CN106370839A - Rapid detection quantum dot immunochromatographic test strip, kit and preparation method - Google Patents
Rapid detection quantum dot immunochromatographic test strip, kit and preparation method Download PDFInfo
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Abstract
The invention discloses a rapid detection quantum dot immunochromatographic test strip, a kit, a preparation method and a use method. Phosphorylated VASP protein murine monoclonal antibodies and quantum dot conjugates are sprayed onto glass fiber membranes to obtain a probe fixation mat, the VASP protein murine monoclonal antibodies are enveloped on a detection membrane detection line, goat-anti-mouse polyclonal antibodies are enveloped on a detection membrane quality control line, a detection membrane, the probe fixation mat, a sample mat and absorbent paper are sequentially assembled on a PVC (polyvinyl chloride) substrate, and the substrate is cut into small strips to obtain the test strip. The quantum dot immunochromatographic test strip is simple and convenient to operate, high in sensitivity and accuracy, low in cost and suitable for clinical rapid detection.
Description
Technical field
The present invention relates to clinical diagnose technical field, especially relate to a kind of amount of quick detection clopidogrel curative effect
Son point immuno-chromatographic test paper strip, test kit and preparation method thereof and using method.
Background technology
Enter 21 century, China's Patients with Cardiovascular/Cerebrovascular Diseases alreadys exceed 2.7 hundred million people, dies from cardiovascular and cerebrovascular disease people every year
Nearly 3,000,000 people of number, bring heavy economy and burden on society to family and society.Cardiovascular and cerebrovascular disease is (as coronary heart disease, cardiac muscle stalk
Extremely, apoplexy) it is all due to long-term atherosclerosiss, then cause platelet function abnormality, finally with thrombosis or bleeding shape
Formula and fall ill.Point out in 2010 " Chinese hypertension guideline of prevention and treatment ", with the treatment of the hyperpietic of stable angina pectoris
Should there are aspirin or clopidogrel, current clinical patients pci are postoperative and acute coronary syndrome (acs), Ah Si in scheme
, as the Main Preventive Measures of Antiplatelet therapy, clopidogrel has become anti-in clinical position for woods and Clopidogrel medication
The important drugs of anti-platelet therapy.
Research in recent years shows, patient has the difference to Effect of Clopidogrel in Treating reactivity, and about 2% patient uses chlorine pyrrole lattice
After thunder, platelet residual activity is higher, and many reasons cause patient insensitive to Effect of Clopidogrel in Treating, i.e. so-called clopidogrel
Opposing.Document report remnants platelet aggregation rate (rpa) defines clopidogrel Resistant, there is no unified diagnostic criteria, has been reported that
Claim to take clopidogrel 24 hours, clopidogrel Resistant incidence rate is in 4%～30%, acs patient, and clopidogrel Resistant occurs
Rate 25%～40%.Clopidogrel Resistant, as aspirin resistance, may result in cardiovascular event.
Due to the reactive obvious difference to clopidogrel for the individuality, need a kind of reliable way analysis individual to chlorine pyrrole lattice
The specific reaction of thunder, adjuvant clinical worker determines patient the need of change clinical treatment or adjustment clinical application agent
Amount.At present, measure the individual detection mode to clopidogrel Resistant and be mainly platelet aggregation inhibition rate, based on detection platelet
The poct detecting instrument assembling suppression ratio has verifynow, teg etc.；The enzyme linked immunological kit cy- that biocytex company produces
Quant vasp/p2y12 also can detect clopidogrel Resistant；Separately have, according to cytochrome enzyme p450 systemic drug metabolic enzyme
Cyp2c19 genotype is predicted the clinical treatment that clopidogrel Resistant and guides clopidogrel.
At present, the poct detecting instrument based on platelet aggregation inhibition rate, needs professional, cost intensive.
The cy-quant vasp/p2y12 enzyme that biocytex company produces exempts from product operation and detection cycle is long, is unsuitable as quick
Detection meanss.
Quantum dot immune chromatographic technique is a kind of rapid immunoassay technology of rising in recent years, has high precision, dynamic
The advantages of detection range is wide, sensitivity is high, needs supporting detection by quantitative instrument.Quantum dot immune chromatography quantitative measurement technology is comprehensive
The subject knowledge such as biology, clinical medicine, optics, mechanics, electronics, computer, quantitative by quantum dot fluorescence intensity
Target protein in detection clinical sample, judges that to clinical workers conditions of patients, prognosis, monitoring treatment process etc. are respectively provided with very
Important meaning.This technology have simple to operate quick it is not necessary to professional, sensitivity height, good stability, accurately reliably etc.
Advantage, the rapid in-vitro diagnostic detection of suitable different medical unit and hospital.
In sum, platelet aggregation inhibition rate detects that the poct machine of clopidogrel Resistant has the disadvantage in that specialty
Testing equipment, single detectable high cost；ELISA measuring reagent kit detection has the disadvantage in that detection cycle is long, no
It is suitable for clinic quick diagnosis；So being clinically badly in need of the detectable of energy quick detection clopidogrel Resistant.
Content of the invention
The present invention is directed to the deficiencies in the prior art, proposes a kind of quantum dot immune chromatography of quick detection clopidogrel curative effect
Test strips, clever structure, easy to use.
In order to realize foregoing invention purpose, the present invention provides a kind of technical scheme below: quantum dot immune of quick detection
Chromatograph test strip, including pvc liner plate, sample pad, the fixing pad of probe, detection film and absorbent paper, described detection film is provided with detection
Line and nature controlling line, detection line is coated vasp albumen Mus resource monoclonal antibody, and nature controlling line is coated sheep anti mouse polyclonal antibody, and probe is solid
Spraying phosphorylation vasp albumen Mus resource monoclonal antibody and quantum point coupling thing on fixed pad；Detection film is fitted in pvc liner plate side
On middle part；The fixing pad of probe is fitted on pvc liner plate, and side covers on detection film one lateral edges；Sample pad is fitted in pvc lining
On plate, side covers on probe fixing pad edge, and another side is alignd with pvc liner plate；Absorbent paper side covers
On detection another lateral edges of film, another side is alignd with the another side of pvc liner plate.
Preferably, the fixing pad of probe covers the edge 2～4mm in detection film；Sample pad pushes down the edge 2 of the fixing pad of probe
～4mm；Edge 2～the 4mm of detection film is pushed down in absorbent paper.
Preferably, the present invention provides the quantum dot immune chromatography reagent card of quick detection clopidogrel curative effect, including quantum
Point immuno-chromatographic test paper strip and housing, housing is provided with handle, well, detection window, product coding region and the information area
Domain, is covered with sample pad, the pvc liner plate of the fixing pad of probe, detection film and absorbent paper is located in housing, handle is located at well one
Side, well is located at sample pad, and detection window is located at detection line and nature controlling line, product coding region be located at detection window and
Between information area, information area is located at the side being located on housing near absorbent paper.
Preferably, the present invention also provides a kind of quantum dot immune of quick detection clopidogrel curative effect to chromatograph test kit, bag
Include described quantum dot immune chromatograph test strip, sample stimulus agent, sample inhibitor and sample dissociation liquid.
The present invention provides the preparation method of the quantum dot immune chromatograph test strip of above-mentioned quick detection clopidogrel curative effect, bag
Include following steps:
(1), the preparation of the fixing pad of probe: the quantum dot of activation and phosphorylation vasp albumen Mus resource monoclonal antibody are carried out
Crosslinking prepares quantum dot and phosphorylation vasp albumen Mus resource monoclonal antibody conjugate, with diluted quantum dot and phosphorus
Acidifying vasp albumen Mus resource monoclonal antibody conjugate, is sprayed on pretreatment fluid immersion and dried glass fibre element film,
Fully dry；
(2), detection film detection line and nature controlling line preparation: paste on detection film and be coated vasp albumen Mus resource monoclonal antibody
Detection line and be coated the nature controlling line of sheep anti mouse polyclonal antibody, fully dry；
(3), sample pad preparation: sample pad is soaked with pretreatment fluid, fully dry；
(4), the preparation of immuno-chromatographic test paper strip: assembling detection film, the fixing pad of probe, sample pad and suction successively on pcv liner plate
Water paper, is cut into little bar.
Preferably, step (1) in, mixed using quantum dot and the phosphorylation vasp albumen Mus resource monoclonal antibody after nhs activation
Close, the conjugate preparing under cross-linking agent edc effect, wherein in reaction system, the mol ratio of key component is quantum dot:
Edc: antibody=20:20:1.
Preferably, step (1) in, quantum dot and phosphorylation vasp albumen Mus resource monoclonal antibody conjugate are diluted to 5 μm,
Uniformly being sprayed on width with the speed of 1～2 μ l/cm is on the glass fibre membrane of 1.0～1.2cm, 24h is dried at 37 DEG C.
Preferably, step (1) in, pretreatment fluid contains in peg20000, bsa, tween-20 and trion x-100
Plant or several.
Preferably, step (1) in, diluent is the buffer containing sucrose, bovine serum albumin and surfactant, institute
State buffer and include one or more of pbs buffer, tris buffer, mes buffer, hepes buffer, described surface
Activating agent is polysorbas20 or Tween 80.
Preferably, step (3) in, pretreatment fluid is containing sucrose, bovine serum albumin, surfactant and dispersant
Buffer, described buffer includes one of pbs buffer, tris buffer, mes buffer, hepes buffer or several
Kind, described surfactant is polysorbas20 or Tween 80, and described dispersant is Polyvinylpyrrolidone 10k, polyethylene pyrrole
One or more of pyrrolidone 40k or polyvinyl alcohol.
The present invention also provides the using method of the quantum dot immune chromatograph test strip of described quick detection clopidogrel curative effect,
Comprise the following steps:
1., sample stimulus agent and inhibitor preparation: prostaglandin and adenosine diphosphate (ADP) mixture are diluted with buffer, system
Standby one-tenth lyophilized powder, is redissolved with front pure water；
2., sample dissociation liquid preparation: the mixture buffer of Tissue lysates and preservative is diluted；
3., sample survey: take clinical sodium citrate anticoagulation to be added separately to sample stimulus agent and the inhibitor redissolving, top
Fall to mix standing 5 minutes, be added to step 2. sample dissociation liquid, overturn and mix standing 5 minutes；It is added dropwise to immuno-chromatographic test paper strip
Carry out immunochromatography reaction, chromatograph through fluorescence detector detectable card detection line and nature controlling line fluorescence intensity after terminating, calculate
Pri value.
Preferably, step 1. in, sample stimulus agent and inhibitor buffer include pbs buffer, tris buffer, sweet ammonia
One or more of acid buffer, mes buffer, hepes buffer.
Preferably, step 2. in, sample dissociation liquid buffer includes pbs buffer, tris buffer, glycine buffer
One or more of liquid, mes buffer, hepes buffer, described Tissue lysates contain triton-x 100, sds, np-
One or more of 40, described preservative is sodium azide or proclin300.
Preferably, step 3. in, pri value computing formula is:
Compared with prior art, the invention has the advantages that
1. adopt quantum dot immune chromatography method of the present invention, realize external quick detection clopidogrel curative effect, the method has
Have that sensitivity is high, accuracy is high, simple to operate, low cost and other advantages.
2. quantum dot immune chromatography method of the present invention, detection line region is using the Mus source Dan Ke that can form complex with vasp
Grand antibody, partner probe is then using quantum dot-labeled phosphorylation vasp Mus resource monoclonal antibody.Detection line coated antibody with join
Set probe antibody is monoclonal antibody, increases sensitivity and the accuracy of the method.
3. quantum dot immune chromatography method of the present invention, quantum dot and phosphorylation vasp albumen Mus resource monoclonal antibody conjugate
In quantum dot be new fluorescent material, possess advantages below:
A the wide ranges of the excitation spectrum of () quantum dot, the scope of emission spectrum is narrower and is symmetric.By adjustment amount
Son point size obtains the quantum dot of different emission, need not change composition and the surface nature of quantum dot.Make the quantum dot can
For the multi-color marking of various ingredients, realize detection while many components.
B () quantum dot fluorescence intensity is strong, good stability.
C the compatibility of () quanta point biological is good, through chemical modification, can connect functional group by specificity.
D () quantum dot small toxicity, can carry out biological living labelling and detection.
4. the quantum dot immune chromatography detection kit of the quick detection clopidogrel curative effect of present invention preparation, sample preparation
Cycle is short, detection cycle is short, simple to operate, without professional operator.
Brief description
Fig. 1 is the structural representation of immuno-chromatographic test paper strip in the present invention；
Fig. 2 is the structural representation of reagent card in the present invention.
The implication of in figure labelling is as follows: 1pvc liner plate, 2 sample pad, 3 probes fix pad, 4 nitrocellulose filters, 5 water suctions
Paper, 6 detection lines, 7 nature controlling lines, 8 handles, 9 wells, 10 detection windows, 11 housings, 12 product coding regions, 13 company informations
Region.
Specific embodiment
Below the technical scheme in the embodiment of the present invention is carried out with clear, complete description it is clear that described embodiment
It is only a part of embodiment of the present invention, rather than whole embodiments, it is not limited to the scope of the present invention.Based on the present invention
Embodiment, the every other embodiment that those of ordinary skill in the art are obtained under the premise of not making creative work,
Broadly fall into the scope of protection of the invention.
As shown in figure 1, a kind of quantum dot immune chromatograph test strip of quick detection clopidogrel curative effect, including pvc liner plate
1st, sample pad 2, probe fix pad 3, detection film 4 and absorbent paper 5, and described detection film 4 is provided with detection line 6 and nature controlling line 7, detection
Line 6 is coated vasp albumen Mus resource monoclonal antibody, and nature controlling line 7 is coated sheep anti mouse polyclonal antibody, and the fixing pad 3 of probe sprays phosphorus
Acidifying vasp albumen Mus resource monoclonal antibody and quantum point coupling thing；Detection film 4 is fitted in the middle part of pvc liner plate 1 side；Probe
Fixing pad 3 is fitted on pvc liner plate 1, and side covers on detection film 4 one lateral edges；Sample pad 2 is fitted on pvc liner plate 1,
Side covers on fixing pad 3 edge of probe, and another side is alignd with pvc liner plate 1；Absorbent paper 5 side covers in inspection
Survey on another lateral edges of film 4, another side is alignd with the another side of pvc liner plate 1.
The fixing pad 3 of probe covers the edge 2～4mm in detection film 4；Sample pad 2 push down the edge 2 of the fixing pad 3 of probe～
4mm；Edge 2～the 4mm of detection film 4 is pushed down in absorbent paper 5.
The quantum dot immune chromatography reagent card of quick detection clopidogrel curative effect includes test strips, also includes housing 11, shell
Body 11 is provided with handle 8, well 9, detection window 10, product coding region 12 and information area 13, be covered with sample pad 2,
The pvc liner plate 1 that probe fixes pad 3, detection film 4 and absorbent paper 5 is located in housing 11, and handle 8 is located at well 9 side, sample-adding
Hole 9 is located at sample pad 2, and detection window 10 is located at detection line 6 and nature controlling line 7, and product coding region 12 is located at detection window
Between 10 and information area 13, information area 13 is located at the side being located on housing 11 near absorbent paper 5.
The preparation method of the quantum dot immune chromatograph test strip of above-mentioned quick detection clopidogrel curative effect, walks including following
Rapid:
Step 1) prepared by the fixing pad 3 of probe: described quantum dot is coupled with phosphorylation vasp albumen Mus resource monoclonal antibody
Prepared by thing: be the quantum dot after activator nhs activation, mix with a certain proportion of phosphorylation vasp albumen Mus resource monoclonal antibody
Close.The conjugate that crosslinking prepares under cross-linking agent edc effect, conjugate diluted is standby to 1 μm.Glass fibre
Plain film pretreatment: with the treatment fluid pretreatment containing peg20000, bsa, tween-20 and trion x-100, after being dried, 1 μm
Conjugate be uniformly sprayed on the glass fibre membrane that width is 1.0～1.2cm pretreatment with the speed of 1～2 μ l/cm, at 37 DEG C
Under be dried after 24h probe fixing pad be obtained.
Step 2) prepared by detection film detection line 6 and nature controlling line 7: coated antibody diluent be containing 1～3% methanol and 1～
The buffer of 5% sucrose, described buffer include pbs buffer, tris buffer, mes buffer, in hepes buffer
One or more.
Step 3) prepared by sample pad 2: sample pad 2 steeps 30 minutes with processing immersion, 37 DEG C of dryings 24 hours, and sample pad is processed
Liquid be containing peg20000, bsa, sucrose, tween-20 0.01m pbs buffer.
Step 4) immuno-chromatographic test paper strip preparation: on pcv liner plate 1, assembling detection film 4, probe fix pad 3, sample pad successively
2 and absorbent paper 5.
The using method of the quantum dot immune chromatograph test strip of above-mentioned quick detection clopidogrel curative effect, walks including following
Rapid:
1. sample stimulus agent and inhibitor preparation: described sample stimulus agent and inhibitor buffer include pbs buffer,
One or more of tris buffer, glycine buffer, mes buffer, hepes buffer.
2. sample dissociation liquid preparation: described sample dissociation liquid buffer includes pbs buffer, tris buffer, sweet ammonia
One or more of acid buffer, mes buffer, hepes buffer, described Tissue lysates be triton-x 100,
One or more of sds, np-40, described preservative is sodium azide or proclin300.
3. sample survey: take the clinical sodium citrate anticoagulation of certain volume be added separately to redissolve sample stimulus agent and
Inhibitor, overturns and mixes standing stimulation 5 minutes, takes the post-stimulatory sample of certain volume to be added in sample dissociation liquid and mixes,
After taking cracking, sample adds immuno-chromatographic test paper strip to carry out immunochromatography reaction, chromatographs after terminating through fluorescence detector detection sample
Middle phosphorylation vasp fluorescence intensity.Calculate pri value, pri value adjuvant clinical doctor judges patient's clopidogrel curative effect.
Further, step 1) in, described quantum dot and phosphorylation vasp albumen Mus resource monoclonal antibody conjugate system
Standby: to be changed quantum dot and antibody to be marked to 0.1m mes buffer using millipore filter membrane (30kd), adjust respectively
Quantum dot and antibody concentration are to 5 μm and 2mg/ml.Take 100 μ l quantum dots, add n- hydroxysuccinimide (nhs) to activate 15 points
Clock, adds 100 μ l phosphorylation vasp albumen Mus resource monoclonal antibodies, adds 1- ethyl -3- [3- dimethylaminopropyl] carbon
Change diimmonium salt acidulants (edc), crosslinked 2 hours, obtain quantum dot and be coupled with phosphorylation vasp albumen Mus resource monoclonal antibody
Thing.With the hepes buffer containing 1～5% sucrose, 0.1～2% bovine serum albumin, 0.5% polysorbas20 and 0.1% Tween 80
Dilution quantum dot is standby to 1 μm.
Further, step 1) in, described quantum dot is used with phosphorylation vasp albumen Mus resource monoclonal antibody conjugate
To 1 μm, glass fibre element film is soaked 30 minutes diluted with pretreatment fluid, 37 DEG C of dryings 24 hours, and pretreatment fluid is to contain
There are 0.1～0.5%peg20000,0.5～5%bsa, 0.1～0.5%tween-20 and 0.01～0.5%trion x-100
Phosphate buffer.With a stroke film metal spraying all-in-one, with the speed of 1～2 μ l/cm, 1 μm of quantum point coupling thing being uniformly sprayed on width is
On the glass fibre membrane of 1.0～1.2cm pretreatment, probe fixing pad after being dried 24 hours at 37 DEG C, is obtained.
Further, step 2) in, described detection film detection line and nature controlling line, it is containing 1～3% that its antibody is coated liquid
The 0.1m pbs buffer of methanol and 1～5% sucrose.Dilute vasp Mus resource monoclonal antibody using being coated liquid to 2～4mg/ml,
Standby；Using being coated liquid dilution sheep anti mouse polyclonal antibody to 1～2mg/ml, standby.To be detected using drawing film metal spraying all-in-one
Line draws film together with the antibody of nature controlling line on detection film, if detection line and nature controlling line are spaced about 0.6cm, detection line and Quality Control
Line is about 0.6cm with detection film back gauge respectively.Detection film is obtained after being dried 24 hours at 37 DEG C of film of detection after being coated end.
Further, step 3) in, described sample pad steeps 30 minutes with processing immersion, 37 DEG C of dryings 24 hours, sample
Pad treatment fluid is containing 0.1～0.5%peg20000,0.5～5%bsa, 1～5% sucrose, 0.1～0.5%tween-20
0.01m pbs buffer.
Further, step 4) in, described immuno-chromatographic test paper strip preparation: on pcv liner plate 1 successively assembling detection film 4,
Probe fixes pad 3, sample pad 2 and absorbent paper 5.Idiographic flow:
A the middle adhesive sticker bar of () pvc liner plate 1 tears off, dried detection film 4 is affixed on pvc liner plate 1, reversely light curved pvc
Liner plate 1, makes detection film 4 fit with pvc liner plate 1 consolidation.
B the adhesive sticker bar of () pvc liner plate 1 side tears off, fixing for dried probe pad 3 is affixed on pvc liner plate 1 it is desirable to
Detection film 4 edge 2～4mm pushed down by the fixing pad 3 of probe, reversely light curved pvc liner plate 1, so that the fixing pad 3 of probe is fitted with pvc liner plate 1
Consolidation.
C () dried sample pad 2 is affixed on pvc liner plate 1 it is desirable to sample pad 2 fixes pad 3 edge 2 while pushing down probe
～4mm, sample pad 2 another side is alignd with pvc liner plate 1, reversely light curved pvc liner plate 1, so that sample pad 2 is pasted with pvc liner plate 1
Close consolidation.
D the adhesive sticker bar of () pvc liner plate 1 opposite side tears off, absorbent paper 5 is affixed on pvc liner plate 1 it is desirable to absorbent paper 5
Push down detection film edge 2～4mm, absorbent paper 5 another side alignd with pvc liner plate 1, reversely light curved pvc liner plate 1, make water suction
Paper 5 is fitted with pvc liner plate 1 consolidation.
E by pcv liner plate 1, be cut into width is the little bar of 3.6～4.0mm to () cutting cutter, prepares Quantitative detection chlorine pyrrole
The quantum dot immune chromatograph test strip of Gray's curative effect.
Further, step 1. in, described sample stimulus agent and inhibitor configuration flow: before the dilution of hepes buffer
Row parathyrine to 20-40 μm, subpackage to cillin bottle, 0.5ml/ bottle, be lyophilized into powder, make sample stimulus agent；Hepes buffer is dilute
Release prostaglandin to 20-40 μm, add final concentration of 5-20 μm of adenosine diphosphate (ADP), mix, subpackage to cillin bottle, 0.5ml/
Bottle, is lyophilized into powder, makes sample stimulus agent.With front, redissolved with 0.5ml distilled water or deionized water, can make after 10 minutes
With.
Further, step 2. in, described sample dissociation liquid buffer is pbs buffer, contains in Tissue lysates
Triton-x 100, sds, np-40 and proclin300, final concentration of 0.1～5%triton-x 100,0.01～2%sds,
0.01～2%np-40 and 0.1%proclin300, mixes, is packed as 12ml/ bottle.
Further, step 3. in, described sample survey, idiographic flow: take the clinical sodium citrate of certain volume to resist
Blood coagulation is added separately to sample stimulus agent and the inhibitor redissolving, and overturns and mixes standing stimulation 5 minutes, takes the stimulation of certain volume
Sample afterwards is added in sample dissociation liquid and mixes, and after taking cracking, sample adds immuno-chromatographic test paper strip to carry out immunochromatography anti-
Should, chromatograph after terminating through phosphorylation vasp fluorescence intensity in fluorescence detector detection sample.As depicted in figs. 1 and 2, mixing liquid
Penetrate in sample pad 2, mixing liquid fixes pad 3, phosphorylation vasp egg therein through impurity screening with after processing to tat probe
Phosphorylation vasp monoclonal antibody conjugates in vain and on probe are combined into complex, and complex penetrates into inspection along detection film 4
5 on survey line, form the new complex of double-antibody sandwich with the vasp monoclonal antibody being fixed in detection line 5, in detection line 5
Vasp monoclonal antibody is different for vasp protein epitope from the phosphorylation vasp monoclonal antibody in probe.Remaining phosphorylation
The complex that phosphorylation vasp monoclonal antibody conjugates on vasp albumen and probe are formed continues to penetrate into be controlled on detection film
Line 6 processed, the phosphorylation vasp Mus resource monoclonal antibody on probe and sheep anti mouse polyclonal antibody form new complex.Fluorescence is examined
When surveying device detection, if can't detect signal on nature controlling line 6 it was demonstrated that testing result is invalid.Calculate pri value, pri value auxiliary is faced
Bed doctor judges patient's clopidogrel curative effect.Described calculating pri value, computing formula is:
As shown in Fig. 1 is to 2, the present invention also provides a kind of quantum dot of quick detection clopidogrel curative effect based on said method
Immune chromatography reagent kit, including sample stimulus agent bottle, sample inhibitor bottle, sample dissociation liquid bottle and immunochromatographydetection detection card,
Pvc liner plate 1 and the sample pad 2 being fixed on pvc liner plate, probe fix pad 3, detection film 4 and absorbent paper 5, and sample pad overlaps
On the fixing pad of probe, the fixing pad 3 of probe and absorbent paper 5 are overlapped on the both sides detecting film respectively, and detection film 4 is provided with detection
It is coated vasp albumen Mus resource monoclonal antibody and sheep anti mouse monoclonal anti respectively in line 6 and nature controlling line 7, detection line 6 and control line 7
Body；The fixing pad 3 of probe is to be dried through spray film by the conjugate of phosphorylation vasp Mus resource monoclonal antibody and quantum dot to be obtained, sample
It is sample stimulus agent lyophilized powder in stimulant bottle, be sample inhibitor lyophilized powder in sample inhibitor bottle, in sample dissociation liquid bottle
It is sample dissociation liquid.
When detection kit of the present invention uses, the clinical sodium citrate anticoagulation of certain volume is taken to be added separately to redissolution
Sample stimulus agent and inhibitor, overturn and mix standing stimulation 5 minutes, take the post-stimulatory sample of certain volume to be added to sample and split
Mix in solution liquid, after taking cracking, sample adds immuno-chromatographic test paper strip to carry out immunochromatography reaction, and chromatography is examined through fluorescence after terminating
Survey phosphorylation vasp fluorescence intensity in device detection sample.As depicted in figs. 1 and 2, mixing liquid penetrates in sample pad 2, mixing
Liquid fixes pad 3, the phosphorylation on phosphorylation vasp albumen therein and probe through impurity screening with after processing to tat probe
Vasp monoclonal antibody conjugates are combined into complex, and complex penetrates in detection line 6 along detection film 4, and is fixed on inspection
Vasp monoclonal antibody on survey line 6 forms new complex, the vasp monoclonal antibody in detection line 6 and the spy of double-antibody sandwich
It is different that phosphorylation vasp monoclonal antibody in pin is directed to vasp protein epitope.On remaining phosphorylation vasp albumen and probe
The complex that phosphorylation vasp monoclonal antibody conjugates are formed continues to penetrate into nature controlling line 7 on detection film, the phosphoric acid on probe
Change vasp Mus resource monoclonal antibody and form new complex with sheep anti mouse polyclonal antibody.During fluorescence detector detection, nature controlling line 7
If upper can't detect signal it was demonstrated that testing result is invalid.Calculate pri value, pri value adjuvant clinical doctor judges patient's chlorine pyrrole
Gray's curative effect.
The detection fluorescence detector of the invention described above, comprises excitation-detection module, pre-amplifying module, controls analysis mould
Block and software system.The light emitting diode that the light source of wherein excitation-detection module is 400～600nm for launch wavelength, preposition
Amplification module is a pre-amplification circuit.
The quantum dot immune chromatography detection of embodiment 1 clopidogrel curative effect
The first step: the preparation of the fixing pad of probe
Take quantum dot in super filter tube (30kd), 8000 revs/min, be centrifuged 10 minutes, by quantum dot dilution fluid exchange to 0.1m
Mes buffer, adjustment concentration is to 5 μm, standby；Take phosphorylation vasp albumen Mus resource monoclonal antibody in super filter tube (30kd),
8000 revs/min, it is centrifuged 10 minutes, antibody diluent is changed to 0.1m mes buffer, adjust concentration to 2mg/ml, standby.
Take 100 μ l quantum dots in apyrogeneity centrifuge tube, add the n- hydroxysuccinimide that 10 μ l concentration are 10mm
(nhs), mix, room temperature stands, activation quantum dot 15 minutes.Add the phosphorylation vasp albumen Mus that 100 μ l concentration are 2mg/ml
Resource monoclonal antibody, mixes, and adds 1- ethyl -3- [3- dimethylaminopropyl] the carbodiimides salt that 10 μ l concentration are 10mm
Acidulants (edc), mix, and room temperature stands, crosslinked 2 hours.
Quantum dot and phosphorylation vasp albumen Mus resource monoclonal antibody conjugate.With pure containing 2% sucrose, 0.2% Sanguis Bovis seu Bubali
The hepes buffer dilution quantum dot of albumen, 0.5% polysorbas20 and 0.1% Tween 80 is standby to 1 μm.
Second step: the preparation of immunochromatographydetection detection card
Detection film detection line and nature controlling line, it is the buffering of the 0.1m pbs containing 2% methanol and 3% sucrose that its antibody is coated liquid
Liquid.Using being coated liquid dilution vasp Mus resource monoclonal antibody to 4mg/ml, standby；Dilute sheep anti mouse Anti-TNF-α using being coated liquid
Body is to 2mg/ml, standby.Detection line is drawn together with the antibody of nature controlling line film on detection film using drawing film metal spraying all-in-one, if
Detection line and nature controlling line are spaced about 0.6cm, and detection line and nature controlling line are about 0.6cm with detection film back gauge respectively.It is coated end
Afterwards, humidity < dries prepared detection film after 24h for 37 DEG C under the conditions of 30%.
Sample pad is steeped 30 minutes with processing immersion, and humidity < dries prepared sample pad after 24h for 37 DEG C under the conditions of 30%.
Immunochromatographydetection detection card assembles: on pcv liner plate (1), assembling detection film (4), probe fix pad (3), sample pad successively
And absorbent paper (5) (2).Idiographic flow:
A in the middle of () pvc liner plate, adhesive sticker bar tears off, dried detection film is affixed on pvc liner plate, and reversely gently curved pvc serves as a contrast
Plate, makes detection film fit with pvc liner plate consolidation.
B () pvc liner plate side adhesive sticker bar tears off, fixing for dried probe pad is affixed on pvc liner plate it is desirable to probe is solid
Determine pad and push down detection film edge 2～4mm, reversely light curved pvc liner plate, so that the fixing pad of probe is fitted with pvc liner plate consolidation.
(c) dried sample pad is affixed on pvc liner plate it is desirable to sample pad while push down probe fixing pad edge 2～
4mm, sample pad another side is alignd with pvc liner plate, reversely light curved pvc liner plate, so that sample pad is fitted with pvc liner plate consolidation.
D () pvc liner plate another side adhesive sticker bar tears off, absorbent paper is affixed on pvc liner plate it is desirable to absorbent paper is pushed down
Detection film edge 2～4mm, absorbent paper another side alignd with pvc liner plate, reversely light curved pvc liner plate, makes absorbent paper and pvc
Liner plate laminating consolidation.
E by pcv liner plate, be cut into width is the little bar of 3.6～4.0mm to () cutting cutter, prepares Quantitative detection chlorine pyrrole
The quantum dot immune chromatograph test strip of Gray's curative effect.
3rd step: sample stimulus agent and inhibitor preparation
Hepes buffer dilutes prostaglandin to 40 μm, subpackage to cillin bottle, and 0.5ml/ bottle is lyophilized into powder, makes sample
Product stimulant；Hepes buffer dilution prostaglandin, to 40 μm, adds final concentration of 20 μm of adenosine diphosphate (ADP)s, mixes, subpackage
To cillin bottle, 0.5ml/ bottle, it is lyophilized into powder, make sample stimulus agent.With front, redissolved with 0.5ml distilled water or deionized water,
Can use after 10 minutes.
4th step: sample dissociation liquid preparation
Following components: triton-x 100, sds, np-40 and proclin300 is added in 0.01m pbs buffer, dense eventually
Degree is respectively 0.1%, 0.01%, 0.01% and 0.1%, mixes, is packed as 12ml/ bottle.
5th step: pattern detection
The present invention and oneself listing 47 pci of ce certified product (cy-quant vasp/p2y12, biocytex) synchronous detecting
The sodium citrate anticoagulation of postoperative clinical patients, in True Positive Rate (sensitivity), true negative rate (specificity) and overall coincidence rate
Evaluated on clinical performance.
True negative rate (specificity)=93.75%
True Positive Rate (sensitivity)=86.67%
Overall coincidence rate=91.49%
The above is only the preferred embodiment of the present invention it is noted that ordinary skill people for the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of quick detection quantum dot immune chromatograph test strip it is characterised in that: include pvc liner plate (1), sample pad (2), visit
The fixing pad (3) of pin, detection film (4) and absorbent paper (5), described detection film (4) is provided with detection line (6) and nature controlling line (7), detection
Line (6) is coated vasp albumen Mus resource monoclonal antibody, and nature controlling line (7) is coated sheep anti mouse polyclonal antibody, on the fixing pad (3) of probe
Spraying phosphorylation vasp albumen Mus resource monoclonal antibody and quantum point coupling thing；Detection film (4) is fitted in pvc liner plate (1) side
On middle part；The fixing pad (3) of probe is fitted on pvc liner plate (1), and side covers on detection film (4) lateral edges；Sample pad
(2) it is fitted on pvc liner plate (1), side covers on the fixing pad of probe (3) edge, another side and the one of pvc liner plate (1)
Align in side；Absorbent paper (5) side covers on another lateral edges of detection film (4), the another side of another side and pvc liner plate (1)
Alignment.
2. quick detection quantum dot immune chromatograph test strip as claimed in claim 1 it is characterised in that: probe fixing pad (3) pressure
Overlay on the edge 2～4mm of detection film (4)；Sample pad (2) pushes down the edge 2～4mm of the fixing pad (3) of probe；Absorbent paper (5) is pressed
Live in the edge 2～4mm of detection film (4).
3. a kind of quick detection quantum dot immune chromatography reagent card it is characterised in that: include quick detection amount described in claim 1
Son point immuno-chromatographic test paper strip, and housing (11), housing (11) be provided with handle (8), well (9), detection window (10),
Product coding region (12) and information area (13), are covered with sample pad (2), the fixing pad (3) of probe, detection film (4) and water suction
The pvc liner plate (1) of paper (5) is located in housing (11), and handle (8) is located at well (9) side, and well (9) is located at sample pad
(2) place, detection window (10) is located at detection line (6) and nature controlling line (7) place, and product coding region (12) are located at detection window (10)
And information area (13) between, information area (13) is located at the upper side being located near absorbent paper (5) of housing (11).
4. a kind of quick detection quantum dot immune chromatography test kit it is characterised in that: include test strips or power described in claim 1
Profit requires reagent card described in 3, also includes sample stimulus agent, sample inhibitor and sample dissociation liquid.
5. a kind of preparation method of quick detection quantum dot immune chromatograph test strip as claimed in claim 1, comprises the following steps:
(1), the preparation of the fixing pad (3) of probe: the quantum dot of activation and phosphorylation vasp albumen Mus resource monoclonal antibody are handed over
Connection prepares quantum dot and phosphorylation vasp albumen Mus resource monoclonal antibody conjugate, with diluted quantum dot and phosphoric acid
Change vasp albumen Mus resource monoclonal antibody conjugate, be sprayed on pretreatment fluid immersion and dried glass fibre element film, fill
Divide drying；Described diluent is the buffer containing sucrose, bovine serum albumin and surfactant, and described buffer includes
One or more of pbs buffer, tris buffer, mes buffer, hepes buffer, described surfactant is tween
20 or Tween 80；Described pretreatment fluid contains one or more of peg20000, bsa, tween-20 and trion x-100；
(2), detection film (4) detection line and nature controlling line preparation: paste in detection film (4) and be coated vasp albumen Mus resource monoclonal and resist
The detection line (6) of body and the nature controlling line (7) being coated sheep anti mouse polyclonal antibody, fully dry；
(3), sample pad (2) preparation: sample pad (2) is soaked with pretreatment fluid, fully dry；Described pretreatment fluid be containing sucrose,
The buffer of bovine serum albumin, surfactant and dispersant, described buffer include pbs buffer, tris buffer,
One or more of mes buffer, hepes buffer, described surfactant is polysorbas20 or Tween 80, and described divides
Powder is one or more of Polyvinylpyrrolidone 10k, PVP k or polyvinyl alcohol；
(4), immuno-chromatographic test paper strip preparation: on pcv liner plate (1), assembling detection film (4), probe fix pad (3), sample pad successively
(2) and absorbent paper (5), it is cut into little bar.
6. preparation method as claimed in claim 5 it is characterised in that: step (1) in, using nhs activation after quantum dot and phosphorus
Acidifying vasp albumen Mus resource monoclonal antibody mixing, the conjugate preparing under cross-linking agent edc effect, wherein reaction system
The mol ratio of middle key component is quantum dot: edc: antibody=20:20:1.
7. a kind of using method of quick detection quantum dot immune chromatograph test strip as claimed in claim 1, comprises the following steps:
1., sample stimulus agent and inhibitor preparation: prostaglandin and adenosine diphosphate (ADP) mixture are diluted with buffer, is prepared into
Lyophilized powder, is redissolved with front pure water；
2., sample dissociation liquid preparation: the mixture buffer of Tissue lysates and preservative is diluted；
3., sample survey: take clinical sodium citrate anticoagulation to be added separately to sample stimulus agent and the inhibitor redissolving, overturn mixed
Even standing 5 minutes, is added to step 2. sample dissociation liquid, overturns and mixes standing 5 minutes；It is added dropwise to immuno-chromatographic test paper strip to carry out
Immunochromatography reacts, and chromatographs through fluorescence detector detectable card detection line and nature controlling line fluorescence intensity after terminating, calculates pri
Value.
8. using method as claimed in claim 7 it is characterised in that: step 1. in, sample stimulus agent and inhibitor buffer bag
Include one or more of pbs buffer, tris buffer, glycine buffer, mes buffer, hepes buffer.
9. using method as claimed in claim 7 it is characterised in that: step 2. in, sample dissociation liquid buffer include pbs buffering
One or more of liquid, tris buffer, glycine buffer, mes buffer, hepes buffer, described Tissue lysates
Containing triton-x 100, one or more of sds, np-40, described preservative is sodium azide or proclin300.
10. using method as claimed in claim 7 it is characterised in that: step 3. in, pri value computing formula is:
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| CN108956989A (en) * | 2018-09-27 | 2018-12-07 | 重庆新赛亚生物科技有限公司 | A kind of classification of helicobacter pylori detection detection reagent card and preparation method thereof |
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