CN117447337A - Tapentadol artificial hapten and artificial antigen as well as preparation methods and application thereof - Google Patents
Tapentadol artificial hapten and artificial antigen as well as preparation methods and application thereof Download PDFInfo
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- CN117447337A CN117447337A CN202311391724.9A CN202311391724A CN117447337A CN 117447337 A CN117447337 A CN 117447337A CN 202311391724 A CN202311391724 A CN 202311391724A CN 117447337 A CN117447337 A CN 117447337A
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- tapentadol
- artificial
- hapten
- artificial antigen
- serum albumin
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- 229960005126 tapentadol Drugs 0.000 title claims abstract description 130
- KWTWDQCKEHXFFR-SMDDNHRTSA-N tapentadol Chemical compound CN(C)C[C@H](C)[C@@H](CC)C1=CC=CC(O)=C1 KWTWDQCKEHXFFR-SMDDNHRTSA-N 0.000 title claims abstract description 120
- 239000000427 antigen Substances 0.000 title claims abstract description 65
- 102000036639 antigens Human genes 0.000 title claims abstract description 65
- 108091007433 antigens Proteins 0.000 title claims abstract description 65
- 238000002360 preparation method Methods 0.000 title claims abstract description 37
- 229940098773 bovine serum albumin Drugs 0.000 claims abstract description 40
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims abstract description 38
- 230000008878 coupling Effects 0.000 claims abstract description 16
- 238000010168 coupling process Methods 0.000 claims abstract description 16
- 238000005859 coupling reaction Methods 0.000 claims abstract description 16
- 230000003053 immunization Effects 0.000 claims abstract description 10
- JKVBTSJLQLSTHJ-SWLSCSKDSA-N (2r,3r)-3-(3-methoxyphenyl)-n,n,2-trimethylpentan-1-amine Chemical compound CN(C)C[C@H](C)[C@@H](CC)C1=CC=CC(OC)=C1 JKVBTSJLQLSTHJ-SWLSCSKDSA-N 0.000 claims abstract description 6
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 42
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 39
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 28
- 239000000243 solution Substances 0.000 claims description 27
- 238000006243 chemical reaction Methods 0.000 claims description 24
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 21
- 239000006228 supernatant Substances 0.000 claims description 18
- 238000000034 method Methods 0.000 claims description 17
- 239000000203 mixture Substances 0.000 claims description 16
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 14
- 238000004809 thin layer chromatography Methods 0.000 claims description 14
- 238000003756 stirring Methods 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 8
- 239000008367 deionised water Substances 0.000 claims description 8
- 229910021641 deionized water Inorganic materials 0.000 claims description 8
- 239000011259 mixed solution Substances 0.000 claims description 8
- 239000000047 product Substances 0.000 claims description 8
- 238000002156 mixing Methods 0.000 claims description 7
- 239000002904 solvent Substances 0.000 claims description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 6
- 238000000502 dialysis Methods 0.000 claims description 6
- 238000001035 drying Methods 0.000 claims description 6
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 6
- 241001465754 Metazoa Species 0.000 claims description 5
- PQJJJMRNHATNKG-UHFFFAOYSA-N ethyl bromoacetate Chemical compound CCOC(=O)CBr PQJJJMRNHATNKG-UHFFFAOYSA-N 0.000 claims description 5
- 238000010992 reflux Methods 0.000 claims description 5
- BMYNFMYTOJXKLE-UHFFFAOYSA-N 3-azaniumyl-2-hydroxypropanoate Chemical compound NCC(O)C(O)=O BMYNFMYTOJXKLE-UHFFFAOYSA-N 0.000 claims description 4
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 4
- OMOVVBIIQSXZSZ-UHFFFAOYSA-N [6-(4-acetyloxy-5,9a-dimethyl-2,7-dioxo-4,5a,6,9-tetrahydro-3h-pyrano[3,4-b]oxepin-5-yl)-5-formyloxy-3-(furan-3-yl)-3a-methyl-7-methylidene-1a,2,3,4,5,6-hexahydroindeno[1,7a-b]oxiren-4-yl] 2-hydroxy-3-methylpentanoate Chemical compound CC12C(OC(=O)C(O)C(C)CC)C(OC=O)C(C3(C)C(CC(=O)OC4(C)COC(=O)CC43)OC(C)=O)C(=C)C32OC3CC1C=1C=COC=1 OMOVVBIIQSXZSZ-UHFFFAOYSA-N 0.000 claims description 4
- 238000007514 turning Methods 0.000 claims description 4
- KEAYESYHFKHZAL-UHFFFAOYSA-N Sodium Chemical compound [Na] KEAYESYHFKHZAL-UHFFFAOYSA-N 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 239000012312 sodium hydride Substances 0.000 claims description 3
- 229910000104 sodium hydride Inorganic materials 0.000 claims description 3
- 239000003054 catalyst Substances 0.000 claims description 2
- 238000005119 centrifugation Methods 0.000 claims description 2
- 238000001816 cooling Methods 0.000 claims description 2
- 239000000463 material Substances 0.000 claims description 2
- PBMIETCUUSQZCG-UHFFFAOYSA-N n'-cyclohexylmethanediimine Chemical compound N=C=NC1CCCCC1 PBMIETCUUSQZCG-UHFFFAOYSA-N 0.000 claims description 2
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims description 2
- 239000000126 substance Substances 0.000 claims description 2
- 239000007822 coupling agent Substances 0.000 claims 1
- 102000014914 Carrier Proteins Human genes 0.000 abstract description 5
- 108010078791 Carrier Proteins Proteins 0.000 abstract description 5
- 210000002966 serum Anatomy 0.000 abstract description 5
- 238000003018 immunoassay Methods 0.000 abstract description 4
- 238000011587 new zealand white rabbit Methods 0.000 abstract description 4
- 230000000890 antigenic effect Effects 0.000 abstract description 3
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 230000002194 synthesizing effect Effects 0.000 abstract description 2
- 239000007858 starting material Substances 0.000 abstract 1
- 230000000052 comparative effect Effects 0.000 description 23
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 12
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 11
- 241000283690 Bos taurus Species 0.000 description 10
- 230000009102 absorption Effects 0.000 description 9
- 238000010521 absorption reaction Methods 0.000 description 9
- 238000001514 detection method Methods 0.000 description 9
- 108010074605 gamma-Globulins Proteins 0.000 description 9
- 238000002835 absorbance Methods 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 7
- 102000004169 proteins and genes Human genes 0.000 description 7
- 108090000623 proteins and genes Proteins 0.000 description 7
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 description 6
- 239000000872 buffer Substances 0.000 description 5
- 238000002649 immunization Methods 0.000 description 5
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 4
- 208000002193 Pain Diseases 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 235000011114 ammonium hydroxide Nutrition 0.000 description 4
- 239000007864 aqueous solution Substances 0.000 description 4
- 239000007853 buffer solution Substances 0.000 description 4
- 239000007795 chemical reaction product Substances 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 3
- 101000662009 Homo sapiens UDP-N-acetylglucosamine pyrophosphorylase Proteins 0.000 description 3
- 102100037921 UDP-N-acetylglucosamine pyrophosphorylase Human genes 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000013213 metal-organic polyhedra Substances 0.000 description 3
- 239000012071 phase Substances 0.000 description 3
- OOSZCNKVJAVHJI-UHFFFAOYSA-N 1-[(4-fluorophenyl)methyl]piperazine Chemical compound C1=CC(F)=CC=C1CN1CCNCC1 OOSZCNKVJAVHJI-UHFFFAOYSA-N 0.000 description 2
- YOETUEMZNOLGDB-UHFFFAOYSA-N 2-methylpropyl carbonochloridate Chemical compound CC(C)COC(Cl)=O YOETUEMZNOLGDB-UHFFFAOYSA-N 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- 239000008346 aqueous phase Substances 0.000 description 2
- 210000003169 central nervous system Anatomy 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- DGLRDKLJZLEJCY-UHFFFAOYSA-L disodium hydrogenphosphate dodecahydrate Chemical compound O.O.O.O.O.O.O.O.O.O.O.O.[Na+].[Na+].OP([O-])([O-])=O DGLRDKLJZLEJCY-UHFFFAOYSA-L 0.000 description 2
- 239000003480 eluent Substances 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- 238000001819 mass spectrum Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 229940074545 sodium dihydrogen phosphate dihydrate Drugs 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 1
- FALRKNHUBBKYCC-UHFFFAOYSA-N 2-(chloromethyl)pyridine-3-carbonitrile Chemical compound ClCC1=NC=CC=C1C#N FALRKNHUBBKYCC-UHFFFAOYSA-N 0.000 description 1
- 229960000549 4-dimethylaminophenol Drugs 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 208000032131 Diabetic Neuropathies Diseases 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 238000000862 absorption spectrum Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 208000005298 acute pain Diseases 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 1
- 230000000202 analgesic effect Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000010364 biochemical engineering Methods 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 210000003710 cerebral cortex Anatomy 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- 238000003810 ethyl acetate extraction Methods 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 210000004201 immune sera Anatomy 0.000 description 1
- 229940042743 immune sera Drugs 0.000 description 1
- 238000002329 infrared spectrum Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 230000014725 late viral mRNA transcription Effects 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 1
- 239000012046 mixed solvent Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000009871 nonspecific binding Effects 0.000 description 1
- 230000008058 pain sensation Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000009103 reabsorption Effects 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 210000000278 spinal cord Anatomy 0.000 description 1
- 239000012192 staining solution Substances 0.000 description 1
- 229940014800 succinic anhydride Drugs 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
-
- 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
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C217/00—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton
- C07C217/54—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups bound to carbon atoms of at least one six-membered aromatic ring and amino groups bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings of the same carbon skeleton
- C07C217/56—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups bound to carbon atoms of at least one six-membered aromatic ring and amino groups bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings of the same carbon skeleton with amino groups linked to the six-membered aromatic ring, or to the condensed ring system containing that ring, by carbon chains not further substituted by singly-bound oxygen atoms
- C07C217/62—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups bound to carbon atoms of at least one six-membered aromatic ring and amino groups bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings of the same carbon skeleton with amino groups linked to the six-membered aromatic ring, or to the condensed ring system containing that ring, by carbon chains not further substituted by singly-bound oxygen atoms linked by carbon chains having at least three carbon atoms between the amino groups and the six-membered aromatic ring or the condensed ring system containing that ring
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
- C07K16/065—Purification, fragmentation
-
- 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
-
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Genetics & Genomics (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Food Science & Technology (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Zoology (AREA)
- Microbiology (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a tapentadol artificial hapten, an artificial antigen and a preparation method and application thereof, wherein (2R, 3R) -3- (3-methoxyphenyl) -N, N, 2-trimethylpentylamine is used as a starting material for synthesizing the artificial hapten to obtain the tapentadol artificial hapten shown as a formula (I), and the artificial antigen is obtained after coupling bovine serum albumin with the hapten. The artificial hapten of the tapentadol of the invention furthest maintains the characteristic structure of the tapentadol andhas active groups which can be coupled with carrier protein and can be used as antigenic determinants; the anti-tapentadol artificial antigen obtained by further preparation can be immunized to obtain an anti-tapentadol artificial antibody with high affinity, high sensitivity and strong specificity, and the titer of immune serum obtained by immunizing New Zealand white rabbits is as high as 1:80000, can be used for rapid and accurate immunodetection and immunoassay of tapentadol.
Description
Technical Field
The invention belongs to the technical field of biochemical engineering, and particularly relates to a tapentadol artificial hapten and an artificial antigen as well as a preparation method and application thereof.
Background
Tapentadol (Tapentadol) is a novel oral analgesic acting on the central nervous system and has two mechanisms of action: one is to inhibit the transmission of pain in the spinal cord by improving pain sensation and affective factors, thereby affecting and controlling the movements of the cerebral cortex sites where pain is perceived; one is the inhibition of noradrenal hormone reabsorption, which is generally used clinically in the treatment of diabetic peripheral neuropathy pain and in the relief of moderately severe acute pain in the adult central nervous system.
At present, high performance liquid chromatography, gas chromatography, mass spectrum, nuclear magnetic resonance hydrogen spectrum, nuclear magnetic resonance carbon spectrum, infrared spectrum and the like are mostly adopted for analysis and measurement, and the artificial synthesized antigen is not suitable for mass sampling analysis due to higher use and maintenance cost, and the artificial synthesized antigen is utilized for carrying out the immune analysis, so that the artificial hapten and the artificial antigen of the tapentadol are very necessary to be synthesized to meet the requirements of the immune analysis.
It is therefore necessary to develop an economical, simple and rapid assay for detecting the level of tapentadol in humans, the preparation of artificial antigen of tapentadol being the basis for the implementation of this method.
Disclosure of Invention
A first object of the present invention is to provide an artificial hapten for tapentadol which addresses the deficiencies of the prior art.
The molecular structural formula of the tapentadol artificial hapten is shown as a formula (I):
the second object of the invention is to provide a preparation method of the artificial hapten of tapentadol. The invention takes (2R, 3R) -3- (3-methoxyphenyl) -N, N, 2-trimethyl pentylamine as the initial raw material for synthesizing artificial hapten, not only maintains the characteristic structure of tapentadol to the greatest extent, but also has active group which can be coupled with carrier protein and can be used as antigenic determinant.
The preparation method of the tapentadol artificial hapten comprises the following steps:
step (1), mixing (2R, 3R) -3- (3-methoxyphenyl) -N, N, 2-trimethylpentylamine and hydrobromic acid with the mass fraction of 48 percent according to (100-110) mg:5ml of the mixture is mixed and stirred at 100 to 110 ℃ for reflux reaction for 20 to 21 hours. After the reaction was completed, the mixture was cooled to room temperature, dried, added with 15ml of deionized water, and the solution was adjusted to be alkaline, extracted with ethyl acetate several times, and the ethyl acetate phase was collected, dried, filtered and dried to give yellow oil A.
Preferably, the step (1) of adjusting the pH specifically includes: adding 1N NaOH aqueous solution, adjusting ph=9;
preferably, the ethyl acetate extraction in the step (1) is performed three times, under the reaction condition, the yield of the yellow oily product A is higher, the post-treatment procedure is simpler, and the purification is easier.
Step (2), the yellow oily matter A obtained in the step (1) and ethyl bromoacetate are mixed according to a molar ratio of 1: (2.5-3.5) mixing the above materials in a solvent, adding 60% sodium hydride as a catalyst, stirring and refluxing at 65-70 ℃ for 18-20 h, performing thin layer chromatography analysis, directly drying the reaction, extracting with 20ml ethyl acetate, filtering, and drying to obtain yellow oily matter B.
Preferably, the solvent in the step (2) is pyridine or acetonitrile or one of N, N-dimethylformamide.
Preferably, the molar ratio of yellow oil A to ethyl bromoacetate in step (2) is 1:3.
preferably, the developing agent used in the thin layer chromatography in the step (2) is a mixed solvent of ethyl acetate and concentrated ammonia water.
And (3) adding tetrahydrofuran, methanol and NaOH aqueous solution into the yellow oily matter B obtained in the step (2), and stirring and reacting for 18-20 h at 25-30 ℃. And (3) finishing the reaction, turning to dryness, extracting with a proper amount of absolute ethyl alcohol, turning to dryness, and separating by thin layer chromatography to obtain the tapentadol artificial hapten.
Preferably, in the step (3), the ratio of the yellow oily substance B, tetrahydrofuran, methanol and NaOH aqueous solution is (100-110) mg:2ml:1.5ml:5ml, more preferably 100mg:2ml:1.5ml:5ml.
Preferably, the aqueous NaOH solution in step (3) has an equivalent concentration of 1N.
By the method, the connecting arm is introduced at the methoxy position of the tapentadol, and the connecting arm is introduced at the modification position, so that the characteristic structure of the tapentadol can be reserved to the greatest extent.
Compared with the cyclic connecting arm, the linear chain is adopted as the connecting arm, so that nonspecific binding during immunoassay can be reduced, immune response to the connecting arm generated during immunization can be reduced, and the probability of generating specific antibodies is improved.
The third object of the present invention is to provide an artificial antigen of tapentadol, the molecular structural formula of which is shown in formula (II):
wherein BSA is bovine serum albumin.
A fourth object of the present invention is to provide a method for preparing the artificial antigen of tapentadol, comprising: and combining the tapentadol artificial hapten with bovine serum albumin by an active ester method to obtain the tapentadol artificial antigen.
Specifically, the method for preparing the artificial antigen of the tapentadol by adopting the active ester method comprises the following steps of:
step (1), tapentadol artificial hapten, N-dicyclohexylcarbodiimide and N-hydroxysuccinimide are mixed according to a molar ratio of 1: (1.35-1.5): (1.35-1.5) mixing the mixture with N, N-dimethylformamide, stirring the mixture at 20-30 ℃ for reaction for 18-20 hours, centrifuging the mixture after the reaction is finished, and taking supernatant;
and (2) adding the supernatant into a bovine serum albumin solution, standing the obtained mixed solution at 3-5 ℃ overnight, and obtaining the supernatant through dialysis and centrifugation to obtain the tapentadol artificial antigen.
The bovine serum albumin solution of the present invention is prepared by dissolving bovine serum albumin in 0.01M PBS buffer (pH 7.2-7.4), unless otherwise specified.
Preferably, in the step (2), the concentration of the bovine serum albumin solution is 5mg/mL, and the volume ratio of the supernatant to the bovine serum albumin solution is 1:5.
the invention selects Bovine Serum Albumin (BSA) as a macromolecular carrier, and has the following advantages compared with Bovine Gamma Globulin (BGG): (1) bovine serum albumin can better and more bind to the artificial hapten of tapentadol, so as to prepare artificial antigens with different coupling ratios; (2) from experiments, after bovine gamma globulin is combined with the artificial hapten of tapentadol, the artificial hapten of tapentadol is greatly influenced by external environment, and protein separation phenomenon is easy to occur in subsequent treatment, so that the stability is poor. The combination of bovine serum albumin and tapentadol artificial hapten can be preserved for a long time under different pH values and temperatures, and the self protein is not easy to separate and has stability; (3) after adopting bovine serum albumin to combine with artificial hapten of tapentadol, compared with bovine gamma protein, the specific recognition is stronger; (4) compared with Niu Bing proteins, the bovine serum albumin adopted in the experiment is low in price and easy to obtain, and the cost can be reduced to a certain extent.
A fifth object of the present invention is to provide the use of said artificial antigen of tapentadol for the preparation of anti-tapentadol antibodies.
It is a sixth object of the present invention to provide an anti-tapentadol artificial antibody, which is obtained by immunizing animals with the tapentadol artificial antigen and can specifically react with tapentadol.
Experiments show that the titer of immune serum obtained by immunizing New Zealand white rabbits with the tapentadol artificial antigen is 1:80000. the anti-tapentadol artificial antigen provided by the invention can be used for immunization to obtain the anti-tapentadol antibody with high affinity, high sensitivity and strong specificity, and the anti-tapentadol antibody can be used for immunodetection and analysis of tapentadol.
Compared with the prior art, the invention has the beneficial effects that:
the artificial hapten of tapentadol of the invention furthest maintains the characteristic structure of tapentadol, has active groups which can be coupled with carrier protein and can be used as antigenic determinants; the obtained artificial antigen of tapentadol can be immunized to obtain the anti-tapentadol antibody with high price, high sensitivity and strong specificity, and the polyclonal antibody obtained by immunizing New Zealand white rabbits can be used for quick and accurate immunodetection and analysis of tapentadol.
Drawings
FIG. 1 is a liquid chromatogram of a tapentadol artificial hapten I of the present invention; wherein mV represents signal intensity and min represents minutes;
FIG. 2 is a mass spectrum of artificial hapten I of tapentadol; wherein Intins represents intensity, and m/z represents mass-to-charge ratio;
FIG. 3 is an ultraviolet scan of bovine serum albumin, tapentadol artificial hapten I, tapentadol artificial antigen II; where Abs represents the ultraviolet-visible absorption spectrum and WL (nm) represents the wavelength (nm).
Detailed Description
The invention is described in further detail below with reference to the drawings and the detailed description.
In the embodiment of the invention, the MOP developing agent comprises the following formula:
the volume ratio is 8:10:1:1, 95% ethanol, dichloromethane, 1, 4-dioxane and concentrated ammonia water.
Example 1
The embodiment provides a preparation method of tapentadol artificial antigen II, which comprises the following steps:
(1) Preparation of tapentadol artificial hapten I:
(1) 8g of sodium hydroxide solids were dissolved in 200ml of deionized water to give a 1N aqueous NaOH solution.
(2) 100mg (0.426 mmol) of (2R, 3R) -3- (3-methoxyphenyl) -N, N, 2-trimethylpentylamine are weighed, reacted for 20h at 100℃in a 50ml single-necked flask, 5ml of 48% hydrobromic acid are added, TLC detection shows that the reaction is substantially complete, cooled to room temperature, dried, 15ml of deionized water are added, pH=9 is adjusted by 1N of NaOH aqueous solution in (1), at this time, a large amount of precipitation is generated, the aqueous phase is extracted with 15ml of X3 ethyl acetate, the ethyl acetate phase is collected, dried with anhydrous magnesium sulfate, filtered and dried to obtain 89mg (0.403 mmol) of yellow transparent oil A, which is subjected to TLC detection, and the chromatography liquid is a mixture of ethyl acetate and concentrated ammonia water in a volume ratio of 10ml:3 drops, product R f ＝0.3～0.4；
(3) The yellow oil A obtained in (2) was dissolved with 5ml of N, N-dimethylformamide, 55mg (1.375 mmol) of 60% sodium hydride was added, stirred at 25℃for 0.5h, 149. Mu.L (1.343 mmol) of ethyl bromoacetate was added, stirred at 65℃under reflux for 18h, TLC detection showed that the reaction was essentially complete, and the chromatography liquid was identical to (2), product R f =0.5 to 0.6, and is dried, extracted with 20ml of ethyl acetate, dried over anhydrous sodium sulfate, filtered, and dried to give 100mg (0.325 mol) of yellow oil B;
(4) dissolving yellow oily substance B obtained in step (3) in 1.5ml of methanol and 2ml of tetrahydrofuran in a 50ml single-neck flask, adding 5ml of 1N sodium hydroxide solution in step (1), wherein a large amount of precipitate is generated, reacting the solution in a milky suspension at 25 ℃ for 18 hours, wherein TLC detection shows that the reaction is basically complete, and obtaining a product R f Directly drying the mixture, extracting the mixture with 5ml multiplied by 3 absolute ethyl alcohol, drying the mixture, and separating the mixture by TLC (developing agent MOP, solvent and eluent are absolute ethyl alcohol) to obtain 60mg (0.215 mmol) of tapentadol artificial hapten I shown in a formula I.
The liquid chromatogram of the tapentadol artificial hapten I is shown in fig. 1, and the mass chromatogram of the tapentadol artificial hapten I is shown in fig. 2.
It can be seen from fig. 1 that the purity of the purified tapentadol artificial hapten I reaches more than 99%, and from fig. 2, the mass-to-charge ratio (M/z) of the m+h ion peak of the tapentadol artificial hapten I obtained in this example 1 is 280.18, which is consistent with the theoretical relative molecular mass 279, so that the final compound obtained in the step (2) is the tapentadol artificial hapten I designed by the invention.
(2) Preparation of tapentadol artificial antigen II:
(5) in a 50ml single-neck round bottom flask, 60mg (0.215 mmol) of tapentadol artificial hapten I is dissolved in 3ml DMF, 33mg (0.287 mmol) of NHS and 59mg (0.287 mmol) of DCC are added, stirring is carried out at 20-25 ℃ for 18h, the reaction is finished, and the reaction product is centrifuged, and the supernatant is taken for later use.
(6) 14.5g (40.503 mol) of disodium hydrogen phosphate dodecahydrate, 43.875g (750 mol) of sodium chloride, 1.495g (9.583 mol) of sodium dihydrogen phosphate dihydrate were weighed out and dissolved in deionized water to a constant volume of 5.0L, to obtain 0.01M, pH as a PBS buffer of 7.4.
(7) 75mg of bovine serum albumin was weighed and dissolved in 15ml of the PBS buffer of step (6), to obtain a bovine serum albumin solution having a concentration of 5 mg/ml.
(8) Slowly dropwise adding the supernatant obtained in the step (5) into the bovine serum albumin solution obtained in the step (7) under the condition of rapid stirring, wherein the volume ratio of the supernatant to the bovine serum albumin solution is 1:5, standing and preserving the obtained mixed solution at the temperature of 4 ℃ overnight to obtain the artificial antigen mixed solution.
(9) And (3) transferring the artificial antigen mixed solution into a dialysis bag, dialyzing for 7 times by using the PBS buffer solution in the step (6), centrifuging after the dialysis is finished, and taking the supernatant to obtain the tapentadol artificial antigen II (tapentadol-bovine serum albumin conjugate) shown in the formula II. The synthetic route is as follows:
wherein MeOH represents anhydrous methanol, THF represents tetrahydrofuran, DMF represents N, N-dimethylformamide, DCC represents cyclohexylcarbodiimide, BSA represents bovine serum albumin, and the same applies below.
The ultraviolet scanning patterns before and after the preparation of the tapentadol artificial antigen II are shown in figure 3.
In fig. 3, curve a is the uv scan of tapentadol artificial hapten I, curve b is the uv scan of tapentadol artificial hapten II, and curve c is the uv scan of bovine serum albumin. The maximum absorption wavelength of the tapentadol artificial hapten I is 256nm, the maximum absorption wavelength of the tapentadol artificial hapten II is 342nm, and compared with the tapentadol artificial hapten I and bovine serum albumin, the maximum absorption wavelength of the tapentadol artificial hapten II is obviously changed, so that the coupling of the tapentadol artificial hapten I and the bovine serum albumin is successful.
Comparative example 1
The embodiment provides a preparation method of tapentadol artificial antigen III, which comprises the following steps:
(1) Preparation of tapentadol artificial hapten I:
(1) - (4) the same as in example 1.
(2) Preparation of tapentadol artificial antigen III:
the bovine gamma globulin is used as a carrier and is coupled with the artificial hapten I of the tapentadol, and the coupling step is the same as that of the example 1, so as to obtain the artificial hapten III of the tapentadol.
The synthetic route is as follows:
wherein BGG represents bovine gamma globulin, the same applies below.
Comparative example 2
The embodiment provides a preparation method of tapentadol artificial antigen V, which comprises the following steps:
(1) Preparation of tapentadol artificial hapten IV:
(1) 8g of sodium hydroxide solids were dissolved in 200ml of deionized water to give a 1N aqueous sodium hydroxide solution.
(2) Weighing and weighing100mg (0.426 mmol) of (2R, 3R) -3- (3-methoxyphenyl) -N, N, 2-trimethylpentylamine are reacted in a 50ml single-necked flask with the addition of 5ml of 48% hydrobromic acid at 100℃for 20h, TLC detection indicates that the reaction is substantially complete, cooling to room temperature, drying by transfer, adding 15ml of deionized water, adjusting the pH=9 with 1N of sodium hydroxide solution in (1) to give a large amount of precipitate, the solution being a milky suspension, the aqueous phase being extracted with 15ml of ethyl acetate, the ethyl acetate phase being collected, dried over anhydrous magnesium sulfate, filtered and transferred to give 91mg (0.411 mmol) of yellow transparent oil A which is subjected to TLC detection as a mixture of ethyl acetate and concentrated aqueous ammonia in a volume ratio of 10ml:3 drops, product R f ＝0.3～0.4；
(3) The yellow oily substance A was dissolved in a 50ml single-necked flask with 10ml of methylene chloride, 82mg (0.82 mmol) of succinic anhydride, 171. Mu.L (1.185 mmol) of triethylamine, 9mg (0.073 mmol) of 4-dimethylaminopyridine were added and reacted at 42℃for 24 hours, and the reaction was substantially complete as indicated by TLC detection, product point R f After TLC (developing solvent MOP, solvent and eluent absolute ethanol) separation, 33mg (0.066 mmol) of tapentadol artificial hapten IV was obtained.
(2) Preparation of tapentadol artificial antigen V:
(4) 33mg (0.066 mmol) of tapentadol artificial hapten IV is dissolved in a 50ml single-neck flask by 1.65ml of DMF, 12.76mg (0.0.090 mmol) of NHS and 18.51mg (0.0.090 mmol) of DCC are added, the reaction is stirred for 18h at 25 ℃, the reaction is ended, and the reaction product is centrifuged to obtain a supernatant for later use.
(5) 14.5g (40.503 mol) of disodium hydrogen phosphate dodecahydrate, 43.875g (750 mol) of sodium chloride, 1.495g (9.583 mol) of sodium dihydrogen phosphate dihydrate were weighed out and dissolved in deionized water to a constant volume of 5.0L, to obtain 0.01M, pH as a PBS buffer of 7.4.
(6) 50mg of bovine serum albumin was weighed and dissolved in 10ml of PBS buffer of step (5), to obtain a bovine serum albumin solution with a concentration of 5 mg/ml.
(7) Slowly dropwise adding the supernatant obtained in the step (4) into the bovine serum albumin solution obtained in the step (6) under the condition of rapid stirring, wherein the volume ratio of the supernatant to the bovine serum albumin solution is 1:5, standing and preserving the obtained mixed solution at the temperature of 4 ℃ overnight to obtain the artificial antigen mixed solution.
Transferring the artificial antigen mixed solution into a dialysis bag, dialyzing for 7 times by using the PBS buffer solution in the step (5), centrifuging after the dialysis is finished, and taking the supernatant to obtain the artificial antigen: tapentadol-bovine serum albumin conjugate (formula V)
The synthetic route is as follows:
wherein DCM represents dichloromethane, DMAP represents 4-dimethylaminopyridine, and the same applies below.
Comparative example 3
The embodiment provides a preparation method of tapentadol artificial antigen VI, which comprises the following steps:
(1) Preparation of tapentadol artificial hapten IV:
(1) - (3) is the same as in comparative example 2.
(2) Preparation of tapentadol artificial antigen VI:
bovine gamma globulin is used as a carrier and is coupled with tapentadol hapten IV, and the coupling step is the same as that of comparative example 2, so that tapentadol artificial antigen VI is obtained.
The synthetic route is as follows:
comparative example 4
The embodiment provides a preparation method of tapentadol artificial antigen VII, which comprises the following steps:
(1) Preparation of tapentadol artificial hapten I:
(1) - (4) the same as in example 1.
(2) Preparation of tapentadol artificial antigen VII:
(5) in a 50ml single neck round bottom flask, 60mg (0.215 mmol) of tapentadol artificial hapten I is dissolved in 3ml DMF, 36ul (0.258 mmol) of triethylamine and 33ul (0.258 mmol) of isobutyl chloroformate are then added, the reaction is stirred for 18h at 20-25 ℃, the reaction is ended, and the reaction product is centrifuged to obtain a supernatant for later use. (6) - (9) the same as in example 1, the artificial antigen VII of tapentadol was obtained.
The synthetic route is as follows:
wherein Et 3 N represents triethylamine, and is the same as below.
Comparative example 5
The embodiment provides a preparation method of tapentadol artificial antigen VIII, which comprises the following steps:
(1) Preparation of tapentadol artificial hapten I:
(1) - (4) the same as in example 1.
(2) Preparation of tapentadol artificial antigen VIII:
(5) the same procedures as in comparative example 4 were carried out using bovine gamma globulin as a carrier and coupling with tapentadol artificial hapten I, and the coupling procedure was the same as in example 1, to obtain tapentadol artificial antigen VIII.
The synthetic route is as follows:
comparative example 6
The embodiment provides a preparation method of tapentadol artificial antigen IX, which comprises the following steps:
(1) Preparation of tapentadol artificial hapten IV:
(1) - (3) is the same as in comparative example 2.
(2) Preparation of tapentadol artificial antigen IX:
(4) in a 50ml single-neck flask, 33mg (0.066 mmol) of tapentadol artificial hapten IV is dissolved in 1.65ml of DMF, then 11.83ul (0.082 mmol) of triethylamine and 10.56ul (0.082 mmol) of isobutyl chloroformate are added, the reaction is stirred for 18h at 20-25 ℃, the reaction is ended, and the reaction product is centrifuged, and the supernatant is taken for later use.
(5) - (8) is the same as comparative example 2; the tapentadol artificial antigen IX is obtained.
The synthetic route is as follows:
comparative example 7
The embodiment provides a preparation method of tapentadol artificial antigen X, which comprises the following steps:
(1) Preparation of tapentadol artificial hapten IV:
(1) - (3) is the same as in comparative example 2.
(2) Preparation of tapentadol artificial antigen X:
(4) the same as in comparative example 6.
(5) - (8) coupling with tapentadol hapten IV using bovine gamma globulin as a carrier, the coupling procedure being the same as comparative example 6, to give tapentadol artificial antigen X.
The synthetic route is as follows:
test example 1: performance measurement of Tapentadol Artificial antigen
(1) Identification of tapentadol artificial antigen:
molar absorption coefficient ε: the maximum absorption wavelength of the tapentadol hapten is 256nm, the absorbance is measured at 256nm, and the concentrations are parallel. The molar absorption coefficient (i.e., molar absorption coefficient) is calculated as: epsilon = absorbance/molarity.
Determination of conjugate protein concentration: PBS buffer solution is prepared into 0 mu g/ml, 10 mu g/ml, 20 mu g/ml, 30 mu g/ml, 40 mu g/ml, 60 mu g/ml, 80 mu g/ml, 100 mu g/ml and 120 mu g/ml bovine serum albumin solution, 3ml coomassie brilliant blue staining solution is added into each 1ml, the mixture is immediately mixed, the mixture is warmed for 5 minutes in a water bath at 30 ℃, each concentration is used as a parallel sample, the absorbance value is measured at 655nm, and a relation curve of the protein concentration and the absorbance value is drawn. The artificial antigen solution (prepared by PBS buffer solution) is diluted according to a certain proportion, the absorbance value of the artificial antigen is measured at 655nm, and the corresponding protein concentration value of the artificial antigen solution is read from the curve.
Coupling ratio determination: a PBS solution of 100. Mu.g/ml bovine serum albumin was prepared, the conjugate (i.e., tapentadol artificial antigen) was diluted to 100. Mu.g/ml with PBS and absorbance A was measured at 342nm 1 The absorbance A2 was measured with PBS as blank, and the coupling ratio γ was: gamma= [ (a) 1 －A 2 )/ε]/(100×10 -3 /66400)。
Wherein ε is the molar absorptivity (L/mol), 66400 is the molecular weight of bovine serum albumin, 100×10 -3 Is bovine serum albumin concentration (g/l).
When bovine gamma globulin is used as a carrier, the coupling ratio is calculated as: gamma= [ (a) 1 －A 2 )/ε]/(100×10 -3 43000); wherein 43000 is the molecular weight of bovine gamma globulin.
The results of the above detection are shown in Table 1.
TABLE 1 coupling ratio and molar absorption coefficient of Tapentadol artificial antigen
| Numbering device | Artificial antigen | Coupling ratio | Conjugate protein concentration | Molar absorption coefficient |
| Example 1 | II | 28 | 3.576mg/ml | 5856.86 |
| Comparative example 1 | III | 15 | 2.865mg/ml | 5856.86 |
| Comparative example 2 | V | 22 | 3.013mg/ml | 5749.18 |
| Comparative example 3 | VI | 17 | 2.466mg/ml | 5749.18 |
| Comparative example 4 | VII | 23 | 3.066mg/ml | 5856.86 |
| Comparative example 5 | VIII | 18 | 2.545mg/ml | 5856.86 |
| Comparative example 6 | IX | 21 | 2.977mg/ml | 5749.18 |
| Comparative example 7 | X | 2 | 0.584mg/ml | 5749.18 |
As can be seen from Table 1, the structure of the artificial hapten, the method of activating the artificial hapten and the type of carrier protein all have an influence on the coupling ratio of the artificial hapten to the carrier protein when cross-linked.
(2) Immunization of animals
The prepared tapentadol artificial antigen is immunized with New Zealand white rabbits, and the obtained immune serum is tested for titer by ELISA method, and the test results are shown in Table 2.
TABLE 2 results of potency detection of various immune sera
As can be seen from Table 2, the immune serum obtained by animal immunization with the artificial antigen of tapentadol of each comparative example was lower in titer than that of example 1, and could not be used in immunoassay. The immune serum obtained by animal immunization with the tapentadol artificial antigen II has a titer of 1:80000 can be used in immunoassay completely, and can provide a more convenient, rapid and accurate way for detecting tapentadol.
Claims (10)
1. The tapentadol artificial hapten is characterized in that the molecular structural formula is shown as a formula (I):
2. a process for the preparation of the tapentadol artificial hapten according to claim 1, characterized in that it comprises the following steps:
step (1), mixing (2R, 3R) -3- (3-methoxyphenyl) -N, N, 2-trimethylpentylamine and hydrobromic acid with the mass fraction of 48 percent according to (100-110) mg: mixing 5ml of the materials in a mass-volume ratio, and stirring and refluxing at 100-110 ℃ for 20-21 h; cooling to room temperature after the reaction is finished, turning to dry, adding deionized water, adjusting the pH to be alkaline, extracting for a plurality of times by using ethyl acetate, collecting an ethyl acetate phase, drying, filtering, and turning to dry to obtain oily matter A;
mixing the oily substance A obtained in the step (1) with ethyl bromoacetate in a solvent, adding sodium hydride with the mass fraction of 60% as a catalyst, stirring and refluxing at 65-70 ℃ for 18-20 h, directly transferring to dryness after the reaction is completed by thin layer chromatography, extracting with 20ml of ethyl acetate, filtering, and transferring to dryness to obtain oily substance B;
step (3), adding tetrahydrofuran, methanol and NaOH solution into the oily matter B obtained in the step (2), and stirring and reacting for 18-20 h at 25-30 ℃; and (3) after the reaction is finished, converting to a dry product, extracting with a proper amount of absolute ethyl alcohol, converting to a dry product again, and separating by thin-layer chromatography to obtain the tapentadol artificial hapten.
3. The process according to claim 2, wherein the molar ratio of oil a to ethyl bromoacetate in step (2) is 1: (2.5-3.5).
4. The process according to claim 2, wherein the solvent in step (2) is pyridine or acetonitrile or N, N-dimethylformamide.
5. The preparation method according to claim 2, wherein the ratio of the oily substance B to the tetrahydrofuran, the methanol and the NaOH solution in the step (3) is (100-110) mg:2ml:1.5ml:5ml.
6. The artificial antigen of tapentadol is characterized in that the artificial antigen is obtained by coupling the tapentadol hapten with bovine serum albumin and purifying the coupling agent, and the molecular structural formula of the artificial antigen is shown as a formula (II):
wherein BSA is bovine serum albumin.
7. A method for preparing the artificial antigen of tapentadol according to claim 6, comprising the steps of:
step (1), tapentadol artificial hapten, N-dicyclohexylcarbodiimide and cyclohexyl carbodiimide are mixed according to a molar ratio of 1: (1.35-1.5): (1.35-1.5) mixing the mixture with N, N-dimethylformamide, stirring the mixture at 20-30 ℃ for reaction for 18-20 hours, centrifuging the mixture after the reaction is finished, and taking supernatant;
and (2) adding the supernatant into a bovine serum albumin solution, standing the obtained mixed solution at 3-5 ℃ overnight, and obtaining the supernatant through dialysis and centrifugation to obtain the tapentadol artificial antigen.
8. The method according to claim 7, wherein in the step (2), the concentration of the bovine serum albumin solution is 5mg/mL, and the volume ratio of the supernatant to the bovine serum albumin solution is 1:5.
9. use of the artificial antigen of tapentadol according to claim 6 for the preparation of an anti-tapentadol antibody.
10. An anti-tapentadol artificial antibody, which is obtained by immunizing animals with the tapentadol artificial antigen according to claim 6 and can specifically react with tapentadol.
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