CN105572372A - Immunity colloid gold detecting card of imidacloprid and preparing method of immunity colloid gold detecting card - Google Patents

Immunity colloid gold detecting card of imidacloprid and preparing method of immunity colloid gold detecting card Download PDF

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Publication number
CN105572372A
CN105572372A CN201410551751.2A CN201410551751A CN105572372A CN 105572372 A CN105572372 A CN 105572372A CN 201410551751 A CN201410551751 A CN 201410551751A CN 105572372 A CN105572372 A CN 105572372A
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China
Prior art keywords
imidacloprid
gold
colloid gold
solution
antibody
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Pending
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CN201410551751.2A
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Chinese (zh)
Inventor
洪霞
杜霞
刘静
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Nanjing Yite Biological Technology Co Ltd
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Nanjing Yite Biological Technology Co Ltd
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Priority to CN201410551751.2A priority Critical patent/CN105572372A/en
Publication of CN105572372A publication Critical patent/CN105572372A/en
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Abstract

The invention discloses an immunity colloid gold detecting card of imidacloprid and a preparing method of the immunity colloid gold detecting card, and relates to the technical field of animal-derived food residue of veterinary drug detection. A test strip in a shell of the detecting card is composed of a PVC rubber slab, a sample cushion, a colloid gold combination cushion, a coating membrane and a water absorbing cushion. A colloid gold membrane is a glass cellulose membrane containing an imidacloprid monoclonal antibody. The coating membrane is a nitrocellulose membrane and is provided with T lines and C lines. The T lines are coated with imidacloprid protein conjugates, and the C lines are coated with a goat anti-mouse IgG antibody. The immunity colloid gold detecting card is effectively used for quickly detecting imidacloprid, convenience and fastness are achieved, and the result is accurate.

Description

Immune colloid gold test card of Imidacloprid and preparation method thereof
Technical field
The present invention relates to cereal foods Pesticide Residues detection technique field, immune colloid gold test card particularly relating to Imidacloprid and preparation method thereof.
Background technology
Imidacloprid (imidacloprid), chemical name 1-(6-chloro-3-pyridyl ylmethyl)-N-Nitroimidazoline-2-base amine.Imidacloprid has broad-spectrum insecticidal activity, has special efficacy, also have good prevention effect to the insect of other kinds to suckings pest such as plant hopper, aphid, leafhopper.Imidacloprid is agricultural chemicals conventional in growing vegetables process, and detecting Determination of Imidacloprid Residue becomes the necessary means of country to the monitoring of vegetable plants Pesticide Residues.Japan's regulation maximum residue limit(MRL) of Imidacloprid in vegetables (MRL) is 0.5mg/kg.
Prior art is for the detection mainly detection method such as high performance liquid chromatography of Imidacloprid, 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 Imidacloprid is provided and preparation method thereof, effectively can solve the problem that can detect Imidacloprid fast, easily.
Imidacloprid colloidal-gold detecting-card of the present invention, comprise and being formed by the nitrocellulose filter of Imidacloprid-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 Imidacloprid 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 Imidacloprid 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 Imidacloprid monoclonal antibody colloid gold label thing;
(4) preparation of colloidal gold film: step (3) Imidacloprid 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 Imidacloprid protein conjugate monoclonal antibody colloid gold label thing;
(5) coated film preparation: sheep anti-mouse igg antibody, Imidacloprid 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 Imidacloprid, 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 Imidacloprid 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 Imidacloprid 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 Imidacloprid-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 Imidacloprid-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 Imidacloprid 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 Imidacloprid 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 Imidacloprid monoclonal antibody colloid gold label thing;
The preparation of colloidal gold film: step (3) Imidacloprid 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 Imidacloprid protein conjugate monoclonal antibody colloid gold label thing;
Prepared by coated film: sheep anti-mouse igg antibody, Imidacloprid 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. Imidacloprid 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 Imidacloprid 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 Imidacloprid monoclonal antibody colloid gold label thing;
(4) preparation of colloidal gold film: step (3) Imidacloprid 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 Imidacloprid protein conjugate monoclonal antibody colloid gold label thing;
(5) coated film preparation: sheep anti-mouse igg antibody, Imidacloprid 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. Imidacloprid 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.
CN201410551751.2A 2014-10-17 2014-10-17 Immunity colloid gold detecting card of imidacloprid and preparing method of immunity colloid gold detecting card Pending CN105572372A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
CN108226486A (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 Madumycin 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

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
CN103018467A (en) * 2012-09-27 2013-04-03 江苏维赛科技生物发展有限公司 Colloidal gold immunochromatographic card for detecting bisphenol A (BPA) and preparation method thereof
CN103048455A (en) * 2012-09-27 2013-04-17 江苏维赛科技生物发展有限公司 Herbicide 2, 4-D and pesticide CHI bigeminy detection card and preparation method thereof
CN103439487A (en) * 2013-09-18 2013-12-11 南京农业大学 Simple pesticide three-channel semi-quantitative detection gold-labeled test strip sensor

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
CN103018467A (en) * 2012-09-27 2013-04-03 江苏维赛科技生物发展有限公司 Colloidal gold immunochromatographic card for detecting bisphenol A (BPA) and preparation method thereof
CN103048455A (en) * 2012-09-27 2013-04-17 江苏维赛科技生物发展有限公司 Herbicide 2, 4-D and pesticide CHI bigeminy detection card and preparation method thereof
CN103439487A (en) * 2013-09-18 2013-12-11 南京农业大学 Simple pesticide three-channel semi-quantitative detection gold-labeled test strip sensor

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
CN108226486A (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 Madumycin 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

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Application publication date: 20160511