CN106970216A - A kind of ELISA detection kit and detection method for detecting pyrimethanil residual - Google Patents
A kind of ELISA detection kit and detection method for detecting pyrimethanil residual Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The invention discloses a kind of ELISA detection kit and detection method for detecting pyrimethanil residual, detection kit is coated with the ELISA Plate of pyrimethanil envelope antigen, pyrimethanil standard items, pyrimethanil antibody, concentrated cleaning solution, ELIAS secondary antibody, nitrite ion, reaction terminating liquid.Detection method key step includes：Sample pre-treatments, kit detection, Analysis of test results.With at present more compared with the detection method such as conventional high performance liquid chromatography, the preparation method that the present invention is provided can effectively obtain the higher polyclonal antibody of potency, enzyme linked immunological kit and detection method have the advantages that cost is low, detection sensitivity is high, and effective method is provided for convenience of the residues of pesticides of, quick detection pyrimethanil.
Description
Technical field
It is one more specifically the present invention relates to a kind of enzyme-linked immunologic detecting kit and pesticide residue detection method
The residual using pyrimethanil in ELISA technology for detection fruit box vegetables is planted, belongs to enzyme linked immunosorbent detection（ELISA）Skill
Art field.
Technical background
Pyrimethanil（Pyrimethanil）It is a kind of anilino-pyrimidine wide spectrum low toxicity bactericide developed in recent years, to fruit
Gray molds of crop such as droop and strawberry, cucumber in vegetable etc. have fabulous prevention effect, in addition, being transported in fruits and vegetables
Cheng Zhongke is as mould inhibitor, so as to play a part of preservation and antisepsis.Pyrimethanil has unique mechanism of action, can suppress pathogen
The generation of middle invasive enzyme is to prevent pathogen from invading, and there is blade to penetrate and root systemic activity for it, and crop can be transferred to rapidly
Internal each position.Growing with diseases and pests of agronomic crop incidence, the usage amount of agricultural chemicals is consequently increased, and residues of pesticides are asked
Topic is derived from the extensive concern of domestic and international researcher.Pyrimethanil in China mandatory national standards regulation fruits and vegetables
Highest residual quantity be 7 mg/kg（Citrus fruit etc.）With 3 mg/kg（Green onion, Kidney bean etc.）, Japan and other countries are also to me in addition
The residual quantity of pyrimethanil has formulated strict standard in the fruits and vegetables of state outlet.
The analysis method for the relevant pyrimethanil residual reported at present has gas chromatography（NPD detectors）, high-efficient liquid phase color
Spectrometry, gas（Liquid）Phase chromatography-mass spectroscopy is used in conjunction, but these methods all exist the rate of recovery it is low, detection sample pre-treatments program it is cumbersome,
The shortcomings of detection cycle is long.Therefore, pyrimethanil is detected in the urgent need to a kind of fast and convenient new method.ELISA
（ELISA）Be a kind of sensitive, quick and ultrahigh in efficiency determination method compared with traditional detection method, it has sample
The advantages of processing is simple, detection speed is fast, method is easy.It is related to the ELISA kit of fruits and vegetables Pesticide Residues detection and analysis
The report of method is a lot, but has not yet to see the report that pyrimethanil residual is detected using enzyme-linked immunoassay method.
The content of the invention
The invention aims to provide a kind of enzyme linked immunosorbent detection examination for detecting that pyrimethanil is remained in fruits and vegetables
Agent box and detection method, wherein, pyrimethanil polyclonal antibody is composition main in the kit, and the kit has
The advantages of high sensitivity, high specific, pyrimethanil residual that can be efficiently, in quick detection fruits and vegetables, can batch making be applied to agriculture
In industry production practices.
A kind of preparation method for pyrimethanil polyclonal antibody that the present invention is provided, it is characterised in that：By acetonitrile, 2- chloro- 4,
6- dimethyl pyrimidines, pyridine are added in three-necked flask, and beaker is placed on magnetic stirring apparatus, and in placing temperature in reactor
Meter, is added dropwise equal amido phenenyl acid at 50 ~ 60 DEG C, after reaction, and TLC tracking disappears up to raw material, is then cooled to 30 DEG C, will
Reaction product crosses chromatography pillar, collects haptens product；Haptens is coupled with carrier protein, pyrimethanil is obtained and manually resists
It is former；Small-sized mammalian is immunized using pyrimethanil artificial antigen, resulting polyclonal antibody is many grams of pyrimethanil
Grand antibody.
Wherein, it is that pyrimethanil resists to be coated with the envelope antigen used in the ELISA Plate preparation process of pyrimethanil envelope antigen
The former coupled complex with ovalbumin, coating buffer solution buffers for 0.05 mol/L PH 9.6 sodium carbonate-bicarbonate
Liquid, confining liquid is 1 % BSA/PBS buffer solutions.
Wherein, cleaning solution is PH 4.7 PBST solution, and its preparation steps is as follows：Take Na2HPO4The g of 12H2O 68.8,
NaH2PO4The g of 6.9 g, NaCl 45, plus distilled water, to 1000 mL, PH is adjusted to 7.4.
Wherein, enzyme marker is ELIAS secondary antibody, and marker enzyme is horseradish peroxidase.
Wherein, substrate nitrite ion is TMB- hydrogen peroxide urea solutions, and preparation steps are as follows：Substrate solution A：Take anhydrous sodium acetate
8.2 g, the g of powder-beta-dextrin 2.5, the mg of carbamide peroxide 428.6, plus distilled water is to 1000 mL, adjusts PH to 5.0,4 DEG C of preservations；Bottom
Thing liquid B：100 mg TMB are taken to be dissolved in 10 mL DMSO, brown bottle is preserved；14.6 mL substrate solutions A and 0.45 mL are taken before use
Substrate solution B mixes 15 min.Terminate liquid is 1.25 mol/L H2SO4。
The method that the utilization kit that the present invention is provided detects pyrimethanil residues of pesticides, step is as follows：
1st, weigh the g of edible portion 10 in fruits and vegetables to be put into mortar, add 10 mL extract solutions, be fully ground filtering, collection
Filtrate is testing sample.
2nd, take testing sample and coating buffer solution to add in hole, and positive control is set（Pyrimethanil standard items are coated with）, 37
DEG C coating 30 min.Washing, is drained, wherein, wash 3 times with cleaning solution, every time 3 min.
3rd, 0.4 mL confining liquids closing 30min is added per hole, is drained after cleaning.
4th, 0.1 mL pyrimethanil antibody is added per hole, 37 DEG C are incubated washing 3 times after 40min.
5th, 0.1 mL ELIAS secondary antibody goat-anti rabbits are added per hole（HRP）Antibody, 37 DEG C are incubated washing 3 times after 40min.
6th, 0.1 mL substrate nitrite ions are added per hole（Matching while using）37 DEG C of incubation 20min.
7th, 0.05 mL terminate liquids are added per hole.
8th, each hole OD values are determined in 450nm, 655nm wavelength using ELIASA.By pyrimethanil testing sample OD values and standard
Product are contrasted.
The analysis principle of kit of the present invention is：
The pyrimethanil artificial antigen being coated on microwell plate is coupled to form coupled complex with bovine serum albumin, and pyrimethanil treats test sample
Product are combined with pyrimethanil artificial antigen, then with ELIAS secondary antibody（HRP）With reference to " pyrimethanil artificial antigen-pyrimethanil is treated for formation
Test sample product-ELIAS secondary antibody " compound, adds substrate solution, chromogenic reaction result is observed, when pyrimethanil concentration is higher in testing sample
When, its colour developing is deeper, and ELIASA testing result OD values are higher；Conversely, when pyrimethanil concentration is relatively low in testing sample, it develops the color anti-
Should be shallower, OD values are relatively low.
The positive effect of the present invention：The pyrimethanil remained in fruits and vegetables, sample pretreatment process letter can be detected rapidly and sensitively
Single, processing time is few, and convenient while detecting a large amount of samples, and its cost is less than traditional high performance liquid chromatography, gas-chromatography etc.
Method.
Embodiment
The present invention is further elaborated below with reference to specific embodiment, these embodiments are merely to illustrate this hair
It is bright, but do not limit the scope of the invention.
Embodiment 1
It is prepared by pyrimethanil haptens
30 mL acetonitrile is taken respectively, and 1.5 g chloro- 4, the 6- dimethyl pyrimidines of 2-, 3 g pyridine is added in three-necked flask,
Beaker is placed on magnetic stirring apparatus, and in placing thermometer in reactor, 1.9 g p-aminophenyl second are added dropwise at 50 ~ 60 DEG C
The h of back flow reaction 5 after acid, completion of dropping, TLC tracking are until raw material disappearance, is then cooled to 30 DEG C, reaction product is crossed into chromatography
Pillar, the product after collection obtains pure material 1.5g after revolving.
Embodiment 2
It is prepared by pyrimethanil artificial antigen
1.5 g pyrimethanil haptens are weighed, are dissolved in 2 mL DMF, uniform stirring, 30 μ L tri-n-butylamines, ice bath, plus 30 μ are added
L is dissolved in DMF isobutyl chlorocarbonate, stirs 1h.3.2 g BSA are weighed, the mol/L citric acids of 5 mL pH 9.6 0.1 are dissolved in
In salt buffer.Haptens, DMF mixed solutions are added dropwise in BSA solution, 5 h, 5000 r/min centrifugations are stirred at 4 DEG C
After 20 min, precipitation is discarded, supernatant is dialysed three days, is changed a dialyzate every 8h during dialysis, is finally given pyrimethanil
Artificial antigen.
Embodiment 3
It is prepared by pyrimethanil polyclonal antibody
3 mg pyrimethanil artificial antigens are taken to be dissolved in 1 mL physiological saline, by the pyrimethanil artificial antigen after dilution and in equal volume
Freund's complete adjuvant emulsified, the pyrimethanil artificial antigen after emulsification is injected in followed by subcutaneous multi-point injection method
In new zealand white rabbit body, every White Rabbit ampoule is 0.5 mg/kg.After 15 days, equally normal saline dilution mistake will be used
Pyrimethanil artificial antigen emulsified with isometric incomplete Freund's adjuvant, booster immunization, dosage be 0.01 mg/kg, 15 days
Third time is carried out afterwards using pyrimethanil artificial antigen was diluted with carrying out booster immunization after incomplete Freund's adjuvant emulsification, from this
Booster immunization starts, and blood is taken after being immunized 7 days, and determines its antiserum titre, until its potency reaches certain numerical value, will prepare
Polyclonal antibody be stored in -20 DEG C it is standby.
Embodiment 4
Pyrimethanil Enzyme Linked Immunoadsorbent Assay detection kit, it includes following components：
（1）It is coated with the ELISA Plate of pyrimethanil envelope antigen；
（2）Pyrimethanil standard items；
（3）Pyrimethanil polyclonal antibody；
（4）The goat anti-rabbit antibody of horseradish peroxidase-labeled；
（5）Cleaning solution：Na2HPO412H2O 68.8 g, NaH2PO4The g of 6.9 g, NaCl 45；
（6）Substrate solution：Substrate solution A：The g of anhydrous sodium acetate 8.2, the g of powder-beta-dextrin 2.5, the mg of carbamide peroxide 428.6；Substrate solution B：
100 mg TMB, 10 mL DMSO；
（7）Reaction terminating liquid：1.25 mol/L H2SO4；
（8）Specification is a.
Embodiment 5
Utilize kit detection pyrimethanil residual
（1）Sample pre-treatments：Weigh the g of edible portion 10 in fruits and vegetables to be put into mortar, add 10 mL extract solutions, be fully ground
Filtering, the filtrate of collection is testing sample.
（2）Take testing sample and coating buffer solution to add in hole, and positive control is set（Pyrimethanil standard items are coated with）, 37
DEG C coating 30 min.Washing, is drained, wherein, wash 3 times with cleaning solution, every time 3 min.
0.4 mL confining liquids closing 30min is added per hole, is drained after cleaning.
（3）In the ELISA Plate hole for being coated with antigen and having closed, add the pyrimethanil antibody of same volume and treat test sample
Washed after product, 37 DEG C of 40 min of incubation with cleaning solution 3 times.
（4）Washed after adding horseradish peroxidase-labeled goat anti-rabbit antibody 0.1 mL, 37 DEG C of incubation 40min with cleaning solution
Wash 3 times.
（5）Add bottom ELISA Plate thing nitrite ion 0.1 mL, 37 DEG C of incubation 20min. of Fresh
（6）Add the mL terminating reactions of terminate liquid 0.05.
（7）ELISA Plate is placed in ELIASA, testing result is determined at 490 nm wavelength.
Claims (5)
1. a kind of preparation method of pyrimethanil polyclonal antibody, it is characterised in that：By acetonitrile, the chloro- 4,6- dimethyl pyrimidines of 2-, pyrrole
Pyridine is added in three-necked flask, and beaker is placed on magnetic stirring apparatus, and in placing thermometer in reactor, is dripped at 50 ~ 60 DEG C
Plus equal amido phenenyl acid, after reaction, TLC tracking is until raw material disappearance, is then cooled to 30 DEG C, reaction product is crossed into chromatographic column
Son, collects haptens product；Haptens is coupled with carrier protein, pyrimethanil artificial antigen is obtained；Utilize pyrimethanil people
Small-sized mammalian is immunized work antigen, and resulting polyclonal antibody is pyrimethanil polyclonal antibody.
2. a kind of Enzyme-linked Immunosorbent Assay ELISA detection kit for detecting pyrimethanil residual, it is characterised in that:It is phonetic including being coated with
The ELISA Plate of mould amine envelope antigen, pyrimethanil standard items, pyrimethanil antibody, cleaning solution, enzyme mark thing, substrate nitrite ion, reaction are whole
Only liquid.
3. the Enzyme-linked Immunosorbent Assay ELISA detection kit of detection pyrimethanil residual according to claim 2, its feature exists
In：Enzyme mark thing is the goat anti-rabbit antibody of horseradish peroxidase-labeled.
4. the Enzyme-linked Immunosorbent Assay ELISA detection kit of detection pyrimethanil residual according to claim 2, its feature exists
In：Cleaning solution is Na2HPO412H2O 68.8 g, NaH2PO4The g of 6.9 g, NaCl 45；
Substrate nitrite ion is TMB- hydrogen peroxide urea solutions, and preparation steps are as follows：Substrate solution A：Take anhydrous sodium acetate 8.2 g, β-
The g of dextrin 2.5, the mg of carbamide peroxide 428.6, plus distilled water is to 1000 mL, adjusts PH to 5.0；Substrate solution B：Take 100 mg
TMB is dissolved in 10 mL DMSO；Reaction terminating liquid is 1.25 mol/L H2SO4。
5. a kind of method remained using pyrimethanil in the kit detection fruits and vegetables as described in Claims 2 or 3 or 4, its feature is existed
In comprising the following steps：
（1）Weigh edible portion in fruits and vegetables；10 mL extract solutions are added, filtering is fully ground, the filtrate of collection is to treat test sample
Product；
（2）In the enzyme-linked immuno sorbent assay kit target hole for the enzyme detection pyrimethanil residual for being coated with antigen and having closed
In, washed after adding the pyrimethanil antibody and testing sample of same volume, 37 DEG C of 40 min of incubation with cleaning solution 3 times；
（3）After adding horseradish peroxidase-labeled goat anti-rabbit antibody 0.1 mL, 37 DEG C of incubation 40min 3 are washed with cleaning solution
It is secondary；
（4）Add bottom ELISA Plate thing nitrite ion 0.1 mL, 37 DEG C of incubation 20min of Fresh；
（5）Add the mL terminating reactions of terminate liquid 0.05；
（6）ELISA Plate is placed in ELIASA, testing result is determined at 490 nm wavelength.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201611267775.0A CN106970216A (en) | 2016-12-31 | 2016-12-31 | A kind of ELISA detection kit and detection method for detecting pyrimethanil residual |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201611267775.0A CN106970216A (en) | 2016-12-31 | 2016-12-31 | A kind of ELISA detection kit and detection method for detecting pyrimethanil residual |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110117575A (en) * | 2019-05-22 | 2019-08-13 | 江南大学 | One plant of pyrimethanil monoclonal antibody hybridoma cell strain HFG and its application |
| CN111879938A (en) * | 2020-06-16 | 2020-11-03 | 烟台市疾病预防控制中心 | Quantum dot immunochromatography test strip for rapidly detecting pyrimethanil residues and preparation method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013110837A1 (en) * | 2012-01-25 | 2013-08-01 | Consejo Superior De Investigaciones Científicas (Csic) | Haptens, conjugates and antibodies for pyrimethanil fungicide |
| CN105548060A (en) * | 2016-01-30 | 2016-05-04 | 华中师范大学 | Colorimetric detection reagent for pyrimethanil and preparation method and application of colorimetric detection reagent |
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2016
- 2016-12-31 CN CN201611267775.0A patent/CN106970216A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013110837A1 (en) * | 2012-01-25 | 2013-08-01 | Consejo Superior De Investigaciones Científicas (Csic) | Haptens, conjugates and antibodies for pyrimethanil fungicide |
| CN105548060A (en) * | 2016-01-30 | 2016-05-04 | 华中师范大学 | Colorimetric detection reagent for pyrimethanil and preparation method and application of colorimetric detection reagent |
Non-Patent Citations (1)
| Title |
|---|
| JOSEP V.MERCADER,ET AL.: "Antibody generation and immunoassay development in diverse formats for pyrimethanil specific and sensitive analysis", 《ANALYST》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110117575A (en) * | 2019-05-22 | 2019-08-13 | 江南大学 | One plant of pyrimethanil monoclonal antibody hybridoma cell strain HFG and its application |
| CN110117575B (en) * | 2019-05-22 | 2022-03-15 | 江南大学 | Pyrimidinemonoclonal antibody hybridoma cell strain HFG and application thereof |
| CN111879938A (en) * | 2020-06-16 | 2020-11-03 | 烟台市疾病预防控制中心 | Quantum dot immunochromatography test strip for rapidly detecting pyrimethanil residues and preparation method |
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Application publication date: 20170721 |