CN107118159A - Cistofuran metabolite AHD derivatizations haptens, the preparation method and applications of artificial antigen - Google Patents
Cistofuran metabolite AHD derivatizations haptens, the preparation method and applications of artificial antigen Download PDFInfo
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- CN107118159A CN107118159A CN201710022433.0A CN201710022433A CN107118159A CN 107118159 A CN107118159 A CN 107118159A CN 201710022433 A CN201710022433 A CN 201710022433A CN 107118159 A CN107118159 A CN 107118159A
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- haptens
- cistofuran metabolite
- artificial antigen
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- NXFQHRVNIOXGAQ-YCRREMRBSA-N nitrofurantoin Chemical class O1C([N+](=O)[O-])=CC=C1\C=N\N1C(=O)NC(=O)C1 NXFQHRVNIOXGAQ-YCRREMRBSA-N 0.000 title claims abstract description 70
- 239000000427 antigen Substances 0.000 title claims abstract description 32
- 102000036639 antigens Human genes 0.000 title claims abstract description 32
- 108091007433 antigens Proteins 0.000 title claims abstract description 32
- 238000002360 preparation method Methods 0.000 title claims abstract description 20
- 238000001212 derivatisation Methods 0.000 title abstract description 7
- 238000001514 detection method Methods 0.000 claims abstract description 23
- 102000014914 Carrier Proteins Human genes 0.000 claims abstract description 20
- 108010078791 Carrier Proteins Proteins 0.000 claims abstract description 20
- 238000006243 chemical reaction Methods 0.000 claims abstract description 11
- 150000001875 compounds Chemical class 0.000 claims description 25
- 239000000243 solution Substances 0.000 claims description 24
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 20
- WMFOQBRAJBCJND-UHFFFAOYSA-M Lithium hydroxide Chemical compound [Li+].[OH-] WMFOQBRAJBCJND-UHFFFAOYSA-M 0.000 claims description 15
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 15
- 210000004027 cell Anatomy 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
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 12
- 238000002965 ELISA Methods 0.000 claims description 10
- 108010058846 Ovalbumin Proteins 0.000 claims description 10
- 230000003053 immunization Effects 0.000 claims description 10
- 229940092253 ovalbumin Drugs 0.000 claims description 10
- 241000699666 Mus <mouse, genus> Species 0.000 claims description 9
- 150000001718 carbodiimides Chemical class 0.000 claims description 9
- 238000002649 immunization Methods 0.000 claims description 8
- 239000012460 protein solution Substances 0.000 claims description 8
- 210000002966 serum Anatomy 0.000 claims description 7
- 206010003445 Ascites Diseases 0.000 claims description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
- CGIHPACLZJDCBQ-UHFFFAOYSA-N acibenzolar Chemical compound SC(=O)C1=CC=CC2=C1SN=N2 CGIHPACLZJDCBQ-UHFFFAOYSA-N 0.000 claims description 6
- 210000004408 hybridoma Anatomy 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 6
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 5
- 239000000126 substance Substances 0.000 claims description 5
- 238000012360 testing method Methods 0.000 claims description 5
- 238000013019 agitation Methods 0.000 claims description 4
- 238000010790 dilution Methods 0.000 claims description 4
- 239000012895 dilution Substances 0.000 claims description 4
- 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 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 108010088751 Albumins Proteins 0.000 claims description 3
- 102000009027 Albumins Human genes 0.000 claims description 3
- 241000581650 Ivesia Species 0.000 claims description 3
- 239000002671 adjuvant Substances 0.000 claims description 3
- 230000003321 amplification Effects 0.000 claims description 3
- 238000003556 assay Methods 0.000 claims description 3
- 210000004369 blood Anatomy 0.000 claims description 3
- 239000008280 blood Substances 0.000 claims description 3
- 230000007910 cell fusion Effects 0.000 claims description 3
- 239000003636 conditioned culture medium Substances 0.000 claims description 3
- 150000002240 furans Chemical class 0.000 claims description 3
- 108060003552 hemocyanin Proteins 0.000 claims description 3
- 230000002163 immunogen Effects 0.000 claims description 3
- 238000007689 inspection Methods 0.000 claims description 3
- 239000002609 medium Substances 0.000 claims description 3
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 3
- 238000012545 processing Methods 0.000 claims description 3
- 238000007920 subcutaneous administration Methods 0.000 claims description 3
- 241000699670 Mus sp. Species 0.000 claims description 2
- 238000004458 analytical method Methods 0.000 claims description 2
- 230000021615 conjugation Effects 0.000 claims description 2
- 238000001914 filtration Methods 0.000 claims description 2
- 230000014759 maintenance of location Effects 0.000 claims description 2
- 230000028327 secretion Effects 0.000 claims description 2
- 210000000952 spleen Anatomy 0.000 claims description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 claims 1
- KCCZJTJZWUNGHI-UHFFFAOYSA-N octanoic acid;sulfuric acid Chemical compound OS(O)(=O)=O.CCCCCCCC(O)=O KCCZJTJZWUNGHI-UHFFFAOYSA-N 0.000 claims 1
- 238000001556 precipitation Methods 0.000 claims 1
- 102000004169 proteins and genes Human genes 0.000 claims 1
- 108090000623 proteins and genes Proteins 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 6
- 238000011017 operating method Methods 0.000 abstract 1
- 241001465754 Metazoa Species 0.000 description 6
- 238000000576 coating method Methods 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 239000002953 phosphate buffered saline Substances 0.000 description 6
- 239000011248 coating agent Substances 0.000 description 5
- 0 CCOC(CNCCOC(C)C(CCC1)C=CCCCC(*C=O)C1[N+]([O-])=O)=O Chemical compound CCOC(CNCCOC(C)C(CCC1)C=CCCCC(*C=O)C1[N+]([O-])=O)=O 0.000 description 4
- 230000007812 deficiency Effects 0.000 description 4
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- PLHJDBGFXBMTGZ-WEVVVXLNSA-N furazolidone Chemical compound O1C([N+](=O)[O-])=CC=C1\C=N\N1C(=O)OCC1 PLHJDBGFXBMTGZ-WEVVVXLNSA-N 0.000 description 4
- 229960001625 furazolidone Drugs 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 230000005847 immunogenicity Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 2
- 241000251468 Actinopterygii Species 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 2
- JCXJVPUVTGWSNB-UHFFFAOYSA-N Nitrogen dioxide Chemical class O=[N]=O JCXJVPUVTGWSNB-UHFFFAOYSA-N 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 2
- MUUGCDGGIVCSDT-UHFFFAOYSA-N [NH4+].[NH4+].[O-]S([O-])(=O)=O.CCCCCCCC(O)=O Chemical compound [NH4+].[NH4+].[O-]S([O-])(=O)=O.CCCCCCCC(O)=O MUUGCDGGIVCSDT-UHFFFAOYSA-N 0.000 description 2
- 238000003453 ammonium sulfate precipitation method Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 239000000385 dialysis solution Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000004945 emulsification Methods 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 229940005654 nitrite ion Drugs 0.000 description 2
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 239000008213 purified water Substances 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- FUBFWTUFPGFHOJ-UHFFFAOYSA-N 2-nitrofuran Chemical group [O-][N+](=O)C1=CC=CO1 FUBFWTUFPGFHOJ-UHFFFAOYSA-N 0.000 description 1
- YVQVOQKFMFRVGR-VGOFMYFVSA-N 5-(morpholin-4-ylmethyl)-3-[(e)-(5-nitrofuran-2-yl)methylideneamino]-1,3-oxazolidin-2-one Chemical compound O1C([N+](=O)[O-])=CC=C1\C=N\N1C(=O)OC(CN2CCOCC2)C1 YVQVOQKFMFRVGR-VGOFMYFVSA-N 0.000 description 1
- 241000143060 Americamysis bahia Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- PLZXYQKNBVQROH-UHFFFAOYSA-N CC(C)(CCC(O)=O)Oc(cc1)cc(C=O)c1[N+]([O-])=O Chemical compound CC(C)(CCC(O)=O)Oc(cc1)cc(C=O)c1[N+]([O-])=O PLZXYQKNBVQROH-UHFFFAOYSA-N 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 208000004232 Enteritis Diseases 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241001597008 Nomeidae Species 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- 208000031320 Teratogenesis Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- CNEWFSOPZHKDHX-UHFFFAOYSA-N [O-][N+](C(C=CC(C1)OCCCC(O)=O)=C1C=O)=O Chemical compound [O-][N+](C(C=CC(C1)OCCCC(O)=O)=C1C=O)=O CNEWFSOPZHKDHX-UHFFFAOYSA-N 0.000 description 1
- WWWZHQWLSLRDQP-VIZOYTHASA-N [O-][N+](c(cc1)c(/C=N/N(CC(N2)=O)C2=O)cc1OCCCC(O)=O)=O Chemical compound [O-][N+](c(cc1)c(/C=N/N(CC(N2)=O)C2=O)cc1OCCCC(O)=O)=O WWWZHQWLSLRDQP-VIZOYTHASA-N 0.000 description 1
- 125000002252 acyl group Chemical group 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 231100000315 carcinogenic Toxicity 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical class P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000009313 farming Methods 0.000 description 1
- 235000021393 food security Nutrition 0.000 description 1
- 229950000337 furaltadone Drugs 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 1
- 239000007952 growth promoter Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 230000000984 immunochemical effect Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 239000010985 leather Substances 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 239000006210 lotion Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- IAIWVQXQOWNYOU-FPYGCLRLSA-N nitrofural Chemical compound NC(=O)N\N=C\C1=CC=C([N+]([O-])=O)O1 IAIWVQXQOWNYOU-FPYGCLRLSA-N 0.000 description 1
- 229960001907 nitrofurazone Drugs 0.000 description 1
- 230000001473 noxious effect Effects 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- GOLXNESZZPUPJE-UHFFFAOYSA-N spiromesifen Chemical compound CC1=CC(C)=CC(C)=C1C(C(O1)=O)=C(OC(=O)CC(C)(C)C)C11CCCC1 GOLXNESZZPUPJE-UHFFFAOYSA-N 0.000 description 1
- 210000004988 splenocyte Anatomy 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- -1 sulphonyl amine Chemical class 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
- C07D233/54—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
- C07D233/66—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members 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 ring carbon atoms
- C07D233/72—Two oxygen atoms, e.g. hydantoin
- C07D233/80—Two oxygen atoms, e.g. hydantoin with hetero atoms or acyl radicals directly attached to ring nitrogen atoms
-
- 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
-
- 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
-
- 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
-
- 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Engineering & Computer Science (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a kind of Cistofuran metabolite AHD derivatizations haptens, the preparation method and applications of artificial antigen, the active arm that described haptens is introduced, the complete feature structure for remaining AHD derivatives, on its cloud density without influence, again have can be with carrier protein couplet active group, reaction condition is gentle, and operating procedure is simple；The artificial antigen of preparation can and antibody specificity combination, the features such as with high-titer, high sensitivity, high precision, can in quick detection various product Cistofuran metabolite residual.
Description
Technical field
The present invention relates to immunochemical technique field, more particularly to a kind of Cistofuran metabolite AHD derivatizations haptens,
The preparation method and applications of artificial antigen.
Background technology
Furans are the antibiotic that an artificial synthesized class has 5- nitrofuran ring structures, to gram-positive bacteria, leather
Lan Shi negative bacteriums, some fungi all have antibacterial effect.Because cheap, it is widely used in livestock and poultry and fish culture,
For treating the enteritis as caused by Salmonella, Escherichia coli, global-worm illness etc., and frequently as farming animals and aquatic products fish
The growth promoter of shrimps is inserted in feed.Itrofurans mainly includes：Furaltadone, nitrofurazone, furantoin and
Furazolidone.But in food security, a large amount of or continuous of furantoin are used not only to the toxic effect of cultivated animals, and
With carcinogenic, teratogenesis, mutagenesis, its metabolin also has malicious influences to human health.Therefore, many national explicit orders
Forbid using furantoin during letting animals feed derived food, and define the inspection and quarantine mark in animal derived food
It is accurate.
Mainly there are traditional detection method, such as spectrophotometric currently used for the method for detection furantoin and its metabolin
Method, high performance liquid chromatography and its GC-MS etc., although above method can with accurate quantification, because equipment and instrument is expensive,
Detection time is long, and needs professional to operate, therefore can not realize Site Detection truly；And immunology detection skill
Art, the characteristics of the method has high specific and high selectivity is highly suitable for the separation or detection of complex matrices trace components,
Interpenetrated with interdisciplinary, its detection method emerges in an endless stream, and application also expands day by day.With traditional detection method phase
Than, immunology detection technology have economic, quick, technical essential it is low, it is easy to operate the features such as.Immunoassay detection technique is near
A kind of new quick and precisely detection method developed over year in the field such as environment and food inspection, has been increasingly becoming the world
One of main method of the quick selective mechanisms of various countries' noxious residual chemicals, also provides new way for the detection of furantoin
Footpath.
Although Cistofuran metabolite active group in itself, because its molecular weight is smaller, space structure is not protruded,
There is the low great deficiency of sensitivity in the artificial antigen directly prepared, it is impossible to meet the demand of existing market.Therefore it need to first pass through
Chemical method designs its haptens, is further combined with carrier protein and obtains the comlete antigen with immunogenicity.
In patent announcement number in the patent document of " CN200910085648.2 ", its object detected is furazolidone
Bulk drug, but AHD can be metabolized as after entering due to furazolidone in animal body, and AHD has huge injury to human body, therefore
Only there is significant deficiency in detection furazolidone bulk drug, it is impossible to meet market demands.
In patent announcement number for " CN200810019047.7, CN201210049810.7, CN201210098138.0's "
In patent document, its haptens structure designed is traditional 4-CP derivant structures, can not retain its object to be measured
Nitro end so that the antibody that haptens, antigen are prepared has the low deficiency of poor specificity, affinity.
In patent announcement number in the patent document of " CN201410593519.5 ", its haptens structure designed can not be protected
Nitro end is stayed, and between haptens and AHD is connected by sulphonyl amine key, is not conjugated with phenyl ring, makes the electron cloud of haptens
Density and the object difference of detection are big, reduce sensitivity.
The content of the invention
The technical problem to be solved in the present invention is to overcome traditional AHD haptens can not completely retain competitor feature structure
Defect there is provided a kind of Cistofuran metabolite AHD derivatizations haptens, the preparation method and applications of artificial antigen, it can
Retain the haptens at competitor nitro end, artificial antigen improves the specificity of competitor.
To reach above-mentioned purpose, offer technology of the present invention is as follows：A kind of haptens of Cistofuran metabolite, is following knot
Structure formula III：
The present invention solves another technical scheme for being used of the technical problem, a kind of haptens of Cistofuran metabolite
Preparation method, step is as follows：
(a) chemical compounds I is dissolved in ethanol, resulting solution is adjusted pH value by adding 6mol/L lithium hydroxide solution
To 10-11, reaction obtains compound ii；
The structural formula of the chemical compounds I is as follows：
The structural formula of the compound ii is as follows：
(b) compound ii and Cistofuran metabolite are reacted under the conditions of methanol, both the furantoin generation
Thank to the haptens of thing；It is following structural formula III：
The present invention solves another technical scheme for being used of the technical problem, a kind of haptens of Cistofuran metabolite
Preparation method, step is as follows：
Chemical compounds I is dissolved in ethanol by (a '), and resulting solution is adjusted pH value by adding 6mol/L lithium hydroxide solution
To 10-12,15~26h is reacted at room temperature, purified water is added, resulting solution is adjusted by 1M watery hydrochloric acid pH value being adjusted to 4-5, filtered
Compound ii is obtained, the process of the chemical compounds I reaction generation compound ii is as follows：
The compound ii is dissolved in methanol by (b '), plus 1.2-1.5 molar equivalents Cistofuran metabolite AHD,
React 2h in 60~70 DEG C, reaction finishes, filter, both the Cistofuran metabolite haptens, the knot of the haptens
Structure formula III and synthetic route are as follows：
Another technical scheme that the present invention solution technical problem is used is that a kind of the artificial of Cistofuran metabolite resists
The conjugate that original is carrier protein and the hapten conjugation of described Cistofuran metabolite is obtained, its structural formula IV is：
Another technical scheme that the present invention solution technical problem is used is that a kind of the artificial of Cistofuran metabolite resists
Former preparation method, step is as follows：
(1) haptens of the Cistofuran metabolite is taken, is dissolved in dimethylformamide (DMF), after stirring fully,
Carbodiimide (EDC) and n-hydroxysuccinimide (NHS) are added, the haptens, carbodiimide and N- hydroxysuccinimidyls acyl are sub-
The weight ratio of amine is 5：4：3,4-8h is stirred at room temperature, produces haptens Acibenzolar；
(2) carrier protein is weighed, the molar equivalent ratio of the carrier protein and the haptens is 30：1, make the carrier
Albumen is substantially dissolved in 0.01mol/L PBS solution, is formed carrier protein solution, is under agitation activated the haptens
Ester is slowly added dropwise into the load protein solution dropwise, and 16-24h is stirred at room temperature；
(3) the PBS solution room temperature by step (2) resulting solution in 0.01mol/L is dialysed 3 days, and 3 dialyzates are changed daily；
(4) dispense, saved backup in 4 DEG C.
Wherein, the carrier protein is bovine serum albumin(BSA) (BSA), ovalbumin (OVA), human serum albumins (HAS)
Or any one in hemocyanin (KLH).
The present invention solves another technical scheme for being used of the technical problem, the monoclonal of consistent Cistofuran metabolite
The preparation method of antibody, step is as follows：
(i) using the artificial antigen of Cistofuran metabolite as immunogene, after the emulsification of isometric Freund's adjuvant, it is immunized
BALB/C mice, every mouse immunizing dose is 50~100 μ g, and immunization interval 3 weeks is immunized 3 times；
(ii) adopt step (i) processing after mouse tail venous blood detection serum titer, such as antibody titer not up to 60000,
Booster immunization is needed, after antibody titer is no longer raised, subcutaneous booster immunization is carried out with 100 μ g immunogenes, the spleen of mouse is taken after 5 days
Cell and SP20 cell fusions；
(iii) cell after merging is screened in HAT culture mediums, and being substituted for HAT culture mediums after 5 days with complete medium enters
Row culture；
(iv) cell conditioned medium after step (iii) processing is detected with ELISA, by the hole that testing result is strong positive
Inner cell carries out limiting dilution assay clone's culture, after being detected through 3 time cloning cultures, and the hole inner cell being positive is that secretion is single
The hybridoma of clonal antibody；
(v) by after hybridoma amplification culture, be seeded to mouse peritoneal, produce the ascites containing antibody, with octanoic acid-
Ammonium sulfate precipitation method purifies ascites, produces.
The preparation method can obtain high-purity, the monoclonal antibody of high specific, and the monoclonal antibody is in detection water
Applied in product in Cistofuran metabolite retention analysis.
The present invention solves another technical scheme for being used of the technical problem, the artificial antigen of Cistofuran metabolite and
Application of the monoclonal antibody of Cistofuran metabolite in detection Cistofuran metabolite AHD, by described furantoin generation
The artificial antigen for thanking to thing is coated on ELISA Plate as coating antigen, and the monoclonal antibody of described Cistofuran metabolite is added
In microwell plate；The content of Cistofuran metabolite AHD in sample to be checked is detected using competition law.
Compared with prior art, with following good effect：The AHD haptens structure of traditional scheme can not retain to be measured
The nitro end of thing so that the antibody that haptens, antigen are prepared has the low deficiency of poor specificity, affinity.The present invention
In provide a kind of new A HD derivative haptens, and based on the haptens prepare a kind of artificial antigen, elaborate both
Preparation method and application.Its thinking is the derivative that AHD derivatizations are obtained to active group and double bond rigid support arm
Thing, while the also complete feature structure for remaining AHD derivatives, on its cloud density without influence, while in order to strengthen half
The immunogenicity of antigen prepares high-quality antibody, and the antibody can be with specific recognition AHD derivatives.
The thinking has the beneficial effect that：(1) the double bond arm acted on rigid support is introduced so that AHD knot
Structure feature is remarkably reinforced, beneficial to the good presentation of determinant；(2) spy for remaining AHD derivatives of the haptens prepared completely
Structure is levied, on its cloud density without influence so that the immunogenicity of small haptens is remarkably reinforced, and is conducive to stimulating animal
Immune response produces the higher antibody of specific stronger, sensitivity；(3) quick determination method for the AHD that the present invention is provided is minimum
Test limit is up to 0.05ng/g.
The invention will be further described with reference to the accompanying drawings and examples.
Brief description of the drawings
Fig. 1 is the synthetic route of the haptens of the Cistofuran metabolite of the present invention.
Fig. 2 is the ESI-MS collection of illustrative plates of compound ii in the present embodiment 1.
Fig. 3 is the hydrogen nuclear magnetic resonance spectrogram spectrum of compound ii in the present embodiment 1.
Fig. 4 is the ESI-MS collection of illustrative plates of the haptens of Cistofuran metabolite in the present embodiment 1.
Fig. 5 is the hydrogen nuclear magnetic resonance spectrogram spectrum of the haptens of Cistofuran metabolite in the present embodiment 1.
Fig. 6 is the antibody foundation that the artificial antigen of Cistofuran metabolite in the present embodiment 4 prepares for immunogene to AHD
The suppression curve of derivative.
Embodiment
Embodiment 1
As Figure 1-5, the Cistofuran metabolite AHD derivatization haptens of the present embodiment 1 is prepared by the following method,
Step is as follows：
The chemical compounds I of 2.0g (i.e. 7.1mmol) is dissolved in 10ml ethanol by (a '), and the lithium hydroxide for adding 6mol/L is molten
Liquid, is adjusted to pH value for 10-12 by the ethanol solution of chemical compounds I, reacts at room temperature 15~26h, add 40ml purified waters, resulting solution
Adjust pH value to 4~5 with 1M watery hydrochloric acid, filtering, drying obtains 1.1g compound ii.
ESI-MS：167 [M-CH2CH2CH2COOH-1], 252 [M-1], 288 [M+2H2O-1], 315 [2 × 167-H2O-
1], 505 [2M-1], 537 [2M+MeOH-1]；
1H NMR (600MHz, CDCl3, TMS)：δ 10.50 (s, 1H), 8.20 (d, 1H), 7.35 (d, 1H), 7.15-7.19
(dd, 1H), 4.23 (t, 2H), 2.66 (t, 2H), 2.26 (m, 2H).
The compound ii of 0.5g (i.e. 2.0mmol) is dissolved in 10ml methanol by (b '), adds 0.27g (i.e. 2.4mmol)
Cistofuran metabolite AHD, react 2h in 60~70 DEG C, reaction finishes, be cooled to room temperature, filter, the furans for obtaining 0.26g is appropriate
Because of the haptens of metabolin, the structural formula III and its synthetic route of the haptens are shown in Fig. 1.
ESI-MS：351[M+1]；
1H NMR (600MHz, DMSO, TMS)：δ 11.39 (s, 1H), 8.16 (t, 2H), 7.40 (d, 1H), 7.22 (dd,
1H), 4.37 (s, 2H), 4.19 (t, 2H), 2.42 (t, 2H), 2.04 (m, 2H).
Embodiment 2
The present embodiment 2 is the artificial antigen of Cistofuran metabolite, and the artificial antigen includes immunogene and coating antigen, two
The difference of person is that the carrier protein species being coupled in preparation process is different, and the immunogene is prepared using embodiment 1
Haptens, carrier protein uses bovine serum albumin (BSA)；The coating antigen uses haptens prepared by embodiment 1, carrier protein
Using ovalbumin (OVA).
The coating antigen of the Cistofuran metabolite of the present embodiment 2, is prepared by the following method, and its step is as follows successively：
(1) haptens of 10.0mg embodiments 1 is taken, is dissolved in 0.5ml dimethylformamides (DMF), after stirring fully,
8.0mg carbodiimides (EDC) and 6.0mg n-hydroxysuccinimide (NHS) are added, 4h is stirred at room temperature, you can half is obtained
Antigen-activated ester；
(2) 38.1mg ovalbumin (OVA) is weighed, it is substantially dissolved in the PBS that 4ml concentration is 0.01mol/L
In solution, carrier protein solution is formed, haptens Acibenzolar is slowly added dropwise into carrier protein solution dropwise under agitation, and
24h is stirred at room temperature；
(3) solution made from step (2) is dialysed 3 days with 0.01mol/L PBS solution room temperature, and 3 dialyzates are changed daily,
To remove unreacted small-molecule substance；
(4) dispense, saved backup in 4 DEG C.
Wherein, the mol ratio of the haptens of embodiment 1 and ovalbumin (OVA) is 30：1.
The immunogene of the Cistofuran metabolite of the present embodiment 2, is prepared by the following method, and its step is as follows successively：
(1 ') takes the haptens of 10.0mg embodiments 1, is dissolved in 0.5ml dimethylformamides (DMF), and stirring is abundant
Afterwards, 8.0mg carbodiimides (EDC) and 6.0mg n-hydroxysuccinimide (NHS) are added, 4h is stirred at room temperature, you can obtain
Haptens Acibenzolar；
(2 ') weigh 62.8mg bovine serum albumin(BSA) (BSA), it is substantially dissolved in 4ml concentration for 0.01mol/L
PBS solution in, formed carrier protein solution, haptens Acibenzolar is slowly added dropwise to carrier protein solution dropwise under agitation
In, and 24h is stirred at room temperature；
Solution made from (3 ') step (2 ') is dialysed 3 days with 0.01mol/L PBS solution room temperature, and 3 dialysis are changed daily
Liquid, to remove unreacted small-molecule substance；
(4 ') dispense, saved backup in 4 DEG C.
Wherein, the mol ratio of the haptens of embodiment 1 and bovine serum albumin(BSA) (BSA) is 30：1.
Wherein, immune former formula (III)-BSA mentioned below is the Cistofuran metabolite of the present embodiment 2, is coated with former formula
(III)-OVA is the Cistofuran metabolite of the present embodiment 2.
Embodiment 3
The monoclonal antibody of the Cistofuran metabolite of the present embodiment 3, is prepared by the following method, and its step is：
After immune former formula (III)-BSA prepared by embodiment 2, with the emulsification of isometric Freund's adjuvant, BALB/C is immunized small
Mouse.Every mouse immunizing dose is 50~100 μ g, and immunization interval 3 weeks after being immunized 3 times, adopts mouse tail venous blood detection serum effect
Valency.Such as antibody titer needs booster immunization not up to 60000, after antibody titer is no longer raised, and is carried out with 100 μ g immunogenes subcutaneous
Booster immunization, takes the splenocyte and SP20 cell fusions of mouse after 5 days.Cell after fusion is screened in HAT culture mediums, 5 days
HAT culture mediums are substituted for afterwards with complete medium to be cultivated.Cell conditioned medium is detected with ELISA, is by testing result
The hole inner cell of strong positive carries out limiting dilution assay clone's culture, after being detected through 3 time cloning cultures, the hole inner cell being positive
The as hybridoma of secrete monoclonal antibody.After hybridoma amplification culture, mouse peritoneal is seeded to, is produced containing anti-
The ascites of body.Purify ascites with octanoic acid-ammonium sulfate precipitation method, you can obtain high-purity, the monoclonal antibody of high specific.
ELISA indirect competitives are determined antibody positive titre and are defined by the measured value of 3 times of negative serums, as a result show bag
It is 1 by the corresponding antiserums of former formula (III)-OVA (Ab- formulas (III)) potency：72000.
Embodiment 4
The specificity of monoclonal antibody and sensitivity.
The preparation method of 4.1 ELISA Plates
Make coating dilution with PH9.6 carbonate buffer solution, formula (III)-OVA prepared by embodiment 2 is diluted to 0.2 μ
G/ml, is added in polystyrene micropore plate, 4 DEG C of coatings are stayed overnight by 100 μ L/ holes, is dried, and is added by 250 μ L/ holes and is contained 1%BSA,
37 DEG C of closing 1h, are dried in phosphate buffer, are vacuum-packed and are preserved after drying.
The preparation of 4.2 antibody
With containing 0.05% Sodium azide, PH7.4 phosphate buffer, monoclonal antibody prepared by embodiment 3 is diluted to
0.05 μ g/ml, 4 DEG C of preservations.
The preparation of 4.3 standard liquids
Cistofuran metabolite AHD derivative standard liquid is accurate weighing Cistofuran metabolite AHD derivative
Standard items 10mg, first with DMF and each 1ml of purified water dissolvings, then with PH7.4 phosphate buffered saline series concentration is 0 μ g/
L, 0.05 μ g/L, 0.15 μ g/L, 0.45 μ g/L, 1.35 μ g/L, and 4.05 μ g/L Cistofuran metabolite AHD derivative
Standard liquid.
4.4 evaluation result
The derivative standard liquid 50 that Cistofuran metabolite AHD is added in (III)-OVA micropore ELISA Plate to being coated with
μ L/ holes, add the μ L/ holes of monoclonal antibody solution 50 prepared, 37 DEG C of reaction 0.5h；After drying, the μ L/ of washing lotion 300 are added
Hole, is patted dry after washing 3 times；Add the μ L/ holes of ELIAS secondary antibody 100,37 DEG C of reaction 0.5h；Pat dry, add respectively after washing 3 times again
Enter the nitrite ion A and nitrite ion B, 37 DEG C of reaction 15min in 50 μ L/ holes；The μ L/ holes of 2M sulfuric acid 50 are added to terminate, setting ELIASA in
450nm wavelength determines the OD values per hole.As a result such as table 1 and Fig. 6.
Table 1：ELISA test various concentrations Cistofuran metabolites AHD derivative standard items OD values
Wherein, B/B0 refers to the standard items instrument connection OD values of various concentrations and content is 0 standard items instrument connection OD values
Ratio.As can be seen from Table 1, when Cistofuran metabolite AHD derivative content is 0.05 μ g/L, its B/B0 value is
74.54%, illustrate that it detects that OD and the Cistofuran metabolite AHD containing 0 μ g/L concentration derivative instrument connection OD values have substantially
Difference, therefore ELISA is to the Cistofuran metabolite AHD reachable 0.05 μ g/L of derivative detection sensitivity.
The reagent and instrument and other terms that the present invention is used are referring to prior art and common knowledge.
The invention is not limited in above-mentioned embodiment, if the various changes or modification to the present invention do not depart from the present invention
Spirit and scope, if these are changed and modification belongs within the scope of the claim and equivalent technologies of the present invention, then this hair
It is bright to be also intended to comprising these changes and modification.
Claims (10)
1. a kind of haptens of Cistofuran metabolite, it is characterised in that be following structural formula III：
2. a kind of preparation method of the haptens of Cistofuran metabolite, it is characterised in that step is as follows：
(a) chemical compounds I is dissolved in ethanol, is adjusted to 10-11 by adding lithium hydroxide solution by pH value, reaction obtains chemical combination
Thing II；
The structural formula of the chemical compounds I is as follows：
The structural formula of the compound ii is as follows：
(b) compound ii and Cistofuran metabolite are reacted under the conditions of methanol, both the Cistofuran metabolite
Haptens；It is following structural formula III：
3. a kind of preparation method of the haptens of Cistofuran metabolite, it is characterised in that step is as follows：
Chemical compounds I is dissolved in ethanol by (a '), and pH value is adjusted into 10-12 by lithium hydroxide solution, reacted, and adds purifying
Water, 4-5 is adjusted to by watery hydrochloric acid by PH, is filtrated to get compound ii, the mistake of the chemical compounds I reaction generation compound ii
Journey is as follows：
The compound ii is dissolved in methanol by (b '), plus 1.2-1.5 molar equivalents Cistofuran metabolite AHD, in 60
~70 DEG C are reacted, and reaction is finished, filtering, both the Cistofuran metabolite haptens, the structure of the haptens
Formula III and synthetic route are as follows：
4. a kind of artificial antigen of Cistofuran metabolite, it is characterised in that：It is carrier protein and the furans described in claim 1
The conjugate that the appropriate hapten conjugation because of metabolin is obtained, it is following structural formula IV：
5. the artificial antigen of Cistofuran metabolite according to claim 4, it is characterised in that：The carrier protein is ox
Any one in seralbumin, ovalbumin, human serum albumins or hemocyanin.
6. a kind of preparation method of the artificial antigen of Cistofuran metabolite, it is characterised in that step is as follows：
(1) haptens of the Cistofuran metabolite described in claim 1 is taken, is dissolved in dimethylformamide, after stirring, plus
Enter carbodiimide and n-hydroxysuccinimide, the weight ratio of the haptens, carbodiimide and n-hydroxysuccinimide is 5
: 4: 3, stir at room temperature, produce haptens Acibenzolar；
(2) carrier protein is weighed, the mole ratio of the carrier protein and the haptens is 30: 1, fills the carrier protein
Divide and be dissolved in PBS solution, form carrier protein solution, the haptens Acibenzolar is slowly added dropwise to institute dropwise under agitation
State in load protein solution, stirring；
(3) step (2) resulting solution is dialysed by PBS solution, both.
7. the preparation method of the artificial antigen of Cistofuran metabolite according to claim 6, it is characterised in that：It is described to carry
Body protein is any one in bovine serum albumin(BSA), ovalbumin, human serum albumins or hemocyanin.
8. the method for monoclonal antibody prepared by a kind of artificial antigen of Cistofuran metabolite by described in claim 4,
Characterized in that, step is as follows successively：
(i) using the artificial antigen of the Cistofuran metabolite described in claim 4 as immunogene, emulsified with isometric Freund's adjuvant
Afterwards, immune balb/c mice；
(ii) take the step (i) handle after mouse tail venous blood detection serum titer, such as antibody titer not up to 60000,
Booster immunization is needed, after antibody titer is no longer raised, subcutaneous booster immunization is carried out with 100 μ g immunogenes, the spleen of mouse is taken after 5 days
Cell and SP20 cell fusions；
(iii) cell after merging is screened in HAT culture mediums, and being then substituted for HAT culture mediums with complete medium is trained
Support；
(iv) cell conditioned medium after the step (iii) processing is detected with ELISA, by the hole that testing result is strong positive
Inner cell carries out limiting dilution assay clone's culture, after clone's culture detection, and the hole inner cell being positive is secretion Dan Ke
The hybridoma of grand antibody；
(v) by after hybridoma amplification culture, mouse peritoneal is seeded to, the ascites containing antibody is produced, with octanoic acid-sulfuric acid
The ammonium precipitation method purify ascites, produce.
9. the monoclonal antibody described in the artificial antigen of the Cistofuran metabolite described in claim 4 and claim 8 is in inspection
The application surveyed in Cistofuran metabolite, it is characterised in that step is as follows：It regard the artificial antigen described in claim 4 as bag
By primordial covering on ELISA Plate, the monoclonal antibody described in claim 8 is added in microwell plate；Detect to be checked using competition law
The content of Cistofuran metabolite in sample.
10. the monoclonal antibody described in claim 8 is applied in detection aquatic products in Cistofuran metabolite retention analysis.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108530373A (en) * | 2018-05-14 | 2018-09-14 | 广东达元绿洲食品安全科技股份有限公司 | A kind of furans metabolite hapten, artificial antigen and its application in fluorescent quantitation immunochromatography |
| CN110683965A (en) * | 2019-10-18 | 2020-01-14 | 中国农业大学 | Nifurtida hapten and artificial antigen as well as preparation methods and application thereof |
| CN111285786A (en) * | 2020-03-09 | 2020-06-16 | 中国农业大学 | Nifuroxazone hapten and artificial antigen as well as preparation method and application thereof |
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|---|---|---|---|---|
| CN101412697A (en) * | 2008-11-21 | 2009-04-22 | 华南农业大学 | Preparation of 1-amino hydantoin derivative hapten, antigen and antibody |
| CN105131121A (en) * | 2015-08-02 | 2015-12-09 | 武汉上成生物科技有限公司 | Monoclonal antibody for detecting furazolidone metabolites, ELISA method, and kit |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101412697A (en) * | 2008-11-21 | 2009-04-22 | 华南农业大学 | Preparation of 1-amino hydantoin derivative hapten, antigen and antibody |
| CN105131121A (en) * | 2015-08-02 | 2015-12-09 | 武汉上成生物科技有限公司 | Monoclonal antibody for detecting furazolidone metabolites, ELISA method, and kit |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108530373A (en) * | 2018-05-14 | 2018-09-14 | 广东达元绿洲食品安全科技股份有限公司 | A kind of furans metabolite hapten, artificial antigen and its application in fluorescent quantitation immunochromatography |
| CN110683965A (en) * | 2019-10-18 | 2020-01-14 | 中国农业大学 | Nifurtida hapten and artificial antigen as well as preparation methods and application thereof |
| CN111285786A (en) * | 2020-03-09 | 2020-06-16 | 中国农业大学 | Nifuroxazone hapten and artificial antigen as well as preparation method and application thereof |
| CN111285786B (en) * | 2020-03-09 | 2021-04-06 | 中国农业大学 | Nifuroxazone hapten and artificial antigen as well as preparation method and application thereof |
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