CN106568960A - Immunocolloidal gold test card of methomyl and preparation method thereof - Google Patents
Immunocolloidal gold test card of methomyl and preparation method thereof Download PDFInfo
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- CN106568960A CN106568960A CN201510656071.1A CN201510656071A CN106568960A CN 106568960 A CN106568960 A CN 106568960A CN 201510656071 A CN201510656071 A CN 201510656071A CN 106568960 A CN106568960 A CN 106568960A
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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Abstract
The invention discloses an immunocolloidal gold test card of methomyl and a preparation method thereof, and relates to the technical field of veterinary drug residue detection of animal-derived food. A test strip in the shell of the test card is composed of a PVC rubber sheet, a sample pat, a colloidal gold conjugate pad, a coating membrane and an absorbent pad. The colloidal gold membrane is a glassfiber membrane containing a methomyl monoclonal antibody. The coating membrane is a nitrocellulose membrane, on which a T line and a C line are arranged. The T line is coated with a methomyl protein conjugate, and the C line is coated with a goat anti-mouse IgG antibody. The test card of the invention is effectively used for rapid detection of methomyl, is convenient and fast, and has accurate results.
Description
Technical field
The present invention relates to immune colloid gold detection card of cereal foods Pesticide Residues detection technique field, more particularly to Methomyl and preparation method thereof.
Background technology
Methomyl is a kind of broad spectrum activity carbamate insecticidess, with characteristics such as high volatility, toxicity on inhalation height, is mainly used in preventing and treating the class pests such as striped rice borer, plant hopper, Prodenia litura.Active compound is white crystalline powder, and commodity state is wettable powder, soluble powder or cream.Methomyl be a kind of absorbability have tag, the broad spectrum pesticide of the carbamatess of stomach poison function.Recommend as parasite killing, nematicide at first.Suitable for Cotton Gossypii, Nicotiana tabacum L., fruit tree, vegetable it is anti-eliminate aphis, moth, cutworm etc..Therefore periodically methomyl residue is carried out detecting the necessary means of the monitoring for becoming many countries.
Prior art is mainly the detection methods such as gas chromatography-mass spectrography, high performance liquid chromatography for the detection of Methomyl, but the defect of these technologies is obvious：Apparatus expensive, complex operation, it is difficult to promote.So, set up it is a kind of it is simple, effectively, be adapted to basic unit using assay method be extremely necessary.The purpose of the present invention is to develop a kind of enterprise that is more suitable for carry out Site Detection and fast and simple, with low cost qualitative checking method.
The content of the invention
For case above, the purpose of the present invention is exactly to overcome the prior art defect for existing and immune colloid gold detection card for providing a kind of Methomyl and preparation method thereof, can effectively solving can quick, easily detect the problem of Methomyl.
The Methomyl colloidal-gold detecting-card of the present invention, constitute including the gold conjugation pad, the nitrocellulose filter, sample pad, adsorptive pads, PVC offset plates and the mould of plastics that have been coated with Methomyl-BSA and sheep anti-mouse igg for being coated with monoclonal antibody colloid gold label thing, sample pad, pad are adhered to successively in one end of PVC offset plates, paste nitrocellulose filter, other end adhesion adsorptive pads in centre.
The preparation method of the Methomyl colloidal-gold detecting-card of the present invention, is realized by following steps：
(1) prepared by colloidal gold solution：1 L conical flasks 1 are taken, 495 mL of ultra-pure water is added, is then added 1% gold chloride (HAuCl4·3H2O) 5 mL, is configured to 500 mL, 0.01% aqueous solution of chloraurate, adds 1% trisodium citrate (Na in the case where persistently stirring after heated and boiled3C6H5O7·2H2O) solution 5-7 mL, continue agitating heating, when the color of solution is changed into transparent aubergine completely, stop heating after maintaining 5 min, and moisturizing is cooled to room temperature to original volume, and 2-8 DEG C saves backup；
(2) pretreatment of antibody：Will labelling Methomyl antibody in 1000 r/min, under the conditions of 4 DEG C, be centrifuged 20 min, take supernatant, be diluted to 1 mg/mL with 0.01 mol/L PBS；Or 1 mg/ml is diluted to 0.01 mol/L PBS, cross 0.22 m filter membranes；
(3) preparation of colloid gold label thing：40 mL of colloidal gold solution in step (1) being taken, colloidal gold solution pH to 8.5 being adjusted with 0.25 mol/L K2CO3,250 r/min of magnetic stirrer stirrings are added dropwise over 4 mL
Protein solution containing 0.3 mg antibody proteins, reacts 10 min；It is added dropwise over 4 mL
10% BSA, continues 10 min of stirring reaction；Gold labeling antibody solution room temperature low speed（1500 r/min）10 min are centrifuged, the precipitation formed by the gold grain for condensing is discarded；4 DEG C of red supernatant solution, 12000 r/min are centrifuged 20 min, abandon supernatant, collect precipitation, precipitation gold labeling antibody diluent is settled to 1 mL, Methomyl monoclonal antibody colloid gold label thing is prepared into；
(4) preparation of colloidal gold film：Step (3) Methomyl protein conjugate monoclonal antibody colloid gold label thing is sprayed on carrier glass cellulose membrane with the even concentration of 5 L/cm with film instrument is drawn, room temperature is dried or 37 DEG C of 3 h of drying naturally, makes the colloidal gold film of the monoclonal antibody colloid gold label thing of protein conjugate containing Methomyl；
(5) it is coated with film preparation：Sheep anti-mouse igg antibody, Methomyl protein conjugate are diluted to into 1 mg/mL, are sprayed on nitrocellulose filter with the concentration of 1 L/cm with film instrument is drawn successively, be prepared into coated film, after 37 DEG C of 2 h of coating, room temperature is dried or 37 DEG C of drying naturally；
(6) sample pad pre-treatment：Sample pad treatment fluid is uniformly coated on glass fibre element film, under conditions of air humidity is less than 60%, room temperature is dried naturally；
(7) assembling of detection card：PVC offset plates paste sample pad, colloidal gold film, coated film and absorbent cotton from top to bottom successively, are assembled into test strips, cut into the strip of one fixed width, then test strips are arranged in the detection card shell of the flat shelly of strip.
In the step (5) coated detection line T be located at the lower section of nature controlling line C；
Sample pad treatment fluid in the step (6) is, by 1 g bovin serum albumin (BSA) and 0.8 g Sodium Chloride (NaCl), to be settled to 100 mL with the 0.01 mol/L PBS containing 0.5%TRITON-100；
The present invention can be effectively used for determining Methomyl, and method is simple, convenient, fast, as a result accurately.
Description of the drawings
Fig. 1 is the structure chart of the immune colloid gold detection card of Methomyl of the present invention：The detection of 1. well, 2. detection line, 3. nature controlling line 4. 5. test strips of hole 6. detection card shell in figure.
Fig. 2 be Methomyl of the present invention immune colloid gold detection card in test strips sectional structure chart, 7. sample pad, 8. 10. nitrocellulose filter of gold conjugation pad 9.PVC offset plates, 11. adsorptive pads in figure.
Specific embodiment
Embodiment 1
Embodiment shown in Fig. 1, Fig. 2：In figure, 9 is PVC offset plates；7 is sample pad；8 is gold conjugation pad, and monoclonal antibody colloid gold label thing has been coated with the gold conjugation pad；10 is coated film, i.e. nitrocellulose filter, and Methomyl-BSA and sheep anti-mouse igg have been coated with the nitrocellulose filter；11 is adsorptive pads, is made up of absorbent material such as filter paper.
(sample end) adhesion sample pad 7, pad 8 on one end of PVC offset plates 9, sample pad 7 and pad 8 are side by side configuration.
In the intermediate adhesion nitrocellulose filter 10 of PVC offset plates 9.Sheep anti-mouse igg nature controlling line 3 and Methomyl-BSA detection lines 2 are provided with nitrocellulose filter 10.
In the other end adhesion adsorptive pads 11 of PVC offset plates 9.One end of nitrocellulose filter 10 is slightly intersected with pad 8, and the other end is slightly intersected with adsorptive pads 11.The test strips 5 can be incorporated with detecting card shell 6 made by mould of plastics, make detection card, and well 1 and detection hole 4 are provided with the upper lid of detection card shell 6, and, just to well 1, nitrocellulose filter 10 is just to detecting hole 4 for sample pad 7.
Embodiment 2
Prepared by Methomyl colloidal-gold detecting-card, implemented by following steps：
It is prepared by colloidal gold solution：1 L conical flasks 1 are taken, 495 mL of ultra-pure water is added, is then added 1% gold chloride (HAuCl4·3H2O) 5 mL, is configured to 500 mL, 0.01% aqueous solution of chloraurate, adds 1% trisodium citrate (Na in the case where persistently stirring after heated and boiled3C6H5O7·2H2O) solution 5-7 mL, continue agitating heating, when the color of solution is changed into transparent aubergine completely, stop heating after maintaining 5 min, and moisturizing is cooled to room temperature to original volume, and 2-8 DEG C saves backup；
The pretreatment of antibody：Will labelling Methomyl antibody in 1000 r/min, under the conditions of 4 DEG C, be centrifuged 20 min, take supernatant, be diluted to 1 mg/mL with 0.01 mol/L PBS；Or 1 mg/ml is diluted to 0.01 mol/L PBS, cross 0.22 m filter membranes；
The preparation of colloid gold label thing：40 mL of colloidal gold solution in step (1) being taken, colloidal gold solution pH to 8.5 being adjusted with 0.25 mol/L K2CO3,250 r/min of magnetic stirrer stirrings are added dropwise over 4 mL
Protein solution containing 0.3 mg antibody proteins, reacts 10 min；It is added dropwise over 4 mL
10% BSA, continues 10 min of stirring reaction；Gold labeling antibody solution room temperature low speed（1500 r/min）10 min are centrifuged, the precipitation formed by the gold grain for condensing is discarded；4 DEG C of red supernatant solution, 12000 r/min are centrifuged 20 min, abandon supernatant, collect precipitation, precipitation gold labeling antibody diluent is settled to 1 mL, Methomyl monoclonal antibody colloid gold label thing is prepared into；
The preparation of colloidal gold film：Step (3) Methomyl protein conjugate monoclonal antibody colloid gold label thing is sprayed on carrier glass cellulose membrane with the even concentration of 5 L/cm with film instrument is drawn, room temperature is dried or 37 DEG C of 3 h of drying naturally, makes the colloidal gold film of the monoclonal antibody colloid gold label thing of protein conjugate containing Methomyl；
Coating film preparation：Sheep anti-mouse igg antibody, Methomyl protein conjugate are diluted to into 1 mg/mL, are sprayed on nitrocellulose filter with the concentration of 1 L/cm with film instrument is drawn successively, be prepared into coated film, after 37 DEG C of 2 h of coating, room temperature is dried or 37 DEG C of drying naturally；
Sample pad pre-treatment：Sample pad treatment fluid is uniformly coated on glass fibre element film, under conditions of air humidity is less than 60%, room temperature is dried naturally；
The assembling of detection card：PVC offset plates paste sample pad, colloidal gold film, coated film and absorbent cotton from top to bottom successively, are assembled into test strips, cut into one fixed width strip, then test strips are arranged in the detection card shell of the flat shelly of strip.
Claims (2)
1. Methomyl colloidal-gold detecting-card, it is characterised in that prepare as steps described below：
(1) prepared by colloidal gold solution：1 L conical flasks 1 are taken, 495 mL of ultra-pure water is added, is then added 1% gold chloride HAuCl4·3H2O) 5 mL, is configured to 500 mL, 0.01% aqueous solution of chloraurate, adds 1% trisodium citrate Na in the case where persistently stirring after heated and boiled3C6H5O7·2H2O solution 5-7 mL, continue agitating heating, when the color of solution is changed into transparent aubergine completely, stop heating after maintaining 5 min, and moisturizing is cooled to room temperature to original volume, and 2-8 DEG C saves backup；
(2) pretreatment of antibody：Will labelling Methomyl antibody 1000
R/min, under the conditions of 4 DEG C, is centrifuged 20 min, takes supernatant, be diluted to 1 mg/mL with 0.01 mol/L PBS；Or 1 mg/ml is diluted to 0.01 mol/L PBS, cross 0.22 m filter membranes；
(3) preparation of colloid gold label thing：40 mL of colloidal gold solution in step (1) is taken, 0.25 mol/L is used
K2CO3Colloidal gold solution pH to 8.5 is adjusted, 250 r/min of magnetic stirrer stirrings are added dropwise over protein solutions of 4 mL containing 0.3 mg antibody proteins, react 10 min；It is added dropwise over 4 mL
10% BSA, continues 10 min of stirring reaction；Gold labeling antibody solution room temperature 1500
R/min is centrifuged 10 min, discards the precipitation formed by the gold grain for condensing；4 DEG C of red supernatant solution, 12000 r/min centrifugations 20
Min, abandons supernatant, collects precipitation, precipitation gold labeling antibody diluent is settled to 1 mL, Methomyl monoclonal antibody colloid gold label thing is prepared into；
(4) preparation of colloidal gold film：Step (3) Methomyl protein conjugate monoclonal antibody colloid gold label thing is sprayed on carrier glass cellulose membrane with the even concentration of 5 L/cm with film instrument is drawn, room temperature is dried or 37 DEG C of 3 h of drying naturally, makes the colloidal gold film of the monoclonal antibody colloid gold label thing of protein conjugate containing Methomyl；
(5) it is coated with film preparation：Sheep anti-mouse igg antibody, Methomyl protein conjugate are diluted to into 1 mg/mL, are sprayed on nitrocellulose filter with the concentration of 1 L/cm with film instrument is drawn successively, be prepared into coated film, after 37 DEG C of 2 h of coating, room temperature is dried or 37 DEG C of drying naturally；
(6) sample pad pre-treatment：Sample pad treatment fluid is uniformly coated on glass fibre element film, under conditions of air humidity is less than 60%, room temperature is dried naturally；
(7) assembling of detection card：PVC offset plates paste sample pad, colloidal gold film, coated film and absorbent cotton from top to bottom successively, are assembled into test strips, cut into the strip of one fixed width, then test strips are arranged in the detection card shell of the flat shelly of strip.
2. Methomyl colloidal-gold detecting-card according to claim 1, it is characterised in that in the step (5) coated detection line T be located at the lower section of nature controlling line C；
Sample pad treatment fluid in the step (6) is, by 1 g bovin serum albumin (BSA) and 0.8 g Sodium Chloride (NaCl), to be settled to 100 mL with the 0.01 mol/L PBS containing 0.5%TRITON-100.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510656071.1A CN106568960A (en) | 2015-10-13 | 2015-10-13 | Immunocolloidal gold test card of methomyl and preparation method thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510656071.1A CN106568960A (en) | 2015-10-13 | 2015-10-13 | Immunocolloidal gold test card of methomyl and preparation method thereof |
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| CN106568960A true CN106568960A (en) | 2017-04-19 |
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| CN201510656071.1A Withdrawn CN106568960A (en) | 2015-10-13 | 2015-10-13 | Immunocolloidal gold test card of methomyl and preparation method thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109813899A (en) * | 2017-11-18 | 2019-05-28 | 丹阳亿太生物科技发展有限公司 | Ceftiofur time-resolved fluoroimmunoassay chromatographs quantitative testing test paper item |
| CN115073337A (en) * | 2022-05-16 | 2022-09-20 | 江南大学 | Methomyl hapten, holoantigen, monoclonal antibody, preparation method and application thereof |
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| JP2002372537A (en) * | 2001-06-15 | 2002-12-26 | Nitto Denko Corp | Immuno-chromatograph test piece and detection method of test substance |
| 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 |
| CN103808930A (en) * | 2012-11-06 | 2014-05-21 | 江苏维赛科技生物发展有限公司 | Zilpaterol immune colloidal gold detection card and preparation method thereof |
| CN104655835A (en) * | 2013-11-20 | 2015-05-27 | 南京亿特生物科技有限公司 | Immune colloidal gold detect card of chlorpromazine, and preparation method thereof |
-
2015
- 2015-10-13 CN CN201510656071.1A patent/CN106568960A/en not_active Withdrawn
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002372537A (en) * | 2001-06-15 | 2002-12-26 | Nitto Denko Corp | Immuno-chromatograph test piece and detection method of test substance |
| 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 |
| CN103808930A (en) * | 2012-11-06 | 2014-05-21 | 江苏维赛科技生物发展有限公司 | Zilpaterol immune colloidal gold detection card and preparation method thereof |
| CN104655835A (en) * | 2013-11-20 | 2015-05-27 | 南京亿特生物科技有限公司 | Immune colloidal gold detect card of chlorpromazine, and preparation method thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109813899A (en) * | 2017-11-18 | 2019-05-28 | 丹阳亿太生物科技发展有限公司 | Ceftiofur time-resolved fluoroimmunoassay chromatographs quantitative testing test paper item |
| CN115073337A (en) * | 2022-05-16 | 2022-09-20 | 江南大学 | Methomyl hapten, holoantigen, monoclonal antibody, preparation method and application thereof |
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Application publication date: 20170419 |