing of AS vulnerable plaque, and by effective prevention, before vulnerable plaque breaks, to be identified in early days and to make it trend stable, is expected to reach the object reducing ACS generation.
But existing diagnostic kit focuses mostly in the diagnostic sensitivity improved by Peripheral Blood mark ACS and specificity aspect, related kit in the risk stratification improving patient and Index for diagnosis or a blank, and this respect exactly at present clinical patients administrative institute to be badly in need of.
Liquid-phase chip technology (xMAP) is also referred to as streaming fluorescent technique, that one can be widely used in albumen, gene, the biochip technology platform of the multiple biological respinse such as receptor/ligand, mainly comprise microballoon, probe molecule, detected material and reporter molecules four kinds of compositions, its microballoon uses two of different ratio kinds of redness to divide fluorochrome in manufacturing process, and by microballoon, (microballoon is divided into diameter to be the polystyrene microsphere of 5.6 μm and the magnetic microsphere of 6.5 μm, two kinds of microballoons mainly the washing in later stage and applicable instrument different, principle is consistent) dye different iridescent, thus obtain nearly 100 kinds of fluorescence-encoded microballoons.Being attached on specific coding microball for the antibody molecule of different determinand or gene probe in the mode of covalent cross-linking, the corresponding corresponding test item of each coding microball.First the fluorescence-encoded micro-beads for different determinand being mixed, then add test substance or amplified fragments to be measured, there is association reaction with mark fluorescent element in the compound formed again.Microballoon places an order leu time by red green laser in the drive of flowing sheath fluid, and red laser is used for judging the fluorescence-encoded of microballoon, and green laser is used for measuring the fluorescence intensity of reporter molecules on microballoon.Thus reach quantitative testing goal fast and accurately.Due to respond and be all in liquid phase environment, be more conducive to the natural conception keeping protein, make the reaction of probe and detected material faster more complete, therefore detection sensitivity and the range of linearity are all greatly improved.
Summary of the invention:
The object of the present invention is to provide a kind of liquid phase chip reagent box for acute coronary syndrome of improvement with its preparation method, it can overcome diagnostic kit in prior art and focus mostly in the diagnostic sensitivity improved by Peripheral Blood mark ACS and specificity aspect, related kit in the risk stratification improving patient and Index for diagnosis or a blank, the some shortcomings that simultaneously preparation method's complexity of available reagent box, poor stability, effect are not good.
To achieve these goals, technical scheme of the present invention is: a kind of liquid phase chip reagent box for acute coronary syndrome, is characterized in that: described liquid phase chip reagent box includes following component:
1) bag is by microballoon: microballoon mainly comprises two classes, a kind of diameter is the polystyrene microsphere of 5.6 μm and the magnetic microsphere of another kind of 6.5 μm, above-mentioned two class microballoons wrap by the microballoon of ox-LDL capture antibody respectively, wrap the quilt microballoon of sCD40L capture antibody, wrap the quilt microballoon of MMP-9 capture antibody, wrap the quilt microballoon of TF capture antibody, wrap the microballoon of quilt omentin capture antibody and the bag quilt microballoon of esRAGE capture antibody, and above-mentioned microballoon has different colours respectively and encodes;
2) biotin labeling detects antibody: contain respectively with the detection antibody of biotin labeled ox-LDL, sCD40L, MMP-9, TF, omentin and esRAGE;
3) streptavidin phycoerythrin.
For a preparation method for the liquid phase chip reagent box of acute coronary syndrome, it is characterized in that: described preparation method includes following steps:
1) according to the composition of mentioned reagent box, corresponding antibody bag is prepared by microballoon:
Choose different microballoons respectively, through vortex or ultrasound suspending 20-25 second;
After 50 μ l microballoon activation, centrifugal condition is >=8000g, centrifugal 1-2 minute;
Suck supernatant, be resuspended in by microballoon in the solution of the MES of 250 μ l50mM, vortex 25-30 second, ultrasonic 25-30 second, the microballoon after collected by centrifugation activation, centrifugal condition is >=8000g, centrifugal 1-2 minute; Repeat this step once;
Suck supernatant, microballoon is resuspended in the solution of the MES of 100 μ l50mM, vortex 25-30 second, ultrasonic 25-30 second, add the antibody of 1 μ g, with the solution of the MES of 50mM, cumulative volume is mended to 500 μ l; Through vortex concussion mixing, room temperature lucifuge is vibrated 3 hours;
The microballoon of collected by centrifugation coupled antibody, centrifugal condition is >=8000g, centrifugal 2-3 minute; Suck supernatant, be resuspended in by microballoon in the PBS-TBN solution of 500 μ l, vortex 25-30 second, ultrasonic 25-30 second, room temperature lucifuge shakes 30 minutes, collected by centrifugation microballoon, and centrifugal condition is >=8000g, centrifugal 1-2 minute;
Suck supernatant, be resuspended in by microballoon in the PBS-TBN solution of 1ml, vortex 25-30 second, ultrasonic 25-30 second, collected by centrifugation microballoon, centrifugal condition is >=8000g, and centrifugal 1-2 minute repeats this step once;
Suck supernatant, microballoon is resuspended in the PBS-TBN solution of 600 μ l, bag is sent to Luminex instrument or Flow cytometry by good microballoon;
Bag is placed in 2-8 degree by the microballoon of good antibody and keeps in Dark Place, and the microballoon of often kind of antibody coupling is preserved separately, during use, selects equal proportion mixing according to test item.
2) biotin labeling that often kind is detected antibody is carried out:
Reaction system is calculated: calculate volume antibody-solutions being diluted to 1mg/ml, be target volume (V), preparation NHS-Biotin reactant liquor (dissolve with DMSO, final concentration is 10mg/ml) according to protein concentration; The ratio of 1: 100 adds detection antibody and N-Biotin reactant liquor respectively in reaction tube in molar ratio; Add the sodium bicarbonate solution (pH8.9) of 1/10V; Add PBS solution to mend to target volume;
Wrap aluminium foil or put into lucifuge magazine, be placed on circumference oscillator by reaction tube or lucifuge magazine, in 25 degree of constant temperature ovens, lucifuge hatches 5 hours, and rotating speed is 1000rpm;
By the liquid rotating in reaction tube in dialysis card, dialysed overnight twice in the PBS damping fluid of 1000 times of volumes, to remove unreacted NHS-Biotin, measures protein concentration.
During use, disclosed liquid phase chip reagent box of the present invention can complete the quantitative detection of multiple blood serum designated object simultaneously, thus the risk stratification reached Acute Coronary Syndrome patient and Index for diagnosis, also there is detection efficiency high, required sample size is few, high specificity, sensitivity advantages of higher, the blood serum designated object simultaneously detected with ox-LDL, TF, sCD40L for core, by carrying out different collocation from remaining esRAGE, MMP-9 and omentin, the prognosis situation from injuring and repairing two angle Comprehensive assess patient can be realized.Preparation method of the present invention is simple simultaneously, and good stability, the amount, course of reaction etc. of the various technological parameters in its technical scheme as microballoon and antibody all draw on great many of experiments basis, are the parameter value of preparation process the best.
Accompanying drawing explanation
Fig. 1 is the table of comparisons of normal control population of the present invention and ACS peripheral blood in patients ox-LDL level (μ g/ml).
Fig. 2 is the table of comparisons of normal control population of the present invention and ACS peripheral blood in patients sCD40L level (ng/ml).
Fig. 3 is the table of comparisons of normal control population of the present invention and ACS peripheral blood in patients TF level (ng/ml).
Fig. 4 is the table of comparisons of normal control population of the present invention and ACS peripheral blood in patients MMP-9 level (ng/ml).
Fig. 5 is the table of comparisons of normal control population of the present invention and ACS peripheral blood in patients omentin level (ng/ml).
Fig. 6 is the table of comparisons of normal control population of the present invention and ACS peripheral blood in patients esRAGE level (ng/ml).
Fig. 7 is that the present invention carries out the chart of the risk stratification of ACS patient according to blood serum designated object.
Embodiment
Below in conjunction with chart and embodiment, the invention will be further described.
A kind of liquid phase chip reagent box for acute coronary syndrome of the present invention, is characterized in that: described liquid phase chip reagent box includes following component:
1) bag is by microballoon: microballoon mainly comprises two classes, a kind of diameter is the polystyrene microsphere of 5.6 μm and the magnetic microsphere of another kind of 6.5 μm, above-mentioned two class microballoons wrap by the microballoon of ox-LDL capture antibody respectively, wrap the quilt microballoon of sCD40L capture antibody, wrap the quilt microballoon of MMP-9 capture antibody, wrap the quilt microballoon of TF capture antibody, wrap the microballoon of quilt omentin capture antibody and the bag quilt microballoon of esRAGE capture antibody, and above-mentioned microballoon has different colours respectively and encodes;
2) biotin labeling detects antibody: contain respectively with the detection antibody of biotin labeled ox-LDL, sCD40L, MMP-9, TF, omentin and esRAGE;
3) streptavidin phycoerythrin.
The working concentration of the microballoon of often kind of captured antibody of bag is 120-130/μ l; The working concentration of often kind of biotin labeling detection antibody is 1-4 μ g/ml.
For a preparation method for the liquid phase chip reagent box of acute coronary syndrome, it is characterized in that: described preparation method includes following steps:
1) according to the composition of mentioned reagent box, corresponding antibody bag is prepared by microballoon:
Choose different microballoons respectively, through vortex or ultrasound suspending 20-25 second;
After 50 μ l microballoon activation, centrifugal condition is >=8000g, centrifugal 1-2 minute;
Suck supernatant, be resuspended in by microballoon in the solution of the MES of 250 μ l50mM, vortex 25-30 second, ultrasonic 25-30 second, the microballoon after collected by centrifugation activation, centrifugal condition is >=8000g, centrifugal 1-2 minute; Repeat this step once;
Suck supernatant, microballoon is resuspended in the solution of the MES of 100 μ l50mM, vortex 25-30 second, ultrasonic 25-30 second, add the antibody of 1 μ g, with the solution of the MES of 50mM, cumulative volume is mended to 500 μ l; Through vortex concussion mixing, room temperature lucifuge is vibrated 3 hours;
The microballoon of collected by centrifugation coupled antibody, centrifugal condition is >=8000g, centrifugal 2-3 minute; Suck supernatant, be resuspended in by microballoon in the PBS-TBN solution of 500 μ l, vortex 25-30 second, ultrasonic 25-30 second, room temperature lucifuge shakes 30 minutes, collected by centrifugation microballoon, and centrifugal condition is >=8000g, centrifugal 1-2 minute;
Suck supernatant, be resuspended in by microballoon in the PBS-TBN solution of 1ml, vortex 25-30 second, ultrasonic 25-30 second, collected by centrifugation microballoon, centrifugal condition is >=8000g, and centrifugal 1-2 minute repeats this step once;
Suck supernatant, microballoon is resuspended in the PBS-TBN solution of 600 μ l, bag is sent to Luminex instrument or Flow cytometry by good microballoon;
Bag is placed in 2-8 degree by the microballoon of good antibody and keeps in Dark Place, and the microballoon of often kind of antibody coupling is preserved separately, during use, selects equal proportion mixing according to test item.
2) biotin labeling that often kind is detected antibody is carried out:
Reaction system is calculated: calculate volume antibody-solutions being diluted to 1mg/ml, be target volume (V), preparation NHS-Biotin reactant liquor (dissolve with DMSO, final concentration is 10mg/ml) according to protein concentration; The ratio of 1: 100 adds detection antibody and N-Biotin reactant liquor respectively in reaction tube in molar ratio; Add the sodium bicarbonate solution (pH8.9) of 1/10V; Add PBS solution to mend to target volume;
Wrap aluminium foil or put into lucifuge magazine, be placed on circumference oscillator by reaction tube or lucifuge magazine, in 25 degree of constant temperature ovens, lucifuge hatches 5 hours, and rotating speed is 1000rpm;
By the liquid rotating in reaction tube in dialysis card, dialysed overnight twice in the PBS damping fluid of 1000 times of volumes, to remove unreacted NHS-Biotin, measures protein concentration.
The step of described activation microballoon is as follows:
With turbula shaker or supersonic wave suspended microballoon;
Get 50 μ l microballoon centrifugal collecting precipitations, centrifugal condition is >=8000g, centrifugal 1-2 minute;
Remove supernatant, be suspended from by microballoon in the distilled water of 100 μ l, with turbula shaker or supersonic wave suspended microballoon 20-25 second, centrifugal collecting precipitation, centrifugal condition is >=8000g, centrifugal 1-2 minute;
Supernatant is abandoned in suction, adds the phosphate buffer (pH6.2) of 80 μ l, vortex oscillation 20-25 second, supersonic wave suspended microballoon 20-25 second;
Add the N-hydroxy thiosuccinimide (S-NHS) of 10 μ l50mg/ml, mix lightly with turbula shaker;
Add 1-ethyl-(3-dimethylamino third class)-3-ethyl-carbodiimide hydrochloride (EDC) of 10 μ l50mg/ml, mix lightly with turbula shaker;
Room temperature lucifuge leaves standstill 20min, within every 10 minutes, uses turbula shaker to mix lightly.
The formula of various solution is as follows:
The MES damping fluid of 50mM, pH5.0,500ml formula is as follows: be dissolved in 450ml distilled water by the MES4.88g of available from Sigma, add the sodium hydroxide solution adjust ph to 5.0 of the good 0.1M of configured in advance, after 0.22 μm of sterile filters is filtered, 4 degree of refrigerators are for subsequent use;
PBS fills a prescription: by 10, the PBS tablet purchased from Takara company, be dissolved in 1000ml DDW, then add NaCl8,000mg, KCl200mg, Na
2hPO
41150mg, KH
2pO
4200mg, after autoclaving, normal temperature is for subsequent use.
PBS-TBN fills a prescription, and pH is 7.4, and by the BSA1g purchased from Amresco company, the Tween-20 of available from Sigma, 0.2ml and Sodium azide 500mg, be dissolved in the PBS solution that the 1L purchased from takara company prepares, stir and evenly mix.
Different microballoons refers to wrap the different microballoons by ox-LDL, sCD40L, MMP-9, TF, omentin and esRAGE capture antibody respectively, and above-mentioned different microballoon is purchased in Luminex company of the U.S..
Screening to more than 30 kinds of blood serum designated objects before the present invention combines according to a large amount of documents and materials, therefrom determine the serum biomarkers that ox-LDL, sCD40L, MMP-9, TF, omentin and esRAGE these six kinds contributes to ACS patient risk layering and Index for diagnosis, utilize liquid-phase chip platform to carry out the detection of running simultaneously of these six kinds of indexs.
Ox-LDL, sCD40L, MMP-9 and TF is the serum biological markers of closely-related several mediated vascular damage with the ACS course of disease, Omentin and esRAGE is the factor of newfound two kinds of mediate protection effects in the recent period, large quantity research shows that the order of severity of the vascular lesion of itself and ACS patient is negative correlation, but existing research adopts one or both above-mentioned factors to carry out related experiment more, overall condition for ACS patient lacks a understanding in all directions, therefore we intend adopting above-mentioned agents to be applied to risk stratification and the clinical prognosis judgement of ACS patient, wherein ox-LDL, sCD40L, MMP-9 and TF is for assessment of the degree of impairment of patient by paathogenic factor, the self-protective mechanism that Omentin and esRAGE is used for patient inherence activates situation, both can carry out combination mutually according to actual conditions and apply.
At present, mensuration for above-mentioned blood serum designated object has multiple detection method, comprise immunofluorescence analysis, enzyme-linked immuno assay (ELISA), radiommunoassay (RIA) etc., but these technology once can only detect for a mark, and complex operation, susceptibility is different, can not really meet clinical needs, in addition because various blood serum designated object has different expression in the course of disease of ACS, the problems referred to above make the value of each index in the risk stratification and Index for diagnosis of ACS patient different, if can detect simultaneously, then can increase substantially the accuracy of ACS patient risk layering and Index for diagnosis.And solid phase biological chip technology also exists the shortcoming of the not high and complex operation of poor repeatability, susceptibility.
Embodiment 1
The preparation of the liquid-phase chip of Acute Coronary Syndrome Patients risk stratification and Index for diagnosis and the detection of antigen
Capture antibody of the present invention is the monoclonal antibody of energy correspondence and ox-LDL, sCD40L, MMP-9, TF, omentin and esRAGE specific binding respectively; Described detection antibody be respectively can with the monoclonal of ox-LDL, sCD40L, MMP-9, TF, omentin and esRAGE specific binding or polyclonal antibody.
The capture antibody of " ox-LDL, sCD40L, MMP-9, TF, omentin and esRAGE " used in the present embodiment and detection antibody are respectively purchased from Abcam, Santacruz, Invitrogen and Millipore company.
Microballoon (surface carboxyl groups modification), the SA-PE of different numbering are all purchased in Luminex company of the U.S..
EDC, S-NHS and S-NHS-Biotin all purchase in Pierce company.
In the present embodiment, the formula of described various solution is as follows:
1, the MES damping fluid (pH5.0) formula (500ml) of 50mM: MES (article No. the is M5287) 4.88g of available from Sigma is dissolved in 450ml distilled water, add the sodium hydroxide solution adjust ph to 5.0 of the good 0.1M of configured in advance, after 0.22 μm of sterile filters is filtered, 4 degree of refrigerators are for subsequent use.
2, PBS formula: by 10, the PBS tablet (article No. T900) purchased from Takara company, be dissolved in (NaCl8,000mg, KCl200mg, Na in 1000ml DDW
2hPO
41150mg, KH
2pO
4200mg), after autoclaving, normal temperature is for subsequent use.
3, PBS-TBN formula (namely contains the BSA of 0.1% in PBS, the Tween-20 of 0.02%, the Sodium azide of 0.05%, pH is 7.4), by BSA (article No. the is 0903) 1g purchased from Amresco company, Tween-20 (article No. the is P2287) 0.2ml and Sodium azide 500mg (article No. is S8032) of available from Sigma, is dissolved in the PBS solution (purchased from takara company, article No. T900) that 1L realizes preparing and stirs and evenly mixs.
4, the liquid phase chip reagent box of Acute Coronary Syndrome Patients risk stratification and Index for diagnosis, includes: 1) bag is by microballoon: containing wrapping by the different microballoons of ox-LDL, sCD40L, MMP-9, TF, omentin and esRAGE capture antibody respectively; 2) biotin labeling detects antibody: contain respectively with the detection antibody of biotin labeled ox-LDL, sCD40L, MMP-9, TF, omentin and esRAGE; 3) streptavidin phycoerythrin (SA-PE, 10 μ g/ml); 4) supporting analysis buffer; 5) microballoon standard items; 6) control liquid one; 7) control liquid two; 8) serum matrix liquid; 9) sealed membrane; 10) 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 bag by microballoon, (often kind of capture antibody bag is identical by the preparation method of microballoon):
Choose different microballoons (purchasing in Luminex company of the U.S.) respectively, through vortex or ultrasound suspending 20-25 second;
Get 50 μ l microballoons in 1.5ml centrifuge tube, centrifugal condition is >=8000g, centrifugal 1-2 minute;
Suck supernatant, be resuspended in by microballoon in the distilled water of 100 μ l, with vortex or ultrasound suspending 20-25 second, centrifugal collecting precipitation, centrifugal condition is >=8000g, centrifugal 1-2 minute;
Successively add S-NHS and EDC of each 10 μ l50mg/ml, mix through vortex;
Room temperature lucifuge leaves standstill 25-30 minute, within every 10 minutes, uses vortex mixing once;
Microballoon after collected by centrifugation activation, centrifugal condition is >=8000g, centrifugal 1-2 minute;
Suck supernatant, be resuspended in by microballoon in the solution of MES (pH5.0) of 250 μ l50mM, vortex 25-30 second, ultrasonic 25-30 second, the microballoon after collected by centrifugation activation, centrifugal condition is >=8000g, centrifugal 1-2 minute; Repeat this step once;
Suck supernatant, microballoon is resuspended in the solution of MES (pH5.0) of 100 μ l50mM, vortex 25-30 second, ultrasonic 25-30 second, add the antibody of 1 μ g, with the solution of the MES (pH5.0) of 50mM, cumulative volume is mended to 500 μ l; Through vortex concussion mixing, room temperature lucifuge is vibrated 3 hours;
The microballoon of collected by centrifugation coupled antibody, centrifugal condition is >=8000g, centrifugal 2-3 minute; Suck supernatant, be resuspended in by microballoon in the PBS-TBN solution of 500 μ l, vortex 25-30 second, ultrasonic 25-30 second, room temperature lucifuge shakes 30 minutes, collected by centrifugation microballoon, and centrifugal condition is >=8000g, centrifugal 1-2 minute;
Suck supernatant, be resuspended in by microballoon in the PBS-TBN solution of 1ml, vortex 25-30 second, ultrasonic 25-30 second, collected by centrifugation microballoon, centrifugal condition is >=8000g, and centrifugal 1-2 minute repeats this step once;
Suck supernatant, microballoon is resuspended in the PBS-TBN solution of 600 μ l, bag is sent to Luminex instrument or Flow cytometry by good microballoon;
Bag is placed in 2-8 degree by the microballoon of good antibody and keeps in Dark Place, and the microballoon of often kind of antibody coupling is preserved separately, during use, selects equal proportion mixing according to test item.
2) according to the composition of mentioned reagent box, the biotin labeling that often kind is detected antibody is carried out:
Reaction system is calculated: calculate volume antibody-solutions being diluted to 1mg/ml, be target volume (V), preparation NHS-Biotin reactant liquor (dissolve with DMSO, final concentration is 10mg/ml) according to protein concentration; The ratio of 1: 100 adds detection antibody and N-Biotin reactant liquor respectively in reaction tube in molar ratio; Add the sodium bicarbonate solution (pH8.9) of 1/10V; Add PBS solution to mend to target volume;
Wrap aluminium foil or put into lucifuge magazine, be placed on circumference oscillator by reaction tube or lucifuge magazine, in 25 degree of constant temperature ovens, lucifuge hatches 5 hours, and rotating speed is 1000rpm;
By the liquid rotating in reaction tube in dialysis card, dialysed overnight twice in the PBS damping fluid of 1000 times of volumes, to remove unreacted NHS-Biotin, measures protein concentration.
6, detect, comprise the following steps:
1) take out all reagent before using, place balance to room temperature 15-30 minute;
2) dilution of standard items: each standard items arrange 6 dilutabilitys, then equal proportion mixing, respective final concentration is made to be required concentration, just can be used for the dilution of next concentration after the standard items of each concentration must thoroughly mix three times with vortex after having diluted, high speed centrifugation in blending process, can be used to reduce the impact of issuable foam on subsequent dilution.The dilution process of each pipe and theoretical concentration:
3) according to the position of the order confirmed standard product of the layout of different orifice plate (96 or 384 hole filter opening plate) and instrument readings, quality-control product, testing sample and blank well; In the orifice plate layout set, every hole adds the analysis buffer of 25 μ l, then adds the standard items of 25 μ l, quality-control product and testing sample respectively in respective hole, and blank well adds the analysis buffer of 25 μ l;
4) take out six kinds of capture antibody coupling microballoons of above-mentioned preparation respectively, the equal proportion mixing after 30 seconds through vortex and ultrasonic process respectively, makes the concentration of microballoon in mixed liquor be 125/μ l.The mixing suspension 25 μ l of the six kinds of capture antibody coupling microballoons added in every hole.Microballoon should mix before use, and should at once apply after mixing, avoids microballoon to precipitate;
5) with all wells of ParafilmTM, and encase orifice plate with tinfoil paper or put into lucifuge magazine with lucifuge, 25 degree are placed on microwell plate oscillator with 800 rotary speed concussions, hatch 1 hour, then damping fluid is sucked by Vacuum filtration device, use lavation buffer solution washes twice, then adds 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, use ParafilmTM entire plate, and wrap up lucifuge with tinfoil paper, 25 degree are placed on microwell plate oscillator with 800 rotary speed concussions, hatch 1 hour again, suck damping fluid by Vacuum filtration device after having hatched, use lavation buffer solution to wash twice, then add damping fluid;
7) every hole adds 25 μ l streptavidin phycoerythrin, use ParafilmTM entire plate, and wrap up with tinfoil paper, 25 degree are placed on microwell plate oscillator with 800 rotary speed concussions, hatch half an hour again, suck damping fluid by Vacuum filtration device after having hatched, use lavation buffer solution to wash twice, then add damping fluid;
8) on liquid-phase chip analyser, read result, drawing standard curve also calculates the numerical value of testing sample.
9) aforesaid operations is for coordinating polystyrene microsphere, use filter opening plate and manually or automatically vacuum suction apparatus realize the quick wash operation of entire plate, when coordinate be magnetic microsphere time, then both can use aforesaid operations flow process, also can use magnetic attached plate and automatic or manual instead and wash panel assembly and complete quick wash operation.
7, interpretation:
Specifically see table 1-2 and Fig. 1-Fig. 7.
Table 1. Normal group Peripheral Blood marker level concentration
Table 2.ACS group Peripheral Blood marker level concentration
The ACS patient peripheral serum marker level concentration of the different risk stratification of table 3.
When patient risk is layered as low danger, its prognosis is relatively better, along with risk stratification is from low danger to high-risk upgrading, the clinical prognosis of patient also worse and worse, thus realizing according to Peripheral Blood mark to the risk stratification of ACS patient and prognosis interpretation, above-mentioned data are through the checking of the Follow-up results of the clinical data of 5 years.
Above result shows, can carry out associating parallel detection by the inventive method to 6 kinds of blood serum designated objects simultaneously, but also can carry out the joint-detection of various combination to above-mentioned 6 kinds of marks according to reagent demand.Mean concentration as can be seen from 6 kinds of marks in table 1-2 and Fig. 1-6: 1) this 6 kinds of biomarker concentrations and Normal group significant difference of ACS patient; 2) above-mentioned 6 kinds of biomarkers can help clinically to carry out better risk stratification and prognosis evaluation to ACS patient.
The present invention relates to a kind of liquid phase chip reagent box for acute coronary syndrome and preparation method thereof.Liquid-phase chip mainly includes: bag is by oxidative low density lipoprotein (oxidizedlowdensitylipoprotein respectively, ox-LDL), soluble CD 40 ligand (solubleCD40ligand, sCD40L), GELB (matrixmetallopeptidase9, MMP-9), tissue factor (tissuefactor, TF), the microballoon of nethike embrane element (omentin) and endogenous solubility RAGE (endogenoussecretoryreceptorofadvancedglycationendproduct s, esRAGE) capture antibody; Biotin labeled detection antibody and streptavidin phycoerythrin.Liquid-phase chip provided by the present invention has that high, the required sample size of detection efficiency is few, high specificity, highly sensitive, detect the advantage such as quick and precisely.Meanwhile, this chip reflects the height of machine vivo protective and damaging factor level simultaneously, so that the risk stratification of comprehensive assessment patient and prognosis situation more comprehensively.Preparation method of the present invention is simple simultaneously, and good stability, the amount, course of reaction etc. of the various technological parameters in its technical scheme as microballoon and antibody all draw on great many of experiments basis, are the parameter value of preparation process the best.
1, for a liquid phase chip reagent box for acute coronary syndrome, it is characterized in that: described liquid phase chip reagent box includes following component:
1) bag is by microballoon: microballoon mainly comprises two classes, a kind of diameter is the polystyrene microsphere of 5.6 μm and the magnetic microsphere of another kind of 6.5 μm, above-mentioned two class microballoons wrap by the microballoon of ox-LDL capture antibody respectively, wrap the quilt microballoon of sCD40L capture antibody, wrap the quilt microballoon of MMP-9 capture antibody, wrap the quilt microballoon of TF capture antibody, wrap the microballoon of quilt omentin capture antibody and the bag quilt microballoon of esRAGE capture antibody, and above-mentioned microballoon has different colours respectively and encodes;
2) biotin labeling detects antibody: contain respectively with the detection antibody of biotin labeled ox-LDL, sCD40L, MMP-9, TF, omentin and esRAGE;
3) streptavidin phycoerythrin.
2, a kind of liquid phase chip reagent box for acute coronary syndrome according to claim 1, is characterized in that: the working concentration of the microballoon of often kind of captured antibody of bag is 120-130/μ l; The working concentration of often kind of biotin labeling detection antibody is 1-4 μ g/ml.
3, the preparation method of a kind of liquid phase chip reagent box for acute coronary syndrome according to claim 1, is characterized in that: described preparation method includes following steps:
1) according to the composition of mentioned reagent box, corresponding antibody bag is prepared by microballoon:
Choose different microballoons respectively, through vortex or ultrasound suspending 20-25 second;
After 50 μ l microballoon activation, centrifugal condition is >=8000g, centrifugal 1-2 minute;
Suck supernatant, be resuspended in by microballoon in the solution of the MES of 250 μ l50mM, vortex 25-30 second, ultrasonic 25-30 second, the microballoon after collected by centrifugation activation, centrifugal condition is >=8000g, centrifugal 1-2 minute; Repeat this step once;
Suck supernatant, microballoon is resuspended in the solution of the MES of 100 μ l50mM, vortex 25-30 second, ultrasonic 25-30 second, add the antibody of 1 μ g, with the solution of the MES of 50mM, cumulative volume is mended to 500 μ l; Through vortex concussion mixing, room temperature lucifuge is vibrated 3 hours;
The microballoon of collected by centrifugation coupled antibody, centrifugal condition is >=8000g, centrifugal 2-3 minute; Suck supernatant, be resuspended in by microballoon in the PBS-TBN solution of 500 μ l, vortex 25-30 second, ultrasonic 25-30 second, room temperature lucifuge shakes 30 minutes, collected by centrifugation microballoon, and centrifugal condition is >=8000g, centrifugal 1-2 minute;
Suck supernatant, be resuspended in by microballoon in the PBS-TBN solution of 1ml, vortex 25-30 second, ultrasonic 25-30 second, collected by centrifugation microballoon, centrifugal condition is >=8000g, and centrifugal 1-2 minute repeats this step once;
Suck supernatant, microballoon is resuspended in the PBS-TBN solution of 600 μ l, bag is sent to Luminex instrument or Flow cytometry by good microballoon;
Bag is placed in 2-8 degree by the microballoon of good antibody and keeps in Dark Place, and the microballoon of often kind of antibody coupling is preserved separately, during use, selects equal proportion mixing according to test item.
2) biotin labeling that often kind is detected antibody is carried out:
Reaction system is calculated: calculate volume antibody-solutions being diluted to 1mg/ml, be target volume (V), preparation NHS-Biotin reactant liquor (dissolve with DMSO, final concentration is 10mg/ml) according to protein concentration; The ratio of 1: 100 adds detection antibody and N-Biotin reactant liquor respectively in reaction tube in molar ratio; Add the sodium bicarbonate solution (pH8.9) of 1/10V; Add PBS solution to mend to target volume;
Wrap aluminium foil or put into lucifuge magazine, be placed on circumference oscillator by reaction tube or lucifuge magazine, in 25 degree of constant temperature ovens, lucifuge hatches 5 hours, and rotating speed is 1000rpm;
By the liquid rotating in reaction tube in dialysis card, dialysed overnight twice in the PBS damping fluid of 1000 times of volumes, to remove unreacted NHS-Biotin, measures protein concentration.
4, the preparation method of a kind of liquid phase chip reagent box for acute coronary syndrome according to claim 3, is characterized in that: the step of described activation microballoon is as follows:
With turbula shaker or supersonic wave suspended microballoon;
Get 50 μ l microballoon centrifugal collecting precipitations, centrifugal condition is >=8000g, centrifugal 1-2 minute;
Remove supernatant, be suspended from by microballoon in the distilled water of 100 μ l, with turbula shaker or supersonic wave suspended microballoon 20-25 second, centrifugal collecting precipitation, centrifugal condition is >=8000g, centrifugal 1-2 minute;
Supernatant is abandoned in suction, adds the phosphate buffer (pH6.2) of 80 μ l, vortex oscillation 20-25 second, supersonic wave suspended microballoon 20-25 second;
Add the N-hydroxy thiosuccinimide (S-NHS) of 10 μ l50mg/ml, mix lightly with turbula shaker;
Add 1-ethyl-(3-dimethylamino third class)-3-ethyl-carbodiimide hydrochloride (EDC) of 10 μ l50mg/ml, mix lightly with turbula shaker;
Room temperature lucifuge leaves standstill 20min, within every 10 minutes, uses turbula shaker to mix lightly.
5, the preparation method of a kind of liquid phase chip reagent box for acute coronary syndrome according to claim 3, is characterized in that: the formula of various solution is as follows:
The MES damping fluid of 50mM, pH5.0,500ml formula is as follows: be dissolved in 450ml distilled water by the MES4.88g of available from Sigma, add the sodium hydroxide solution adjust ph to 5.0 of the good 0.1M of configured in advance, after 0.22 μm of sterile filters is filtered, 4 degree of refrigerators are for subsequent use;
PBS fills a prescription: by 10, the PBS tablet purchased from Takara company, be dissolved in 1000ml DDW, then add NaCl8,000mg, KCl200mg, Na
2hPO
41150mg, KH
2pO
4200mg, after autoclaving, normal temperature is for subsequent use.
PBS-TBN fills a prescription, and pH is 7.4, and by the BSA1g purchased from Amresco company, the Tween-20 of available from Sigma, 0.2ml and Sodium azide 500mg, be dissolved in the PBS solution that the 1L purchased from takara company prepares, stir and evenly mix.
6, the preparation method of a kind of liquid phase chip reagent box for acute coronary syndrome according to claim 3, it is characterized in that: different microballoons refers to wrap the different microballoons by ox-LDL, sCD40L, MMP-9, TF, omentin and esRAGE capture antibody respectively, and above-mentioned different microballoon is purchased in Luminex company of the U.S..
Claims (1)
1. for a preparation method for the liquid phase chip reagent box of acute coronary syndrome, it is characterized in that: described preparation method includes following steps:
1) according to the composition of mentioned reagent box, corresponding antibody bag is prepared by microballoon:
Choose different microballoons respectively, through vortex or ultrasound suspending 20-25 second;
The step of activation microballoon is as follows:
With turbula shaker or supersonic wave suspended microballoon;
Get 50 μ l microballoon centrifugal collecting precipitations, centrifugal condition is >=8000g, centrifugal 1-2 minute;
Remove supernatant, be suspended from by microballoon in the distilled water of 100 μ l, with turbula shaker or supersonic wave suspended microballoon 20-25 second, centrifugal collecting precipitation, centrifugal condition is >=8000g, centrifugal 1-2 minute;
Supernatant is abandoned in suction, adds phosphate buffer, the pH6.2 of 80 μ l; Vortex oscillation 20-25 second, supersonic wave suspended microballoon 20-25 second;
Add the N-hydroxy thiosuccinimide (S-NHS) of 10 μ l50mg/ml, mix lightly with turbula shaker;
Add 1-ethyl-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDC) of 10 μ l50mg/ml, mix lightly with turbula shaker;
Room temperature lucifuge leaves standstill 20min, within every 10 minutes, uses turbula shaker to mix lightly;
After 50 μ l microballoon activation, centrifugal condition is >=8000g, centrifugal 1-2 minute;
Suck supernatant, be resuspended in by microballoon in the solution of the MES of 250 μ l50mM, vortex 25-30 second, ultrasonic 25-30 second, the microballoon after collected by centrifugation activation, centrifugal condition is >=8000g, centrifugal 1-2 minute; Repeat this step once;
Suck supernatant, microballoon is resuspended in the solution of the MES of 100 μ l50mM, vortex 25-30 second, ultrasonic 25-30 second, add the antibody of 1 μ g, with the solution of the MES of 50mM, cumulative volume is mended to 500 μ l; Through vortex concussion mixing, room temperature lucifuge is vibrated 3 hours;
The microballoon of collected by centrifugation coupled antibody, centrifugal condition is >=8000g, centrifugal 2-3 minute; Suck supernatant, be resuspended in by microballoon in the PBS-TBN solution of 500 μ l, vortex 25-30 second, ultrasonic 25-30 second, room temperature lucifuge shakes 30 minutes, collected by centrifugation microballoon, and centrifugal condition is >=8000g, centrifugal 1-2 minute;
Suck supernatant, be resuspended in by microballoon in the PBS-TBN solution of 1ml, vortex 25-30 second, ultrasonic 25-30 second, collected by centrifugation microballoon, centrifugal condition is >=8000g, and centrifugal 1-2 minute repeats this step once;
Suck supernatant, microballoon is resuspended in the PBS-TBN solution of 600 μ l, bag is sent to Luminex instrument or Flow cytometry by good microballoon;
Bag is placed in 2-8 degree by the microballoon of good antibody and keeps in Dark Place, and the microballoon of often kind of antibody coupling is preserved separately, during use, selects equal proportion mixing according to test item;
The MES damping fluid of 50mM, pH5.0,500ml formula is as follows: be dissolved in 450ml distilled water by the MES4.88g of available from Sigma, add the sodium hydroxide solution adjust ph to 5.0 of the good 0.1M of configured in advance, after 0.22 μm of sterile filters is filtered, 4 degree of refrigerators are for subsequent use;
PBS fills a prescription: by 10, the PBS tablet purchased from Takara company, be dissolved in 1000ml DDW, then add NaCl8,000mg, KCl200mg, Na
2hPO
41150mg, KH
2pO
4200mg, after autoclaving, normal temperature is for subsequent use;
PBS-TBN fills a prescription, and pH is 7.4, and by the BSA1g purchased from Amresco company, the Tween-20 of available from Sigma, 0.2ml and Sodium azide 500mg, be dissolved in the PBS solution that the 1L purchased from takara company prepares, stir and evenly mix;
2) biotin labeling that often kind is detected antibody is carried out:
Calculate reaction system according to protein concentration: calculating volume antibody-solutions being diluted to 1mg/ml, is target volume V, preparation NHS-Biotin reactant liquor, dissolve with DMSO, final concentration is 10mg/ml; The ratio of 1:100 adds detection antibody and NHS-Biotin reactant liquor respectively in reaction tube in molar ratio; Add the sodium bicarbonate solution of 1/10V, pH8.9; Add PBS solution to mend to target volume;
Wrap aluminium foil or put into lucifuge magazine, be placed on circumference oscillator by reaction tube or lucifuge magazine, in 25 degree of constant temperature ovens, lucifuge hatches 5 hours, and rotating speed is 1000rpm;
By the liquid rotating in reaction tube in dialysis card, dialysed overnight twice in the PBS damping fluid of 1000 times of volumes, to remove unreacted NHS-Biotin, measures protein concentration;
Different microballoons refers to wrap the different microballoons by ox-LDL, sCD40L, MMP-9, TF, omentin and esRAGE capture antibody respectively, and above-mentioned different microballoon is purchased in Luminex company of the U.S..
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Citations (17)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002089657A2 (en) * | 2001-05-04 | 2002-11-14 | Biosite, Inc. | Diagnostic markers of acute coronary syndromes and methods of use thereof |
| CA2457775A1 (en) * | 2001-08-20 | 2003-02-27 | Biosite Incorporated | Diagnostic markers of stroke and cerebral injury and methods of use thereof |
| WO2003103711A1 (en) * | 2002-06-07 | 2003-12-18 | Genentech, Inc. | Combinations of anti-tissue factor antibodies and anticoagulant and/or antiplatelet agents |
| CN1613012A (en) * | 2001-11-05 | 2005-05-04 | 布赖汉姆妇女医院 | Soluble cd40l (cd154) as a prognostic marker of atherosclerotic diseases |
| WO2006105439A2 (en) * | 2005-03-30 | 2006-10-05 | Decode Genetics Ehf. | Susceptibility gene for myocardial infarction, stroke. and paod; methods of treatment |
| WO2007110104A1 (en) * | 2006-03-28 | 2007-10-04 | Universität Zürich | Drug-eluting clinical device |
| WO2008020013A2 (en) * | 2006-08-17 | 2008-02-21 | F. Hoffmann La-Roche Ag | Methods and means for determining platelet function and diagnosing platelet- related and cardiovascular disorders |
| CN101195659A (en) * | 2006-12-05 | 2008-06-11 | 复旦大学 | High anticoagulating active antihuman tissue factor monoclone antibody, preparation method and application thereof |
| CN101271108A (en) * | 2008-01-11 | 2008-09-24 | 广州益善生物技术有限公司 | Myocardial infarct early diagnosis liquid phase chip and method for producing the same |
| CN101327214A (en) * | 2008-07-18 | 2008-12-24 | 中国药科大学 | Medicine use of isoquinoline alkaloids as tissue factor inhibitor |
| CN101526535A (en) * | 2009-04-14 | 2009-09-09 | 河南省豫康生物工程技术有限公司 | Liquid phase chip for joint detection of multiple tumor markers and preparation method thereof |
| CN101568837A (en) * | 2006-08-07 | 2009-10-28 | 比奥-拉德巴斯德公司 | Method for the prediction of vascular events and the diagnosis of acute coronary syndrome |
| CN101792746A (en) * | 2010-01-28 | 2010-08-04 | 中南大学 | Vascular peroxidase ELISA kit used for coronary disease diagnosis |
| WO2011026017A1 (en) * | 2009-08-31 | 2011-03-03 | Abbott Laboratories | Biomarkers for prediction of major adverse cardiac events and uses thereof |
| CN102062735A (en) * | 2009-11-18 | 2011-05-18 | 江苏迈迪基因生物科技有限公司 | Biomarker detection method and diagnostic kit for acute coronary syndrome |
| CN102109517A (en) * | 2010-12-14 | 2011-06-29 | 邵棠 | Joint detection method of cardio-cerebral-vascular disease (CCVD) biomarkers and diagnostic kit |
| CN102159723A (en) * | 2008-07-17 | 2011-08-17 | 斯塔戈诊断公司 | Method for in vitro assay of the circulating tissue factor, and use in the detection of coagulation diseases |
-
2011
- 2011-12-08 CN CN201110407416.1A patent/CN103163295B/en not_active Expired - Fee Related
Patent Citations (17)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002089657A2 (en) * | 2001-05-04 | 2002-11-14 | Biosite, Inc. | Diagnostic markers of acute coronary syndromes and methods of use thereof |
| CA2457775A1 (en) * | 2001-08-20 | 2003-02-27 | Biosite Incorporated | Diagnostic markers of stroke and cerebral injury and methods of use thereof |
| CN1613012A (en) * | 2001-11-05 | 2005-05-04 | 布赖汉姆妇女医院 | Soluble cd40l (cd154) as a prognostic marker of atherosclerotic diseases |
| WO2003103711A1 (en) * | 2002-06-07 | 2003-12-18 | Genentech, Inc. | Combinations of anti-tissue factor antibodies and anticoagulant and/or antiplatelet agents |
| WO2006105439A2 (en) * | 2005-03-30 | 2006-10-05 | Decode Genetics Ehf. | Susceptibility gene for myocardial infarction, stroke. and paod; methods of treatment |
| WO2007110104A1 (en) * | 2006-03-28 | 2007-10-04 | Universität Zürich | Drug-eluting clinical device |
| CN101568837A (en) * | 2006-08-07 | 2009-10-28 | 比奥-拉德巴斯德公司 | Method for the prediction of vascular events and the diagnosis of acute coronary syndrome |
| WO2008020013A2 (en) * | 2006-08-17 | 2008-02-21 | F. Hoffmann La-Roche Ag | Methods and means for determining platelet function and diagnosing platelet- related and cardiovascular disorders |
| CN101195659A (en) * | 2006-12-05 | 2008-06-11 | 复旦大学 | High anticoagulating active antihuman tissue factor monoclone antibody, preparation method and application thereof |
| CN101271108A (en) * | 2008-01-11 | 2008-09-24 | 广州益善生物技术有限公司 | Myocardial infarct early diagnosis liquid phase chip and method for producing the same |
| CN102159723A (en) * | 2008-07-17 | 2011-08-17 | 斯塔戈诊断公司 | Method for in vitro assay of the circulating tissue factor, and use in the detection of coagulation diseases |
| CN101327214A (en) * | 2008-07-18 | 2008-12-24 | 中国药科大学 | Medicine use of isoquinoline alkaloids as tissue factor inhibitor |
| CN101526535A (en) * | 2009-04-14 | 2009-09-09 | 河南省豫康生物工程技术有限公司 | Liquid phase chip for joint detection of multiple tumor markers and preparation method thereof |
| WO2011026017A1 (en) * | 2009-08-31 | 2011-03-03 | Abbott Laboratories | Biomarkers for prediction of major adverse cardiac events and uses thereof |
| CN102062735A (en) * | 2009-11-18 | 2011-05-18 | 江苏迈迪基因生物科技有限公司 | Biomarker detection method and diagnostic kit for acute coronary syndrome |
| CN101792746A (en) * | 2010-01-28 | 2010-08-04 | 中南大学 | Vascular peroxidase ELISA kit used for coronary disease diagnosis |
| CN102109517A (en) * | 2010-12-14 | 2011-06-29 | 邵棠 | Joint detection method of cardio-cerebral-vascular disease (CCVD) biomarkers and diagnostic kit |
Non-Patent Citations (4)
| Title |
|---|
| Development of an optimized multimarker strategy for early risk assessment of patients with acute coronary syndromes;Martin Möckel et al.;《Clinica Chimica Acta》;20080327;第393卷;103-109 * |
| The long-term prognostic value of multiple biomarkers following a myocardial infarction;Thrombosis Research et al.;《Thrombosis Research》;20080402;第123卷;60-66 * |
| 应用蛋 白芯片联合检测急性冠状动脉综合征患者;毛华 等;《中国循环杂志》;20061231;第21卷(第6期);426-430 * |
| 联合检测生化标志物在急性冠脉综合征患者早期危险分层中的意义;刘北;《中国优秀硕士学位论文全文数据库（医药卫生科技辑）》;20110415;E062-76 * |
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