CN105510587A - Neomycin immuno-colloidal gold detection card and preparation method thereof - Google Patents
Neomycin immuno-colloidal gold detection card and preparation method thereof Download PDFInfo
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- CN105510587A CN105510587A CN201410547766.1A CN201410547766A CN105510587A CN 105510587 A CN105510587 A CN 105510587A CN 201410547766 A CN201410547766 A CN 201410547766A CN 105510587 A CN105510587 A CN 105510587A
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- neomycin
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Abstract
The invention discloses a neomycin immuno-colloidal gold detection card and a preparation method thereof, and relates to the technical field of beta-adrenergic receptor agonist detection. The test strip on the shell of the detection card is composed of a PVC rubber sheet, a sample pad, a colloidal gold combined pad, a coating membrane, and a water absorbing pad; the colloidal gold membrane is a glass cellulose membrane containing a neomycin monoclonal antibody; the coating membrane is a nitrocellulose membrane and is provided with a T line and C line, neomycin protein conjugate is coated in the T line, and goat anti mouse IgG antibody is coated in the C line. The provided detection card is used to detect neomycin in a short time, and has the advantages of convenience, quickness, and precise results.
Description
Technical field
The present invention relates to animal-derived food microbiotic detection technique field, immune colloid gold test card particularly relating to neomycin and preparation method thereof.
Background technology
Neomycin is a kind of broad-spectrum antibiotic, is usually used in the treatment of the various infectious disease of animal, has stronger inhibiting effect for multiple pathogen, to Gram-positive and negative bacterium all effective.There is serious spinoff in neomycin, has renal toxicity and interior ototoxicity, the injury of its Ear, irreversible often, and the health of the Detection of neomycin residues therefore in animal foodstuff to the mankind forms grave danger.The content detecting neomycin in animal-derived food seems more direct, convenient, and can realize killing front detection, and for getting rid of food security hidden danger in time, ensure the quality safety of food, JP opens up this Test Research.
Prior art is for the detection mainly detection method such as high performance liquid chromatography-tandem mass method, high performance liquid chromatography of neomycin, but the defect of these technology is obvious: apparatus expensive, complicated operation, be difficult to promote.So it is extremely necessary for setting up a kind of assay method that is simple, effective, that be applicable to basic unit's use.The object of the invention is a kind of enterprise that is more suitable for of development 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 in order to overcome prior art exist defect and immune colloid gold test card that a kind of neomycin is provided and preparation method thereof, effectively can solve the problem that can detect receptor,β activator neomycin fast, easily.
Neomycin colloidal-gold detecting-card of the present invention, comprise and being formed by the nitrocellulose filter of neomycin-BSA and sheep anti-mouse igg, sample pad, adsorptive pads, PVC offset plate and mould of plastics by the gold conjugation pad of monoclonal antibody colloid gold label thing, bag, sample pad, pad is adhered to successively in one end of PVC offset plate, nitrocellulose filter is pasted in centre, and the other end adheres to adsorptive pads.
The preparation method of neomycin colloidal-gold detecting-card of the present invention is realized by following steps:
(1) colloidal gold solution preparation: get 1L Erlenmeyer flask 1, add ultrapure water 495mL, then add 1% gold chloride (HAuCl
43H
2o) 5mL, is mixed with 500mL0.01% aqueous solution of chloraurate, and heating adds 1% trisodium citrate (Na after boiling when Keep agitation
3c
6h
5o
72H
2o) solution 5-7mL, continues agitating heating, and when the color of solution becomes transparent aubergine completely, stop heating after maintaining 5min, moisturizing, to original volume, is cooled to room temperature, and 2-8 DEG C saves backup;
(2) pre-service of antibody: the neomycin antibody that will mark is at 1000r/min, and under 4 DEG C of conditions, centrifugal 20min, gets supernatant, is diluted to 1mg/mL with 0.01mol/LPBS; Or be diluted to 1mg/ml with 0.01mol/LPBS, cross 0.22 μm of filter membrane;
(3) preparation of colloid gold label thing: get the colloidal gold solution 40mL in step (1), colloidal gold solution pH to 8.5 is regulated with 0.25mol/LK2CO3, magnetic stirrer 250r/min stirs, and dropwise adds the protein solution of 4mL containing 0.3mg antibody protein, reaction 10min; Dropwise add 4mL10%BSA, continue stirring reaction 10min; Gold labeling antibody solution normal temperature low speed (1500r/min) centrifugal 10min, discards the precipitation formed by the gold grain condensed; Red supernatant solution 4 DEG C, the centrifugal 20min of 12000r/min, abandons supernatant, collecting precipitation, and the golden labeling antibody dilution of precipitation is settled to 1mL, is prepared into neomycin monoclonal antibody colloid gold label thing;
(4) preparation of colloidal gold film: step (3) neomycin protein conjugate monoclonal antibody colloid gold label thing is sprayed on carrier glass cellulose membrane with drawing film instrument with the even concentration of 5 μ L/cm, room temperature is dried or 37 DEG C of oven dry 3h naturally, makes the colloidal gold film containing neomycin protein conjugate monoclonal antibody colloid gold label thing;
(5) coated film preparation: sheep anti-mouse igg antibody, neomycin protein conjugate are diluted to 1mg/mL, be sprayed on nitrocellulose filter with the concentration of 1 μ L/cm successively with drawing film instrument, be prepared into coated film, 37 DEG C of bags are by after 2h, and room temperature is dried or 37 DEG C of oven dry naturally;
(6) sample pad pre-treatment: be uniformly coated on by sample pad treating fluid on glass fibre element film, under the condition of air humidity lower than 60%, room temperature is dried naturally;
(7) assembling of test card: PVC offset plate pastes sample pad, colloidal gold film, coated film and absorbent wool from top to bottom successively, is assembled into test strips, cuts into the rectangular of one fixed width, then test strips is arranged in the test card shell of rectangular flat shelly.
In described step (5), the detection line T of institute's bag quilt is located at the below of nature controlling line C;
Sample pad treating fluid in described step (6) is by 1g Bovine serum albumin (BSA) and 0.8g sodium chloride (NaCl), is settled to 100mL with the 0.01mol/LPBS containing 0.5%TRITON-100;
The present invention can be effective to measure receptor,β activator neomycin, and method is simple, and convenient, fast, result is accurate.
Accompanying drawing explanation
Fig. 1 is the structural drawing of the immune colloid gold test card of neomycin of the present invention: 1. well 2. detection line 3. nature controlling line 4. detect aperture 5. test strips 6. test card shells in figure.
Fig. 2 is the sectional structure chart of the test strips in the immune colloid gold test card of neomycin of the present invention, 7. sample pad 8. gold conjugation pad 9.PVC offset plate 10. nitrocellulose filter 11. adsorptive pads in figure.
Embodiment
Embodiment 1
Embodiment shown in Fig. 1, Fig. 2: in figure, 9 is PVC offset plate; 7 is sample pad; 8 is gold conjugation pad, and on this gold conjugation pad, bag is by monoclonal antibody colloid gold label thing; 10 is coated film, i.e. nitrocellulose filter, and on this nitrocellulose filter, bag is by neomycin-BSA and sheep anti-mouse igg; 11 is adsorptive pads, by absorbent material as filter paper is made.
On one end of PVC offset plate 9, (sample end) adheres to sample pad 7, pad 8, and sample pad 7 and pad 8 are side by side configuration.
At the intermediate adhesion nitrocellulose filter 10 of PVC offset plate 9.Nitrocellulose filter 10 is provided with sheep anti-mouse igg nature controlling line 3 and neomycin-BSA detection line 2.
Adsorptive pads 11 is adhered at the other end of PVC offset plate 9.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 make test card, test card shell 6 covers and is provided with well 1 and detect aperture 4, sample pad 7 is just to well 1, and nitrocellulose filter 10 is just to detect aperture 4.
Embodiment 2
Prepared by neomycin colloidal-gold detecting-card, be by following steps specific implementation:
Prepared by colloidal gold solution: get 1L Erlenmeyer flask 1, add ultrapure water 495mL, then add 1% gold chloride (HAuCl
43H
2o) 5mL, is mixed with 500mL0.01% aqueous solution of chloraurate, and heating adds 1% trisodium citrate (Na after boiling when Keep agitation
3c
6h
5o
72H
2o) solution 5-7mL, continues agitating heating, and when the color of solution becomes transparent aubergine completely, stop heating after maintaining 5min, moisturizing, to original volume, is cooled to room temperature, and 2-8 DEG C saves backup;
The pre-service of antibody: the neomycin antibody that will mark is at 1000r/min, and under 4 DEG C of conditions, centrifugal 20min, gets supernatant, is diluted to 1mg/mL with 0.01mol/LPBS; Or be diluted to 1mg/ml with 0.01mol/LPBS, cross 0.22 μm of filter membrane;
The preparation of colloid gold label thing: get the colloidal gold solution 40mL in step (1), colloidal gold solution pH to 8.5 is regulated with 0.25mol/LK2CO3, magnetic stirrer 250r/min stirs, and dropwise adds the protein solution of 4mL containing 0.3mg antibody protein, reaction 10min; Dropwise add 4mL10%BSA, continue stirring reaction 10min; Gold labeling antibody solution normal temperature low speed (1500r/min) centrifugal 10min, discards the precipitation formed by the gold grain condensed; Red supernatant solution 4 DEG C, the centrifugal 20min of 12000r/min, abandons supernatant, collecting precipitation, and the golden labeling antibody dilution of precipitation is settled to 1mL, is prepared into neomycin monoclonal antibody colloid gold label thing;
The preparation of colloidal gold film: step (3) neomycin protein conjugate monoclonal antibody colloid gold label thing is sprayed on carrier glass cellulose membrane with drawing film instrument with the even concentration of 5 μ L/cm, room temperature is dried or 37 DEG C of oven dry 3h naturally, makes the colloidal gold film containing neomycin protein conjugate monoclonal antibody colloid gold label thing;
Prepared by coated film: sheep anti-mouse igg antibody, neomycin protein conjugate are diluted to 1mg/mL, be sprayed on nitrocellulose filter with the concentration of 1 μ L/cm successively with drawing film instrument, be prepared into coated film, 37 DEG C of bags are by after 2h, and room temperature is dried or 37 DEG C of oven dry naturally;
Sample pad pre-treatment: be uniformly coated on by sample pad treating fluid on glass fibre element film, under the condition of air humidity lower than 60%, room temperature is dried naturally;
The assembling of test card: PVC offset plate pastes sample pad, colloidal gold film, coated film and absorbent wool from top to bottom successively, is assembled into test strips, cuts into one fixed width rectangular, then test strips is arranged in the test card shell of rectangular flat shelly.
Embodiment 3
Neomycin minimum detectability amount is tested
Precise 0.500g neomycin antigen is dissolved in ultrapure water and is settled to 100mL, is designated as A
1solution, gets A
1solution 1mL adds ultrapure water in volumetric flask and is settled to 1000mL, forms A
2solution, gets A
2solution 1mL adds ultrapure water in volumetric flask and is settled to 1000mL, forms A
3solution, then the concentration of A3 solution is 5ng/mL (5ppb).Get A
3solution 1mL is in 4 test tubes, and difference label 1 ~ 4, add respectively in test tube toward 9mL, 4mL, 1mL, 0mL ultrapure water, be made into neomycin antigen solution concentration respectively: 0.5ng/mL, 1ng/mL, 2.5ng/mL, 5ng/mL; Separately get an empty test tube, add 5mL ultrapure water, be designated as No. 5 test tubes.Getting liquid in above-mentioned 1 ~ No. 5 test tube is added in neomycin test card as sample drop, and 5min observations after application of sample, the colour developing of detection line 2 and nature controlling line 3 the results are shown in Table 1.
Table 1 neomycin minimum detectability amount is tested
Through repeatedly checking, the recall rate of neomycin reaches 99%, and minimum detectability amount is 5ng/mL.
Claims (2)
1. neomycin colloidal-gold detecting-card, is characterized in that preparing according to following step:
(1) colloidal gold solution preparation: get 1L Erlenmeyer flask 1, add ultrapure water 495mL, then add 1% gold chloride HAuCl
43H
2o) 5mL, is mixed with 500mL0.01% aqueous solution of chloraurate, and heating adds 1% trisodium citrate Na after boiling when Keep agitation
3c
6h
5o
72H
2o solution 5-7mL, continues agitating heating, and when the color of solution becomes transparent aubergine completely, stop heating after maintaining 5min, moisturizing, to original volume, is cooled to room temperature, and 2-8 DEG C saves backup;
(2) pre-service of antibody: the neomycin antibody that will mark is at 1000r/min, and under 4 DEG C of conditions, centrifugal 20min, gets supernatant, is diluted to 1mg/mL with 0.01mol/LPBS; Or be diluted to 1mg/ml with 0.01mol/LPBS, cross 0.22 μm of filter membrane;
(3) preparation of colloid gold label thing: get the colloidal gold solution 40mL in step (1), use 0.25mol/LK
2cO
3regulate colloidal gold solution pH to 8.5, magnetic stirrer 250r/min stirs, and dropwise adds the protein solution of 4mL containing 0.3mg antibody protein, reaction 10min; Dropwise add 4mL10%BSA, continue stirring reaction 10min; The centrifugal 10min of gold labeling antibody solution normal temperature 1500r/min, discards the precipitation formed by the gold grain condensed; Red supernatant solution 4 DEG C, the centrifugal 20min of 12000r/min, abandons supernatant, collecting precipitation, and the golden labeling antibody dilution of precipitation is settled to 1mL, is prepared into neomycin monoclonal antibody colloid gold label thing;
(4) preparation of colloidal gold film: step (3) neomycin protein conjugate monoclonal antibody colloid gold label thing is sprayed on carrier glass cellulose membrane with drawing film instrument with the even concentration of 5 μ L/cm, room temperature is dried or 37 DEG C of oven dry 3h naturally, makes the colloidal gold film containing neomycin protein conjugate monoclonal antibody colloid gold label thing;
(5) coated film preparation: sheep anti-mouse igg antibody, neomycin protein conjugate are diluted to 1mg/mL, be sprayed on nitrocellulose filter with the concentration of 1 μ L/cm successively with drawing film instrument, be prepared into coated film, 37 DEG C of bags are by after 2h, and room temperature is dried or 37 DEG C of oven dry naturally;
(6) sample pad pre-treatment: be uniformly coated on by sample pad treating fluid on glass fibre element film, under the condition of air humidity lower than 60%, room temperature is dried naturally;
(7) assembling of test card: PVC offset plate pastes sample pad, colloidal gold film, coated film and absorbent wool from top to bottom successively, is assembled into test strips, cuts into the rectangular of one fixed width, then test strips is arranged in the test card shell of rectangular flat shelly.
2. neomycin colloidal-gold detecting-card according to claim 1, is characterized in that the detection line T of institute's bag quilt in described step (5) is located at the below of nature controlling line C;
Sample pad treating fluid in described step (6) is by 1g Bovine serum albumin (BSA) and 0.8g sodium chloride (NaCl), is settled to 100mL with the 0.01mol/LPBS containing 0.5%TRITON-100.
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| CN201410547766.1A CN105510587A (en) | 2014-10-16 | 2014-10-16 | Neomycin immuno-colloidal gold detection card and preparation method thereof |
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| CN105510587A true CN105510587A (en) | 2016-04-20 |
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Cited By (9)
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| CN106526190A (en) * | 2016-11-23 | 2017-03-22 | 百奥森（江苏）食品安全科技有限公司 | Detection card for detecting neomycin in feed |
| CN108226500A (en) * | 2016-12-15 | 2018-06-29 | 江苏维赛科技生物发展有限公司 | The immune colloid gold detection blocking of fluoroquinolones Ciprofloxacin is standby |
| 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 |
| CN108226503A (en) * | 2016-12-15 | 2018-06-29 | 江苏维赛科技生物发展有限公司 | Immune colloid gold detection card of aflatoxin G 1 and preparation method thereof |
| CN108226502A (en) * | 2016-12-15 | 2018-06-29 | 江苏维赛科技生物发展有限公司 | Immune colloid gold detection card of Enrofloxacin and preparation method thereof |
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| CN108226500A (en) * | 2016-12-15 | 2018-06-29 | 江苏维赛科技生物发展有限公司 | The immune colloid gold detection blocking of fluoroquinolones Ciprofloxacin is standby |
| 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 |
| CN108226503A (en) * | 2016-12-15 | 2018-06-29 | 江苏维赛科技生物发展有限公司 | Immune colloid gold detection card of aflatoxin G 1 and preparation method thereof |
| CN108226502A (en) * | 2016-12-15 | 2018-06-29 | 江苏维赛科技生物发展有限公司 | Immune colloid gold detection card of Enrofloxacin and preparation method thereof |
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| CN108226501A (en) * | 2016-12-15 | 2018-06-29 | 江苏维赛科技生物发展有限公司 | Immune colloid gold detection card of aflatoxin B1 and preparation method thereof |
| CN109813901A (en) * | 2017-11-21 | 2019-05-28 | 南京亿特生物科技有限公司 | A kind of immune colloid gold detection card and preparation method thereof detecting ethiprole |
| CN109596604A (en) * | 2018-12-24 | 2019-04-09 | 东北师范大学 | A kind of adjustable chemiluminescence Fiber imunosensor of the range of linearity and application |
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