CN102636642A - Quick quantitative kit for hepatic fibrosis diagnosis - Google Patents
Quick quantitative kit for hepatic fibrosis diagnosis Download PDFInfo
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- CN102636642A CN102636642A CN2012100981766A CN201210098176A CN102636642A CN 102636642 A CN102636642 A CN 102636642A CN 2012100981766 A CN2012100981766 A CN 2012100981766A CN 201210098176 A CN201210098176 A CN 201210098176A CN 102636642 A CN102636642 A CN 102636642A
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- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The invention relates to immune chromatography test paper based on an up-conversion luminescence technology, a test kit and a manufacturing method of the test paper. The test paper comprises a sticky substrate (5), wherein an analysis film (3) is arranged on one side of the sticky substrate (5) while a water absorbing pad (4) is arranged on the other side; a combining pad (2) is arranged on the analysis film (3); and an up-converting particle (UCP) ligature (6) of a tissue inhibitor of metalloproteinase TIMP-1 antibody is arranged in the combining pad (2). The test paper is quickly, conveniently and quantitatively applied, and can provide support for preventing, diagnosing and treating hepatic fibrosis.
Description
Technical field
The invention belongs to the medical product technical field, the particularly a kind of immuno-chromatographic test paper strip that changes luminescence method, manufacturing approach of detection kit and said test strips of going up.
Background technology
Some organic or inorganic materials that occurring in nature exists can be excited by electromagnetic radiation, and emitting fluorescence or phosphorescence are all observed the Stokes rule in its luminous process, and promptly radiative wavelength is longer than and is excited light wavelength.The seventies in 20th century; Scientist has found one type of special material that can anti-Stokes rule produces phosphorescence; (wavelength>780nm) is excited this material in the infrared light district; But can the long-range visible light (wavelength 475nm-670nm) that is shorter than exciting light of transmitted wave, promptly to change on the energy, this phenomenon is called as transmits light.
Be doped in the material that constitutes in the lattice of crystal by some thulium and have the forwarding of going up optical phenomenon, three kinds of major ingredients are arranged: main matrix (host matrix), absorption (absorber) and emission (emitter) in this material.Crystalline material as main matrix has: oxysulfide (like Y2O2S, GdO2S, La2O2S etc.), fluoride (like YF3, GdF3, LaF3 etc.), gallate (like YGaO3, Y3Ga5O12 etc.) and silicate (like YSi2O5, YSi3O7 etc.) etc.; The rare earth ion of making absorption commonly used has: ytterbium ion (Yb3+), erbium ion (Er3+), samarium ion (Sm3+) etc.; The rare earth ion of making emission commonly used has: erbium ion (Er3+), holmium ion (Ho3+), thulium ion (Tm3+), terbium ion (Tb3+) etc.Absorption and spatial orientation and distance that this ion pair of emission suits in the main matrix lattice are to produce to go up the basis of transmitting light.The generation of last forwarding light is a two-phonon process that relates to a plurality of photons (at least two).In this process; Absorption (like Yb3+) in the last forwarding luminescent material will absorb two energy photons (infrared light districts at least; Like 970nm), then through a series of internal energy conversions, with radiationless form (A1 → A2; A2 → A3) energy with these two photons passes to emission (like Er3+) continuously, so that it is in excited state (A3).A transition of returning ground state level then takes place in the latter, and discharging on a high-energy photon (visible region is like 525nm or 540nm) the completion energy changes.
Transmit the process (see figure 1) of light in two photon excitation UCP generation.
Differing absorption, emission, main matrix combine to make forwarding light particle (up-converting particle UCP) to have different optical properties.⑴ under the identical situation of main matrix; A series of UCP adopt identical absorption; Distinct transmit; What then it can be by identical wavelength is infrared ray excited, produces the emission light (as: the infrared ray excited absorption-Yb3+ of 980nm-emission-Er3+, absorb son-Yb3+-emission-Tm3+, launch 550nm green visible light, 475nm blue visible light respectively) of different wave length; Employing differing absorption, identical emission is though then via different exciting lights, can produce identical emission light.⑵ under the main matrix condition of different, the UCP spectral signature also will change (as: absorb son-Yb3+-emission-Er3+, in main matrix YF3, GdF3, launch red with green visible light respectively).The diversity of forming has determined the diversity of UCP spectrum (excitation spectrum, emission spectrum), and this becomes the basis of dirigibility in its use.
This unique optical properties that last forwarding light particle UCP is had makes it will bring into play huge potential as the novel markings thing in field of biological detection:
⑴ sensitivity highly: exclusive going up transmitted optical phenomenon and guaranteed that never there is the background interference that comes from the external world in UCP in the process that detects;
⑵ the stability of height: the luminescence phenomenon of UCP is the pure physical process that results from inside configuration, thereby has avoided the way of luminescence quenching from test sample is corroded and self decays and causes fully;
⑶ the simplification of height: utilize UCP directly to detect to whole blood, urine, saliva, tissue secretion thing etc., and need not sample pretreatment;
⑷ the dirigibility of height: but the diversified characteristic spectrum of the independent assortment that UCP has (absorption spectrum and emission spectrum) makes it be applicable to multiple analysis;
⑸ the security of height: inorganic inertia, infrared ray excited, VISIBLE LIGHT EMISSION makes does not all have any harm based on the detection of UCP for tester, article to be detected, environment.
These characteristics makes UCP as biomarker very application prospects arranged just, and for example in area of medical diagnostics, particularly the other fast detecting of bed (Point Of Care Testing, POCT) use by the field.On the basis of UCP labelling technique platform that with the antigen-antibody is representative, binding immunoassay chromatography Fast Detection Technique, utilize again microminiaturized photoelectron material can develop a kind of sensitivity, fast, quantitatively accurately, control easy immunodiagnosis analyser
(Point Of Care Testing POCT) is the trend of the times of detection technique development to the other fast detecting of bed.The distinguishing feature of POCT is quick (requiring to obtain data in 15 minutes), portable, the simple and easy medical diagnosis result that obtains of operation, except large-scale medical centre or laboratory use, can also walk out hospital, comes into community, towards basic unit.
Because the liver fiber is the reversible stage, so early diagnosis and effective especially key of treating.The main pathology of liver fibrosis is changed to extracellular matrix (ECM) and in liver, too much deposits.At normal liver, the synthetic and degraded maintenance mobile equilibrium of ECM is because matrix metalloproteinase (MMPs) and its specific inhibitor---the result that NMPI (TIMPs) etc. are accurately regulated.In the liver fibrosis forming process, TIMPs reduces the ECM degraded through the activity that suppresses MMPs, causes the over-deposit of ECM, causes liver fibrosis.
Found TIMPs family at present by 4 member compositions, TIMP 1,2,3,4, and wherein TIMP-1 is the strongest to the MMPs activity inhibition in liver fibrosis process.TIMP-1 combines with MMP-1 specifically, and therefore, TIMP-1 not only is formed with certain facilitation to liver fibrosis, cirrhosis, and can be used as one of index of liver fibrosis, liver cirrhosis diagnosis and judging prognosis.Research shows that the TIMP that expresses in TIMP in the serum and the hepatic tissue has tangible correlativity.The TIMP-1 that detects in the serum is all more satisfied to accuracy and the specificity of diagnosing liver fibrosis unusually, be in recent years in the world clinical research think preferably liver fibrosis non-invasive diagnosis and follow up a case by regular visits to New Set.
Therefore, using going up of China's independent development transmits light immuno analytical method platform development TIMP-1 quick detection reagent and is applied to clinical POCT field and have the important clinical using value.
Summary of the invention
The present invention provides the immunochromatography quantitative test paper bar of a kind of mensuration NMPI TIMP-1 based on last commentaries on classics luminescence method, has characteristics accurately and rapidly.
Its detection principle of test strips of the present invention is through a series of finishinges and activation; Up-conversion luminescent material UCP (Up-Converting Phosphor; UCP) particle can combine with bioactive molecule; Utilize UPT to go up and transmit light immunity analysis instrument (or being called the UPT biology sensor); UCP particle in the chromatography process, (detecting band and quality control band) through the specific immune reaction bonded in the specific region carries out scanning analysis, realizes accurate quantification or qualitative thereby utilize by UPT rapidly and quantitatively testing system.
Particularly, the present 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 has combined NMPI TIMP-1 antibody.
In a preferred embodiment, the analyzing film of said immuno-chromatographic test paper strip is a nitrocellulose filter.
In another preferred embodiment, said test strips comprises a shell, comprises well, interpretation window and terminal point indication window as a result on the said shell.
In another preferred embodiment, through said well, biological sample is added on the sample pad; The interpretation window is corresponding to detection band and quality control band on the analyzing film as a result; The terminal point indication window is corresponding to the terminal point index strip on the adsorptive pads.
The present invention also provides a kind of method for preparing above-mentioned immuno-chromatographic test paper strip, and its characteristic has combined NMPI TIMP-1 antibody at last forwarding light particle surface.
The present invention also provides a kind of immunochromatography based on up-converting phosphor technology quick kit, comprises above-mentioned immuno-chromatographic test paper strip of the present invention.
The present invention can carry out quick and precisely quantitative to pattern detection, makes NMPI TIMP-1 go up the immuno-chromatographic test paper strip of transmitting light and can be applied to clinical.
Description of drawings
Fig. 1 is a process of transmitting light in two photon excitation UCP generation.
Fig. 2 transmits the cross-sectional view that the light immune quantitative detects immuno-chromatographic test paper strip for NMPI TIMP-1 of the present invention goes up.Wherein 1 is that sample pad, 2 is quality control band C line for detecting band T line, 8 for NMPI TIMP-1 antibody-UCP bond, 7 for viscosity end liner, 6 for adsorptive pads, 5 for analyzing film, 4 for pad, 3.
Embodiment
Further specify the present invention with embodiment below.It should be understood that embodiments of the invention are to be used to explain the present invention rather than limitation of the present invention.The concrete improvement that essence according to the present invention is carried out the present invention all belongs to the present invention and requires the scope protected.
It is following that NMPI TIMP-1 goes up the technology of transmitting the production of light kit:
(1) encapsulates: TIMP-1 monoclonal antibody (buying from Finland Hytest company) is diluted to 2mg/ml,, sheep anti-mouse igg (buying from Beijing the Suo Laibao bio tech ltd) is diluted to 2mg/ml, as C line coating buffer as T line coating buffer.Through Membrane jetter, T line coating buffer and C line coating buffer are encapsulated on the nitrocellulose filter.Dry, promptly obtain monoclonal antibody coated film bar.
(2) UCP mark monoclonal antibody: the UCP labeled monoclonal antibody in this kit is to obtain with following step, and step is following:
A) take by weighing 10 mg UCP particles and place conical flask;
B) add 10 ml pH=7.2 0.20M PB;
C) add 0. 5 mg TIMP-1 antibody (purchase) in the UCP particle suspension, add 1%, 37 ℃ of stirred overnight of anhydrous glutaraldehyde to final concentration again from Finland Hytest company;
D) 12000rpm, 4 ℃, centrifugal 15min abandons supernatant;
E) add 10ml pH=7.2 0.20M PB, piping and druming mixing in the UCP sediment;
F) 12000rpm, 4 ℃, centrifugal 15min abandons supernatant;
G) the UCP sediment is collected for use;
(3) preparation freeze-drying pad:
A) with 50ml UCP label suspension, 12000rpm, 4 ℃, centrifugal 30min abandons supernatant;
B) the freeze-drying liquid (pH=7.2 0.20M PB contains 2%BSA, 3% sucrose) of adding 45ml;
C) UCP label suspension is added on the spun glass by 5cm2/ml;
D) 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 the base plate, are cut into the wide paper slip of 4.0mm.
(6) dress plastic clip; The test strips that 4.0mm is wide installs in the plastic clip drain pan, covers the plastic clip upper casing, compresses.
(7) sample diluent preparing; Sample dilution composition is the 0.20M PB of pH=7.2, and 1%Tween 20.
The composition of the last forwarding of TIMP-1 light quantification kit:
1, TIMP-1-UPT quick detection test paper bar (40 person-portion)
2, the sample dilution (1 bottle, 45ml)
3, sample hose (40)
The concrete improvement of the present invention being carried out with AN all belongs to the present invention and requires the scope protected.4, operation instructions (1 part)
The principle of TIMP-1-UPT immuno-chromatographic test paper strip
TIMP-1 in the TIMP-1-UPT quick detection test paper strip adoption double-antibody sandwich immune chromatography method detection by quantitative sample.Test section on the analyzing film (T) encapsulates anti-TIMP-1 monoclonal antibody in advance, and Quality Control district (C) encapsulates sheep anti-mouse igg in advance, and the anti-TIMP-1 monoclonal antibody of another strain of UCP particle mark is arranged on the pad.TIMP-1 albumen and two strain antibody reaction bonded in the sample during reaction; Form the anti-TIMP-1 protein antibodies compound of anti-TIMP-1 protein antibodies-TIMP-1 proteantigen-UCP mark in the T district; The UCP particle is in infrared ray excited visible emitting down, and radiative intensity is directly proportional with the concentration of TIMP-1 albumen in the sample.No matter whether there is TIMP-1 albumen in the sample, all can forms the anti-TIMP-1 protein antibodies compound of sheep anti mouse-UCP mark in the C district.
Adopting going up of detection provided by the invention TIMP-1-UPT to change the luminescence method detection kit carries out the step of TIMP-1 content detection and is:
A. first complete reading operation instructions before test,
B. sample to be tested is taken out numbering and balance to room temperature (20 ℃-30 ℃) from storage condition.
C. draw whole blood sample 0.5 ml, add in the dilution tube, splash into dilution 0.5ml, mixing.
D. test strips is placed on the clean smooth plane, get the well that 60 μ l samples add to the UPT immune chromatography test paper, add 100 μ l dilutions again, pick up counting simultaneously.
D. after placing 15 min, utilize UPT to go up forwarding light immunity analysis instrument test strips is carried out scanning analysis.
E. UPT goes up and transmits light immunity analysis instrument demonstration measurement result, and the Y value is the concentration of TIMP-1, and system default unit is pg/ml.
Detect index:
1) accuracy: adopt the calibration object (deriving from Finland Hytest company) of kit calibration object and respective concentration to carry out assay determination simultaneously; With the match of double-log (or other is suitable) mathematical model; Require the not remarkable parallel deviate (t check) of two dose-response curves; With the import standard items is contrast, and the mensuration concentration of kit calibration object and the ratio of theoretical concentration are tiring of kit calibration object.Measure the result and should meet following provisions, promptly 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 TIMP-1 standard items (4ng/ml) by specification operation steps and carry out replication.Calculate each result of mensuration, obtain average, standard deviation (SD) and coefficient of variation CV.Variation within batch coefficient (CV) should not be higher than 15%.
Concrete detection method is following:
A. with test strips, dilution and sample to be tested balance to room temperature (20～25 ℃).
B. take the packaging of aluminium foil bag of test strips apart, test strips is placed on the even curface.
C. on the test strips shell, write the numbering of sample to be tested.
D. get 60 μ l samples, add in the well.
E. get 100 μ l dilutions, add in the well.
F. room temperature was placed 15 minutes.
G. the parameter of this batch of input test strips in last forwarding light immunity analysis instrument is measured.
H. data processing: transmit the light immunity analysis instrument with supporting the going up of TIMP-1 quantitative determination reagent kit and have the calculating concentration function.Instrument is presented at T/C value (the concentration ratio of TEST line/CONTROL line) on the screen through the concentration value that calculates automatically after measuring completion.
3) detection sensitivity: according to TIMP-1 standard items dilution metering result, the detection sensitivity of this kit is 20ng-4000 ng/ml.
Measure 20 0 pg/ml points, calculating mean value and standard deviation are sensitivity with (mean value+2 times standard deviation) corresponding concentration on typical curve.
Table 1 sensitivity detects data
Table 2 sensitivity testing result
The TIMP-1 chemical luminescence reagent kit of getting TIMP-1-UPT kit of the present invention and U.S. De Ling company has carried out the comparison and detection experiment.
Two kinds of TIMP-1 testing result contrasts of table 3
| Numbering | Moral spirit (ng/ml) | Hot scape (ng/ml) |
| 1# | 431.3 | 443.11 |
| 2# | 1090.2 | 799.3 |
| 3# | 2218.4 | 1970.49 |
| 4# | 7818.5 | 4961.55 |
| 5# | 254.6 | 202.12 |
| 6# | 364.2 | 492.87 |
| 7# | 241.9 | 258.08 |
| 8# | 2191.9 | 2588.41 |
| 9# | 183 | 151.74 |
| 10# | 4045 | 5635.99 |
| 11# | 481.9 | 385.85 |
| 12# | 1680 | 1790.82 |
| 13# | 621.7 | 581.43 |
| 14# | 823.8 | 489.92 |
| 15# | 654.5 | 862.96 |
| 16# | 489.9 | 335.59 |
| 17# | 1408.1 | 1682.31 |
| 18# | 945.9 | 691.74 |
| 19# | 358.4 | 460.94 |
| 20# | 598.9 | 445.77 |
The result shows like table 3, and two kinds of kit sensitivity do not have difference, and TIMP-1-UPT kit promptly of the present invention is consistent with the performance of the TIMP-1 kit of u s company, does not have difference.
Claims (6)
1. immuno-chromatographic test paper strip based on up-converting phosphor technology; Comprise viscosity end liner (5); Side at viscosity end liner (5) is provided with analyzing film (3); Opposite side is provided with adsorptive pads (4), and pad (2) is arranged on the analyzing film (3), and up-conversion luminescent material (UCP) bond (6) of NMPI TIMP-1 antibody is arranged in the pad (2).
2. immuno-chromatographic test paper strip according to claim 1 is characterized in that analyzing film (3) is a nitrocellulose filter.
3. immuno-chromatographic test paper strip according to claim 1 wherein through said well, adds biological sample on the sample pad (1) on the pad (2) to; The interpretation window is corresponding to detection band (7) on the analyzing film (3) and quality control band (8) as a result; The terminal point indication window is corresponding to the terminal point index strip on the adsorptive pads (4).
4. immuno-chromatographic test paper strip according to claim 1, this test strips is used to detect blood sample.
5. a TIMP-1 goes up and transmits light detection by quantitative kit, comprises any said immuno-chromatographic test paper strip of claim 1-4, sample dilution and sample hose.
6. according to the manufacturing approach of any said immuno-chromatographic test paper strip of claim 1-4, may further comprise the steps:
(1) encapsulates
The TIMP-1 monoclonal antibody is diluted to 2mg/ml,, sheep anti-mouse igg is diluted to 2mg/ml, as C line coating buffer as T line coating buffer; Through Membrane jetter, T line coating buffer and C line coating buffer encapsulated on the nitrocellulose filter dry, promptly obtain monoclonal antibody coated film bar;
(2) preparation UCP mark monoclonal antibody
A) take by weighing 10 mg UCP particles and place conical flask,
B) add 10 ml pH=7.2 0.20M PB,
C) in UCP particle suspension, add 0. 5 mg TIMP-1 antibody, add 1%, 37 ℃ of stirred overnight of anhydrous glutaraldehyde to final concentration again,
D) 12000rpm, 4 ℃, centrifugal 15min abandons supernatant,
E) add 10ml pH=7.2 0.20M PB in the UCP sediment, mixing,
F) 12000rpm, 4 ℃, centrifugal 15min abandons supernatant,
G) collection UCP sediment is for use;
(3) preparation freeze-drying pad
A) with 50ml UCP label suspension, 12000rpm, 4 ℃, centrifugal 30min abandons supernatant,
B) the freeze-drying liquid of adding 45ml, pH=7.2 0.20M PB contains 2%BSA, 3% sucrose,
C) UCP label suspension is added on the spun glass by 5cm2/ml,
D) 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 the base plate, are cut into the wide paper slip of 4.0mm.
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