WO2008099968A1 - 酸化亜鉛結合性抗体及びその用途 - Google Patents
酸化亜鉛結合性抗体及びその用途 Download PDFInfo
- Publication number
- WO2008099968A1 WO2008099968A1 PCT/JP2008/052917 JP2008052917W WO2008099968A1 WO 2008099968 A1 WO2008099968 A1 WO 2008099968A1 JP 2008052917 W JP2008052917 W JP 2008052917W WO 2008099968 A1 WO2008099968 A1 WO 2008099968A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- zinc oxide
- antibody
- binding
- peptide
- cdr
- Prior art date
Links
- XLOMVQKBTHCTTD-UHFFFAOYSA-N Zinc monoxide Chemical compound [Zn]=O XLOMVQKBTHCTTD-UHFFFAOYSA-N 0.000 title claims abstract description 434
- 239000011787 zinc oxide Substances 0.000 title claims abstract description 217
- 230000027455 binding Effects 0.000 title claims abstract description 150
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 91
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 87
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 65
- 241000282836 Camelus dromedarius Species 0.000 claims abstract description 57
- 239000007787 solid Substances 0.000 claims abstract description 4
- 239000013604 expression vector Substances 0.000 claims description 27
- 238000010494 dissociation reaction Methods 0.000 claims description 26
- 230000005593 dissociations Effects 0.000 claims description 26
- 239000010931 gold Substances 0.000 claims description 25
- 102000037865 fusion proteins Human genes 0.000 claims description 21
- 108020001507 fusion proteins Proteins 0.000 claims description 21
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 19
- 229910052737 gold Inorganic materials 0.000 claims description 19
- 238000004519 manufacturing process Methods 0.000 claims description 15
- 239000007790 solid phase Substances 0.000 claims description 7
- 230000009878 intermolecular interaction Effects 0.000 claims description 5
- 238000012258 culturing Methods 0.000 claims description 3
- 125000003275 alpha amino acid group Chemical group 0.000 claims 3
- 238000000034 method Methods 0.000 abstract description 42
- 239000000758 substrate Substances 0.000 abstract description 22
- 229910052725 zinc Inorganic materials 0.000 abstract description 19
- 239000011701 zinc Substances 0.000 abstract description 19
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 abstract description 14
- 108010047041 Complementarity Determining Regions Proteins 0.000 description 60
- 235000018102 proteins Nutrition 0.000 description 59
- 210000004027 cell Anatomy 0.000 description 36
- 101100203200 Danio rerio shha gene Proteins 0.000 description 33
- 239000000463 material Substances 0.000 description 30
- 102100033400 4F2 cell-surface antigen heavy chain Human genes 0.000 description 27
- 239000002245 particle Substances 0.000 description 24
- 150000001413 amino acids Chemical class 0.000 description 23
- 239000013598 vector Substances 0.000 description 23
- 238000005516 engineering process Methods 0.000 description 17
- 239000005090 green fluorescent protein Substances 0.000 description 15
- 230000000694 effects Effects 0.000 description 14
- 230000002441 reversible effect Effects 0.000 description 14
- 239000000427 antigen Substances 0.000 description 13
- 102000036639 antigens Human genes 0.000 description 13
- 108091007433 antigens Proteins 0.000 description 13
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 13
- 238000002054 transplantation Methods 0.000 description 13
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 12
- 238000011156 evaluation Methods 0.000 description 12
- 239000000243 solution Substances 0.000 description 12
- 241000588724 Escherichia coli Species 0.000 description 11
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 11
- 238000006243 chemical reaction Methods 0.000 description 11
- 239000010408 film Substances 0.000 description 11
- 102000004196 processed proteins & peptides Human genes 0.000 description 11
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 10
- 235000001014 amino acid Nutrition 0.000 description 10
- 238000002310 reflectometry Methods 0.000 description 10
- 238000001179 sorption measurement Methods 0.000 description 10
- 239000002105 nanoparticle Substances 0.000 description 9
- 239000008363 phosphate buffer Substances 0.000 description 9
- 108020004414 DNA Proteins 0.000 description 8
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 239000007864 aqueous solution Substances 0.000 description 8
- 238000001514 detection method Methods 0.000 description 8
- 238000010586 diagram Methods 0.000 description 8
- 239000013612 plasmid Substances 0.000 description 8
- 239000002253 acid Substances 0.000 description 7
- 238000002983 circular dichroism Methods 0.000 description 7
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 6
- 102000002322 Egg Proteins Human genes 0.000 description 6
- 108010000912 Egg Proteins Proteins 0.000 description 6
- 102000016943 Muramidase Human genes 0.000 description 6
- 108010014251 Muramidase Proteins 0.000 description 6
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 6
- 235000014103 egg white Nutrition 0.000 description 6
- 210000000969 egg white Anatomy 0.000 description 6
- 230000014509 gene expression Effects 0.000 description 6
- 229960000274 lysozyme Drugs 0.000 description 6
- 239000004325 lysozyme Substances 0.000 description 6
- 235000010335 lysozyme Nutrition 0.000 description 6
- 238000000059 patterning Methods 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 102000014914 Carrier Proteins Human genes 0.000 description 5
- 229910019142 PO4 Inorganic materials 0.000 description 5
- 230000008901 benefit Effects 0.000 description 5
- 108091008324 binding proteins Proteins 0.000 description 5
- 235000014304 histidine Nutrition 0.000 description 5
- 238000005259 measurement Methods 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 239000000178 monomer Substances 0.000 description 5
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 5
- 239000010452 phosphate Substances 0.000 description 5
- 239000004065 semiconductor Substances 0.000 description 5
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 5
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 4
- 108010076504 Protein Sorting Signals Proteins 0.000 description 4
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 4
- 239000012491 analyte Substances 0.000 description 4
- 238000001142 circular dichroism spectrum Methods 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 229910010272 inorganic material Inorganic materials 0.000 description 4
- 239000011147 inorganic material Substances 0.000 description 4
- 230000003993 interaction Effects 0.000 description 4
- 229910044991 metal oxide Inorganic materials 0.000 description 4
- 150000004706 metal oxides Chemical class 0.000 description 4
- 230000035772 mutation Effects 0.000 description 4
- 238000007857 nested PCR Methods 0.000 description 4
- 238000001742 protein purification Methods 0.000 description 4
- 230000004850 protein–protein interaction Effects 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 230000004572 zinc-binding Effects 0.000 description 4
- 239000004475 Arginine Substances 0.000 description 3
- 244000063299 Bacillus subtilis Species 0.000 description 3
- 235000014469 Bacillus subtilis Nutrition 0.000 description 3
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 3
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 3
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 3
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 3
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 3
- 230000009830 antibody antigen interaction Effects 0.000 description 3
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 3
- 238000004925 denaturation Methods 0.000 description 3
- 230000036425 denaturation Effects 0.000 description 3
- 238000003795 desorption Methods 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 238000001641 gel filtration chromatography Methods 0.000 description 3
- 238000002523 gelfiltration Methods 0.000 description 3
- 230000003100 immobilizing effect Effects 0.000 description 3
- 229910052751 metal Inorganic materials 0.000 description 3
- 239000002184 metal Substances 0.000 description 3
- 239000003208 petroleum Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 108091008146 restriction endonucleases Proteins 0.000 description 3
- 229920006395 saturated elastomer Polymers 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 230000003248 secreting effect Effects 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 244000303258 Annona diversifolia Species 0.000 description 2
- 235000002198 Annona diversifolia Nutrition 0.000 description 2
- 101000616562 Danio rerio Sonic hedgehog protein A Proteins 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- 241000238631 Hexapoda Species 0.000 description 2
- UQSXHKLRYXJYBZ-UHFFFAOYSA-N Iron oxide Chemical compound [Fe]=O UQSXHKLRYXJYBZ-UHFFFAOYSA-N 0.000 description 2
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 2
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 2
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 2
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 2
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 2
- 102000001400 Tryptase Human genes 0.000 description 2
- 108060005989 Tryptase Proteins 0.000 description 2
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 2
- 101000616493 Xenopus laevis Sonic hedgehog protein Proteins 0.000 description 2
- 238000001042 affinity chromatography Methods 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 239000004599 antimicrobial Substances 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 239000000539 dimer Substances 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000010419 fine particle Substances 0.000 description 2
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 description 2
- 229960000789 guanidine hydrochloride Drugs 0.000 description 2
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 238000012482 interaction analysis Methods 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 238000002703 mutagenesis Methods 0.000 description 2
- 231100000350 mutagenesis Toxicity 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 238000002823 phage display Methods 0.000 description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 230000004952 protein activity Effects 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 230000000717 retained effect Effects 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 239000010703 silicon Substances 0.000 description 2
- 229910052710 silicon Inorganic materials 0.000 description 2
- 238000004544 sputter deposition Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 230000005030 transcription termination Effects 0.000 description 2
- 241000701447 unidentified baculovirus Species 0.000 description 2
- 125000002987 valine group Chemical group [H]N([H])C([H])(C(*)=O)C([H])(C([H])([H])[H])C([H])([H])[H] 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- BRZYSWJRSDMWLG-DJWUNRQOSA-N (2r,3r,4r,5r)-2-[(1s,2s,3r,4s,6r)-4,6-diamino-3-[(2s,3r,4r,5s,6r)-3-amino-4,5-dihydroxy-6-[(1r)-1-hydroxyethyl]oxan-2-yl]oxy-2-hydroxycyclohexyl]oxy-5-methyl-4-(methylamino)oxane-3,5-diol Chemical compound O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H]([C@@H](C)O)O2)N)[C@@H](N)C[C@H]1N BRZYSWJRSDMWLG-DJWUNRQOSA-N 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- DLZKEQQWXODGGZ-KCJUWKMLSA-N 2-[[(2r)-2-[[(2s)-2-amino-3-(4-hydroxyphenyl)propanoyl]amino]propanoyl]amino]acetic acid Chemical compound OC(=O)CNC(=O)[C@@H](C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 DLZKEQQWXODGGZ-KCJUWKMLSA-N 0.000 description 1
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 description 1
- 108010051457 Acid Phosphatase Proteins 0.000 description 1
- 108010021809 Alcohol dehydrogenase Proteins 0.000 description 1
- 101100335652 Autographa californica nuclear polyhedrosis virus GP64 gene Proteins 0.000 description 1
- 101900315840 Bacillus subtilis Alpha-amylase Proteins 0.000 description 1
- 108700040077 Baculovirus p10 Proteins 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 241001000171 Chira Species 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 241000699800 Cricetinae Species 0.000 description 1
- 241000701022 Cytomegalovirus Species 0.000 description 1
- -1 EAHVMHK Chemical class 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 description 1
- 241001524679 Escherichia virus M13 Species 0.000 description 1
- 101150010917 GP67 gene Proteins 0.000 description 1
- 108700007698 Genetic Terminator Regions Proteins 0.000 description 1
- 201000005569 Gout Diseases 0.000 description 1
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 1
- 241000235058 Komagataella pastoris Species 0.000 description 1
- 125000002059 L-arginyl group Chemical group O=C([*])[C@](N([H])[H])([H])C([H])([H])C([H])([H])C([H])([H])N([H])C(=N[H])N([H])[H] 0.000 description 1
- 125000003290 L-leucino group Chemical group [H]OC(=O)[C@@]([H])(N([H])[*])C([H])([H])C(C([H])([H])[H])([H])C([H])([H])[H] 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- 241000255777 Lepidoptera Species 0.000 description 1
- 102000004895 Lipoproteins Human genes 0.000 description 1
- 108090001030 Lipoproteins Proteins 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 108091007491 NSP3 Papain-like protease domains Proteins 0.000 description 1
- 241001045988 Neogene Species 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 102000008153 Peptide Elongation Factor Tu Human genes 0.000 description 1
- 108010049977 Peptide Elongation Factor Tu Proteins 0.000 description 1
- 108010033276 Peptide Fragments Proteins 0.000 description 1
- 102000007079 Peptide Fragments Human genes 0.000 description 1
- 108010067902 Peptide Library Proteins 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 101710093543 Probable non-specific lipid-transfer protein Proteins 0.000 description 1
- 108020005067 RNA Splice Sites Proteins 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 101100434317 Rattus norvegicus Ada gene Proteins 0.000 description 1
- 108020005091 Replication Origin Proteins 0.000 description 1
- 241000235070 Saccharomyces Species 0.000 description 1
- 241000124033 Salix Species 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 239000006087 Silane Coupling Agent Substances 0.000 description 1
- 241000256251 Spodoptera frugiperda Species 0.000 description 1
- 102400000368 Surface protein Human genes 0.000 description 1
- 101150056072 TUFB gene Proteins 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- 108010022394 Threonine synthase Proteins 0.000 description 1
- GWEVSGVZZGPLCZ-UHFFFAOYSA-N Titan oxide Chemical compound O=[Ti]=O GWEVSGVZZGPLCZ-UHFFFAOYSA-N 0.000 description 1
- 241000255993 Trichoplusia ni Species 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 235000010724 Wisteria floribunda Nutrition 0.000 description 1
- DXWQDVZGROCFPG-UHFFFAOYSA-N [O--].[Zn++].[Au+3] Chemical compound [O--].[Zn++].[Au+3] DXWQDVZGROCFPG-UHFFFAOYSA-N 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 238000005377 adsorption chromatography Methods 0.000 description 1
- 230000003042 antagnostic effect Effects 0.000 description 1
- 229940125644 antibody drug Drugs 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 239000000919 ceramic Substances 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 229940096384 chicken egg white lysozyme Drugs 0.000 description 1
- 230000004186 co-expression Effects 0.000 description 1
- 238000000975 co-precipitation Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 239000007822 coupling agent Substances 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 102000004419 dihydrofolate reductase Human genes 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 230000005669 field effect Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000002189 fluorescence spectrum Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000000833 heterodimer Substances 0.000 description 1
- 150000002411 histidines Chemical class 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000001114 immunoprecipitation Methods 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 239000012212 insulator Substances 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000000873 masking effect Effects 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 239000011859 microparticle Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- 101150091879 neo gene Proteins 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 230000002611 ovarian Effects 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 210000004976 peripheral blood cell Anatomy 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 238000011158 quantitative evaluation Methods 0.000 description 1
- 238000003380 quartz crystal microbalance Methods 0.000 description 1
- 238000002708 random mutagenesis Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000010187 selection method Methods 0.000 description 1
- 238000010206 sensitivity analysis Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 239000012134 supernatant fraction Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 239000013076 target substance Substances 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 239000010409 thin film Substances 0.000 description 1
- OGIDPMRJRNCKJF-UHFFFAOYSA-N titanium oxide Inorganic materials [Ti]=O OGIDPMRJRNCKJF-UHFFFAOYSA-N 0.000 description 1
- 238000006257 total synthesis reaction Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 101150099542 tuf gene Proteins 0.000 description 1
- 101150071165 tuf1 gene Proteins 0.000 description 1
- 101150010742 tuf2 gene Proteins 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 241000701366 unidentified nuclear polyhedrosis viruses Species 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 238000001262 western blot Methods 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2318/00—Antibody mimetics or scaffolds
- C07K2318/10—Immunoglobulin or domain(s) thereof as scaffolds for inserted non-Ig peptide sequences, e.g. for vaccination purposes
Definitions
- Zinc oxide-binding antibodies and their uses
- the present invention relates to a zinc oxide-binding camel antibody capable of specifically binding to zinc oxide, the antibody expression vector, and a solid phase support (biosensor, protein chip, etc.) comprising a zinc oxide layer on which the antibody is immobilized.
- a solid phase support biosensor, protein chip, etc.
- zinc oxide is transparent and can be used as both an insulator and a semiconductor, so it can be said that it can be used for various biosensing applications. If zinc oxide can easily and directly immobilize biomolecules (proteins), it is extremely useful for developing high-throughput sensing technologies.
- the inventors screened the phage display library to obtain a peptide that is specific to oxidized suboxide and has high binding activity (Non-patent Document 1), and succeeded in patterning zinc oxide particles using this ( Patent Document 3).
- the inventors used the CDR grafting method used for antibody humanization technology to convert the above-mentioned material recognition peptides (gold-binding proteins and zinc oxide-binding peptides) into anti-hen egg white lysozyme (HEL).
- HEL anti-hen egg white lysozyme
- the zinc oxide-binding antibody fused to the HEL antibody is unstable and unsuitable for practical use such as biosensors and protein chips.
- the camel antibody was thought to be unsuitable for peptide CDR grafting because it does not have a force-null structure or has a disulfide bond in the complementarity determining region (CDR).
- Non-Patent Document 4 a camel antibody suitable for CDR transplantation was developed by Saerens et al.
- camel antibody is expected to be used in the pharmaceutical field such as targeting to cancer cells.
- An object of the present invention is to provide a zinc oxide-binding antibody having high stability and binding activity, and by using this to stably bind the antibody to zinc oxide, high-throughput sensing such as an immunobiosensor It is to enable the development of technology.
- the inventors have intensively studied to achieve the above object, and have found that a stable zinc oxide-binding antibody can be prepared by using a camel antibody. Furthermore, we have succeeded in producing a zinc oxide-binding antibody with high stability and binding activity suitable for practical use by further increasing the affinity of this antibody using in vitro affinity maturing.
- the present invention provides an amino acid containing EAHVMHK in the CDR H-1 region of a camel antibody.
- a peptide-grafted antibody containing a zinc oxide-recognizing peptide consisting of a sequence, or a mutation having a further mutation in the CDR H-3 region of the peptide-grafted antibody and having a higher zinc oxide affinity than the peptide-grafted antibody It relates to a zinc oxide-binding antibody consisting of the body.
- mutants include a peptide comprising HXXHXXHXXH or HXXHXXHXR force (where X is G, L, R, or V) in the CDR H-3 region, for example, HLGHGGHRLH, HLGHGGHGLH in the CDR H-3 region. , HVGHGLHGVR, or HLGHGLHRVH.
- Zinc oxide-binding antibody of the present invention is preferably a dissociation equilibrium constant K d for the zinc oxide 1. smaller than 7xl0- 7 [M], more preferably 9. smaller than 5xlO_ 9 [M].
- the zinc oxide recognition peptide is preferably a peptide consisting of 7 to 20 amino acids including EAHVMHK, particularly a peptide of about 7 to 12 amino acids, such as EAHVMHKVAPRP.
- the camel antibody CDR H-1 region contains a zinc oxide recognition peptide consisting of EAHVMHKVAPRP, and the CDR H-3 region contains a peptide consisting of HLGHGLHRVH. Things can be mentioned.
- the camel antibody is preferably one having no disulfide bond between CDRs.
- the present invention also provides an expression vector comprising a gene encoding the aforementioned zinc oxide-binding antibody.
- an expression vector containing a gene encoding the amino acid sequence shown in SEQ ID NO: 14 can be mentioned.
- the present invention also provides a method for producing a zinc oxide-binding antibody, comprising culturing a host cell into which the expression vector has been introduced, and obtaining a zinc oxide-binding antibody from the culture.
- the present invention provides a solid support (for example, a substrate such as a particle or a protein chip) comprising a zinc oxide layer on which the zinc oxide-binding antibody is immobilized.
- a solid support for example, a substrate such as a particle or a protein chip
- a zinc oxide layer on which the zinc oxide-binding antibody is immobilized.
- the present invention also provides a biosensor having a solid phase support including a zinc oxide layer on which the zinc oxide-binding antibody is immobilized, and means for detecting an intermolecular interaction via the antibody.
- the present invention also provides a protein obtained by fusing a second protein having a material recognition sequence to the zinc oxide-binding antibody.
- the second protein having the material recognition sequence is a sequence that specifically recognizes and binds to a certain material (for example, metal or metal oxide) like the zinc oxide recognition peptide of the present invention.
- a gold recognition peptide having the sequence LKAHLPPSRLPS Examples include metal-binding antibodies (Japanese Patent Laid-Open No. 2 0 0 5-3 1 2 4 4 6, Japanese Patent Laid-Open No. 2 0 6-2 2 5 2 94 4).
- an antibody necessary for detection of a target substance can be easily immobilized on zinc oxide. This makes it possible to develop high-throughput sensing technologies such as immune biosensors.
- FIG. 1A is a schematic diagram of a VHH1 or 2 or 3 expression vector
- FIG. 1B is a schematic diagram of pTZ-VHH
- FIG. 1C is a schematic diagram of pRA 4F2-AuVHHl.
- Fig. 2A shows the protein purification method
- Fig. 2B shows the results of confirming the expression of VHH1, VHH2, and VHH3 in E. coli.
- Fig. 3 is a graph showing the results of gel filtration chromatography of the peptide-grafted antibody (VHH 2 (yellowish green), VHH3 (blue), WT VHH1 (black), VHH1 (red)).
- Fig. 4 is a graph showing the results of circular dichroism spectrum measurements (from the top of the graph, WT VHH1 (black), VHH3 (blue), VHH1 (red), VHH2 (yellowish green)).
- Figure 5 shows the process for assessing binding between zinc oxide particles and peptide-grafted antibodies.
- Figure 6A shows the results of SDS-PAGE analysis of the supernatant, Wash, and Elute fractions shown in Figure 5 (1: Total, 2: Supernatant fraction, 3-5: Wash fraction, 6:
- Figure 6B shows the results of material identification evaluation tests by SDS-PAGE.
- Fig. 7 shows the results of evaluating the binding activity of VHH3 to zinc oxide (lmM phosphate buffer).
- Figure 8 shows adsorption hot springs (A) and Langmuir plots (B) for the adsorption of VHH1 on acid-zinc.
- FIG. 9 is a schematic diagram showing a production protocol for a peptide library in which CDR H-3 is mutated.
- Figure 10 shows the results of material identification evaluation by SDS-PAGE.
- Fig. 11 is a graph showing the results of 4F2 gel filtration chromatography (WT VHH1 ( ⁇ ), VHH1 (blue), F2 (orange)).
- Figure 12 shows the 4F2 circular dichroism spectra (WT VHH1 (black), VHH1 (blue), 4F2 (orange)).
- FIG. 13 is a photograph showing the results of evaluation of dispersibility of oxidized dumbbell particles (A: antibody 3 ⁇ , ⁇ : antibody 0.5 3 ⁇ 41).
- FIG. 14 shows the results of the desorption test of zinc oxide-binding antibody immobilized on zinc oxide particles.
- FIG. 15 is a schematic diagram of a co-expression vector of Fv (H-2), (H-3), (L-2), and (L-3).
- FIG. 16 shows the results of CDR grafting of zinc oxide recognition peptide to mouse-derived anti-hen egg white lysozyme antibody HyHEL-10 Fv.
- A shows the three-dimensional structure of HyHEL-10 Fv.
- B shows the gel filtration production process of antibody grafted with heavy chain CDR-2.
- C shows the result of circular dichroism spectrum of VH alone (VH2sd) with peptide grafted on heavy chain CDR-2.
- FIG. 17 shows the results of A: Zinc oxide binding evaluation with Fv alone (Fv (H-2): V-chain CDR-2, Fv (H-3): V-chain Transplanted to CDR-3, Fv (H-2): transplanted to CDR-2 of L chain, Fv (L-3): transplanted to CDR-3 of L chain), B: purified peptide transplantation oxidation of VH chain
- A Zinc oxide binding evaluation with Fv alone (Fv (H-2): V-chain CDR-2
- Fv (H-3) V-chain Transplanted to CDR-3
- Fv (H-2) transplanted to CDR-2 of L chain
- Fv (L-3) transplanted to CDR-3 of L chain
- B purified peptide transplantation oxidation of VH chain
- Fig. 18 shows the results of evaluating the binding of zinc oxide-recognizing peptide fragments to zinc oxide.
- FIG. 19 shows a conceptual diagram of a biosensor using the zinc oxide-binding antibody of the present invention.
- Figure 21 shows the immobilization of zinc oxide-binding camel antibody 4F2 on the flow system (saturated binding: 130 nmol / m 2 ).
- Figure 22 shows the immobilization of anti-GFP camel antibody 4F2 in the flow system (camel antibody 4F2 (bottom): 134 nmol / m 2 , anti-GFP camel antibody 4F2 (top): 134 nmol I m 2 , Immobilization amount of GFP: 110 nmol / m 2 ).
- This specification includes the contents described in the specification of Japanese Patent Application No. 2 073 _3 5 073 which is the basis of the priority of the present application.
- the zinc oxide recognition peptide used in the present invention is a peptide that specifically recognizes zinc oxide and can bind to zinc oxide with high affinity.
- the inventors have so far conducted a screening using the fuzzy presentation method, and peptides having binding properties to five kinds of acid zinc: EAHVMHKVAPRP (ZnOl: SEQ ID NO: 1), QNTATAVSRLSP (Zn02: SEQ ID NO: 2), ATHTNQTHALYR (Zn03: SEQ ID NO: 3), VSNHKALDYPTR (Zn04: SEQ ID NO: 4), DSGRYSMTNHYS (Zn05: SEQ ID NO: 5) have been obtained (Japanese Patent Laid-Open No.
- the peptide of SEQ ID NO: 1 is 10_ 7 M against zinc oxide It has a high binding activity.
- the inventors further analyzed the sequence of this peptide and identified that the 7 peptide of EAHVMHK (SEQ ID NO: 6) is important for zinc oxide recognition. See Reference Example 1).
- histidine which is a basic amino acid, has a large effect on the binding constant, and the HVMH region between two histidines is considered to be highly likely to be important as a binding motif.
- the above peptide comprising an amino acid sequence containing the 7 peptides of EAHVMHK is used as an acid zinc recognition peptide.
- the zinc oxide recognition peptide preferably has a size of about 7 to 20 amino acids, particularly about 7 to 12 amino acids.
- each amino acid constituting the peptide may be appropriately modified or derivatized, and such a modified or derivatized peptide is also used in the present invention. Included in zinc oxide recognition peptides.
- the antibody used in the present invention is a camel-derived antibody molecule (latada antibody).
- the variable region of an antibody has a heterodimeric structure consisting of a heavy chain and a light chain.
- FIG. 16A shows a three-dimensional structure of mouse-derived anti-hen egg white lysozyme antibody HyHEL-IOFv, which is a dimer composed of heavy chain (VH) and light chain (VL) subunits.
- HSHEL-IOFv mouse-derived anti-hen egg white lysozyme antibody
- VH heavy chain
- VL light chain
- force Latada antibody does not have a light chain and shows antigen-binding ability in a VH single domain. Therefore, it has a smaller molecular weight than conventional antibodies, has a simple structure, and can be easily mass-produced using yeast or the like.
- camel antibodies have many antibodies that do not have a canonical structure and have CDR H-1-H-3 disulfide bonds to stabilize the complementarity-determining region (CDR) loop. It was thought that it was not suitable for peptide transplantation into the CDR region.
- an anti-lactamase camel antibody cAb BCII-10 suitable for CDR-grafting has been isolated and named "Universal VHH framework" (J. Mol. Biol. 352 (2005) 597).
- This cAb BCII-10 has no disulfide bond between CDRs, and the expression amount of the recombinant antibody after CDR grafting is large, so it is extremely useful as a truncated antibody for peptide grafting.
- a mouse-derived anti-chicken egg white lysozyme antibody having a heterodimer structure that has been used in the past is used, the fusion protein in which the acid-zinc recognition peptide is transplanted to the CDR portion (CDR H-2) is oxidized. Although it exhibits zinc binding activity, the phenomenon that VH and VL dissociate during the purification process could not be avoided (Fig. 16B).
- camel antibodies originally have a single domain, so domain dissociation does not occur, and a fusion protein in which a zinc oxide-recognizing peptide is grafted onto its CDR portion maintains the antibody structure stably.
- a fusion protein in which a zinc oxide-recognizing peptide is grafted onto its CDR portion maintains the antibody structure stably.
- camel antibodies are monomers, so their molecular weight is small, and all molecules are linked by covalent bonds, so there is no phenomenon of dissociation, so it is easy to create fusion proteins with other domains.
- camel antibodies are monomers, so their molecular weight is small, and all molecules are linked by covalent bonds, so there is no phenomenon of dissociation, so it is easy to create fusion proteins with other domains.
- the stability of the antibody structure is extremely important, but the camel antibody, which is a monomer, is extremely stable compared to conventional antibodies having a dimer structure and is actually used.
- Zinc oxide-binding antibodies suitable for the above can be provided.
- the entire amino acid sequence of the camel antibody is known, and the sequence of cAb BCII-10 is described in Antimicrobial Agents and Chemotherapy, 2807-2812 (2001).
- the entire amino acid sequence of camel antibody cAb BCII-10 having no disulfide bond in the CDR region is shown in SEQ ID NO: 7 in the sequence listing.
- residues 26-38 correspond to the CDR H-1 region
- residues 53-69 correspond to the CDR H-2 region
- residues 102-117 correspond to the CDR H-3 region.
- Zinc oxide-binding antibody (latada antibody dumbbell recognition peptide fusion protein)
- the above-mentioned zinc oxide recognition peptide is added to the CDR region of a camel antibody (in particular,
- a zinc oxide-binding antibody (camel antibody, zinc monoxide-recognizing peptide fusion protein) is prepared by transplanting to CDR H-1 region.
- an antigen binding site is called a complementary determining region (CDR) or a hypervariable region.
- CDR complementary determining region
- a hypervariable region In the production of antibody drugs CDR grafting, in which only this CDR is replaced with a heterologous (mouse) -derived one, is used for the production of human-type antibodies.
- an acid zinc-recognizing peptide is grafted to the Latada antibody.
- the inventors have exposed the CDR from the wild-type conformation, Regions that were not directly linked (residues 29-35, 53-69, 102-117) were determined and a zinc oxide recognition peptide was transplanted.
- the zinc oxide-binding antibody of the present invention must maintain the original antibody structure even by CDR grafting, and should have a zinc oxide-binding property equivalent to or higher than that of the grafted zinc oxide-recognizing peptide.
- the three-dimensional structure of the above-mentioned camel antibody zinc monoxide recognition peptide fusion protein can be determined by measuring X-ray crystal structure analysis, nuclear magnetic resonance spectrum, fluorescence spectrum, circular dichroism spectrum, etc. You can investigate. As a result, when completely different results were obtained between the wild-type camel antibody and the fusion protein, it can be seen that transplantation at the site 8 changes the antibody structure and impairs the antibody activity.
- the zinc oxide binding activity of the camel antibody zinc monoxide recognition peptide fusion protein can be evaluated by measuring the binding rate, dissociation rate, dissociation equilibrium constant, and amount of saturated binding.
- the dissociation equilibrium constant can be calculated by adding acid 0
- transplantation to the H-2 region changes the structure unique to the camel antibody, and its activity is reduced. It was confirmed that it could be lost.
- Kd dissociation equilibrium constant
- a zinc oxide-binding protein having a high binding activity can be obtained by transplanting a zinc oxide recognition peptide into the CDR H-1 region, particularly the antibody surface exposed region.
- a zinc oxide recognition peptide an antibody in which a zinc oxide-recognizing peptide is grafted on the latada antibody CDR H-1 region is referred to as “VHH1J”.
- In vitro affinity maturing consists of two steps: the creation of a mutation library by mutagenesis and the screening of high-affinity antibodies from the library.
- the mutagenesis method a method well known in the art such as chain shuffling method, random mutagenesis method, and CDR walking can be used.
- a selection method from the mutation library thus prepared a method of selecting a high affinity antibody using a low concentration of antigen, or a method of recovering only antibodies that are strongly bound to the antigen under strict washing conditions. And a method of selecting only high-affinity antibodies using an antagonistic reaction. Since the zinc oxide-binding antibody of the present invention is a low molecular weight single chain antibody, mass production is easy, and the above-described affinity process can be easily performed.
- HXXH H may be R: SEQ ID NO: 8
- HXXHXHXXH H may be R, X is G (glycine), L (leucine), R (arginine: SEQ ID NO: 9), or V (valine)), in which HXXH is repeated 3 times, Introduced into the aforementioned VHH1 and evaluated its zinc oxide bond 1 "life.
- amino acid X in the motif histidine, tryptophan, arginine, Considering the importance of lysine and glycine residues for binding to inorganic materials, the theoretically calculated library size from the codons encoding amino acids, and the library size that can be prepared, histidine, glycine, arginine, and leucine Limited to valine residues.
- HLGHGGHRLH Self sequence number 10
- HLGHGGHGLH SEQ ID NO: 1 1
- HVGHGLHGVR SEQ ID NO: 12
- HLGHGLHRVH SEQ ID NO: 1 3
- the present invention also provides a vector containing the gene encoding the zinc oxide-binding antibody and capable of expressing the zinc oxide-binding antibody.
- the expression vector of the present invention can be obtained by linking (inserting) a gene encoding the zinc oxide-binding antibody of the present invention to a known vector such as a plasmid.
- the base sequence may be appropriately optimized according to the host cell to be introduced.
- the vector is not particularly limited as long as it can replicate in the host, and for example, plasmid DNA, phage DNA, etc. can be used.
- plasmid DNA As the plasmid DNA, plasmid from E.coli (e.g. P BR322, pBR325, pUC18, pUC119 , pTrcHis, pBlueBacHis , etc.), plasmids derived from Bacillus subtilis (e.g. pUB110, pTP5, etc.), plasmid derived from yeast (e.g. Upushiron'ipushironro13, ⁇ 24, YCp50, pYE52, etc.) and phage DNA include fly phage.
- E.coli e.g. P BR322, pBR325, pUC18, pUC119 , pTrcHis, pBlueBacHis , etc.
- Bacillus subtilis e.g. pUB110, pTP5, etc.
- yeast e.g. Upushiron'ipushironro13, ⁇ 24, YC
- a method is employed in which the purified DNA is cleaved with a suitable restriction enzyme, inserted into a suitable restriction enzyme site or a multicloning site of the vector DNA, and ligated to the vector.
- the promoter is not particularly limited, and any promoter known to function in the host can be used. The promoters will be described in detail for each host in the next section. If necessary, a cis element such as an enhancer, a splicing signal, a poly A addition signal, a ribosome binding sequence (SD sequence), a terminator sequence, etc. may be arranged.
- the vector may contain genes for other enzymes that function in the host cell.
- the zinc oxide-binding antibody of the present invention can be expressed as a fusion protein with another material recognition peptide. In that case, a gene encoding a fusion protein may be introduced into the vector.
- the zinc oxide-binding '1 ⁇ live antibody of the present invention can be produced in a suitable manner by introducing the expression vector described in the previous section into an appropriate host so that the gene can be expressed and culturing.
- a zinc oxide-binding antibody using the Latada antibody of the present invention is a low-molecular monomer antibody and does not have a disulfide bond, so that it can be efficiently produced recombinantly, and cAb BCII-10 is highly produced by yeast. Production has been confirmed (see above).
- host cells include prokaryotic cells such as Escherichia coli and Bacillus subtilis.
- a host cell is transformed with a plasmid vector containing a replicon derived from a species compatible with the host, ie, an origin of replication, and regulatory sequences.
- the vector preferably has a sequence capable of imparting phenotypic (phenotypic) selectivity to transformed cells.
- the K12 strain is often used, and the vector is generally a PBR322 or pUC-type plasmid, but is not limited thereto, and various known strains and vectors can be used.
- promoters used in E. coli include tryptophan (trp) promoter, ratatose (lac) promoter, tryptophan-lactose (tac) promoter, lipoprotein (lpp) promoter, polypeptide chain elongation factor Tu (tufB) promoter, etc. Any of these can be preferably used.
- strain 207-25 is preferable, and pTUB228 (Ohmura, K. et al. (1984) J. Biochem. 95, 87-93) is used as the vector, but it is not limited to this. Is not to be done.
- pTUB228 Ohmura, K. et al. (1984) J. Biochem. 95, 87-93
- secretion expression outside the cell can also be achieved.
- eukaryotic host cells include cells of vertebrates, insects and yeasts.
- Vertebrate cells include, for example, monkey COS cells (Gluzman, Y. (1981) Cell 23, 175-182, ATCC CRL-1650) and Chinese 'hamster ovary cells (CH0 cells, ATCC CCL-61)
- a dihydrofolate reductase-deficient strain (Urlaub, G. and Chasin, LA (1980) Proc. Natl. Acad. Sci. USA 77, 4126-4220) is often used, but is not limited thereto.
- an expression vector for a vertebrate cell a vector having a promoter located upstream of a gene to be normally expressed, an RNA splice site, a polyaduration site, a transcription termination sequence, and the like can be used. In addition, it may have a replication origin if necessary.
- expression vectors include pCR3.1 (Invitrogen) having the cytomegalovirus early promoter, pSV2dhfr (Subramani, S. et al. (1981) Mol. Cell. Biol having the SV40 early promoter. 1, 854-864), etc., but is not limited to these.
- the expression vector when using COS cells as host cells, the expression vector has an SV40 origin of replication, and can grow independently in COS cells.
- a transcription promoter, transcription termination signal, and RNA splice Those provided with a part can be preferably used.
- the expression vector is: Jetylaminoethyl (DEAE) -tex lan run method (Luthman, H. and Magnus son, G. (1983) Nucleic Acids Res, 11, 1295-1308), calcium phosphate-DNA coprecipitation method (Graham, FL and van der W
- a vector capable of expressing a neo gene functioning as an antibiotic G418 resistance marker together with an expression vector, such as pRSVneo (Sambrook, J. et al. (1989): Molecular Cloning A Laboratory Manual Cold Spring Harbor Laboratory, NY) and SV2neo (Southern, PJ and Berg, P. (1982) J. Mol. Appl. Genet. 1, 327-341) etc.
- an expression vector such as pRSVneo (Sambrook, J. et al. (1989): Molecular Cloning A Laboratory Manual Cold Spring Harbor Laboratory, NY) and SV2neo (Southern, PJ and Berg, P. (1982) J. Mol. Appl. Genet. 1, 327-341) etc.
- baculovirus transfer vector is pVL1392 /, which utilizes the promoter of the polygraphin protein of autographer nuclear polyhedrosis virus (AcNPV). 1393 is often used (Kidd, 1. M. and V. Emery (1993) The use of baculoviruses as expression vectors.
- vectors using the promoter of baculovirus P10 or the basic protein can also be used.
- a thread-replaceable protein as a secreted protein (Zhemei Wang, et al. ( 1998) Biol. Chem., 379, 167-174).
- yeast As an expression system using a eukaryotic microorganism as a host cell, yeast is generally well known. Among them, yeast of the genus Saccharomyces, such as baker's yeast Saccharomyces cerevisiae and petroleum yeast Pichia pastoris are preferable. Examples of expression vectors for eukaryotic microorganisms such as yeast include, for example, the alcohol dehydrogenase gene promoter (Bennetzen, JL and Hall, BD (1982) J. Biol. Chem. 257, 3018-3025) and the acid phosphatase gene. (Miyanohara, A. et al. (1983) Proc. Natl. Acad. Sci.
- a secreted protein when expressed as a secreted protein, it can also be expressed as a recombinant having a secretory signal sequence and the endogenous protease possessed by the host cell or a known protease cleavage site at the N-terminal side.
- a secretory signal sequence for example, in a system in which human mast cell tryptase, a trypsin-type serine mouth therase, is expressed in petroleum yeast, the secretory signal sequence of yeast hypoactor and the KEX 2 protein possessed by petroleum yeast are located on the N-terminal side.
- the transformant obtained as described above can be cultured according to a conventional method, and the target zinc oxide-binding antibody is produced intracellularly or extracellularly by the culture.
- a medium used for the culture various media commonly used according to the employed host cells can be appropriately selected.
- a medium such as RPMI1640 medium or Dulbecco's modified Eagle medium (hereinafter referred to as “DMEM”) supplemented with serum components such as fetal bovine serum as necessary can be used.
- Recombinant protein produced inside or outside the transformed host cell by the above culture should be separated and purified by a known separation method utilizing the physical and chemical properties of the protein.
- a known separation method utilizing the physical and chemical properties of the protein.
- the method include treatment with a protein precipitant, ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, high performance liquid chromatography.
- Various liquid chromatography such as (HPLC) and dialysis methods can be used alone or in combination.
- histidine consisting of 6 residues is linked to the recombinant protein to be expressed, it can be efficiently purified with nickel affinity ram.
- the target zinc oxide-binding antibody protein can be easily produced in high yield and high purity by appropriately combining the methods described above.
- camel antibodies are monomeric antibodies, so their molecular weight is small, and each molecule is all covalently linked, so there is no phenomenon of dissociation. Therefore, a recombinant protein in which the zinc oxide-binding antibody of the present invention is fused with another protein can be easily prepared.
- the present invention also provides such a fusion protein.
- second protein having a material recognition sequence specifically refers to a specific material such as a metal oxide or a metal oxide such as a zinc oxide recognition peptide of the present invention. Contain sequences that bind automatically Means protein.
- proteins include, for example, W02004 / 031381, TO2005 / 010031, W02006 / 064639, W02006 / 068250, TO2006 / 126595, Brown et al., J. Mol. Biol. (2000), 299, 725-735, Naik et al., Nature materials, (2002), Vol.
- fusion proteins can be used for nanoparticle array technology and nanoparticle bonding technology.
- LKAHLPPSRLPS SEQ ID NO: 20
- a gold-binding antibody JP 2 0 0 5-3 1 2 4 4 6 and JP 2 0 0 6-2 2 5 2 9 4
- the zinc oxide-binding antibody of the present invention can be used to immobilize zinc oxide particles on the surface of the gold material, and to bind the zinc oxide particles to the gold nanoparticles.
- the zinc oxide-binding antibody of the present invention can be used for protein purification by fusing a His tag peptide, a GST protein, and various binding proteins.
- the present invention provides a solid support including a zinc oxide layer on which a zinc oxide-binding antibody is immobilized.
- the shape of the support is not particularly limited as long as it includes an acid zinc layer, and may be a substrate (such as a protein chip) or a particle (such as a nanoparticle).
- the acid semiconductor is a semiconductor Substrates with various structures can be manufactured by using processing technology (microfabrication technology). Furthermore, as already reported, the patterning technology has been established (Japanese Patent Laid-Open No. 2 0 06-2 2 5 2 94), which can be said to be useful for the production of solid phase substrates such as protein chips.
- the material of the solid phase substrate is not particularly limited as long as the zinc oxide layer can be formed, and a substrate generally used in the field such as metal, glass, silicon, etc. can be used as appropriate.
- Particles having a zinc oxide layer on which the zinc oxide-binding antibody of the present invention is immobilized can be applied to various analyzes such as protein purification.
- nanoparticles are useful for high-sensitivity analysis in microchannels by taking advantage of their surface area.
- zinc oxide particles with immobilized zinc oxide-binding antibodies are well-dispersed in aqueous solutions because they are modified with proteins, and react efficiently with active substances. When complete dispersion is achieved, the particles aggregate when protein-protein interactions occur, making it easy to analyze protein-protein binding. Such good dispersibility is also important when patterning nanoparticles using the inkjet method or spray method.
- the present invention also provides a pyosensor (immunobiosensor) having a solid phase support including a zinc oxide layer on which a zinc oxide-binding antibody is immobilized, and means for detecting an intermolecular interaction via the antibody.
- the intermolecular interaction through the antibody is not limited to the interaction between the antibody and the antigen, but the binding between the antibody and the analyte through the second antibody, etc. ⁇ Includes all of the dissociation.
- the detection means may be any of well-known detection means such as an electrical detection means such as an FET and an optical detection such as surface plasmon resonance.
- Fig. 19 shows a conceptual diagram of a biosensor using the zinc oxide-binding antibody of the present invention.
- FETs Field-effect transistors
- the principle is that if the current flowing from the source electrode to the drain electrode is controlled by the third electrode, the gate electrode, and the protein-protein interaction occurs on the surface of the gate electrode, the charge of the gate electrode changes. The current response is changed (Anal. Chira. Acta 136, 93 (1982)).
- zinc oxide for this gate electrode, protein-protein interaction using FET technology is achieved. The effect can be measured. That is, it can be used as a biosensor.
- a surface plasmon resonance sensor is a biosensor that uses surface plasmon resonance to analyze intermolecular interactions in real time on a sensor chip.
- surface plasmon resonance sensors for example, BIAcore's SPR measurement gold film sensor chip Sensor Chip Au
- a zinc oxide layer is formed on the sensor chip, the zinc oxide-binding antibody of the present invention is stably adsorbed with high binding properties.
- a microchannel is provided on a sensor chip to which an acid-zinc binding antibody is bound, and a sample containing an analyte (antigen) is fed at a constant flow rate.
- the surface plasmon resonance sensor has the advantage that it can detect a very small amount of sample with high sensitivity and can analyze the kinetics of interaction in real time. Therefore, the surface plasmon resonance sensor to which the zinc oxide-binding antibody of the present invention is bound is useful as a biosensor for antigen-antibody interaction analysis.
- the zinc oxide-binding antibody of the present invention can be applied to fine processing of zinc oxide and two-dimensional surface plasmon resonance (SPR) using zinc oxide dots (about several hundreds / m). Formation of a zinc oxide film on a microfabricated inorganic material such as zinc (semiconductor material) can be easily performed using semiconductor fabrication technology. For example, patterning at the ⁇ ⁇ level can be performed by the lift-off method or sputtering method using masking, and patterning at room temperature using a Pd catalyst (Adv. Mater. 14, 418-421 (2002 )) Is also possible.
- SPR surface plasmon resonance
- Reflectometry is a technique for measuring the amount of protein adsorbed on the surface of a sample plate by reflecting the laser beam onto the sample plate and measuring the reflected light.
- This reflectometry principle and a microfluidic chip combined with a reflectometry meter biosensor are readily available (Fluidware Technologies' Reflectmet V-Biosensor Array System Fluid— RIfS, etc.) ).
- the zinc oxide-binding antibody of the present invention has a high V, binding. Compatible and stable adsorption.
- a microchannel is provided on a sensor chip to which a zinc oxide-binding antibody is bound, and a sample containing an analyte (antigen) is fed at a constant flow rate.
- the reflectometry biosensor has the advantage of being able to detect minute samples with high sensitivity and to analyze the kinetics of interactions in real time. Therefore, the reflectometry biosensor to which the oxidized sub-binding antibody of the present invention is bound is useful as a biosensor for antigen-antibody interaction analysis.
- the zinc oxide-binding antibody of the present invention can also be applied to a biosensor using transmitted light, reflected light, near-field light, quartz crystal microbalance of an electromagnetic wave such as infrared rays.
- other well-known techniques used for immunodetection For example, development of biosensors using solid-phase immunization methods such as immunoprecipitation, Western blotting, dot plotting, slot blotting, ELISA, and RIA Is also possible.
- the zinc oxide-binding antibody of the present invention immobilized on a zinc oxide substrate can be easily detached by setting appropriate conditions. That is, by changing the conditions, the immobilized zinc oxide-binding antibody can be recovered and analyzed.
- the solution used for desorption may be an ordinary protein preparation aqueous solution (phosphate buffer solution, etc.), and the protein can be desorbed while maintaining its active structure without denaturation. It is.
- the camel antibody originally has a disulfide bond between CDR H-1-H-3 that stabilizes the complementarity-determining region (CDR) loop. In this example, it does not have a disulfide bond between CDRs.
- “Universal VHH Anti- / 3-lactamase camel antibody cAb BCII-10 (J. Mol. Biol. 352 (2005) 597) named “framework” was used as a peptide-type antibody to be used for peptide transplantation.
- cAb BCII-10 was prepared according to the sequence described previously (Antimicrobial Agents and Chemotheraphy, 2807-2812 (2001)).
- CDR of anti-] 3-Lactamase antibody cAbBCII-10 A gene encoding the antibody VHH3 in which the zinc oxide recognition peptide (SEQ ID NO: 1) consisting of EAHVMHKVAPRP was grafted at the 103-114 site in the H-3 region was completely synthesized. Subsequently, wild-type cAb BCII-10 (WT VHH) was prepared using a primer that replaces CDRH-3, which is a ZnO-recognizing peptide sequence, with a wild-type CDR. Similarly, zinc oxide-recognizing peptides were transplanted into CDR H-1 and H-2 to produce peptide-grafted ratada antibodies VHH1 and VHH2.
- 12 amino acid residues (29-35 residues, 53-69 residues, 102-117 residues) exposed on the surface of CDR are determined from the three-dimensional structure of wild-type camel antibody. Selected. A schematic diagram of the prepared expression vector is shown in FIG. 1A.
- the PCR reaction solution composition and reaction conditions (Table 1), and the PCR primers used and amino acid sequences of peptide-grafted camel antibodies (Table 2) are shown below.
- VHH3-NcoI (Reverse) 5 -NN CCATGGCCCAGGTTCAGCTGGTTGAAAG-3, (SEQ ID NO: 2 1)
- VHH3-1 (Reverse) 5 '-CAGGTTCAGCTGGTTGAAAGCGGCGGCGGCAGCGTGCAGGCGGGCGGCA
- VHH3-2 (Forward) 5, -AATTGCTGCCACTGCTTCGCGTTCCTGGCCCGGCGCCTGGCGAAACCAG
- VHH3-3 (Reverse) 5 '-GCGAAGCAGTGGCAGCAATTGCGAGCATGGGCGGCCTGACCTATTATGC
- VHH3-4 (Forward) 5, -GCTTCCACTGCTGCGCAATAATAAATCGCGGTATCTTCCGGTTTCAGGT
- VHH3-5 (Reverse) 5 '-TATTGCGCAGCAGTGGAAGCGCATGTGATGCATAAAGTGGCGCCGCGCC
- VHH3- sacll (Forward) 5 '-NNNCCGCGGATGAAACGGTAACCTGGG-3' (SEQ ID NO: 2 7)
- VHH-1 (Forward) 5 '-GCGCCACTTTATGCATCACATGCGCTTCGCTACCACCGCTCGCGGTGCAGC TCAG-3' (SEQ ID NO: 30)
- VHH-1 (Reverse) 5, -TGTGATGCATAAAGTGGCGCCGCGTCCGAGCCTGGGCTGGTTTCGCCAG-3 '(SEQ ID NO: 3 1)
- VHH-2 (Forward) 5 '-GCGCCACTTTATGCATCACATGCGCTTCCGCAATTGCTGCCACTGCTTC-3' (SEQ ID NO: 3 2)
- VHH-2 (Reverse) 5 '-TGTGATGCATAAAGTGGCGCCGCGTCCGGATAGCGTGAAAGGCCGCTTT-3' (SEQ ID NO: 3 3)
- ZnOtag-VHH (B) 5 '-NNNCCATGGCCGAAGCGCATGTGATGCATAAAGTGGCGCCGCGTCCGCAGGT TCAGCTGGTTGAAAG-3, (SEQ ID NO: 3 4)
- VHH1, VHH2, and VHH3 expression vectors prepared in the previous section E. coli BL21 (DE3) was transformed to express the protein.
- the protein purification method is shown in Figure 2A.
- VHH1, VHH2, and VHH3 could all be prepared as soluble fractions ( Figure 2B).
- the prepared protein was purified using Genore filtration chromatography (Superdex75; 50 mM Tris-HC1 (pH 8.0) / 200 mM NaCl) and circular dichroism (AVIV circular dichroism spectrometer (Proterion Co., New jersey, USA).
- VHH1 and VHH3 are single components of the monomer (red and blue lines in Fig. 3) ) And the circular dichroism peculiar to the camel antibody (red and blue lines in Fig. 4), VHH2 does not show a uniform gel filtration chromatogram, and the circular dichroism is also easy. It was not unique to the antibody (yellow-green line in Figs. 3 and 4).
- the activity of the prepared VHH1, VHH2, and VHH3 with zinc oxide particles was evaluated. Specifically, it was mixed with zinc oxide with a particle size of 100 nm in 10 phosphate buffer (containing a surfactant), and separated into a supernatant and a precipitate (zinc oxide) after centrifugation (Fig. 5). The precipitate was washed several times with the above phosphate buffer, and finally the adsorbed protein was solubilized with 6M aqueous guanidine hydrochloride (GdnHCl) solution and evaluated by SDS-PAGE (Figure 6). . SDS-PAGE results showed that VHH1 bound to zinc oxide (Figure 6A) and retained material specificity (Figure 6A).
- VHH3 binding properties were not observed when 10 mM phosphate buffer was used, but it was bound to ZnO when 1 mM phosphate buffer was used (Fig. 7). This shows that VHH3 also has a binding function to ZnO, but its binding activity is weaker than VHH1.
- VHH1 was evaluated for binding using Langmuir plots (Fig. 8).
- the dissociation equilibrium constant of VHH1 for zinc oxide there 176 nM, which fused zinc oxide recognition downy peptide to the N-terminus of WT VHH (3. 30 X 10- 7 M) and evaluation the same level of activity It was done. From this, it was confirmed that even when a peptide was transplanted into the CDR H-1 region, the three-dimensional structure of the Latada antibody itself was retained and the activity of the peptide was not reduced.
- Example 2 Production of high-affinity camel antibodies by in vitro affinity chromatography
- VHH1 prepared in Example 1 as a base material, an attempt was made to further improve the activity. Specifically, we focused on the CDR H-3 region to which no zinc oxide-recognizing peptide was transplanted, and constructed a library by randomizing the amino acid sequence. The CDR H-2 region was not used for modification because the results of Example 1 showed that the structure of the camel antibody was denatured and lost activity when the sequence of this part was changed.
- the library 1 was prepared by library-forming the 105 to 114 residues of the CDR-H3 region with the sequence shown in FIG.
- the gene in the H (R) part in the figure is designed so that the DNA base sequence is “(C or A or G) (T or C or A)”, and the His or Arg residue I tried to become.
- VHH1 library in which the antibody fragment library was presented on the M13 phage, was prepared by introducing the gene library into the phage media vector ⁇ , that is, the peptide-grafted antibody VHH1 was used as a cage to form CDR by Overlap extension PCR. After amplifying the gene fragment with randomized H-3, it was introduced into the phage media vector pTZ (Fig. 1B)
- the PCR reaction solution composition and reaction conditions were the same as in Table 1 of Example 1, and the mutation-introducing primers The sequence is as follows.
- VHH3-sacII (Forward) 5, -NNNCCGCGGATGAAACGGTAACCTGGG-3 '(SEQ ID NO: 3 8)
- the phage midvector pTZ used in this study has the lac promoter arranged in the opposite direction to the target gene, and transcription of mRNA Is initiated from the gVI promoter derived from the fuji located at the g3p end.
- the production of a fusion protein with g3p is moderately suppressed, and more types of clones can be presented without causing a gene defect.
- coli DH12S strain was transformed by electroporation method using the prepared phagemid vector, transferred to a 20 ml LB medium, cultured at 37 ° C, 0 / N, and extracted with plasmid. It was used for the operation of the phage display method as a phage mid library.
- the scale of the prepared library was 6.5 ⁇ 10 4
- the transformant was 1.2 ⁇ 10 5 .
- phage antibody library binds to the surface of zinc oxide and has been confirmed in previous experiments to prevent protein adsorption, so a phage antibody library does not bind to zinc oxide or is weak after mixing a solution with zinc oxide particles.
- the antibody-displayed phage that binds is phosphoric acid (10-50raM) and detergent (0.05% It was removed with an aqueous solution containing Tween-20) (washing operation). The strongly bound phage was removed using a high-concentration phosphoric acid aqueous solution (200), and the removed phage was reamplified.
- 840 ⁇ of the prepared phage antibody library was added to ZnO powder (0.2 mg ) and mixed for 1 hr.
- Nonspecifically adsorbed molecules for ZnO were washed with 10 mM phosphate (pH 7.4) I 200 mM NaCl I 0.05% Tween20 solution, and the bound phage antibody was eluted with 0.2 M phosphate buffer (pH 7.4, 200 mM NaCl).
- a re-fuzzy antibody was prepared and repeatedly subjected to a banning operation.
- the phosphate concentration in the wash was increased for each round. The above operation was repeated 4 rounds to concentrate the antibody capable of binding to ZnO.
- Table 3 shows the various conditions.
- camel antibodies having the amino acid sequences shown in Table 4 in CDR-H3 were selected.
- the affinity to zinc oxide was evaluated by Langmuir plot for each camel antibody, the results in Table 5 were obtained, and it was confirmed that 4F2 had the highest affinity.
- Example 5 Preparation of a zinc oxide-gold linked protein in which 4F2 and a gold recognition antibody are fused Bispecificity is achieved by fusing a 4F2 antibody that exhibits high affinity to ZnO and Au VHHl that binds to Au with a hinge linker. An antibody is produced.
- T7 promoter 5 5-GAAATTAATACGACTCACTATAGGG-3, (SEQ ID NO: 40)
- VHH- Eagl-Hinge (Forward) 5, -TTGCGGTTGTGGTTGCGGTTGCGGTTTCGGCTGCGGCTGCG GAATTTTCGGTTCGGCCGATGAAACGGTAACCTGGGTG-3 '(SEQ ID NO: 4 1)
- Hinge-Bam-VHH (Reverse) 5 '-CAACCGCAACCACAACCGCAACCGCAGCCGAAACCGCAGCCG AAACCGGAACCGGGATCCCAGGTTCAGCTGGTTGAAAGC-3' (SEQ ID NO: 4 2)
- Example 6 Evaluation of binding activity using surface plasmon resonance
- a zinc oxide film is prepared on the sensor chip Au of BIAcore's SPR measurement gold sensor chip, and the affinity of the antibody for the zinc oxide film is measured using BIAcore (registered trademark) 2000. Specifically, a few nn zinc oxide films are deposited on the gold surface of the Sensor Chip Au by sputtering. Fabricate with a film thickness of ⁇ 50nm. Then, the plate is set in BIAcore (registered trademark) 2000, and an antibody aqueous solution is allowed to flow to measure the adsorption of the zinc oxide film of the antibody.
- the crystal structure of the zinc oxide film can be changed by changing the preparation conditions, the adsorption characteristics of the antibody due to the difference in crystallinity of the oxide film (the following binding rate (ka), dissociation rate ( kd), dissociation constant (KD) and saturation bond amount).
- the quantitative evaluation is performed by determining the binding rate (ka), dissociation rate (kd), and dissociation constant (KD) of the antibody to zinc oxide from the binding curve obtained according to the analysis software attached to the apparatus.
- a microchannel is provided on the sensor chip (substrate) having the zinc oxide film adsorbed with the zinc oxide-binding antibody in the previous section, and a sample containing the analyte (antigen) is fed at a constant speed to the antibody and sample.
- the interaction of antigens in the medium is monitored in real time by surface plasmon phenomenon.
- the sensor chip to which the zinc oxide-binding antibody of the present invention is adsorbed can be used as a sensor chip for analyzing the kinetics of antigen-antibody interaction.
- Example 7 Adsorption of zinc oxide-binding antibody to zinc oxide using reflectometry and application to protein chip flow sensor
- Reflectometry is a technique for measuring the amount of protein adsorbed on the surface of a sample plate by reflecting the laser beam onto the sample plate and measuring the reflected light.
- a reflectometry method biosensor array system Fluid—RIfS manufactured by Firmware Technologies Co., Ltd.
- RIfS manufactured by Firmware Technologies Co., Ltd.
- Example 1 instead of the camel antibody, the anti-hen egg white lysozyme antibody HyHEL-10 Fv (Protein Data Bank: 1C08, J. Biol. Chem. 274 27623-27631 (1999), three-dimensional structure: FIG. 16A) The six CDR regions were each implanted with a zinc oxide recognition peptide.
- a PCR primer was designed based on the sequence of the ZnO-binding peptide (EAHVMHKVAPRP), and the vector pRA-WT VH (or VL) containing the HyHEL-10 VH (or VL) gene existing in our laboratory was selected.
- overlap extension PCR was performed using the following primers, and a peptide sequence was transplanted to each CDR.
- VH-2 (CDRH-2), VH-3 (CDRH-3) primer
- VH-NcoI 5 '-NNNCCATGGCCGATATCCAGCTGCAG-3' (SEQ ID NO: 4 4)
- VL-2 (CDR L-2), VL-3 (CDR L-3) primer
- VL-NcoI 5 '-NNNCCATGGCCGATATCGTCCTGACCC-3' (SEQ ID NO: 50)
- VL-SacII 5 -Image CCGCGGCCTTGATCTCCAGCTTGG-3, (SEQ ID NO: 5 1)
- PCR-amplified DNA fragments and our existing expression vector pRA-scFv (HyHEL-10 scFv expression vector) were digested with I and Ssc ⁇ , ligated, and VH-2, VH- 3, VL-2 and VL-3 single expression vectors were prepared.
- antibody molecules can be bound to zinc oxide in a stable and specific orientation.
- This technology can be used to develop high-throughput sensing technologies such as biosensors and protein chips. Sequence listing free text
- SEQ ID NO: 5 Zinc oxide recognition peptide ( ⁇ 05)
- SEQ ID NO: 6 Zinc oxide recognition region sequence
- SEQ ID NO: 8 Zinc oxide binding motif
- SEQ ID NO: 10 High affinity mutant H-3 region introduction sequence (3D2)
- SEQ ID NO: 1 1 High affinity mutant H-3 region introduction sequence (3E2)
- SEQ ID NO: 12 High affinity mutant H-3 region introduction sequence (4D4)
- SEQ ID NO: 13 High affinity mutant H-3 region introduction sequence (4F2)
- SEQ ID NO: 14 All amino acid sequences of 4F2
- SEQ ID NO: 15 All amino acid sequences of VHH1
- SEQ ID NO: 16 Full amino acid sequence of VHH2
- SEQ ID NO: 17 Complete amino acid sequence of VHH3
- SEQ ID NO: 20 Gold recognition, recognition peptide
- SEQ ID NO: 39 Linker sequence (Llama IgG2 upper hinge)
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
Claims
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP08711706A EP2128249A4 (en) | 2007-02-15 | 2008-02-14 | ZINC OXIDE FIXING ANTIBODIES AND USE THEREOF |
US12/527,129 US20100173360A1 (en) | 2007-02-15 | 2008-02-14 | Zinc oxide-binding antibody and use thereof |
JP2008558164A JP5029997B2 (ja) | 2007-02-15 | 2008-02-14 | 酸化亜鉛結合性抗体及びその用途 |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2007035073 | 2007-02-15 | ||
JP2007-035073 | 2007-02-15 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2008099968A1 true WO2008099968A1 (ja) | 2008-08-21 |
Family
ID=39690188
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2008/052917 WO2008099968A1 (ja) | 2007-02-15 | 2008-02-14 | 酸化亜鉛結合性抗体及びその用途 |
Country Status (4)
Country | Link |
---|---|
US (1) | US20100173360A1 (ja) |
EP (1) | EP2128249A4 (ja) |
JP (1) | JP5029997B2 (ja) |
WO (1) | WO2008099968A1 (ja) |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2011158369A (ja) * | 2010-02-02 | 2011-08-18 | National Institute Of Advanced Industrial Science & Technology | バイオチップ、抗原抗体反応検出用キット、及び抗原抗体反応の検出方法 |
US20120219504A1 (en) * | 2009-11-03 | 2012-08-30 | Snu R&Db Foundation | Complex of a protein comprising zinc oxide-binding peptides and zinc oxide nanoparticles, and use thereof |
JP2013029372A (ja) * | 2011-07-27 | 2013-02-07 | Konica Minolta Advanced Layers Inc | イオン性官能基修飾センサーチップおよびリガンド担持荷電微粒子を使用する分子間相互作用測定方法 |
WO2013146249A1 (ja) * | 2012-03-27 | 2013-10-03 | コニカミノルタ株式会社 | 等電点の測定方法、ならびにそのためのセンサーチップ、測定装置、測定システムおよびプログラム |
WO2021039574A1 (ja) * | 2019-08-23 | 2021-03-04 | 株式会社カネカ | O結合型糖鎖修飾が抑制された重鎖抗体 |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP6507969B2 (ja) * | 2015-09-25 | 2019-05-08 | コニカミノルタ株式会社 | ガス検知方法及びガス検知装置 |
CN114478803B (zh) * | 2022-02-11 | 2023-09-26 | 北京大学深圳研究生院 | 一种新型双特异性嵌合抗原受体的构建及其应用 |
Citations (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2004031381A1 (ja) | 2002-10-04 | 2004-04-15 | Japanese Foundation For Cancer Research | ナノ黒鉛構造体に結合能を有するペプチド |
WO2005010031A1 (ja) | 2003-07-30 | 2005-02-03 | Japan Science And Technology Agency | チタン、銀、シリコンに結合能を有するペプチド |
JP2005314411A (ja) | 2004-03-31 | 2005-11-10 | Canon Inc | 構造体及び該構造体からの物質の放出方法 |
JP2005312446A (ja) | 2004-03-31 | 2005-11-10 | Canon Inc | 金結合性タンパク質及びその用途 |
JP2006052201A (ja) * | 2004-07-13 | 2006-02-23 | National Institute Of Advanced Industrial & Technology | ラクダ科動物の変異vhh抗体及びその製造法 |
WO2006045076A2 (en) * | 2004-10-19 | 2006-04-27 | Massachusetts Institute Of Technology | Virus scaffold for self-assembled, flexible and light lithium battery |
WO2006064639A1 (ja) | 2004-12-14 | 2006-06-22 | Matsushita Electric Industrial Co., Ltd. | チタン結合性フェリチン及び無機粒子の配置方法 |
WO2006068250A1 (ja) | 2004-12-24 | 2006-06-29 | Japan Science And Technology Agency | ナノ黒鉛構造体-金属ナノ粒子複合体 |
JP2006225294A (ja) | 2005-02-16 | 2006-08-31 | Tohoku Univ | 酸化亜鉛特異的認識ペプチド、酸化亜鉛の固定化方法およびその製膜方法 |
WO2006126595A1 (ja) | 2005-05-27 | 2006-11-30 | Japan Science And Technology Agency | 機能性材料の三次元構造体 |
JP2007035073A (ja) | 2006-11-09 | 2007-02-08 | Toshiba Tec Corp | 貨幣払出装置 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8796544B2 (en) * | 2005-12-14 | 2014-08-05 | Massachusetts Institute Of Technology | Bio-sensitized solar cells (BSSC) |
-
2008
- 2008-02-14 JP JP2008558164A patent/JP5029997B2/ja not_active Expired - Fee Related
- 2008-02-14 US US12/527,129 patent/US20100173360A1/en not_active Abandoned
- 2008-02-14 EP EP08711706A patent/EP2128249A4/en not_active Withdrawn
- 2008-02-14 WO PCT/JP2008/052917 patent/WO2008099968A1/ja active Application Filing
Patent Citations (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2004031381A1 (ja) | 2002-10-04 | 2004-04-15 | Japanese Foundation For Cancer Research | ナノ黒鉛構造体に結合能を有するペプチド |
WO2005010031A1 (ja) | 2003-07-30 | 2005-02-03 | Japan Science And Technology Agency | チタン、銀、シリコンに結合能を有するペプチド |
JP2005314411A (ja) | 2004-03-31 | 2005-11-10 | Canon Inc | 構造体及び該構造体からの物質の放出方法 |
JP2005312446A (ja) | 2004-03-31 | 2005-11-10 | Canon Inc | 金結合性タンパク質及びその用途 |
JP2006052201A (ja) * | 2004-07-13 | 2006-02-23 | National Institute Of Advanced Industrial & Technology | ラクダ科動物の変異vhh抗体及びその製造法 |
WO2006045076A2 (en) * | 2004-10-19 | 2006-04-27 | Massachusetts Institute Of Technology | Virus scaffold for self-assembled, flexible and light lithium battery |
WO2006064639A1 (ja) | 2004-12-14 | 2006-06-22 | Matsushita Electric Industrial Co., Ltd. | チタン結合性フェリチン及び無機粒子の配置方法 |
WO2006068250A1 (ja) | 2004-12-24 | 2006-06-29 | Japan Science And Technology Agency | ナノ黒鉛構造体-金属ナノ粒子複合体 |
JP2006225294A (ja) | 2005-02-16 | 2006-08-31 | Tohoku Univ | 酸化亜鉛特異的認識ペプチド、酸化亜鉛の固定化方法およびその製膜方法 |
WO2006126595A1 (ja) | 2005-05-27 | 2006-11-30 | Japan Science And Technology Agency | 機能性材料の三次元構造体 |
JP2007035073A (ja) | 2006-11-09 | 2007-02-08 | Toshiba Tec Corp | 貨幣払出装置 |
Non-Patent Citations (39)
Title |
---|
"The 3d Tohoku University Bioscience Symposium", LECTURE SUMMARIES, 2006, pages 240 PS - 189 |
ADV. MATER., vol. 14, 2002, pages 418 - 421 |
ANDREW, L. NILES ET AL., BIOTECHNOL. APPL. BIOCHEM., vol. 28, 1998, pages 125 - 131 |
ANTIMICROBIAL AGENTS AND CHEMOTHERAPHY, 2001, pages 2807 - 2812 |
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, 2001, pages 2807 - 2812 |
ARNOLD F.H.: "Metal-affinity separations: a new dimension in protein processing", BIO./TECHNOLOGY, vol. 9, no. 2, 1991, pages 151 - 156, XP009003669 * |
BENNETZEN, J. L.; HALL, B. D., J. BIOL. CHEM., vol. 257, 1982, pages 3018 - 3025 |
BIOCHEM. BIOPHYS. RES. COMMUN., vol. 328, 2005, pages 98 - 105 |
CANCER IMMUNOL. IMMUNOTHER., vol. 53, 2004, pages 497 - 509 |
GLUZMAN, Y., CELL, vol. 23, 1981, pages 175 - 182 |
GRAHAM, F. L.; VAN DER EB, A. J., VIROLOGY, vol. 52, 1973, pages 456 - 457 |
J. BIOL. CHEM., vol. 274, 1999, pages 27623 - 27631 |
J. MOL. BIOL., vol. 299, 2000, pages 725 - 735 |
J. MOL. BIOL., vol. 352, 2005, pages 597 |
KIDD, I. M.; V.C. EMERY: "The use of baculoviruses as expression vectors", APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY, vol. 420, 1993, pages 137 - 159, XP009055324, DOI: doi:10.1007/BF02788049 |
LUTHMAN, H.; MAGNUSSON, G., NUCLEIC ACIDS RES, vol. 11, 1983, pages 1295 - 1308 |
MAO ET AL., PNAS, vol. 100, no. 12, 2003, pages 6946 - 6951 |
MAO ET AL., SCIENCE, vol. 303, 2004, pages 213 - 217 |
MIYANOHARA, A. ET AL., PROC. NATL. ACAD. SCI. U.S.A., vol. 80, 1983, pages 1 - 5 |
NAIK ET AL., J. NANOSCI. NANOTECH., vol. 2, no. 1, 2002, pages 95 - 100 |
NAIK ET AL., NATURE MATERIALS, vol. 1, November 2002 (2002-11-01), pages 169 - 172 |
NATURE BIOTECH., vol. 23, pages 1126 - 1136 |
NEUMANN, E. ET AL., EMBO J., vol. 1, 1982, pages 841 - 845 |
OHMURA, K. ET AL., J. BIOCHEM., vol. 95, 1984, pages 87 - 93 |
PEELLE B.R. ET AL.: "Design criteria for engineering inorganic material-specific peptides", LANGMUIR, vol. 21, no. 15, 2005, pages 6929 - 6933, XP008115098 * |
SAERENS D. ET AL.: "Identification of a Universal VHH Framework to Graft Non-canonical Antigen-binding Loops of Camel Single-domain Antibodies", JOURNAL OF MOLECULAR BIOLOGY, vol. 352, no. 3, 2005, pages 597 - 607, XP005052664 * |
SAERENS ET AL., J. MOL. BIOL., vol. 352, 2005, pages 597 - 607 |
SAMBROOK, J. ET AL.: "Molecular Cloning A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY |
See also references of EP2128249A4 |
SOUTHERN, P. J.; BERG, P., J. MOL. APPL. GENET., vol. 1, 1982, pages 327 - 341 |
STOCKER W. ET AL.: "The metzincins - Topological and sequential relations between the astacins, adamlysins, serralysins, and matrixins (collagenases) define a superfamily of zinc-peptidases", PROTEIN SCIENCE, vol. 4, 1995, pages 823 - 840, XP008005305 * |
SUBRAMANI, S. ET AL., MOL. CELL. BIOL., vol. 1, 1981, pages 854 - 864 |
THAI C.K. ET AL.: "Identification and characterization of Cu(2)-O- and ZnO-binding polypeptides by Escherichia coli cell surface display: toward an understanding of metal oxide binding", BIOTECHNOLOGY AND BIOENGINEERING, vol. 87, no. 2, 2004, pages 129 - 137, XP002387068 * |
THE 3RD TOHOKU UNIVERSITY BIOSCIENCE SYMPOSIUM, LECTURE SUMMARIES, 2006, pages 241 PS - 190 |
UMETSU ET AL., ADVANCED MATERIALS, vol. 17, 2005, pages 2571 - 2575 |
UMEZU K. ET AL.: "Zairyo Tokuisei Peptide.Kotai Bunshi o Riyo shita Shink Zairyo Kogaku", ABSTRACTS OF MEETING ON BIOMOLECULAR, vol. 34, 24 August 2007 (2007-08-24), pages 36 - 37, XP008142302 * |
URLAUB, G.; CHASIN, L. A., PROC. NATL. ACAD. SCI. U.S.A., vol. 77, 1980, pages 4126 - 4220 |
WICKHAM, T. J. ET AL., BIOTECHNOL. PROG.I, 1992, pages 391 - 396 |
ZHE-MEI WANG ET AL., BIOL. CHEM., vol. 379, 1998, pages 167 - 174 |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20120219504A1 (en) * | 2009-11-03 | 2012-08-30 | Snu R&Db Foundation | Complex of a protein comprising zinc oxide-binding peptides and zinc oxide nanoparticles, and use thereof |
JP2011158369A (ja) * | 2010-02-02 | 2011-08-18 | National Institute Of Advanced Industrial Science & Technology | バイオチップ、抗原抗体反応検出用キット、及び抗原抗体反応の検出方法 |
JP2013029372A (ja) * | 2011-07-27 | 2013-02-07 | Konica Minolta Advanced Layers Inc | イオン性官能基修飾センサーチップおよびリガンド担持荷電微粒子を使用する分子間相互作用測定方法 |
WO2013146249A1 (ja) * | 2012-03-27 | 2013-10-03 | コニカミノルタ株式会社 | 等電点の測定方法、ならびにそのためのセンサーチップ、測定装置、測定システムおよびプログラム |
WO2021039574A1 (ja) * | 2019-08-23 | 2021-03-04 | 株式会社カネカ | O結合型糖鎖修飾が抑制された重鎖抗体 |
Also Published As
Publication number | Publication date |
---|---|
JP5029997B2 (ja) | 2012-09-19 |
JPWO2008099968A1 (ja) | 2010-05-27 |
EP2128249A1 (en) | 2009-12-02 |
EP2128249A4 (en) | 2010-07-14 |
US20100173360A1 (en) | 2010-07-08 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US9714941B2 (en) | Bio-sensing nanodevice | |
JP5029997B2 (ja) | 酸化亜鉛結合性抗体及びその用途 | |
JP6475630B2 (ja) | ストレプトアビジン突然変異タンパク質およびそれらを使用する方法 | |
Jason‐Moller et al. | Overview of Biacore systems and their applications | |
US8748111B2 (en) | Multiplexed olfactory receptor-based microsurface plasmon polariton detector | |
JP4208942B2 (ja) | 金結合性タンパク質 | |
EP3203233A2 (en) | Methods for using antibodies and analogs thereof | |
Kim et al. | Engineering of anti-human interleukin-4 receptor alpha antibodies with potent antagonistic activity | |
WO2019046699A1 (en) | NON-DETERGENT GPCR BIOELECTRONIC INTERFACES COUPLED TO THE 2D S-PROTEIN NETWORK, DEVICES AND METHODS OF USE THEREOF | |
JP2005312446A5 (ja) | ||
Gonzalez | Protein microarrays, biosensors, and cell-based methods for secretome-wide extracellular protein–protein interaction mapping | |
JP2009534035A (ja) | 細胞系のハイスループットスクリーニング方法 | |
KR101243379B1 (ko) | 구리 결합펩타이드를 이용한 탐침단백질이 고정된 다중 스팟 구리 증착형 나노구조체 배열 바이오칩 및 이의 제조방법 | |
Gronewold et al. | Selection process generating peptide aptamers and analysis of their binding to the TiO2 surface of a surface acoustic wave sensor | |
CN116120440A (zh) | 抗SARS-CoV-2刺突蛋白S1的单克隆抗体及其应用 | |
JP2007121276A (ja) | 基板およびその製造方法 | |
JP2007107913A (ja) | 反応素子及びその製造方法 | |
KR100979282B1 (ko) | 실리카 결합단백질을 이용한 바이오-실리카 칩 및 그제조방법 | |
KR101300187B1 (ko) | 융합 단백질 및 이를 포함하는 바이오센서 | |
KR100965480B1 (ko) | 재조합 융합단백질, 및 이를 이용한 바이오센서 및 이를이용한 목적물질의 면역검출법 | |
US20220154174A1 (en) | Method of Selecting for Antibodies | |
CN114751985A (zh) | Tim-3抗体、制备方法及其应用 | |
Shwetha | ESCORTing proteins directly from whole cell-lysate for single molecule studies | |
Bong et al. | Refolding of autodisplayed anti NEF scFv | |
JP2007289198A (ja) | 金結合性タンパク質 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 08711706 Country of ref document: EP Kind code of ref document: A1 |
|
DPE2 | Request for preliminary examination filed before expiration of 19th month from priority date (pct application filed from 20040101) | ||
ENP | Entry into the national phase |
Ref document number: 2008558164 Country of ref document: JP Kind code of ref document: A |
|
WWE | Wipo information: entry into national phase |
Ref document number: 12527129 Country of ref document: US |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2008711706 Country of ref document: EP |