CN104744285A - Synthesis method for dioctyl phthalate hapten and artificial antigen and application thereof - Google Patents
Synthesis method for dioctyl phthalate hapten and artificial antigen and application thereof Download PDFInfo
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- CN104744285A CN104744285A CN201410336147.8A CN201410336147A CN104744285A CN 104744285 A CN104744285 A CN 104744285A CN 201410336147 A CN201410336147 A CN 201410336147A CN 104744285 A CN104744285 A CN 104744285A
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Abstract
The invention discloses a synthesis method for a dioctyl phthalate hapten and an artificial antigen and application thereof, and belongs to the technical field of immunological detection. The synthesis method disclosed by the invention comprises the following steps: preparing an intermediate product containing an amino group from DEHP used as a raw material through a nitration reaction and a reduction reaction, and then reacting with a reagent such as a halogenated acid salt to generate the hapten with a carboxyl group; and then coupling with proteins to prepare the artificial antigen through an active lipid method or a mixed anhydride method. The artificial antigen can be used for preparing an anti-DEHP specific antibody in animal immunization, and has important practical significance on establishment for an ELISA analysis method for detecting DEHP.
Description
Technical field
The present invention relates to technical field of immunological detection, in particular, the present invention relates to a kind of dioctyl phthalate (DOP) haptens and synthesizing artificial antigen and application thereof.
Background technology
Phthalic acid two (α-ethyl hexyl) ester (Di (2-ethylhexyl) phthalate), english abbreviation DEHP, have another name called dioctyl phthalate (DOP), molecular weight is 390.5, these product solubleness <0.01% in water when 25 DEG C, be dissolved in most of organic solvent and hydro carbons, its structural formula is:
DEHP belongs to phthalate fluidizer, is a kind of industrial raw material, is to use the widest and that output is maximum fluidizer at present.DEHP can by food and water absorb, the food contacted with containing DEHP container also can be polluted, and also has illegal manufacturer to it can be used as additive for foodstuff production.
The World Health Organization points out that phthalate enters in human body and animal body and has similar estrogenic effect, meeting disturbance endocrine, endocrine disturbance can be caused, encumber organism Reproductive Performance, comprise reproduction rate reduction, miscarriage, congenital defect, abnormal sperm count, damage of testis, also can cause malignant tumour or cause teratogenesis.Clinical study shows to take or long-term exposure also can have significant impact for myocardial cell in DEHP, causes heart irregular rhythm.Therefore, DEHP belongs to violated industrial raw material in food.
To the detection of the phthalate fluidizer in food mainly based on physico-chemical analysis method (as GC-MS, HPLC).But during application instrumental method, its sensitivity is very large by the impact of the purification of sample, the step such as concentrated, and measuring method is complicated, loaded down with trivial details, detection flux is little, need cultivate professional personnel to detect, testing cost is expensive, can not realize the rapid detection analysis of batch samples.Therefore, simple and fast detection method easily is more necessary to develop.
Immunologic detection method, be one of important method of current food safety rapid detection, testing cost is very low, uses simple, is applicable to the selective mechanisms of a large amount of sample, in food safety detection, has played vital role.
Summary of the invention
The technical problem that the present invention solves is to provide a kind of dioctyl phthalate (DOP) haptens and synthesizing artificial antigen and application thereof, provides immunizing antigen and envelope antigen and antibody preparation to lay the foundation for setting up inspection-free detection method.
For solving the problems of the technologies described above, the present invention adopts following technical scheme:
The haptenic molecular structural formula of a kind of DEHP is:
N is the natural number of 1 ~ 6.
Described DEHP haptens is combined with carrier proteins, obtains DEHP artificial antigen, and its molecular structural formula is:
N is the natural number of 1 ~ 6; Described n is preferably 2; Described carrier proteins is bovine serum albumin, ovalbumin or hemocyanin.
The haptenic synthetic method of described DEHP, comprises the following steps:
(1) be that concentrated nitric acid and DEHP are placed in round-bottomed flask by 2:1 ~ 3:1 by feed ratio, be added drop-wise to 2 times under condition of ice bath in the vitriol oil of concentrated nitric acid amount, magnetic agitation, reacts to room temperature, then 60 DEG C of back flow reaction 5h, and period is monitored with TLC.With the sodium hydroxide solution neutralization reaction liquid of 1moL/L to weakly alkaline, extraction into ethyl acetate 3 times, organic phase evaporated under reduced pressure on Rotary Evaporators obtains thick nitration product DEHP-NO
2, cross silicagel column, collect elutriant to be V/V be the chloroform/hexanaphthene wash-out phase of 2:1 ~ 3:1, elutriant is concentrated into dry, obtains the nitration product DEHP-NO of purifying
2;
(2) be that 1:3:3 is by DEHP-NO by feed ratio
2, NH
4cl, iron powder are placed in round-bottomed flask, and add in 70% appropriate ethanolic soln, magnetic agitation, 50 DEG C of back flow reaction 5h, period is monitored with TLC; After reaction terminates, in reaction solution, add 100mL dehydrated alcohol, elimination iron powder, collect filtrate, concentrated near dry in Rotary Evaporators; In concentrated solution, add 30mL water, be extracted with ethyl acetate 3 times, collect organic phase, evaporate to dryness, obtains crude product DEHP-NH
2; Cross silicagel column, collect elutriant to be V/V be the chloroform/methanol wash-out phase of 5:1 ~ 4:1, elutriant is concentrated into dry, obtains the reduzate DEHP-NH of purifying
2;
(3) be that 1:2 ~ 1:3 is by DEHP-NH by feed ratio
2be placed in round-bottomed flask with halo hydrochlorate, and by a small amount of anhydrous alcohol solution of its mixture, add the salt of wormwood with halo hydrochlorate equivalent simultaneously, back flow reaction at 60 DEG C, appropriate potassiumiodide is added as catalyzer in reaction process, after return stirring reaction 5h, stopped reaction.Filter reaction mixture and obtain yellowish brown filtrate, concentrate and obtain brown color grease-like material.Cross silicagel column, collect elutriant to be V/V be the chloroform/methanol wash-out phase of 4:1 ~ 5:1, elutriant is concentrated into dry, obtains product and is target haptens.
Described concentrated nitric acid is the nitric acid of 65%, and halo hydrochlorate is halogenated acid sodium, and its structural formula is R-(CH
2)
n-COONa, wherein R can be Cl or Br, n=1 ~ 6
The preparation method of described DEHP artificial antigen is active fat method and mixed anhydride method.
Described active ester method comprises the following steps:
(1) be dissolved in the DMF of 1mL by the DEHP haptens of 0.1mmol, then in this solution, add equimolar DCC and NHS, the reaction of room temperature lucifuge is spent the night;
(2) 4 DEG C of centrifugal segregation precipitations, get supernatant liquor, and it slowly joined in the carbonic acid buffer of 5mL pH9.5, its damping fluid contains carrier proteins BSA or OVA, and wherein the concentration of carrier proteins is 10 ~ 15mg/mL, reacts and spend the night at 4 DEG C;
(3) reaction solution in step (2) is loaded dialysis tubing, with the normal saline dialysis 3d of 0.9%, obtain artificial antigen.
Described mixed anhydride method comprises the following steps:
(1) be dissolved in 1mL DMF by 0.1mmol DEHP haptens, add the positive Tributylamine of 80 μ L, slowly drip 0.15mmol isobutyl chlorocarbonate under ice bath, 4 DEG C of stirring reaction 1h, this is first liquid;
(2) be dissolved in the sodium bicarbonate buffer liquid of 5mL1mol/L pH9.6 by 60mg carrier proteins BSA or OVA, this is second liquid;
(3) be slowly added drop-wise in second liquid by first liquid, 4 DEG C of magnetic agitation reactions are spent the night, and after question response completes, load dialysis tubing, then use the normal saline dialysis 3d of 0.9%, obtain artificial antigen.
Described DEHP artificial antigen can be applicable to the preparation preparing anti-DEHP polyclonal antibody or monoclonal antibody.
The invention has the beneficial effects as follows:
Because DEHP is small-molecule substance (molecular weight is less than 1000Da), itself does not possess immunogenicity, must be prepared as artificial antigen with high molecular weight protein coupling, and animal just can be made to produce antibody.Because DEHP itself does not possess active group, can not with high molecular weight protein coupling, therefore need to carry out molecular modification to DEHP, introducing-NH
2with-COOH isoreactivity group, synthesis DEHP haptens.The present invention take DEHP as raw material, obtains DEHP haptens with nitric acid, ammonium chloride, halogenated acid reactant salt; By active ester method or mixed anhydride method, DEHP haptens and carrier protein couplet are obtained DEHP artificial antigen, this artificial antigen can be used for the specific antibody that animal immune prepares anti-DEHP, to setting up the elisa assay method detecting DEHP, have important practical significance, be and set up inspection-free detection method and provide immunizing antigen and envelope antigen and antibody preparation to lay the foundation.
Embodiment
Embodiment 1
DEHP hapten synthesis
As n=1, route prepared by DEHP haptens is as follows:
4mL DEHP (10mmoL) and the concentrated nitric acid (20mmoL) of 1mL65% are placed in round-bottomed flask, add the 5mL vitriol oil under condition of ice bath, magnetic agitation, react to room temperature, then 60 DEG C of back flow reaction 5h, period is monitored with TLC.After reaction terminates, with the sodium hydroxide solution neutralization reaction liquid of 1moL/L to weakly alkaline, extraction into ethyl acetate 3 times, organic phase evaporated under reduced pressure on Rotary Evaporators obtains thick nitration product DEHP-NO
2, cross silicagel column, collect elutriant to be V/V be the chloroform/hexanaphthene wash-out phase of 2:1 ~ 3:1, elutriant is concentrated into dry, obtains the nitration product DEHP-NO of purifying
2.
1mmoL DEHP-NO
2, 3mmoLNH
4cl, 3mmoL iron powder is placed in round-bottomed flask, adds in the ethanolic soln of 20mL70%, magnetic agitation, and 50 DEG C of back flow reaction 5h, period is monitored with TLC.After reaction terminates, in reaction solution, add 100mL dehydrated alcohol, elimination iron powder, collect filtrate, concentrated near dry in Rotary Evaporators; In concentrated solution, add 30mL water, be extracted with ethyl acetate 3 times, collect organic phase, evaporate to dryness, obtains crude product DEHP-NH
2; Cross silicagel column, collect elutriant to be V/V be the chloroform/methanol wash-out phase of 5:1 ~ 4:1, elutriant is concentrated into dry, obtains the reduzate DEHP-NH of purifying
2.
Take 1mmoL DEHP-NH
2(406mg) round-bottomed flask is placed in 2mmoL sodium chloroacetate (0.232g), and by the anhydrous alcohol solution of its mixture 10mL, the 0.276g salt of wormwood simultaneously added, back flow reaction at 60 DEG C, appropriate potassiumiodide is added as catalyzer in reaction process, after return stirring reaction 5h, stopped reaction.Filter reaction mixture and obtain yellowish brown filtrate, concentrate and obtain brown color grease-like material.Cross silicagel column, collect elutriant to be V/V be the chloroform/methanol wash-out phase of 4:1 ~ 5:1, elutriant is concentrated into dry, obtains product and is target haptens.
Embodiment 2
The synthesis of artificial antigen
The synthesis of 2.1 immunizing antigens and purifying:
Immunizing antigen adopts active ester method.The DEHP haptens of 50 μm of ol is dissolved in the DMF of 1mL, then in this solution, equimolar DCC and NHS is added, the reaction of room temperature lucifuge is spent the night, 4 DEG C of centrifugal segregation precipitations, get supernatant liquor, and it slowly being joined in the carbonic acid buffer of 5mL pH9.5, its damping fluid contains carrier proteins BSA, and wherein the concentration of carrier proteins is 10mg/mL.Mixture is reacted at 4 DEG C and spends the night, after question response completes, load dialysis tubing, then use the normal saline dialysis 3d of 0.9%.The solution obtaining having dialysed is artificial immunization antigen DEHP-BSA.
The synthesis of 2.2 envelope antigens and purifying:
Envelope antigen adopts mixed anhydride method.Be dissolved in 1mL DMF by 0.1mmol DEHP haptens, add the positive Tributylamine of 80 μ L, slowly drip 0.15mmol isobutyl chlorocarbonate under ice bath, 4 DEG C of stirring reaction 1h, this is first liquid.Be dissolved in the sodium bicarbonate buffer liquid of 5mL1mol/L pH9.6 by 60mg carrier proteins OVA, this is second liquid.Slowly be added drop-wise to by first liquid in second liquid, 4 DEG C of magnetic agitation reactions are spent the night, and after question response completes, load dialysis tubing, then use the normal saline dialysis 3d of 0.9%.The solution obtaining having dialysed is artificial envelope antigen DEHP-OVA.
Embodiment 3:
The qualification of artificial antigen:
According to the obtained the maximum absorption of spectrophotometry haptens, carrier proteins and conjugate, calculate the coupling ratio of conjugate.Scan raw material, BSA, OVA and conjugate ultra-violet absorption spectrum respectively between 200-400nm, whether qualification haptens and carrier proteins there is coupling.Estimate haptens and carrier protein couplet ratio, result is as follows as calculated: DEHP-BSA is 18.5:1, DEHP-OVA is 6.8:1 simultaneously.
Although preferred embodiment of the present invention has disclosed as above, the present invention has been not limited to above-described embodiment, any those skilled in the art, not departing from the scope disclosed by the present invention, when doing a little change and adjustment.
Claims (6)
1. a DEHP haptens, is characterised in that molecular structural formula is:
N is the natural number of 1 ~ 6.
2. a DEHP artificial antigen, is be combined into by DEHP haptens according to claim 1 and carrier proteins, it is characterized in that: described DEHP artificial antigen molecular structural formula is:
N is the natural number of 1 ~ 6; Described carrier proteins is bovine serum albumin, ovalbumin or hemocyanin.
3. the haptenic synthetic method of DEHP as claimed in claim 1, is characterized in that comprising the following steps:
(1) be that concentrated nitric acid and DEHP are placed in round-bottomed flask by 3:1 by feed ratio, be added drop-wise to 2 times under condition of ice bath in the vitriol oil of concentrated nitric acid amount, magnetic agitation, reacts to room temperature, then 60 DEG C of back flow reaction 5h, and period is monitored with TLC.With the sodium hydroxide solution neutralization reaction liquid of 1moL/L to weakly alkaline, extraction into ethyl acetate 3 times, organic phase evaporated under reduced pressure on Rotary Evaporators obtains thick nitration product DEHP-NO
2, cross silicagel column, collect elutriant to be V/V be the chloroform/hexanaphthene wash-out phase of 2:1 ~ 3:1, elutriant is concentrated into dry, obtains the nitration product DEHP-NO of purifying
2;
(2) be that 1:2:2 is by DEHP-NO by feed ratio
2, NH
4cl, iron powder are placed in round-bottomed flask, and add in 70% appropriate ethanolic soln, magnetic agitation, 50 DEG C of back flow reaction 5h, period is monitored with TLC; After reaction terminates, in reaction solution, add 100mL dehydrated alcohol, elimination iron powder, collect filtrate, concentrated near dry in Rotary Evaporators; In concentrated solution, add 30mL water, be extracted with ethyl acetate 3 times, collect organic phase, evaporate to dryness, obtains crude product DEHP-NH
2; Cross silicagel column, collect elutriant to be V/V be the chloroform/methanol wash-out phase of 5:1 ~ 4:1, elutriant is concentrated into dry, obtains the reduzate DEHP-NH of purifying
2;
(3) be that 1:2 ~ 1:3 is by DEHP-NH by feed ratio
2round-bottomed flask is placed in halo hydrochlorate, and by a small amount of anhydrous alcohol solution of its mixture, add the salt of wormwood with halo hydrochlorate equivalent simultaneously, back flow reaction at 60 DEG C, appropriate potassiumiodide is added as catalyzer in reaction process, after return stirring reaction 5h, stopped reaction, filter reaction mixture and obtain yellowish brown filtrate, concentrated obtain brown color grease-like material, cross silicagel column, to collect elutriant be V/V is the chloroform/methanol wash-out phase of 4:1 ~ 5:1, elutriant is concentrated into dry, obtains product and is target haptens.
4. the haptenic synthetic method of DEHP according to claim 3, it is characterized in that: described halo hydrochlorate is halogenated acid sodium, its structural formula is R-(CH
2)
n-COONa, wherein R can be Cl or Br, n=1 ~ 6.
5. the preparation method of DEHP artificial antigen is active fat method and mixed anhydride method as claimed in claim 2, it is characterized in that:
Described active ester method is:
(1) be dissolved in the DMF of 1mL by the DEHP haptens of 0.1mmol, then in this solution, add equimolar DCC and NHS, the reaction of room temperature lucifuge is spent the night;
(2) 4 DEG C of centrifugal segregation precipitations, get supernatant liquor, and it slowly joined in the carbonic acid buffer of 5mL pH9.5, its damping fluid contains carrier proteins BSA or OVA, and wherein the concentration of carrier proteins is 10 ~ 15mg/mL, reacts and spend the night at 4 DEG C;
(3) reaction solution in step (2) is loaded dialysis tubing, with the normal saline dialysis 3d of 0.9%, obtain artificial antigen;
Described mixed anhydride method is:
(1) be dissolved in 1mL DMF by 0.1mmol DEHP haptens, add the positive Tributylamine of 80 μ L, slowly drip 0.15mmol isobutyl chlorocarbonate under ice bath, 4 DEG C of stirring reaction 1h, this is first liquid;
(2) be dissolved in the sodium bicarbonate buffer liquid of 5mL1mol/L pH9.6 by 60mg carrier proteins BSA or OVA, this is second liquid;
(3) be slowly added drop-wise in second liquid by first liquid, 4 DEG C of magnetic agitation reactions are spent the night, and after question response completes, load dialysis tubing, then use the normal saline dialysis 3d of 0.9%, obtain artificial antigen.
6. the application of DEHP artificial antigen as claimed in claim 2, is characterized in that, described DEHP artificial antigen is applied to the preparation of the anti-DEHP polyclonal antibody of preparation or monoclonal antibody.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107098961A (en) * | 2017-06-14 | 2017-08-29 | 重庆师范大学 | Triptolide artificial antigen and preparation method and polyclonal antibody |
| CN110272876A (en) * | 2019-04-15 | 2019-09-24 | 上海交通大学 | Anti- diethyl phthalate monoclonal antibody hybridoma cell strain and its construction method |
| CN114317447A (en) * | 2021-12-10 | 2022-04-12 | 浙江树人学院（浙江树人大学） | Hybridoma cell strain secreting di (2-ethylhexyl) phosphate monoclonal antibody, monoclonal antibody and application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101328218A (en) * | 2008-07-29 | 2008-12-24 | 江南大学 | Preparation of diisooctyl phthalate artificial antigen |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101328218A (en) * | 2008-07-29 | 2008-12-24 | 江南大学 | Preparation of diisooctyl phthalate artificial antigen |
Non-Patent Citations (2)
| Title |
|---|
| CHENXI WEI ET AL: "An Immunoassay for Dibutyl Phthalate Based on Direct Hapten Linkage to the Polystyrene Surface of Microtiter Plates", 《PLOS ONE》 * |
| MING-CUI ZHANG ET AL: "A novel competitive fluorescence immunoassay for the determination of dibutyl phthalate", 《ANAL BIOANAL CHEM》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107098961A (en) * | 2017-06-14 | 2017-08-29 | 重庆师范大学 | Triptolide artificial antigen and preparation method and polyclonal antibody |
| CN110272876A (en) * | 2019-04-15 | 2019-09-24 | 上海交通大学 | Anti- diethyl phthalate monoclonal antibody hybridoma cell strain and its construction method |
| CN114317447A (en) * | 2021-12-10 | 2022-04-12 | 浙江树人学院（浙江树人大学） | Hybridoma cell strain secreting di (2-ethylhexyl) phosphate monoclonal antibody, monoclonal antibody and application |
| CN114317447B (en) * | 2021-12-10 | 2023-09-08 | 浙江树人学院（浙江树人大学） | Hybridoma cell strain secreting di (2-ethylhexyl) phosphate monoclonal antibody, monoclonal antibody and application |
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