CN105319355A - Preparation and application method of dipterex colloidal gold test strip - Google Patents
Preparation and application method of dipterex colloidal gold test strip Download PDFInfo
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- CN105319355A CN105319355A CN201410356194.9A CN201410356194A CN105319355A CN 105319355 A CN105319355 A CN 105319355A CN 201410356194 A CN201410356194 A CN 201410356194A CN 105319355 A CN105319355 A CN 105319355A
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- metrifonate
- detection
- dipterex
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- colloidal gold
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Abstract
The invention belongs to the field of biological detection, and relates to a preparation method and an application method of a dipterex colloidal gold test strip. The dipterex colloidal gold test strip comprises a paper board; one surface of the paper board is bonded with a water absorbent pad, a detection pad, a gold marked pad, and a sample pad successively from top to bottom; adjacent pads are overlapped at connection parts; a nitrocellulose membrane is taken as the base pad of the detection pad; the nitrocellulose membrane is provided with a transverse reference line and a detection line from top to bottom; the detection line is coated with a dipterex-bovine serum albumin conjugate; the reference line is coated with rabbit-anti polyclonal antibodies; and the gold marked pad is transversely sprayed with nano gold labeled anti-dipterex monoclonal antibodies. The dipterex colloidal gold test strip is used for detecting dipterex, detection is rapid, operation is simple, and sensitivity is high.
Description
Technical field
The invention belongs to field of detection of food safety, be specifically related to the detection method of residuals in food, particularly a kind of method of preparation and use of metrifonate colloidal gold strip.
Background technology
Metrifonate is used as pesticide.Be applicable to the pests with chewing mouthparts on the crops such as water paddy and wheat class, vegetables, tea tree, fruit tree, mulberry tree, cotton, and animal parasite, sanitary insect pest control, efficient, low toxicity, low-residual, broad spectrum insecticide, based on stomach poison function, have action of contace poison concurrently, also have osmotically active.Agriculturally be of wide application, for preventing and treating the various pests such as cabbage caterpillar, Amrasca biguttula (Ishida), Sang Yecan, Phellinus, weevil, fruit tree sawfly, fruit bat.Refining dipterex can be used for control pig, ox, horse, mule livestock endoparasite and ectoparasite, to family and public health insect all effective.
The Ministry of Agriculture of China also promulgated the maximum residue limit about metrifonate in 2003, and metrifonate middle maximum residue limit in fruit is respectively 0.1mg/kg.Therefore strengthening the research to metrifonate residue detection technology, is the key measure effectively controlling fruit drug residue, is also one of foundation and the task of improving medicament residue monitoring system.There is gas chromatography to the detection method of metrifonate at present, the online and high performance liquid chromatography of gas-matter, due to above all analytic approach sample pre-treatments more complicated, need special technician, testing cost expensive during operation, be unfavorable for applying.
Summary of the invention
The object of the invention is, in order to overcome the deficiencies in the prior art, to provide a kind of high specificity, highly sensitive, and simple to operation, to sample through simple process and the quick test paper semi-quantitative detection method of detectable metrifonate.
Object of the present invention realizes by following technical scheme:
Its technical essential of metrifonate colloidal gold strip detection method is: the backing of test paper posts successively sample pad, gold mark pad, nitrocellulose filter and thieving paper, the material of the upper mark of gold mark pad is the potpourri of the second kind animal protein and metrifonate detection antigen or the potpourri of the second kind animal protein and metrifonate antibody; Nitrocellulose filter is coated with metrifonate antibody 1 as detection line, the IgG1 bar being simultaneously also coated with anti-second kind animal protein is as reference line or be coated with metrifonate detection antigen 1 bar as detection line, is also coated with the work IgG1 bar of anti-second kind animal protein as reference line simultaneously.When prepared by test paper by regulating the concentration of detection line and reference line encrusting substance, the colour developing depth of detection line and reference line when controlling to detect, and the depth that developed the color is mapped with standard substance concentration, reaches half-quantitative detection.
Described detection antigen refers to that the conjugates that metrifonate and carrier mass gelatin are formed, immunity antigen refer to the conjugates that metrifonate and carrier mass keyhole limpet hemocyanin (KLH) are formed.
Described carrier mass also can select other macromolecular substances, as: lipoprotein, polyamino acid, glucosan, egg white
Albumin etc., but require that the carrier of detection antigen and immunity antigen is without cross-immune reaction.
The second described kind animal protein refers to that non-antibody source belongs to the albumen of animal, and such as, antibody is rabbit source property, then the second kind animal protein can be the chicken such as oralbumin, porcine hemoglobin, duck or other non-rabbit source property animal proteins.
The each several part process of test paper described in the invention and function as follows:
Backing: for one side scribbles the toughness material do not absorbed water of adhesive sticker, as PVC board, plays fixing other ingredients of support test paper.
Sample pad preparation: filter paper or all-glass paper are immersed about 2min in the PBS of pH7.0-8.4, take out, dries or other modes dryings, namely as sample pad, plays a part to absorb sample solution, be convenient to sample solution and move up during detection for 80 DEG C.
The preparation of colloid gold label part: this part plays antigen or the antibody of fixing colloid gold label.
Preparation process comprises the preparation of colloidal gold solution, colloid gold label metrifonate antigen or metrifonate antibody.
The process of colloid gold label part.
(1) the preparation of colloidal gold solution: by gold chloride (HAuCl
4) to be mixed with ultrapure water 1% mother liquor, get the mother liquor of 1mL, be settled to 100mL with ultrapure water, be made into the solution of 0.01%, be heated to boiling, add the trisodium citrate aqueous solution of 1 – 5mL1%, continue to be heated to occur transparent orange red till, be colloidal gold solution.
(2) colloid gold label metrifonate antigen: metrifonate detection antigen and the second kind animal protein are used PBS(0.01mo1/L respectively, pH7.0-7.5) dissolved dilution is to 2-4mg/mL, every 100mL colloidal gold solution adds detection antigen and the 1-1.5mL2-4mg/mL second kind animal protein concussion 2min of 1-3mL2-4mg/mL, with the K of 0.2mol/L
2cO
3:, regulate pH to 8.4, concussion 5min adds 11%PEG-100002mL, concussion 5min, the centrifugal 15min of 8000-15000r/min, removing supernatant, by precipitation PBS(0.0lmol/L, pH7.0-7.5) redissolve, with the centrifugal 15min of 6000-13000r/min, removing supernatant, using precipitation PBS(0.0lmol/L, pH7.0-7.5) dilute 500-2000 times of product as gold mark metrifonate antigen.
(3) colloid gold label metrifonate antibody: metrifonate monoclonal antibody or polyclonal antibody and the second kind albumen are used PBS(0.01mo1/L respectively, pH7.0-7.5) dissolved dilution is to 2-4mg/mL, every 100mL colloidal gold solution adds metrifonate antibody and the 1-1.5mL2-4mg/mL second kind animal protein of 1-3mL2-4mg/mL, concussion 2min, with the K of 0.2mol/L
2cO
3, regulate pH to 8.4, concussion 5min, add 11%PEG-100002mL, the centrifugal 15min of concussion 5min, 8000-15000r/min, except supernatant, by precipitation PBS(0.01mo1/L, pH7.0-7.5) redissolve, the centrifugal 15min of 6000-13000r/min, removing supernatant, by precipitation PBS(0.01mol/L, pH7.0-7.5) dilute 500-2000 doubly, product is as gold mark metrifonate antibody.
(4) gold mark pad process: pour in a groove by metrifonate antigen or metrifonate antibody, immerses 1min by glass fibre or filter paper, takes out, after drying at room temperature, namely as gold mark pad.
Nitrocellulose filter divides preparation: the glutaraldehyde solution with 0.8% or 0.2% Carbodiimide solution soak nitrocellulose membrane or cellulose acetate film or nylon membrane 30min, take out, 37 DEG C of oven dry, top bag by the metrifonate detection of 1 variable concentrations with antigen line or metrifonate antibody line as detection line, wrap by the work IgG line of 1 anti-second kind animal protein as with reference to line simultaneously.When colloid gold label portion markings object be metrifonate detection antigen and the second kind albumen time, detection line then wraps by metrifonate antibody.This is detection reaction part, and this part Main Function is by reaction result with macroscopic characterization out.
Prepared by thieving paper: after all-glass paper or filter paper or thieving paper drying at room temperature, namely as water absorbent portion.This part Main Function is the mobile unnecessary sample solution come up to absorb.
Test paper is assembled: on backing, be pasted with sample pad, gold mark pad, nitrocellulose filter and thieving paper successively, metrifonate Test paper.
Cleaning Principle: Cleaning Principle because of the object of colloid gold label different, and slightly difference.
When colloid gold label portion markings object be metrifonate detection antigen and the second kind animal protein time, if containing metrifonate in sample, sample solution is by the absorption of the sample pad of test paper and by capillary action is moved, the little translational speed of free metrifonate molecular weight in sample is fast, first arrive detection line, the metrifonate antibody first wrapping quilt on detection line is combined, because metrifonate antibody detection line wrapping quilt only has specific binding site, and the metrifonate binding ability of dissociating in sample is stronger than the metrifonate detection antigen of parataxic, so the detection antigen of colloid gold label can not metrifonate antibody capture again on tested survey line, so detection line is colourless, this is the positive, if without metrifonate in sample, then just tested survey line wraps the metrifonate antibody capture of quilt after the metrifonate detection antigen of colloid gold label arrives detection line, form macroscopic redness, this is feminine gender.No matter in sample whether containing metrifonate, the second kind animal protein of colloid gold label to move on to when reaching reference line the work IgG that all referenced line can wrap the anti-second kind animal protein of quilt and catch and form macroscopic redness, this is reference line.
When colloid gold label portion markings object be metrifonate antibody and the second kind animal protein time, if containing metrifonate in sample, sample solution is absorbed by the sample pad of test paper and reaches colloid gold label part by capillary action moves on to, the metrifonate antibody response of the metrifonate in sample solution and colloid gold label forms bond, bond moves on to detection line on continuing, because the metrifonate antibody of colloid gold label only has specific binding site, after metrifonate in sample solution combines with it, detection antigen on detection line just can not be combined with the metrifonate antibody of colloid gold label again, so detection line is colourless, this is the positive, when not having metrifonate in sample, detected antigen capture when the metrifonate antibody of colloid gold label arrives detection line, then form macroscopic redness.No matter in sample whether containing metrifonate, the second kind albumen of colloid gold label to move on to when reaching reference line the work IgG that all referenced line can wrap the anti-second kind animal protein of quilt and catch and form macroscopic redness, this is reference line.
The test paper of above two kinds of modes, when prepared by test paper by regulating detection line and reference line encrusting substance concentration, the colour developing depth of detection line and reference line when controlling to detect, and the depth that developed the color is mapped with standard substance concentration, can reach half-quantitative detection object.
Accompanying drawing explanation
Accompanying drawing is the structural representation of test strip of the present invention:
1 be sample liquid absorption portion, 2 be colloid gold label part in figure, 3 be detection reaction part, 4 be detection line, 5 be reference line, 6 for water absorbent portion.
embodiment
The preparation of metrifonate semi-quantitative rapid detection test paper is as described in technique scheme.
Embodiment: in cereal, beans, metrifonate detects
Pre-treatment: take sample 1.0g, add water 2mL, places 2 hours.Add 10mL acetone, stir after 3 minutes, with the thick diatomaceous filter paper of coating 1cm, suction filtration is in ground decompression concentrator.Take out the residue on filter paper, add 5mL acetone, stir after 3 minutes, by above-mentioned same operation, merging filtrate is in decompression concentrator, and less than 40 DEG C are concentrated into about 3mL.Moved into the separating funnel injecting 10mL10% sodium chloride solution in advance.Wash the eggplant type bottle of above-mentioned decompression concentrator with 10mL ethyl acetate, be incorporated to washing lotion in above-mentioned separating funnel.With the vibration of oscillator fierceness after 5 minutes, leave standstill, ethyl acetate layer moves in 30mL triangular flask bottle.5mL ethyl acetate is added, by above-mentioned same operation in water layer.Combined ethyl acetate layer is in above-mentioned 30mL triangular flask.Add appropriate anhydrous sodium sulfate, frequently vibrate, mix, place after 15 minutes, filter in ground decompression concentrator.Above-mentioned triangular flask is washed again with 2mL ethyl acetate, with the residue on this cleansing solution washing filter paper, repetitive operation secondary.Merge two cleansing solutions in above-mentioned decompression concentrator, less than 40 DEG C remove ethyl acetate.Add 3mL normal hexane in Liquid Residue, move in 10mL separating funnel.Add the saturated acetonitrile of 3mL normal hexane, with the vibration of oscillator fierceness after 5 minutes, leave standstill, acetonitrile layer moves in ground decompression concentrator.The saturated acetonitrile of 3mL normal hexane is added again in normal hexane layer, by above-mentioned same operation, repetitive operation twice.Merge acetonitrile layer in decompression concentrator, less than 40 DEG C are concentrated into about 1mL, at room temperature dry up further with nitrogen.1:19 dilution proportion is pressed with deionized water; After getting dilution, liquid is to be measured.
The using method of metrifonate semi-quantitative rapid detection test paper product: sample thief liquid 100 μ g slowly drips on the glass fibre membrane of test strips, observes the colour developing situation of detection line in test strips (T line), reference line (C line) after 15 minutes.Limit the quantity according to the Cleaning Principle of test strips and judge the content of metrifonate in sample extracting solution: if only have a red ribbon to occur, judge that sample liquid is as the positive, metrifonate exceeds standard; If there are two red ribbons, judge that sample liquid is as feminine gender, does not exceed standard; If colourless band occurs, this test strips lost efficacy.
Claims (4)
1. metrifonate colloidal gold strip, is characterized in that: on the backing of test paper, post sample pad, gold mark pad, nitrocellulose filter and thieving paper successively, and the material of the upper mark of gold mark pad is the potpourri of the second kind animal protein and metrifonate antibody; Nitrocellulose filter is coated with detection metrifonate antigen 1 bar as detection line, is also coated with the IgG1 bar of anti-second kind animal protein as line of reference simultaneously.
2. metrifonate colloidal gold strip according to claim 1, is characterized in that: metrifonate detection antigen is the conjugates that metrifonate and carrier mass are formed.
3. metrifonate colloidal gold strip according to claim 2, is characterized in that: described carrier mass, is protein, protein fragments, improvement on synthesis, semi-synthetic polypeptide or polysaccharide.
4. metrifonate colloidal gold strip according to claim 1, is characterized in that: the second described kind animal protein is the protein of non-antibody source animal.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201410356194.9A CN105319355A (en) | 2014-07-25 | 2014-07-25 | Preparation and application method of dipterex colloidal gold test strip |
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| CN201410356194.9A CN105319355A (en) | 2014-07-25 | 2014-07-25 | Preparation and application method of dipterex colloidal gold test strip |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106290833A (en) * | 2016-08-28 | 2017-01-04 | 周江红 | A kind of nanometer gold immune chromatography test paper detecting strongyloidiasis and preparation method |
| CN109709254A (en) * | 2019-03-07 | 2019-05-03 | 厦门泓益检测有限公司 | Liquid chromatography tandem mass spectrometry measures the detection method of metrifonate in meat product |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000004031A1 (en) * | 1998-07-15 | 2000-01-27 | Hassan Jomaa | Phosphorous organic compounds and their use |
| CN1804631A (en) * | 2005-12-02 | 2006-07-19 | 广东工业大学 | Immune colloidal god reagent for detecting methamidophos pesticide residue |
| CN101042405A (en) * | 2007-04-26 | 2007-09-26 | 浙江省农业科学院 | Immune colloidal gold test strip for detecting organophosphorus pesticide and method for making same |
| CN103808939A (en) * | 2012-11-06 | 2014-05-21 | 江苏维赛科技生物发展有限公司 | Spectinomycin colloidal gold detection card |
-
2014
- 2014-07-25 CN CN201410356194.9A patent/CN105319355A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000004031A1 (en) * | 1998-07-15 | 2000-01-27 | Hassan Jomaa | Phosphorous organic compounds and their use |
| CN1804631A (en) * | 2005-12-02 | 2006-07-19 | 广东工业大学 | Immune colloidal god reagent for detecting methamidophos pesticide residue |
| CN101042405A (en) * | 2007-04-26 | 2007-09-26 | 浙江省农业科学院 | Immune colloidal gold test strip for detecting organophosphorus pesticide and method for making same |
| CN103808939A (en) * | 2012-11-06 | 2014-05-21 | 江苏维赛科技生物发展有限公司 | Spectinomycin colloidal gold detection card |
Non-Patent Citations (2)
| Title |
|---|
| 杨代凤等: "标记免疫分析在农兽药残留检测中的应用研究进展", 《中国农学通报》 * |
| 范世奇等: "兔抗敌百虫多克隆抗体的制备", 《湖北农业科学》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106290833A (en) * | 2016-08-28 | 2017-01-04 | 周江红 | A kind of nanometer gold immune chromatography test paper detecting strongyloidiasis and preparation method |
| CN106290833B (en) * | 2016-08-28 | 2019-05-10 | 青岛果子科技服务平台有限公司 | A kind of nanogold immune chromatography test paper and preparation method detecting strongyloidosis |
| CN109709254A (en) * | 2019-03-07 | 2019-05-03 | 厦门泓益检测有限公司 | Liquid chromatography tandem mass spectrometry measures the detection method of metrifonate in meat product |
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