CN116283885A - Paroxetine hapten and artificial antigen as well as preparation methods and application thereof - Google Patents
Paroxetine hapten and artificial antigen as well as preparation methods and application thereof Download PDFInfo
- Publication number
- CN116283885A CN116283885A CN202211533425.XA CN202211533425A CN116283885A CN 116283885 A CN116283885 A CN 116283885A CN 202211533425 A CN202211533425 A CN 202211533425A CN 116283885 A CN116283885 A CN 116283885A
- Authority
- CN
- China
- Prior art keywords
- paroxetine
- solution
- hapten
- artificial
- reaction
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- AHOUBRCZNHFOSL-UHFFFAOYSA-N Paroxetine hydrochloride Natural products C1=CC(F)=CC=C1C1C(COC=2C=C3OCOC3=CC=2)CNCC1 AHOUBRCZNHFOSL-UHFFFAOYSA-N 0.000 title claims abstract description 134
- 229960002296 paroxetine Drugs 0.000 title claims abstract description 134
- AHOUBRCZNHFOSL-YOEHRIQHSA-N (+)-Casbol Chemical compound C1=CC(F)=CC=C1[C@H]1[C@H](COC=2C=C3OCOC3=CC=2)CNCC1 AHOUBRCZNHFOSL-YOEHRIQHSA-N 0.000 title claims abstract description 124
- 239000000427 antigen Substances 0.000 title claims abstract description 45
- 102000036639 antigens Human genes 0.000 title claims abstract description 45
- 108091007433 antigens Proteins 0.000 title claims abstract description 45
- 238000002360 preparation method Methods 0.000 title claims abstract description 31
- 238000001514 detection method Methods 0.000 claims abstract description 33
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 25
- 238000000034 method Methods 0.000 claims abstract description 16
- 239000000126 substance Substances 0.000 claims abstract description 15
- 241001465754 Metazoa Species 0.000 claims abstract description 6
- 239000000243 solution Substances 0.000 claims description 35
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 27
- 238000006243 chemical reaction Methods 0.000 claims description 26
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 claims description 21
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 21
- 238000003756 stirring Methods 0.000 claims description 19
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 claims description 16
- 238000012360 testing method Methods 0.000 claims description 16
- 102000014914 Carrier Proteins Human genes 0.000 claims description 14
- 108010078791 Carrier Proteins Proteins 0.000 claims description 14
- DYHSDKLCOJIUFX-UHFFFAOYSA-N tert-butoxycarbonyl anhydride Chemical compound CC(C)(C)OC(=O)OC(=O)OC(C)(C)C DYHSDKLCOJIUFX-UHFFFAOYSA-N 0.000 claims description 14
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 13
- 229940098773 bovine serum albumin Drugs 0.000 claims description 13
- 238000010992 reflux Methods 0.000 claims description 13
- 239000011259 mixed solution Substances 0.000 claims description 12
- 239000007853 buffer solution Substances 0.000 claims description 10
- 238000010438 heat treatment Methods 0.000 claims description 10
- QAEDZJGFFMLHHQ-UHFFFAOYSA-N trifluoroacetic anhydride Chemical compound FC(F)(F)C(=O)OC(=O)C(F)(F)F QAEDZJGFFMLHHQ-UHFFFAOYSA-N 0.000 claims description 10
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 9
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 9
- PBMIETCUUSQZCG-UHFFFAOYSA-N n'-cyclohexylmethanediimine Chemical compound N=C=NC1CCCCC1 PBMIETCUUSQZCG-UHFFFAOYSA-N 0.000 claims description 9
- 239000002904 solvent Substances 0.000 claims description 9
- 238000001704 evaporation Methods 0.000 claims description 8
- 238000003908 quality control method Methods 0.000 claims description 8
- 239000006228 supernatant Substances 0.000 claims description 7
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 6
- 230000003053 immunization Effects 0.000 claims description 6
- 238000002649 immunization Methods 0.000 claims description 6
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 claims description 5
- 239000012295 chemical reaction liquid Substances 0.000 claims description 5
- VANNPISTIUFMLH-UHFFFAOYSA-N glutaric anhydride Chemical compound O=C1CCCC(=O)O1 VANNPISTIUFMLH-UHFFFAOYSA-N 0.000 claims description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 4
- 238000001816 cooling Methods 0.000 claims description 4
- 230000008569 process Effects 0.000 claims description 4
- 239000002994 raw material Substances 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-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 3
- 239000005457 ice water Substances 0.000 claims description 3
- 239000008213 purified water Substances 0.000 claims description 3
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims description 3
- 241000283690 Bos taurus Species 0.000 claims description 2
- 101000800130 Bos taurus Thyroglobulin Proteins 0.000 claims description 2
- 101000609762 Gallus gallus Ovalbumin Proteins 0.000 claims description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 2
- UXGNZZKBCMGWAZ-UHFFFAOYSA-N dimethylformamide dmf Chemical compound CN(C)C=O.CN(C)C=O UXGNZZKBCMGWAZ-UHFFFAOYSA-N 0.000 claims description 2
- 238000001035 drying Methods 0.000 claims description 2
- 238000001914 filtration Methods 0.000 claims description 2
- 239000012530 fluid Substances 0.000 claims description 2
- 108010074605 gamma-Globulins Proteins 0.000 claims description 2
- 108010045069 keyhole-limpet hemocyanin Proteins 0.000 claims description 2
- 238000005406 washing Methods 0.000 claims description 2
- 230000005847 immunogenicity Effects 0.000 abstract description 3
- 230000009257 reactivity Effects 0.000 abstract description 3
- 239000002243 precursor Substances 0.000 abstract description 2
- 230000000875 corresponding effect Effects 0.000 abstract 1
- 239000000872 buffer Substances 0.000 description 8
- QZAYGJVTTNCVMB-UHFFFAOYSA-N serotonin Chemical compound C1=C(O)C=C2C(CCN)=CNC2=C1 QZAYGJVTTNCVMB-UHFFFAOYSA-N 0.000 description 8
- 238000000502 dialysis Methods 0.000 description 7
- 238000011161 development Methods 0.000 description 6
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- 239000003814 drug Substances 0.000 description 5
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- 102000004169 proteins and genes Human genes 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 210000002700 urine Anatomy 0.000 description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 239000000020 Nitrocellulose Substances 0.000 description 4
- 241001494479 Pecora Species 0.000 description 4
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 4
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 4
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 description 4
- 229920001220 nitrocellulos Polymers 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 229930006000 Sucrose Natural products 0.000 description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 3
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 3
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 238000007865 diluting Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 230000035484 reaction time Effects 0.000 description 3
- 239000005720 sucrose Substances 0.000 description 3
- 241000283707 Capra Species 0.000 description 2
- 206010013654 Drug abuse Diseases 0.000 description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- 230000001430 anti-depressive effect Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- VYFYYTLLBUKUHU-UHFFFAOYSA-N dopamine Chemical compound NCCC1=CC=C(O)C(O)=C1 VYFYYTLLBUKUHU-UHFFFAOYSA-N 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 230000001804 emulsifying effect Effects 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- 238000002523 gelfiltration Methods 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 229940124834 selective serotonin reuptake inhibitor Drugs 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 229910000029 sodium carbonate Inorganic materials 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 238000010254 subcutaneous injection Methods 0.000 description 2
- 239000007929 subcutaneous injection Substances 0.000 description 2
- 208000011117 substance-related disease Diseases 0.000 description 2
- SFLSHLFXELFNJZ-QMMMGPOBSA-N (-)-norepinephrine Chemical compound NC[C@H](O)C1=CC=C(O)C(O)=C1 SFLSHLFXELFNJZ-QMMMGPOBSA-N 0.000 description 1
- OOSZCNKVJAVHJI-UHFFFAOYSA-N 1-[(4-fluorophenyl)methyl]piperazine Chemical compound C1=CC(F)=CC=C1CN1CCNCC1 OOSZCNKVJAVHJI-UHFFFAOYSA-N 0.000 description 1
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 102000004980 Dopamine D2 Receptors Human genes 0.000 description 1
- 108090001111 Dopamine D2 Receptors Proteins 0.000 description 1
- 102000003834 Histamine H1 Receptors Human genes 0.000 description 1
- 108090000110 Histamine H1 Receptors Proteins 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 108010044467 Isoenzymes Proteins 0.000 description 1
- 102000010909 Monoamine Oxidase Human genes 0.000 description 1
- 108010062431 Monoamine oxidase Proteins 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
- 239000002250 absorbent Substances 0.000 description 1
- 230000002745 absorbent Effects 0.000 description 1
- 230000001800 adrenalinergic effect Effects 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000001078 anti-cholinergic effect Effects 0.000 description 1
- 239000000935 antidepressant agent Substances 0.000 description 1
- 229940005513 antidepressants Drugs 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 102000012740 beta Adrenergic Receptors Human genes 0.000 description 1
- 108010079452 beta Adrenergic Receptors Proteins 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 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 1
- 238000004090 dissolution Methods 0.000 description 1
- 229960003638 dopamine Drugs 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 238000002825 functional assay Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 229960002748 norepinephrine Drugs 0.000 description 1
- SFLSHLFXELFNJZ-UHFFFAOYSA-N norepinephrine Natural products NCC(O)C1=CC=C(O)C(O)=C1 SFLSHLFXELFNJZ-UHFFFAOYSA-N 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 239000012896 selective serotonin reuptake inhibitor Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 229940074545 sodium dihydrogen phosphate dihydrate Drugs 0.000 description 1
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical class O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 1
- 125000006850 spacer group Chemical group 0.000 description 1
- 230000000946 synaptic effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000001839 systemic circulation Effects 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D317/00—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms
- C07D317/08—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3
- C07D317/44—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D317/46—Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 ortho- or peri-condensed with carbocyclic rings or ring systems condensed with one six-membered ring
- C07D317/48—Methylenedioxybenzenes or hydrogenated methylenedioxybenzenes, unsubstituted on the hetero ring
- C07D317/62—Methylenedioxybenzenes or hydrogenated methylenedioxybenzenes, unsubstituted on the hetero ring with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to atoms of the carbocyclic ring
- C07D317/64—Oxygen atoms
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
-
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
-
- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
-
- 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
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
- G01N33/9466—Antidepressants
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B2200/00—Indexing scheme relating to specific properties of organic compounds
- C07B2200/07—Optical isomers
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Analytical Chemistry (AREA)
- Food Science & Technology (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- General Chemical & Material Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pain & Pain Management (AREA)
- Psychiatry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Pharmacology & Pharmacy (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a paroxetine hapten and an artificial antigen as well as preparation methods and applications thereof. The 2-hydroxy paroxetine is used as a precursor to synthesize a paroxetine hapten with a chemical structural formula (I) and a paroxetine artificial antigen with a chemical structural formula (II), the obtained antigen has good immunogenicity and reactivity, corresponding antibodies are easy to obtain when animals are immunized, and the method can be used for preparing paroxetine colloidal gold detection products.
Description
Technical Field
The invention relates to the field of paroxetine detection, in particular to a paroxetine hapten, an artificial antigen, a preparation method and application thereof.
Background
Paroxetine (Paroxetine) is an antidepressant (SSRIs) of the 5-HT reuptake inhibitor, and can selectively inhibit reuptake of 5-hydroxytryptamine (5-HT) and increase the concentration of 5-HT in the synaptic cleft, thereby producing antidepressant effect. It only weakly inhibits reuptake of norepinephrine and dopamine. The medicine does not act with adrenergic alpha 1, alpha 2 or beta receptor, and also does not bind with dopamine D2 or histamine H1 receptor, and does not inhibit monoamine oxidase. The medicine can be widely distributed in various tissues and organs, and only 1% of the medicine appears in the systemic circulation. The protein binding rate is as high as 95%. The drug is metabolized by the liver P450 isoenzyme. Paroxetine is largely excreted with urine via the kidneys; about 36% is excreted by faeces; can also be excreted via milk. Paroxetine is the strongest reuptake agent within the SSRI class, but it is also the only drug with mild anticholinergic effect.
For drug abuse needs, medical personnel can help diagnose paroxetine dependence and paroxetine recovery by detecting paroxetine in urine. Currently, high Performance Liquid Chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS) methods and the like are commonly used for detecting paroxetine-related substances. The method has the advantages of strong separation and analysis capability, high sensitivity, reliable and accurate result, complex structure, high requirements on the temperature and humidity of the environment and high maintenance cost. Mass spectrometers are high-precision instruments that require specially trained technicians to operate and have slow testing speeds, thus being unfavorable for widespread popularization of drug abuse detection. Therefore, there is a need to develop a simple and efficient method for detecting paroxetine substances.
Disclosure of Invention
The invention aims to solve the problems that HPLC and LC-MS are used for detecting paroxetine in the prior art, the requirements on instruments and operators are high, the popularization is difficult, and the detection speed is low, and provides an artificial hapten for paroxetine, which can specifically identify paroxetine.
A paroxetine hapten has a molecular structural formula shown in a formula (I),
no active group in the 2-hydroxy paroxetine molecule can be linked to the carrier protein, and thus it is modified by attaching a suitable spacer to produce a carboxyl or amino group to facilitate coupling of the carrier protein.
The second object of the invention is to provide a preparation method of paroxetine artificial hapten, which adopts the following technical scheme:
dissolving 2-hydroxy paroxetine in N, N-Dimethylformamide (DMF), adding triethylamine and di-tert-butyl dicarbonate, heating and refluxing at 45-50 ℃, cooling the reaction liquid to normal temperature after the reaction is completed, and evaporating the solvent under reduced pressure to obtain oily matter A;
dissolving an oil phase substance A in pyridine, then adding glutaric anhydride and 4-Dimethylaminopyridine (DMAP), heating and refluxing at 98-100 ℃, cooling the reaction liquid to normal temperature after the reaction is completed, and evaporating the solvent under reduced pressure to obtain oily residues; adding dichloromethane into oily residues, respectively washing with purified water and saturated NaCl, collecting dichloromethane phase, drying, filtering, and evaporating under reduced pressure to obtain oily substance B;
step (3), dissolving the oily matter B in a mixed solution of HCl and 1, 4-dioxane, heating, stirring and refluxing at 98-100 ℃, and evaporating the solvent under reduced pressure to obtain oily matter C;
and (4) dissolving the oily substance C in benzene, placing the benzene in an ice-water bath, adding trifluoroacetic anhydride, stirring at normal temperature for a period of time, heating and stirring at 78-81 ℃ for reflux, and transferring the reaction solution to dryness after the reaction is completed, so as to obtain the oily substance D, namely the paroxetine artificial hapten.
Preferably, in step (1), the molar ratio of 2-hydroxy paroxetine to triethylamine and di-tert-butyl dicarbonate is 1:2:2;
preferably, in step (1), the reaction temperature is 45 ℃, and the reaction time is 14.5-16h, more preferably 15h;
preferably, in step (2), the molar ratio of oil A, glutaric anhydride and 4-Dimethylaminopyridine (DMAP) is (10-12): (4-5.5): (1-2), more preferably 10:5:1, a step of;
preferably, in step (2), the reaction temperature is 100 ℃, and the reaction time is 16-19h, more preferably 18h;
preferably, in step (3), 7.5nmol of oil B is added per mL of the mixed solution;
preferably, in step (3), the heating temperature is 100 ℃, and the stirring reflux reaction time is 5-6.5h, more preferably 6h;
preferably, in the step (3), the concentration of HCl in the mixed solution is 2mol/L;
preferably, in step (4), 0.056mmol of oily C is added per mL of benzene;
preferably, in step (4), the stirring time is from 0.8 to 1.2 hours, more preferably 1 hour;
preferably, in step (4), the molar ratio of oil C to trifluoroacetic anhydride is (1-1.3): (1.8-2.4), more preferably 1:2;
preferably, in step (4), the heating temperature is 80℃and the reflux reaction is stirred for a period of 2.6 to 3.0 hours, more preferably 3 hours.
In the synthesis reaction of paroxetine hapten, thin layer chromatography can be used for monitoring the formation of brown oily matters to judge whether the reaction is complete, and the volume ratio of each component in the used chromatographic liquid is 95% ethanol: dichloromethane: 1, 4-dioxane: ammonia = 8:10:1:1, product rf=0.4-0.5.
The third object of the invention is to provide an artificial paroxetine antigen which is obtained by coupling hapten and carrier protein of formula (I); it has the molecular structure characteristics of paroxetine, can be recognized by animal immunocompetent cells, and can generate antibodies capable of specifically binding to paroxetine. The molecular structural formula is shown as formula (II):
wherein R represents a carrier protein;
preferably, the carrier protein is one of bovine serum albumin, bovine gamma globulin, bovine thyroglobulin, keyhole limpet hemocyanin and chicken ovalbumin.
The fourth object of the present invention is to provide a preparation method of paroxetine artificial antigen, which adopts the following technical scheme:
dissolving paroxetine artificial hapten in N, N-dimethylformamide DMF, adding N-hydroxysuccinimide NHS and cyclohexyl carbodiimide, stirring at normal temperature for reaction for 15-18 hours, centrifuging, and taking supernatant to obtain solution A;
step (2), dissolving carrier protein in PBS buffer solution to obtain solution B, wherein the concentration of the carrier protein in the solution B is 4-9mg/ml;
step (3), dropwise adding the solution A into the solution B under the stirring condition, and standing overnight at a low temperature to obtain an artificial antigen mixed solution;
and (4) dialyzing the artificial antigen mixed solution obtained in the step (3) in alkaline dialyzate for a plurality of times, transferring into PBS buffer solution for dialyzing for a plurality of times, and centrifuging after the dialyzate is finished to obtain supernatant fluid, thus obtaining the paroxetine artificial antigen.
Preferably, in the step (1), the molar ratio of the paroxetine artificial hapten, the N-hydroxysuccinimide and the cyclohexylcarbodiimide is (1.3 to 1.4): 1.9:1.9;
preferably, in the step (2), the concentration of the carrier protein in the solution B is 5mg/ml;
preferably, in the step (3), the volume ratio of the liquid A to the liquid B is 1:5;
preferably, in the step (3), the low temperature is 4 ℃;
preferably, in step (4), the alkaline dialysate is a sodium carbonate solution having a ph=11.05-12.05; the concentration of PBS buffer solution is 0.1mol/L, and the pH value is 7.2-7.4;
preferably, in the step (4), the alkaline dialysate is dialyzed for 2 to 3 times, and the dialysis time is 24 hours each time;
preferably, in step (4), the dialysis is performed 6-7 times in PBS buffer, each time for more than 2 hours.
The paroxetine hapten reacts with N-hydroxysuccinimide and N, N-cyclohexyl carbodiimide to obtain active ester, and the active ester is coupled with carrier protein to obtain the paroxetine artificial antigen, the active site of the paroxetine is protected in the reaction process, the immunogenicity and the reactivities of the antigen are improved, corresponding antibodies are easy to obtain when animals are immunized, and the preparation of subsequent detection reagents is ensured. The supernatant obtained by centrifugation after the end of ultraviolet scanning dialysis can be used for identifying whether the hapten and BSA are successfully coupled.
A fifth object of the present invention is to provide a paroxetine polyclonal antibody obtained by animal immunization with an artificial antigen of formula (II).
The preparation method of the paroxetine polyclonal antibody comprises the following steps: diluting paroxetine artificial antigen to 1.67mg/mL by PBS buffer solution, emulsifying with Freund's adjuvant according to the volume ratio of 1:1, then carrying out multi-point subcutaneous injection on the back of a rabbit, taking little blood after each injection for 500ug and 7 days for potency detection, confirming whether immunization is successful or not, and collecting serum for purification after the immunization is successful to prepare the paroxetine polyclonal antibody.
The sixth object of the invention is to provide a paroxetine colloidal gold test paper, which comprises a detection line, a quality control line and a bonding pad, wherein the detection line is obtained by taking a paroxetine artificial antigen as a raw material spot film;
preferably, the quality control line is obtained by spotting a membrane with sheep anti-mouse IgG or sheep anti-rabbit IgG as a raw material, and the binding pad is obtained by spraying paroxetine polyclonal antibody-colloidal gold complex on the binding pad.
The paroxetine colloidal gold test paper has high detection sensitivity, and the detectable concentration reaches 500ng/mL, and the color development intensity of the detection line is inversely related to the concentration of paroxetine in a sample because the detection principle of the paroxetine detection kit disclosed by the invention uses a competition method. Meanwhile, colloidal gold marks are used, so that qualitative detection can be performed. The user visually observes the aggregation color development condition of the colloidal gold on the NC film, and under the condition that the quality control line appears, the T line appears as negative, and the T line does not appear as positive.
Preferably, the quality control line and the detection line are manufactured through the following processes: a1.0 mg/mL sheep anti-rabbit IgG and a 0.1mg/mL sheep anti-mouse IgG were used as a quality control line on the nitrocellulose membrane at a spot of 1.0. Mu.L/cm, and a 0.3mg/mL paroxetine antigen was used as a detection line on the nitrocellulose membrane at a spot of 1.0. Mu.L/cm.
Preferably, the preparation method of the paroxetine polyclonal antibody-colloidal gold complex comprises the following steps: adjusting the pH value of the colloidal gold solution to 7.6, adding the paroxetine polyclonal antibody solution, stirring, adding 1% PEG2000 buffer solution, centrifuging for 60min at 100000g at 4 ℃, removing precipitate, re-suspending with the PEG2000 buffer solution, repeating for 2-3 times to obtain colloidal gold labeled protein, purifying the colloidal gold labeled protein by a gel filtration method, eluting with PBS buffer solution containing BSA and sodium azide, and collecting to obtain the paroxetine polyclonal antibody-colloidal gold complex.
Preferably, the preparation step of the bonding pad includes: the paroxetine polyclonal antibody-colloidal gold complex is diluted in PBS buffer containing bovine serum albumin to 1/4-1/3 of the initial OD value of the complex, and the complex is uniformly sprayed on the binding pad in a spraying amount of 1.0 mu L/cm.
Preferably, the conjugate pad is prepared by adding 50mg/mL trehalose and 200mg/mL sucrose to PBS buffer containing bovine serum albumin.
The activity of the paroxetine polyclonal antibody-colloidal gold complex is effectively maintained by adding sucrose and trehalose, the validity period of the binding pad is prolonged, and the product performance is improved.
The invention has the following beneficial effects:
(1) 2-hydroxy paroxetine is used as a precursor to synthesize paroxetine hapten and paroxetine artificial antigen, the immunogenicity and the reactivity of the obtained antigen are good, corresponding antibodies are easy to obtain when animals are immunized, and the paroxetine hapten and paroxetine artificial antigen can be used for preparing paroxetine colloidal gold-detection test paper;
(2) The paroxetine colloidal gold-detection test paper provided by the invention has high sensitivity, the detection limit of the colloidal gold is 500ng/mL, the color development intensity of the detection line and the concentration of the paroxetine show negative correlation, the paroxetine in urine, blood and saliva can be rapidly detected, and the paroxetine can be qualitatively detected by naked eyes.
Detailed Description
The invention is further described below in connection with specific embodiments.
The PBS buffer used in the following examples was prepared from 14.5g of disodium hydrogen phosphate dodecahydrate, 43.875g of sodium chloride, 1.495g of sodium dihydrogen phosphate dihydrate, and dissolved in double distilled water to a constant volume of 5.0L, and 0.1M, pH was 7.4; the preparation process of the alkaline dialysate comprises the following steps: the sodium carbonate aqueous solution with the mass fraction of 0.5% was adjusted to ph=12.00 with 2mol/L NaOH solution.
Example 1
(1) Preparation of paroxetine artificial hapten:
A. 200mg (0.58 mmol) of 2-hydroxy paroxetine is weighed and placed in a 50mL single-neck round bottom flask, 5mL of LDMF is added for dissolution, 160 mu L (1.16 mmol) of triethylamine and 266 mu L (1.16 mmol) of di-tert-butyl dicarbonate are added, and the mixture is placed in an oil bath at 45 ℃ for stirring and refluxing for 15 hours, and TLC detection shows that the raw materials are completely reacted; after the reaction is finished, the reaction liquid is cooled to normal temperature, and is pumped by an oil pump in a water bath at 60 ℃ to obtain a pale yellow oily substance A:267mg, directly into the next reaction.
B. The above pale yellow oil a:267mg was placed in a 50mL single neck round bottom flask, 15mL pyridine was added to dissolve, 132mg (1.16 mmol) glutaric anhydride and 27mg DMAP were added, the mixture was stirred in an oil bath at 100deg.C and stirred for 18 hours under reflux, and TLC showed that most of the reaction was complete; the reaction was stopped, the reaction mixture was cooled to room temperature, the solvent was evaporated under reduced pressure to give a brown oily residue, 30mL of dichloromethane was added, the dichloromethane phases were washed with 30mL of purified water and 30mL of saturated brine, respectively, the dichloromethane phases were collected, dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure to give a brown oil. The brown oil was subjected to TLC separation (developing solvent 95% ethanol: dichloromethane: 1, 4-dioxane: ammonia=8:10:1:1) and the product spot with Rf=0.4-0.5 was collected to give 168mg (0.30 mmol) of brown oil B.
C. The above 168mg (0.30 mmol) of brown oily substance B was dissolved in 20mL of a 2mol/LHCl solution of 1, 4-dioxane, and the mixture was placed in an oil bath at 100℃with stirring and refluxed for 6 hours, and the reaction was stopped by TLC, and the solvent was evaporated under reduced pressure to give 130mg (0.28 mmol) of brown oily substance C.
D. 130mg (0.28 mmol) of brown oily substance C is dissolved in 5mL of benzene, the benzene is placed in an ice-water bath, 78 mu L (0.56 mmol) of trifluoroacetic anhydride is slowly added, the mixture is stirred at normal temperature for reaction for 1 hour, the mixture is transferred into an oil bath at 80 ℃ for stirring and refluxing reaction for 3 hours, the reaction is stopped, the reaction solution is dried, and TLC (thin layer chromatography) is carried out to obtain 146mg (0.26 mmol) of brown oily substance, namely paroxetine artificial hapten.
Nuclear magnetic resonance hydrogen spectrum data:
1H-NMR(400MHz,CDCl3):δ1.79-1.95(m,4H)，δ2.23-2.38(m,5H)，δ2.77(m,1H)，δ3.3-3.34(t,4H)，δ3.9(d,2H)，δ6.17-6.22(t,2H)，δ6.55(d,1H)，δ6.89-7.11(m,4H)，δ8.37(s,1H)，δ10.52(s,1H)。
nuclear magnetic resonance carbon spectrum data:
13C-NMR(100MHz)：δ20.2(CH2)，δ29.7(CH2)，δ33.0(CH)，δ33.3(CH2)，δ35.1(CH)，δ35.2(CH2)，δ43.6(CH2)，δ44.3(CH2)，δ71.5(CH2)，δ100.8(CH)，δ107.5(CH)，δ112.7(CH)，δ114.6(2CH)，δ115.7(CH)，δ123.1(C)，δ130.3(2CH)，δ136.7(C)，δ139.2(C)，δ148.2(C)，δ152.1(C)，δ158.9(C)，δ172.0(C)，δ172.7(C)，δ177.0(C)。
(2) Preparation of paroxetine artificial antigen:
A. 130mg of paroxetine artificial hapten (0.28 mmol) is placed in a 25mL single-neck round bottom flask, N-Dimethylformamide (DMF) is added, 43mg (0.38 mmol) of N-hydroxysuccinimide (NHS) and 78mg (0.38 mmol) of cyclohexyl carbodiimide (DCC) are added, the reaction is stirred at normal temperature for 16 hours, the reaction is finished, the supernatant is centrifuged at 8000r/min and 4 ℃ for 15min and is recorded as A solution.
B. 0.160g of Bovine Serum Albumin (BSA) was weighed and dissolved in 32mL of BSA solution to give 5mg/mL of BSA solution, which was designated as solution B.
C. Slowly dripping the solution A into the solution B under the condition of rapid stirring, wherein the volume ratio of the solution A to the solution B is 1:5, standing and preserving the obtained mixed solution at 4 ℃ overnight to obtain an artificial antigen mixed solution, and identifying whether the hapten and BSA are successfully coupled or not through ultraviolet scanning;
D. transferring the artificial antigen mixed solution which is successfully coupled into a dialysis bag, dialyzing in alkaline dialyzate for 24 hours under the condition of stirring, and repeating the dialysis twice;
E. dialyzing the alkaline dialyzed artificial antigen mixed solution in PBS solution for 7 times, wherein each time of dialysis is 2 hours, centrifuging after the dialysis is finished, and taking supernatant to obtain the paroxetine artificial antigen.
Example 2
(1) Preparation of paroxetine artificial hapten: the molar ratio of 2-hydroxy paroxetine to triethylamine and di-tert-butyl dicarbonate was changed to 1:2:2, preparing paroxetine artificial hapten by the rest preparation conditions in the same way as in the example 1;
(2) Preparation of paroxetine artificial antigen: the molar ratio of paroxetine hapten, N-hydroxysuccinimide and cyclohexyl carbodiimide was changed to 1.3:1.9:1.9, the rest of the preparation conditions are the same as in example 1, and paroxetine artificial antigen is prepared.
Example 3
(1) Preparation of paroxetine artificial hapten: the molar ratio of 2-hydroxy paroxetine to triethylamine and di-tert-butyl dicarbonate was changed to 1.2:2:2, preparing paroxetine artificial hapten by the rest preparation conditions in the same way as in the example 1;
(2) Preparation of paroxetine artificial antigen: the molar ratio of paroxetine hapten, N-hydroxysuccinimide and cyclohexyl carbodiimide was changed to 1.4:1.9:1.9, the rest of the preparation conditions are the same as in example 1, and paroxetine artificial antigen is prepared.
Example 4: preparation of paroxetine polyclonal antibody:
diluting the paroxetine antigen obtained in the example 1 to 1.67mg/mL by using PBS buffer solution, emulsifying with Freund's adjuvant according to the volume ratio of 1:1, performing multi-point subcutaneous injection on the back of a rabbit, taking little blood after each injection with 500ug for 7 days for potency detection, confirming whether immunization is successful, and collecting serum for purification after the immunization is successful to obtain the paroxetine polyclonal antibody.
Example 5: preparation of paroxetine polyclonal antibody:
paroxetine polyclonal antibodies were prepared using the Paroxetine antigen obtained in example 2, and the Paroxetine polyclonal antibodies were prepared in the same manner as in example 4.
Example 6: preparation of paroxetine polyclonal antibody:
paroxetine polyclonal antibodies were prepared using the Paroxetine antigen obtained in example 3, and the Paroxetine polyclonal antibodies were prepared in the same manner as in example 4.
Example 7: preparation of paroxetine colloidal gold test paper:
A. detection line and quality control line: using 0.2mg/mL of paroxetine antigen obtained in example 1, spraying 1.0 mu L/cm on a nitrocellulose membrane as a detection line, using 1.0mg/mL of goat anti-rabbit IgG and 0.1mg/mL of goat anti-mouse IgG, and spraying 1.0 mu L/cm on a nitrocellulose membrane as a quality control line;
B. paroxetine polyclonal antibody-colloidal gold complex: dissolving the paroxetine polyclonal antibody obtained in the example 4 in a colloidal gold solution to obtain a paroxetine polyclonal antibody solution, regulating the pH value of the colloidal gold solution to 7.6, adding 1mL of the paroxetine polyclonal antibody solution, stirring, adding 10mL of 1% PEG2000 buffer, centrifuging at the temperature of 4 ℃ for 60min with the centrifugal force of 100000g, removing precipitate, re-suspending with PEG2000 buffer, repeating for 3 times to obtain colloidal gold labeled protein, purifying the colloidal gold labeled protein by a gel filtration method, eluting with PBS buffer containing 1% BSA and 0.02% sodium azide, and collecting to obtain a paroxetine polyclonal antibody-colloidal gold complex;
C. and (3) a bonding pad: diluting the paroxetine polyclonal antibody-colloidal gold complex in PBS buffer containing 1% BSA, 50mg/mL trehalose and 200mg/mL sucrose to 1/3 of the initial OD value of the complex, and uniformly spraying the complex on a binding pad in a spraying amount of 1.0 mu L/cm;
D. and (3) assembling: and assembling the sheet bottom plate with the spotted film, the combination pad, the sample pad and the absorbent paper into a large card, and cutting the large card to obtain the paroxetine colloidal gold detection test strip.
Example 8: preparation of paroxetine colloidal gold test paper:
the paroxetine antigen obtained in example 2 was paroxetine antigen, and the paroxetine polyclonal antibody obtained in example 5 was paroxetine polyclonal antibody, and the other preparation procedures were the same as in example 7.
Example 9: preparation of paroxetine colloidal gold test paper:
the paroxetine antigen obtained in example 3 was paroxetine antigen, and the paroxetine monoclonal antibody obtained in example 6 was paroxetine monoclonal antibody, and the other preparation procedures were the same as in example 7.
Paroxetine solutions with different concentrations are prepared, a Paroxetine colloidal gold detection reagent strip prepared in example 7 is used for carrying out functional detection on a sample, a colloidal gold colorimetric card is used for judging the color development intensity of a detection line, each concentration is repeatedly measured for 5 times, and an average value is obtained, so that the result is shown in table 1.
TABLE 1 color development intensity of Paroxetine colloidal gold test paper
| Sample concentration (ng/mL) | T line strength |
| 0 | G9 |
| 250 | G5 |
| 500 | G3 |
| 750 | G2 |
| 1000 | G1 |
| 1500 | G1 |
The result interpretation of the colloidal gold test paper adopts a colorimetric card method, G1-G10 represent the color development degree of the T-line colloidal gold, wherein the G1 is observed by naked eyes to be that the T-line is not developed, and the strong positive is represented; g10 is dark in the T line and shows strong negative. As can be seen from Table 1, the detection concentration of the detection test paper prepared by the invention can be as low as 500ng/mL, the sensitivity is high, and the result can be obtained through visual observation. Functional assays were performed on 101 clinical urine samples using the paroxetine colloidal gold test strips prepared in examples 7-9. In the clinical urine samples, the concentration of paroxetine is distributed between 500 and 1500ng/mL in 70 cases of negative and 31 cases of positive. The detection results are shown in Table 2.
TABLE 2 clinical sample test results
As can be seen from Table 2, the paroxetine colloidal gold test paper has higher detection accuracy on clinical samples, and the overall accuracy is higher than 90%.
Claims (10)
1. The paroxetine hapten is characterized in that the molecular structural formula is shown as a formula (I):
2. a process for the preparation of paroxetine hapten according to claim 1, which comprises the steps of:
dissolving 2-hydroxy paroxetine in N, N-dimethylformamide, adding triethylamine and di-tert-butyl dicarbonate, heating and refluxing at 45-50 ℃, cooling the reaction liquid to normal temperature after the reaction is completed, and evaporating the solvent under reduced pressure to obtain oily matter A;
step (2), dissolving an oil phase substance A in pyridine, then adding glutaric anhydride and 4-dimethylaminopyridine, heating and refluxing at 98-100 ℃, cooling the reaction liquid to normal temperature after the reaction is completed, and evaporating the solvent under reduced pressure to obtain oily residues; adding dichloromethane into oily residues, respectively washing with purified water and saturated NaCl, collecting dichloromethane phase, drying, filtering, and evaporating under reduced pressure to obtain oily substance B;
step (3), dissolving the oily matter B in a mixed solution of HCl and 1, 4-dioxane, heating, stirring and refluxing at 98-100 ℃, and evaporating the solvent under reduced pressure to obtain oily matter C;
and (4) dissolving the oily substance C in benzene, placing the benzene in an ice-water bath, adding trifluoroacetic anhydride, stirring at normal temperature for a period of time, heating and stirring at 78-81 ℃ for reflux, and transferring the reaction solution to dryness after the reaction is completed, so as to obtain the oily substance D, namely the paroxetine artificial hapten.
3. The process according to claim 2, characterized in that in step (1), the molar ratio of 2-hydroxy paroxetine to triethylamine and di-tert-butyl dicarbonate is 1:2:2; in the step (2), the molar ratio of the oily matter A, glutaric anhydride and 4-Dimethylaminopyridine (DMAP) is (10-12): (4-5.5): (1-2); in the step (4), the mole ratio of the oily substance C to the trifluoroacetic anhydride is (1-1.3): (1.8-2.4).
4. A paroxetine artificial antigen, which is characterized in that the paroxetine hapten is conjugated with a carrier protein according to claim 1; the molecular structural formula is shown as formula (II):
wherein R represents a carrier protein.
5. The artificial paroxetine antigen of claim 4, wherein the carrier protein is one of bovine serum albumin, bovine gamma globulin, bovine thyroglobulin, keyhole limpet hemocyanin, and chicken ovalbumin.
6. A process for the preparation of paroxetine artificial antigen according to claim 4 or 5, comprising the steps of:
dissolving paroxetine artificial hapten in N, N-dimethylformamide DMF, adding N-hydroxysuccinimide NHS and cyclohexyl carbodiimide, stirring at normal temperature for reaction for 15-18 hours, centrifuging, and taking supernatant to obtain solution A;
step (2), dissolving carrier protein in PBS buffer solution to obtain solution B, wherein the concentration of the carrier protein in the solution B is 4-9mg/ml;
step (3), dropwise adding the solution A into the solution B under the stirring condition, and standing overnight at a low temperature to obtain an artificial antigen mixed solution;
and (4) dialyzing the artificial antigen mixed solution obtained in the step (3) in alkaline dialyzate for a plurality of times, transferring into PBS buffer solution for dialyzing for a plurality of times, and centrifuging after the dialyzate is finished to obtain supernatant fluid, thus obtaining the paroxetine artificial antigen.
7. The method according to claim 6, wherein in the step (1), the molar ratio of paroxetine artificial hapten, N-hydroxysuccinimide and cyclohexylcarbodiimide is (1.3 to 1.4): 1.9:1.9.
8. the method of claim 7, wherein in step (3), the volume ratio of the solution a to the solution B is 1:5.
9. a paroxetine polyclonal antibody, which is obtained by animal immunization with a paroxetine artificial antigen according to claim 4 or 5.
10. A paroxetine colloidal gold test paper comprises a detection line, a quality control line and a combination pad; the detection line is characterized in that the paroxetine artificial antigen as claimed in claim 4 or 5 is adopted as a raw material spot film.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1668331A (en) * | 2002-07-18 | 2005-09-14 | 赛托斯生物技术公司 | Hapten-carrier conjugates and uses thereof |
| CN109824673A (en) * | 2019-01-25 | 2019-05-31 | 杭州同舟生物技术有限公司 | A kind of zopiclone artificial semiantigen, artificial antigen and its preparation method and application |
| CN109824599A (en) * | 2019-02-28 | 2019-05-31 | 中国农业大学 | A kind of albendazole haptens and its preparation method and application |
| CN113105538A (en) * | 2021-04-21 | 2021-07-13 | 杭州奥泰生物技术股份有限公司 | Citalopram antigen and citalopram colloidal gold detection test paper |
| CN113135823A (en) * | 2021-04-27 | 2021-07-20 | 华南农业大学 | Simvastatin hapten, artificial antigen and antibody and application thereof |
| US20210403425A1 (en) * | 2020-06-30 | 2021-12-30 | Field Trip Psychedelics Inc. | Tryptamine prodrugs |
-
2022
- 2022-12-01 CN CN202211533425.XA patent/CN116283885A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1668331A (en) * | 2002-07-18 | 2005-09-14 | 赛托斯生物技术公司 | Hapten-carrier conjugates and uses thereof |
| CN109824673A (en) * | 2019-01-25 | 2019-05-31 | 杭州同舟生物技术有限公司 | A kind of zopiclone artificial semiantigen, artificial antigen and its preparation method and application |
| CN109824599A (en) * | 2019-02-28 | 2019-05-31 | 中国农业大学 | A kind of albendazole haptens and its preparation method and application |
| US20210403425A1 (en) * | 2020-06-30 | 2021-12-30 | Field Trip Psychedelics Inc. | Tryptamine prodrugs |
| CN113105538A (en) * | 2021-04-21 | 2021-07-13 | 杭州奥泰生物技术股份有限公司 | Citalopram antigen and citalopram colloidal gold detection test paper |
| CN113135823A (en) * | 2021-04-27 | 2021-07-20 | 华南农业大学 | Simvastatin hapten, artificial antigen and antibody and application thereof |
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| CN116478060A (en) * | 2023-03-07 | 2023-07-25 | 杭州同舟生物技术有限公司 | Dezocine hapten and artificial antigen as well as preparation methods and applications thereof |
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