CN105273003A - Isocarbophos semiantigen, and preparation method and application thereof - Google Patents
Isocarbophos semiantigen, and preparation method and application thereof Download PDFInfo
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- CN105273003A CN105273003A CN201510658709.5A CN201510658709A CN105273003A CN 105273003 A CN105273003 A CN 105273003A CN 201510658709 A CN201510658709 A CN 201510658709A CN 105273003 A CN105273003 A CN 105273003A
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Abstract
A disclosed isocarbophos semiantigen possesses a structural formula shown in the specification. In the formula, -R is -(CH2)5COOH or -PhCOOH. The isocarbophos semiantigen is designed aiming at isocarbophos and is applicable to prepare an isocarbophos antibody. Experiments prove that the prepared isocarbophos antibody is capable of specifically identifying isocarbophos with both relatively good accuracy and sensitivity. The invention also discloses a preparation method and application of the above isocarbophos semiantigen.
Description
Technical field
The present invention relates to immunological technique field, particularly a kind of isocarbophos haptens and its preparation method and application.
Background technology
Isocarbophos (isocarbophos, ICP), be a kind of broad spectrum organophosphorus Insecticidal and acaricidal agent, insecticide efficiency is high, easily decomposes, and is once once becoming one of main agricultural chemicals of disease and pest control in agriculture production; But a large amount of ICP that uses can cause the residual of agricultural chemicals, and by the enrichment of food chain, make animal and human poisoning, affect the metabolic balance of people's hormone in vivo, especially even more serious impact is produced on the normal development of nervous system in children.The ICP reported detects and mainly contains polarography and chromatography etc.But these methods need professional and technical personnel to operate, expensive equipment, not portable, be difficult to popularize.
In recent years, immuno analytical method has more and more been widely used in agricultural and veterinary chemicals retention analysis and has mixed pseudo-food qualification field, simple, cheap, quick, highly sensitive with it, be applicable to the advantages such as batch samples detection and become a kind of important analytical procedure.The current report not yet occurring the immunochemistry method of inspection for isocarbophos and product.
Summary of the invention
Based on this, be necessary to provide a kind of isocarbophos haptens for isocarbophos design and its preparation method and application.
A kind of isocarbophos haptens, has following structural formula:
-R is-(CH
2)
5cOOH or
The haptenic preparation method of a kind of isocarbophos, comprises the steps:
Exist at catalyzer and under alkaline condition, by isopropyl salicylate and O-methyl thio-phosphoryl dichloride hybrid reaction, after purifying, obtain O-methyl-O-2-isopropyl salicylate thiophosphoryl one chlorine; And
Under dioxane existent condition, described O-methyl-O-2-isopropyl salicylate thiophosphoryl one chlorine is stirred and dropwise adds 6-aminocaprolc acid or para-amino benzoic acid at the temperature of-5 DEG C ~ 0 DEG C, be warming up to room temperature after having added fully to react, after purifying, obtain the isocarbophos haptens with following structural formula:
-R is-(CH
2)
5cOOH or
In one embodiment, described catalyzer is quaternary ammonium salt phase transformation catalyzer.
A kind of isocarbophos complete antigen, has following structural formula:
Wherein ,-R is-(CH
2)
5cONH-Pro or
pro is carrier proteins.
In one embodiment, described carrier proteins is bovine serum albumin, human serum albumin, hemocyanin or ovalbumin.
A preparation method for isocarbophos complete antigen, comprises the steps:
The haptenic preparation method of isocarbophos according to any one of claim 3 ~ 4 is adopted to obtain isocarbophos haptens;
Described isocarbophos haptens is dissolved in organic solvent, then dicyclohexylcarbodiimide and N-hydroxy-succinamide is added under stirring, 6 ~ 12h is stirred at 0 ~ 4 DEG C, then centrifugal and retain supernatant liquor, described supernatant liquor is A liquid, wherein, the mol ratio of described isocarbophos haptens, described dicyclohexylcarbodiimide and described N-hydroxy-succinamide is 1:1.5 ~ 4:1.5 ~ 4;
Carrier proteins being dissolved in pH value is in the PBS damping fluid of 7.4 ~ 9.6, obtains B liquid; And
Be added in described B liquid by described A drop under stirring at 0 ~ 4 DEG C, fully after reaction, purifying obtains the isocarbophos complete antigen with following structural formula:
Wherein ,-R is-(CH
2)
5cONH-Pro or
pro is bovine serum albumin, human serum albumin, hemocyanin or ovalbumin.
In one embodiment, in the described operation be dissolved in by carrier proteins in PBS damping fluid, described carrier proteins is bovine serum albumin, human serum albumin, hemocyanin or ovalbumin;
The mol ratio of the described isocarbophos haptens in described A liquid and the described carrier proteins in described B liquid is 20 ~ 100:1.
A kind of isocarbophos antibody, described isocarbophos antibody adopts above-mentioned isocarbophos haptens or above-mentioned isocarbophos complete antigen to prepare.
In one embodiment, described isocarbophos antibody is polyclonal antibody, monoclonal antibody or genetic engineering antibody.
For detecting a preparation for isocarbophos, comprise above-mentioned isocarbophos haptens, above-mentioned isocarbophos complete antigen or above-mentioned isocarbophos antibody.
This isocarbophos haptens designs for isocarbophos, may be used for preparing isocarbophos antibody, can specific identification isocarbophos through the obtained isocarbophos antibody of experimental verification, and accuracy and sensitivity are all better.
Accompanying drawing explanation
Fig. 1 is the schema of the haptenic preparation method of isocarbophos of an embodiment;
Fig. 2 is the schema of the preparation method of the isocarbophos complete antigen of an embodiment;
Fig. 3 is the ELISA competition test graphic representation of the isocarbophos antibody obtained in embodiment 3.
Embodiment
Mainly in conjunction with the drawings and the specific embodiments isocarbophos haptens and its preparation method and application is described in further detail below.
A kind of isocarbophos haptens of one embodiment, has following structural formula:
-R is-(CH
2)
5cOOH or
The integrally-built hapten design of this reservation isocarbophos, may be used for preparing isocarbophos antibody.Can specific identification isocarbophos through the obtained isocarbophos antibody of experimental verification, accuracy and sensitivity are all better.
The haptenic preparation method of above-mentioned isocarbophos as shown in Figure 1, comprises the steps:
S110, catalyzer exist and alkaline condition under, by isopropyl salicylate and O-methyl thio-phosphoryl dichloride hybrid reaction, after purifying, obtain O-methyl-O-2-isopropyl salicylate thiophosphoryl one chlorine.
In S110, concrete reaction formula is as follows:
Wherein, catalyzer is quaternary ammonium salt phase transformation catalyzer.Quaternary ammonium salt phase transformation catalyzer can be Tetrabutyl amonium bromide.
Concrete, S110 is operating as: dissolved by isopropyl salicylate methylene dichloride, add appropriate quaternary ammonium salt-type phase transfer catalyst, be cooled to about 0 DEG C, the O-methyl thio-phosphoryl dichloride of amount such as dropwise add under stirring, finish and rise to room temperature, drip the sodium hydroxide solution of 20% of 4 ~ 6 times of amounts again, make reaction be alkaline environment, after reaction 4h, isolate methylene dichloride phase evaporate to dryness, cross column purification and obtain product O-methyl-O-2-isopropyl salicylate thiophosphoryl one chlorine.
Isopropyl salicylate can directly be bought, also can adopt and operate preparation as follows: appropriate Whitfield's ointment is put into round-bottomed flask, then the vitriol oil of 5 ~ 10 times of amounts and excessive Virahol is added successively, oil bath is cooled to room temperature thin up after being heated to 100 DEG C of condensing reflux 12h, be extracted with ethyl acetate, merge organic layer successively with saturated sodium bicarbonate, distilled water wash, organic over anhydrous sodium sulfate dewaters, and dry rear evaporate to dryness is concentrated obtains isopropyl salicylate.
O-methyl thio-phosphoryl dichloride can directly be bought, also can adopt and operate preparation as follows: measure phosphorus thiochloride in round-bottomed flask with graduated cylinder, the anhydrous methanol reaction 2 ~ 5h of 3 ~ 5 times of volumes is slowly added at-5 DEG C ~ 0 DEG C, leave standstill for 2 ~ 3 times with frozen water jolting, isolate organic over anhydrous sodium sulfate to dewater after drying and obtain colourless transparent liquid O-methyl thio-phosphoryl dichloride, cryopreservation is for subsequent use.
S120, under dioxane existent condition, O-methyl-O-2-isopropyl salicylate thiophosphoryl one chlorine obtained by S110 stirs and dropwise adds 6-aminocaprolc acid or para-amino benzoic acid at the temperature of-5 DEG C ~ 0 DEG C, be warming up to room temperature after having added fully to react, after purifying, obtain isocarbophos haptens.
The haptenic structural formula of isocarbophos is as follows:
-R is-(CH
2)
5cOOH or
The isocarbophos complete antigen of one embodiment is also provided, there is following structural formula:
Wherein ,-R is-(CH
2)
5cONH-Pro or
pro is carrier proteins.
Carrier proteins can be bovine serum albumin (BSA), human serum albumin, hemocyanin or ovalbumin (OVA).
This isocarbophos complete antigen adopts the isocarbophos haptens for isocarbophos design to obtain, and may be used for preparing isocarbophos antibody.Can specific identification isocarbophos through the obtained isocarbophos antibody of experimental verification, accuracy and sensitivity are all better.
The preparation method of above-mentioned isocarbophos complete antigen as shown in Figure 2, comprises the steps:
S210, the haptenic preparation method of above-mentioned isocarbophos is adopted to obtain isocarbophos haptens.
S220, the isocarbophos haptens obtained by S210 are dissolved in organic solvent, add dicyclohexylcarbodiimide and N-hydroxy-succinamide under then stirring, and stir 6 ~ 12h at 0 ~ 4 DEG C, and then centrifugal and retain supernatant liquor, supernatant liquor is A liquid.
In S220, the mol ratio of isocarbophos haptens, dicyclohexylcarbodiimide and N-hydroxy-succinamide is 1:1.5 ~ 4:1.5 ~ 4.
Organic solvent can be DMF (DMF).
In A liquid, the haptenic concentration of isocarbophos is 1 × 10
-5mol/L ~ 9 × 10
-5mol/L.
S230, carrier proteins is dissolved in pH value is in the PBS damping fluid of 7.4 ~ 9.6, obtains B liquid.
Carrier proteins can be bovine serum albumin, human serum albumin, hemocyanin or ovalbumin.
The pH of PBS damping fluid can be 7.4.
In B liquid, the concentration of carrier proteins can be 0.5 × 10
-6mol/L ~ 0.5 × 10
-5mol/L.
S240, stir at 0 ~ 4 DEG C under A drop that S220 is obtained be added in the B liquid that S230 obtains, fully after reaction, purifying obtains isocarbophos complete antigen.
Isocarbophos complete antigen has following structural formula:
Wherein ,-R is-(CH
2)
5cONH-Pro or
pro is bovine serum albumin, human serum albumin, hemocyanin or ovalbumin.
In S240, the mol ratio of the isocarbophos haptens in A liquid and the carrier proteins in B liquid is 20 ~ 100:1.
The isocarbophos antibody of one embodiment being also provided, preparing by adopting above-mentioned isocarbophos haptens or above-mentioned isocarbophos complete antigen.
Concrete, isocarbophos antibody is polyclonal antibody, monoclonal antibody or genetic engineering antibody.
Concrete, isocarbophos antibody can be that above-mentioned isocarbophos haptens or above-mentioned isocarbophos complete antigen are obtained by methods such as ascites immunity, myeloma cell fusion.
In S240, the purifying of isocarbophos complete antigen adopts the mode of normal saline dialysis to complete.
A kind of for detecting the preparation of isocarbophos (as ELISA method, colloidal gold method etc.), comprise above-mentioned isocarbophos haptens, above-mentioned isocarbophos complete antigen or above-mentioned isocarbophos antibody.
Be below specific embodiment, the various instrument occurred in embodiment and reagent if not otherwise specified, all adopt this area conventional instrument or reagent.Below in conjunction with specific embodiment, isocarbophos haptens and its preparation method and application is described in further detail.
The haptenic preparation of embodiment 1 isocarbophos
The haptenic synthesis of isocarbophos:
(1) 6g Whitfield's ointment is put into round-bottomed flask, then the Virahol of the 10mL vitriol oil and 15g is added successively, oil bath is cooled to room temperature thin up after being heated to 100 DEG C of condensing reflux 12h, be extracted with ethyl acetate, merge organic layer successively with saturated sodium bicarbonate, distilled water wash, organic over anhydrous sodium sulfate dewaters, and dry rear evaporate to dryness is concentrated obtains isopropyl salicylate.
(2) 10mL phosphorus thiochloride is measured in round-bottomed flask with graduated cylinder, the anhydrous methanol reaction 3h of 3 times of volumes is slowly added at-5 DEG C ~ 0 DEG C, leave standstill for 2 ~ 3 times with frozen water jolting, isolate organic over anhydrous sodium sulfate to dewater after drying and obtain colourless transparent liquid O-methyl thio-phosphoryl dichloride, cryopreservation is for subsequent use.
(3) 1.8g isopropyl salicylate dichloromethane solvent is taken, add 200mg Tetrabutyl amonium bromide, be cooled to about 0 DEG C, the O-methyl thio-phosphoryl dichloride of 1.65g is dropwise added under stirring, finish and rise to room temperature, then drip the sodium hydroxide solution that 10mL concentration is 4mol/L, make reacting liquid pH value be about 10, isolate methylene dichloride phase evaporate to dryness after reaction 4h, cross column purification and obtain product O-methyl-O-2-isopropyl salicylate thiophosphoryl one chlorine.
(4) 0.616mgO-methyl-O-2-isopropyl salicylate thiophosphoryl one chlorine is taken, add 10mL dioxane, stir at being placed in 0 DEG C, dropwise add the 6-aminocaprolc acid (or para-amino benzoic acid) that 4 are dissolved with NaOH and 300mg of 500mg under stirring, finish and be warming up to room temperature; TLC monitoring after reaction 4h, is adjusted to acidity with dilute hydrochloric acid by the pH value of solution after question response is complete, obtains light yellow oil with chloroform extraction evaporate to dryness, crosses column purification and obtains required isocarbophos haptens.
In embodiment, the isocarbophos haptens that O-methyl-O-2-isopropyl salicylate thiophosphoryl one chlorine and 6-aminocaprolc acid reaction obtain is designated as ICP-H1, the isocarbophos haptens that O-methyl-O-2-isopropyl salicylate thiophosphoryl one chlorine and para-amino benzoic acid reaction obtain is designated as ICP-H2.
The preparation of embodiment 2 isocarbophos artificial antigen
Antigen I CP-H1-BSA: by 15mg haptens ICP-H1, (N-hydroxy-succinamide and 15mgDCC are dissolved in 1mL dry DMF (DMF) 8.8mgNHS, stirred at ambient temperature reaction 18h, the centrifugal 5min of reaction solution 4000 turns/min, draws supernatant activation solution..Weigh the carbonate buffer solution (0.05mol/L that 120mgBSA is dissolved in 8mL again, pH9.6) in, slow gradation drips 400 μ L activation solutions, then stirred at ambient temperature reaction 3h, then yellow reaction liquid is loaded dialysis tubing, with PBS (phosphate buffered saline buffer 0.01mol/L, pH7.4) dialyse, every 4h changes liquid 1 time, changes liquid 7 ~ 8 times, dialyse the centrifugal 5min of rear 4000 turns/min, supernatant liquor-20 DEG C preservation.
Antigen I CP-H2-OVA: by 18mg haptens ICP-H2, (N-hydroxy-succinamide and 13.5mgDCC are dissolved in 1mL dry DMF (DMF) 8.2mgNHS, stirred at ambient temperature reaction 18h, the centrifugal 5min of reaction solution 4000 turns/min, draws supernatant activation solution..Weigh the carbonate buffer solution (0.05mol/L that 1150mgOVA is dissolved in 8mL again, pH9.6) in, slow gradation drips 400 μ L activation solutions, then stirred at ambient temperature reaction 3h, then yellow reaction liquid is loaded dialysis tubing, with PBS (phosphate buffered saline buffer 0.01mol/L, pH7.4) dialyse, every 4h changes liquid 1 time, changes liquid 7 ~ 8 times, dialyse the centrifugal 5min of rear 4000 turns/min, supernatant liquor-20 DEG C preservation.
The preparation of embodiment 3 isocarbophos monoclonal antibody.
1, animal immune.
Get Balb/c female mice in 7 ~ 8 week age (Guangdong Province's Experimental Animal Center), by the immunizing antigen of preparation and Freund's complete adjuvant balanced mix, after complete emulsification, abdominal part hypodermic, dosage is for containing ICP-H1-BSA100 μ g, subcutaneous multi-point injection after later getting artificial antigen and Freund's incomplete adjuvant mixing and emulsifying with same dosage every three weeks, measure it after immune four times to tire and inhibiting rate, choose the mouse all higher with inhibiting rate of tiring and carry out cytogamy, merge first 3 days doubling dose reinforced immunologicals once.
2, cytogamy and monoclonal antibody preparation.
Mouse myeloma SP2/0 cell is mixed with the ratio of 5:1 with splenocyte, merges under 50%PEG, washing, centrifugal after with HAT substratum suspend, be inoculated in 96 well culture plates containing feeder cell, at the CO of 37 DEG C 5%
2cultivate in incubator after 3 days and change liquid with HAT substratum, within the 10th day, change HT substratum into.When plate inner cell grows to 1/3 of culture hole area, indirect elisa method screening cell positive hole, using ICP-H2-OVA as envelope antigen during screening.Indirect ELISA evaluation and screening is used in positive hole further, and limiting dilution assay is cloned into approximately every hole <1 cell, test positive after 10 days and compete the cell strain that good mono-clonal hole gained cell strain is secrete monoclonal antibody.After hybridoma enlarged culturing, prepare for ascites.
3, antiserum(antisera) lowest detectable limit (LOD value) and half amount of suppression (IC
50value) detection.
With the working concentration of square formation volumetry determination coating antigen ICP-H2-OVA and isocarbophos antibody, the working concentration of coating antigen is 0.15 μ g/mL, and the working concentration of isocarbophos antibody is 1/32000.
Do experimental solutions with the ICP solution of different concns, its concentration is as follows: 0,0.3,1.2,4.8,9.6,19.2ng/mL, adopt 9 groups of parallel laboratory tests (n=3).
Indirect Competitive ELISA method: with the coated elisa plate of above-mentioned working concentration, experimental solutions and antibody-solutions are joined in enzyme plate aperture simultaneously, blank well and negative control hole are set simultaneously, 37 DEG C of incubation 40min, pour out liquid in hole, 5 times are washed with washings, enzyme plate is upside down on thieving paper and pats: add the ELIAS secondary antibody solution diluted, 37 DEG C of incubation 20min, 5 times are washed with washings, pat dry: add substrate chromophoric solution, 37 DEG C of incubation 10min, add stop buffer color development stopping, light absorption value OD is measured at wavelength 450nm place by microplate reader, with light absorption value OD for ordinate zou, with the log of ICP experimental solutions concentration
10value is X-coordinate, draws semilog canonical plotting, obtains Fig. 3.
As seen from Figure 3, typical curve has complete reverse-s shape shape, and has upper mounting plate and lower platform, the replicate(determination) number of times of typical curve 3 times, and experimental repeatability is good, and relative standard deviation is but within 15%.
Obtaining the lowest detectable limit of isocarbophos antibody in buffered soln (LOD value) by formulae discovery is 0.3ng/mL, half amount of suppression (IC
50value) be 5.2ng/mL, linearity range is 1.8 ~ 10.1ng/mL.The inventive method accuracy and sensitivity are described better, can be used for the detection of ICP in agricultural-food and food samples.
The above embodiment only have expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but therefore can not be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (10)
1. an isocarbophos haptens, is characterized in that, has following structural formula:
-R is-(CH
2)
5cOOH or
2. the haptenic preparation method of isocarbophos, is characterized in that, comprise the steps:
Exist at catalyzer and under alkaline condition, by isopropyl salicylate and O-methyl thio-phosphoryl dichloride hybrid reaction, after purifying, obtain O-methyl-O-2-isopropyl salicylate thiophosphoryl one chlorine; And
Under dioxane existent condition, described O-methyl-O-2-isopropyl salicylate thiophosphoryl one chlorine is stirred and dropwise adds 6-aminocaprolc acid or para-amino benzoic acid at the temperature of-5 DEG C ~ 0 DEG C, be warming up to room temperature after having added fully to react, after purifying, obtain the isocarbophos haptens with following structural formula:
-R is-(CH
2)
5cOOH or
3. the haptenic preparation method of isocarbophos according to claim 2, is characterized in that, described catalyzer is quaternary ammonium salt phase transformation catalyzer.
4. an isocarbophos complete antigen, is characterized in that, has following structural formula:
Wherein ,-R is-(CH
2)
5cONH-Pro or
pro is carrier proteins.
5. isocarbophos complete antigen according to claim 4, is characterized in that, described carrier proteins is bovine serum albumin, human serum albumin, hemocyanin or ovalbumin.
6. a preparation method for isocarbophos complete antigen, is characterized in that, comprises the steps:
The haptenic preparation method of isocarbophos according to any one of claim 3 ~ 4 is adopted to obtain isocarbophos haptens;
Described isocarbophos haptens is dissolved in organic solvent, then dicyclohexylcarbodiimide and N-hydroxy-succinamide is added under stirring, 6 ~ 12h is stirred at 0 ~ 4 DEG C, then centrifugal and retain supernatant liquor, described supernatant liquor is A liquid, wherein, the mol ratio of described isocarbophos haptens, described dicyclohexylcarbodiimide and described N-hydroxy-succinamide is 1:1.5 ~ 4:1.5 ~ 4;
Carrier proteins being dissolved in pH value is in the PBS damping fluid of 7.4 ~ 9.6, obtains B liquid; And
Be added in described B liquid by described A drop under stirring at 0 ~ 4 DEG C, fully after reaction, purifying obtains the isocarbophos complete antigen with following structural formula:
Wherein ,-R is-(CH
2)
5cONH-Pro or
pro is bovine serum albumin, human serum albumin, hemocyanin or ovalbumin.
7. the preparation method of isocarbophos complete antigen according to claim 6, it is characterized in that, in the described operation be dissolved in by carrier proteins in PBS damping fluid, described carrier proteins is bovine serum albumin, human serum albumin, hemocyanin or ovalbumin;
The mol ratio of the described isocarbophos haptens in described A liquid and the described carrier proteins in described B liquid is 20 ~ 100:1.
8. an isocarbophos antibody, is characterized in that, described isocarbophos antibody adopts isocarbophos haptens according to claim 1 or the isocarbophos complete antigen according to any one of claim 4 ~ 5 to prepare.
9. isocarbophos antibody according to claim 8, is characterized in that, described isocarbophos antibody is polyclonal antibody, monoclonal antibody or genetic engineering antibody.
10. one kind for detecting the preparation of isocarbophos, it is characterized in that, comprise isocarbophos haptens according to claim 1, the isocarbophos complete antigen according to any one of claim 4 ~ 5 or the isocarbophos antibody according to any one of claim 8 ~ 9.
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| US11198700B2 (en) | 2018-07-19 | 2021-12-14 | Arysta Lifescience Inc | Process for preparation of O,O dimethyl phosphoramidothioate and N-(methoxymethylsulfanylphosphoryl) acetamide |
| CN112595844A (en) * | 2020-11-17 | 2021-04-02 | 北京勤邦生物技术有限公司 | Test strip and method for detecting isocarbophos |
| CN112595844B (en) * | 2020-11-17 | 2023-07-07 | 北京勤邦科技股份有限公司 | Test strip and method for detecting fenpyrazamine |
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