CN106093322A - A kind of quantum dot immune chromatograph test strip method for quick of the many residuals of beta-agonist - Google Patents
A kind of quantum dot immune chromatograph test strip method for quick of the many residuals of beta-agonist Download PDFInfo
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Abstract
The invention discloses the quantum dot immune chromatograph test strip method for quick of the many residuals of a kind of beta-agonist.It is first by quantum dot and beta-agonist monoclonal antibody coupling, obtains the labeled complex of quantum dot and beta-agonist monoclonal antibody, be quantum dot fluorescence probe；Again using this labeled complex as after quantum dot fluorescence probe and testing sample blending incubation, immuno-chromatographic test paper strip is utilized to carry out the detection of beta-agonist residual.The present invention is capable of rapid screening while multiple beta-agonist class medicine, and detection method is easy, quick, result is accurate, highly sensitive, can do qualitative and quantitative analysis, cheap, applied widely, has good popularizing application prospect.
Description
Technical field
The invention belongs to food safety technical field of immunoassay.More particularly, to the many residuals of a kind of beta-stimulants
Quantum dot immune chromatograph test strip method for quick.
Background technology
Beta-stimulants is a class chemical constitution epinephrine similar with physiological function and the phenethylamine class medicine of adrenalectomy element
Thing.Beta-stimulants is usually used in the diseases such as bronchospasm and the asthma of preventing and treating people, animal clinically.Heavy dose of beta-stimulants energy
Affect nutrient substance redistributing in animal body, accelerate the catabolism of fat, increase the synthesis of protein, significantly improve
Lean meat percentage.Therefore, beta-stimulants is once widely used in husbandry sector as feed additive.But, beta-stimulants is animal
Internal organs accumulation is serious, can enter human body by food chain, make people the symptoms such as cardiopalmus, dizziness, arrhythmia occur, time serious even
Death can be caused.No. 235 bulletin " animal food herbal medicine MRL " that the Ministry of Agriculture of China issues for 2002 is bright
Really specify that Clenbuterol, albuterol, cimaterol and salt thereof, esters must not detect.Owing to sending out the most successively
Eat the poisoning with the animal food remaining Clenbuterol raw so that various countries all strengthen the inspection of the supervision to Clenbuterol
Surveying, its illegal use is greatly limited, and lawless person has to look for new succedaneum.Ractopamine, sand
Butylamine alcohol etc. becomes the most frequently used succedaneum of Clenbuterol, and other beta-stimulants such as Mabuterol and bromine Boot sieve etc. are in 20th century
After the nineties, just listing, is used less relatively.
The emphasis of the detection of beta-stimulants residual always food safety, at present about the multi-residue determination side of beta-stimulants
Method is a lot, including gas chromatography-mass spectrography (GC-MS), HPLC-MS (HPLC-MS), euzymelinked immunosorbent assay (ELISA)
(ELISA) method such as.These methods, while highly sensitive, specificity good, degree of accuracy is high, but these methods need Specialty Experiment
Personnel, large-scale experiment instrument and loaded down with trivial details sample pretreatment, it is difficult to meet the needs of high flux, field quick detection, be difficult to suitable
Agree the needs that many occasions quickly detect.In recent years, immuno-chromatographic assay technology is because of it quickly, conveniently, it is adaptable to field screening,
It is widely used in the quick detection of beta-stimulants.But, the immuno-chromatographic test paper strip the most reported all can only be examined
Survey one to two kinds of beta-stimulants medicines, it is impossible to multiple beta-stimulants is carried out examination simultaneously, and qualitative detection can only be realized, very
Difficulty carries out quantitative analysis to detection sample.
Summary of the invention
The technical problem to be solved in the present invention is defect and the deficiency overcoming above-mentioned prior art, it is provided that a kind of detection limit
Low, highly sensitive, it is possible to realize the method for rapid screening while multiple beta-stimulants；Simultaneously improve immunochromatography
System, it is thus achieved that highly sensitive, good stability, low cost, can be used for on-site quick screening and measure beta-stimulants in food
Detection method.
It is an object of the invention to provide the quantum dot immune chromatograph test strip quickly side of detection of the many residuals of a kind of beta-stimulants
Method.
Another object of the present invention is to provide the application in terms of detection beta-stimulants residual of the described method.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
The quantum dot immune chromatograph test strip method for quick of the many residuals of a kind of beta-stimulants, is first by quantum dot and β-emerging
Put forth energy agent monoclonal antibody coupling, obtain the labeled complex of quantum dot and beta-stimulants monoclonal antibody, be quantum dot fluorescence
Probe；Again using this labeled complex as after quantum dot fluorescence probe and testing sample blending incubation, standing and reacting 1min, utilize
Immuno-chromatographic test paper strip carries out the detection of beta-stimulants residual.
Preferably, described quantum dot is water-soluble carboxyl based quantum dot.
It is highly preferred that described quantum dot is water-soluble carboxylated nuclear shell structure quantum point, nucleocapsid structure is CdTe/ZnS.
Preferably, described beta-stimulants monoclonal antibody is beta-stimulants class wide spectrum monoclonal antibody.
It is highly preferred that the preparation method of described beta-stimulants class wide spectrum monoclonal antibody is as follows:
(1) preparation of antigen:
The most haptenic synthesis
Clenbuterol and glutaric anhydride are carried out acylation reaction, make the alcoholic extract hydroxyl group on Clenbuterol molecular structure acylated become containing
The carboxyl spacerarm of 5 carbon, and referred to as beta-stimulants hapten；
The most immunogenic preparation
Beta-stimulants hapten and ovalbumin (OVA) coupling are obtained immunogen；
C. the preparation of coating antigen
Use mixed anhydride method to carry out coupling beta-stimulants hapten and bovine serum albumin (BSA) to be coated
Former；
The envelope antigen of above-mentioned beta-stimulants is CL-BSA.
(2) animal immune: by the artificial antigen immunity Balb/C mice of above-mentioned preparation, and use indirect ELISA method to survey
Its titer fixed；
(3) cell merges: use PEG fusion method be able to enter with the oncocyte of energy Immortalization by the Mouse spleen cells of secretory antibody
Row merges；
(4) filtering hybridoma: by the specific antibody in indirect ELISA detection culture fluid, carry out titer and suppression ratio
Measure, filter out the positive hybridoma cell that titer is high and strong to Drug inhibition；
(5) hybridoma sub-clone and build strain: utilize limiting dilution assay to carry out sub-clone, the most repeatedly 3 ~ 4 take turns, obtaining can be steady
Surely the hybridoma cell strain of homogeneous antibody is secreted；
(6) prepared by monoclonal antibody: induces monoclonal antibody method in using animal body and prepares monoclonal antibody.
It addition, as one preferably can embodiment, the preparation method of described quantum dot fluorescence probe is as follows:
S1. take quantum dot to be dissolved in phosphate buffer, add coupling agent and N-hydroxy-succinamide solution (NHS), room
Temperature stirring 10～30min；Described coupling agent is 1-(3-dimethyl propyl)-3-ethyl-carbodiimide hydrochloride (EDC)；
S2. add beta-stimulants monoclonal antibody, reaction 1～2h is stirred at room temperature；
S3. continuously add 10% BSA solution capping 0.8～1.2h, reactant liquor is centrifuged 30 with 10000～14000r/min
～50min；
S4. remove supernatant after being centrifuged, with redissolving liquid redissolution precipitation, obtain the labelling of quantum dot and beta-stimulants monoclonal antibody
Complex, 4 DEG C keep in Dark Place.
Wherein it is preferred to, quantum dot described in step S1: 1-(3-dimethyl propyl)-3-ethyl-carbodiimide hydrochloride
(EDC): the molar concentration rate of N-hydroxy-succinamide solution (NHS) is 1:3～6:13～17.
It is highly preferred that quantum dot described in step S1: 1-(3-dimethyl propyl)-3-ethyl-carbodiimide hydrochloride (EDC):
The molar concentration rate of N-hydroxy-succinamide solution (NHS) is 1:5:15.
The i.e. optimal proportion of quantum dot and coupling agent is: n (QDs): n (EDC): n (NHS)=1:5.
Preferably, 15min is stirred at room temperature described in step S1.
Preferably, quantum dot described in step S1 is 1:3 with beta-stimulants monoclonal antibody molar concentration rate described in step S2
～6.
It is highly preferred that quantum dot described in step S1: beta-stimulants monoclonal antibody=1:4 described in step S2.
It is highly preferred that reaction 1.5h is stirred at room temperature described in step S2.
Furthermore it is preferred that phosphate buffer described in step S1 is the phosphate buffer of 0.01mol/L, pH7.4.
Preferably, described quantum dot: phosphate buffer: the volume ratio of the consumption of 10% BSA solution is 1:150～170:
15～25.
It is highly preferred that described quantum dot: phosphate buffer: the volume ratio of the consumption of 10% BSA solution is 1:160:20.
Preferably, capping 1h described in step S3.
Preferably, it is centrifuged described in step S3 and is centrifuged 40min for 12000r/min.
Preferably, described in step S4, redissolution liquid is the Tris-HCl of 0.01mol/L, pH 7.4, including 0.5%
The BSA of Tween-20 and 0.5%.
Further, described immuno-chromatographic test paper strip includes base plate, sample pad, reaction film, adsorptive pads, described sample pad,
Reaction film and adsorptive pads are attached on base plate successively, and described reaction film is provided with the detection line comprising beta-stimulants envelope antigen
With comprise the nature controlling line of sheep anti-mouse igg antibody, and detection line is positioned at sample pad side, and nature controlling line is positioned at adsorptive pads side.
Wherein it is preferred to, on detection line, the concentration of beta-stimulants envelope antigen is 0.85mg/mL, sheep on described nature controlling line
The concentration of dynamics is 0.8 mg/mL.
Preferably, described detection line and nature controlling line are separated by 5mm.
I.e. as one preferably can embodiment, described detection line and the preparation of nature controlling line: be with nitrocellulose filter
Reaction film, is attached to fixing for reaction film on base plate, with being coated solution, the envelope antigen of beta-stimulants is configured to 0.85mg/
ML, sheep anti-mouse igg antibody (be sheep anti-mouse igg two resist) is configured to 0.8 mg/mL, by beta-stimulants coating antigen with being coated solution
Be sprayed on reaction film corresponding detection zone with sheep anti-mouse igg antibody and control zone form detection line and nature controlling line, detection line and
Nature controlling line is separated by 5mm, is positioned in 37 DEG C of baking ovens after being dried 2h standby, is wherein coated the phosphorus that solution is 0.01mol/L, pH 7.4
Phthalate buffer (PBS).
Preferably, the envelope antigen of described beta-stimulants is CL-BSA.
Furthermore it is preferred that described base plate is PVC.
Preferably, described sample pad is glass fibre element film.
Preferably, described adsorptive pads is common absorbent paper.
Preferably, described reaction film is nitrocellulose filter.
It is highly preferred that the width of described test strips is 4mm, sample pad, nitrocellulose filter, the width of adsorptive pads are respectively
2.0cm, 2.5cm, 1.7cm, lap one another and be attached on PVC base plate；Wherein, sample pad and the overlap width of nitrocellulose filter
For 2mm, nitrocellulose filter is 2mm with the overlap width of sample pad.
Further, the quantum dot immune chromatograph test strip quickly side of detection of the many residuals of beta-stimulants that the present invention is above-mentioned
Method, described detection can be qualitative detection, half-quantitative detection or detection by quantitative, and concrete outcome decision method is as follows:
A. qualitative detection or half-quantitative detection: use ultra violet lamp reacted reaction film region, observe by the naked eye band
Luminous situation, if there is fluorescence signal in detection line, it is determined that this index is negative；If detection line occurs without fluorescence signal, sentence
This index fixed is positive；Meanwhile, at either case, it is invalid that control line occurs without fluorescence signal then testing result；
B. detection by quantitative: configure the standard curve of the test substance of a series of variable concentrations, then utilizes fluorescence immune chromatography to read
Number instrument detects respectively, does standard curve by the T/C value that the standard solution of variable concentrations is corresponding, then is carried out by testing sample
Same process, obtains corresponding T/C value, learns test substance content in sample according to standard curve.
Said method of the present invention can be to rapid screening while multiple beta-stimulants, therefore, above-mentioned beta-stimulants
The application in terms of detection beta-stimulants residual of the quantum dot immune chromatograph test strip method for quick of many residuals, also at this
Within bright protection domain.
Preferably, described application refers to the quantum dot immune chromatograph test strip quickly side of detection of the many residuals of above-mentioned beta-stimulants
Method the most quickly detects or application in terms of examination multiple beta-stimulants residual.
Wherein it is preferred to, described multiple beta-stimulants is clenobuterol hydrochloride, albuterol, Mabuterol, bromine Boot
Appointing in sieve, isoproterenol sulfate or cimaterol is several.
Quantum dot used by the present invention has special skin effect and dimensional effect so that it is have unique spectrochemical property,
As quantum becomes rate height, excitation wavelength range width, launching wavelength narrow and symmetrical, Stokes shift is big, therefore can use same
Exciting light excites the quantum dot of different-grain diameter to carry out multivariate detection simultaneously.The transmitting range of wavelengths of quantum dot is relatively big, can be cross-domain
Ultraviolet is to infrared interval, and launches what wavelength can change according to the size and chemical composition changing quantum dot.Quantum
The fluorescence intensity of point is high, good stability, and fluorescence lifetime is long, through repeatedly exciting all without by photobleaching and chemical degradation, and amount
Son point has preferable biocompatibility, is suitable as fluorescent marker.
The method have the advantages that
The present invention provides a kind of beta-stimulants many residuals quantum dot immuno-chromatographic test paper strip, and the many residuals of a kind of beta-stimulants
Quantum dot immune chromatograph test strip method for quick, utilizes quantum dot as fluorescent probe, has been combined with immunochromatography technique
Come, can realize rapid screening while multiple beta-stimulants.
And, the fluorescence intensity of quantum dot is high, and good stability, fluorescence lifetime is long, floats all without by light through repeatedly exciting
White and chemical degradation, compares gold colloidal, organic fluorescence materials etc., can be greatly improved sensitivity.
It addition, the present invention is easy to operate, result is accurate, preferably resolves existing detection method and can only detect one simultaneously
To two kinds of problems such as beta-stimulants, poor stability.
The present invention is capable of rapid screening while multiple beta-stimulants, and detection method is easy, quick, knot
The most accurate, highly sensitive, qualitative and quantitative analysis can be done, cheap, applied widely, there is good popularizing application prospect.
Accompanying drawing explanation
Fig. 1 is beta-stimulants many residuals quantum dot immuno-chromatographic test paper strip structural representation of the present invention.
Fig. 2 is beta-stimulants many residuals quantum dot immuno-chromatographic test paper strip Competitive assays curve of the present invention.
Fig. 3 is beta-stimulants many residuals quantum dot immuno-chromatographic test paper strip immunoreaction kineties curve of the present invention.
Detailed description of the invention
Further illustrate the present invention below in conjunction with Figure of description and specific embodiment, but embodiment is not to the present invention
Limit in any form.Unless stated otherwise, the present invention uses reagent, method and apparatus are the examination of the art routine
Agent, method and apparatus.
Unless stated otherwise, following example agents useful for same and material are commercial.
The quantum dot immune chromatograph test strip method for quick of the many residuals of embodiment 1 beta-stimulants
The quantum dot immune chromatograph test strip method for quick of the many residuals of a kind of beta-stimulants, comprises the following steps:
(1) quantum dot fluorescence probe is prepared: take 20 μ L quantum dots and be dissolved in the phosphate-buffered of 3.2mL 0.01mol/L pH7.4
In liquid, add coupling agent 1-(3-dimethyl propyl)-3-ethyl-carbodiimide hydrochloride, N-hydroxy-succinamide solution, room
Temperature stirring 15min, adds 50 μ g beta-stimulants monoclonal antibodies, reaction 1.5h is stirred at room temperature, continuously adds 0.4mL 10%
BSA solution capping 1h, is centrifuged 40min by reaction solution with 12000r/min, removes supernatant, and with redissolving, liquid redissolution is heavy
Forming sediment, obtain the labeled complex of quantum dot and beta-stimulants monoclonal antibody, 4 DEG C keep in Dark Place；
(2) detection zone and the preparation of control zone: with nitrocellulose filter as reaction film, is attached to fixing for reaction film on base plate,
With being coated solution, the envelope antigen of beta-stimulants is configured to 0.85 mg/mL, and sheep anti mouse is configured to 0.8 mg/ with being coated solution
ML, is sprayed on reaction film corresponding detection zone and control zone, detection zone by anti-to beta-stimulants envelope antigen and sheep anti-mouse igg two
Being separated by 5 mm with control zone, be positioned in 37 DEG C of baking ovens after being dried 2 h standby, being wherein coated solution is 0.01 mol/L pH
7.4 phosphate buffer.
(3) on above-mentioned base plate, attach the reaction film in sample pad, step (2), adsorptive pads successively, and detection line is positioned at sample
Product pad side, nature controlling line is positioned at adsorptive pads side, test paper plate is cut into test strips, test strips is loaded on during test strips gets stuck,
Composition immuno-chromatographic test paper strip；
(4) qualitatively or quantitatively detection: quantum dot fluorescence probe hatched with sample blending, standing and reacting 1 min, draws addition and exempts from
Detect in the sample pad of epidemic disease chromatograph test strip；
A. qualitative detection or half-quantitative detection: use ultra violet lamp reacted reaction film region, observe by the naked eye band
Luminous situation, if detection line occurs judging during fluorescence signal that this index, as feminine gender, judges this index when occurring without fluorescence signal
For the positive, at either case, it is invalid that control line occurs without fluorescence signal then testing result；
B. detection by quantitative: configure the standard curve of the test substance of a series of variable concentrations, then utilizes fluorescence immune chromatography to read
Number instrument detects respectively, does standard curve by the T/C value that the standard solution of variable concentrations is corresponding, then by unknown sample to be measured
Process equally, obtain corresponding T/C value, learn test substance content in sample according to standard curve.
Wherein, described quantum dot is water-soluble carboxylated nuclear shell structure quantum point, and nucleocapsid structure is CdTe/ZnS.
Wherein, the amount of the coupling agent described in step (1) is proportional to the molar concentration of quantum dot, n (QDs): n (EDC): n
(NHS)=1:5:15.
Wherein, the liquid that redissolves described in step (1) is the Tris-HCl of 0.01mol/L, pH7.4, including 0.5%
Tween-20 and 0.5% BSA.
Wherein, described base plate is PVC, and sample pad is glass fibre element film, and absorbent paper is common absorbent paper.
Wherein, the width of described test strips is 4 mm, and sample pad, nitrocellulose filter, the width of adsorptive pads are respectively
2.0,2.5,1.7 cm, laps one another and is attached on PVC；Wherein, sample pad is 2 mm with the overlap width of nitrocellulose filter,
The overlap width of nitrocellulose filter and sample pad be 2 mm(as shown in Figure 1).
It addition, above-mentioned beta-stimulants monoclonal antibody is beta-stimulants class wide spectrum monoclonal antibody, concrete preparation method
As follows:
(1) preparation of antigen:
The most haptenic synthesis
Clenbuterol and glutaric anhydride are carried out acylation reaction, make the alcoholic extract hydroxyl group on Clenbuterol molecular structure acylated become containing
The carboxyl spacerarm of 5 carbon, and referred to as beta-stimulants hapten；
The most immunogenic preparation
Beta-stimulants hapten and ovalbumin (OVA) coupling are obtained immunogen；
C. the preparation of coating antigen
Use mixed anhydride method to carry out coupling beta-stimulants hapten and bovine serum albumin (BSA) to be coated
Former；
The envelope antigen of above-mentioned beta-stimulants is CL-BSA.
(2) animal immune: by the artificial antigen immunity Balb/C mice of above-mentioned preparation, and use indirect ELISA method to survey
Its titer fixed；
(3) cell merges: use PEG fusion method be able to enter with the oncocyte of energy Immortalization by the Mouse spleen cells of secretory antibody
Row merges；
(4) filtering hybridoma: by the specific antibody in indirect ELISA detection culture fluid, carry out titer and suppression ratio
Measure, filter out the positive hybridoma cell that titer is high and strong to Drug inhibition；
(5) hybridoma sub-clone and build strain: utilize limiting dilution assay to carry out sub-clone, the most repeatedly 3 ~ 4 take turns, obtaining can be steady
Surely the hybridoma cell strain of homogeneous antibody is secreted；
(6) prepared by monoclonal antibody: induces monoclonal antibody method in using animal body and prepares monoclonal antibody.
The quantum dot immune chromatograph test strip of the above-mentioned preparation of the present invention can detect multiple beta-stimulants simultaneously.
The standard that embodiment 2 sets up quantum dot immune chromatograph test strip as a example by standard substance clenobuterol hydrochloride (CL) is bent
Line
1, experiment material
(1) beta-stimulants class wide spectrum monoclonal antibody, preparation method is with embodiment 1.
(2) sheep anti-mouse igg two resists for commercialization reagent, purchased from Guangzhou You Kangduo Bioisystech Co., Ltd.
The preparation of immuno-chromatographic test paper strip is with embodiment 1.
2, the configuration of CL standard solution, with Tris-HCl compound concentration be respectively 0,0.0156,0.0625,0.250,
1.00, the series standard solution of 4.00,16.00 and 64.0 μ g/L, for ELISA test strip.
3, the mensuration of test strips
Each concentration duplicate detection 3 times, reads test strips FI with fluorescence immune chromatography readout instrument after 10 minT/FICRatio.With
FIT/FICFor vertical coordinate, with standard concentration logarithm as abscissa, draw test strips Competitive assays curve, calculate test strips 50%
Competitive assays rate concentration and determine the ELISA test strip range of linearity.
4, result is as in figure 2 it is shown, the detection range of linearity of standard curve of quantum dot immune chromatograph test strip is (IC20-
IC80) it is 0.14～2.28 μ g/L, 50% Competitive assays rate concentration (IC50) it is 0.56 μ g/L, detection limit (IC10) it is 0.06 μ g/L.
The precision of embodiment 3 quantum dot immune chromatograph test strip
1, as a example by CL, testing the standard solution of different CL content respectively with the test strips of different batches, each concentration repeats inspection
Survey 3 times, measure 3 different batches, calculate actual detection value.
2, as shown in table 1, ELISA test strip result batch between, variation within batch coefficient be respectively less than 15%, the weight of the method is described
Renaturation is good.
Table 1 quantum dot immune chromatograph test strip detection method criticizes interior, batch variation
The crossing-over rate of embodiment 4 various beta-stimulants medicine
1, with the cross reacting rate (CR) of CL for 100%, the cross reacting rate of the similar beta-stimulants of 10 kinds of structure functions is determined
(CR), as shown in table 2, be respectively as follows: clenobuterol hydrochloride (Clenbuterol, CL), albuterol (Salbutamol, SAL),
Mabuterol (Mabuterol), bromine Boot sieve (Brombuterol), cimaterol (Cimaterol), clorprenaline (
Clorprenaline), isoproterenol sulfate (Isoprenaline sulphate), Ractopamine (
Ractopamine), adrenalin hydrochloride (L-epinephrine hydrochloride) and noradrenaline bitartrate
(L-noradrenaline bitartrate).
2, result is as shown in table 2, albuterol, Mabuterol, bromine Boot sieve, isoproterenol sulfate, cimaterol
Clenbuterol there is is crossing-over rate, illustrates that the method is capable of rapid screening while multiple beta-stimulants.
Table 2 cross reaction measurement result
Quantum dot immune chromatograph test strip accuracy is measured as a example by CL, SAL by embodiment 5
1, sample pre-treatments
(1) pre-treatment of pig urine: the urine sample taking clear is standby, if any precipitation, centrifuging and taking supernatant.
(2) pre-treatment of Carnis Sus domestica: using literature method, concrete operation step is as follows:
2 g minced pork meat add 4mL 0.02mol/L HCl-2.8% extract with NaCl liquid, even tissue, and 13000r/min is centrifuged
4min, takes supernatant.Adding 4mL 0.02mol/L HCl-2.8% extracting solution second extraction, 13000r/min is centrifuged 4min.
Twice supernatant merges, and is centrifuged 4min with sodium hydroxide solution regulation pH to 7.0,13000r/min, and supernatant is to be checked.
2, result is as shown in table 3, the response rate that pig is urinated by constructed quantum dot immune chromatograph test strip 83.6～
Between 102.19%, the response rate of Carnis Sus domestica is between 64.27～87.98%, and the coefficient of variation of each sample is respectively less than 15%, explanation
The method has preferable accuracy.
Table 3 sample adds recovery testing result (n=3)
The preparation condition of embodiment 6 quantum dot fluorescence probe optimizes quantum dot and the optimization of coupling agent amount ratio
1, with reference to embodiment 1, with the quantum dot of different gradients and coupling agent amount ratio as variable, other conditions, with embodiment 1, are entered
The preparation of row quantum dot fluorescence probe, further prepares described immuno-chromatographic test paper strip, and the fluorescence observing test strips T line is strong
Degree.
2, result is as shown in table 4, when the molar concentration rate of quantum dot and coupling agent amount ratio is 1:3～6, test strips
Better performances, optimum molar concentration is than for 1:5.
Table 4 quantum dot and the optimization of coupling agent amount ratio
| QDs:EDC | 1:0 | 1:1 | 1:5 | 1:10 | 1:50 | 1:100 |
| Fluorescence intensity | - | + | +++ | ++ | ++ | + |
The preparation condition of embodiment 7 quantum dot fluorescence probe optimizes quantum dot and beta-stimulants monoclonal antibody amount ratio
Optimization
1, with reference to embodiment 1, with the quantum dot of different gradients and beta-stimulants monoclonal antibody amount ratio as variable, other conditions
With embodiment 1, carry out the preparation of quantum dot fluorescence probe, further prepare described immuno-chromatographic test paper strip, observe reagent paper
The fluorescence intensity of bar T line.
2, result is as shown in table 5, when quantum dot and beta-stimulants monoclonal antibody molar concentration rate are 1:3～6, and reagent paper
The better performances of bar, optimum amount is than for 1:4.
Table 5 quantum dot and the optimization of beta-stimulants monoclonal antibody amount ratio
| QDs:McAb | 1:0.5 | 1:1 | 1:2 | 1:4 | 1:8 |
| Fluorescence intensity | - | + | + | ++ | ++ |
The preparation condition of embodiment 8 quantum dot fluorescence probe optimizes the optimization of labelling buffer
1, with reference to embodiment 1, with not isolabeling buffer, other conditions, with embodiment 1, carry out the system of quantum dot fluorescence probe
Standby, further prepare described immuno-chromatographic test paper strip, observe the fluorescence intensity of test strips T line.
2, result is as shown in table 6, when labelling buffer is phosphate buffer, and the better performances of test strips,
The good phosphate buffer that labelling buffer is 0.01mol/L, pH7.4.
The optimization of table 6 labelling buffer
| Buffer | BB | MES | PBS | PB | CB |
| Fluorescence intensity | ++ | ++ | +++ | + | - |
Embodiment 9 chessboard method optimizes QDs-mAbs consumption volume and CL-BSA sprays concentration
1, with reference to embodiment 1, determine that the QDs-mAbs consumption volume of optimum and CL-BSA spray concentration by Checkerboard titration experiment,
Choosing negative sample T, the colour developing of C line normally, positive sample (c (CL)=0.5 μ g/L) suppression ratio is up to optimal conditions.Other
Part, with embodiment 1, is further prepared described immuno-chromatographic test paper strip and is carried out the preparation of beta-stimulants.
2, result is as shown in table 7, when the consumption of QDs-mAbs is 3 μ L, when CL-BSA spraying concentration is 0.85 mg/mL,
Test strips is that suppression ratio during 0.5 ng/mL reaches 54.98 %, and test strips T, C line in the mark product concentration of Clenbuterol (CL)
Fluorescence intensity the strongest.
Table 7 Checkerboard titration optimizes QDs-mAbs volume and CL-BSA spraying concentration (n=3)
Embodiment 10 QDs test strips immunoreaction kineties is analyzed
1, the determination in QDs fluorescence immune chromatography test paper bar response time is the premise of test strips detection by quantitative.To QDs fluorescence immunoassay
Chromatograph test strip T, C line immunoreaction kineties curve is measured, and step is as follows: drip 50 μ L feminine gender Quality Controls in test strips
Well in, read the fluorescent value FI of T, C line in test strips every 1min fluorescence immune chromatography readout instrumentT、FICAnd FIT/
FICValue, and tracing record 30min.
2, result as it is shown on figure 3, in 25 min fluorescence intensity FI of T, C lineT、FICThe most constantly raise,
Tend towards stability after 25 min；But FIT/FICRatio tends to stable after 10 min, and 10 ~ 30 min become without obvious subsequently
Change, show FIT/FICThe quantitative approach of value can eliminate the difference of immunoreaction kineties within a certain period of time, when shortening interpretation
Between.Therefore, the QDs fluorescence immune chromatography test paper bar detection by quantitative time is 10 min.
The optimization of the anti-extension rate of embodiment 11 nature controlling line two
1, with reference to embodiment 1, with the diluted concentration of two different anti-sheep anti mouses as variable, other conditions, with embodiment 1, prepare institute
The immuno-chromatographic test paper strip stated, compares the color of T line and C line.
2, result shows, along with the increase of two anti-concentration, the concentration of T line is gradually increased, when two anti-concentration are 0.8mg/mL
Time, C line is the depth as T line color.Therefore, select two anti-concentration 0.8mg/mL as the anti-optium concentration of nature controlling line two.
The anti-concentration optimization (n=3) of table 8 nature controlling line two
| Two anti-concentration (mg/mL) | 0.2 | 0.4 | 0.8 | 1.6 |
| FI<sub>T</sub>/FI<sub>C</sub> | 2.38 | 1.62 | 1.08 | 0.64 |
Claims (10)
1. the quantum dot immune chromatograph test strip method for quick of the many residuals of beta-stimulants, it is characterised in that be first
By quantum dot and beta-stimulants monoclonal antibody coupling, obtain the labeled complex of quantum dot and beta-stimulants monoclonal antibody,
It is quantum dot fluorescence probe；Again using this labeled complex as after quantum dot fluorescence probe and testing sample blending incubation, profit
The detection of beta-stimulants residual is carried out with immuno-chromatographic test paper strip.
The quantum dot immune chromatograph test strip method for quick of the many residuals of beta-stimulants the most according to claim 1, its
Being characterised by, described quantum dot is water-soluble carboxyl based quantum dot.
The quantum dot immune chromatograph test strip method for quick of the many residuals of beta-stimulants the most according to claim 1, its
Being characterised by, described quantum dot is water-soluble carboxylated nuclear shell structure quantum point, and nucleocapsid structure is CdTe/ZnS.
The quantum dot immune chromatograph test strip method for quick of the many residuals of beta-stimulants the most according to claim 1, its
Being characterised by, described beta-stimulants monoclonal antibody is beta-stimulants class wide spectrum monoclonal antibody.
The quantum dot immune chromatograph test strip method for quick of the many residuals of beta-stimulants the most according to claim 1, its
Being characterised by, the preparation method of described quantum dot fluorescence probe is as follows:
S1. take quantum dot to be dissolved in phosphate buffer, add coupling agent and N-hydroxy-succinamide solution, be stirred at room temperature
10～30min；Described coupling agent is 1-(3-dimethyl propyl)-3-ethyl-carbodiimide hydrochloride；
S2. add beta-stimulants monoclonal antibody, reaction 1～2h is stirred at room temperature；
S3. continuously add 10% BSA solution capping 1h, reactant liquor is centrifuged 40min with 12000r/min；
S4. remove supernatant after being centrifuged, with redissolving liquid redissolution precipitation, obtain the labelling of quantum dot and beta-stimulants monoclonal antibody
Complex.
The quantum dot immune chromatograph test strip method for quick of the many residuals of beta-stimulants the most according to claim 5, its
It is characterised by, quantum dot described in step S1: coupling agent: the molar concentration rate of N-hydroxy-succinamide solution is 1:3～6:13
～17；Quantum dot described in step S1 is 1:3～6 with the molar concentration rate of beta-stimulants monoclonal antibody described in step S2.
The quantum dot immune chromatograph test strip method for quick of the many residuals of beta-stimulants the most according to claim 5, its
Being characterised by, described in step S4, redissolution liquid is the Tris-HCl of 0.01mol/L, pH 7.4, including the Tween-20 of 0.5%
With 0.5% BSA.
The quantum dot immune chromatograph test strip method for quick of the many residuals of beta-stimulants the most according to claim 1, its
Being characterised by, described immuno-chromatographic test paper strip includes base plate, sample pad, reaction film, adsorptive pads, described sample pad, reaction film and
Adsorptive pads is attached on base plate successively, described reaction film is provided with the detection line comprising beta-stimulants envelope antigen and comprises sheep
The nature controlling line of dynamics, and detection line be positioned at sample pad side, nature controlling line is positioned at adsorptive pads side；Wherein, described β-emerging
Putting forth energy agent envelope antigen is CL-BSA.
The quantum dot immune chromatograph test strip method for quick of the many residuals of beta-stimulants the most according to claim 1, its
Being characterised by, described detection can be qualitative detection, half-quantitative detection or detection by quantitative, and concrete outcome decision method is as follows:
A. qualitative detection or half-quantitative detection: use ultra violet lamp reacted reaction film region, observe by the naked eye band
Luminous situation, if there is fluorescence signal in detection line, it is determined that this index is negative；If detection line occurs without fluorescence signal, sentence
This index fixed is positive；Meanwhile, at either case, it is invalid that control line occurs without fluorescence signal then testing result；
B. detection by quantitative: configure the standard curve of the test substance of a series of variable concentrations, then utilizes fluorescence immune chromatography to read
Number instrument detects respectively, does standard curve by the T/C value that the standard solution of variable concentrations is corresponding, then is carried out by testing sample
Same process, obtains corresponding T/C value, learns test substance content in sample according to standard curve.
10. the quantum dot immune chromatograph test strip method for quick of the many residuals of the arbitrary described beta-stimulants of claim 1～9 exists
Application in terms of detection beta-stimulants residual.
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