CN101246163B - Pyemia early diagnosis liquid phase chip and method for producing the same - Google Patents
Pyemia early diagnosis liquid phase chip and method for producing the same Download PDFInfo
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Abstract
The invention discloses a sepsis early diagnosis liquid chip which mainly includes the following components: microsphere coated with PCT capturing antibody, microsphere coated with CRP capturing antibody, microsphere coated with IL-6 capturing antibody, microsphere coated with Neopterin capturing antibody, these microspheres have codes with different colors; biotin-labeled test antibody; avidin-linked phycoerythrin. The invention sepsis early diagnosis liquid chip has advantages of high detection efficiency, a small amount of sample, high specificity and high sensitivity. And at the same time, the invention can test four kinds of specific markers of sepsis simultaneously, improve sensitivity and specificity of sepsis early diagnosis, distinguish sepsis caused by bacterial and viral, judge severity and poor prognosis of sepsis and monitor continuously for judging reflection of patient to certain kind of treatment.
Description
Technical field
The present invention relates to the medical science vitro diagnostic techniques, the concrete pyemia early diagnosis liquid phase chip and preparation method thereof that relates to.
Background technology
Pyemia (Sepsis) is a common complication after severe infections, serious wound (burning) wound, shock, the operation; Be owing to microorganism (like bacterium, virus, fungi, parasite etc.) is invaded the fierce systemic inflammatory response that human body brings out, and tissue is had one group of clinical manifestation of abrasive pathophysiological process.Severe sepsis can cause septic shock, multiple organ dysfunction (MODS), and human health and economic development are constituted threat greatly and challenge, has become the main cause of non-deaths from heart disease.
Current, pyemia exists the three high phenomenons that morbidity rate height, case fatality rate are high, medical expense is high.According to statistics, pyemic morbidity rate is about 0.3% of total population, and the whole world etesian total case load about 1,800 ten thousand examples are equivalent to the summation of Denmark, Finland, Ireland and Norway's population, and only the annual number of patients of the U.S. just has 750,000 examples.Though China does not have definite statistics, calculate be not less than annual 4000000 examples.Moreover, data shows that also the pyemia case load just increases with annual 1.5% ratio, expects the year two thousand fifty, and U.S. population increases about 30% (reaching 400,000,000), but the pyemia case load will increase more than 1 times and (reach 1,600,000 examples).Pyemic case fatality rate is about 28%～50%; Average 40%, the domestic incomplete report data of China is close therewith, and whole world death toll surpasses 1.4 ten thousand examples every day; The U.S. reaches 21.5 ten thousand examples every year, still has suitable death not count and is attributed to protopathy.Pyemia produces serious threat to human health, for economic development brings great burden.In the U.S., each patient's mean treatment expense is about 2.2 ten thousand dollars, expensive nearly 20,000,000,000 dollars of year, expensive nearly 10,000,000,000 dollars of Europe year.China does not have the definite data of this respect, estimates that by rule of thumb each patient's mean treatment expense can not be lower than the U.S., and therefore treatment is total expensive considerable.
Accurately and timely diagnosing early stage pyemia, instructing pyemic treatment and judging prognosis is the thorny difficult problem that present ICU (CICU) is faced.Although modern medical service technology and Intensive Care Therapy level obviously improve, the case fatality rate of severe sepsis, septic shock or MODS complication such as (MODS) is still up to 50%～70%.At present clinically severe sepsis or MODS are still lacked effective warning index and monitoring method.Because urgent patient's systemic inflammatory reaction; Infect with non-infection clinical manifestation and obscure each other; Pyemic traditional diagnosis index such as body temperature (TEMP), white blood cell count(WBC) (WBC), erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) etc. all are nonspecific, and reliability is not strong.
The current experiments diagnostic method comprises that early stage pathogen diagnosis and serological index detect.The method bacterial detection of early stage pathogen utilization bacterium 16srRNA gene magnification, thus judge whether the existence of bacterium reaches the kind of common bacteria.Advantage such as that this technology has is quick, highly sensitive, do not receive that antibiotic therapy influences.Its weak point is false positive to occur, and price is expensive at present.Serological index detects mark commonly used at present has Procalcitonin (PCT), c reactive protein (CRP), interleukin-6 (IL-6) and mopterin etc.As an identification system property inflammatory reaction syndrome (SIRS) and a pyemic index, and there is relevant commercial reagent production marketing in some European countries with PCT, but also have the people that this index is held differing views, and think that it is too responsive.CRP also is used as diagnosis and monitors a pyemic parameter.Regrettably to exist specificity all the time not strong for the unique identification thing, positive rate is low wait not enough, can only be as the reference of sepsis diagnosis and monitoring, can not be as the real specific index of diagnosis of sepsis disease.
The PCT specificity is good, and stable chemical performance predict that in early days pyemia and prognosis thereof have big potentiality, but its susceptibility is poor slightly, cooperates good mopterin and the c reactive protein of susceptibility, can further improve its forecasting efficiency.The rising of IL-6 raises than these acute phase proteins and occurs more early during pyemia.These indexs of joint-detection can improve the sensitivity and the accuracy of detection, can be used for pyemia is carried out early diagnosis and judging prognosis.
The present invention is according to amount of literature data; Confirm that Combined application Procalcitonin (PCT), c reactive protein (CRP), interleukin-6 (IL-6) and this four indices of mopterin come comprehensive assessment pyemia and prognosis thereof; And then carry out early intervention in time, prevent pyemic further deterioration and improve prognosis of patients.
(1) c reactive protein (CRP): CRP found in the pneumonia disease human serum in nineteen thirty, can react with pneumococcal polysaccharide segment C and precipitate.Normal population CRP level is below 10mg/L, and sepsis patient CRP in infection back 4～6h takes place promptly to begin to raise, and 36～50h peaks, and peak value can reach hundreds of times of normal reference value, infects the hurried decline of elimination its content of back, can return to normal in 1 week.CRP does not have remarkable rising when virus infections, can be used for distinguishing the inflammation of infective inflammation and other types, and detection sensitivity is 92.1%, and specificity is 82.1%.CRP CONCENTRATION DISTRIBUTION in the whole process of disease surpasses down normal value, but irrelevant with the degree of being in a bad way.
(2) Procalcitonin (PCT): PCT is found in 1986 the earliest, is the preceding peptide material of calcitonin (CT) of no hormonal activity.Under the normal condition in the blood PCT less than 0.5ng/ml or detect less than, infect the back and rise rapidly, can in blood, detect about 2 hours, reached peak value in 12～24 hours, can rise to more than 2000 times of normal value during severe infections.
PCT can raise at systemic inflammatory reaction (after 2-3 hour) in early days, has early diagnosis and is worth.PCT concentration does not increase or slightly increases when diseases such as local infection, virus infections, chronic nonspecific inflammation, carcinous heating, graft host rejection or LADA; Only when serious whole body systemic infection, just obviously increase; Have high degree of specificity, can be used for the antidiastole of various clinical settings.PCT can monitor pyemic incidence and development process, and its PC and infection and pyemia disease severity and active level are directly proportional.PCT can confirm prognosis and curative effect, and it is the bad strong sign of prognosis that its level continues to increase, and level decline indicates the prognosis bona carries out blood plasma PCT assay continuously, helps the generation of early prediction and identification MODS.PCT has excellent specificity at differentiation bacterial infection and non-infectious aspect of inflammation, reaches 81%; Severe sepsis and septic shock had the specificity up to 100%.
(3) interleukin-6 (IL-6): IL-6 is the core member of cell factor, is found in 1980.IL-6 metabolism in blood is slower, detects easily, thereby is used as pyemic important symbol thing.The rising of IL-6 occurs more early than other acute phase protein rising, and therefore, IL-6 has the early diagnosis advantage in the detection blood.Simultaneously, IL-6 has susceptibility and specificity preferably, is respectively 81.1% and 78.9%.Many researchs show that pyemia patient blood IL-6 level obviously increases, and the amplitude that increases is relevant with the pyemic order of severity, septic shock and poor prognosis.The IL-6 level continues to increase patient's multi-organ function, and (MODS) and mortality ratio obviously do not increase entirely.
(4) mopterin: mopterin (neopterin) is the GTP metabolism is derived in the body the low-molecular-weight pyridine compounds of talking endlessly.The pathologic process that mopterin and pyemia etc. infect is closely related, has shock tendency person serum levels obviously to change in 24 hours, to the significant prediction significance of having of septic shock.Susceptibility, specificity and the accuracy of mopterin diagnosis MODS are 83.0%, 88.5% and 86.6%, and its positive predictive value and negative predictive value then are 79.6% and 90.6%.The content of METHOD FOR CONTINUOUS DETERMINATION mopterin has the generation that helps predict early and discern MODS, for early intervention lays the foundation.Pyemic generation is closely related with development after the lasting rising of mopterin and the serious burn, and burn can cause that the serum mopterin raises.Mopterin can be predicted neonate's pyemia, and can distinguish bacterial infection and virus infections, and continuous monitoring can the early diagnosis NEC.
Traditional antigen detection method mainly contains radioimmunology, ELISA and electrochemiluminescence immunization etc.Yet the each test of these methods can only obtain a kind of concentration of determinand.Simultaneously, traditional immunologic detection method is limited by sensitivity, and therefore there are defectives such as testing result is inaccurate in the haemocyanin material for some low concentrations.
Summary of the invention
The objective of the invention is to the susceptibility and the specificity of present pyemic detection, prognosis and curative effect appraisal procedure not high; Can not distinguish the defective that bacterium and virus infections and primary first-order equation can only detect a kind of mark, a kind of pyemia early diagnosis liquid phase chip that can four kinds of mark joint inspections is provided.
The technical scheme that realizes above-mentioned purpose is following:
A kind of pyemia early diagnosis liquid phase chip includes
1) encapsulate microballoon:
Contain the microballoon that has encapsulated the PCT capture antibody respectively, encapsulated the microballoon of CRP capture antibody, encapsulated the microballoon of IL-6 capture antibody and encapsulated the microballoon of new dish purine capture antibody, above-mentioned microballoon has the different colours coding respectively;
2) biotin labeling detects antibody: contain respectively the detection antibody with biotin labeled PCT, CRP, IL-6 and new dish purine;
3) streptavidin phycoerythrin.
Be preferably, every kind of working concentration that encapsulates the microballoon of capture 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, and as reaction carriers, process with polystyrene material by microballoon by a kind of microballoon for this technology, diameter 5.6um, and the surface has pendant carboxylic group can supply chemical even logotype.Biomacromolecule such as antigen, antibody can be through amino and microsphere surface carboxyl through chemical reaction covalent bond (promptly encapsulating 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, earlier the capture antibody with PCT, CRP, IL-6 and new dish purine encapsulates respectively on the microballoon of different colours coding, simultaneously respectively with the detection antibody of biotin labeling PCT, CRP, IL-6 and new dish purine.Four kinds of microballoons that encapsulate are mixed; Be suspended in liquid phase; Add sample to be detected again, some epi-positions of relevant detection thing combine different in naturely in the capture antibody of microballoon marked and the sample in suspension, add biotin labeled detection antibody afterwards and combine 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, so four kinds of compounds of formation " microballoon-capture antibody+thing to be detected+biotin labeled detection antibody+SA-PE " at last in the reaction system, be carrier with the microballoon; Detect through 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; Through measuring PCT, CRP, IL-6 and the fluorescent value of new dish purine 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 PCT, CRP, IL-6 and new dish purine 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 more complete sooner, 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 pyemia early diagnosis liquid phase chip.A kind of preparation method of pyemia early diagnosis liquid phase chip mainly may further comprise the steps:
(1) corresponding capture antibody encapsulates microballoon:
-with after the 50 μ L microballoon activation, >=15000rpm 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 30s of vortex vibration, sonicated 1min, after with microballoon in >=15000rpm, centrifugal 8-12min; 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 30s of vortex vibration, sonicated 1min;
-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 1.5-2.5hr;
-coupling the speed of the microballoon behind the antibody with >=12000g, centrifugal 8-12min;
-inhale and abandon supernatant, microballoon is resuspended in the 500 μ L PBS-TBN solution the about 30s of vortex vibration, sonicated 1min;
-room temperature lucifuge vibration 30min; Again in >=12000g, centrifugal 8-12min;
-inhale and abandon supernatant, microballoon is resuspended in the 1mL PBS-TBN solution the about 30s of vortex vibration, sonicated 1min; Microballoon >=12000g, centrifugal 4-6min; Repeat this step once;
-inhale and abandon supernatant, microballoon is resuspended in the 500-1000 μ L PBS-TBN solution;
-every kind microballoon that encapsulates is placed on 2-8 ℃ of lucifuge through luminex instrument counting and preserves separately, during use, needs equal proportion to mix according to detection, 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 AC, be regarded as target volume;
-with DMSO (Dimethyl sulfoxide) dissolving, the NHS-Biotin reactant liquor of configuration 10mg/ml;
In reaction tube, added respectively in-1: 100 in molar ratio and detect antibody and NHS-Biotin reactant liquor;
-add volume and be the NaHCO of 1/10 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 said activation microballoon is following:
-with vortex oscillator or ultrasound wave suspension microballoon;
-get the centrifugal 8-10min of 50 μ L microballoons;
-remove supernatant, microballoon is resuspended in the distilled water of 100 μ L, with vortex oscillator or the about 30s of ultrasound wave suspension microballoon, the centrifugal 1-2min of >=15000rpm;
-inhale and abandon supernatant, add the phosphate buffer (pH6.2) of 80 μ L, the about 20s of vortex vibration, the about 1min of ultrasound wave suspension microballoon;
-add 10 μ L 50mg/mL Sulfo-NHS, with vortex oscillator mixing lightly, sonicated 1min;
-add 10 μ L50mg/mL EDC, with vortex oscillator mixing lightly;
The about 20min of-room temperature concussion reaction.
But the disclosed multinomial sepsis markers parallel detection liquid-phase chip primary first-order equation of the present invention is accomplished the qualitative and quantitative detection of multiple sepsis markers simultaneously, thereby reaches early stage pyemic accurate diagnosis and prognosis evaluation.Multinomial sepsis markers parallel detection liquid-phase chip provided by the present invention has the detection efficiency height, and required sample size is few, high specificity, advantages such as sensitivity height.
The inventive method combines liquid-phase chip platform high flux, accurate sensitive characteristic and the highly sensitive characteristic of double antibody sandwich method; 4 kinds of pyemia specificity markers of synchronous detection thing; Improve the susceptibility and the specificity of pyemia early diagnosis; Distinguish the pyemia that bacterium and virus infections cause, judge the pyemic order of severity and poor prognosis, continuous monitoring is to judge the reaction of patient to certain methods of treatment.
In addition, preparation method of the present invention is simple, good stability, and the various technological parameters in its technical scheme such as the concentration of microballoon and antibody, course of reaction etc. all draw on a large amount of experimental basis, are the best parameter value of preparation process.
Description of drawings
Fig. 1 is the typical curve synoptic diagram that detects PCT;
Fig. 2 is the typical curve synoptic diagram that detects CRP;
Fig. 3 is the typical curve synoptic diagram that detects IL-6;
Fig. 4 is the typical curve synoptic diagram that detects new dish purine.
Embodiment
The preparation and the detection of antigens of embodiment 1 pyemia early diagnosis liquid phase chip
Capture antibody of the present invention for respectively can be corresponding with CRP, PCT, IL-6, reach the monoclonal antibody that the mopterin specificity combines;
The used CRP capture antibody of present embodiment is the monoclonal antibody of CRP, and the clone number be C2, and detecting antibody is the monoclonal antibody of CRP, and cloning number is C6, all available from Hytest company; The PCT capture antibody is the monoclonal antibody of PCT, and the clone number be 14C12, and detecting antibody is the monoclonal antibody of PCT, and cloning number is 38F11, all available from Hytest company; The IL-6 capture antibody is the monoclonal antibody of IL-6, and the clone number is 6708, and available from R&D company, detecting antibody is the polyclonal antibody of IL-6, available from abcam company; The capture antibody of mopterin is the monoclonal antibody of mopterin, and the clone number be 117/14E10, and available from Alexis company, detection antibody is the polyclonal antibody of mopterin, available from abcam company.
In the present embodiment, the prescription of said various solution is following:
1.50mM MES damping fluid (pH5.0) prescription (250ml):
2.PBS prescription:
3.PBS-TBN prescription (be to contain 0.1%BSA among the PBS, 0.02%Tween-20,0.05%Na3N, pH7.4)
1. pyemia early diagnosis liquid phase chip kit includes:
1) 4-plex encapsulates microballoon: contain No. 20 microballoons that encapsulated the PCT capture antibody respectively, encapsulated No. 38 microballoons of CRP capture antibody, encapsulated No. 53 microballoons of IL-6 capture antibody, encapsulated No. 72 microballoons of new dish purine capture antibody.
2) the 4-plex biotin labeling detects antibody: contain respectively the detection antibody with biotin labeled PCT, CRP, IL-6 and new dish purine;
3) the streptavidin phycoerythrin (SA-PE, 10ug/ml); Also have according to prior art is supporting
4) analysis buffer;
5) 4-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 encapsulates corresponding microballoon, and the preparation method is identical:
-choose No. 20, No. 38, No. 53, No. 72 microballoons (U.S. Luminex company) respectively, with vortex oscillator or ultrasound wave suspension microballoon, about 30s;
-respectively get 50 μ L microballoons in the centrifuge tube of 1.5ml, the centrifugal 10min of 15000rpm;
-carefully remove supernatant, microballoon is resuspended in the distilled water of 100 μ L the about 30s of vortex vibration, sonicated 1min; The centrifugal 10min of 15000rpm 12000g;
-inhale and abandon supernatant, add the phosphate buffer (pH6.2) of 80 μ L, the about 30s of vortex vibration, sonicated 1min;
-add 10 μ L 50mg/mL Sulfo-NHS, with vortex oscillator mixing lightly, sonicated 1min;
-add 10 μ L 50mg/mL EDC, with vortex oscillator mixing lightly;
The about 20min of-room temperature concussion reaction;
Microballoon after the-activation, >=15000rpm, centrifugal 10min;
-inhale and to abandon supernatant, microballoon is resuspended in the solution of MES (pH5.0) of 250 μ L 50mM the about 30s of vortex vibration, sonicated 1min; Microballoon >=15000rpm, centrifugal 10min; 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 30s of vortex vibration, sonicated 1min;
-in the microballoon of every kind of suspension, add corresponding 1 μ g antibody, with MES (pH5.0) solution of 50mM cumulative volume is mended to 500 μ L;
-with vortex oscillator mixing, 25 ℃ of lucifuge vibration 2hr (rotating speed 900rpm);
Microballoon behind the-coupling antibody is with the speed of >=12000g, centrifugal 10min;
-inhale and abandon supernatant, microballoon is resuspended in the 500 μ L PBS-TBN solution the about 30s of vortex vibration, sonicated 1min;
-room temperature lucifuge vibration 30min;
-microballoon >=12000g, centrifugal 10min;
-inhale and abandon supernatant, microballoon is resuspended in the 1mL PBS-TBN solution the about 30s of vortex vibration, sonicated 1min; Microballoon >=12000g, centrifugal 5min; Repeat this step once;
-inhale and abandon supernatant, microballoon is resuspended in the 600 μ L PBS-TBN solution;
-the microballoon that encapsulates is through luminex instrument counting;
-the microballoon that encapsulates places 2-8 ℃ 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) 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 regarded 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 after each concentration standard article has diluted with the dilution that just can be used for next concentration behind the thorough mixing of vortex mixed appearance.Blending process avoids producing foam.
3) 96 orifice plate layouts are set, confirm 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 of surveying to four kinds of pyemia specificity marker quality testings of above-mentioned preparation respectively; With vortex mixed appearance 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 four kinds of capture antibody coupling microballoons that add in every hole.Encapsulate microballoon and should use preceding mixing facing, and should use immediately behind the mixing, can precipitate again otherwise place microballoon of a specified duration.
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) (wherein said four kinds of biotin labeled detection antibody are in advance by the equal proportion mixing to hatch the mixed liquor of accomplishing every hole, back and adding the biotin labeled detection antibody of the above-mentioned preparation of 25ul; 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 and accomplish every hole, back and add 25ul streptavidin phycoerythrin, 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:
Experimental result such as table 1, table 2 (seeing also Fig. 1 to Fig. 4)
The disclosed multinomial sepsis markers parallel detection liquid-phase chip of the present invention only needs the serum sample of 25 μ L to get final product the detection by quantitative that primary first-order equation is accomplished four kinds of sepsis markers 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 more complete sooner, so detection sensitivity and the range of linearity all are greatly improved.
Through the pairing to CRP, PCT, IL-6 and mopterin capture antibody and detection antibody, we have found the optimum antibody that is applicable to these four kinds of sepsis markers detections right.Through the analysis of typical curve, can see that pyemia early diagnosis liquid phase chip provided by the present invention has very high detection sensitivity and specificity to the detection of four kinds of sepsis markers.Wherein, the linearity of PCT and IL-6 typical curve R in the concentration range of 9.77pg/ml～10ng/ml
2>=0.99, LDL can reach 9.77pg/ml.The typical curve of CRP is R in the concentration range of 97.7pg/ml～100ng/ml
2>=0.99, LDL can reach 97.7pg/ml.The typical curve of mopterin is R in the concentration range of 2.44pmol/ml～2.5nmol/ml
2>=0.99, LDL can reach 2.44pmol/ml, and the relative error of the measured concentration of each standard point and expection concentration is no more than 8%.Therefore; Pyemia early diagnosis liquid phase chip provided by the invention can detect the variation of four kinds of sepsis markers deniers in the serum; And four kinds of different blood serum designated objects of diagnostic value of the disposable detection of ability, thereby improve the accuracy rate of early diagnosis and antidiastole.
Simultaneously; Can distinguish the SIRS that pyemia and other reasons cause, distinguish the pyemia that bacterium and virus infections cause, for treatment targetedly provides foundation; Judge the pyemic order of severity and poor prognosis, continuous monitoring is to judge the reaction of patient to certain methods of treatment.
Table 1
Table 2
Claims (4)
1. a pyemia early diagnosis liquid phase chip is characterized in that, mainly includes:
1) encapsulates microballoon: contain the microballoon that has encapsulated the Procalcitonin capture antibody respectively; Encapsulated the microballoon of c reactive protein capture antibody; Encapsulated the microballoon of interleukin-6 capture antibody and encapsulated the microballoon of mopterin capture antibody, above-mentioned microballoon has the different colours coding respectively;
2) biotin labeling detects antibody: contain the detection antibody of using biotin labeled Procalcitonin, c reactive protein, interleukin-6 and mopterin respectively;
3) streptavidin phycoerythrin;
(1) preparation that encapsulates microballoon of every kind of corresponding capture antibody is following:
-with after the 50 μ L microballoon activation, >=15000rpm is centrifugal;
-inhale and to abandon supernatant, microballoon is resuspended in the solution of MES of 230-280 μ L 50mM of pH5.0, vortex vibration 30s, sonicated 1min, after with microballoon in >=15000rpm, centrifugal; Repeat this step;
-inhale and to abandon supernatant, microballoon is resuspended in the solution of MES of 100 μ L 50mM pH5.0 vortex vibration 30s, sonicated 1min;
-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 1.5-2.5hr;
-coupling the speed of the microballoon behind the antibody with >=12000g, centrifugal 8-12min;
-inhale and abandon supernatant, microballoon is resuspended in the 500 μ L PBS-TBN solution vortex vibration 30s, sonicated 1min;
-room temperature lucifuge vibration 30min; Again in >=12000g, centrifugal 8-12min;
-inhale and abandon supernatant, microballoon is resuspended in the 1mL PBS-TBN solution vortex vibration 30s, sonicated 1min; Microballoon >=12000g, centrifugal 4-6min; Repeat this step once;
-inhale and abandon supernatant, microballoon is resuspended in the 500-1000 μ L PBS-TBN solution;
-every kind microballoon that encapsulates is placed on 2-8 ℃ of lucifuge through luminex instrument counting and preserves separately, during use, needs equal proportion to mix according to detection, 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 labeled preparation that detects antibody is following:
-calculate the volume that the detection antibody-solutions is diluted to 1mg/ml according to detecting AC, be regarded as target volume;
-with the DMSO dissolving, the NHS-Biotin solution of preparation 10mg/ml;
In reaction tube, added respectively in-1: 100 in molar ratio and detect antibody and NHS-Biotin solution;
-add volume and be the NaHCO of 1/10 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 1-3 μ g/ml.
2. pyemia early diagnosis liquid phase chip according to claim 1 is characterized in that, every kind of working concentration that encapsulates the microballoon of capture antibody is 120/μ l; The working concentration of every kind of biotin labeling detection antibody is 2 μ g/ml.
3. one kind prepares the method for pyemia early diagnosis liquid phase chip according to claim 1, it is characterized in that, mainly may further comprise the steps:
(1) every kind of corresponding capture antibody encapsulates microballoon:
-with after the 50 μ L microballoon activation, >=15000rpm is centrifugal;
-inhale and to abandon supernatant, microballoon is resuspended in the solution of MES of 230-280 μ L 50mM of pH5.0, vortex vibration 30s, sonicated 1min, after with microballoon in >=15000rpm, centrifugal; Repeat this step;
-inhale and to abandon supernatant, microballoon is resuspended in the solution of MES of 100 μ L 50mM pH5.0 vortex vibration 30s, sonicated 1min;
-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 1.5-2.5hr;
-coupling the speed of the microballoon behind the antibody with >=12000g, centrifugal 8-12min;
-inhale and abandon supernatant, microballoon is resuspended in the 500 μ L PBS-TBN solution vortex vibration 30s, sonicated 1min;
-room temperature lucifuge vibration 30min; Again in >=12000g, centrifugal 8-12min;
-inhale and abandon supernatant, microballoon is resuspended in the 1mL PBS-TBN solution vortex vibration 30s, sonicated 1min; Microballoon >=12000g, centrifugal 4-6min; Repeat this step once;
-inhale and abandon supernatant, microballoon is resuspended in the 500-1000 μ L PBS-TBN solution;
-every kind microballoon that encapsulates is placed on 2-8 ℃ of lucifuge through luminex instrument counting and preserves separately, during use, needs equal proportion to mix according to detection, 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 AC, be regarded as target volume;
-with the DMSO dissolving, the NHS-Biotin solution of preparation 10mg/ml;
In reaction tube, added respectively in-1: 100 in molar ratio and detect antibody and NHS-Biotin solution;
-add volume and be the NaHCO of 1/10 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 1-3 μ g/ml.
4. preparation method according to claim 3 is characterized in that: the step of said activation microballoon is following:
-with vortex oscillator or ultrasound wave suspension microballoon;
-get the centrifugal 8-10min of 50 μ L microballoons;
-remove supernatant, microballoon is resuspended in the distilled water of 100 μ L, with vortex oscillator or ultrasound wave suspension microballoon 30s, sonicated 1min, the centrifugal 8-12min of >=15000rpm;
-inhale and abandon supernatant, add the phosphate buffer of 80 μ L, vortex vibration 20s, ultrasound wave suspension microballoon 1min;
-add 10 μ L50mg/mL Sulfo-NHS, with vortex oscillator mixing lightly, sonicated 1min;
-add 10 μ L50mg/mL EDC, with vortex oscillator mixing lightly;
-room temperature concussion reaction 20min.
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| CN101487051B (en) * | 2009-02-24 | 2011-07-20 | 广州益善生物技术有限公司 | Detecting probe and liquid phase chip for BRAF gene mutation and detecting method thereof |
| WO2011023813A1 (en) * | 2009-08-28 | 2011-03-03 | Brahms Gmbh | Procalcitonin for the prognosis of adverse events |
| CN102507949B (en) * | 2011-11-15 | 2013-12-25 | 吉林出入境检验检疫局检验检疫技术中心 | Method using liquid phase chip to detect staphylococcus aureus |
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| CN105388292A (en) * | 2015-10-19 | 2016-03-09 | 清华大学深圳研究生院 | Reagent kit and method for joint detection of PCT, CRP and IL-6 |
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| CN109884320A (en) * | 2019-03-29 | 2019-06-14 | 重庆医科大学 | Application of the oncostatinM as biomarker in preparation sepsis diagnosis reagent |
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| CN113049835A (en) * | 2019-12-27 | 2021-06-29 | 深圳市帝迈生物技术有限公司 | Combined detection kit, detection method and immunoassay system |
| CN110988364A (en) * | 2019-12-31 | 2020-04-10 | 天津美瑞特医疗科技有限公司 | Method for detecting GVHD (GVHD-associated cytokine) after transplantation by using flow cytometry and detection kit |
| CN111721941B (en) * | 2020-07-17 | 2023-08-25 | 南方科技大学 | Device for judging sepsis infection condition and application thereof |
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