CN101957367B - Up-converting luminescence rapid quantitative kit for diagnosing heart failure - Google Patents
Up-converting luminescence rapid quantitative kit for diagnosing heart failure Download PDFInfo
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- CN101957367B CN101957367B CN201010241048.3A CN201010241048A CN101957367B CN 101957367 B CN101957367 B CN 101957367B CN 201010241048 A CN201010241048 A CN 201010241048A CN 101957367 B CN101957367 B CN 101957367B
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Abstract
The invention relates to the test use of an immunochromatographic test strip and an amino terminal-pro brain natriuretic peptide (NT-proBNP) rapid quantitative reagent using the test method. A chromatographic reagent uses an up-converting phosphor (UCP) as a biomarker on which an amino terminal-pro brain natriuretic peptide (NT-proBNP) antibody is bonded. The invention relates to a test using method of an up-converting luminescence reagent. The test method can be used for quickly, conveniently, simply and quantitatively detecting the detected object content and providing a support for preventing, diagnosing and treating the heart failure.
Description
Technical field
The present invention relates to turn on a kind of the preparation method of the immunochromatographytest test kit of luminescence method, relate to the preparation of the NT-proBNP NT-proBNP immunochromatography immue quantitative detection reagent box that adopting said method tests, belong to medical product technical field, for the immunodiagnosis of carrying out to heart failure.
Background technology
Occurring in nature exists some organic or inorganic materials, and they can be excited by electromagnetic radiation, emitting fluorescence or phosphorescence.In its luminous process, all observe Stokes rule, radiative wavelength is longer than and is excited light wavelength.And at 20 century 70s, some scientist has but found that a class can anti-Stokes rule produces the special material of phosphorescence, in infrared light district, (wavelength > 780nm's this material) is excited, but can the long-range visible ray (wavelength 475nm-670nm) that is shorter than exciting light of transmitted wave, be to turn on energy, this phenomenon is called as forwarding light.
Be doped in by some thulium the material forming in the lattice of crystal and there is upper forwarding optical phenomenon, in this material, have three kinds of main compositions: main matrix (host matrix), absorption (absorber) and transmitting (emitter).Crystalline material as main matrix has: oxysulfide (as Y2O2S, GdO2S, La2O2S etc.), fluoride (as YF3, GdF3, LaF3 etc.), gallate (as YGaO3, Y3Ga5O12 etc.) and silicate (as YSi2O5, YSi3O7 etc.) etc.; Being commonly used for the sub rare earth ion of absorption has: ytterbium ion (Yb3+), erbium ion (Er3+), samarium ion (Sm3+) etc.; Being commonly used for the sub rare earth ion of transmitting has: erbium ion (Er3+), holmium ion (Ho3+), thulium ion (Tm3+), terbium ion (Tb3+) etc.Absorbing son and this ion pair of transmitting son suitable spatial orientation and distance in main matrix lattice, is to produce the upper basis that forwards light.The generation of upper forwarding light is a two-phonon process that relates to multiple photons (at least two).In this process, absorption (as Yb3+) in upper forwarding luminescent material at least will absorb two energy photons (infrared light districts, as 970nm), then through a series of internal energy conversions, with radiationless form (A1 → A2, A2 → A3) energy of these two photons is passed to transmitting (as Er3+) continuously, so that it is in excited state (A3).Then there is a transition of returning to ground state level in the latter, discharges a high-energy photon (visible region, as 525nm or 540nm) and complete on energy and turn.
In two photon excitation UCP generation, forward the process (seeing figure) of light
Different absorption, transmitting, main matrix have different optical properties in conjunction with making to turn light-emitting particles (up-converting particle UCP).In situation as identical in: (1) main matrix, a series of UCP adopt identical absorption, different transmitting, what it can be by identical wavelength is infrared ray excited, produce the utilizing emitted light (as: infrared ray excited absorption of 980nm-Yb3+-transmitting-Er3+, absorption son-Yb3+-launch son-Tm3+, launch respectively the green visible ray of 550nm, 475nm blue visible light) of different wave length; Adopt different absorption, identical transmitting, though via different exciting lights, can produce identical utilizing emitted light.(2), in the different situation of main matrix, UCP spectral signature also will change (as: absorb son-Yb3+-and launch son-Er3+, in main matrix YF3, GdF3, transmitting redness respectively and green visible ray).The diversity of composition has determined the diversity of UCP spectrum (excitation spectrum, emission spectrum), and this becomes the basis of dirigibility in its use.
On turn this unique optical properties that light-emitting particles UCP has, make it will the huge potential of performance in field of biological detection as novel markings thing:
(1) sensitivity of height: exclusive upper forwarding optical phenomenon has guaranteed that UCP never exists and comes from extraneous background interference in the process detecting;
(2) stability of height: the luminescence phenomenon of UCP is the pure physical process that results from inside configuration, thereby avoided the luminous cancellation causing from detecting sample corrosion and self decay completely;
(3) simplification of height: utilize UCP to carry out direct-detection to whole blood, urine, saliva, tissue secretion thing etc., and need not sample pretreatment;
(4) what the dirigibility of height: UCP had can independent assortment diversified characteristic spectrum (absorption spectrum and emission spectrum), make it be applicable to multiple analysis;
(5) security of height: inorganic inertia, infrared ray excited, VISIBLE LIGHT EMISSION make detection based on UCP for tester, detected product, environment all without any harm.
These characteristics makes UCP have application prospect very widely as biomarker just, particularly uses in the other fast detecting (Point Of Care Testing, POCT) of bed field in area of medical diagnostics.Thereby on the basis of the UCP labelling technique platform taking antigen-antibody as representative, binding immunoassay chromatography Fast Detection Technique, recycle microminiaturized photoelectron material can develop a kind of sensitive, quick, quantitatively accurately, manipulate easy immunodiagnosis analyser
The other fast detecting of bed (Point Of Care Testing, POCT) is the trend of the times of detection technique development.The distinguishing feature of POCT be fast (in 15 minutes, require to obtain data), portable, operate the simple and easy medical diagnosis result that obtains.Outside large-scale medical centre or laboratory are used, can also Go out of Hospital, come into community, towards basic unit.
In heart failure (Heart Failure) is a kind of clinical syndrome of the S&S producing due to heart dysfunction.Although some present medicines can obviously improve patient's life quality as Angiotensin-Converting (ACE) inhibitor, receptor blocking agent etc., delay the deterioration of the state of an illness, unless but carry out heart transplant, otherwise in heart failure cannot healing.Early stage diagnosis in heart failure and to avoid the deterioration of the state of an illness be the key issue that current health care faces, more early begin treatment is just more useful to patient.
But (especially early stage at heart failure) in heart failure is difficult to make a definite diagnosis.The classical symptom of heart failure, as be short of breath, ankle swelling, fatigue etc. are not had specificity, the slight patient of some state of an illness does not show classical symptom yet, especially gerontal patient and obese patient, even if there are these symptoms to be also difficult to explain.Therefore, primary medical care mechanism only relies on clinical criteria as diagnosis basis, and the false positive rate of diagnosis of heart failure can be up to 50%.The most frequently used effective method of current diagnosis heart failure mainly contains echocardiogram, radionuclide imaging and heart nuclear magnetic resonance check; But when daily actual diagnosis left ventricular function exhaustion, above-mentioned detection methods does not have one to be to trust completely, and have good reproducible.
Studies show that, the risk of the elevated levels of patients with heart failure NT-proBNP and the rising of mortality ratio and reentry institute is closely related.It is the independent tag thing of patient's curative effect that NT-proBNP raises, more meaningful than left ventricular ejection fraction (LVEF) and VO2 peak.In Acute Coronary Syndrome Patients, detection NT-proBNP can predict myocardial damage patient (being with or without ST section raises) or unstable angina patient's mortality prediction; In addition, NT-proBNP can also indicate the danger that the dangerous of heart failure or carrying out property heart failure and generation heart stalk occurs or heart stalk occurs again.Therefore, NT-proBNP can be used for differentiating patient whether need strengthen treatment, and follow the tracks of detect there is high death or the dangerous patient of reentry academic and work atmosphere, with this, patients with heart failure is carried out to the classification of risks.
Have a large amount of first-hand datas to show, NT-proBNP can be used for instructing the treatment of heart failure.After strong dose thing treatment heart failure, the NT-proBNP level of serum or blood plasma can decline, and instructs drug therapy with NT-proBNP level, has not only reduced total generation number of cardiovascular event, and know compared with therapeutic scheme with clinical, postpone to occur first the time of cardiovascular event.Therefore, according to the horizontal guiding clinical treatment of NT-proBNP, be, an effective heart failure treatment measure.
The patient of some performance symptoms of heart failure may think that oneself does not obtain enough treatments.But, if these patients' NT-proBNP level is normal, may just mean that these patients have been subjected to suitable treatment and need not intensive treatment (avoiding " overmedication "), because their symptom may be irrelevant with heart failure.
NT-proBNP is highly stable in serum and plasma, can adopt transmission and the disposal route of conventional blood sample, without sample pretreatment.Almost not variation in the daytime of NT-proBNP content under normal circumstances, this means and can sample at any time detection; But also be not subject to the impact of patient body position and moving situation, in the time extracting blood sample, patient is without having a rest or lying low.
Therefore, the upper forwarding light immuno analytical method platform development NT-proBNP precursor NT-proBNP quick detection reagent of application China independent development be applied to clinical POCT field and there is important clinical using value.
Summary of the invention
In order to overcome above-mentioned deficiency of the prior art, the invention provides a kind of immunochromatography quantitative test paper bar of mensuration NT-proBNP precursor NT-proBNP based on above turning luminescence method, there is feature accurately and rapidly.
Its detection principle of test strips of the present invention is by a series of finishinges and activation, up-conversion luminescent material UCP (Up-Converting Phosphor, UCP) particle can combine with bioactive molecule, utilize the upper light immunity analysis instrument (or being called UPT biology sensor) that forwards of UPT, the UCP particle that is incorporated into specific region (detect band and quality control band) by Immunel response in chromatography process is carried out to scanning analysis, realize accurate quantification or qualitative thereby utilize by UPT rapidly and quantitatively testing system.
Particularly, the invention provides a kind of immuno-chromatographic test paper strip based on up-converting phosphor technology, it is characterized in that containing up-conversion luminescent material (UCP) particle that combines NT-proBNP precursor NT-proBNP antibody.
In a preferred embodiment, the analyzing film of described immuno-chromatographic test paper strip is nitrocellulose filter.
In another preferred embodiment, described test strips comprises a shell, comprises well, result interpretation window and End point indication window on described shell.
In another preferred embodiment, by described well, biological sample is added in sample pad; Result interpretation window is corresponding to detection band and quality control band on analyzing film; End point indication window is corresponding to the End point indication band on adsorptive pads.
The present invention also provides a kind of method of preparing above-mentioned immuno-chromatographic test paper strip, and its feature combines NT-proBNP precursor NT-proBNP antibody at upper forwarding light particle surface.
The present invention also provides a kind of immunochromatography Quick kit based on up-converting phosphor technology, comprises above-mentioned immuno-chromatographic test paper strip of the present invention.
The present invention can carry out pattern detection quick and precisely quantitative, makes to turn luminescence method immuno-chromatographic test paper strip on NT-proBNP precursor NT-proBNP and can be applied to clinical.
Brief description of the drawings
Fig. 1 is the process that forwards light in two photon excitation UCP generation.
Fig. 2 is that accompanying drawing is the upper cross-sectional view that forwards light immune quantitative detection immuno-chromatographic test paper strip immuno-chromatographic test paper strip of NT-proBNP precursor NT-proBNP of the present invention.Wherein 1 be sample pad, 2 for pad, 3 for analyzing film, 4 for adsorptive pads, 5 for the low lining of viscosity, 6 for NT-proBNP precursor NT-proBNP antibody-UCP bond, 7 be quality control band C line for detecting band T line, 8.
Fig. 3 is kit testing result of the present invention and the contrast of import reagent result.
Embodiment
Further illustrate the present invention with embodiment below.It should be understood that embodiments of the invention are for the present invention instead of limitation of the present invention are described.The concrete improvement that essence according to the present invention is carried out the present invention all belongs to the scope of protection of present invention.
Embodiment 1: the technique that the upper forwarding of NT-proBNP precursor NT-proBNP light kit is produced is as follows:
(1) coated: NT-proBNP monoclonal antibody (buying from Hytest company of Finland) is diluted to 2mg/ml, as T line coating buffer, sheep anti-mouse igg (buying from Suo Laibao bio tech ltd, Beijing) is diluted to 2mg/ml, as C line coating buffer.By Membrane jetter, T line coating buffer and C line coating buffer are coated with on nitrocellulose filter.Dry, obtain monoclonal antibody coated film bar.
(2) UCP mark monoclonal antibody: the UCP labeled monoclonal antibody in this kit is to obtain by following steps, and step is as follows:
A) take 10mg UCP particle and be placed in conical flask;
B) add 10ml pH=7.2 0.20M PB;
C) in UCP particle suspension, add 0.5mg NT-proBNP antibody (buying from Hytest company of Finland), then add anhydrous glutaraldehyde to 1%, 37 DEG C of stirring of final concentration to spend the night;
D) 12000rpm, 4 DEG C, centrifugal 15min, abandons supernatant;
E) in UCP sediment, add 10ml pH=7.20.20M PB, piping and druming mixes;
F) 12000rpm, 4 DEG C, centrifugal 15min, abandons supernatant; ;
G) UCP sediment is collected stand-by;
(3) preparation of freeze-drying pad:
A) by 50ml UCP label suspension, 12000rpm, 4 DEG C, centrifugal 30min, abandons supernatant;
B) add the freeze-drying liquid (pH=7.20.20M PB, containing 2%BSA, 3% sucrose) of 45ml;
D) UCP label suspension is added on glass fibre by 5cm2/ml;
E) vacuum freeze-drying 11h;
(4) cutting pad; The pad of freeze-drying is cut into wide rectangular of 1cm.
(5) assembling test strips; Successively nitrocellulose filter, thieving paper, pad, sample pad are attached on base plate, are cut into the wide paper slip of 4.0mm.
(6) dress plastic clip; Test strips wide 4.0mm is installed in plastic clip drain pan, cover plastic clip upper casing, compress.
(7) sample diluent preparing; Sample diluent ingredient is the 0.20M PB of pH=7.2,1%Tween20.
On embodiment 2.NT-proBNP, forward the composition of light quantification kit:
1, NT-proBNP-UPT Rapid detection test strip (40 person-portion)
2, sample dilution (1 bottle, 45ml)
3, sample hose (40)
4, operation instructions (1 part)
The principle of embodiment 3:NT-proBNP-UPT immuno-chromatographic test paper strip
The principle of this test strips: NT-proBNP-UPT Rapid detection test strip adopts double-antibody sandwich immune chromatography method quantitatively to detect NT-proBNP in sample.The pre-coated anti-NT-proBNP monoclonal antibody in test section on analyzing film (T), the pre-coated sheep anti-mouse igg in Quality Control district (C), has the anti-NT-proBNP monoclonal antibody of another strain of UCP particle marker on pad.NT-proBNP albumen and two strain antibody reaction bonded in sample when reaction, form the anti-NT-proBNP protein antibodies compound of anti-NT-proBNP protein antibodies-NT-proBNP proteantigen-UCP mark in T district, UCP particle is at infrared ray excited lower transmitting visible ray, and radiative intensity is directly proportional to the concentration of NT-proBNP albumen in sample.No matter in sample, whether there is NT-proBNP albumen, all can form the anti-NT-proBNP protein antibodies compound of sheep anti mouse-UCP mark in C district.
Embodiment 4: the quality testing of NT-proBNP-UPT kit of the present invention
Adopt detection provided by the invention NT-proBNP-UPT on turn luminescence method detection kit and carry out the step of NT-proBNP content detection and be:
A. first complete reading operation instructions before test,
B. sample to be tested is taken out from storage condition, numbering and balance are to room temperature (20 DEG C-30 DEG C).
C. draw whole blood sample 0.5ml, add in dilution tube, splash into dilution 0.5ml, mix.
D. test strips is placed in clean smooth plane, gets 60 μ l samples and add to the well of UPT immune chromatography test paper, then add 100 μ l dilutions, start timing simultaneously.
D. place after 15min, utilize the upper light immunity analysis instrument that forwards of UPT to carry out scanning analysis to test strips.
The upper forwarding of e.UPT light immunity analysis instrument display measurement result, the concentration that Y value is NT-proBNP, system default unit is pg/ml.
Detect index:
1) accuracy: adopt the calibration object (deriving from Hytest company of Finland) of kit calibration object and respective concentration simultaneously to analyze mensuration, with double-log (or other is suitable) Model fitting, require the not remarkable parallel deviate (t inspection) of two dose-response curves, taking import standard items as contrast, the mensuration concentration of kit calibration object and the ratio of theoretical concentration are tiring of kit calibration object.Measurement result should meet following provisions, and tiring of kit calibration object should be in 0.900～1.100 scope.
2) precision: randomly draw 20 person-portion test strips, use with a NT-proBNP standard items (4ng/ml) by specification operation steps and carry out replication.Calculate each measurement result, obtain average, standard deviation (SD) and coefficient of variation CV.Variation within batch coefficient (CV) should be higher than 15%.
Concrete detection method is as follows:
A. by test strips, dilution and sample to be tested balance to room temperature (20～25 DEG C).
B. the packaging of aluminium foil bag of taking test strips apart, is placed on test strips on even curface.
C. on test strips shell, write the numbering of sample to be tested.
D. get 60 μ l samples, add in well.
E. get 100 μ l dilutions, add in well.
F. room temperature is placed 15 minutes.
G. in upper forwarding light immunity analysis instrument, input the parameter of this batch of test strips, measure.
H. data processing: the upper forwarding light immunity analysis instrument supporting with NT-proBNP quantitative determination reagent kit has calculating concentration function.After measurement completes, instrument is presented at T/C value (the concentration ratio of TEST line/CONTROL line) on screen through the concentration value calculating automatically.
3) detection sensitivity: according to NT-proBNP standard items dilution metering result, the detection sensitivity of this kit is 200pg-32ng/ml.
Measure 20 0pg/ml points, calculating mean value and standard deviation, taking (mean value+2 times standard deviation) on typical curve corresponding concentration as sensitivity.
Table 1 sensitivity detects data
Table 2 sensitivity testing result
Embodiment 5: the performance comparison of NT-proBNP-UPT kit of the present invention and fFN colloidal gold kit:
The NT-proBNP chemical luminescence reagent kit of getting NT-proBNP-UPT kit of the present invention and De Ling company of the U.S. has carried out contrast test experience.
Two kinds of NT-proBNP testing result contrasts of table 3
As Fig. 3 result shows, these 2 kinds of method sensitivity relations do not have difference, and NT-proBNP-UPT kit of the present invention is consistent with the performance of the NT-proBNP kit of u s company, there is no difference.
Claims (3)
1. the immuno-chromatographic test paper strip based on up-converting phosphor technology, it is characterized in that: immuno-chromatographic test paper strip comprises well, result interpretation window, End point indication window, wherein by described well, biological sample is added in sample pad, result interpretation window is corresponding to detection band and the quality control band of analyzing film; End point indication window is corresponding to the End point indication band on adsorptive pads; Wherein on pad, there is the NT-proBNP monoclonal antibody of UCP particle marker;
The preparation method of above-mentioned immuno-chromatographic test paper strip comprises that (1) is coated, (2) UCP mark monoclonal antibody, (3) freeze-drying pad, (4) cutting pad, (5) assembling test strips, (6) dress plastic clip and the preparation of (7) sample dilution.
2. a kind of immuno-chromatographic test paper strip based on up-converting phosphor technology according to claim 1, is characterized in that: described analyzing film is coated NT-proBNP NT-proBNP antibody on nitrocellulose filter, analyzing film.
3. a kind of immuno-chromatographic test paper strip based on up-converting phosphor technology claimed in claim 1, is characterized in that: the sample of detection is blood.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201010241048.3A CN101957367B (en) | 2010-07-30 | 2010-07-30 | Up-converting luminescence rapid quantitative kit for diagnosing heart failure |
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| CN201010241048.3A CN101957367B (en) | 2010-07-30 | 2010-07-30 | Up-converting luminescence rapid quantitative kit for diagnosing heart failure |
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| CN101957367B true CN101957367B (en) | 2014-10-22 |
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| CN1690711B (en) * | 2004-04-23 | 2010-04-14 | 中国人民解放军军事医学科学院微生物流行病研究所 | Immune chromatographic test paper bar based on up conversion luminescence technology |
| ES2431358T3 (en) * | 2008-11-11 | 2013-11-26 | B.R.A.H.M.S Gmbh | Prognosis and risk assessment in patients suffering from heart failure by determining the concentration of ADM |
| US20110270530A1 (en) * | 2008-12-08 | 2011-11-03 | Seok-Won Lee | Combined natriuretic peptide assays |
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Address after: 102600 Daxing District, Zhongguancun science and Technology Park Daxing biomedical industry base, Fu Street, No. 9, building 9, No. Patentee after: Beijing hot King biotechnology Limited by Share Ltd Address before: 102600 Beijing City, Daxing District Daxing 27 Keyuan Road, building 3 layer. Patentee before: Beijing Hotgen Biotechnology Co., Ltd. |