CN110408600A - One plant of hybridoma cell strain for secreting preventing from heavy metal copper ion monoclonal antibody and its application - Google Patents
One plant of hybridoma cell strain for secreting preventing from heavy metal copper ion monoclonal antibody and its application Download PDFInfo
- Publication number
- CN110408600A CN110408600A CN201811506878.7A CN201811506878A CN110408600A CN 110408600 A CN110408600 A CN 110408600A CN 201811506878 A CN201811506878 A CN 201811506878A CN 110408600 A CN110408600 A CN 110408600A
- Authority
- CN
- China
- Prior art keywords
- liquid
- solution
- heavy metal
- copper ion
- monoclonal antibody
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 229910001431 copper ion Inorganic materials 0.000 title claims abstract description 81
- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 title claims abstract description 77
- 229910001385 heavy metal Inorganic materials 0.000 title claims abstract description 77
- 210000004408 hybridoma Anatomy 0.000 title claims abstract description 28
- 230000003248 secreting effect Effects 0.000 title claims abstract description 4
- 238000002360 preparation method Methods 0.000 claims abstract description 23
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 claims abstract description 16
- 229910052802 copper Inorganic materials 0.000 claims abstract description 16
- 239000010949 copper Substances 0.000 claims abstract description 16
- 238000001514 detection method Methods 0.000 claims abstract description 16
- 239000000243 solution Substances 0.000 claims description 149
- 239000007788 liquid Substances 0.000 claims description 126
- 239000000427 antigen Substances 0.000 claims description 85
- 102000036639 antigens Human genes 0.000 claims description 85
- 108091007433 antigens Proteins 0.000 claims description 85
- 238000006243 chemical reaction Methods 0.000 claims description 54
- 238000005406 washing Methods 0.000 claims description 48
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims description 40
- 210000004027 cell Anatomy 0.000 claims description 34
- 238000000034 method Methods 0.000 claims description 34
- 239000007995 HEPES buffer Substances 0.000 claims description 30
- 239000002738 chelating agent Substances 0.000 claims description 29
- 241000699666 Mus <mouse, genus> Species 0.000 claims description 23
- 238000002965 ELISA Methods 0.000 claims description 22
- 230000015572 biosynthetic process Effects 0.000 claims description 22
- 238000003786 synthesis reaction Methods 0.000 claims description 22
- XTVVROIMIGLXTD-UHFFFAOYSA-N copper(II) nitrate Chemical compound [Cu+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O XTVVROIMIGLXTD-UHFFFAOYSA-N 0.000 claims description 20
- 238000005259 measurement Methods 0.000 claims description 18
- PAYRUJLWNCNPSJ-UHFFFAOYSA-N Aniline Chemical compound NC1=CC=CC=C1 PAYRUJLWNCNPSJ-UHFFFAOYSA-N 0.000 claims description 16
- 238000010790 dilution Methods 0.000 claims description 15
- 239000012895 dilution Substances 0.000 claims description 15
- 230000003053 immunization Effects 0.000 claims description 13
- 206010003445 Ascites Diseases 0.000 claims description 12
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 12
- 229940098773 bovine serum albumin Drugs 0.000 claims description 12
- 238000002649 immunization Methods 0.000 claims description 12
- 238000005138 cryopreservation Methods 0.000 claims description 11
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 10
- 241000699670 Mus sp. Species 0.000 claims description 10
- 210000000991 chicken egg Anatomy 0.000 claims description 10
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 10
- 238000000108 ultra-filtration Methods 0.000 claims description 10
- 239000012498 ultrapure water Substances 0.000 claims description 10
- 238000013019 agitation Methods 0.000 claims description 9
- 238000012216 screening Methods 0.000 claims description 9
- 230000028327 secretion Effects 0.000 claims description 8
- 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 claims description 7
- 229910000033 sodium borohydride Inorganic materials 0.000 claims description 7
- 239000012279 sodium borohydride Substances 0.000 claims description 7
- 238000010241 blood sampling Methods 0.000 claims description 6
- 230000007910 cell fusion Effects 0.000 claims description 6
- XOJVVFBFDXDTEG-UHFFFAOYSA-N pristane Chemical compound CC(C)CCCC(C)CCCC(C)CCCC(C)C XOJVVFBFDXDTEG-UHFFFAOYSA-N 0.000 claims description 6
- 238000000746 purification Methods 0.000 claims description 6
- 230000002401 inhibitory effect Effects 0.000 claims description 5
- 239000007924 injection Substances 0.000 claims description 5
- 238000002347 injection Methods 0.000 claims description 5
- 229920001223 polyethylene glycol Polymers 0.000 claims description 5
- 239000002202 Polyethylene glycol Substances 0.000 claims description 4
- 210000001015 abdomen Anatomy 0.000 claims description 4
- 238000005119 centrifugation Methods 0.000 claims description 4
- 239000001963 growth medium Substances 0.000 claims description 4
- 206010035226 Plasma cell myeloma Diseases 0.000 claims description 3
- 238000011091 antibody purification Methods 0.000 claims description 3
- 238000003556 assay Methods 0.000 claims description 3
- 239000004519 grease Substances 0.000 claims description 3
- 244000005700 microbiome Species 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 201000000050 myeloid neoplasm Diseases 0.000 claims description 3
- 238000001556 precipitation Methods 0.000 claims description 3
- 241001529936 Murinae Species 0.000 claims description 2
- 238000010367 cloning Methods 0.000 claims description 2
- 238000009396 hybridization Methods 0.000 claims description 2
- 239000007928 intraperitoneal injection Substances 0.000 claims description 2
- 238000004321 preservation Methods 0.000 claims description 2
- 210000004881 tumor cell Anatomy 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims 1
- 230000001804 emulsifying effect Effects 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 6
- 230000007613 environmental effect Effects 0.000 abstract description 3
- 102000004169 proteins and genes Human genes 0.000 description 32
- 108090000623 proteins and genes Proteins 0.000 description 32
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 25
- 150000002500 ions Chemical class 0.000 description 24
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 20
- 239000006210 lotion Substances 0.000 description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 17
- 238000010521 absorption reaction Methods 0.000 description 16
- 238000004458 analytical method Methods 0.000 description 16
- 239000003292 glue Substances 0.000 description 16
- 238000011534 incubation Methods 0.000 description 16
- 238000000926 separation method Methods 0.000 description 16
- 230000001588 bifunctional effect Effects 0.000 description 13
- 239000011248 coating agent Substances 0.000 description 13
- 238000000576 coating method Methods 0.000 description 13
- 230000008878 coupling Effects 0.000 description 12
- 238000010168 coupling process Methods 0.000 description 12
- 238000005859 coupling reaction Methods 0.000 description 12
- 125000000623 heterocyclic group Chemical group 0.000 description 12
- 239000002671 adjuvant Substances 0.000 description 10
- 239000000872 buffer Substances 0.000 description 10
- 239000011734 sodium Substances 0.000 description 10
- 238000009010 Bradford assay Methods 0.000 description 9
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 9
- 230000001808 coupling effect Effects 0.000 description 9
- 238000001095 inductively coupled plasma mass spectrometry Methods 0.000 description 9
- 210000002966 serum Anatomy 0.000 description 9
- 229910052708 sodium Inorganic materials 0.000 description 9
- 239000003643 water by type Substances 0.000 description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 8
- 238000002835 absorbance Methods 0.000 description 8
- 238000004364 calculation method Methods 0.000 description 8
- 230000002860 competitive effect Effects 0.000 description 8
- 239000012141 concentrate Substances 0.000 description 8
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 8
- 238000001962 electrophoresis Methods 0.000 description 8
- 229910052751 metal Inorganic materials 0.000 description 8
- 239000002184 metal Substances 0.000 description 8
- 238000010186 staining Methods 0.000 description 8
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 7
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 241001494479 Pecora Species 0.000 description 5
- UHOVQNZJYSORNB-UHFFFAOYSA-N benzene Substances C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 5
- 239000013642 negative control Substances 0.000 description 5
- 239000008399 tap water Substances 0.000 description 5
- 235000020679 tap water Nutrition 0.000 description 5
- 238000010586 diagram Methods 0.000 description 4
- 230000014759 maintenance of location Effects 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 4
- 238000012856 packing Methods 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Inorganic materials [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 4
- 238000003860 storage Methods 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- 238000003968 anodic stripping voltammetry Methods 0.000 description 3
- 238000001479 atomic absorption spectroscopy Methods 0.000 description 3
- XYOVOXDWRFGKEX-UHFFFAOYSA-N azepine Chemical compound N1C=CC=CC=C1 XYOVOXDWRFGKEX-UHFFFAOYSA-N 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000004945 emulsification Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- WGYKZJWCGVVSQN-UHFFFAOYSA-N propylamine Chemical group CCCN WGYKZJWCGVVSQN-UHFFFAOYSA-N 0.000 description 3
- 238000004448 titration Methods 0.000 description 3
- 102000014914 Carrier Proteins Human genes 0.000 description 2
- 108010078791 Carrier Proteins Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 2
- 239000012980 RPMI-1640 medium Substances 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 239000007640 basal medium Substances 0.000 description 2
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 2
- 229910000397 disodium phosphate Inorganic materials 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 230000037189 immune system physiology Effects 0.000 description 2
- 238000002354 inductively-coupled plasma atomic emission spectroscopy Methods 0.000 description 2
- 108010045069 keyhole-limpet hemocyanin Proteins 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000002203 pretreatment Methods 0.000 description 2
- 210000004988 splenocyte Anatomy 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- YRNWIFYIFSBPAU-UHFFFAOYSA-N 4-[4-(dimethylamino)phenyl]-n,n-dimethylaniline Chemical compound C1=CC(N(C)C)=CC=C1C1=CC=C(N(C)C)C=C1 YRNWIFYIFSBPAU-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- VYZAMTAEIAYCRO-UHFFFAOYSA-N Chromium Chemical compound [Cr] VYZAMTAEIAYCRO-UHFFFAOYSA-N 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 101100203936 Mus musculus Srpra gene Proteins 0.000 description 1
- 208000031320 Teratogenesis Diseases 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 239000008351 acetate buffer Substances 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 229910052785 arsenic Inorganic materials 0.000 description 1
- RQNWIZPPADIBDY-UHFFFAOYSA-N arsenic atom Chemical compound [As] RQNWIZPPADIBDY-UHFFFAOYSA-N 0.000 description 1
- GEWYFWXMYWWLHW-UHFFFAOYSA-N azanium;octanoate Chemical compound [NH4+].CCCCCCCC([O-])=O GEWYFWXMYWWLHW-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 210000005252 bulbus oculi Anatomy 0.000 description 1
- 229910052793 cadmium Inorganic materials 0.000 description 1
- BDOSMKKIYDKNTQ-UHFFFAOYSA-N cadmium atom Chemical compound [Cd] BDOSMKKIYDKNTQ-UHFFFAOYSA-N 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 231100000315 carcinogenic Toxicity 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 229910052804 chromium Inorganic materials 0.000 description 1
- 239000011651 chromium Substances 0.000 description 1
- 230000009514 concussion Effects 0.000 description 1
- 239000003636 conditioned culture medium Substances 0.000 description 1
- 239000003431 cross linking reagent Substances 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 229960000935 dehydrated alcohol Drugs 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 238000000295 emission spectrum Methods 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000003760 magnetic stirring Methods 0.000 description 1
- 239000000155 melt Substances 0.000 description 1
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 description 1
- 229910052753 mercury Inorganic materials 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 231100000572 poisoning Toxicity 0.000 description 1
- 230000000607 poisoning effect Effects 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000002787 reinforcement Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000006152 selective media Substances 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
- 239000011780 sodium chloride Substances 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
Classifications
-
- 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
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/35—Valency
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Food Science & Technology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Tropical Medicine & Parasitology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The hybridoma cell strain for secreting preventing from heavy metal copper ion monoclonal antibody the present invention provides one plant and its application；The hybridoma cell strain can secrete preventing from heavy metal copper ion monoclonal antibody, and the present invention also provides the preparation methods of the preparation method of hybridoma cell strain and preventing from heavy metal copper ion monoclonal antibody；The monoclonal antibody specificity that the present invention is prepared is stronger, has preferably sensitivity, IC to copper ion20Value is 0.14ngmL‑1, can be used in quickly detecting heavy metal copper ion.The anti-copper ion monoclonal antibody can be used for the residue detection of copper ion in environmental sample.
Description
Technical field
The invention belongs to immunochemistry detection technique fields, and in particular to one plant of secretion preventing from heavy metal copper ion monoclonal is anti-
The hybridoma cell strain of body and its application.
Background technique
Heavy metal pollution refers mainly to pollution of the pollutants such as copper, cadmium, mercury, nickel, chromium, arsenic, zinc, copper to environment.Heavy metal point
Cloth is extensive, is difficult to degrade, and can enter human body by big gas and water, food chain, have an effect, make with vivo protein and various enzymes
They lose activity, and are enriched in certain organs, if it exceeds the limit that human body is resistant to, it is acute or slow to will cause human body
Property poisoning, have carcinogenic, teratogenesis and mutagenesis, to human body have very big harm.Therefore, reinforce heavy metal environment,
Important means of the residue detection as guarantee heavy metal safety in agricultural product and food, and the research of new detecting technique under the new situation
Seem especially urgent with exploitation.
Traditional heavy metal detection method mainly uses atomic absorption spectroscopy (Atomic Absorption
Spectroscopy, AAS), inductive coupling plasma emission spectrum (InductiveLy CoupLedpLasma Atomic
Emission Spectroscopy, ICP-AES), anodic stripping voltammetry (Anodic Stripping VoLtammetry,
ASV), chromatography and various combination detection methods.Although these methods can carry out the heavy metal ion in various environmental samples
Effectively analysis, but large-scale instrument is needed mostly, analysis method is at high cost, and sample is needed by resolution, and analysis time is long, is unsuitable for
The field quick detection of heavy metal, it is difficult to adapt to the spot check of environment and market product, manufacturing enterprise checks oneself and product disengaging
The requirement that mouth speeds passage through customs.
Immunology detection technology is fast with detection speed, analysis capacity is big, low-cost, the simple portable, user of instrument
Member's technical requirements are not high, are easy universal and promote, the advantages that high sensitivity and selectivity are strong, are especially suitable for scene screening and a large amount of
The quick analysis of sample, it has also become the 21 century most competitive detection and analysis technology with challenge.
The key of heavy metal ion immunology detection is the preparation of preventing from heavy metal specific antibody, and specific antibody system
Standby key is therefore how the synthesis of high-quality heavy metal immunogene prepares high-quality heavy metal artificial antigen, how to prepare again
Preventing from heavy metal specific antibody, the immunoassay detection heavy metal ion established more rapidly, more economical become what needs solved
Problem.
Summary of the invention
To overcome the shortcomings of existing technologies, the present invention provides the hybridization of one plant of secretion preventing from heavy metal copper ion monoclonal antibody
Tumor cell strain and its application.
The hybridoma cell strain of one plant of secretion preventing from heavy metal copper ion monoclonal antibody, has been preserved in China Microbiological bacterium
Kind preservation administration committee common micro-organisms center, deposit number are CGMCC No.16388.
Preventing from heavy metal copper ion monoclonal antibody, it is by the hybridoma cell strain point that deposit number is CGMCC No.16388
Secrete generation.
Hybridoma cell strain CGMCC No.16388 or the monoclonal antibody of its secretion are in copper ion immune detection
Using.
The invention adopts the following technical scheme:
A kind of preparation method of hybridoma cell strain that secreting preventing from heavy metal copper ion monoclonal antibody, this method is for making
Standby above-described hybridoma cell strain, comprising the following steps:
(1) synthesis and identification of heterocyclic bifunctional chelating agent copper ion artificial antigen:
5~8mg 2-S- (3- aminobenzene)-Isosorbide-5-Nitrae, 7 7-triazacyclononanes-Isosorbide-5-Nitrae are weighed, 7- triacetic acid (is abbreviated as o-NH2-
Bn-NOTA), it is dissolved in 2mL 0.01molL-1, pH7.4 4- hydroxyethyl piperazineethanesulfonic acid HEPES solution, be made chelating agent it is molten
Liquid, this solution are A liquid；
It weighs 70.33mg copper nitrate to be dissolved in 5mL ultrapure water, it is 7.5 × 10 that concentration, which is made,-2mol·L-1Copper nitrate
Solution, this solution are B liquid；
The B liquid of 150~200 μ L is added in A liquid, is protected from light 3~5h at room temperature, this reaction solution is C liquid；
500~800 μ L, 20mmolL are added dropwise into C liquid-1Glutaraldehyde solution, room temperature be protected from light overnight, this
Reaction solution is D liquid；
It weighs 20~30mg bovine serum albumin(BSA) BSA or chicken egg white OVA is dissolved in 3mL HEPES, at room temperature magnetic force
It stirs and evenly mixs, this reaction solution is E liquid；
D liquid is added dropwise in E liquid, is protected from light at room temperature for 24 hours, after that 150~200 μ L sodium borohydrides are added dropwise is molten
Liquid (20mg is dissolved in 200 μ L ultrapure waters), is protected from light 1h at room temperature；
Reaction solution first uses the bag filter of 8KD to dialyse 3~5 times, then with 7000~9000rmin of ultra-filtration centrifuge tube of 30KD-1
Centrifuge washing 3~5 times, 15~20min, is redissolved with the HEPES solution of the 0.01M of 5~10mL, pH7.4 every time, rear to dispense, and -20
Artificial antigen of heavy metal copper Cu-L is made in DEG C cryo-conservation2- BSA or Cu-L2- OVA, L2Indicate 2-S- (3- aminobenzene) -1,
4,7 7-triazacyclononanes-Isosorbide-5-Nitrae, 7- triacetic acid
Synthetic route are as follows:
It takes UV scanning and SDS-PAGE to identify its coupling effect, is measured respectively using ICP-MS method and Bradford method
Heavy metal ion and protein concentration, calculate artificial antigen coupling combine than.
(2) mouse is immune:
Female BAl BIc/C mice of 6~8 week old of health is selected to be immunized.By the artificial antigen of heavy metal copper of preparation
Cu-L2After-BSA and isometric Freund's complete adjuvant are sufficiently mixed emulsification using syringe pressurization, pass through abdomen and oxter multiple spot
Injection.Booster immunization is carried out at interval of 21d (day) afterwards, blood sampling measurement potency, potency use indirect ELISA after 3 booster immunizations
Method measurement.It carries out last end when potency is no longer significantly raised to exempt from, end carries out cell fusion after exempting from 3d.First in immunologic process
Secondary immune using Freund's complete adjuvant, booster immunization uses incomplete Freund's adjuvant, and end is exempted from without using adjuvant, direct immunization original annotation
It penetrates immune.
(3) cell fusion and cell strain screen:
Mouse boosting cell and murine myeloma cell SP2/0 are carried out according to the ratio of 5~10:1 by polyethylene glycol method
Fusion, HAT selective medium culture, indirect ELISA detects positive hole, and further utilizes indirect competition to positive hole
ELISA measures the inhibitory effect of positive cell hole, and then the positive hole good to inhibitory effect carries out limiting dilution assay and carry out 3~4
Time cloning, screening obtains hybridoma cell strain 6F9 after finally being verified with indirect competitive ELISA method.
The preparation method of preventing from heavy metal copper ion monoclonal antibody, comprising the following steps:
It is mono- that the 6F9 screened is injected with method in BALB/C mice intraperitoneal injection norphytane 0.3ml/, after 7~10d (day)
Clonal cell line cell, ascites is extracted in sterile working after mouse peritoneal obviously swells, and is centrifuged off after precipitation of grease to get small
Mouse ascites fluid McAb；
Ascites after purification, obtains monoclonal antibody purification.
Further, by heterocyclic bifunctional chelating agent L2It is substituted for 2-S- (2- aminobenzene)-Isosorbide-5-Nitrae, 7 three azacyclo-s respectively
Nonane-Isosorbide-5-Nitrae, 7- triacetic acid (are abbreviated as o-NH2- Bn-NOTA) or 2-S- (4- aminobenzene)-Isosorbide-5-Nitrae, 7 7-triazacyclononane -1,
4,7- triacetic acids (are abbreviated as p-NH2- Bn-NOTA) or 2-S- [4- (3- aminopropyl)-benzene]-Isosorbide-5-Nitrae, 7 7-triazacyclononanes-
Isosorbide-5-Nitrae, 7- triacetic acid, using the above identical preparation method can be made different copper ion artificial antigen, hybridoma cell strain,
Preventing from heavy metal copper ion monoclonal antibody.
Further, bovine serum albumin(BSA) BSA is replaced with into keyhole limpet hemocyanin KLH or other carrier proteins, used
The above identical preparation method, can prepare different copper ion artificial antigens, hybridoma cell strain, monoclonal antibody.
The beneficial effects of the present invention are:
(1) present invention uses heterocyclic bifunctional chelating agent m-NH2- Bn-NOTA prepares copper ion artificial antigen, the chelating
Agent can preferably combine heavy metal ion in structure, while can be preferably by the structure of heavy metal copper ion on space structure
It shows, the copper ion artificial antigen being prepared has stronger specificity.
(2) the hybridoma cell strain 6F9 that the present invention is prepared can secrete heavy metal copper ion monoclonal antibody, the list
Clonal antibody specificity is stronger, has preferably sensitivity, IC to copper ion chelator compound20Value is 0.14ngmL-1,
It can be used in quickly detecting heavy metal copper ion.The anti-copper ion monoclonal antibody can be used for the residual of copper ion in environmental sample
Stay detection.
Detailed description of the invention
Fig. 1 is that (wherein, Protein represents bovine serum albumin(BSA) to copper ion artificial antigen synthesis schematic diagram in embodiment 1,2
BSA or chicken egg white OVA).
Fig. 2 is that (wherein, Protein represents bovine serum albumin(BSA) to copper ion artificial antigen synthesis schematic diagram in embodiment 3,4
BSA or chicken egg white OVA).
Fig. 3 is that (wherein, Protein represents bovine serum albumin(BSA) to copper ion artificial antigen synthesis schematic diagram in embodiment 5,6
BSA or chicken egg white OVA).
Fig. 4 is that (wherein, Protein represents bovine serum albumin(BSA) to copper ion artificial antigen synthesis schematic diagram in embodiment 7,8
BSA or chicken egg white OVA).
Specific embodiment
Further details of the technical solution of the present invention below, it is noted that specific embodiment is
Detailed description of the invention is not construed as limitation of the invention.
Heterocyclic bifunctional chelating agent used in the present invention indicates that heterocyclic bifunctional chelating agent is distinguished in embodiment with L
Are as follows: 2-S- (2- aminobenzene)-Isosorbide-5-Nitrae, 7 7-triazacyclononanes-Isosorbide-5-Nitrae, 7- triacetic acid (are abbreviated as o-NH2- Bn-NOTA) use L1It indicates,
2-S- (3- aminobenzene)-Isosorbide-5-Nitrae, 7 7-triazacyclononanes-Isosorbide-5-Nitrae, 7- triacetic acid (are abbreviated as m-NH2- Bn-NOTA) use L2It indicates, 2-
S- (4- aminobenzene)-Isosorbide-5-Nitrae, 7 7-triazacyclononanes-Isosorbide-5-Nitrae, 7- triacetic acid (are abbreviated as p-NH2- Bn-NOTA) use L3It indicates, 2-S-
[4- (3- aminopropyl)-benzene]-Isosorbide-5-Nitrae, 7 7-triazacyclononanes-Isosorbide-5-Nitrae, 7- triacetic acid (are abbreviated as p-CH2CH2CH2NH2-Bn-
NOTA L) is used4It indicates, these chelating agents are commercially available by commercial sources.
1. the preparation and authentication of heterocyclic bifunctional chelating agent copper ion artificial antigen
Embodiment 1
(1) synthesis of copper ion artificial antigen:
Weigh 6mg L1, it is dissolved in 2mL 0.01molL-1, 7.4 4- hydroxyethyl piperazineethanesulfonic acid HEPES solution of pH,
L is made1Chelating agent solution, this solution are A liquid；
It weighs 70.33mg copper nitrate to be dissolved in 5mL ultrapure water, it is 7.5 × 10 that concentration, which is made,-2mol·L-1Copper nitrate
Solution, this solution are B liquid；
The B liquid of 180 μ L is added in A liquid, is protected from light 3~5h at room temperature, this reaction solution is C liquid；
700 μ L, 20mmolL are added dropwise into C liquid-1Glutaraldehyde water solution, room temperature be protected from light overnight, this reaction
Liquid is D liquid；
It weighs 20mg bovine serum albumin(BSA) BSA and is dissolved in 3mL HEPES, magnetic agitation mixes at room temperature, this reaction solution is E
Liquid；
D liquid is added dropwise in E liquid, is protected from light at room temperature for 24 hours, after that 150~200 μ L sodium borohydrides are added dropwise is molten
Liquid (20mg sodium borohydride is dissolved in 200 μ L ultrapure waters), is protected from light 1h at room temperature；
Reaction solution first uses the bag filter of 8KD to dialyse 3~5 times, then with 7000~9000rmin of ultra-filtration centrifuge tube of 30KD-1
Centrifuge washing 3~5 times, every time 15~20min, with the 0.01molL of 5~10mL-1, the HEPES solution redissolution of pH 7.4, after
Artificial antigen of heavy metal copper Cu-L is made in packing, -20 DEG C of cryo-conservations1- BSA, L1Expression 2-S- (2- aminobenzene)-Isosorbide-5-Nitrae, 7 three
Azepine cyclononane-Isosorbide-5-Nitrae, 7- triacetic acid (are abbreviated as o-NH2-Bn-NOTA)。
(2) identification of copper ion artificial antigen:
It takes UV scanning and SDS-PAGE to identify its coupling effect, is measured respectively using ICP-MS method and Bradford method
Heavy metal ion and protein concentration, calculate artificial antigen coupling combine than.
UV scanning scheme: BSA, Cu-L1- BSA and Cu-L1Being configured to concentration is 1~5mgmL-1Solution in range,
The absorbance in 200~400nm wave-length coverage is measured, and establishes ultraviolet scanning atlas, it is bent by comparing the absorption of every kind of solution
Whether line succeeds to identify to synthesize.
In ultraviolet scanning atlas, Cu-L1- BSA solution is compared with BSA solution, Cu-L1The maximum absorption wavelength of-BSA solution
It changes, illustrates to be coupled successfully.
SDS-PAGE electrophoresis protocols: selecting volume fraction for 5% concentration glue, select volume fraction for 10% separation gel, on
The 10 every hole μ L of sample amount, concentrate glue voltage 75V, separation gel voltage 100V, coomassie brilliant blue staining 1h decolourize 4 times, gel imager
Photographic analysis.
Show that conjugate electrophoretic band has hysteresis, the molecular weight of conjugate than single albumen one through SDS-PAGE
It is bigger than single molecular weight of albumen, illustrate to be coupled successfully.
It is 24:1 by measurement content of beary metal and conjugate protein concentration calculations incorporated ratio.In conjunction with than for 24:1, then table
Show and combines 24 heavy metal ion on a protein molecular.
Embodiment 2
(1) synthesis of copper ion artificial antigen:
Weigh 7mg L1, it is dissolved in 2mL0.01molL-1, 7.4 4- hydroxyethyl piperazineethanesulfonic acid HEPES solution of pH, system
Obtain L1Chelating agent solution, this solution are A liquid；
It weighs 70.33mg copper nitrate to be dissolved in 5mL ultrapure water, it is 7.5 × 10 that concentration, which is made,-2mol·L-1Copper nitrate
Solution, this solution are B liquid；
The B liquid of 195 μ L is added in A liquid, is protected from light 3~5h at room temperature, this reaction solution is C liquid；
720 μ L, 20mmolL are added dropwise into C liquid-1Glutaraldehyde solution, room temperature be protected from light overnight, this reaction solution
For D liquid；
It weighs 20mg chicken egg white OVA and is dissolved in 3mL HEPES, magnetic agitation mixes at room temperature, this reaction solution is E
Liquid；
D liquid is added dropwise in E liquid, is protected from light at room temperature for 24 hours, after that 150~200 μ L sodium borohydrides are added dropwise is molten
Liquid (20mg sodium borohydride is dissolved in 200 μ L ultrapure waters), is protected from light 1h at room temperature；
Reaction solution first uses the bag filter of 8KD to dialyse 3~5 times, then with 7000~9000rmin of ultra-filtration centrifuge tube of 30KD-1
Centrifuge washing 3~5 times, every time 15~20min, with the 0.01molL of 5~10mL-1, the HEPES solution redissolution of pH 7.4, after
Artificial antigen of heavy metal copper Cu-L is made in packing, -20 DEG C of cryo-conservations1- OVA, L1Expression 2-S- (2- aminobenzene)-Isosorbide-5-Nitrae, 7 three
Azepine cyclononane-Isosorbide-5-Nitrae, 7- triacetic acid (are abbreviated as o-NH2-Bn-NOTA)。
(2) identification of artificial antigen:
It takes UV scanning and SDS-PAGE to identify its coupling effect, is measured respectively using ICP-MS method and Bradford method
Heavy metal ion and protein concentration, calculate artificial antigen coupling combine than.
UV scanning scheme: OVA, Cu-L1- OVA and Cu-L1Being configured to concentration is 1~5mgmL-1Solution in range,
The absorbance in 200~400nm wave-length coverage is measured, and establishes ultraviolet scanning atlas, it is bent by comparing the absorption of every kind of solution
Whether line succeeds to identify to synthesize.
In ultraviolet scanning atlas, Cu-L1Compared with OVA solution, maximum absorption wavelength changes-OVA solution, illustrates to be coupled
Success.
SDS-PAGE electrophoresis protocols: selecting volume fraction for 5% concentration glue, select volume fraction for 10% separation gel, on
The 10 every hole μ L of sample amount, concentrate glue voltage 75V, separation gel voltage 100V, coomassie brilliant blue staining 1h decolourize 4 times, gel imager
Photographic analysis.
Show that conjugate electrophoretic band has hysteresis, the molecular weight of conjugate than single albumen one through SDS-PAGE
It is bigger than single molecular weight of albumen, illustrate to be coupled successfully.
It is 21:1 by measurement content of beary metal and conjugate protein concentration calculations incorporated ratio.In conjunction with than for 21:1, then table
Show and combines 21 heavy metal ion on a protein molecular.
Embodiment 3
The heterocyclic bifunctional chelating agent that the present embodiment is selected is L2, L in alternative embodiment 11；According to 1 phase of embodiment
Copper ion artificial antigen Cu-L is made in same step2- BSA, copper ion artificial antigen synthetic route is as shown in Fig. 2, specific synthesis
Steps are as follows.
(1) synthesis of copper ion artificial antigen:
Weigh 6mg L2, it is dissolved in 2mL 0.01molL-1, 7.4 4- hydroxyethyl piperazineethanesulfonic acid HEPES solution of pH,
L is made2Chelating agent solution, this solution are A liquid；
It weighs 70.33mg copper nitrate to be dissolved in 5mL ultrapure water, it is 7.5 × 10 that concentration, which is made,-2mol·L-1Copper nitrate
Solution, this solution are B liquid；
The B liquid of 180 μ L is added in A liquid, is protected from light 3~5h at room temperature, this reaction solution is C liquid；
700 μ L, 20mmolL are added dropwise into C liquid-1Glutaraldehyde solution, room temperature be protected from light overnight, this reaction solution
For D liquid；
It weighs 20mg bovine serum albumin(BSA) BSA and is dissolved in 3mL HEPES, magnetic agitation mixes at room temperature, this reaction solution is E
Liquid；
D liquid is added dropwise in E liquid, is protected from light at room temperature for 24 hours, after that 150~200 μ L sodium borohydrides are added dropwise is molten
Liquid (20mg sodium borohydride is dissolved in 200 μ L ultrapure waters), is protected from light 1h at room temperature；
Reaction solution first uses the bag filter of 8KD to dialyse 3-5 times, then with 7000~9000rmin of ultra-filtration centrifuge tube of 30KD-1
Centrifuge washing 3~5 times, every time 15~20min, with the 0.01molL of 5~10mL-1, the HEPES solution of pH7.4 redissolves, rear to divide
Artificial antigen of heavy metal copper Cu-L is made in dress, -20 DEG C of cryo-conservations2- BSA, L2Indicate 2-S- (3- aminobenzene)-Isosorbide-5-Nitrae, 7 three nitrogen
Ononane-Isosorbide-5-Nitrae, 7- triacetic acid (are abbreviated as m-NH2-Bn-NOTA)。
(2) identification of artificial antigen:
It takes UV scanning and SDS-PAGE to identify its coupling effect, is measured respectively using ICP-MS method and Bradford method
Heavy metal ion and protein concentration, calculate artificial antigen coupling combine than.
UV scanning scheme: BSA, Cu-L2- BSA and Cu-L2Being configured to concentration is 1~5mgmL-1Solution in range,
The absorbance in 200~400nm wave-length coverage is measured, and establishes ultraviolet scanning atlas, it is bent by comparing the absorption of every kind of solution
Whether line succeeds to identify to synthesize.
In ultraviolet scanning atlas, Cu-L2Compared with BSA solution, maximum absorption wavelength changes-BSA solution, illustrates to be coupled
Success.
SDS-PAGE electrophoresis protocols: selecting volume fraction for 5% concentration glue, select volume fraction for 10% separation gel, on
The 10 every hole μ L of sample amount, concentrate glue voltage 75V, separation gel voltage 100V, coomassie brilliant blue staining 1h decolourize 4 times, gel imager
Photographic analysis.
Show that conjugate electrophoretic band has hysteresis, the molecular weight of conjugate than single albumen one through SDS-PAGE
It is bigger than single molecular weight of albumen, illustrate to be coupled successfully.
It is 46:1 by measurement content of beary metal and conjugate protein concentration calculations incorporated ratio, coupling efficiency is higher.In conjunction with
Than higher, illustrate that the quantity that heavy metal ion is coupled on a protein molecular is more, coupling efficiency is also higher, in conjunction with than being 46:
1, then it represents that 46 heavy metal ion are combined on a protein molecular.
Embodiment 4
The heterocyclic bifunctional chelating agent that the present embodiment is selected is L2, L in alternative embodiment 21；According to 2 phase of embodiment
Copper ion artificial antigen Cu-L is made in same step2- OVA, copper ion artificial antigen synthetic route is as shown in Fig. 2, specific synthesis
Steps are as follows.
(1) synthesis of copper ion artificial antigen:
Weigh 7mg L2, it is dissolved in 2mL 0.01molL-1, pH7.4 4- hydroxyethyl piperazineethanesulfonic acid HEPES solution, system
Obtain L2Chelating agent solution, this solution are A liquid；
It weighs 70.33mg copper nitrate to be dissolved in 5mL ultrapure water, it is 7.5 × 10 that concentration, which is made,-2mol·L-1Copper nitrate
Solution, this solution are B liquid；
The B liquid of 195 μ L is added in A liquid, is protected from light 3~5h at room temperature, this reaction solution is C liquid；
720 μ L, 20mmolL are added dropwise into C liquid-1Glutaraldehyde solution, room temperature be protected from light overnight, this reaction solution
For D liquid；
It weighs 20mg chicken egg white OVA and is dissolved in 3mL HEPES, magnetic agitation mixes at room temperature, this reaction solution is E
Liquid；
D liquid is added dropwise in E liquid, is protected from light at room temperature for 24 hours, after that 150~200 μ L sodium borohydrides are added dropwise is molten
Liquid (20mg sodium borohydride is dissolved in 200 μ L ultrapure waters), is protected from light 1h at room temperature；
Reaction solution first uses the bag filter of 8KD to dialyse 3-5 times, then with 7000~9000rmin of ultra-filtration centrifuge tube of 30KD-1
Centrifuge washing 3~5 times, every time 15~20min, with the 0.01molL of 5~10mL-1, the HEPES solution of pH7.4 redissolves, rear to divide
Artificial antigen of heavy metal copper Cu-L is made in dress, -20 DEG C of cryo-conservations2- OVA, L2Indicate 2-S- (3- aminobenzene)-Isosorbide-5-Nitrae, 7 three nitrogen
Ononane-Isosorbide-5-Nitrae, 7- triacetic acid (are abbreviated as m-NH2-Bn-NOTA)。
(2) identification of artificial antigen:
It takes UV scanning and SDS-PAGE to identify its coupling effect, is measured respectively using ICP-MS method and Bradford method
Heavy metal ion and protein concentration, calculate artificial antigen coupling combine than.
UV scanning scheme: OVA, Cu-L2- OVA and Cu-L2Being configured to concentration is 1~5mgmL-1Solution, measurement
Absorbance in 200~400nm wave-length coverage, and ultraviolet scanning atlas is established, come by comparing the absorption curve of every kind of solution
Identify whether synthesis succeeds.
In ultraviolet scanning atlas, Cu-L2Compared with OVA solution, maximum absorption wavelength changes-OVA solution, illustrates to be coupled
Success.
SDS-PAGE electrophoresis protocols: selecting volume fraction for 5% concentration glue, select volume fraction for 10% separation gel, on
The 10 every hole μ L of sample amount, concentrate glue voltage 75V, separation gel voltage 100V, coomassie brilliant blue staining 1h decolourize 4 times, gel imager
Photographic analysis.
Show that conjugate electrophoretic band has hysteresis, the molecular weight of conjugate than single albumen one through SDS-PAGE
It is bigger than single molecular weight of albumen, illustrate to be coupled successfully.
It is 40:1 by measurement content of beary metal and conjugate protein concentration calculations incorporated ratio, in conjunction with than then being indicated for 40:1
40 heavy metal ion are combined on one protein molecular.
Embodiment 5
The heterocyclic bifunctional chelating agent that the present embodiment is selected is L3, L in alternative embodiment 11；According to 1 phase of embodiment
Copper ion artificial antigen Cu-L is made in same step3- BSA, copper ion artificial antigen synthetic route is as shown in figure 3, specific synthesis
Steps are as follows.
(1) synthesis of copper ion artificial antigen:
Weigh 6mg L3, it is dissolved in 2mL 0.01molL-1, 7.4 4- hydroxyethyl piperazineethanesulfonic acid HEPES solution of pH,
L is made3Chelating agent solution, this solution are A liquid；
It weighs 70.33mg copper nitrate to be dissolved in 5mL ultrapure water, it is 7.5 × 10 that concentration, which is made,-2mol·L-1Copper nitrate
Solution, this solution are B liquid；
The B liquid of 180 μ L is added in A liquid, is protected from light 3~5h at room temperature, this reaction solution is C liquid；
700 μ L, 20mmolL are added dropwise into C liquid-1Glutaraldehyde solution, room temperature be protected from light overnight, this reaction solution
For D liquid；
It weighs 20mg bovine serum albumin(BSA) BSA and is dissolved in 3mL HEPES, magnetic agitation mixes at room temperature, this reaction solution is E
Liquid；
D liquid is added dropwise in E liquid, is protected from light at room temperature for 24 hours, after that 150~200 μ L sodium borohydrides are added dropwise is molten
Liquid (20mg sodium borohydride is dissolved in 200 μ L ultrapure waters), is protected from light 1h at room temperature；
Reaction solution first uses the bag filter of 8KD to dialyse 3~5 times, then with 7000~9000rmin of ultra-filtration centrifuge tube of 30KD-1
Centrifuge washing 3~5 times, every time 15~20min, with the 0.01molL of 5~10mL-1, the HEPES solution of pH7.4 redissolves, rear to divide
Artificial antigen of heavy metal copper Cu-L is made in dress, -20 DEG C of cryo-conservations3- BSA, L3Indicate 2-S- (4- aminobenzene)-Isosorbide-5-Nitrae, 7 three nitrogen
Ononane-Isosorbide-5-Nitrae, 7- triacetic acid (are abbreviated as p-NH2-Bn-NOTA)。
(2) identification of artificial antigen:
It takes UV scanning and SDS-PAGE to identify its coupling effect, is measured respectively using ICP-MS method and Bradford method
Heavy metal ion and protein concentration, calculate artificial antigen coupling combine than.
UV scanning scheme: BSA, Cu-L3- BSA and Cu-L3Being configured to concentration is 1~5mgmL-1Solution in range,
The absorbance in 200~400nm wave-length coverage is measured, and establishes ultraviolet scanning atlas, it is bent by comparing the absorption of every kind of solution
Whether line succeeds to identify to synthesize.
In ultraviolet scanning atlas, Cu-L3Compared with BSA solution, maximum absorption wavelength changes-BSA solution, illustrates to be coupled
Success.
SDS-PAGE electrophoresis protocols: selecting volume fraction for 5% concentration glue, select volume fraction for 10% separation gel, on
The 10 every hole μ L of sample amount, concentrate glue voltage 75V, separation gel voltage 100V, coomassie brilliant blue staining 1h decolourize 4 times, gel imager
Photographic analysis.
Show that conjugate electrophoretic band has hysteresis, the molecular weight of conjugate than single albumen one through SDS-PAGE
It is bigger than single molecular weight of albumen, illustrate to be coupled successfully.
It is 39:1 by measurement content of beary metal and conjugate protein concentration calculations incorporated ratio, in conjunction with than for 39:1, then table
Show and combines 39 heavy metal ion on a protein molecular.
Embodiment 6
The heterocyclic bifunctional chelating agent that the present embodiment is selected is L3, L in alternative embodiment 11；According to 2 phase of embodiment
Copper ion artificial antigen Cu-L is made in same step3- OVA, copper ion artificial antigen synthetic route is as shown in figure 3, specific synthesis
Steps are as follows.
(1) synthesis of copper ion artificial antigen:
Weigh 7mg L3, it is dissolved in 2mL 0.01molL-1, 7.4 4- hydroxyethyl piperazineethanesulfonic acid HEPES solution of pH,
L is made3Chelating agent solution, this solution are A liquid；
It weighs 70.33mg copper nitrate to be dissolved in 5mL ultrapure water, it is 7.5 × 10 that concentration, which is made,-2mol·L-1Copper nitrate
Solution, this solution are B liquid；
The B liquid of 195 μ L is added in A liquid, is protected from light 3~5h at room temperature, this reaction solution is C liquid；
720 μ L, 20mmolL are added dropwise into C liquid-1Glutaraldehyde solution, room temperature be protected from light overnight, this reaction solution
For D liquid；
It weighs 20mg chicken egg white OVA and is dissolved in 3mL HEPES, magnetic agitation mixes at room temperature, this reaction solution is E
Liquid；
D liquid is added dropwise in E liquid, is protected from light at room temperature for 24 hours, after that 150~200 μ L sodium borohydrides are added dropwise is molten
Liquid (20mg sodium borohydride is dissolved in 200 μ L ultrapure waters), is protected from light 1h at room temperature；
Reaction solution first uses the bag filter of 8KD to dialyse 3~5 times, then with 7000~9000rmin of ultra-filtration centrifuge tube of 30KD-1
Centrifuge washing 3~5 times, every time 15~20min, with the 0.01molL of 5~10mL-1, the HEPES solution redissolution of pH 7.4, after
Artificial antigen of heavy metal copper Cu-L is made in packing, -20 DEG C of cryo-conservations3- OVA, L3Expression 2-S- (4- aminobenzene)-Isosorbide-5-Nitrae, 7 three
Azepine cyclononane-Isosorbide-5-Nitrae, 7- triacetic acid (are abbreviated as p-NH2-Bn-NOTA)。
(2) identification of artificial antigen:
It takes UV scanning and SDS-PAGE to identify its coupling effect, is measured respectively using ICP-MS method and Bradford method
Heavy metal ion and protein concentration, calculate artificial antigen coupling combine than.
UV scanning scheme: OVA, Cu-L3- OVA and Cu-L3Being configured to concentration is 1~5mgmL-1Solution in range,
The absorbance in 200~400nm wave-length coverage is measured, and establishes ultraviolet scanning atlas, it is bent by comparing the absorption of every kind of solution
Whether line succeeds to identify to synthesize.
In ultraviolet scanning atlas, Cu-L3Compared with OVA solution, maximum absorption wavelength changes-OVA solution, illustrates to be coupled
Success.
SDS-PAGE electrophoresis protocols: selecting volume fraction for 5% concentration glue, select volume fraction for 10% separation gel, on
The 10 every hole μ L of sample amount, concentrate glue voltage 75V, separation gel voltage 100V, coomassie brilliant blue staining 1h decolourize 4 times, gel imager
Photographic analysis.
Show that conjugate electrophoretic band has hysteresis, the molecular weight of conjugate than single albumen one through SDS-PAGE
It is bigger than single molecular weight of albumen, illustrate to be coupled successfully.
It is 36:1 by measurement content of beary metal and conjugate protein concentration calculations incorporated ratio, in conjunction with than for 36:1, then table
Show and combines 36 heavy metal ion on a protein molecular.
Embodiment 7
The heterocyclic bifunctional chelating agent that the present embodiment is selected is L4, L in alternative embodiment 11；According to 1 phase of embodiment
Copper ion artificial antigen Cu-L is made in same step4- BSA, copper ion artificial antigen synthetic route is as shown in figure 4, specific synthesis
Steps are as follows.
(1) synthesis of copper ion artificial antigen:
Weigh 6mg L4, it is dissolved in 2mL 0.01molL-1, pH7.4 4- hydroxyethyl piperazineethanesulfonic acid HEPES solution, system
Obtain L4Chelating agent solution, this solution are A liquid；
It weighs 70.33mg copper nitrate to be dissolved in 5mL ultrapure water, it is 7.5 × 10 that concentration, which is made,-2mol·L-1Copper nitrate
Solution, this solution are B liquid；
The B liquid of 180 μ L is added in A liquid, is protected from light 3~5h at room temperature, this reaction solution is C liquid；
700 μ L, 20mmolL are added dropwise into C liquid-1Glutaraldehyde solution, room temperature be protected from light overnight, this reaction solution
For D liquid；
It weighs 20mg bovine serum albumin(BSA) BSA and is dissolved in 3mL HEPES, magnetic agitation mixes at room temperature, this reaction solution is E
Liquid；
D liquid is added dropwise in E liquid, is protected from light at room temperature for 24 hours, after that 150~200 μ L sodium borohydrides are added dropwise is molten
Liquid (20mg is dissolved in 200 μ L ultrapure waters), is protected from light 1h at room temperature；
Reaction solution first uses the bag filter of 8KD to dialyse 3~5 times, then with 7000~9000rmin of ultra-filtration centrifuge tube of 30KD-1
Centrifuge washing 3~5 times, every time 15~20min, with the 0.01molL of 5~10mL-1, the HEPES solution of pH7.4 redissolves, rear to divide
Artificial antigen of heavy metal copper Cu-L is made in dress, -20 DEG C of cryo-conservations4- BSA, L4It indicates 2-S- [4- (3- aminopropyl)-benzene]-
Isosorbide-5-Nitrae, 7 7-triazacyclononanes-Isosorbide-5-Nitrae, 7- triacetic acid (are abbreviated as p-CH2CH2CH2NH2-Bn-NOTA)。
(2) identification of artificial antigen:
It takes UV scanning and SDS-PAGE to identify its coupling effect, is measured respectively using ICP-MS method and Bradford method
Heavy metal ion and protein concentration, calculate artificial antigen coupling combine than.
UV scanning scheme: BSA, Cu-L4- BSA and Cu-L4Being configured to concentration is 1~5mgmL-1Solution in range,
The absorbance in 200~400nm wave-length coverage is measured, and establishes ultraviolet scanning atlas, it is bent by comparing the absorption of every kind of solution
Whether line succeeds to identify to synthesize.
In ultraviolet scanning atlas, Cu-L4Compared with BSA solution, maximum absorption wavelength changes-BSA solution, illustrates to be coupled
Success.
SDS-PAGE electrophoresis protocols: selecting volume fraction for 5% concentration glue, select volume fraction for 10% separation gel, on
The 10 every hole μ L of sample amount, concentrate glue voltage 75V, separation gel voltage 100V, coomassie brilliant blue staining 1h decolourize 4 times, gel imager
Photographic analysis.
Show that conjugate electrophoretic band has hysteresis, the molecular weight of conjugate than single albumen one through SDS-PAGE
It is bigger than single molecular weight of albumen, illustrate to be coupled successfully.
It is 42:1 by measurement content of beary metal and conjugate protein concentration calculations incorporated ratio, in conjunction with than for 42:1, then table
Show and combines 42 heavy metal ion on a protein molecular.
Embodiment 8
The heterocyclic bifunctional chelating agent that the present embodiment is selected is L4, L in alternative embodiment 11；According to 2 phase of embodiment
Copper ion artificial antigen Cu-L is made in same step4- OVA, copper ion artificial antigen synthetic route is as shown in figure 4, specific synthesis
Steps are as follows.
(1) synthesis of copper ion artificial antigen:
Weigh 7mg L4, it is dissolved in 2mL 0.01molL-1, 7.4 4- hydroxyethyl piperazineethanesulfonic acid HEPES solution of pH,
L is made4Chelating agent solution, this solution are A liquid；
It weighs 70.33mg copper nitrate to be dissolved in 5mL ultrapure water, it is 7.5 × 10 that concentration, which is made,-2mol·L-1Copper nitrate
Solution, this solution are B liquid；
The B liquid of 195 μ L is added in A liquid, is protected from light 3~5h at room temperature, this reaction solution is C liquid；
720 μ L, 20mmolL are added dropwise into C liquid-1Glutaraldehyde solution, room temperature be protected from light overnight, this reaction solution
For D liquid；
It weighs 20mg chicken egg white OVA and is dissolved in 4mL HEPES, magnetic agitation mixes at room temperature, this reaction solution is E
Liquid；
D liquid is added dropwise in E liquid, is protected from light at room temperature for 24 hours, after that 150~200 μ l sodium borohydrides are added dropwise is molten
Liquid (20mg is dissolved in 200 μ l ultrapure waters), is protected from light 1h at room temperature；
Reaction solution first uses the bag filter of 8KD to dialyse 3~5 times, then with 7000~9000rmin of ultra-filtration centrifuge tube of 30KD-1
Centrifuge washing 3~5 times, every time 15~20min, with the 0.01molL of 5~10mL-1, the HEPES solution redissolution of pH 7.4, after
Artificial antigen of heavy metal copper Cu-L is made in packing, -20 DEG C of cryo-conservations4- OVA, L4Indicate 2-S- [4- (3- aminopropyl)-
Benzene]-Isosorbide-5-Nitrae, 7 7-triazacyclononanes-Isosorbide-5-Nitrae, 7- triacetic acid (abbreviation p-CH2CH2CH2NH2-Bn-NOTA)。
(2) identification of artificial antigen:
It takes UV scanning and SDS-PAGE to identify its coupling effect, is measured respectively using ICP-MS method and Bradford method
Heavy metal ion and protein concentration, calculate artificial antigen coupling combine than.
UV scanning scheme: OVA, Cu-L4- OVA and Cu-L4Being configured to concentration is 1~5mgmL-1Solution in range,
The absorbance in 200~400nm wave-length coverage is measured, and establishes ultraviolet scanning atlas, it is bent by comparing the absorption of every kind of solution
Whether line succeeds to identify to synthesize.
In ultraviolet scanning atlas, Cu-L4Compared with OVA solution, maximum absorption wavelength changes-OVA solution, illustrates to be coupled
Success.
SDS-PAGE electrophoresis protocols: selecting volume fraction for 5% concentration glue, select volume fraction for 10% separation gel, on
The 10 every hole μ L of sample amount, concentrate glue voltage 75V, separation gel voltage 100V, coomassie brilliant blue staining 1h decolourize 4 times, gel imager
Photographic analysis.
Show that conjugate electrophoretic band has hysteresis, the molecular weight of conjugate than single albumen one through SDS-PAGE
It is bigger than single molecular weight of albumen, illustrate to be coupled successfully.
It is 38:1 by measurement content of beary metal and conjugate protein concentration calculations incorporated ratio, in conjunction with than for 38:1, then table
Show and combines 38 heavy metal ion on a protein molecular.
Embodiment 9
The measurement of antiserum titre:
Artificial antigen prepared by embodiment 1,3,5 and 7 is respectively immunized BALB/C mice, initial immunity is using not
Family name's Freund's complete adjuvant emulsifies artificial antigen, and after emulsification, injection is measured as 250 μ g/ mouse, after at interval of 21d booster immunization, altogether plus
3 times strong immune, booster immunization is emulsified using Freund's incomplete adjuvant, and it is 150 μ g/ mouse that metering, which is immunized, small after booster immunization 14d
Mouse docking blood sampling carries out the titration of polyvalent antibody, and ELISA method measures antiserum titre after serum confining liquid doubling dilution,
Using the mice serum before being immunized as negative control, with positive serum OD450nmValue and negative serum OD450nmIt is worth ratio and is greater than 2.1
Place dilution is antiserum titre, and the results are shown in Table 1.It finally carries out end to exempt from, end is exempted from using the direct abdominal cavity note of artificial antigen
The mode penetrated carries out, and it is 300 μ g/ mouse that metering, which is immunized,.
Titration uses indirect ELISA method, and steps are as follows for specific experiment:
A. it is coated with: being respectively packet with the artificial antigen in embodiment 2 or embodiment 4 or embodiment 6 or embodiment 8
It is former, pH 9.6,0.05molL-1CBS is coating buffer, and primary concentration of envelope is 10 μ gmL-1, package amount is 100 μ L/
Hole, is coated with 2h in 37 DEG C, and latter PBST washing lotion board-washing 4 times；
B. it closes: 250 hole μ L/ of confining liquid, after 37 DEG C of incubation 30min, PBST washing lotion board-washing 4 times is added；
C. plus antiserum: the antiserum 10000rmin that mouse blood sampling is obtained-1After being centrifuged 5min, draws 10 μ L and be added
To 2mL confining liquid, (initial dilution multiple is 200 times) uses 11 gradients of confining liquid doubling dilution and 1 negative control afterwards,
100 holes μ L/, 4 repetitions of each gradient, 37 DEG C of incubation 1h are rear to use PBST washing lotion board-washing 4 times；
Plus ELIAS secondary antibody d.: PBST board-washing 4 times after reaction, after 10000 times of sheep anti mouse secondary antibody dilutions of HRP label plus
Enter ELISA Plate, 100 holes μ L/, 37 DEG C of incubation 1h, latter PBST washing lotion board-washing 4 times；
E. plus substrate reactions liquid: tmb substrate buffer, 100 holes μ L/, 37 DEG C of incubation 15min are added；
F. it terminates reading: 2molL is added-1OD is read with microplate reader in 50 hole μ L/ of sulfuric acid450nmValue.
Combination ratio and antiserum titre measurement result in 1 embodiment 1-8 of table
| Immunizing antigen | In conjunction with than | Detect antigen | In conjunction with than | Antiserum titre |
| Cu-L1- BSA embodiment 1 | 24:1 | Cu-L1- OVA embodiment 2 | 21:1 | 256000 |
| Cu-L2- BSA embodiment 3 | 46:1 | Cu-L2- OVA embodiment 4 | 40:1 | 650000 |
| Cu-L3- BSA embodiment 5 | 39:1 | Cu-L3- OVA embodiment 6 | 36:1 | 550000 |
| Cu-L4- BSA embodiment 7 | 42:1 | Cu-L4- OVA embodiment 8 | 38:1 | 600000 |
Show antiserum titre sequence with antiserum titre measurement result by the combination ratio of table 1 are as follows: embodiment 3 > reality
7 > embodiment of example, 5 > embodiment 1 is applied, in conjunction with the sequence that compares so as to see who is superior are as follows: 5 > embodiment of embodiment 3 > embodiment, 7 > embodiment 1, wherein
The antiserum titre highest of embodiment 3, the antiserum titre of embodiment 7 are only below embodiment 3.This illustrates to prepare in embodiment 3
Copper ion artificial antigen Cu-L2- BSA can preferably show heavy-metal antigen determinant, and antigentic specificity is stronger, together
When embodiment 3 and embodiment 7 prepared by artificial antigen its combine relatively high, height is combined than being easier to cause strong immune answers
Reaction is answered, is conducive to prepare preventing from heavy metal copper ion monoclonal antibody.
Specific preparation method, the measuring method of the use of embodiment 1,3,5,7 are identical, still, the bifunctional chelating agent of use
Difference is respectively as follows: o-NH2- Bn-NOTA, m-NH2- Bn-NOTA, p-NH2- Bn-NOTA, p-CH2CH2CH2NH2-Bn-NOTA.This
Illustrate-NH in chelating agent2Meta position in phenyl ring, chelating agent m-NH2After-Bn-NOTA combines heavy metal ion, in space structure
On preferably the structure of heavy metal copper ion can be shown, and from the experimental results, the artificial of bit architecture preparation resists
Former combination is than also highest, as antigenic determinant, is more advantageous to preparation affinity and sensitivity is higher, specific stronger anti-
Heavy metal monoclonal antibody.
Embodiment 10
The screening of hybridoma cell strain and the preparation purifying of monoclonal antibody
(1) mouse immune:
Select 6~8 week old, weight 18~20g BALB/C female mice.By the immunogene of preparation, (immunogene is used herein
: copper ion artificial antigen Cu-L2- BSA) being pressurizeed with isometric Freund's complete adjuvant using syringe is sufficiently mixed emulsification
Afterwards, by abdomen and oxter multi-point injection, dosage be 50~100 μ g/ only, after at interval of 21d carry out booster immunization, 3 reinforcements
Blood sampling measurement potency, potency are measured using indirect ELISA method after immune, and ELISA method is surveyed after serum confining liquid doubling dilution
Antiserum titre is determined, using the mice serum before being immunized as negative control, with positive serum OD450nmIt is worth big with negative serum ratio
In 2.1 place dilutions be antiserum titre.It carries out end when potency is no longer significantly raised to exempt from, progress cell melts after 3d is exempted from end
It closes.It is immunized for the first time in immunologic process and uses Freund's complete adjuvant, booster immunization uses incomplete Freund's adjuvant, and end exempts from not using
Adjuvant, direct immunization original injecting immune.
Titration uses indirect ELISA method, and steps are as follows for specific experiment:
A. it is coated with: with Cu-L2- OVA is coating antigen, pH 9.6,0.05molL-1CBS is coating buffer, and coating antigen is dense
Degree is 10 μ gmL-1, package amount is 100 holes μ L/, after being coated with 2h in 37 DEG C, PBST washing lotion board-washing 4 times；
B. it closes: 250 hole μ L/ of confining liquid is added, after 37 DEG C of incubation 30min, PBST board-washing 4 times, every hole adds 300 μ of PBST
L, latter PBST washing lotion board-washing 4 times；
C. plus antiserum: the antiserum 10000rmin that mouse blood sampling is obtained-1After being centrifuged 5min, draws 10 μ L and be added
To 2mL confining liquid (initial dilution multiple is 200 times), 11 gradients of confining liquid doubling dilution and 1 negative control are used afterwards,
100 holes μ L/, 4 repetitions of each gradient, 37 DEG C of incubation 1h, latter PBST washing lotion board-washing 4 times；
Plus ELIAS secondary antibody d.: enzyme is added after 10000 times of sheep anti mouse secondary antibody dilutions of HRP label in board-washing 4 times after reaction
Target, 100 holes μ L/, 37 DEG C of incubation 1h, latter PBST washing lotion board-washing 4 times；
E. plus substrate reactions liquid: tmb substrate buffer, 100 holes μ L/, 37 DEG C of incubation 15min are added；
F. it terminates reading: 2molL is added-1OD is read with microplate reader in 50 hole μ L/ of sulfuric acid450nmValue.
(2) cell fusion and culture:
After 3d is exempted from end, cell fusion is carried out according to conventional polyethylene glycol (PEG1500) method, the specific steps are as follows:
A. the eyeball of mouse bloodletting after end is exempted from, centrifugation sucts clear spare after serum collection, and neck execution is drawn to be placed on 70% wine
3~5min in essence takes mouse spleen under aseptic condition, is placed in sterile grinding bowl and mills after being shredded with aseptic operation, uses RPMI-
After 1640 basal mediums blow outstanding cell, crosses 200 mesh cell screen clothes and obtain splenocyte suspension, and carry out cell count；
B. SP2/0 cell (myeloma cell) is collected, it is desirable that cell growth state is good, and cell activity is greater than 90%, sucks
New RPMI-1640 basal medium is added after cell conditioned medium and blows and beats cell and suspends, carries out cell count afterwards；
C. according to cell counts, after splenocyte and SP2/0 cell are mixed according to the ratio of 5~10:1,1800r
min-1Supernatant is sucked after centrifugation 5min, remaining cell is added 0.5mL PEG and is gently mixed mixing in 1min inner edge edged, after adding
RPMI-1640 basal medium 45mL, rear 1500rmin are added from slow to fast after standing 1min-1Supernatant is removed after centrifugation 5min,
96 porocyte culture plates are plated on after selectivity HAT culture medium is added, every 250 μ L of hole is placed in 37 DEG C, 5%CO2Incubator in
Culture.
D. it after cultivating 3~5d (day), is changed liquid 1 time with HAT culture medium, 10d is changed to HT culture medium culture.
(3) cell screening and cell strain are established:
When cell to be fused grows into covering 10%~30% hole floor space of culture hole, supernatant is taken to be screened with indirect ELISA
Antibody positive wells, envelope antigen is Cu-L when screening2- OVA cross-linking agent, and negative control is made with OVA, BSA.The positive filtered out
Reacting hole is further with the detection sensitivity of competitive ELISA analysis antibody.
According to the standard curve of foundation, IC is calculated50Value and IC20Value, (abscissa is copper to selection standard slope of curve range
The natural logrithm of ion standard concentration, ordinate are the competition test curve that inhibiting rate is established) between 8~13, IC50It is worth low
In 200ngml-1, IC20Value is lower than 20ngml-1Cell strain further cloned and screened, connected with limiting dilution assay
Continuous clone 3~4 times, final to obtain a strain of hybridoma strain 6F9, deposit number is CGMCC No.16388.Comparatively,
IC20Numerical value is lower, IC50The inhibitory effect of the lower cell strain of numerical value is preferable, is conducive to prepare the preferable hybridoma of characteristic
Strain.
Hybridoma cell strain 6F9 was preserved in China Committee for Culture Collection of Microorganisms on 09 25th, 2018
Common micro-organisms center (abbreviation CGMCC, address: Institute of Microorganism, Academia Sinica, BeiChen West Road, Chaoyang District, BeiJing City 1
Institute 3, postcode 100101).
On the one hand the cell strain is moved into cell cryopreservation tube after expanding culture by the hybridoma cell strain 6F9 finally obtained
And be put into liquid nitrogen and save for a long time, the cell strain is on the other hand used for ascites preparation, the purifying and application of monoclonal antibody.
(3) preparation, purifying and identification of monoclonal antibody
Monoclonal antibody is prepared using method is induced in animal body.
6~8 week old health BALB/C female mices are selected, norphytane 0.3mL/ is injected intraperitoneally only in BALB/C mice, 7~
After 10d with method injection screen 6F9 monoclonal cell strain cell (0.4mL/ only, every ml cell strain quantity is 2.5 × 106~1
×107Between), ascites is extracted after mouse peritoneal obviously swells after 5~7d, is centrifuged off after precipitation of grease to get mouse abdomen
Water McAb.
After ascites uses caprylic acid-ammonium preliminary purification, crossing affinity chromatographic column, (filler is the production of TOSOH company
TOYOPEARL AF-rProtein A HC-650F) it is further purified, Purified monoclonal is measured respectively with ultraviolet specrophotometer
The ultraviolet 260nm of antibody and the optical density of 280nm, calculating MAb concentration with Lowry-kalokar formula is
2.43mg·mL-1, remaining purifying -70 DEG C of monoclonal antibody save backup.
Specific step is as follows for ascites purifying:
1) by ascites 60mmolL-1The acetate buffer solution of pH 4.0 is diluted by 1:4 volume ratio, uses 0.1mol
L-1NaOH adjusts pH value to 4.5；
2) according to 33 μ lmL under magnetic agitation-1Octanoic acid is added dropwise in the ratio of ascites, after stir 30min at room temperature, it is quiet in 4 DEG C
Set 2h, rear 10000rmin-1It is centrifuged 30min；
3) after supernatant is filtered with qualitative filter paper, 0.1molL is added according to 1:10 (PBS: filtered supernatant) ratio- 1PBS uses 1molL-1NaOH adjusts pH to 7.4, is placed in 4 DEG C of 30min；
4) under magnetic stirring according to 0.277gmL-1Ammonium sulfate is added portionwise in (ammonium sulfate: mixed liquor) ratio, after adding
It is stirred to react 30min, 4 DEG C of standings 3h, 12000rmin-1It is centrifuged 30min, abandons supernatant；
5) a small amount of 0.01molL of precipitating-1PBS dissolution is placed in bag filter, with pH 7.4,0.01molL-1's
PBS dialysis, centre is changed liquid 4~6 times；
6) monoclonal antibody solution of preliminary purification crosses Protein A (TOYOPEARL AF-rProtein AHC-650F)
Affinity chromatographic column is purified, and is saved backup after being after purification freeze-dried antibody in -70 DEG C.
Embodiment 11
The foundation of monoclonal antibody indirect competitive ELISA method
The measurement of monoclonal antibody working concentration is carried out using orthogonal test, and steps are as follows for specific experiment:
1) it is coated with: with pH 9.6,0.05molL-1CBS is coating buffer, Cu-L2- OVA is coating antigen, and package amount is pressed
According to 10 μ gmL-1Starting doubling dilution, coating volume is 100 holes μ L/, after 37 DEG C of incubation 2h, PBST washing lotion board-washing 4 times；
2) it closes: 250 hole μ L/ of confining liquid, after 37 DEG C of incubation 30min, PBST washing lotion board-washing 4 times is added；
3) it is loaded: monoclonal antibody after purification is added, according to 10 μ gmL-1Coating hole is added after starting doubling dilution
In, 100 holes μ L/, after 37 DEG C of reaction 1h, PBST washing lotion board-washing 4 times；
4) add ELIAS secondary antibody: board-washing 4 times after reaction, the sheep anti mouse secondary antibody of HRP label is diluted with 10000 times of confining liquid
After be added ELISA Plate, 100 holes μ L/, after 37 DEG C of reaction 1h, PBST washing lotion board-washing 4 times；
5) add substrate reactions liquid: tmb substrate buffer, 100 holes μ L/, 37 DEG C of incubation 15min are added；
6) it terminates reading: 2molL is added-150 hole μ L/ of sulfuric acid, microplate reader read OD450nmValue.
7) OD is chosen450nmBeing worth the antigen-antibody concentration combination in the section 1.0-1.2 is working concentration, chooses 4
Working concentration combination, concentration combination respectively (the 0.625 μ gmL of antigen, antibody-1, 5 μ gmL-1)、
(1.25μg·mL-1, 2. μ gmL-1)、(2.5μg·mL-1, 1.25 μ gmL-1) and (5 μ gmL-1,
0.625μg·mL-1), the 4 working concentrations combination selected using indirect competitive ELISA method carries out sensitive
Analysis and Screening is spent, 2 is the results are shown in Table, passes through the slope of standard curve of foundation, IC50Value and standard curve r value
Etc. parameters carry out overall merit, determine optimal working concentration be 1.25 μ gmL of antigen coat concentration-1,
Antibody concentration is 2.5 μ gmL-1。
2 working concentration of table analyzes result
Using indirect ELISA measurement monoclonal antibody purification to the sensitivity for analysis IC of copper ion chelator compound20For
0.14ng·mL-1, this illustrates that the preventing from heavy metal copper ion monoclonal antibody being prepared has very high detection sensitive copper ion
Degree and well specificity, can be used in quickly detecting heavy metal copper ion.
Specific step is as follows for indirect competitive ELISA:
A. it is coated with: with pH 9.6,0.05molL-1CBS is coating buffer, Cu-L2- OVA is coating antigen, is coated with volume
For 100 holes μ L/, peridium concentration is the best effort concentration of aforementioned screening, after 37 DEG C of incubation 2h, PBST washing lotion board-washing 4 times；
B. it closes: 250 hole μ L/ of confining liquid, after 37 DEG C of incubation 30min, PBST washing lotion board-washing 4 times is added；
C. sample pre-treatments: 5mmolL-12-S- (2- aminobenzene)-Isosorbide-5-Nitrae, 7 7-triazacyclononanes-Isosorbide-5-Nitrae, 7- triacetic acid
That is L1It is mixed in equal volume with various concentration gradient heavy metal copper ion standard specimen, 37 DEG C of incubation 1h；
D. it is loaded: pre- mixed 50 hole μ L/ of sample is added, twice of best effort concentration primary antibody (i.e. above embodiments is added
The anti-copper ion monoclonal antibody of 10 preparations) 50 holes μ L/, after concussion mixes after 37 DEG C of reaction 1h, PBST washing lotion board-washing 4 times；
Plus ELIAS secondary antibody e.: after reaction, board-washing 4 times, the sheep anti mouse secondary antibody of HRP label is dilute with 10000 times of confining liquid
It is added ELISA Plate after releasing, 100 holes μ L/, after 37 DEG C of reaction 1h, PBST washing lotion board-washing 4 times；
F. plus substrate reactions liquid: tmb substrate buffer, 100 holes μ L/, 37 DEG C of incubation 15min are added；
G. it terminates reading: 2molL is added-150 hole μ L/ of sulfuric acid, microplate reader read OD450nmValue.
Solution is prepared:
Carbonate buffer solution (CBS): Na is weighed2CO31.59g NaHCO32.93g is added pure water to 990mL, adjusts pH extremely
9.6, then it is settled to 1000mL with pure water, 4 DEG C of storages are spare.
Phosphate buffer (PBS): 8.5g NaCl, 2.2g Na2HPO4·12H2O, 0.2g NaH2PO4·2H2O is dissolved in
In 900mL pure water, pH to 7.4 is adjusted, 1000mL is settled to.
PBST washing lotion: 500mL 0.01molL is taken-10.25mL polysorbas20 is added in PBS, mixes spare.
Confining liquid: 1g skimmed milk power is dissolved in 50mL pH 7.4,0.01molL-1PBS。
For tmb substrate buffer by A liquid, B liquid and C liquid composition, specific formula and use volume ratio are as follows:
Developing solution A:2.35g citric acid, 9.2g Na2HPO4·12H2O adds pure water to be settled to 490mL, adjusts pH value 5.0
~5.4, then it is settled to 500mL with pure water, 4 DEG C of storages are spare.
Developing solution B: taking 30% hydrogen peroxide 1.25mL, is settled to 50mL with pure water, 4 DEG C of storages are spare.
Developing solution C: taking 200mg tetramethyl benzidine (TMB) to be dissolved in 100ml dehydrated alcohol, and 4 DEG C of storages are standby after dissolution
With.
Tmb substrate buffer uses preceding interim adapted, and adapted ratio is 42 μ L, C liquid 0.5mL of A liquid 9.5mL, B liquid, mixes
After use.
Sheep anti mouse HRP marks ELIAS secondary antibody to be purchased from Sigma company.
Embodiment 12
The experiment of TIANZHU XINGNAO Capsul in water sample sample
Recovery experiment is added to actual sample, respectively with tap water, Lake Water is experimental subjects, heavy metal copper ion
Addition concentration be respectively 10ngmL-1And 50ngmL-1, sample pre-treatments step are as follows:
Lake water sample: after Lake Water is first filtered with qualitative filter paper, copper ion standard solution is added, tap water directly adds copper
Ion standard solution, copper ion ultimate density is 10ngmL after addition-1And 50ngmL-1, after addition mixes, draw respectively
Tap water, lake water sample and 5mmolL after 100 μ L mixing-1L2The 50 above-mentioned premixs of μ L are added in isometric premix, rear every hole
Tap water, lake water sample after conjunction is into reacting hole, 3 repeating holes, adds the primary antibody of 50 twice of best effort concentration of μ L
(i.e. the anti-copper ion monoclonal antibody of the preparation of above embodiments 10), mixing is placed on 37 DEG C of incubations.Subsequent experimental procedure with it is upper
It is consistent to state indirect competitive ELISA reaction step in embodiment 11.In tap water, lake water sample TIANZHU XINGNAO Capsul experimental result is shown in
Table 4.
4 water sample sample TIANZHU XINGNAO Capsul result of table
By TIANZHU XINGNAO Capsul experiment it can be concluded that the accuracy of testing result, above TIANZHU XINGNAO Capsul value can illustrate to adopt
The testing result accuracy measured with the method is higher, and the testing result coefficient of variation is low, illustrates that testing result stability is good.
The preventing from heavy metal copper ion monoclonal antibody that this explanation is prepared can be used in quickly and accurately detecting heavy metal copper ion.
In above-described embodiment 10-12, by artificial antigen Cu-L2- BSA replaces with Cu-L1- BSA, coating antigen Cu-L2- OVA is replaced
It is changed to Cu-L1- OVA, or by artificial antigen Cu-L2- BSA replaces with Cu-L3- BSA, coating antigen Cu-L2- OVA replaces with Cu-
L3- OVA, or by artificial antigen Cu-L2- BSA replaces with Cu-L4- BSA, coating antigen Cu-L2- OVA replaces with Cu-L4- OVA is adopted
Different hybridoma cell strain, preventing from heavy metal copper ion monoclonal antibody can be made with the above identical preparation method and make
Copper ion is detected with indirect competitive ELISA method.
In above-described embodiment 1-12, by bovine serum albumin(BSA) BSA therein be substituted for keyhole limpet hemocyanin KLH or other
Carrier protein can also prepare copper ion artificial antigen, hybridoma cell strain, monoclonal using the above identical preparation method
Antibody and copper ion is detected using indirect ELISA.
Claims (5)
1. the hybridoma cell strain of one plant of secretion preventing from heavy metal copper ion monoclonal antibody, is preserved in Chinese microorganism strain preservation
Administration committee's common micro-organisms center, deposit number are CGMCC No.16388.
2. preventing from heavy metal copper ion monoclonal antibody, which is characterized in that the hybridization that it is CGMCC No.16388 by deposit number
Tumor cell strain secretion generates.
3. hybridoma cell strain CGMCC No.16388 or the preventing from heavy metal copper ion monoclonal antibody of its secretion are in copper ion
Application in immune detection.
4. a kind of preparation method for the hybridoma cell strain for secreting preventing from heavy metal copper ion monoclonal antibody, which is characterized in that should
Method is used to prepare hybridoma cell strain described in claim 1, comprising the following steps:
(1) synthesis of copper ion artificial antigen:
5-8mg 2-S- (3- aminobenzene)-Isosorbide-5-Nitrae, 7 7-triazacyclononanes-Isosorbide-5-Nitrae are weighed, 7- triacetic acid is dissolved in 2mL
0.01mol·L-1, 7.4 4- hydroxyethyl piperazineethanesulfonic acid HEPES solution of pH, be made chelating agent solution, this solution be A liquid；
It weighs 70.33mg copper nitrate to be dissolved in 5mL ultrapure water, it is 7.5 × 10 that concentration, which is made,-2mol·L-1Copper nitrate solution,
This solution is B liquid；
The B liquid of 150~200 μ L is added in A liquid, is protected from light 3~5h at room temperature, this reaction solution is C liquid；
500~800 μ L, 20mmolL are added into C liquid-1Glutaraldehyde solution, room temperature be protected from light overnight, this reaction solution be D
Liquid；
It weighs 20~30mg bovine serum albumin(BSA) BSA or chicken egg white OVA is dissolved in 3mL HEPES, at room temperature magnetic agitation
It mixes, this reaction solution is E liquid；
D liquid is added in E liquid, is protected from light at room temperature for 24 hours, 150~200 μ L sodium borohydride solutions are added afterwards, keep away at room temperature
Light reaction 1h；
Reaction solution first uses the bag filter of 8KD to dialyse 3~5 times, then with 7000~9000rmin of ultra-filtration centrifuge tube of 30KD-1Centrifugation
Washing 3~5 times, with the 0.01molL of 5~10mL-1, the HEPES solution of pH 7.4 redissolves, rear to dispense, -20 DEG C of cryo-conservations,
Artificial antigen of heavy metal copper Cu-L is made2- BSA or Cu-L2- OVA, L2Indicate 2-S- (3- aminobenzene)-Isosorbide-5-Nitrae, 7 three azacyclo-s
Nonane-Isosorbide-5-Nitrae, 7- triacetic acid；
Synthetic route are as follows:
(2) mouse is immune:
Selection BALB/C mice is immunized, by the artificial antigen of heavy metal copper Cu-L of preparation2- BSA is helped completely with isometric Freund
After agent mixing and emulsifying, by abdomen and oxter multi-point injection, after at interval of certain time carry out booster immunization, 3 booster immunizations
Blood sampling measurement potency afterwards, carries out last end when potency is no longer significantly raised and exempts from, end carries out cell fusion after exempting from 3 days；
(3) cell fusion and cell strain screen:
Mouse boosting cell and murine myeloma cell SP2/0 are merged according to the ratio of 5~10:1 by polyethylene glycol method,
Be added culture medium culture, indirect ELISA screening antibodies positive hole, the hybridoma limiting dilution assay good to inhibitory effect into
3~4 time cloning of row obtains hybridoma cell strain 6F9 after screening.
5. the preparation method of preventing from heavy metal copper ion monoclonal antibody, which is characterized in that this method is used to prepare claim 2 institute
The monoclonal antibody stated, comprising the following steps:
In BALB/C mice intraperitoneal injection norphytane 0.3ml/, the 6F9 monoclonal cell screened is injected after 7~10 days with method
Strain cell, ascites is extracted in sterile working after mouse peritoneal obviously swells, and is centrifuged off after precipitation of grease to get mouse ascites
McAb；
Ascites after purification, obtains monoclonal antibody purification.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811506878.7A CN110408600A (en) | 2018-12-10 | 2018-12-10 | One plant of hybridoma cell strain for secreting preventing from heavy metal copper ion monoclonal antibody and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811506878.7A CN110408600A (en) | 2018-12-10 | 2018-12-10 | One plant of hybridoma cell strain for secreting preventing from heavy metal copper ion monoclonal antibody and its application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110408600A true CN110408600A (en) | 2019-11-05 |
Family
ID=68357494
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811506878.7A Pending CN110408600A (en) | 2018-12-10 | 2018-12-10 | One plant of hybridoma cell strain for secreting preventing from heavy metal copper ion monoclonal antibody and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110408600A (en) |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1993008837A1 (en) * | 1991-10-28 | 1993-05-13 | The Wellcome Foundation Limited | Stabilised antibodies |
| CN101560257A (en) * | 2009-04-27 | 2009-10-21 | 南京农业大学 | Heavy metal copper monoclonal antibody and preparation method thereof |
| CN101914153A (en) * | 2010-07-05 | 2010-12-15 | 吉林大学 | Pb<2+> antigen and corresponding monoclonal antibody and preparation method thereof |
| CN101968481A (en) * | 2010-08-13 | 2011-02-09 | 中国农业大学 | Method for detecting existence of heavy metal copper ion in sample and special kit thereof |
| CN101979511A (en) * | 2010-11-05 | 2011-02-23 | 北京市农林科学院 | Heavy metal zinc-resistant monoclonal antibody |
| CN102071170A (en) * | 2010-11-05 | 2011-05-25 | 北京市农林科学院 | Monoclonal antibody capable of resisting heavy metal copper and preparation method thereof |
| CN107955070A (en) * | 2017-12-07 | 2018-04-24 | 浙江工商大学 | The purposes of a kind of synthetic method and NOTA of improved artificial antigen of heavy metal copper in artificial antigen of heavy metal copper reagent is prepared |
-
2018
- 2018-12-10 CN CN201811506878.7A patent/CN110408600A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1993008837A1 (en) * | 1991-10-28 | 1993-05-13 | The Wellcome Foundation Limited | Stabilised antibodies |
| CN101560257A (en) * | 2009-04-27 | 2009-10-21 | 南京农业大学 | Heavy metal copper monoclonal antibody and preparation method thereof |
| CN101914153A (en) * | 2010-07-05 | 2010-12-15 | 吉林大学 | Pb<2+> antigen and corresponding monoclonal antibody and preparation method thereof |
| CN101968481A (en) * | 2010-08-13 | 2011-02-09 | 中国农业大学 | Method for detecting existence of heavy metal copper ion in sample and special kit thereof |
| CN101979511A (en) * | 2010-11-05 | 2011-02-23 | 北京市农林科学院 | Heavy metal zinc-resistant monoclonal antibody |
| CN102071170A (en) * | 2010-11-05 | 2011-05-25 | 北京市农林科学院 | Monoclonal antibody capable of resisting heavy metal copper and preparation method thereof |
| CN107955070A (en) * | 2017-12-07 | 2018-04-24 | 浙江工商大学 | The purposes of a kind of synthetic method and NOTA of improved artificial antigen of heavy metal copper in artificial antigen of heavy metal copper reagent is prepared |
Non-Patent Citations (1)
| Title |
|---|
| FENGQUAN LIU 等: "Preparation and characterization of monoclonal antibody specific for copper-chelate complex", 《J IMMUNOL METHODS》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101477126A (en) | Hepatitis C virus antigen-antibody combined detection method | |
| CN101863981A (en) | Preparation method of anti-bisphenol A monoclonal antibody | |
| CN101363850A (en) | Melamine rapid immune detecting kit | |
| CN103756973B (en) | A kind of indirect ELISA testing kit of GCRV | |
| CN112877296B (en) | Anti-phenacetin monoclonal antibody hybridoma cell strain AD and preparation method and application thereof | |
| CN104762267B (en) | Hybridoma AFB1 2A4 and its caused aflatoxin B1 monoclonal antibody | |
| CN110408599A (en) | One plant of hybridoma cell strain for secreting preventing from heavy metal lead ion monoclonal antibody and its application | |
| CN101551393B (en) | Immunodiagnosis kit for detecting IV-type dengue virus NS1 antigen | |
| CN110407931A (en) | A kind of lead ion artificial antigen and its application | |
| CN101914153A (en) | Pb<2+> antigen and corresponding monoclonal antibody and preparation method thereof | |
| CN109320608A (en) | A kind of preparation method of hemoglobin antibodies | |
| CN114058594B (en) | Hybridoma cell strain secreting vitamin A monoclonal antibody and application thereof | |
| CN107012128A (en) | A kind of hybridoma cell strain for secreting aspergillus flavus resisting toxin B1 monoclonal antibodies and its application | |
| CN110408598A (en) | One plant of hybridoma cell strain for secreting preventing from heavy metal lead ion monoclonal antibody and its application | |
| CN110407932A (en) | A kind of lead ion artificial antigen and its application | |
| CN108998424A (en) | One plant of aristolochic acid A monoclonal antibody hybridoma cell strain and its application | |
| CN103288965A (en) | Polychlorobiphenyl monoclonal antibody preparation method | |
| CN109112114A (en) | Anti-human igg monoclonal antibody, its hybridoma cell strain and application | |
| CN108103002A (en) | Preparation of mdck cell host's residual protein and application thereof | |
| CN107955070A (en) | The purposes of a kind of synthetic method and NOTA of improved artificial antigen of heavy metal copper in artificial antigen of heavy metal copper reagent is prepared | |
| CN104977419A (en) | Purifying preparation method of autoimmuno antigen positive serum | |
| CN110408600A (en) | One plant of hybridoma cell strain for secreting preventing from heavy metal copper ion monoclonal antibody and its application | |
| CN110407930A (en) | A kind of copper ion artificial antigen and its application | |
| CN110408601A (en) | One plant of hybridoma cell strain for secreting preventing from heavy metal cadmium ion monoclonal antibody and its application | |
| CN110408597A (en) | One plant of hybridoma cell strain for secreting preventing from heavy metal cadmium ion monoclonal antibody and its application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20191105 |
|
| RJ01 | Rejection of invention patent application after publication |