CN101915841B - Ternary detection test bar of beta-stimulants albuterol, clenobuterol hydrochloride and ractopamine and preparation method thereof - Google Patents
Ternary detection test bar of beta-stimulants albuterol, clenobuterol hydrochloride and ractopamine and preparation method thereof Download PDFInfo
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- CN101915841B CN101915841B CN 201010235565 CN201010235565A CN101915841B CN 101915841 B CN101915841 B CN 101915841B CN 201010235565 CN201010235565 CN 201010235565 CN 201010235565 A CN201010235565 A CN 201010235565A CN 101915841 B CN101915841 B CN 101915841B
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- protein conjugate
- monoclonal antibody
- ractopamine
- clenobuterol hydrochloride
- salbutamol
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- 229960002052 salbutamol Drugs 0.000 title claims abstract description 57
- NDAUXUAQIAJITI-UHFFFAOYSA-N albuterol Chemical compound CC(C)(C)NCC(O)C1=CC=C(O)C(CO)=C1 NDAUXUAQIAJITI-UHFFFAOYSA-N 0.000 title claims abstract description 34
- YJQZYXCXBBCEAQ-UHFFFAOYSA-N ractopamine Chemical compound C=1C=C(O)C=CC=1C(O)CNC(C)CCC1=CC=C(O)C=C1 YJQZYXCXBBCEAQ-UHFFFAOYSA-N 0.000 title claims abstract description 34
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- 206010003445 Ascites Diseases 0.000 claims description 13
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 9
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 9
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- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 6
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- STJMRWALKKWQGH-UHFFFAOYSA-N clenbuterol Chemical compound CC(C)(C)NCC(O)C1=CC(Cl)=C(N)C(Cl)=C1 STJMRWALKKWQGH-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention relates to a ternary detection test bar of beta-stimulants albuterol, clenobuterol hydrochloride and ractopamine and a preparation method thereof. The problem of detecting the beta-stimulants the albuterol, the clenobuterol hydrochloride and the ractopamine simultaneously is solved. A parent material PVC plate is sequentially provided with a diversion glass fiber, a carrier glass fiber, a nitrocellulose membrane and a suction cotton pulp plate from front to back; the diversion glass fiber and the suction cotton pulp plate are covered with membranes; the carrier glass fiber absorbs a monoclonal antibody colloidal gold marker resistant to albuterol protein conjugates, clenobuterol hydrochloride protein conjugates and ractopamine protein conjugates; and the intermediate nitrocellulose membrane is provided with a rabbit anti-mouse polyclonal antibody quality control line and three detection lines which are respectively coated with the albuterol protein conjugates, the clenobuterol hydrochloride protein conjugates and the ractopamine protein conjugates. By the invention, the beta-stimulants the albuterol, the clenobuterol hydrochloride and the ractopamine can be effectively detected simultaneously with convenience, quickness and accurate result.
Description
One, technical field
The present invention relates to beta-stimulants detection technique field, particularly relate to a kind of beta-stimulants albuterol, clenobuterol hydrochloride, Ractopamine ternary detection test bar and preparation method thereof.
Two, background technology
Beta-stimulants is a class adrenomimetic drug, because of its can make the beta receptor excited (excitement) in the animal and human soma thus the function of different tissues is gained the name in the control agent, its chemical constitution is Phenethanolamine derivative, such medicine has the livestock and poultry of promotion lipolysis, increases the muscle growth effect, as the heavy partitioning agent of nutrition, to improve lean meat percentage, reduce fat deposition and promote growth of animal, illegally used as cultivation promoter by some livestock-raising persons.The most frequently used in aquaculture is Clenizole Hydrochloride (Clenbuterol), salbutamol (Salbutamol) and Ractopamine (ractopamine), uses beta-stimulants and cause consumer's food poisoning to happen occasionally in feed stripped.Beta-stimulants is as the feed addictive that promotes growth of animal, raising lean meat percentage; but long-term use can make such medicine accumulate in animal foodstuff; the people eaten feed contain the livestock products of beta-stimulants as the adjuvant feed after; toxicity symptom will appear; muscular tremor, palpitaition, stress, headache, myalgia, dizzy, the symptom such as feel sick, vomit, have a fever, tremble can appear usually, in addition dead.Therefore, world many countries forbids increasing with beta-stimulants such as clenobuterol hydrochlorides the lean meat percentage of animal in feed.The Chinese government has prohibited beta-stimulants such as using clenobuterol hydrochloride in herding is produced.
At present, prior art mainly relies on chemical apparatuses analysis, enzyme immunoassay kit and the independent detection test-strips that detects Clenizole Hydrochloride, salbutamol and Ractopamine for the detection of Clenizole Hydrochloride, salbutamol and Ractopamine.Supervision department does not also know which kind of beta-agonist the raiser has used, and detects to have certain blindness, usually causes undetected.Therefore, market is in the urgent need to there being a kind of reagent can detect simultaneously common beta-agonist, to make things convenient for routine monitoring work.
Three, summary of the invention
For above-mentioned situation, for overcoming the prior art defective, the present invention's purpose just provides a kind of beta-stimulants albuterol, clenobuterol hydrochloride, Ractopamine ternary detection test bar and preparation method thereof, can effectively solve can be fast, detect simultaneously easily beta-stimulants albuterol, clenobuterol hydrochloride, the problem of Ractopamine, the technical scheme of its solution is, this test-strips comprises base material PVC plate and overlay film, be disposed with from front to back the diversion glass fibre above the base material PVC plate, the carrier glass fibre, nitrocellulose membrane and absorbent wool pulpboard, diversion glass fibre and be coated with the overlay film that is formed by anterior coverlay and rear portion coverlay above the absorbent wool pulpboard, absorption has anti-salbutamol protein conjugate on the carrier glass fibre, anti-clenobuterol hydrochloride protein conjugate, anti-Ractopamine protein conjugate monoclonal antibody colloid gold label thing; The middle part is distributed with from back to front one on the nitrocellulose membrane and is coated with that the anti-mouse of rabbit is how anti-(to be known commercially available prod, product such as Zhengzhou Lan Sen Bioisystech Co., Ltd) nature controlling line is (before said, after refer to the diversion direction to the suction direction, before the diversion glass fibre is, the absorbent wool pulpboard is rear), article three, be coated with respectively the salbutamol protein conjugate, the clenobuterol hydrochloride protein conjugate, the detection line of Ractopamine protein conjugate, the nitrocellulose membrane two ends respectively with carrier glass fibre rear end face and absorbent wool pulpboard front end face close contact, carrier glass fibre front end face and diversion glass fibre rear end face close contact;
The preparation method of test-strips of the present invention is, realized by following steps: prepare at first respectively colloid gold label monoclonal antibody, colloidal gold solution, colloid gold label thing, solidify gold mark thing, coated film, again the above-mentioned coated film for preparing, curing gold mark thing, diversion glass fibre, absorbent wool pulpboard fixedly are bonded on the base material PVC plate, overlay film in the covering cuts into rectangular getting final product.
The present invention can be effective to detect simultaneously beta-stimulants albuterol, clenobuterol hydrochloride, Ractopamine, and method is simple, and is convenient, fast, and the result is accurate.
Four, description of drawings
Accompanying drawing is test-strips three-dimensional structure diagram of the present invention (overlay film starts).
Five, embodiment
Below in conjunction with accompanying drawing the specific embodiment of the present invention is elaborated.
Shown in accompanying drawing, ternary detection test bar of the present invention comprises base material PVC plate and overlay film, be disposed with from front to back diversion glass fibre 3, carrier glass fibre 4, nitrocellulose membrane 2 and absorbent wool pulpboard 5 above the base material PVC plate 1, diversion glass fibre 3, carrier glass fibre 4 and be coated with the overlay film that is formed by anterior coverlay 7 and rear portion coverlay 6 above the absorbent wool pulpboard 5, absorption has anti-salbutamol protein conjugate, anti-clenobuterol hydrochloride protein conjugate, anti-Ractopamine protein conjugate monoclonal antibody colloid gold label thing on the carrier glass fibre; Be distributed with from back to front one on the nitrocellulose membrane of middle part and be coated with nature controlling line (said forward and backward the referring to the diversion direction to the suction direction that the anti-mouse of rabbit resists more, before the diversion glass fibre is, the absorbent wool pulpboard is rear), article three, be coated with respectively the detection line of salbutamol protein conjugate, clenobuterol hydrochloride protein conjugate, Ractopamine protein conjugate, the nitrocellulose membrane two ends respectively with carrier glass fibre rear end face and absorbent wool pulpboard front end face close contact, carrier glass fibre front end face and diversion glass fibre rear end face close contact.
In order to guarantee result of use, said overlay film is pressure sensitive membrane.
The preparation method of ternary detection test bar of the present invention is, realized by following steps: prepare at first respectively colloid gold label monoclonal antibody, colloidal gold solution, colloid gold label thing, solidify gold mark thing, coated film, again the above-mentioned coated film for preparing, curing gold mark thing, diversion glass fibre, absorbent wool pulpboard fixedly are bonded on the base material PVC plate, overlay film in the covering cuts into rectangular getting final product; Specifically:
1, the preparation of colloid gold label monoclonal antibody: get 1 part of mouse ascites that contains anti-salbutamol protein conjugate or anti-clenobuterol hydrochloride protein conjugate or anti-Ractopamine protein conjugate monoclonal antibody and mix with 2 parts of 60mM acetate buffer solutions, dropwise add sad 33 μ l/ml ascites (being that sad addition is to add the sad of 33 μ l in every 1ml mouse ascites) under the 18-25 ℃ of stirring at room, behind the mixing, room temperature was placed 30 minutes, 15000 rev/mins, 4 ℃ centrifugal 20 minutes, use the glass wool filtering supernatant, abandon sediment, supernatant after the filtration is transferred pH value to 7.2 with NaOH, add again ammonium sulfate, the ammonium sulfate addition is to add ammonium sulfate 0.277g (ammonium sulfate 0.277g/ml pH value 7.2 supernatants) in the supernatant of every 1ml pH value 7.2, make fast the ammonium sulfate dissolving, 18-25 ℃ of stirring at room 30 minutes, 15000 rev/mins, 4 ℃ centrifugal 20 minutes, abandon supernatant, sediment dissolves with 0.01M PBS pH 7.2 solution of former mouse ascites 1/2 volume, put into again 0.01M PBS pH 7.2 solution submergences, dialysed 48 hours for 4 ℃, measure protein content with ultraviolet spectrophotometer, namely be prepared into the anti-salbutamol protein conjugate monoclonal antibody that colloid gold label is used, anti-clenobuterol hydrochloride protein conjugate monoclonal antibody, anti-Ractopamine protein conjugate monoclonal antibody; Said mouse ascites, its preparation method is, in whiteruss injection Balb/c mouse peritoneal in 7 age in week, injection volume is 500 μ l/, after 10 days, the hybridoma of the anti-Ractopamine protein conjugate of secretion or anti-salbutamol protein conjugate or anti-clenobuterol hydrochloride protein conjugate monoclonal antibody is expelled in the mouse peritoneal, and the quantity of injection hybridoma is 2 * 10
6/ mouse is injected in mouse peritoneal with asepsis injector after 10 days and gathers ascites, is the mouse ascites of anti-Ractopamine protein conjugate or anti-salbutamol protein conjugate or anti-clenobuterol hydrochloride protein conjugate monoclonal antibody;
2, preparation colloidal gold solution: in beaker, add 1000ml distilled water, 100 ℃ were boiled about 5 minutes, add again mass concentration and be 1% chlorauric acid solution 10ml and mass concentration and be citric acid three sodium solution 15～20ml of 1%, be stirred to solution and be peony, be chilled to 18-25 ℃ of room temperature, sal tartari (K
2CO
3) transfer pH value to the isoelectric point of monoclonal antibody to be marked or omit meta-alkali, be colloidal gold solution to be marked;
3, the preparation of colloid gold label thing: get the colloidal gold solution that mixes up PH in the 100ml step 2, add anti-salbutamol protein conjugate monoclonal antibody or anti-clenobuterol hydrochloride protein conjugate monoclonal antibody or anti-Ractopamine protein conjugate monoclonal antibody 1～10 μ g/ml colloidal gold solution, stirred 2 minutes, the adding mass concentration is 10% bovine serum albumin(BSA) (BSA) 15 μ l/ml colloidal gold solution cessation reactions, centrifugal 50 minutes, get sediment, taking precipitate PH is 30% of 7.4 the 0.01M PBS former colloid gold solution volume that is diluted to step 2, is prepared into anti-salbutamol protein conjugate monoclonal antibody or anti-clenobuterol hydrochloride protein conjugate monoclonal antibody or anti-Ractopamine protein conjugate monoclonal antibody colloid gold label thing;
4, solidify the preparation of gold mark thing: draw anti-salbutamol protein conjugate monoclonal antibody or anti-clenobuterol hydrochloride protein conjugate monoclonal antibody or anti-Ractopamine protein conjugate monoclonal antibody colloid gold label thing with the carrier glass fibre, drying is prepared into anti-salbutamol protein conjugate monoclonal antibody or anti-clenobuterol hydrochloride protein conjugate monoclonal antibody or anti-Ractopamine protein conjugate monoclonal antibody and solidifies gold mark thing;
5, coated film preparation: get how anti-the anti-mouse of rabbit is, salbutamol protein conjugate, clenobuterol hydrochloride protein conjugate, Ractopamine protein conjugate, be coated in respectively on the nitrocellulose membrane, be prepared into coated film, the coated concentration of the anti-mouse multispecific antibody of rabbit is 0.5～5mg/ml, and salbutamol protein conjugate, clenobuterol hydrochloride protein conjugate, the coated concentration of Ractopamine protein conjugate are 0.05～2mg/ml;
6, test-strips equipment: get base material PVC plate 1, nitrocellulose membrane 2, the absorbent wool pulpboard 5 of diversion glass fibre 3, the carrier glass fibre 4 that solidifies gold mark thing, formation coated film are pasted on the base material PVC plate 1 from front to back successively, the overlay film that is comprised of anterior coverlay 7 and rear portion coverlay 6 in the covering cuts into rectangular getting final product (as shown in drawings).
The present invention also can be provided by following examples in implementation:
Embodiment 1: beta-stimulants albuterol, clenobuterol hydrochloride, the Ractopamine ternary detection test bar is, be stained with successively from front to back diversion glass fibre 3 on the base material PVC plate 1, carrier glass fibre 4, nitrocellulose membrane 2 and absorbent wool pulpboard 5, diversion glass fibre 3, carrier glass fibre 4 and being coated with above the absorbent wool pulpboard 5 by rear portion coverlay 6, the overlay film that anterior coverlay 7 forms, diversion glass fibre 3 rear end faces and carrier glass fibre 4 front end face close proximity, nitrocellulose membrane 2 both ends of the surface respectively with rear end face and the absorbent wool pulpboard 5 front end face close contacts of carrier glass fibre 4, absorption has anti-salbutamol protein conjugate monoclonal antibody on the anterior carrier glass fibre 4, anti-clenobuterol hydrochloride protein conjugate monoclonal antibody and anti-Ractopamine protein conjugate monoclonal antibody colloid gold label thing, mass concentration is that 1% citric acid three sodium solution amount is 17ml in the colloidal gold solution preparation, and colloid gold label anti-salbutamol protein conjugate monoclonal antibody or anti-clenobuterol hydrochloride protein conjugate monoclonal antibody or anti-Ractopamine protein conjugate monoclonal anti bulk concentration are 5 μ g/ml; Be coated with a nature controlling line and two p-wires on the nitrocellulose membrane 2 of middle part, it is how anti-that nature controlling line is coated with the anti-mouse of rabbit, p-wire is coated with salbutamol protein conjugate, clenobuterol hydrochloride protein conjugate, Ractopamine protein conjugate, how anti-coated concentration is 1mg/ml to the anti-mouse of rabbit, and salbutamol protein conjugate, clenobuterol hydrochloride protein conjugate, the coated concentration of Ractopamine protein conjugate are 0.2mg/ml.
Embodiment 2: in the present embodiment, in the colloidal gold solution preparation, mass concentration is that 1% citric acid three sodium solution amount is 15ml, and colloid gold label anti-salbutamol protein conjugate monoclonal antibody or anti-clenobuterol hydrochloride protein conjugate monoclonal antibody or anti-Ractopamine protein conjugate monoclonal anti bulk concentration are 4 μ g/ml; How anti-coated concentration is 0.9mg/ml to the anti-mouse of rabbit, and salbutamol protein conjugate, clenobuterol hydrochloride protein conjugate, the coated concentration of Ractopamine protein conjugate are 0.1mg/ml.The other the same as in Example 1.
Embodiment 3: in the present embodiment, in the colloidal gold solution preparation, mass concentration is that 1% citric acid three sodium solution amount is 19ml, and collaurum anti-salbutamol protein conjugate monoclonal antibody or anti-clenobuterol hydrochloride protein conjugate monoclonal antibody or anti-Ractopamine protein conjugate monoclonal anti bulk concentration are 8 μ g/ml; How anti-coated concentration is 2mg/ml to the anti-mouse of rabbit, and salbutamol protein conjugate, clenobuterol hydrochloride protein conjugate, the coated concentration of Ractopamine protein conjugate are 0.5mg/ml.The other the same as in Example 1.
Test-strips of the present invention is vertically inserted tunica fibrosa one end in the specimen samples in use, or specimen samples is dropped on the test-strips bottom diversion glass fibre.Sample is under capillary action, along test-strips to the swimming of absorbent wool pulpboard direction, if contain beta-stimulants albuterol, clenobuterol hydrochloride and Ractopamine or two kinds, a kind of in the specimen samples, they and respective markers antibody (colloid gold label monoclonal antibody) reaction form labelled antibody-antigenic compound.When the specimen samples swimming does not then react when salbutamol protein conjugate, clenobuterol hydrochloride protein conjugate, Ractopamine protein conjugate detect band to being coated with, and with nature controlling line reaction, a red line (nature controlling line) or two red lines or three only appear in the result; If in the specimen samples without beta-stimulants albuterol, clenobuterol hydrochloride and Ractopamine, labelled antibody (anti-salbutamol protein conjugate monoclonal antibody or anti-clenobuterol hydrochloride protein conjugate monoclonal antibody or anti-Ractopamine protein conjugate monoclonal antibody) respectively on coated film salbutamol protein conjugate, clenobuterol hydrochloride protein conjugate, Ractopamine protein conjugate and nature controlling line be combined and form the red line band, n+1 bar red line appears in the result; Red line do not occur such as nature controlling line, show that then test-strips lost efficacy.Therefore, utilize this test-strips, only need an application of sample, can detect three kinds of beta-stimulants in 5～10 minutes.The method is simple, convenient, fast, the result is accurate.
The present invention is through test, utilize this test-strips to test simultaneously beta-stimulants albuterol, clenobuterol hydrochloride, Ractopamine, only need an application of sample, can test beta-stimulants albuterol, clenobuterol hydrochloride, Ractopamine in 5-10 minute, on probation through 158 times, except 1 example is indefinite, all the other 157 examples have all reached promising result, rate of accuracy reached is more than 99%, therefore, it is simple that the present invention has method, convenient and swift, the characteristics that the result is accurate are the innovations in beta-stimulants albuterol, clenobuterol hydrochloride, the Ractopamine detection.
Claims (1)
1. the preparation method of a beta-stimulants albuterol, clenobuterol hydrochloride, Ractopamine ternary detection test bar is characterized in that, is realized by following steps:
(1), the preparation of colloid gold label monoclonal antibody: get 1 part of mouse ascites that contains anti-salbutamol protein conjugate or anti-clenobuterol hydrochloride protein conjugate or anti-Ractopamine protein conjugate monoclonal antibody and mix with 2 parts of 60mM acetate buffer solutions, dropwise add sad 33 μ l/ml ascites under the 18-25 ℃ of stirring at room, behind the mixing, room temperature was placed 30 minutes, 15000 rev/mins, 4 ℃ centrifugal 20 minutes, use the glass wool filtering supernatant, abandon sediment, supernatant after the filtration is transferred pH value to 7.2 with NaOH, add again ammonium sulfate, the ammonium sulfate addition is to add ammonium sulfate 0.277g in the supernatant of every 1ml pH value 7.2, make fast the ammonium sulfate dissolving, 18-25 ℃ of stirring at room 30 minutes, 15000 rev/mins, 4 ℃ centrifugal 20 minutes, abandon supernatant, sediment dissolves with 0.01M PBS pH 7.2 solution of former mouse ascites 1/2 volume, put into again 0.01M PBS pH 7.2 solution submergences, dialysed 48 hours for 4 ℃, measure protein content with ultraviolet spectrophotometer, namely be prepared into the anti-salbutamol protein conjugate monoclonal antibody that colloid gold label is used, anti-clenobuterol hydrochloride protein conjugate monoclonal antibody, anti-Ractopamine protein conjugate monoclonal antibody; Said mouse ascites, its preparation method is, in whiteruss injection Balb/c mouse peritoneal in 7 age in week, injection volume is 500 μ l/, after 10 days, the hybridoma of the anti-Ractopamine protein conjugate of secretion or anti-salbutamol protein conjugate or anti-clenobuterol hydrochloride protein conjugate monoclonal antibody is expelled in the mouse peritoneal, and the quantity of injection hybridoma is 2 * 10
6/ mouse is injected in mouse peritoneal with asepsis injector after 10 days and gathers ascites, is the mouse ascites of anti-Ractopamine protein conjugate or anti-salbutamol protein conjugate or anti-clenobuterol hydrochloride protein conjugate monoclonal antibody;
(2), preparation colloidal gold solution: in beaker, add 1000ml distilled water, 100 ℃ were boiled 5 minutes, add again mass concentration and be 1% chlorauric acid solution 10ml and mass concentration and be citric acid three sodium solution 15～20ml of 1%, be stirred to solution and be peony, be chilled to 18-25 ℃ of room temperature, sal tartari is transferred pH value to the isoelectric point of monoclonal antibody to be marked or is omited meta-alkali, is colloidal gold solution to be marked;
(3), the preparation of colloid gold label thing: get the colloidal gold solution that mixes up PH in the 100ml step 2, add anti-salbutamol protein conjugate monoclonal antibody or anti-clenobuterol hydrochloride protein conjugate monoclonal antibody or anti-Ractopamine protein conjugate monoclonal antibody 1～10 μ g/ml colloidal gold solution, stirred 2 minutes, the adding mass concentration is 10% bovine serum albumin(BSA) 15 μ l/ml colloidal gold solution cessation reactions, centrifugal 50 minutes, get sediment, taking precipitate PH is 30% of 7.4 the 0.01M PBS former colloid gold solution volume that is diluted to step 2, is prepared into anti-salbutamol protein conjugate monoclonal antibody or anti-clenobuterol hydrochloride protein conjugate monoclonal antibody or anti-Ractopamine protein conjugate monoclonal antibody colloid gold label thing;
(4), solidify the preparation of gold mark thing: draw anti-salbutamol protein conjugate monoclonal antibody or anti-clenobuterol hydrochloride protein conjugate monoclonal antibody or anti-Ractopamine protein conjugate monoclonal antibody colloid gold label thing with the carrier glass fibre, drying is prepared into anti-salbutamol protein conjugate monoclonal antibody or anti-clenobuterol hydrochloride protein conjugate monoclonal antibody or anti-Ractopamine protein conjugate monoclonal antibody and solidifies gold mark thing;
(5), coated film preparation: get how anti-the anti-mouse of rabbit is, salbutamol protein conjugate, clenobuterol hydrochloride protein conjugate, Ractopamine protein conjugate, be coated in respectively on the nitrocellulose membrane, be prepared into coated film, how anti-coated concentration is 0.5～5mg/ml to the anti-mouse of rabbit, and salbutamol protein conjugate, clenobuterol hydrochloride protein conjugate, the coated concentration of Ractopamine protein conjugate are 0.05～2mg/ml;
(6), test-strips equipment: get base material PVC plate (1), nitrocellulose membrane (2), the absorbent wool pulpboard (5) of diversion glass fibre (3), the carrier glass fibre (4) that solidifies gold mark thing, formation coated film are pasted on the base material PVC plate (1) from front to back successively, the overlay film that is comprised of anterior coverlay (7) and rear portion coverlay (6) in the covering cuts into rectangular getting final product.
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| CN102636640A (en) * | 2011-03-17 | 2012-08-15 | 南通戴尔诺斯生物科技有限公司 | Clenbuterol type detection test paper and preparation method thereof |
| CN102230937A (en) * | 2011-06-17 | 2011-11-02 | 河南省农业科学院 | Brown meat essence multi-residue combined detection test paper card and preparation method thereof |
| CN102967707A (en) * | 2012-03-30 | 2013-03-13 | 无锡福阳生物科技有限公司 | Triple immune colloidal gold rapid detection card and its preparation and use method |
| CN102901818A (en) * | 2012-09-27 | 2013-01-30 | 江苏维赛科技生物发展有限公司 | Five-conjugate testing card for detecting beta-stimulant drugs and preparation method thereof |
| CN103257226B (en) * | 2013-04-09 | 2015-12-09 | 江西中德生物工程有限公司 | Ractopamine, salbutamol, Cimaterol three colloidal gold strip and preparation method thereof and purposes |
| CN105092567B (en) * | 2015-07-15 | 2019-01-29 | 宁波高新区绿邦科技发展有限公司 | Three link detection reagent card of clenobuterol hydrochloride-Ractopamine-salbutamol |
| CN105203766B (en) * | 2015-09-29 | 2017-01-11 | 河南省科学院生物研究所有限责任公司 | Preparation method for pathogenic yersinia enterocolitica test strips |
| CN109061197A (en) * | 2018-09-27 | 2018-12-21 | 河南省科学院生物研究所有限责任公司 | The preparation method of three colloid gold immune test paper item of cTnI, NT-proBNP and D-dimer pectoralgia |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003020257A2 (en) * | 2001-08-29 | 2003-03-13 | Eucro European Contract Research Gmbh & Co. Kg | USE OF β-ADRENOCEPTOR AGONISTS FOR THE TREATMENT OF NEURODEGENERATIVE DISEASES |
| CN101231293A (en) * | 2008-02-14 | 2008-07-30 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | Method for detecting vestigial clenbuterol |
| CN101566629A (en) * | 2009-05-22 | 2009-10-28 | 无锡安迪生物工程有限公司 | Clenobuterol hydrochloride, salbutamol and paylean three joint inspection card and method for processing detecting sample |
| CN101706506A (en) * | 2009-12-03 | 2010-05-12 | 杭州迪恩科技有限公司 | Immunochromatographic test paper used for detecting beta stimulant and detection method thereof |
| CN201828562U (en) * | 2010-07-26 | 2011-05-11 | 河南省科学院生物研究所有限责任公司 | Test strip for salbutamol, clenbuterol and ractopamine ternary-detection of Beta-stimulant |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1613266A4 (en) * | 2003-04-15 | 2009-05-06 | Theraquest Biosciences Llc | Methods of treating pain and compositions for use therefor |
-
2010
- 2010-07-26 CN CN 201010235565 patent/CN101915841B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003020257A2 (en) * | 2001-08-29 | 2003-03-13 | Eucro European Contract Research Gmbh & Co. Kg | USE OF β-ADRENOCEPTOR AGONISTS FOR THE TREATMENT OF NEURODEGENERATIVE DISEASES |
| CN101231293A (en) * | 2008-02-14 | 2008-07-30 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | Method for detecting vestigial clenbuterol |
| CN101566629A (en) * | 2009-05-22 | 2009-10-28 | 无锡安迪生物工程有限公司 | Clenobuterol hydrochloride, salbutamol and paylean three joint inspection card and method for processing detecting sample |
| CN101706506A (en) * | 2009-12-03 | 2010-05-12 | 杭州迪恩科技有限公司 | Immunochromatographic test paper used for detecting beta stimulant and detection method thereof |
| CN201828562U (en) * | 2010-07-26 | 2011-05-11 | 河南省科学院生物研究所有限责任公司 | Test strip for salbutamol, clenbuterol and ractopamine ternary-detection of Beta-stimulant |
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