CN104849467A - Fluorescent microsphere immunochromatography test paper strip for detecting clenbuterol hydrochloride residue, preparation method and applications thereof - Google Patents
Fluorescent microsphere immunochromatography test paper strip for detecting clenbuterol hydrochloride residue, preparation method and applications thereof Download PDFInfo
- Publication number
- CN104849467A CN104849467A CN201410052593.6A CN201410052593A CN104849467A CN 104849467 A CN104849467 A CN 104849467A CN 201410052593 A CN201410052593 A CN 201410052593A CN 104849467 A CN104849467 A CN 104849467A
- Authority
- CN
- China
- Prior art keywords
- clenizole hydrochloride
- test paper
- paper strip
- fluorescent
- clenizole
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention discloses a fluorescent microsphere immunochromatography test paper strip for detecting clenbuterol hydrochloride residue, a preparation method and applications thereof. The test paper strip comprises a bottom plate, a sample binding pad, a nitrocellulose membrane, and a water absorbing pad, wherein the sample binding pad, the nitrocellulose membrane, and the water absorbing pad are sequentially adhered on the bottom plate in an overlapping manner, fluorescent microsphere-labeled anti-clenbuterol hydrochloride monoclonal antibodies are embedded on the binding pad, a detection zone and a quality control zone are immobilized on the nitrocellulose membrane, a clenbuterol hydrochloride hapten-carrier protein conjugate is sprayed on the detection zone, and goat anti-mouse anti-antibodies are sprayed on the quality control zone. The test paper strip of the present invention is mainly used for the clenbuterol hydrochloride residue detection, and has advantages of high sensitivity, rapid detection, easy operation, economy, practicality, and the like.
Description
Technical field
The invention belongs to food security herbal medicine detection field, be specifically related to a kind ofly detect residual fluorescent micro-ball immune chromatography test paper strip of Clenizole Hydrochloride in sample and its preparation method and application.
Technical background
Clenbuterol (Clebuterol) is a kind of beta-receptor activator belonging to phenyl amines, mainly exist with hydrochloride form, have and redistribute body nutrition, suppress fat deposition, improve protein content, increase ketoboidies lean meat percentage, improve meat and promote the effect of growth of animal.Therefore, in recent years under the ordering about of economic interests, a large amount of clenbuterol is misused in animal husbandry and aquaculture.When the amount of human body absorption CL is excessive, a series of bad reaction can be caused, mainly contain muscular tremor, heartbeat and accelerated breathing etc., serious meeting life threatening.Given this, many countries have forbidden clenbuterol to use as feed and feed addictive, and the Ministry of Agriculture of China was also clearly listed in 2002 " forbidding the types of drugs catalogue used in feed and animal drinking water ".
At present, the method detecting Clenizole Hydrochloride more classical has tablets by HPLC-MS (HPLC-MS), GC-MS(gas chromatography-mass spectrography) (GC-MS), high performance liquid chromatography (HPLC), these are all more traditional methods, are also current confirmation methods.In addition, conventional at present euzymelinked immunosorbent assay (ELISA) (ELISA) in addition.But because above method all needs advanced detecting instrument, testing cost costliness, complex steps, consuming time, and higher to the professional requirement of operating personnel, the large flux rapid screening not being suitable for enterprises and institutions of basic unit detects.Colloidal gold immunity chromatography has that detection speed is fast, low price, simple operation and other advantages, it is the main method for supervising detecting Clenizole Hydrochloride at present both at home and abroad, but still there are some defects, as less stable, sensitivity compared with low, quantitative detection cannot be realized, the obvious background interference of matrix effect is large, and color is single, be difficult to realize many inspections and joint inspection.
Fluorescent micro-ball immune chromatography technology is after colloidal gold immunochromatographimethod technology, technically grow up at fluorochrome label, as a kind of immunological detection method, it is the combination of immune affine technology, immunolabelling technique, immunochromatography technique, and the same with colloidal gold immunochromatographimethod technology have the advantages such as quick, easy and simple to handle.But compare the conventional tag things such as collaurum, the luminous intensity of fluorescent microsphere can strengthen with the strength-enhanced of exciting light, so fluorescent microsphere mark is expected to the detectability improving immunochromatography technique; And under the effect of microballoon shell structure, fluorescent microsphere has metastable morphosis, homogeneous grain diameter, monodispersity are good, good stability, luminescence efficiency are high, reproducible, have good biocompatibility; And dyestuff fluorescent quenching greatly reduces after formation microballoon, launch strong and stablize, and substantially not by the impact of external environment media variations.Therefore compare above-mentioned detection method, fluorescent micro-ball immune chromatography technology has that detection sensitivity is high, easy and simple to handle, the advantage of good stability simultaneously.
Summary of the invention
An object of the present invention is the defect for above-mentioned prior art, provide a kind of highly sensitive, easy and simple to handle, detect the residual fluorescent micro-ball immune chromatography test paper strip of quick, cheap detection Clenizole Hydrochloride; Another object of the present invention is to provide the preparation method of above-mentioned test strips; Another object of the present invention is to provide above-mentioned test strips and is detecting the application in Clenizole Hydrochloride.
To achieve these goals, the technical scheme that the present invention takes is:
There is provided a kind of and detect the residual fluorescent micro-ball immune chromatography test paper strip of Clenizole Hydrochloride, it comprises base plate and on base plate, overlaps the sample pad of stickup, nitrocellulose filter and adsorptive pads successively, described sample pad is embedded with the anti-Clenizole Hydrochloride monoclonal antibody of fluorescent microsphere mark, described nitrocellulose filter is fixed with detection zone and quality control region, detection zone is coated with Clenizole Hydrochloride hapten-carrier protein conjugate, and quality control region is coated with sheep anti mouse antiantibody.
Described Clenizole Hydrochloride hapten-carrier protein conjugate is obtained by Clenizole Hydrochloride haptens and carrier protein couplet, described Clenizole Hydrochloride haptens be by Clenizole Hydrochloride through pyridinium chlorochromate be oxidized after, be obtained by reacting with propane diamine, described carrier protein can be bovine serum albumin(BSA), ovalbumin, hemocyanin, thyroprotein, human serum albumins again.
The microballoon with polystyrene parcel fluorescent material of described fluorescent microsphere to be diameter be 100 ~ 300nm, its surface is connected with-COOH group, and described fluorescent material is fluorescein isothiocynate.
Another technical scheme that the present invention takes is to provide a kind of method preparing the fluorescent micro-ball immune chromatography test paper strip that above-mentioned detection Clenizole Hydrochloride remains, and it comprises the steps:
1) preparation of sample pad: mark anti-Clenizole Hydrochloride monoclonal antibody with commercially available fluorescent microsphere, and by it with after specific buffer system dilution, sample pad is soaked in dilution buffer, prepares after vacuum freeze drying;
2) preparation of nitrocellulose filter: Clenizole Hydrochloride hapten-carrier protein conjugate is sprayed to the detection zone scope on nitrocellulose filter, make detection zone; Sheep anti mouse antiantibody is sprayed to the quality control region scope on nitrocellulose filter, make quality control region;
3) assembling and shearing: paste with overlapping successively on base plate and be embedded with fluorescent microsphere and mark the sample pad of anti-Clenizole Hydrochloride monoclonal antibody, the nitrocellulose filter being fixed with detection zone and quality control region and adsorptive pads, and cut into required width and be fluorescent micro-ball immune chromatography test paper strip.
Specifically, step comprises:
1) by Clenizole Hydrochloride after pyridinium chlorochromate oxidation, then to react with propane diamine, prepare Clenizole Hydrochloride haptens;
2) by Clenizole Hydrochloride haptens and carrier protein couplet, Clenizole Hydrochloride hapten-carrier protein conjugate is prepared;
3) with Clenizole Hydrochloride hapten-carrier protein conjugate immune mouse, mouse boosting cell and murine myeloma cell are passed through to merge, screen, obtains the hybridoma cell strain secreting Clenizole Hydrochloride monoclonal antibody;
4) extract mouse IgG immune health goat, obtain sheep anti mouse antiantibody;
5) respectively Clenizole Hydrochloride hapten-carrier protein conjugate and sheep anti mouse antiantibody are sprayed to detection zone scope (T) and the quality control region scope (C) of nitrocellulose filter;
6) phosphate buffer of sample pad containing bovine serum albumin(BSA) (final concentration of bovine serum albumin(BSA) in buffer system is 0.5% volumn concentration), pH7.2,0.1mol/L is evenly soaked 2h, at 37 DEG C, dry 2h;
7) anti-Clenizole Hydrochloride monoclonal antibody is marked with commercially available fluorescent microsphere, and by it with after the dilution of specific buffer system, by 6) the sample pad that processed is soaked in dilution buffer, for subsequent use after vacuum freeze drying;
8) paste with overlapping successively on base plate and be embedded with fluorescent microsphere and mark the sample pad of anti-Clenizole Hydrochloride monoclonal antibody, the nitrocellulose filter being fixed with detection zone and quality control region and adsorptive pads, and cut into required width and be fluorescent micro-ball immune chromatography test paper strip.
Another technical scheme that the present invention takes is to provide the application of fluorescent micro-ball immune chromatography test paper strip in detection Clenizole Hydrochloride that a kind of above-mentioned detection Clenizole Hydrochloride remains, and it comprises the steps:
1) sample pre-treatments;
2) detect with the fluorescent micro-ball immune chromatography test paper strip that described detection Clenizole Hydrochloride is residual;
3) testing result is analyzed with fluorescence detector.
Compared with prior art, the present invention has following beneficial effect:
(1) high specificity, highly sensitive: this test strips adopts the anti-Clenizole Hydrochloride monoclonal antibody by fluorescent microsphere marks to be embedded on sample pad, have water wettability good, can Large Copacity adsorb antibodies conjugate, the advantage such as rapidly heavy moistening, antibody conjugates release fully, performance is good, release is fast, form is good, thus minimizing error, reduce costs, increase the reaction sensitivity of whole system;
(2) polystyrene has been wrapped up on fluorescent microsphere surface, achieves the protection to fluorescent material fluorescein isothiocynate, decreases the interference of external environment, adds stability and the fluorescence lifetime of fluorescent microsphere;
(3) fluorescent microsphere surface modified active group-COOH, adopts the method for chemical coupling to carry out labelled antibody, forms the stable bond of antibody and microballoon.
Accompanying drawing explanation
Fig. 1 is fluorescent micro-ball immune chromatography test paper strip cross-sectional view;
Fig. 2 is fluorescent micro-ball immune chromatography test paper strip vertical view;
Fig. 3 is Clenizole Hydrochloride hapten synthesis route map;
Fig. 4 is Clenizole Hydrochloride haptens hydrogen nuclear magnetic resonance spectrogram.
Embodiment
Below in conjunction with embodiment and accompanying drawing, further detailed description is done to the present invention, but embodiments of the present invention are not limited thereto.
Embodiment 1 detects the formation of the fluorescent micro-ball immune chromatography test paper strip that Clenizole Hydrochloride remains
One, test strips (Fig. 1, Fig. 2)
Described test strips is made up of base plate, sample pad, nitrocellulose filter and adsorptive pads;
Described sample pad 1, nitrocellulose filter 2 and adsorptive pads 3 overlap in order successively and are pasted onto on base plate 6, the end of sample pad is connected with the top of nitrocellulose filter, the end of nitrocellulose filter is connected with the top of adsorptive pads, the top of sample pad aligns with the top of base plate, and the end of adsorptive pads aligns with the end of base plate;
Described nitrocellulose filter is fixed with detection zone 4 and quality control region 5, detection zone is coated with Clenizole Hydrochloride hapten-carrier protein conjugate (Clenizole Hydrochloride haptens-ovalbumin conjugate), and quality control region is coated with sheep anti mouse antiantibody;
Described base plate is PVC base plate; Described sample pad is glass wool; Described adsorptive pads is thieving paper.
Embodiment 2 detects the preparation of the fluorescent micro-ball immune chromatography test paper strip that Clenizole Hydrochloride remains
The preparation method detecting the fluorescent micro-ball immune chromatography test paper strip that Clenizole Hydrochloride remains mainly comprises the following steps:
1) preparation of sample pad: mark anti-Clenizole Hydrochloride monoclonal antibody with commercially available fluorescent microsphere, and by it with after specific buffer system dilution, sample pad is soaked in dilution buffer, prepares after vacuum freeze drying;
2) preparation of nitrocellulose filter: Clenizole Hydrochloride hapten-carrier protein conjugate is sprayed to the detection zone scope on nitrocellulose filter, make detection zone; Sheep anti mouse antiantibody is sprayed to the quality control region scope on nitrocellulose filter, make quality control region;
3) assembling and shearing: paste with overlapping successively on base plate and be embedded with fluorescent microsphere and mark the sample pad of anti-Clenizole Hydrochloride monoclonal antibody, the nitrocellulose filter being fixed with detection zone and quality control region and adsorptive pads, and cut into required width and be fluorescent micro-ball immune chromatography test paper strip.
Substep describes in detail below:
(1) preparation of each parts
1, the synthesis of Clenizole Hydrochloride hapten-carrier protein conjugate and qualification
Clenizole Hydrochloride is small-molecule substance, only has immunoreactivity, does not have immunogenicity, can not bring out body produce immune response, must with macromolecular carrier albumen coupling after just there is immunogenicity.
(1) the haptenic preparation of Clenizole Hydrochloride (Fig. 3)
Step one: get 1.4g Clenizole Hydrochloride, dissolve in 25mL dimethyl formamide (DMF), divide at 0 DEG C and add 1.8g pyridinium chlorochromate (PCC) for 3 times, be progressively warmed up to room temperature, after continuing reaction 3h, remove most of solvent, extraction into ethyl acetate, washing, dry, except desolventizing rear pillar chromatographic purifying, obtaining white solid, is Clenizole Hydrochloride oxide;
Step 2: the Clenizole Hydrochloride oxide getting 1.0g step one gained, dissolves in 20mL DMF, adds 1mL propane diamine, 60 DEG C of reaction 10h, except desolventizing, recrystallization in alcohol-water, obtain white solid, be the propane diamine list condensation product of Clenizole Hydrochloride, be Clenizole Hydrochloride haptens.
Get above-mentioned product through nuclear magnetic resonance hydrogen spectruming determining, as shown in Figure 4, the 3 groups of propane diamine fragment methylene signals peaks increased between 1.8 ~ 3.7ppm, illustrate hapten synthesis success.
(2) immunogenic preparation
Get the water-soluble solution of bovine serum albumin(BSA) (BSA) 50mg 4mL; Get carbodiimides (EDC) and the water-soluble solution of N-hydroxy-succinamide (NHS) each 50mg 2mL completely after add in protein solution, stirring at room temperature reaction 30min, can obtain reactant liquor A; After getting haptens 15mg 2mL DMF dissolving, slowly join in reactant liquor A, stirring at room temperature reaction 24h; With the PBS dialysis 3d of 0.02mol/L, change 3 dislysates every day, to remove unreacted small-molecule substance; Packing, saves backup in-20 DEG C.
(3) preparation of coating antigen
BSA, with immunogenic preparation, is replaced with ovalbumin (OVA) and namely obtains coating antigen by preparation method.
(4) qualification of Clenizole Hydrochloride hapten-carrier protein conjugate
The PBS damping fluid of Clenizole Hydrochloride haptens, carrier protein, Clenizole Hydrochloride hapten-carrier protein conjugate pH7.4 is made into the solution of 0.5mg/mL, return to zero with the PBS damping fluid of 0.01mol/L pH7.4, with ultraviolet spectrophotometer in the interscan of wavelength 200 ~ 800nm scope, obtain the absorption curve of Clenizole Hydrochloride haptens, carrier protein, Clenizole Hydrochloride hapten-carrier protein conjugate.There is different absorption curves in three, shows Clenizole Hydrochloride haptens and carrier protein couplet success.
2, the preparation of Clenizole Hydrochloride monoclonal antibody
(1) animal immune
Immunogene step 1 obtained is injected in Balb/c Mice Body, and immunizing dose is 150 μ g/, makes it produce antiserum.
(2) Fusion of Cells and cloning
The Balb/c mouse boosting cell and the myeloma cell SP20 that get generation specific antibody merge, and adopt indirect competitive enzyme-linked immunosorbent analytical approach to measure cell conditioned medium liquid, screen positive hole.Utilize limiting dilution assay to carry out cloning to positive hole, obtain and set up the hybridoma cell strain producing monoclonal antibody.
(3) cell cryopreservation and recovery
Get the hybridoma cryopreserving liquid being in exponential phase and make cell suspension, be sub-packed in cryopreservation tube, preserve for a long time in liquid nitrogen.Take out cryopreservation tube during recovery, put into 37 DEG C of water-bath middling speeds immediately and melt, after centrifugal segregation cryopreserving liquid, move into and cultivate culture in glassware.
(4) preparation and purification of monoclonal antibody
Adopt in body and induce method, by Balb/c mouse (8 week age) Intraperitoneal injection sterilizing paraffin oil, 7 ~ 14 days pneumoretroperitoneum injection hybridomas, gathered ascites after 7 ~ 10 days.Carry out ascites purifying through sad-saturated ammonium sulfate method, purity through SDS-PAGE electroresis appraisal, bottle packing ,-20 DEG C of preservations.
3, the preparation of sheep anti mouse antiantibody
Using sheep as immune animal, for immunogene, immunity is carried out to pathogen-free domestic sheep with mouse source antibody, obtain sheep anti mouse antiantibody.
4, fluorescent microsphere marks the preparation of anti-Clenizole Hydrochloride monoclonal antibody
(1) activate: get commercially available inside embedding fluorescent dye, finishing has the microsphere suspensions 100 μ L of carboxyl functional group to be suspended in 900 μ L activation buffer, supernatant is abandoned after 4 DEG C of centrifugal 10min of 10000r/min, resuspended microballoon is in 1mL activation buffer, wash microballoon 2 times in this way, add appropriate activator, room temperature concussion activation 10min after mixing;
(2) coupling: described to (1) suspension is abandoned supernatant after 4 DEG C of centrifugal 10min of 10000r/min, be resuspended in coupling buffer, wash microballoon 2 times in this way, add the anti-Clenizole Hydrochloride monoclonal antibody solution of 10 ~ 20 μ L (protein concentration 1mg/mL), room temperature concussion coupling 120min after mixing;
(3) close: described to (2) suspension is abandoned supernatant after 4 DEG C of centrifugal 10min of 10000r/min, is resuspended in Block buffer, wash microballoon 1 time in this way, after mixing, 30min is closed in room temperature concussion;
(4) store: described to (3) suspension is abandoned supernatant after 4 DEG C of centrifugal 10min of 10000r/min, is resuspended in storage buffer, wash microballoon 1 time in this way, keep in Dark Place in 4 DEG C after mixing.
Described activation buffer is 2-(N-morpholine) ethyl sulfonic acid (MES) damping fluid of pH5.5 ~ 6.5,0.05mol/L.
Described activator is water-soluble carbodiimide, and wherein molal weight is than EDC: NHS: COOH=(1.5 ~ 3): (8 ~ 20): 1, is diluted to desired concn before use with activation buffer.
Described coupling buffer is the borate buffer solution (avoiding using the solvent that there is unhindered amina) of pH7.5 ~ 8.50.05mol/L.
Described Block buffer is the PB damping fluid of the pH7.4 containing 0.1 ~ 0.4mol/L primary amine (oxammonium hydrochloride, monoethanolamine or ethylaminoethanol), 1% ~ 10%BSA.
Described storage buffer is for containing 0.01%NaN
3, 0.1%BSA the PB damping fluid of pH7.4.
5, the preparation of sample pad
(1) phosphate buffer of sample pad containing bovine serum albumin(BSA) (final concentration of BSA in buffer system is 0.5% volumn concentration), pH7.2,0.1mol/L is evenly soaked 2h, at 37 DEG C, dry 2h;
(2), after the anti-Clenizole Hydrochloride monoclonal antibody of the fluorescent microsphere of storage mark being diluted with storage buffer, the sample pad (1) processed is soaked in dilution buffer, for subsequent use after vacuum freeze drying.
6, the preparation of cellulose nitrate (NC) film
With the PBS damping fluid of 0.05mol/L, pH7.2, Clenizole Hydrochloride haptens-ovalbumin conjugate is diluted to 100 μ g/mL, is sprayed at the detection zone (T) on NC film with Isoflow point film instrument, spray film amount is 1.0 μ L/cm; With the PBS damping fluid of 0.01mol/L, pH7.4, sheep anti mouse antiantibody is diluted to 200 μ g/mL, is sprayed at the quality control region (C) on NC film with Isoflow point film instrument, spray film amount is 1.0 μ L/cm.The NC film prepared is placed in dry 2h under 37 DEG C of conditions, for subsequent use.
(2) assembling of test strips
Sample pad, nitrocellulose filter, adsorptive pads being overlapped successively is from left to right pasted and fixed on base plate, the end of sample pad is connected with the top of nitrocellulose filter, the end of nitrocellulose filter is connected with the top of adsorptive pads, the top of sample pad aligns with the top of base plate, the end of adsorptive pads aligns with the end of base plate, then be cut into the wide little bar of 3.96mm with machine, be contained in special plastics fabrication, form test card.Clenizole Hydrochloride fluorescent micro-ball immune chromatography test paper is stuck in 2 ~ 8 DEG C of shady and cool lucifuge kept dry, and the term of validity is 12 months.
Embodiment 3 detects the application of the fluorescent micro-ball immune chromatography test paper strip that Clenizole Hydrochloride remains
1, sample pre-treatments
(1) urine specimen
Get limpid urine specimen directly to measure, sample must be collected in clean dried, the plastics urine cup not containing any antiseptic or glass container; If can not censorship in time, can 24h be preserved 2 ~ 8 DEG C of refrigerations, preserve if long-term need be placed in-20 DEG C freezing, must guard against multigelation.
(2) animal tissue's sample
Get animal tissue's sample 10000r/min homogeneous 1min in homogenizer of degrease; Take the good tissue samples of 2.0g ± 0.05g homogeneous in 4mL polystyrene centrifuge tube, add 500 μ L sample extraction liquid (the PB damping fluid of 0.35mol/L), whirling motion 1min; After putting into 80 DEG C of water-bath water-bath 10min, the centrifugal 5min of 4000r/min, is cooled to room temperature, to be checked.
2, ELISA test strip is used
Draw 100 μ L sample solution to be checked vertically to drip in test card well, during liquid flow, start timing, reaction 10min; Inserted by test card in the carrier of KFT-100A type fluorescence detector, select project to be checked by touch display screen, press " starting to detect " button, fluorescence detector will carry out sweep test to test card automatically; By the display screen of instrument reading or printing testing result.
3, Analysis of test results
After having tested, instrument, by according to the power detecting the fluorescence signal obtained, calculates the concentration value of Clenizole Hydrochloride in urine, animal tissue's sample automatically, and provides yin and yang attribute judgement according to the threshold value preset.
Negative (-): if result is shown as feminine gender on the display screen of fluorescence detector, to represent in sample containing Clenizole Hydrochloride or its concentration lower than detectability.
Positive (+): if result is shown as the positive on the display screen of fluorescence detector, represents that in sample, Clenizole Hydrochloride concentration is equal to or higher than detectability.
Invalid: if quality control region does not detect fluorescence signal intensity, show that incorrect operating process or test card lost efficacy.
Embodiment 4 detects the determination of the fluorescent micro-ball immune chromatography test paper strip technical parameter that Clenizole Hydrochloride remains
1, sensitivity test
Clenizole Hydrochloride standard items are diluted to respectively 0.5,1,2 μ g/L, used diluent is the phosphate buffer of pH7.2,0.2mol/L.
Detect with fluorescent micro-ball immune chromatography test paper strip, result is: when Clenizole Hydrochloride standard concentration is 0.5 μ g/L, fluorescence detector is detected as feminine gender; When Clenizole Hydrochloride standard concentration is 1,2 μ g/L, fluorescence detector test positive, shows that the sensitivity of this ELISA test strip Clenizole Hydrochloride is 1 μ g/L.
2, false positive rate, false negative rate test
Get positive pig urine sample and each 20 parts of positive pork sample that known Clenizole Hydrochloride content is greater than 1 μ g/L, concentration is less than negative pig urine sample and each 20 parts of the negative pork sample of 1 μ g/L, the fluorescent micro-ball immune chromatography test paper strip produced with 3 batches detects respectively, calculates its yin and yang attribute rate.The results are shown in Table 1.
Table 1 detects the positive, negative sample result
Result shows: during the ELISA test strip positive sample of producing with 3 batches, and result is positive entirely, and known positive coincidence rate is 100%, and false negative rate is 0; When detecting negative sample, result is negative entirely, and known negative match-rate is 100%, and false positive rate is 0.Illustrate that the fluorescent micro-ball immune chromatography test paper strip that detection Clenizole Hydrochloride of the present invention remains can detect fast to Clenizole Hydrochloride in animals urine and animal tissue is residual.
Claims (5)
1. one kind is detected the residual fluorescent micro-ball immune chromatography test paper strip of Clenizole Hydrochloride, it is characterized in that comprising base plate and overlap successively on base plate the sample pad of stickup, nitrocellulose filter and adsorptive pads, described sample pad is embedded with the anti-Clenizole Hydrochloride monoclonal antibody of fluorescent microsphere mark, described nitrocellulose filter is fixed with detection zone and quality control region, detection zone is coated with Clenizole Hydrochloride hapten-carrier protein conjugate, and quality control region is coated with sheep anti mouse antiantibody.
2. test strips according to claim 1, it is characterized in that described Clenizole Hydrochloride hapten-carrier protein conjugate is obtained by Clenizole Hydrochloride haptens and carrier protein couplet, described Clenizole Hydrochloride haptens be by Clenizole Hydrochloride through pyridinium chlorochromate be oxidized after, be obtained by reacting with propane diamine, molecular structural formula is again:
Described carrier protein can be bovine serum albumin(BSA), ovalbumin, hemocyanin, thyroprotein, human serum albumins.
3. test strips according to claim 1, it is characterized in that described fluorescent microsphere to be diameter be the microballoon with polystyrene parcel fluorescent material of 100 ~ 300nm, its surface is connected with-COOH group, and described fluorescent material is fluorescein isothiocynate.
4. a preparation method for the fluorescent micro-ball immune chromatography test paper strip that the detection Clenizole Hydrochloride described in any one of claim 1-3 remains, is characterized in that comprising step:
1) preparation of sample pad: mark anti-Clenizole Hydrochloride monoclonal antibody with commercially available fluorescent microsphere, and by it with after specific buffer system dilution, sample pad is soaked in dilution buffer, prepares after vacuum freeze drying;
2) preparation of nitrocellulose filter: Clenizole Hydrochloride hapten-carrier protein conjugate is sprayed to the detection zone scope on nitrocellulose filter, make detection zone; Sheep anti mouse antiantibody is sprayed to the quality control region scope on nitrocellulose filter, make quality control region;
3) assembling and shearing: paste with overlapping successively on base plate and be embedded with fluorescent microsphere and mark the sample pad of anti-Clenizole Hydrochloride monoclonal antibody, the nitrocellulose filter being fixed with detection zone and quality control region and adsorptive pads, and cut into required width and be fluorescent micro-ball immune chromatography test paper strip.
5. the fluorescent micro-ball immune chromatography test paper strip that the detection Clenizole Hydrochloride described in any one of claim 1-3 remains is detecting the application in Clenizole Hydrochloride, it is characterized in that comprising step:
1) sample pre-treatments;
2) detect with the fluorescent micro-ball immune chromatography test paper strip described in any one of claim 1-3;
3) testing result is analyzed with fluorescence detector.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410052593.6A CN104849467B (en) | 2014-02-15 | 2014-02-15 | Fluorescent micro-ball immune chromatography test paper strip of detection clenbuterol hydrochloride residual and its preparation method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410052593.6A CN104849467B (en) | 2014-02-15 | 2014-02-15 | Fluorescent micro-ball immune chromatography test paper strip of detection clenbuterol hydrochloride residual and its preparation method and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104849467A true CN104849467A (en) | 2015-08-19 |
| CN104849467B CN104849467B (en) | 2016-08-17 |
Family
ID=53849265
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410052593.6A Active CN104849467B (en) | 2014-02-15 | 2014-02-15 | Fluorescent micro-ball immune chromatography test paper strip of detection clenbuterol hydrochloride residual and its preparation method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104849467B (en) |
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105301253A (en) * | 2015-09-07 | 2016-02-03 | 北京勤邦生物技术有限公司 | T-2 toxin-enriched immunomagnetic bead as well as preparation method and application thereof |
| CN105527439A (en) * | 2015-12-30 | 2016-04-27 | 厦门依柯利斯医疗科技有限公司 | An NGAL time-resolved fluoroimmunoassay nanometer immunochromatographic quantitative detection test paper strip and a preparing method thereof |
| CN108181465A (en) * | 2018-02-09 | 2018-06-19 | 广东优尼德生物科技有限公司 | Detect the fluorescent chromatographic kit and its quantitative detecting method of 25-hydroxy-vitamin D |
| CN108303549A (en) * | 2018-02-09 | 2018-07-20 | 广东优尼德生物科技有限公司 | A kind of fluorescence immune chromatography test paper bar and its quantitative detecting method of detection adiponectin |
| CN109061157A (en) * | 2018-09-21 | 2018-12-21 | 中国烟草总公司郑州烟草研究院 | A kind of time-resolved fluoroimmunoassay chromatograph test strip and its preparation method and application detecting flumetralim |
| CN109061158A (en) * | 2018-09-21 | 2018-12-21 | 中国烟草总公司郑州烟草研究院 | A kind of time-resolved fluoroimmunoassay chromatograph test strip and its preparation method and application detecting Acetamiprid |
| CN109061153A (en) * | 2018-09-21 | 2018-12-21 | 中国烟草总公司郑州烟草研究院 | A kind of time-resolved fluoroimmunoassay chromatograph test strip and its preparation method and application detecting iprodione |
| CN109116027A (en) * | 2018-08-09 | 2019-01-01 | 江西中德生物工程股份有限公司 | Detect the double colour developing qualitative, quantitative immuno-chromatographic test paper strips of fluorescent microsphere-colloidal gold and preparation method thereof of clenobuterol hydrochloride |
| CN110927375A (en) * | 2019-10-08 | 2020-03-27 | 杭州佰昕科技有限公司 | Fluorescent microsphere immunochromatography test strip for detecting olaquindox residue and application thereof |
| CN111638363A (en) * | 2020-04-30 | 2020-09-08 | 吉林省格瑞斯特生物技术有限公司 | 3-deoxyfructose rapid quantitative fluorescence detection device and preparation method thereof |
| CN111855999A (en) * | 2020-06-02 | 2020-10-30 | 广东省药品检验所（广东省药品质量研究所、广东省口岸药品检验所） | rhEGF rapid detection test paper card, kit and detection method thereof in cosmetics |
| CN112698027A (en) * | 2020-12-14 | 2021-04-23 | 广东唯实生物技术有限公司 | Hapten immunochromatography detection reagent |
| CN114236122A (en) * | 2021-11-22 | 2022-03-25 | 深圳市雅为泓源生物科技有限公司 | Kit and preparation method and application thereof |
| CN117343346A (en) * | 2023-12-04 | 2024-01-05 | 内蒙古大学 | Ultrasensitive europium-based MOFs sensor for visually identifying clenbuterol hydrochloride, and preparation method and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1793108A (en) * | 2006-01-06 | 2006-06-28 | 浙江大学 | Haptenic compound of clenbuterol synthesizing process and application thereof |
| CN101435823A (en) * | 2007-11-12 | 2009-05-20 | 无锡中德伯尔生物技术有限公司 | Fluorescent micro-ball immune chromatography test paper strip for detecting residual animal medicine and preparing method thereof |
| CN101993486A (en) * | 2009-08-27 | 2011-03-30 | 深圳市三方圆生物科技有限公司 | Clenbuterol immunogen, coatingen and application thereof in colloidal gold test paper |
| CN103159852A (en) * | 2013-03-28 | 2013-06-19 | 江南大学 | Method for synthesizing specific artificial antigen of clenbuterol |
| CN103508910A (en) * | 2012-06-19 | 2014-01-15 | 北京勤邦生物技术有限公司 | Beta-agonist semi-antigen, and preparation method and application thereof |
-
2014
- 2014-02-15 CN CN201410052593.6A patent/CN104849467B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1793108A (en) * | 2006-01-06 | 2006-06-28 | 浙江大学 | Haptenic compound of clenbuterol synthesizing process and application thereof |
| CN101435823A (en) * | 2007-11-12 | 2009-05-20 | 无锡中德伯尔生物技术有限公司 | Fluorescent micro-ball immune chromatography test paper strip for detecting residual animal medicine and preparing method thereof |
| CN101993486A (en) * | 2009-08-27 | 2011-03-30 | 深圳市三方圆生物科技有限公司 | Clenbuterol immunogen, coatingen and application thereof in colloidal gold test paper |
| CN103508910A (en) * | 2012-06-19 | 2014-01-15 | 北京勤邦生物技术有限公司 | Beta-agonist semi-antigen, and preparation method and application thereof |
| CN103159852A (en) * | 2013-03-28 | 2013-06-19 | 江南大学 | Method for synthesizing specific artificial antigen of clenbuterol |
Cited By (17)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105301253A (en) * | 2015-09-07 | 2016-02-03 | 北京勤邦生物技术有限公司 | T-2 toxin-enriched immunomagnetic bead as well as preparation method and application thereof |
| CN105527439A (en) * | 2015-12-30 | 2016-04-27 | 厦门依柯利斯医疗科技有限公司 | An NGAL time-resolved fluoroimmunoassay nanometer immunochromatographic quantitative detection test paper strip and a preparing method thereof |
| CN108181465A (en) * | 2018-02-09 | 2018-06-19 | 广东优尼德生物科技有限公司 | Detect the fluorescent chromatographic kit and its quantitative detecting method of 25-hydroxy-vitamin D |
| CN108303549A (en) * | 2018-02-09 | 2018-07-20 | 广东优尼德生物科技有限公司 | A kind of fluorescence immune chromatography test paper bar and its quantitative detecting method of detection adiponectin |
| CN108181465B (en) * | 2018-02-09 | 2024-01-23 | 广东优尼德生物科技有限公司 | Fluorescent chromatography kit for detecting 25-hydroxyvitamin D and quantitative detection method thereof |
| CN109116027B (en) * | 2018-08-09 | 2020-11-03 | 江西中德生物工程股份有限公司 | Fluorescent microsphere-colloidal gold double-color-development qualitative and quantitative immunochromatographic test strip for detecting clenbuterol hydrochloride and preparation method thereof |
| CN109116027A (en) * | 2018-08-09 | 2019-01-01 | 江西中德生物工程股份有限公司 | Detect the double colour developing qualitative, quantitative immuno-chromatographic test paper strips of fluorescent microsphere-colloidal gold and preparation method thereof of clenobuterol hydrochloride |
| CN109061157A (en) * | 2018-09-21 | 2018-12-21 | 中国烟草总公司郑州烟草研究院 | A kind of time-resolved fluoroimmunoassay chromatograph test strip and its preparation method and application detecting flumetralim |
| CN109061158A (en) * | 2018-09-21 | 2018-12-21 | 中国烟草总公司郑州烟草研究院 | A kind of time-resolved fluoroimmunoassay chromatograph test strip and its preparation method and application detecting Acetamiprid |
| CN109061153A (en) * | 2018-09-21 | 2018-12-21 | 中国烟草总公司郑州烟草研究院 | A kind of time-resolved fluoroimmunoassay chromatograph test strip and its preparation method and application detecting iprodione |
| CN110927375A (en) * | 2019-10-08 | 2020-03-27 | 杭州佰昕科技有限公司 | Fluorescent microsphere immunochromatography test strip for detecting olaquindox residue and application thereof |
| CN111638363A (en) * | 2020-04-30 | 2020-09-08 | 吉林省格瑞斯特生物技术有限公司 | 3-deoxyfructose rapid quantitative fluorescence detection device and preparation method thereof |
| CN111855999A (en) * | 2020-06-02 | 2020-10-30 | 广东省药品检验所（广东省药品质量研究所、广东省口岸药品检验所） | rhEGF rapid detection test paper card, kit and detection method thereof in cosmetics |
| CN112698027A (en) * | 2020-12-14 | 2021-04-23 | 广东唯实生物技术有限公司 | Hapten immunochromatography detection reagent |
| CN114236122A (en) * | 2021-11-22 | 2022-03-25 | 深圳市雅为泓源生物科技有限公司 | Kit and preparation method and application thereof |
| CN117343346A (en) * | 2023-12-04 | 2024-01-05 | 内蒙古大学 | Ultrasensitive europium-based MOFs sensor for visually identifying clenbuterol hydrochloride, and preparation method and application thereof |
| CN117343346B (en) * | 2023-12-04 | 2024-03-08 | 内蒙古大学 | Ultrasensitive europium-based MOFs sensor for visually identifying clenbuterol hydrochloride, and preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104849467B (en) | 2016-08-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104849467A (en) | Fluorescent microsphere immunochromatography test paper strip for detecting clenbuterol hydrochloride residue, preparation method and applications thereof | |
| CN101226194B (en) | Malachite green vestigial ELISA detection kit and usage method thereof | |
| CN104897863A (en) | Fluorescent microsphere immunochromatographic test strip for detecting aflatoxin M1, and method thereof | |
| CN106771238B (en) | Detect the fluorescent micro-ball immune chromatography test paper strip and its preparation method and application of melamine residual | |
| CN109324182B (en) | Fluorescent microsphere immunochromatography test strip for detecting pendimethalin and preparation method and application thereof | |
| CN105424939B (en) | A kind of test strips for detecting carbendazim and its preparation method and application | |
| CN108508215A (en) | A kind of time-resolved fluoroimmunoassay chromatograph test strip and its preparation method and application of detection tetracycline medication | |
| CN103018452B (en) | A kind of colloidal gold test paper card and detection method detecting salbutamol medicine | |
| CN109239336B (en) | Test strip for detecting isoprocarb and application thereof | |
| CN109061152A (en) | A kind of time-resolved fluoroimmunoassay chromatograph test strip and its preparation method and application detecting carbendazim | |
| CN105785011B (en) | A kind of test strips for detecting maleic hydrazide and its preparation method and application | |
| CN107271665B (en) | Test strip for detecting salbutamol and application thereof | |
| CN103575887B (en) | A kind of test card and application thereof detecting aflatoxin B1 | |
| CN203720183U (en) | Clebuterol hydrochloride fluorescent microsphere immunochromatography test strip | |
| CN105004844B (en) | A kind of gentamicin residue test strip and application thereof | |
| CN103513035A (en) | Test strip and method for detecting aflatoxin M1 | |
| CN104977410A (en) | Melamine fluorescent microsphere immunochromatography test paper strip, preparation method and application thereof | |
| CN103728449A (en) | Test paper and method for detecting florfenicol and thiamphenicol | |
| CN105301233A (en) | Enzyme linked immunosorbent assay kit for detecting imidacloprid residue in tea leaves and application method of enzyme linked immunosorbent assay kit | |
| CN103105490B (en) | A kind of kit and method detecting tetracycline medication | |
| CN101387644A (en) | Melamine immune chromatography test paper detecting method | |
| CN108931640B (en) | Test strip for detecting organophosphorus pesticide and application thereof | |
| CN103940999B (en) | A kind of test strip detecting Betamethasone Valerate and its preparation method and application | |
| CN109061149A (en) | A kind of time-resolved fluoroimmunoassay chromatograph test strip and its preparation method and application detecting butralin | |
| CN111735951B (en) | Test strip for detecting fenpropathrin and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| EXSB | Decision made by sipo to initiate substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |