CN101017165B - Method for preparing fenpropathrin artificial antigen - Google Patents
Method for preparing fenpropathrin artificial antigen Download PDFInfo
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- CN101017165B CN101017165B CN 200710020231 CN200710020231A CN101017165B CN 101017165 B CN101017165 B CN 101017165B CN 200710020231 CN200710020231 CN 200710020231 CN 200710020231 A CN200710020231 A CN 200710020231A CN 101017165 B CN101017165 B CN 101017165B
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- fenpropathrin
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Abstract
This invention relates to one cypermethrin human antigen process method in immune chemical analysis technique field, which comprises chrysanthemumic acid into alcohol liquid for centrifugation and taking for upper clear liquid for use; then taking cow blood serum protein and carbodiimide into sterilized water; then dripping by mixing into the protein liquid with chrysanthemumic acid and carbodiimide liquid for light shield reaction in ice; dripping remaining carbodiimide liquid into the above mixture liquid for continuing shield reaction; through centrifugation to take dialysis through cooling to get human antigen.
Description
Technical field
The present invention relates to a kind of preparation method of fenpropathrin artificial antigen, specifically utilize carbodlimide method, with Fenpropathrin [Fenpropathrin, (RS)-a-cyano group-3-benzyloxy phenoxy base 2,2,3, the 3-4-methyl cyclopropane carboxylic acid ester] a kind of very important precursor substance first cyanogen chrysanthemumic acid (2,2,3 in the production run, 3-Tetramethylcycloprop-ne-ne carboxylic acid) with the direct crosslinked method for preparing fenpropathrin artificial antigen of carrier molecule (protein), belongs to fields of immunochemistry analysis.
Background technology
In field of immunology, for the small-molecule substance that makes non-immunogenicity (often is called haptens, Hapten) can bring out generation antibody in animal body, to be artificial antigen often with this haptens and certain macromolecular carrier such as crosslinked be combined into such as protein or polypeptide, be injected into again and produce antibody in the animal body, after the purification antibody, promptly can be used for immunoassay, it is one of most active fields in current clinical medicine, food inspection, the pesticide residue analysis.
As everyone knows, the pyrethroid insectide stable in properties, insecticidal spectrum is wide, and the insecticidal activity height belongs to nerve toxicant, and application in the world is very extensive, also is one of fastest-rising agricultural chemicals of consumption in China.Wherein Fenpropathrin be use more a kind of.Fenpropathrin is medium to the toxicity of higher mammal, but to hydrobionts such as fish is high poison, and 1-2ug/L can cause fish death, and under the normal temperature half life period in bed mud longer, can there be certain potential risk to the aquatic ecological environment at water body environment and hydrobiont body accumulation and residual.Therefore the content detection of Fenpropathrin seems very important in water body environment or the aquatic organism.
The habitual in the world at present residual standard method of analysis of Fenpropathrin is gas chromatography (GC) method and high performance liquid chromatography (HPLC) method.But these methods need expensive instrument and reagent, and tediously long sample pretreatment process can't satisfy the needs of gross sample field quick detection.Attach great importance to enzyme linked immunological (ELISA) detection technique in recent years in the world, this method is quick, sensitive, has good selectivity and selectivity, and has saved the complicated sample pre-treatment step.
In order to set up the method for immunity of Fenpropathrin, at first must obtain the antibody of anti-Fenpropathrin.The Fenpropathrin molecular structure is simple, and molecular weight (349.4) is little, belongs to haptens, can not direct immunization produce antibody.Must at first itself and carrier protein couplet be prepared its comlete antigen, just can bring out animal and produce antibody.In prior art, once adopted carbodlimide method that Fenpropathrin and bovine serum albumin(BSA) (BSA) have been carried out direct coupling, but unsuccessful, reason is the no free carboxy of Fenpropathrin itself, can't with the amino dehydrated crosslinking on the BSA.So far there is not successful precedent owing to employing ELISA method mensuration Fenpropathrin is domestic, external document also only has the report of Wengatz, and its aromatic rings end at Fenpropathrin is introduced carboxyl and BSA coupling, but reaction conditions is relatively harsher, production efficiency is low, and the experimentation more complicated possesses.Therefore fenpropathrin artificial antigen is synthetic very important.
Summary of the invention
The objective of the invention is to overcome above-mentioned weak point, thereby a kind of preparation method of fenpropathrin artificial antigen is provided, it uses carbodiimide with first cyanogen chrysanthemumic acid and bovine serum albumin(BSA) coupling, constructs artificial conjugated antigen; This method is easy and simple to handle, and a step is finished reaction, has overcome shortcomings such as other method is loaded down with trivial details, throughput rate is low, poor specificity, for the immune analysis technology of setting up Fenpropathrin is laid a good foundation.
Main solution of the present invention is achieved in that
The preparation method of fenpropathrin artificial antigen of the present invention adopts following processing step:
1, get 50～200mg first cyanogen chrysanthemumic acid, be dissolved in the ethanolic solution of 1～5mL25%, centrifugal after dissolving 4～6h, get supernatant;
2, get 100～400mg bovine serum albumin(BSA) (BSA) and 50～200mg carbodiimide (EDC), be dissolved in respectively in the sterilized water of 5～10mL and 1～6mL;
3, in bovine serum albumin(BSA) (BSA) solution, slowly drip the first cyanogen chrysanthemumic acid supernatant of 1～5mL and carbodiimide (EDC) solution of 1～3mL while stirring, shading reaction 5～7h under 3.8～4.2 ℃ of ice baths;
4, in above-mentioned mixed liquor, drip remaining 1～3mL carbodiimide (EDC) solution again, continue shading reaction 23～25h down in 3.8～4.2 ℃ of ice baths;
5, after reaction finishes, after centrifugal, get supernatant in bag filter, in the PBS of 0.01mol/L, pH7.4 damping fluid, the 4～6d that dialyses under 3～5 ℃ of temperature changes dislysate every day 2 times, packing after dialysis finishes stays little part to identify, all the other are frozen in-18～-22 ℃.
Bovine serum albumin(BSA) of the present invention (BSA) can adopt ovalbumin (OVA).
Centrifugal speed of the present invention is: 3800～4200r/min, centrifugation time is: 8～12min.
The frozen time of the present invention: within six months.
Compared with the prior art the present invention has the following advantages:
The present invention selects the precursor substance first cyanogen chrysanthemumic acid of synthetic Fenpropathrin for use, under the effect of carbodiimide with the direct coupling of bovine serum albumin(BSA), through dialysis and freezing after obtain the artificial antigen of Fenpropathrin, the micromolecule that is connected on the product has haptenic structure, does not change its original configuration at all; By the method for uv scan, sds gel electrophoresis and immune animal, artificial antigen is synthesized successfully in confirmation; This method is easy and simple to handle, and a step is finished reaction, has overcome shortcomings such as other method is loaded down with trivial details, throughput rate is low, poor specificity, for the specific antibody that filters out it later on set up immune analysis method the basis is provided.
Embodiment
The preparation method of following fenpropathrin artificial antigen of the present invention will be further described in conjunction with the embodiments:
Embodiment one:
The preparation method of fenpropathrin artificial antigen of the present invention adopts following processing step:
Take by weighing 50mg first cyanogen chrysanthemumic acid [Fenpropathrin, (RS)-a-cyano group-3-benzyloxy phenoxy base 2,2,3, the 3-4-methyl cyclopropane carboxylic acid ester], be dissolved in the ethanolic solution of 2mL25% (percent by weight), dissolving is carried out behind the 4h centrifugal, (going up the 800 type centrifugation devices that industrial corporation of Nereid section produces), centrifugal speed: 4000r/min, centrifugation time: 10min, it is stand-by to get supernatant.Get 100mg bovine serum albumin(BSA) (BSA) and 50mg carbodiimide (EDC) again, be dissolved in respectively in the sterilized water of 5mL and 3mL, in bovine serum albumin(BSA) BSA solution, slowly drip the first cyanogen chrysanthemumic acid supernatant of 2mL and carbodiimide (EDC) solution of 2mL while stirring, shading reaction 6h under 4 ℃ of ice baths, in above-mentioned mixed liquor, drip remaining 1mL carbodiimide (EDC) solution again, continue shading reaction 24h down in 4 ℃ of ice baths.After reaction finishes, with the centrifugal 10min of 4000r/min, get supernatant (feed liquid of surplus is removed) in bag filter, 4 ℃ of dialysis 5d in the PBS of 0.01mol/L, pH7.4 damping fluid, change dislysate every day 2 times, packing after dialysis finishes stays little part to identify, all the other in-20 ℃ frozen, the frozen time: within six months.Obtain artificial antigen through freezing.
The evaluation of artificial antigen: ultraviolet spectroscopy, sds polyacrylamide electrophoresis and immune animal method are adopted in the evaluation of artificial immunity antigen.According to the synthetic ratio of immune complex, get a certain amount of 2mg/mL first cyanogen chrysanthemumic acid solution, 4mg/mL bovine serum albumin(BSA) (BSA) solution, the first cyanogen chrysanthemumic acid compound dilution that is equivalent to 4mg/mL albumen, with the PBS that contains 25% (percent by weight) ethanol is reference, bovine serum albumin(BSA) (BSA), first cyanogen chrysanthemumic acid and first cyanogen chrysanthemumic acid-BSA conjugates are carried out the uv scan of 200nm-800nm, and bovine serum albumin(BSA) (BSA) has absorption maximum at the 280nm place, and obtained the maximum absorption is 2.8; First cyanogen chrysanthemumic acid has absorption maximum at the 230nm place, and obtained the maximum absorption is 1.9; First cyanogen chrysanthemumic acid-BSA conjugates has absorption maximum at the 254nm place, and obtained the maximum absorption is 2.2.Obviously intersecting appears in three's UV scanning collection of illustrative plates, protein macromolecule contains different separately ultraviolet and visible absorption peak with chemical micromolecule, but in conjugate UV, visible light scanning optical spectrum, show the character of spectrogram superposition separately, Fenpropathrin and bovine serum albumin(BSA) (BSA) successful connection is described.
The SDS-polyacrylamide gel electrophoresis can be understood the size of molecular weight analyte from the swimming rate of sample.Molecular weight analyte is more little, and the swimming rate is big more.First cyanogen chrysanthemumic acid and bovine serum albumin(BSA) (BSA) if successful connection, molecular weight will be greatly, difference to some extent on the bands of a spectrum during electrophoresis.The visible bovine serum albumin(BSA) of electrophoresis result (BSA) and Fenpropathrin-BSA have comparatively approaching electrophoretic band, but the swimming rate of bovine serum albumin(BSA) (BSA) is slightly faster than conjugate, because the molecular weight of conjugate is slightly larger than bovine serum albumin(BSA) (BSA).
The immune animal proof has produced the antibody of anti-Fenpropathrin: (1) selects healthy new zealand white rabbit, immunizing antigen is diluted to 1.0mg/mL with physiological saline, suck 1mL in syringe, add the Freund's complete adjuvant (the 1st immunity) or the incomplete Freund (the 2nd to the 5th immunity) of equivalent, lead to valves with antigen and the abundant mixing and emulsifying of adjuvant by medical 3.The 1-5 time subcutaneous multiple spot immunity in back; The 6th time directly with immunizing antigen 0.3mL ear vein injecting immune.Be 7d each immune interval time, amounts to immune 42d.Immunity finishes the back and gets blood (about 0.5mL) from the ear vein of rabbit, is placed on 4 ℃ of refrigerator overnight.Took out in the 2nd day, 4000r/min is centrifugal, the antiserum about can about 0.2mL.Also can in refrigerator, place after 2 hours centrifugal.The antiserum assay method adopts ELISA microplate reader method.(2) indirect competitive ELISA test adds the envelope antigen of 100 μ L with the carbonate buffer solution dilution of pH9.6 in every hole of the ELISA Plate in every 12 hole, includes first cyanogen chrysanthemumic acid-OVA of 2.5 μ g, 37 ℃ hatch 1h after, moves into 4 ℃ of refrigerators placements and spends the night.(pH7.5 0.01mol/L) dries after the washing three times with the PBS damping fluid that contains 0.5%Tween-20 to take out the back.Preceding 1～2 hole at every adds the pH7.5 that 50 μ L contain 0.1% gelatin, 0.01mol/L the PBS dilution, 3～4 holes add the standard Fenpropathrin solution 50 μ L with diluent preparing, include 5 μ g Fenpropathrin standard items, 5～6 holes are with 3～4 holes, include 10 μ g Fenpropathrin standard items, 7～8 holes include 20 μ g Fenpropathrin standard items, 9～10 holes include 100 μ g Fenpropathrin standard items, 11～12 holes add 50 μ L dilutions, in preceding 10 holes, add antiserum 50 μ L then with the 1:400 of diluent preparing, 11～12 holes still add the dilution of 50 μ L, put into 37 ℃ of waters bath with thermostatic control after adding and hatch 1h, take out, the same with Tween-PBS damping fluid washing three times, every then hole adds the goat-anti rabbit ELIAS secondary antibody of 100 μ L with the 1:5000 of diluent preparing, hatch 1h in 37 ℃ of waters bath with thermostatic control, take out with Tween-PBS damping fluid washing three times, add o-phenylenediamine and the hydrogen peroxide substrate solution of 100 μ L with the preparation of pH5.0 citric acid, place 15min in 37 ℃ of waters bath with thermostatic control, every hole adds 50 μ L10% sulfuric acid, reads the OD value with the 490nm wavelength then on microplate reader, gets 6 hole average computation results.
Competition suppresses the ELISA test findings
| Hole count | 1-2 | 3-4 | 5-6 | 7-8 | 9-10 | 11-12 |
| OD 490Value | 1.62 | 1.16 | 0.98 | 0.78 | 0.42 | 0.10 |
Last table data declaration: the color that adds the Fenpropathrin hole is all than not adding the of light color of Fenpropathrin hole, comparison is according to hole depth, and along with the increase of the Fenpropathrin standard items that added, the color of enzyme-to-substrate reaction becomes more and more shallow, illustrate in the antiserum exist can with the antibody of Fenpropathrin reaction, thereby proof is exempted from the specific antibody that son has produced anti-Fenpropathrin, proves that further artificial antigen synthesizes successfully.
Embodiment two:
The preparation method of fenpropathrin artificial antigen of the present invention adopts following processing step:
Take by weighing 100mg first cyanogen chrysanthemumic acid [Fenpropathrin, (RS)-a-cyano group-3-benzyloxy phenoxy base 2,2,3,3-4-methyl cyclopropane carboxylic acid ester], be dissolved in the ethanolic solution of 3mL25%, centrifugal behind the dissolving 4h, get supernatant.Get 200mg ovalbumin (OVA) and 100mg carbodiimide (EDC), be dissolved in respectively in the sterilized water of 8mL and 4mL, in ovalbumin (OVA) solution, slowly drip the first cyanogen chrysanthemumic acid supernatant of 3mL and carbodiimide (EDC) solution of 3mL while stirring, shading reaction 6h under 4 ℃ of ice baths, in mixed liquor, drip remaining 1mL carbodiimide (EDC) solution again, continue shading reaction 24h down in 4 ℃ of ice baths.Reaction with the centrifugal 10min of 4000r/min, is got supernatant in bag filter after finishing, and 4 ℃ of dialysis 5d change dislysate every day 2 times in the PBS of 0.01mol/L, pH7.4 damping fluid, can obtain artificial antigen in freezing six months after dialysis finishes.Above-mentioned artificial antigen is adopted the authentication method of artificial antigen among the embodiment 1, method by uv scan, sds gel electrophoresis and immune animal, confirm that artificial antigen synthesize successfully, for the specific antibody that filters out it later on set up immune analysis method and provide basic.
Embodiment three:
The preparation method of fenpropathrin artificial antigen of the present invention adopts following processing step:
Take by weighing 50mg first cyanogen chrysanthemumic acid [Fenpropathrin, (RS)-a-cyano group-3-benzyloxy phenoxy base 2,2,3,3-4-methyl cyclopropane carboxylic acid ester], be dissolved in the ethanolic solution of 2mL25%, centrifugal behind the dissolving 4h, get supernatant.Get 100mg bovine serum albumin(BSA) (BSA) and 100mg carbodiimide (EDC), be dissolved in respectively in the sterilized water of 5mL and 3mL, in bovine serum albumin(BSA) (BSA) solution, slowly drip the first cyanogen chrysanthemumic acid supernatant of 2mL and carbodiimide (EDC) solution of 2mL while stirring, shading reaction 6h under 4 ℃ of ice baths, in mixed liquor, drip remaining 1mL carbodiimide (EDC) solution again, continue shading reaction 24h down in 4 ℃ of ice baths.After reaction finishes, with the centrifugal 10min of 4000r/min, get supernatant in bag filter, 4 ℃ of dialysis 5d change dislysate every day 2 times in the PBS of 0.01mol/L, pH7.4 damping fluid, and dialysis finishes, and the back is freezing to obtain artificial antigen.Replace BSA with ovalbumin (OVA) and can make envelope antigen equally.The artificial antigen that obtains in the present embodiment is adopted the authentication method of artificial antigen among the embodiment 1, and by the method for uv scan, sds gel electrophoresis and immune animal, same susceptible of proof artificial antigen is synthesized successfully.Shortcomings such as other method is loaded down with trivial details, throughput rate is low, poor specificity have been overcome, for the immune analysis technology of setting up Fenpropathrin is laid a good foundation.
Claims (4)
1. the preparation method of a fenpropathrin artificial antigen is characterized in that: adopt following processing step:
(1), get 50～200mg first cyanogen chrysanthemumic acid, be dissolved in the ethanolic solution of 1～5mL 25%, centrifugal after dissolving 4～6h, get supernatant;
(2), get 100～400mg bovine serum albumin(BSA) and 50～200mg carbodiimide, be dissolved in respectively in the sterilized water of 5～10mL and 1～6mL;
(3), in bovine serum albumin solution, slowly drip the carbodiimide solution of the supernatant described in 1～5mL step (1) and 1～3mL while stirring, shading reaction 5～7h under 3.8～4.2 ℃ of ice baths;
(4), again in above-mentioned mixed liquor, drip remaining carbodiimide solution, continue shading reaction 23～25h down in 3.8～4.2 ℃ of ice baths;
(5), after reaction finishes, after centrifugal, get supernatant in bag filter, in the PBS of 0.01mol/L, pH7.4 damping fluid, the 4～6d that under 3～5 ℃ of temperature, dialyses, the dialysis back is frozen in-18～-22 ℃.
2. the preparation method of fenpropathrin artificial antigen according to claim 1 is characterized in that described bovine serum albumin(BSA) can adopt ovalbumin to replace.
3. the preparation method of fenpropathrin artificial antigen according to claim 1, it is characterized in that described centrifugal speed is: 3800～4200r/min, centrifugation time is: 8～12min.
4. the preparation method of fenpropathrin artificial antigen according to claim 1 is characterized in that the described frozen time: within six months.
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| CN102363634A (en) * | 2011-11-16 | 2012-02-29 | 厦门出入境检验检疫局检验检疫技术中心 | Method for preparing pyrethroid pesticide polyclonal antibody |
| CN106918705B (en) * | 2017-01-22 | 2023-06-13 | 贵州勤邦食品安全科学技术有限公司 | Test paper for detecting fenpropathrin and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1062348A (en) * | 1990-12-13 | 1992-07-01 | 中国科学院大连化学物理研究所 | From the Tetramethylcycloprop-ne-ne carboxylic acid cyanhydrin process for preparing methyl-cyanide chrysanthester |
| CN1070186A (en) * | 1991-08-30 | 1993-03-24 | 中国科学院大连化学物理研究所 | A kind of synthetic method of Fenvalerate |
| CN1357538A (en) * | 2000-12-06 | 2002-07-10 | 南开大学 | Prepn. of fenpropathrin |
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- 2007-03-09 CN CN 200710020231 patent/CN101017165B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1062348A (en) * | 1990-12-13 | 1992-07-01 | 中国科学院大连化学物理研究所 | From the Tetramethylcycloprop-ne-ne carboxylic acid cyanhydrin process for preparing methyl-cyanide chrysanthester |
| CN1070186A (en) * | 1991-08-30 | 1993-03-24 | 中国科学院大连化学物理研究所 | A kind of synthetic method of Fenvalerate |
| CN1357538A (en) * | 2000-12-06 | 2002-07-10 | 南开大学 | Prepn. of fenpropathrin |
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