CN102901818A - Five-conjugate testing card for detecting beta-stimulant drugs and preparation method thereof - Google Patents
Five-conjugate testing card for detecting beta-stimulant drugs and preparation method thereof Download PDFInfo
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- CN102901818A CN102901818A CN2012103663799A CN201210366379A CN102901818A CN 102901818 A CN102901818 A CN 102901818A CN 2012103663799 A CN2012103663799 A CN 2012103663799A CN 201210366379 A CN201210366379 A CN 201210366379A CN 102901818 A CN102901818 A CN 102901818A
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- monoclonal antibody
- clenbuterol
- terbutaline
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Abstract
The invention relates to a five-conjugate testing card for detecting beta-stimulant drugs and a preparation method thereof and belongs to the technical field of beta-adrenergic receptor stimulant detection. A test strip is arranged in a casing of the testing card which is composed of a polyvinyl chloride (PVC) glue board, a sample pad, a colloidal gold combination pad, a coated film and absorbent cotton. A colloidal gold film is a glass cellulose film which contains colloidal gold markers of a clenbuterol monoclonal antibody, a ractopamine monoclonal antibody, a salbutamol monoclonal antibody, a phenylethanolamine A monoclonal antibody and a terbutaline monoclonal antibody. The coated film is a nitrocellulose film, wherein five detection lines T and a quality control line C are arranged on the coated film, and the quality control line C is coated with a rabbit anti-mouse immunoglobulin G (IgG) antibody. The five-conjugate testing card can simultaneously detect out clenbuterol, ractopamine, salbutamol, phenylethanolamine A and terbutaline, and is simple, convenient and fast in method, and accurate in result.
Description
Technical field
The present invention relates to receptor,β activator detection technique field, particularly relate to a kind of Clenbuterol, Ractopamine, salbutamol, phenolethanolamine A and Terbutaline 5-linked test card and preparation method thereof.
Background technology
Along with animal husbandry scale, business-like development, veterinary drug and feed addictive are widely used in animal husbandry is produced, and veterinary drug is widely used in effective control of various diseases, has reduced mortality of animals; Improve food conversion ratio, improved the animal rate of body weight gain and shortened the animal feeding cycle, improve leather quality, reduce the discarded rate of condemned carcass and animal tissue; The growth of animal derived product yield and the development of animal intensive culture have been promoted.Present veterinary drug not only is used for preventing and treating Animal diseases, and increasing medicine is used for promoting growth, and the non-therapeutical uses such as synchronization of estrus has caused more serious residue of veterinary drug problem.
Food-safety problem was constantly upgraded in recent years, " clenbuterol hydrochloride " abuse in the edible animal feed is subject to the more and more higher attention of people, because this excitant residues in the food substrate, enter human body by food chain after, can cause great harm to health.
" clenbuterol hydrochloride " is the general designation of a class animal-use drug.It is as a class chemical article, rather than a kind of specific material, refers to promote the medicated premix of lean meat growth.Any material of lean meat growth, the growth of inhibition fat meat that can promote can be called " clenbuterol hydrochloride ".At present, the material that can realize this function is that a class is called the beta-stimulants (medicine of β-agonist), Ractopamine (Ractopamine) such as the Clenbuterol (clenbuterol) that causes poisoning in China and U.S.'s permission use, other similar medicines also have salbutamol (Salbutamol) and Terbutaline (Terbutaline) etc., and last year the phenolethanolamine A that never occurred in the feed, China's food security alarm bell has been beaten in the appearance of this clenbuterol hydrochloride new varieties again.
The at present detection for common beta-stimulants Clenbuterol, Ractopamine, salbutamol, phenolethanolamine A and Terbutaline mainly contains the detection methods such as enzyme linked immunological (ELISA), gas chromatography-mass spectrography (GC-MC), high performance liquid chromatography (HPLC), but the apparatus expensive that these methods need, complicated operation is difficult to promote.Thereby be necessary to set up a kind of simple and efficient, reliable, and and can detect simultaneously the detection method of these five kinds of materials, the residue of veterinary drug in the monitor animal derived food is guaranteed the safety of animal derived food.
Summary of the invention
The object of the present invention is to provide a kind of test card that can detect simultaneously Clenbuterol, Ractopamine, salbutamol, phenolethanolamine A and Terbutaline, suitable enterprise carries out accurately detection method of Site Detection and fast and simple, with low cost, result.
Clenbuterol of the present invention, Ractopamine, salbutamol, phenolethanolamine A and Terbutaline 5-linked colloidal-gold detecting-card, by being coated with clenbuterol monoclonal antibody, the Ractopamine monoclonal antibody, the salbutamol monoclonal antibody, the collaurum pad of phenolethanolamine A monoclonal antibody and Terbutaline monoclonal antibody colloid gold label thing, be coated with Clenbuterol, Ractopamine, salbutamol, phenolethanolamine A and Terbutaline be the nitrocellulose filter of totally 5 kinds of protein conjugates and rabbit anti-mouse igg, sample pad, adsorptive pads, the compositions such as PVC offset plate and mould of plastics, end at the PVC offset plate adheres to sample pad successively, pad, nitrocellulose filter is pasted in the centre, and the other end adheres to adsorptive pads.
The preparation method of Clenbuterol of the present invention, Ractopamine, salbutamol, phenolethanolamine A and Terbutaline 5-linked colloidal-gold detecting-card is to be realized by following steps:
(1) colloidal gold solution preparation: get 1 of 1 L Erlenmeyer flask, add ultrapure water 495 mL, then adding mass concentration is 1% gold chloride (HAuCl
43H
2O) 5 mL, being mixed with 500 mL mass concentrations is 0.01% aqueous solution of chloraurate, heating is boiled rear in the situation that lasting stirring adding mass concentration is 1% trisodium citrate (Na
3C
6H
5O
72H
2O) solution 5-7 mL continues agitating heating, when the color of the solution becomes transparent aubergine fully, keeps stopped heating behind 5 min, and moisturizing is cooled to room temperature to original volume, and 2-8 ℃ saves backup;
(2) pre-service of antibody: Clenbuterol, Ractopamine, salbutamol, phenolethanolamine A and Terbutaline monoclonal antibody that will mark be at 1000 r/min, under 4 ℃ of conditions, centrifugal 20 min get supernatant, are diluted to 1 mg/mL with 0.01 mol/L PBS; Perhaps with 0.01 mol/L PBS will mark Clenbuterol, Ractopamine, salbutamol, phenolethanolamine A and Terbutaline monoclonal antibody be diluted to 1 mg/mL, cross 0.22 μ m filter membrane;
(3) preparation of colloid gold label thing: get colloidal gold solution 40 mL in the step (1), with 0.25 mol/L K
2CO
3Regulate colloidal gold solution pH to 8.5, magnetic stirrer 250 r/min stir, and dropwise add the protein solution that 4 mL contain 0.3 mg Clenbuterol or Ractopamine or salbutamol or phenolethanolamine A or Terbutaline monoclonal antibody, react 10 min; Dropwise add 4 mL, 10% BSA, continue stirring reaction 10 min; Centrifugal 20 min of gold labeling antibody solution normal temperature low speed 1500 r/min discard the precipitation that is formed by the gold grain that condenses; 4 ℃ of red supernatant solution, centrifugal 20 min of 12000 r/min, abandon supernatant, collecting precipitation, to precipitate with golden labeling antibody dilution and be settled to 1 mL, be prepared into the colloid gold label thing of clenbuterol monoclonal antibody protein conjugate or Ractopamine monoclonal antibody protein matter conjugate or salbutamol monoclonal antibody protein matter conjugate or phenolethanolamine A monoclonal antibody protein matter conjugate or Terbutaline monoclonal antibody protein matter conjugate;
(4) preparation of collaurum film: with step (3) clenbuterol monoclonal antibody protein conjugate, Ractopamine monoclonal antibody protein matter conjugate, salbutamol monoclonal antibody protein matter conjugate, the colloid gold label thing of phenolethanolamine A monoclonal antibody protein matter conjugate and Terbutaline monoclonal antibody protein matter conjugate evenly is sprayed on the carrier glass fibre element film with drawing the concentration of film instrument with 5 μ L/cm successively, room temperature is dried or 37 ℃ of oven dry 3 h naturally, makes to contain the clenbuterol monoclonal antibody protein conjugate, Ractopamine monoclonal antibody protein matter conjugate, salbutamol monoclonal antibody protein matter conjugate, the collaurum film of phenolethanolamine A monoclonal antibody protein matter conjugate and Terbutaline monoclonal antibody protein matter conjugate;
(5) coated film preparation: become 1 mg/mL, Clenbuterol antigen diluent to become 0.3 mg/mL, Ractopamine antigen diluent to become 0.5 mg/mL, salbutamol antigen diluent to become 1 mg/mL, phenolethanolamine antigen diluent to become 1.5 mg/mLA to become 2.0 mg/mL with the Terbutaline antigen diluent rabbit anti-mouse igg antibody dilution, be sprayed on the nitrocellulose filter with the concentration of 1 μ L/cm successively with drawing the film instrument, be prepared into coated film, behind 37 ℃ of coated 2 h, room temperature is dried or 37 ℃ of oven dry naturally;
(6) sample pad pre-treatment: the sample pad treating fluid is uniformly coated on the glass fibre element film, is lower than in air humidity under 60% the condition, room temperature is dried naturally;
(7) assembling of test card: the PVC offset plate is pasted sample pad, collaurum film, coated film and adsorptive pads from top to bottom successively, is assembled into test strips, cuts into the rectangular of certain width, test strips is installed in the test card shell of rectangular flat shelly again.
Golden labeling antibody dilution in the described step (3) is by 5 g Bovine serum albumins (BSA), and 10 g sucrose add the dissolving of 100 mL, 0.01 mol/L PBS solution formulated, cross 0.22 μ m filter membrane, and are now with the current; Gold labeling antibody lavation buffer solution is by 10 g Bovine serum albumins (BSA) and 1 g PEG-20000, is settled to 100 mL gained with 0.01 mol/L PBS pH7.4.
Sample pad treating fluid in the described step (6) is by 1 g Bovine serum albumin (BSA) and 0.8 g sodium chloride (NaCl), is settled to 100 mL with the 0.01 mol/L PBS that contains 0.5%TRITON-100.
The present invention can be effective to measure simultaneously beta-stimulants Clenbuterol, Ractopamine, salbutamol, phenolethanolamine A and five kinds of materials of Terbutaline, and method is simple, and is convenient, fast, and the result is accurate.
Description of drawings
Fig. 1 is the structural drawing of 5-linked colloidal-gold detecting-card of the present invention: 1. wells, 2. Clenbuterol detection lines, 3. Ractopamine detection lines, 4. salbutamol detection lines, 5. phenolethanolamine A detection lines, 6. Terbutaline detection lines, 7. nature controlling lines 8. detect hole 9. test strips, 10. test card shells among the figure;
Fig. 2 is the sectional structure chart of the test strips in the 5-linked colloidal-gold detecting-card of the present invention, 11. sample pad, 12. collaurum pad 13.PVC offset plates, 14. coated films, 15. adsorptive pads among the figure.
Embodiment
Embodiment 1
Fig. 1 is that structural drawing, Fig. 2 of 5-linked colloidal-gold detecting-card of the present invention is the sectional structure chart of the test strips in the 5-linked colloidal-gold detecting-card of the present invention: 13 are the PVC offset plate among the figure; 11 is sample pad; 12 is the collaurum pad, has been coated with monoclonal antibody colloid gold label thing on this collaurum pad; 14 is coated film, and namely nitrocellulose filter has been coated with Clenbuterol-BSA, Ractopamine-BSA, salbutamol-BSA, phenolethanolamine A-BSA, Terbutaline-BSA and rabbit anti-mouse igg on this nitrocellulose filter; 15 is adsorptive pads.
(sample pipetting volume end) adheres to sample pad 11, collaurum pad 12 on an end of PVC offset plate 13, and sample pad 11 is overlapped on the collaurum pad 12.
In the middle of PVC offset plate 13, adhere to nitrocellulose filter 14.Be provided with Clenbuterol-BSA detection line 2, Ractopamine-BSA detection line 3, salbutamol-BSA detection line 4, phenolethanolamine A-BSA detection line 5, Terbutaline detection line 6 and rabbit anti-mouse igg nature controlling line 7 at nitrocellulose filter 14.
The other end at PVC offset plate 13 adheres to adsorptive pads 15.One end of nitrocellulose filter 14 slightly intersects with pad 9, and the other end slightly intersects with adsorptive pads 15.This test strips 9 can be incorporated with in the test card shell 10 that mould of plastics makes, and is provided with well 1 and detects hole 5 covering of test card shell 10, and sample pad 8 is over against well 1, and nitrocellulose filter 14 is over against detecting hole 8.
Embodiment 2
Clenbuterol, Ractopamine, salbutamol, phenolethanolamine A and the preparation of Terbutaline 5-linked colloidal-gold detecting-card are by the following steps specific implementation:
One, colloidal gold solution preparation: get 1 of 1 L Erlenmeyer flask, add ultrapure water 495 mL, then add 1% gold chloride (HAuCl
43H
2O) 5 mL are mixed with 500 mL, 0.01% aqueous solution of chloraurate, add 1% trisodium citrate (Na in the situation that continue stirring after heating is boiled
3C
6H
5O
72H
2O) solution 5-7 mL continues agitating heating, when the color of the solution becomes transparent aubergine fully, keeps stopped heating behind 5 min, and moisturizing is cooled to room temperature to original volume, and 2-8 ℃ saves backup;
Two, the pre-service of antibody: Clenbuterol, Ractopamine, salbutamol, phenolethanolamine A and Terbutaline monoclonal antibody that will mark be at 1000 r/min, under 4 ℃ of conditions, centrifugal 20 min get supernatant, are diluted to 1 mg/mL with 0.01 mol/L PBS; Perhaps with 0.01 mol/L PBS will mark Clenbuterol, Ractopamine, salbutamol, phenolethanolamine A and Terbutaline monoclonal antibody be diluted to 1 mg/mL, cross 0.22 μ m filter membrane;
Three, the preparation of colloid gold label thing: get colloidal gold solution 40 mL in the step (), with 0.25 mol/L K
2CO
3Regulate colloidal gold solution pH to 8.5, magnetic stirrer 250 r/min stir, and dropwise add the protein solution that 4 mL contain 0.3 mg Clenbuterol or Ractopamine or salbutamol or phenolethanolamine A or Terbutaline monoclonal antibody, react 10 min; Dropwise add 4 mL, 10% BSA, continue stirring reaction 10 min; Centrifugal 20 min of gold labeling antibody solution normal temperature low speed 1500 r/min discard the precipitation that is formed by the gold grain that condenses; 4 ℃ of red supernatant solution, centrifugal 20 min of 12000 r/min, abandon supernatant, collecting precipitation, to precipitate with golden labeling antibody dilution and be settled to 1 mL, be prepared into the colloid gold label thing of human relations Te Luokelong antibody protein conjugate or Ractopamine clonal antibody protein conjugate or salbutamol clonal antibody protein conjugate or phenolethanolamine A clonal antibody protein conjugate or Terbutaline monoclonal antibody protein matter conjugate;
Four, the preparation of collaurum film: with step (three) Clenbuterol clonal antibody protein conjugate, Ractopamine clonal antibody protein conjugate, salbutamol clonal antibody protein conjugate, the colloid gold label thing of phenolethanolamine A clonal antibody protein conjugate and Terbutaline monoclonal antibody protein matter conjugate evenly is sprayed on the carrier glass fibre element film with drawing the concentration of film instrument with 5 μ L/cm successively, room temperature is dried or 37 ℃ of oven dry 3 h naturally, makes to contain Clenbuterol clonal antibody protein conjugate, Ractopamine clonal antibody protein conjugate, salbutamol clonal antibody protein conjugate, the collaurum film of phenolethanolamine A clonal antibody protein conjugate and Terbutaline monoclonal antibody protein matter conjugate;
Five, coated film preparation: become 1 mg/mL, Clenbuterol antigen diluent to become 0.3 mg/mL, Ractopamine antigen diluent to become 0.5 mg/mL, salbutamol antigen diluent to become 1 mg/mL, phenolethanolamine antigen diluent to become 1.5 mg/mLA to become 2.0 mg/mL with the Terbutaline antigen diluent rabbit anti-mouse igg antibody dilution, be sprayed on the nitrocellulose filter with the concentration of 1 μ L/cm successively with drawing the film instrument, be prepared into coated film, behind 37 ℃ of coated 2 h, room temperature is dried or 37 ℃ of oven dry naturally;
Six, sample pad pre-treatment: the sample pad treating fluid is uniformly coated on the glass fibre element film, is lower than in air humidity under 60% the condition, room temperature is dried naturally;
Seven, the assembling of test card: the PVC offset plate is pasted sample pad, collaurum film, coated film and adsorptive pads from top to bottom successively, is assembled into test strips, cuts into the rectangular of certain width, test strips is installed in the test card shell of rectangular flat shelly again.
Embodiment 3
The test of minimum detectability amount
Clenbuterol, Ractopamine, salbutamol, phenolethanolamine A and the Terbutaline standard solution concentration of preparing respectively standard are: 0,0.5 ng/mL, 1 ng/mL, 2.5 ng/mL, 5 ng/mL; Drip 2 samples to the test card well, observe the colour developing result of detection line T and nature controlling line C behind 5 min.Through repeatedly check, the recall rate of Clenbuterol, Ractopamine, salbutamol, phenolethanolamine A and Terbutaline reaches more than 99%, and the minimum detectability amount is less than 5 ng/mL.
。
Claims (4)
1. detect the 5-linked test card of beta-stimulants, it is characterized in that by the collaurum pad that has been coated with clenbuterol monoclonal antibody, Ractopamine monoclonal antibody, salbutamol monoclonal antibody, phenolethanolamine A monoclonal antibody and Terbutaline monoclonal antibody colloid gold label thing, be coated with Clenbuterol, Ractopamine, salbutamol, phenolethanolamine A and Terbutaline nitrocellulose filter, sample pad, absorbent wool, PVC offset plate and the mould of plastics of totally 5 kinds of protein conjugates and rabbit anti-mouse igg form, at an end of PVC offset plate
Adhere to successively sample pad, pad, nitrocellulose filter is pasted in the centre, and the other end adheres to absorbent wool.
2. the preparation method of the 5-linked test card of detection beta-stimulants according to claim 1 is characterized in that being prepared by following steps:
Colloidal gold solution preparation: get 1 of 1 L Erlenmeyer flask, add ultrapure water 495 mL, then adding mass concentration is 1% gold chloride, 5 mL, and being mixed with 500 mL mass concentrations is 0.01% aqueous solution of chloraurate, and heating is boiled rear in the situation that lasting stirring adding mass concentration is 1% citric acid three sodium solution 5-7 mL, continue agitating heating, when the color of the solution becomes transparent aubergine fully, keep stopped heating behind 5 min, moisturizing is to original volume, be cooled to room temperature, 2-8 ℃ saves backup;
The pre-service of antibody: Clenbuterol, Ractopamine, salbutamol, phenolethanolamine A and Terbutaline monoclonal antibody that will mark be at 1000 r/min, and under 4 ℃ of conditions, centrifugal 20 min get supernatant, are diluted to 1 mg/mL with 0.01 mol/L PBS; Perhaps with 0.01 mol/L PBS will mark Clenbuterol, Ractopamine, salbutamol, phenolethanolamine A and Terbutaline monoclonal antibody be diluted to 1 mg/mL, cross 0.22 μ m filter membrane;
The preparation of colloid gold label thing: get colloidal gold solution 40 mL in the step (1), with 0.25 mol/L K
2CO
3Regulate colloidal gold solution pH to 8.5, magnetic stirrer 250 r/min stir, and dropwise add the protein solution that 4 mL contain 0.3 mg Clenbuterol or Ractopamine or salbutamol or phenolethanolamine A or Terbutaline monoclonal antibody, react 10 min; Dropwise add 4 mL, 10% BSA, continue stirring reaction 10 min; Centrifugal 20 min of gold labeling antibody solution normal temperature 1500 r/min discard the precipitation that is formed by the gold grain that condenses; 4 ℃ of red supernatant solution, centrifugal 20 min of 12000 r/min, abandon supernatant, collecting precipitation, to precipitate with golden labeling antibody dilution and be settled to 1 mL, be prepared into the colloid gold label thing of clenbuterol monoclonal antibody protein conjugate or Ractopamine monoclonal antibody protein matter conjugate or salbutamol monoclonal antibody protein matter conjugate or phenolethanolamine A monoclonal antibody protein matter conjugate or Terbutaline monoclonal antibody protein matter conjugate;
The preparation of collaurum film: with step (3) clenbuterol monoclonal antibody protein conjugate, Ractopamine monoclonal antibody protein matter conjugate, salbutamol monoclonal antibody protein matter conjugate, the colloid gold label thing of phenolethanolamine A monoclonal antibody protein matter conjugate and Terbutaline monoclonal antibody protein matter conjugate evenly is sprayed on the carrier glass fibre element film with drawing the concentration of film instrument with 5 μ L/cm successively, room temperature is dried or 37 ℃ of oven dry 3 h naturally, makes to contain the clenbuterol monoclonal antibody protein conjugate, Ractopamine monoclonal antibody protein matter conjugate, salbutamol monoclonal antibody protein matter conjugate, the collaurum film of phenolethanolamine A monoclonal antibody protein matter conjugate and Terbutaline monoclonal antibody protein matter conjugate;
Coated film preparation: become 1 mg/mL, Clenbuterol antigen diluent to become 0.3 mg/mL, Ractopamine antigen diluent to become 0.5 mg/mL, salbutamol antigen diluent to become 1 mg/mL, phenolethanolamine antigen diluent to become 1.5 mg/mLA to become 2.0 mg/mL with the Terbutaline antigen diluent rabbit anti-mouse igg antibody dilution, be sprayed on the nitrocellulose filter with the concentration of 1 μ L/cm successively with drawing the film instrument, be prepared into coated film, behind 37 ℃ of coated 2 h, room temperature is dried or 37 ℃ of oven dry naturally;
The sample pad pre-treatment: the sample pad treating fluid is uniformly coated on the glass fibre element film, is lower than in air humidity under 60% the condition, room temperature is dried naturally;
The assembling of test card: the PVC offset plate is pasted sample pad, collaurum film, coated film and absorbent wool from top to bottom successively, is assembled into test strips, cuts into the rectangular of certain width, test strips is installed in the test card shell of rectangular flat shelly again.
3. the preparation method of the 5-linked test card of detection beta-stimulants according to claim 2, it is characterized in that golden labeling antibody dilution in the described step (3) is by 5 g Bovine serum albumins, 10 g sucrose, add the dissolving of 100 mL, 0.01 mol/L PBS solution formulated, cross 0.22 μ m filter membrane, now with the current; Gold labeling antibody lavation buffer solution is by 10 g Bovine serum albumins and 1 g PEG-20000, is settled to 100 mL gained with 0.01 mol/L PBS pH7.4.
4. the preparation method of the 5-linked test card of detection beta-stimulants according to claim 2, it is characterized in that the sample pad treating fluid in the described step (6) is by 1 g Bovine serum albumin and 0.8 g sodium chloride, be settled to 100 mL with the 0.01 mol/L PBS that contains 0.5%TRITON-100.
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| CN105510587A (en) * | 2014-10-16 | 2016-04-20 | 镇江先创生物科技有限公司 | Neomycin immuno-colloidal gold detection card and preparation method thereof |
| CN106568889A (en) * | 2016-10-31 | 2017-04-19 | 刘沛明 | Pesticide residue detection card |
| CN106568890A (en) * | 2016-11-07 | 2017-04-19 | 吴文华 | Portable pesticide residue detection device |
| CN108226505A (en) * | 2016-12-15 | 2018-06-29 | 南京亿特生物科技有限公司 | Immune colloid gold detection card of sodium sulfocyanate and preparation method thereof |
| CN115356475A (en) * | 2022-07-11 | 2022-11-18 | 黄淮学院 | Rapid detection test paper strip for beta receptor agonist and preparation method and application thereof |
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Application publication date: 20130130 |