Chinese Patent Translated in Google

06/09/2020 CN105085299A - Plasticizing agent diethyl phthalate hapten and preparation method thereof - Google Patents
Patents
Plasticizing agent diethyl phthalate hapten and preparation method thereof
Abstract
CN105085299A
The invention provides a plasticizing agent diethyl phthalate (DEP) hapten and a preparation method
China
thereof. The plasticizing agent diethyl phthalate hapten has a structural formula as shown in the
speci cation, and the preparation method of the diethyl phthalate hapten comprises the following
steps: taking 4-nitrophthalic acid as a raw material, and performing two-step reaction to prepare the Download PDF Find Prior Art Similar
DEP hapten with amino active groups. The preparation method of the diethyl phthalate hapten is
simple and convenient, is good in stability, low in cost and easy for realization of industrial Other languages: Chinese
production; the diethyl phthalate hapten is coupled with a carrier protein by virtue of a
Inventor: 庄惠生, 孙瑞艳
glutaraldehyde method, a diazotization method or a diimine method to prepare a diethyl phthalate
arti cial holoantigen, and provides a guarantee for preparing an antibody with good speci city and
high titer and establishing an avidin-biotin enzyme linked immunosorbent assay. Worldwide applications
2015 CN
Application CN201510515294.6A events
2015-08-20 Application led by 上海交通大学
2015-08-20 Priority to CN201510515294.6A
2015-11-25 Publication of CN105085299A
Info: Patent citations (4), Legal events, Similar documents,

Priority and Related Applications
External links: Espacenet, Global Dossier, Discuss
Claims (10) Hide Dependent
1. a diethyl phthalate haptens, is characterized in that, structural formula is such as formula shown in I:
2. the haptenic preparation method of diethyl phthalate according to claim 1, is characterized in that, described preparation method comprises step:
Haptens Intermediate Preparation: in strong acid environment, 4-nitrophthalic acid and ethanol synthesis, generate 4-nitrophthalic acid diethyl ester, i.e. haptens
intermediate;
Prepared by haptens: in non-proton organic solvent environment, described haptens intermediate and zinc powder react, and generates diethyl phthalate haptens.
3. the haptenic preparation method of diethyl phthalate according to claim 2, it is characterized in that, the step of described haptens Intermediate Preparation is
specially: be dissolved in ethanolic soln by 4-nitrophthalic acid, be added dropwise to strong acid, the reaction system generation esteri cation of formation.
4. the haptenic preparation method of the diethyl phthalate according to Claims 2 or 3, is characterized in that, the mol ratio of described 4-nitrophthalic acid, ethanol,
strong acid is 1:(5 ~ 7): (0.6 ~ 0.8); The temperature of described reaction is 86 ~ 90 DEG C, and the time of described reaction is 8 ~ 10 hours; Described strong acid is
the vitriol oil or concentrated hydrochloric acid.
5. the haptenic preparation method of the diethyl phthalate according to Claims 2 or 3, is characterized in that, described haptens Intermediate Preparation also
comprises the steps:
A1, remove the water of unreacted ethanol and generation in described reaction system, then remainder to be poured in frozen water to separate out solid, is crude
intermediate.
A2, to wash described crude intermediate to washings be colourless, recrystallization, dry, obtains the diethyl phthalate haptens of purifying.
6. the haptenic preparation method of diethyl phthalate according to claim 2, is characterized in that, step prepared by described haptens is specially: be dissolved in non-
proton organic solvent environment by described haptens intermediate; Then add zinc powder, mix; Be added dropwise to strong acid again, after mixing, add zinc
powder again, the reaction system isothermal reaction of formation.
7. the haptenic preparation method of the diethyl phthalate according to claim 2 or 6, it is characterized in that, in step prepared by described haptens, the mol ratio of
described haptens intermediate, non-proton organic solvent, strong acid and zinc powder is 1:(550 ~ 580): (20 ~ 22): (18 ~ 20); The temperature of described reaction is
25 ~ 35 DEG C, and the time of described reaction is 10 ~ 12 hours; Described non-proton organic solvent is benzene or toluene or acetone.
8. the haptenic preparation method of the diethyl phthalate according to claim 2 or 6, is characterized in that, described haptens preparation also comprises the steps:
After B1, described reaction terminate, cool described reaction system, adjustment pH is weakly alkaline;
B2, leave standstill, extract to obtain extraction liquid; Wash described extraction liquid, dry;
B3, the organic solvent constituent removed in described dried extraction liquid, recrystallization;
B4, the product after described recrystallization is carried out silica gel column chromatography, then through underpressure distillation, obtain diethyl phthalate haptens.
9. the haptenic purposes of diethyl phthalate according to claim 1, is characterized in that, for the detection of trace uidizer diethyl phthalate.
10. one kind based on the haptenic arti cial holoantigen of diethyl phthalate described in claim 1, it is characterized in that, being prepared as follows of described
holoantigen: by diethyl phthalate haptens by glutaraldehyde method or diazotization method or carbodlimide method and carrier protein couplet, obtain the arti cial
https://patents.google.com/patent/CN105085299A/en 1/10
holoantigen of diethyl phthalate.
Description
Fluidizer diethyl phthalate haptens and preparation method thereof
Technical eld
The present invention relates to a kind of haptens and preparation method thereof, especially relate to a kind of uidizer diethyl phthalate haptens and preparation
method thereof.
Background technology
Phthalate uidizer (being abbreviated as PAEs), also known as phthalate, is the general designation of the important derivatives of the ester that a class is formed with
alcohol generation Fischer (Fischer) esteri cation containing 4-15 carbon by phthalic acid.This kind of material has speci c characteristics smell, and toxicity is
comparatively large, and generally state is thick liquid; Under liquid condition, temperature range is wide, and mobility is large, and volatility is low, water insoluble, is
soluble in most of organic solvent.This kind of substance use is extensive, it not only can be used as the production starting material of nearly thousand kinds of
products such as packaging material for food, toy, farm chemical carrier, wormer, lubricant, vinyl ooring and wallpaper, defoaming agents, sanitising agent, medical
material arti cial organs, blood bag, syringe and sebi c ducts such as (as:) heart valve prosthesises and personal-care supplies (mainly containing makeup, fragrance
product, nail varnish, hair spray, perfumed soap and shampoo), and also Chang Zuowei plastic plasticizer is used to transformation plastics performance.
Wherein, diethyl phthalate (English name: Diethylphthalate, vehicle economy P), has another name called Unimoll DA, and No. CAS is 84-66-2, and molecular formula is C
12h 14o 4, molecular weight is 222.24, and colourless extremely micro-yellow clear oil liquid, is very easily dissolved in ethanol, water-soluble hardly; Meet naked light, high
hot incendivity, there is pungency.This material low toxicity, has hormesis to skin, eyes, the upper respiratory tract; Absorb through skin or headache, dizzy and vomiting
after absorption, can be caused; Phototoxicity, slight sensitization that tool is potential; There is impact to womb, fetal anomaly or death may be caused ].Because it can
be relatively good with the consistency of most of resin, therefore, it is widespread use mainly as the softening agent of resin; In addition, also can be used as the
auxiliary material of lubricant, pore forming material, perfume and pharmaceutical coating material.In the last few years, there is researcher nd the too high levels of this
material in food (as: wine, beverage etc.) thus jeopardize food safety, and therefore just enjoyed people to pay close attention to.
Along with the fast development of plastics industry and the widespread use of plastic prod, just there is many chances be in direct contact with it in our health, as the
easiliest in: most people touches PAEs by plasthetics packaging (preservative lm, plastic containers etc.), but food (box lunch, vegetables, wine, beverage etc.) DEP very
likely stripping thus enter DEP in air, food and even soil, water source from pack in the process of processing, heating, packaging or splendid attire; Doctors and patients
can touch therapeutic medical plastic gloves or transfusion bags etc., and such DEP just can in ltrate inside of human body; Containing DEP in the fragrance ingredient of
the makeup that women commonly uses, the DEP content wherein in nail varnish is the highest, and these substances enter in body by respiratory system and skin;
Children are the easiest touches DEP in plastic toy and articles for children, the stripping quantity of DEP just can be caused to exceed security level if children put it in
mouth for a long time.Therefore, DEP ubiquity in the natures such as air, water body, soil, biology and even human body and human environment, all generally can detect
in each major industrial country's environment of the whole world, oneself becomes the most general global ubiquitous pollutent, is also referred to as the 2nd class
global " PCB pollutent " simultaneously.
At present the instrument such as gas-chromatography and high performance liquid chromatography detection method is mainly to the detection means of diethyl
phthalate, although these methods accurately and reliably, has very high requirement to the pretreatment process of sample and the professional of operator.Just
because of the process of these methods is complicated, consuming time, instrument price is expensive and be not suitable for promoting the use of, be also
unfavorable in environmental pollution accident eld quick detection.For overcoming these shortcomings, seek the main direction of studying that a kind of quick, easy,
sensitive and economical and practical analytical procedure just becomes environmental monitoring eld.
The immunoassay (Immunoassay, IA) that the sixties in 20th century grows up is based on antigen and the speci city of antibody, the analytical technology of
reversibility association reaction.Immunoassay has the unrivaled selectivity of conventional physical and chemical analysis technology and high sensitivity, is applicable
to very much the analysis of trace components in complex dielectrics.Therefore the high speci city, the advantage such as highly sensitive, method is quick and easy,
analysis throughput is large, testing cost is low that have of immunoassay, makes these class methods can meet simply, detects the requirement of persistence organic
pollutant fast, delicately.Within 1971, Engvail, VanWeerman etc. report the solid-phase immunoassay technology detecting micro substance in body uid, i.e. enzyme
linked immunosorbent assay analysis method (enzyme-linkedimmunosorbentassay, ELISA).At present, ELISA method has become integral part important in immune
analysis method.ELISA method be by Ag-Ab between immune response and the e cient catalytic characteristic of enzyme organically combine and a kind of immune
analysis method grown up.Wherein, the Avidin-Biotin enzyme linked immunosorbent assay analysis method based on Avidin-Biotin signal amplifying system, with biotin
labelled antibodies (antigen), and replaces enzyme labelled antibody in ELISA method with enzyme mark avidin.Avidin is a kind of alkaline glycoprotein in Protalbinic acid,
and molecular weight is about 68kDa.An avidin molecule is made up of 4 subunits, each subunit can with a biotin molecule (molecular weight is 244) speci c binding.By
force, its avidity is more much bigger than antigen antibody reaction, and a nity costant is up to 1015M for vitamin H and avidin binding speci city -1.Because an avidin
can be combined with 4 biotin molecules, therefore can improve by the quantity of solid phase desmoenzyme in the detection, and then improve the sensitivity of
detection method.
Avidin-Biotin enzyme linked immunosorbent assay analysis method is not still had to detect the relevant report of diethyl phthalate at present.For the foundation of
diethyl phthalate immunologic surveillance method, the haptens that high purity is suitable is that preparation has the basic of highly sensitive and speci c immunogens
and high-titer antibody, sets up the key of diethyl phthalate immunologic detection method especially.Therefore, the haptenic preparation of diethyl phthalate is the basis
adopting immunization method to measure diethyl phthalate, for the monitoring method setting up Polybrominated biphenyl monomer, has important using value and
theoretical signi cance.
Summary of the invention
For defect of the prior art, the object of this invention is to provide a kind of uidizer diethyl phthalate haptens and preparation method thereof, namely provide a kind of
step simple rst, speed is fast, diethyl phthalate haptens that productive rate is high and preparation method thereof.
The object of the invention is to be achieved through the following technical solutions:
First aspect, the invention provides a kind of diethyl phthalate haptens, and structural formula is such as formula shown in I:
Described formula I is faint yellow acicular crystals, and fusing point is 90 ~ 92 DEG C.
Second aspect, the invention still further relates to the haptenic preparation method of a kind of described diethyl phthalate, described preparation method comprises
step:
Haptens Intermediate Preparation: in strong acid environment, 4-nitrophthalic acid and ethanol synthesis, generate 4-nitrophthalic acid diethyl ester, i.e. haptens
intermediate;
Prepared by haptens: in non-proton organic solvent environment, described haptens intermediate and zinc powder reacts, generation diethyl phthalate haptens, is also
DEP haptens, with amino active group, can with carrier protein couplet.
Preferably, in haptens Intermediate Preparation, described strong acid is the vitriol oil or concentrated hydrochloric acid, analytical pure rank.
Preferably, the step of described haptens Intermediate Preparation is specially: be dissolved in ethanolic soln by 4-nitrophthalic acid, be added dropwise to strong acid,
the reaction system generation esteri cation of formation;
Described dropping strong acid solution is specially and dropwise drips, and be the routine operation of esteri cation, strong acid can not add fast, dropwise add, and
prevents local release of heat from too much causing bulk temperature uneven, easily like this sets off an explosion; The degree of described generation esteri cation is
till raw material point disappears; Described zinc powder is pure zinc powder, i.e. chemical rank analytical pure, and traditional Chinese medicines Reagent Company
produces.
Preferably, in described haptens Intermediate Preparation, the mol ratio of described 4-nitrophthalic acid, ethanol, strong acid is 1:(5 ~ 7): (0.6 ~ 0.8).As shown in
chemical reaction equation, ideally the mol ratio of 4-nitrophthalic acid, ethanol is 1:2, but carry out to the right smoothly to react, the consumption of general ethanol is
more than 5 times of the molar weight of 4-nitrophthalic acid, but can not more than 7 of the molar weight of 4-nitrophthalic acid times; In addition, strong acid is as the
catalyzer of esteri cation, and amount used is generally more than 0.6 times of raw material 4-nitrophthalic acid molar weight, but is no more than 0.8 times of raw
material 4-nitrophthalic acid molar weight.
Preferably, in described haptens Intermediate Preparation, the temperature of reaction of described reaction system is 86 ~ 90 DEG C, and the reaction times of
described reaction system is 8 ~ 10 hours.This temperature is more conducive to the carrying out reacted, and detects with thin-layer chromatography, and constant
temperature back ow can react completely for 8 ~ 10 hours.
Preferably, described haptens Intermediate Preparation also comprises the steps:
A1, remove the water of unreacted ethanol and generation in described reaction system, then remainder to be poured in frozen water to separate out solid, is crude
intermediate, is conducive to termination reaction like this, also prevent the generation of reversed reaction.
A2, to wash described crude intermediate to washings be colourless, recrystallization, dry, obtains the diethyl phthalate haptens of purifying.
Preferably, in described A1 step, the concrete employing distillation of the water of unreacted ethanol and generation in the described reaction system of described
removing.
Preferably, in described A2 step, the washings that described washing adopts is the Na of 10% 2cO 3solution, when washings is colourless, its pH is 7.0-8.0; Described
recrystallization adopts dehydrated alcohol; Described drying speci cally refers to-20 DEG C of freeze-drying; Finally obtain the 4-nitrophthalic acid diethyl ester after light
yellow needles and purifying, namely diethyl phthalate haptens intermediate, i.e. formula II compound.Diethyl phthalate haptens intermediate molecule formula: C 12h
13nO 6, molecular weight: 267.23; Fusing point: 29 ~ 31 DEG C.
Preferably, the step that prepared by described haptens is specially: be dissolved in non-proton organic solvent environment by described haptens intermediate; Then add
zinc powder, mix; Be added dropwise to strong acid again, after mixing, add zinc powder again, the reaction system isothermal reaction of formation;
Described dropping dropwise adds, and this operation is the routine operation of chemical reaction, and strong acid can not add fast, dropwise add, and prevents local
release of heat from too much causing bulk temperature uneven, easily like this sets off an explosion; Till described isothermal reaction speci cally refers to that reaction
continues to the disappearance of raw material point; Described zinc powder is pure zinc powder, i.e. chemical rank analytical pure, and traditional Chinese medicines
Reagent Company produces.
Preferably, in step prepared by described haptens, described non-proton organic solvent is benzene or toluene or acetone; Described strong acid is the vitriol oil or
concentrated hydrochloric acid; Described mixing is specially magnetic agitation mixing.
Preferably, in step prepared by described haptens, the mol ratio of described haptens intermediate, organic solvent, strong acid and zinc powder is 1:(550 ~ 580): (20 ~
22): (18 ~ 20).As shown in chemical reaction equation, ideally the mol ratio of 4-nitrophthalic acid diethyl ester, strong acid and pure zinc powder is 1:10:10, but carry out
to the right smoothly to react, the consumption of general pure zinc powder is more than 20 times of raw material 4-nitrophthalic acid diethyl ester molar weight, but can
not exceed raw material 4-nitrophthalic acid diethyl ester, 22 times of molar weight; In addition, strong acid is as the strong oxidizer of reduction reaction, amount used is
generally more than 1 times of the pure zinc powder mole number of raw material, but be no more than 1.1 times of the pure zinc powder mole number of raw material,
and the mole number of aprotic organic solvent-benzene needs to be more than 25 times of strong oxidizer mole number, but be no more than 28 times of strong oxidizer
mole number.
Preferably, in step prepared by described haptens, the temperature of described reaction is 25 ~ 35 DEG C, and the time of described reaction is 10 ~ 12 hours.This
temperature is more conducive to the carrying out reacted, and detects with thin-layer chromatography, and constant temperature back ow can react completely for 10 ~
12 hours.
Preferably, described haptens preparation also comprises the steps:
After B1, described reaction terminate, cool described reaction system, adjustment pH is weakly alkaline;
B2, leave standstill, extract to obtain extraction liquid; Wash described extraction liquid, dry;
B3, the organic solvent constituent removed in dried extraction liquid, recrystallization;
B4, the product after described recrystallization is carried out silica gel column chromatography, then through underpressure distillation, obtain diethyl phthalate haptens,
be 4-aminophthalic acid diethyl ester; Light yellow crystal, i.e. type I compound.Diethyl phthalate hapten molecule formula: C 12h 15nO 4, molecular weight: 237.25; Fusing
point: 92 ~ 94 DEG C.
Preferably, in step B1, being operating as of described cooling adds cold water; Described adjustment pH adopts strong alkali solution, and described strong alkali
solution is sodium hydroxide or potassium hydroxide.
Preferably, in step B2, the described standing time is 1h; Described extraction adopts benzene; Described drying speci cally refers to anhydrous Na 2sO 4dehydrate;
Described washing is specially washing.
Preferably, in step B3, the organic solvent constituent removed in described dried extraction liquid speci cally adopts distillation under vacuum; Described
recrystallization adopts dehydrated alcohol.
Preferably, in step B4, described silica gel column chromatography elutriant used adopts volume ratio to be the acetic acid of 1:8 and the mixing solutions of normal
hexane composition.Because haptens is organic phase material, the present invention surprisingly nds to adopt this developping agent to be more conducive to
haptenic purifying, makes the haptens purity of acquisition higher.
Its chemical equation is as follows:
The third aspect, present invention also offers the haptenic purposes of a kind of diethyl phthalate, for the detection of trace uidizer diethyl phthalate.
Fourth aspect, the invention provides a kind of based on the haptenic arti cial holoantigen of diethyl phthalate, being prepared as follows of described holoantigen: by
diethyl phthalate haptens by glutaraldehyde method or diazotization method or carbodlimide method and carrier protein couplet, obtain the arti cial holoantigen of
diethyl phthalate.
Preferably, described carrier proteins comprises bovine serum albumin or oralbumin.
Preferably, the arti cial holoantigen of described diethyl phthalate is diethyl phthalate arti cial immunogen DEP-BSA or the arti cial coating antigen DEP-OVA of diethyl
phthalate
Wherein, BSA is bovine serum albumin, and OVA is oralbumin.
The present invention relates to a kind of diethyl phthalate haptens and preparation method thereof, because diethyl phthalate is small-molecule substance, only there is
reactionogenicity and there is no immunogenicity, and this molecule there is no the functional group such as amino, carboxyl that directly can be combined with protein
molecule, therefore need to make its molecule to be brought an amino to diethyl phthalate molecule derivatize.In order to the realization of immune analysis method, with
glutaraldehyde method or diazotization method or carbodlimide method, this carboxylic haptens and protein molecule coupling are prepared arti cial holoantigen
(comprising arti cial immunogen and arti cial coating antigen), wherein arti cial immunogen immunize New Zealand White Rabbit, by the immunne response in
organism and then the speci c antibody preparing anti-diethyl phthalate, and arti cial coating antigen and diethyl phthalate can with the reaction of anti-diethyl phthalate
speci c antibody immunoglobulin IgG generation speci c recognition, namely arti cial coating antigen and diethyl phthalate are the relations of direct competitive, thus
set up immune analysis method for water body, soil, the detection of trace uidizer diethyl phthalate in the environmental samples such as air and varieties of food items.
Compared with prior art, the present invention has following bene cial effect:
1, haptens is practical: the preparation of diethyl phthalate haptens and antibody preparation have important practical value and realistic meaning; This haptens remains
the structure of diethyl phthalate, recycling glutaraldehyde method or diazotization method or carbodlimide method coupling protein matter make it have the antigenic
determinant for diethyl phthalate, successfully can prepare diethyl phthalate arti cial immunogen DEP-BSA or the arti cial coating antigen DEP-OVA of diethyl phthalate,
for preparation speci city is good, the high antibody and set up Novel immune analytical procedure and provide guarantee of tiring.
2, haptens good stability: the present invention has prepared diethyl phthalate haptens rst, and the diethyl phthalate haptens structure of this method synthesis has
good stability, can preserve for many years, lost e cacy hardly under room temperature state.
3, haptens technology of preparing is simple and feasible: haptenic whole preparation process is without the need to special plant and instrument, with low cost, and
preparation speed is fast, and productive rate is high, easy commercial scale production.
Accompanying drawing explanation
By reading the detailed description done non-limiting example with reference to the following drawings, other features, objects and advantages of the present invention
will become more obvious:
Fig. 1 is the infrared spectrogram of diethyl phthalate haptens intermediate;
Fig. 2 is the haptenic infrared spectra of diethyl phthalate;
Fig. 3 is the haptenic nmr spectrum of diethyl phthalate;
Fig. 4 is the ultraviolet spectrogram of diethyl phthalate haptens DEP-hapten, carrier proteins BSA and arti cial holoantigen DEP-BSA;
Fig. 5 is the ultraviolet spectrogram of diethyl phthalate haptens DEP-hapten, carrier proteins OVA and arti cial holoantigen DEP-OVA.
Fig. 6 is the standard working curve that indirect competition BA-ELISA immune analysis method detects diethyl phthalate.
Embodiment
Below in conjunction with speci c embodiment, the present invention is described in detail.Following examples will contribute to those skilled in the art and understand
the present invention further, but not limit the present invention in any form.It should be pointed out that to those skilled in the art, without departing from the inventive
concept of the premise, some distortion and improvement can also be made.These all belong to protection scope of the present invention.
the Preparation and characterization of embodiment 1, diethyl phthalate haptens intermediate
(1) preparation of DEP haptens intermediate
Getting clean volume is 100mL tri-mouthfuls of round-bottomed asks, loads 10g (0.0474mol) 4-nitrophthalic acid, slowly add 16.6mL (0.2850mol) dehydrated alcohol
subsequently, add the dense H of 1.65mL while stirring at the bottom of bottle 2sO 4(0.0304mol) catalysis, 86 DEG C of stirring and re uxing, detect (developer: dissolve
a small amount of potassium permanganate in acetone solvent till raw material point disappears until thin plate chromatography (TLC); Developping agent, normal
hexane: ethyl acetate=5:1); React after stirring and re uxing 9h;
The water of the distillation unreacted ethanol of removing and generation; Now liquid is poured in frozen water while hot, separate out solid; The solid crude product
10%Na obtained after ltration 2cO 3solution washing is to water layer colourless (pH7.0-8.0); Finally, crude oil dehydrated alcohol recrystallization purifying, obtains light
yellow needles 4-nitrophthalic acid diethyl ester and DEP haptens intermediate after-20 DEG C of freeze-drying.4-nitrophthalic acid diethyl ester molecular formula: C 12h
13nO 6, molecular weight: 267.23; Productive rate: 73.6%; Fusing point: 29-31 DEG C, purity 97%.
(2) sign of DEP haptens intermediate
The DEP haptens intermediate 4-nitrophthalic acid diethyl ester obtained, its structure is by the detection means such as infrared spectra, UV spectrum Analysis and
Identi cation, as shown in Figure 1, its feature is as follows for the infrared spectrogram of DEP haptens intermediate: IR (KBr) ν/cm -1: 1536.55,1352.77 (-NO 2), 1728.04
(C=O), 1288.11,1131.01 (C-O-C), 2986.02,2875.40 (-CH 3), 1474.52 (d-OCH 2-), 1612.73,1589.33,1446.66 (C=C), 3049.64,1726.04,837.89 (C-H, Ar).Analytical results is
known ,-the NO of 4-nitrophthalic acid diethyl ester 2asymmetrical stretching vibration (ν as- nO2) absorption band appear at 1536.55cm -1near, symmetrical stretching
vibration (ν s- nO2) absorption band appear at 1352.77cm -1near; In addition, aryl esters ν c=oabsorption peak obviously appears at 1728.04cm -1place, the biobelt of the
stretching vibration of phthalic ester appears at 1288.11,1131.01cm -1place; The ν of virtue core c=cabsorption band appears at 1612.73,1589.33,1446.66cm -1place.All in
all, above-mentioned crest has absolutely proved in reaction product the constitutional features possessing phenyl ring, oxyethyl group, nitro base functional group.
10%Na obtained after ltration 2cO 3solution washing is to water layer colourless (pH7.0-8.0).Finally, crude oil dehydrated alcohol recrystallization purifying, obtains light
Identi cation, and the infrared spectrogram feature of DEP haptens intermediate is as follows: IR (KBr) ν/cm - 1: 1536.55,1352.77 (-NO 2), 1728.04 (C=O),
1288.11,1131.01 (C-O-C), 2986.02,2875.40 (-CH 3), 1474.52 (d-OCH 2-), 1612.73,1589.33,1446.66 (C=C), 3049.64,1726.04,837.89 (C-H, Ar).Analytical results is known ,-the
NO of 4-nitrophthalic acid diethyl ester 2asymmetrical stretching vibration (ν as- nO2) absorption band appear at 1536.55cm - 1near, symmetrical stretching vibration (ν s-
1near; In addition, aryl esters ν -1place, the biobelt of the stretching
nO2) absorption band appear at 1352.77cm - c=oabsorption peak obviously appears at 1728.04cm
vibration of phthalic ester appears at 1288.11,1131.01cm -1place; The ν of virtue core c=cabsorption band appears at 1612.73,1589.33,1446.66cm - 1place.All in all,
above-mentioned crest has absolutely proved in reaction product the constitutional features possessing phenyl ring, oxyethyl group, nitro base functional group.
10%Na obtained after ltration 2cO 3solution washing is to water layer colourless (pH7.0-8.0).Finally, crude oil dehydrated alcohol recrystallization purifying, obtains light
Identi cation, and the infrared spectrogram feature of DEP haptens intermediate is as follows: IR (KBr) ν/cm -1: 1536.55,1352.77 (-NO 2), 1728.04 (C=O), 1288.11,1131.01
(C-O-C), 2986.02,2875.40 (-CH 3), 1474.52 (d-OCH 2-), 1612.73,1589.33,1446.66 (C=C), 3049.64,1726.04,837.89 (C-H, Ar).Analytical results is known ,-the NO of 4-
nitrophthalic acid diethyl ester 2asymmetrical stretching vibration (ν as- nO2) absorption band appear at 1536.55cm -1near, symmetrical stretching vibration (ν s- nO2)
absorption band appear at 1352.77cm -1near; In addition, aryl esters ν c=oabsorption peak obviously appears at 1728.04cm -1place, the biobelt of the stretching vibration
of phthalic ester appears at 1288.11,1131.01cm -1place; The ν of virtue core c=cabsorption band appears at 1612.73,1589.33,1446.66cm - 1place.All in all, above-
mentioned crest has absolutely proved in reaction product the constitutional features possessing phenyl ring, oxyethyl group, nitro base functional group.
the haptenic Preparation and characterization of embodiment 4, diethyl phthalate
(1) the haptenic preparation of DEP
It is bottom 500mL tri-mouthfuls of round-bottomed asks that 1.5g (0.0056mol) DEP haptens intermediate (4-nitrophthalic acid diethyl ester) is loaded volume,
subsequently to adding 280mL (3.1495mol) benzene at the bottom of bottle while stirring, the pure zinc powder of 3.4g (0.0520mol) is added again after haptens is
dissolved by benzene, after stirring, gradation adds the dense HCl of 10mL (0.1200mol), the pure zinc powder of 3.4g (0.0520mol) is again added, 35 DEG C of stirring
reaction 10h after stirring at room temperature 15min;
After reaction terminates, 340mL cold water is added reaction system, and is neutralized to weakly alkaline (pH7.0-8.5) with 1M sodium hydroxide solution; Isolate
benzene layer after leaving standstill 1h, then use benzene aqueous layer extracted, combining extraction liquid, after washing, use anhydrous Na 2sO 4dehydrate,
extraction liquid underpressure distillation is to remove benzene, the light yellow solid obtained uses dehydrated alcohol recrystallization again, (eluent is silica gel
column chromatography, the mixed solution (V/V=8:1) of acetic acid and normal hexane) underpressure distillation afterwards, obtain light yellow crystal 4-aminophthalic
acid diethyl ester, i.e. DEP haptens.Diethyl phthalate hapten molecule formula: C 12h 15nO 4; Molecular weight 237.25; Productive rate 76.2%; Fusing point: 90 ~ 92 DEG C,
purity 98%.
(2) the haptenic sign of DEP
The DEP haptens 4-aminophthalic acid diethyl ester obtained, its structure is by detection means Analysis and Identi cation such as nucleus magnetic resonance,
infrared spectra, UV spectrum, and as shown in Figure 2, its feature is as follows for the haptenic infrared spectrogram of DEP: IR (KBr) ν/cm -1: 3466.06,3369.16 (-NH 2),
1720.18 (C=O), 1257.81,1068.87 (C-O-C), 2980.89,2927.48 (-CH 3), 1449.97 (d-OCH 2-), 1630.07,1564.19,1449.97 (C=C), 3227.98,1604.33,833.75 (C-H, Ar).Analytical
results is known, the asymmetrical stretching vibration (ν of the N-H key of the primary amine of 4-aminophthalic acid diethyl ester asN-H) and symmetrical stretching
vibration (ν sN-H) absorption band appear at 3466.06 and 3369.16cm respectively -1near, this has con rmed that nitro is reduced into amino; In addition, aryl esters ν
-1place, the biobelt of the stretching vibration of phthalic ester appears at 1257.81,1068.87cm respectively -1place;
c=oabsorption peak obviously appears at 1720.18cm
The ν of virtue core c=cabsorption band appears at 1630.07,1564.19,1449.97cm -1place.All in all, above-mentioned crest has absolutely proved the constitutional features
possessing phenyl ring, oxyethyl group, amido functional group in reaction product.
The data results of its nucleus magnetic hydrogen spectrum (Fig. 3) is as follows: 1hNMR (400MHz, CDCl 3): δ 7.73 (d, 1H, ArH), 6.73 (d, 1H, ArH), 6.67 (d, 1H, ArH), 4.36
(q, 2H, OCH 2), 4.30 (q, 2H, OCH 2), 4.26 (b, 2H ,-NH 2), 1.39 (t, 3H, OCH 2), 1.33 (t, 3H, OCH 2) ppm.Therefrom can obviously nd out: after phenyl ring being introduced
amino coupled arm, there is amino hydrogen in δ 4.17 place, and the position of the hydrogen atom occurred in wave spectrogram conforms to synthesized haptens 4-
aminophthalic acid diethyl ester structure with number.
On the whole, the result of infrared spectra and proton nmr spectra quali cation is all presented in Benzene Molecule structure and successfully introduces amino, by
itself and protein generation coupling and then can synthesize the arti cial holoantigen of DEP.
dissolved by benzene, after stirring, gradation adds the dense HCl of 10mL (0.1232mol), the pure zinc powder of 3.3g (0.0504mol) is again added, 30 DEG C of stirring
After reaction terminates, 330mL cold water is added reaction system, and is neutralized to weakly alkaline (pH7.0-8.5) with 1M potassium hydroxide solution; Isolate
benzene layer after leaving standstill 1h, then use benzene aqueous layer extracted, combining extraction liquid, after washing, use anhydrous Na 2sO 4dehydrate,
extraction liquid underpressure distillation is to remove benzene, the light yellow solid obtained uses dehydrated alcohol recrystallization again, (eluent is silica gel
column chromatography, the mixed solution (V/V=8:1) of acetic acid and normal hexane) underpressure distillation afterwards, obtain light yellow crystal 4-aminophthalic
acid diethyl ester, i.e. DEP haptens.Diethyl phthalate hapten molecule formula: C 12h 15nO 4; Molecular weight 237.25; Productive rate 76.2%; Fusing point: 90 ~ 92 DEG C,
purity 98%.
infrared spectra, UV spectrum, and DEP haptenic infrared spectrogram feature is as follows: IR (KBr) ν/cm -1: 3466.06,3369.16 (-NH 2), 1720.18 (C=O), 1257.81,1068.87
(C-O-C), 2980.89,2927.48 (-CH 3), 1449.97 (d-OCH 2-), 1630.07,1564.19,1449.97 (C=C), 3227.98,1604.33,833.75 (C-H, Ar).Analytical results is known, the asymmetrical
stretching vibration (ν of the N-H key of the primary amine of 4-aminophthalic acid diethyl ester asN-H) and symmetrical stretching vibration (ν sN-H) absorption band
appear at 3466.06 and 3369.16cm respectively -1near, this has con rmed that nitro is reduced into amino; In addition, aryl esters ν c=oabsorption peak obviously appears
at 1720.18cm -1place, the biobelt of the stretching vibration of phthalic ester appears at 1257.81,1068.87cm respectively -1place; The ν of virtue core c=cabsorption band
appears at 1630.07,1564.19,1449.97cm -1place.All in all, above-mentioned crest has absolutely proved the constitutional features possessing phenyl ring, oxyethyl group,
amido functional group in reaction product.
The data results of its nucleus magnetic hydrogen spectrum is as follows: 1hNMR (400MHz, CDCl 3): δ 7.73 (d, 1H, ArH), 6.73 (d, 1H, ArH), 6.67 (d, 1H, ArH), 4.36 (q, 2H,
OCH 2), 4.30 (q, 2H, OCH 2), 4.26 (b, 2H ,-NH 2), 1.39 (t, 3H, OCH 2), 1.33 (t, 3H, OCH 2) ppm.Therefrom can obviously nd out: after phenyl ring being introduced amino
coupled arm, there is amino hydrogen in δ 4.17 place, and the position of the hydrogen atom occurred in wave spectrogram conforms to synthesized haptens 4-
dissolved by benzene, after stirring, gradation adds the dense HCl of 9.3mL (0.112mol), the pure zinc powder of 3.7g (0.0560mol) is again added, 25 DEG C of stirring
After reaction terminates, 370mL cold water is added reaction system, and is neutralized to weakly alkaline (pH7.0-8.5) with 1M potassium hydroxide solution; Benzene
layer is isolated after leaving standstill 1h, use benzene aqueous layer extracted again, combining extraction liquid, dry with anhydrous Na2SO4 dehydration after
washing, extraction liquid underpressure distillation is to remove benzene, and the light yellow solid obtained uses dehydrated alcohol recrystallization again, (eluent is
silica gel column chromatography, the mixed solution (V/V=8:1) of acetic acid and normal hexane) underpressure distillation afterwards, obtain light yellow crystal 4-
aminophthalic acid diethyl ester, i.e. DEP haptens.Diethyl phthalate hapten molecule formula: C12H15NO4; Molecular weight 237.25; Productive rate 76.2%; Fusing point:
90 ~ 92 DEG C, purity 98%.
infrared spectra, UV spectrum, DEP haptenic infrared spectrogram feature is as follows: IR (KBr) ν/cm-1:3466.06, (3369.16-NH2), 1720.18 (C=O), 1257.81,1068.87 (C-O-
C), 2980.89,2927.48 (-CH3), 1449.97 (d-OCH2-), 1630.07,1564.19,1449.97 (C=C), 3227.98,1604.33,833.75 (C-H, Ar).Analytical results is known, the asymmetrical
stretching vibration (ν asN-H) of the N-H key of the primary amine of 4-aminophthalic acid diethyl ester and the absorption band of symmetrical stretching vibration (ν sN-
H) appear near 3466.06 and 3369.16cm-1 respectively, and this has con rmed that nitro is reduced into amino; In addition, aryl esters ν c=o absorption peak obviously
appears at 1720.18cm-1 place, and the biobelt of the stretching vibration of phthalic ester appears at 1257.81,1068.87cm-1 place respectively; The ν c=c absorption band
of virtue core appears at 1630.07,1564.19,1449.97cm-1 place.All in all, above-mentioned crest has absolutely proved the constitutional features possessing phenyl ring,
oxyethyl group, amido functional group in reaction product.
The data results of its nucleus magnetic hydrogen spectrum is as follows: 1HNMR (400MHz, CDCl3): δ 7.73 (d, 1H, ArH), 6.73 (d, 1H, ArH), 6.67 (d, 1H, ArH), 4.36 (q, 2H,
OCH2), 4.30 (q, 2H, OCH2), 4.26 (b, 2H ,-NH2), 1.39 (t, 3H, OCH2), 1.33 (t, 3H, OCH2) ppm.Therefrom can obviously nd out: after phenyl ring being introduced amino
coupled arm, there is amino hydrogen in δ 4.17 place, and the position of the hydrogen atom occurred in wave spectrogram conforms to synthesized haptens 4-
the preparation of the arti cial holoantigen of embodiment 7, diethyl phthalate
Diethyl phthalate haptens prepared by the present invention, is mainly used in the immunodetection of diethyl phthalate in environment.One of its main application, can
be used for exactly directly and protein macromolecule coupling, prepare the immunogen for immune animal, and then prepare corresponding mono-clonal or polyclonal
antibody.Set up the method for immunity of diethyl phthalate on this basis.Following applicating example is used as below with regard to haptenic the making of diethyl
phthalate:
Adopt diazotization legal system for DEP immunogen DEP-BSA, concrete synthesis step: it is in the Erlenmeyer ask of 25mL that 0.0238g (0.1mM) DEP haptens is
loaded volume, limit drips 0.75mLH 2o limit is stirred, and adds the dense HCl of 0.025mL again, cool in the rearmounted ice bath of thing heating for dissolving to be
mixed after mixing; Subsequently, when 4 DEG C of low temperature stir, dropwise drip 1M sodium nitrite solution, controlling such as reaction acidity with pH test paper
is 2.0-3.0, simultaneously with starch potassium iodide paper colour developing, developing time is the 1-3s after dripping, and stops dripping when test paper becomes
dusty blue instantaneously by white, continue reaction 30min again, add 2.0g urea to remove unreacted Sodium Nitrite; Then, 0.08mMBSA solution (dissolving of
0.01MpH9.18 sodium borate buffer liquid) is dropwise added in above-mentioned diazonium salt, until solution is gradually in orange red, after continuing reaction 2h, the
antigen crude product obtained is loaded dialysis tubing, be placed in 0.01MpH7.40 phosphate buffered saline buffer to dialyse 3d, change water 1 time, nally by end
product 4000rmin every 8h -1off-line 15min, gets its supernatant liquor and DEP immunogen DEP-BSA.Immunogen after ultraviolet-visible spectrophotometer
quali cation, in a small amount packing ,-20 DEG C of lyophilizes, packing in-20 DEG C of preservations.
DEP coating antigen DEP-OVA is prepared by glutaraldehyde method, concrete synthesis step: it is in the Erlenmeyer ask of 25mL that 0.0238g (0.1mM) DEP haptens is
loaded volume, limit drips 1mLN, dinethylformamide (DMF) limit is stirred, dropwise join in 0.08mMOVA solution (dissolving of 0.01MpH7.40 phosphate buffered saline
buffer) 10mL after stirring again, 0.036mL25% glutaraldehyde is slowly added subsequently in this mixed system, after 4 DEG C of low temperature black out stirring
reaction 24h, the antigen crude product obtained is loaded dialysis tubing, be placed in 0.01MpH7.40 phosphate buffered saline buffer low temperature 4 DEG C dialysis
3d, water is changed 1 time every 8h, nally by end product 4000rmin -1off-line 15min, gets its supernatant liquor and DEP coating antigen DEP-OVA.Coating antigen after
ultraviolet-visible spectrophotometer quali cation, lyophilize, in a small amount packing ,-20 DEG C of freezen protective.
Conjugate is after ultraviolet-visible pectrophotometer scanning quali cation, and as can be seen from the DEP haptens of Fig. 4 and Fig. 5, carrier proteins and arti cial
holoantigen ultraviolet spectrogram, the haptenic charateristic avsorption band of DEP is positioned at 295nm; The charateristic avsorption band of carrier proteins BSA
is positioned at 227nm and 278nm place, and wherein the absorption value at 278nm ultraviolet absorption peak place is very low; Carrier proteins OVA has two
ultraviolet absorption peaks, lays respectively at 234nm and 279nm place, and wherein the absorption value at 279nm ultraviolet absorption peak place is lower.Not only
comprise the characteristic peak of BSA and OVA in the UV spectrum of conjugate DEP-BSA and DEP-OVA respectively, also occur new absorption peak in 336 and
360nm place.DEP-BSA and DEP-OVA haptens charateristic avsorption band there occurs red shift, and this may be caused by the impact of the absorption peak being
subject to carrier proteins.The appearance of absorption peak Red Shift Phenomena, above information shows DEP-BSA and DEP-OVA coupling success.
These holoantigens both may be used for immune animal, and the immune response produced by animal obtains the mono-clonal that can be used for doing
immunoassay or polyclonal antibody, can use again as Immune competition object.
the preparation of embodiment 8, phthalic acid two (2-ethyl) own ester polyclonal antibody
Select 2 adult healthy male New Zealand rabbits, the arti cial immunogen DEP-BSA synthesized is after fully mixing with Freund's complete adjuvant, neck and back
point-like injecting immune are carried out to White Rabbit, after 7 booster immunizations, adopt indirect enzyme-linked immunosorbent assay antiserum titre, its
antiserum titre reaches 120000 all.Test display cross reaction is all not obvious, illustrates that its speci city is better.
diethyl phthalate in embodiment 9, indirect competition BA-ELISA immune analysis method testing environment sample
Concrete steps are as follows: be buffered liquid (0.05MpH9.60 carbonate buffer solution) suitably dilution coating antigen solution (DEP-OVA) with bag, 100 μ L/ holes,
add in 96 hole enzyme plates, and 4 DEG C of bags are spent the night; Next day outwells coating buffer, and every hole adds 200 μ L washingss (0.01MpH7.40PBST),
repeated washing three times, each 3min; After patting dry enzyme plate with thieving paper, every hole adds con ning liquid 200 μ L, 37 DEG C of incubations 1 hour;
Outwell con ning liquid, repeat above-mentioned washing process three times, the anti-DEP polyclonal antibody (Bio-pAb-DEP) of biotinylation is diluted to proper concn,
every hole adds 50 μ L biotinylated antibodies and 50 μ LDEP standard models (PBS (v/v) gradient dilution with containing 5% methyl-sulphoxide (DMSO)) successively,
the every hole of blank well adds 100 μ LPBS damping uids, 37 DEG C of incubations 30 minutes; Outwell antigen-antibody reaction liquid, repeated washing three times,
suitably dilute with PBST the Streptavidin (HRP-SA) that horseradish peroxidase (HRP) marks, every hole adds 100 μ L, 37 DEG C of incubations 1 hour; Outwell HRP-SA
solution, repeated washing ve times, every hole adds freshly prepared nitrite ion (400 μ L2.5mg/mL3,3 ', 5, the H of 5 '-tetramethyl benzidine substrate solution and
10mL phosphoric acid salt-citric acid substrate buffer solution, 10 μ L30% 2o 2be mixed to form nitrite ion) 100 μ L/ holes, lucifuge reaction 15min under room
temperature; Every hole adds 50 μ L stop buffers (2mol/L sulphuric acid soln) with color development stopping, then measures each hole in 450nm and 630nm place
absorbance (OD value), with OD=OD by microplate reader 450-OD 630as nal reading.Experimental result inhibiting rate represents, and is ordinate zou with inhibiting rate,
and the logarithmic value of standard model concentration is X-coordinate drawing standard curve, and the standard working curve of foundation as shown in Figure
6.Wherein, inhibiting rate (%)=(1-A/A 0) × 100, wherein A is the OD value in the hole having sample to exist, A 0for there is no the OD value in the hole of sample.
Being prepared as follows of biotinylated antibody: with 0.05MpH9.60 carbonate buffer solution, the anti-DEP polyclonal antibody after purifying is diluted to about
1.0mg/mL, gets 2mL and add in Erlenmeyer ask; Prepare 1.0mg/mL vitamin H-N-succinimido ester (BNHS) solution with DMSO, in above-mentioned Erlenmeyer ask,
add BNHS solution, make BNHS: the mass ratio of anti-DEP polyclonal antibody is 1:10, and stirred at ambient temperature reacts 4 hours; After reaction terminates,
reaction solution is loaded in dialysis tubing, with PBS damping uid dialysis 3d, change water every day 3 times.Centrifugation obtains corresponding biotinylated
antibody solution (Bio-pAb-DEP), after the packing of antibody-solutions small volume, in-20 DEG C of freezen protective after removing a small amount of precipitation.
The typical curve equation that indirect competition BA-ELISA immune analysis method detects diethyl phthalate is Y=22.53LgC dEP+ 57.96, coe cient R=0.9832,
wherein IC 50represent the sensitivity of detection method, analyte concentration 0.443 μ g/L corresponding when namely inhibiting rate is 50%; IC 10the Monitoring
lower-cut of method for expressing, analyte concentration 0.0079 μ g/L corresponding when namely inhibiting rate is 10%; IC 20-IC 80represent linearity range, i.e. 0.021 μ
g/L ~ 9.512 μ g/L.
In addition, we determine the cross reacting rate of 7 kinds of DEP analogs, its result is as shown in following table 4-6, DEP and dimethyl phthalate (DMP), dipropyl
phthalate (DPrP), dibutyl phthalate (DBP), diisobutyl phthalate (DIBP), phthalic acid two (ethyl) own ester (DEHP), the cross reacting rate relatively little (all below 5%) of
the analogs such as diisononyl phthalate (DINP), then higher with the cross reacting rate of 4-DENP and 4-DEAP, analyzing its reason may be due to this two kinds of
material system DEP haptens precursors and haptens, with DEP, there is similar spatial con guration of molecules, cause their cross reaction stronger thus.On the whole,
anti-DEP polyclonal antibody has good speci city, more feasible with DEP in indirect BA-ELISA method testing environment sample.
The cross reacting rate of table 1DEP analog
Above speci c embodiments of the invention are described.It is to be appreciated that the present invention is not limited to above-mentioned particular implementation,
those skilled in the art can make various distortion or amendment within the scope of the claims, and this does not affect esh and blood of the present invention.
Patent Citations (4)
Publication number Priority date Publication date Assignee Title
WO2009119858A1 * 2008-03-27 2009-10-01 田辺三菱製薬株式会 Benzene compound, and use thereof for medical purposes
社
CN102539743A * 2011-12-15 2012-07-04 安徽师范大学 Detection method of diethyl phthalate
CN102590493A * 2012-02-17 2012-07-18 南开大学 Kit for diethyl phthalate uorescence polarization immunoassay
CN104130144A * 2014-07-30 2014-11-05 新疆农垦科学院 Synthetic method and application of universal hapten and antigen for phthalate
plasticizer
Family To Family Citations
* Cited by examiner, † Cited by third party
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Priority And Related Applications
Priority Applications (1)
Application Priority date Filing date Title
CN201510515294.6A 2015-08-20 2015-08-20 Plasticizing agent diethyl phthalate hapten and preparation method thereof
Applications Claiming Priority (1)
Application Filing date Title
CN201510515294.6A 2015-08-20 Plasticizing agent diethyl phthalate hapten and preparation method thereof
Legal Events
Date Code Title Description
2015-11-25 C06 Publication
2015-11-25 PB01 Publication
2016-03-23 C10 Entry into substantive examination
2016-03-23 SE01 Entry into force of request for substantive examination
2018-07-20 RJ01 Rejection of invention patent application after publication
2018-07-20 RJ01 Rejection of invention patent application after publication Application publication date: 20151125
Concepts
machine-extracted Download Filter table
Name Image Sections Count Query match
Diethyl phthalate title,claims,abstract,description 90 0.000
O O
O O
preparation methods title,claims,abstract,description 52 0.000
4-Nitrophthalic acid OH
claims,abstract,description 15 0.000
-
O O
+
N O
OH
Carrier Proteins claims,abstract,description 9 0.000
Carrier Proteins claims,abstract,description 9 0.000
glutaraldehyde claims,abstract,description 7 0.000
O O
diazotization reactions claims,abstract,description 6 0.000
chemical reactions claims,description 48 0.000
ethanol claims,description 25 0.000
OH
water claims,description 25 0.000
liquids claims,description 23 0.000
benzene claims,description 22 0.000
zinc claims,description 22 0.000
Zn
acids claims,description 20 0.000
diethyl 4-nitrobenzene-1,2-dicarboxylate O claims,description 18 0.000

N+
O
O-
O
O
extraction claims,description 14 0.000
recrystallisation claims,description 14 0.000
EtOH claims,description 13 0.000
distillation claims,description 13 0.000
organic solvents claims,description 12 0.000
phenyl group claims,description 11 0.000
solids claims,description 11 0.000
biosynthetic process claims,description 9 0.000
HCl claims,description 7 0.000
HCl
esteri cation reactions claims,description 6 0.000
silica gel chromatography claims,description 6 0.000
acetone claims,description 5 0.000

O
acetone claims,description 5 0.000
synthesis reactions claims,description 5 0.000
synthesizing claims,description 5 0.000
formation reactions claims,description 4 0.000
oils claims,description 4 0.000
products claims,description 4 0.000
constituents claims,description 3 0.000
hydrochloric acid claims,description 3 0.000
mixtures claims,description 3 0.000
toluene claims,description 2 0.000
toluene claims,description 2 0.000
ELISA abstract,description 14 0.000
antibodies abstract,description 13 0.000
chemical substances by application abstract,description 7 0.000
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06/09/2020 CN105085299A - Plasticizing agent diethyl phthalate hapten and preparation method thereof - Google Patents

Plasticizing agent diethyl phthalate hapten and preparation method thereof

Application CN201510515294.6A events

2015-08-20 Application led by 上海交通大学

2015-08-20 Priority to CN201510515294.6A

2015-11-25 Publication of CN105085299A

Info: Patent citations (4), Legal events, Similar documents,

External links: Espacenet, Global Dossier, Discuss

Claims (10) Hide Dependent

Fluidizer diethyl phthalate haptens and preparation method thereof

Summary of the invention

Preferably, described haptens Intermediate Preparation also comprises the steps:

13nO 6, molecular weight: 267.23; Fusing point: 29 ~ 31 DEG C.

Preferably, described haptens preparation also comprises the steps:

Preferably, described carrier proteins comprises bovine serum albumin or oralbumin.

Wherein, BSA is bovine serum albumin, and OVA is oralbumin.

Accompanying drawing explanation

Fig. 1 is the infrared spectrogram of diethyl phthalate haptens intermediate;

Fig. 2 is the haptenic infrared spectra of diethyl phthalate;

Fig. 3 is the haptenic nmr spectrum of diethyl phthalate;

the Preparation and characterization of embodiment 1, diethyl phthalate haptens intermediate

(1) preparation of DEP haptens intermediate

(2) sign of DEP haptens intermediate

the Preparation and characterization of embodiment 2, diethyl phthalate haptens intermediate

(2) sign of DEP haptens intermediate

the Preparation and characterization of embodiment 3, diethyl phthalate haptens intermediate

(1) preparation of DEP haptens intermediate

(2) sign of DEP haptens intermediate

the haptenic Preparation and characterization of embodiment 4, diethyl phthalate

(1) the haptenic preparation of DEP

(2) the haptenic sign of DEP

the haptenic Preparation and characterization of embodiment 5, diethyl phthalate

(1) the haptenic preparation of DEP

(2) the haptenic sign of DEP

the haptenic Preparation and characterization of embodiment 6, diethyl phthalate

(1) the haptenic preparation of DEP

(2) the haptenic sign of DEP

the preparation of the arti cial holoantigen of embodiment 7, diethyl phthalate

The cross reacting rate of table 1DEP analog

Patent Citations (4)

Publication number Priority date Publication date Assignee Title

CN102539743A * 2011-12-15 2012-07-04 安徽师范大学 Detection method of diethyl phthalate

Family To Family Citations

* Cited by examiner, † Cited by third party

Publication Publication Date Title

JP2731739B2 1998-03-25 Quaternary ammonium immunogen conjugates and immunoassay reagents

EP0327163B1 1995-04-12 Detection of chemicals by immunoassay

AU614733B2 1991-09-12 Novel derivatives and use thereof

US6153442A 2000-11-28 Reagents and methods for speci c binding assays

CN110054694A 2019-07-26 The antibody and application thereof of Aripiprazole haptens

JP4570601B2 2010-10-27 Tricyclic antidepressant derivatives and immunoassays

ES2731254T3 2019-11-14 Essays for vitamin D epimers

Su et al. 2004 Chromatic immunoassay based on polydiacetylene vesicles

Priority And Related Applications

Priority Applications (1)

Application Priority date Filing date Title

Applications Claiming Priority (1)

Application Filing date Title

Date Code Title Description

2015-11-25 C06 Publication

2015-11-25 PB01 Publication

2016-03-23 C10 Entry into substantive examination

2016-03-23 SE01 Entry into force of request for substantive examination