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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- 229960001399 clenbuterol hydrochloride Drugs 0.000 title claims abstract 12
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- 238000010998 test method Methods 0.000 title claims abstract 11
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- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
一种检测技术领域的检测动物性食品中盐酸克伦特罗的快速测试方法，首先合成酶标记克伦特罗偶联物，然后在硝酸纤维素膜上固定辣根过氧化物酶与抗克伦特罗的单克隆抗体，经脱脂奶粉封闭及冷冻干燥后将这种包被有抗体及酶的纤维素膜放置在一次性聚氟乙烯或玻璃材料制成的反应孔内，并用聚四氟乙烯膜封装后贮存待用；配制显色底物液，同时装配检测克伦特罗快速反应板，在现场检测时，将预处理过的待检样品和酶标记克伦特罗偶联物混合后滴加于反应板的反应孔内的抗体膜上，室温反应后用水冲洗，再将底物显色液加入反应孔内，观察结果，根据显色程度判定克伦特罗的含量。本发明灵敏度高，操作简便省时，有广阔的应用前景。
A rapid test method for detecting clenbuterol hydrochloride in animal food in the field of detection technology, first synthesizing an enzyme-labeled clenbuterol conjugate, and then immobilizing horseradish peroxidase and anti-gram on a nitrocellulose membrane The monoclonal antibody of Renbuterol is blocked by skimmed milk powder and freeze-dried. The cellulose membrane coated with antibodies and enzymes is placed in a reaction well made of disposable polyvinyl fluoride or glass material, and polytetrafluoroethylene Sealed in vinyl film and stored for use; prepare chromogenic substrate solution, and assemble detection clenbuterol rapid reaction plate at the same time, mix the pretreated sample to be tested and the enzyme-labeled clenbuterol conjugate during on-site detection Then drop it on the antibody membrane in the reaction well of the reaction plate, rinse with water after reacting at room temperature, then add the substrate chromogenic solution into the reaction well, observe the results, and determine the content of clenbuterol according to the degree of color development. The invention has high sensitivity, simple and time-saving operation and broad 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.
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| US7732153B2 (en) * | 2006-05-09 | 2010-06-08 | Beckman Coulter, Inc. | Nonseparation assay methods |
| US9588111B2 (en) * | 2009-11-24 | 2017-03-07 | Ingibio, Ltd. | Membrane biosensor having multi-hole film attached thereto and method for measuring immunological reaction or enzymatic 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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| 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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| 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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