CN102636642B - Preparation method of quick quantitative kit for hepatic fibrosis diagnosis - Google Patents
Preparation method of quick quantitative kit for hepatic fibrosis diagnosis Download PDFInfo
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- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
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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 medical product technical field, turn the immuno-chromatographic test paper strip of luminescence method on particularly a kind of, the manufacture method of detection kit and described test strips.
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 Stokes rule in its luminous process, and radiative wavelength is longer than and is excited light wavelength.20 century 70s, scientist has 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.
The material forming in the lattice that is doped in crystal by some thulium has upper forwarding optical phenomenon, has three kinds of main compositions in this material: 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 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 a plurality of 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 continuously to transmitting (as Er3+), 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 (see figure 1) of light.
Different absorption, transmitting, main matrix have different optical properties in conjunction with making to turn light-emitting particles (up-converting particle UCP).(1) in the situation that main matrix is identical, 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: the infrared ray excited absorption-Yb3+-transmitting of 980nm-Er3+, absorption son-Yb3+-transmitting-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+-transmitting-Er3+, in main matrix YF3, GdF3, transmitting redness respectively and green visible ray).The diversity forming 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) the 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) the stability of height: the luminescence phenomenon of UCP is the pure physical process that results from inside configuration, thereby avoided the luminous cancellation that causes from detecting sample corrosion and self decay completely;
(3) the 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) the dirigibility of height: UCP has can independent assortment diversified characteristic spectrum (absorption spectrum and emission spectrum), make it be applicable to multiple analysis;
(5) the 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, and for example, in area of medical diagnostics, particularly the other fast detecting (Point Of Care Testing, POCT) of bed field is used.Take on the basis of the UCP labelling technique platform that antigen-antibody is representative, binding immunoassay chromatography Fast Detection Technique, recycle microminiaturized photoelectron material can develop a kind of sensitive, quick, quantitatively accurately, control 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, except large-scale medical centre or laboratory are used, can also Go out of Hospital, come into community, towards basic unit.
Because liver fiber is the reversible stage, so early diagnosis effective especially key for the treatment of.The main pathology of liver fibrosis is changed to extracellular matrix (ECM) and too much deposits in liver.At normal liver, ECM synthesizes and degraded keeps mobile equilibrium, is due to matrix metalloproteinase (MMPs) and its specific inhibitor---the result of the fine adjustments such as NMPI (TIMPs).In process of hepatic fibrosis, TIMPs reduces ECM degraded by suppressing the activity of MMPs, causes the over-deposit of ECM, causes liver fibrosis.
Found that at present TIMPs family is by 4 member compositions, TIMP 1,2,3,4, and wherein TIMP-1 is the strongest to MMPs activity inhibition in liver fibrosis process.TIMP-1 is combined with MMP-1 specifically, and therefore, TIMP-1 is not only 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.Studies show that, the TIMP expressing in the TIMP in serum and hepatic tissue has obvious correlativity.The TIMP-1 detecting in serum is extremely all more satisfied to the accuracy of diagnosing liver fibrosis and specificity, be in recent years in the world clinical research think good liver fibrosis non-invasive diagnosis and follow up a case by regular visits to New Set.
Therefore, the upper forwarding light immuno analytical method platform development TIMP-1 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
The invention provides a kind of immunochromatography quantitative test paper bar based on above turning the mensuration NMPI TIMP-1 of 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, thereby utilize, by UPT rapidly and quantitatively testing system, realize accurate quantification or qualitative.
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 NMPI TIMP-1 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 NMPI TIMP-1 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 the upper immuno-chromatographic test paper strip that forwards light of NMPI TIMP-1 can be applied to clinical.
Accompanying drawing explanation
Fig. 1 is the process of light that forwards in two photon excitation UCP generation.
Fig. 2 is the upper cross-sectional view that forwards light immune quantitative detection immuno-chromatographic test paper strip of NMPI TIMP-1 of the present invention.Wherein 1 be sample pad, 2 for pad, 3 for analyzing film, 4 for adsorptive pads, 5 for viscosity end liner, 6 for NMPI TIMP-1 antibody-UCP bond, 7 for detecting band T line, 8, be quality control band C line.
Embodiment
With embodiment, further illustrate the present invention below.It should be understood that embodiments of the invention are for the present invention rather than 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 NMPI TIMP-1 light kit is produced is as follows:
(1) coated: TIMP-1 monoclonal antibody (buying from Finland Hytest company) to be diluted to 2mg/ml, as T line coating buffer, sheep anti-mouse igg (buying from Suo Laibao bio tech ltd, Beijing) to be 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 10 mg UCP particles and be placed in conical flask;
B) add 10 ml pH=7.2 0.20M PB;
C) in UCP particle suspension, add 0. 5 mg TIMP-1 antibody (buying from Finland Hytest company), then add anhydrous glutaraldehyde to 1%, 37 ℃ of stirring of final concentration to spend the night;
D) 12000rpm, 4 ℃, centrifugal 15min, abandons supernatant;
E) in UCP sediment, add 10ml pH=7.2 0.20M PB, piping and druming mixes;
F) 12000rpm, 4 ℃, centrifugal 15min, abandons supernatant; ;
G) UCP sediment is collected stand-by;
(3) prepare freeze-drying pad:
A) by 50ml UCP label suspension, 12000rpm, 4 ℃, centrifugal 30min, abandons supernatant;
B) add the freeze-drying liquid (pH=7.2 0.20M PB contains 2%BSA, 3% sucrose) of 45ml;
C) UCP label suspension is added on glass fibre 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 base plate, are cut into the wide paper slip of 4.0mm.
(6) dress plastic clip; The wide test strips of 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, and 1%Tween 20.
The composition of the upper forwarding of TIMP-1 light quantification kit:
1, TIMP-1-UPT Rapid detection test strip (40 person-portion)
2, 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 scope of protection of present invention.4, operation instructions (1 part)
The principle of TIMP-1-UPT immuno-chromatographic test paper strip
TIMP-1-UPT Rapid detection test strip adopts double-antibody sandwich immune chromatography method quantitatively to detect TIMP-1 in sample.The pre-coated anti-TIMP-1 monoclonal antibody in test section on analyzing film (T), the pre-coated sheep anti-mouse igg in Quality Control district (C), has the anti-TIMP-1 monoclonal antibody of another strain of UCP particle marker on pad.TIMP-1 albumen and two strain antibody reaction bonded in sample during reaction, in T district, form the anti-TIMP-1 protein antibodies compound of anti-TIMP-1 protein antibodies-TIMP-1 proteantigen-UCP mark, UCP particle is at infrared ray excited lower transmitting visible ray, and radiative intensity is directly proportional to the concentration of TIMP-1 albumen in sample.No matter in sample, whether there is TIMP-1 albumen, in C district, all can form the anti-TIMP-1 protein antibodies compound of sheep anti mouse-UCP mark.
Embodiment 4
Adopt detection provided by the invention TIMP-1-UPT on turn luminescence method detection kit and carry out the step of TIMP-1 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 ℃-30 ℃).
C. draw whole blood sample 0.5 ml, 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 15 min, utilize the upper light immunity analysis instrument that forwards of UPT to carry out scanning analysis to test strips.
E. UPT above forwards light immunity analysis instrument display measurement result, the concentration that Y value is 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 simultaneously to analyze mensuration, with double-log (or other is suitable) Model fitting, require the not remarkable parallel deviate (t check) of two dose-response curves, take import standard items as contrast, and 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 TIMP-1 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 ℃).
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 TIMP-1 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 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, (mean value+2 times standard deviation) the corresponding concentration on typical curve of take is sensitivity.
Table 1 sensitivity detects data
Table 2 sensitivity testing result
Embodiment 5
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 contrast test experience.
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 |
As table 3 result shows, two kinds of kit sensitivity do not have difference, and TIMP-1-UPT kit of the present invention is consistent with the performance of the TIMP-1 kit of u s company, there is no difference.
Claims (1)
1. the manufacture method of an immuno-chromatographic test paper strip, described immuno-chromatographic test paper strip comprises viscosity end liner (5), a side at viscosity end liner (5) arranges analyzing film (3), opposite side arranges adsorptive pads (4), on analyzing film (3), there is pad (2), in pad (2), have the up-conversion luminescent material UCP bond (6) of NMPI TIMP-1 antibody;
Comprise the following steps:
(1) coated
TIMP-1 monoclonal antibody is diluted to 2mg/ml, as T line coating buffer, sheep anti-mouse igg 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 and are dried, obtain monoclonal antibody coated film bar;
(2) prepare UCP mark monoclonal antibody
A) take 10 mg UCP particles and be placed in conical flask,
B) add 10 ml pH=7.2 0.20M PB,
C) in UCP particle suspension, add 0. 5 mg TIMP-1 antibody, then add anhydrous glutaraldehyde to 1%, 37 ℃ of stirring of final concentration to spend the night,
D) 12000rpm, 4 ℃, centrifugal 15min, abandons supernatant,
E) in UCP sediment, add 10ml pH=7.2 0.20M PB, mix,
F) 12000rpm, 4 ℃, centrifugal 15min, abandons supernatant,
G) collection UCP sediment is stand-by;
(3) prepare freeze-drying pad
A) by 50ml UCP label suspension, 12000rpm, 4 ℃, centrifugal 30min, abandons supernatant,
B) add the freeze-drying liquid of 45ml, pH=7.2 0.20M PB, containing 2%BSA, 3% sucrose,
C) UCP label suspension is pressed to 5cm
2/ ml is added on glass fibre,
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 base plate, are cut into the wide paper slip of 4.0mm.
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| CN101023356A (en) * | 2004-08-12 | 2007-08-22 | 霍夫曼-拉罗奇有限公司 | Method for diagnosing liver fibrosis |
| CN101551388A (en) * | 2009-05-22 | 2009-10-07 | 上海师范大学 | Immunochromatographic test paper for quantitatively detecting AFP based on up-converting phosphor technology |
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| EP1697543A2 (en) * | 2003-11-21 | 2006-09-06 | ANP Technologies, Inc. | Asymmetrically branched polymer conjugates and microarray assays |
| CN101788559A (en) * | 2004-04-23 | 2010-07-28 | 中国人民解放军军事医学科学院微生物流行病研究所 | Immune chromatography test paper strip based on up-conversion luminescence technology |
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| WO2009140757A1 (en) * | 2008-05-20 | 2009-11-26 | University Health Network | Device and method for fluorescence-based imaging and monitoring |
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