CN118724807B - A decoquinate hapten, antigen, antibody and preparation and application thereof - Google Patents
A decoquinate hapten, antigen, antibody and preparation and application thereof Download PDFInfo
- Publication number
- CN118724807B CN118724807B CN202411207661.1A CN202411207661A CN118724807B CN 118724807 B CN118724807 B CN 118724807B CN 202411207661 A CN202411207661 A CN 202411207661A CN 118724807 B CN118724807 B CN 118724807B
- Authority
- CN
- China
- Prior art keywords
- decoquinate
- formula
- hapten
- antigen
- antibody
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- JHAYEQICABJSTP-UHFFFAOYSA-N decoquinate Chemical compound N1C=C(C(=O)OCC)C(=O)C2=C1C=C(OCC)C(OCCCCCCCCCC)=C2 JHAYEQICABJSTP-UHFFFAOYSA-N 0.000 title claims abstract description 106
- 229960001878 decoquinate Drugs 0.000 title claims abstract description 103
- 239000000427 antigen Substances 0.000 title claims abstract description 30
- 102000036639 antigens Human genes 0.000 title claims abstract description 30
- 108091007433 antigens Proteins 0.000 title claims abstract description 30
- 238000002360 preparation method Methods 0.000 title abstract description 13
- 238000006243 chemical reaction Methods 0.000 claims description 20
- 238000000034 method Methods 0.000 claims description 16
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 16
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 12
- 238000005406 washing Methods 0.000 claims description 11
- PLIKAWJENQZMHA-UHFFFAOYSA-N 4-aminophenol Chemical compound NC1=CC=C(O)C=C1 PLIKAWJENQZMHA-UHFFFAOYSA-N 0.000 claims description 10
- 238000001704 evaporation Methods 0.000 claims description 10
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 9
- -1 ethoxymethylene diethyl Chemical group 0.000 claims description 9
- 229910000027 potassium carbonate Inorganic materials 0.000 claims description 8
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 6
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 6
- USIUVYZYUHIAEV-UHFFFAOYSA-N diphenyl ether Chemical compound C=1C=CC=CC=1OC1=CC=CC=C1 USIUVYZYUHIAEV-UHFFFAOYSA-N 0.000 claims description 6
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 6
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 5
- 229940098773 bovine serum albumin Drugs 0.000 claims description 5
- 239000002904 solvent Substances 0.000 claims description 5
- 102000014914 Carrier Proteins Human genes 0.000 claims description 4
- 108010078791 Carrier Proteins Proteins 0.000 claims description 4
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 4
- 201000010099 disease Diseases 0.000 claims description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 4
- 239000012044 organic layer Substances 0.000 claims description 4
- 238000004440 column chromatography Methods 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 3
- 101000609762 Gallus gallus Ovalbumin Proteins 0.000 claims description 2
- 108091006905 Human Serum Albumin Proteins 0.000 claims description 2
- 102000008100 Human Serum Albumin Human genes 0.000 claims description 2
- 108060003552 hemocyanin Proteins 0.000 claims description 2
- 238000010324 immunological assay Methods 0.000 claims description 2
- 230000001376 precipitating effect Effects 0.000 claims description 2
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 2
- 239000003814 drug Substances 0.000 abstract description 18
- 229940079593 drug Drugs 0.000 abstract description 17
- 238000001514 detection method Methods 0.000 abstract description 12
- 230000005764 inhibitory process Effects 0.000 abstract description 12
- 230000037029 cross reaction Effects 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 18
- 230000003053 immunization Effects 0.000 description 17
- 238000002649 immunization Methods 0.000 description 17
- 239000000243 solution Substances 0.000 description 15
- 239000011248 coating agent Substances 0.000 description 13
- 238000000576 coating method Methods 0.000 description 13
- 238000002965 ELISA Methods 0.000 description 10
- 230000001165 anti-coccidial effect Effects 0.000 description 9
- 239000007853 buffer solution Substances 0.000 description 8
- 239000007788 liquid Substances 0.000 description 8
- 238000003756 stirring Methods 0.000 description 8
- 230000008878 coupling Effects 0.000 description 7
- 238000010168 coupling process Methods 0.000 description 7
- 238000005859 coupling reaction Methods 0.000 description 7
- 239000001963 growth medium Substances 0.000 description 7
- 241000699666 Mus <mouse, genus> Species 0.000 description 6
- 238000010009 beating Methods 0.000 description 6
- 241000699670 Mus sp. Species 0.000 description 5
- 238000001035 drying Methods 0.000 description 5
- 230000004927 fusion Effects 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 238000007789 sealing Methods 0.000 description 5
- 210000004989 spleen cell Anatomy 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 206010035226 Plasma cell myeloma Diseases 0.000 description 4
- 239000002202 Polyethylene glycol Substances 0.000 description 4
- SMWDFEZZVXVKRB-UHFFFAOYSA-N Quinoline Chemical compound N1=CC=CC2=CC=CC=C21 SMWDFEZZVXVKRB-UHFFFAOYSA-N 0.000 description 4
- 230000003213 activating effect Effects 0.000 description 4
- 239000003085 diluting agent Substances 0.000 description 4
- 239000003640 drug residue Substances 0.000 description 4
- 235000013305 food Nutrition 0.000 description 4
- 201000000050 myeloid neoplasm Diseases 0.000 description 4
- 229920001223 polyethylene glycol Polymers 0.000 description 4
- 210000000952 spleen Anatomy 0.000 description 4
- 206010003445 Ascites Diseases 0.000 description 3
- 241000287828 Gallus gallus Species 0.000 description 3
- 108010063045 Lactoferrin Proteins 0.000 description 3
- 102100032241 Lactotransferrin Human genes 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 210000000683 abdominal cavity Anatomy 0.000 description 3
- 239000002671 adjuvant Substances 0.000 description 3
- 230000007910 cell fusion Effects 0.000 description 3
- 235000013330 chicken meat Nutrition 0.000 description 3
- 238000007865 diluting Methods 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- CSSYQJWUGATIHM-IKGCZBKSSA-N l-phenylalanyl-l-lysyl-l-cysteinyl-l-arginyl-l-arginyl-l-tryptophyl-l-glutaminyl-l-tryptophyl-l-arginyl-l-methionyl-l-lysyl-l-lysyl-l-leucylglycyl-l-alanyl-l-prolyl-l-seryl-l-isoleucyl-l-threonyl-l-cysteinyl-l-valyl-l-arginyl-l-arginyl-l-alanyl-l-phenylal Chemical compound C([C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 CSSYQJWUGATIHM-IKGCZBKSSA-N 0.000 description 3
- 229940078795 lactoferrin Drugs 0.000 description 3
- 235000021242 lactoferrin Nutrition 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 241000283707 Capra Species 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 238000003018 immunoassay Methods 0.000 description 2
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 2
- 238000001819 mass spectrum Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 239000000273 veterinary drug Substances 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 241000224483 Coccidia Species 0.000 description 1
- 208000003495 Coccidiosis Diseases 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 206010023076 Isosporiasis Diseases 0.000 description 1
- 241000699667 Mus spretus Species 0.000 description 1
- 101710120037 Toxin CcdB Proteins 0.000 description 1
- 208000020560 abdominal swelling Diseases 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
- 230000002745 absorbent Effects 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 239000007640 basal medium Substances 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 150000001793 charged compounds Chemical class 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 230000009260 cross reactivity Effects 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- LTMHNWPUDSTBKD-UHFFFAOYSA-N diethyl 2-(ethoxymethylidene)propanedioate Chemical compound CCOC=C(C(=O)OCC)C(=O)OCC LTMHNWPUDSTBKD-UHFFFAOYSA-N 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 229920001971 elastomer Polymers 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 230000001804 emulsifying effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 210000004408 hybridoma Anatomy 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 229940057995 liquid paraffin Drugs 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 125000002943 quinolinyl group Chemical group N1=C(C=CC2=CC=CC=C12)* 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000007363 ring formation reaction Methods 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000012089 stop solution Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- WMOVHXAZOJBABW-UHFFFAOYSA-N tert-butyl acetate Chemical compound CC(=O)OC(C)(C)C WMOVHXAZOJBABW-UHFFFAOYSA-N 0.000 description 1
- IEDVJHCEMCRBQM-UHFFFAOYSA-N trimethoprim Chemical compound COC1=C(OC)C(OC)=CC(CC=2C(=NC(N)=NC=2)N)=C1 IEDVJHCEMCRBQM-UHFFFAOYSA-N 0.000 description 1
- 229960001082 trimethoprim Drugs 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
- C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
- C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom 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
- C07D215/48—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen
- C07D215/54—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen attached in position 3
- C07D215/56—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen attached in position 3 with oxygen atoms in position 4
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C227/00—Preparation of compounds containing amino and carboxyl groups bound to the same carbon skeleton
- C07C227/04—Formation of amino groups in compounds containing carboxyl groups
- C07C227/06—Formation of amino groups in compounds containing carboxyl groups by addition or substitution reactions, without increasing the number of carbon atoms in the carbon skeleton of the acid
- C07C227/08—Formation of amino groups in compounds containing carboxyl groups by addition or substitution reactions, without increasing the number of carbon atoms in the carbon skeleton of the acid by reaction of ammonia or amines with acids containing functional groups
-
- 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
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biomedical Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Gastroenterology & Hepatology (AREA)
- Food Science & Technology (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Pharmacology & Pharmacy (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明公开了一种癸氧喹酯半抗原、抗原、抗体及其制备与应用，涉及癸氧喹酯半抗原、抗原、抗体及其制备和检测癸氧喹酯残留的应用。本发明通过使用水溶性强的手臂制备得到了结构式如式（I）所示癸氧喹酯半抗原及人工抗原，并进一步制备得到用于检测癸氧喹酯的特异性抗体，对癸氧喹酯的半抑制浓度IC50值为12.3ng/mL，检测限LOD（IC10）为3.41 ng/mL，线性范围（IC20~IC80）为7.4~20.9ng/mL，对常见结构类似物的交叉反应率均低于10%，说明该抗体对癸氧喹酯具有良好的特异性，可有效的排除其他结构类似物药物的干扰。
The present invention discloses a decoquinate hapten, antigen, antibody and preparation and application thereof, and relates to a decoquinate hapten, antigen, antibody and preparation thereof and application thereof for detecting decoquinate residues. The present invention prepares a decoquinate hapten and an artificial antigen having a structural formula as shown in formula (I) by using a highly water-soluble arm, and further prepares a specific antibody for detecting decoquinate, with a half inhibition concentration IC 50 value of 12.3 ng/mL for decoquinate, a detection limit LOD (IC 10 ) of 3.41 ng/mL, a linear range (IC 20 ~IC 80 ) of 7.4~20.9 ng/mL, and a cross-reaction rate to common structural analogs of less than 10%, indicating that the antibody has good specificity for decoquinate and can effectively eliminate the interference of other structural analog drugs.
Description
Technical Field
The invention relates to the technical field of food safety detection, in particular to decoquinate hapten, antigen and antibody as well as preparation and application thereof.
Background
Decoquinate (Decoquinate) is a quinoline anticoccidial drug. Quinoline anticoccidial drugs are early developed in anticoccidial drugs, have good efficacy, and several drugs are added into feeds so far to prevent and treat coccidiosis. Although these anticoccidial drugs have low toxicity, the use of anticoccidial drugs tends to cause the emergence of coccidian resistance, and the use of anticoccidial drugs in livestock production tends to increase the dosage.
In recent years, a liquid chromatography-tandem mass spectrometry detection technology has become one of the main means for detecting veterinary drug residues, and a few reports on the detection of anticoccidial drug residues by adopting a liquid chromatography-tandem mass spectrometry method are also reported, mainly aiming at chicken, eggs and beef, and also reports on the detection of chicken skin and fat, anticoccidial drug residues in chicken excreta and the like. But less research is conducted on the detection of anticoccidial drug residues in complex matrixes such as livers, kidneys and the like.
The immunoassay method based on antigen-antibody specificity recognition is one of the main technologies of the current veterinary drug residue detection, but the related reports are not available at present because of the high fat solubility and poor solubility of the drugs, and hapten design and antibody preparation are difficult. Therefore, a monoclonal antibody capable of specifically recognizing decoquinate is urgently needed, and a foundation is laid for developing an immunity rapid detection technology and a product aiming at decoquinate.
Disclosure of Invention
The invention aims to obtain a decoquinate hapten with increased polarity and enhanced water solubility by connecting an arm with strong water solubility to a decoquinate analogue, so that the immune effect of the antigen is enhanced.
According to one aspect of the present invention, there is provided decoquinate hapten having the structure as shown in formula (I):
Formula (I).
According to another aspect of the present invention, there is provided a method for preparing decoquinate hapten, comprising the steps of:
S1, dissolving 4-aminophenol and ethoxymethylene diethyl malonate in isopropanol, reacting at room temperature under alkaline condition, filtering to remove insoluble substances, evaporating the solvent to obtain an intermediate 1, dissolving the intermediate 1 in diphenyl ether, reacting at 250 ℃ for 30min, cooling to room temperature, precipitating a product, washing with n-hexane to obtain an intermediate 2, wherein the reaction formula of the step is shown in the formula (II),
A formula (II);
S2, dissolving the intermediate 2 in DMF, adding bromotriethylene glycol tert-butyl acetate and potassium carbonate, wherein the mol ratio of the intermediate 2 to the bromotriethylene glycol tert-butyl acetate to the potassium carbonate is 1:1:1, heating to 90 ℃ for reaction overnight, adding a large amount of saturated saline solution, extracting with ethyl acetate, evaporating an organic layer, passing through column chromatography to obtain an intermediate 3, wherein the reaction formula is shown in the formula (III),
A formula (III);
S3, dissolving the intermediate 3 by using dichloromethane, then reacting with trifluoroacetic acid, evaporating the solvent after the reaction is finished to obtain decoquinate hapten, wherein the structural formula is shown as a formula (I), the reaction formula of the step is shown as a formula (IV),
Formula (IV).
In some embodiments, the molar ratio of 4-aminophenol to diethyl ethoxymethylene malonate in step S1 is 1 (1-1.2), with the basic condition being the addition of potassium carbonate or sodium carbonate to the reaction system.
According to a further aspect of the invention, there is provided a decoquinate antigen which is a conjugate of a decoquinate hapten and a carrier protein.
In some embodiments, the carrier protein is bovine serum albumin, human serum albumin, chicken egg albumin, or hemocyanin.
According to a fourth aspect of the present invention, there is provided decoquinate antibodies, which are prepared from decoquinate antigens by animal immunization, and which are decoquinate monoclonal antibodies.
According to a fifth aspect of the present invention there is provided use of decoquinate hapten, decoquinate antigen in immunological detection of decoquinate for non-disease diagnostic purposes.
According to a sixth aspect of the invention there is provided the use of an antibody to decoquinate in an immunological assay for the purpose of non-disease diagnosis of decoquinate.
According to a seventh aspect of the present invention, there is provided a method for detecting decoquinate for non-disease diagnosis, wherein decoquinate antigen and decoquinate antibody are used to detect decoquinate drug residues in food, and the food is animal-derived food.
The invention has the beneficial effects that: according to the invention, the decoquinate hapten and the artificial antigen with the structural formula shown in the formula (I) are prepared by using arms with strong water solubility, and the specific antibody for detecting the decoquinate is further prepared, wherein the value of the semi-inhibition concentration IC 50 of the decoquinate is 12.3ng/mL, the detection limit LOD (IC 10) is 3.41 ng/mL, the linear range (IC 20~IC80) is 7.4-20.9 ng/mL, and the cross reaction rate of the decoquinate hapten and the decoquinate hapten is lower than 10% for common structural analogues, so that the antibody has good specificity for the decoquinate, the interference of other structural analogues can be effectively eliminated, and a core reagent is provided for establishing an immunodetection method of the decoquinate.
Drawings
Fig. 1 is a mass spectrum of decoquinate hapten of an embodiment of the present invention.
Fig. 2 is a graph of an indirect competition ELISA standard established based on decoquinate monoclonal antibodies of one embodiment of the present invention.
Detailed Description
The application will be described in further detail with reference to specific embodiments thereof, it being understood that these embodiments are for purposes of illustration only and not for purposes of limiting the scope of the application, as various equivalent modifications of the application will occur to those skilled in the art upon reading the application, as defined in the appended claims. Unless otherwise specified, all materials and reagents of the application are those commercially available in the conventional market.
Example 1 preparation of decoquinate hapten
The preparation method of decoquinate hapten comprises the following steps:
S1, respectively weighing 4.36 g of 4-aminophenol, 9.0 g ethoxy methylene diethyl malonate and 6.5 g potassium carbonate, dissolving in 50mL isopropanol, reacting overnight at room temperature, filtering after the reaction is finished, and evaporating filtrate to dryness to obtain an intermediate 1 (the subsequent experiment can be directly carried out without purification); dissolving the intermediate 1 in 40 mL diphenyl ether, reacting at 250 ℃ for 30min for cyclization reaction, cooling to room temperature, separating out a product, washing with n-hexane, and recrystallizing to obtain 6.25g of intermediate 2 with the total yield of 67%;
S2, dissolving 2.4 g of the intermediate 2 in 30mL of DMF, adding 2.6 g bromo-tri-polyethylene glycol tert-butyl acetate and 2.5 g potassium carbonate, heating to 90 ℃ for reaction overnight, adding a large amount of saturated saline after the reaction is finished, extracting for 2-3 times by using 50 mL ethyl acetate, evaporating an organic layer by using anhydrous sodium sulfate, and performing column chromatography to obtain 3.2 g of intermediate 3;
S3, dissolving the 1.5 g intermediate 3 by using 10 mL dichloromethane, slowly adding 5 mL trifluoroacetic acid to react for 30 min, evaporating the solvent after the reaction is finished, adding saturated saline water to wash, extracting for 2-3 times by using 30 mL ethyl acetate, and evaporating the organic layer by using anhydrous sodium sulfate to obtain decoquinate hapten.
The decoquinate hapten is identified by mass spectrometry, and the obtained mass spectrum is shown in figure 1. As can be seen from FIG. 1, the molecular ion peak of decoquinate hapten is ESI- [ M-H ] -: 422.26, and is the highest peak, which is consistent with molecular weight 423.42 of decoquinate hapten, indicating successful synthesis of decoquinate hapten represented by formula (I).
EXAMPLE 2 preparation of antigen for decoquinate immunization and antigen for coating
2.1 Preparation of antigen for decoquinate immunization
Using decoquinate hapten prepared in example 1, coupling Lactoferrin (LF) by active ester method to prepare antigen for decoquinate immunization, the method is as follows:
Dissolving 16.5 mg of decoquinate hapten prepared in the example 1 in 0.5 mL DMF solution, stirring and adding 12 mg of EDC (EDC) HCl and 10 mg NHS, and stirring and reacting overnight at room temperature in a dark place to obtain decoquinate hapten activating solution; 16mg of LF is taken and dissolved in 1.6 mL of BB buffer solution with pH of 9.0, 150 mu L of decoquinate hapten activating solution is added by stirring, and after uniform stirring, 4 h is coupled at room temperature and in a dark place, so as to obtain a coupling mixture; dialyzing the coupling mixture with PBS buffer solution at 4deg.C for 3 days, and changing the dialysate for 2 times per day to obtain antigen for decaquindox immunization (decaquindox hapten-LF), subpackaging at 1 mg/mL concentration, and freezing in a refrigerator at-20deg.C.
2.2 Preparation of antigen for decoquinate coating
Using decoquinate hapten prepared in example 1, decoquinate coating antigen is prepared by coupling Bovine Serum Albumin (BSA) by an active ester method, and the method is as follows:
Dissolving 15.7 mg of decoquinate hapten prepared in the example 1 in 0.5 mL DMF solution, stirring and adding 12 mg EDC and 10 mg NHS, and stirring and reacting overnight at room temperature in dark to obtain decoquinate hapten activating solution; dissolving 24 mg BSA into 2.4 mL BB buffer solution with pH of 9.0, adding 225 mu L decoquinate hapten activating solution under stirring, and coupling 4 h at room temperature and in a dark place after stirring uniformly to obtain a coupling mixture; the coupling mixture was dialyzed against PBS buffer solution at 4deg.C for 3 days, the dialysate was changed 2 times per day, and the obtained decoquinate coating antigen (decoquinate hapten-BSA) was sub-packaged at a concentration of 1 mg/mL and frozen in a-20deg.C refrigerator.
Example 3 preparation and identification of decoquinate monoclonal antibodies
3.1 Immunization of mice
Taking 250 mu L of decoquinate immunization antigen (decoquinate hapten-LF) prepared in the example 2 and diluted to be 1 mg/mL, and uniformly emulsifying the antigen and an equivalent amount of immune adjuvant (Freund complete adjuvant for the first immunization and Freund incomplete adjuvant for the later booster immunization) to immunize animals; bal b/c mice with the sizes of 6-7 weeks are immunized by adopting various injection modes of subcutaneous back, subcutaneous parts, abdominal cavity and feet respectively, the immunization is metered to be 100 mu L/mouse, the immunization is performed for the second time after 2 weeks, the immunization is performed every 2 weeks, the immunization is enhanced every other time, the tail of the mice is taken out after 1 week of the third-time enhancement immunization, and the serum titer is determined by utilizing indirect competition ELISA. When the potency no longer rises, 100 mu L of antigen for immunization of decoquinate with the concentration of 1 mg/mL is taken for intraperitoneal injection, and impact immunization is carried out;
3.2 cell fusion
Three days after the impact immunization, cell fusion was performed using PEG (polyethylene glycol) as follows:
a. Mouse spleen cells were collected: killing a mouse by a cervical dislocation method, immediately soaking the mouse in 75% alcohol for sterilization, taking out the spleen of the mouse by aseptic operation, putting the spleen into a 200-mesh cell screen, moderately grinding the spleen by a rubber head of an aseptic injector, flushing the spleen with a basic culture medium to obtain spleen cell suspension, collecting, centrifuging (1000 rpm,7 min), washing the spleen cells with the basic culture medium for three times, and diluting the spleen cells to a certain volume and counting after the last centrifuging for later use;
b. Collecting SP2/0 cells: culturing SP2/0 myeloma cells in a complete culture medium in a 5% CO 2 incubator 7-10 days before fusion, wherein the number of the SP2/0 myeloma cells required to reach 1-4×10 7 before fusion, and ensuring that the SP2/0 myeloma cells before fusion are in logarithmic growth phase. During fusion, myeloma cells are collected and suspended in a basal medium for cell counting;
c. according to spleen cells: SP 2/0=5: 1, mixing the two cells in proportion, centrifuging, and discarding the supernatant to obtain mixed cells deposited at the bottom of the centrifuge tube;
d. Fusion: slowly dripping 1 mL PEG into cells at the bottom of the centrifuge tube in the first minute; the second minute, keep the centrifuge tube to shake at a constant speed; thirdly, dropwise adding 1 mL of preheated basic culture medium; dripping 3 mL of preheated basic culture medium in the fourth minute; in fifth minute, 8 mL of preheated basic culture medium is dripped; in sixth minute, 8 mL of preheated basic culture medium is dripped; centrifuging (1000 rpm,7 min), discarding supernatant, re-suspending into HAT-containing screening culture solution, adding into 96-well cell plate at 200 μl/well, and culturing in 5% CO 2 incubator at 37deg.C;
3.3 cell Screen and cell line establishment
Half-changing the fused cells with HT medium on day 5 of cell fusion, full-changing on day 8, taking cell supernatant on day 10, screening by ic-ELISA, and determining the potency inhibition effect of positive cells. Selecting cell holes with better inhibition on decoquinate standard substances, subcloning by adopting a limiting dilution method, detecting by the same method, and repeating for 4-5 times to obtain cell strains;
3.4 preparation and identification of monoclonal antibodies
Taking a plurality of Bal b/c mice over 10 weeks old, and injecting 500 mu L of liquid paraffin into the abdominal cavity of each mouse; after 7 days, about 1×10 6 hybridoma cells were injected into the abdominal cavity of each mouse, and after 7 days, the ascites was collected after the abdominal swelling of the mice, and the collected ascites was purified by chromatography. Filling a column by using 1 mL protein G packing, adding ascites 50 mL diluted by PBS buffer solution, repeatedly loading the flow-through solution for 7-8 times, eluting by using glycine, and timely regulating the eluent to be neutral by using Tris-HCl. And (3) dialyzing and desalting to finally obtain the purified decoquinate monoclonal antibody A4B1, subpackaging and then preserving at-20 ℃.
EXAMPLE 4 ELISA Performance evaluation of decoquinate monoclonal antibodies
The potency and inhibition rate of the antibodies were determined by indirect competition ELISA, the specific method was:
(1) Coating: diluting the coating antigen to 1 mug/mL by using coating liquid, and coating the coating antigen in a water bath at 37 ℃ for overnight according to 100 mug/Kong Jiaru;
(2) Closing: the coating solution was discarded, the plate was washed 2 times, the plate was dried by beating on absorbent paper, 120. Mu.L of blocking solution was added to each well, and blocking was performed by incubation in a 37℃water bath for 3 h. Spin-drying the dried powder, drying in a baking oven with the temperature of 37 ℃ inverted 1 h;
(3) Adding an antibody and a drug: the monoclonal antibody is diluted to be 1K (1000), 2K, 4K, 8K, 16K, 32K, 64K and other multiples by PBS, and decoquinate standard substance is diluted to be 100 ng/mL by PBS for standby;
the titers are as follows: adding 50 mu L of PBS buffer solution into each hole, sequentially adding the diluted monoclonal antibodies into the holes according to 50 mu L/hole, and adding PBS into the last hole to serve as blank control;
inhibition column: adding 50 mu L of a medicament diluted by PBS buffer solution into each hole, sequentially adding 50 mu L of antibody diluted by gradient into the holes, and adding PBS into the last hole to serve as a blank control;
37. Incubating at 40 min deg.c, washing the plate 5 times;
(4) Adding a secondary antibody: adding goat anti-mouse secondary antibody (100 mu L/hole) diluted 5000 times by PBST buffer solution, incubating at 37 ℃ for 30 min times, and washing the plate for 5 times;
(5) Color development: mixing TMB substrate buffer A, B in equal volume to obtain substrate solution, adding substrate solution (100 μl/well), and incubating at 37deg.C for 10 min;
(6) And (3) terminating: adding 10% H 2SO4 stop solution (50 mu L/hole) on the ELISA plate to stop the reaction;
(7) Reading: the absorbance (OD) was read with a microplate reader at a wavelength of 450 nm. The dilution multiple of the antibody with the absorbance value within the range of 1.0-1.5 is selected as the antibody titer, and the drug recognition performance of the antibody is obtained by the inhibition rate. The inhibition rate is calculated in the mode shown in the formula (V), the specific potency and the inhibition rate result are shown in the table 1,
Formula (V).
TABLE 1 antiserum titers and inhibition ratios Table
As is clear from Table 1, the higher potency was 45K at a drug concentration of 100 ng/mL, at which time the inhibition was 93.45%. The serum has high sensitivity and good potency, and the reaction condition can be further optimized, so that an immunodetection method aiming at decoquinate is developed.
EXAMPLE 5 evaluation of specificity of decoquinate monoclonal antibodies
This example uses the icELISA procedure to select several analogues similar in structure to decoquinate under already optimized icELISA conditions and examine the specificity of the decoquinate monoclonal antibody prepared in example 3. The decoquinate analogue competition inhibition curves were sequentially made, the standard mass concentration at 50% inhibition rate was obtained, and then the cross-reactivity of each structural analogue with the antibody was calculated by a formula after conversion to molar concentration, see table 2 below, and 3 replicates were set for each treatment.
As shown in Table 2, the decoquinate monoclonal antibody prepared in example 3 has high sensitivity recognition capability to decoquinate, and only has higher crossing rate to Ding Kuizhi and trimethoprim; other drugs contain quinoline groups, but the crossover rate of decoquinate monoclonal antibodies to other structural analogues is less than 10% because of the large difference of side chain groups. The decoquinate monoclonal antibody prepared in example 3 has better specificity to decoquinate.
Example 6
6.1 Indirect competition ELISA method for detecting decoquinate
(1) Coating decoquinate prepared in example 2 with antigen, diluting to 4 mug/mL with carbonate buffer (CB, 0.1M pH=9.8), coating 96-well ELISA plates, adding 100 mug of the 96-well ELISA plates, incubating overnight at 37 ℃ (12 h), pouring out the liquid in the wells, washing 2 times with washing liquid, each time for 30 s, beating dry, adding 200 mug of sealing liquid in each well, incubating 2h at 37 ℃ in a dark place, pouring out the liquid in the wells, beating dry, and vacuum sealing and storing by an aluminum film after drying;
(2) Removing the coating liquid, washing twice, and beating to dryness;
(3) 120 mu L of sealing liquid (namely, 5% of skimmed milk powder by mass) is added into each hole, and the mixture is sealed at 37 ℃ for 3 h;
(4) Discarding the sealing liquid, beating the plate, drying at 37 ℃ for 30 min, taking out, and bagging for standby by self-sealing;
(5) Phosphate buffer (PBS, 0.01m, ph=7.4) at 1: 16000-fold dilution of the decoquinate monoclonal antibody prepared in example 3, and 4-fold gradient dilution of decoquinate drug to be detected to 125 μg/L, 31.25 μg/L, 7.8 μg/L, 1.95 μg/L, 0.49 μg/L, 0.12 μg/L;
(6) Adding 50 mu L of decoquinate drug diluent to be detected (three groups are parallel) into each row, adding the diluent of the decoquinate monoclonal antibody according to the adding proportion of 50 mu L/hole, incubating at 37 ℃ for 40min, washing for five times, and beating to dry;
(7) Adding goat anti-mouse secondary antibody-HRP diluted 5000 times with Tween phosphate buffer (PBST, 0.01M), incubating at 37deg.C for 30 min times, washing for five times, and drying;
(8) Adding a developing solution into each well, wherein 100 mu L of the developing solution is used for developing 10 min;
(9) The reaction was stopped by adding 50 μl of 10% H 2SO4 solution and the OD was read at 450 nm;
(10) And (3) operating according to the steps (1) to (9), replacing the decoquinate diluent to be detected in the step (6) with the extracted diluent of the sample to be detected, and determining the content of the actual decoquinate drug in the unknown sample by combining the drawn standard curve.
6.2 Detection result
As shown in the figure 2, the standard curve of the indirect competitive ELISA of the antibody for detecting decoquinate drug shows that the IC 50 value of the decoquinate antibody for detecting decoquinate is 12.3ng/mL, the detection limit LOD (IC 10) is 3.41 ng/mL, the linear range (IC 20~IC80) is 7.4-20.9 ng/mL, and the crossing rate of the antibody for detecting decoquinate to other structural analogues is not more than 10%, thus the antibody can be used for establishing a high-sensitivity immunoassay method for detecting decoquinate.
The foregoing is merely illustrative of some embodiments of the invention, and it will be apparent to those skilled in the art that various modifications and improvements can be made without departing from the inventive concept.
Claims (6)
1. Decoquinate hapten is characterized in that the structure is shown as a formula (I):
， （Ⅰ）。
2.A method of preparing the decoquinate hapten of claim 1, comprising the steps of:
S1, dissolving 4-aminophenol and ethoxymethylene diethyl malonate in isopropanol, reacting at room temperature under alkaline condition, filtering to remove insoluble matters, evaporating the solvent to obtain an intermediate 1, dissolving the intermediate 1 in diphenyl ether, reacting at 250 ℃ for 30min, cooling to room temperature, precipitating a product, washing with n-hexane to obtain an intermediate 2, wherein the reaction formula of the step is shown in the formula (II),
A formula (II);
S2, dissolving the intermediate 2 in DMF, adding bromotriethylene glycol tert-butyl acetate and potassium carbonate, wherein the mol ratio of the intermediate 2 to the bromotriethylene glycol tert-butyl acetate to the potassium carbonate is 1:1:1, heating to 90 ℃ for reaction overnight, adding a large amount of saturated saline solution, extracting with ethyl acetate, evaporating an organic layer, passing through column chromatography to obtain an intermediate 3, wherein the reaction formula is shown in the formula (III),
A formula (III);
S3, dissolving the intermediate 3 by using dichloromethane, then reacting with trifluoroacetic acid, evaporating the solvent after the reaction is finished to obtain the decoquinate hapten, wherein the structural formula is shown as a formula (I), the reaction formula of the step is shown as a formula (IV),
Formula (IV).
3. The method for preparing decoquinate hapten according to claim 2, wherein the molar ratio of 4-aminophenol to ethoxymethylene diethyl malonate in step S1 is 1 (1-1.2), and the alkaline condition is adding potassium carbonate or sodium carbonate into the reaction system.
4. Decoquinate antigen, wherein the decoquinate antigen is the conjugate of the decoquinate hapten and carrier protein of claim 1.
5. The decoquinate antigen of claim 4, wherein the carrier protein is bovine serum albumin, human serum albumin, chicken ovalbumin, or hemocyanin.
6. Use of the decoquinate hapten of claim 1, the decoquinate antigen of claim 4 in an immunological assay for non-disease diagnostic purposes of decoquinate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202411207661.1A CN118724807B (en) | 2024-08-30 | 2024-08-30 | A decoquinate hapten, antigen, antibody and preparation and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202411207661.1A CN118724807B (en) | 2024-08-30 | 2024-08-30 | A decoquinate hapten, antigen, antibody and preparation and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN118724807A CN118724807A (en) | 2024-10-01 |
| CN118724807B true CN118724807B (en) | 2024-11-15 |
Family
ID=92847822
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202411207661.1A Active CN118724807B (en) | 2024-08-30 | 2024-08-30 | A decoquinate hapten, antigen, antibody and preparation and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN118724807B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN120172910A (en) * | 2025-05-22 | 2025-06-20 | 北京纳百生物科技有限公司 | Decoquinate hapten and synthesis and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107382855A (en) * | 2017-06-28 | 2017-11-24 | 浙江荣耀生物科技股份有限公司 | A kind of synthetic method of deccox |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104569398B (en) * | 2013-10-22 | 2016-06-22 | 北京勤邦生物技术有限公司 | The enzyme linked immunological kit of detection ethoxy quinoline and application thereof |
-
2024
- 2024-08-30 CN CN202411207661.1A patent/CN118724807B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107382855A (en) * | 2017-06-28 | 2017-11-24 | 浙江荣耀生物科技股份有限公司 | A kind of synthetic method of deccox |
Non-Patent Citations (1)
| Title |
|---|
| 喹啉类抗球虫药物半抗原的理性设计及血清学验证研究;申晴 等，;《中国毒理学会第九次全国青年科技大会暨第二届生物技术药物毒理与安全评价委员会学术会议论文集》;20231029;第102-103页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN118724807A (en) | 2024-10-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN118724807B (en) | A decoquinate hapten, antigen, antibody and preparation and application thereof | |
| CN101413955A (en) | ELISA test box for detecting zearalenone and preparing and detecting method thereof | |
| CN113736744B (en) | Digoxigenin monoclonal antibody hybridoma cell line and its application | |
| CN107904209B (en) | H7 subtype avian influenza virus monoclonal antibody and application thereof | |
| CN101429250A (en) | Mold toxin penicillic acid monoclone antibody and preparation method thereof | |
| CN104388392A (en) | Enrofloxacin monoclonal antibody as well as preparation method and application thereof | |
| CN111269262B (en) | Profenofos hapten, artificial antigen and antibody, and preparation method and application thereof | |
| CN106565737B (en) | A kind of aflatoxin B1The preparation method of haptens, artificial antigen and its Yolk antibody | |
| CN107255716A (en) | The monoclonal antibody and its enzyme-linked immune detection method of a kind of tenuazonic acid | |
| CN112574957B (en) | A hybridoma cell line secreting clomazone monoclonal antibody and its application | |
| CN108218747A (en) | Florfenicol haptens, artificial antigen and monoclonal antibody and its enzyme-linked immune detection method | |
| CN116813537B (en) | Preparation method and application of novel flunixin hapten | |
| CN112778420A (en) | Pyridaben monoclonal antibody and application thereof | |
| CN112625135A (en) | Dicofol monoclonal antibody and application thereof | |
| CN118146088A (en) | Hapten for detecting content of metrafenone, preparation method and application thereof | |
| CN114031528B (en) | Florfenicol hapten, artificial antigen, antibody and synthetic method and application thereof | |
| WO2018103630A1 (en) | Hybridoma cell strain c1 for secreting anti-paromomycin monoclonal antibody and use thereof | |
| CN108794507B (en) | Rifaximin hapten, artificial antigen, preparation method and application thereof | |
| CN116840473A (en) | Double-antibody sandwich ELISA detection method and kit for detecting Cryptosporidium parvum | |
| CN110927376B (en) | Magnetic immunochemistry luminescence detection kit of olaquindox and application thereof | |
| CN116199652A (en) | Anti-5-hydroxymethylfurfural hapten, complete antigen and antibody and preparation method thereof | |
| CN109734675B (en) | Method and product suitable for detecting olaquindox content in veterinary drug preparation | |
| CN114752568A (en) | Furosemide monoclonal antibody, hybridoma cell strain and application | |
| CN102154218B (en) | Hybridoma cell strain 4e9 and monoclonal antibody produced by using hybridoma cell strain 4e9 | |
| CN113754568A (en) | Acid orange I hapten, artificial antigen and its synthesis and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |