CN102520173B - Fluorescence immunochromatographic assay and kit for quantitative detection of creatine kinase isoenzyme (CK-MB) - Google Patents
Fluorescence immunochromatographic assay and kit for quantitative detection of creatine kinase isoenzyme (CK-MB) Download PDFInfo
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- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
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Abstract
The invention discloses a fluorescence immunochromatographic assay and kit for quantitative detection of acute myocardial infarction marker-creatine kinase isoenzyme (CK-MB). The fluorescence immunochromatographic assay for quantitative detection of the CK-MB realizes fluorescent quantitative detection by utilizing excellent fluorescent properties of quantum dots and combining a bicolour marking technology and an immunochromatographic assay on the basis of optimizing each component of a test strip. Compared with the common collaurum immunochromatographic assay, the fluorescence immunochromatographic assay has the advantages of good mark stability, low nonspecificity, high sensitivity, wide linear range and accuracy in quantification. The kit disclosed by the invention is used for carrying out quantification detection on the CK-MB, is suitable for detection of whole blood, blood serum and plasma samples, can provide a reference for cardiovascular and cerebrovascular disease diagnosis, and is widely applied to primary hospitals and clinics.
Description
Technical field
The present invention relates to field of medical examination, particularly a kind of fluorescence immune chromatography method and kit thereof of quantitative detection acute myocardial infarction mark creatine kinase isozyme.
Background technology
Current, angiocardiopathy has worldwide become the potential killer of adult's maximum, only just have and exceed 7,000 ten thousand people and have a heart disease in the U.S., annual nearly 6,000,000 people enter emergency ward because of pectoralgia, the death toll of annual acute myocardial infarction AMI (AMI) has exceeded 500,000, in China, the morbidity rate of angiocardiopathy, the incidence of disease and risk factor levels thereof are all continuous ascendant trend.Statistics in recent years, the mortality ratio of cardiovascular disease accounts for 40% in human mortality, higher than American-European countries, belongs to cardiovascular disease state occurred frequently.Therefore, only accomplish early prevention, early find treatment early, could prevent to greatest extent disabling, lethal effect, improve to greatest extent prognosis and the quality of life of cardiovascular patient.
To angiocardiopathy, conventional detection index is myocardial enzymes series.Myocardium enzyme refers to the enzyme material in cardiac muscle cell, has the effect of catalysis cardiac muscle cell metabolism and adjusting Electrical Activity of Myocardial Cells.Myocardial enzymes comprises aspartate transaminase (AST), lactic dehydrogenase (LDH), creatine kinase (CK), CK-MB (CK-MB) etc.If cardiac muscle cell necroses, breaks, these enzymes will discharge in the middle of human blood, make it to raise.Therefore, the rising degree of myocardial enzymes can be weighed cardiac muscle cell's degree of damage indirectly.Wherein CK and CK-MB are the higher indexs of sensitivity, can when not yet large area is downright bad, in blood samples of patients, detect at the early stage cardiac muscle of morbidity, and the very first time provides reliable data for clinical.
The dimer that CK is made up of two kinds of different subunits (M and B), normal human tissue is mainly containing 3 kinds of isodynamic enzymes, i.e. CK-MM, CK-BB, CK-MB like this.CK-MM is mainly present in muscle cell, and CK-BB is mainly present in brain cell, and CK-MB is mainly present in cardiac muscle cell.
CK-MB is current conventional myocardial injury markers, is once once being regarded as diagnosing " goldstandard " of AMI.CK-MB increases in 4～8h after acute myocardial infarction, 24h peaking, and it is normal that couple of days recovers.CK-MB has important physiological function and clinical value has caused that people pay attention to widely and deep research because of it.
Multiplex enzyme linked immunosorbent assay, chemoluminescence method and colloidal gold immunity chromatography etc. are measured the CK-MB in serum traditionally.But enzyme linked immunosorbent assay complicated operation, detects length consuming time; Chemoluminescence method is high to technical requirement, is difficult for carrying out routine and carries out in clinical labororatory.Although it is few that colloidal gold immunity chromatography has sample consumption, easy to be quick, cheap advantage, but when running in some sample antigen or antibody content when extremely low, the color of collaurum will be very shallow, be difficult to the naked eye judged result, easily occur erroneous judgement, sensitivity is lower.
Chinese Patent Application No. CN200610114997.9 has announced the chemical luminescent detecting method of creatine kinase isozyme in a kind of serum.This application patent is mainly utilized creatine kinase catalysis phosphocreatine to react with adenosine diphosphate (ADP) and is generated atriphos and creatine, generate glycerol 3-phosphate with glycerokinase catalysis atriphos and glycerine again, glycerol 3-phosphate is oxidized and produces hydrogen peroxide by GPO, again through peroxidase effect, hydrogen peroxide makes luminol oxidation and luminous.Chinese Patent Application No. 200520041211.6 has disclosed a kind of Cardiovascular Disease Diagnosis And Predicting Multi-index Albumen Chip Measuring Kit Vessel, this kit adopts chemoluminescence method (as horseradish peroxidase and luminol) to detect, and can detect eight kinds of antibody such as c reactive protein, myoglobins, cardiac muscle troponin I, creatine kinase isozyme simultaneously.Although chemoluminescence method sensitivity is higher, repeatability and stability are all poor, and instrument complexity, costliness, can not single part of detection, limit their widespread use.
U.S. Pat 60/576327 and 60/592202, and all colloidal gold immunochromatographimethod is detected and discussed in the patent 200580025931.6 of China's application.But than fluoroscopic examination, absorbance detection has the defects such as sensitivity is low, the range of linearity is narrow.
Quantum dot (quantum dots, QDs) claim again semiconductor nano, stable, the nanocrystals of size between 1～20nm that are normally made up of II～VI family or III～V group element.It has many good optical characteristics: the fluorescence emission spectrum of (1) quantum dot is narrow and symmetrical, and fluorescent emission wavelength is adjustable, and coverage can be from ultraviolet to near-infrared region.(2) absorption spectrum of quantum dot is wide and continuous, can realize an elementary excitation, and polynary transmitting, is suitable for multi-color marking.(3) quantum dot has stronger optical stability.The light stability of CdSe/ZnS quantum dot is the more than 100 times of rhodamine 6G.(4) quantum dot molar absorptivity can be up to 106L/ (molcm), and fluorescence quantum yield high (50～80%), thereby can produce compared with hyperfluorescenceZeng Yongminggaoyingguang signal.(5) quantum dot Stokes shift is larger, and fluorescence lifetime long (20～50ns) makes signal can significantly distinguish over background and other fluorophor.Quantum dot is a kind of desirable hypersensitive and chemico-analytic fluorescence probe of multicomponent biological of being applied to.Quantum dot is combined with antibody and is can be applicable to fluoroimmunoassay and detection.
Low for the sensitivity of existing CK-MB detection method, sensing range is narrow and the quantitative deficiency such as inaccurate, quantum dot fluorescence quantitative detecting method utilizes quantum dot multi-wavelength excitation, high strength fluorescence intensity, emission peak is narrow, peak shape is symmetrical, stability of photoluminescence is good fluorescent characteristic, the detection method that a kind of quantum dot mark fast immune chromatographic test paper bar can be provided, quantitatively detects CK-MB.
Summary of the invention
The technical problem to be solved in the present invention, for for the shortcoming such as detect in prior art that creatine kinase isozyme sensitivity is low, the range of linearity is narrow and instrument is expensive, provides the fluorescence immune chromatography method of quantitative detection CK-MB a kind of.
For solving the problems of the technologies described above, technical scheme provided by the invention is:
A fluorescence immune chromatography method of quantitative detection creatine kinase isozyme CK-MB, comprises the following steps:
Step 1) specific antibody of creatine kinase isozyme CK-MB is connected to quantum dot surface with specific effect between chemical crosslinking or biomolecule, obtain the quantum dot of antibody modification, the emission wavelength ranges of the quantum dot of described antibody modification is 550～1300nm;
Step 2) by step 1) quantum dot of the antibody modification that obtains is fixed in label pad, and in label pad, be fixed with the quantum dot of Quality Control molecular modification simultaneously, the emission wavelength and step 1 of the quantum dot of described Quality Control molecular modification) emission wavelength of quantum dot of the antibody modification that obtains is different, and wavelength coverage is 550～1300nm, in chromatographic film, be respectively equipped with quantitative band and two quality control bands, wherein quality control band is fixed with the biomolecule that can be combined with described Quality Control molecular specificity, quantitatively band be fixed with corresponding CK-MB from step 1) specific antibodies of the different antigenic determinants of described specific antibody,
Step 3) be built into fluorescence immune chromatography test paper bar with sample pad, label pad, chromatographic film, adsorptive pads and base plate, wherein said chromatographic film is hypofluorescence chromatographic film, the low fluorescent characteristic of base plate tool;
Step 4) after test strips immunochromatography, detect quantitatively band and quality control band fluorescence signal intensity, and proofread and correct and be quantitatively with fluorescence signal intensity with quality control band fluorescence signal intensity, and then realize the quantitative detection of CK-MB.
The method of the fluorescence immune chromatography of a kind of quantitative detection CK-MB provided by the invention, is a kind of take quantum dot as fluorescence signal, adopts Bicolor-code technology, realizes the method that CK-MB rapid sensitive detects on immuno-chromatographic test paper strip.
The fluorescence immune chromatography method that the present invention quantitatively detects CK-MB can solve in existing fluorescence immune chromatography technology that background and signal are difficultly distinguished, sensitivity is low, the indefinite deficiency of fluorescent quantitation method and defect, can realize the detection to trace sample.Because the interference of quantum dot fluorescence stimulated luminescence is very little, sensitivity improves greatly, and its sensitivity is 10～1000 times by conventional dyes and coloured label detection method.
The present invention adopts specific effect between chemical crosslinking or biomolecule that the specific antibody of CK-MB is connected to quantum dot surface, obtains the quantum dot of antibody modification, the antibody of one or more epitopes that wherein specific antibody is corresponding CK-MB.
Chemical crosslinking in the present invention is: when there is reactive group on quantum dot surface, can directly react with antibody or Quality Control molecule time, do not need with chemical cross-linking agent, otherwise antibody modification is arrived to quantum dot surface with chemical cross-linking agent.
The method that adopts in an embodiment of the present invention chemical crosslink technique to carry out protein molecule modification to quantum dot is: utilize the crosslinking chemicals such as 1-ethyl-3-(3-dimethyl amine propyl group) carbodiimides (EDC)/N-hydroxy-succinamide (NHS), glutaraldehyde that the functional group on quantum dot surface (as carboxyl, amino) is connected with functional group's (as amino, carboxyl, aldehyde radical etc.) on protein molecule (as antigen, antibody etc.) surface.
As preferably, in one embodiment of the invention, adopt EDC/NHS cross-linking method to modify quantum dot.The general step that wherein EDC/NHS cross-linking method carries out protein molecule modification to quantum dot is: quantum dot solution is mixed with EDC and NHS, then add a certain amount of protein, take damping fluid as reaction medium, cultivate 4h, add the sealing of L-glycocoll, with the mode purifying such as chromatogram, chromatographic column or ultrafiltration be centrifugal, thereby obtain the quantum dot of protein modification.
Between biomolecule, specific effect comprises biotin-avidin system and Ag-Ab system.As preferably, in another embodiment of the present invention, adopt the combination of biotin-avidin system to carry out quantum dot modification to protein, this combination has the effect of amplifying signal.Be specially: with EDC, Streptavidin is connected to quantum dot surface, biotin is connected to protein molecule surface, by the interaction between Avidin-Biotin, protein molecule be connected to quantum dot surface.
In order to improve and the discrimination of background, it is the quantum dot of 550～1300nm that the present invention selects wavelength of transmitted light.Because under ultraviolet irradiation, more than the fluorescence intensity of chromatographic film, base plate and button card is far better than 550nm below 550nm, thereby when being detected, low concentration CK-MB produces certain impact, therefore can preferred emission wavelength be greater than the quantum dot of 550nm.In addition chromatographic film, base plate and button be stuck near infrared region (750～1300nm) fluorescence intensity extremely a little less than, and in conjunction with quantum dot synthetic technology, preferably the quantum dot (750～1300nm) of near-infrared wavelength can further improve sensitivity.
The compound quantum dot that the quantum dot that the present invention's quantum dot used comprises single compound formation or several compound are assembled into, and quantum dot has stronger light stability.Forming the compound of quantum dot is to select from the group of ZnS, CdS, HgS, MgS, CdSe, MgSe, ZnSe, PbSe, PbSe, CdTe, MgTe, ZnTe, HgTe, InAs, InP composition, and can doped with Cu, Mn and Hg.
The quantum dot of the quantum dot of Quality Control molecular modification and CK-MB antibody modification can be selected the quantum dot of same emission wavelength.But in the present invention, for the impact on quantitative band and background signal of the quantum dot that weakens Quality Control molecular modification, adopt Bicolor-code technology, select two kinds of quantum dots mark Quality Control molecule and antibody respectively that emission wavelength is different.
As preferably, in an embodiment of the present invention, use the CdSe/ZnS of organic phase method synthetic 570nm, 600nm and 650nm, and quantum dot is transferred to water-solublely by fat-soluble, in this process, quantum dot fluorescence is without significant change; Use the InAs of the synthetic 900nm of water method.
As preferably, in one embodiment of the invention, in label pad, the quantum dot emission wavelengths of the antibody modification of CK-MB is 650nm, and the quantum dot emission wavelengths of Quality Control molecular modification is 570nm.
As preferably, in another embodiment of the present invention, in label pad, the quantum dot emission wavelengths of the antibody modification of CK-MB is 900nm, and the quantum dot emission wavelengths of Quality Control molecular modification is 600nm.
In order to reduce the impact on quantum dot fluorescence signal, the present invention adopts hypofluorescence chromatographic film, low fluorescence base plate and low fluorescence button card, thereby guarantees to obtain high fluorescence signal-to-background ratio, energy good discrimination signal and background, and then improve detection sensitivity.
As preferably, in embodiments of the present invention, base plate (8) is black, and surface is with adhesive sticker, and chromatographic film (4), button card (11), base plate (8) and adhesive sticker all do not contain fluorescer.As when detaining card and containing wetting hole (13), wetting hole can be in nearly sample pad one end of chromatographic film or in nearly adsorptive pads one end.
For being different from the detection band of colloidal gold immuno-chromatography test paper strip, in the present invention, in chromatographic film, the region of fixation of C K-MB antibody is called quantitative band, detect the concentration of CK-MB for accurate quantitative analysis, and in doing with quality control band, quantitatively band is proofreaied and correct in Quality Control, to weaken the impact of sample, environmental factor etc.
Immuno-chromatographic test paper strip of the present invention can be taked conventional chromatography mode, and as preferably, the present invention adopts pre-profit immunochromatography to carry out CK-MB detection.Wherein moistening in advance immuno-chromatographic test paper strip has direct and indirectly moistens two kinds in advance, in one embodiment, directly pre-profit immuno-chromatographic test paper strip is made up of sample pad (1), filtering membrane (2), label pad (3), chromatographic film (4), adsorptive pads (7) and base plate (8), as shown in Figure 1.In label pad, be fixed with the quantum dot of the specific antibody modification of CK-MB, and the quantum dot of Quality Control molecular modification.In test strips chromatographic film, be respectively equipped with two quantitatively band and two quality control bands, wherein quality control band is fixed with the biomolecule that can be combined with Quality Control molecular specificity, and quantitatively band is fixed with corresponding CK-MB and the specific antibodies different antigenic determinants of specific antibody in above-mentioned label pad.CK-MB can form double-antibody sandwich structure with the antibody on quantum dot surface and the antibody of quantitatively bringing.And can expand sensing range by the number that increases quantitative band, avoid HOOK effect.
In another embodiment of the present invention, pre-profit immuno-chromatographic test paper strip is made up of sample pad (1), label pad (3), chromatographic film (4), wetting pad (9), connection gasket (10), adsorptive pads (7) and base plate (8) indirectly, as shown in Figure 2.
In the embodiment of the present invention, the sample that inventor selects is respectively whole blood and serum, and sample further comprises blood plasma.In the time that sample is whole blood, fluorescence immune chromatography test paper bar is also included in the filtering membrane arranging between sample pad and chromatographic film, be used for solidifying, filtration cell (being mainly red blood cell), this filtering membrane can contact with the direct capillary action of sample pad and label pad respectively, as shown in Fig. 1, also can contact with the direct capillary action of label pad and chromatographic film.
The embodiment of the present invention adopts pre-profit immune chromatography method quantitatively to detect the CK-MB in sample.Wherein moistening in advance immunochromatography is: add the damping fluid of certain volume on chromatographic film, after wetting certain hour, sample is added in sample pad, sample flows along chromatographic film to adsorptive pads direction.The object of pre-profit is to soak in advance chromatographic film, and sealing nonspecific binding site so that quantum dot-labeled thing evenly passes through chromatographic film, and reduces the non-specific adsorption in chromatographic film, and slows down sample flowing velocity, thereby increases specific binding.Wherein damping fluid volume is generally 20～80 μ l, and wetting time is generally 30 seconds～and 2 minutes, and the sample chromatography time is generally 8～25 minutes.
Pre-profit immunochromatography of the present invention can be damping fluid is directly or indirectly dripped in chromatographic film.And damping fluid is dripped when the chromatographic film indirectly, fluorescence immune chromatography test paper bar also comprises wetting pad and connection gasket, and wherein wetting pad contacts with capillary action with connection gasket with chromatographic film respectively, and connection gasket contacts with capillary action with adsorptive pads with wetting pad respectively.Damping fluid arrives chromatographic film by wetting pad.
The damping fluid that the present invention adopts is the alkaline buffer of pH 7.2～11, and this damping fluid can comprise bovine serum albumin(BSA), casein and surfactant.Wherein surfactant can comprise polysorbas20, Tween 80, triton x-100, polyglycol and polyvinyl pyrrolidone etc.
As preferably, in the embodiment of the present invention, using damping fluid is the phosphate buffer of the pH9.0 that comprises bovine serum albumin(BSA) and polysorbas20.
Detection of the present invention comprises excitation source module, optical filtering module, photoelectric conversion module, control analysis module and software systems with fluorescent quantitation instrument.Wherein excitation source module comprises light source and beam condensing unit, and this light source is light emitting diode or laser diode, and wavelength is selected between 300～400nm or between 500～600nm.Optical filtering module comprises optical filter wheel, and this optical filter wheel comprises optical filter not of the same race, to obtain the fluorescence signal of corresponding quantum dot.Photoelectric conversion module comprises imageing sensor or photomultiplier.
After chromatography finishes, under light source activation, the fluorescence signal that test strips produces, optical module filtering parasitic light and background fluorescence after filtration, arrives photoelectric conversion module, is converted to digital signal.Wherein the quality control band that obtains have certain correlativity with the quantitative fluorescence intensity of band, major influence factors has temperature, humidity, matrix etc.
The decision method of testing result of the present invention is: if quality control band fluorescence signal intensity value exceedes the acceptable value of fluorescent quantitation instrument inner setting, illustrate that testing result is invalid; Under the effective prerequisite of testing result, be quantitatively with fluorescence intensity and quality control band ratio higher, represent that CK-MB concentration is higher, on the contrary lower.
The present invention adopts fluorescent quantitation instrument to detect the standard items of a series of variable concentrations, drawing standard curve.Wherein typical curve is the relation curve of standard items series concentration (c) and corresponding correction fluorescence signal intensity (F), and relational expression is F=f (c).Proofreading and correct fluorescence signal intensity is F=α F
quantitatively band/ F
quality control band, wherein α is correction coefficient, affected by temperature, humidity and matrix etc.Then detect sample, establishing criteria curve obtains CK-MB concentration in sample.
The present invention also provides a kind of fluorescence immune chromatography kit of quantitative detection creatine kinase isozyme, comprises damping fluid, button card (11) and fluorescence immune chromatography test paper bar, as shown in Figure 3.This kit adopts pre-profit immune chromatography method indirectly.Button card (11) is the external shell structure of fluorescence immune chromatography test paper bar, comprises loading hole (12), form (14) and wetting hole (13), and the low fluorescent characteristic of tool, is preferably not containing fluorescer.
As shown in Figure 2, it comprises sample pad (1), label pad (3), chromatographic film (4), wetting pad (9), connection gasket (10), adsorptive pads (7) and base plate (8) to fluorescence immune chromatography test paper bar structure.Chromatographic film (4) comprises quality control band (5) and is quantitatively with (6).Wherein sample pad (1), label pad (3), chromatographic film (4), wetting pad (9), connection gasket (10) and adsorptive pads (7) are equipped on base plate (8), sample pad (1) is connected in label pad (3), and be positioned at the below of loading hole (12), label pad (3) is connected in chromatographic film (4), in chromatographic film (4), be fixed with two quality control bands (5) and two quantitative bands (6), and quality control band (5) is positioned at the both sides of quantitative band (6), chromatographic film (4) is positioned at the below of form (14), chromatographic film (4) is connected in wetting pad (9), and wetting pad (9) is positioned at the below of wetting hole (13), wetting pad (9) is connected in connection gasket (10), connection gasket (10) is connected in adsorptive pads (7).
While carrying out whole blood sample chromatography, test strips also comprises filtering membrane (2), as shown in Figure 1, wherein test strips comprises sample pad (1), label pad (3), chromatographic film (4), adsorptive pads (7), base plate (8) and filtering membrane (2) to concrete structure.Filtering membrane (2) contacts with chromatographic film (4) capillary action with sample pad (1), be that filtering membrane can be connected between sample pad (1) and label pad (3), also can be connected in label pad (3) and chromatographic film (4).
The pre-profit immuno-chromatographic test paper strip that the present invention builds, it is characterized in that, increase wetting pad (9) and connection gasket (10), wherein wetting pad contacts with connection gasket (10) capillary action with chromatographic film (4) respectively, while being used for reducing to drip damping fluid, the impact of damping fluid to chromatographic film, to soak chromatographic film (4), and reduces non-specific adsorption.
And connection gasket (10) contacts with adsorptive pads (7) capillary action with wetting pad (9) respectively, there is absorption speed slowly, water absorbing capacity when wetting to reduce, impel the fully wetting chromatographic film (4) of damping fluid, in the time adding sample chromatography, adsorptive pads (7) can pass through connection gasket (10) water suction, impels sample chromatography.
The chromatographic film (4) of test strips has hypofluorescence characteristic, and preferably, not containing fluorescer, its fluorescence noise is very weak in the time being greater than 550nm, to reduce the impact that low concentration object is detected.
Major advantage of the present invention is as follows:
1) the present invention quantitatively detects the fluorescence immune chromatography method employing quantum dot of CK-MB as the label of specific antibody, compared with organic fluorescent dye, have that luminous intensity is high, an exciting light spectrum width, the advantage such as emission spectrum is narrow, fluorescence lifetime is long, finishing multifunction and good stability.And the preferred quantum dot of 550～1300nm, to improve and the discrimination of background.
2) button card, base plate and the chromatographic film in the building block of kit of the present invention all has low fluorescent characteristic in the time being greater than 550nm, to reduce the impact on quantum dot fluorescence signal acquisition, thereby guarantee to obtain high fluorescence signal-to-background ratio, and then reach and put forward highly sensitive object.
3) the present invention adopts fluorescent quantitation instrument to detect quantitatively band and quality control band fluorescence signal intensity, and proofreaies and correct quantitatively band fluorescence signal intensity with quality control band, and then the typical curve obtaining according to fluorescent quantitation instrument is realized the quantitative detection of CK-MB.Wherein typical curve is the relation curve of standard items series concentration (c) and corresponding correction fluorescence signal intensity (F), and relational expression is F=f (c).Proofreading and correct fluorescence signal intensity is F=α F
quantitatively band/ F
quality control band, wherein α is correction coefficient, affected by temperature, humidity and matrix etc.Then detect sample, establishing criteria curve obtains CK-MB concentration in sample.
4) the present invention utilizes quantum dot Bicolor-code technology, reduce the impact that Quality Control molecule detects CK-MB, the impact of the quantum dot that also can reduce Quality Control molecular modification on quantitative band and chromatographic film background, method is simple and quick, highly sensitive, be conducive to highly sensitive quantitative detection.
5) the present invention can utilize pre-profit immunochromatography technique, strengthens specific binding, reduces non-specific adsorption, and the detection sensitivity of Contrast agent box is conducive in sample accurate quantitative analysis when CK-MB content is extremely low.
6) the inventive method is simple, quick, accurate, cost is low, and sensitive very high.Detect creatine kinase isozyme with traditional chemoluminescence method and euzymelinked immunosorbent assay (ELISA) and compare, have easy and simple to handle, fast, do not need expensive checkout equipment, be applicable to the advantages such as single part or short run detection; Compared with conventional colloidal gold immunochromatographimethod method, the present invention have mark good stability, non-specific low, highly sensitive, the range of linearity is wide and the advantage such as quantitatively accurate.
Accompanying drawing explanation
Fig. 1 is the assembling schematic diagram that directly moistens in advance immuno-chromatographic test paper strip, and wherein 1 is sample pad, and 2 is filtering membrane, and 3 is label pad, and 4 is chromatographic film, and 5 is quality control band, and 6 is to be quantitatively with, and 7 is adsorptive pads, and 8 is base plate;
Fig. 2 is the assembling schematic diagram that indirectly moistens in advance immuno-chromatographic test paper strip, and wherein 1 is sample pad, and 3 is label pad, and 4 is chromatographic film, and 5 is quality control band, and 6 is to be quantitatively with, and 7 is adsorptive pads, and 8 is base plate, and 9 pad for soaking, and 10 is connection gasket;
Fig. 3 is fluorescence immune chromatography kit schematic diagram, and wherein 11 for detaining card, and 12 is loading hole, and 13 is wetting hole, and 14 is form, and 5 is quality control band, and 6 is to be quantitatively with.
Embodiment
The invention discloses the fluorescence immune chromatography method of quantitative detection creatine kinase isozyme CK-MB a kind of, those skilled in the art can use for reference content herein, suitably improve technological parameter and realize.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the artly, they are all deemed to be included in the present invention.Method of the present invention and kit are described by preferred embodiment, related personnel obviously can, not departing from content of the present invention, spirit and scope method as herein described and kit is changed or suitably change and combination, realize and apply the technology of the present invention.
Technical scheme provided by the invention is:
Step 1) specific antibody of creatine kinase isozyme CK-MB is connected to quantum dot surface with specific effect between chemical crosslinking or biomolecule, obtain the quantum dot of antibody modification, the emission wavelength ranges of the quantum dot of described antibody modification is 550～1300nm;
Step 2) by step 1) quantum dot of the antibody modification that obtains is fixed in label pad, and in label pad, be fixed with the quantum dot of Quality Control molecular modification simultaneously, the emission wavelength and step 1 of the quantum dot of described Quality Control molecular modification) emission wavelength of quantum dot of the antibody modification that obtains is different, and wavelength coverage is 550～1300nm, in chromatographic film, be respectively equipped with quantitative band and two quality control bands, wherein quality control band is fixed with the biomolecule that can be combined with described Quality Control molecular specificity, quantitatively band be fixed with corresponding CK-MB from step 1) specific antibodies of the different antigenic determinants of described specific antibody,
Step 3) be built into fluorescence immune chromatography test paper bar with sample pad, label pad, chromatographic film, adsorptive pads and base plate, wherein said chromatographic film is hypofluorescence chromatographic film, the low fluorescent characteristic of base plate tool;
Step 4) after test strips immunochromatography, detect quantitatively band and quality control band fluorescence signal intensity, and proofread and correct and be quantitatively with fluorescence signal intensity with quality control band fluorescence signal intensity, and then realize the quantitative detection of CK-MB.
The principle of fluorescence immune chromatography of the present invention and detection method are: adopt fluorescence immune chromatography technology and double antibody sandwich method principle quantitatively to detect the content of the creatine kinase isozyme (CK-MB) in people's sample (whole blood, serum or blood plasma).When detection, first in wetting hole, drip damping fluid, then sample drop is added in loading hole, sample first mixes mutually with the quantum dot-labeled thing in label pad, and along chromatographic film to adsorptive pads direction capillary moving, quantitative band and the quality control band of flowing through respectively in chromatographic film.If contain CK-MB in sample, combine with the CK-MB antibody on quantum dot surface, in the time that chromatography is extremely quantitatively with, can be coated in advance the antibody capture of this band, thereby be formed double-antibody sandwich compound, quantum dot stays in quantitative band.Under light source activation, employing fluorescent quantitation instrument obtains the fluorescence signal intensity of quantitative band and quality control band, the typical curve obtaining according to fluorescent quantitation instrument, and then CK-MB concentration in analyzing samples.
In order to make those skilled in the art understand better technical scheme of the present invention, below in conjunction with specific embodiment, the present invention is described in further detail.
Embodiment 1: antibody modification is directly moistened to the quantitative detection of immunochromatography to CK-MB in advance in quantum dot employing with chemical crosslink technique
(1) modification of quantum dot and antibody
Take people CK-MM and CK-BB as antigen, can immunity obtain goat-anti people CK-MM antibody and CK-BB antibody respectively.Because CK-MB has M and two subunits of B, CK-MM antibody and CK-BB antibody can be combined with M and the B subunit of CK-MB respectively, form double-antibody sandwich structure.，
Be quantum dot, 10 μ g EDC and 15 μ g NHS solution and 10～30 μ g goat-anti people CK-BB antibody-solutions of 650nm to adding 60pmol emission wavelength in pH 7.4 phosphate buffers, mix and react 4h under room temperature, add the sealing of 1mg glycocoll.With chromatographic column or chromatographic column separation and purification, obtain the quantum dot of CK-BB antibody modification.In like manner obtain the quantum dot that goat anti-rabbit antibody is modified.Wherein the fluorescent emission wavelength of the quantum dot of CK-BB antibody modification is 650nm, and the fluorescent emission wavelength of the quantum dot that goat anti-rabbit antibody is modified is 570nm.
(2) structure of kit
Mix above-mentioned two kinds of quantum dot-labeled things with the mol ratio ratio of 1: 1, wherein in mixed liquor, contain the surfactants such as 1～15% sucrose, 0.01～2% bovine serum albumin(BSA) (BSA) and polysorbas20, Tween 80, triton x-100, wherein surface-active contents is between 0.01～2%, then be evenly sprayed in label pad, 37 ℃ dry after sealing, preserve at 4 ℃.
In hypofluorescence chromatographic film, draw four parallel bands to draw film instrument, band interval 5mm, bar bandwidth is all about 1mm.Wherein two of both sides is quality control band, and middle two is to be quantitatively with.On quality control band, spray rabbit immunoglobulin (rabbit igg), concentration is respectively 0.2～1mg/ml and 1～3mg/ml; Quantitatively bring spraying CK-MM how anti-, concentration is respectively 0.2～1mg/ml and 0.5～2mg/ml.37 ℃ dry after sealing, preserve at 4 ℃.
In black floor, paste successively chromatographic film (long 30cm, wide 2.5cm), label pad (long 30cm, wide 8mm), filtering membrane (long 30cm, wide 16mm), sample pad (long 30cm, wide 16mm), connection gasket (long 30cm, wide 10mm), wetting pad (long 30cm, wide 10mm) and adsorptive pads (long 30cm, wide 16mm).Between film and film, all will closely be connected, make the large plate of pre-profit fluorescence immune chromatography test paper, schematic diagram as shown in Figure 1.Large the test paper pasting plate is longitudinally cut into the wide test strips of 4mm with cutting cutter, puts into low fluorescence button card and be assembled into the not immune chromatography reagent kit containing damping fluid, dry after sealing, be jointly configured to fluorescence immune chromatography kit with damping fluid, 4 ℃ keep in Dark Place.
(3) pattern detection
Make dilution with normal person's whole blood, CK-MB standard items are prepared to series concentration standard solution as follows: 0.125ng/mL, 0.25ng/mL, 0.5ng/mL, 1.0ng/mL, 2.0ng/mL, 4.0ng/mL, 8.0ng/mL, 16ng/mL, 32ng/mL, 64ng/mL, 128ng/mL and 256ng/mL.
Damping fluid is the pH9.0 phosphate buffer that contains 0.1～0.5%BSA and 0.05～1% polysorbas20.When chromatography, first 50 μ l damping fluids are added drop-wise in chromatographic film, after 1 minute, negative 100 μ l whole blood or standard solution are added drop-wise in loading hole, after 13 minutes, detect (each sample is measured 3 times by 3 test strips respectively, averages), drawing standard curve with fluorescent quantitation instrument.
50 μ l damping fluids are added drop-wise in chromatographic film, after 1 minute, 100 μ l whole blood samples are added drop-wise in loading hole, after 13 minutes, detect with fluorescent quantitation instrument, establishing criteria curve obtains the concentration of CK-MB in sample.
Result shows, its lowest detection is limited to 0.5ng/mL, the minimum 1.0ng/mL that is quantitatively limited to, in quantitative sensing range,, there is not HOOK effect in coefficient R 2 > 0.99, and batch in criticize between repeatability all better, can be cardiovascular disease diagnosis reference be provided.
Embodiment 2: antibody modification is moistened to the quantitative detection of immunochromatography to CK-MB indirectly in advance in quantum dot employing with avidin-biotin complex technology
(1) quantum dot synthesizes and modifies
According to the method for embodiment 1, prepare the quantum dot that Streptavidin is modified.In like manner obtain the quantum dot that goat anti-rabbit antibody is modified.
0.1mol/L sodium bicarbonate buffer liquid (pH 8.0) for CK-BB monoclonal antibody is diluted to 1mg/ml, with 1ml dimethyl sulfoxide (DMSO) dissolving 1mg N-hydroxy-succinamide biotin (NHSB), get 1ml monoclonal antibody solution and add 20～120 μ l NHSB solution, under room temperature, react after 4 hours, add 1mol/L NH
4cl, at room temperature stirs 10 minutes.With ultrafiltration centrifugal purification, to remove free biotin, obtain biotinylated monoclonal antibody.
Mix with the ratio of 1: 3～1: 12 quantum dot and the biotinylated monoclonal antibody that Streptavidin is modified, obtain the quantum dot that CK-BB monoclonal antibody is modified.Wherein the fluorescent emission wavelength of the quantum dot of CK-BB antibody modification is 900nm, and the fluorescent emission wavelength of the quantum dot that goat anti-rabbit antibody is modified is 600nm.
(2) structure of kit and pattern detection
According to the method for embodiment 1, quantum dot-labeled thing is sprayed to label pad, antibody and Quality Control molecule is sprayed to quantitative band and the quality control band of chromatographic film.
In black floor, paste successively upper chromatographic film (long 30cm, wide 2.5cm), label pad (long 30cm, wide 8mm), sample pad (long 30cm, wide 16mm), connection gasket (long 30cm, wide 10mm), wetting pad (long 30cm, wide 10mm) and adsorptive pads (long 30cm, wide 16mm).Between film and film, all will closely be connected, make the large plate of fluorescence immune chromatography test paper, schematic diagram as shown in Figure 2.Large the test paper pasting plate is longitudinally cut into the wide test strips of 4mm with cutting cutter, put into low fluorescence button card and be assembled into the not immune chromatography reagent kit containing damping fluid, as shown in Figure 3, dry rear sealing, jointly be configured to fluorescence immune chromatography kit with damping fluid, 4 ℃ keep in Dark Place.
(3) pattern detection
Carry out standard items preparation according to embodiment 1.
Damping fluid is the pH9.0 phosphate buffer that contains 0.05～0.2%BSA and 0.05～1%Tween-20.When chromatography, first 50 μ l damping fluids are added drop-wise in wetting hole, after 1 minute, 100 μ l negative serums or standard solution are added drop-wise in loading hole, after 13 minutes, with the detection of fluorescent quantitation instrument, (each sample is measured 3 times by 3 test strips respectively, average), drawing standard curve.
50 μ l damping fluids are added drop-wise in wetting hole, after 1 minute, 100 μ l serum samples are added drop-wise in loading hole, after 13 minutes, detect with fluorescent quantitation instrument, establishing criteria curve obtains the concentration of CK-MB in sample.
Result shows, its lowest detection is limited to 0.1ng/mL, the minimum 0.25ng/mL that is quantitatively limited to, and in quantitative sensing range, coefficient R
2, there is not HOOK effect in > 0.99, and batch in criticize between repeatability all better, can be cardiovascular disease diagnosis reference be provided.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (3)
1. one kind is quantitatively detected the fluorescence immune chromatography kit of creatine kinase isozyme CK-MB, comprise damping fluid, button card (11) and fluorescence immune chromatography test paper bar, it is characterized in that, described button card (11) is the external shell structure of fluorescence immune chromatography test paper bar, comprises loading hole (12), form (14) and wetting hole (13), described fluorescence immune chromatography test paper bar comprises sample pad (1), label pad (3), chromatographic film (4), wetting pad (9), connection gasket (10), adsorptive pads (7) and base plate (8), wherein sample pad (1), label pad (3), chromatographic film (4), wetting pad (9), connection gasket (10) and adsorptive pads (7) are equipped on base plate (8), sample pad (1) is connected in label pad (3), and be positioned at the below of loading hole (12), label pad (3) is connected in chromatographic film (4), in chromatographic film (4), be fixed with two quality control bands (5) and two quantitative bands (6), and quality control band (5) is positioned at the both sides of quantitative band (6), chromatographic film (4) is positioned at the below of form (14), chromatographic film (4) is connected in wetting pad (9), and wetting pad (9) is positioned at the below of wetting hole (13), wetting pad (9) is connected in connection gasket (10), connection gasket (10) is connected in adsorptive pads (7),
In described label pad, be fixed with the quantum dot of antibody modification and the quantum dot of Quality Control molecular modification, the quantum dot of described antibody modification is prepared in accordance with the following methods:
Step 1) specific antibody of creatine kinase isozyme CK-MB is connected to quantum dot surface with specific effect between chemical crosslinking or biomolecule, obtain the quantum dot of antibody modification, the emission wavelength ranges of the quantum dot of described antibody modification is 550～1300nm;
The emission wavelength and step 1 of the quantum dot of described Quality Control molecular modification) emission wavelength of quantum dot of the antibody modification that obtains is different, and wavelength coverage is 550～1300nm, quality control band in described chromatographic film is fixed with the biomolecule that can be combined with described Quality Control molecular specificity, the quantitative band in described chromatographic film be fixed with corresponding CK-MB from step 1) specific antibodies of the different antigenic determinants of described specific antibody.
2. fluorescence immune chromatography kit according to claim 1, it is characterized in that, described fluorescence immune chromatography test paper bar also comprises filtering membrane (2), and wherein said filtering membrane (2) contacts with sample pad (1) or chromatographic film (4) capillary action respectively.
3. fluorescence immune chromatography kit according to claim 1, is characterized in that, described button card (11) has low fluorescent characteristic or do not contain fluorescer.
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| CN103487578A (en) * | 2013-10-16 | 2014-01-01 | 深圳市大爱医疗科技有限公司 | CRP (C-reactive protein)/PCT (procalcitonin) combined diagnosis test paper and preparation method thereof |
| CN108982828A (en) * | 2018-06-28 | 2018-12-11 | 长春万成生物电子工程有限公司 | A kind of quantum dot examines the preparation and application of test paper fastly |
| CN109738630B (en) * | 2019-01-14 | 2023-08-22 | 复旦大学附属华山医院 | High-sensitivity magnetic bead sorting combined colloidal gold marked exosome immune electron microscope method |
| CN110082523A (en) * | 2019-04-12 | 2019-08-02 | 西安交通大学苏州研究院 | A kind of quantum dot-preparation method of myoglobins antibody immune complex and the preparation method of test strips |
| CN110082521A (en) * | 2019-04-12 | 2019-08-02 | 西安交通大学苏州研究院 | A kind of quantum dot-preparation method of Creatine Kinase MB antibody immune complex and the preparation method of test strips |
| CN113189337B (en) * | 2021-04-28 | 2022-01-11 | 北京丹大生物技术有限公司 | Kit for specifically detecting creatine kinase isoenzyme |
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| CN101031798A (en) * | 2004-07-29 | 2007-09-05 | 瑞莱诊断体系有限公司 | Quantitative lateral flow system and assay |
| CN1811449A (en) * | 2006-02-23 | 2006-08-02 | 上海交通大学 | Method for detecting quantum dot mark fast immune chromatographic test paper bar |
| CN201087837Y (en) * | 2007-03-30 | 2008-07-16 | 万华普曼生物工程有限公司 | Human myohemoglobin/creatine kinase isoenzyme/myocardium calcium protein I diagnosis test paper |
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