CN101271108A - Myocardial infarct early diagnosis liquid phase chip and method for producing the same - Google Patents
Myocardial infarct early diagnosis liquid phase chip and method for producing the same Download PDFInfo
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Abstract
The invention discloses a liquid-phase chip for the early diagnosis of myocardial infarction, which mainly comprises coated microspheres, wherein, the coated microspheres comprise the microspheres which are coated by CK-MB capture antibodies, the microspheres which are coated by Mb capture antibodies, the microspheres which are coated by cTnI capture antibodies, the microspheres which are coated by GPBB capture antibodies, the microspheres which are coated by H-FABP capture bodies, detection antibodies which are respectively labeled by biotin, and a streptavidin phycoerythrin. The liquid-phase chip for the early diagnosis of the myocardial infarction which is provided by the invention has the advantages of high detection efficiency, a small number of the needed samples, strong specificity, high sensitivity, etc. At the same time, all the myocardial damage markers can be freely combined, thus the usage is convenient. Moreover, all the reactions are in the liquid-phase environment, which is better for keeping the natural conformation of the protein, and leads the reaction of a probe and an object to be detected to be faster and more complete, thus greatly improving the detection sensitivity and the linear range.
Description
Technical field
The present invention relates to the medical science vitro diagnostic techniques, the concrete liquid-phase chip of early diagnosis of acute myocardial infarction and preparation method thereof that relates to.
Background technology
Angiocardiopathy has developed into one of underlying cause of death of China people, wherein case fatality rate is the highest be acute myocardial infarction (Acute Myocardial Infarction, AMI).When acute myocardial infarction takes place, owing to the coronary artery acute infarct, causing lasting and serious myocardium acute ischemia, and the part cardiac muscle is necrosed, is a very serious type of coronary heart disease, and current case fatality rate can reach 10%～15%.Even acute stage survivor, because after the myocardial necrosis, ejection function is impaired, its prognosis at a specified future date is still had a strong impact on.The size of infarct size is all depended in incidence, the order of severity and the prognosis of acute myocardial infarction three big severe complications (as malignant arrhythmia, heart failure and cardiogenic shock).The initial stage of acute myocardial infarction, if can find and give timely processing as early as possible, can save more dying cardiac muscle, its prognosis can get a new look.The thromboembolism treatment of acute myocardial infarction also requires the patient is made early stage diagnosis.Because the time of vascular occlusion is longer, the cardiac muscle that can save is just fewer, generally advocates to carry out in 4～6 hours falling ill, and the time heals morning, and the coronary artery recanalization rate is high more.In general, only in AMI morbidity 6 hours, through clear and definite diagnosis and suitable treatment, the cardiac muscle cell of pathology reversibility just might occur and change.
The AMI diagnostic criteria of WHO suggestion has three, promptly clinical symptoms, cardiogram (ECG) is unusual and sero-enzyme unusually.Yet a large amount of clinical practice finds, has 25% patient AMI to fall ill approximately and do not have typical clinical symptoms in early days; About patient AMI of about 50% lacks Electrocardiographic special change.If rely on ECG change and clinical symptoms separately, the diagnostic accordance rate of AMI only is 75%.In this case, the detection of myocardial damage biochemical marker is particularly important when diagnosis AMI.The early diagnosis reagent of development high sensitivity, high specific is to improve the Diagnosis of Acute Myocardial Infarction rate at present and improve pressing for of result of treatment, reduction case fatality rate.The goldstandard that CK-MB Ceng Zuowei morbidity was diagnosed in 24 hours.But because the CK-MB specificity is poor slightly, many new detection indexs have appearred again in recent years.These detect index and are playing vital role aspect early diagnosis, state of illness monitoring, prognosis judgement and the minimizing complication of AMI.
The present invention has determined creatine kinase isozyme (CK-MB) according to a large amount of documents and materials, myoglobins (Myoglobin, Mb), cardiac muscle troponin I (cTnI), glycogen phosphorylase isoenzyme BB (GPBB), these five kinds of cardic fatty acid binding proteins (H-FABP) are expressed in the different courses of disease of acute myocardial infarction, and the myocardial damage mark that respectively has superiority of diagnostic sensitivity and specificity, utilize the liquid-phase chip platform to carry out the detection of running simultaneously of these five kinds of indexs.
(1) CK-MB is the clinical the most frequently used index that is used for the diagnosing acute miocardial infarction, the goldstandard that the Ceng Zuowei morbidity was diagnosed in 24 hours.CK-MB mainly is present in cardiac muscle cell's ectoplasm layer, is considered to enzyme the most special in the myocardial enzymes always.CK-MB increases in 4 hours in onset, reaches the peak in 16-24 hour, recovers normal in 3-4 days.But because CK-MB is not myocardium proprietary, specificity is poor; Simultaneously, this albumen occurs later in blood, so early diagnosis sensitivity is not high.The CK-MB retention time is shorter in addition, has brought certain difficulty also for the AMI early diagnosis.Have report to show, the specificity of CK-MB diagnosis AMI is 76.25%, and sensitivity is 50.25% (horse is strong, Li Yanmei etc., 2006).
(2) myoglobins (Mb) myoglobins is a kind of chromoprotein that is present among skeletal muscle and the cardiac muscle cell, because molecular weight is little, so can discharge into blood circulation from the ischemic injuries position rapidly when the minor injury.Mb is the impaired sensitive indicator of cardiac muscle, and myoglobin concentration obviously increases in the blood in miocardial infarction was shown effect 1.5 hours, occurs in 3 hours at least in advance than CK-MB, and peaking in the morbidity back in 4-8 hour, can recover normal in 12-24 hour.There were significant differences more with healthy group to detect myoglobins after morbidity in 1-3 hour.Therefore, myoglobin concentration is measured in the blood, can be used as AMI and rejects index, and early diagnosis is worth high.But the Mb specificity is not strong, can raise when muscle damage and kidney disease yet.Simultaneously, the Mb retention time is short, still has weak point, so can not use the sign of the absolute value of Mb as diagnosis AMI separately.
(3) the cardiac muscle troponin I troponin is the adjusting albumen of myocardial contraction, is present on the actin filament of myocardial contraction albumen, forms (TnT, TnI, TnC) by 3 subunits.More deep to the former two's research clinically.CTnT, cTnI are more special impaired indexs.Under the normal condition, cTnT and cTnI can not be released into blood by complete myocardial cell membrane, when the cardiac muscle cell is impaired, go into blood by the cell membrane of breakage.Many data show: the value of cTnI in the AMI early diagnosis is identical with cTnT, but specificity is higher than cTnT.CTnI and skeletal muscle no cross reaction are to find the longest index of retention time in blood at present, AMI diagnosis state of illness monitoring and unstable angina pectoris patient's prognosis are monitored play an important role.CTnI can detect in blood in 1 hour behind paresthesia epilepsy the earliest, and its susceptibility can reach 50% in the time of 3-4 hour.Its duration in blood is longer, continues to reach 7-14 days.There is report to think that its specificity of diagnosing AMI is up to 96.7%.Han Rui equality report, AMI morbidity 3 hours, the cTnI positive rate is 52%, and CK-MB just raise gradually at 4～8 hours.Wang Min etc. are reported in AMI morbidity 2.5 hours, and cTnI promptly raises, and CK-MB sees rising at 8 hours rears.1997, Zhang Ji south etc. are found the not thrombolytic group cTnI among the 61 routine AMI patients and its variation of CK-MB dynamic measurement: patient AMI fell ill back 3～6 hours, serum cTnI rises, the peak was at 18～24 hours, rise time and peak occur similar to CK-MB, after the pectoralgia the 5th day still very high, returned to reference range on the 7th day.This shows that the susceptibility of cTnI diagnosis AMI and time and CK-MB are approaching.But CK-MB reduced to normal after pectoralgia in 3 days rapidly, promptly cTnI curve after arriving the peak descend not as CB-MB steep like that.Some patient still increases at back 10 days cTnI of infraction, and this just provides strong foundation for the patient that AMI just was admitted to hospital after 48～72 hours in diagnosis.Simultaneously, cTnI and skeletal muscle no cross reaction, therefore, specificity is stronger than CK-MB.
(4) glycogen phosphorylase isoenzyme BB (GPBB) glycogen phosphorylase has 3 kinds of isodynamic enzymes: brain type BB, skeletal muscle type MM, liver type LL.GPBB also is present among the cardiac muscle cell except that being present in brain tissue, is the glycogenolytic key enzyme of cardiac muscle cell, is cardiac muscle cell's material composition the most responsive to ischemic, anoxic.Early stage when AMI outbreak, because the cardiac muscle cell is to the high susceptibility of ischemic, anoxic, ATP minimizing and ADP and inorganic phosphate increase in the cell, start and quicken decomposition of glycogen, owing to the change of permeability of cell membrane, the GPBB disperse enters in the blood in extracellular fluid simultaneously, (Mair J, 1998 obviously raise in pectoralgia was shown effect 4 hours; Ma Yili, 2003).GPBB is a kind of very promising diagnosis index, and the specificity of its diagnosis and susceptibility have caused domestic and international cardiovascular specialist's extensive concern all above 80%.The positive rate of GPBB in 4 hours is higher than 75%, and CK-MB 50% (Wang Guanyu, 2006 only; Gao Weihong, 2006; Wang Tao, 2006).Rabitzsch etc., to 116 routine healthy people, 14 routine stable angina pectoris patients, 107 routine atraumatic pectoralgias (45 routine AMI, 49 routine unstable anginas, 13 routine other diseases) patient measures CK, CK-MB, MB, cTnT and GPBB in the serum.The result shows that 116 routine normal healthy people GPBB are less than 7ng/ml.Pectoralgia begins that GPBB concentration begins to raise in the 2-4 hour blood plasma in back, reaches peak value after 8 hours, recovers normal after 40 hours.All indexs all raise during AMI, but raise the most obvious with GPBB.In addition, the unstable angina patient who changes with ST-T, GPBB raises, and other indexs are normal, and do not have ST-T variation person, and all index is normal.Illustrate that GPBB can indicate the generation of special mess heart stalk.
(5) cardic fatty acid binding protein (H-FABP) is the carrier of long-chain fatty acid, has the effect of regulating fatty acid metabolism.H-FABP is a large amount of exist with cardiac muscular tissue in, be soluble protein, molecular weight is little, is the early stage index of the myocardial damage of just using in recent years.Than hanging down 10 times in the cardiac muscle, content is also very low in kidney, liver and small intestine in skeletal muscle for H-FABP content.When cardiac muscle was impaired, H-FABP discharged from the cardiac muscle cell, entered blood circulation rapidly, and it can not only find the situation of myocardial damage rapidly, the amount of reflecting myocardium infarct, and can infer the scope of myocardial infarction.Many studies confirm that, during early diagnosis AMI, H-FABP has superior ageing and susceptibility than CK-MB, Mb.Diagnose total susceptibility of early stage AMI, H-FABP is 78%, and CK-MB is 57%, and Mb is 53% (Glatz JF, 1998); Diagnose super acute stage of AMI, the susceptibility of H-FABP is 93.1%, and specificity is 64.3%; Total susceptibility of H-FABP diagnosis AMI was to be 84.38% (TanakaT, Sohmiya K, et al, 2006 in 64.29%, 6 hour in 3 hours; Chen L, Guo X, et al, 2004).Simultaneously, H-FABP can detect the small cardiac damage of CK-MB when having no to change, and can early monitoring AMI (Glatz JF, 1998) for the second time.
Because various myocardium marks have different expression in the course of disease of AMI, the diagnostic sensitivity of various marks and specific difference make that the value of each index in judging AMI is different simultaneously.If can detect simultaneously, sensitivity, specificity and the accuracy of diagnosis AMI will improve greatly.
Traditional myocardium marker detection method mainly contains radioimmunology, euzymelinked immunosorbent assay (ELISA) and electrochemiluminescence immunization etc.Yet the each test of these methods can only obtain a kind of concentration of mark.Simultaneously, traditional immunologic detection method is limited by sensitivity, and therefore there are defectives such as testing result is inaccurate in the mark for some low concentrations.
Summary of the invention
The objective of the invention is the method susceptibility that detects at present acute myocardial infarction and specificity is not high and primary first-order equation can only detect a kind of defective of myocardium mark, the liquid-phase chip of the myocardial infarct early diagnosis of the multiple myocardial damage mark of a kind of synchronous detection is provided.
The technical scheme that realizes above-mentioned purpose is as follows:
A kind of liquid-phase chip of myocardial infarct early diagnosis includes
1) wrap by microballoon:
Contain and wrap respectively by the microballoon of CK-MB capture antibody, bag is by the microballoon of Mb capture antibody, and bag is by the microballoon of cTnI capture antibody, and bag is by the microballoon of GPBB capture antibody, with bag by the microballoon of H-FABP capture antibody, above-mentioned microballoon has different colours coding respectively;
2) biotin labeling detects antibody: contain the detection antibody of using biotin labeled CK-MB, Mb, cTnI, GPBB and H-FABP respectively; With
3) streptavidin phycoerythrin.
Be preferably, every kind of bag be hunted down the working concentration of microballoon of antibody be 110-140/μ l, more preferably 120/μ l; The working concentration of every kind of biotin labeling detection antibody is 1-3ug/ml, more preferably 2ug/ml.
Liquid-phase chip technology also is the streaming fluorescent technique, this technology by a kind of microballoon as reaction carriers, microballoon is made with polystyrene material, diameter 5.6um, the surface has pendant carboxylic group can supply chemical coupled usefulness, and the carboxyl that biomacromolecules such as antigen, antibody can be by amino and microsphere surface is by chemical reaction covalent bond (promptly wrapping by process).In the microballoon manufacture process, add infrared and two kinds of fluorescent dyes of far infrared, microballoon is encoded, can distinguish the microballoon of hundreds of different coding according to the difference of two kinds of dyestuff blending ratios.During use, the capture antibody with CK-MB, Mb, cTnI, GPBB and H-FABP wraps respectively by on the microballoon of different colours coding earlier, uses the detection antibody of biotin labeling CK-MB, Mb, cTnI, GPBB and H-FABP simultaneously respectively.Bag is mixed by five kinds of good microballoons, be suspended in liquid phase, add sample again, in suspension on the microballoon in the capture antibody of mark and the sample some epi-positions of relevant detection thing combine different in naturely, adding biotin labeled detection antibody afterwards combines with another epitope specificity of respective detection thing in the sample, back adding fluorescent material---the streptavidin of phycoerythrin mark reacts completely, because streptavidin phycoerythrin (SA-PE) can combine with the biotin high degree of specificity, therefore form five kinds of compounds of " microballoon-capture antibody+thing to be detected+biotin labeled detection antibody+SA-PE " in the reaction system at last, with the microballoon is carrier, detect by Luminex series liquid-phase chip analytical instrument, read the color numbers of microballoon and the fluorescent value of SA-PE.The microballoon color numbers can be distinguished test item, SA-PE fluorescent value and each detect substrate concentration and are proportionate, by measuring CK-MB, Mb, cTnI, GPBB and the fluorescent value of H-FABP standard items under variable concentrations, can obtain each and detect index standard items concentration-fluorescent value typical curve and typical curve equation.Test serum pattern detection gained fluorescent value substitution typical curve equation can be tried to achieve the amount of CK-MB, Mb, cTnI, GPBB and H-FABP in the sample to be tested respectively.
Because institute responds and all is in liquid phase environment, more helps keeping the native conformation 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 above-mentioned myocardial infarct early diagnosis liquid phase chip.A kind of method for preparing myocardial infarct early diagnosis liquid phase chip mainly may further comprise the steps:
(1) corresponding capture antibody bag is by microballoon:
-with after the activation of 50 μ L microballoons, 〉=8000g is centrifugal;
-inhale and to abandon supernatant, microballoon is resuspended in the solution of MES (2[N-Morpholino] ethanesulfonic acid) of the 230-280 μ L 50mM of pH5.0, the about 20s of vortex vibration, ultrasonic about 20s, after with microballoon in 〉=8000g, centrifugal 1-2min; Repeat this step;
-inhale and to abandon supernatant, microballoon is resuspended in the solution of MES of 100 μ L 50mM pH5.0 the about 20s of vortex vibration, ultrasonic about 20s;
-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;
-usefulness vortex oscillator mixing, room temperature lucifuge vibration 1.5-2.5hr;
-coupling the speed of the microballoon behind the antibody with 〉=8000g, centrifugal 1-2min;
-inhale and abandon supernatant, microballoon is resuspended in the 500 μ L PBS-TBN solution the about 20s of vortex vibration, the about 20s of sonic oscillation;
-room temperature lucifuge vibration 30min; Again in 〉=8000g, centrifugal 1-2min;
-inhale and abandon supernatant, microballoon is resuspended in the 1mL PBS-TBN solution the about 20s of vortex vibration, ultrasonic about 20s; Microballoon 〉=8000g, centrifugal 1-2min; Repeat this step once;
-inhale and abandon supernatant, microballoon is resuspended in the 500-1000 μ L PBS-TBN solution;
-every kind bag is placed on 2-8 ℃ of lucifuge by good microballoon by luminex instrument counting and preserves separately, during use, needs equal proportion to mix according to detecting, and makes the concentration of every kind of capture antibody coupling microballoon in the mixed liquor be 120/μ l;
(2) 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, be considered as target volume;
-dissolve the NHS-Biotin reactant liquor of configuration 10mg/ml with DMSO (Dimethyl sulfoxide);
In reaction tube, added respectively in-1: 100 in molar ratio and detect antibody and NHS-Biotin reactant liquor;
-add volume and be 1/10 the NaHCO of the pH8.9 of target volume
3Solution;
The PBS of-adding pH7.4 mends to target volume;
-wrap aluminium foil, reaction tube is placed on the oscillator, lucifuge was hatched 3-5 hour in 25 ℃ of constant temperature ovens, and rotating speed is 800rpm-1000rpm;
-reactant liquor is gone in the dialysis cassette, dialysed overnight in the PBS damping fluid is to remove unreacted NHS-Biotin;
-every kind of as required that mark is good when using detection antibody equal proportion is mixed.
The step of described activation microballoon is as follows:
-usefulness vortex oscillator or ultrasound wave suspension microballoon;
-get the centrifugal 1-2min of 50 μ L microballoons;
-remove supernatant, microballoon is resuspended in the distilled water of 100 μ L, with vortex oscillator or the about 20s of ultrasound wave suspension microballoon, the centrifugal 1-2min of 〉=8000g;
-inhale and abandon supernatant, add the phosphate buffer (pH6.2) of 80 μ L, the about 20s of vortex vibration, the about 20s of ultrasound wave suspension microballoon;
-add 10 μ L 50mg/mL Sulfo-NHS, use vortex oscillator mixing lightly;
-add 10 μ L 50mg/mL EDC, use vortex oscillator mixing lightly;
-room temperature lucifuge leaves standstill 20min, every 10min vortex oscillator mixing lightly.
The inventive method is in conjunction with the characteristic and the highly sensitive characteristic of double antibody sandwich method of the parallel detection of many indexs of liquid-phase chip platform high flux, 5 kinds of myocardial damage marks of synchronous detection, can improve the accuracy rate of early diagnosis of acute myocardial infarction, state of illness monitoring and the prognosis judgement for acute myocardial infarction simultaneously provides foundation.
But multinomial myocardial damage mark parallel detection liquid-phase chip primary first-order equation disclosed in this invention is finished the detection by quantitative of multiple myocardial damage mark simultaneously, thereby reaches the early stage accurately diagnosis to acute myocardial infarction.Multinomial myocardial damage mark parallel detection liquid-phase chip provided by the present invention also has the detection efficiency height, and required sample size is few, high specificity, 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 a large amount of experimental basis, are the parameter value of preparation process the best.
Description of drawings
Fig. 1 is the typical curve synoptic diagram that detects CK-MB;
Fig. 2 is the typical curve synoptic diagram that detects Mb;
Fig. 3 is the typical curve synoptic diagram that detects cTnI;
Fig. 4 is the typical curve synoptic diagram that detects GPBB;
Fig. 5 is the typical curve synoptic diagram that detects H-FABP.
Embodiment
The preparation and the detection of antigens of embodiment 1 myocardial infarct early diagnosis liquid phase chip
The monoclonal antibody of capture antibody of the present invention for corresponding combining respectively with CK-MB, Mb, cTnI, GPBB and H-FABP specificity; Monoclonal antibody or the polyclonal antibody of described detection antibody for combining with CK-MB, Mb, cTnI, GPBB and H-FABP specificity respectively.
The capture antibody of " CK-MB, Mb, cTnI, GPBB and H-FABP " that present embodiment is used and detection antibody are available from abcam company.
In the present embodiment, the prescription of described various solution is as follows:
1.50mM MES damping fluid (pH5.0) prescription (250ml):
| Reagent | The source | Final concentration | The consumption of every 250ml |
| MES (2[N-Morpholino] ethanesulfonic acid) | Sigma M-2933 | 0.05M | 2.44g |
| 5MNaOH | Fisher SS256-500 | --- | 5 |
2.PBS prescription:
| Reagent | Catalog number (Cat.No.) | Final concentration | The consumption of every 1L |
| PBS | Sigma-3813 | 138mM NaCl 2.7mM KCl | 1 bag |
3.PBS-TBN prescription (be to contain 0.1%BSA among the PBS, 0.02%Tween-20,0.05%Na3N, pH7.4)
| Reagent | Catalog number (Cat.No.) | Final concentration | The consumption of every 1L |
| PBS pH7.4 | Sigma P-3813 | 138mM NaCl 2.7mM KCl | 1 bag |
| BSA | Sigma A-9647 | 0.1％ | 1g |
| Tween-20 | Sigma P-9416 | 0.02％ | 0.2ml |
| Sodium Azide | Sigma S-8032 | 0.05％Azide | 500mg |
1. myocardial infarct early diagnosis liquid phase chip kit includes:
1) 5-plex wraps by microballoon: contain and wrap respectively by No. 20 microballoons of CK-MB capture antibody, bag is by the couplet of No. 34 microballoons of Mb capture antibody, bag is by No. 38 microballoons of cTnI capture antibody, bag is by No. 51 microballoons of GPBB capture antibody, and bag is by No. 53 microballoons of H-FABP capture antibody.
2) the 5-plex biotin labeling detects antibody: contain the detection antibody of using biotin labeled CK-MB, Mb, cTnI, GPBB and H-FABP respectively;
3) the streptavidin phycoerythrin (SA-PE, 10ug/ml); Also have according to prior art is supporting
4) analysis buffer;
5) 5-plex standard items;
6) control liquid I;
7) control liquid II;
8) serum matrix liquid;
9) seal film;
10) filter plate.
2. prepare above-mentioned liquid phase chip reagent box, include following steps:
(1) by the composition of mentioned reagent box, every kind of capture antibody bag is by corresponding microballoon, and the preparation method is identical:
-choose No. 20, No. 34, No. 38, No. 51, No. 53 microballoons (U.S. Luminex company) respectively, with vortex oscillator or ultrasound wave suspension microballoon, about 20s;
-respectively get 50 μ L microballoons in the centrifuge tube of 1.5ml, 8, the centrifugal 2min of the above speed of 000g;
-remove supernatant, microballoon is resuspended in the distilled water of 100 μ L, with vortex oscillator or the about 20s of ultrasound wave suspension microballoon, 8000g (or more than) the centrifugal 2min of speed;
-inhale and abandon supernatant, add the phosphate buffer (pH6.2) of 80 μ L, the about 20s of vortex vibration, the about 20s of ultrasound wave suspension microballoon;
-add 10 μ L 50mg/mL Sulfo-NHS, use vortex oscillator mixing lightly;
-add 10 μ L 50mg/mLEDC, use vortex oscillator mixing lightly;
-room temperature lucifuge leaves standstill 20min, every 10min vortex oscillator mixing lightly;
Microballoon after the-activation, 〉=8000g, centrifugal 2min;
-inhale and to abandon supernatant, microballoon is resuspended in the solution of MES (pH5.0) of 250 μ L 50mM the about 20s of vortex vibration, ultrasonic about 20s; Microballoon 〉=8000g, centrifugal 1-2min; Repeat this step once;
-inhale and to abandon supernatant, microballoon is resuspended in the solution of MES (pH5.0) of 100 μ L 50mM the about 20s of vortex vibration, ultrasonic about 20s;
-in the microballoon that suspends, add 1 μ g antibody, with MES (pH5.0) solution of 50mM cumulative volume is mended to 500 μ L;
-usefulness vortex oscillator mixing, room temperature lucifuge vibration 2hr;
Microballoon behind the-coupling antibody is with the speed of 〉=8000g, centrifugal 1-2min;
-inhale and abandon supernatant, microballoon is resuspended in the 500 μ L PBS-TBN solution the about 20s of vortex vibration, ultrasonic about 20s;
-room temperature lucifuge vibration 30min;
-microballoon 〉=8000g, centrifugal 1-2min;
-inhale and abandon supernatant, microballoon is resuspended in the 1mL PBS-TBN solution the about 20s of vortex vibration, ultrasonic about 20s; Microballoon 〉=8000g, centrifugal 1-2min; Repeat this step once;
-inhale and abandon supernatant, microballoon is resuspended in the 600 μ L PBS-TBN solution;
-Bao is counted by the luminex instrument by good microballoon;
-Bao is placed 2-8 ℃ to keep in Dark Place by good microballoon, and the microballoon of every kind of antibody coupling is preserved separately, during use, selects equal proportion to mix according to test item.
(2) by the composition of mentioned reagent box, every kind of biotin labeling that detects antibody:
-calculate reaction system according to protein concentration;
-according to the volume of protein concentration calculating antibody solution dilution, be considered as target volume (V) to 1mg/ml;
-configuration NHS-Biotin reactant liquor (with the DMSO dissolving, making its concentration is 10mg/ml);
In reaction tube, added respectively in-1: 100 in molar ratio and detect antibody and NHS-Biotin (10mg/ml) reactant liquor;
The NaHCO of-adding 1/10 target volume
3(pH8.9) solution;
-add PBS (pH7.4) to mend to target volume;
-wrap aluminium foil, reaction tube is placed on the oscillator, lucifuge is hatched 4h in 25 ℃ of constant temperature ovens, and rotating speed is 900rpm;
-reactant liquor is gone in the dialysis cassette, dialysed overnight in the PBS damping fluid is to remove unreacted NHS-Biotin;
-mensuration protein concentration.
3. be used for detecting, comprise the steps:
1) uses preceding all reagent that take out earlier, place balance to room temperature.
2) dilution of standard items: each standard items is established 6 dilutabilitys, and equal proportion is mixed then, and making each contented final concentration is required concentration.Each manages dilution process and theoretical concentration (with the expection concentration in the following table):
Attention: all must be with the dilution that just can be used for next concentration behind the thorough mixing of vortex mixed instrument after each concentration standard product has diluted.Blending process avoids producing foam.
3) 96 orifice plate layouts are set, determine the position of standard items, quality-control product, testing sample and blank well on 96 orifice plates.The order of considering instrument readings be according to from the 1st be listed as the 12nd row, capable from A to the vertical reading of the capable order of H, when 96 orifice plate layouts are set, should follow from the 1st be listed as the 12nd row, capable from A to the capable series arrangement of H.
4) according to the 96 orifice plate layouts that set, every hole adds 25 μ l analysis buffer, adds 25ul standard items, quality-control product more respectively in hole separately, and blank well adds the 25ul analysis buffer.
5) take out the capture antibody coupling microballoon at five kinds of myocardium marker detection of above-mentioned preparation respectively, with vortex mixed instrument mixing 30 paper money, sonicated 30 seconds is mixed by equal proportion, makes the microballoon concentration of every kind of capture antibody coupling in the mixed liquor be 120/μ l.The mixing suspension 25 μ l of five kinds of capture antibody coupling microballoons that add in every hole.Bag should be used preceding mixing facing by microballoon, and should use immediately behind the mixing, can precipitate otherwise place microballoon of a specified duration again.
6) seal all application of sample apertures with sealing film, and encase 96 orifice plates with lucifuge with masking foil, 25 ℃ are placed on the microwell plate oscillator with the concussion of 600 rotary speeds, hatched 60 minutes.
7) hatching every hole after finishing adds the mixed liquor of the biotin labeled detection antibody of the above-mentioned preparation of 25ul (wherein said five kinds of biotin labeled detection antibody mixes by equal proportion in advance, make every kind of detection antibody ultimate density reach 2ug/ml, shake up), seal all application of sample apertures with sealing film, and encase 96 orifice plates with lucifuge with masking foil, 25 ℃ are placed on the microwell plate oscillator with the concussion of 600 rotary speeds, hatched 60 minutes again.
8) hatch that every hole adds 25ul streptavidin phycoerythrin after finishing, seal all application of sample apertures with sealing film, and encase 96 orifice plates with lucifuge with masking foil, 25 ℃ are placed on the microwell plate oscillator and shake with 600 rotary speeds, hatch 30 minutes again.
9) on Luminex series liquid-phase chip analyser, read the result.Instrument is the drawing standard curve automatically, and calculates the measured value of testing sample.
4. interpretation:
See also table 1-table 3 and Fig. 1-Fig. 5.
Table 1
Table 2
Table 3
Myocardial infarct early diagnosis liquid phase chip disclosed in this invention only needs the serum sample of 25 μ L to get final product the detection by quantitative that primary first-order equation is finished five kinds of myocardial damage marks simultaneously.Simultaneously, with radio-immunity, methods such as chemiluminescence detection and enzyme linked immunological absorption are compared, and liquid-phase chip platform sensing range is wideer, and sensitivity is higher, and repeatability is better.Because institute responds and all is in liquid phase environment, more helps keeping the native conformation 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.
By the pairing to CK-MB, Mb, cTnI, GPBB and H-FABP capture antibody and detection antibody, we have found the optimum antibody that is applicable to these five kinds of myocardial damage marker detection right.By the analysis of typical curve, can see that myocardial infarct early diagnosis liquid phase chip provided by the present invention has very high detection sensitivity and specificity to the detection of myocardial damage mark.Wherein, the linearity of CK-MB, Mb, GPBB and H-FABP typical curve R in the concentration range of 97.7pg/ml～100ng/ml
2〉=0.99, lowest detectable limit can reach 97.7pg/ml.The typical curve of cTnI is R in the concentration range of 9.77pg/ml～10ng/ml
2〉=0.99, lowest detectable limit can reach 9.77pg/ml, and the relative error of the measured concentration of each standard point and expection concentration is no more than 8%.Therefore, myocardial infarct early diagnosis liquid phase chip provided by the invention can detect the variation of myocardial damage mark denier in the serum, and can disposable detection in early diagnosis of acute myocardial infarction, be worth different myocardium mark for five kinds, thereby improve the acute myocardial infarction accurate rate of diagnosis, state of illness monitoring and the prognosis judgement for acute myocardial infarction simultaneously provides important evidence.
Claims (5)
1. a myocardial infarct early diagnosis liquid phase chip is characterized in that, mainly includes:
1) wraps by microballoon: contain and wrap respectively by the microballoon of CK-MB capture antibody, bag is by the microballoon of Mb capture antibody, and bag is by the microballoon of cTnI capture antibody, and bag is by the microballoon of GPBB capture antibody, with bag by the microballoon of H-FABP capture antibody, above-mentioned microballoon has different colours coding respectively;
2) biotin labeling detects antibody: contain the detection antibody of using biotin labeled CK-MB, Mb, cTnI, GPBB and H-FABP respectively; With
3) streptavidin phycoerythrin.
2. myocardial infarct early diagnosis liquid phase chip according to claim 1 is characterized in that, the be hunted down working concentration of microballoon of antibody of every kind of bag is 110-140/μ l; The working concentration of every kind of biotin labeling detection antibody is 1-3ug/ml.
3. myocardial infarct early diagnosis liquid phase chip according to claim 2 is characterized in that, the be hunted down working concentration of microballoon of antibody of every kind of bag is 120/μ l; The working concentration of every kind of biotin labeling detection antibody is 2ug/ml.
4. method for preparing the described myocardial infarct early diagnosis liquid phase chip of claim 1 mainly may further comprise the steps:
(1) every kind of corresponding capture antibody bag is by microballoon:
-with after the activation of 50 μ L microballoons, 〉=8000g is centrifugal;
-inhale and to abandon supernatant, microballoon is resuspended in the solution of MES of 230-280 μ L 50mM of pH5.0, the about 20s of vortex vibration, ultrasonic about 20s, after with microballoon in 〉=8000g, centrifugal 1-2min; Repeat this step;
-inhale and to abandon supernatant, microballoon is resuspended in the solution of MES of 100 μ L 50mM pH5.0 the about 20s of vortex vibration, ultrasonic about 20s;
-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;
-usefulness vortex oscillator mixing, room temperature lucifuge vibration 1.5-2.5hr;
-coupling the speed of the microballoon behind the antibody with 〉=8000g, centrifugal 1-2min;
-inhale and abandon supernatant, microballoon is resuspended in the 500 μ L PBS-TBN solution the about 20s of vortex vibration, the about 20s of sonic oscillation;
-room temperature lucifuge vibration 30min; Again in 〉=8000g, centrifugal 1-2min;
-inhale and abandon supernatant, microballoon is resuspended in the 1mL PBS-TBN solution the about 20s of vortex vibration, ultrasonic about 20s; Microballoon 〉=8000g, centrifugal 1-2min; Repeat this step once;
-inhale and abandon supernatant, microballoon is resuspended in the 500-1000 μ L PBS-TBN solution;
-every kind bag is placed on 2-8 ℃ of lucifuge by good microballoon by luminex instrument counting and preserves separately, during use, needs equal proportion to mix according to detecting, and makes the concentration of every kind of capture antibody coupling microballoon in the mixed liquor be 110-140/μ l;
(2) 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, be considered as target volume;
-dissolve the NHS-Biotin reactant liquor of configuration 10mg/ml with DMSO;
In reaction tube, added respectively in-1: 100 in molar ratio and detect antibody and NHS-Biotin reactant liquor;
-add volume and be 1/10 the NaHCO of the pH8.9 of target volume
3Solution;
The PBS of-adding pH7.4 mends to target volume;
-wrap aluminium foil, reaction tube is placed on the oscillator, lucifuge was hatched 3-5 hour in 25 ℃ of constant temperature ovens, and rotating speed is 800rpm-1000rpm;
-reactant liquor is gone in the dialysis cassette, dialysed overnight in the PBS damping fluid is to remove unreacted NHS-Biotin;
-every kind of as required that mark is good when using detection antibody equal proportion is mixed, and makes every kind of detection antibody ultimate density reach 1-3ug/ml.
5. preparation method according to claim 4 is characterized in that: the step of described activation microballoon is as follows:
-usefulness vortex oscillator or ultrasound wave suspension microballoon;
-get the centrifugal 1-2min of 50 μ L microballoons;
-remove supernatant, microballoon is resuspended in the distilled water of 100 μ L, with vortex oscillator or the about 20s of ultrasound wave suspension microballoon, the centrifugal 1-2min of 〉=8000g;
-inhale and abandon supernatant, add the phosphate buffer (pH6.2) of 80 μ L, the about 20s of vortex vibration, the about 20s of ultrasound wave suspension microballoon;
-add 10 μ L 50mg/mL Sulfo-NHS, use vortex oscillator mixing lightly;
-add 10 μ l 50mg/mL EDC, use vortex oscillator mixing lightly;
-room temperature lucifuge leaves standstill 20min, every 10min vortex oscillator mixing lightly.
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