CN107163081A - A kind of chlopyrifos antigen and preparation method thereof - Google Patents
A kind of chlopyrifos antigen and preparation method thereof Download PDFInfo
- Publication number
- CN107163081A CN107163081A CN201710383757.7A CN201710383757A CN107163081A CN 107163081 A CN107163081 A CN 107163081A CN 201710383757 A CN201710383757 A CN 201710383757A CN 107163081 A CN107163081 A CN 107163081A
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- Prior art keywords
- chlopyrifos
- haptens
- antigen
- carrier protein
- reaction
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- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- BMGGTWMJGOWATJ-PPVWGBJPSA-N CCOP(OCC)(OC([C@H](C1)Cl)=NC(SCCC2O[C@@H]2O)=C1Cl)S Chemical compound CCOP(OCC)(OC([C@H](C1)Cl)=NC(SCCC2O[C@@H]2O)=C1Cl)S BMGGTWMJGOWATJ-PPVWGBJPSA-N 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
- C07F9/02—Phosphorus compounds
- C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
- C07F9/553—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having one nitrogen atom as the only ring hetero atom
- C07F9/576—Six-membered rings
- C07F9/58—Pyridine rings
-
- 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
Abstract
The present invention relates to a kind of chlopyrifos antigen and preparation method thereof, the chlopyrifos antigen is obtained by chlopyrifos haptens with carrier protein by amido link coupling.The synthetic method of chlopyrifos antigen provided by the present invention is simple, and purity is high, and yield is high, and its preparation and chlorpyrifos pesticide residue detection for chlopyrifos antibody has important value.
Description
Technical field
The present invention relates to technical field of biochemical industry, more particularly to a kind of chlopyrifos antigen and preparation method thereof.
Background technology
Chlopyrifos [chlorpyrifos, O, O- diethyl-O- (3,5,6- trichloro-2-pyridyl) phosphorothionate], business
The name of an article is that chlopyrifos, Le Siben, termite are clear etc., and chlopyrifos active compound is white granular crystal, stablizes at room temperature, there is mercaptan gas
Taste, proportion 1.398 (43.5 DEG C), 41.5~43.5 DEG C of fusing point, the solubility in water is 1.2 × 10-5G/kg, is dissolved in most of
Organic solvent.By tagging, stomach toxicity and stifling three kinds of modes of action preventing and treating grains, fruit tree, vegetables and the evil of other industrial crops
Worm, after the approach such as skin, respiratory tract and stomach absorption enter human body, can rapidly be combined with cholinesterase, form phosphinylidyne
Change cholinesterase, the ability for making it lose catalyzing hydrolysis acetylcholine causes substantial amounts of acetylcholine to be accumulated in vivo, and suppress
Only Acetylcholinesterasein, makes central nervous system and cholinergic nerve be overexcited and poisoning symptom occur, for a long time
Effect on human body can also cause teratogenesis, carcinogenic, mutagenesis harm.
Chlopyrifos is widely used today in agricultural production, as a kind of broad spectrum type organophosphorus ester insecticides, is
One of the new and effective of the high-toxic pesticide such as acephatemet and parathion-methyl, low toxicity substitute species, are also that Safe Agricultural Product agricultural chemicals is residual
Stay the important kind of monitoring；Moreover, finding that the situation of chlopyrifos residue is also on the rise in environmental sample, to the healthy structure of people
Into potential threat.Residues of pesticides are food-safe to cause serious threat, the Fast Detection Technique of persticide residue by
People more and more widely pay close attention to.
The residual method of domestic detection agriculture is generally gas-chromatography and liquid chromatography at present, and these methods not only need costliness
Instrument, and sample pretreatment is cumbersome, it is impossible to meet fast-field evaluation agriculture it is residual the need for.Therefore, a kind of residues of pesticides are studied
Rapid assay methods have great importance.Immuno analytical method is the detection residual new method of agriculture developed in recent years, tool
There is easy, quick, sensitive and high specificity.Wherein, enzyme linked immunosorbent assay (ELISA) technology Enzyme-Linked
Immunosorbent Assay (ELISA), are a kind of ultramicron that immunological technique is combined and set up with modern means of testing
Assay method, with the characteristics of cost is low, speed is fast, sensitivity is high, instrument and equipment is simple, be adapted to the quick of batch samples
Analysis.And the basis of immunological detection method is corresponding high specific, the preparation of the antibody of high-affinity, and chlopyrifos conduct
Micromolecular compound, itself does not possess immunogenicity, therefore, and structure of modification and the holoantigen synthesis of its haptens are to set up immune
One of key and difficult point of quick test technique.
The content of the invention
In view of problems of the prior art, an object of the present invention is to provide a kind of chlopyrifos antigen and its system
Preparation Method, the synthetic method of chlopyrifos antigen provided by the present invention is simple, and purity is high, yield is high, for chlopyrifos antibody
Prepare and chlorpyrifos pesticide residue detection has important value.
For up to this purpose, the present invention uses following technical scheme：
In a first aspect, the invention provides a kind of chlopyrifos haptens, the chemical structural formula of the chlopyrifos haptens is such as
Shown in formula (I)：
Second aspect, present invention also offers the preparation method of chlopyrifos haptens as described in relation to the first aspect, it includes
Following steps：
By chlopyrifos and mercaptopropionic acid according to 1:The molar ratio reaction of (2~2.5), obtains the chlopyrifos haptens.
The reaction scheme of heretofore described chlopyrifos haptens is as follows：
In the present invention, the mol ratio of the chlopyrifos and mercaptopropionic acid is 1:(2~2.5), such as 1:2、1:2.05、1:
2.1、1:2.15、1:2.2、1:2.25、1:2.3、1:2.35、1:2.4、1:2.45 or 1:2.5.
Preferably, the reaction of the chlopyrifos and mercaptopropionic acid is carried out in organic solvent.
Preferably, the organic solvent is the ethanol solution containing NaOH.
Preferably, the time of the reaction is 2~4h, such as 2h, 2.2h, 2.5h, 3h, 3.2h, 3.5h or 4h.
Preferably, the preparation method of the chlopyrifos haptens can specifically include following steps：
(1) NaOH is added while stirring into the ethanol solution of chlopyrifos, add the ethanol solution of mercaptopropionic acid, poison with poison
The mol ratio of tick and mercaptopropionic acid is 1:(2~2.5), 2~4h of back flow reaction；
(2) after reacting completely, suction filtration goes out solid, and filtrate rotary evaporation is done, and 5% NaHCO is added in residue3Solution,
With n-hexane extraction, organic phase is discarded, aqueous phase adjusts pH value to be extracted three times to 3~4, then with dichloromethane with hydrochloric acid, and organic phase is used
Anhydrous Na SO4After being dried overnight, rotary evaporation is done, and is crossed silicagel column purifying, is obtained the chlopyrifos haptens.
The third aspect, present invention also offers a kind of chlopyrifos antigen, the chlopyrifos antigen is as described in first aspect
Chlopyrifos haptens is obtained with carrier protein couplet.
Preferably, the chlopyrifos haptens is coupled with carrier protein by amido link.
Preferably, the mol ratio of the chlopyrifos haptens and carrier protein couplet is (25~30):1, such as 25:1、
25.5:1、26:1、26.5:1、27:1、27.5:1、28:1、28.5:1、29:1、29.5:1 or 30:1, preferably 30:1.
Preferably, the carrier protein is bovine serum albumin(BSA) (BSA).
Preferably, shown in the chemical structural formula such as formula (II) of the chlopyrifos antigen：
Fourth aspect, present invention also offers the preparation method of the chlopyrifos antigen as described in the third aspect, it include with
Lower step：
By the chlopyrifos haptens, carrier protein, tri-n-butylamine and isobutyl chlorocarbonate in borate buffer solution it is anti-
Should, obtain the chlopyrifos antigen.
Preferably, the carrier protein, chlopyrifos haptens, tri-n-butylamine, isobutyl chlorocarbonate and borate buffer solution
Proportioning be：100mg:0.09mol:23μl:11μl:5ml.
Preferably, the pH value of the borate buffer solution is 8.5, and its concentration is 0.1mol/L.
Compared with prior art, the present invention at least has the advantages that：
The synthetic method of chlopyrifos antigen provided by the present invention is simple, and single step reaction can complete to prepare, and be made
Chlopyrifos antigen purity it is high, yield is high, and its preparation and chlorpyrifos pesticide residue detection for chlopyrifos antibody has weight
It is worth.
Brief description of the drawings
Fig. 1 is the ESI collection of illustrative plates for the chlopyrifos haptens that embodiment 1 is prepared；
Fig. 2 is that ultraviolet after the chlopyrifos haptens that embodiment 1 is prepared is coupled with carrier protein by amido link is swept
Tracing.
The present invention is described in more detail below.But following examples is only the simple example of the present invention, not generation
Table or limitation the scope of the present invention, protection scope of the present invention are defined by claims.
Embodiment
Further illustrate technical scheme below in conjunction with the accompanying drawings and by embodiment.
For the present invention is better described, technical scheme is readily appreciated, of the invention is typical but non-limiting
Embodiment is as follows：
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material, reagent used in following embodiments etc..Unless otherwise specified, commercially obtain.
PH value used in following embodiments is that the formula that 8.5, concentration is 0.1mol/L borate buffer solutions is as follows：Claim
2.84g boric acid, 5.15g boraxs, 1L is settled to distilled water.
PH value used in following embodiments is that 7.4, concentration is that 0.01M phosphate-buffered formula of liquid is as follows：Claim 7.9g
NaCl、0.2g KCl、0.24g KH2PO4With 1.8g K2HPO4, it is dissolved in 800ml distilled water, the pH value of solution is adjusted with HCl
To 7.4, finally distilled water is added to be settled to 1L.
The preparation of the chlopyrifos haptens of embodiment 1
The building-up process of chlopyrifos haptens is as follows：
The synthetic method of chlopyrifos haptens, comprises the following steps：
In 100ml reaction bulbs, weigh 1.75g (5mmol) chlopyrifos and be dissolved in 25ml ethanol, weigh 0.53g (5mmol) mercapto
Base propionic acid is dissolved in 25ml ethanol, is added drop-wise in chlopyrifos solution, and adds 0.71g NaOH, back flow reaction 3h, TLC monitoring reaction
Process.After the completion of reaction, suction filtration goes out white solid, and filtrate is spin-dried for after solvent, adds 25ml 5%NaHCO3Dissolving, uses 2*50ml
N-hexane extraction, collects aqueous phase.Aqueous phase adjusts pH value to be extracted to 2~3, then with dichloromethane 3*50ml with 6M HCl, collects organic
Phase, organic phase is stayed overnight with anhydrous sodium sulfate drying.Suction filtration goes out solid, is spin-dried for organic phase, crosses silicagel column.Eluent is n-hexane:
Ethyl acetate=8:1, product section is collected, collection liquid is spin-dried for, obtained product is chlopyrifos haptens, its chemical structural formula
As shown in formula (I).
Fig. 1 is the ESI collection of illustrative plates for the chlopyrifos haptens that the present embodiment is prepared, the ESI molecular ions of the synthetic product
Peak is 442.1 (M+23), and 402.3 is remove the quasi-molecular ions after hydroxyl, it can be seen that chlopyrifos haptens is successfully closed
Into.
The preparation of the chlorpyrifos artificial antigen of embodiment 2
1st, the preparation of chlorpyrifos artificial antigen
The chlopyrifos haptens 30mg (0.071mmol) for taking 30mg to be prepared by embodiment 1, is dissolved in 1.5ml DMF, 4 DEG C
Stirring is lower to be added after 18 μ l tri-n-butylamines, reaction 30min, is added 9 μ l isobutyl chlorocarbonates, is reacted at room temperature 1h, after completion of the reaction
As A liquid；BSA 79mg are weighed, are dissolved in borate buffer solution, referred to as B liquid；A liquid is added dropwise in B liquid, side edged is stirred
Mix, put magnetic agitation reaction under room temperature (25 DEG C) and stay overnight (12h), supernatant, 4 DEG C of 0.01M, pH7.4 phosphate-buffereds are taken after centrifugation
Dialysed 3 days in liquid, dialyzate is changed daily 2 times, obtain chlorpyrifos artificial antigen, freezen protective.
2nd, the identification of chlorpyrifos artificial antigen
By above-mentioned product 0.01M, pH7.4 phosphate buffers wiring solution-forming carries out ultraviolet (200~400nm) scanning,
Comlete antigen ultraviolet spectra has characteristic absorption peak at 277.5nm, and bovine serum albumin(BSA) (BSA) has characteristic absorption in 264.5nm
Peak, and haptens has characteristic absorption peak at 250nm, the ultraviolet absorpting spectrum of comlete antigen is different from body protein published originally and half and resisted
Former UV scanning collection of illustrative plates, illustrates that chlopyrifos haptens is successfully coupled (see Fig. 2) with carrier protein.
By the above results as can be seen that the present invention has successfully prepared chlorpyrifos artificial antigen, the letter of its synthetic method
Single, single step reaction can complete to prepare, and obtained chlopyrifos antigen purity is high, and yield is high, and it is for chlopyrifos antibody
Prepare and chlorpyrifos pesticide residue detection has important value.
Applicant states that the present invention illustrates the detailed construction feature of the present invention by above-described embodiment, but the present invention is simultaneously
Above-mentioned detailed construction feature is not limited to, that is, does not mean that the present invention has to rely on above-mentioned detailed construction feature and could implemented.Institute
Belong to those skilled in the art it will be clearly understood that any improvement in the present invention, to the equivalence replacement of part selected by the present invention
And increase, the selection of concrete mode of accessory etc., within the scope of all falling within protection scope of the present invention and being open.
The preferred embodiment of the present invention described in detail above, still, the present invention are not limited in above-mentioned embodiment
Detail, in the range of the technology design of the present invention, a variety of simple variants can be carried out to technical scheme, this
A little simple variants belong to protection scope of the present invention.
It is further to note that each particular technique feature described in above-mentioned embodiment, in not lance
In the case of shield, can be combined by any suitable means, in order to avoid unnecessary repetition, the present invention to it is various can
The combination of energy no longer separately illustrates.
In addition, various embodiments of the present invention can be combined randomly, as long as it is without prejudice to originally
The thought of invention, it should equally be considered as content disclosed in this invention.
Claims (9)
1. a kind of chlopyrifos haptens, it is characterised in that shown in the chemical structural formula such as formula (I) of the chlopyrifos haptens：
2. the preparation method of chlopyrifos haptens as claimed in claim 1, it is characterised in that comprise the following steps：
By chlopyrifos and mercaptopropionic acid according to 1:The molar ratio reaction of (2~2.5), obtains the chlopyrifos haptens.
3. method as claimed in claim 2, it is characterised in that the reaction is carried out in organic solvent；
Preferably, the organic solvent is the ethanol solution containing NaOH；
Preferably, the time of the reaction is 2~4h.
4. method as claimed in claim 2 or claim 3, it is characterised in that methods described comprises the following steps：
(1) add NaOH while stirring into the ethanol solution of chlopyrifos, add the ethanol solution of mercaptopropionic acid, chlopyrifos with
The mol ratio of mercaptopropionic acid is 1:(2~2.5), 2~4h of back flow reaction；
(2) after reacting completely, suction filtration goes out solid, and filtrate rotary evaporation is done, and 5% NaHCO is added in residue3Solution, with just oneself
Alkane is extracted, and discards organic phase, and aqueous phase adjusts pH value to be extracted three times to 3~4, then with dichloromethane with hydrochloric acid, and organic phase is with anhydrous
NaSO4After being dried overnight, rotary evaporation is done, and is crossed silicagel column purifying, is obtained the chlopyrifos haptens.
5. a kind of chlopyrifos antigen, it is characterised in that the chlopyrifos antigen as the chlopyrifos haptens described in claim 1 with
Carrier protein couplet is obtained.
6. chlopyrifos antigen as claimed in claim 5, it is characterised in that the chlopyrifos haptens passes through acyl with carrier protein
Amine key is coupled；
Preferably, the mol ratio of the chlopyrifos haptens and carrier protein couplet is (25~30):1.
7. the chlopyrifos antigen as described in claim 5 or 6, it is characterised in that the carrier protein is bovine serum albumin(BSA)；
Preferably, shown in the chemical structural formula such as formula (II) of the chlopyrifos antigen：
8. the preparation method of the chlopyrifos antigen as described in one of claim 5-7, it is characterised in that comprise the following steps：
The chlopyrifos haptens, carrier protein, tri-n-butylamine and isobutyl chlorocarbonate are reacted in borate buffer solution, obtained
To the chlopyrifos antigen.
9. method as claimed in claim 8, it is characterised in that the carrier protein, chlopyrifos haptens, tri-n-butylamine, chlorine
The proportioning of iso-butyl formate and borate buffer solution is：100mg:0.09mol:23μl:11μl:5ml；
Preferably, the pH value of the borate buffer solution is 8.5, and its concentration is 0.1mol/L.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710383757.7A CN107163081A (en) | 2017-05-26 | 2017-05-26 | A kind of chlopyrifos antigen and preparation method thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710383757.7A CN107163081A (en) | 2017-05-26 | 2017-05-26 | A kind of chlopyrifos antigen and preparation method thereof |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1431213A (en) * | 2003-01-13 | 2003-07-23 | 浙江大学 | Method for preparing artificial hapten, artificial antigen and specific antibody of chlorpyrifos and its usage |
| CN101477115A (en) * | 2008-12-18 | 2009-07-08 | 孙家隆 | Preparation of chlorpyrifos and methyl parathion universal antibody and universal envelope antigen |
| CN102053155A (en) * | 2010-06-29 | 2011-05-11 | 华中农业大学 | Homogeneous chemiluminescence immunoassay method for measuring for organophosphorus pesticide Dursban |
-
2017
- 2017-05-26 CN CN201710383757.7A patent/CN107163081A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1431213A (en) * | 2003-01-13 | 2003-07-23 | 浙江大学 | Method for preparing artificial hapten, artificial antigen and specific antibody of chlorpyrifos and its usage |
| CN101477115A (en) * | 2008-12-18 | 2009-07-08 | 孙家隆 | Preparation of chlorpyrifos and methyl parathion universal antibody and universal envelope antigen |
| CN102053155A (en) * | 2010-06-29 | 2011-05-11 | 华中农业大学 | Homogeneous chemiluminescence immunoassay method for measuring for organophosphorus pesticide Dursban |
Non-Patent Citations (4)
| Title |
|---|
| ANGEL MONTOYA ET AL.: "Development of a Chlorpyrifos Immunoassay Using Antibodies Obtained from a Simple Hapten Design", 《J. AGRIC. FOOD CHEM.》 * |
| ROSA PUCHADES ET AL.: "Highly Sensitive Enzyme-Linked Immunosorbent Assay for Chlorpyrifos. Application to Olive Oil Analysis", 《J. AGRIC. FOOD CHEM.》 * |
| 刘冰等: "毒死蜱人工抗原的合成及多克隆抗体制备", 《现代农药》 * |
| 朱国念等: "有机磷杀虫剂毒死蜱人工抗原的合成与鉴定", 《中国农业科学》 * |
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