CN112812006A - P-hydroxyacetophenone hapten, artificial antigen, and synthesis method and application thereof - Google Patents
P-hydroxyacetophenone hapten, artificial antigen, and synthesis method and application thereof Download PDFInfo
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- CN112812006A CN112812006A CN202011644034.6A CN202011644034A CN112812006A CN 112812006 A CN112812006 A CN 112812006A CN 202011644034 A CN202011644034 A CN 202011644034A CN 112812006 A CN112812006 A CN 112812006A
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- hydroxyacetophenone
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- TXFPEBPIARQUIG-UHFFFAOYSA-N 4'-hydroxyacetophenone Chemical compound CC(=O)C1=CC=C(O)C=C1 TXFPEBPIARQUIG-UHFFFAOYSA-N 0.000 title claims abstract description 153
- 239000000427 antigen Substances 0.000 title claims abstract description 32
- 102000036639 antigens Human genes 0.000 title claims abstract description 32
- 108091007433 antigens Proteins 0.000 title claims abstract description 32
- 238000001308 synthesis method Methods 0.000 title description 3
- 238000000034 method Methods 0.000 claims abstract description 29
- 102000014914 Carrier Proteins Human genes 0.000 claims abstract description 14
- 108010078791 Carrier Proteins Proteins 0.000 claims abstract description 14
- 150000003839 salts Chemical class 0.000 claims abstract description 12
- 239000002253 acid Substances 0.000 claims abstract description 6
- 238000002360 preparation method Methods 0.000 claims abstract description 4
- 238000006243 chemical reaction Methods 0.000 claims description 17
- 239000012043 crude product Substances 0.000 claims description 8
- 239000003960 organic solvent Substances 0.000 claims description 8
- 230000002194 synthesizing effect Effects 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 239000002585 base Substances 0.000 claims description 3
- 239000012074 organic phase Substances 0.000 claims description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical group C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 2
- 108010058846 Ovalbumin Proteins 0.000 claims description 2
- NPYPAHLBTDXSSS-UHFFFAOYSA-N Potassium ion Chemical group [K+] NPYPAHLBTDXSSS-UHFFFAOYSA-N 0.000 claims description 2
- 239000003513 alkali Substances 0.000 claims description 2
- 229940098773 bovine serum albumin Drugs 0.000 claims description 2
- 150000002632 lipids Chemical class 0.000 claims description 2
- 229940092253 ovalbumin Drugs 0.000 claims description 2
- 229910001414 potassium ion Chemical group 0.000 claims description 2
- 230000035484 reaction time Effects 0.000 claims description 2
- 238000010898 silica gel chromatography Methods 0.000 claims description 2
- 239000011734 sodium Substances 0.000 claims description 2
- 229910001415 sodium ion Inorganic materials 0.000 claims description 2
- 150000008065 acid anhydrides Chemical class 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 10
- 150000008064 anhydrides Chemical class 0.000 abstract description 6
- 150000002148 esters Chemical class 0.000 abstract description 6
- 238000010189 synthetic method Methods 0.000 abstract description 5
- 239000002994 raw material Substances 0.000 abstract description 4
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 abstract description 2
- 238000003018 immunoassay Methods 0.000 abstract description 2
- 238000012216 screening Methods 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 20
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 18
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical group CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 11
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 10
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 10
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical group [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 239000007853 buffer solution Substances 0.000 description 6
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 4
- 150000001793 charged compounds Chemical class 0.000 description 4
- 239000011248 coating agent Substances 0.000 description 4
- 238000000576 coating method Methods 0.000 description 4
- 238000000502 dialysis Methods 0.000 description 4
- 239000002504 physiological saline solution Substances 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- YOETUEMZNOLGDB-UHFFFAOYSA-N 2-methylpropyl carbonochloridate Chemical compound CC(C)COC(Cl)=O YOETUEMZNOLGDB-UHFFFAOYSA-N 0.000 description 3
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 3
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 3
- 230000008878 coupling Effects 0.000 description 3
- 238000010168 coupling process Methods 0.000 description 3
- 238000005859 coupling reaction Methods 0.000 description 3
- 239000003480 eluent Substances 0.000 description 3
- 235000019441 ethanol Nutrition 0.000 description 3
- 238000011049 filling Methods 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 2
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical group [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-N carbonic acid Chemical compound OC(O)=O BVKZGUZCCUSVTD-UHFFFAOYSA-N 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 235000017557 sodium bicarbonate Nutrition 0.000 description 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- MVNVIVWSAFEIII-UHFFFAOYSA-N 1-(4-hydroxyphenyl)ethanone Chemical compound CC(=O)C1=CC=C(O)C=C1.CC(=O)C1=CC=C(O)C=C1 MVNVIVWSAFEIII-UHFFFAOYSA-N 0.000 description 1
- 229940073735 4-hydroxy acetophenone Drugs 0.000 description 1
- 241000208327 Apocynaceae Species 0.000 description 1
- 235000008658 Artemisia capillaris Nutrition 0.000 description 1
- 241000092668 Artemisia capillaris Species 0.000 description 1
- 235000003069 Artemisia scoparia Nutrition 0.000 description 1
- 241001249148 Artemisia scoparia Species 0.000 description 1
- 241000208838 Asteraceae Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 241000511967 Tylophora Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000000862 absorption spectrum Methods 0.000 description 1
- NDAUXUAQIAJITI-UHFFFAOYSA-N albuterol Chemical compound CC(C)(C)NCC(O)C1=CC=C(O)C(CO)=C1 NDAUXUAQIAJITI-UHFFFAOYSA-N 0.000 description 1
- 230000002421 anti-septic effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 239000008845 cholagoga Substances 0.000 description 1
- 229940124571 cholagogue Drugs 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000002740 effect on eyes Effects 0.000 description 1
- 230000001626 effect on respiratory system Effects 0.000 description 1
- 230000002500 effect on skin Effects 0.000 description 1
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 1
- 238000002101 electrospray ionisation tandem mass spectrometry Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 208000014951 hematologic disease Diseases 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 239000012770 industrial material Substances 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 230000007794 irritation Effects 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- -1 p-hydroxyacetophenone-chloroacetic acid Chemical compound 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- YJQZYXCXBBCEAQ-UHFFFAOYSA-N ractopamine Chemical compound C=1C=C(O)C=CC=1C(O)CNC(C)CCC1=CC=C(O)C=C1 YJQZYXCXBBCEAQ-UHFFFAOYSA-N 0.000 description 1
- 229940074095 ractopamine Drugs 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 229960002052 salbutamol Drugs 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- FDRCDNZGSXJAFP-UHFFFAOYSA-M sodium chloroacetate Chemical compound [Na+].[O-]C(=O)CCl FDRCDNZGSXJAFP-UHFFFAOYSA-M 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 229920002994 synthetic fiber Polymers 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C59/00—Compounds having carboxyl groups bound to acyclic carbon atoms and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
- C07C59/40—Unsaturated compounds
- C07C59/76—Unsaturated compounds containing keto groups
- C07C59/90—Unsaturated compounds containing keto groups containing singly bound oxygen-containing groups
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
- C07C51/347—Preparation of carboxylic acids or their salts, halides or anhydrides by reactions not involving formation of carboxyl groups
- C07C51/367—Preparation of carboxylic acids or their salts, halides or anhydrides by reactions not involving formation of carboxyl groups by introduction of functional groups containing oxygen only in singly bound form
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/77—Ovalbumin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
Abstract
The invention discloses a p-hydroxyacetophenone hapten, an artificial antigen, a synthetic method and application thereof. The method takes p-hydroxyacetophenone as a raw material, the p-hydroxyacetophenone reacts with a halogenated acid salt to generate p-hydroxyacetophenone hapten containing-COOH active groups, and the p-hydroxyacetophenone hapten and carrier protein are coupled to synthesize the p-hydroxyacetophenone artificial antigen by an active ester method or a mixed anhydride method. The p-hydroxyacetophenone artificial antigen prepared by the invention can be applied to the preparation of p-hydroxyacetophenone polyclonal antibodies or monoclonal antibodies. The p-hydroxyacetophenone artificial antigen immunoassay method is low in detection cost, simple to use and suitable for screening and detecting a large number of samples.
Description
Technical Field
The invention belongs to the technical field of immunological detection, and relates to a p-hydroxyacetophenone hapten, an artificial antigen, a synthetic method and application thereof.
Background
p-Hydroxyacetophenone (p-Hydroxyacetophenone), also known as 4-Hydroxyacetophenone and 4-acetylphenol, commonly known as conidinol, has a molecular formula C8H8O2The compound has molecular weight of 136.15, is white needle-shaped crystal at normal temperature, is flammable, is easy to dissolve in hot water, methanol, ethanol, ether, acetone and benzene, is difficult to dissolve in petroleum ether, is a common synthetic drug intermediate, and has a structural formula as follows:
a ketonic compound belonging to the genus of p-hydroxyacetophenone is an industrial material used for cholagogue, ractopamine, salbutamol and other organic synthetic materials, and naturally exists in stems and leaves of Artemisia scoparia of Compositae, and roots of plants such as Artemisia capillaris and Tylophora rostrata of Asclepiadaceae. P-hydroxyacetophenone can be used as antiseptic of cosmetic, and has irritation effect on eyes, respiratory system and skin; chronic hazards may occur when used in large quantities; prolonged exposure may lead to hematologic disease.
The detection of p-hydroxyacetophenone is mainly based on physicochemical analysis (such as LC-MS and HPLC). However, when the instrument method is used, the sensitivity of the method is greatly influenced by the steps of purifying, concentrating and the like of the sample, the determination method is complex and tedious, the detection flux is small, the detection needs to be carried out by culture professionals, the detection cost is high, and the rapid detection and analysis of a large batch of samples cannot be realized. Therefore, it is necessary to develop a detection method which is simpler, faster and more convenient.
The immunoassay method is one of important methods for rapidly detecting trace residues of small molecular substances at present, has low detection cost and simple use, and is suitable for screening and detecting a large number of samples.
Disclosure of Invention
Aiming at the problems, the invention aims to provide a p-hydroxyacetophenone hapten, an artificial antigen, a synthetic method and an application thereof, and lays a foundation for establishing a non-detection method to provide an immune antigen, a coating antigen and an antibody preparation.
In order to achieve the purpose, the invention adopts the technical scheme that: the molecular structural formula of p-hydroxyacetophenone hapten is as follows:
n is a natural number of 1 to 6.
As a preferred embodiment of the present invention, n ═ 2.
The invention claims a synthetic method of p-hydroxyacetophenone hapten, which comprises the following steps: reacting p-hydroxyacetophenone with a halogenated acid salt in an organic solvent in the presence of a base to generate the p-hydroxyacetophenone hapten.
As a preferred embodiment of the invention, the reaction temperature is 70-80 ℃ and the reaction time is 3.5-4.5 h.
As a preferred embodiment of the present invention, TLC monitoring was performed using chloroform/methanol developing agent during the reaction; the volume ratio of chloroform to methanol is 5: 1.
As a preferred embodiment of the invention, the reaction solution obtained from the reaction is adjusted to pH value of 1.0-2.5, is extracted by organic solvent, the organic phase is collected and added with salt to remove water, then the organic solvent is removed to obtain crude product, and the crude product is purified by silica gel column chromatography to obtain purified p-hydroxyacetophenone hapten.
More preferably, the pH value of the obtained reaction liquid is adjusted by using a hydrochloric acid solution with the concentration of 2 moL/L; the organic solvent is ethyl acetate; the salt is anhydrous sodium sulfate; the crude product was purified using chloroform/methanol elution at a volume ratio of 5:1 to 6: 1.
As a preferred embodiment of the present invention, the formula of the haloacid salt is R- (CH)2) n-COOM, wherein R is Cl or Br, and M is sodium or potassium ion.
In a preferred embodiment of the invention, the molar ratio of the p-hydroxyacetophenone to the alkali is 1:4 to 1:5, and the molar amount of the halogenated acid salt is 1.5 to 2.0 times of the molar amount of the p-hydroxyacetophenone.
As a preferred embodiment of the present invention, the haloate salt is added to the reaction in 3 portions with a 10min interval between each addition.
As a preferred embodiment of the present invention, the base is sodium hydroxide or potassium hydroxide; the organic solvent is ethanol.
In addition, the p-hydroxyacetophenone hapten is combined with carrier protein to obtain the p-hydroxyacetophenone artificial antigen, and the molecular structural formula is as follows:
the carrier protein is bovine serum albumin and ovalbumin.
As a preferred embodiment of the invention, the synthetic method of the p-hydroxyacetophenone artificial antigen is an active lipid method or a mixed anhydride method.
The active ester process comprises the steps of:
s1: dissolving p-hydroxyacetophenone hapten in N, N-dimethylformamide, adding N, N-dicyclohexylcarbodiimide and N-hydroxysuccinimide, and reacting at room temperature in a dark place overnight;
s2: centrifuging at 0-4 deg.C, collecting supernatant, slowly adding into carbonic acid buffer solution with pH of 9.0-9.5, and reacting at 0-4 deg.C overnight;
s3: and (5) filling the reaction solution obtained in the step two into a dialysis bag, and dialyzing with physiological saline to obtain the p-hydroxyacetophenone artificial antigen.
More preferably, in the active ester process S1, the molar amounts of hydroxyacetophenone hapten, N-dicyclohexylcarbodiimide and N-hydroxysuccinimide are the same.
More preferably, in the active ester method S2, a buffer solution contains carrier protein BSA or OVA, and the mass concentration of the carrier protein in the buffer solution is 10-15 mg/mL.
The mixed anhydride process comprises the steps of:
the method comprises the following steps: dissolving p-hydroxyacetophenone hapten in N, N-dimethylformamide, adding N-tributylamine, dropwise adding isobutyl chloroformate at 0-4 ℃, and stirring for reacting for 1h to obtain a solution A;
step two: dissolving carrier protein in 1mol/L sodium bicarbonate buffer solution with pH of 9.0-9.6 to obtain solution B;
step three: slowly dripping the solution A into the solution B, stirring at 0-4 ℃ for reaction overnight, filling into a dialysis bag after the reaction is finished, and dialyzing with physiological saline to obtain the artificial antigen p-hydroxyacetophenone.
More preferably, in the first step of the mixed anhydride method, the molar ratio of p-hydroxyacetophenone hapten, n-tributylamine to isobutyl chloroformate is 2: 6: 3.
in addition, the invention also claims that the p-hydroxyacetophenone artificial antigen is applied to the preparation of p-hydroxyacetophenone polyclonal antibodies or monoclonal antibodies.
The principle of the invention is as follows: because the p-hydroxyacetophenone is a small molecular substance (the molecular weight is less than 1000Da) and has no immunogenicity, the p-hydroxyacetophenone must be coupled with a macromolecular protein to prepare an artificial antigen so as to enable an animal to produce an antibody. However, as the p-hydroxyacetophenone does not contain active groups and cannot be coupled with macromolecular protein, the molecular modification of the p-hydroxyacetophenone is needed, and-COOH active groups are introduced to synthesize the p-hydroxyacetophenone hapten. The invention takes p-hydroxyacetophenone as raw material, and reacts with sodium hydroxide, ethanol and haloid salt to synthesize p-hydroxyacetophenone hapten; then coupling the p-hydroxyacetophenone hapten and carrier protein by an active ester method or a mixed anhydride method to synthesize the p-hydroxyacetophenone artificial antigen.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples.
Example 1
The invention relates to an embodiment of p-hydroxyacetophenone hapten, in particular to a method for synthesizing the p-hydroxyacetophenone hapten, which comprises the following steps:
(1) placing 6.81g of p-hydroxyacetophenone (50mmoL) and 8g of sodium hydroxide (200mmoL) in a round-bottom flask, adding 100mL of absolute ethyl alcohol, and magnetically stirring in a water bath at 75 ℃; adding 8.7g of sodium chloroacetate (75mmoL) into the anhydrous ethanol reaction solution in batches within 30min, and continuing reflux reaction for 4h, wherein TLC monitoring is carried out by using a developing agent with the mass ratio of chloroform to methanol being 5: 1;
(2) after the reaction is finished, extracting with 150mL ethyl acetate for 5 times, removing unreacted raw materials, adjusting the pH of the water phase to 1.5-2.5 by using 50% hydrochloric acid solution in mass concentration, separating out oily substances, and extracting with ethyl acetate for 3 times; removing water in an organic phase by using anhydrous sodium sulfate, evaporating ethyl acetate on a rotary evaporator to obtain a crude product, passing the crude product through a silica gel column, eluting the crude product by using eluent with the volume ratio of chloroform to methanol being 5:1-6:1, and concentrating the eluent on the rotary evaporator at reduced pressure and low temperature until the eluent is dried to obtain the p-hydroxyacetophenone hapten.
Example 2
The invention relates to an embodiment of an artificial antigen of p-hydroxyacetophenone, and a synthesis method of the artificial antigen of p-hydroxyacetophenone comprises the following specific steps:
synthesis and purification of immune antigen:
the immune antigen adopts active ester method. Mu. mol of p-hydroxyacetophenone hapten prepared in example 1 was dissolved in 1mL of DMF, and then N, N-dicyclohexylcarbodiimide and N-hydroxysuccinimide were added in equimolar amounts to this solution, and reacted overnight at room temperature in the absence of light, and the precipitate was removed by centrifugation at 4 ℃ to take the supernatant, and this was slowly added to 5mL of a carbonic acid buffer pH 9.5 containing the carrier protein BSA at a carrier protein concentration of 10 mg/mL. The mixture was allowed to react overnight at 4 ℃ and, after completion of the reaction, filled into dialysis bags, which were then dialyzed with 0.9% physiological saline for 3 d. The dialyzed solution is the artificial immune antigen p-hydroxyacetophenone-BSA.
Synthesis and purification of coating antigen:
the coating antigen adopts a mixed anhydride method. 0.1mmol of p-hydroxyacetophenone hapten prepared in example 1 was dissolved in 1mL of DMF, 0.3mmol of n-tributylamine was added, 0.15mmol of isobutyl chloroformate was slowly dropped in an ice bath, and the mixture was stirred at 4 ℃ for 1 hour to obtain solution A. 60mg of the carrier protein OVA was dissolved in 5mL of 1mol/L sodium bicarbonate buffer solution at pH 9.6 to obtain solution B. Slowly dripping the solution A into the solution B, magnetically stirring at 4 ℃ for reaction overnight, filling into a dialysis bag after the reaction is finished, and dialyzing with 0.9% physiological saline for 3 d. Obtaining the dialyzed solution, namely the artificial coating antigen p-hydroxyacetophenone-OVA.
Test example 1:
when n is 1, the route for preparing p-hydroxyacetophenone hapten is as follows:
the (-) ESI-MS full scan mass spectrum of p-hydroxyacetophenone hapten (p-hydroxyacetophenone-chloroacetic acid) prepared in example 1 shows a molecular ion peak of m/z 208, which corresponds to the molecular mass of the target. In order to further determine the structure of the p-hydroxyacetophenone hapten prepared in example 1, the molecular ions of m/z 208 are analyzed by (-) ESI-MS2, fragment ion peaks of m/z 206.3, 191.3, 148.5 and the like appear after the molecular ions of m/z 208 are cracked, and all the fragments can be reasonably assigned, so the molecular ions of m/z 208 can be assigned as the p-hydroxyacetophenone derivative.
Test example 2: identification of Artificial antigens
And (3) measuring the maximum absorption values of the hapten, the carrier protein and the conjugate according to a spectrophotometry method, and calculating the coupling ratio of the conjugate. Scanning the ultraviolet absorption spectra of the raw material, the carrier protein BSA, the carrier protein OVA and the conjugate respectively between 200 and 400nm to identify whether the hapten and the carrier protein are coupled. The hapten and carrier protein coupling ratio was simultaneously estimated and calculated as follows: the ratio of p-hydroxyacetophenone-BSA is 15.5:1, and the ratio of p-hydroxyacetophenone-OVA is 6.3: 1.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention, and not for limiting the protection scope of the present invention, although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (10)
1. A p-hydroxyacetophenone hapten is characterized in that the molecular structural formula of the p-hydroxyacetophenone hapten is as follows:
n is a natural number of 1 to 6.
2. The p-hydroxyacetophenone hapten according to claim 1 wherein n is 2.
3. The method for synthesizing p-hydroxyacetophenone hapten as claimed in claim 1 or 2, characterized by comprising the steps of: reacting p-hydroxyacetophenone with a halogenated acid salt in an organic solvent in the presence of a base to generate the p-hydroxyacetophenone hapten.
4. The method for synthesizing p-hydroxyacetophenone hapten as claimed in claim 3, wherein the reaction temperature is 70-80 ℃ and the reaction time is 3.5-4.5 h.
5. The method for synthesizing p-hydroxyacetophenone hapten as claimed in claim 3, characterized in that the reaction solution obtained by the reaction is adjusted to pH 1.0-2.5, extracted with organic solvent, the organic phase is collected and added with salt to remove water, then the organic solvent is removed to obtain crude product, and the crude product is purified by silica gel column chromatography to obtain purified p-hydroxyacetophenone hapten.
6. The method for synthesizing p-hydroxyacetophenone hapten as claimed in claim 3, wherein the structural formula of the said haloid acid salt is R- (CH)2) n-COOM, wherein R is Cl or Br, and M is sodium or potassium ion.
7. The method for synthesizing p-hydroxyacetophenone hapten according to claim 3, wherein the molar ratio of the p-hydroxyacetophenone to the alkali is 1:4 to 1:5, and the molar amount of the halogenated acid salt is 1.5 to 2.0 times that of the p-hydroxyacetophenone.
8. An artificial antigen of p-hydroxyacetophenone, which is prepared by combining the hapten of p-hydroxyacetophenone according to claim 1 with carrier protein, wherein the molecular structural formula of the artificial antigen of p-hydroxyacetophenone is as follows:
the carrier protein is bovine serum albumin or ovalbumin.
9. The method for synthesizing p-hydroxyacetophenone artificial antigen according to claim 8, wherein the method for synthesizing p-hydroxyacetophenone artificial antigen is an active lipid method or a mixed acid anhydride method.
10. The use of p-hydroxyacetophenone artificial antigen as claimed in claim 8, characterized in that the p-hydroxyacetophenone artificial antigen is used for the preparation of a polyclonal antibody or a monoclonal antibody against p-hydroxyacetophenone.
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| CN101489544A (en) * | 2006-05-09 | 2009-07-22 | 海玛奎斯特医药公司 | Methods for treating blood disorders |
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| CN101489544A (en) * | 2006-05-09 | 2009-07-22 | 海玛奎斯特医药公司 | Methods for treating blood disorders |
| US20120094316A1 (en) * | 2010-10-19 | 2012-04-19 | Mcconnell Robert Ivan | Drug Detection |
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