CN115232119A - Preparation method of vitamin B1 hapten, immunogen, coating antigen and monoclonal antibody - Google Patents
Preparation method of vitamin B1 hapten, immunogen, coating antigen and monoclonal antibody Download PDFInfo
- Publication number
- CN115232119A CN115232119A CN202110450632.8A CN202110450632A CN115232119A CN 115232119 A CN115232119 A CN 115232119A CN 202110450632 A CN202110450632 A CN 202110450632A CN 115232119 A CN115232119 A CN 115232119A
- Authority
- CN
- China
- Prior art keywords
- vitamin
- mouse
- solution
- monoclonal antibody
- hapten
- 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.)
- Pending
Links
- 229960003495 thiamine Drugs 0.000 title claims abstract description 179
- 239000011691 vitamin B1 Substances 0.000 title claims abstract description 164
- 229930003451 Vitamin B1 Natural products 0.000 title claims abstract description 163
- 235000010374 vitamin B1 Nutrition 0.000 title claims abstract description 163
- 230000002163 immunogen Effects 0.000 title claims abstract description 35
- 239000000427 antigen Substances 0.000 title claims abstract description 28
- 102000036639 antigens Human genes 0.000 title claims abstract description 28
- 108091007433 antigens Proteins 0.000 title claims abstract description 28
- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 title claims abstract 49
- 239000011248 coating agent Substances 0.000 title claims description 30
- 238000000576 coating method Methods 0.000 title claims description 30
- 210000004027 cell Anatomy 0.000 claims abstract description 47
- 210000004408 hybridoma Anatomy 0.000 claims abstract description 27
- 238000001514 detection method Methods 0.000 claims abstract description 25
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims abstract description 17
- 229940098773 bovine serum albumin Drugs 0.000 claims abstract description 17
- 210000004989 spleen cell Anatomy 0.000 claims abstract description 17
- 206010035226 Plasma cell myeloma Diseases 0.000 claims abstract description 15
- 201000000050 myeloid neoplasm Diseases 0.000 claims abstract description 15
- 108010058846 Ovalbumin Proteins 0.000 claims abstract description 7
- 229940092253 ovalbumin Drugs 0.000 claims abstract description 7
- 230000003248 secreting effect Effects 0.000 claims abstract description 7
- 239000000243 solution Substances 0.000 claims description 60
- 241000699666 Mus <mouse, genus> Species 0.000 claims description 39
- 238000000034 method Methods 0.000 claims description 35
- 238000002965 ELISA Methods 0.000 claims description 29
- 210000000683 abdominal cavity Anatomy 0.000 claims description 23
- 238000006243 chemical reaction Methods 0.000 claims description 20
- 239000006285 cell suspension Substances 0.000 claims description 19
- 230000003053 immunization Effects 0.000 claims description 19
- 238000012216 screening Methods 0.000 claims description 17
- 238000002649 immunization Methods 0.000 claims description 14
- 206010003445 Ascites Diseases 0.000 claims description 13
- 239000007853 buffer solution Substances 0.000 claims description 11
- 230000007910 cell fusion Effects 0.000 claims description 11
- 238000002156 mixing Methods 0.000 claims description 10
- 238000003756 stirring Methods 0.000 claims description 9
- XOJVVFBFDXDTEG-UHFFFAOYSA-N pristane Chemical compound CC(C)CCCC(C)CCCC(C)CCCC(C)C XOJVVFBFDXDTEG-UHFFFAOYSA-N 0.000 claims description 8
- 241000283707 Capra Species 0.000 claims description 7
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 claims description 7
- 238000002835 absorbance Methods 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 7
- 239000007787 solid Substances 0.000 claims description 7
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 6
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 6
- 239000002671 adjuvant Substances 0.000 claims description 6
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 6
- 230000008569 process Effects 0.000 claims description 6
- WMFATTFQNRPXBQ-UHFFFAOYSA-N 2-bromopentanoic acid Chemical compound CCCC(Br)C(O)=O WMFATTFQNRPXBQ-UHFFFAOYSA-N 0.000 claims description 5
- 241000699670 Mus sp. Species 0.000 claims description 5
- 210000004988 splenocyte Anatomy 0.000 claims description 5
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 4
- 238000011161 development Methods 0.000 claims description 4
- 230000018109 developmental process Effects 0.000 claims description 4
- 230000004927 fusion Effects 0.000 claims description 4
- 238000011534 incubation Methods 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 229920001223 polyethylene glycol Polymers 0.000 claims description 4
- 239000006228 supernatant Substances 0.000 claims description 4
- 230000003698 anagen phase Effects 0.000 claims description 3
- 239000000872 buffer Substances 0.000 claims description 3
- 229910000027 potassium carbonate Inorganic materials 0.000 claims description 3
- 239000000725 suspension Substances 0.000 claims description 3
- YOETUEMZNOLGDB-UHFFFAOYSA-N 2-methylpropyl carbonochloridate Chemical compound CC(C)COC(Cl)=O YOETUEMZNOLGDB-UHFFFAOYSA-N 0.000 claims description 2
- 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 claims description 2
- 238000012286 ELISA Assay Methods 0.000 claims description 2
- 229920001030 Polyethylene Glycol 4000 Polymers 0.000 claims description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical class N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 2
- 238000010276 construction Methods 0.000 claims description 2
- 238000011081 inoculation Methods 0.000 claims description 2
- 238000007789 sealing Methods 0.000 claims description 2
- 230000001804 emulsifying effect Effects 0.000 claims 2
- 230000008878 coupling Effects 0.000 claims 1
- 238000010168 coupling process Methods 0.000 claims 1
- 238000005859 coupling reaction Methods 0.000 claims 1
- 239000012530 fluid Substances 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 claims 1
- 230000000638 stimulation Effects 0.000 claims 1
- 238000011725 BALB/c mouse Methods 0.000 abstract description 17
- JZRWCGZRTZMZEH-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N JZRWCGZRTZMZEH-UHFFFAOYSA-N 0.000 description 146
- 238000005406 washing Methods 0.000 description 18
- 235000019157 thiamine Nutrition 0.000 description 15
- 239000011721 thiamine Substances 0.000 description 15
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 14
- 210000002966 serum Anatomy 0.000 description 12
- 102000004190 Enzymes Human genes 0.000 description 11
- 108090000790 Enzymes Proteins 0.000 description 11
- GEYOCULIXLDCMW-UHFFFAOYSA-N 1,2-phenylenediamine Chemical compound NC1=CC=CC=C1N GEYOCULIXLDCMW-UHFFFAOYSA-N 0.000 description 10
- 238000012360 testing method Methods 0.000 description 8
- 229940088594 vitamin Drugs 0.000 description 8
- 229930003231 vitamin Natural products 0.000 description 8
- 235000013343 vitamin Nutrition 0.000 description 8
- 239000011782 vitamin Substances 0.000 description 8
- 150000003722 vitamin derivatives Chemical class 0.000 description 8
- 239000011259 mixed solution Substances 0.000 description 7
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 208000024827 Alzheimer disease Diseases 0.000 description 5
- 102000014914 Carrier Proteins Human genes 0.000 description 5
- 108010078791 Carrier Proteins Proteins 0.000 description 5
- 238000004113 cell culture Methods 0.000 description 5
- 238000010367 cloning Methods 0.000 description 5
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 5
- 238000007865 diluting Methods 0.000 description 5
- 238000012986 modification Methods 0.000 description 5
- 230000004048 modification Effects 0.000 description 5
- 239000012089 stop solution Substances 0.000 description 5
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 235000008170 thiamine pyrophosphate Nutrition 0.000 description 4
- 239000011678 thiamine pyrophosphate Substances 0.000 description 4
- 229960002363 thiamine pyrophosphate Drugs 0.000 description 4
- YXVCLPJQTZXJLH-UHFFFAOYSA-N thiamine(1+) diphosphate chloride Chemical group [Cl-].CC1=C(CCOP(O)(=O)OP(O)(O)=O)SC=[N+]1CC1=CN=C(C)N=C1N YXVCLPJQTZXJLH-UHFFFAOYSA-N 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 230000004913 activation Effects 0.000 description 3
- 230000002860 competitive effect Effects 0.000 description 3
- 239000012228 culture supernatant Substances 0.000 description 3
- 235000020955 thiamine monophosphate Nutrition 0.000 description 3
- 239000011621 thiamine monophosphate Substances 0.000 description 3
- GUGWNSHJDUEHNJ-UHFFFAOYSA-N thiamine(1+) monophosphate chloride Chemical compound [Cl-].CC1=C(CCOP(O)(O)=O)SC=[N+]1CC1=CN=C(C)N=C1N GUGWNSHJDUEHNJ-UHFFFAOYSA-N 0.000 description 3
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-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
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 125000003277 amino group Chemical group 0.000 description 2
- 210000004958 brain cell Anatomy 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 238000005034 decoration Methods 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 230000037149 energy metabolism Effects 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 210000002540 macrophage Anatomy 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 208000030159 metabolic disease Diseases 0.000 description 2
- NQPDZGIKBAWPEJ-UHFFFAOYSA-N pentanoic acid group Chemical group C(CCCC)(=O)O NQPDZGIKBAWPEJ-UHFFFAOYSA-N 0.000 description 2
- 239000003208 petroleum Substances 0.000 description 2
- 238000010791 quenching Methods 0.000 description 2
- 230000000171 quenching effect Effects 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 238000010992 reflux Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- IWLROWZYZPNOFC-UHFFFAOYSA-O thiamine triphosphate Chemical compound CC1=C(CCOP(O)(=O)OP(O)(=O)OP(O)(O)=O)SC=[N+]1CC1=CN=C(C)N=C1N IWLROWZYZPNOFC-UHFFFAOYSA-O 0.000 description 2
- 206010000060 Abdominal distension Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 102000018251 Hypoxanthine Phosphoribosyltransferase Human genes 0.000 description 1
- 108010091358 Hypoxanthine Phosphoribosyltransferase Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- FZWLAAWBMGSTSO-UHFFFAOYSA-N Thiazole Chemical group C1=CSC=N1 FZWLAAWBMGSTSO-UHFFFAOYSA-N 0.000 description 1
- 102000006601 Thymidine Kinase Human genes 0.000 description 1
- 108020004440 Thymidine kinase Proteins 0.000 description 1
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
- MUUGCDGGIVCSDT-UHFFFAOYSA-N [NH4+].[NH4+].[O-]S([O-])(=O)=O.CCCCCCCC(O)=O Chemical compound [NH4+].[NH4+].[O-]S([O-])(=O)=O.CCCCCCCC(O)=O MUUGCDGGIVCSDT-UHFFFAOYSA-N 0.000 description 1
- 230000003187 abdominal effect Effects 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000003167 anti-vitamin Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000010370 cell cloning Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 239000005515 coenzyme Substances 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- XPPKVPWEQAFLFU-UHFFFAOYSA-J diphosphate(4-) Chemical compound [O-]P([O-])(=O)OP([O-])([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-J 0.000 description 1
- 235000011180 diphosphates Nutrition 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 238000012921 fluorescence analysis Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000037406 food intake Effects 0.000 description 1
- 235000012631 food intake Nutrition 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000013048 microbiological method Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 210000003200 peritoneal cavity Anatomy 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 125000000714 pyrimidinyl group Chemical group 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 229940005605 valeric acid Drugs 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
- C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
- C07D417/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/77—Ovalbumin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
-
- 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
- G01N33/82—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving vitamins or their receptors
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Food Science & Technology (AREA)
- Biotechnology (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a preparation method of a vitamin B1 hapten, an immunogen, an envelope antigen and a monoclonal antibody, wherein the vitamin B1 hapten is coupled with bovine serum albumin to prepare the vitamin B1 immunogen, the vitamin B1 hapten is coupled with ovalbumin to prepare the vitamin B1 envelope antigen, a BALB/c mouse is immunized by using the prepared vitamin B1 immunogen to obtain spleen cells, then the spleen cells and myeloma cells are fused to obtain hybridoma cells capable of secreting the vitamin B1 monoclonal antibody at high titer, the vitamin B1 monoclonal antibody secreted by the hybridoma cells is detected by the vitamin B1 envelope antigen, and the detection result shows that the prepared hybridoma cells secreting the vitamin B1 monoclonal antibody can stably secrete the vitamin B1 monoclonal antibody.
Description
Technical Field
The invention relates to the technical field of monoclonal antibody detection, in particular to a preparation method of a vitamin B1 hapten, an immunogen, a coating antigen and a monoclonal antibody.
Background
Vitamin B1, also known as "thiamine", the thiamine molecule comprises a pyrimidine ring and a thiazole ring linked by methylene bridges. In the animal body, it exists in the form of phosphate, wherein the main form is thiamine pyrophosphate (TPP) formed with pyrophosphate, and the minor portions are Thiamine Monophosphate (TMP), thiamine Triphosphate (TTP) and free thiamine. Plants and some lower animals can synthesize thiamine, and bacteria in the digestive tract of mammals can also synthesize some thiamine in amounts related to many factors such as food intake. In most cases, mammals rely almost entirely on thiamine in food. After thiamine enters human cells, the thiamine is phosphorylated into phospholipides under the participation of various enzymes, wherein 80% of the phospholipides are TPP,10% of the phospholipides are TPP, TMP and thiamine, and researches show that in Alzheimer Disease (AD) patients, the idle reduction of brain cell sugar and energy metabolism of AD patients and animal models is reduced and obviously earlier than the clinical symptoms and the formation of characteristic pathological damage, and sugar metabolism disorder is considered to be an independent risk factor of AD, and further researches show that in AD patients, the activity of KGDH, PDH, TK and the like only taking thiamine (vitamin B1) as a coenzyme is reduced most obviously in enzyme activity abnormality related to brain cell sugar and energy metabolism disorder, so that the research on the content change of thiamine (vitamin B1) in bodies is particularly important. In order to establish an immunoassay method for serum thiamine (vitamin B1) in vivo, it is important to develop an antibody against thiamine (vitamin B1).
At present, instruments and detection methods for detecting the thiamine (vitamin B1) content in a human body are different, and a microbiological method, an ultraviolet spectrophotometry method, a fluorescence analysis method, a high performance liquid chromatography method and the like are mainly adopted. The pretreatment technology in the high performance liquid chromatography is tedious and time-consuming, needs more organic solvents, needs a large amount of serum, needs special technical personnel for operation, is complex in detection method, and is long in detection time, so that the method is not easy to popularize. The establishment of an immunization method aiming at thiamine (vitamin B1) monoclonal antibody is more convenient and accurate.
Disclosure of Invention
In order to solve the problems, the application provides a preparation method of a vitamin B1 hapten, an immunogen, a coating antigen and a monoclonal antibody. Specifically, a conjugate of vitamin B1 and carrier protein is used as an immunogen and a detection source to immunize a BALB/c mouse, an immune spleen cell is fused with a mouse myeloma cell line SP2/0 by a conventional cell fusion technology, and an improved method is utilized to carry out positive screening and negative screening on an antibody secreted by a fusion cell; obtaining 1 hybridoma cell strain capable of stably secreting anti-vitamin B1 by cloning for 3 times, wherein the antibody secreted by the hybridoma cell strain belongs to IgG1 subclass, the relative molecular mass is about 150kD, and the antibody titer is 3.90 multiplied by 10 6 Antibody affinity of 5.0X 10 8 L/mol, and the obtained antibody can be used for establishing an immunological detection method of the vitamin B1.
In order to realize the purpose of the invention, the technical scheme of the invention is as follows:
in a first aspect, a vitamin B1 hapten has the formula (I):
further, the preparation method of the vitamin B1 hapten comprises the following steps:
s1, using a key to take 7.38g of vitamin B1 and 5.34g of bromovaleric acid;
s2, adding a 15mLN solution into a test tube, and adding an N-dimethylformamide solution into the test tube;
s3, adding the measured vitamin B1 and bromovaleric acid into an N, N-dimethylformamide solution in a test tube to obtain a vitamin B1 reaction solution;
s4, adding 170mg of potassium carbonate into the vitamin B1 reaction solution to obtain a vitamin B1 hapten reaction mixed solution;
s5, carrying out reflux treatment on the vitamin B1 hapten reaction mixed solution, quenching the reaction mixture by using water, and then extracting the vitamin B1 hapten reaction mixed solution by using toluene to extract a vitamin B1 hapten organic mixed layer;
s6, washing the vitamin B1 hapten organic mixed layer by using saline water, and drying by using anhydrous sodium sulfate to obtain a high-purity vitamin B1 hapten solid;
and S7, concentrating the high-purity vitamin B1 hapten solid in vacuum, and concentrating the vitamin B1 hapten solid by using petroleum to obtain the high-purity vitamin B1 hapten.
It should be noted that, compared with conventional vitamin B1, the vitamin B1 hapten provided in the present application is mainly different in that the vitamin B1 hapten lengthens and modifies the amino terminal of the vitamin B1, and a hydrogen atom on the amino group is substituted by valeric acid, so that the carboxyl terminal of the vitamin B1 hapten can be combined with the amino group on bovine serum albumin under different experimental conditions to form a vitamin B1 immunogen; and the hydroxyl end of the protein can be combined with the carboxyl end of the ovalbumin to form the vitamin B1 coating antigen.
Furthermore, in order to obtain the vitamin B1 immunogen and the vitamin B1 coating antigen, the vitamin B1 hapten needs to be respectively combined with the bovine serum albumin and the ovalbumin under different experimental conditions, wherein the preparation method of the vitamin B1 hapten comprises the following steps:
(1) dissolving 20mg of vitamin B1 hapten in a test tube filled with 4ml of N, N-dimethylformamide, adding 7.5mg of water-soluble carbodiimide and 9mg of N-hydroxysuccinimide, and stirring at room temperature for 5 hours to obtain a vitamin B1 hapten activation solution A;
(2) taking 50mg of Bovine Serum Albumin (BSA), and dissolving the BSA in 3ml of 0.05mol/L PB buffer solution to obtain immunogen carrier protein B solution;
(3) and dropwise adding the A liquid into the B liquid, stirring for 24 hours, and dialyzing for 3 days by using 0.02mol/LPBS to obtain the immunogen of the vitamin B1.
Further, the preparation method of the vitamin B1 coating antigen comprises the following steps:
(1) dissolving 17mg of vitamin B1 hapten in a centrifuge tube filled with 4ml of DMSO solution, adding 79 mu L of triethylamine and 46 mu L of isobutyl chloroformate into the test tube, and shaking the centrifuge tube filled with the mixed solution at room temperature for 5 hours to obtain vitamin B1 coated original activation solution C;
(2) dissolving 50mg of Ovalbumin (OVA) in 3ml of 0.05mol/L PB buffer solution to obtain coating original carrier protein D solution;
(3) and dropwise adding the C solution into the D solution, stirring for 24 hours, and dialyzing for 3 days by using 0.02mol/LPBS to obtain the vitamin B1 coating antigen.
It should be noted that, the vitamin B1 immunogen solution and the vitamin B1 coating original solution prepared by the present application are stored under the condition of 20 ℃ below zero.
In a second aspect, the present application also includes a method for preparing a monoclonal antibody to vitamin B1 by immunizing a BALB/c mouse with a vitamin B1 immunogen, comprising the steps of:
(1) performing interval immunization on a BALB/c mouse by using the vitamin B1 immunogen so as to activate spleen cells in the abdominal cavity of the BALB/c mouse to generate a vitamin B1 antibody;
(2) carrying out cell fusion on spleen cells of the BALB/c mouse generating the vitamin B1 antibody and a mouse myeloma cell line SP2/0, and obtaining hybridoma cells capable of stably secreting the vitamin B1 monoclonal antibody after screening;
(3) injecting pristane into the abdominal cavity of the BALB/c mouse, collecting ascites after 10-14 days, purifying the collected ascites by an octanoic acid-saturated ammonium sulfate method, and calculating the molecular weight and the monoclonal antibody subclass.
(4) After the third immunization, the abdominal cavity of the mouse is reminiscent stimulated, and the vitamin B1 immunogen is dissolved in PBS buffer solution and injected into the abdominal cavity of the mouse.
Specifically, the step of performing interval immunization on a BALB/c mouse by using a vitamin B1 immunogen to activate spleen cells in the BALB/c mouse to produce a vitamin B1 monoclonal antibody comprises the following steps:
(1) mixing 100 μ g of the vitamin B1 immunogen and Freund's complete adjuvant at a ratio of 1: 1, and injecting the mixture into abdominal cavity of BALB/c mouse for 3 times at an interval of 2 days;
(2) after 3 weeks, uniformly mixing 100 mu g of the vitamin B1 immunogen and Freund's incomplete adjuvant according to the ratio of 1: 1, injecting the mixture into the abdominal cavity of a BALB/c mouse for 3 times at an interval of 2 days;
(3) thereafter, the abdominal cavity of BALB/c mice was injected every 2 weeks with 50. Mu.g of vitamin B1 immunogen, during which the serum titer was determined by the carrier competition ELISA method.
Specifically, the carrier competition ELISA method for determining the serum titer comprises the following steps:
(1) diluting the vitamin B1 coating antigen to 1 mu g/mL by using a carbonate buffer solution, coating a 96-well enzyme label plate by using 100 mu L of each well, standing overnight at 4 ℃, and cleaning the 96-well enzyme label plate by using PBST for 3 times, wherein each time lasts for 3 minutes;
(2) adding 100 μ L of Bovine Serum Albumin (BSA) and equivalent serial diluted serum, reacting at 37 deg.C for 40 min, washing the plate 3 times, each time for 3 min;
(3) adding enzyme-labeled goat anti-mouse IgG, washing the plate for 3 times, wherein each well is 100 mu L,37 ℃ and 30 minutes;
(4) adding o-phenylenediamine (OPD) color development liquid, wherein each hole is 100 mu L,37 ℃ and 10-15 minutes;
(5) adding 50 μ L of stop solution, measuring absorbance at 490nm with ELISA detector, and using negative serum as control.
Further, the construction process of the vitamin B1 hybridoma cell strain comprises the following steps:
(1) preparation of myeloma cell suspension: taking mouse myeloma cells SP2/0 in logarithmic growth phase, centrifuging for 5 minutes at 1000rpm, discarding supernatant, and suspending the cells by using incomplete culture solution to obtain myeloma cell suspension;
(2) preparing immune spleen cell suspension: centrifuging immune splenocytes of the mouse at 1000rpm for 5 minutes, discarding supernatant, and suspending the cells by using incomplete culture solution to obtain an immune splenocyte suspension;
(3) mixing cell suspension: mixing the myeloma cell suspension and the immune spleen cell suspension in a centrifugal tube according to the ratio of 1: 10, and centrifuging for 8 minutes at the rotating speed of 1200 rpm;
(4) fusion of PEG cells: adding PEG 4000 into the centrifugal tube to perform cell fusion reaction for 90 seconds, and then adding preheated incomplete culture solution to terminate the cell fusion reaction;
(5) incubation culture: and adding HAT selective culture solution to culture for 3-7 days after 24-hour fusion to obtain the hybridoma cell strain.
Further, the steps of carrying out cell fusion on spleen cells of a BALB/c mouse producing the vitamin B1 antibody and a mouse myeloma cell line SP2/0, and obtaining hybridoma cells capable of stably secreting the vitamin B1 monoclonal antibody after screening comprise:
(1) fusing the spleen cells with a mouse myeloma cell line SP2/0 by adopting a PEG cell fusion technology to obtain fused cells;
(2) negative screening, removing a large amount of positive holes aiming at the carrier protein to obtain vitamin B1 negative clone;
(3) positive screening, using negative and positive serum as contrast, selecting positive clone to enter next screening to obtain vitamin B1 positive clone;
(4) competing for the preferred hybridoma cells; and screening the vitamin B1 positive clone by an antigen competition method again to obtain an antibody with antigen competition activity, namely the optimized hybridoma cell.
Specifically, the carbonate buffer used in the present invention is 0.05mol/LpH9.6 carbonate buffer.
Further, the process of identifying the vitamin B1 monoclonal antibody contained in the mouse ascites comprises the following steps:
(1) preparing an antigen coating solution: preparing the vitamin B1 coating antigen into a vitamin B1 coating solution by using a carbonate buffer solution with the pH value of 9.6, adding 96-hole ELISA detection plates, adding 100ul of the vitamin B1 coating solution into each hole, and placing the mixture in a refrigerator at the temperature of 4 ℃ for overnight;
(2) adding 200ul of sealing liquid into each hole of the 96-hole ELISA detection plate, and placing the 96-hole ELISA detection plate in a 37 ℃ incubator for incubation for 2 hours to obtain a sealed ELISA detection plate;
(3) adding 100ul of high-purity vitamin B1 monoclonal antibody to each well of the blocked ELISA detection plate, and incubating at 37 ℃ in an incubator for 2 hours;
(4) adding 100ul of a tasted goat anti-mouse Ig-HRP to each well in the blocked ELISA assay plate and incubating for 1 hour at 37 ℃;
(5) and (3) color development reaction: adding a TMB substrate into each hole of an ELISA detection plate, developing for 5-10 min, and then adding 100ul of 2M sulfuric acid per hole to terminate the color reaction;
(6) and (3) detection: and detecting the absorbance OD value of the solution at 450nm by using a microplate reader.
The invention has the beneficial effects that:
the invention provides a vitamin B1 hapten for the first time, the vitamin B1 hapten is prepared into a vitamin B1 immunogen and a vitamin B1 coating antigen, a mouse is immunized by the vitamin B1 immunogen to obtain a vitamin B1 monoclonal antibody, and the prepared vitamin B1 monoclonal antibody is detected by utilizing the vitamin B1 coating antigen, so that the accurate detection of the vitamin B1 monoclonal antibody is realized. No related technical research on the preparation of the vitamin B1 monoclonal antibody exists before, the activity and accuracy of the original vitamin B1 monoclonal antibody detection by coating the vitamin B1 are avoided, and the preparation process is simple and convenient, economic, high in titer, high in sensitivity and large in practical prospect.
Drawings
FIG. 1 is a structural diagram of vitamin B1 hapten.
Detailed Description
The invention discloses a preparation and detection method of vitamin B1 hapten, antigen and monoclonal antibody, and the technical personnel in the field can use the contents for reference and appropriately improve the process parameters for realization. It is specifically noted that all such substitutions and modifications will be apparent to those skilled in the art and are intended to be included herein. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
The materials or reagents used in the preparation method of the vitamin B1 monoclonal antibody provided by the invention can be purchased from the market.
Examples
This example provides a method for preparing a vitamin B1 monoclonal antibody.
The vitamin B1 hapten is prepared by the following method:
a. measuring 24.6mmol of vitamin B1 and 29.52mmol of bromovaleric acid;
b. adding 15mLN, N-dimethylformamide solution to the test tube;
c. adding a measured vitamin B1 hapten and bromovaleric acid into an N, N-dimethylformamide solution in a test tube to obtain a vitamin B1 reaction solution;
d. adding 170mg of potassium carbonate into the vitamin B1 reaction solution to obtain a vitamin B1 hapten reaction mixed solution;
e. refluxing the vitamin B1 hapten reaction mixed solution, quenching the reaction mixture by using water, and then extracting the vitamin B1 hapten reaction mixed solution by using toluene to extract a vitamin B1 hapten organic mixed layer;
f. washing the vitamin B1 hapten organic mixed layer by using saline water, and drying by using anhydrous sodium sulfate to obtain a high-purity vitamin B1 hapten solid;
g. and concentrating the vitamin B1 hapten solid with higher purity in vacuum, and concentrating the vitamin B1 hapten solid with petroleum to obtain the high-purity vitamin B1 hapten.
The preparation method of the immunogen of the vitamin B1 comprises the following steps:
a. dissolving 20mg of vitamin B1 hapten into a test tube filled with 4ml of N, N-dimethylformamide, adding 7.5mg of water-soluble carbodiimide and 9mg of N-hydroxysuccinimide, and stirring at room temperature for 5 hours to obtain a vitamin B1 hapten activation solution A;
b. taking 50mg of Bovine Serum Albumin (BSA), and dissolving the BSA in 3ml of 0.05mol/L PB buffer solution to obtain immunogen carrier protein B solution;
c. dripping the A solution into the B solution, stirring for 24 hours, and dialyzing with 0.02mol/LPBS for 3 days to obtain the immunogen vitamin B1, namely vitamin B1-BSA.
d. And (3) refrigerating the prepared vitamin B1 immunogen in a refrigerator at the temperature of 20 ℃ below zero for later use.
Immunizing BALB/c mice with a vitamin B1 immunogen to obtain a vitamin B1 monoclonal antibody, comprising the following steps:
the first immunization: mixing 100 μ g vitamin B1-BSA with equivalent Freund's complete adjuvant, and performing intraperitoneal injection for 3 times at intervals of 2 days;
and (3) secondary immunization: after 3 weeks, uniformly mixing 100 mu g of vitamin B1-BSA and equivalent Freund incomplete adjuvant, and immunizing in abdominal cavity for 3 times;
three times of immunization: the intraperitoneal booster immunization was repeated every 2 weeks with half the amount of vitamin B1-BSA.
After completion of the immunization, the serum titer is determined by a competitive ELISA method, the specific ELISA procedure of which comprises the steps of:
A. diluting vitamin B1-BSA with 0.05mol/LpH9.6 carbonate buffer solution to 1 μ g/mL, coating 96-well enzyme label plate with 100 μ L per well, and placing in a refrigerator at 4 ℃ overnight;
B. washing the diluted vitamin B1-BSA with PBST for 3 times, and washing the plate for 3 minutes each time;
C. add 1 u g/mL BSA and equivalent serial dilution of serum total 100u L, at 37 degrees C temperature reaction for 40 minutes, PBST plate washing 3 times;
D. adding enzyme-labeled goat anti-mouse IgG (1: 4000), reacting at 37 deg.C for 30 min with 100 μ L per well, and washing the plate with PBST plate for 3 times;
E. adding o-phenylenediamine (OPD) color developing solution, adding 100 mu L of the solution into each hole, and reacting for 10-15 minutes at 37 ℃;
F. adding 50 mu L of stop solution, measuring the absorbance value at 490nm by an enzyme linked immunosorbent assay detector, using negative serum as a control, and detecting that the antibody titer in the serum is 1 5 。
After obtaining spleen cells of healthy BALB/c female mice capable of producing vitamin B1 monoclonal antibodies, cell fusion and screening experiments were performed.
The process of monoclonal cloning hybridoma cells includes:
a. preparing a feeder cell suspension: extracting macrophage, thymocyte or splenocyte from abdominal cavity of mouse, diluting said cell with cell culture solution, if it is macrophage, its concentration is 6-8X 10 6 Per ml; in the case of thymocytes, the concentration thereof is 6X 10 6 Per ml; in the case of mouse fibroblasts, the concentration thereof is 1X 10 5 Per ml; all cells obtained from mice were added to wells of a 96-well ELISA plate in an amount of 100 μ l/well.
b. Counting the number of cells in each positive well, and diluting and adjusting the cell concentration to 1-5 × 10 by using a cell culture solution 3 /ml；
c. Taking 130 cells, putting the 130 cells into a centrifuge tube, putting 6.5ml of complete culture solution containing feeder cells, namely 20 cells/ml, and then averagely adding the diluted cell suspension into each hole of three rows of an ELISA plate A, a plate B and a plate C with 96 holes, wherein each hole is provided with 2 cells;
d. the remaining 2.9ml of cell suspension was supplemented with 2.9ml of complete medium containing feeder cells, i.e. 10 cells/ml, and the diluted cell suspension was added on average to each well of three rows D, E, F of 96-well ELISA plates, 1 cell per well;
e. the remaining 2.2ml of cell suspension was supplemented with 2.2ml of complete medium containing feeder cells, i.e. 5 cells/ml, and the diluted cell suspension was added on average to each well of both rows G, H of 96-well ELISA plates, 0.5 cells per well;
f. static culture of cells obtained on 96-well ELISA plates for 4-5 days to observe small cell clones in complete culture medium of feeder cells, and adding complete culture medium of feeder cells to 200 μ l/well;
g. after 8-9 days of culture, the condition of cell cloning can be observed by naked eyes, and then the antibody detection link can be entered.
It should be noted that: the above is a culture medium for screening hypoxanthine phosphoribosyl transferase and thymidine kinase activity deficient cells by primarily cloning mouse abdominal cells, and adding HT to the complete culture medium is required for the primarily cloned hybridoma cells.
h. And (3) re-immunization: continuously cloning vitamin B1 positive hybridoma cells for 3 times by adopting a limiting dilution method to construct a stable cell strain, then carrying out amplification culture and placing in a refrigerator for freezing storage.
The tail vein of a BALB/c female mouse is strengthened and immunized 1 time, the spleen of the BALB/c female mouse is taken after 3 days, the immune spleen cell and a mouse myeloma cell line SP2/0 are fused by adopting a PEG cell fusion technology, and hybridoma cells are screened by adopting the positive screening and negative screening combined method as follows:
(1) Negative selection removes clones directed to the vector: the fused cells are firstly screened by using a carrier: diluting the carrier BSA to 1 μ g/mL with 0.05mol/LpH9.6 carbonate buffer solution, coating a 96-well enzyme label plate overnight at 4 ℃, and washing the plate 3 times for 3 minutes by PBST; adding 100 mu L of cell culture supernatant, and reacting at 37 ℃ for 40 minutes; washing the plate for 3 times; adding enzyme-labeled goat anti-mouse IgG (1: 4000), reacting at 37 ℃ for 30 minutes in each well with 100 mu L of IgG, and washing the plate for 3 times; adding o-phenylenediamine (OPD) color developing solution, adding 100 mu L of the solution into each hole, and reacting for 10-15 minutes at 37 ℃; adding 50 mu L of stop solution, measuring the absorbance value at 490nm by using an enzyme-linked immunosorbent assay detector, removing a large amount of positive holes aiming at the carrier, and allowing negative clones to enter the next round of screening;
(2) The positive screen selects the target clone of vitamin B1: screening of hybridoma cells against small molecule antibodies with vitamin B1: coating the ELISA plate with 1. Mu.g/mL vitamin B1-OVA (vitamin B1 coating antigen) overnight, washing, adding 100. Mu.L cell culture supernatant, reacting at 37 ℃ for 40 min, and washing the plate for 3 times; adding enzyme-labeled goat anti-mouse IgG (1: 4000), reacting at 37 deg.C for 30 min with each well of 100 μ L, and washing the plate for 3 times; adding o-phenylenediamine (OPD) color development liquid, reacting for 10-15 minutes at 37 ℃ with 100 mu L of each hole; adding 50 μ L of stop solution, measuring absorbance at 490nm with enzyme linked immunosorbent assay, and comparing with negative and positive serum; selecting positive clones to enter the next round of screening;
(3) Competing preferred hybridoma cells: the vitamin B1 positive clone obtained by the screening is screened for an antibody with antigen competition activity by an antigen competition method again: coating an ELISA plate with vitamin B1-OVA (1 mu g/mL), washing, adding a vitamin B1 standard substance (20 ng/mL) and 50 mu L of each cell culture supernatant, and carrying out in-plate competitive reaction at 37 ℃ for 40 minutes; washing the plate for 3 times; adding enzyme-labeled goat anti-mouse IgG (1: 4000), reacting at 37 deg.C for 30 min with each well of 100 μ L, and washing the plate for 3 times; adding o-phenylenediamine (OPD) color developing solution, wherein each well is 100 mu L,37 ℃ and 10-15 minutes; adding 50 mu L of stop solution, measuring the absorbance value at 490nm by an enzyme linked immunosorbent assay detector, wherein the smallest OD value is the antibody with the most competitive activity, and continuously cloning the cell strain until all the cell strain is positive, namely the preferred hybridoma;
when obtaining hybridoma cells capable of stably secreting vitamin B1 monoclonal antibodies, BALB/c female mice need to be subjected to ascites preparation and antibody characteristic study, and the method comprises the following steps:
A. injecting 0.5-1.0 mL of autoclaved pristanane into the abdominal cavities of a plurality of female BALB/c mice aged 10 weeks;
B. 2X 10 times of the solution is injected after 1 to 9 weeks 6 Hybridoma cells to the peritoneal cavity of female BALB/c mice;
C. collecting hybridoma cells in logarithmic growth phase in abdominal cavity of mouse, washing with incomplete culture solution once, and centrifuging with 1000r/min centrifuge for 10min;
D. sampling, extracting the cells in abdominal cavity of mouse, staining the cells in abdominal cavity of mouse with trypan blue, counting the number of living cells, and preparing 1.0X 10 with incomplete culture solution 7 Cell/ml suspension;
E. mice inoculated with pristane were inoculated with hybridoma cells and 1ml of cell suspension was injected per abdominal cavity. (containing 1.0X 107 cells)
F. After 10 days of inoculation, according to the abdominal distension condition of the mouse, ascites of the abdominal cavity of a female BALB/c mouse is collected after 10 to 14 days, the titer of the ascites monoclonal antibody is indirectly determined by adopting an ELISA method, the ascites is purified by adopting an octanoic acid-ammonium sulfate method, and the ascites is analyzed by SDS-PAGE electrophoresis;
G. calculating molecular weight by using a regression equation of relative mobility and relative molecular mass in gel, identifying the subclasses of the monoclonal antibodies by using a monoclonal antibody subclass identification kit, and calculating an affinity constant.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A vitamin B1 hapten is characterized in that the molecular formula structure is shown as formula (I):
2. the vitamin B1 hapten as claimed in claim 1, wherein said vitamin B1 hapten is prepared by a process comprising: dissolving solid vitamin B1 and bromovaleric acid in a DMF solution to obtain an activated vitamin B1 solution, and adding potassium carbonate into the activated vitamin B1 solution to obtain the vitamin B1 hapten.
3. A method for preparing an immunogen of vitamin B1, which is characterized in that the vitamin B1 antigen is obtained by coupling the vitamin B1 hapten as claimed in claim 1 with Bovine Serum Albumin (BSA).
4. The method for preparing according to claim 3, characterized by comprising the steps of:
(1) dissolving the vitamin B1 hapten in DMF, adding EDC and NHS, and stirring for 5 hours at room temperature to obtain solution A;
(2) dissolving Bovine Serum Albumin (BSA) in 3ml PB buffer solution to obtain solution B;
(3) and dropwise adding the A liquid into the B liquid, and stirring for 24 hours to obtain the vitamin B1 immunogen.
5. A preparation method of a vitamin B1 coating antigen is characterized by comprising the following steps:
(1) dissolving the vitamin B1 hapten in a DMSO solution, adding triethylamine and isobutyl chloroformate, and stirring at room temperature for 5 hours to obtain solution C;
(2) dissolving Ovalbumin (OVA) in PB buffer solution to obtain solution D;
(3) and dropwise adding the C solution into the D solution, and stirring for 24 hours to obtain the vitamin B1 coating antigen.
6. A method for preparing a vitamin B1 monoclonal antibody, wherein the vitamin B1 monoclonal antibody is prepared by immunizing a mouse with the vitamin B1 immunogen of claim 3, comprising the steps of:
(1) performing interval immunization on the mouse by using the vitamin B1 immunogen so as to activate spleen cells in the abdominal cavity of the mouse to produce a vitamin B1 antibody;
(2) carrying out cell fusion on spleen cells of the mouse generating the vitamin B1 antibody and a mouse myeloma cell line SP2/0, and screening to obtain hybridoma cells capable of stably secreting the vitamin B1 monoclonal antibody;
(3) injecting pristane into the abdominal cavity of the mouse, collecting ascites after 10-14 days, purifying the collected ascites by an octanoic acid-saturated ammonium sulfate method, and calculating the molecular weight and the monoclonal antibody subclass.
7. The method for preparing a vitamin B1 monoclonal antibody according to claim 6, wherein the step of immunizing a mouse with the vitamin B1 antigen at intervals to activate spleen cells in the mouse to produce the vitamin B1 monoclonal antibody comprises:
(1) first immunization: mixing vitamin B1 immunogen and Freund's complete adjuvant at a ratio of 1: 1, emulsifying, injecting into abdominal cavity of the mouse, wherein the amount of vitamin B1 immunogen injected into the mouse per time is 0.15 mg/time/mouse,
(2) and (3) immunization again: mixing vitamin B1 immunogen and Freund's incomplete adjuvant at a ratio of 1: 1, emulsifying, injecting into the mouse, and immunizing at an interval of 3 weeks for 3 weeks,
(3) memory stimulation: after the third immunization, the vitamin B1 immunogen was dissolved in PBS buffer and injected into the abdominal cavity of the mice.
8. The method of claim 7, wherein the construction of the hybridoma cell line comprises the steps of:
(1) preparation of myeloma cell suspension: taking mouse myeloma cells SP2/0 in logarithmic growth phase, centrifuging for 5 minutes at 1000rpm, discarding supernatant, and suspending the cells by using incomplete culture solution to obtain myeloma cell suspension;
(2) preparing immune spleen cell suspension: centrifuging immune splenocytes of the mouse at 1000rpm for 5 minutes, discarding supernatant, and suspending the cells by using incomplete culture solution to obtain an immune splenocyte suspension;
(3) mixing cell suspension: mixing the myeloma cell suspension and the immune spleen cell suspension in a centrifugal tube according to the ratio of 1: 10, and centrifuging for 8 minutes at the rotating speed of 1200 rpm;
(4) fusion of PEG cells: adding PEG 4000 into the centrifugal tube to perform cell fusion reaction for 90 seconds, and then adding preheated incomplete culture solution to terminate the cell fusion reaction;
(5) incubation culture: and after fusing for 24 hours, adding HAT selective culture solution to culture for 3-7 days to obtain the hybridoma cell strain.
9. The method for preparing a vitamin B1 monoclonal antibody according to claim 8, further comprising the step of monoclonal inoculating a mouse with the obtained hybridoma cell line, comprising the steps of:
(1) injecting pristane into abdominal cavity of mouse;
(2) inoculating hybridoma cells into the abdominal cavity of the mouse;
(3) after 10 days of inoculation, ascites in the abdominal cavity of the mouse is extracted;
(4) and purifying the ascites in the abdominal cavity of the mouse to obtain the high-purity vitamin B1 monoclonal antibody.
10. The method for producing a vitamin B1 monoclonal antibody according to claim 9, wherein the process of identifying the vitamin B1 monoclonal antibody contained in the ascites fluid comprises the steps of:
(1) preparing an antigen coating solution: preparing the vitamin B1 coating antigen into a vitamin B1 coating solution by using a carbonate buffer solution with the pH value of 9.6, adding 96-hole ELISA detection plates, adding 100ul of the vitamin B1 coating solution into each hole, and placing the mixture in a refrigerator at the temperature of 4 ℃ for overnight;
(2) adding 200ul of sealing solution into each well of the 96-well ELISA detection plate, and placing the 96-well ELISA detection plate in an incubator at 37 ℃ for incubation for 2 hours to obtain a sealed ELISA detection plate;
(3) adding 100ul of high-purity vitamin B1 monoclonal antibody to each well of the blocked ELISA detection plate, and incubating at 37 ℃ in an incubator for 2 hours;
(4) adding 100ul of a tenninated goat anti-mouse Ig-HRP to each well in the blocked ELISA assay plate and incubating for 1 hour at 37 ℃;
(5) and (3) color development reaction: adding a TMB substrate into each hole of an ELISA detection plate, developing for 5-10 min, and then adding 100ul of 2M sulfuric acid per hole to terminate the color reaction;
(6) and (3) detection: and detecting the absorbance OD value of the solution at 450nm by using a microplate reader.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110450632.8A CN115232119A (en) | 2021-04-25 | 2021-04-25 | Preparation method of vitamin B1 hapten, immunogen, coating antigen and monoclonal antibody |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110450632.8A CN115232119A (en) | 2021-04-25 | 2021-04-25 | Preparation method of vitamin B1 hapten, immunogen, coating antigen and monoclonal antibody |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115232119A true CN115232119A (en) | 2022-10-25 |
Family
ID=83666948
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110450632.8A Pending CN115232119A (en) | 2021-04-25 | 2021-04-25 | Preparation method of vitamin B1 hapten, immunogen, coating antigen and monoclonal antibody |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115232119A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106467571A (en) * | 2015-08-21 | 2017-03-01 | 天津三箭生物技术有限公司 | Conjugate of vitamin B1 and preparation method and application |
| CN108047071A (en) * | 2017-12-07 | 2018-05-18 | 杭州同舟生物技术有限公司 | A kind of Cathinone artificial semiantigen, artificial antigen and its preparation method and application |
| CN110713986A (en) * | 2019-11-12 | 2020-01-21 | 江南大学 | Vitamin B1Monoclonal antibody hybridoma cell strain CBDD and application thereof |
-
2021
- 2021-04-25 CN CN202110450632.8A patent/CN115232119A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106467571A (en) * | 2015-08-21 | 2017-03-01 | 天津三箭生物技术有限公司 | Conjugate of vitamin B1 and preparation method and application |
| CN108047071A (en) * | 2017-12-07 | 2018-05-18 | 杭州同舟生物技术有限公司 | A kind of Cathinone artificial semiantigen, artificial antigen and its preparation method and application |
| CN110713986A (en) * | 2019-11-12 | 2020-01-21 | 江南大学 | Vitamin B1Monoclonal antibody hybridoma cell strain CBDD and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108998422A (en) | It is a kind of secrete Diacloden monoclonal antibody hybridoma cell strain and its application | |
| CN108997499A (en) | A kind of anti human PD-L 1 antibody and its application | |
| JPH02426A (en) | Hybridoma cell line | |
| CN108330103B (en) | Hybridoma cell strain secreting acyclovir monoclonal antibody and preparation method thereof | |
| CN108997500A (en) | A kind of anti human PD-L 1 antibody and its application | |
| CN110713986B (en) | Vitamin B strain 1 Monoclonal antibody hybridoma cell strain CBDD and application thereof | |
| CN110343669B (en) | Hybridoma cell strain DNC secreting anti-triclabendazole monoclonal antibody and application thereof | |
| CN115232119A (en) | Preparation method of vitamin B1 hapten, immunogen, coating antigen and monoclonal antibody | |
| CN112280744A (en) | Hybridoma cell strain secreting monoclonal antibody of hypnone and application thereof | |
| CN111763658A (en) | Hybridoma cell strain secreting anti-dinitrotolamine monoclonal antibody and application thereof | |
| CN114181911B (en) | Hybridoma cell strain secreting spirolactone and metabolite monoclonal antibody thereof and application of hybridoma cell strain | |
| CN112574957B (en) | Hybridoma cell strain secreting clomazone monoclonal antibody and application thereof | |
| CN106929479B (en) | Vitamin B2 monoclonal antibody hybridoma cell strain GZ-4 and application thereof | |
| CN113637642B (en) | Hybridoma cell strain secreting chlorfenapyr monoclonal antibody and application thereof | |
| CN111454912B (en) | Cyperazine monoclonal antibody hybridoma cell strain and application thereof | |
| CN114752568A (en) | Furosemide monoclonal antibody, hybridoma cell strain and application | |
| CN109765360B (en) | CMPF homogeneous enzyme immunoassay kit, preparation method and detection method | |
| CN112521293B (en) | Betaine hapten, betaine antigen, preparation methods of betaine hapten and betaine antigen and preparation method of monoclonal antibody | |
| CN113151188A (en) | Hybridoma cell strain capable of secreting diphenhydramine monoclonal antibody and application thereof | |
| CN112029731A (en) | Tacrolimus monoclonal antibody hybridoma cell strain and application thereof | |
| CN106701686B (en) | hybridoma cell strain 1A5, secreted anti-sulbactam monoclonal antibody and application | |
| JP6630116B2 (en) | Monoclonal anti-AGEs antibody and method for producing the same | |
| CN112552300B (en) | Preparation method of 5-methyltetrahydrofolate hapten, antigen and monoclonal antibody | |
| CN114277000B (en) | Hybridoma cell strain secreting isoprothiolane monoclonal antibody and application thereof | |
| CN113493434B (en) | Synthesis method and application of T2 toxin hapten and artificial antigen |
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 |