CN105572360A - Immune colloidal gold detection card of pretilachlor, and making method thereof - Google Patents
Immune colloidal gold detection card of pretilachlor, and making method thereof Download PDFInfo
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- CN105572360A CN105572360A CN201410534011.8A CN201410534011A CN105572360A CN 105572360 A CN105572360 A CN 105572360A CN 201410534011 A CN201410534011 A CN 201410534011A CN 105572360 A CN105572360 A CN 105572360A
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- gold
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- colloidal gold
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Abstract
The invention relates to an immune colloidal gold detection card of pretilachlor, and a making method thereof, and relates to the technical field of detection of veterinary drug residual of animal-derived foods. A test strip in the shell of the detection card is composed of a PVC rubber sheet, a sample pad, a colloidal gold binding pad, a coated film and an absorbent pad; and a colloidal gold film is a glass cellulose film containing a pretilachlor monoclonal antibody, the coated film is a cellulose nitrate film, the coated film is provided with a T line and a C line, the T line is coated with a pretilachlor protein conjugate, and the C line is coated with a sheep anti-mouse IgG antibody. The immune colloidal gold detection card is effectively used for rapidly detecting pretilachlor, and has the advantages of convenience, fastness and accurate result.
Description
Technical field
The present invention relates to cereal foods Pesticide Residues detection technique field, particularly relate to immune colloid gold test card of the third careless amine and preparation method thereof.
Background technology
Third careless amine is the rice terrace herbicides special with high-temperature selective.To rice safety, broad weed-killing spectrum.Weed seed absorbs medicament in germination process, and root absorption is poor.These product are preemergence herbicide, are mainly used in preventing and kill off grassy weed.Product belongs to 2-acetyl chloride for phenyl amines herbicide, is cell division inhibitor, can prevents and kill off barnyard grass in paddy field, difformed galingale herb, needle spikesedge herb, Monochoria vaginalis, alisma canaliculatum etc. for soil treatment.Have certain toxicity, therefore safety problem is paid much attention to.Specify to be 0.1mg/kg in rice in GB.Therefore regular the necessary means detecting the monitoring becoming many countries is remained to the third careless amine.
Prior art is for the detection mainly detection method such as gas chromatography-mass spectrography, high performance liquid chromatography of the third careless amine, 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 of a kind of third careless amine and preparation method thereof is provided, effectively can solve the problem that can detect the third careless amine fast, easily.
Third careless amine colloidal-gold detecting-card of the present invention, comprise and being formed by the nitrocellulose filter of the third careless amine-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 the third careless amine 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 will mark third careless amine antibody 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 the third careless amine monoclonal antibody colloid gold label thing;
(4) preparation of colloidal gold film: careless for step (3) third amine 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 the third careless amine protein conjugate monoclonal antibody colloid gold label thing;
(5) coated film preparation: sheep anti-mouse igg antibody, the third careless amine 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 the careless amine of mensuration third, 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 the careless amine of the present invention third: 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 the careless amine of the present invention third, 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 the third careless amine-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 the third careless amine-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
Third careless amine colloidal-gold detecting-card preparation is 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 will mark third careless amine antibody 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 the third careless amine monoclonal antibody colloid gold label thing;
The preparation of colloidal gold film: careless for step (3) third amine 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 the third careless amine protein conjugate monoclonal antibody colloid gold label thing;
Prepared by coated film: sheep anti-mouse igg antibody, the third careless amine 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.
Claims (2)
1. the third careless amine 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 will mark third careless amine antibody 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 the third careless amine monoclonal antibody colloid gold label thing;
(4) preparation of colloidal gold film: careless for step (3) third amine 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 the third careless amine protein conjugate monoclonal antibody colloid gold label thing;
(5) coated film preparation: sheep anti-mouse igg antibody, the third careless amine 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. the third careless amine 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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410534011.8A CN105572360A (en) | 2014-10-12 | 2014-10-12 | Immune colloidal gold detection card of pretilachlor, and making method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410534011.8A CN105572360A (en) | 2014-10-12 | 2014-10-12 | Immune colloidal gold detection card of pretilachlor, and making method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105572360A true CN105572360A (en) | 2016-05-11 |
Family
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410534011.8A Withdrawn CN105572360A (en) | 2014-10-12 | 2014-10-12 | Immune colloidal gold detection card of pretilachlor, and making method thereof |
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|---|---|
| CN (1) | CN105572360A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105572366A (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 pretilachlor 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 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN2869867Y (en) * | 2005-12-06 | 2007-02-14 | 万积成 | Colloid gold test-paper for quick detecting propisochlor and metolachlor residue |
| CN102901819A (en) * | 2012-09-27 | 2013-01-30 | 江苏维赛科技生物发展有限公司 | Immune colloidal gold detection card for phenylethanolamine A and preparation method thereof |
-
2014
- 2014-10-12 CN CN201410534011.8A patent/CN105572360A/en not_active Withdrawn
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN2869867Y (en) * | 2005-12-06 | 2007-02-14 | 万积成 | Colloid gold test-paper for quick detecting propisochlor and metolachlor residue |
| CN102901819A (en) * | 2012-09-27 | 2013-01-30 | 江苏维赛科技生物发展有限公司 | Immune colloidal gold detection card for phenylethanolamine A and preparation method thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105572366A (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 pretilachlor 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 |
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Application publication date: 20160511 |