CN104407146A - Preparation method of abamectin colloidal gold test strip - Google Patents
Preparation method of abamectin colloidal gold test strip Download PDFInfo
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- CN104407146A CN104407146A CN201410727583.8A CN201410727583A CN104407146A CN 104407146 A CN104407146 A CN 104407146A CN 201410727583 A CN201410727583 A CN 201410727583A CN 104407146 A CN104407146 A CN 104407146A
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- avermectin
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N33/531—Production of immunochemical test materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2430/00—Assays, e.g. immunoassays or enzyme assays, involving synthetic organic compounds as analytes
- G01N2430/10—Insecticides
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Abstract
The invention relates to a preparation method of an abamectin colloidal gold test strip, which can effectively achieve quick testing of abamectin and comprises the steps of covering a water diversion glass fiber layer, a carrier glass fiber layer, a nitric acid fiber layer and an absorbent cotton pulp board layer on a PVC (polyvinyl chloride) substrate from left to right, covering a right film on a 1/2 part of the absorbent cotton pulp board layer and the nitric acid fiber layer, covering a left film on a 1/2 part of the water diversion glass fiber layer, the carrier glass fiber layer and the nitric acid fiber layer, coating an anti-mouse immune globulin antibody quality control line and an abamectin protein conjugate testing line on the nitric acid fiber layer from the top down, and coating a colloidal gold marker on the carrier glass fiber layer in an adsorption manner. The strip is simple in structure, easy to produce and prepare, convenient to use, high in accuracy rate and good in effect, can be effectively used for quickly testing the abamectin, is an innovation in testing the abamectin and has huge economic and social benefits.
Description
Technical field
The present invention relates to drug test, particularly a kind of preparation method of Avermectin colloidal gold colloidal gold detection test paper strip.
Background technology
Avermectin is as a kind of ten hexa-atomic macrolide novel pesticides, fermented by Avid kyowamycin in streptomycete and produce, because chemical constitution is novel, unique (the nervous physiology activity of interference polypide of mechanism of action, stimulate release γ-aminobutyric acid, thus suppress arthropodan nerve conduction), insecticidal activity is strong, there is wide spectrum, efficient, holding effect, the feature such as comparatively safe, meet the requirement of modern ecological agricultural chemicals, be widely used in the various nematode in inside and outside, killing of arthropod class parasite and insect etc.The positive large area in the current whole world promotes the use of Avermectin product.But along with pest resistance improves, Avermectin dosage constantly increases, environment and the mankind, animals and plants harm are also manifested day by day, main manifestations is the medicament residue of field soil, humans and animals maincenter and peripheral nerve symptom, as trembled, incoordination, mental depression, highly stupor and even dead, and produce embryotoxicity.By the World Health Organization (WHO) (WTO) 5 grades of criteria for classifications, be classified as high cytotoxic compound.
Given this, in soil and animals and plants product, Avermectin medicament residue detects becomes state key monitor control index, and Avermectin medicament residue detection method also becomes primary study content.At present, chromatographic detection and immunodetection is mainly about Avermectin method for detecting residue, chromatographic detection has thin-layered chromatography, liquid phase chromatography, Liquid Chromatography-Mass Spectrometry etc., there is sample pre-treatments complexity, need professional operator, need expensive instrument to spend more high deficiency in these detection methods, is usually used in the confirmatory analysis of Avermectin medicament residue; Immunodetection has euzymelinked immunosorbent assay (ELISA), and the method is simple, quick, avoids the deficiency of chromatographic detection, is suitable for quick, a large amount of sample examination, very easily promotes.Colloidal gold immunochromatographimethod technology (gold immunochromatographic assay, GICA) be another the novel tachysynthesis detection technique developed on euzymelinked immunosorbent assay (ELISA) basis, namely Avermectin colloidal gold colloidal gold detection test paper strip is merge modern monoclonal antibody technique in colloidal gold immunochromatographimethod technical foundation, the one that new material technology is set up is simple, fast, economic immunologic detection method, it is simpler that this test strip operates comparatively euzymelinked immunosorbent assay (ELISA), fast, easier judged result, that another is novel, economical, the technical method of the detection Avermectin of environmental protection.Current Avermectin colloidal gold colloidal gold detection test paper strip is still at home and abroad blank.
Summary of the invention
For above-mentioned situation, for overcoming the defect of prior art, the object of the present invention is just to provide a kind of preparation method of Avermectin colloidal gold colloidal gold detection test paper strip, effectively can solve the quick test problems to Avermectin.
The technical scheme that the present invention solves is, this test strips be by, PVC substrate is coated with diversion glass layer from left to right, carrier glass fibrage, cellulose nitrate layer and absorbent wool pulp layer, right part overlay film (also known as top overlay film) is coated with above 1/2 place of absorbent wool pulp layer and cellulose nitrate layer, diversion glass layer, left part overlay film (also known as bottom overlay film) is coated with above 1/2 place of carrier glass fibrage and cellulose nitrate layer, on cellulose nitrate layer, (right-to-left) is coated with the nature controlling line of an against murine immune globulin antibody from top to bottom, , a detection line for Avermectin protein conjugate, on carrier glass fibrage, absorption is coated with colloid gold label thing, its preparation method is realized by following steps:
1, monoclonal antibody colloid gold label thing is prepared:
Mouse ascites containing anti-Avermectin protein conjugate monoclonal antibody is mixed with acetate buffer solution, it is sad to add again, centrifugal, obtain supernatant, filter, after adjust pH, add ammonium sulfate to dissolve, centrifugation, sediment phosphate buffer (PBS) dissolves, again with phosphate buffer dialysis, become monoclonal antibody colloid gold label thing;
2, colloidal gold solution is prepared:
By chlorauric acid solution and citric acid three sodium solution in the distilled water that boils, stir into peony, cooling, adjust pH, to isoelectric point or alkalescent, becomes colloidal gold solution;
3, prepare colloid gold label thing: get colloidal gold solution and add monoclonal antibody colloid gold label thing, stirring reaction, adds bovine serum albumin(BSA), centrifugal, obtains precipitation, with PBS dilution, become collaurum label;
4, colloid gold label thing is solidified: absorption bag is by colloid gold label thing on carrier glass fibrage, dry, is solidificated in by colloid gold label thing on carrier glass fibrage;
5, coated film preparation: get against murine immune globulin antibody, Avermectin protein conjugate, be coated on cellulose nitrate layer respectively, (right-to-left) forms the nature controlling line of an against murine immune globulin antibody of bag quilt, the detection line of an Avermectin protein conjugate from top to bottom;
6, cut: by comprise be adsorbed with colloid gold label thing carrier glass fibrage, wrap the test strips carrier after by the cellulose nitrate layer of against murine immune globulin antibody, Avermectin protein conjugate and cut into rectangular, test strips of the present invention.
Structure of the present invention is simple, easy manufacture, and easy to use, accuracy rate is high, effective, is effective to detect Avermectin fast, is to detect the innovation in Avermectin, and economic and social benefit is huge.
Accompanying drawing explanation
Fig. 1 is structural perspective of the present invention.
Embodiment
Below in conjunction with accompanying drawing and concrete condition, the specific embodiment of the present invention is elaborated.
The present invention is in concrete enforcement, this test strips is coated with diversion glass layer 3 from left to right by PVC substrate 1, carrier glass fibrage 4, cellulose nitrate layer 2 and absorbent wool pulp layer 5, right part overlay film (also known as top overlay film) 6 is coated with above 1/2 place of absorbent wool pulp layer 5 and cellulose nitrate layer 2, diversion glass layer 3, left part overlay film (also known as bottom overlay film) 7 is coated with above 1/2 place of carrier glass fibrage 4 and cellulose nitrate layer 2, on cellulose nitrate layer 2, (right-to-left) is coated with the nature controlling line of an against murine immune globulin antibody from top to bottom, , a detection line for Avermectin protein conjugate, on carrier glass fibrage 4, absorption is coated with colloid gold label thing, its preparation method is realized by following steps:
1, monoclonal antibody colloid gold label thing is prepared:
By by volume: 1 part of mouse ascites containing anti-Avermectin protein conjugate monoclonal antibody mixes with 2 parts of 60mM acetate buffer solutions, it is sad that room temperature (18 ~ 25 DEG C) dropwise adds under stirring, it is sad that every 1ml mouse ascites adds 33 μ l, mixing, room temperature places 30 minutes, 15000 revs/min 4 DEG C centrifugal 20 minutes, obtain supernatant, supernatant glass wool filters, abandon precipitation, supernatant after filtration NaOH adjust pH to 7.2, add ammonium sulfate, every 1ml pH7.2 supernatant adds 0.277g ammonium sulfate, ammonium sulfate is made to dissolve fast, stirring at room temperature 30 minutes, 15000 revs/min 4 DEG C centrifugal 20 minutes, abandon supernatant, obtain sediment, sediment 0.01M phosphate buffer (PBS) solubilize of former mouse ascites 1/2 volume, 0.01M phosphate buffer (PBS) 4 DEG C is used to dialyse again 48 hours, become monoclonal antibody colloid gold label thing,
2, colloidal gold solution is prepared:
Distilled water is boiled 4 ~ 6 minutes, the chlorauric acid solution 10ml of the mass concentration 1% and citric acid three sodium solution 15 ~ 20ml of mass concentration 1% is added in every 1000mL distilled water, being stirred to solution is peony, be chilled to room temperature, adjust pH, to the isoelectric point of anti-Avermectin protein conjugate monoclonal antibody or alkalescent (pH value 7.35 ~ 7.44), becomes the colloidal gold solution of anti-Avermectin protein conjugate monoclonal antibody preliminary making;
3, colloid gold label thing is prepared: get colloidal gold solution 100ml prepared by step (2), add monoclonal antibody colloid gold label thing 1 ~ 10 μ g/ml prepared by step (1), stirring reaction 2 minutes, add the bovine serum albumin(BSA) (BSA) of mass concentration 10%, the bovine serum albumin(BSA) of 15 μ l mass concentrations 10% is added in every 1ml colloidal gold solution, at 4 DEG C 15000 revs/min centrifugal 40 minutes, abandon supernatant, must precipitate, precipitation PBS is diluted to 30% ~ 40% of former colloidal gold solution volume, becomes collaurum label;
4, colloid gold label thing is solidified: absorption bag is by colloid gold label thing on carrier glass fibrage 4, dry, is solidificated in by colloid gold label thing on carrier glass fibrage;
5, coated film preparation: get against murine immune globulin antibody, Avermectin protein conjugate, be coated on cellulose nitrate layer 2 respectively, (right-to-left) forms the nature controlling line of an against murine immune globulin antibody of bag quilt, the detection line of an Avermectin protein conjugate from top to bottom, wherein, against murine immune globulin antibody bag is 0.5 ~ 5mg/ml by concentration, and Avermectin protein conjugate bag is 0.05 ~ 2mg/ml by concentration;
6, cut: by comprise be adsorbed with colloid gold label thing carrier glass fibrage 4, wrap the test strips carrier after by the cellulose nitrate layer 2 of against murine immune globulin antibody, Avermectin protein conjugate and cut into rectangular, test strips of the present invention.
The present invention is in concrete enforcement, and described test strips is the rectangular bar of long 6.5 ~ 8.0 cm, wide 0.3 ~ 0.4 cm;
In described step (2), the addition of the citric acid three sodium solution of mass concentration 1% can be 16 ~ 18ml or 17ml;
In described step (3), the addition of monoclonal antibody colloid gold label thing is 4 ~ 8 μ g/ml or 6 μ g/ml;
In described step (5), against murine immune globulin antibody bag is 1 ~ 4mg/ml or 3 ~ 4 mg/ml by concentration; Avermectin protein conjugate bag is 1 ~ 1.5mg/ml or 1.2 ~ 1.8mg/ml by concentration.
From the above, test strips of the present invention comprises base material PVC board 1, the nitrocellulose membrane adhered in the middle part of base material (is coated with the nature controlling line of an against murine immune globulin antibody from top to bottom, , a detection line for Avermectin protein conjugate) 2, adhere to base material bottom (in the present embodiment, when bottom uses with test strips, sample swimming direction is as the criterion) diversion glass fibre 3, carrier glass fiber (wrapping by anti-Avermectin protein conjugate monoclonal antibody colloid gold label thing) 4, adhere to the absorbent wool pulpboard 5 on base material top, top overlay film 6 and bottom overlay film 7.Nitrocellulose membrane (being coated with the detection line of the nature controlling line of an against murine immune globulin antibody, an Avermectin protein conjugate from top to bottom) 2, diversion glass fibre 3, carrier glass fiber (wrapping by anti-Avermectin protein conjugate monoclonal antibody colloid gold label thing) 4 and absorbent wool pulpboard 5 end face close contact.In embodiment, in colloidal gold solution preparation, 1% citric acid three sodium solution amount is 18ml, and in carrier glass fiber (wrapping by anti-Avermectin protein conjugate monoclonal antibody colloid gold label thing) 4, anti-Avermectin protein conjugate monoclonal antibody colloid gold label substrate concentration is 5 μ g/ml; In middle part nitrocellulose membrane (being coated with the detection line of the nature controlling line of an against murine immune globulin antibody, an Avermectin protein conjugate from top to bottom) 2, the against murine immune globulin antibody bag of nature controlling line bag quilt is 1mg/ml by concentration, and the Avermectin protein conjugate bag of detection line bag quilt is 0.5mg/ml by concentration.
When the present invention uses, tunica fibrosa one end is vertically inserted diversion glass fibre in submergence bottom in sample, or by sample drop on test strips bottom diversion glass fibre, under the transudation or capillary siphoning effect of microporous barrier, sample is along test strips to the direction swimming of absorbent wool pulpboard.Cleaning Principle of the present invention is that Avermectin and anti-Avermectin protein conjugate monoclonal antibody colloid gold label thing react and form labelled antigen--antibody complex, what adopt is A competitive inhibition method, if containing Avermectin in sample, sample swimming is to then both competitive reactions simultaneously anti-Avermectin protein conjugate monoclonal antibody colloid gold label thing when being coated with the detection line of Avermectin protein conjugate, swimming reacts to anti-Avermectin protein conjugate monoclonal antibody colloid gold label thing and against murine immune globulin antibody when being coated with the nature controlling line of against murine immune globulin antibody, show a red lines (nature controlling line) or two red lines (response lines, , a nature controlling line, but comparatively the red lines of response line are dark for the red lines of nature controlling line), result is judged as the positive, if not containing Avermectin in sample, sample swimming only has Avermectin protein conjugate and anti-Avermectin protein conjugate monoclonal antibody colloid gold label thing to react to when being coated with the detection line of Avermectin protein conjugate, swimming reacts to anti-Avermectin protein conjugate monoclonal antibody colloid gold label thing and against murine immune globulin antibody when being coated with the nature controlling line of against murine immune globulin antibody, show two red lines (response line, , a nature controlling line, but the red lines of nature controlling line are shallow compared with the red lines look of response line or red color is consistent), result is judged as feminine gender.If after application of sample, there are not red lines in nature controlling line place, then show that the invalid or test strips of ELISA test strip result lost efficacy.The present invention is by Avermectin medicament residue in the colorimetric half-quantitative detection animals and plants food of detection line in test strips and nature controlling line or soil.Utilize this test strips, within 5 ~ 10 minutes, can carry out simply, fast detecting to Avermectin medicament residue in animals and plants food or soil, be applicable to relevant quarantine detection department, import and export the quick detection of Avermectin medicament residue in quarantine detection department and laboratory or extensive examination.
The present invention not only can fill up the blank of Avermectin in field of fast detection, and there is incomparable simple of chromatographic detection method, fast, be convenient to the advantages such as a large amount of samples of examination, a kind of simple in animals and plants food or soil, the new technique of quick detection Avermectin medicament residue, be applicable to relevant quarantine detection department, import and export the quick detection of Avermectin medicament residue in quarantine detection department and laboratory or extensive examination, simply, fast, accurately, through detecting 58 batches of insecticidal materials and 38 field, place soil, rate of accuracy reached more than 99%, show that test strips of the present invention has very strong actual application value, economic and social benefit is huge.
Claims (5)
1. the preparation method of an Avermectin colloidal gold colloidal gold detection test paper strip, it is characterized in that, this test strips is that PVC substrate (1) is coated with diversion glass layer (3) from left to right, carrier glass fibrage (4), cellulose nitrate layer (2) and absorbent wool pulp layer (5), right part overlay film (6) is coated with above 1/2 place of absorbent wool pulp layer (5) and cellulose nitrate layer (2), diversion glass layer (3), left part overlay film (7) is coated with above 1/2 place of carrier glass fibrage (4) and cellulose nitrate layer (2), cellulose nitrate layer (2) is coated with the nature controlling line of an against murine immune globulin antibody from top to bottom, , a detection line for Avermectin protein conjugate, the upper absorption of carrier glass fibrage (4) is coated with colloid gold label thing, its preparation method is realized by following steps:
(1) monoclonal antibody colloid gold label thing, is prepared:
By by volume: 1 part of mouse ascites containing anti-Avermectin protein conjugate monoclonal antibody mixes with 2 parts of 60mM acetate buffer solutions, it is sad dropwise to add under stirring at room temperature, it is sad that every 1ml mouse ascites adds 33 μ l, mixing, room temperature places 30 minutes, 15000 revs/min 4 DEG C centrifugal 20 minutes, obtain supernatant, supernatant glass wool filters, abandon precipitation, supernatant after filtration NaOH adjust pH to 7.2, add ammonium sulfate, every 1ml pH7.2 supernatant adds 0.277g ammonium sulfate, ammonium sulfate is made to dissolve fast, stirring at room temperature 30 minutes, 15000 revs/min 4 DEG C centrifugal 20 minutes, abandon supernatant, obtain sediment, the sediment 0.01M phosphate buffer of former mouse ascites 1/2 volume dissolves, again with 0.01M phosphate buffer 4 DEG C dialysis 48 hours, become monoclonal antibody colloid gold label thing,
(2), colloidal gold solution is prepared:
Distilled water is boiled 4 ~ 6 minutes, the chlorauric acid solution 10ml of the mass concentration 1% and citric acid three sodium solution 15 ~ 20ml of mass concentration 1% is added in every 1000mL distilled water, being stirred to solution is peony, be chilled to room temperature, adjust pH, to the isoelectric point of anti-Avermectin protein conjugate monoclonal antibody or alkalescent, becomes the colloidal gold solution of anti-Avermectin protein conjugate monoclonal antibody preliminary making;
(3) colloid gold label thing, is prepared: get colloidal gold solution 100ml prepared by step (3), add monoclonal antibody colloid gold label thing 1 ~ 10 μ g/ml prepared by step (2), stirring reaction 2 minutes, add the bovine serum albumin(BSA) of mass concentration 10%, the bovine serum albumin(BSA) of 15 μ l mass concentrations 10% is added in every 1ml colloidal gold solution, at 4 DEG C 15000 revs/min centrifugal 40 minutes, abandon supernatant, must precipitate, precipitation PBS is diluted to 30% ~ 40% of former colloidal gold solution volume, becomes collaurum label;
(4), solidification colloid gold label thing: at the upper absorption bag of carrier glass fibrage (4) by colloid gold label thing, drying, is solidificated in colloid gold label thing on carrier glass fibrage;
(5), coated film preparation: get against murine immune globulin antibody, Avermectin protein conjugate, be coated on cellulose nitrate layer (2) respectively, form the nature controlling line of an against murine immune globulin antibody, the detection line of an Avermectin protein conjugate of bag quilt from top to bottom, wherein, against murine immune globulin antibody bag is 0.5 ~ 5mg/ml by concentration, and Avermectin protein conjugate bag is 0.05 ~ 2mg/ml by concentration;
(6), cutting: by comprise be adsorbed with colloid gold label thing carrier glass fibrage (4), wrap the test strips carrier after by the cellulose nitrate layer (2) of against murine immune globulin antibody, Avermectin protein conjugate and cut into rectangular, test strips of the present invention.
2. the preparation method of Avermectin colloidal gold colloidal gold detection test paper strip according to claim 1, is characterized in that, described test strips is the rectangular bar of long 6.5 ~ 8.0 cm, wide 0.3 ~ 0.4 cm.
3. the preparation method of Avermectin colloidal gold colloidal gold detection test paper strip according to claim 1, is characterized in that, in described step (2), the addition of the citric acid three sodium solution of mass concentration 1% can be 16 ~ 18ml or 17ml.
4. the preparation method of Avermectin colloidal gold colloidal gold detection test paper strip according to claim 1, is characterized in that, in described step (3), the addition of monoclonal antibody colloid gold label thing is 4 ~ 8 μ g/ml or 6 μ g/ml.
5. the preparation method of Avermectin colloidal gold colloidal gold detection test paper strip according to claim 1, is characterized in that, in described step (5), against murine immune globulin antibody bag is 1 ~ 4mg/ml or 3 ~ 4 mg/ml by concentration; Avermectin protein conjugate bag is 1 ~ 1.5mg/ml or 1.2 ~ 1.8mg/ml by concentration.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106771154A (en) * | 2016-11-29 | 2017-05-31 | 百奥森（江苏）食品安全科技有限公司 | The detection card and its detection method of AVM in a kind of detection tableware |
| CN106771157A (en) * | 2016-11-30 | 2017-05-31 | 百奥森（江苏）食品安全科技有限公司 | A kind of fortimicin detection method and detection card |
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| CN202383142U (en) * | 2011-11-15 | 2012-08-15 | 黑龙江八一农垦大学 | Colloidal gold test paper for detecting abamectin (AVM) residual |
| CN102928407A (en) * | 2011-08-09 | 2013-02-13 | 北京勤邦生物技术有限公司 | Magnetic particle chemiluminescence kit for detecting avermectins, and applications thereof |
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| EP0450936A1 (en) * | 1990-04-05 | 1991-10-09 | Merck & Co. Inc. | Monoclonal antibody to avermectins |
| RU2416094C1 (en) * | 2009-12-30 | 2011-04-10 | Автономная некоммерческая организация "Научно-исследовательский институт диагностики и профилактики болезней человека и животных" (АНО "НИИ ДПБ") | Set for quantitative determination of avermectins via single-step competitive enzyme-immunoassay |
| CN102928407A (en) * | 2011-08-09 | 2013-02-13 | 北京勤邦生物技术有限公司 | Magnetic particle chemiluminescence kit for detecting avermectins, and applications thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106771154A (en) * | 2016-11-29 | 2017-05-31 | 百奥森（江苏）食品安全科技有限公司 | The detection card and its detection method of AVM in a kind of detection tableware |
| CN106771157A (en) * | 2016-11-30 | 2017-05-31 | 百奥森（江苏）食品安全科技有限公司 | A kind of fortimicin detection method and detection card |
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| Publication number | Publication date |
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| CN104407146B (en) | 2016-05-25 |
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