CN101915842A - Binary detection test strip of beta-exhilarant ractopamine and salbutamol and preparation method thereof - Google Patents
Binary detection test strip of beta-exhilarant ractopamine and salbutamol and preparation method thereof Download PDFInfo
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- CN101915842A CN101915842A CN2010102355927A CN201010235592A CN101915842A CN 101915842 A CN101915842 A CN 101915842A CN 2010102355927 A CN2010102355927 A CN 2010102355927A CN 201010235592 A CN201010235592 A CN 201010235592A CN 101915842 A CN101915842 A CN 101915842A
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- protein conjugate
- ractopamine
- monoclonal antibody
- salbutamol
- glass fibre
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Abstract
The invention relates to a binary detection test strip of beta-exhilarant ractopamine and salbutamol and a preparation method thereof, which can effectively solve the problem that the beta-exhilarant ractopamine and salbutamol can not be detected quickly, easily and simultaneously. The preparation method comprises the following steps of: arranging diversion glass fiber, carrier glass fiber, a nitrocellulose membrane and an absorbent cotton pulp board a on a substrate PVC (Polyvinyl Chloride) board from back to front in order; covering a coating comprising a front coating and a rear coating on the diversion glass fiber, the carrier glass fiber and the absorbent cotton pulp board, wherein a monoclonal antibody for resisting ractopamine protein conjugates and a monoclonal antibody colloid gold marker for resisting salbutamol protein conjugates are adsorbed in the carrier glass fiber; and coating the nitrocellulose membrane with a quality control line and two test lines, wherein the quality control line is coated with rabbit antimouse multi-resistant, and the test lines are coated with ractopamine protein conjugates and salbutamol protein conjugates. The invention can be used for detecting the beta-exhilarant ractopamine and the salbutamol simultaneously, effectively, conveniently and quickly and has accurate result.
Description
One, technical field
The present invention relates to a kind of beta-stimulants Ractopamine, salbutamol binary detection test-strips and preparation method thereof.
Two, background technology
Beta-stimulants is a class adrenomimetic drug, because of its can make the beta receptor excitement (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 a 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 culturing 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, symptom such as feel sick, vomit, have a fever, tremble can appear usually, in addition dead.Therefore, world many countries forbids in feed using beta-stimulants such as clenobuterol hydrochloride to increase the lean meat percentage of animal.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 omission.Therefore, market presses for a kind of reagent can detect common beta-agonist simultaneously, to make things convenient for routine monitoring work.
Three, summary of the invention
At above-mentioned situation, for overcoming the prior art defective, the object of the invention is to provide a kind of energy quick, detect the beta-stimulants Ractopamine easily simultaneously, the salbutamol binary detects test-strips and preparation method thereof, for achieving the above object, the present invention adopts following technical scheme: comprise base material PVC plate and overlay film, be disposed with the diversion glass fibre from back to front above the base material PVC plate, the carrier glass fibre, nitrocellulose membrane and absorbent wool pulpboard, the diversion glass fibre, carrier glass fibre and be coated with the overlay film of forming by anterior coverlay and rear portion coverlay above the absorbent wool pulpboard, be adsorbed with anti-Ractopamine protein conjugate monoclonal antibody and desertification butylamine alcohol protein conjugate monoclonal antibody colloid gold label thing in the carrier glass fibre, be coated with a nature controlling line and two p-wires on the nitrocellulose membrane, nature controlling line is coated with that the anti-mouse of rabbit is how anti-(to be known commercially available prod, product as Zhengzhou Lan Sen Bioisystech Co., Ltd), p-wire is coated with the Ractopamine protein conjugate, the salbutamol protein conjugate; The nitrocellulose membrane two ends closely contact with absorbent wool pulpboard front end face with carrier glass fibre rear end face respectively, and carrier glass fibre front end face closely contacts with diversion glass fibre rear end face; Its preparation method is, realize by following steps: prepare colloid gold label monoclonal antibody, colloidal gold solution, colloid gold label thing at first respectively, 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 Ractopamine, salbutamol, test-strips of the present invention in use, vertically insert diversion glass fibre (film) end in the specimen samples, or specimen samples dripped to the anterior diversion glass fibre of test-strips, sample is under capillary action, along test-strips to rear portion absorbent wool pulpboard direction swimming, if contain beta-stimulants Ractopamine and salbutamol or 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 Ractopamine protein conjugate, salbutamol protein conjugate detect band to being coated with, and with the nature controlling line reaction, a red line (nature controlling line) or two red lines only appear in the result; If no beta-stimulants Ractopamine and salbutamol in the specimen samples, labelled antibody (desertification butylamine alcohol protein conjugate monoclonal antibody or anti-Ractopamine protein conjugate monoclonal antibody) combines with Ractopamine protein conjugate, salbutamol protein conjugate and nature controlling line on the coated film respectively and forms the red line band, and n+1 bar red line appears in the result; Red line do not occur as nature controlling line, show that then test-strips lost efficacy.Therefore, utilize this test-strips, only need an application of sample, can detect two kinds of beta-stimulants in 5～10 minutes.This method is simple, convenient, fast, the result is accurate.
Four, description of drawings
Accompanying drawing is a 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.
Provide by accompanying drawing, binary of the present invention detects its structure of test-strips, comprise base material PVC plate and overlay film, be disposed with diversion glass fibre 3 from front to back above the base material PVC plate 1, carrier glass fibre 4, nitrocellulose membrane 2 and absorbent wool pulpboard 5, the diversion glass fibre, carrier glass fibre and be coated with overlay film (the alleged anterior coverlay of forming by anterior coverlay 7 and rear portion coverlay 67 above the absorbent wool pulpboard, rear portion coverlay 6 is that specimen samples swimming direction is as the criterion when using with test-strips, before the diversion end is, the suction side is the back), be adsorbed with anti-Ractopamine protein conjugate monoclonal antibody and desertification butylamine alcohol protein conjugate monoclonal antibody colloid gold label thing in the carrier glass fibre, be coated with a nature controlling line and two p-wires on the nitrocellulose membrane, it is how anti-that nature controlling line is coated with the anti-mouse of rabbit, and p-wire is coated with the Ractopamine protein conjugate, the salbutamol protein conjugate; The nitrocellulose membrane two ends closely contact with absorbent wool pulpboard front end face with carrier glass fibre rear end face respectively, and carrier glass fibre front end face closely contacts with diversion glass fibre rear end face.
In order to guarantee result of use, said overlay film is a pressure sensitive membrane.
The preparation method that binary of the present invention detects test-strips is, realize by following steps: prepare colloid gold label monoclonal antibody, colloidal gold solution, colloid gold label thing at first respectively, 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. colloid gold label MONOCLONAL ANTIBODIES SPECIFIC FOR: adopt sad-ammonium sulfate method, getting 1 part of mouse ascites that contains anti-Ractopamine protein conjugate or desertification butylamine alcohol protein conjugate monoclonal antibody mixes with 2 parts of 60mM acetate buffer solutions, under 18-25 ℃ of stirring, dropwise add sad 33 μ l/ml mouse ascites (being to add the sad of 33 μ l in the mouse ascites of every 1ml), behind the mixing, room temperature (18-25 ℃) 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, in supernatant, add ammonium sulfate, its addition is to add 0.277g (0.277g/ml) in the supernatant of every 1mlPH value 7.2, make the ammonium sulfate dissolving fast, in 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 0.01M PBS pH 7.2 solution submergences again, dialysed 48 hours for 4 ℃, measure protein content with ultraviolet spectrophotometer, promptly be prepared into the anti-Ractopamine protein conjugate monoclonal antibody that colloid gold label is used, desertification butylamine alcohol 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 anti-Ractopamine protein conjugate of secretion or desertification butylamine alcohol 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 desertification butylamine alcohol protein conjugate monoclonal antibody;
2. preparation colloidal gold solution: in beaker, add 1000ml distilled water, 100 ℃ were boiled about 5 minutes, add mass concentration again 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, with sal tartari (K
2CO
3) transfer isoelectric point or the slightly inclined to one side alkali of pH value to monoclonal antibody to be marked, 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-Ractopamine protein conjugate monoclonal antibody or desertification butylamine alcohol protein conjugate monoclonal antibody 1～10 μ g/ml colloidal gold solution (being to add monoclonal antibody 1-10 μ g in every 1ml 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 0.01M PBS (being phosphate buffer) the former colloid gold solution volume that is diluted to step 2, is prepared into anti-Ractopamine protein conjugate monoclonal antibody or desertification butylamine alcohol protein conjugate monoclonal antibody colloid gold label thing;
4. solidify the preparation of gold mark thing: draw anti-Ractopamine protein conjugate monoclonal antibody or desertification butylamine alcohol protein conjugate monoclonal antibody colloid gold label thing with the carrier glass fibre, drying is prepared into anti-Ractopamine protein conjugate monoclonal antibody or desertification butylamine alcohol protein conjugate monoclonal antibody and solidifies gold mark thing;
5. coated film preparation: get how anti-the anti-mouse of rabbit is, Ractopamine protein conjugate, salbutamol protein conjugate, bag is by on nitrocellulose membrane respectively, be prepared into coated film, wherein: how anti-bag is 0.5～5mg/ml by concentration to the anti-mouse of rabbit, and Ractopamine protein conjugate, salbutamol protein conjugate bag are 0.05～2mg/ml by concentration;
6. test-strips equipment: from preceding then successively with diversion glass fibre 3, the carrier glass fibre 4 that adsorbs anti-Ractopamine protein conjugate monoclonal antibody or salbutamol protein conjugate monoclonal antibody colloid gold label thing, be coated with that the anti-mouse of rabbit is how anti-, Ractopamine protein conjugate, the nitrocellulose membrane 2 of salbutamol protein conjugate, absorbent wool pulpboard 5 fixedly be pasted on the base material PVC plate 1, overlay film in the covering cuts into rectangular getting final product.
Embodiment 1: the present invention is in concrete enforcement, also can be and comprise base material PVC plate 1, adhere to the nitrocellulose membrane 2 at base material middle part, adhere to the diversion glass fibre 3 and the carrier glass fibre 4 of base material PVC plate 1 front portion, adhere to the absorbent wool pulpboard 5 at base material rear portion, top overlay film 6 and bottom overlay film 7, 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 reach with the rear end face of carrier glass fibre 4 respectively and closely contact with absorbent wool pulpboard 5 front end faces, lower carrier glass fibre 4 is that anti-Ractopamine protein conjugate monoclonal antibody and desertification butylamine alcohol protein conjugate monoclonal antibody are solidified gold mark 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-Ractopamine protein conjugate monoclonal antibody or desertification butylamine alcohol 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 Ractopamine protein conjugate, salbutamol protein conjugate, how anti-bag is 1mg/ml by concentration to the anti-mouse of rabbit, and Ractopamine protein conjugate, salbutamol protein conjugate bag are 0.2mg/ml by concentration.
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-Ractopamine protein conjugate monoclonal antibody or desertification butylamine alcohol protein conjugate monoclonal anti bulk concentration are 4 μ g/ml; How anti-bag is 0.9mg/ml by concentration to the anti-mouse of rabbit, and Ractopamine protein conjugate, salbutamol protein conjugate bag are 0.1mg/ml by concentration.Other is with embodiment 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 colloid gold label anti-Ractopamine protein conjugate monoclonal antibody or desertification butylamine alcohol protein conjugate monoclonal anti bulk concentration are 8 μ g/ml; How anti-bag is 2mg/ml by concentration to the anti-mouse of rabbit, and Ractopamine protein conjugate, salbutamol protein conjugate bag are 0.5mg/ml by concentration.Other is with embodiment 1.
The present invention utilizes this test-strips to test beta-stimulants Ractopamine, salbutamol simultaneously through test, only needs an application of sample, can test beta-stimulants Ractopamine, salbutamol in 5-10 minute, through 158 times on probation, except that 1 example was indeterminate, all the other 157 examples had 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 on beta-stimulants Ractopamine, salbutamol detect.
Claims (3)
1. beta-stimulants Ractopamine, the salbutamol binary detects test-strips, comprise base material PVC plate and overlay film, it is characterized in that, be disposed with diversion glass fibre (3) above the base material PVC plate (1) from front to back, carrier glass fibre (4), nitrocellulose membrane (2) and absorbent wool pulpboard (5), the diversion glass fibre, carrier glass fibre and be coated with the overlay film of forming by anterior coverlay (7) and rear portion coverlay (6) above the absorbent wool pulpboard, be adsorbed with anti-Ractopamine protein conjugate monoclonal antibody and desertification butylamine alcohol protein conjugate monoclonal antibody colloid gold label thing in the carrier glass fibre, be coated with a nature controlling line and two p-wires on the nitrocellulose membrane, it is how anti-that nature controlling line is coated with the anti-mouse of rabbit, and p-wire is coated with the Ractopamine protein conjugate, the salbutamol protein conjugate; The nitrocellulose membrane two ends closely contact with absorbent wool pulpboard front end face with carrier glass fibre rear end face respectively, and carrier glass fibre front end face closely contacts with diversion glass fibre rear end face.
2. beta-stimulants Ractopamine according to claim 1, salbutamol binary detect test-strips, it is characterized in that said overlay film is a pressure sensitive membrane.
3. the described beta-stimulants Ractopamine of claim 1, salbutamol binary detect the preparation method of test-strips, it is characterized in that, are realized by following steps:
(1) colloid gold label MONOCLONAL ANTIBODIES SPECIFIC FOR: get 1 part of mouse ascites that contains anti-Ractopamine protein conjugate or desertification butylamine alcohol protein conjugate monoclonal antibody and mix with 2 parts of 60mM acetate buffer solutions, under 18-25 ℃ of stirring, dropwise add sad 33 μ l/ml mouse ascites, behind the mixing, placed 30 minutes for 18-25 ℃, 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, in supernatant, add ammonium sulfate, its addition is to add 0.277g in the supernatant of every 1ml pH value 7.2, make the ammonium sulfate dissolving fast, stirred 30 minutes in 18-25 ℃, 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 0.01M PBS pH 7.2 solution submergences again, dialysed 48 hours for 4 ℃, measure colloid gold label thing protein content with ultraviolet spectrophotometer, promptly be prepared into the anti-Ractopamine protein conjugate monoclonal antibody that colloid gold label is used, desertification butylamine alcohol 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 anti-Ractopamine protein conjugate of secretion or desertification butylamine alcohol 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 desertification butylamine alcohol protein conjugate monoclonal antibody;
(2) preparation colloidal gold solution: in beaker, add 1000ml distilled water, 100 ℃ were boiled 5 minutes, add mass concentration again 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 ℃, transfer isoelectric point or the slightly inclined to one side alkali of pH value with sal tartari, be colloidal gold solution to be marked to monoclonal antibody to be marked;
(3) preparation of colloid gold label thing: get the colloidal gold solution that mixes up PH in the 100ml step 2, add anti-Ractopamine protein conjugate monoclonal antibody or desertification butylamine alcohol 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 colloidal gold solution volume that is diluted to step 2, is prepared into anti-Ractopamine protein conjugate monoclonal antibody or desertification butylamine alcohol protein conjugate monoclonal antibody colloid gold label thing;
(4) solidify the preparation that gold is marked thing: draw anti-Ractopamine protein conjugate monoclonal antibody or desertification butylamine alcohol protein conjugate monoclonal antibody colloid gold label thing with the carrier glass fibre, drying is prepared into anti-Ractopamine protein conjugate monoclonal antibody or desertification butylamine alcohol protein conjugate monoclonal antibody and solidifies gold mark thing;
(5) coated film preparation: get how anti-the anti-mouse of rabbit is, Ractopamine protein conjugate, salbutamol protein conjugate, bag is by on nitrocellulose membrane respectively, be prepared into coated film, wherein: how anti-bag is 0.5～5mg/ml by concentration to the anti-mouse of rabbit, and Ractopamine protein conjugate, salbutamol protein conjugate bag are 0.05～2mg/ml by concentration;
(6) test-strips equipment: from preceding then successively with diversion glass fibre 3, the carrier glass fibre 4 that adsorbs anti-Ractopamine protein conjugate monoclonal antibody or salbutamol protein conjugate monoclonal antibody colloid gold label thing, be coated with that the anti-mouse of rabbit is how anti-, Ractopamine protein conjugate, the nitrocellulose membrane (2) of salbutamol protein conjugate, absorbent wool pulpboard (5) fixedly be pasted on the base material PVC plate (1), overlay film in the covering cuts into rectangular getting final product.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010235592 CN101915842B (en) | 2010-07-26 | 2010-07-26 | Binary detection test strip of beta-exhilarant ractopamine and salbutamol and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN 201010235592 CN101915842B (en) | 2010-07-26 | 2010-07-26 | Binary detection test strip of beta-exhilarant ractopamine and salbutamol and preparation method thereof |
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| CN105738582A (en) * | 2016-04-21 | 2016-07-06 | 天津科技大学 | Immunochromatography test strip capable of simultaneously detecting salbutamol and flumequine and preparation method of test strip |
| CN107860911A (en) * | 2017-11-17 | 2018-03-30 | 河南省科学院生物研究所有限责任公司 | A kind of preparation method of pathogenic salmonella test-strips |
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| 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 |
| CN103235125A (en) * | 2013-04-09 | 2013-08-07 | 江西中德生物工程有限公司 | Ractopamine and cimaterolm combined colloidal gold test strip, and preparation method and application thereof |
| CN105738582A (en) * | 2016-04-21 | 2016-07-06 | 天津科技大学 | Immunochromatography test strip capable of simultaneously detecting salbutamol and flumequine and preparation method of test strip |
| CN105738582B (en) * | 2016-04-21 | 2017-11-21 | 天津科技大学 | Immuno-chromatographic test paper strip that is a kind of while detecting salbutamol and flumequine and preparation method thereof |
| CN107860911A (en) * | 2017-11-17 | 2018-03-30 | 河南省科学院生物研究所有限责任公司 | A kind of preparation method of pathogenic salmonella test-strips |
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| CN101915842B (en) | 2013-04-03 |
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