CN102520173A - 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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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 detection by quantitative acute myocardial infarction mark creatine kinase isozyme.
Background technology
Current; Angiocardiopathy has worldwide become the maximum potential killer of adult; Only just have 7,000 ten thousand people of surpassing to have a heart disease in the U.S., annual nearly 6,000,000 people advance emergency ward because of pectoralgia, and the death toll of annual acute myocardial infarction AMI (AMI) has surpassed 500,000; In China, the morbidity rate of angiocardiopathy, the incidence of disease and hazards level thereof all are continuous ascendant trend.Add up in recent years, the mortality ratio of cardiovascular disease accounts for 40% in the human mortality, be higher than American-European countries, belongs to cardiovascular disease state occurred frequently.Therefore, only accomplish prevention early, early find treatment early, could prevent to greatest extent to disable, lethal effect, improve cardiovascular patient's prognosis and quality of life to greatest extent.
To angiocardiopathy, detection index commonly used is a myocardial enzymes series.Myocardium enzyme is meant the enzyme material in the cardiac muscle cell, has catalysis cardiac muscle cell metabolism and the effect of regulating cardiac muscle cell's electrical activity.Myocardial enzymes comprises aspartate transaminase (AST), lactic dehydrogenase (LDH), creatine kinase (CK), isoenzymes of creatine kinase MB (CK-MB) etc.If the cardiac muscle cell necroses, breaks, these enzymes will discharge in the middle of the 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 large tracts of land is downright bad as yet, in blood samples of patients, detect at the early stage cardiac muscle of morbidity, and the very first time is the clinical reliable data that provides.
The dimer that CK is made up of two kinds of different subunits (M and B), normal human tissue mainly contains 3 kinds of isodynamic enzymes, i.e. CK-MM, CK-BB, CK-MB like this.CK-MM mainly is present in the muscle cell, and CK-BB mainly is present in the brain cell, and CK-MB mainly is present among the cardiac muscle cell.
CK-MB is a myocardial damage mark commonly used at present, once once is being regarded as " goldstandard " of diagnosis of AMI.CK-MB increases in acute myocardial infarction AMI morbidity back 4～8h, and 24h peaking, couple of days recover normal.CK-MB has the important physical function because of it and clinical value has caused that people pay attention to widely and deep research.
Many traditionally with the CK-MB in the mensuration serum such as enzyme linked immunosorbent assay, chemoluminescence method and colloidal gold immunity chromatography.But the enzyme linked immunosorbent assay complicated operation detects length consuming time; Chemoluminescence method is high to technical requirement, is difficult in clinical labororatory, carrying out routine and carries out.Though it is few that colloidal gold immunity chromatography has a sample consumption, easy fast, cheap advantage; Yet when antigen or antibody content are extremely low in running into some sample, the color of collaurum will be very shallow, be difficult to judged result with the naked eye; Occur erroneous judgement easily, sensitivity is lower.
One Chinese patent application CN200610114997.9 has announced the chemical luminescent detecting method of creatine kinase isozyme in a kind of serum.This application patent mainly utilizes creatine kinase catalysis phosphocreatine and adenosine diphosphate (ADP) reaction to generate atriphos and creatine; Generate glycerol 3-phosphate with glycerokinase catalysis atriphos and glycerine again; Glycerol 3-phosphate is by the GPO oxidation and produce hydrogen peroxide; Through the effect of superoxide enzyme, hydrogen peroxide makes the luminol oxidation and is luminous again.One Chinese patent application numbers 200520041211.6 has disclosed a kind of Cardiovascular Disease Diagnosis And Predicting Multi-index Albumen Chip Measuring Kit Vessel; This kit adopts chemoluminescence method (like horseradish hydrogen 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.Though chemoluminescence method sensitivity is higher, repeatability and stability are all relatively poor, and instrument is complicated, expensive, can not single part detect, and have limited their widespread use.
U.S. Pat 60/576327 and 60/592202, and all colloidal gold immunochromatographimethod is detected in Chinese patents 200580025931.6 and to discuss.But than fluoroscopic examination, absorbance detection has defectives such as sensitivity is low, the range of linearity is narrow.
(quantum dots QDs) claims semiconductor nano again to quantum dot, stable, the nano microcrystalline body of size between 1～20nm normally be 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, the fluorescent emission wavelength-tunable, and coverage can be from the ultraviolet to the near-infrared region.(2) the absorbing light spectrum width of quantum dot and continuous can be realized an elementary excitation, and polynary emission is suitable for multi-color marking.(3) quantum dot has stronger optical stability.The light stability of CdSe/ZnS quantum dot is more than 100 times of rhodamine 6G.(4) the quantum dot molar absorptivity can be up to 106L/ (molcm), and fluorescence quantum yield high (50～80%), thereby can produce than the hyperfluorescence signal.(5) the quantum dot Stokes shift is bigger, and fluorescence lifetime length (20～50ns), make 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 and antibodies can be applicable to fluoroimmunoassay and detection.
Low to existing CK-MB detection method sensitivity, sensing range is narrow and quantitative deficiency such as inaccurate; The 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; A kind of detection method of quantum dot mark fast immune chromatographic test paper bar can be provided, CK-MB is carried out detection by quantitative.
Summary of the invention
The technical matters that the present invention will solve provides the fluorescence immune chromatography method of a kind of detection by quantitative CK-MB for to detecting that creatine kinase isozyme sensitivity is low, the range of linearity is narrow in the prior art and shortcoming such as instrument costliness.
For solving the problems of the technologies described above, technical scheme provided by the invention is:
The fluorescence immune chromatography method of a kind of detection by quantitative creatine kinase isozyme CK-MB may further comprise the steps:
Step 1) is connected to the quantum dot surface with specific effect between chemical crosslinking or biomolecule with the specific antibody of creatine kinase isozyme CK-MB, obtains the quantum dot of antibody modification, and the emission wavelength ranges of the quantum dot of said antibody modification is 550～1300nm;
The quantum dot of the antibody modification that step 2) step 1) is obtained is fixed on the label pad; And be fixed with the quantum dot of Quality Control molecular modification on the label pad simultaneously; The emission wavelength of the quantum dot of the antibody modification that the emission wavelength of the quantum dot of said Quality Control molecular modification and step 1) obtain is different; And wavelength coverage is 550～1300nm; On chromatographic film, be respectively equipped with quantitative band and two quality control bands, wherein quality control band is fixed with the biomolecule that can combine with said Quality Control molecular specificity, and quantitatively band is fixed with corresponding CK-MB and the specific antibodies different antigenic determinants of the said specific antibody of step 1);
Step 3) is built into the fluorescence immune chromatography test paper bar with sample pad, label pad, chromatographic film, adsorptive pads and base plate, and wherein said chromatographic film is the hypofluorescence chromatographic film, and the base plate tool hangs down fluorescent characteristic;
Behind the step 4) test strips immunochromatography, detect quantitatively band and quality control band fluorescence signal intensity, and proofread and correct quantitatively band fluorescence signal intensity, and then realize the detection by quantitative of CK-MB with the quality control band fluorescence signal intensity.
The method of the fluorescence immune chromatography of a kind of detection by quantitative CK-MB provided by the invention, be a kind of be fluorescence signal with the quantum dot, adopt double-colored labelling technique, on immuno-chromatographic test paper strip, realize the method that the CK-MB rapid sensitive detects.
The fluorescence immune chromatography method of detection by quantitative CK-MB of the present invention can solve that background and the difficult differentiation of signal in the existing fluorescence immune chromatography technology, sensitivity are low, the indefinite deficiency of fluorescent quantitation method and defective, can realize the detection to micro-sample.Because it is very little that the quantum dot fluorescence stimulated luminescence disturbs, sensitivity improves greatly, and its sensitivity is 10～1000 times with conventional dyes and coloured markers tests method.
The present invention adopts specific effect between chemical crosslinking or biomolecule that the specific antibody of CK-MB is connected to the quantum dot surface, obtains the quantum dot of antibody modification, and wherein specific antibody is the antibody of one or more epitopes of corresponding CK-MB.
Chemical crosslinking among the present invention is: when there is reactive group on the quantum dot surface, in the time of can directly reacting with antibody or Quality Control molecule, need not use chemical cross-linking agent, on the contrary surperficial to quantum dot with chemical cross-linking agent antibody modification.
Adopt chemical crosslink technique to be in an embodiment of the present invention: to utilize crosslinking chemicals such as 1-ethyl-3-(3-dimethyl amine propyl group) carbodiimides (EDC)/N-hydroxy-succinamide (NHS), glutaraldehyde that functional group's (like carboxyl, amino) on quantum dot surface is connected with functional group's (like amino, carboxyl, aldehyde radical etc.) on protein molecule (like antigen, antibody etc.) surface to the method that quantum dot carries out the protein molecule modification.
As preferably, in one embodiment of the invention, adopt the EDC/NHS cross-linking method that quantum dot is modified.Wherein the EDC/NHS cross-linking method to the general step that quantum dot carries out the protein molecule modification is: quantum dot solution is mixed with EDC and NHS; Add a certain amount of protein then; With the damping fluid is reaction medium, cultivates 4h, adds the sealing of L-glycocoll; With mode purifying such as chromatogram, chromatographic column or ultrafiltration be centrifugal, thereby obtain the quantum dot of protein modification.
Specific effect comprises biotin-avidin system and Ag-Ab system between biomolecule.As preferably, in another embodiment of the present invention, adopt the combination of biotin-avidin system that protein is carried out the quantum dot modification, this combination has the effect of amplifying signal.Be specially: with EDC Streptavidin is connected to the quantum dot surface, biotin is connected to the protein molecule surface, through the interaction between Avidin-biotin, is connected to the quantum dot surface to protein molecule.
In order to improve the discrimination with background, it is the quantum dot of 550～1300nm that the present invention selects wavelength of transmitted light for use.Because under ultraviolet irradiation, the fluorescence intensity of chromatographic film, base plate and button card far is being better than more than the 550nm below the 550nm, thereby produces certain influence when low concentration CK-MB detected, so but the preferred emission wavelength greater than the quantum dot of 550nm.In addition chromatographic film, base plate and button be stuck near infrared region (750～1300nm) fluorescence intensities extremely a little less than, and combine the quantum dot synthetic technology, (750～1300nm) can further improve sensitivity to the quantum dot of preferred near-infrared wavelength.
The used quantum dot of the present invention comprises quantum dot or several kinds of compound quantum dots that compound is assembled into that the simplification compound forms, and quantum dot has stronger light stability.The compound that forms quantum dot is from the group that ZnS, CdS, HgS, MgS, CdSe, MgSe, ZnSe, PbSe, PbSe, CdTe, MgTe, ZnTe, HgTe, InAs, InP form, to select, but and 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 for use.But in the present invention,, adopt double-colored labelling technique, promptly select the different quantum dots of two kinds of emission wavelengths mark Quality Control molecule and antibody respectively for use for the influence of the quantum dot that weakens the Quality Control molecular modification to quantitative band and background signal.
As preferably, in an embodiment of the present invention, to use the CdSe/ZnS of synthetic 570nm, 600nm and 650nm of organic phase method, and transfer to quantum dot water-solublely by fat-soluble, quantum dot fluorescence does not have significant change in this process; Use the InAs of the synthetic 900nm of water method.
As preferably, in one embodiment of the invention, the quantum dot emission wavelengths of the antibody modification of CK-MB is 650nm in the label pad, and the quantum dot emission wavelengths of Quality Control molecular modification is 570nm.
As preferably, in another embodiment of the present invention, the quantum dot emission wavelengths of the antibody modification of CK-MB is 900nm in the label pad, and the quantum dot emission wavelengths of Quality Control molecular modification is 600nm.
In order to reduce the influence to the 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, ability good discrimination signal and background, and then improve detection sensitivity.
As preferably, in embodiments of the present invention, base plate (8) is a black, and the 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), then wetting hole can be at nearly sample pad one end of chromatographic film or at nearly adsorptive pads one end.
For being different from the detection band of colloidal gold immuno-chromatography test paper strip; Among the present invention on the chromatographic film zone of fixation of C K-MB antibody be called quantitative band; The concentration that is used for accurate detection by quantitative CK-MB, and do interior Quality Control with quality control band and proofread and correct quantitatively band, to weaken the influence 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 preparatory profit immunochromatography to carry out CK-MB and detects.Wherein moisten immuno-chromatographic test paper strip in advance two kinds of direct and indirect profits are in advance arranged; In one embodiment; Directly the 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) in advance, and is as shown in Figure 1.On 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.On the 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 combine with the Quality Control molecular specificity, quantitatively band be fixed with corresponding CK-MB with above-mentioned label pad in the specific antibodies of the different antigenic determinants of specific antibody.CK-MB can with the antibody on quantum dot surface with quantitatively with on antibody formation double-antibody sandwich structure.And the bar number through increasing quantitative band can enlarge sensing range, avoids the HOOK effect.
In another embodiment of the present invention, the 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) in advance indirectly, and is as shown in Figure 2.
In the embodiment of the invention, the sample that the inventor selects for use respectively is whole blood and serum, and sample further comprises blood plasma.When sample is whole blood; The fluorescence immune chromatography test paper bar also is included in the filtering membrane that is provided with between sample pad and the chromatographic film; Be used to solidify, filtration cell (being mainly red blood cell); This filtering membrane can contact with the direct capillary action of sample pad and label pad respectively, and is as shown in Figure 1, also can contact with the direct capillary action of label pad and chromatographic film.
The embodiment of the invention adopts in advance, and the profit immune chromatography method carries out detection by quantitative to the CK-MB in the sample.Wherein moistening immunochromatography in advance is: the damping fluid that adds certain volume behind the wetting certain hour, adds sample in the sample pad on chromatographic film, and sample flows to the adsorptive pads direction along chromatographic film.The purpose of profit is preparatory wetting chromatographic film in advance, and the sealing nonspecific binding site so that quantum dot-labeled thing evenly passes through chromatographic film, and reduces the non-specific adsorption on chromatographic film, and slows down the sample flowing velocity, combines thereby increase specificity.Wherein the damping fluid volume is generally 20～80 μ l, and wetting time is generally 30 seconds～2 minutes, and the sample chromatography time was generally 8～25 minutes.
Preparatory 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, the 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 through 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 invention, using damping fluid is the phosphate buffer that comprises the pH9.0 of 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 the fluorescent quantitation appearance.Wherein the 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.The 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 finished, under light source activation, the fluorescence signal that test strips produces through filter light module filtering parasitic light and background fluorescence, arrived photoelectric conversion module, converts digital signal into.Wherein the quality control band that obtains with the quantitative fluorescence intensity of band certain correlativity is arranged, 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 surpasses the acceptable value of fluorescent quantitation appearance inner setting, explain that testing result is invalid; Under the effective prerequisite of testing result, quantitatively be with fluorescence intensity and quality control band ratio high more, expression CK-MB concentration is high more, otherwise low more.
The present invention adopts the fluorescent quantitation appearance 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 pairing correction fluorescence signal intensity (F), and relational expression is F=f (c).Proofreading and correct fluorescence signal intensity is F=α F
Quantitatively be with/ F
Quality control band, wherein α is a correction coefficient, influenced by temperature, humidity and matrix etc.Detect sample then, the establishing criteria curve obtains CK-MB concentration in the sample.
The present invention also provides a kind of fluorescence immune chromatography kit of detection by quantitative creatine kinase isozyme, comprises damping fluid, button card (11) and fluorescence immune chromatography test paper bar, and is as shown in Figure 3.This kit adopts indirectly profit immune chromatography method in advance.Button card (11) is the external shell structure of fluorescence immune chromatography test paper bar, comprises appearance hole (12), form (14) and wetting hole (13), and the low fluorescent characteristic of tool, is preferably not contain fluorescer.
Fluorescence immune chromatography test paper bar structure is as shown in Figure 2, and it comprises sample pad (1), label pad (3), chromatographic film (4), wetting pad (9), connection gasket (10), adsorptive pads (7) and base plate (8).Chromatographic film (4) comprises quality control band (5) and quantitatively is 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 the base plate (8), and sample pad (1) is connected in label pad (3), and are positioned at the below in appearance hole (12); Label pad (3) is connected in chromatographic film (4); Be fixed with quantitatively band (6) of two quality control bands (5) and two on the chromatographic film (4), 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), and wetting pad (9) is connected in connection gasket (10), and connection gasket (10) is connected in adsorptive pads (7).
When carrying out the whole blood sample chromatography, test strips also comprises filtering membrane (2), and concrete structure is as shown in Figure 1, and wherein test strips comprises sample pad (1), label pad (3), chromatographic film (4), adsorptive pads (7), base plate (8) and filtering membrane (2).Filtering membrane (2) contacts with chromatographic film (4) capillary action with sample pad (1), and promptly filtering membrane can be connected between sample pad (1) and the label pad (3), also can be connected in label pad (3) and chromatographic film (4).
The preparatory profit immuno-chromatographic test paper strip that the present invention makes up; 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; When being used to reduce to drip damping fluid; Damping fluid with wetting chromatographic film (4), and reduces non-specific adsorption to the impact of chromatographic film.
And connection gasket (10) contacts with adsorptive pads (7) capillary action with wetting pad (9) respectively, has absorption speed slowly, the water absorbing capacity to reduce when wetting; Impel the abundant wetting chromatographic film of damping fluid (4); When adding the sample chromatography, adsorptive pads (7) can pass through connection gasket (10) suction, impels the sample chromatography.
The chromatographic film of test strips (4) has the hypofluorescence characteristic, does not preferably contain fluorescer, its fluorescence noise greater than 550nm the time very a little less than, to reduce the influence that the low concentration object is detected.
Major advantage of the present invention is following:
1) the fluorescence immune chromatography method of detection by quantitative CK-MB of the present invention adopts the label of quantum dot as specific antibody; Compare with organic fluorescent dye, have luminous intensity height, exciting light spectrum width, an 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 the discrimination with 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 greater than 550nm the time; To reduce the influence that the quantum dot fluorescence signal is obtained; Thereby guarantee to obtain high fluorescence signal-to-background ratio, and then reach the purpose that improves sensitivity.
3) the present invention adopts the fluorescent quantitation appearance 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 realizes the detection by quantitative of CK-MB according to the typical curve that the fluorescent quantitation appearance obtains.Wherein typical curve is the relation curve of standard items series concentration (c) and pairing correction fluorescence signal intensity (F), and relational expression is F=f (c).Proofreading and correct fluorescence signal intensity is F=α F
Quantitatively be with/ F
Quality control band, wherein α is a correction coefficient, influenced by temperature, humidity and matrix etc.Detect sample then, the establishing criteria curve obtains CK-MB concentration in the sample.
4) the present invention utilizes the double-colored labelling technique of quantum dot; Reduce the Quality Control molecule to the influence that CK-MB detects, also can reduce the influence of the quantum dot of Quality Control molecular modification to quantitative band and chromatographic film background, method is simply quick; Highly sensitive, help highly sensitive detection by quantitative.
5) the present invention's preparatory profit immunochromatography technique capable of using strengthens specificity and combines, and reduces non-specific adsorption, strengthens the detection sensitivity of kit, help when CK-MB content is extremely low in the sample accurately quantitatively.
6) the inventive method is simple, quick, accurate, cost is low, and sensitivity is very high.Detect creatine kinase isozyme with traditional chemoluminescence method and ELISA and compare, have easy and simple to handle, fast, do not need expensive detection equipment, be fit to advantages such as single part or short run detection; Compare with conventional colloidal gold immunochromatographimethod method, the present invention have the mark good stability, non-specific low, highly sensitive, the range of linearity is wide and advantage such as quantitatively accurate.
Description of drawings
Fig. 1 is the assembling synoptic diagram that directly moistens immuno-chromatographic test paper strip in advance, 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 quantitatively to be with, and 7 is adsorptive pads, and 8 is base plate;
Fig. 2 is the assembling synoptic diagram that moistens immuno-chromatographic test paper strip indirectly in advance, 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 quantitatively to be with, and 7 is adsorptive pads, and 8 is base plate, and 9 is wetting pad, and 10 is connection gasket;
Fig. 3 is a fluorescence immune chromatography kit synoptic diagram, and wherein 11 are the button card, and 12 is last appearance hole, and 13 is wetting hole, and 14 is form, and 5 is quality control band, and 6 is quantitative band.
Embodiment
The invention discloses the fluorescence immune chromatography method of a kind of detection by quantitative creatine kinase isozyme CK-MB, those skilled in the art can use for reference this paper content, suitably improve technological parameter and realize.Special needs to be pointed out is that all similarly replace and change apparent to those skilled in the art, they all are regarded as and are included in the present invention.Method of the present invention and kit are described through preferred embodiment; The related personnel obviously can be in not breaking away from content of the present invention, spirit and scope to method as herein described with kit is changed or suitably change and combination, realize and use technology of the present invention.
Technical scheme provided by the invention is:
Step 1) is connected to the quantum dot surface with specific effect between chemical crosslinking or biomolecule with the specific antibody of creatine kinase isozyme CK-MB, obtains the quantum dot of antibody modification, and the emission wavelength ranges of the quantum dot of said antibody modification is 550～1300nm;
The quantum dot of the antibody modification that step 2) step 1) is obtained is fixed on the label pad; And be fixed with the quantum dot of Quality Control molecular modification on the label pad simultaneously; The emission wavelength of the quantum dot of the antibody modification that the emission wavelength of the quantum dot of said Quality Control molecular modification and step 1) obtain is different; And wavelength coverage is 550～1300nm; On chromatographic film, be respectively equipped with quantitative band and two quality control bands, wherein quality control band is fixed with the biomolecule that can combine with said Quality Control molecular specificity, and quantitatively band is fixed with corresponding CK-MB and the specific antibodies different antigenic determinants of the said specific antibody of step 1);
Step 3) is built into the fluorescence immune chromatography test paper bar with sample pad, label pad, chromatographic film, adsorptive pads and base plate, and wherein said chromatographic film is the hypofluorescence chromatographic film, and the base plate tool hangs down fluorescent characteristic;
Behind the step 4) test strips immunochromatography, detect quantitatively band and quality control band fluorescence signal intensity, and proofread and correct quantitatively band fluorescence signal intensity, and then realize the detection by quantitative of CK-MB with the quality control band fluorescence signal intensity.
The principle of fluorescence immune chromatography of the present invention and detection method are: the content that adopts the creatine kinase isozyme (CK-MB) in fluorescence immune chromatography technology and the double antibody sandwich method principle detection by quantitative people sample (whole blood, serum or blood plasma).During detection; At first in wetting hole, drip damping fluid, then sample drop be added in the appearance hole, sample earlier with label pad in quantum dot-labeled thing mix mutually; And along chromatographic film to adsorptive pads direction capillary moving, quantitative band and quality control band on the chromatographic film of flowing through respectively.If contain CK-MB in the sample, then the CK-MB antibody with the quantum dot surface combines, and when chromatography to quantitative band, can be encapsulated the antibody capture in this band in advance, thereby constitute the double-antibody sandwich compound, and quantum dot stays in quantitative band.Under the light source activation, adopt the fluorescent quantitation appearance to obtain the fluorescence signal intensity of quantitative band and quality control band, the typical curve that obtains according to the fluorescent quantitation appearance, and then CK-MB concentration in the analyzing samples.
In order to make those skilled in the art understand technical scheme of the present invention better, the present invention is done further detailed description below in conjunction with specific embodiment.
Embodiment 1: with chemical crosslink technique antibody modification is directly moistened the detection by quantitative of immunochromatography to CK-MB in advance in quantum dot and employing
(1) modification of quantum dot and antibody
Be antigen with people CK-MM and CK-BB respectively, can immunity obtain goat-anti people CK-MM antibody and CK-BB antibody.Because CK-MB has M and two subunits of B, then CK-MM antibody and CK-BB antibody can combine with M and the B subunit of CK-MB respectively, form the double-antibody sandwich structure.，
In pH 7.4 phosphate buffers, adding the 60pmol emission wavelength is 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, mixes and under room temperature, reacts 4h, adds 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
With the even above-mentioned two kinds of quantum dot-labeled things of 1: 1 mixed of mol ratio; Wherein contain surfactants such as 1～15% sucrose, 0.01～2% bovine serum albumin(BSA) (BSA) and polysorbas20, Tween 80, triton x-100 in the mixed liquor; Wherein surface-active contents is between 0.01～2%; Evenly be sprayed on then on the label pad, 37 ℃ of dry back sealings are preserved down for 4 ℃.
On the hypofluorescence chromatographic film, draw four parallel bands to draw the film appearance, band is 5mm at interval, and the bar bandwidth all is about 1mm.Wherein two of both sides is quality control band, and middle two is quantitatively to be with.Spray rabbit immunoglobulin (rabbit igg) on the quality control band, concentration is respectively 0.2～1mg/ml and 1～3mg/ml; Quantitatively be with spraying CK-MM how anti-, concentration is respectively 0.2～1mg/ml and 0.5～2mg/ml.37 ℃ of dry back sealings are preserved down for 4 ℃.
On black floor, paste chromatographic film (long 30cm, wide 2.5cm), label pad (long 30cm successively; 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).All will closely link to each other between film and the film, process the big plate of preparatory profit fluorescence immune chromatography test paper, synoptic diagram is as shown in Figure 1.With cutting cutter the big plate of the test paper that pastes vertically is cut into the wide test strips of 4mm, puts into low fluorescence button card and be assembled into the immune chromatography reagent kit that does not contain damping fluid, the sealing of dry back is configured to the fluorescence immune chromatography kit jointly with damping fluid, and 4 ℃ keep in Dark Place.
(3) pattern detection
Make dilution with normal person's whole blood, CK-MB standard items preparation series concentration standard solution is following: 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.Earlier 50 μ l damping fluids are added drop-wise on the chromatographic film during chromatography, after 1 minute, negative whole blood of 100 μ l or standard solution are added drop-wise in the appearance hole; After 13 minutes; Detect (each sample is measured 3 times with 3 test strips respectively, averages), drawing standard curve with the fluorescent quantitation appearance.
50 μ l damping fluids are added drop-wise on the chromatographic film, after 1 minute, 100 μ l whole blood samples are added drop-wise in the appearance hole, detect with the fluorescent quantitation appearance after 13 minutes, the establishing criteria curve obtains the concentration of CK-MB in the sample.
The result shows that its lowest detection is limited to 0.5ng/mL, the minimum 1.0ng/mL that quantitatively is limited to, in the detection by quantitative scope, the HOOK effect does not appear in coefficient R 2＞0.99, and batch in criticize between repeatability all better, can be cardiovascular disease diagnosis reference be provided.
Embodiment 2: with avidin-biotin complex technology antibody modification is moistened the detection by quantitative of immunochromatography to CK-MB indirectly in advance in quantum dot and employing
(1) the synthetic and modification of quantum dot
According to the method for embodiment 1, the quantum dot that the preparation Streptavidin is modified.In like manner obtain the quantum dot that goat anti-rabbit antibody is modified.
The CK-BB monoclonal antibody is diluted to 1mg/ml with 0.1mol/L sodium bicarbonate buffer liquid (pH 8.0); 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; Reaction added 1mol/L NH after 4 hours under the room temperature
4Cl at room temperature stirred 10 minutes.With the ultrafiltration centrifugal purification,, obtain biotinylated monoclonal antibody to remove free biotin.
With quantum dot and the biotinylated monoclonal antibody that 1: 3～1: 12 mixed Streptavidin is modified, obtain the quantum dot that the 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
Method according to embodiment 1 is sprayed at label pad to quantum dot-labeled thing, is sprayed at antibody and Quality Control molecule the quantitative band and the quality control band of chromatographic film.
On black floor, paste successively and go up 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).All will closely link to each other between film and the film, process the big plate of fluorescence immune chromatography test paper, synoptic diagram is as shown in Figure 2.With cutting cutter the big plate of the test paper that pastes vertically is cut into the wide test strips of 4mm; Put into low fluorescence button card and be assembled into the immune chromatography reagent kit that does not contain damping fluid, as shown in Figure 3, the sealing of dry back; Be configured to the fluorescence immune chromatography kit jointly with damping fluid, 4 ℃ keep in Dark Place.
(3) pattern detection
Carry out the 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.Earlier 50 μ l damping fluids are added drop-wise in the wetting hole during chromatography; After 1 minute, 100 μ l negative serums or standard solution are added drop-wise in the appearance hole, (each sample is measured 3 times with 3 test strips respectively with the detection of fluorescent quantitation appearance after 13 minutes; Average), the drawing standard curve.
50 μ l damping fluids are added drop-wise in the wetting hole, after 1 minute, 100 μ l serum samples are added drop-wise in the appearance hole, detect with the fluorescent quantitation appearance after 13 minutes, the establishing criteria curve obtains the concentration of CK-MB in the sample.
The result shows that its lowest detection is limited to 0.1ng/mL, the minimum 0.25ng/mL that quantitatively is limited to, and in the detection by quantitative scope, coefficient R
2＞0.99, the HOOK effect does not appear, and batch in criticize between repeatability all better, can be cardiovascular disease diagnosis reference be provided.
The above only is a preferred implementation of the present invention; Should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention; Can also make some improvement and retouching, these improvement and retouching also should be regarded as protection scope of the present invention.
Claims (17)
1. the fluorescence immune chromatography method of a detection by quantitative creatine kinase isozyme CK-MB is characterized in that, may further comprise the steps:
Step 1) is connected to the quantum dot surface with specific effect between chemical crosslinking or biomolecule with the specific antibody of creatine kinase isozyme CK-MB, obtains the quantum dot of antibody modification, and the emission wavelength ranges of the quantum dot of said antibody modification is 550～1300nm;
The quantum dot of the antibody modification that step 2) step 1) is obtained is fixed on the label pad; And be fixed with the quantum dot of Quality Control molecular modification on the label pad simultaneously; The emission wavelength of the quantum dot of the antibody modification that the emission wavelength of the quantum dot of said Quality Control molecular modification and step 1) obtain is different; And wavelength coverage is 550～1300nm; On chromatographic film, be respectively equipped with quantitative band and quality control band, wherein quality control band is fixed with the biomolecule that can combine with said Quality Control molecular specificity, and quantitatively band is fixed with corresponding CK-MB and the specific antibodies different antigenic determinants of the said specific antibody of step 1);
Step 3) is built into the fluorescence immune chromatography test paper bar with sample pad, label pad, chromatographic film, adsorptive pads and base plate, and wherein said chromatographic film is the hypofluorescence chromatographic film, and the base plate tool hangs down fluorescent characteristic;
Behind the step 4) test strips immunochromatography, detect quantitatively band and quality control band fluorescence signal intensity, and proofread and correct quantitatively band fluorescence signal intensity, and then realize the detection by quantitative of CK-MB with the quality control band fluorescence signal intensity.
2. fluorescence immune chromatography method according to claim 1 is characterized in that, the emission wavelength of the quantum dot of the said antibody modification of step 1) is 650nm, step 2) emission wavelength of the quantum dot of said Quality Control molecular modification is 570nm.
3. fluorescence immune chromatography method according to claim 1 is characterized in that, the emission wavelength of the quantum dot of the said antibody modification of step 1) is 900nm, step 2) emission wavelength of the quantum dot of said Quality Control molecular modification is 600nm.
4. fluorescence immune chromatography method according to claim 1 is characterized in that, quantum dot or several kinds of composite quantum dots that compound is formed that the said quantum dot of step 1) forms for the simplification compound.
5. fluorescence immune chromatography method according to claim 4; It is characterized in that; Said compound is to be selected among ZnS, CdS, HgS, MgS, CdSe, MgSe, ZnSe, PbSe, PbSe, CdTe, MgTe, ZnTe, HgTe, InAs, the InP any one or a few, but and doped with Cu, Mn and Hg.
6. fluorescence immune chromatography method according to claim 1 is characterized in that, the said chromatographic film of step 3) fluorescence greater than 550nm the time is very weak or do not contain fluorescer.
7. fluorescence immune chromatography method according to claim 1 is characterized in that, the said base plate of step 3) is a black, and the surface is with adhesive sticker, and both all do not contain fluorescer.
8. fluorescence immune chromatography method according to claim 1 is characterized in that, the said fluorescence immune chromatography test paper bar of step 3) also comprises the filtering membrane that can make liquid and cell separation in the sample.
9. fluorescence immune chromatography method according to claim 1; It is characterized in that; The said immunochromatography of step 4) is to moisten immunochromatography in advance, wherein moistens immunochromatography in advance for moistening chromatographic film in advance with damping fluid earlier, adds sample again in sample pad; Sample flow is carried out fluorescent quantitation and is detected behind quantitative band and quality control band.
10. fluorescence immune chromatography method according to claim 9 is characterized in that, said preparatory profit chromatographic film is for directly to drip damping fluid in chromatographic film.
11. fluorescence immune chromatography method according to claim 9; It is characterized in that; When said preparatory profit chromatographic film is when dripping damping fluid in chromatographic film indirectly; The said fluorescence immune chromatography test paper bar of step 3) 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.
12. fluorescence immune chromatography method according to claim 9 is characterized in that, said damping fluid is the alkaline buffer of pH 7.2～11.
13. fluorescence immune chromatography method according to claim 12 is characterized in that, said alkaline buffer is the alkaline buffer that comprises bovine serum albumin(BSA), casein and surfactant.
14. fluorescence immune chromatography method according to claim 12 is characterized in that, said alkaline buffer is the phosphate buffer that comprises the pH9.0 of bovine serum albumin(BSA) and polysorbas20.
15. the fluorescence immune chromatography kit of a detection by quantitative creatine kinase isozyme CK-MB; Comprise damping fluid, button card (11) and fluorescence immune chromatography test paper bar; It is characterized in that; Said button card (11) is the external shell structure of fluorescence immune chromatography test paper bar, comprises appearance hole (12), form (14) and wetting hole (13); Said 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 the base plate (8), and sample pad (1) is connected in label pad (3), and are positioned at the below in appearance hole (12); Label pad (3) is connected in chromatographic film (4); Be fixed with quantitatively band (6) of two quality control bands (5) and two on the chromatographic film (4), 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), and wetting pad (9) is connected in connection gasket (10), and connection gasket (10) is connected in adsorptive pads (7).
16. fluorescence immune chromatography kit according to claim 15 is characterized in that, said 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.
17. fluorescence immune chromatography kit according to claim 15 is characterized in that, said button card (11) has low fluorescent characteristic or does not contain fluorescer.
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