CN108690129A - A kind of hemp antigen and preparation method - Google Patents
A kind of hemp antigen and preparation method Download PDFInfo
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- CN108690129A CN108690129A CN201710232481.2A CN201710232481A CN108690129A CN 108690129 A CN108690129 A CN 108690129A CN 201710232481 A CN201710232481 A CN 201710232481A CN 108690129 A CN108690129 A CN 108690129A
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- 244000025254 Cannabis sativa Species 0.000 title claims abstract description 92
- 235000012766 Cannabis sativa ssp. sativa var. sativa Nutrition 0.000 title claims abstract description 91
- 235000012765 Cannabis sativa ssp. sativa var. spontanea Nutrition 0.000 title claims abstract description 91
- 235000009120 camo Nutrition 0.000 title claims abstract description 91
- 235000005607 chanvre indien Nutrition 0.000 title claims abstract description 91
- 239000011487 hemp Substances 0.000 title claims abstract description 91
- 239000000427 antigen Substances 0.000 title claims abstract description 84
- 102000036639 antigens Human genes 0.000 title claims abstract description 84
- 108091007433 antigens Proteins 0.000 title claims abstract description 78
- 238000002360 preparation method Methods 0.000 title abstract description 51
- ZTGXAWYVTLUPDT-UHFFFAOYSA-N cannabidiol Natural products OC1=CC(CCCCC)=CC(O)=C1C1C(C(C)=C)CC=C(C)C1 ZTGXAWYVTLUPDT-UHFFFAOYSA-N 0.000 claims abstract description 31
- QHMBSVQNZZTUGM-UHFFFAOYSA-N Trans-Cannabidiol Natural products OC1=CC(CCCCC)=CC(O)=C1C1C(C(C)=C)CCC(C)=C1 QHMBSVQNZZTUGM-UHFFFAOYSA-N 0.000 claims abstract description 30
- QHMBSVQNZZTUGM-ZWKOTPCHSA-N cannabidiol Chemical compound OC1=CC(CCCCC)=CC(O)=C1[C@H]1[C@H](C(C)=C)CCC(C)=C1 QHMBSVQNZZTUGM-ZWKOTPCHSA-N 0.000 claims abstract description 30
- 229950011318 cannabidiol Drugs 0.000 claims abstract description 30
- PCXRACLQFPRCBB-ZWKOTPCHSA-N dihydrocannabidiol Natural products OC1=CC(CCCCC)=CC(O)=C1[C@H]1[C@H](C(C)C)CCC(C)=C1 PCXRACLQFPRCBB-ZWKOTPCHSA-N 0.000 claims abstract description 30
- 238000001514 detection method Methods 0.000 claims abstract description 19
- FALRKNHUBBKYCC-UHFFFAOYSA-N 2-(chloromethyl)pyridine-3-carbonitrile Chemical compound ClCC1=NC=CC=C1C#N FALRKNHUBBKYCC-UHFFFAOYSA-N 0.000 claims abstract description 10
- 229940014800 succinic anhydride Drugs 0.000 claims abstract description 10
- 239000002994 raw material Substances 0.000 claims abstract description 9
- 230000008878 coupling Effects 0.000 claims abstract description 7
- 238000010168 coupling process Methods 0.000 claims abstract description 7
- 238000005859 coupling reaction Methods 0.000 claims abstract description 7
- 230000028993 immune response Effects 0.000 claims abstract description 7
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 54
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 24
- 238000006243 chemical reaction Methods 0.000 claims description 24
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 21
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- 238000000034 method Methods 0.000 claims description 20
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- 239000006228 supernatant Substances 0.000 claims description 19
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 18
- 229910052757 nitrogen Inorganic materials 0.000 claims description 16
- BDNKZNFMNDZQMI-UHFFFAOYSA-N 1,3-diisopropylcarbodiimide Chemical compound CC(C)N=C=NC(C)C BDNKZNFMNDZQMI-UHFFFAOYSA-N 0.000 claims description 14
- 239000008363 phosphate buffer Substances 0.000 claims description 13
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 12
- 150000001718 carbodiimides Chemical class 0.000 claims description 12
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 11
- 238000005119 centrifugation Methods 0.000 claims description 10
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- 239000002904 solvent Substances 0.000 claims description 7
- 239000012043 crude product Substances 0.000 claims description 6
- 239000003960 organic solvent Substances 0.000 claims description 6
- 229910052799 carbon Inorganic materials 0.000 claims description 5
- 238000003756 stirring Methods 0.000 claims description 5
- 238000005406 washing Methods 0.000 claims description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 4
- 230000006837 decompression Effects 0.000 claims description 4
- 238000004090 dissolution Methods 0.000 claims description 4
- 239000000284 extract Substances 0.000 claims description 4
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- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 3
- 108010044091 Globulins Proteins 0.000 claims description 3
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- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims 1
- 150000008064 anhydrides Chemical class 0.000 claims 1
- RAABOESOVLLHRU-UHFFFAOYSA-N diazene Chemical compound N=N RAABOESOVLLHRU-UHFFFAOYSA-N 0.000 claims 1
- 229910000071 diazene Inorganic materials 0.000 claims 1
- CYQFCXCEBYINGO-UHFFFAOYSA-N THC Natural products C1=C(C)CCC2C(C)(C)OC3=CC(CCCCC)=CC(O)=C3C21 CYQFCXCEBYINGO-UHFFFAOYSA-N 0.000 abstract description 14
- CYQFCXCEBYINGO-IAGOWNOFSA-N delta1-THC Chemical compound C1=C(C)CC[C@H]2C(C)(C)OC3=CC(CCCCC)=CC(O)=C3[C@@H]21 CYQFCXCEBYINGO-IAGOWNOFSA-N 0.000 abstract description 14
- 229960004242 dronabinol Drugs 0.000 abstract description 14
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- 239000003557 cannabinoid Substances 0.000 abstract description 4
- 229940065144 cannabinoids Drugs 0.000 abstract description 3
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 230000015572 biosynthetic process Effects 0.000 abstract description 2
- 239000003054 catalyst Substances 0.000 abstract description 2
- 230000036039 immunity Effects 0.000 abstract description 2
- 238000009776 industrial production Methods 0.000 abstract description 2
- 239000000463 material Substances 0.000 abstract description 2
- 238000003786 synthesis reaction Methods 0.000 abstract description 2
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical group C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 26
- 239000003814 drug Substances 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- BGRWYRAHAFMIBJ-UHFFFAOYSA-N diisopropylcarbodiimide Natural products CC(C)NC(=O)NC(C)C BGRWYRAHAFMIBJ-UHFFFAOYSA-N 0.000 description 7
- 239000000126 substance Substances 0.000 description 7
- 229940079593 drug Drugs 0.000 description 6
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 description 6
- 230000000694 effects Effects 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 241000218236 Cannabis Species 0.000 description 4
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- -1 Diisopropyl carbon Chemical compound 0.000 description 4
- 239000000835 fiber Substances 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 239000002207 metabolite Substances 0.000 description 3
- 238000003127 radioimmunoassay Methods 0.000 description 3
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
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- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 208000012902 Nervous system disease Diseases 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
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- 206010063409 Acarodermatitis Diseases 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- VBGLYOIFKLUMQG-UHFFFAOYSA-N Cannabinol Chemical compound C1=C(C)C=C2C3=C(O)C=C(CCCCC)C=C3OC(C)(C)C2=C1 VBGLYOIFKLUMQG-UHFFFAOYSA-N 0.000 description 1
- 206010010774 Constipation Diseases 0.000 description 1
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- 206010018612 Gonorrhoea Diseases 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
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- 241001465754 Metazoa Species 0.000 description 1
- 241000218231 Moraceae Species 0.000 description 1
- 208000025966 Neurological disease Diseases 0.000 description 1
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- 241000447727 Scabies Species 0.000 description 1
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- 208000005392 Spasm Diseases 0.000 description 1
- 208000002474 Tinea Diseases 0.000 description 1
- 241000130764 Tinea Species 0.000 description 1
- 206010047700 Vomiting Diseases 0.000 description 1
- 150000008065 acid anhydrides Chemical class 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 229940025084 amphetamine Drugs 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 230000003444 anaesthetic effect Effects 0.000 description 1
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- 230000017531 blood circulation Effects 0.000 description 1
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- 239000004202 carbamide Substances 0.000 description 1
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- 108010074605 gamma-Globulins Proteins 0.000 description 1
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- 230000003400 hallucinatory effect Effects 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 238000009434 installation Methods 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 239000002398 materia medica Substances 0.000 description 1
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- 210000000653 nervous system Anatomy 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 239000012299 nitrogen atmosphere Substances 0.000 description 1
- 229940127240 opiate Drugs 0.000 description 1
- 239000003973 paint Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
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- 238000000746 purification Methods 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 206010039073 rheumatoid arthritis Diseases 0.000 description 1
- 230000005070 ripening Effects 0.000 description 1
- 208000005687 scabies Diseases 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 235000002639 sodium chloride Nutrition 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- GOLXNESZZPUPJE-UHFFFAOYSA-N spiromesifen Chemical compound CC1=CC(C)=CC(C)=C1C(C(O1)=O)=C(OC(=O)CC(C)(C)C)C11CCCC1 GOLXNESZZPUPJE-UHFFFAOYSA-N 0.000 description 1
- 208000011117 substance-related disease Diseases 0.000 description 1
- RINCXYDBBGOEEQ-UHFFFAOYSA-N succinic anhydride Chemical compound O=C1CCC(=O)O1 RINCXYDBBGOEEQ-UHFFFAOYSA-N 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- 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
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4717—Plasma globulins, lactoglobulin
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/30—Psychoses; Psychiatry
- G01N2800/307—Drug dependency, e.g. alcoholism
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Immunology (AREA)
- Urology & Nephrology (AREA)
- Genetics & Genomics (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Microbiology (AREA)
- Gastroenterology & Hepatology (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention relates to a kind of hemp antigens and preparation method thereof, first using cannabidiol as raw material, are reacted with succinic anhydride, generate carboxylic haptens;Again by haptens and bovineγ-globulin (BGG) albumen coupling, hemp holoantigen is formed.The present invention, which selects in Cannabinoids, does not have the cannabidiol of addiction feature as raw material, cannabidiol is high with addiction feature tetrahydrocannabinol structural similarity, the antigen of preparation, which can stimulate, generates stronger immune response, it is safe and efficient in product preparation process, using the hemp antigen of the application as raw material, the hemp of subsequent preparation sucks immunity detection reagent high sensitivity.And the material and catalyst used in hemp antigen synthesis and preparation process are common product, therefore more practicability, suitable for industrial production and can generate economic value.
Description
Technical field
The present invention relates to technical field of biochemical industry more particularly to a kind of preparation methods of hemp artificial antigen.
Background technology
Hemp Cannabis sativa L. are Moraceae annual herb plant, You Mingma, Chinese fiber crops, fire fiber crops, mountain silk seedling, Huang
Fiber crops have important agricultural and medical value, there is extensive cultivation all over the world.As a kind of ancient cultivated plant, greatly
Fiber crops have relevant record very early, before 4000,"The Yellow Emperor's Canon of Internal Medicine"In about the description of hemp.2nd century of Christian era, I
Name of the country doctor Huatuo once used hemp to be used for clinical treatment as anaesthetic;"Compendium of Materia Medica"In also have the record that hemp is used as medicine;Its
Ripening fruits fructus cannabis also record in"Chinese Pharmacopoeia"Version in 2005.Because its have moisturize, laxation, treating stranguria, promoting blood circulation effect, Chinese medicine
With treatment dry constipation of intestines, quench one's thirst, the diseases such as heat gonorrhea, wandering arthritis, dysentery, irregular menstruation, scabies, tinea leprosy.In China, hemp is to forbid certainly
By what is planted and cultivate, however due to its special economic value and the characteristics such as cultivation are easy, worldwide hemp is planted
Area is growing on and on always.
The ingredient of hemp is extremely complex, and main active is Cannabinoids compound (cannabionids), at present
Knowing natural cannabinoid has 70 kinds, tetrahydrocannabinol (tetrahydrocannabional, THC) and cannabidiol
(cannabidiol, CBD) is the main chemical compositions contained in hemp, is mainly used for multiple in certain the nervous system diseases
Property sclerosis, motility neurological disease, intractable pain and drug induccd vomiting.In addition, to glaucoma, asthma and angiocarpy
Disease also has certain effect.However since tetrahydrocannabinol (THC) has anesthesia and hallucinogenic action, the mankind after sucking or taking orally
The history to smoke cannabis has reached over thousands of year, or even is once disclosed using hemp in certain religions or certain religious sect's history.With tetrahydrochysene
Unlike cannabinol (THC), cannabidiol (CBD) is non-additive ingredient in hemp, and can hinder tetrahydrocannabinol
(THC) to the influence of human's nervous system, there are the pharmaceutical activity such as anti-spasm, resisting rheumatoid arthritis, antianxiety.Tetrahydrocannabinol
(THC) and cannabidiol (CBD) molecular structure is as follows:
Since cannabis plants have certain economic value, worldwide plantation is extensive, thus hemp is relatively inexpensive,
Intake is convenient, is referred to as most popular drugs in the world.According to drugs control roller office of the United Nations (United Nations
Office for Drug Control Program) statistics, worldwide, the Life prevalence (life- of various drugs
Time prevalence, LTP) be respectively:Opiates 1.0%, cocaine 1.9%, hemp 13.5%, amphetamine 2.6%.
Therefore, hemp to suck number very big.
Currently, commonly having radio immunoassay, enzyme-linked immunosorbent assay, immune layer to the prescreening method to smoke cannabis
Analysis technology etc..Confirmation method mainly uses the Physico-chemical tests such as high performance liquid chromatography (HPLC), gas chromatography-mass spectrography (GC/MS)
Method.HPLC and GC/MS tests must all be completed in laboratory, need equipment and operated by professional technician, somewhat expensive.
Radio immunoassay sensitivity and specificity are high, but expensive equipment, thus cannot achieve popularization and application.Enzyme-linked immunosorbent assay
The advantages of remaining with radio immunoassay in terms of sensitivity and specificity, but it is complicated for operation, time-consuming, it is necessary to professional technique people
Member's operation.Immunochromatographic method due to its with simple and efficient, result is clear, without complex operations skill and special installation, sensitive
The advantages that degree and higher specificity, is very suitable for the drug abuse group suspicious to high-volume and carries out field screening work, it has also become faces
One new direction of bed diagnostic field development, is widely used in flourishing state.
Immunochromatographic method may be used competition law and measure in human body fluid sample whether contain object.Due to tetrahydrochysene hemp
Phenol and cannabidiol have approximate structure, simultaneously as cannabidiol can hinder tetrahydrocannabinol to nerve system of human body
Influence, and be different from tetrahydrocannabinol, have it is non-additive.Therefore, using cannabidiol as haptens, immunocyte is stimulated
Immune response is generated, filters out to tetrahydrocannabinol and cannabidiol while having combinative antibody.Made with the antibody of label
For indicant, cannabidiol antigen is coated at the detection line on nitrocellulose filter.When detection, sample is in capillary effect lower layer
Analysis, if analysans in sample is less than preset concentrations, labelled antibody cannot all with the tetrahydrochysene hemp in sample
Phenol combines, and unbonded labelled antibody, with antigen binding at detection line, shows detection line in chromatography process.When being waited in sample point
When analysing object higher than default concentrations, labelled antibody is all combined, and will not show detection line.Therefore, accurate in immunochromatographic method
Really the existing key of measurement hemp metabolin is to prepare high specificity, and the cannabidiol that purity is high, binding force is big manually resists
It is former.
Invention content
The present invention generates carboxylic haptens by using cannabidiol as raw material, being reacted with succinic anhydride;Again by partly resisting
Former and bovineγ-globulin (BGG) albumen coupling prepares hemp artificial antigen, i.e. cannabidiol-bovineγ-globulin antigen.
Specifically, the present invention provides a kind of hemp antigen, and structural formula is:
On the other hand, the present invention also provides the preparation methods of the hemp antigen, i.e., using cannabidiol as raw material, with succinic acid
Anhydride reactant generates carboxylic haptens;Again by the haptens and bovineγ-globulin (BGG) albumen coupling, forms hemp and resist entirely
It is former.Its reaction equation is as follows:
Further, the method for preparation hemp antigen of the invention, includes the following steps:
A) haptens is prepared:After cannabidiol is dissolved in organic solvent, triethylamine react is added, adds fourth two later
Acid anhydrides fully reacts;Organic solvent extracts, and washs organic phase, dry, filters, concentration;
B) coupling protein:Product in step a is dissolved in organic solvent, n-hydroxysuccinimide is added and carbon two is sub-
Amine, stirred under nitrogen atmosphere react, after the completion centrifuging and taking supernatant;Bovineγ-globulin is dissolved in phosphate buffer, and in centrifugation
After clear liquid is sufficiently mixed, 2~8 DEG C of standings;
C) artificial antigen purifies:Product in step b is dialysed in phosphate buffer, dialyzate centrifuging and taking supernatant is
For hemp artificial antigen.
More specifically, in the method for present invention preparation hemp antigen, specifically comprise the following steps:
(1) cannabidiol 1eq is weighed, is dissolved with n,N-Dimethylformamide, 4~10eq of triethylamine is added and stirs at room temperature
10~60min of reaction is mixed, 1.5~4eq of succinic anhydride is added, it is stirred to react 6 at room temperature~for 24 hours;
(2) decompression boils off n,N-Dimethylformamide, and water dissolution is added, is extracted with ethyl acetate, after merging organic phase, according to
Secondary washing, saturated sodium-chloride washing, organic phase drying, filtering, evaporated under reduced pressure obtain crude product;
(3) by back reaction product, it is dissolved in n,N-Dimethylformamide, 1.5~3eq of n-hydroxysuccinimide is added
With 1.5~3eq of carbodiimide, lead to nitrogen protection, it is stirred to react 6 at room temperature~for 24 hours, centrifugation after reaction obtains supernatant;
(4) bovineγ-globulin is dissolved in 10mg/ml phosphate buffers, under fast stirring, will be upper in step (3)
Clear liquid and the ox-gamma globulin phosphate buffer are uniformly mixed, stand 6 under the conditions of mixed liquor is placed on 2~8 DEG C~for 24 hours, it obtains
To artificial antigen mixed liquor;
(5) artificial antigen mixed liquor is transferred to bag filter, is dialysed with phosphate buffer, after dialysis in centrifuging and taking
Clear liquid obtains hemp artificial antigen.
In addition, in the preparation of hemp antigen, in step (2), further includes being purified to going out product, particularly, adopt
Crude product purification is carried out with TLC methods, solvent is that volume ratio is (7~12):1 dichloromethane and the mixed liquor of methanol.
Further, in stepb, carbodiimide is dicyclohexylcarbodiimide DCC or N, N'Diisopropyl carbon two
One kind in imines DIC or 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides EDC or arbitrary two kinds of mixed liquor
Or three kinds of mixed liquor.Preferably, it is DCC.The addition equivalent of carbodiimide is 1.5~3 times of amounts of haptens.
As the further supplement to hemp antigen preparation procedure, in step (3), solvent may be used most of organic molten
Agent, it is only necessary to meet dissolubility and inertness requirement, such as dichloromethane, DMF, ethyl alcohol, isopropanol, acetic acid, urea, DMSO, even
Water can also be used as solvent.
The present invention also provides a kind of detection hemp and/or the kit of hemp metabolin, contain hemp in the kit
Diphenol-bovineγ-globulin antigen.
The kit of detection hemp provided by the invention and/or hemp metabolin is the examination for detecting hemp and sucking situation
Agent box, the kit contain cannabidiol-bovineγ-globulin antigen.
Further, the kit of detection hemp of the invention and/or hemp metabolin, including cannabidiol-ox γ-ball
Proteantigen captures hemp prototype substance or the metabolite of hemp by the way that association reaction is immunized.
More specifically, present invention detection hemp sucks situation, anti-using cannabidiol-bovineγ-globulin antigen capture
Body, the antibody are that hemp produces in human body metabolism's object, the antibody generated by immune response or hemp by immune response
Raw antibody.
Advantageous effect
The invention has the advantages that:
1, the present invention selects in Cannabinoids the cannabidiol for not having addiction feature as raw material, cannabidiol and cause addiction
Property tetrahydrocannabinol structural similarity it is high, the antigen of preparation, which can stimulate, generates stronger immune response, in product preparation process
Safe and efficient, using the hemp antigen of the application as raw material, it is sensitive that the hemp of subsequent preparation sucks immunity detection reagent
Degree is high.
2, the material used in hemp antigen synthesis and preparation process of the invention and catalyst are common product, therefore
More practicability suitable for industrial production and can generate economic value.
3, hemp antigen of the invention is applied to kit detection, accuracy of reading and convenient and efficient.
Specific implementation mode
The preparation of hemp artificial antigen is divided into two steps:1. prepared by haptens:It is anti-with succinic anhydride using cannabidiol as raw material
It answers, generates carboxylic haptens;2. the preparation of antigen:By haptens and bovineγ-globulin (BGG) albumen coupling, prepare big
Numb artificial antigen, i.e. cannabidiol-bovineγ-globulin antigen.
Embodiment 1:The preparation of hemp antigen
1. the preparation of haptens:
Cannabidiol 500mg (1.61mmol, 1eq) is weighed, is dissolved with 25ml n,N-Dimethylformamide, is then added
Reaction half an hour is stirred at room temperature in 1.78ml (12.9mmol, 8eq) triethylamine, adds 403mg (4.03mmol, 2.5eq)
Succinic anhydride is stirred to react overnight (6~for 24 hours) at room temperature.
TLC tracing detections response situation (methylene chloride/methanol=9/1) to reaction terminates.
After reaction, decompression boils off n,N-Dimethylformamide.25ml is added and purifies water dissolution, then uses ethyl acetate
30ml extracts solution three times;The organic phase extracted first uses 20ml water washings 2 times, then washs one with 20ml saturated sodium-chlorides
It is secondary;Then organic phase is dried 15 minutes with anhydrous sodium sulfate, and filtering, filtrate is light yellow transparent solution, and evaporated under reduced pressure is obtained and slightly produced
Object.
The crude product is purified with TLC methods, wherein solvent is methylene chloride/methanol=9/1, and it is (anti-to obtain 340mg products 2
2 substances of Ying Shizhong), i.e. haptens, Rf=0.58, yield (53.8%).
Using chloroform as solvent, nucleus magnetic hydrogen spectrum is products therefrom (2):1H NMR (1H-NMR, 400MHz, CHCl3) δ 0.96
(t,3H),1.25-2.22(m,16H),2.34-2.63(m,6H),2.70-2.73(m,1H),3.10-3.43(m,1H),4.70-
5.12 (m, 3H), 5.30-5.50 (m, 1H), 6.20-6.53 (m, 2H), 11.0 (5,1H) the above results meet the spy of product (2)
Sign.
2. the preparation of antigen:
2.1 take 200mg (0.51mmol) product (2), are dissolved in 15ml n,N-Dimethylformamide, add 117mg
(1.02mmol, 2eq) n-hydroxysuccinimide (NHS) and 210mg (1.02mmol, 2.0eq) dicyclohexylcarbodiimide
(DCC), lead to nitrogen protection, be stirred to react at room temperature overnight (6~for 24 hours), centrifugation after reaction obtains supernatant, the supernatant
For dimethylformamide (DMF) solution of compound 3 (3 substance in reaction equation).
2.2 are dissolved in bovineγ-globulin solution in the phosphate buffer of 10mg/ml, under fast stirring, by supernatant
Liquid (dimethylformamide (DMF) solution of compound 3) and the ox-gamma globulin phosphate buffer are uniformly mixed, and what is obtained is mixed
It closes under the conditions of liquid is placed on 2~8 DEG C and stands overnight (6~for 24 hours), obtain artificial antigen mixed liquor.
Artificial antigen mixed liquor is transferred to bag filter by 2.3, is dialysed 9 times with phosphate buffer, is centrifuged after dialysis
Supernatant is taken to obtain hemp artificial antigen.
Embodiment 2:The preparation of hemp antigen
The embodiment is identical as preparation process in embodiment 1, wherein cannabidiol, triethylamine and the adjustment of succinic anhydride eq ratios
It is 1:4:1.5, last 500mg cannabidiols prepare 318mg products 2, yield (50.3%).It is specific as follows:
1. the preparation of haptens:
Cannabidiol 500mg (1.61mmol, 1eq) is weighed, with 25ml, then n,N-Dimethylformamide dissolving is added
Reaction half an hour is stirred at room temperature in 0.89ml (6.45mmol, 4eq) triethylamine, adds 242mg (2.42mmol, 1.5eq)
Succinic anhydride is stirred to react overnight (6~for 24 hours) at room temperature.
TLC tracing detections response situation (methylene chloride/methanol=9/1) to reaction terminates.
After reaction, decompression boils off n,N-Dimethylformamide.25ml is added and purifies water dissolution, then uses ethyl acetate
30ml extracts solution three times;The organic phase extracted first uses 20ml water washings 2 times, then washs one with 20ml saturated sodium-chlorides
It is secondary;Then organic phase is dried 15 minutes with anhydrous sodium sulfate, and filtering, filtrate is light yellow transparent solution, and evaporated under reduced pressure is obtained and slightly produced
Object.
The crude product is purified with TLC methods, wherein solvent is methylene chloride/methanol=9/1, and it is (anti-to obtain 318mg products 2
2 substances of Ying Shizhong), i.e. haptens, Rf=0.58, yield (50.3%).
Embodiment 3:The preparation of hemp antigen
The preparation process of hemp antigen is same as Example 1, and cannabidiol is with the eq of triethylamine and succinic anhydride ratios
1:10:4,500mg cannabidiols prepare 302mg products 2, yield (47.9%).
Embodiment 4:The preparation of hemp antigen
The preparation process of hemp antigen is same as Example 1, wherein by the dicyclohexyl of the addition in antigen preparation process
Carbodiimide (DCC) is changed to N, N'Diisopropylcarbodiimide (DIC) (1.5~3eq).
That is in the 2nd step antigen preparation process:
200mg (0.51mmol) product (2) is taken, 15ml n,N-Dimethylformamide is dissolved in, adds 117mg
(1.02mmol, 2eq) n-hydroxysuccinimide (NHS) and N, N'Diisopropylcarbodiimide (DIC) 161mg
(1.275mmol, 2.5eq) leads to nitrogen protection, is stirred to react at room temperature overnight, and centrifugation after reaction obtains supernatant.
Embodiment 5:The preparation of hemp antigen
The preparation process of hemp antigen is same as Example 1, wherein by the dicyclohexyl of the addition in antigen preparation process
Carbodiimide (DCC) is changed to 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides (EDC) (1.5~3eq).
That is in the 2nd step antigen preparation process:
200mg (0.51mmol) product (2) is taken, 15ml n,N-Dimethylformamide is dissolved in, adds 117mg
(1.02mmol, 2eq) n-hydroxysuccinimide (NHS) and 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides
(EDC) 146mg (0.765mmol, 1.5eq) leads to nitrogen protection, is stirred to react at room temperature overnight, and centrifugation after reaction obtains
Supernatant.
Embodiment 6:The preparation of hemp antigen
The preparation process of hemp antigen is same as Example 1, wherein by the dicyclohexyl of the addition in antigen preparation process
Carbodiimide (DCC) is changed to dicyclohexylcarbodiimide (DCC) and N, N'The mixed liquor of diisopropylcarbodiimide (DIC)
(1.5~3eq).
That is in the 2nd step antigen preparation process:
200mg (0.51mmol) product (2) is taken, is dissolved in 15mlN, dinethylformamide adds 117mg
(1.02mmol, 2eq) n-hydroxysuccinimide (NHS) and dicyclohexylcarbodiimide (DCC) and N, N'Diisopropyl carbon two
The mixed liquor 1.53mmol (3eq) of imines (DIC), wherein the molar ratio of DCC and DIC is 2:1, lead to nitrogen protection, stirs at room temperature
Mix reaction overnight, centrifugation after reaction obtains supernatant.
Embodiment 7:The preparation of hemp antigen
The preparation process of hemp antigen is same as Example 1, wherein by the dicyclohexyl of the addition in antigen preparation process
Carbodiimide (DCC) is changed to dicyclohexylcarbodiimide (DCC) and 1- (3- dimethylamino-propyls) -3- ethyl carbodiimide salt
The mixed liquor (1.5~3eq) of hydrochlorate (EDC).
That is in the 2nd step antigen preparation process:
200mg (0.51mmol) product (2) is taken, is dissolved in 15mlN, dinethylformamide adds 117mg
(1.02mmol, 2eq) n-hydroxysuccinimide (NHS) and dicyclohexylcarbodiimide (DCC) and 1- (3- dimethylaminos third
Base) -3- ethyl-carbodiimide hydrochlorides (EDC) mixed liquor 1.02mmol (2eq), wherein the molar ratio of DCC and EDC be 1:
1, lead to nitrogen protection, be stirred to react at room temperature overnight, centrifugation after reaction obtains supernatant.
Embodiment 7:The preparation of hemp antigen
The preparation process of hemp antigen is same as Example 1, wherein by the dicyclohexyl of the addition in antigen preparation process
Carbodiimide (DCC) is changed to N, N'Diisopropylcarbodiimide (DIC) and 1- (3- dimethylamino-propyls) -3- ethyls carbon two
The mixed liquor (1.5~3eq) of inferior amine salt hydrochlorate (EDC).
That is in the 2nd step antigen preparation process:
200mg (0.51mmol) product (2) is taken, is dissolved in 15mlN, dinethylformamide adds 117mg
(1.02mmol, 2eq) n-hydroxysuccinimide (NHS) and N, N'Diisopropylcarbodiimide (DIC) and 1- (3- diformazan ammonia
Base propyl) -3- ethyl-carbodiimide hydrochlorides (EDC) mixed liquor 1.02mmol (2eq), wherein the molar ratio of EDC and DIC
It is 3:1, lead to nitrogen protection, be stirred to react at room temperature overnight, centrifugation after reaction obtains supernatant.
Embodiment 9:The preparation of hemp antigen
The preparation process of hemp antigen is same as Example 1, wherein by the dicyclohexyl of the addition in antigen preparation process
Carbodiimide (DCC) is changed to dicyclohexylcarbodiimide (DCC) and N, N'Diisopropylcarbodiimide (DIC) and 1- (3-
Dimethylamino-propyl) three kinds of -3- ethyl-carbodiimide hydrochlorides (EDC) mixed liquor (1.5~3eq).
That is in the 2nd step antigen preparation process:
200mg (0.51mmol) product (2) is taken, 15mlN is dissolved in, dinethylformamide adds N- hydroxysuccinimidyl acyls
Imines (NHS) 117mg (1.02mmol, 2eq) and dicyclohexylcarbodiimide (DCC) and N, N'Diisopropylcarbodiimide
(DIC) and the mixed liquor 1.02mmol of three kinds of 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides (EDC)
(2eq), wherein the molar ratio of DCC, DIC and EDC are 2:1;1, lead to nitrogen protection, is stirred to react at room temperature overnight, reaction terminates
Centrifugation obtains supernatant afterwards.
Embodiment 10:The concentration mensuration of hemp antigen
The measurement of conjugate albumen concentration:Configuration concentration is 0,0.2,0.4,0.6,0.8,1.0,1.2,1.6,2.0mg/ml
Bovineγ-globulin each 1ml of 0.01MPBS solution, be uniformly mixed.Each concentration standard protein solutions of 50ul are taken, disposable ratio is added
In color ware, 250ul Reagent A (being purchased from DC Protein assay outside) are added and are uniformly mixed, add 2ml Reagent
B (is purchased from DC Protein assay) outside, and reaction measures the absorption value at 655nm with ultraviolet specrophotometer after ten minutes, paints
The relation curve of albumen concentration and light absorption value processed.Sample 50ul is precisely measured, is added in disposable cuvette, 250ul is added
Reagent A are uniformly mixed, and add 2ml Reagent B, and reaction is measured with ultraviolet specrophotometer in 655nm after ten minutes
Locate absorption value, sample concentration is calculated according to gained standard curve.A concentration of 7.16mg/ of the hemp artificial antigen of gained of the invention
ml。
Embodiment 11:Application of the hemp antigen in detection hemp and/or the kit of hemp metabolin
The antibody prepared using cannabidiol-bovineγ-globulin antigen-immunized animal, by the antibody and cannabidiol-ox
Gamma globulin antigen is handled respectively in the kit of detection, using the kit come measure in human body fluid sample whether
Metabolite containing hemp prototype substance or hemp.
Specifically, immune association reaction is carried out by the test strips in kit capture hemp prototype substance or big
Fiber crops metabolite mode come realize hemp antigen detection hemp and/or hemp metabolin kit application.
Claims (8)
1. a kind of hemp antigen, which is characterized in that its structural formula is:
2. a kind of method for the hemp antigen preparing claim 1, which is characterized in that first using cannabidiol as raw material, with fourth
Diacid anhydride reactant generates carboxylic haptens;Again by haptens and bovineγ-globulin (BGG) albumen coupling, it is complete to form hemp
Antigen.
3. a kind of method for the hemp antigen preparing claim 1, which is characterized in that include the following steps:
A) haptens is prepared:After cannabidiol is dissolved in organic solvent, triethylamine react is added, adds succinic anhydride later
Fully reaction;Organic solvent extracts, and washs organic phase, dry, filters, concentration;
B) coupling protein:Product in step a is dissolved in organic solvent, n-hydroxysuccinimide and carbodiimide, nitrogen is added
It is stirred to react under gas shielded, after the completion centrifuging and taking supernatant;Bovineγ-globulin is dissolved in phosphate buffer, with centrifuged supernatant
After being sufficiently mixed, 2~8 DEG C of standings;
C) artificial antigen purifies:Product in step b is dialysed in phosphate buffer, dialyzate centrifuging and taking supernatant is as big
Numb artificial antigen.
4. a kind of method for the hemp antigen preparing claim 3, which is characterized in that include the following steps:
(1) cannabidiol 1eq is weighed, is dissolved with n,N-Dimethylformamide, 4~10eq of triethylamine is added and is stirred at room temperature instead
10~60min is answered, 1.5~4eq of succinic anhydride is added, it is stirred to react 6 at room temperature~for 24 hours;
(2) decompression boils off n,N-Dimethylformamide, and water dissolution is added, is extracted with ethyl acetate, after merging organic phase, water successively
It washes, saturated sodium-chloride washing, organic phase drying, filtering, evaporated under reduced pressure obtains crude product;
(3) by back reaction product, it is dissolved in n,N-Dimethylformamide, 1.5~3eq of n-hydroxysuccinimide and carbon is added
1.5~3eq of diimine leads to nitrogen protection, it is stirred to react 6 at room temperature~for 24 hours, centrifugation after reaction obtains supernatant;
(4) bovineγ-globulin is dissolved in 10mg/ml phosphate buffers, under fast stirring, by the supernatant in step (3)
It is uniformly mixed with the ox-gamma globulin phosphate buffer, by mixed liquor in 2~8 DEG C of standings
6~for 24 hours, obtain artificial antigen mixed liquor;
(5) artificial antigen mixed liquor is transferred to bag filter, is dialysed with phosphate buffer, centrifuging and taking supernatant after dialysis
Obtain hemp artificial antigen.
5. the method according to claim 3 for preparing hemp antigen, which is characterized in that in step b, carbodiimide is two rings
Base carbodiimide DCC or N, N'Diisopropylcarbodiimide DIC or 1- (3- dimethylamino-propyls) -3- ethyl carbodiimides
One kind or arbitrary two kinds of mixed liquor in hydrochloride EDC or three kinds of mixed liquor.
6. the method according to claim 4 for preparing hemp antigen, which is characterized in that further include to slightly producing in step (2)
Object is purified:TLC methods purify crude product, and solvent is that volume ratio is (7~12):1 dichloromethane and the mixed liquor of methanol.
7. a kind of detection hemp sucks the kit of situation, which is characterized in that the kit contains described in claim 1
Antigen.
8. kit according to claim 7, which is characterized in that the detection hemp sucks situation, utilizes claim 1
In antigen capture antibody, which is the antibody or hemp that hemp is generated by immune response in human body metabolism's object
The antibody generated by immune response.
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| CN111517997A (en) * | 2020-04-17 | 2020-08-11 | 杭州博拓生物科技股份有限公司 | Ethyl sulfate artificial antigen, preparation method and application |
| CN113979914A (en) * | 2021-11-03 | 2022-01-28 | 公安部第三研究所 | Synthetic cannabinoid hapten compounds, methods of making and uses thereof |
| CN115160135A (en) * | 2022-08-10 | 2022-10-11 | 郑州左安检测科技有限公司 | Preparation method and application of cannabidiol hapten and complete antigen |
| CN116041183A (en) * | 2023-01-03 | 2023-05-02 | 中国农业科学院农产品加工研究所 | Cannabidiol hapten, artificial antigen, monoclonal antibody and application |
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