CN106543163A - A kind of haptenic preparation method and applications of fluoxastrobin - Google Patents
A kind of haptenic preparation method and applications of fluoxastrobin Download PDFInfo
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- CN106543163A CN106543163A CN201610980256.2A CN201610980256A CN106543163A CN 106543163 A CN106543163 A CN 106543163A CN 201610980256 A CN201610980256 A CN 201610980256A CN 106543163 A CN106543163 A CN 106543163A
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- fluoxastrobin
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
- C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
- C07D413/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/77—Ovalbumin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
-
- 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
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
Abstract
The invention discloses a kind of haptenic preparation method and applications of fluoxastrobin, the preparation method is with 3 [1 (2 hydroxy phenyl) 1 (methoxyimino) methyl] 5 of key intermediate in fluoxastrobin synthesis route, 6 dihydro Isosorbide-5-Nitraes, 2 dioxazines are raw material, first with 4, there is etherification reaction in 5,6 trifluoropyrimidines, then obtain fluoxastrobin hapten with o-methyl hydroxyphenylacetate reaction, the method of synthesis is simple, and product purity and yield are high；Immune mouse after be directly coupled with bovine serum albumin by hapten, produces the specific antibody for fluoxastrobin；The test kit of the detection fluoxastrobin of preparation can be used to, in competitive ELISA analysis, meet the domestic detection needs to fluoxastrobin residual.The fluoxastrobin hapten that the present invention is provided provides new material base for the detection method for setting up quick, easy, inexpensive, sensitive, special fluoxastrobin.
Description
Technical field：
The invention belongs to technical field of biochemical industry, and in particular to the haptenic preparation method of a kind of fluoxastrobin and its should
With.
Background technology：
Fluoxastrobin is that Beyer Co., Ltd develops the methoxy acrylic bactericide for listing in 2004, and its mechanism of action is
Mitochondrial respiratory inhibitor, i.e., suppress mitochondrial breathing by the electron transfer between cytochrome b and C1.Fluoxastrobin belongs to
Absorbability cauline leaf process antibacterial, the bactericidal activity with wide spectrum, to nearly all Mycophytes disease for example rust, glume blight,
The tens of kinds of diseases such as net blotch, powdery mildew, downy mildew have good activity, under recommended dose to cereal crop, Rhizoma Solani tuber osi,
The crop safety such as vegetable and coffee, to subsoil water, Environmental security.Fluoxastrobin is entered the market in Britain first within 2004, is subsequently entered
Holland, France, Mexico, Italy, Argentina, America ＆ Canada market, after entering the market, market increases very fast, sells within 2012
Volume reaches 1.65 hundred million dollars.
The analysis method of existing fluoxastrobin preparation adopts high performance liquid chromatography, and the analysis method of residual quantity is using high
Effect liquid phase chromatogram-mass spectrography, high performance liquid chromatography and gas chromatography-mass spectrography, above-mentioned instrumental method need expensive
Instrument and equipment and professional and technical personnel, sample pre-treatments are complicated, analysis time is long, it is impossible to meet field monitoring easily and fast
Require.Enzyme linked immunosorbent assay based on immunoreation is less demanding to sample, and high specificity, sensitivity it is high, easy to operate,
Safety is cheap, measurement result reliability, can carry out the quick detection of batch samples in outdoor.The premise of enzyme-linked immunosorbent assay is
The preparation of specific antibody, and fluoxastrobin is micromolecular compound, and the functional group that can be coupled is not contained in itself, it is necessary to enter
The haptenic synthesis of row, and then prepare artificial antigen.Report be there is no with regard to the haptenic preparation method of fluoxastrobin both at home and abroad at present
Road, therefore, the present invention devises a kind of haptenic preparation method of fluoxastrobin, and is prepared for artificial antigen, the detection of preparation
The test kit of fluoxastrobin can be used in competitive ELISA analysis.
The content of the invention
It is an object of the invention to provide a kind of haptenic preparation method of fluoxastrobin, with 3- [1- (2- hydroxy phenyls) -1-
(methoxyimino)-methyl] -5,6- dihydros-Isosorbide-5-Nitrae, 2- dioxazines are raw material, etherificate occur with 4,5,6- trifluoropyrimidines first anti-
Should, then fluoxastrobin hapten is obtained with o-methyl hydroxyphenylacetate reaction, the fluoxastrobin hapten of preparation can be applicable to system
Standby artificial antigen and Duo Long gram of antibody, it can also be used to further prepare a kind of phonetic using direct competive ELISA standard measure detection fluorine
The test kit of bacterium ester.
To solve above-mentioned technical problem, the technical solution used in the present invention is as follows：
There is provided a kind of fluoxastrobin haptenic preparation method, [(methoxy is sub- for 1- (2- hydroxy phenyls) -1- with 3- for the method
Amino)-methyl] -5,6- dihydros-Isosorbide-5-Nitrae, 2- dioxazines are raw material, and etherification reaction first occurs with 4,5,6- trifluoropyrimidines, then with neighbour
Hydroxyphenylacetic acid methyl ester reaction obtains fluoxastrobin hapten；
The haptenic preparation method of the fluoxastrobin is comprised the following steps:
(1) at 0 DEG C, by 3- [1- (2- hydroxy phenyls) -1- (methoxyimino)-methyl] -5, the 6- bis- of equimolar ratio
Hydrogen-Isosorbide-5-Nitrae, 2- dioxazines and 4,5,6- trifluoropyrimidines are dissolved in 1L tetrahydrofurans, and the hydrogenation of 7.0g 80% is added in the solution
Sodium plant oil suspension obtains mixed liquor, the magnetic agitation reaction 3h at 0 DEG C, and rotating speed is 1500-2000r/min, subsequently no longer
Cooled down, continued stirring 12-14 hours, reactant liquor ethyl acetate is digested and uses water cyclic washing, organic phase with sodium sulfate
It is dried and concentrating under reduced pressure, obtains sticky grease, treats that the grease forms crystallization, obtain 3- { 1- [2- (4,5- fluoropyrimidine -6-
Base epoxide)-phenyl] -1- (methoxyimino)-methyl } -5,6- dihydro -1,4,2- dioxazines；
(2) 3- { 1- [2- (4,5- fluoropyrimidine -6- base epoxides)-phenyl] -1- (the methoxy imido that just prepared by step (1)
Base)-methyl } o-methyl hydroxyphenylacetate of -5,6- dihydro -1,4,2- dioxazines and equimolar ratio is dissolved in 1L dimethylformamides
In, 32g potassium carbonate and 2.2g Cu-lyt .s are added, the mixed liquor is heated to into 100 DEG C of reaction 12-14 hours；
(3) mixed liquor obtained after terminate the reaction of step (2) returns to room temperature, adds the salt of 10-30% concentration
Acid, heats 5-10h, stands and be cooled to room temperature, filters, the rinsing of filter cake deionized water, obtains fluoxastrobin half anti-after drying
It is former.
A kind of application of fluoxastrobin hapten in terms of fluoxastrobin artificial antigen is prepared, will the fluoxastrobin half
After antigen and carrier protein couplet, fluoxastrobin artificial antigen is prepared using carbodlimide method, comprised the following steps：
(1) fluoxastrobin hapten is first dissolved into DMSO the storing liquid of 50mg/mL, the above-mentioned storages of 50-100 μ L are taken
Liquid, 4- hydroxyethyl piperazine ethanesulfonic acid coupling buffers (HEPES) dilutions for adding 1mL concentration to be that 0.1mol/L, pH are 7.2, obtains
To fluoxastrobin hapten diluent；
(2) weigh 2.5-5mg carrier proteins to be dissolved in during 0.4mL concentration is 7.2 HEPES buffer solution for 0.1mol/L, pH,
Obtain carrier protein solution；
(3) under room temperature magnetic agitation, rotating speed is 1500-2000r/min, and the fluoxastrobin that step (1) is obtained half is anti-
During former diluent adds the carrier protein solution that step (2) is obtained, 4mg (1- (3- dimethylamino-propyls) -3- ethyl carbon is added
Diimmonium salt hydrochlorate) (EDC), continue stirring reaction 2h, then reactant liquor is loaded in bag filter, in phosphate-buffered salt at 4 DEG C
Dialysis 72h in solution (PBS), changes three dialysis solution daily, and the product for obtaining is centrifuged 10min with 5000rpm, abandons precipitation, take
Clear liquid, supernatant are fluoxastrobin artificial antigen, are stored in -20 DEG C of refrigerators.
When described carrier protein is bovine serum albumin (BSA), fluoxastrobin hapten is 30-50 with the mol ratio of BSA:
1, the artificial antigen of preparation is immunogen；When described carrier protein is ovalbumin (OVA), fluoxastrobin hapten and OVA
Mol ratio be 15-20:1, the artificial antigen of preparation is coating antigen.
A kind of application of fluoxastrobin hapten in terms of fluoxastrobin polyclonal antibody is prepared, the phonetic bacterium of fluorine that will be described
Ester hapten is coupled the 8 week old female mice of immunogen immune for preparing and obtains fluoxastrobin polyclonal antibody with bovine serum albumin,
Specifically include following steps：
(1) choosing 8 week old female mices carries out initial immunity, and immunizing dose is 1.0mg/kg, immunogen normal saline
Dilution, adds equal-volume Freund's complete adjuvant, after emulsifying, carries out back multiple intradermal injections；
After (2) three weeks, booster immunization is carried out, immunizing dose is 1.0mg/kg, add equal-volume incomplete Freund's adjuvant, the back of the body
Portion's multiple spot subcutaneous injection, pressed same dosage later per three weeks and method is immune once；
(3) after the 4th is immune one week, from the ear edge vein exploitating blood of mice, separate antiserum；
(4) antiserum obtains polyclonal antibody using ammonium sulfate precipitation method purification, after dialysis lyophilization into powder, in-
Save backup at 20 DEG C.
A kind of application of fluoxastrobin hapten in terms of detection fluoxastrobin test kit is prepared, the test kit is using straight
Inhibition ELISA detection by quantitative fluoxastrobin is connect, is made up of reagent set and coating plate.
Described reagent set includes:
(1) phosphate buffer (PBS)：Concentration is 0.01moI/L, and pH is 7.4；
(2) cleaning mixture (PBST)：PBS buffer solution containing 1% tween 20；
(3) substrate buffer solution：3.67g/L Na2The citric acid mixed solution of HPO4 and 2.56g/L；
(4) terminate liquid：2mol/L H2SO4 solution；
(5) fluoxastrobin standard solution：Fluoxastrobin standard solution mother solution is 1g/L, dilute with PBS buffer solution during use
Release the series mass concentration of 5000,1000,500,100,10,1 μ g/L；
(6) the fluoxastrobin antigenic solution of enzyme labelling：Fluoxastrobin hapten and the conjugate of horseradish peroxidase, make
Used time dilutes 10,000 times with PBS；
(7) developer：The dimethyl sulphoxide solution of the 3,3' of 96.15mg/L, 5,5'- tetramethyl benzidine (TMB).
Described coating plate is 96 holes or 48 hole elisa Plates, is that fluoxastrobin polyclonal antibody is coated in ELISA Plate
On, concrete preparation process is as follows：
It is 9.6 by fluoxastrobin polyclonal antibody pH, concentration is molten for the Na2CO3-NaHCO3 coating bufferings of 0.1mol/L
Liquid is diluted to 10 μ g/mL, is then coated in coating plate with 100 μ L of every hole, and 4 DEG C overnight, after discarding coating buffer, with washing
Liquid board-washing three times, adds the PBST that 200 μ L mass concentrations are 5%OVA, closes 1 hour, finally with cleaning mixture board-washing four times,
Lyophilizing is vacuum-packed, and preserves in 4 DEG C.
Compared with prior art, the present invention has the advantages that：
[(methoxy is sub- for 1- (2- hydroxy phenyls) -1- with the key intermediate 3- in fluoxastrobin synthesis route for the present invention
Amino)-methyl] -5,6- dihydros-Isosorbide-5-Nitrae, 2- dioxazines are raw material, and etherification reaction first occurs with 4,5,6- trifluoropyrimidines, then with neighbour
Hydroxyphenylacetic acid methyl ester reaction obtains fluoxastrobin hapten, and the method for synthesis is simple, and product purity and yield are high；
Immune mouse after be directly coupled with bovine serum albumin by hapten, is produced for fluoxastrobin
Specific antibody；
The test kit of the detection fluoxastrobin of preparation can be used in competitive ELISA analysis, meet domestic phonetic to fluorine
The detection of bacterium ester residual needs.
Specific embodiment
Embodiment 1：
The haptenic preparation of fluoxastrobin, step are as follows：
(1) at 0 DEG C, by 3- [1- (2- hydroxy phenyls) -1- (methoxyimino)-methyl] -5, the 6- bis- of 0.25mol
4,5, the 6- trifluoropyrimidines of hydrogen-Isosorbide-5-Nitrae, 2- dioxazines and 0.25mol are dissolved in 1L tetrahydrofurans, in the solution add 7.0g
80% sodium hydride plant oil suspension obtains mixed liquor, the magnetic agitation reaction 3h at 0 DEG C, and rotating speed is 1500r/min, subsequently
No longer cooled down, continued stirring 13 hours, reactant liquor ethyl acetate is digested and uses water cyclic washing, organic phase with sodium sulfate
It is dried and concentrating under reduced pressure, obtains sticky grease, treats that the grease forms crystallization, obtain 3- { 1- [2- (4,5- fluoropyrimidine -6-
Base epoxide)-phenyl] -1- (methoxyimino)-methyl } -5,6- dihydro -1,4,2- dioxazines.
(2) by 3- { 1- [2- (4,5- fluoropyrimidine -6- base epoxides)-the phenyl] -1- (methoxyimino)-first of 0.2mol
Base } o-methyl hydroxyphenylacetate of -5,6- dihydros-Isosorbide-5-Nitrae, 2- dioxazines and 0.2mol is dissolved in 1L dimethylformamides, then plus
Enter 32g potassium carbonate and 2.2g Cu-lyt .s, the mixed liquor is heated to into 100 DEG C and is reacted 13 hours.
(3) mixed liquor that the reaction of step (2) terminates is returned to into room temperature, adds the hydrochloric acid of 20% concentration, heat 8h,
Room temperature is stood and be cooled to, is filtered, the rinsing of filter cake deionized water obtains fluoxastrobin hapten after drying.
Embodiment 2：
The preparation of fluoxastrobin artificial antigen
Using carbodlimide method by fluoxastrobin hapten and carrier protein couplet after, prepare fluoxastrobin artificial antigen,
Comprise the following steps：
(1) fluoxastrobin hapten is first dissolved into DMSO the storing liquid of 50mg/mL, the above-mentioned storing liquids of 80 μ L are taken, plus
Enter 1mL concentration and dilute for 4- hydroxyethyl piperazine ethanesulfonic acid coupling buffers (HEPES) that 0.1mol/L, pH are 7.2, obtain fluorine phonetic
Bacterium ester hapten diluent.
(2) 4mg carrier proteins are weighed to be dissolved in during 0.4mL concentration is 7.2 HEPES buffer solution for 0.1mol/L, pH, is obtained
Carrier protein solution.
(3) under room temperature magnetic agitation, rotating speed is 2000r/min, the fluoxastrobin hapten dilution that step (1) is obtained
During liquid adds the carrier protein solution that step (2) is obtained, 4mg (1- (3- dimethylamino-propyls) -3- ethyl carbodiimides are added
Hydrochlorate) (EDC), continue stirring reaction 2h, then reactant liquor is loaded in bag filter, in phosphate buffered saline(PBS) at 4 DEG C
(PBS) dialysis 72h in, changes three dialysis solution daily, and the product for obtaining is centrifuged 10min with 5000rpm, abandons precipitation, take supernatant,
Supernatant is fluoxastrobin artificial antigen, is stored in -20 DEG C of refrigerators.
When carrier protein is bovine serum albumin (BSA), fluoxastrobin hapten is 40 with the mol ratio of BSA:1, preparation
Fluoxastrobin artificial antigen is immunogen；When described carrier protein is ovalbumin (OVA), fluoxastrobin hapten and OVA
Mol ratio be 20:1, the fluoxastrobin artificial antigen of preparation is coating antigen.
Immunogen is identified：To carrier protein BSA, hapten and immunogen carry out UV scanning measure (200～400nm),
It was found that immunogen is compared with BSA with hapten, absorption curve has substantially change, illustrates hapten and the coupled successes of BSA.Trinitro-
It is coupled than being 40 with BSA that benzene yellow acid method measures hapten:1.
Coating antigen is identified：To carrier protein OVA, hapten and coating antigen carry out UV scanning measure (200～400nm),
It was found that coating antigen is compared with OVA with hapten, absorption curve has substantially change, illustrates hapten and the coupled successes of OVA.Trinitro-
It is coupled than being 20 with KLH that benzene yellow acid method measures hapten:1.
Embodiment 3：
The preparation of fluoxastrobin polyclonal antibody
Immunogen described in embodiment 2 is obtained into fluoxastrobin polyclonal antibody by immune 8 week old female mice, specifically
Comprise the following steps：
(1) choosing 8 week old female mices carries out initial immunity, and immunizing dose is 1.0mg/kg, immunogen normal saline
Dilution, adds equal-volume Freund's complete adjuvant, after emulsifying, carries out back multiple intradermal injections.
After (2) three weeks, booster immunization is carried out, immunizing dose is 1.0mg/kg, add equal-volume incomplete Freund's adjuvant, the back of the body
Portion's multiple spot subcutaneous injection, pressed same dosage later per three weeks and method is immune once.
(3) after the 4th is immune one week, from the ear edge vein exploitating blood of mice, separate antiserum；
(4) antiserum obtains polyclonal antibody using ammonium sulfate precipitation method purification, after dialysis lyophilization into powder, in-
Save backup at 20 DEG C.
Embodiment 4：
The test kit of fluoxastrobin is detected using direct competive ELISA standard measure
Test kit is made up of reagent set and coating plate, and described reagent set includes:
(1) phosphate buffer (PBS)：Concentration is 0.01moI/L, and pH is 7.4；
(2) cleaning mixture (PBST)：PBS buffer solution containing 1% tween 20；
(3) substrate buffer solution：3.67g/L Na2The citric acid mixed solution of HPO4 and 2.56g/L；
(4) terminate liquid：2mol/L H2SO4 solution；
(5) fluoxastrobin standard solution：Fluoxastrobin standard solution mother solution is 1g/L, dilute with PBS buffer solution during use
Release the series mass concentration of 5000,1000,500,100,10,1 μ g/L；
(6) the fluoxastrobin antigenic solution of enzyme labelling：Fluoxastrobin hapten and the conjugate of horseradish peroxidase, make
Used time dilutes 10,000 times with PBS；
(7) developer：The dimethyl sulphoxide solution of the 3,3' of 96.15mg/L, 5,5'- tetramethyl benzidine (TMB).
Coating plate is 96 holes, is that fluoxastrobin polyclonal antibody is coated in ELISA Plate, and concrete preparation process is as follows：
It is 9.6 by fluoxastrobin polyclonal antibody pH, concentration is molten for the Na2CO3-NaHCO3 coating bufferings of 0.1mol/L
Liquid is diluted to 10 μ g/mL, is then coated in coating plate with 100 μ L of every hole, and 4 DEG C overnight, after discarding coating buffer, with washing
Liquid board-washing three times, adds the PBST that 200 μ L mass concentrations are 5%OVA, closes 1 hour, finally with cleaning mixture board-washing four times,
Lyophilizing is vacuum-packed, and preserves in 4 DEG C.
Claims (8)
1. the haptenic preparation method of a kind of fluoxastrobin, it is characterised in that：With 3- [1- (2- hydroxy phenyls) -1- (methoxy imido
Base)-methyl] -5,6- dihydros-Isosorbide-5-Nitrae, 2- dioxazines are raw material, and etherification reaction first occurs with 4,5,6- trifluoropyrimidines, then with adjacent hydroxyl
The reaction of base methyl phenylacetate obtains fluoxastrobin hapten；The haptenic preparation method of the fluoxastrobin is comprised the following steps:
(1) at 0 DEG C, by 3- [1- (2- hydroxy phenyls) -1- (methoxyimino)-methyl] -5,6- dihydro -1 of equimolar ratio,
4,2- dioxazines and 4,5,6- trifluoropyrimidines are dissolved in 1L tetrahydrofurans, and the sodium hydride vegetable oil for adding 7.0g 80% suspends
Liquid obtains mixed liquor, the magnetic agitation reaction 3h at 0 DEG C, and rotating speed is 1500-2000r/min, is subsequently no longer cooled down, and is continued
Stirring 12-14 hours, reactant liquor ethyl acetate digest and use water cyclic washing, organic phase with sodium sulfate to be dried and reduce pressure dense
Contracting, obtains sticky grease, treats that the grease forms crystallization, obtains 3- { 1- [2- (4,5- fluoropyrimidine -6- base epoxides)-benzene
Base] -1- (methoxyimino)-methyl } -5,6- dihydro -1,4,2- dioxazines；
(2) 3- { 1- [2- (4,5- fluoropyrimidine -6- base epoxides)-the phenyl] -1- (methoxyimino)-first for being prepared by step (1)
Base } o-methyl hydroxyphenylacetate of -5,6- dihydros-Isosorbide-5-Nitrae, 2- dioxazines and equimolar ratio is dissolved in 1L dimethylformamides, then
32g potassium carbonate and 2.2g Cu-lyt .s are added, the mixed liquor is heated to into 100 DEG C of reaction 12-14 hours；
(3) mixed liquor obtained after terminate the reaction of step (2) returns to room temperature, adds the hydrochloric acid of 10-30% concentration, plus
Hot 5-10h, stands and is cooled to room temperature, filters, the rinsing of filter cake deionized water, and fluoxastrobin hapten is obtained after drying.
2. application of a kind of fluoxastrobin hapten in terms of fluoxastrobin artificial antigen is prepared, it is characterised in that：By the fluorine
After Fluoxastrobin hapten and carrier protein couplet, fluoxastrobin artificial antigen is prepared using carbodlimide method, comprised the following steps：
(1) fluoxastrobin hapten is first dissolved into DMSO the storing liquid of 50mg/mL, the above-mentioned storing liquids of 50-100 μ L are taken, plus
Enter 1mL concentration and dilute for the 4- hydroxyethyl piperazine ethanesulfonic acid coupling buffer that 0.1mol/L, pH are 7.2, obtain fluoxastrobin half
Antigenic dilution；
(2) 2.5-5mg carrier proteins are weighed and is dissolved in the 4- hydroxyethyl piperazine ethanesulfonic acid that 0.4mL concentration is that 0.1mol/L, pH are 7.2
In coupling buffer, carrier protein solution is obtained；
(3) under room temperature magnetic agitation, rotating speed is 1500-2000r/min, and the fluoxastrobin hapten that step (1) is obtained is dilute
Release in the carrier protein solution that liquid addition step (2) is obtained, (1- (3- dimethylamino-propyls) -3- ethyls carbon two is sub- to add 4mg
Amine hydrochlorate), continue stirring reaction 2h, then load reactant liquor in bag filter, dialyse in phosphate buffered saline(PBS) at 4 DEG C
72h, changes three dialysis solution daily, and the product for obtaining is centrifuged 10min with 5000rpm, abandons precipitation, take supernatant, and supernatant is fluorine
Fluoxastrobin artificial antigen, is stored in -20 DEG C of refrigerators.
3. application of the fluoxastrobin hapten according to claim 2 in terms of fluoxastrobin artificial antigen is prepared, which is special
Levy and be：Described carrier protein is bovine serum albumin, and fluoxastrobin hapten is 30-50 with the mol ratio of bovine serum albumin:
1, described fluoxastrobin artificial antigen is immunogen.
4. application of the fluoxastrobin hapten according to claim 2 in terms of fluoxastrobin artificial antigen is prepared, which is special
Levy and be：Described carrier protein is ovalbumin, and fluoxastrobin hapten is 15-20 with the mol ratio of ovalbumin:1, institute
The fluoxastrobin artificial antigen for stating is coating antigen.
5. application of a kind of fluoxastrobin hapten in terms of fluoxastrobin polyclonal antibody is prepared, it is characterised in that：By inciting somebody to action
Described fluoxastrobin hapten is coupled the 8 week old female mice of immunogen immune for preparing and obtains fluoxastrobin with bovine serum albumin
Polyclonal antibody, specifically includes following steps：
(1) choosing 8 week old female mices carries out initial immunity, and immunizing dose is 1.0mg/kg, immunogen normal saline dilution,
Equal-volume Freund's complete adjuvant is added, after emulsifying, back multiple intradermal injections is carried out；
After (2) three weeks, booster immunization is carried out, immunizing dose is 1.0mg/kg, add equal-volume incomplete Freund's adjuvant, back is more
Point subcutaneous injection, pressed same dosage later per three weeks and method is immune once；
(3) after the 4th is immune one week, from the ear edge vein exploitating blood of mice, separate antiserum；
(4) antiserum obtains polyclonal antibody using ammonium sulfate precipitation method purification, after dialysis lyophilization into powder, in -20 DEG C
Under save backup.
6. the haptenic application of fluoxastrobin for being prepared with claim 1 methods described, it is characterised in that：The fluoxastrobin half
Antigen is used to prepare the test kit that a kind of employing direct competive ELISA standard measure detects fluoxastrobin, and the test kit is by reagent
Group and coating plate are constituted.
7. the haptenic application of fluoxastrobin according to claim 6, it is characterised in that：Described reagent set includes:
(1) phosphate buffer：Concentration is 0.01moI/L, and pH is 7.4；
(2) cleaning mixture：Phosphate buffered solution containing 1% tween 20；
(3) substrate buffer solution：3.67g/L Na2The citric acid mixed solution of HPO4 and 2.56g/L；
(4) terminate liquid：2mol/L H2SO4 solution；
(5) fluoxastrobin standard solution：Fluoxastrobin standard solution mother solution is 1g/L, is diluted to PBS buffer solution during use
5000th, the series mass concentration of 1000,500,100,10,1 μ g/L；
(6) the fluoxastrobin antigenic solution of enzyme labelling：The conjugate of fluoxastrobin hapten and horseradish peroxidase, during use
10,000 times are diluted with PBS；
(7) developer：The dimethyl sulphoxide solution of the 3,3' of 96.15mg/L, 5,5'- tetramethyl benzidine.
8. the haptenic application of fluoxastrobin according to claim 6, it is characterised in that：Described coating plate is 96 holes
Or 48 hole elisa Plates, the coating plate is that fluoxastrobin polyclonal antibody is coated in ELISA Plate, and concrete preparation process is as follows：
It is 9.6 by fluoxastrobin polyclonal antibody pH, Na of the concentration for 0.1mol/L2CO3-NaHCO3Coating buffer solution is dilute
10 μ g/mL are released, is then coated in coating plate with 100 μ L of every hole, 4 DEG C overnight, after discarding coating buffer, washed with cleaning mixture
Plate three times, adds the cleaning mixture that 200 μ L mass concentrations are 5%OVA, closes 1 hour, finally with cleaning mixture board-washing four times, freezes
Dry vacuum is packed, and is preserved in 4 DEG C.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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| CN110642793A (en) * | 2019-09-24 | 2020-01-03 | 北京勤邦生物技术有限公司 | Azoxystrobin hapten, artificial antigen and antibody, and preparation method and application thereof |
| CN111333570A (en) * | 2020-03-05 | 2020-06-26 | 北京望尔生物技术有限公司 | Haloxyfop hapten, artificial antigen and antibody as well as preparation method and application thereof |
| CN113406239A (en) * | 2021-06-30 | 2021-09-17 | 浙江省农业科学院 | Method for measuring fluoxastrobin and metabolite thereof in water |
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| CN110642793A (en) * | 2019-09-24 | 2020-01-03 | 北京勤邦生物技术有限公司 | Azoxystrobin hapten, artificial antigen and antibody, and preparation method and application thereof |
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| CN113406239A (en) * | 2021-06-30 | 2021-09-17 | 浙江省农业科学院 | Method for measuring fluoxastrobin and metabolite thereof in water |
| CN113406239B (en) * | 2021-06-30 | 2023-03-10 | 浙江省农业科学院 | Method for measuring fluoxastrobin and metabolite thereof in water |
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