CN108484752B - Spiral shell worm ethyl ester antigen and the preparation method and application thereof - Google Patents
Spiral shell worm ethyl ester antigen and the preparation method and application thereof Download PDFInfo
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- CN108484752B CN108484752B CN201810570441.3A CN201810570441A CN108484752B CN 108484752 B CN108484752 B CN 108484752B CN 201810570441 A CN201810570441 A CN 201810570441A CN 108484752 B CN108484752 B CN 108484752B
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- spiral shell
- ethyl ester
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- shell worm
- worm ethyl
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- 125000004494 ethyl ester group Chemical group 0.000 title claims abstract description 77
- 239000000427 antigen Substances 0.000 title claims abstract description 41
- 102000036639 antigens Human genes 0.000 title claims abstract description 41
- 108091007433 antigens Proteins 0.000 title claims abstract description 41
- 238000002360 preparation method Methods 0.000 title claims abstract description 30
- 150000001875 compounds Chemical class 0.000 claims abstract description 39
- 102000014914 Carrier Proteins Human genes 0.000 claims abstract description 23
- 108010078791 Carrier Proteins Proteins 0.000 claims abstract description 23
- 229940098773 bovine serum albumin Drugs 0.000 claims abstract description 9
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims abstract description 6
- 238000012360 testing method Methods 0.000 claims abstract description 6
- 108010058846 Ovalbumin Proteins 0.000 claims abstract description 5
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims abstract description 5
- 108060003552 hemocyanin Proteins 0.000 claims abstract description 5
- 229940092253 ovalbumin Drugs 0.000 claims abstract description 5
- 102000004190 Enzymes Human genes 0.000 claims abstract description 4
- 108090000790 Enzymes Proteins 0.000 claims abstract description 4
- 238000003018 immunoassay Methods 0.000 claims abstract description 4
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 4
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 4
- 230000001900 immune effect Effects 0.000 claims abstract 2
- 239000000243 solution Substances 0.000 claims description 38
- 238000005859 coupling reaction Methods 0.000 claims description 14
- 238000006243 chemical reaction Methods 0.000 claims description 11
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 10
- 239000000872 buffer Substances 0.000 claims description 10
- 239000007853 buffer solution Substances 0.000 claims description 10
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 9
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 claims description 7
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 6
- 238000000502 dialysis Methods 0.000 claims description 6
- 150000003839 salts Chemical class 0.000 claims description 6
- 239000008363 phosphate buffer Substances 0.000 claims description 5
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 4
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 4
- -1 1- ethyl-(3- dimethyl Aminopropyl) Chemical group 0.000 claims description 3
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 claims description 3
- 229910052794 bromium Inorganic materials 0.000 claims description 3
- WBJINCZRORDGAQ-UHFFFAOYSA-N ethyl formate Chemical compound CCOC=O WBJINCZRORDGAQ-UHFFFAOYSA-N 0.000 claims description 3
- GAWAYYRQGQZKCR-REOHCLBHSA-N (S)-2-chloropropanoic acid Chemical compound C[C@H](Cl)C(O)=O GAWAYYRQGQZKCR-REOHCLBHSA-N 0.000 claims description 2
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims description 2
- YAQLSKVCTLCIIE-UHFFFAOYSA-N 2-bromobutyric acid Chemical compound CCC(Br)C(O)=O YAQLSKVCTLCIIE-UHFFFAOYSA-N 0.000 claims description 2
- MONMFXREYOKQTI-UHFFFAOYSA-N 2-bromopropanoic acid Chemical compound CC(Br)C(O)=O MONMFXREYOKQTI-UHFFFAOYSA-N 0.000 claims description 2
- RVBUZBPJAGZHSQ-UHFFFAOYSA-N 2-chlorobutanoic acid Chemical compound CCC(Cl)C(O)=O RVBUZBPJAGZHSQ-UHFFFAOYSA-N 0.000 claims description 2
- KZLYQYPURWXOEW-UHFFFAOYSA-N 2-iodopropanoic acid Chemical compound CC(I)C(O)=O KZLYQYPURWXOEW-UHFFFAOYSA-N 0.000 claims description 2
- RCUGMVMZCPXQCL-UHFFFAOYSA-N C(CCC)(=O)O.[I] Chemical compound C(CCC)(=O)O.[I] RCUGMVMZCPXQCL-UHFFFAOYSA-N 0.000 claims description 2
- 101710204837 Envelope small membrane protein Proteins 0.000 claims description 2
- 101710145006 Lysis protein Proteins 0.000 claims description 2
- 229910052801 chlorine Inorganic materials 0.000 claims description 2
- 229910052740 iodine Inorganic materials 0.000 claims description 2
- JDNTWHVOXJZDSN-UHFFFAOYSA-N iodoacetic acid Chemical compound OC(=O)CI JDNTWHVOXJZDSN-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
- FDRCDNZGSXJAFP-UHFFFAOYSA-M sodium chloroacetate Chemical compound [Na+].[O-]C(=O)CCl FDRCDNZGSXJAFP-UHFFFAOYSA-M 0.000 claims description 2
- 239000002904 solvent Substances 0.000 claims description 2
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 2
- ZOXJGFHDIHLPTG-UHFFFAOYSA-N Boron Chemical compound [B] ZOXJGFHDIHLPTG-UHFFFAOYSA-N 0.000 claims 1
- 229910052796 boron Inorganic materials 0.000 claims 1
- 239000008055 phosphate buffer solution Substances 0.000 claims 1
- XNGIFLGASWRNHJ-UHFFFAOYSA-L phthalate(2-) Chemical compound [O-]C(=O)C1=CC=CC=C1C([O-])=O XNGIFLGASWRNHJ-UHFFFAOYSA-L 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 16
- 230000015572 biosynthetic process Effects 0.000 abstract description 9
- 150000002148 esters Chemical class 0.000 abstract description 8
- 238000003786 synthesis reaction Methods 0.000 abstract description 7
- 230000000694 effects Effects 0.000 abstract description 6
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 abstract description 3
- 150000001408 amides Chemical class 0.000 abstract 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 29
- 239000007788 liquid Substances 0.000 description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 13
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 10
- 238000010790 dilution Methods 0.000 description 10
- 239000012895 dilution Substances 0.000 description 10
- 239000012086 standard solution Substances 0.000 description 10
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 9
- 238000004140 cleaning Methods 0.000 description 9
- 238000002965 ELISA Methods 0.000 description 8
- 238000001514 detection method Methods 0.000 description 8
- 238000007865 diluting Methods 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 241000699666 Mus <mouse, genus> Species 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 239000011248 coating agent Substances 0.000 description 5
- 238000000576 coating method Methods 0.000 description 5
- 239000011780 sodium chloride Substances 0.000 description 5
- 238000005406 washing Methods 0.000 description 5
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 239000007836 KH2PO4 Substances 0.000 description 4
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 4
- 239000002671 adjuvant Substances 0.000 description 4
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 4
- 229910000397 disodium phosphate Inorganic materials 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 239000002689 soil Substances 0.000 description 4
- 230000000890 antigenic effect Effects 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 238000004945 emulsification Methods 0.000 description 3
- 238000002649 immunization Methods 0.000 description 3
- 230000003053 immunization Effects 0.000 description 3
- 239000002917 insecticide Substances 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- DTDSAWVUFPGDMX-UHFFFAOYSA-N spirodiclofen Chemical compound CCC(C)(C)C(=O)OC1=C(C=2C(=CC(Cl)=CC=2)Cl)C(=O)OC11CCCCC1 DTDSAWVUFPGDMX-UHFFFAOYSA-N 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- 241000607479 Yersinia pestis Species 0.000 description 2
- 210000000683 abdominal cavity Anatomy 0.000 description 2
- 238000010241 blood sampling Methods 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 238000003760 magnetic stirring Methods 0.000 description 2
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 238000002203 pretreatment Methods 0.000 description 2
- 239000012460 protein solution Substances 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 241000676705 Aulacosternum nigrorubrum Species 0.000 description 1
- WKBOTKDWSSQWDR-UHFFFAOYSA-N Bromine atom Chemical compound [Br] WKBOTKDWSSQWDR-UHFFFAOYSA-N 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 241000258937 Hemiptera Species 0.000 description 1
- 206010021703 Indifference Diseases 0.000 description 1
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 241000220317 Rosa Species 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
- 239000005931 Spirotetramat Substances 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Substances BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 description 1
- 210000005252 bulbus oculi Anatomy 0.000 description 1
- 238000001460 carbon-13 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000012864 cross contamination Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 210000001508 eye Anatomy 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- PEQJBOMPGWYIRO-UHFFFAOYSA-N n-ethyl-3,4-dimethoxyaniline Chemical class CCNC1=CC=C(OC)C(OC)=C1 PEQJBOMPGWYIRO-UHFFFAOYSA-N 0.000 description 1
- 238000006303 photolysis reaction Methods 0.000 description 1
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 239000012898 sample dilution Substances 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- CLSVJBIHYWPGQY-GGYDESQDSA-N spirotetramat Chemical compound CCOC(=O)OC1=C(C=2C(=CC=C(C)C=2)C)C(=O)N[C@@]11CC[C@H](OC)CC1 CLSVJBIHYWPGQY-GGYDESQDSA-N 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 150000003457 sulfones Chemical class 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 239000002023 wood Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/77—Ovalbumin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Hematology (AREA)
- Zoology (AREA)
- Toxicology (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention relates to compound preparation and application technical field, in particular to a kind of spiral shell worm ethyl ester antigen and the preparation method and application thereof, general structureX is CH2, one of CO group, n is the integer of 0-6；Protein indicates that carrier protein, the carrier protein are selected from least one of bovine serum albumin, ovalbumin and hemocyanin.It is that spiral shell worm ethyl ester and carrier protein are keyed the conjugate to be formed by amide；The amido bond is that the carboxyl on formula C passes through the amino formation on active ester and carrier protein；Preparation is for the application in the enzyme linked immunological kit of spiral shell worm ethyl ester, the electrochemiluminescent immunoassay kit of spiral shell worm ethyl ester or immune affinity chromatographic column in test sample.This method can quickly and easily obtain spiral shell worm ethyl ester antigen, and synthesis step is concise, synthesis cost is low, and effect is good.
Description
Technical field
The present invention relates to compound preparation and application technical field, in particular to a kind of spiral shell worm ethyl ester antigen and its preparation side
Method and application.
Background technique
Spiral shell worm ethyl ester (spirotetramat) is that the novel internal-suction type tetronic acid class developed by Bayer A.G is killed
Worm agent, belongs to high-efficient broad-spectrum insecticide, can effectively prevent various suckings pest.Spiral shell worm ethyl ester is low toxic insecticide,
It is degradable in the soil, the pests such as wood louse, aleyrodid, scale insect, cotton plant bug can be effectively prevented.Its chemical structural formula is as follows:
In October, 2007, Beyer Co., Ltd describe spiral shell worm ethyl ester, the end of the year 2007, spiral shell in Glasgow, United Kingdom plant protection meeting
Worm ethyl ester is approved the first registration in the whole world in Tunisia.2008, Bayer registered in America & Canada and has listed spiral shell worm
Ethyl ester, then in the registration listing of the country such as Colombia, Mexico, Turkey and New Zealand.On March 16th, 2011, spiral shell worm second
Ester is listed in Chinese formal registration.The sales volume of spiral shell worm ethyl ester rose year by year, from risen less than 0.3 hundred million dollars in 2008
It was 1.40 hundred million dollars by 2012.With a large amount of uses of spiral shell worm ethyl ester, residue problem also causes the concern of people.Although spiral shell
Worm ethyl ester belongs to low toxic insecticide, but for environmental safety evaluation, the volatilization of spiral shell worm ethyl ester difficulty, difficult photodissociation, in soil
In can be degraded, but its catabolite has certain mobility in the soil, it is easy to cross contamination.
Currently, method for detecting residue of the spiral shell worm ethyl ester in agricultural product be mainly high performance liquid chromatography (HPLC) and efficiently
Liquid Chromatography-Tandem Mass Spectrometry (HPLC-MS) is although instrumental method detection has many advantages, such as high sensitivity, accurate and reliable, sample
Pre-treatment is cumbersome, and detection time is long, and cost is big, and technical requirement is high, and expensive equipment, is not suitable for the quick inspection of a large amount of samples
It surveys, does not also have the ability of field quick detection.Compared with instrumental method, immunoassay have quickly, it is easy, in real time,
It is easy to carry out that on-site test, sample pre-treatments simple, high sensitivity, selectivity is strong, is suitable for the advantages that high throughput analysis, and
Testing cost can also be greatly lowered.
Summary of the invention
The present invention in view of the drawbacks of the prior art, provides a kind of spiral shell worm ethyl ester antigen and the preparation method and application thereof, energy
Effective solution the above-mentioned problems of the prior art.
In order to realize the above goal of the invention, the technical solution adopted by the present invention is as follows:
A kind of spiral shell worm ethyl ester antigen, general structure as shown in formula A,
In the formula A, X CH2, one of CO group, n is the integer of 0-6；Protein indicates carrier protein, described
Carrier protein is selected from least one of bovine serum albumin, ovalbumin and hemocyanin.
The preparation method of the spiral shell worm ethyl ester antigen, spiral shell worm ethyl ester antigen are that spiral shell worm ethyl ester and carrier protein are connected by amido bond
Connect the conjugate to be formed；The amido bond is that the carboxyl on formula C passes through the amino formation on active ester and carrier protein.Including
Following steps:
Step 1, spiral shell worm ethyl ester is hydrolyzed to obtain compound shown in formula B；
Step 2, formula B compound is reacted to obtain formula D institute with the compound shown in formula C simultaneously containing amino and carboxyl
Show compound；
Y-X-(CH2)n-COOH
Formula C-structure formula
In the formula C and formula D, X CH2, one of CO group, one of Y Cl, Br and I group, n is 0-6's
Integer；
Step 3, compound shown in formula D and n-hydroxysuccinimide are in dicyclohexylcarbodiimide or 1- ethyl-(3- bis-
Dimethylaminopropyl) coupling reaction is carried out under the conditions of phosphinylidyne diimmonium salt hydrochlorate is existing obtains compound shown in formula E；
In the formula E, X CH2, one of CO group, n is the integer of 0-6；
Step 4, compound shown in formula E and carrier protein are through coupling reaction up to spiral shell worm ethyl ester antigen shown in formula A；The load
Body protein is selected from least one of bovine serum albumin, ovalbumin and hemocyanin.
Preferably, in step 2, compound shown in formula C be selected from monoxone, bromoacetic acid, iodoacetic acid, chloropropionic acid, bromo-propionic acid,
At least one of iodopropionic acid, chloro-butyric acid, bromo-butyric acid, iodine butyric acid.
Preferably, the mol ratio that feeds intake of formula B Formula and C compound is 0.1-10:1, instead in the step 2
The temperature answered is 0~100 DEG C, and the time is 3~48 hours；It is sub- that the solvent of reaction is selected from pyridine, N,N-dimethylformamide, diformazan
At least one of sulfone and tetrahydrofuran.
Preferably, in the step 3, compound shown in formula D, n-hydroxysuccinimide and dicyclohexylcarbodiimide
Molfraction ratio be 1:1~5:1~5；Compound shown in formula D, n-hydroxysuccinimide and 1- ethyl-(3- dimethylamino
Base propyl) phosphinylidyne diimmonium salt hydrochlorate molfraction ratio be 1:1~5:1~5；The temperature of the coupling reaction is 0~50 DEG C,
Time is 4~24 hours.
Preferably, the molfraction ratio of compound shown in formula E and the carrier protein is 5~30:1 in step 3；Institute
The temperature for stating coupling reaction is 0~50 DEG C, and the time is 4~36 hours；The coupling reaction pH value be 5~9 under conditions of into
Row；Compound shown in formula E carries out coupling reaction in the solution of the carrier protein, and the solution of the carrier protein is by described
Carrier protein is added to obtained in buffer solution, and the buffer solution is selected from carbonate buffer solution, phosphate buffer, boric acid
At least one of salt buffer and 4- hydroxyethyl piperazineethanesulfonic acid buffer, the pH value of the buffer is 7.4.
Preferably, after step 4, the step that the method also includes the reaction system of the coupling reaction is dialysed
Suddenly；In the dialysis step, dialyzate used is that the phosphate-buffered that pH value is 4~10, concentration is 0.01~0.2mol/L is molten
Liquid.
Application based on above-mentioned spiral shell worm ethyl ester antigen, comprising: exempt from preparation for the enzyme-linked of spiral shell worm ethyl ester in test sample
Epidemic disease kit, the electrochemiluminescent immunoassay kit of spiral shell worm ethyl ester or the application in immune affinity chromatographic column.The test sample be water body,
Drug, food or soil.
Compared with prior art the present invention has the advantages that spiral shell worm ethyl ester antigen can be obtained quickly and easily, synthesis step
It is rapid it is concise, synthesis cost is low, effect is good.The antibody that the spiral shell worm ethyl ester antigen of the method for the present invention preparation is immunized
Specificity is good, lowest detection limit value is low.The method of preparation spiral shell worm ethyl ester antigen of the invention and the spiral shell worm second obtained by this method
Ester antigen will hold out broad prospects in the tachysynthesis detection application of spiral shell worm ethyl ester.
Detailed description of the invention
Fig. 1 is the spiral shell worm ethyl ester indirect elisa method standard curve that embodiment is established.
Specific embodiment
To make the objectives, technical solutions, and advantages of the present invention more comprehensible, below in conjunction with attached drawing and embodiment is enumerated,
The present invention is described in further details.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Dicyclohexylcarbodiimide (DCC), 1- ethyl-(3- dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDC),
N-hydroxysuccinimide (NHS), Freund's complete adjuvant, incomplete Freund's adjuvant, bovine serum albumin(BSA) and oralbumin are equal
It is purchased from Sigma company, goat anti-mouse igg-HRP is purchased from Jackson company, one pack system TMB developing solution, bromoacetic acid, spiral shell worm ethyl ester
Shanghai Aladdin biochemical technology limited liability company is purchased from Deng remaining conventional reagent.
The preparation of embodiment 1, spiral shell worm ethyl ester-oralbumin (spiral shell worm ethyl ester-OVA) antigen
Synthetic route:
1) synthesis of compound shown in Formulas I
Addition 1.5g spiral shell worm ethyl ester and 0.65g NaOH in 100mL round-bottomed flask, addition 20mL methanol and 4mL water, 25
After stirring 4h at DEG C, reaction solution is poured into 100mL water, adjusting pH value with concentrated hydrochloric acid is 3, is extracted with ethyl acetate (3 × 50mL)
It takes, anhydrous sodium sulfate is dry.Precipitation is rotated, obtained product n-hexane and ethyl acetate (3:1, v/v) are recrystallized, obtained white
Color solid 0.88g, yield 73%.
1H NMR (600MHz, dmso) δ 7.96 (s, 1H), 7.04 (d, J=7.7Hz, 1H), 6.94 (d, J=7.5Hz,
1H), 6.87 (s, 1H), 3.24 (s, 3H), 3.09 (t, J=10.7Hz, 1H), 2.22 (s, 3H), 2.07 (s, 3H), 1.92 (m,
4H), 1.49 (q, J=11.3Hz, 2H), 1.38 (d, J=12.4Hz, 2H)
13C NMR(151MHz,dmso)δ178.56,172.39,134.64,133.93,131.99,131.54,129.71,
127.76,93.27,78.10,59.64,55.34,32.61,28.05,20.97,19.69.
ESI-MS m/z 302.13[M+H]+。
Product warp1H-NMR、13C-NMR and mass spectrum confirmation are compound shown in Formulas I.
2) synthesis of compound shown in formula II
Compound 0.45g shown in Formulas I is added in 50mL there-necked flask, 20mL DMF is added and makes it dissolve, is added dropwise to bromine second
NaOH 0.2g is added in sour 0.3g, is heated with stirring to 100 degree, reaction is for 24 hours.Reaction liquid cooling is gone to room temperature, 100mL is subsequently poured into
In water, after stirring 4h, adjusting pH value with concentrated hydrochloric acid is 3, is extracted with ethyl acetate (3 × 50mL), anhydrous magnesium sulfate is dry.Rotation
Precipitation, column chromatograph to obtain product 0.3g, yield 56%.
1H NMR(600MHz,dmso)δ12.92(s,1H),8.37(s,1H),7.08(m,2H),6.87(s,1H),4.31
(dd,,2H),3.24(m,3H),3.15(s,1H),2.25(s,3H),2.05(s,3H),1.99(m,2H),1.89(m,2H),
1.52(m,4H).
13C NMR(151MHz,dmso)δ171.19,171.12,169.02,135.05,134.46,131.97,131.27,
129.72,128.89,106.95,77.93,66.87,60.15,55.36,33.31,27.89,20.89,19.47.
ESI-MS m/z 360.31[M+H]+,382.28[M+Na]+。
3) coupling reaction
Method (1): spiral shell worm ethyl ester haptens (0.036mmol), NHS shown in the formula II that step 2) obtains are weighed
(0.047mmol) and DCC (0.040mmol) 1mL anhydrous DMF dissolve, and after 25 DEG C of room temperature are stirred to react 6 hours, will react
Liquid is centrifuged 5 minutes under 8000 turns, takes supernatant, obtains compound shown in formula III；
Method (2): spiral shell worm ethyl ester haptens (0.036mmol), NHS shown in the formula II that step 2) obtains are weighed
(0.047mmol) and EDC (0.040mmol) 1mL water dissolve, and after 25 DEG C of room temperature are stirred to react 6 hours, obtain III institute of formula
Show compound；
4) compound shown in formula III obtained by step 3) is slowly added dropwise in carrier protein OVA solution, the carrier protein is molten
Liquid is to be dissolved in phosphate (PBS) buffer that 10mL pH value is 7.4 by 105mg OVA to mix and obtain；III compound of formula and carrier
The molar ratio of albumen is 15:1, is stirred overnight at 4 DEG C.
5) it dialyses: by reaction solution obtained by step 4) with pH value being 7.4, the PBS solution dialysis three that concentration is 0.01mol/L
It, the complete reaction product solution (spiral shell worm ethyl ester-OVA) that will dialyse is diluted to the solution of 1mg/mL, as -40 DEG C freeze to
With.The effect of dialysis is to remove unreacted spiral shell worm ethyl ester haptens and other small molecules, obtains spiral shell worm second shown in formula IV -1
Spiral shell worm ethyl ester antigen shown in the conjugate namely formula A of ester and OVA.
Wherein, which is prepared as follows and obtains: by NaCl, KH2PO4And Na2HPO4·12H2O is with quality
It is more soluble in water than the ratio of 8.0:0.2:2.96, with water constant volume to 1L.
The preparation of embodiment 2, spiral shell worm ethyl ester-bovine serum albumin(BSA) (spiral shell worm ethyl ester-BSA) antigen
1) synthesis and its activation of spiral shell worm ethyl ester haptens shown in formula II and indifference in embodiment 1, details are not described herein.
2) compound shown in formula III obtained by step 3) in embodiment 1 is slowly dropped in carrier protein solution, the carrier
Protein solution is the phosphate buffer (PBS) for being dissolved in 10mL pH value by 157.5mg BSA and being 7.4, III compound of formula and carrier
The molar ratio of albumen is 15:1, is stirred overnight at 4 DEG C.
3) it dialyses: by reaction solution obtained by step 2) with pH value being 7.4, the PBS solution dialysis three that concentration is 0.01mol/L
It, the complete reaction product solution (spiral shell worm ethyl ester-BSA) that will dialyse is diluted to the solution of 1mg/mL, as -40 DEG C freeze to
With.The effect of dialysis is to remove unreacted spiral shell worm ethyl ester haptens and other small molecules, obtains spiral shell worm second shown in formula IV -2
Spiral shell worm ethyl ester antigen shown in the conjugate namely formula A of ester and OVA.
Wherein, which is prepared as follows and obtains: by NaCl, KH2PO4 and Na2HPO412H2O with matter
Amount is more soluble in water than the ratio of 8.0:0.2:2.96, with water constant volume to 1L.
The application of embodiment 3, spiral shell worm ethyl ester-bovine serum albumin(BSA) (spiral shell worm ethyl ester-BSA) antigen
One, antibody is prepared using spiral shell worm ethyl ester-bovine serum albumin(BSA) (spiral shell worm ethyl ester-BSA) antigen
(1) take the Bal b/c small white mouse of 8-10 week old as experimental animal.
(2) fundamental immunity: by spiral shell worm ethyl ester-BSA antigenic solution (the concentration 1mg/ for obtaining having diluted in embodiment 2
ML), isometric Freund's complete adjuvant is added after sterilizing filter filters, emulsification is sufficiently stirred with magnetic stirring apparatus, until drop
Enter indiffusion in water.Abdominal cavity and dorsal sc multi-point injection Bal b/c mouse, injection dosage are used with the comlete antigen emulsified
For 0.1mg emulsify antigen/only.
(3) booster immunization: after fundamental immunity 2 weeks, the spiral shell worm ethyl ester-BSA antigenic solution that the above-mentioned dilution of 1mL is good is taken, then
1mL incomplete Freund's adjuvant is added, emulsification is sufficiently stirred with magnetic stirring apparatus, until instilling indiffusion in water.By what is emulsified
Antigen uses abdominal cavity and dorsal sc multi-point injection Bal b/c mouse, and the injection dosage of every mouse is that 0.1mg emulsification dilution is anti-
Former (the Bal b/C Mice Body of 8 week old weighs about 23-25g).
Booster immunization is immune primary every 15 days, since third time booster immunization, it is immune every time after the 3-5 days, from small
The blood sampling of rathole socket of the eye measures antibody titer, and coating antigen is that 1mg/mL spiral shell worm ethyl ester-OVA dilutes 500 times of use, is greater than 1 to potency:
After 8000 (potency be defined as zero hole colour developing value be 1 when, the extension rate of serum), eyeball excise blood sampling, it is small that blood is stored at room temperature 1
Shi Hou stands 2 hours in 4 DEG C of refrigerators, and then 8000r/min centrifugation after five minutes, isolates antiserum in centrifuge,
Obtain spiral shell worm ethyl ester-BSA antibody.For following each experiments.
Two, antibody effects detect
Various buffers used in following experiments are as follows:
(1) it is coated with buffer: the carbonate buffer solution of 0.05M, pH 9.6；
(2) 4.0g NaCl, 0.1g KH phosphate buffer PBS (pH 7.4): are weighed2PO4、1.48g Na2HPO4·
12H2O distilled water constant volume to 500mL, concentration 0.01M, pH are 7.4 phosphate buffers；
(3) sample diluting liquid PBSTG: be 0.1M, pH by 0.5mL polysorbas20,0.5g gelatin and 500mL concentration being 7.4
PBS buffer solution is mixed to get；
(4) stop buffer: the aqueous sulfuric acid of 2.0M；
(5) cleaning solution: by NaCl, KH2PO4、Na2HPO4·12H2O, Tween-20 and water composition；NaCl is in cleaning solution
Concentration be 8.0g/L, KH2PO4Concentration in cleaning solution is 0.2g/L, Na2HPO4·12H2Concentration of the O in cleaning solution is
2.96g/L, the Tween-20 volumn concentration in cleaning solution are 1:1000.
(1) antibody inhibition experiments
1, the preparation of spiral shell worm ethyl ester-OVA envelope antigen solution
After the spiral shell worm ethyl ester-OVA antigen of 1mg/mL after dilution prepared by above-described embodiment 1 thaws completely, with coating
Buffer carries out gradient dilution by 1:500,1:1000,1:2000,1:4000, obtains the packet of the spiral shell worm ethyl ester-OVA of various concentration
By antigenic solution.
2, the preparation of spiral shell worm ethyl ester standard solution
(1) 10mg spiral shell worm ethyl ester standard specimen is weighed, is completely dissolved in 10mL anhydrous methanol to get 1.0mg/mL spiral shell worm second is arrived
Ester standard solution；
(2) the 1.0mg/mL spiral shell worm ethyl ester standard solution of above-mentioned steps (1) is made into concentration with sample diluting liquid is
The spiral shell worm ethyl ester standard solution of 1000ng/mL.
3, the preparation of spiral shell worm ethyl ester-BSA antiserum dilution
Spiral shell worm ethyl ester-BSA antibody sample diluting liquid prepared by above-mentioned steps one by 1:1000,1:2000,1:4000,
1:8000 carries out gradient dilution, obtains spiral shell worm ethyl ester-BSA antiserum dilution.
4, the gridiron pattern experiment of antigen, antibody
Coating: it is molten that the spiral shell worm ethyl ester-OVA envelope antigen that 100 μ L steps 1 are prepared is added in every hole in 96 hole elisa Plates
Liquid, 37 DEG C are coated with 3 hours, are washed 4 times with cleaning solution.
Closing: weighing 5g skimmed milk power, is completely dissolved in 100mL sample diluting liquid to get to 5% confining liquid.?
150 μ L confining liquids are added in every hole in 96 hole elisa Plates, and 1h is closed in 37 DEG C of wet box, abandon confining liquid, wash 3 times.
Competition: the every hole in zero hole adds 50 μ l sample diluting liquids, inhibits the every hole in hole that the spiral shell worm second that 50 μ l steps 2 are prepared is added
Ester standard solution.Spiral shell worm ethyl ester-BSA antiserum the dilution that above-mentioned steps 3 are obtained is (from 1 × 103Again to 8 × 103Times) plus
Enter in ELISA Plate (50 hole μ l/), sets in wet box 30min under the conditions of 37 DEG C, board-washing 4 times.
Add ELIAS secondary antibody: by sheep anti mouse ELIAS secondary antibody (IgG-HRP, Jackson company, catalog number 79556)
(0.1mg/mL) dilutes 1000 times, and dilution is 0.01M, and the PBSTG that pH is 7.4, every hole adds 100 μ L, sets 37 DEG C of items in wet box
30min under part, board-washing 4 times.
Colour developing: one pack system TMB developing solution is added in ELISA Plate, every 100 μ l of hole.It is protected from light colour developing 15min.
Terminate: 50 μ L stop buffers are added in every hole, with the OD value for measuring each hole at microplate reader 450nm.
The serum diluting multiple that the definition of potency is zero hole OD value when being 1.
The results are shown in Table 1.
The serum titer detection of table 1, anti-spiral shell worm ethyl ester mouse
(inhibition of one pack system TMB developing solution color development at room temperature 15min, 1000ng standard specimen)
Note: I indicates the inhibition hole in ELISA Plate, and C indicates the control wells in ELISA Plate.
As a result illustrate that the spiral shell worm ethyl ester-BSA of the preparation of above-described embodiment 2 can be used as immunogene and prepare detection spiral shell worm ethyl ester
Antibody.
(2) foundation of spiral shell worm ethyl ester standard curve
The spiral shell worm ethyl ester standard solution of above-mentioned preparation is diluted to following different concentration respectively with sample diluting liquid:
1000ng/mL、500ng/mL、250ng/mL、125ng/mL、62.5ng/mL、31.25ng/mL、15.6ng/mL。
(1) coating of coating antigen: enzyme is added to after the spiral shell worm ethyl ester-OVA antigen of above-mentioned preparation is diluted according to 1:8000
In target, every 100 μ L of hole, 37 DEG C are incubated 3 hours；The solution in ELISA Plate is removed, with cleaning solution board-washing 4 times, drying；
(2) the spiral shell worm ethyl ester standard solution (experiment of above-mentioned various concentration is separately added into the ELISA Plate of step (1)
Hole), every 50 μ L of hole does not add spiral shell worm ethyl ester standard solution and 50 μ L sample dilutions is added in control wells；
(3) spiral shell worm ethyl ester-BSA antiserum that extension rate is 1:8000 is added into above-mentioned experimental port and control wells respectively
Dilution, every 50 μ L of hole；37 DEG C incubate 30 minutes；The solution in ELISA Plate is outwelled, with cleaning solution board-washing 4 times, drying；
(4) IgG-HRP that 100 μ L extension rates are 1:1000 is separately added into experimental port and control wells, and (Jackson is public
Department, catalog number 79556) (0.1mg/mL), 37 DEG C incubate 30 minutes；With cleaning solution board-washing 4 times, outwell in ELISA Plate
Solution, drying；
(5) 100 μ L one pack system TMB developing solutions are separately added into experimental port and control wells, be protected from light colour developing 15min, then to
The sulfuric acid solution that 50 μ L 2.0M are added in every hole terminates reaction；
(6) light absorption value is measured at 450 nm；
(7) standard curve is drawn: using the spiral shell worm ethyl ester standard solution of various concentration (ng/mL) as X-axis, with absorbance
Value is used as Y-axis, draws canonical plotting.
Experiment sets 3 repetitions, takes the average value of experimental result three times, obtained canonical plotting is as shown in Figure 1.As a result table
It is bright, sensitivity (IC50) it is 60.0ng/mL, detection range is 17ng/mL-232ng/mL.Illustrate prepared by above-described embodiment 1
Spiral shell worm ethyl ester-BSA has good effect as the antibody that mice immunized with antigen obtains.
(3) antibody specificity detects
1, the preparation of spiral shell worm ethyl ester analog standard solution
The preparation of spiral shell worm ethyl ester analog standard items
Referring to the preparation method of spiral shell worm ethyl ester standard items in step (1), Envidor, spiral shell worm ethyl ester metabolite are prepared
(BYI08330-enol-glucoside, BYI08330-mono-hydroxy, BYI08330-enol and BYI08330-
Ketohydroxy standard sample).
With sample diluting liquid by above-mentioned Envidor, spiral shell worm ethyl ester metabolite (BYI08330-enol-glucoside,
BYI08330-mono-hydroxy, BYI08330-enol and BYI08330-ketohydroxy) it is diluted to following concentration respectively:
20000ng/mL、10000ng/mL、5000ng/mL、2500ng/mL、1250ng/mL、625ng/mL、312.5ng/mL。
2, standard curve, concentration IC in measurement inhibition are established50(inhibiting rate reaches 50% standard specimen concentration value).
The method for building up of standard curve is identical as the method for building up of above-mentioned spiral shell worm ethyl ester standard curve.
Cross reacting rate (%)=(spiral shell worm ethyl ester IC50)/(spiral shell worm ethyl ester similar compound IC50) × 100%.
Experiment sets 3 repetitions, takes the average value of experimental result three times, the results are shown in Table 2.
Table 2, by spiral shell worm ethyl ester-BSA preparation antibody specific detection
| Analyte | IC50(ng/mL) | Cross reacting rate (%) |
| Spiral shell worm ethyl ester | 60.0 | 100 |
| BYI08330-enol | 82.3 | 72.9 |
| BYI08330-enol-glucoside | 393 | 15.3 |
| BYI08330-mono-hydroxy | >20000 | - |
| BYI08330-ketohydroxy | >20000 | - |
| Envidor | >20000 | - |
The result shows that the above-mentioned antibody being prepared by spiral shell worm ethyl ester-BSA can not only identify spiral shell worm ethyl ester, while can know
The metabolin BYI08330-enol and BYI08330-enol-glucoside of other spiral shell worm ethyl ester, but handed over its analog Envidor
Reactivity very little is pitched, antibody prepared by explanation spiral shell worm ethyl ester-BSA has specificity well to spiral shell worm ethyl ester.
Those of ordinary skill in the art will understand that the embodiments described herein, which is to help reader, understands this hair
Bright implementation method, it should be understood that protection scope of the present invention is not limited to such specific embodiments and embodiments.Ability
The those of ordinary skill in domain disclosed the technical disclosures can make its various for not departing from essence of the invention according to the present invention
Its various specific variations and combinations, these variations and combinations are still within the scope of the present invention.
Claims (8)
1. a kind of spiral shell worm ethyl ester antigen, which is characterized in that general structure as shown in formula A,
In the formula A, X CH2, one of CO group, n is the integer of 0-6；Protein indicates carrier protein, the carrier
Albumen is a kind of in bovine serum albumin, ovalbumin and hemocyanin.
2. a kind of preparation method of spiral shell worm ethyl ester antigen according to claim 1, which comprises the steps of:
Step 1, spiral shell worm ethyl ester is hydrolyzed to obtain compound shown in formula B；
Step 2, formula B compound is reacted to obtain compound shown in formula D with compound shown in formula C；
Y-X-(CH2)n-COOH
Formula C-structure formula
In the formula C and formula D, X CH2, one of CO group, one of Y Cl, Br and I group, n is the integer of 0-6；
Step 3, compound shown in formula D and n-hydroxysuccinimide are in dicyclohexylcarbodiimide or 1- ethyl-(3- dimethyl
Aminopropyl) coupling reaction is carried out under the conditions of phosphinylidyne diimmonium salt hydrochlorate is existing obtains compound shown in formula E；
In the formula E, X CH2, one of CO group, n is the integer of 0-6；
Step 4, compound shown in formula E and carrier protein are through coupling reaction up to spiral shell worm ethyl ester antigen shown in formula A；The carrier egg
It is white a kind of in bovine serum albumin, ovalbumin and hemocyanin.
3. a kind of preparation method of spiral shell worm ethyl ester antigen according to claim 2, which is characterized in that in step 2, formula C institute
Show compound in monoxone, bromoacetic acid, iodoacetic acid, chloropropionic acid, bromo-propionic acid, iodopropionic acid, chloro-butyric acid, bromo-butyric acid, iodine butyric acid
It is at least one.
4. a kind of preparation method of spiral shell worm ethyl ester antigen according to claim 2, which is characterized in that in the step 2, formula
The mol ratio that feeds intake of B Formula and C compound is 0.1-10:1；The temperature of reaction is 0~100 DEG C, and the time is 3~48 small
When；The solvent of reaction is selected from least one of pyridine, N,N-dimethylformamide, dimethyl sulfoxide and tetrahydrofuran.
5. a kind of preparation method of spiral shell worm ethyl ester antigen according to claim 2, which is characterized in that in the step 3, formula
The molfraction of compound, n-hydroxysuccinimide shown in D and dicyclohexylcarbodiimide ratio is 1:1~5:1~5；
Compound shown in formula D, n-hydroxysuccinimide and 1- ethyl-(3- dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate
Molfraction ratio be 1:1~5:1~5；
The temperature of the coupling reaction is 0~50 DEG C, and the time is 4~24 hours.
6. a kind of preparation method of spiral shell worm ethyl ester antigen according to claim 2, which is characterized in that in step 4, formula E institute
The molfraction ratio for showing compound and the carrier protein is 5~30:1；
The temperature of the coupling reaction is 0~50 DEG C, and the time is 4~36 hours；The item that the coupling reaction is 5~9 in pH value
It is carried out under part；
Compound shown in formula E carries out coupling reaction in the solution of the carrier protein, and the solution of the carrier protein is by institute
It states carrier protein to be added to obtained in buffer solution, the buffer solution is selected from carbonate buffer solution, phosphate buffer, boron
At least one of phthalate buffer and 4- hydroxyethyl piperazineethanesulfonic acid buffer, the pH value of the buffer is 7.4.
7. a kind of preparation method of spiral shell worm ethyl ester antigen according to claim 2, which is characterized in that further include by the idol
The reaction system of connection reaction is dialysed；In the dialysis step, dialyzate used be pH value be 4~10, concentration be 0.01~
The phosphate buffer solution of 0.2mol/L.
8. a kind of application of spiral shell worm ethyl ester antigen according to claim 1, which is characterized in that be used to prepare in test sample
Application in the enzyme linked immunological kit of spiral shell worm ethyl ester, the electrochemiluminescent immunoassay kit of spiral shell worm ethyl ester or immune affinity chromatographic column.
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