CN116813537A - Preparation method and application of novel flunixin hapten - Google Patents
Preparation method and application of novel flunixin hapten Download PDFInfo
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- CN116813537A CN116813537A CN202310683563.4A CN202310683563A CN116813537A CN 116813537 A CN116813537 A CN 116813537A CN 202310683563 A CN202310683563 A CN 202310683563A CN 116813537 A CN116813537 A CN 116813537A
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- hapten
- flunixin
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- 229960000588 flunixin Drugs 0.000 title claims abstract description 56
- NOOCSNJCXJYGPE-UHFFFAOYSA-N flunixin Chemical compound C1=CC=C(C(F)(F)F)C(C)=C1NC1=NC=CC=C1C(O)=O NOOCSNJCXJYGPE-UHFFFAOYSA-N 0.000 title claims abstract description 49
- 238000002360 preparation method Methods 0.000 title claims abstract description 21
- 239000000427 antigen Substances 0.000 claims abstract description 32
- 102000036639 antigens Human genes 0.000 claims abstract description 32
- 108091007433 antigens Proteins 0.000 claims abstract description 32
- 238000006243 chemical reaction Methods 0.000 claims abstract description 31
- 238000000034 method Methods 0.000 claims abstract description 20
- JSXNJGKWSWRIGA-UHFFFAOYSA-N 5-hydroxy-2-[2-methyl-3-(trifluoromethyl)anilino]pyridine-3-carboxylic acid Chemical compound C1=CC=C(C(F)(F)F)C(C)=C1NC1=NC=C(O)C=C1C(O)=O JSXNJGKWSWRIGA-UHFFFAOYSA-N 0.000 claims abstract description 14
- 102000014914 Carrier Proteins Human genes 0.000 claims abstract description 9
- 108010078791 Carrier Proteins Proteins 0.000 claims abstract description 9
- 239000002207 metabolite Substances 0.000 claims abstract description 7
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- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 4
- 239000000126 substance Substances 0.000 claims abstract description 4
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 claims description 36
- 210000004027 cell Anatomy 0.000 claims description 32
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 26
- 238000005406 washing Methods 0.000 claims description 23
- 230000003053 immunization Effects 0.000 claims description 19
- 238000002649 immunization Methods 0.000 claims description 19
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 18
- 241000699670 Mus sp. Species 0.000 claims description 18
- 238000001035 drying Methods 0.000 claims description 16
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- 206010035226 Plasma cell myeloma Diseases 0.000 claims description 13
- 201000000050 myeloid neoplasm Diseases 0.000 claims description 13
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- 238000000576 coating method Methods 0.000 claims description 12
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- 238000002156 mixing Methods 0.000 claims description 10
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- 239000012074 organic phase Substances 0.000 claims description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 8
- -1 4-nitrofluorooctyl Chemical group 0.000 claims description 7
- 239000000047 product Substances 0.000 claims description 7
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 6
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 claims description 6
- 230000007910 cell fusion Effects 0.000 claims description 6
- 239000012043 crude product Substances 0.000 claims description 6
- 238000000502 dialysis Methods 0.000 claims description 6
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- 210000004989 spleen cell Anatomy 0.000 claims description 5
- WOFZRBCITDVRON-UHFFFAOYSA-N 2-chloro-5-nitronicotinic acid Chemical compound OC(=O)C1=CC([N+]([O-])=O)=CN=C1Cl WOFZRBCITDVRON-UHFFFAOYSA-N 0.000 claims description 4
- 206010003445 Ascites Diseases 0.000 claims description 4
- 238000012216 screening Methods 0.000 claims description 4
- 230000002194 synthesizing effect Effects 0.000 claims description 4
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Substances CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 claims description 3
- TWLDBACVSHADLI-UHFFFAOYSA-N 2-methyl-3-(trifluoromethyl)aniline Chemical compound CC1=C(N)C=CC=C1C(F)(F)F TWLDBACVSHADLI-UHFFFAOYSA-N 0.000 claims description 3
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims description 3
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 claims description 3
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 3
- 125000003277 amino group Chemical group 0.000 claims description 3
- 125000003118 aryl group Chemical group 0.000 claims description 3
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- 125000000487 histidyl group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C([H])=N1 0.000 claims description 3
- 239000005457 ice water Substances 0.000 claims description 3
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- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 claims description 3
- 108091003079 Bovine Serum Albumin 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
- 108010058846 Ovalbumin Proteins 0.000 claims description 2
- 229940098773 bovine serum albumin Drugs 0.000 claims description 2
- 230000002708 enhancing effect Effects 0.000 claims description 2
- 229940092253 ovalbumin Drugs 0.000 claims description 2
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- 239000000243 solution Substances 0.000 description 34
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- 229940079593 drug Drugs 0.000 description 3
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- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 3
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
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- 241000283690 Bos taurus Species 0.000 description 1
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- 206010015719 Exsanguination Diseases 0.000 description 1
- 102000009123 Fibrin Human genes 0.000 description 1
- 108010073385 Fibrin Proteins 0.000 description 1
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- MBBZMMPHUWSWHV-BDVNFPICSA-N N-methylglucamine Chemical compound CNC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO MBBZMMPHUWSWHV-BDVNFPICSA-N 0.000 description 1
- 102000004005 Prostaglandin-endoperoxide synthases Human genes 0.000 description 1
- 108090000459 Prostaglandin-endoperoxide synthases Proteins 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
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- 230000006022 acute inflammation Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
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- 229940088710 antibiotic agent Drugs 0.000 description 1
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- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- CVXBEEMKQHEXEN-UHFFFAOYSA-N carbaryl Chemical compound C1=CC=C2C(OC(=O)NC)=CC=CC2=C1 CVXBEEMKQHEXEN-UHFFFAOYSA-N 0.000 description 1
- 229960005286 carbaryl Drugs 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
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- 230000003203 everyday effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 229950003499 fibrin Drugs 0.000 description 1
- 229960000469 flunixin meglumine Drugs 0.000 description 1
- MGCCHNLNRBULBU-WZTVWXICSA-N flunixin meglumine Chemical compound CNC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO.C1=CC=C(C(F)(F)F)C(C)=C1NC1=NC=CC=C1C(O)=O MGCCHNLNRBULBU-WZTVWXICSA-N 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
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- 230000004054 inflammatory process Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
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- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 229960003194 meglumine Drugs 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
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- 230000000144 pharmacologic effect Effects 0.000 description 1
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- 150000003180 prostaglandins Chemical class 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
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- 238000011897 real-time detection Methods 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- UIIMBOGNXHQVGW-UHFFFAOYSA-M sodium bicarbonate Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
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- 208000024891 symptom Diseases 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
- C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
- C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen 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
- C07D213/78—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, e.g. ester or nitrile radicals
- C07D213/79—Acids; Esters
- C07D213/80—Acids; Esters in position 3
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
- C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
- C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen 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
- C07D213/78—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, e.g. ester or nitrile radicals
- C07D213/79—Acids; Esters
- C07D213/803—Processes of preparation
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
- G01N33/9486—Analgesics, e.g. opiates, aspirine
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Gastroenterology & Hepatology (AREA)
- Food Science & Technology (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Pharmacology & Pharmacy (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Emergency Medicine (AREA)
- Pain & Pain Management (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a preparation method and application of a novel flunixin hapten, which relates to the technical field of chemistry, designs and synthesizes a small molecular target analyte hapten, is coupled with carrier protein to prepare an effective artificial antigen, and an immune animal prepares a specific antibody for the small molecular analyte, so that the specific immunological reaction of the antigen antibody and the amplification effect of a marker which is easy to detect and identify are utilized to qualitatively and quantitatively detect the ultra-micro small molecular target in a sample, the method can be used for sample determination, and a monoclonal antibody prepared by the hapten can specifically identify flunixin and 5-hydroxyflunixin serving as a metabolite thereof, so that the hapten furthest maintains the common chemical structure of the two, improves the specificity and sensitivity of the hapten, and also adds a reaction site for antigen preparation.
Description
Technical Field
The invention relates to the technical field of chemistry, in particular to a preparation method and application of a novel flunixin hapten.
Background
Flunixin is a special non-steroidal anti-inflammatory drug for animals, and is usually mixed with meglumine serving as a stabilizer in an equal proportion to form a stable flunixin Xin Pujia amine salt. The pharmacological action mechanism is mainly to reduce the generation of inflammatory mediators such as prostaglandin and the like by inhibiting cyclooxygenase so as to play roles of relieving fever, easing pain and resisting inflammation. Clinically, the traditional Chinese medicine composition is often used for acute inflammation of large animals such as pigs, cows, horses and the like caused by various disease infections; in addition, the flunixin meglumine is combined with antibiotics, has remarkable drug synergistic effect, can effectively improve clinical symptoms of diseases, has been widely applied in a plurality of countries including China since the successful development of the 90 th century, and becomes a non-steroidal anti-inflammatory drug with the largest clinical use amount for veterinarians at home and abroad.
At present, liquid chromatography-mass spectrometry/mass spectrometry is mainly adopted for detecting the residue of flunixin in animal-derived foods in the national industry standard. The method has strong specificity and high sensitivity, but the sample pretreatment is complex and complicated, the detection time is long, the cost is high, and the popularization and the use are limited. The immunoassay method is a qualitative and quantitative analysis method based on the specific reaction of antigen and antibody, and the method has the advantages of strong specificity, high sensitivity, quick reaction, simple operation and suitability for real-time detection of a large number of samples on site.
In limited studies involving the preparation of flunixin Xin Rengong antigens and antibodies, either free flunixin was used as hapten or the metabolite 5-hydroxyflunixin was used as hapten; the two have carboxyl groups, and can be coupled with protein without modification. However, the antibodies immunized by the two antibodies cannot recognize flunixin and 5-hydroxyflunixin simultaneously or have low cross-reaction rate.
Aiming at the problems, the prior device is improved, and a preparation method and application of the novel flunixin hapten are provided.
Disclosure of Invention
The invention aims to provide a preparation method and application of a novel flunixin hapten, aiming at detecting the residual total amount of flunixin in food, aiming at flunixin and 5-hydroxyflunixin, a hapten which maintains the complete structure of the flunixin and the 5-hydroxyflunixin and has aromatic amino is synthesized from the beginning, and different carrier proteins are further coupled to be respectively used as artificial immunogens and coating sources, so that a flunixin detection antibody is prepared, and the preparation method has the remarkable advantages of high sensitivity, good specificity, simultaneous identification of the flunixin and the 5-hydroxyflunixin and the like, and can be used for rapidly detecting the residual flunixin and metabolites thereof in animal food.
In order to achieve the above purpose, the present invention provides the following technical solutions: a preparation method of a novel flunixin hapten comprises the following steps:
s1: 2.1g (10 mmoL) of 2-chloro-5-nitronicotinic acid and 2.6g (15 mmoL) of 2-methyl-3-trifluoromethylaniline are taken, and 20mL of DMF is added and stirred for dissolution; then adding 0.8g of sodium hydroxide (20 mmoL), heating to 60 ℃ for reaction for 6-8 hours, cooling to room temperature, adding 50mL of ice water, extracting with ethyl acetate, merging organic phases, drying with anhydrous sodium sulfate, spin-drying the ethyl acetate to obtain a crude product of 5-nitroflunixin, and recrystallizing with ethanol to obtain a pure product;
s2: weighing 1.7g of 4-nitrofluorooctyl (5 mmoL), loading into the bottom of a three-neck round bottom flask with the volume of 250mL, adding 100mL of the three-neck round bottom flask while stirring, adding 2.6g (40 mmoL) of pure zinc powder after the 4-nitrofluorooctyl is dissolved by benzene, adding 8mL of concentrated HCl after stirring uniformly, adding 2.6g (40 mmoL) of pure zinc powder again after stirring at room temperature for 30min, stirring at room temperature for reaction for 8h, and monitoring the reaction by a thin-plate chromatography (TLC) until the reaction is complete;
s3: after the reaction was completed, 100mL of cold water was added to the reaction system, and neutralized to weak acidity (ph=6-7) with 2m noh solution;
s4: standing for 1h, separating and retaining benzene layer, extracting water layer with benzene, mixing the extracts, washing organic phase with water, dehydrating and drying with anhydrous sodium sulfate to remove excessive water, distilling the organic phase under reduced pressure to remove benzene, and purifying the obtained crude product with silica gel column chromatography to obtain light yellow solid 5-amino flunixin hapten.
Further, the flunixin Xin Wanquan antigen (flunixin artificial antigen) is a conjugate formed by reacting an aromatic amino group in the hapten with tyrosine or histidine residues in the carrier protein to form a diazonium bond connection, and the carrier protein is at least one of bovine serum albumin, human serum albumin, hemocyanin and ovalbumin.
A preparation method of a novel flunixin complete antigen comprises the following steps:
s01: synthesizing hapten 5-amino flunixin;
s02: synthesizing hapten activated ester;
s03: synthesis of artificial antigen.
Further, S02 includes the steps of:
s021: in a 5mL glass bottle, 30mg of 5-amino flunixin is taken and dissolved by 1mL of N, N-dimethylformamide to prepare solution A;
s022: respectively weighing 20mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and 12mg of N-hydroxysuccinimide, mixing the two, and dissolving the two with 0.5mL of N, N-dimethylformamide to prepare solution B;
s023: and (3) dropwise adding the solution B into the solution A at normal temperature, stirring overnight at room temperature, and stopping the reaction to obtain the hapten activated ester.
Further, S03 includes the steps of:
s031: hapten activated ester 4mg is taken and added into PBS (0.01 mol/L, pH=7.4) solution containing 5mg of hemocyanin, and the mixture is reacted overnight under the condition of 4 ℃;
s032: then, the reaction solution is filled into a dialysis bag, and is fully dialyzed by 0.01mol/LPBS solution to obtain artificial antigen (immunogen);
s033: 25mg of hapten activated ester is taken and added into a PBS (0.01 mol/L, pH=7.4) solution containing 100mgBSA, and the mixture is reacted overnight under the condition of 4 ℃;
s034: then, the reaction solution was packed into a dialysis bag, and was thoroughly dialyzed with a 0.01mol/LPBS solution to obtain an artificial antigen (coating antigen).
The application of the novel flunixin hapten and the preparation method for preparing the antibody by using the novel flunixin hapten comprise the following steps:
s001: selecting female Balb/C mice of proper age for immunization, measuring the titer and the inhibition rate of antiserum after the 4 th immunization and the 5 th immunization, and enhancing the immunization 3 days before cell fusion;
s002: fusing the spleen cells and myeloma cells of the mice after the immunity enhancement, screening out hybridoma cells capable of secreting specific antibodies, and performing amplification culture;
s003: injecting the expanded hybridoma cells into mice injected with paraffin in advance, and collecting ascites to obtain monoclonal antibodies.
Furthermore, the monoclonal antibody prepared from the hapten can specifically identify flunixin and a metabolite 5-hydroxyflunixin thereof, the hapten furthest maintains the common chemical structure of the flunixin and the metabolite 5-hydroxyflunixin, the hapten is used as a basis, is coupled with carrier protein and is used for preparing antibodies by immunized mice, and a kit product is developed based on the antigen and the antibodies.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a preparation method and application of a novel flunixin hapten, which is characterized in that a small molecular target analyte hapten is designed and synthesized and coupled with carrier protein to prepare an effective artificial antigen, an immune animal is used for preparing a specific antibody for the small molecular analyte, and the specific immunological reaction of the antigen antibody and the amplification effect of a marker which is easy to detect and identify are utilized, so that the ultra-micro small molecular target in a qualitative and quantitative detection sample can be used for sample determination. The kit product is developed based on antigen and antibody, and provides a high-efficiency and reliable detection method for detecting the residue of flunixin in food.
Drawings
FIG. 1 is a flow chart of the preparation method of the novel flunixin hapten.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
Referring to FIG. 1, hapten synthesis is shown
The hapten with a common structure of flunixin and 5-hydroxyflunixin is prepared by taking 2-chloro-5-nitronicotinic acid as an initial raw material through 2-step chemical synthesis, the molecular weight of the hapten is 311.26, and the specific preparation method is as follows:
(1) 2.1g (about 10 mmoL) of 2-chloro-5-nitronicotinic acid and 2.6g (about 15 mmoL) of 2-methyl-3-trifluoromethylaniline are taken, and 20mL of DMF is added and stirred for dissolution; then adding 0.8g of sodium hydroxide (about 20 mmoL), heating to 60 ℃ for reaction for 6-8 hours, cooling to room temperature, adding 50mL of ice water, extracting with ethyl acetate, combining organic phases, drying with anhydrous sodium sulfate, spin-drying the ethyl acetate to obtain a crude product of 5-nitrofluoronixin, and recrystallizing with ethanol to obtain a pure product;
(2) Weighing 1.7g of 4-nitrofluorooctyl (5 mmoL), loading into the bottom of a three-neck round bottom flask with the volume of 250mL, adding 100mL of the three-neck round bottom flask while stirring, adding 2.6g (40 mmoL) of pure zinc powder after the 4-nitrofluorooctyl is dissolved by benzene, adding 8mL of concentrated HCl after stirring uniformly, adding 2.6g (40 mmoL) of pure zinc powder again after stirring at room temperature for 30min, stirring at room temperature for reaction for 8h, and monitoring the reaction by a thin-plate chromatography (TLC) until the reaction is complete; after the reaction was completed, 100mL of cold water was added to the reaction system, and neutralized to weak acidity (ph=6-7) with 2m noh solution; standing for 1h, separating and retaining benzene layer, extracting water layer with benzene, mixing the extracts, washing organic phase with water, dehydrating with anhydrous sodium sulfate, and drying to remove excessive water; the organic phase is distilled under reduced pressure to remove benzene, and the obtained crude product is purified by silica gel column chromatography to obtain light yellow solid 5-amino flunixin hapten.
Synthesis of hapten activated esters
In a 5mL glass bottle, 30mg of hapten is taken and dissolved by 1mL of N, N-dimethylformamide to prepare solution A; respectively weighing 20mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and 12mg of N-hydroxysuccinimide, mixing the two, and dissolving the two with 0.5mL of N, N-dimethylformamide to prepare solution B; solution B was added dropwise to solution A under ice-bath conditions and then stirred overnight at room temperature. And stopping the reaction to obtain hapten activated ester.
Synthesis of artificial antigen
Adding hapten activated ester 4mg into PBS (0.01 mol/L, pH=7.4) solution containing 5mg hemocyanin, and reacting overnight under the condition of 4 ℃; then, the reaction solution is filled into a dialysis bag, and is fully dialyzed by 0.01mol/LPBS solution to obtain artificial antigen (immunogen); 16mg of hapten activated ester is taken and added into a PBS (0.01 mol/L, pH=7.4) solution containing 100mgBSA, and the mixture is reacted overnight under the condition of 4 ℃; then, the reaction solution was packed into a dialysis bag, and was thoroughly dialyzed with a 0.01mol/LPBS solution to obtain an artificial antigen (coating antigen).
Example 2
Preparation of monoclonal antibodies
1. Immunization of animals
Balb/C female mice were selected for immunization and were numbered 1, 2, 3 and 4, respectively, about 8 weeks old. Immunization was performed by subcutaneous multipoint injection of the back of the neck.
Immunization procedure: primary immunization: to improve the immunity of the antigen, the immunogen emulsified by Freund's complete adjuvant is mixed with the same volume of the immunogen and Freund's complete adjuvant for emulsification, and the dosage is 0.1mg. Boosting: boosting was performed at days 14 and 35 after primary immunization at a dose of 0.1mg, and the emulsification method was the same. The animals were then boosted once every 21 days for 6 total immunizations, and serum titers and specificity assays were performed on the test blood samples 7 days after each immunization, starting with the 2 nd boost.
Wherein, the serum potency detection method comprises the following steps:
1) Dissolving the prepared fluorous Xin Bao in a buffer solution of pH9.6Na2CO3-NaHCO3, diluting the fluorous Xin Bao to 1.0 mug/mL as a coating solution, adding 100 mug of the fluorous Xin Bao into each well of a 96-well microplate, standing overnight at 4 ℃ or incubating for 2-3 hours at 37 ℃, and washing for 1 time by using PBST (phosphate buffer solution) of 0.05% (V/V) and Tween20 washing liquid;
2) 200 mu L of PBS blocking solution of 3% skimmed milk powder is added into each hole, the mixture is blocked for 2 hours, and washing is carried out for 1 time by using washing liquid;
3) Adding antiserum and standard substance: gradient dilution of antisera; the carbaryl standard is dissolved in a proper amount of DMF to prepare a 1.0mg/mL standard solution, and the solution is preserved at 4 ℃ for standby. The titers are as follows: adding 50uL of blank diluent, 50uL of antiserum subjected to gradient dilution and 50uL of goat anti-mouse enzyme-labeled secondary antibody into each hole; inhibition column: 50uL of standard solution, 50uL of gradient diluted antiserum and 50uL of goat anti-mouse enzyme-labeled secondary antibody are added into each hole, and the mixture is incubated for 30 minutes at 37 ℃;
4) Washing the microplate with washing liquid for 3 times, throwing away liquid in the microplate, spin-drying by a microplate dehydrator, adding 50uL of substrate A and 50uL of substrate B, and reacting for 15 minutes at room temperature;
5) Reading and measuring: absorbance at two wavelengths of 450nm and 630 nm; the dilution factor of the antiserum with the absorbance value within the range of 1.5-2.0 is selected as the antiserum titer, and the effect of the antiserum is obtained by the inhibition rate.
2. Antiserum efficacy analysis
(1) Starting from the 3 rd booster immunization, taking 30uL of blood from the tail of the mice on the 8 th day after each immunization, centrifuging to obtain antiserum, and preserving at-20 ℃ for later use;
(2) The potency and specificity of antisera were determined by indirect enzyme-linked immunosorbent assay as follows:
s1, coating: diluting 5mg/mL of coating antigen with coating buffer solution for 5000 times, preparing a blank control group, adding 100 uL/hole into an ELISA plate, placing the ELISA plate at a refrigerator at 4 ℃ for incubation overnight, and washing with PBST, namely phosphate buffer solution 0.05% (V/V), tween20 washing liquid for 1 time;
s2, closing: pouring out the liquid in the holes, washing the plate for 1 time, adding 200 mu L of washing liquid into each hole, spin-drying, adding 200 mu L of sealing liquid into each hole, sealing for 2 hours at 37 ℃, spin-drying, and placing in a refrigerator at 4 ℃ for standby;
s3, sample adding: adding 50uL of standard dilution, diluted enzyme-labeled secondary antibody and diluted serum into each control hole; adding 50uL of diluted standard, diluted enzyme-labeled secondary antibody and diluted serum into each sample hole; shaking and mixing uniformly, reacting for 30min at 37 ℃, washing the plate 3 times by a plate washing machine, adding 200 mu L of washing liquid into each hole, and spin-drying;
s4, color development: 50 mu L of each of the developing solutions A and B is added to each well, and after the mixture is placed in a 37 ℃ oven for developing for 15min, 50 mu L of stop solution (1 moL/L H2SO 4) is added to each well;
s5, measuring: the absorbance at A450nm was measured for each well using an ELISA. The dilution multiple of the antiserum with the absorbance value within the range of 1.5-2.0 is selected as the antiserum titer, the effect of the antiserum is obtained by the inhibition rate, and the higher the inhibition rate is, the higher the sensitivity of the antibody to the drug is.
3. Cell fusion and selection of positive hybridomas
(1) Resuscitates myeloma cells: taking myeloma cells out of liquid nitrogen, quickly placing the myeloma cells into a water bath at 37 ℃ for thawing, centrifuging at 1000r/min for 5min after thawing, pouring the supernatant in an ultra-clean workbench, adding about 1mL of complete culture solution into cell sediment, blowing off the cells, taking out the myeloma cells by a pipette, mixing the myeloma cells with the complete culture solution, placing the myeloma cells into a 25cm < 2 > culture flask, expanding the myeloma cells into 4-6 flasks, and replacing the myeloma cells with the solution for many times, wherein when the bottom of the cells of each culture flask are paved, the myeloma cells can be used for cell fusion;
(2) Feeder cell preparation: balb/C mice were cervical removed and sacrificed 1 day prior to cell fusion, and were immersed in 75% alcohol for 5min before being transferred to an ultra clean bench for dissection. Cutting abdomen, peeling abdomen skin, cutting peritoneum, taking out spleen, transferring into 9cm culture dish, adding DMEM basic culture medium, repeatedly sucking and flushing with syringe, transferring flushing liquid into 50mL centrifuge tube, centrifuging at 1200r/min for 5min, discarding supernatant, resuspending bottom cell sediment with complete culture medium, and adding 96-well cell culture plate with 100uL per well;
(3) Spleen cell preparation: balb/c mice with qualified blood after multiple immunization are subjected to orbital exsanguination, serum is collected, soaked in 75% alcohol for 5min for sterilization, and then transferred to an ultra-clean workbench for dissection. The spleens were removed aseptically, rinsed with DMEM basal medium and placed in petri dishes for use. The culture medium was aspirated with a disposable syringe, the removed spleen was gripped with forceps by the left hand, the syringe was inserted into the spleen by the right hand, and the culture medium was slowly injected to wash out cells in the spleen. The procedure was repeated until the spleen changed from dark red to colorless and transparent, and the spleen was discarded. The mixed culture medium is collected into a 50mL centrifuge tube, and is centrifuged after sealing, and the rotation speed is 1200r/min for 6min. Centrifuging, and removing supernatant for later use;
(4) Cell fusion: the myeloma cells and the immune spleen cells which are centrifuged to remove the supernatant are mixed in a centrifuge tube according to the ratio of about 1:6, 25mL of basic culture medium is added, and after sealing, the mixture is centrifuged at 1500r/min for 8min. The supernatant was discarded after centrifugation for use. The supernatant was discarded from the centrifugation-mixed myeloma cells and immune spleen cells, and the excess medium was sucked down by a centrifuge nozzle with a gun. The precipitated cells were flicked with fingers, the centrifuge tube was placed in 37℃warm water, 1mL of PEG preheated to 37℃was sucked with the gun head, PEG was slowly added to the precipitated cells within 1min, each drop of PEG was gently stirred with the gun head for mixing, left stand for 1min, the complete medium was preheated, 10mL was added within 2min with gentle stirring, and PEG was split along the wall. Centrifuging at 1000r/min for 10min, pouring out the supernatant, adding HAT complete culture medium, gently sucking liquid with elbow pipette, stirring gently, and adding into 10 96-well culture plates containing feeder cells prepared the day before. Maintaining the same area of HAT medium with feeder cells added to each well as HAT medium containing fusion cells;
(5) Screening of positive hybridomas: and (3) changing the liquid by using the HAT culture medium once within 3-4 days after fusion, extracting the supernatant in the porous culture plate after 7 days, detecting the specific antibody in the culture solution by using indirect ELISA, selecting positive hybridoma cells with high titer and strong drug inhibition, screening out positive holes with the best fusion effect, and making a mark. Under the aseptic condition, picking cells growing in positive holes by using a microscope, transferring the cells into a 96-hole culture plate which is plated with feeder cells in advance, cloning each original hole into 96 holes, taking supernatant after the cells are adhered to the walls and grow to the bottoms of 1/4 holes, performing ICELISA detection, taking positive strong people as a measurement index, subcloning by using a limiting dilution method, repeating the steps for 3-4 rounds (note that the positive hole cells picked out in each round need to be expanded for culture and then frozen for standby), until each hole of each plate is positive and the detection titer and inhibition are similar, and establishing a hybridoma cell line successfully until the hybridoma cell line can stably secrete uniform antibodies. And (3) selecting single cell clones, and transferring the single cell clones detected to be positive to a 24-hole cell culture plate or a cell culture dish for expansion culture, and timely freezing.
4. Large-scale preparation of monoclonal antibodies
After obtaining hybridoma cell clones that secrete a specific monoclonal antibody, the monoclonal antibody is generally prepared in large quantities by an in vitro culture method and an in vivo animal induction monoclonal antibody method. The conventional practice is as follows: the liquid Dan is injected into the abdominal cavity of more than ten Balb/c mice with the age of more than 8 weeks in advance, the dosage is 0.5 mL/mouse, and the hybridoma cells are injected into the abdominal cavity of the mice after 1-2 weeks. The state of the mice is observed every day after the cells are inoculated, particularly, the abdominal cavity of the mice is swelled from day 7, the ascites is aseptically collected by a disposable injector before the mice die, the collected ascites is centrifuged for 10min at 12000r/min, the upper fat and the lower fibrin are removed, the middle layer is collected, the titer and the inhibition rate are measured by an icELISA method, and the mice are preserved at the temperature of minus 20 ℃ for standby after purification.
Example 3
Establishment of indirect competitive enzyme-linked immunoassay flunixin method
1. The indirect competitive enzyme linked immunosorbent assay (iceelisa) reaction specifically comprises the following steps:
s1, coating: diluting 5mg/mL of coating antigen with coating buffer solution for 5000 times, preparing a blank control group, adding 100 uL/hole into an ELISA plate, placing the ELISA plate at a refrigerator at 4 ℃ for incubation overnight, and washing with PBST, namely phosphate buffer solution 0.05% (V/V), tween20 washing liquid for 1 time;
s2, closing: pouring out the liquid in the holes, washing the plate for 1 time, adding 200 mu L of washing liquid into each hole, spin-drying, adding 200 mu L of sealing liquid into each hole, sealing for 2 hours at 37 ℃, spin-drying, and placing in a refrigerator at 4 ℃ for standby;
s3, sample adding: adding 50uL of standard dilution, diluted enzyme-labeled secondary antibody and diluted serum into each control hole; adding 50uL of diluted standard, diluted enzyme-labeled secondary antibody and diluted serum into each sample hole; shaking and mixing uniformly, reacting for 30min at 37 ℃, washing the plate 3 times by a plate washing machine, adding 200 mu L of washing liquid into each hole, and spin-drying;
s4, color development: 50 mu L of each of the developing solutions A and B is added to each well, and after the mixture is placed in a 37 ℃ oven for developing for 15min, 50 mu L of stop solution (1 moL/L H2SO 4) is added to each well;
s5, measuring: measuring the absorbance of each hole A450nm by using an enzyme-linked immunosorbent assay instrument;
s6, calculating: IC50 values of the inhibition curves were calculated using graphpatrism 7.0 fitting module.
2. 2.43ng/mL, 0.81ng/mL, 0.27ng/mL, 0.09ng/mL, 0.03ng/mL of flunixin and 5-hydroxyflunixin Xin Biaozhun product solutions are respectively prepared, and a standard curve of an indirect ELISA method is established. After condition optimization, the method aims at flunixin IC50=0.32 ng/mL, and the linear detection range is 0.03-2.43 ng/mL.
Example 4
Indirect competition ELISA experiments were performed with flunixin and 5-hydroxy as competition standards according to the optimal coating antigen concentration and optimal antiserum dilution obtained in example 3, and the specificity of the flunixin Xin Shan clone antibody was tested, and the half maximal inhibitory concentration (IC 50) and cross-reactivity (CR) values are shown in Table 1.
TABLE 1
Experimental results show that the cloned antibody of the flunixin Xin Shan can simultaneously identify the flunixin and the 5-hydroxyflunixin, has high cross reaction rate, and can be used for detecting the residual total amount of the flunixin and the 5-hydroxyflunixin in food.
It is noted that relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art, who is within the scope of the present invention, should be covered by the protection scope of the present invention by making equivalents and modifications to the technical solution and the inventive concept thereof.
Claims (7)
1. The preparation method of the novel flunixin hapten is characterized by comprising the following steps of:
s1: 2.1g (10 mmoL) of 2-chloro-5-nitronicotinic acid and 2.6g (15 mmoL) of 2-methyl-3-trifluoromethylaniline are taken, and 20mL of DMF is added and stirred for dissolution; then adding 0.8g of sodium hydroxide (20 mmoL), heating to 60 ℃ for reaction for 6-8 hours, cooling to room temperature, adding 50mL of ice water, extracting with ethyl acetate, merging organic phases, drying with anhydrous sodium sulfate, spin-drying the ethyl acetate to obtain a crude product of 5-nitroflunixin, and recrystallizing with ethanol to obtain a pure product;
s2: weighing 1.7g of 4-nitrofluorooctyl (5 mmoL), loading into the bottom of a three-neck round bottom flask with the volume of 250mL, adding 100mL of the three-neck round bottom flask while stirring, adding 2.6g (40 mmoL) of pure zinc powder after the 4-nitrofluorooctyl is dissolved by benzene, adding 8mL of concentrated HCl after stirring uniformly, adding 2.6g (40 mmoL) of pure zinc powder again after stirring at room temperature for 30min, stirring at room temperature for reaction for 8h, and monitoring the reaction by a thin-plate chromatography (TLC) until the reaction is complete;
s3: after the reaction was completed, 100mL of cold water was added to the reaction system, and neutralized to weak acidity (ph=6-7) with 2m noh solution;
s4: standing for 1h, separating and retaining benzene layer, extracting water layer with benzene, mixing the extracts, washing organic phase with water, dehydrating and drying with anhydrous sodium sulfate to remove excessive water, distilling the organic phase under reduced pressure to remove benzene, and purifying the obtained crude product with silica gel column chromatography to obtain light yellow solid 5-amino flunixin hapten.
2. The method of claim 1, wherein the flunixin hapten is a conjugate of an aromatic amino group in the hapten and a tyrosine or histidine residue in the carrier protein, wherein the carrier protein is selected from at least one of bovine serum albumin, human serum albumin, hemocyanin and ovalbumin, and the aromatic amino group in the hapten reacts with the tyrosine or histidine residue to form a diazo bond.
3. The method for preparing the novel flunixin complete antigen as claimed in claim 2, which comprises the following steps:
s01: synthesizing hapten 5-amino flunixin;
s02: synthesizing hapten activated ester;
s03: synthesis of artificial antigen.
4. A method for preparing a novel flunixin complete antigen according to claim 3, wherein S02 comprises the steps of:
s021: in a 5mL glass bottle, 30mg of 5-amino flunixin is taken and dissolved by 1mL of N, N-dimethylformamide to prepare solution A;
s022: respectively weighing 20mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and 12mg of N-hydroxysuccinimide, mixing the two, and dissolving the two with 0.5mL of N, N-dimethylformamide to prepare solution B;
s023: and (3) dropwise adding the solution B into the solution A at normal temperature, stirring overnight at room temperature, and stopping the reaction to obtain the hapten activated ester.
5. A method for preparing a novel flunixin complete antigen according to claim 3, wherein S03 comprises the steps of:
s031: hapten activated ester 4mg is taken and added into PBS (0.01 mol/L, pH=7.4) solution containing 5mg of hemocyanin, and the mixture is reacted overnight under the condition of 4 ℃;
s032: then, the reaction solution is filled into a dialysis bag, and is fully dialyzed by 0.01mol/LPBS solution to obtain artificial antigen (immunogen);
s033: 25mg of hapten activated ester is taken and added into a PBS (0.01 mol/L, pH=7.4) solution containing 100mgBSA, and the mixture is reacted overnight under the condition of 4 ℃;
s034: then, the reaction solution was packed into a dialysis bag, and was thoroughly dialyzed with a 0.01mol/LPBS solution to obtain an artificial antigen (coating antigen).
6. The application of the novel flunixin hapten is characterized in that the preparation method for preparing the antibody by utilizing the novel flunixin hapten comprises the following steps of:
s001: selecting female Balb/C mice of proper age for immunization, measuring the titer and the inhibition rate of antiserum after the 4 th immunization and the 5 th immunization, and enhancing the immunization 3 days before cell fusion;
s002: fusing the spleen cells and myeloma cells of the mice after the immunity enhancement, screening out hybridoma cells capable of secreting specific antibodies, and performing amplification culture;
s003: injecting the expanded hybridoma cells into mice injected with paraffin in advance, and collecting ascites to obtain monoclonal antibodies.
7. The use of a novel flunixin hapten as defined in claim 6, wherein: the monoclonal antibody prepared from the hapten can specifically identify flunixin and a metabolite 5-hydroxyflunixin thereof, the hapten furthest maintains the common chemical structure of the flunixin and the metabolite 5-hydroxyflunixin, the hapten is used as a basis, is coupled with carrier protein, is used for preparing antibodies by immunized mice, and a kit product is developed based on the antigen antibodies.
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|---|---|---|---|---|
| CN102442944A (en) * | 2011-12-14 | 2012-05-09 | 齐鲁动物保健品有限公司 | Preparation method of flunixin |
| CN103694167A (en) * | 2013-12-11 | 2014-04-02 | 威海雅瑞生物科技有限公司 | Method for synthesizing flunixin meglumine |
| CN110498766A (en) * | 2019-08-26 | 2019-11-26 | 北京勤邦生物技术有限公司 | Fluazinam haptens, artificial antigen and antibody and its preparation method and application |
| CN113372264A (en) * | 2021-06-16 | 2021-09-10 | 宁夏常晟药业有限公司 | Synthetic method of 2- [ 2-methyl-3- (trifluoromethyl) phenylamino ] nicotinic acid |
| CN114316027A (en) * | 2020-10-10 | 2022-04-12 | 中国农业大学 | Flunixin artificial antigen and preparation method and application thereof |
-
2023
- 2023-06-09 CN CN202310683563.4A patent/CN116813537A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102442944A (en) * | 2011-12-14 | 2012-05-09 | 齐鲁动物保健品有限公司 | Preparation method of flunixin |
| CN103694167A (en) * | 2013-12-11 | 2014-04-02 | 威海雅瑞生物科技有限公司 | Method for synthesizing flunixin meglumine |
| CN110498766A (en) * | 2019-08-26 | 2019-11-26 | 北京勤邦生物技术有限公司 | Fluazinam haptens, artificial antigen and antibody and its preparation method and application |
| CN114316027A (en) * | 2020-10-10 | 2022-04-12 | 中国农业大学 | Flunixin artificial antigen and preparation method and application thereof |
| CN113372264A (en) * | 2021-06-16 | 2021-09-10 | 宁夏常晟药业有限公司 | Synthetic method of 2- [ 2-methyl-3- (trifluoromethyl) phenylamino ] nicotinic acid |
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