CN104130144A - Synthetic method and application of universal hapten and antigen for phthalate plasticizer - Google Patents
Synthetic method and application of universal hapten and antigen for phthalate plasticizer Download PDFInfo
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- CN104130144A CN104130144A CN201410368522.7A CN201410368522A CN104130144A CN 104130144 A CN104130144 A CN 104130144A CN 201410368522 A CN201410368522 A CN 201410368522A CN 104130144 A CN104130144 A CN 104130144A
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Abstract
The present invention provides a synthetic method and application of a universal hapten and antigen for phthalate plasticizers and relates to biological detection technology. The invention uses 4-nitrophthalic acid as a raw material, which is subjected to an esterification and reduction reaction for the formation of a hapten; the hapten is coupled with a carrier protein by a glutaraldehyde method to obtain the artificial general antigen for phthalate ester plasticizer. The successful synthesis of artificial antigen is confirmed by UV scanning, and immune animal experiments show that the artificial antigen prepared by the invention has the advantages of good immunogenicity, good selectivity of generated antibodies and high sensitivity. The antigen or antibody of the invention can be used for immune analysis of phthalate plasticizer, and satisfy the need of rapid detection of phthalate plasticizer residual in food or environmental medium, and has broad application prospects.
Description
Technical field
The present invention relates to Measurement for Biotechnique, be specially preparation method and the application of phthalate fluidizer general artificial antigen.
Background technology
Phthalate (Phthalic Acid Esters, PAEs) compounds is a class fluidizer the most frequently used in plastics-production, account for 88% of the softening agent market share, approximately comprise 30 kinds of compounds, the most frequently used as dimethyl phthalate (DMP), diethyl phthalate (DEP), dibutyl phthalate (DBP), phthalic acid two (2-ethylhexyl) ester (DEHP) etc., mainly for the production of medical article, plastic toy, makeup, food product pack, agricultural mulching etc., owing to being non-covalent combination between phthalate and product matrix, cause it producing, in processing and use procedure, constantly in environment, discharge, the most general global class environmental pollutant have been become.Toxicologic study shows: phthalate is a class endocrine disrupter, has estrogen effect, and shows certain biological accumulation, is a kind of environmental estrogens with genotoxicity and development toxicity, and relevant with the generation of obesity and diabetes.U.S. EPA and China environmental monitoring general headquarters all classify phthalate compound as the preferential organic pollutant of controlling in environment, and the Ministry of Health is also listed 16 kinds of phthalates in may the non-edible material from soybeans of illegal interpolation and foodstuff additive list (the 6th batch) the > > of easy abuse in < < food.
The detection of phthalate at present adopts instrumental method more, needs expensive plant and instrument and professional and technical personnel, and sample is had relatively high expectations, and sample pre-treatments and purification process are wasted time and energy, and can not meet modern measure to convenient, requirement fast and accurately.Enzyme-linked immunosorbent assay and colloidal gold strip method are easy and simple to handle, less demanding to sample, can realize the requirement of field quick detection.But phthalate classes of compounds is various, is usually detected for a plurality of compounds simultaneously.Existing immunoassay, mainly for specific phthalate compound, can not be realized many residue detection of phthalate fluidizer.Therefore, the present invention be take 4-nitrophthalic acid as raw material, designs a kind of general artificial antigen of phthalate fluidizer, for setting up the how residual immunoassay technology of phthalate fluidizer, provides basis.
Summary of the invention
The present invention is directed to demand and the deficiency of current phthalate fluidizer immunoassay detection field, the synthetic method of a kind of phthalate fluidizer universal hapten and artificial antigen is provided.Testing data shows, this general antigen has higher immunogenicity, and the antiserum(antisera) of preparation and phthalate fluidizer have good affinity, and antibody versatility is good, highly sensitive.
A universal hapten for phthalate fluidizer, is characterized in that: its molecular structural formula as the formula (1):
formula (1).
A general artificial antigen for phthalate fluidizer, is characterized in that: be the molecule that coupling carrier albumen obtains on the amino group of universal hapten formula (1).
Described carrier proteins is a kind of in bovine serum albumin BSA, chicken ovalbumin OVA, human serum albumin HSA, keyhole limpet hemocyanin KLH.
The synthetic route of the general artificial antigen of described phthalate fluidizer is:
Processing step is as follows.
1. the preparation of phthalate fluidizer universal hapten.
(1) get the 4-nitrophthalic acid of 10-20g, join 30mL dehydrated alcohol, slowly add while stirring H
2sO
4, 120 ℃ of reaction 7h, distillation is except the water of dereaction generation and unnecessary ethanol.Go to while hot in frozen water, get and separate out 10% Na for solid
2cO
3(pH7-8) wash colourless to water layer after, with dehydrated alcohol recrystallization, obtain pale yellow crystals.
(2) pale yellow crystals of getting 0.7-1.5g is dissolved in 200mL benzene, adds pure zinc powder, and gradation adds dense HCL, under room temperature, stir in the backward reaction solution of 12h and add cold water, with NaOH neutralization reaction liquid, to alkalescence, with benzene, carry out separating and extracting, extraction liquid is used anhydrous Na after washing
2sO
4dry, after removing benzene, distillation uses dehydrated alcohol recrystallization, obtain light yellow crystal, be phthalate fluidizer universal hapten.
2. the preparation of phthalate fluidizer general artificial antigen.
(1) prepare A liquid: get phthalate fluidizer universal hapten and add N, in dinethylformamide (DMF), in room temperature magnetic agitation after abundant dissolving, at 4 ℃, dropwise add 2.5% glutaraldehyde solution, haptens: glutaraldehyde=1:2(mol ratio), lucifuge mixes and react 2h under magnetic agitation, obtains A liquid.
(2) prepare B liquid: get bovine serum albumin, haptens: bovine serum albumin=80:1(mol ratio), with pH9.6, the carbonate buffer solution of 0.05M dissolves, cryopreservation.This liquid is B liquid.
(3) in ice-water bath, A liquid is slowly added drop-wise in B liquid, and under agitation condition, continues reaction and spend the night, obtain phthalate fluidizer general artificial antigen mixed solution.
(4) mixed solution is moved in dialysis tubing, with PBS damping fluid dialysis 3-4 days, change PBS damping fluid every day 2 times.Use lyophilization that the liquid in dialysis tubing is made to powder, obtain phthalate fluidizer general artificial antigen.
The application in preparing phthalate fluidizer mass selection antibody of described universal hapten or artificial antigen also belongs to protection scope of the present invention.
Antiserum(antisera), antibody or hybridoma that described general artificial antigen immune animal obtains also belong to protection scope of the present invention.
The application in detecting phthalate fluidizer of described universal hapten or artificial antigen or antibody also belongs to protection scope of the present invention.
Beneficial effect of the present invention: the present invention is the synthetic method of novel phthalate fluidizer general artificial antigen, take 4-nitrophthalic acid as initial substance, by esterification and reduction reaction, introduce two side chains and an amino, thereby acquisition universal hapten, by glutaraldehyde method and carrier protein couplet, introduces one containing the arm chain of 5 carbon, farthest expose phthalate fluidizer antigenic determinant, as immunogen, be used for the effect of immune animal better.
Experimental data proves, artificial antigen immune animal of the present invention, and four exempt from rear blood sampling mensuration antiserum titre can reach 128000, illustrates that the synthetic antigen of the present invention has good immunogenicity.Indirect ELISA experiment showed, that obtained antiserum(antisera) and DMP, DEP, tri-kinds of phthalate fluidizers of DBP have good specificity, and antibody versatility is good, highly sensitive, can be used for detecting the residual of phthalate fluidizer in food and surrounding medium.
Accompanying drawing explanation
The synthetic route of Fig. 1 phthalate fluidizer general artificial antigen.
Fig. 2 phthalate fluidizer general artificial antigen UV scanning figure
Embodiment
For a better understanding of the present invention, below in conjunction with specific embodiment, further illustrate, should be understood that these embodiment are only for the present invention is described, and be not used for limiting the scope of the invention.
The preparation of embodiment 1 phthalate fluidizer universal hapten
(1) get the 4-nitrophthalic acid of 15g, join 30mL dehydrated alcohol, slowly add while stirring H
2sO
4, 120 ℃ of reaction 7h, distillation is except the water of dereaction generation and unnecessary ethanol.Go to while hot in frozen water, get and separate out 10% Na for solid
2cO
3(pH7-8) wash colourless to water layer after, with dehydrated alcohol recrystallization, obtain pale yellow crystals.
(2) pale yellow crystals of getting 1.5g is dissolved in 200mL benzene, adds pure zinc powder, and gradation adds dense HCL, stirs in the backward reaction solution of 12h and add cold water under room temperature, with NaOH neutralization reaction liquid, to alkalescence, with benzene, carries out separating and extracting, and extraction liquid is used anhydrous Na after washing
2sO
4dry, after removing benzene, distillation uses dehydrated alcohol recrystallization, obtain light yellow crystal, be phthalate fluidizer universal hapten.
The preparation of embodiment 2 phthalate fluidizer general artificial antigens.
(1) prepare A liquid: get 12mg phthalate fluidizer universal hapten and add 2mL N, in dinethylformamide (DMF), in room temperature magnetic agitation after abundant dissolving, at 4 ℃, dropwise add 2.5% glutaraldehyde solution 500 μ L, lucifuge mixes and react 2h under magnetic agitation, obtains A liquid.
(2) prepare B liquid: take 38.8mg bovine serum albumin, be dissolved in 6mL carbonate buffer solution (pH9.6,0.05M) cryopreservation.This liquid is B liquid.
(3) in ice-water bath, A liquid is slowly added drop-wise in B liquid, and under agitation condition, continues reaction and spend the night, obtain phthalate fluidizer general artificial antigen mixed solution.
(4) mixed solution is moved in dialysis tubing, with PBS damping fluid dialysis 3-4 days, change PBS damping fluid every day 2 times.Use lyophilization that the liquid in dialysis tubing is made to powder, obtain phthalate fluidizer general artificial antigen.
The sero-fast preparation of embodiment 3 phthalate fluidizer.
With the general artificial antigen of embodiment 2 preparation, as immunogen, with Freund's complete adjuvant balanced mix, after emulsification completely, choose the Balb/c female mice in 6-8 age in week in the subcutaneous multi-point injection of nape portion, at interval of 3 weeks booster immunizations once.Four exempt from latter the 10th day to mouse docking blood sampling, the centrifugal antiserum(antisera) that obtains.
The sero-fast mensuration of embodiment 4 phthalate fluidizer.
1, sero-fast titration.
Adopt indirect non-competing ELISA method to measure serum titer, concrete steps are.
(1) coated: by the phthalate fluidizer general artificial antigen 0.05M of embodiment 2 preparations, the carbonate buffer solution of pH9.6 is made the coated 96 hole enzyme plates of doubling dilution, 100 μ L/ holes, 4 ℃ are spent the night.
(2) washing: solution in the plate that inclines, dry, with the PBS damping fluid of 0.5% Tween-20, wash 3 times, 3 min that at every turn vibrate, firmly get rid of washings, on thieving paper, pat dry.
(3) sealing: add the sealing damping fluid containing 1% gelatin, 200 μ L/ holes, 37 ℃ of incubation 2 h, taking-up washing post-drying is stand-by.
(4) increase serum: the phthalate fluidizer antiserum(antisera) that embodiment 3 is made starts doubling dilution from 1:1000, correspondence joins front 7 ranks of enzyme plate, and eighth row adds negative serum, and 50 μ L/ holes are hatched washing after 30min, are patted dry for 37 ℃.
(5) adding two resists: add the HRP-sheep anti-mouse igg of 1:3000 dilution, 50 μ L/ holes, 37 ℃ of reaction 30min.
(6) colour developing: take out and wash after 3 times, add TMB nitrite ion, 100 μ L/ holes, 37 ℃ of lucifuges reaction 15min.
(7) stop and measure: enzyme plate being taken out, add stop buffer (2M H
2sO
4), 50 μ L/ holes, then measure each hole at the light absorption value at 450nm place by microplate reader.
(8) result interpretation: with OD
450value is more than or equal to the ELISA that the highly diluted multiple of 2.1 times of corresponding serum of negative control hole is serum and tires.
2, sero-fast specific assay.
Adopt indirect competitive ELISA method antagonistic Serum to carry out specific assay, concrete steps are as follows.
(1), according to above-mentioned indirect non-competing ELISA method, select OD
450value when 1.5 left and right corresponding antigen and antibody concentration as the suitableeest working concentration.
(2) coated: with coated damping fluid, coating antigen is diluted to the suitableeest working concentration, 100 μ L/holes, 4 ℃ are spent the night.
(3) washing and sealing: with the operation of above-mentioned indirect non-competing ELISA method.
(4) standardized solution preparation: the phthalate fluidizer (DBP, DIBP, DNP, DMP, DEHP) of take is research object, is mixed with respectively the mother liquor of 1mg/mL with methanol solution, becomes to need concentration with PBS solution (0.01M, pH7.4) doubling dilution during use.
(5) competition: every row first add each concentration standard product, 50 μ L/holes successively; Every hole adds the 50 μ L antiserum(antisera) of suitable extension rate again, in 37 ℃ of incubation l h.
(6) add ELIAS secondary antibody: fully, after washing, add the HRP-sheep anti-mouse igg of 1:3000 dilution, 100 μ L/holes, 37 ℃ of incubation l h.
(7) colour developing: with the operation of above-mentioned indirect non-competing ELISA method.
(8) stop and measure: with the operation of above-mentioned indirect non-competing ELISA method.
(9) data processing: the logarithm of each concentration of phthalate fluidizer (DBP, DIBP, DNP, DMP, DEHP) of take is X-coordinate, corresponding OD value is ordinate zou, drawing standard curve calculates respectively the half-inhibition concentration (IC of each standard substance
50), judge whether antiserum(antisera) has specificity to each phthalate fluidizer.
Experimental result shows, the antiserum(antisera) that general artificial antigen immune mouse prepared by the present invention obtains, and four exempt to tire can reach 128000, and DMP, DEP, tri-kinds of phthalate fluidizers of DBP are all had to good recognition capability, and antibody versatility is good.
Claims (7)
1. a universal hapten for phthalate fluidizer, its molecular structural formula as the formula (1):
。
2. a general artificial antigen for phthalate fluidizer, is characterized in that: be the molecule that coupling carrier albumen obtains on the amino group of the universal hapten formula (1) described in claim 1.
3. carrier proteins claimed in claim 2 is a kind of in bovine serum albumin BSA, chicken ovalbumin OVA, human seralbumin egg HSA, keyhole limpet hemocyanin KLH.
4. the synthetic method of universal hapten claimed in claim 1, its synthesis step is:
(1) get the 4-nitrophthalic acid of 10-20g, join 30mL dehydrated alcohol, slowly add while stirring H
2sO
4, 120 ℃ of reaction 7h, distillation is except the water of dereaction generation and unnecessary ethanol; Go to while hot in frozen water, get and separate out 10% Na for solid
2cO
3(pH7-8) wash colourless to water layer after, with dehydrated alcohol recrystallization, obtain pale yellow crystals;
(2) pale yellow crystals of getting 0.7-1.5g is dissolved in 200mL benzene, adds pure zinc powder, and gradation adds dense HCL, under room temperature, stir in the backward reaction solution of 12h and add cold water, with NaOH neutralization reaction liquid, to alkalescence, with benzene, carry out separating and extracting, extraction liquid is used anhydrous Na after washing
2sO
4dry, after removing benzene, distillation uses dehydrated alcohol recrystallization, obtain light yellow crystal, be phthalate fluidizer universal hapten.
5. the synthetic method of general artificial antigen claimed in claim 2, its synthesis step is:
(1) get phthalate fluidizer universal hapten and add N, in dinethylformamide (DMF), in room temperature magnetic agitation after abundant dissolving, at 4 ℃, dropwise add 2.5% glutaraldehyde solution, haptens: glutaraldehyde=1:2(mol ratio), lucifuge mixes and react 2h under magnetic agitation, obtains A liquid;
(2) get bovine serum albumin, haptens: bovine serum albumin=80:1(mol ratio), with pH9.6, the carbonate buffer solution of 0.05M dissolves, cryopreservation, this liquid is B liquid;
(3) in ice-water bath, A liquid is slowly added drop-wise in B liquid, and under agitation condition, continues reaction and spend the night, mixed solution is moved in dialysis tubing, with PBS damping fluid dialysis 3-4 days, change PBS damping fluid every day 2 times; Use lyophilization that the liquid in dialysis tubing is made to powder, obtain phthalate fluidizer general artificial antigen.
6. the application of the general artificial antigen described in claim 2 and 3, is characterized in that the application in preparing antiserum(antisera), antibody or hybridoma.
7. right will be gone the application of the general artificial antigen described in 2 and 3, it is characterized in that the application in residual of phthalate fluidizer in detecting phthalate fluidizer, food or surrounding medium.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104530221A (en) * | 2014-12-17 | 2015-04-22 | 西南大学 | Synthetic method of phthalic acid ester compound general artificial antigen |
| CN105085299A (en) * | 2015-08-20 | 2015-11-25 | 上海交通大学 | Plasticizing agent diethyl phthalate hapten and preparation method thereof |
| CN105085298A (en) * | 2015-08-20 | 2015-11-25 | 上海交通大学 | Dibutyl phthalate hapten as well as preparation method and application thereof |
| CN108033890A (en) * | 2017-12-07 | 2018-05-15 | 新疆农垦科学院 | A kind of design method of phthalate environmental hormone universal architecture haptens and application |
| CN108148130A (en) * | 2018-02-09 | 2018-06-12 | 浙江工商大学 | A kind of application of the preparation method and NOTA of heavy metal Hg artificial antigen in heavy metal Hg artificial antigen reagent is prepared |
| CN108148129A (en) * | 2018-02-09 | 2018-06-12 | 浙江工商大学 | A kind of application of the preparation method and NOTA of heavy metal zinc artificial antigen in heavy metal zinc artificial antigen reagent is prepared |
| CN108148128A (en) * | 2018-02-09 | 2018-06-12 | 浙江工商大学 | A kind of application of the preparation method and DOTA of heavy metal cadmium artificial antigen in heavy metal cadmium artificial antigen reagent is prepared |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104530221A (en) * | 2014-12-17 | 2015-04-22 | 西南大学 | Synthetic method of phthalic acid ester compound general artificial antigen |
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| CN105085299A (en) * | 2015-08-20 | 2015-11-25 | 上海交通大学 | Plasticizing agent diethyl phthalate hapten and preparation method thereof |
| CN105085298A (en) * | 2015-08-20 | 2015-11-25 | 上海交通大学 | Dibutyl phthalate hapten as well as preparation method and application thereof |
| CN108033890A (en) * | 2017-12-07 | 2018-05-15 | 新疆农垦科学院 | A kind of design method of phthalate environmental hormone universal architecture haptens and application |
| CN108033890B (en) * | 2017-12-07 | 2020-04-21 | 石河子大学 | Design method and application of phthalate ester environmental hormone universal structure hapten |
| CN108148130A (en) * | 2018-02-09 | 2018-06-12 | 浙江工商大学 | A kind of application of the preparation method and NOTA of heavy metal Hg artificial antigen in heavy metal Hg artificial antigen reagent is prepared |
| CN108148129A (en) * | 2018-02-09 | 2018-06-12 | 浙江工商大学 | A kind of application of the preparation method and NOTA of heavy metal zinc artificial antigen in heavy metal zinc artificial antigen reagent is prepared |
| CN108148128A (en) * | 2018-02-09 | 2018-06-12 | 浙江工商大学 | A kind of application of the preparation method and DOTA of heavy metal cadmium artificial antigen in heavy metal cadmium artificial antigen reagent is prepared |
| CN108148129B (en) * | 2018-02-09 | 2020-10-27 | 浙江工商大学 | Preparation method of heavy metal zinc artificial antigen and application of NOTA in preparation of heavy metal zinc artificial antigen reagent |
| CN108148128B (en) * | 2018-02-09 | 2020-11-20 | 浙江工商大学 | Preparation method of heavy metal cadmium artificial antigen and application of DOTA in preparation of heavy metal cadmium artificial antigen reagent |
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Application publication date: 20141105 |