CN103808930A - Zilpaterol immune colloidal gold detection card and preparation method thereof - Google Patents
Zilpaterol immune colloidal gold detection card and preparation method thereof Download PDFInfo
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- CN103808930A CN103808930A CN201210438039.2A CN201210438039A CN103808930A CN 103808930 A CN103808930 A CN 103808930A CN 201210438039 A CN201210438039 A CN 201210438039A CN 103808930 A CN103808930 A CN 103808930A
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54386—Analytical elements
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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Abstract
The invention discloses a zilpaterol immune colloidal gold detection card and a preparation method thereof, and relates to the beta-epinephrine receptor agonist detection technical field. A test strip in an outer shell of the detection card is composed of a PVC rubber plate, a sample pad, a colloidal gold combined pad, a coating film and a water-absorbing pad; a colloidal gold film is a glass cellulose film containing a zilpaterol monoclonal antibody, and the coating film is a nitrocellulose film; a T line and a C line are arranged on the nitrocellulose film, the T line is coated with a zilpaterol-protein conjugate, and the C line is coated with a sheep anti-mouse IgG antibody. The detection card is effectively used for rapid detection of zilpaterol, is convenient and fast, and has accurate results.
Description
Technical field
The present invention relates to receptor,β activator detection technique field, particularly relate to immune colloid gold test card of Zilpaterol and preparation method thereof.
Background technology
β
2-2-adrenergic agonist components is the common growth promoter of a class, when this type of material is used in animal-breeding, can increase feed conversion rate, improves muscle and fatty ratio in tissue.β
2-2-adrenergic agonist components belongs to catecholamines, can be divided into phenyl amines, resorcinol class and phenol according to chemical constitution.Zilpaterol (Zilpaterol) is a kind of novel β
2-2-adrenergic agonist components, its chemical constitution (seeing formula 1) is diverse with common phenyl amines-adrenaline excitomotor (clenbuterol and salbutamol) and phenol beta-adrenaline excitomotor (Ractopamine), its action effect is equivalent to 1/10th of clenbuterol, but more effective than Ractopamine.Be allowed to use as the feed addictive of slaughtering phase ox in Mexico and South Africa Zilpaterol, but prohibited use in European Union.
The chemical constitution of formula 1 Zilpaterol
At present, European Union member countries relate to GC-MS(gas chromatography-mass spectrography), high performance liquid chromatography-tandem mass method etc. to the more experimental technique of research of residual aspect in Zilpaterol use and animal tissue.Gas chromatography mass spectrometry method all needs sample to derive, and operation steps is many, and experimental period is long, and high performance liquid chromatography-series winding mass spectroscopy is high to instrument and equipment requirement, conventionally need to utilize deuterated internal standard compound matter, and experimental procedure is more complicated.Therefore, a kind of simple, quick, accurate, reliable detection method of foundation, monitors and is very important the use of Zilpaterol.The object of the invention is to develop a kind of enterprise that is more suitable for and carry out Site Detection and qualitative checking method fast and simple, with low cost.
Summary of the invention
For above situation, object of the present invention be exactly for overcome prior art exist defect and immune colloid gold test card of a kind of Zilpaterol and preparation method thereof is provided, can effectively solve the problem that can detect fast, easily receptor,β activator Zilpaterol.
Zilpaterol colloidal-gold detecting-card of the present invention, comprise and be coated with the collaurum pad of monoclonal antibody colloid gold label thing, the nitrocellulose filter that has been coated with Zilpaterol-BSA and sheep anti-mouse igg, sample pad, adsorptive pads, PVC offset plate and mould of plastics composition, one end at PVC offset plate adheres to sample pad, pad successively, nitrocellulose filter is pasted in centre, and the other end adheres to adsorptive pads.
The preparation method of Zilpaterol colloidal-gold detecting-card of the present invention 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 add 1% gold chloride (HAuCl
43H
2o) 5 mL, are mixed with 500 mL 0.01% aqueous solution of chloraurate, and heating adds 1% trisodium citrate (Na after boiling in the situation that continuing stirring
3c
6h
5o
72H
2o) solution 5-7 mL, continues agitating heating, in the time that the color of solution becomes transparent aubergine completely, maintains after 5 min and stops heating, and moisturizing, to original volume, is cooled to room temperature, and 2-8 ℃ saves backup;
(2) pre-service of antibody: Zilpaterol antibody that will mark is at 1000 r/min, and under 4 ℃ of conditions, centrifugal 20 min, get supernatant, is diluted to 1 mg/mL with 0.01 mol/L PBS; Or be diluted to 1 mg/ml with 0.01 mol/L PBS, cross 0.22 μ m filter membrane;
(3) preparation of colloid gold label thing: get colloidal gold solution 40 mL in 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 of 4 mL containing 0.3 mg antibody protein, reaction 10 min; Dropwise add 4 mL 10% BSA, continue stirring reaction 10 min; Centrifugal 10 min of gold labeling antibody solution normal temperature low speed (1500 r/min), discard the precipitation being formed by the gold grain condensing; 4 ℃ of red supernatant solution, centrifugal 20 min of 12000 r/min, abandon supernatant, and collecting precipitation is settled to 1 mL by precipitation with golden labeling antibody dilution, is prepared into Zilpaterol monoclonal antibody colloid gold label thing;
(4) preparation of collaurum film: step (3) Zilpaterol monoclonal antibody colloid gold label thing is evenly sprayed on carrier glass fibre element film with the concentration of 5 μ L/cm with drawing film instrument, room temperature is dried or 37 ℃ of oven dry 3 h naturally, makes the collaurum film containing Zilpaterol monoclonal antibody colloid gold label thing;
(5) coated film preparation: sheep anti-mouse igg antibody, Zilpaterol protein conjugate are diluted to 1 mg/mL, be sprayed on nitrocellulose filter with the concentration of 1 μ L/cm successively with drawing film instrument, be prepared into coated film, after 37 ℃ of coated 2 h, room temperature is dried or 37 ℃ of oven dry naturally;
(6) sample pad pre-treatment: sample pad treating fluid is uniformly coated on glass fibre element film, air humidity lower than 60% condition under, room temperature is dried naturally;
(7) assembling of test card: 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, then test strips is arranged in the test card shell of rectangular flat shelly.
In described step (5), coated detection line T is located at the below of nature controlling line C;
Sample pad treating fluid in described step (6) is by 1 g Bovine serum albumin (BSA) and 0.8 g sodium chloride (NaCl), uses the 0.01 mol/L PBS that contains 0.5%TRITON-100 to be settled to 100 mL;
The present invention can be effective to measure receptor,β activator Zilpaterol, and method is simple, convenient, fast, and result is accurate.
Accompanying drawing explanation
Fig. 1 is the structural drawing of the immune colloid gold test card of Zilpaterol of the present invention: in figure, 1. well 2. detection line 3. nature controlling lines 4. detect hole 5. test strips 6. test card shells;
Fig. 2 is the sectional structure chart of the test strips in the immune colloid gold test card of Zilpaterol of the present invention, 7. sample pad 8. collaurum pad 9.PVC offset plate 10. nitrocellulose filter 11. adsorptive pads in figure.
Embodiment
Embodiment 1
Fig. 1 is the sectional structure chart of the test strips in structural drawing, the immune colloid gold test card that Fig. 2 is Zilpaterol of the present invention of the immune colloid gold test card of Zilpaterol of the present invention: in figure, 9 is PVC offset plate; 7 is sample pad; 8 is collaurum pad, has been coated with monoclonal antibody colloid gold label thing on this collaurum pad; 10 is coated film, i.e. nitrocellulose filter, has been coated with Zilpaterol-BSA and sheep anti-mouse igg on this nitrocellulose filter; 11 is adsorptive pads, is made up as filter paper of absorbent material.
On one end of PVC offset plate 9, (sample end) adheres to sample pad 7, collaurum pad 8, and sample pad 7 and collaurum pad 8 are structure side by side.
In the middle of PVC offset plate 9, adhere to nitrocellulose filter 10.On nitrocellulose filter 10, be provided with sheep anti-mouse igg nature controlling line 3 and Zilpaterol-BSA detection line 2.
The other end at PVC offset plate 9 adheres to adsorptive pads 11.One end of nitrocellulose filter 10 slightly intersects with pad 8, and the other end slightly intersects with adsorptive pads 11.This test strips 5 can be incorporated with in the test card shell 6 that mould of plastics makes, and covers and be provided with well 1 and detect hole 4 on test card shell 6, and sample pad 7 is just to well 1, and nitrocellulose filter 10 is just to detecting hole 4.
Embodiment 2
The preparation of Zilpaterol colloidal-gold detecting-card is by following steps specific implementation:
(1) 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, and heating adds 1% trisodium citrate (Na after boiling in the situation that continuing stirring
3c
6h
5o
72H
2o) solution 5-7 mL, continues agitating heating, in the time that the color of solution becomes transparent aubergine completely, maintains after 5 min and stops heating, and moisturizing, to original volume, is cooled to room temperature, and 2-8 ℃ saves backup;
(2) pre-service of antibody: Zilpaterol antibody that will mark is at 1000 r/min, and under 4 ℃ of conditions, centrifugal 20 min, get supernatant, is diluted to 1 mg/mL with 0.01 mol/L PBS; Or be diluted to 1 mg/ml with 0.01 mol/L PBS, cross 0.22 μ m filter membrane;
(3) preparation of colloid gold label thing: get colloidal gold solution 40 mL in 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 of 4 mL containing 0.3 mg antibody protein, reaction 10 min; Dropwise add 4 mL 10% BSA, continue stirring reaction 10 min; Centrifugal 10 min of gold labeling antibody solution normal temperature low speed (1500 r/min), discard the precipitation being formed by the gold grain condensing; 4 ℃ of red supernatant solution, centrifugal 20 min of 12000 r/min, abandon supernatant, and collecting precipitation is settled to 1 mL by precipitation with golden labeling antibody dilution, is prepared into Zilpaterol monoclonal antibody colloid gold label thing;
(4) preparation of collaurum film: step (3) Zilpaterol monoclonal antibody colloid gold label thing is evenly sprayed on carrier glass fibre element film with the concentration of 5 μ L/cm with drawing film instrument, room temperature is dried or 37 ℃ of oven dry 3 h naturally, makes the collaurum film containing Zilpaterol monoclonal antibody colloid gold label thing;
(5) coated film preparation: sheep anti-mouse igg antibody, Zilpaterol protein conjugate are diluted to 1 mg/mL, be sprayed on nitrocellulose filter with the concentration of 1 μ L/cm successively with drawing film instrument, be prepared into coated film, after 37 ℃ of coated 2 h, room temperature is dried or 37 ℃ of oven dry naturally;
(6) sample pad pre-treatment: sample pad treating fluid is uniformly coated on glass fibre element film, air humidity lower than 60% condition under, room temperature is dried naturally;
(7) assembling of test card: 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 certain width rectangular, then test strips is arranged in the test card shell of rectangular flat shelly.
Embodiment 3
The test of Zilpaterol minimum detectability amount
Accurately weigh 0.500 g Zilpaterol standard items and be dissolved in ultrapure water and be settled to 100 mL, be designated as A
1solution, gets A
1solution 1 mL adds ultrapure water and is settled to 1000 mL in volumetric flask, forms A
2solution, gets A
2solution 1 mL adds ultrapure water and is settled to 1000 mL in volumetric flask, forms A
3solution, the concentration of A3 solution is 5 ng/mL (5ppb).Get A
3solution 1 mL is in 4 test tubes, and difference label 1 ~ 4, in test tube, adds respectively toward 9 mL, 4 mL, 1 mL, 0 mL ultrapure water, is made into Zilpaterol standard solution concentration respectively: 0.5 ng/mL, 1 ng/mL, 2.5 ng/mL, 5 ng/mL; Separately get an empty test tube, add 5 mL ultrapure waters, be designated as test tube No. 5.Get liquid in above-mentioned 1 ~ No. 5 test tube and be added in Zilpaterol test card as sample drop, 5 min observationss after application of sample, the colour developing of detection line 2 and nature controlling line 3 the results are shown in Table 1.
The test of table 1 Zilpaterol minimum detectability amount
| Test tube numbering | Zilpaterol solution concentration (ng/mL) | Measurement result |
| 1 | 0.5 | Detection line T colour developing is slightly lighter than nature controlling line C |
| 2 | 1 | Detection line T colour developing is slightly light |
| 3 | 2.5 | Detection line T colour developing is lighter |
| 4 | 5 | Detection line T does not develop the color |
| 5 | 0 | Detection line T and nature controlling line colour developing are basic identical |
Through repeatedly check, the recall rate of Zilpaterol reaches 99%, and minimum detectability amount is 5 ng/mL.
Claims (2)
1. Zilpaterol colloidal-gold detecting-card, is characterized in that preparing according to following step:
(1) 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, and heating adds 1% trisodium citrate Na after boiling in the situation that continuing stirring
3c
6h
5o
72H
2o solution 5-7 mL, continues agitating heating, in the time that the color of solution becomes transparent aubergine completely, maintains after 5 min and stops heating, and moisturizing, to original volume, is cooled to room temperature, and 2-8 ℃ saves backup;
(2) pre-service of antibody: Zilpaterol antibody that will mark is at 1000 r/min, and under 4 ℃ of conditions, centrifugal 20 min, get supernatant, is diluted to 1 mg/mL with 0.01 mol/L PBS; Or be diluted to 1 mg/ml with 0.01 mol/L PBS, cross 0.22 μ m filter membrane;
(3) preparation of colloid gold label thing: get colloidal gold solution 40 mL in 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 of 4 mL containing 0.3 mg antibody protein, reaction 10 min; Dropwise add 4 mL 10% BSA, continue stirring reaction 10 min; Centrifugal 10 min of gold labeling antibody solution normal temperature 1500 r/min, discard the precipitation being formed by the gold grain condensing; 4 ℃ of red supernatant solution, centrifugal 20 min of 12000 r/min, abandon supernatant, and collecting precipitation is settled to 1 mL by precipitation with golden labeling antibody dilution, is prepared into Zilpaterol monoclonal antibody colloid gold label thing;
(4) preparation of collaurum film: step (3) Zilpaterol monoclonal antibody colloid gold label thing is evenly sprayed on carrier glass fibre element film with the concentration of 5 μ L/cm with drawing film instrument, room temperature is dried or 37 ℃ of oven dry 3 h naturally, makes the collaurum film containing Zilpaterol monoclonal antibody colloid gold label thing;
(5) coated film preparation: sheep anti-mouse igg antibody, Zilpaterol protein conjugate are diluted to 1 mg/mL, be sprayed on nitrocellulose filter with the concentration of 1 μ L/cm successively with drawing film instrument, be prepared into coated film, after 37 ℃ of coated 2 h, room temperature is dried or 37 ℃ of oven dry naturally;
(6) sample pad pre-treatment: sample pad treating fluid is uniformly coated on glass fibre element film, air humidity lower than 60% condition under, room temperature is dried naturally;
(7) assembling of test card: 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, then test strips is arranged in the test card shell of rectangular flat shelly.
2. Zilpaterol colloidal-gold detecting-card according to claim 1, is characterized in that detection line T coated in described step (5) is located at the below of nature controlling line C;
Sample pad treating fluid in described step (6) is by 1 g Bovine serum albumin (BSA) and 0.8 g sodium chloride (NaCl), uses the 0.01 mol/L PBS that contains 0.5%TRITON-100 to be settled to 100 mL.
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105334323A (en) * | 2014-08-07 | 2016-02-17 | 暨南大学 | Method and test strip for detecting zilpaterol, and application of test strip |
| 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 |
| CN105572365A (en) * | 2014-10-13 | 2016-05-11 | 江苏维赛科技生物发展有限公司 | Immunocolloidal gold detection card for pesticide CHI and preparation method thereof |
| CN106568947A (en) * | 2015-10-13 | 2017-04-19 | 镇江亿特生物科技发展有限公司 | Immune colloidal gold detection card of DDVP and preparation method thereof |
| CN106568946A (en) * | 2015-10-13 | 2017-04-19 | 镇江亿特生物科技发展有限公司 | immunogold detection card of propiconazole, and preparation method thereof |
| CN106568958A (en) * | 2015-10-13 | 2017-04-19 | 丹阳亿太生物科技发展有限公司 | Immunocolloidal-gold detection cassette for trifuralin and preparation method thereof |
| CN106568960A (en) * | 2015-10-13 | 2017-04-19 | 丹阳亿太生物科技发展有限公司 | Immunocolloidal gold test card of methomyl and preparation method thereof |
| 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 |
| CN109085353A (en) * | 2018-06-13 | 2018-12-25 | 比杭生物科技（杭州）有限公司 | A kind of Candida albicans colloidal-gold detecting-card and its application |
| CN114184782A (en) * | 2021-12-13 | 2022-03-15 | 深圳容金科技有限公司 | Colloidal gold detection kit and detection method for zilpaterol |
| CN115112888A (en) * | 2022-08-25 | 2022-09-27 | 山东博科快速检测技术有限公司 | Preparation method of novel coronavirus antigen detection reagent card |
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| CN105334323A (en) * | 2014-08-07 | 2016-02-17 | 暨南大学 | Method and test strip for detecting zilpaterol, and application of test strip |
| CN105572365A (en) * | 2014-10-13 | 2016-05-11 | 江苏维赛科技生物发展有限公司 | Immunocolloidal gold detection card for pesticide CHI and preparation method thereof |
| 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 |
| CN106568947A (en) * | 2015-10-13 | 2017-04-19 | 镇江亿特生物科技发展有限公司 | Immune colloidal gold detection card of DDVP and preparation method thereof |
| CN106568946A (en) * | 2015-10-13 | 2017-04-19 | 镇江亿特生物科技发展有限公司 | immunogold detection card of propiconazole, and preparation method thereof |
| CN106568958A (en) * | 2015-10-13 | 2017-04-19 | 丹阳亿太生物科技发展有限公司 | Immunocolloidal-gold detection cassette for trifuralin and preparation method thereof |
| CN106568960A (en) * | 2015-10-13 | 2017-04-19 | 丹阳亿太生物科技发展有限公司 | Immunocolloidal gold test card of methomyl and preparation method thereof |
| 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 |
| CN109085353A (en) * | 2018-06-13 | 2018-12-25 | 比杭生物科技（杭州）有限公司 | A kind of Candida albicans colloidal-gold detecting-card and its application |
| CN114184782A (en) * | 2021-12-13 | 2022-03-15 | 深圳容金科技有限公司 | Colloidal gold detection kit and detection method for zilpaterol |
| CN115112888A (en) * | 2022-08-25 | 2022-09-27 | 山东博科快速检测技术有限公司 | Preparation method of novel coronavirus antigen detection reagent card |
| CN115112888B (en) * | 2022-08-25 | 2022-12-13 | 山东博科快速检测技术有限公司 | Preparation method of novel coronavirus antigen detection reagent card |
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Application publication date: 20140521 |