CN104655844A - Chemiluminescence enzyme-linked immunoassay method used for detecting thiophanate - Google Patents
Chemiluminescence enzyme-linked immunoassay method used for detecting thiophanate Download PDFInfo
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Abstract
The invention discloses a chemiluminescence enzyme-linked immunoassay detection kit used for detecting thiophanate. The chemiluminescence enzyme-linked immunoassay detection kit comprises a kit body, and an ELISA plate and reagents arranged in the kit body. The chemiluminescence enzyme-linked immunoassay detection kit is characterized in that: wells of the ELISA plate are coated with an envelope antigen which is a conjugate of thiophanate parent nucleus with a carrier protein; the reagents comprise thiophanate monoclonal antibody, horseradish peroxidase labeled goat anti mouse antibody, thiophanate series stock solutions, concentrated phosphate buffer, a concentrate washing agent, and a chemiluminescence liquid. The chemiluminescence enzyme-linked immunoassay detection kit is high in sensitivity and accuracy, is simple and convenient for using, and can be used for detecting a plurality of kinds of drugs. Compared with conventional colorimetric ELISA, operation time is shortened greatly.
Description
Technical field
The present invention relates to a kind of enzyme-linked immunologic detecting kit, particularly relate to a kind of chemical luminescence ELISA detection kit of thiophanate.
Background technology
Thiophanate is a kind of substituted beneze-group fungicides of efficient, low toxicity, wide spectrum, and have interior suction bactericidal action, have protection and therapeutic action concurrently, have carcinogenicity and teratogenesis, therefore safety problem is paid much attention to.Japan pesticide registration retention criteria regulation meter Zhong Wei 2 mg/kg, wheat, cereal, beans are 1 mg/kg, and fruit, vegetables are 5 mg/kg.
At present, the method detecting thiophanate mainly contains: radioimmunology, high performance liquid chromatography (HPLC), look/matter combination analysis method (LC-MS), liquid/matter combination analysis method (LC-MS/MS).The defect of thin-layered chromatography is: operating process is complicated, and the time is long; Operating personnel need through professional training; The disturbing factor of impact analysis is more, result poor repeatability.Radioimmunology, high performance liquid chromatography, look/matter are used in conjunction analytic approach, the defect of liquid/these physico-chemical methods of matter combination analysis method is that instrument and equipment is expensive, sample pre-treatments is complicated, time-consuming, effort, not easily popularize, testing cost is high, particularly radioimmunology also needs to be equipped with radioactive source, has certain risk.Given this, a kind of method setting up effective, quick, simple, sensitive detection thiophanate is significant.
Summary of the invention
The object of this invention is to provide a kind of chemical luminescence ELISA detection kit of thiophanate.This kit has that detection sensitivity is high, applying flexible, easily feature.
The chemical luminescence ELISA detection kit of thiophanate of the present invention, comprise box body, be located at ELISA Plate in box body and be located at thiophanate series standard solution in box body, enzyme mark goat anti-rabbit antibody, thiophanate antibody, luminescent solution, wash solution, bag by solution and lock solution; It is characterized in that:
Each hole of described ELISA Plate is coated with the envelope antigen made with thiophanate and ovalbumin coupling, wherein envelope antigen concentration preferably 10 μ g/mL.
Preferred 6.7KDa ~ the 6.8KDa of molecular weight ranges of described ovalbumin.
Described thiophanate series standard solution is that described in 0mg/kg, 0.1mg/kg, 0.3mg/kg, 0.9mg/kg, 2.7mg/kg and 8.1mg/kg, enzyme mark goat anti-rabbit antibody is horseradish peroxidase-goat anti-rabbit igg stoste respectively, and its working concentration is preferably 1:1000.
Described thiophanate antibody is the obtained polyclonal antibody of the bovine serum albumin coupling being 6.7KDa ~ 6.8KDa by thiophanate and the molecular weight ranges artificial immunogen immune animal of making, and its working concentration is preferably 1:1000.
Described luminescent solution is three (methylol) aminomethane solution (pH=8.8) and 3/10000 (volume ratio) H of 0.01M luminol and 0.001M p-cresol
2o
2mixed liquor.Described luminol is luminous substrate, and p-cresol is luminescence enhancer.
Described wash solution is pH7.5,0.1mol/L phosphate buffer containing volume fraction 0.05% Tween-20.
Described bag is the solution containing 1.59g sodium carbonate and 2.53g sodium bicarbonate in every premium on currency by solution, and pH is 9.5.
Described lock solution contains 10g ovalbumin (OVA, ovalbumin, also claim chicken ovalbumin or chicken ovalbumin, be made up of 386aa, molecular weight is about 43Kd) in often liter of wash solution and adds weight fraction 0.5%NaN
3solution.
Kit maximum detection range of the present invention is 0.1mg/kg ~ 8.1mg/kg.
The sensitivity impact that the thiophanate standard solution related in kit of the present invention, enzyme mark goat anti-rabbit antibody solution, thiophanate antibody-solutions, luminescent solution and wash solution and formula thereof detect kit of the present invention is very large; Wherein the principal ingredient of each solution and compound method thereof are:
1, thiophanate standard solution: compound concentration is respectively 0mg/kg in conventional manner, the thiophanate standard solution of 0.1mg/kg, 0.3mg/kg, 0.9mg/kg, 2.7mg/kg and 8.1mg/kg;
2, enzyme mark goat anti-rabbit antibody solution: enzyme mark goat anti-rabbit antibody is horseradish peroxidase-goat anti-rabbit igg stoste, is mixed with the working concentration of 1:1000 during use with wash solution;
3, thiophanate antibody-solutions: thiophanate antibody is with the obtained polyclonal antibody of artificial immunizing antigen immune animal, gained thiophanate antibody wash solution is diluted to the working concentration of 1:1000;
4, luminescent solution: three (methylol) aminomethane solution (pH=8.8) and 3/10000 (volume ratio) H of being 0.01M luminol and 0.001M p-cresol
2o
2mixed liquor;
5, wash solution: refer to pH7.5,0.1mol/L phosphate buffer containing volume fraction 0.05% Tween-20;
6, wrap by solution: 1.59g sodium carbonate and 2.53g sodium bicarbonate are dissolved in 1L water, regulate pH to be 9.5;
7, lock solution preparation: 10g OVA is dissolved in 1L wash solution, then adds the NaN that weight ratio is 0.05%
3.
The preparation of ELISA Plate of the present invention:
The method for coating of ELISA Plate of the present invention adopts thiophanate-OVA at the bag of setting by solution, with the concentration set, and reaction overnight bag quilt in 4 DEG C.
The present invention adopt to be pH be 9.5 sodium carbonate-bicarbonate buffer solution.In ELISA Plate of the present invention, the thiophanate-ovalbumin (OVA) of institute's bag quilt can well be combined on microwell plate frosting under alkaline environment, can stand repeatedly to wash plate, and the envelope antigen concentration of employing is 10 μ g/mL.
Bag can be closed by lock solution by good ELISA Plate, and in confining liquid, the preferred ovalbumin of inert protein (OVA), need add NaN
3prevent from going bad.
The preparation of thiophanate antibody-solutions and enzyme mark goat anti-rabbit antibody solution:
In the present invention, thiophanate antibody-solutions, enzyme mark goat anti-rabbit antibody solution concentration are the key factors determining Ractopamine enzyme linked immunological test kit measurement range and sensitivity in the present invention.
It is 0.1 ~ 8.1mg/kg solution that the thiophanate antibody-solutions related in the present invention can be mixed with concentration with wash solution; Or the working concentration of 1:1000 is diluted to wash solution.
The enzyme mark goat anti-rabbit antibody solution related in the present invention preferably with wash solution preparation concentration be 1:1000.
The sensitivity (0.1mg/kg) that the kit prepared according to above-mentioned thiophanate antibody-solutions concentration and enzyme mark goat anti-rabbit antibody solution concentration can reach the good range of linearity (standard lines scope can reach 0.1mg/kg ~ 8.1mg/kg) and become reconciled.
The preparation of luminescent solution:
The present invention adopts horseradish peroxidase labeled substrate luminescent system, mainly luminol-hydrogen peroxide system.
Described luminescent solution is three (methylol) aminomethane solution (pH=8.8) and 3/10000 (volume ratio) H of 0.01M luminol and 0.001M p-cresol
2o
2mixed liquor.Described luminol is luminous substrate, and p-cresol is luminescence enhancer.
Principle of the present invention is combined with enzymatic high sensitivity by the high degree of specificity that antibody-antigene reacts, and utilizes the chemiluminescence reaction of substrate for enzymatic activity to detect production concentration.
Chemical luminescence ELISA detection kit of the present invention has highly sensitive, easy point fast and accurately, compares with traditional colorimetric ELISA method, and sensitivity can improve an order of magnitude.Be expected to play a significant role in the thiophanate residue detection in crop products.
Accompanying drawing explanation
Fig. 1 is the canonical plotting of thiophanate.
Embodiment
Embodiment 1, immunogene, envelope antigen and the preparation of antibody
(1) immunogenic synthesis
Adopt p-aminobenzoic acid method to carry out coupling thiophanate and bovine serum albumin (BSA) and obtain immunogene.Specifically comprise the following steps:
A, take 14mg (100 μm of ol) p-aminobenzoic acid (ABA) and dissolve in 1.5mL0.2M hydrochloric acid, then take the sodium nitrite (NaNO of 8.3mg (120 μm of ol)
2) be dissolved in the distilled water of 0.24mL, 0-4 DEG C of stirrings, by sodium nitrite (NaNO
2) dropwise joins in p-aminobenzoic acid solution, lucifuge reacts 1 hour, obtains solution A;
B, take 34mg (100 μm of ol) thiophanate be dissolved in 5mL ice-cold borax buffering (0.05M) (pH8.5, NaCl containing 0.15M) in, 0-4 DEG C of stirring, above-mentioned solution A 2mL is dropwise joined in this solution, lucifuge reacts 2 hours, obtains orange solution;
C, solution is added a small amount of boric acid crystal adjust pH to 7.4, then 136mg (2 μm of ol) cBSA (bovine serum albumin(BSA)) is added, add 160mg (834 μm of ol) water-soluble carbodiimide (EDC) simultaneously, 48mg (417 μm of ol) N-hydroxy-succinamide (NHS), stirring at room temperature 3 hours, obtains orange solution;
D, reactant liquor is transferred in semi-permeable diaphragm, dialyse 3 days by phosphate buffered solution (PBS) (0.01M, pH7.4) at 0-4 DEG C, often within 4-6 hour, change a dislysate therebetween; Dialyse 3 days with high purity water subsequently, often within 4-6 hour, change a dislysate therebetween; Dialyse complete, use freeze drier freeze-drying, obtain yellow orange solid powder and be immunogene (conjugate of thiophanate and bovine serum albumin) ,-20 DEG C of preservations, for subsequent use.
(2) synthesis of envelope antigen
Adopt Isosorbide-5-Nitrae-Ding diether method to carry out coupling thiophanate and ovalbumin (OVA) and obtain envelope antigen.Specifically comprise the following steps:
A. taking 66mg ovalbumin (OVA) is dissolved in 5mL50mM carbonate (pH10.7) damping fluid, then in solution, add 1 of 28 μ L (147.9 μm of ol), 4-fourth diether (BDE), room temperature reaction 24 hours, obtains solution A;
B. taking 76mg (277.1 μm of ol) thiophanate is dissolved in 1ml DMF (anhydrous N-N dimethyl formamide), add in 1mL50mM carbonate (pH10.7) damping fluid again, solution A is passed into nitrogen, subsequently thiophanate dropwise is added in solution A, room temperature reaction 24 hours, obtains yellow solution;
C. reactant liquor is transferred in semi-permeable diaphragm, dialyse 3 days by phosphate buffered solution (PBS) (0.01M, pH7.4) at 0-4 DEG C, often within 4-6 hour, change a dislysate therebetween; Dialyse 3 days with high purity water subsequently, often within 4-6 hour, change a dislysate therebetween; Dialyse complete, use freeze drier freeze-drying, obtain white solid powder and be envelope antigen (conjugate of thiophanate and ovalbumin) ,-20 DEG C of preservations, for subsequent use.
(3) preparation of thiophanate polyclonal antibody
Adopt new zealand white rabbit as immune animal, be the conjugate of the bovine serum albumin of 6.7KDa ~ 6.8KDa with thiophanate and molecular weight ranges be immunogene, first immunisation dosage is 500 μ g/mL, when head exempts from, immunogene is dissolved in and makes emulsifying agent into isopyknic physiological saline and Freund's complete adjuvant, nape portion multi-point injection, later immunity is got the immunogene that dosage reduces by half and is dissolved in isopyknic physiological saline and incomplete Freund's adjuvant mixing and emulsifying, head exempts from and two exempts from interval 20 days, once be total to immunity five times every immunity in two weeks later, do not add adjuvant for the last time.Culling heart blood after last immune 7 days, centrifugal antiserum, obtains thiophanate polyclonal antibody.
The foundation of embodiment 2, CL-ELISA detection method
(1) preferred (square formation) of antibody and envelope antigen concentration
Longitudinally with the dilution series coated elisa plate of often kind of envelope antigen by 40.0 μ g/mL, 20.0 μ g/mL, 10.0 μ g/mL, 5.0 μ g/mL, 2.5 μ g/mL, 1.25 μ g/mL, 0.625 μ g/mL, 0.3125 μ g/mL, 100 μ L/ holes, 0-4 DEG C of placement is spent the night, wash plate three times with cleansing solution, pat dry at every turn; 250 μ L/ hole lock solution are closed, and room temperature places 2 hours, washes plate three times, pats dry at every turn; Add the antibody (1:100 to 1:1024000) of the 100 a series of dilutions in μ L/ hole, room temperature places 2.5 hours, washes plate three times, pats dry at every turn; Add the horseradish peroxidase-goat anti-rabbit igg antibody of the 1:1000 in 100 μ L/ holes, room temperature places 1 hour, washes plate three times, pats dry at every turn; Add the luminescent solution in 100 μ L/ holes, measure luminous value.The envelope antigen concentration of obvious graded and antibody dilution is had to carry out specific assay for optium concentration with luminous value with the concentration of envelope antigen.
(2) mensuration of antibody sensitivity
According to the optimization experiment of above-mentioned antagonist and envelope antigen concentration, applicant selects and determines that antibody concentration is 1:1000, and envelope antigen concentration is the mensuration that 10 μ g/mL carry out the sensitivity of antibody:
A, bag quilt: the solution by solution, the envelope antigen of thiophanate being made into 10 μ g/mL with the carbonate bag of 0.05M pH9.6, add 100 μ L in the reacting hole of each polystyrene board, 4 DEG C are spent the night;
Next day, discard solution in hole, wash 3 times with lavation buffer solution, 300 μ L/ holes, each 5 minutes, pat dry; (this step is called for short washing, lower same);
B, close: close above-mentioned ELISA Plate of having wrapped quilt by lock solution, 250 μ L/ holes, room temperature incubates 2-4 hour, then washs;
C, application of sample: add the thiophanate solution 50 μ L/ hole of dilution thiophanate antibody (1:1000) 50 μ L/ hole and variable concentrations in the above-mentioned reacting hole closed, room temperature 2-4 hour, then washs;
D, add enzyme labelled antibody: in each reacting hole, add antibody (1:1000) the 100 μ L/ hole of diluted fresh horseradish peroxidase-goat anti-rabbit igg, 1.5 hours, washing;
E, luminescence: the luminescent solution 100 μ L/ hole adding Extemporaneous in each reacting hole, detect with chemical illumination immunity analysis instrument immediately;
F, testing result calculate with inhibiting rate:
Inhibiting rate (%)=B/Bo%, B is the luminous value of medicine as rival of variable concentrations, and Bo is the luminous value of not dosing
The concentration calculating medicine during 50% inhibiting rate is the sensitivity of this antibody.
The chemiluminescence enzyme linked immunoassay reagent kit of embodiment 3, detection thiophanate
(1) composition of the chemiluminescence enzyme linked immunoassay reagent kit of thiophanate is detected
A, be coated with the solid phase carrier (ELISA Plate) of envelope antigen (conjugate of thiophanate and carrier protein);
B, thiophanate standard solution: 0mg/kg, 0.1mg/kg, 0.3mg/kg, 0.9mg/kg, 2.7mg/kg and 8.1mg/kg;
C, enzyme mark goat anti-rabbit antibody solution: enzyme mark goat anti-rabbit antibody is horseradish peroxidase-goat anti-rabbit igg stoste, load, be mixed with the working concentration of 1:1000 during use with wash solution;
D, thiophanate antibody-solutions: the polyclonal antibody preparing gained with artificial immunizing antigen immune animal, be diluted to 1:1000 working concentration by gained thiophanate antibody wash solution;
E, luminescent solution: use three (methylol) aminomethane solution preparation of 0.0001M p-cresol of pH8.8 to become the luminol solution of 0.01M, then with H
2o
2mix according to the volume ratio of 3:10000;
F, wash solution: pH7.5,0.1mol/L phosphate buffer containing volume fraction 0.05% Tween-20;
G, wrap by solution: 1.59g sodium carbonate and 2.53g sodium bicarbonate are dissolved in 1L water, regulate pH9.5;
H, lock solution are prepared: 10g ovalbumin (OVA) is dissolved in 1L wash solution, then adds the NaN that weight ratio is 0.05%
3.
(2) preparation of ELISA Plate
With coating buffer, envelope antigen is diluted to 10 μ g/mL, every hole adds 100 μ L, and 4 DEG C are spent the night, and incline coating buffer, every hole adds 250 μ L cleansing solutions and washs 3 times, pat dry, then every hole adds confining liquid 250 μ L, hatches 1h for 37 DEG C, incline liquid in hole, cleansing solution washs 3 times, pats dry, and preserves with masking foil vacuum seal.
Embodiment
4, the application of the chemiluminescence enzyme linked immunoassay reagent kit of thiophanate is detected
(1) preparation of reagent
A. sample diluting liquid: use after the concentrated phosphoric acid salt buffer solution distilled water diluting 10 times provided in kit;
B. wash solution: use after the concentrated cleaning solution distilled water diluting 10 times provided in kit;
C. luminescent solution: three (methylol) aminomethane solution (pH8.8)+3/10000 (volume ratio) H of 0.01M luminol+0.001M p-cresol
2o
2.
(2) sample pre-treatments
(1) chloroform-methanol (volume ratio is 1:1) is prepared;
(2) representative sample (make the sample of 50% can by 20 object filter screens) is ground;
(3) sample weighed after 20 g grinding filtrations adds 40 mL chloroform-methanol, and Sample Dilution is than being 1:2 (w/v);
(4) mix in the container of sealing, concussion vortex 1 min;
(5) room temperature centrifugal more than 4000r/min, 10min; Get centrifugal after supernatant or filter after filtrate 1 mL, 50 ~ 60 DEG C of water-bath nitrogen dry up;
(6) add standard dilutions 1 mL and fully mixing, centrifugal 5 min of 4000 r/min or use quantitative test Filter paper filtering, get centrifugal after supernatant or filtrate after filtration analyze.
(3) detecting step
A. application of sample: add thiophanate series standard strength solution or the molten 50 μ L of sample in ELISA Plate micropore, then add thiophanate antibody-solutions 50 μ L, room temperature (25 DEG C) constant-temperature incubation 2.5h;
B. wash: incline the middle liquid that portals, and every hole adds wash solution 250 μ L, washs 3 times, pat dry;
C. enzyme-added mark goat anti-rabbit antibody solution: every hole adds enzyme mark goat anti-rabbit antibody solution 100 μ L, room temperature constant-temperature incubation 1h;
D. wash: incline the middle liquid that portals, and every hole adds wash solution 250 μ L, washs 3 times, pat dry;
E. luminescent solution is added: every hole adds luminescent solution 100 μ L;
F. detect: the luminous intensity measuring every hole with chemical illumination immunity analysis instrument.
(4) result judges
The mean value of the standard items obtained and sample luminous value is multiplied by 100 again divided by the luminous value of first standard (0 standard), take inhibiting rate as ordinate, the logarithm of thiophanate concentration is that horizontal ordinate makes typical curve, and the concentration of each sample can read from typical curve.
Inhibiting rate (%)=standard items luminous value (or sample) × 100%/0 standard items luminous value.
Embodiment 5 kit preci-sion and accuracy is tested
Get 0.1mg/kg, the thiophanate standard specimen of 0.3mg/kg, 0.9mg/kg, 2.7mg/kg and 8.1mg/kg, adds in milk sample, detects the thiophanate recovery.The interassay coefficient of variation of each concentration calculates with 5 repeating datas of different 5 days, and variation within batch coefficient calculates with time repeating data of 5 on the same day.
The quantitative calculating of the recovery is carried out according to the linear equation of the typical curve formulated.
From said determination result, the coefficient of variation is lower than 24.7%, and the recovery is between 78-120%.Show that this kit has well repeatability and accuracy.
Claims (9)
1. a chemical luminescence ELISA detection kit for thiophanate, comprises box body, the reagent being located at the ELISA Plate in box body and being located in box body; It is characterized in that, each hole of described ELISA Plate is coated with the envelope antigen made with thiophanate parent nucleus and ovalbumin coupling; Described reagent comprises: the sheep anti-mouse antibody of thiophanate class monoclonal antibody, horseradish peroxidase-labeled, thiophanate class series standard solution, concentrated phosphoric acid salt buffer, concentrated cleaning solution, chemical luminescence for liquid.
2. the chemical luminescence ELISA detection kit of thiophanate class according to claim 1, is characterized in that: described ELISA Plate is milky opaque polystyrene 96 hole chemiluminescence ELISA Plate.
3. the chemical luminescence ELISA detection kit of thiophanate class according to claim 1, is characterized in that: described envelope antigen concentration is 10 μ g/mL.
4. the chemical luminescence ELISA detection kit of thiophanate class according to claim 1, is characterized in that: the working concentration of described thiophanate monoclonal antibody is 1: 64000.
5. the chemical luminescence ELISA detection kit of thiophanate class according to claim 1, is characterized in that: the monoclonal antibody of described thiophanate is that the conjugate be made up of thiophanate parent nucleus and bovine serum albumin coupling prepares as immunogen immune Balb/c mouse.
6. the chemical luminescence ELISA detection kit of thiophanate according to claim 1, is characterized in that: described thiophanate series standard solution concentration is respectively: 0mg/kg, 0.1mg/kg, 0.3mg/kg, 0.9mg/kg, 2,7mg/kg and 8.1mg/kg.
7. the chemical luminescence ELISA detection kit of thiophanate according to claim 1, is characterized in that: described concentrated phosphoric acid salt buffer be often liter containing NaH
2pO
42H
2o 5.74g, Na
2hPO
412 H
2the aqueous solution of O 32.6g.
8. the chemical luminescence ELISA detection kit of thiophanate according to claim 1, is characterized in that: described thickening and washing solution is the pH=7.4 containing volume fraction 0.05% Tween-20,0.1mol/L phosphate buffer.
9. the chemical luminescence ELISA detection kit of thiophanate class according to claim 1, is characterized in that: three (methylol) aminomethane solution that described chemical luminescence for liquid A liquid is luminol content is 0.01M, p-cresol content is 0.001M pH8.8; B liquid is that 100mL solution contains citric acid 2.1g, anhydrous Na
2hPO
4the solution of 2.82g, the carbamide peroxide 0.64mL of 0.75%, described number percent is mass percent.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105572376A (en) * | 2016-03-07 | 2016-05-11 | 中国烟草总公司贵州省公司 | Test paper for detecting residual thiophanate methyl in crops, and application and preparation method thereof |
| CN108226471A (en) * | 2016-12-15 | 2018-06-29 | 南京亿特生物科技有限公司 | A kind of chemiluminescence enzyme-linked immunoassay for detecting Madumycin |
| CN108226472A (en) * | 2016-12-15 | 2018-06-29 | 镇江亿特生物科技发展有限公司 | A kind of chemiluminescence enzyme-linked immunoassay for detecting sodium sulfocyanate |
| CN108614114A (en) * | 2016-12-12 | 2018-10-02 | 丹阳亿太生物科技发展有限公司 | A kind of chemiluminescence enzyme-linked immunoassay of detection sarafloxacin |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101368953A (en) * | 2008-09-24 | 2009-02-18 | 山东大学 | Chemical luminescence ELISA detection reagent kit of ciprofloxacin |
| US20090270471A1 (en) * | 2005-03-31 | 2009-10-29 | Herbert Buschhaus | Method for inhibiting mycotoxin production |
| CN101830980A (en) * | 2010-04-21 | 2010-09-15 | 大连民族学院 | Chlorothalonil antigen, antibody preparation method and residual chlorothalonil ELISA (Enzyme-Linked Lmmuno Sorbent Assay) detection method |
| CN103018454A (en) * | 2011-09-21 | 2013-04-03 | 北京勤邦生物技术有限公司 | Sulfanilamide drug chemiluminescence enzyme-linked immunodetection kit |
-
2013
- 2013-11-20 CN CN201310581873.1A patent/CN104655844A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090270471A1 (en) * | 2005-03-31 | 2009-10-29 | Herbert Buschhaus | Method for inhibiting mycotoxin production |
| CN101368953A (en) * | 2008-09-24 | 2009-02-18 | 山东大学 | Chemical luminescence ELISA detection reagent kit of ciprofloxacin |
| CN101830980A (en) * | 2010-04-21 | 2010-09-15 | 大连民族学院 | Chlorothalonil antigen, antibody preparation method and residual chlorothalonil ELISA (Enzyme-Linked Lmmuno Sorbent Assay) detection method |
| CN103018454A (en) * | 2011-09-21 | 2013-04-03 | 北京勤邦生物技术有限公司 | Sulfanilamide drug chemiluminescence enzyme-linked immunodetection kit |
Non-Patent Citations (2)
| Title |
|---|
| 曹爱华等: "高效液相色谱法同时测定烟草中吡虫啉、多菌灵、甲基硫菌灵的残留量", 《中国烟草科学》, vol. 30, no. 1, 31 December 2009 (2009-12-31), pages 31 - 34 * |
| 王国霞: "多菌灵酶联免疫分析技术研究", 《中国优秀硕士论文电子期刊网农业科技辑》, no. 10, 15 October 2006 (2006-10-15), pages 046 - 64 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN105572376B (en) * | 2016-03-07 | 2017-07-11 | 中国烟草总公司贵州省公司 | A kind of test paper and its application, preparation method for detecting that thiophanate-methyl is remained in crops |
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Application publication date: 20150527 |