CN104502595A - Troponin detection method and kit thereof - Google Patents
Troponin detection method and kit thereof Download PDFInfo
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- CN104502595A CN104502595A CN201410629661.0A CN201410629661A CN104502595A CN 104502595 A CN104502595 A CN 104502595A CN 201410629661 A CN201410629661 A CN 201410629661A CN 104502595 A CN104502595 A CN 104502595A
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Abstract
The invention relates to a troponin detection method and a kit thereof, and especially relates to a dual wavelength fluorescence immunity chromatography detection method for troponin and its detection kit. The method comprises the following steps: 1)preparing an immunity chromatography test strip; 2)preparing a freeze-drying probe, 3)preparing sample diluents and 4)examining the sample. The troponin detection method and the kit have the advantages of high sensitivity, high accuracy, simple operation and low cost.
Description
Technical field
The present invention relates to a kind of detection method and kit of troponin, particularly relate to a kind of double wave length fluorescent immunochromatography detection method and detection kit thereof of troponin.
Background technology
Cardiovascular and cerebrovascular disease is the first kind major disease threatening human life's health.According to World Health Organization's recent statistics, the whole world has 1,670 ten thousand people to die from all kinds of cardiovascular and cerebrovascular disease every year, and this numeral accounts for 29.2% of all disease deaths, wherein has over halfly to die from acute myocardial infarction AMI (AMI).Over nearly 10 years, the morbidity rate of China's acute myocardial infarction AMI, the incidence of disease and risk factor levels thereof all in obvious ascendant trend, close to international average level.Therefore, only accomplish early prevention, early find to give treatment to early, could prevent to greatest extent disabling, lethal effect, improve prognosis and the quality of life of cardiovascular patient to greatest extent.
Because the symptom of AMI is varied, complication is many to be also existed sizable mistaken diagnosis clinical and fail to pinpoint a disease in diagnosis, the myocardial damage having some to exist, because atypical outbreak is ignored by doctor and patient, is not carried out treatment timely and has been occurred the threat to life that again shows effect.In view of the feature that AMI morbidity is anxious, mortality ratio is high, and the early diagnosis of AMI and in time treatment are for the importance reducing AMI mortality ratio, develop a kind of highly sensitive, specificity good, clinical diagnosis coincidence rate is high, detect the developing direction that acute myocardial infarction detection method fast and easily becomes acute myocardial infarction clinical diagnosis.
In recent years, the research emphasis of diagnosis AMI is transferred on myocardium protein mark by clinical chemistry men gradually, wherein cardiac troponin (cTn) is the contractile protein be uniquely present in cardiac muscle, myocardial necrosis is had to the Sensitivity and Specificity of height.Troponin is made up of the subunit that three structures are different, i.e. TnT (cTnT), Troponin I (cTnI) and TnC (cTnC).T and I subunit structure in cardiac muscle is different from other musculature, and because molecular weight is little, after morbidity, concentration in blood will raise rapidly.Prove through international clinical chemistry and laboratory federation (IFCC) and the sick association (ESC) of heart of Europe: cTnI and cTnT is in clinical diagnosis, and cTnI is higher than cTnT specificity.Its principle is that cTnT and skeletal troponin have cross reaction, and also has unaccountable cTnT to raise phenomenon when kidney injury.Thus, cTnI uniquely exists in cardiac muscle cell to diagnose AMI sensitivity and the highest Biochemical Indices In Serum of specificity up to now, is extensively studied the determinacy mark as myocardial damage, is just becoming " goldstandard " of current diagnosis AMI.In normal human blood, the content of cTnI is generally lower than 0.4ng/mL, after AMI occurs, cTnI can raise fast in 6-8 hour, peak value is reached in 18-24 hour, raise multiple and can reach 30-200 doubly, sustainable 10-15 talent recovers normal, and namely cTnI has time of occurrence early and the advantage that the duration is long in blood, can be used as AMI early diagnosis index, be proved to be one of special, the most responsive blood serum designated object of myocardial damage.
Existing cTnI detection method mainly contains enzyme linked immunosorbent assay, chemoluminescence method and colloid gold immune analytic approach.The lowest detection sensitivity of ELISA can reach 0.2ng/mL, but complicated operation, the professional through screwing up discipline need complete, and detect length consuming time, reach 5-6 hour; Chemoluminescence method is the labelling immunoassay technology of trace antigen or the antibody that luminesceence analysis and immune response are combined and set up, its sensitivity is up to 0.03ng/mL, detection time is also shorter, about 1-2 hour, but need to adopt special main equipment, high to technical requirement, in clinical labororatory, not easily carry out routine carry out; Although it is few that colloidal gold immunity chromatography has amount of samples, fast easy, cheap advantage, but when running into that in some sample, target analytes content is lower, the color of collaurum will be very shallow, be difficult to the naked eye judged result, easily occur erroneous judgement, sensitivity is low.
Chinese patent publication No. is the patent of invention of CN102192986B, although disclose a kind of method of nano-colloid immunochromatographyassay assay troponin, top layer is adopted to introduce organosilicon parcel nm of gold particulate and the antibody response of the functional groups such as amido, carboxyl, hydroxyl, form the mark that stable covalent bond carries out antibody, replace traditional collaurum for immune response, prepare immunoreagent.But the preparation technology of the nm of gold particulate of surface-functionalized organosilicon parcel is more complicated, production cost is high; Detectability only has 0.4ng/ml, can not meet the needs of a part of patient; Discernable by eye is adopted easily to occur erroneous judgement; Detect nm of gold particulate and the shortcomings such as sensitivity is low, quantitatively inaccurate are easily occurred to the scattering of light.Chinese patent publication No. is the patent of invention of CN102565422B, although disclose the kit that a kind of fluorescence immune chromatography method detects troponin, utilizes the excellent fluorescent characteristic of quantum dot, carries out fluorescent quantitation detection in conjunction with Bicolor-code technology.But it is not eliminated the material easily producing fluorescence signal in complicated blood sample, easily produces false Yangxin number in some complicated component samples.Chinese patent publication No. is the patent of invention of CN102539784B, although disclose a kind of immunoturbidimetry to detect troponin, antibody compositions and polystyrene latex microparticles are carried out coupling, form aggregated particle with corresponding antigens generation immune response in sample to be checked, measure the content that its turbidity can draw cTnI.But it detects the impact being vulnerable to the muddy serum such as piarhemia, easily occur quantitatively inaccurate; Detection needs Biochemical Analyzer, and instrument is more expensive, and except needs appointees operate, also need special place to place instrument, operating process is more complicated, and detection time is also longer.
Because above-mentioned defect, the design people, actively in addition research and innovation, to founding a kind of highly sensitive, accuracy is high, simple to operate, cost is low detection method and kit, make it have more value in industry.
Summary of the invention
For solving the problems of the technologies described above, the object of this invention is to provide a kind of detection method and kit of highly sensitive, accuracy is high, simple to operate, cost is low troponin.
The double wave length fluorescent immunochromatography detection method of troponin of the present invention, comprises the following steps:
1) immuno-chromatographic test paper strip preparation: on the detection line that troponin monoclonal antibody and chicken IgY are coated on chromatographic test paper respectively and control line, obtains immuno-chromatographic test paper strip after drying, cutting;
2) freeze-drying probe preparation: the fluorescent latex particles solution of two kinds of different wavelength of transmitted light is mixed in proportion, adds activator after mixing and react a period of time, centrifugal, washing after obtain activate fluorescent latex particles;
By troponin monoclonal antibody and goat-anti chicken IgY respectively in proportion with activation fluorescent latex particles coupling reaction a period of time, obtain troponin monoclonal antibody fluorescent latex particles and goat-anti chicken IgY fluorescent latex particles;
Subsequently troponin monoclonal antibody fluorescent latex particles is mixed to get by a certain percentage with goat-anti chicken IgY fluorescent latex particles and mixes fluorescent latex particles, add the dilution of diatom sugar aqueous solution subsequently, after freeze-drying, save as freeze-drying probe;
3) sample diluting liquid preparation: sample diluting liquid is the damping fluid containing bovine serum albumin(BSA), protein protective agent, surfactant and antiseptic;
4) sample survey: mixed with freeze-drying probe by blood serum sample after disperseing a period of time, takes out and a certain amount ofly adds in sample diluting liquid, to be mixed evenly after drop on immuno-chromatographic test paper strip and carry out immunochromatography reaction; Under fluorescence detector, adopt the two kind wavelength of transmitted light corresponding with two kinds of fluorescent latex particles to carry out fluoroscopic examination subsequently.
Concrete, in described freeze-drying probe preparation steps, the fluorescent latex particles solution of two kinds of different wavelength of transmitted light is mixed in 1:5 ~ 5:1 ratio, 0.5 ~ 2h is reacted under adding activator 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride room temperature after mixing, wherein, the weight ratio of 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and fluorescent latex particles is 0.5:100 ~ 5:100.
Concrete, in described freeze-drying probe preparation steps, the wavelength of transmitted light of described two kinds of fluorescent latex particles is respectively 500 ~ 590nm and 600 ~ 740nm.
Concrete, in described freeze-drying probe preparation steps, troponin monoclonal antibody is 0.01:100 ~ 0.05:100 with the blending ratio of activation fluorescent latex particles, coupling reaction 2 ~ 5h under room temperature after mixing; Goat-anti chicken IgY is 0.01:100 ~ 0.05:100 with the blending ratio of activation fluorescent latex particles, coupling reaction 2 ~ 5h under room temperature after mixing.
Concrete, in described freeze-drying probe preparation steps, in ratio mixing troponin monoclonal antibody fluorescent latex particles and the goat-anti chicken IgY fluorescent latex particles of 3:1 ~ 6:1, obtain mixing fluorescent latex particles, by the diatom sugar aqueous solution dilution 10 ~ 50 times of mixing fluorescent latex particles by sugar content 5 ~ 30%, freezing 2h at-80 DEG C, after taking-up under-30 ~-50 DEG C of condenser temperatures freeze drying 10 ~ 20h, sealing be stored in 4 DEG C.
Concrete, in described sample diluting liquid preparation process, described damping fluid comprises one or more in PBS damping fluid, Tris damping fluid, glycine buffer, MES damping fluid, HEPES damping fluid, described surfactant is polysorbas20, Tween 80, triton x-100 or polyglycol, and described antiseptic is Sodium azide or proclin.
The double wave length fluorescent immunochromatographytest test kit of troponin of the present invention, comprise kit box, cryopreservation tube and dilution bottle, described kit box comprises plastics lining board and is fixed on sample pad, immuno-chromatographic test paper strip and the thieving paper on plastics lining board, described sample pad and thieving paper are overlapped on the both sides of described immuno-chromatographic test paper strip respectively, described immuno-chromatographic test paper strip is provided with detection line and control line, in described detection line and control line, is coated with troponin monoclonal antibody and chicken IgY respectively; Deposited freeze-drying probe in described cryopreservation tube, described freeze-drying probe is react obtained freeze-drying probe by the fluorescent latex particles of troponin monoclonal antibody wavelength of transmitted light different from from two kinds goat-anti chicken IgY, has deposited sample diluting liquid in described dilution bottle.
Further, described kit box also comprises housing, described housing is provided with well, detection window and handle, described well is arranged on described sample pad place, described detection window is arranged on described detection line and control line place, and described handle is arranged on the side described housing being positioned at close thieving paper.
Further, described immuno-chromatographic test paper strip is cellulose nitrate membrane material.
By such scheme, the present invention at least has the following advantages:
1) the present invention utilizes fluorescence immune chromatography method, and the Quantitative in vitro realizing troponin detects.
2) fluorescence immune chromatography method of the present invention, originality uses separate control lines method; Detection line region adopts and can form the troponin monoclonal antibody of compound with troponin, and partner probe then uses the troponin monoclonal antibody of 2 kinds of fluorescent latex particles marks; Control line region is coated with chicken IgY antibody, and partner probe uses 2 kinds of fluorescent latex particles of goat-anti chicken IgY antibody labeling.Control line and detection line independent reaction, be independent of each other and disturb.Cryoprobe is being dispersed in the quality in mixing material to utilize control line signal accurately to judge, is used for correct detection line signal.
3) the present invention adopts dual wavelength Comparison calibration method.By collecting the signal value of control line at different wave length place, can predict that in sample, impurity is on the impact of fluorescence signal, by selecting the utilizing emitted light signal of suitable wavelength, reducing the impact of impurity, more reliable data can be drawn.Double UV check core is the fluorescent latex particles label probe antibody of use two kinds of different emission, then participates in reaction simultaneously, on control line and detection line, finally has the signal of two kinds of fluorescence respectively.Be the equal of that a test card does twice detection to same determinand is simultaneously parallel, on the control line in two all normal situations of wavelength result, do computing to two kinds of wavelength signals on detection line, the concentration results conversed is averaged
4) the present invention is directed to quantitative chromatography requirement, originality adopts probe freeze drying technology, by the probe independence freeze-drying in detection system, and redissolves with sample after dilution before the reaction.This technology avoids Traditional immunochromatographic product technology to adopt metal spraying legal system to obtain the problem detecting precision as sprayed the impacts such as irregular, cutting error of golden mark rim strip.The gold mark pad that uses of traditional chromatographic technique in addition, the heat drying under its drying condition many employings condition of normal pressure, heat-treat condition affects the life-span of probe to a certain extent, reduces detection sensitivity, and increases the inaccuracy of testing result.
5) the troponin detection kit prepared of the present invention, detection sensitivity is high, can detect the troponin of 0.60ng/ml in serum sample; Detecting instrument is simple, simple to operate, without the need to professional operator; Detect fast, within 10 ~ 20 minutes, can testing result be obtained; Kit containment is convenient.
Above-mentioned explanation is only the general introduction of technical solution of the present invention, in order to better understand technological means of the present invention, and can be implemented according to the content of instructions, coordinates accompanying drawing to be described in detail as follows below with preferred embodiment of the present invention.
Accompanying drawing explanation
Fig. 1 is the structural representation of immuno-chromatographic test paper strip in the present invention;
Fig. 2 is the structural representation of kit box in the present invention;
Fig. 3 is Comparability test result schematic diagram in the embodiment of the present invention six.
Embodiment
Below in conjunction with drawings and Examples, the specific embodiment of the present invention is described in further detail.Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.
Embodiment one
The double wave length fluorescent immunochromatography detection method of troponin of the present invention, comprises the following steps:
1) immuno-chromatographic test paper strip preparation: adopt and draw on the film metal spraying machine detection line (T line) that troponin monoclonal antibody and chicken IgY is coated on respectively chromatographic test paper and control line (C line), obtains the immuno-chromatographic test paper strip of 3 ~ 5mm width through cutting cutter cutting after dry 1 ~ 5h;
Should be noted that: a) traditional quantitative immune chromatographic technique adopts bag by sheep anti-mouse antibody as control line, along with the increase of serum cTnT or some against murine source antibody blocking agent, signal on control line decreases, its signal value just can not be used for calculating, and the accuracy of the signal of p-wire is also without reference frame.The present invention adopts chicken IgY antibody and goat-anti chicken antibody to match as control line, and the reaction of C line and T line is independently carried out, without cross influence.C line and T line can be avoided to contend with one other probe particulate and the repeated deviation produced; B) be less than 1h drying time, the joint efficiency of antibody on N controlling diaphragm is low, causes the detectability of troponin to raise; Drying time is greater than 5h, the easy inactivation of antibody, causes the sensitivity decrease detected, and best drying time is 3h; C), when the width of test strips is less than 3mm, cutting machine is comparatively large on the impact of test strips, and significantly reduce the precision of testing result, when the width of test strips is greater than 5mm, can increase the cost of test strips and probe, optimized test strips width is 4mm.
2) freeze-drying probe preparation: the fluorescent latex particles solution with carboxyl or amino wavelength of transmitted light being respectively 500 ~ 590nm and 600 ~ 740nm mixes in 1:5 ~ 5:1 ratio, 0.5 ~ 2h is reacted under adding activator 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) room temperature after mixing, wherein, the weight ratio of 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) and fluorescent latex particles is 0.5:100 ~ 5:100, obtains activating fluorescent latex particles after centrifugal, washing;
Should be noted that: the fluorescent latex that a) wavelength of transmitted light is respectively 500 ~ 590nm and 600 ~ 740nm is easily obtaining on the market, thus under the prerequisite ensureing accuracy of detection, economy and applicability higher; B) volume ratio of fluorescent latex particles is optimized according to actual sample detection signal, and single present latex particulate proportion crosses that I haven't seen you for ages causes sensitivity decrease, and too much increase testing cost, optimization ratio is 1:1; C) when the weight ratio of EDC and fluorescent latex particles is less than 0.5:100, the activation rate of present latex particulate is very low, and on coupling microballoon, the combination of antibody is little, and sensitivity during detection is low; When the weight ratio of EDC and fluorescent latex particles is greater than 5:100, the limited extent that the activation rate of present latex particulate increases with EDC amount and increases, cost raises, and optimal proportion is 1:100; D) soak time is less than 0.5h, and the efficiency of activation is not high, waste starting material; Soak time is greater than 2h, and the speed of activation is less than the speed of activating substance hydrolysis, reduces activation efficiency, optimum activating time 1h.
Mixed by the 0.01:100 ~ 0.05:100 in proportion of troponin monoclonal antibody with activation fluorescent latex particles, coupling reaction 2 ~ 5h under room temperature, obtains troponin monoclonal antibody fluorescent latex particles; By goat-anti chicken IgY with activation fluorescent latex particles in proportion 0.01:100 ~ 0.05:100 mix, coupling reaction 2 ~ 5h under room temperature, obtains goat-anti chicken IgY fluorescent latex particles;
Should be noted that: when a) weight ratio of antibody and fluorescent latex particles is less than 0.01:100, coupling efficiency is high, but waste present latex particulate, the non-binding antibody of major part activation point of present latex particulate; When being greater than 0.05:100, the most activation point of present latex particulate is combined, and the amount continuing to increase antibody has no significant effect coupling efficiency, waste antibody, and optimal proportion is 0.02:100; B) reaction time is relevant with Conjugate ratio, and the time is too short is less than 2 hours, then antibody and present latex particulate in conjunction with less, uneconomical; Overlong time is greater than 5 hours, then Conjugate ratio increases limited, loses time, optimum reacting time 3h.
In ratio mixing troponin monoclonal antibody fluorescent latex particles and the goat-anti chicken IgY fluorescent latex particles of 3:1 ~ 6:1, obtain mixing fluorescent latex particles, by the diatom sugar aqueous solution dilution 10 ~ 50 times of mixing fluorescent latex particles by sugar content 5 ~ 30%, often prop up in cryopreservation tube the mixing fluorescent latex particles after adding 15 ~ 30 μ l dilutions, freezing 2h at-80 DEG C, after taking-up under-30 ~-50 DEG C of condenser temperatures freeze drying 10 ~ 20h, sealing be stored in 4 DEG C.
Should be noted that: a) probe is not easily kept in sample diluting liquid, the one-tenth branch of sample diluting liquid promotes the reunion between probe, can assemble, reduce the sensitivity detected between nitrocellulose filter (NC film) and sample pad; Probe adhesion prepared by freeze-day with constant temperature and vacuum drying is comparatively strong, is unfavorable for dissolving, larger to the testing result deviation of enriched sample; Freeze drying can be very stable preservation probe, avoid the reunion between probe, and the probe of freeze-drying can redissolve in the sample to which very soon, can be evenly distributed in testing sample very soon; B) sugar content is less than 5%, and when extension rate is greater than 50, the solids content in the probe skeleton structure of freeze-drying is few, containing a large amount of pores, loosely organized, can produce a large amount of bubbles, affect application of sample amount when adding sample; Sugar content is higher than 30%, and when extension rate is less than 10, when adding sample, the dissolution time that freeze-drying probe needs is slightly long, and the viscosity of reaction system increases, and is unfavorable for chromatography and application of sample, best sugar content 20%; C) often prop up probe solution that cryopreservation tube adds when being less than 15 μ l, during packing, the requirement of pipettor is strengthened, be not suitable for manual packing; When being greater than 30 μ l, the sugar content in probe solution is more, is unfavorable for chromatography and application of sample, and best dispensed loading amount is 25 μ l; D) condenser temperature is too low, and the energy consumption of needs is large, and the function also with machine is relevant; Condenser temperature is too high, excessively slow to the condensation of moisture, and reduce drying efficiency, best condenser temperature is-45 DEG C; E) cooling time is too short, and moisture removal is inadequate, and the solid of non-freeze-drying under room temperature can get damp again, and affects the quality of probe; Cooling time is long, and moisture is substantially removed and is over, and there is no need to continue waste resource; F) blending ratio of troponin monoclonal antibody fluorescent latex particles carries out adjusting according to the binding ability of antigen on T line, and the too low meeting of concentration causes insufficient sensitivity, and low concentration is distinguished not; Excessive concentration, cost can increase, and the linear upper limit also may decline.
3) sample diluting liquid preparation: sample diluting liquid is the damping fluid containing bovine serum albumin(BSA), protein protective agent, surfactant and antiseptic; Damping fluid comprises one or more in PBS damping fluid, Tris damping fluid, glycine buffer, MES damping fluid, HEPES damping fluid, and surfactant is polysorbas20, Tween 80, triton x-100 or polyglycol, and antiseptic is Sodium azide or proclin;
Should be noted that: a) bovine serum albumin(BSA) plays a part flow sealing, can be combined by the foreign protein in human serum, close the blank site on probe, reduce the non-specific binding of probe, reduce false Yangxin number; B) protein protective agent is for reducing the decomposition of some antigens to be checked, plays a part to protect antigen; C) surfactant can improve the water wettability of hydrophobic substance in immune system, makes probe being more evenly distributed on NC film, avoids the impact of signal fluctuation on signal; D) antiseptic postpones or suppresses microbial growth, avoids the corruption of sample diluting liquid; E) damping fluid is for controlling immunoreactive reaction conditions, reduces the difference of human serum to the impact of reaction system
4) sample survey: mixed with freeze-drying probe by blood serum sample after disperseing a period of time, takes out and a certain amount ofly adds in sample diluting liquid, to be mixed evenly after drop on immuno-chromatographic test paper strip and carry out immunochromatography reaction; Under fluorescence detector, adopt the two kind wavelength of transmitted light corresponding with two kinds of fluorescent latex particles to carry out fluoroscopic examination subsequently.In mixing material, troponin collides to combine with the troponin monoclonal antibody being marked with fluorescent latex particles and forms compound, penetrate in sample pad, mixing material is after glass fiber filter impurity, compound wherein continues to penetrate on detection line along nitrocellulose filter, the new compound of double-antibody sandwich is formed with the troponin monoclonal antibody be fixed on detection line, troponin monoclonal antibody on detection line and the troponin monoclonal antibody in probe, its epi-position on troponin is different; Remaining antigen-fluorescently-labeled antibody complex, the unconjugated troponin monoclonal antibody being marked with fluorescent latex particles and the goat-anti chicken IgY being marked with fluorescent latex particles continue to penetrate into control line, and the chicken IgY that the goat-anti chicken IgY being marked with fluorescent latex particles can fix on control line is combined and forms antigen-antibody complex; Under fluorescence detector during fluoroscopic examination, only signal detected on the control line, could prove that testing result is effective.
Embodiment two
As shown in Fig. 1 to 2, the double wave length fluorescent immunochromatographytest test kit of troponin of the present invention, comprise kit box, cryopreservation tube and dilution bottle, kit box comprises plastics lining board 1 and is fixed on sample pad 2, immuno-chromatographic test paper strip 3 and the thieving paper 6 on plastics lining board, immuno-chromatographic test paper strip is cellulose nitrate membrane material, sample pad and thieving paper are overlapped on the both sides of immuno-chromatographic test paper strip respectively, immuno-chromatographic test paper strip is provided with detection line 4 and control line 5, in detection line and control line, is coated with troponin monoclonal antibody and chicken IgY respectively; Deposited freeze-drying probe in cryopreservation tube, freeze-drying probe is react obtained freeze-drying probe by the fluorescent latex particles of troponin monoclonal antibody wavelength of transmitted light different from from two kinds goat-anti chicken IgY, has deposited sample diluting liquid in dilution bottle; Kit box also comprises housing 7, and housing is provided with well 8, detection window 9 and handle 10, well is arranged on sample pad place, and detection window is arranged on detection line and control line place, and handle is arranged on side housing being positioned at close thieving paper.
During use, blood serum sample is joined in freeze-drying probe cryopreservation tube, take out a certain amount of liquid after probe dispersion and join in sample diluting liquid, join in well after mixing; Under fluorescence detector, adopt the two kind wavelength of transmitted light corresponding with two kinds of fluorescent latex particles to carry out fluoroscopic examination subsequently.In mixing material, troponin collides to combine with the troponin monoclonal antibody being marked with fluorescent latex particles and forms compound, penetrate in sample pad, mixing material is after glass fiber filter impurity, compound wherein continues to penetrate on detection line along nitrocellulose filter, the new compound of double-antibody sandwich is formed with the troponin monoclonal antibody be fixed on detection line, troponin monoclonal antibody on detection line and the troponin monoclonal antibody in probe, its epi-position on troponin is different; Remaining antigen-fluorescently-labeled antibody complex, the unconjugated troponin monoclonal antibody being marked with fluorescent latex particles and the goat-anti chicken IgY being marked with fluorescent latex particles continue to penetrate into control line, and the chicken IgY that the goat-anti chicken IgY being marked with fluorescent latex particles can fix on control line is combined and forms antigen-antibody complex; Under fluorescence detector during fluoroscopic examination, only signal detected on the control line, could prove that testing result is effective.
Detection fluorescence detector of the present invention, comprises excitation-detection module, pre-amplifying module, control analysis module and software systems.The wherein light emitting diode of the light source of excitation-detection module to be emission wavelength be 400 ~ 600nm, pre-amplifying module is a pre-amplification circuit.
Embodiment three
The specific embodiment that the present invention also provides a kind of double wave length fluorescent immunochromatography of troponin to detect, comprises the following steps successively:
The first step: the preparation of freeze-drying probe
1) the fluorescent latex particles solution two kinds of wavelength of transmitted light being respectively 550nm and 700nm mixes according to the ratio of volume ratio 1:1, after mixing, after getting 500 μ l mixing fluorescent latex particles solution (containing carboxyl) pH6.0MES buffer solution centrifuging three times, precipitation pH6.0MES damping fluid dilution, after adding 10mg EDC mixing, at room temperature reaction activation 30min, after centrifuging, precipitation continues to use pH6.0MES buffer solution three times, dilute with postprecipitation pH6.0MES damping fluid, add 125 μ g troponin monoclonal antibodies, 3h is reacted under room temperature, add BSA to close, continue reaction 30min, centrifuged deposit pH7.4PBS buffer solution four times, obtain the troponin antibodies precipitation being marked with fluorescent latex particles, the goat-anti chicken IgY that in like manner can obtain being marked with fluorescent latex particles precipitates, precipitation is resuspended in 500 μ l pH7.4PBS damping fluids, add 15 μ lproclin, preserve at 4 DEG C.
2) coupling is had the present latex particulate of troponin monoclonal antibody and coupling have the present latex particulate of goat-anti chicken IgY by volume 6:1 mix, epoxy glue lactoconium mixes with 5% diatom sugar aqueous solution whirlpool of 50 times of volumes, obtain probe solution, getting 30 μ l probe solutions joins in 500 μ l cryopreservation tubes, at-80 DEG C of freezing 2h, at-25 DEG C of freeze drying 4h after taking-up, sealing is kept at 4 DEG C of refrigerators.
Second step: the preparation of immunochromatographydetection detection card
Liner plate pastes nitrocellulose filter, sample pad and thieving paper successively, and sample pad and thieving paper are overlapped on film, and are closely connected.The antibody coating buffer that another strain troponin monoclonal antibody (coated antibody) of different epi-position and goat-anti chicken IgY coating buffer are mixed with 1mg/ml and 0.5mg/ml is respectively positioned at respectively by from the troponin monoclonal antibody that fluorescent latex particles marks.Troponin monoclonal antibody coating buffer and chicken IgY coating buffer are coated on detection line corresponding on nitrocellulose filter and control line with the linear speed of 1 μ l/cm, detection line and control line interval 8mm, under humidity <30% condition after 37 DEG C of oven dry 1h, make fluorescence immune chromatography test paper plate.
With cutting cutter, the fluorescence immune chromatography test paper plate prepared longitudinally is cut into the wide fluorescence immune chromatography test paper bar of 4mm, putting it into gets stuck interiorly obtains immunochromatographydetection detection card through case pressing machine process.
3rd step: the preparation of sample diluting liquid
Get the PBS damping fluid 1L of 0.01M pH7.5, add 9ml polysorbas20,15g antibody protective agent, 6.1g bovine serum albumin(BSA) and 0.19g Sodium azide, ultrasonic until solid all dissolves, mixing.
Embodiment four
The specific embodiment that the present invention also provides a kind of double wave length fluorescent immunochromatography of troponin to detect, comprises the following steps successively:
The first step: the preparation of freeze-drying probe
1) the fluorescent latex particles solution two kinds of wavelength of transmitted light being respectively 500nm and 600nm mixes according to the ratio of volume ratio 1:5, after mixing, after getting 500 μ l mixing fluorescent latex particles solution (containing carboxyl) pH6.0MES buffer solution centrifuging three times, precipitation pH6.0MES damping fluid dilution, after adding 2mg EDC mixing, at room temperature reaction activation 1h, after centrifuging, precipitation continues to use pH6.0MES buffer solution three times, dilute with postprecipitation pH6.0MES damping fluid, add 62.5 μ g troponin monoclonal antibodies, 2h is reacted under room temperature, add BSA to close, continue reaction 30min, centrifuged deposit pH7.4PBS buffer solution four times, obtain the troponin antibodies precipitation being marked with fluorescent latex particles, the goat-anti chicken IgY that in like manner can obtain being marked with fluorescent latex particles precipitates, precipitation is resuspended in 500 μ l pH7.4PBS damping fluids, add 15 μ lproclin, preserve at 4 DEG C.
2) coupling is had the present latex particulate of troponin monoclonal antibody and coupling have the present latex particulate of goat-anti chicken IgY by volume 5:1 mix, epoxy glue lactoconium mixes with 5% diatom sugar aqueous solution whirlpool of 30 times of volumes, obtain probe solution, getting 25 μ l probe solutions joins in 500 μ l cryopreservation tubes, at-80 DEG C of freezing 2h, at-25 DEG C of freeze drying 4h after taking-up, sealing is kept at 4 DEG C of refrigerators.
Second step: the preparation of immunochromatographydetection detection card
Identical with corresponding steps in embodiment three.
3rd step: the preparation of sample diluting liquid
Get the PBS damping fluid 1L of 0.01M pH7.5, add 8ml triton x-100,15g antibody protective agent, 6.1g bovine serum albumin(BSA) and 0.19g Sodium azide, ultrasonic until solid all dissolves, mixing.
Embodiment five
The specific embodiment that the present invention also provides a kind of double wave length fluorescent immunochromatography of troponin to detect, comprises the following steps successively:
The first step: the preparation of freeze-drying probe
1) the fluorescent latex particles solution two kinds of wavelength of transmitted light being respectively 590nm and 740nm mixes according to the ratio of volume ratio 5:1, after mixing, after getting 500 μ l mixing fluorescent latex particles solution (containing carboxyl) pH6.0MES buffer solution centrifuging three times, precipitation pH6.0MES damping fluid dilution, after adding 20mg EDC mixing, at room temperature reaction activation 1h, after centrifuging, precipitation continues to use pH6.0MES buffer solution three times, dilute with postprecipitation pH6.0MES damping fluid, add 312.5 μ g troponin monoclonal antibodies, 5h is reacted under room temperature, add BSA to close, continue reaction 30min, centrifuged deposit pH7.4PBS buffer solution four times, obtain the troponin antibodies precipitation being marked with fluorescent latex particles, the goat-anti chicken IgY that in like manner can obtain being marked with fluorescent latex particles precipitates, precipitation is resuspended in 500 μ l pH7.4PBS damping fluids, add 15 μ lproclin, preserve at 4 DEG C.
2) coupling is had the present latex particulate of troponin monoclonal antibody and coupling have the present latex particulate of goat-anti chicken IgY by volume 3:1 mix, epoxy glue lactoconium mixes with 5% diatom sugar aqueous solution whirlpool of 10 times of volumes, obtain probe solution, getting 15 μ l probe solutions joins in 500 μ l cryopreservation tubes, at-80 DEG C of freezing 2h, at-25 DEG C of freeze drying 4h after taking-up, sealing is kept at 4 DEG C of refrigerators.
Second step: the preparation of immunochromatographydetection detection card
Identical with corresponding steps in embodiment three.
3rd step: the preparation of sample diluting liquid
Get the PBS damping fluid 1L of 0.01M pH7.5, add 10ml polyglycol, 15g antibody protective agent, 6.1g bovine serum albumin(BSA) and 0.19g Sodium azide, ultrasonic until solid all dissolves, mixing.
Embodiment six
The sample testing method that the present invention also provides a kind of double wave length fluorescent immunochromatography of troponin to detect, comprises the following aspects:
1) linearly, detectability and precision assessment:
Adopt negative cow's serum as dilution, troponin standard items are mixed with the standard solution that concentration is 10000,9000,8000,7000,6000,5000,4000,3000,2000,1000 and 0pg/ml; get standard items 100 μ l and 100 μ l sample diluting liquids join in the cryopreservation tube containing freeze-drying probe; get 100 μ l after blowing and beating 15 times and join in well, after 15 minutes, adopt fluorescence detector to detect.Result shows, 500 ~ 590nm and 600 ~ 740nm, two groups of utilizing emitted lights, often organize linear correlation coefficient r ^2 and be all greater than 0.99, detectability is respectively 30pg/ml and 27pg/ml, and the detection coefficient of variation of each concentration is all less than 8%.
2) checking of linear dimensions:
Adopt low value human serum and high level human serum, compound concentration is the troponin human serum solution of 10000,8000,6000,4000 and 2000pg/ml, each sample repeats 4 times, result shows, employing Single wavelength there will be the situation that match value is greater than or less than actual value, adopts the method for averaging, can remove the impact that Single wavelength brings, r^2 is greater than 0.99, and highest detection scope can reach 10000pg/ml.
3) assessment of accuracy
Adopt Troponin concentration be 10pg/ml human serum sample based on sample, add the troponin reference material human serum of same volume variable concentrations, be mixed with the troponin human serum solution that concentration is 5000,1000,120 and 60pg/ml; Another increment originally adds the negative human serum of same volume, carries out 4 duplicate detection analyses, and calculate recovery sample and basic sample.Result shows, Single wavelength detects the recovery within the scope of 80%-120%, and adopt the method for averaging, the recovery can in 95%-105% scope.Wherein, the recovery=(reclaiming concentration of specimens-basic concentration of specimens)/add concentration * 100%, specifically in table one.
The assessment of table one accuracy
| The recovery concentration | 5000 | 1000 | 120 | 60 |
| 500-590nm match value | 104.5％ | 105.3％ | 107.5％ | 110.0％ |
| 600-740nm match value | 96.3％ | 93.4％ | 92.0％ | 87.0％ |
| Average fit value | 100.4％ | 99.4％ | 99.8％ | 98.5％ |
4) Comparability test
The human serum getting different value detects, and the value that detected value and Roche reagent detect is compared, and carries out Calculation of correlation factor by all sample duplicate determination values.Result as described in Figure 3, adopts Single wavelength to detect, and correlation coefficient r ^2 is all 0.98, and detected value differ with Roche reagent detected value comparatively greatly, and after adopting dual wavelength correction, correlation coefficient r ^2 is all greater than 0.99, detected value and Roche reagent detected value accordance better.
The above is only the preferred embodiment of the present invention; be not limited to the present invention; should be understood that; for those skilled in the art; under the prerequisite not departing from the technology of the present invention principle; can also make some improvement and modification, these improve and modification also should be considered as protection scope of the present invention.
Claims (9)
1. a double wave length fluorescent immunochromatography detection method for troponin, is characterized in that: comprise the following steps:
1) immuno-chromatographic test paper strip preparation: on the detection line that troponin monoclonal antibody and chicken IgY are coated on chromatographic test paper respectively and control line, obtains immuno-chromatographic test paper strip after drying, cutting;
2) freeze-drying probe preparation: the fluorescent latex particles solution of two kinds of different wavelength of transmitted light is mixed in proportion, adds activator after mixing and react a period of time, centrifugal, washing after obtain activate fluorescent latex particles;
By troponin monoclonal antibody and goat-anti chicken IgY respectively in proportion with activation fluorescent latex particles coupling reaction a period of time, obtain troponin monoclonal antibody fluorescent latex particles and goat-anti chicken IgY fluorescent latex particles;
Subsequently troponin monoclonal antibody fluorescent latex particles is mixed to get by a certain percentage with goat-anti chicken IgY fluorescent latex particles and mixes fluorescent latex particles, add the dilution of diatom sugar aqueous solution subsequently, after freeze-drying, save as freeze-drying probe;
3) sample diluting liquid preparation: sample diluting liquid is the damping fluid containing bovine serum albumin(BSA), protein protective agent, surfactant and antiseptic;
4) sample survey: mixed with freeze-drying probe by blood serum sample after disperseing a period of time, takes out and a certain amount ofly adds in sample diluting liquid, to be mixed evenly after drop on immuno-chromatographic test paper strip and carry out immunochromatography reaction; Under fluorescence detector, adopt the two kind wavelength of transmitted light corresponding with two kinds of fluorescent latex particles to carry out fluoroscopic examination subsequently.
2. the double wave length fluorescent immunochromatography detection method of troponin according to claim 1, it is characterized in that: in described freeze-drying probe preparation steps, the fluorescent latex particles solution of two kinds of different wavelength of transmitted light is mixed in 1:5 ~ 5:1 ratio, 0.5 ~ 2h is reacted under adding activator 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride room temperature after mixing, wherein, the weight ratio of 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and fluorescent latex particles is 0.5:100 ~ 5:100.
3. the double wave length fluorescent immunochromatography detection method of troponin according to claim 1, it is characterized in that: in described freeze-drying probe preparation steps, the wavelength of transmitted light of described two kinds of fluorescent latex particles is respectively 500 ~ 590nm and 600 ~ 740nm.
4. the double wave length fluorescent immunochromatography detection method of troponin according to claim 1, it is characterized in that: in described freeze-drying probe preparation steps, troponin monoclonal antibody is 0.01:100 ~ 0.05:100 with the blending ratio of activation fluorescent latex particles, coupling reaction 2 ~ 5h under room temperature after mixing; Goat-anti chicken IgY is 0.01:100 ~ 0.05:100 with the blending ratio of activation fluorescent latex particles, coupling reaction 2 ~ 5h under room temperature after mixing.
5. the double wave length fluorescent immunochromatography detection method of troponin according to claim 1, it is characterized in that: in described freeze-drying probe preparation steps, in ratio mixing troponin monoclonal antibody fluorescent latex particles and the goat-anti chicken IgY fluorescent latex particles of 3:1 ~ 6:1, obtain mixing fluorescent latex particles, by the diatom sugar aqueous solution dilution 10 ~ 50 times of mixing fluorescent latex particles by sugar content 5 ~ 30%, freezing 2h at-80 DEG C, after taking-up under-30 ~-50 DEG C of condenser temperatures freeze drying 10 ~ 20h, sealing be stored in 4 DEG C.
6. the double wave length fluorescent immunochromatography detection method of troponin according to claim 1, it is characterized in that: in described sample diluting liquid preparation process, described damping fluid comprises one or more in PBS damping fluid, Tris damping fluid, glycine buffer, MES damping fluid, HEPES damping fluid, described surfactant is polysorbas20, Tween 80, triton x-100 or polyglycol, and described antiseptic is Sodium azide or proclin.
7. the double wave length fluorescent immunochromatographytest test kit of a troponin, it is characterized in that: comprise kit box, cryopreservation tube and dilution bottle, described kit box comprises plastics lining board and is fixed on sample pad, immuno-chromatographic test paper strip and the thieving paper on plastics lining board, described sample pad and thieving paper are overlapped on the both sides of described immuno-chromatographic test paper strip respectively, described immuno-chromatographic test paper strip is provided with detection line and control line, in described detection line and control line, is coated with troponin monoclonal antibody and chicken IgY respectively; Deposited freeze-drying probe in described cryopreservation tube, described freeze-drying probe is react obtained freeze-drying probe by the fluorescent latex particles of troponin monoclonal antibody wavelength of transmitted light different from from two kinds goat-anti chicken IgY, has deposited sample diluting liquid in described dilution bottle.
8. the double wave length fluorescent immunochromatographytest test kit of troponin according to claim 7, it is characterized in that: described kit box also comprises housing, described housing is provided with well, detection window and handle, described well is arranged on described sample pad place, described detection window is arranged on described detection line and control line place, and described handle is arranged on the side described housing being positioned at close thieving paper.
9. the double wave length fluorescent immunochromatographytest test kit of troponin according to claim 7, is characterized in that: described immuno-chromatographic test paper strip is cellulose nitrate membrane material.
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