CN105758846A - Chemiluminescence enzyme-linked immunosorbent assay reagent kit for detecting clenbuterol - Google Patents
Chemiluminescence enzyme-linked immunosorbent assay reagent kit for detecting clenbuterol Download PDFInfo
- Publication number
- CN105758846A CN105758846A CN201511015852.9A CN201511015852A CN105758846A CN 105758846 A CN105758846 A CN 105758846A CN 201511015852 A CN201511015852 A CN 201511015852A CN 105758846 A CN105758846 A CN 105758846A
- Authority
- CN
- China
- Prior art keywords
- clenbuterol
- solution
- enzyme
- chemiluminescence
- reagent kit
- 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.)
- Pending
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
-
- 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Plasma & Fusion (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention discloses a chemiluminescence enzyme-linked immunosorbent assay reagent kit for detecting clenbuterol.The chemiluminescence enzyme-linked immunosorbent assay reagent kit is characterized by comprising clenbuterol standard solution, chemiluminescence plates, enzyme-labeled antigen concentrated solution, enzyme-labeled antigen diluent, chemiluminescence substrate solution, washing solution and recombination solution; various holes in the chemiluminescence plates are coated with clenbuterol-resistant monoclonal antibodies.The chemiluminescence enzyme-linked immunosorbent assay reagent kit has the advantages that the chemiluminescence enzyme-linked immunosorbent assay reagent kit is high in flux, sensitivity and specificity and low in detection cost, the clenbuterol can be accurately and quickly detected, requirements of quick screening tests of large-scale detection in fields can be met, and the like.
Description
Technical field
The invention belongs to field of detection of food safety, be specifically related to a kind of chemical luminescence ELISA detection kit, particularly detect the chemical luminescence ELISA detection kit of Ke Lunluo residual quantity in animal tissue, urine, liver equal samples.
Background technology
Clenbuterol (Clenbuterol) belongs to beta-stimulants, it is commonly called as clenbuterol hydrochloride, another name: Clenbuterol, amine double; two isoprophenamine, clenbuterol, when clenbuterol is to exceed the consumption of therapeutic dose 5-10 times for cattle breeding, namely there is significant nutrition " redistribution effects ", can promote that animal body is proteins deposited, promote that steatolysis suppresses lipidosis, significantly improve the lean meat percentage of trunk, increase weight and improve food conversion ratio, be therefore once used as the growth promoter of poultry, the feed additives such as cattle, sheep, fowl, pig.Illegally added in recent years to improve the lean meat percentage of lard type animal and to accelerate growth of animal in feedstuff, and extensively made an addition in animal feed, and can be remained in animal body.But people is had serious side effect by this medicine, gently then cause that the heart beating rhythm of the heart is abnormal, heavy then can cardiac trigger sick, China and many countries have put into effect relevant laws and regulations, forbid that it uses as feed additive.
At present, mainly adopt the methods such as high performance liquid chromatography (HPLC), LC/MS (LC-MS), gas chromatography-mass spectrography (GC-MS) and euzymelinked immunosorbent assay (ELISA) (ELISA) for detecting the residual of Clenbuterol in animal derived food.The instrumental methods such as high performance liquid chromatography, liquid chromatograph, gas chromatography, precision is high, highly sensitive, but large-scale instrument is expensive and needs specialty detection technique personnel, it is difficult to realize large sample field screening.Enzyme-linked immunoassay method detection is cheap, quickly, but insufficient sensitivity, it is adaptable to the detection of trace substance and qualification, the detection in trace materials, it is difficult to make the most of the advantage.
Chemiluminescence immune assay (Chemiluminescenceanalysis, CLlA) high-sensitive chemiluminescence is combined by technology with the immunoreation of high specific, has the feature such as highly sensitive, high specificity, range of linearity width, easy and simple to handle, instrument and equipment that need not be sufficiently expensive.CLIA does not need external light source, there is the signal to noise ratio higher than fluorescence immunoassay, stronger than the anti-background interference ability of conventional enzyme-linked immunosorbent assay method, high 1 to 2 order of magnitude of its remolding sensitivity ELISA, detection range is up to 6 orders of magnitude, automaticity is high, improves the precision of analysis method, and CLIA has become as the trace of a kind of advanced person or the detection technique of ultra trace material.CLIA just, in veterinary, medical science, food analysis etc., has a extensive future.
The technical problem to be solved is in that to provide a kind of clenbuterol detection reagent kit, adopts this test kit not only to possess higher sensitivity, specificity when carrying out the detection of Clenbuterol, and it is very fast to have response speed.
Summary of the invention
Further object is that the method for testing that a kind of Clenbuterol is provided, the feature that the method specificity is good, highly sensitive, simple to operate.
Realizing above-mentioned purpose, the present invention provides a kind of clenbuterol detection reagent kit, and its main agents comprised has:
It is coated with the polystyrene Chemiluminescent plate of anti-clenbuterol monoclonal antibody.
Described Clenbuterol standard solution 6 bottles, concentration is 0ug/L, 0.05ug/L, 0.15ug/L, 0.45ug/L, 1.35ug/L, 4.05ug/L respectively.
Enzyme-labelled antigen concentrated solution: the artificial antigen being made up of Clenbuterol and bovine serum albumin coupling and horseradish peroxidase prepare.
Enzyme-labelled antigen diluent: 0.01-0.02M phosphate buffer, pH7.0-8.0, the bovine serum albumin of 0.5-1%.
Luminous substrate liquid.Luminous substrate liquid is divided into A, B liquid.A liquid is chemical luminous substrate-luminol and luminescence enhancer-p-cresol solution, and B liquid is hydrogen peroxide urea solution.
Redissolution liquid.The liquid that redissolves is specially 2 times of concentrated phosphoric acid salt buffers, uses front distilled water to use after being diluted to working concentration, for sample pre-treatments.
Cleaning mixture.Wash solution is specially 20 times of concentrated phosphoric acid salt buffers containing tween 20 (Tween-20) buffer, uses front distilled water to use after being diluted to working concentration, for washing chemistry luminous plaque in experimentation.
The preparation of solution of the present invention:
The sensitivity impact that test kit of the present invention is detected by the enzyme mark Clenbuterol monoclonal anti original solution, chemiluminescent solution and the wash solution that relate in test kit of the present invention and formula thereof is very big；Wherein the main component of each solution and compound method thereof are as follows:
1, enzyme mark Clenbuterol monoclonal anti original solution: the artificial antigen prepared with Clenbuterol and coupling protein coupling prepares with horseradish peroxidase, and gained enzyme mark Clenbuterol antigen diluent becomes the working concentration of 1:5000.
2, enzyme-labelled antigen diluent: 0.01-0.02M phosphate buffer, pH7.0-8.0, the bovine serum albumin of 0.5-1%.
3, luminous substrate liquid: three (methylol) aminomethane solution of A liquid is luminol content to be 0.01M, p-cresol content be 0.001MpH8.8, B liquid is that every 100mL solution is containing citric acid 2.1g, anhydrous Na2HPO42.82g, the aqueous solution of the carbamide peroxide 0.64mL of 0.75%.
4, redissolution working solution: pH value is 7.5-7.8, and containing 2-4% casein, the phosphate buffer of 0.1-0.2mol/L, described percentage ratio is w/v.
5, cleaning mixture: pH value is 7.2-7.5, containing 0.8-1.2% tween 20,0.3-0.6 ‰ Hydrazoic acid,sodium salt, the phosphate buffer of 0.1-0.2mol/L, described percentage ratio is mass volume ratio.
6, being coated buffer: pH value is the phosphate buffer of 9.2-9.6,0.1-0.2mol/L, described percentage ratio is w/v.
7, lock solution preparation: containing 5-8% defatted milk powder, the phosphate buffer of pH value 7.2-7.6,0.1-0.2mol/L, described percentage ratio is mass volume ratio.
Being coated of Chemiluminescent plate of the present invention:
Being coated Chemiluminescent plate in the present invention adopts what anti-clenbuterol monoclonal antibody was placed in setting to be coated in solution, with the concentration set, reacts and be coated in 37 DEG C of calorstats.
What the present invention adopted is pH9.2-9.6 phosphate buffer.In the present invention, in microwell plate, coated anti-clenbuterol can well be combined on microwell plate frosting under alkaline environment, it is possible to stands repeatedly to wash plate, and the antibody of employing is coated concentration can from 5mg/ml-10mg/ml.
The microwell plate being coated can be closed by lock solution, the preferred BSA of inert protein in confining liquid, need to add NaN3Prevent from going bad.
The preparation of enzyme mark Clenbuterol antigenic solution:
In the present invention, enzyme mark Clenbuterol antigen solution concentration is to determine the key factor of Clenbuterol chemical luminescence ELISA detection kit measurement range and sensitivity in the present invention.
The enzyme mark Clenbuterol antigenic solution related in the present invention can become the working concentration of 1:5000 by enzyme mark Clenbuterol diluted.
The test kit prepared according to above-mentioned enzyme mark Clenbuterol antigen solution concentration can reach the good range of linearity (normal line scope can reach 0.05-4.05ug/L).
The preparation of chemiluminescent solution:
The present invention adopts horseradish peroxidase labeled substrate luminescent system, mainly luminol-hydrogen peroxide system.
Three (methylol) aminomethane solution of described luminous substrate liquid A liquid is luminol content to be 0.01M, p-cresol content be 0.001MpH8.8, B liquid is that 100mL solution is containing citric acid 2.1g, anhydrous Na2HPO42.82g, the aqueous solution of the carbamide peroxide 0.64mL of 0.75%.Described luminol is chemical luminous substrate, and p-cresol is luminescence enhancer.
Principles of the invention is to be combined with enzymatic high sensitivity by the high degree of specificity that antibody-antigene reacts, and utilizes the chemiluminescence reaction detection production concentration of substrate for enzymatic activity.
The chemical luminescence ELISA detection kit of the present invention has feature fast and accurately highly sensitive, easy, is expected to the residue detection of Clenbuterol in animal-derived food is played a significant role.
Accompanying drawing explanation
Fig. 1 Clenbuterol hapten synthesis route.
Fig. 2 Clenbuterol hapten identifies figure.
Fig. 3 Clenbuterol chemiluminescence enzyme linked immunoassay reagent kit canonical plotting.
Detailed description of the invention
The synthesis of embodiment 1 Clenbuterol hapten-carrier protein conjugate and qualification
(1) Clenbuterol hapten synthesis
Weigh in the tetrahydrofuran solution that 0.5g-3g clenbuterol is dissolved in after drying; logical nitrogen protection; add 0.25-1.4 succinic anhydrides stirring at normal temperature to dissolve; add 0.1-1gDMAP solid and carry out catalytic reaction; monitor reaction with TLC lamellae to carry out, until not having raw material or raw material point very shallow, stopped reaction; silicagel column purifies, and concentrates to obtain product.Identify through magnetic resonance detection, the success of Clenbuterol hapten synthesis.
(2) immunogenic preparation
Weigh 27.5mg hapten and be dissolved in 4mLDMF solution, add each 60mgEDC and 60mgNHS(to be dissolved in 2mL water) carry out activation 30 minutes, join 110-264mg carrier protein BSA(and be dissolved in 5mL containing in 30%DMF aqueous solution) carry out coupling and prepare immunogen, dialyse 3 days with 0.02mol/LPB buffer, every day changes dialysis solution sooner or later, prepares antibody for animal immune after having dialysed.Subpackage, saves backup in-20 DEG C.
(3) preparation of coating antigen
Weigh 27.5mg hapten and be dissolved in 4mLDMF solution, add each 60mgEDC and 60mgNHS(to be dissolved in 2mL water) carry out activation 30 minutes, join 110-264mg carrier protein OVA(to be dissolved in 5mL water) carry out coupling and prepare immunogen, dialyse 3 days with 0.02mol/LPB buffer, every day changes dialysis solution sooner or later, to remove unreacted small-molecule substance；Antibody is prepared for animal immune after having dialysed.Subpackage, saves backup in-20 DEG C.
(4) qualification of Clenbuterol hapten-carrier protein conjugate
By carrier protein, Clenbuterol hapten, Clenbuterol hapten-carrier protein conjugate pH7.4 PBS be made into the solution of 0.5mg/mL, return to zero with 0.01mol/LpH7.4PBS, with ultraviolet spectrophotometer in wavelength 200 ~ 800nm scope interscan, obtain the absorption curve of carrier protein, Clenbuterol hapten, Clenbuterol hapten-carrier protein conjugate.There is different absorption curves in three, it was shown that Clenbuterol hapten and carrier protein couplet success.
(5) preparation of clenbuterol monoclonal antibody
A. animal immune
Immunogen above-mentioned steps obtained is injected in Balb/c Mice Body, and immunizing dose is 150 μ g/ so that it is produce antiserum.
B. cell fusion and cloning
Take immunity Balb/c mouse boosting cell, in 8:1(quantitative proportion) ratio and SP2/0 myeloma cell fusion, screening obtains the clenbuterol monoclonal antibody hybridoma cell strain of stably excreting clenbuterol monoclonal antibody.
C. cell cryopreservation and recovery
Hybridoma frozen stock solution is made 5 × 106The cell suspension of individual/ml, preserves for a long time in liquid nitrogen.Take out cryopreservation tube during recovery, be immediately placed in 37 DEG C of water-bath middling speeds and melt, after centrifugal segregation frozen stock solution, move into and cultivate culture in glassware.
D. the preparation of monoclonal antibody and purification
Increment culture method: be placed in cell culture medium by hybridoma, cultivates under 37 DEG C of conditions, is purified by the culture fluid obtained by sad-saturated ammonium sulfate method, obtains monoclonal antibody, measures titer, and frozen-20 DEG C save backup.
Embodiment 2: the preparation of enzyme-labelled antigen
A. weigh 2mgHRP to be dissolved in 0.5mL distilled water；Add the 0.5mL 0.06mol/LNaIO newly prepared4Solution, 4 DEG C of lucifuge effect 30min；
B. the ethylene glycol 0.5mL of 160mmol/L, room temperature effect 30min are added；
C. adding Clenbuterol hapten-carrier protein conjugate 2mg, in the bag filter that after mixing, loading processed, put in the 0.05mmol/L sodium phosphate buffer of 1000mL and dialyse, 4 DEG C overnight；
D. dialysis solution is drawn in the centrifuge tube of 10mL, adds the 0.25mL 5g/LNaBH newly joined4Liquid, mixes rearmounted 4 DEG C of 2h；Adding isopyknic saturated ammonium sulfate solution, 4 DEG C of effect 30min, at 4 DEG C, the centrifugal 25min of 3000rpm, abandons supernatant；
E. precipitation is dissolved in 1.5mL0.02mol/LpH7.0-7.50.2-0.4%NaN3In 0.2-0.3MOL/L borate buffer solution, suck in bag filter, at 0.02mol/LpH=7.0-7.50.2-0.4%NaN30.2-0.3mol/L borate buffer solution is dialysed, and 4 DEG C overnight (borate buffer solution is changed 3 times in midway)；
F, is drawn in microcentrifugal tube by liquid in dialysis solution, and at 4 DEG C, the centrifugal 30min of 10000rpm, by supernatant sucking-off, adds equivalent glycerol, and mixing ,-20 DEG C save backup.
The foundation of embodiment 3CLEIA detection method
(1) preferred (the square formation method) of antibody and envelope antigen concentration
Longitudinally it is coated Chemiluminescent plate with every kind of coated antibody by the dilution series of 100.0,50.0,25.0,12.5,6.3,3.2,1.6,0.8 μ g/mL, 100 μ L/ holes, after being placed in 37 DEG C of calorstat 2h, pat dry；Closing with 150 μ L/ hole lock solution, 37 DEG C of calorstats are placed 2 hours, wash plate once, pat dry；Adding enzyme mark Clenbuterol antigen (1:1000 to 1:512000) of the 50 a series of dilutions in μ L/ hole, room temperature (20-25 DEG C) hatches 15min, washes plate five times, pats dry for the last time；It is separately added into chemiluminescence A, B liquid in 50 μ L/ holes, measures luminous intensity values.The envelope antigen concentration of obvious graded and antibody dilution is had to carry out specific assay for optium concentration with luminous intensity values with the concentration of envelope antigen.
(2) mensuration of antibody sensitivity
According to the above-mentioned optimization experiment to coated antibody and enzyme-labelled antigen concentration, selecting and determine that enzyme-labelled antigen concentration is 1:5000, coated antibody concentration is the 5.0 μ g/mL mensuration carrying out the sensitivity of antibody:
A. it is coated: be coated solution with the carbonate of 0.05MpH9.6 and anti-clenbuterol antibody is made into the solution of 5.0 μ g/mL, each polystyrene board Chemiluminescent plate reacting hole adds 100 μ L, 37 DEG C of calorstat 2h.Discard solution in hole, pat dry.
B. close: closing above-mentioned coated Chemiluminescent plate by lock solution, 150 μ L/ holes, then 37 DEG C of calorstat 2h wash plate once, pat dry.
C. application of sample: add the Clenbuterol standard solution 50 μ L/ hole of variable concentrations, add enzyme mark Clenbuterol antigen (1:4000) of 50 μ L/ hole dilutions in the above-mentioned reacting hole closed, room temperature (20-25 DEG C) lucifuge hatches 15min, then washes plate five times, pats dry for the last time.
D. luminous: in each reacting hole, to add the chemiluminescent solution 100 μ L/ hole of Extemporaneous, detect with chemical illumination immunity analysis instrument after reaction 3min.
E. testing result calculates with suppression ratio:
Relative luminous intensity (%)=RLU/RLU0, RLU is standard substance or the luminous intensity values of sample solution mensuration, RLU0It it is the luminous intensity values of blank (concentration is the standard solution of 0).
Calculate the concentration of medicine during 50% suppression ratio and be the sensitivity of this antibody.
Embodiment 4 detects the chemiluminescence enzyme linked immunoassay reagent kit of Clenbuterol
(1) composition of the chemiluminescence enzyme linked immunoassay reagent kit of Clenbuterol is detected
A. the solid phase carrier (Chemiluminescent plate) of clenbuterol monoclonal antibody it is coated with；
B. Clenbuterol standard solution: 0,0.05,0.15,0.45,1.35,4.05ug/L.
C. enzyme mark Clenbuterol hapten-carrier protein conjugate solution: prepare with horseradish peroxidase with artificial antigen, by diluted to working concentration during use.
D. enzyme mark Clenbuterol hapten-carrier protein conjugate diluent: sodium phosphate, NaCl buffer solution.
E. luminescent solution: A liquid is luminol, p-cresol solution, B liquid hydrogen peroxide urea solution, during use, A liquid, the mixing of B liquid equal-volume, now with the current.
F. redissolve liquid, during use, be diluted to working concentration with distilled water.
G. cleaning mixture: be diluted to working concentration with distilled water during use.
(2) preparation of Chemiluminescent plate
With being coated liquid, anti-clenbuterol monoclonal antibody is diluted to 5.0 μ g/mL, every hole adds 100 μ L, 2h placed by 37 DEG C of calorstats, inclines and is coated liquid, pats dry, then every hole adds confining liquid 150 μ L, 37 DEG C of calorstats place 2h, liquid in hole of inclining, and cleaning mixture washs once, pat dry, seal by masking foil vacuum and preserve.
Embodiment 5 detects the application of the chemiluminescence enzyme linked immunoassay reagent kit of Clenbuterol
(1) preparation of reagent
A. cleaning mixture: use after the concentrated cleaning solution deionized water provided in test kit is diluted by 1:19 times.
B. working solution: the concentrated phosphoric acid salt buffer provided in test kit is spent and uses after ionized water dilutes by 1:1 times.
C. chemiluminescent solution: by A liquid and B liquid 1:1 by volume mixing before using.
D. enzyme-labelled antigen working solution: enzyme-labelled antigen diluent and enzyme-labelled antigen concentrated solution are mixed by 10:1 volume ratio and mixes.
(2) sample pre-treatments
A. urine specimen pre-treating method:
Taking the limpid urine sample of 20ml and directly measure (must flow through filtration or more than 3000g such as urine sample muddiness, the centrifugal 10min of room temperature (20-25 DEG C) until limpid), freezen protective answered by the sample that wouldn't use.
B. tissue (muscle, liver) Sample pretreatment method:
Weigh the tissue samples after 2.0 ± 0.05g homogenizing to 50ml polystyrene centrifuge tube；Adding 4ml2%NaCl-0.2MHCl-methyl alcohol mixed liquor (see dosing 3), vibrate 30s；More than 3000g, room temperature (20-25 DEG C) is centrifuged 5min；Liver samples: take 0.5ml supernatant (if upper strata has float to avoid) and add 20ml0.5M sodium hydroxide solution, adds 0.5ml redissolution working solution mixing；
Muscle sample: take 0.5ml supernatant (if upper strata has float to avoid) and add 35ml0.5M sodium hydroxide solution (see dosing 4), adds 0.5ml redissolution working solution mixing；Take 20ml for analyzing.
C. feedstuff Sample pretreatment method
Weigh 1.0 ± 0.05g feedstuff sample to 50ml polystyrene centrifuge tube；Add 10ml methanol, vibrate 1min with agitator；More than 3000g, room temperature (20-25 DEG C) is centrifuged 5min；Pipette 1ml upper organic phase to 10ml clean dried teat glass, flow down dry up in 50-60 DEG C of water-bath nitrogen/air；Add 1ml n-hexane, whirling motion 1min, add 1ml redissolution working solution, whirling motion 30s；More than 3000g, room temperature (20-25 DEG C) is centrifuged 5min；Remove upper organic phase, take 50ml lower layer of water and be added 200ml redissolution working solution mixing；Take 20ml for analyzing.
(3) detecting step
A. application of sample: add standard substance/sample 50 μ L in corresponding micropore, adds enzyme-labelled antigen working solution 50 μ L/ hole, and mixing of vibrating gently, with reacting 15min in the rearmounted 25 DEG C of light protected environment of cover plate membrane cover plate.
B. washing: carefully open cover plate film, dried by liquid in hole, with wash operating solution 250 μ L/ hole, fully washing 7 times, every minor tick 10s, pat dry with absorbent paper.
C. luminescent solution is added: every hole adds the luminous substrate 100 μ L of new preparation, shakes about about 30 seconds, places 3min by room temperature after cover plate membrane cover plate.
D. detection: be directly placed into survey measurements in microwell plate luminescence analyzer.
(4) result judges
The standard substance obtained and the meansigma methods of sample luminous intensity values are multiplied by 100 again divided by the luminous intensity values of first standard (0 standard), with suppression ratio for vertical coordinate, the logarithm of Clenbuterol concentration is that abscissa makes standard curve, and the concentration of each sample can read from standard curve.
Relative luminous intensity (%)=RLU/RLU0, RLU is standard substance or the luminous intensity values of sample solution mensuration, RLU0It it is the luminous intensity values of blank (concentration is the standard solution of 0).
Embodiment 6 test kit preci-sion and accuracy is tested
Accuracy refers to the matching degree between measured value and true value, and the conventional response rate of test kit accuracy represents.Precision is also known as repeatability, and the conventional coefficient of variation represents.
Sample extraction method according to embodiment 5, it is added reclaiming to urine, muscle, feedstuff sample respectively with the Clenbuterol of two concentration of 0.15ug/kg, 0.45ug/kg, every kind of each concentration of sample each 4 parallel, it is measured with three batches of test kits, calculates average recovery rate and the precision of sample.Experimental result is shown in following table.
Table 1 Clenbuterol test kit accuracy and precision measure
As seen from the table, the average recovery rate scope that in urine, muscle, Feed Sample, two concentration of Clenbuterol are all added between 87.4-98.3%, in batch, batch between all the coefficient of variation less than 10%.
Claims (3)
1. the chemical luminescence ELISA detection kit detecting Clenbuterol, including Clenbuterol standard solution, Chemiluminescent plate, enzyme-labelled antigen concentrated solution, enzyme-labelled antigen diluent, luminous substrate liquid, cleaning mixture, redissolution liquid, each hole of described Chemiluminescent plate is coated with anti-clenbuterol monoclonal antibody.
2. the chemiluminescence enzyme linked immunoassay reagent kit detecting Clenbuterol as claimed in claim 1, it is characterised in that: it is 5.0mg/mL that described anti-clenbuterol monoclonal antibody is coated concentration.
3. the as claimed in claim 1 chemiluminescence enzyme linked immunoassay reagent kit detecting Clenbuterol, it is characterised in that: described Clenbuterol standard solution concentration is respectively as follows: 0,0.05,0.15,0.45,1.35,4.05ug/L.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201511015852.9A CN105758846A (en) | 2015-12-31 | 2015-12-31 | Chemiluminescence enzyme-linked immunosorbent assay reagent kit for detecting clenbuterol |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201511015852.9A CN105758846A (en) | 2015-12-31 | 2015-12-31 | Chemiluminescence enzyme-linked immunosorbent assay reagent kit for detecting clenbuterol |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105758846A true CN105758846A (en) | 2016-07-13 |
Family
ID=56342235
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201511015852.9A Pending CN105758846A (en) | 2015-12-31 | 2015-12-31 | Chemiluminescence enzyme-linked immunosorbent assay reagent kit for detecting clenbuterol |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105758846A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106153607A (en) * | 2016-08-23 | 2016-11-23 | 江苏出入境检验检疫局动植物与食品检测中心 | A kind of clenobuterol hydrochloride chemoluminescence method quantitative determination reagent kit |
| CN106680489A (en) * | 2016-11-22 | 2017-05-17 | 云南辉瑞贝尔生物科技有限公司 | Test strip for detecting beta-stimulant drugs and preparation method |
| CN107356745A (en) * | 2017-08-23 | 2017-11-17 | 太原瑞盛生物科技有限公司 | The magnetic microparticle chemiluminescence detection kit and preparation method of a kind of Clenbuterol |
| CN113960305A (en) * | 2021-10-15 | 2022-01-21 | 北京勤邦生物技术有限公司 | Immunomagnetic bead for clenbuterol enrichment and purification and preparation method and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101413951A (en) * | 2008-11-27 | 2009-04-22 | 上海交通大学 | Chemiluminescence immune detection reagent kit for detecting Clenbuterol |
| CN101799469A (en) * | 2010-03-17 | 2010-08-11 | 北京阿匹斯生物技术有限公司 | Enzyme linked immunosorbent assay kit for detecting residual clenbuterol hydrochloride and a detection method thereof |
| CN101936984A (en) * | 2010-08-03 | 2011-01-05 | 中国农业大学 | Method and special chemical luminous immunoassay kit for detecting clenbuterol |
| CN103575890A (en) * | 2012-08-03 | 2014-02-12 | 北京勤邦生物技术有限公司 | Chemiluminescence assay kit of ractopamine (RAC) and application thereof |
-
2015
- 2015-12-31 CN CN201511015852.9A patent/CN105758846A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101413951A (en) * | 2008-11-27 | 2009-04-22 | 上海交通大学 | Chemiluminescence immune detection reagent kit for detecting Clenbuterol |
| CN101799469A (en) * | 2010-03-17 | 2010-08-11 | 北京阿匹斯生物技术有限公司 | Enzyme linked immunosorbent assay kit for detecting residual clenbuterol hydrochloride and a detection method thereof |
| CN101936984A (en) * | 2010-08-03 | 2011-01-05 | 中国农业大学 | Method and special chemical luminous immunoassay kit for detecting clenbuterol |
| CN103575890A (en) * | 2012-08-03 | 2014-02-12 | 北京勤邦生物技术有限公司 | Chemiluminescence assay kit of ractopamine (RAC) and application thereof |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106153607A (en) * | 2016-08-23 | 2016-11-23 | 江苏出入境检验检疫局动植物与食品检测中心 | A kind of clenobuterol hydrochloride chemoluminescence method quantitative determination reagent kit |
| CN106680489A (en) * | 2016-11-22 | 2017-05-17 | 云南辉瑞贝尔生物科技有限公司 | Test strip for detecting beta-stimulant drugs and preparation method |
| CN107356745A (en) * | 2017-08-23 | 2017-11-17 | 太原瑞盛生物科技有限公司 | The magnetic microparticle chemiluminescence detection kit and preparation method of a kind of Clenbuterol |
| CN113960305A (en) * | 2021-10-15 | 2022-01-21 | 北京勤邦生物技术有限公司 | Immunomagnetic bead for clenbuterol enrichment and purification and preparation method and application thereof |
| CN113960305B (en) * | 2021-10-15 | 2023-10-17 | 北京勤邦科技股份有限公司 | Immunomagnetic bead for clenbuterol Luo Fuji purification and preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101256188A (en) | ELISA kit for detecting lincomycin medicine as well as usage thereof | |
| CN102967709B (en) | Detect enzyme linked immunological kit and the application thereof of zearalenone medicine | |
| CN108152499B (en) | Antigen and antibody of amantadine and enzyme-linked immunosorbent assay kit thereof | |
| CN101571539A (en) | Elisa kit for detecting cephalo-type medicine and application thereof | |
| CN103421072B (en) | Dexamethasone hapten and its preparation method and application | |
| CN105758846A (en) | Chemiluminescence enzyme-linked immunosorbent assay reagent kit for detecting clenbuterol | |
| CN101776685B (en) | Enzyme linked immunosorbent assay kit for detecting trimethoprim medicament and application thereof | |
| CN109180519B (en) | Olaquindox metabolite antigen, antibody, enzyme-linked immunosorbent assay kit and detection method | |
| CN104897864A (en) | ELISA (enzyme linked immunosorbent assay) kit for detecting amantadine and application of ELISA kit | |
| CN102367270A (en) | Preparation method of cyclosporin A haptin and enzymelinked immunosorbent quantitative detection kit of cyclosporin A | |
| CN105785012A (en) | Enzyme linked immunosorbent assay kit for detecting ribavirin and application thereof | |
| CN107014993A (en) | Indirect competitive ELISA kit for detecting cephalosporin antibiotics in animal derived food and application thereof | |
| CN100501409C (en) | ELISA kit for detecting chloramphenicols in animal derived food | |
| CN110133306A (en) | Detect enzyme linked immunological kit and its application of Cimaterol | |
| CN103575885A (en) | Enzyme linked immunoassay kit for detecting T-2 toxin, and application thereof | |
| CN103102319B (en) | Melamine hapten and its preparation method and application | |
| CN103288723B (en) | Sulfonamide hapten and its preparation method and application | |
| CN103018447B (en) | Detect enzyme linked immunological kit and the method thereof of salbutamol | |
| CN103792374A (en) | Chemiluminesent immunoassay kit for detection of bisphenol A | |
| CN105301244B (en) | Detect enzyme linked immunological kit and its application of acid orange | |
| CN105017347B (en) | Gentamicin haptens and its preparation method and application | |
| CN106483300A (en) | A kind of colloidal gold immuno-chromatography test paper strip of detection Furaxone metabolite and preparation method and application | |
| CN105758847A (en) | Chemiluminescence enzyme linked immunoassay kit for detecting sulfonamides | |
| CN100501407C (en) | ELISA kit for detecting avermectins and detection method thereof | |
| CN102368072B (en) | Chemiluminescent enzyme-linked immunoassay kit for detecting concentration of cyclosporine A drug |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20160713 |
|
| WD01 | Invention patent application deemed withdrawn after publication |