CN1332203C - Quick test method fro detecting clenbuterol hydrochloride in animal derived food - Google Patents
Quick test method fro detecting clenbuterol hydrochloride in animal derived food Download PDFInfo
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- CN1332203C CN1332203C CNB2005101101941A CN200510110194A CN1332203C CN 1332203 C CN1332203 C CN 1332203C CN B2005101101941 A CNB2005101101941 A CN B2005101101941A CN 200510110194 A CN200510110194 A CN 200510110194A CN 1332203 C CN1332203 C CN 1332203C
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Abstract
The present invention relates to a quick test method for detecting clenbuterol hydrochloride in animal derived food in the field of a detection technique. Firstly, a clenbuterol coupling object labelled by an enzyme is synthesized; then, horseradish peroxidase and a monoclonal antibody of resisting clenbuterol are fixed on a cellulose nitrate film; after being closed by defatted milk powder and dried by freezing, the cellulose film coated with the antibody and the enzyme is put in a reaction hole made of disposable polyvinyl fluoride or glass material; after being encapsulated by a polytetrafluoroethylene film, the cellulose film is stored for standby; chromogenic substrate solution is prepared; simultaneously, a quick reaction plate for detecting clenbuterol is assembled; in the process of site detection, after being mixed with the clenbuterol coupling object labelled by the enzyme, a preprocessed sample to be measured is dripped on an antibody film in the reaction hole of the reaction plate; after the sample reacts at room temperature, the sample is flushed with water; the chromogenic substrate solution is added in the reaction hole for observing a result; clenbuterol content is judged according to chromogenic degree. The present invention has the advantages of high sensitivity, simple and convenient operation, time saving and wide application prospect.
Description
Technical field
What the present invention relates to is the method in a kind of detection technique field, specifically is a kind of method for rapidly testing that detects clenobuterol hydrochloride in the animal food.
Background technology
Relate to the method that detects clenobuterol hydrochloride both at home and abroad and mainly contain chromatographic technique, capillary electrophoresis, immunoassay (radio immunoassay, enzyme immunoassay (EIA)) and electrochemical measuring technique.The modal technology that is used for the detection of fast detecting or rapid semi-quantitative is the immune colloid gold method.Clenbuterol in the sample and the Clenbuterol-protein conjugate that is fixed on the nitrocellulose filter are competitive in conjunction with the golden labeling antibody of being come by the sample pad diffusion, judge the content of checking matter by the depth of color on the detection line.But the defective of this method is: when the antibody on the gold mark pad with after Clenbuterol-protein conjugate on the nitrocellulose filter combines, Clenbuterol in the sample is difficult to make the gold mark antibody of clenbuteral of combination to run off from detection line at short notice, thereby detects insensitive and time lengthening.When reducing the content of golden labeling antibody, though detection sensitivity can improve, the colour developing of control line and detection line is comparatively light, makes result's judgement comparatively difficult.
Find that by prior art documents number of patent application is CN02131321, name is called " a kind of method of immunochromatography single stage method detection beta-adrenin agonist, medicine and the preparation of test paper ".Its gordian technique mainly comprises the preparation method of The pretreatment method to be checked, nitrocellulose filter, the preparation method of collaurum bond, the preparation of collaurum bond pad and the assemble method of test strips etc.It detects principle is the anti-beta-stimulants antibodies of the colloid gold label of limiting the quantity of on beta-stimulants and the collaurum pad in the sample, the anti-beta-stimulants antibody of unnecessary colloid gold label again with nitrocellulose filter on beta-stimulants reaction, reach one or more purpose of easy, quick, single part of examination beta-stimulants class medicine.But what this technology still adopted is traditional colloidal gold immunochromatographimethod technology, and detection time is long, can reach more than 20 minute, and detectability can't reach 0.3ppb (μ g/L), and the specificity that detects is strong inadequately.The lowest detection lower limit that detects clenobuterol hydrochloride with the colloid gold immune test paper bar in the current patent of having applied for generally more than 0.09ppb (ng/ml), thereby can't reach fast, needs that ultramicron detects.
Summary of the invention
The present invention is directed to existing deficiency and the scarce limit that detects the technology of clenobuterol hydrochloride, a kind of method for rapidly testing that detects clenobuterol hydrochloride in the animal food is provided, make it utilize quick, sensitive, the on-the-spot clenobuterol hydrochloride that detects in the animal product (meat sample or urine sample) of dual-enzyme system fast detecting clenobuterol hydrochloride reaction, having overcome needs specific apparatus when conventional ELISA detects and expends time in and the shortcoming of manpower, has also overcome the lower shortcoming of the immune-gold labeled test strips sensitivity of existing detection Clenbuterol simultaneously.
The present invention is achieved by the following technical solutions, and the present invention at first synzyme mark Clenbuterol conjugate is Clenbuterol-glucose oxidase enzyme conjugates, and the dilution back is stored standby.The fixing monoclonal antibody of horseradish peroxidase and anti-clenbuterol on nitrocellulose filter then, after skimmed milk power sealing and freeze drying, this cellulose membrane that is coated with antibody and enzyme is placed in the reacting hole that disposable polyvinyl fluoride or glass material make, and with the back stored for future use of poly tetrafluoroethylene encapsulation.Preparation chromogenic substrate liquid, assembling simultaneously detects Clenbuterol rapid reaction plate, when detecting at the scene, on the antibody membrane that drips after pretreated sample to be checked and the mixing of enzyme labeling Clenbuterol conjugate in the reacting hole of reaction plate, water flushing behind the room temperature reaction, the liquid that again substrate developed the color adds in the reacting hole, and observations is according to the content of colour developing deciding degree Clenbuterol.
Below the present invention is further illustrated, particular content is as follows:
1. Clenbuterol-glucose oxidase enzyme conjugates is synthetic
A) the clenobuterol hydrochloride standard items with 10mg are dissolved in 200mlH
2Among the O, the HCL accent pH to 1.0 with 2mol/l puts into the frozen water compound then and placed 30 minutes.
B) adding contains 10mgNaNO
2Aqueous solution 100ml, do not leave the frozen water compound during operation, place 30min after, add 5ml Amcide Ammate (wherein containing Amcide Ammate 125mg), until no N
2Till emitting, promptly obtain diazotizing Clenbuterol.
C) the pH7.0 PBS liquid that above-mentioned diazotizing Clenbuterol 2ml is joined 19ml 0.01mol/L (contains 0.1M NaCL, 1mM MgCl
2) and 1 ml contain 22.475mg glucose oxidase (GOD) PBS (0.01mol/L, pH7.2) in the potpourri, abundant mixing.
D) K of usefulness 0.2mol/L
2CO
3After transferring pH to 7.5,4 ℃ of dark conditions are placed down and are spent the night.Add 28ml 0.01mol/L PBS liquid next day and (contain 0.1M NaCL, 1mM MgCl
2, pH7.0).
E) above-mentioned solution is packed in the bag filter that soaks in advance and clean, in the pH7.5 of 0.01mol/l PBS liquid, 4 ℃ of dark conditions are 3 days (changing one time PBS liquid) of dialysis down every 4 hours.
F) collect dislysate, mix-20 ℃ of storages with equal-volume glycerine.
To carry out also that enzymatic activity is identified and in conjunction with than evaluation to synthetic Clenbuterol-glucose oxidase enzyme conjugates.The activity of enzyme is subjected to the influence of inorganic reaction thing or reaction conditions and the part inactivation in building-up process, available 4-amino-antipyrine method is identified, the enzymatic activity of Clenbuterol-glucose oxidase enzyme conjugates can not be lower than 20% of original activity, and it is synthetic invalid then to be considered as less than this value.
In addition also will be to the combination of Clenbuterol one glucose oxidase enzyme conjugates than calculating with the UV scanning method, Clenbuterol combines than being controlled between 30: 1 to 120: 1 with the molecule of glucose oxidase enzyme conjugates.
2. the fixing monoclonal antibody of horseradish peroxidase and anti-clenbuterol on nitrocellulose filter
After 4000 times of dilutions of PBS (pH7.2) with Clenbuterol monoclonal anti body and function 0.01M, add horseradish peroxidase (ultimate density is 0.05mg/ml), accurately draw 50 microlitres with sample injector and be added to nitrocellulose filter surface (the film diameter is 1.3cm) in the reacting hole, 4 ℃ of reaction package are spent the night.The PBS of inferior daily 0.01M (pH7.2) washs once, and (0.01M, pH7.2) the room temperature sealing is 30 minutes with the PBS liquid that contains 3% skimmed milk power.0.01M PBS (pH7.2) washing postlyophilization.
3. prepare chromogenic substrate liquid
At first prepare the Brotton-Robbinson damping fluid of 0.2mol/L, promptly get 11.5mL glacial acetic acid, 13.5mL strong phosphoric acid, 12.44g boric acid and be put in the large beaker of 1000ml, add tri-distilled water 700mL.Be settled to 1000mL after the NaOH adjusting pH value to 6.0 with 2M.Get above-mentioned damping fluid then, add therein amino o-phenylendiamine dihydrochloride and β-D-glucose, both final concentrations are respectively 1 * 10
-2Mol/L and 2 * 10
-3Mol/L.Fully filter behind the mixing, 4 ℃ of dark conditions are preserved stand-by down.
4. assembling detects Clenbuterol rapid reaction plate
With rectangular glass sheet or disposable PVC plastic plate is support, and settles two of columned reacting holes at two ends.The support plate specification is 5.2cm * 2.6cm, the reacting hole internal diameter is 1.2cm, the nitrocellulose filter that is fixed with horseradish peroxidase and Clenbuterol monoclonal antibody that will prepare earlier is put at the bottom of the reacting hole, uses poly tetrafluoroethylene package reaction plate and silica-gel desiccant parcel again.
5. on-the-spot the detection
The cellulose membrane that is coated with antibody and enzyme is placed in the reacting hole, and encapsulates back stored for future use under 4 ℃ of conditions with poly tetrafluoroethylene.During on-the-spot the detection, handle sample to be checked earlier, drip after by (volume ratio) ratio mixing in 1: 1 on the antibody membrane in reacting hole with test sample with Clenbuterol-glucose oxidase enzyme conjugates then, room temperature reaction after 10 minutes water wash three times.Liquid 300 microlitres that again substrate developed the color add in the reacting hole, observations in 3 minutes.
6. according to the content of colour developing deciding degree Clenbuterol
Whenever-and overlap reaction plate to be provided with two holes, be respectively instrument connection and control wells.Control wells shows vivid blue after adding filter paper; If instrument connection is near colourless, then in the sample Clenbuterol greater than 0.008ppm; Still have blueness if reacting hole colour developing is shallower than control wells, then in the sample Clenbuterol content less than 0.008ppm and greater than 30ppt; If instrument connection and control wells solid colour, then in the sample no Clenbuterol or Clenbuterol less than 30ppt.
The present invention is on the basis of adopting competitive solid-phase enzyme-linked immune detection technique, utilize two enzyme systems (glucose oxidase and horseradish peroxidase) to strengthen the sub-ability of conducting electricity of redox reaction chain innovatively, strengthened the sensitivity, the accuracy that detect, and the utmost point has shortened detection time significantly and has reduced the detection lower limit.The present invention's fixing monoclonal antibody of horseradish peroxidase and anti-clenbuterol on nitrocellulose filter; After dripping the Clenbuterol of test sample and glucose oxidase enzyme labeling on the reaction plate, the Clenbuterol in the sample combines the anti-clenbuterol monoclonal antibody on the nitrocellulose filter with enzyme mark Clenbuterol competitiveness.Through behind the washing step, unconjugated enzyme mark Clenbuterol is removed.After adding contains developer (o-tolidine hydrochloride) and substrate solution (glucose), specificity is combined in the enzyme mark Clenbuterol and substrate glucose response generation glucuronic acid and hydrogen peroxide on the nitrocellulose filter, and hydrogen peroxide is oxidized to water and oxygen molecule as the substrate of horseradish peroxidase again.In this course of reaction, dual-enzyme system and substrate constitute the good subsystem that conducts electricity, thereby make the developer oxidative color-developing.Because the enzyme amount of specific adsorption is inverse relation with the content of Clenbuterol, thereby according to the content of the degree of colour developing decidable Clenbuterol.
In the present invention, utilize the sub-enlarge-effect of conducting electricity of dual-enzyme system, the lower limit that detects clenobuterol hydrochloride is reached below the 0.03ppb (ng/ml), be no more than 15 minutes detection time, detect cost and will be lower than the gold-marking immunity test strips, and can realize once the detection of a plurality of samples.The inventive method can significantly improve the sensitivity of detection, and easy and simple to handle saving time, thereby has broad application prospects.
Description of drawings
Fig. 1 principle of the invention figure
The schematic diagram of Fig. 2 use test Clenbuterol of the present invention rapid reaction board test
Embodiment
Provide embodiment below in conjunction with content of the present invention and accompanying drawing:
Before concrete the enforcement, earlier 20 parts of porks sampling observation samples are carried out pre-treatment: get the watery hydrochloric acid that 5 gram meat sample tissues are put into 25 milliliters of 50mM, cut fully with ophthalmologic operation and shred, use forced oscillation after 5 minutes, left standstill 3 minutes.Get 1 milliliter of supernatant, add the caustic lye of soda of 300 microlitre 1M, left standstill again after shaking up 5 minutes, get supernatant place 4 ℃ to be measured.
Embodiment
1. synthetic Clenbuterol-glucose oxidase enzyme conjugates, and carry out enzymatic activity in the conjugate and in conjunction with than evaluation.When identifying enzymatic activity, the amino antipyrine of 3.5mg horseradish peroxidase and 3.5mg 4-is dissolved in the 20ml 0.2mol/L pH7.0 phosphate buffer mixes, add the phenol solution wiring solution-forming A of 1ml 3%, solution B is 6.5% D/W.During mensuration with the mixed liquor of 1.5ml solution A and 1.5ml solution B 25 ℃ of insulations, add the Clenbuterol of synthesizing and purifying-glucose oxidase enzyme conjugates, measure A
500Variation, and calculate the vigor of enzyme.Identify in conjunction with than the time, Clenbuterol and Clenbuterol-glucose oxidase enzyme conjugates are made into 200 μ g/ml and 0.4mg/ml respectively, carry out UV scanning then, according to OD
280Calculate molar absorptivity ε separately, measure Clenbuterol and glucose oxygenase combine than, in conjunction with than=[ε 280 (conjugate)-ε (glucose oxidase)]/ε 280 (Clenbuterol).
2. fixedly horseradish peroxidase and anti-clenbuterol monoclonal antibody.Wherein for the antibody dilution multiple determine that the square formation method in the available solid-phase enzyme-linked immune reaction technology is carried out, and positive control, negative control and blank be set identify antibody sandwich effect and sealing effect.
3. prepare chromogenic substrate liquid, the term of validity of chromogenic substrate liquid under above-mentioned preservation condition is 60 days, and guarantees in use not polluted by other chemical substance.
4. assembling detects the rapid reaction plate of Clenbuterol, simultaneously chromogenic substrate liquid is distributed into inner wrapping (10ml), together with the quantitative dropper packing box of packing in the lump, and places 4 ℃ of dark conditions under and preserves, and when detecting, directly carries a cover packing box and carries out the scene detection.
When 5. detecting at the scene, the enzyme of 30 microlitre test samples (the pork sample of having handled well) and 500 times of dilutions is marked in the reacting hole of Clenbuterol 30 microlitres adding check-out console room temperature reaction 10 minutes.With PBS (pH7.2) the washing reaction hole of 0.01M three times, and on thieving paper, pat clean.(the Brotton-Robbinson damping fluid of 0.2M, pH6.0 contains o-tolidine and glucose is respectively 1 * 10 to add substrate colour developing liquid in reacting hole
-2Mol/L and 2.0 * 10
-3) 300 microlitres, shook reaction plate gently several 10 seconds.Wait for 3 minutes, observations.
6. measurement result is as follows: in 20 parts of pork samples, there have the instrument connection of 16 duplicate samples to present to be vivid blue, and comparing with control wells not have has difference, promptly is judged to be feminine gender; The instrument connection of 3 duplicate samples presents colourless, is judged to be the positive, and the content of Clenbuterol is greater than 0.008ppm.There have the instrument connection of 1 duplicate samples to show as to be light blue, significantly is lower than control wells, is judged to be the weak positive, and the content of Clenbuterol is greater than 30ppt but less than 0.008ppm.
Kit with the detection clenobuterol hydrochloride of German Bai Fa Bioisystech Co., Ltd detects above-mentioned 20 parts of pork samples, and operating process is carried out according to the teachings on the kit.Testing result shows have the testing result of 16 duplicate samples still to be 0ppt in 20 parts of pork samples, has the detected value of 3 duplicate samples to be respectively 0.026ppm, 0.018ppm and 0.063ppm; The detected value that one duplicate samples is arranged is 2.630ppb.Be 4 hours detection time.When the Clenbuterol test strips with Henan hundred Australia Bioisystech Co., Ltd detects this 20 duplicate samples, there is the testing result of 18 duplicate samples negative, 1 part of positive, 1 part is suspicious.Every duplicate samples average detected time is 25 minutes.
Show through the comparative test result, the accuracy rate that the present invention detects clenobuterol hydrochloride matches with the competitive ELISA method basically, false positive rate and false negative rate are less than 0.1%, and detectability can reach 30ppt, but are far smaller than the ELISA minute detection time.As method for quick, detection sensitivity and detection lower limit significantly are better than gold-marking immunity test strips and competitive ELISA method, are the on-the-spot very effective instruments that detects and screen clenobuterol hydrochloride in the animal product.
Claims (9)
1, a kind of method for rapidly testing that detects clenobuterol hydrochloride in the animal food, it is characterized in that, at first synzyme mark Clenbuterol conjugate is Clenbuterol-glucose oxidase enzyme conjugates, and the dilution back is stored standby, the fixing monoclonal antibody of horseradish peroxidase and anti-clenbuterol on nitrocellulose filter then, the nitrocellulose filter that will thisly be coated with the monoclonal antibody of anti-clenbuterol and glucose oxidase after skimmed milk power sealing and freeze drying is placed in the reacting hole that disposable polyvinyl fluoride or glass material make, and with the back stored for future use of poly tetrafluoroethylene encapsulation; Preparation chromogenic substrate liquid, assembling simultaneously detects Clenbuterol rapid reaction plate, when detecting at the scene, on the antibody membrane that drips after pretreated sample to be checked and the mixing of enzyme labeling Clenbuterol conjugate in the reacting hole of reaction plate, water flushing behind the room temperature reaction, the liquid that again substrate developed the color adds in the reacting hole, and observations is according to the content of colour developing deciding degree Clenbuterol.
2, the method for rapidly testing of clenobuterol hydrochloride in the detection animal food according to claim 1 is characterized in that, synthesizing of described Clenbuterol-glucose oxidase enzyme conjugates is specific as follows:
A) the clenobuterol hydrochloride standard items with 10mg are dissolved in 200mlH
2Among the O, the HCL accent pH to 1.0 with 2mol/l puts into the frozen water compound then and placed 30 minutes;
B) adding contains 10mgNaNO
2Aqueous solution 100ml, do not leave the frozen water compound during operation, place 30min after, add 2.5% sulfaminic acid aqueous ammonium 5ml, until no N
2Till emitting, promptly obtain diazotizing Clenbuterol;
C) the pH7.0 PBS liquid of preparation 19ml 0.01mol/L, and make and contain 0.1M NaCl, 1mM MgCl in this solution simultaneously
2Preparation 1ml contains the 0.01mol/L pH7.2 PBS solution of 22.475mg glucose oxidase, after these two kinds of solution are mixed fully, above-mentioned diazotizing Clenbuterol 2ml is joined in this mixed liquor, fully mixing;
D) K of usefulness 0.2mol/L
2CO
3After transferring pH to 7.5,4 ℃ of dark conditions are placed down and are spent the night, and add the PBS solution of 28ml 0.01mol/L pH7.0 next day, contain 0.1M NaCl in this PBS liquid simultaneously, 1mM MgCl
2
E) above-mentioned solution is packed in the bag filter that soaks in advance and clean, in the pH7.5 of 0.01mol/l PBS liquid, PBS liquid was changed one time in the dialysis 3 days down of 4 ℃ of dark conditions every 4 hours;
F) collect dislysate, mix-20 ℃ of storages with equal-volume glycerine.
3, according to the method for rapidly testing of clenobuterol hydrochloride in claim 1 or the 2 described detection animal foods, it is characterized in that, to carry out also that enzymatic activity is identified and in conjunction with than evaluation to synthetic Clenbuterol-glucose oxidase enzyme conjugates, the activity of enzyme is subjected to the influence of inorganic reaction thing or reaction conditions and the part inactivation in building-up process, identify with 4-amino-antipyrine method, the enzymatic activity of Clenbuterol-glucose oxidase enzyme conjugates must be higher than or equal 20% of original activity, and it is synthetic invalid then to be considered as less than this value.
4, according to the method for rapidly testing of clenobuterol hydrochloride in claim 1 or the 2 described detection animal foods, it is characterized in that, than calculating with the UV scanning method, Clenbuterol combines than being controlled between 30: 1 to 120: 1 with the molecule of glucose oxidase enzyme conjugates to the combination of Clenbuterol-glucose oxidase enzyme conjugates.
5, the method for rapidly testing of clenobuterol hydrochloride in the detection animal food according to claim 1 is characterized in that, described on nitrocellulose filter the fixing monoclonal antibody of horseradish peroxidase and anti-clenbuterol, specific as follows:
After 4000 times of dilutions of PBS solution with Clenbuterol monoclonal anti body and function 0.01M pH7.2, add horseradish peroxidase, ultimate density is 0.05mg/ml, accurately draw 50 microlitres with sample injector and be added to nitrocellulose filter surface in the reacting hole, the diameter of this film is 1.3cm, 4 ℃ of reaction package are spent the night, the PBS washing of inferior daily 0.01MpH7.2 once, and with the PBS solution room temperature of the 0.01M pH7.2 that contains 3% skimmed milk power sealing 30 minutes, with the PBS solution washing postlyophilization of 0.01M pH7.2.
6, the method for rapidly testing of clenobuterol hydrochloride in the detection animal food according to claim 1 is characterized in that, described preparation chromogenic substrate liquid is specific as follows:
At first prepare the Brotton-Robbinson damping fluid of 0.2mol/L, promptly getting 11.5mL glacial acetic acid, 13.5mL strong phosphoric acid, 12.44g boric acid is put in the large beaker of 1000ml, add tri-distilled water 700mL, be settled to 1000mL after the NaOH adjusting pH value to 6.0 with 2M, get above-mentioned damping fluid then, add therein amino o-phenylendiamine dihydrochloride and β-D-glucose, both final concentrations are respectively 1 * 10
-2Mol/L and 2 * 10
-3Mol/L fully filters behind the mixing, and 4 ℃ of dark conditions are preserved stand-by down.
7, the method for rapidly testing of clenobuterol hydrochloride in the detection animal food according to claim 1 is characterized in that, described assembling detects Clenbuterol rapid reaction plate, and is specific as follows:
With rectangular glass sheet or disposable PVC plastic plate is support, and settle two of columned reacting holes at two ends, the nitrocellulose filter that is fixed with horseradish peroxidase and Clenbuterol monoclonal antibody that will prepare earlier is put at the bottom of the reacting hole, uses poly tetrafluoroethylene package reaction plate and silica-gel desiccant parcel again.
8, the method for rapidly testing of clenobuterol hydrochloride in the detection animal food according to claim 1, it is characterized in that, described on-the-spot the detection, specific as follows: the cellulose membrane that is coated with antibody and enzyme is placed in the reacting hole, and with poly tetrafluoroethylene encapsulation back stored for future use under 4 ℃ of conditions, during on-the-spot the detection, handle sample to be checked earlier, drip after mixing by 1: 1 volume ratio with test sample with Clenbuterol-glucose oxidase enzyme conjugates then on the antibody membrane in reacting hole, room temperature reaction water flushing three times after 10 minutes, liquid 300 microlitres that again substrate developed the color add in the reacting hole, observations in 3 minutes.
9, the method for rapidly testing of clenobuterol hydrochloride in the detection animal food according to claim 1 is characterized in that, described content according to colour developing deciding degree Clenbuterol is specific as follows:
Every suit reaction plate is provided with two holes, is respectively instrument connection and control wells, and control wells shows vivid blue after adding filter paper, if instrument connection near colourless, then in the sample Clenbuterol greater than 0.008ppm; Still have blueness if reacting hole colour developing is shallower than control wells, then in the sample Clenbuterol content less than 0.008ppm and greater than 30ppt; If instrument connection and control wells solid colour, then in the sample no Clenbuterol or Clenbuterol less than 30ppt.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US7732153B2 (en) * | 2006-05-09 | 2010-06-08 | Beckman Coulter, Inc. | Nonseparation assay methods |
| JP2013512429A (en) * | 2009-11-24 | 2013-04-11 | インフォピア カンパニー，リミテッド | Membrane biosensor with porous film and method for measuring immune reaction or enzyme reaction using the same |
| CN102168072B (en) * | 2010-12-20 | 2013-03-06 | 江苏出入境检验检疫局动植物与食品检测中心 | A monoclonal antibody of anti-clenobuterol hydrochloride and a kit for detecting clenobuterol hydrochloride |
| CN102539749A (en) * | 2012-02-16 | 2012-07-04 | 江阴康奈尔生物科技有限公司 | Field high-sensitivity immunoassay method and kit |
| CN103335963A (en) * | 2013-06-14 | 2013-10-02 | 常州大学 | Method for measuring content of clenbuterol by ultraviolet-visible spectrophotometry |
| CN108872215A (en) * | 2018-05-10 | 2018-11-23 | 武汉工程大学 | A kind of cellulose base glucose sensor material and preparation method thereof |
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| JPS57192867A (en) * | 1981-05-25 | 1982-11-27 | Teijin Ltd | Enzyme immunoassay method of clenbuterol and its analog |
| US4943525A (en) * | 1987-11-02 | 1990-07-24 | Bioventures, Inc. | Simultaneous immunoassay for the determination of antigens and antibodies |
| CN1403814A (en) * | 2001-12-15 | 2003-03-19 | 河南省农业科学院生物技术研究所 | Fast clenbuterol hydrochloride detecting test paper strip |
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2005
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| JPS57192867A (en) * | 1981-05-25 | 1982-11-27 | Teijin Ltd | Enzyme immunoassay method of clenbuterol and its analog |
| US4943525A (en) * | 1987-11-02 | 1990-07-24 | Bioventures, Inc. | Simultaneous immunoassay for the determination of antigens and antibodies |
| CN1403814A (en) * | 2001-12-15 | 2003-03-19 | 河南省农业科学院生物技术研究所 | Fast clenbuterol hydrochloride detecting test paper strip |
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