JP7481733B2 - Monoclonal antibody against brain-derived neurotrophic factor and hybridoma producing the antibody - Google Patents
Monoclonal antibody against brain-derived neurotrophic factor and hybridoma producing the antibody Download PDFInfo
- Publication number
- JP7481733B2 JP7481733B2 JP2020029569A JP2020029569A JP7481733B2 JP 7481733 B2 JP7481733 B2 JP 7481733B2 JP 2020029569 A JP2020029569 A JP 2020029569A JP 2020029569 A JP2020029569 A JP 2020029569A JP 7481733 B2 JP7481733 B2 JP 7481733B2
- Authority
- JP
- Japan
- Prior art keywords
- bdnf
- antibody
- antigen
- monoclonal antibody
- binding fragment
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 102000004219 Brain-derived neurotrophic factor Human genes 0.000 title claims description 124
- 108090000715 Brain-derived neurotrophic factor Proteins 0.000 title claims description 124
- 210000004408 hybridoma Anatomy 0.000 title claims description 21
- 229940077737 brain-derived neurotrophic factor Drugs 0.000 title description 116
- 238000009739 binding Methods 0.000 claims description 62
- 230000027455 binding Effects 0.000 claims description 60
- 150000001413 amino acids Chemical class 0.000 claims description 56
- 239000012634 fragment Substances 0.000 claims description 55
- 239000000427 antigen Substances 0.000 claims description 51
- 108091007433 antigens Proteins 0.000 claims description 51
- 102000036639 antigens Human genes 0.000 claims description 51
- 238000000034 method Methods 0.000 claims description 36
- 239000002243 precursor Substances 0.000 claims description 29
- 238000002965 ELISA Methods 0.000 claims description 19
- 238000011156 evaluation Methods 0.000 claims description 9
- 244000005700 microbiome Species 0.000 claims description 5
- 241000124008 Mammalia Species 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 210000004027 cell Anatomy 0.000 description 22
- 229940077456 human brain-derived neurotrophic factor Drugs 0.000 description 17
- 101000739876 Homo sapiens Brain-derived neurotrophic factor Proteins 0.000 description 16
- 102000051542 human BDNF Human genes 0.000 description 16
- 238000001262 western blot Methods 0.000 description 15
- 241000699666 Mus <mouse, genus> Species 0.000 description 14
- 239000000523 sample Substances 0.000 description 14
- 238000006243 chemical reaction Methods 0.000 description 12
- 210000001519 tissue Anatomy 0.000 description 12
- 238000004458 analytical method Methods 0.000 description 11
- 239000006166 lysate Substances 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
- 241000699670 Mus sp. Species 0.000 description 9
- 239000000872 buffer Substances 0.000 description 9
- 108090000623 proteins and genes Proteins 0.000 description 9
- 238000003118 sandwich ELISA Methods 0.000 description 9
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 8
- 210000004556 brain Anatomy 0.000 description 8
- 238000001514 detection method Methods 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 8
- 230000003053 immunization Effects 0.000 description 8
- 239000002609 medium Substances 0.000 description 8
- 102100035080 BDNF/NT-3 growth factors receptor Human genes 0.000 description 7
- 101710101214 BDNF/NT-3 growth factors receptor Proteins 0.000 description 7
- 239000012228 culture supernatant Substances 0.000 description 7
- 230000006870 function Effects 0.000 description 7
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 7
- 108010025020 Nerve Growth Factor Proteins 0.000 description 6
- 238000010790 dilution Methods 0.000 description 6
- 239000012895 dilution Substances 0.000 description 6
- 239000002953 phosphate buffered saline Substances 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- 230000009257 reactivity Effects 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- 230000009870 specific binding Effects 0.000 description 6
- 101150056950 Ntrk2 gene Proteins 0.000 description 5
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 5
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 5
- 239000006180 TBST buffer Substances 0.000 description 5
- 230000007910 cell fusion Effects 0.000 description 5
- 239000013612 plasmid Substances 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 102000007072 Nerve Growth Factors Human genes 0.000 description 4
- 108010076504 Protein Sorting Signals Proteins 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 4
- 239000002671 adjuvant Substances 0.000 description 4
- 229960002685 biotin Drugs 0.000 description 4
- 235000020958 biotin Nutrition 0.000 description 4
- 239000011616 biotin Substances 0.000 description 4
- 210000001124 body fluid Anatomy 0.000 description 4
- 239000010839 body fluid Substances 0.000 description 4
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 4
- 238000010494 dissociation reaction Methods 0.000 description 4
- 230000005593 dissociations Effects 0.000 description 4
- 230000000971 hippocampal effect Effects 0.000 description 4
- 238000002649 immunization Methods 0.000 description 4
- 238000003018 immunoassay Methods 0.000 description 4
- 238000012744 immunostaining Methods 0.000 description 4
- 238000002372 labelling Methods 0.000 description 4
- 229940126619 mouse monoclonal antibody Drugs 0.000 description 4
- 210000002569 neuron Anatomy 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 210000004989 spleen cell Anatomy 0.000 description 4
- 239000013598 vector Substances 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 101150035467 BDNF gene Proteins 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 229920001213 Polysorbate 20 Polymers 0.000 description 3
- 241000700159 Rattus Species 0.000 description 3
- 150000001412 amines Chemical class 0.000 description 3
- 239000012491 analyte Substances 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 210000005013 brain tissue Anatomy 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000010168 coupling process Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 230000016784 immunoglobulin production Effects 0.000 description 3
- 238000001114 immunoprecipitation Methods 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 3
- 238000011813 knockout mouse model Methods 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 239000002504 physiological saline solution Substances 0.000 description 3
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 3
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 3
- 108090000765 processed proteins & peptides Proteins 0.000 description 3
- 238000011002 quantification Methods 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 230000019491 signal transduction Effects 0.000 description 3
- 210000000952 spleen Anatomy 0.000 description 3
- 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 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 101500024763 Homo sapiens BDNF precursor form Proteins 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 102000015336 Nerve Growth Factor Human genes 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 206010035226 Plasma cell myeloma Diseases 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- 230000032683 aging Effects 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 238000011088 calibration curve Methods 0.000 description 2
- 230000005754 cellular signaling Effects 0.000 description 2
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 230000002950 deficient Effects 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000007876 drug discovery Methods 0.000 description 2
- 239000000839 emulsion Substances 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 239000003446 ligand Substances 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 201000000050 myeloid neoplasm Diseases 0.000 description 2
- 210000000653 nervous system Anatomy 0.000 description 2
- 230000001537 neural effect Effects 0.000 description 2
- 239000003900 neurotrophic factor Substances 0.000 description 2
- 210000005259 peripheral blood Anatomy 0.000 description 2
- 239000011886 peripheral blood Substances 0.000 description 2
- 230000026731 phosphorylation Effects 0.000 description 2
- 238000006366 phosphorylation reaction Methods 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 239000012146 running buffer Substances 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- GEYOCULIXLDCMW-UHFFFAOYSA-N 1,2-phenylenediamine Chemical compound NC1=CC=CC=C1N GEYOCULIXLDCMW-UHFFFAOYSA-N 0.000 description 1
- 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 description 1
- GHCZTIFQWKKGSB-UHFFFAOYSA-N 2-hydroxypropane-1,2,3-tricarboxylic acid;phosphoric acid Chemical compound OP(O)(O)=O.OC(=O)CC(O)(C(O)=O)CC(O)=O GHCZTIFQWKKGSB-UHFFFAOYSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 241000251468 Actinopterygii Species 0.000 description 1
- 108700028369 Alleles Proteins 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 239000012583 B-27 Supplement Substances 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 208000003174 Brain Neoplasms Diseases 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 101710098119 Chaperonin GroEL 2 Proteins 0.000 description 1
- 241000938605 Crocodylia Species 0.000 description 1
- 206010012289 Dementia Diseases 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 102100037412 Germinal-center associated nuclear protein Human genes 0.000 description 1
- 102000034615 Glial cell line-derived neurotrophic factor Human genes 0.000 description 1
- 108091010837 Glial cell line-derived neurotrophic factor Proteins 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 1
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 208000019022 Mood disease Diseases 0.000 description 1
- 101000933313 Mus musculus Brain-derived neurotrophic factor Proteins 0.000 description 1
- 102100029268 Neurotrophin-3 Human genes 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 239000002033 PVDF binder Substances 0.000 description 1
- 108090000526 Papain Proteins 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 108010033276 Peptide Fragments Proteins 0.000 description 1
- 102000007079 Peptide Fragments Human genes 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 239000012979 RPMI medium Substances 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 108010003723 Single-Domain Antibodies Proteins 0.000 description 1
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 101710120037 Toxin CcdB Proteins 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003044 adaptive effect Effects 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 230000001430 anti-depressive effect Effects 0.000 description 1
- 210000000628 antibody-producing cell Anatomy 0.000 description 1
- 239000000935 antidepressant agent Substances 0.000 description 1
- 229940005513 antidepressants Drugs 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000012888 bovine serum Substances 0.000 description 1
- 230000003925 brain function Effects 0.000 description 1
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000002490 cerebral effect Effects 0.000 description 1
- 230000037326 chronic stress Effects 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000012875 competitive assay Methods 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 210000003618 cortical neuron Anatomy 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 239000000104 diagnostic biomarker Substances 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 1
- DNJIEGIFACGWOD-UHFFFAOYSA-N ethyl mercaptane Natural products CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 239000012595 freezing medium Substances 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 210000002288 golgi apparatus Anatomy 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000009878 intermolecular interaction Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 210000005171 mammalian brain Anatomy 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 229940055729 papain Drugs 0.000 description 1
- 235000019834 papain Nutrition 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 238000002823 phage display Methods 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000003518 presynaptic effect Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 210000003079 salivary gland Anatomy 0.000 description 1
- 239000012723 sample buffer Substances 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 210000004739 secretory vesicle Anatomy 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 210000004988 splenocyte Anatomy 0.000 description 1
- 230000035882 stress Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 230000033504 synapse organization Effects 0.000 description 1
- 230000000946 synaptic effect Effects 0.000 description 1
- 230000005062 synaptic transmission Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000011282 treatment Methods 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Description
本発明は、脳由来神経栄養因子に対するモノクローナル抗体およびその抗体を産生するハイブリドーマに関する。 The present invention relates to a monoclonal antibody against brain-derived neurotrophic factor and a hybridoma that produces the antibody.
脳はさまざまな外界刺激に対する反応として神経細胞の電気活動さらには神経伝達の伝搬を誘導して、その活動痕が例えば記憶という情報の蓄積につながる。しかしこのとき同時にその情報蓄積を確かにするためにタンパク質が合成され、当該タンパク質が機能することも見出されており、神経細胞の成長を促進する脳内成長因子（brain-derived neurotrophic factor：BDNF）はその代表的因子であることが報告されている(非特許文献１～４)。したがって、BDNFの機能低下は認知症やうつ病といった神経回路ネットワークの機能低下につながることが予想され、これらの疾患との関連研究も増えると同時に創薬の標的分子として注目されている (非特許文献５および６)。
また、BDNFは脳以外に臓器や体液に存在することも報告されている（非特許文献７～９）。BDNFのシグナルと脳腫瘍や肺がんの関係、血液や脳脊髄液に存在するBDNFをうつ病等の診断バイオマーカーの候補とする研究である。
このような背景と近年の研究展開を考えたとき、BDNFに対する抗体は重要な研究試薬としてさらには創薬や診断技術への応用などが期待される。
In response to various external stimuli, the brain induces electrical activity of neurons and the propagation of neurotransmission, and the traces of this activity lead to the accumulation of information, such as memory. However, at the same time, proteins are synthesized to ensure the accumulation of information, and it has been found that these proteins function, and it has been reported that brain-derived neurotrophic factor (BDNF), which promotes the growth of neurons, is a representative factor (Non-Patent Documents 1-4). Therefore, it is predicted that a decrease in the function of BDNF leads to a decrease in the function of neural circuit networks, such as dementia and depression, and there is an increasing amount of research into the relationship with these diseases, and BDNF is also attracting attention as a target molecule for drug discovery (Non-Patent Documents 5 and 6).
It has also been reported that BDNF is present in organs and body fluids other than the brain (Non-Patent Documents 7-9). Research is being conducted on the relationship between BDNF signals and brain tumors and lung cancer, and on the BDNF present in blood and cerebrospinal fluid as a candidate diagnostic biomarker for depression, etc.
Considering this background and recent research developments, antibodies against BDNF are expected to be important research reagents and to have applications in drug discovery and diagnostic technologies.
これまでにBDNFに対する抗体は多数作製されており、市販もされている。しかし、それらのほとんどは、反応特異性について不十分な評価しかされていない。その重要な理由として、BDNFは生体において微量にしか発現していないため、高感度な抗BDNF抗体が必要とされ、一方で、抗BDNF抗体の反応特異性を評価するためには、BDNFを発現しない細胞または組織においては結合反応が見られないことを確認する必要があることが挙げられる。例えば、BDNFノックアウトマウス由来の組織切片または細胞のライセートをウエスタンブロットやELISAに供した場合には、シグナルが検出されないことが必須である。ところが、市販抗体の多くは十分な性能評価がされておらず、当業者により上記基準を満たすものとして認識されている市販の抗BDNF抗体は、本願出願時では、プロメガ社の抗BDNF抗体#G700Bおよび#G164G、ならびにAbcam社のマウスBDNFモノクローナル抗体#3C11およびウサギBDNFモノクローナル抗体#EPR1292のみであり、しかも、プロメガ社の抗BDNF抗体はすでに入手不能である（販売中止）。一般的に、抗体は、そのアプリケーションごとに使用可能なものが異なり、例えば、ウェスタンブロッティング法では使用できる抗体がELISA法では使用できない場合や、その逆の場合も多いことを考えると、信頼できる抗BDNF抗体が明らかに不足しているのが現状である。 Many antibodies against BDNF have been produced and are commercially available. However, most of them have only been insufficiently evaluated for their reaction specificity. The main reason is that BDNF is expressed only in trace amounts in the body, so highly sensitive anti-BDNF antibodies are required, and on the other hand, in order to evaluate the reaction specificity of anti-BDNF antibodies, it is necessary to confirm that no binding reaction is observed in cells or tissues that do not express BDNF. For example, when tissue sections or cell lysates derived from BDNF knockout mice are subjected to Western blot or ELISA, it is essential that no signal is detected. However, many commercially available antibodies have not been sufficiently evaluated for performance, and the only commercially available anti-BDNF antibodies recognized by those skilled in the art as meeting the above criteria at the time of filing this application are Promega's anti-BDNF antibodies #G700B and #G164G, and Abcam's mouse BDNF monoclonal antibody #3C11 and rabbit BDNF monoclonal antibody #EPR1292, and furthermore, Promega's anti-BDNF antibodies are no longer available (discontinued). Generally, different antibodies can be used for different applications. For example, there are many cases where an antibody that can be used for Western blotting cannot be used for ELISA, and vice versa. Considering this, there is a clear lack of reliable anti-BDNF antibodies.
さらに、上記の信頼できる抗BDNF抗体はいずれも、成熟型BDNFのみを認識し、その前駆体であるproBDNFを認識しない。すなわち、現状、細胞において産生されたproBDNFと成熟型BDNFとをあわせたBDNF分子の総量を測定可能な信頼できる抗BDNF抗体は存在しない。また、BDNFの機能を阻害または亢進するような生理活性を有する抗BDNF抗体も、これまでには報告されていない。 Furthermore, all of the reliable anti-BDNF antibodies mentioned above recognize only mature BDNF and do not recognize its precursor, proBDNF. In other words, at present, there are no reliable anti-BDNF antibodies that can measure the total amount of BDNF molecules, including proBDNF and mature BDNF produced in cells. Furthermore, no anti-BDNF antibodies have been reported to date that have physiological activity that inhibits or enhances the function of BDNF.
本発明は、成熟型BDNFおよびその前駆体の両方に対して特異的結合能を有する、信頼できる品質の抗BDNF抗体の提供を課題とする。 The objective of the present invention is to provide an anti-BDNF antibody of reliable quality that has specific binding ability to both mature BDNF and its precursor.
本発明者らは、鋭意検討の結果、成熟型BDNFおよびその前駆体に対して特異的結合能を有するマウスモノクローナル抗体（2D7、3C8）とそのハイブリドーマの作製に成功し、本発明の完成に至った。すなわち、本発明は以下の態様を含む：
〔１〕配列番号１で示されるアミノ酸配列の129～176番目のアミノ酸からなる領域に含まれるエピトープを認識する、成熟型BDNFおよびその前駆体に特異的に結合するモノクローナル抗体またはその抗原結合断片。
〔２〕上記〔１〕に記載のモノクローナル抗体またはその抗原結合断片であって、配列番号１で示されるアミノ酸配列の203～247番目のアミノ酸からなる領域を認識しない、モノクローナル抗体またはその抗原結合断片。
〔３〕上記〔１〕または〔２〕に記載のモノクローナル抗体またはその抗原結合断片であって、前記エピトープが配列番号１で示されるアミノ酸配列の165～176番目のアミノ酸からなる領域に含まれる、モノクローナル抗体またはその抗原結合断片。
〔４〕上記〔１〕～〔３〕のいずれかに記載のモノクローナル抗体またはその抗原結合断片であって、独立行政法人製品評価技術基盤機構特許微生物寄託センターに受託番号：NITE P-02652または受託番号：NITE P-02653として寄託されたハイブリドーマより産生される、モノクローナル抗体またはその抗原結合断片。
〔５〕上記〔１〕～〔３〕のいずれかに記載のモノクローナル抗体またはその抗原結合断片であって、受託番号：NITE P-02652のハイブリドーマ、または、受託番号：NITE P-02653のハイブリドーマより産生されるモノクローナル抗体と同じエピトープと反応する、モノクローナル抗体またはその抗原結合断片。
〔６〕上記〔１〕～〔５〕のいずれかに記載のモノクローナル抗体または抗原結合断片であって、哺乳動物由来の成熟型BDNFおよびその前駆体を認識する、モノクローナル抗体またはその抗原結合断片。
〔７〕成熟型BDNFおよびその前駆体を検出するためのキットであって、上記〔１〕～〔６〕のいずれかに記載のモノクローナル抗体またはその抗原結合断片を含む、キット。
〔８〕独立行政法人製品評価技術基盤機構特許微生物寄託センターに受託番号：NITE P-02652またはNITE P-02653として寄託されたハイブリドーマ。
〔９〕上記〔８〕に記載のハイブリドーマを培養してモノクローナル抗体を産生させる工程を含む、成熟型BDNFおよびその前駆体に特異的に結合するモノクローナル抗体の製造方法。
〔１０〕試料中の成熟型BDNFおよびその前駆体を検出する方法であって、上記〔１〕～〔６〕のいずれかに記載のモノクローナル抗体またはその抗原結合断片と前記試料とを反応させる工程と、前記モノクローナル抗体またはその抗原結合断片と結合した成熟型BDNFおよびその前駆体を検出する工程とを含む方法。
〔１１〕上記〔１０〕に記載の方法であって、前記検出工程がELISAにより行われる方法。
As a result of extensive research, the present inventors have succeeded in producing mouse monoclonal antibodies (2D7, 3C8) and hybridomas thereof that have specific binding ability to mature BDNF and its precursor, thereby completing the present invention. That is, the present invention includes the following aspects:
[1] A monoclonal antibody or an antigen-binding fragment thereof that recognizes an epitope contained in the region consisting of amino acids 129 to 176 of the amino acid sequence shown in SEQ ID NO:1 and specifically binds to mature BDNF and its precursor.
[2] The monoclonal antibody or antigen-binding fragment thereof according to [1] above, which does not recognize the region consisting of amino acids 203 to 247 of the amino acid sequence shown in SEQ ID NO:1.
[3] The monoclonal antibody or antigen-binding fragment thereof according to [1] or [2] above, wherein the epitope is contained in a region consisting of amino acids 165 to 176 of the amino acid sequence shown in SEQ ID NO:1.
[4] The monoclonal antibody or antigen-binding fragment thereof according to any one of [1] to [3] above, which is produced by a hybridoma deposited at the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation under Accession No. NITE P-02652 or Accession No. NITE P-02653.
[5] The monoclonal antibody or antigen-binding fragment thereof according to any one of [1] to [3] above, which reacts with the same epitope as the monoclonal antibody produced by the hybridoma having accession number NITE P-02652 or the hybridoma having accession number NITE P-02653.
[6] The monoclonal antibody or antigen-binding fragment thereof according to any one of [1] to [5] above, which recognizes mammalian-derived mature BDNF and its precursor.
[7] A kit for detecting mature BDNF and its precursor, comprising the monoclonal antibody or antigen-binding fragment thereof described in any one of [1] to [6] above.
[8] A hybridoma deposited at the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation under the accession number NITE P-02652 or NITE P-02653.
[9] A method for producing a monoclonal antibody that specifically binds to mature BDNF and its precursor, comprising the step of culturing the hybridoma according to [8] above to produce the monoclonal antibody.
[10] A method for detecting mature BDNF and its precursor in a sample, comprising the steps of reacting the sample with a monoclonal antibody or antigen-binding fragment thereof described in any one of [1] to [6] above, and detecting mature BDNF and its precursor bound to the monoclonal antibody or antigen-binding fragment thereof.
[11] The method according to [10] above, wherein the detection step is carried out by ELISA.
本発明のモノクローナル抗体またはその抗原結合断片によれば、proBDNFおよび成熟型BDNFの両方をあわせたBDNF分子の総発現量を測定することが可能となる。また、本発明のモノクローナル抗体を、proBDNFのプロドメインを特異的に認識し、かつ、成熟型BDNFを認識する既存の抗体と組み合わせて用いることにより、proBDNFと成熟型BDNFとをそれぞれ定量することが可能となる。 The monoclonal antibody or antigen-binding fragment of the present invention makes it possible to measure the total expression level of BDNF molecules, including both proBDNF and mature BDNF. In addition, by using the monoclonal antibody of the present invention in combination with an existing antibody that specifically recognizes the pro domain of proBDNF and also recognizes mature BDNF, it becomes possible to quantitate both proBDNF and mature BDNF.
また、本発明の抗体またはその抗原結合断片はBDNF/TrkBシグナル経路を阻害効果を有する。そのため、BDNF/TrkBシグナル経路が関与する脳機能や疾患を研究するために有用であり得る。 In addition, the antibody or antigen-binding fragment of the present invention has an inhibitory effect on the BDNF/TrkB signaling pathway. Therefore, it may be useful for studying brain functions and diseases in which the BDNF/TrkB signaling pathway is involved.
以下、本発明を詳細に説明するが、本発明は本明細書中に説明した実施形態に限定されるものではない。 The present invention is described in detail below, but is not limited to the embodiments described in this specification.
本発明は、第一の実施形態によれば、配列番号１で示されるアミノ酸配列の129～176番目のアミノ酸領域に含まれるエピトープを認識する、成熟型BDNFおよびその前駆体に特異的に結合するモノクローナル抗体またはその抗原結合断片である。 According to a first embodiment, the present invention is a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to mature BDNF and its precursor and recognizes an epitope contained in the region of amino acids 129 to 176 of the amino acid sequence shown in SEQ ID NO:1.
本実施形態において、「BDNF」とは、脳由来神経栄養因子（Brain-derived neurotrophic factor）を意味する。BDNFは、シグナルペプチド、プロドメイン（プロペプチドとも呼ばれる）および成熟ドメインからなるpreproBDNFとして小胞体内腔で合成され、その後、preproBDNFからシグナルペプチドが切り離されてproBDNFとなり、ゴルジ体にてさらにプロドメインが切り離されて成熟型BDNFとなる。本実施形態におけるBDNFの「前駆体」は、preproBDNFおよびproBDNFのどちらも含み得るが、preproBDNFは短時間で速やかにproBDNFへと変換されるため、実質的にはproBDNFである。また、本明細書では、成熟型BDNFを指して、「mBDNF」または単に「BDNF」とも記載する。 In this embodiment, "BDNF" means brain-derived neurotrophic factor. BDNF is synthesized in the lumen of the endoplasmic reticulum as preproBDNF consisting of a signal peptide, a prodomain (also called propeptide), and a mature domain. The signal peptide is then cleaved from preproBDNF to form proBDNF, and the prodomain is further cleaved in the Golgi apparatus to form mature BDNF. In this embodiment, the "precursor" of BDNF may include both preproBDNF and proBDNF, but since preproBDNF is rapidly converted to proBDNF in a short period of time, it is essentially proBDNF. In this specification, mature BDNF is also referred to as "mBDNF" or simply "BDNF".
本実施形態における成熟型BDNFおよびその前駆体は、哺乳動物（ヒト、サル、ウシ、ウマ、ヒツジ、ブタ、マウス、ラット、ウサギ、イヌ、ネコなど）、魚類、両生類、爬虫類、鳥類などの任意の脊椎動物由来のものであってよいが、好ましくは哺乳動物由来、さらに好ましくはヒト由来である。なお、BDNFのアミノ酸配列は優れた種間の保存性を有しており、霊長類および齧歯類の間では100％の配列同一性を有する。ヒトpreproBDNFのアミノ酸配列（アクセッション番号：CAA62632）を図１および配列番号１に示す。1～18番目のアミノ酸からなる領域がシグナルペプチドであり、19～128番目のアミノ酸からなる領域がプロドメインであり、129～247番目のアミノ酸からなる領域（図１、下線部）が成熟ドメインである。 In this embodiment, the mature BDNF and its precursor may be derived from any vertebrate such as mammals (humans, monkeys, cows, horses, sheep, pigs, mice, rats, rabbits, dogs, cats, etc.), fish, amphibians, reptiles, and birds, but is preferably derived from mammals, and more preferably from humans. The amino acid sequence of BDNF is highly conserved between species, and has 100% sequence identity between primates and rodents. The amino acid sequence of human preproBDNF (accession number: CAA62632) is shown in Figure 1 and SEQ ID NO: 1. The region consisting of amino acids 1 to 18 is the signal peptide, the region consisting of amino acids 19 to 128 is the prodomain, and the region consisting of amino acids 129 to 247 (underlined in Figure 1) is the mature domain.
本実施形態の抗体は、モノクローナル抗体である。「モノクローナル」とは、実質的に均一な集団を意味する語であり、したがって、「モノクローナル抗体」とは、その集団を構成する個々の抗体が同一であり、同じエピトープに対して同一の結合特異性および親和性を有するものをいう。本実施形態のモノクローナル抗体は、マウス抗体、キメラ抗体、ヒト化抗体または完全ヒト抗体のいずれであってもよい。 The antibody of this embodiment is a monoclonal antibody. "Monoclonal" is a term meaning a substantially homogeneous population, and therefore a "monoclonal antibody" refers to an antibody whose individual antibodies are identical and have the same binding specificity and affinity for the same epitope. The monoclonal antibody of this embodiment may be a mouse antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.
本実施形態において、「抗原結合断片」とは、抗体の一部であって、少なくとも抗原結合領域を含む断片をいう。抗原結合断片の具体例としては、例えば、Fv、scFv、Fab、F(ab')2、Fab'、Fd、dAb、CDR、scFv-Fc断片、ナノボディ、アフィボディ、ダイアボディ、アビマー、ミニボディ、バーサボディなどが挙げられる。 In this embodiment, the "antigen-binding fragment" refers to a part of an antibody that contains at least an antigen-binding region. Specific examples of antigen-binding fragments include Fv, scFv, Fab, F(ab') 2 , Fab', Fd, dAb, CDR, scFv-Fc fragments, nanobodies, affibodies, diabodies, avimers, minibodies, and versabodies.
本実施形態のモノクローナル抗体またはその抗原結合断片（以下、単に「抗体」とも記載する）は、ビオチン、蛍光色素、化学発光色素、放射性同位元素、酵素などの当該分野で公知の任意の標識物質により標識されていてもよい。抗体またはその抗原結合断片の標識は、公知の手法により適宜行うことができる。 The monoclonal antibody or antigen-binding fragment thereof (hereinafter also simply referred to as "antibody") of this embodiment may be labeled with any labeling substance known in the art, such as biotin, a fluorescent dye, a chemiluminescent dye, a radioisotope, or an enzyme. Labeling of the antibody or antigen-binding fragment thereof can be appropriately performed by known techniques.
本実施形態のモノクローナル抗体またはその抗原結合断片は、配列番号１のアミノ酸配列における129～176番目のアミノ酸からなる領域内のエピトープを認識する。本実施形態のモノクローナル抗体またはその抗原結合断片が認識するエピトープは、配列番号１のアミノ酸配列における129～176番目のアミノ酸からなる領域内に含まれる連続したアミノ酸配列からなってもよいし、立体構造的に近接する不連続なアミノ酸からなってもよい。エピトープを構成するアミノ酸の数は、特に限定はされないが、例えば、4以上、5以上、6以上であり、好ましくは約6～10アミノ酸である。好ましくは、本実施形態のモノクローナル抗体またはその抗原結合断片は、配列番号１のアミノ酸配列における165～176番目のアミノ酸からなる領域（TVLEKVPVSKGQ）に含まれるエピトープを認識する。また、本実施形態のモノクローナル抗体またはその抗原結合断片は、配列番号１のアミノ酸配列における1～18番目のアミノ酸からなる領域（シグナルペプチド）、19～128番目のアミノ酸からなる領域（プロドメイン）および203～247番目のアミノ酸からなる領域を認識しないものであることが好ましい。 The monoclonal antibody or antigen-binding fragment of this embodiment recognizes an epitope in the region consisting of the 129th to 176th amino acids in the amino acid sequence of SEQ ID NO: 1. The epitope recognized by the monoclonal antibody or antigen-binding fragment of this embodiment may consist of a continuous amino acid sequence contained in the region consisting of the 129th to 176th amino acids in the amino acid sequence of SEQ ID NO: 1, or may consist of discontinuous amino acids that are adjacent in terms of three-dimensional structure. The number of amino acids constituting the epitope is not particularly limited, but is, for example, 4 or more, 5 or more, 6 or more, and preferably about 6 to 10 amino acids. Preferably, the monoclonal antibody or antigen-binding fragment of this embodiment recognizes an epitope contained in the region consisting of the 165th to 176th amino acids (TVLEKVPVSKGQ) in the amino acid sequence of SEQ ID NO: 1. In addition, it is preferable that the monoclonal antibody or antigen-binding fragment of this embodiment does not recognize the region consisting of the 1st to 18th amino acids (signal peptide), the region consisting of the 19th to 128th amino acids (prodomain), and the region consisting of the 203rd to 247th amino acids in the amino acid sequence of SEQ ID NO: 1.
モノクローナル抗体またはその抗原結合断片の調製方法は十分に確立されており、本実施形態のモノクローナル抗体またはその抗原結合断片は、上で定義したエピトープを含む抗原ペプチドを用いて、当分野において十分に確立された公知の方法にしたがって調製することができる。例えば、モノクローナル抗体は、ハイブリドーマ法またはファージディスプレイ法などにより調製することができる。また、抗原結合断片は、酵素（例えば、パパイン、ペプシンなど）で抗体を断片化するか、または、抗体断片をコードする遺伝子構築物を発現ベクターに導入し、適当な宿主細胞で発現させることにより調製することができる。 Methods for preparing monoclonal antibodies or antigen-binding fragments thereof are well established, and the monoclonal antibodies or antigen-binding fragments thereof of this embodiment can be prepared according to well-established methods known in the art using antigen peptides containing the epitopes defined above. For example, monoclonal antibodies can be prepared by the hybridoma method or the phage display method. Antigen-binding fragments can also be prepared by fragmenting antibodies with enzymes (e.g., papain, pepsin, etc.) or by introducing a gene construct encoding an antibody fragment into an expression vector and expressing it in a suitable host cell.
本実施形態における好ましいモノクローナル抗体の具体例としては、独立行政法人製品評価技術基盤機構特許微生物寄託センターに受託番号：NITE P-02652または受託番号：NITE P-02653として寄託されたハイブリドーマが生産するモノクローナル抗体を挙げることができる。受託番号：NITE P-02652として寄託されたハイブリドーマからは、配列番号１のアミノ酸配列における129～176番目のアミノ酸からなる領域内のエピトープを認識する抗体（2D7）を得ることができる。また、受託番号：NITE P-02653として寄託されたハイブリドーマからは、配列番号１のアミノ酸配列における165～176番目のアミノ酸からなる領域（TVLEKVPVSKGQ）内のエピトープを認識する抗体（3C8）を得ることができる。 Specific examples of preferred monoclonal antibodies in this embodiment include monoclonal antibodies produced by hybridomas deposited at the National Institute of Technology and Evaluation Patent Microorganisms Depositary Center under accession number NITE P-02652 or accession number NITE P-02653. From the hybridoma deposited under accession number NITE P-02652, an antibody (2D7) that recognizes an epitope within the region consisting of amino acids 129 to 176 in the amino acid sequence of SEQ ID NO: 1 can be obtained. From the hybridoma deposited under accession number NITE P-02653, an antibody (3C8) that recognizes an epitope within the region consisting of amino acids 165 to 176 (TVLEKVPVSKGQ) in the amino acid sequence of SEQ ID NO: 1 can be obtained.
上記抗体2D7または3C8と同じエピトープを認識する抗体も同様に、本実施形態における好ましい抗体である。同じエピトープを認識するものであるかどうかは、例えば、抗体2D7または3C8を用いた競合アッセイにより確認することができる。 Antibodies that recognize the same epitope as the above-mentioned antibody 2D7 or 3C8 are also preferred antibodies in this embodiment. Whether or not they recognize the same epitope can be confirmed, for example, by a competitive assay using the antibody 2D7 or 3C8.
本実施形態のモノクローナル抗体またはその抗原結合断片の、成熟型BDNFおよびその前駆体に対する反応性は、公知の免疫学的方法、例えば、ウェスタンブロッティング法、ELISA（Enzyme-Linked Immuno Sorbent Assay）、表面プラズモン共鳴（Surface plasmon resonance : SPR）、細胞免疫染色、組織免疫染色などにより確認することができる。 The reactivity of the monoclonal antibody or antigen-binding fragment thereof of this embodiment to mature BDNF and its precursor can be confirmed by known immunological methods, such as Western blotting, ELISA (Enzyme-Linked Immunosorbent Assay), surface plasmon resonance (SPR), cell immunostaining, and tissue immunostaining.
本発明は、第二の実施形態によれば、第一の実施形態のモノクローナル抗体またはその抗原結合断片を含んでなる、成熟型BDNFおよびその前駆体を検出するためのキットである。本実施形態における「モノクローナル抗体」、「抗原結合断片」、「成熟型BDNFおよびその前駆体」は、第一の実施形態で定義したものと同様である。 According to a second embodiment, the present invention provides a kit for detecting mature BDNF and its precursor, comprising the monoclonal antibody or antigen-binding fragment thereof of the first embodiment. In this embodiment, the terms "monoclonal antibody," "antigen-binding fragment," and "mature BDNF and its precursor" are the same as those defined in the first embodiment.
本実施形態のキットは、第一の実施形態のモノクローナル抗体またはその抗原結合断片の１種類または複数種類を含んでよい。また、それに加えて、容器、緩衝液、陽性対照、陰性対照、検出プロトコールを記載した説明書などをさらに含んでもよい。 The kit of this embodiment may include one or more types of the monoclonal antibody or antigen-binding fragment thereof of the first embodiment. In addition, the kit may further include a container, a buffer solution, a positive control, a negative control, instructions describing a detection protocol, and the like.
本発明は、第三の実施形態によれば、試料中の成熟型BDNFおよびその前駆体を検出する方法であって、第一の実施形態のモノクローナル抗体またはその抗原結合断片と前記試料とを反応させる工程と、前記モノクローナル抗体またはその抗原結合断片と結合した成熟型BDNFおよびその前駆体を検出する工程とを含む方法である。本実施形態における「モノクローナル抗体」、「抗原結合断片」、「成熟型BDNFおよびその前駆体」は、第一の実施形態で定義したものと同様である。 According to a third embodiment, the present invention provides a method for detecting mature BDNF and its precursor in a sample, the method comprising the steps of reacting the monoclonal antibody or antigen-binding fragment thereof of the first embodiment with the sample, and detecting mature BDNF and its precursor bound to the monoclonal antibody or antigen-binding fragment thereof. In this embodiment, the terms "monoclonal antibody," "antigen-binding fragment," and "mature BDNF and its precursor" are the same as those defined in the first embodiment.
本実施形態の方法では、第一の実施形態のモノクローナル抗体またはその抗原結合断片と、試料とを反応させる。試料は、任意の動物、好ましくは哺乳動物に由来する組織、細胞または体液などであってよいが、特に限定されない。組織試料または細胞試料としては、例えば、脳組織、脳組織から調製された培養神経細胞などが挙げられる。体液試料としては、例えば、血液、血漿、血清、脳脊髄液などが挙げられる。試料は、当業者に周知の方法により被験者から得ることができる。また、上記の試料は、必要に応じて、生理食塩水や適切な緩衝液を用いてホモジネート処理などの任意の処理を行ったものであってもよい。 In the method of this embodiment, the monoclonal antibody or antigen-binding fragment thereof of the first embodiment is reacted with a sample. The sample may be tissue, cells, or body fluid derived from any animal, preferably a mammal, but is not particularly limited thereto. Examples of tissue or cell samples include brain tissue and cultured nerve cells prepared from brain tissue. Examples of body fluid samples include blood, plasma, serum, and cerebrospinal fluid. The sample can be obtained from a subject by a method well known to those skilled in the art. Furthermore, the above sample may be optionally treated, such as homogenized, using physiological saline or an appropriate buffer solution.
抗体またはその抗原結合断片と試料との反応は、抗体またはその抗原結合断片と試料とを、生理食塩水や適切な緩衝液中で一定時間インキュベートすることにより行うことができる。添加される抗体またはその抗原結合断片の濃度は、例えば、0.5～50nMまたは0.1～100μg/mLの範囲で適宜選択することができる。インキュベート時間は、例えば、10分～24時間であってよい。 The reaction between the antibody or its antigen-binding fragment and the sample can be carried out by incubating the antibody or its antigen-binding fragment and the sample in physiological saline or an appropriate buffer for a certain period of time. The concentration of the antibody or its antigen-binding fragment to be added can be appropriately selected from the range of, for example, 0.5 to 50 nM or 0.1 to 100 μg/mL. The incubation time can be, for example, 10 minutes to 24 hours.
次いで、抗体またはその抗原結合断片と結合した成熟型BDNFおよびその前駆体を検出する。結合の検出は、当分野において周知の手法により行うことができ、例えば、ウェスタンブロッティング法、ELISA、SPR、細胞免疫染色、組織免疫染色などの手法により行うことができる。本実施形態の方法における結合検出手法は、好ましくはELISAである。 Then, mature BDNF and its precursor bound to the antibody or its antigen-binding fragment are detected. Binding can be detected by techniques well known in the art, such as Western blotting, ELISA, SPR, cell immunostaining, and tissue immunostaining. The binding detection technique in the method of this embodiment is preferably ELISA.
本発明のモノクローナル抗体またはその抗原結合断片、ならびにそれらを用いたキットおよび第方法は、成熟型BDNFおよびその前駆体をあわせたBDNF分子の総発現量を測定することができ、有用である。 The monoclonal antibody or its antigen-binding fragment, as well as the kit and method using the same, of the present invention are useful in measuring the total expression level of BDNF molecules, including both mature BDNF and its precursor.
以下、本発明を実施例に従いより詳細に説明する。なお、本発明は以下の実施の形態に限定されない。 The present invention will be described in more detail below with reference to examples. Note that the present invention is not limited to the following embodiments.
（実施例１：ヒト成熟型BDNFおよびその前駆体に対するマウスモノクローナル抗体の作製）
目的
従来の抗体作製技術であるハイブリドーマ法を用いて、ヒト成熟型BDNFおよびその前駆体に対するマウスモノクローナル抗体の作製を行った。
(Example 1: Preparation of mouse monoclonal antibodies against human mature BDNF and its precursor)
the purpose
Using the hybridoma method, a conventional antibody production technique, we produced mouse monoclonal antibodies against human mature BDNF and its precursor.
実験
（１）抗原
pET-32b(+)ベクター（Novagen社製）にヒトBDNF（配列番号１のアミノ酸における129番目から247番目のアミノ酸配列）のcDNAを導入したプラスミドを大腸菌に導入し、発現させた組換えヒトBDNFを免疫抗原として用いた。
（２）マウスへの免疫
免疫は以下の方法で行った。PBSを用いて1.0 mg/mLに調製した免疫抗原を完全フロイントアジュバンド（FCA；Freund’s Adjuvant, Complete, SIGMA）と等量混合した。エマルジョンを形成させたものを370 μLずつマウスの背部皮下に投与し、その後2週間間隔で1.0 mg/mLの免疫抗原と不完全フロイントアジュバンド（FIA；Freund’s Adjuvant, Incomplete, SIGMA）と等量混合し、エマルジョンを形成させたものを140 μLずつマウスの背部皮下に投与した。最終的に合計3回抗原を投与した。3回免疫7日後にマウスより血清を回収し、ELISAにより抗体価を確認した。抗体価の高かったものに対して、1.0 mg/mLの免疫抗原50 μLと生理食塩水450 μLを混合したものをマウスの腹腔内に最終投与し、その3日後に細胞融合用に脾臓を摘出した。
Experiment (1) Antigen
A plasmid containing the cDNA for human BDNF (the amino acid sequence from amino acid 129 to amino acid 247 of SEQ ID NO: 1) introduced into the pET-32b(+) vector (Novagen) was introduced into Escherichia coli, and the expressed recombinant human BDNF was used as an immunogen.
(2) Immunization of mice Immunization was performed as follows. The immunizing antigen, prepared at 1.0 mg/mL using PBS, was mixed with an equal amount of Freund's Adjuvant, Complete (FCA; Freund's Adjuvant, Complete, SIGMA). The emulsion was administered subcutaneously to the back of the mouse in 370 μL portions, and then at 2-week intervals, 1.0 mg/mL of the immunizing antigen was mixed with an equal amount of Freund's Adjuvant, Incomplete (FIA; Freund's Adjuvant, Incomplete, SIGMA), and the emulsion was administered subcutaneously to the back of the mouse in 140 μL portions. The antigen was administered three times in total. Seven days after the third immunization, serum was collected from the mouse, and the antibody titer was confirmed by ELISA. For mice with high antibody titers, a mixture of 50 μL of 1.0 mg/mL of the immunizing antigen and 450 μL of physiological saline was administered intraperitoneally to the mouse for the final administration, and the spleen was removed for cell fusion three days later.
（３）脾臓細胞の調製と細胞融合
摘出した脾臓をすりつぶし、抗BDNF抗体産生細胞を含む脾臓細胞を調製したところ、1匹あたり約1×108個の脾臓細胞を調製できた。一方で、ミエローマ細胞であるP3U1を培養し、細胞融合当日に生細胞率が90%以上のP3U1を調製した。これら脾臓細胞とP3U1を5：1で混ぜ、50%濃度の分子量1,450のポリエチレングリコールにより細胞融合を行った。融合後、培地で洗浄し、HAT培地に懸濁したものを、96穴培養プレートの各ウェルに1×105個/ウェルとなるように細胞を播きこんだ。
(3) Preparation of spleen cells and cell fusion The excised spleen was ground to prepare spleen cells containing anti-BDNF antibody-producing cells, and approximately 1 x 10 8 spleen cells were prepared per mouse. On the other hand, myeloma cells, P3U1, were cultured to prepare P3U1 with a viability rate of 90% or more on the day of cell fusion. These spleen cells and P3U1 were mixed at a ratio of 5:1, and cell fusion was performed using 50% concentration of polyethylene glycol with a molecular weight of 1,450. After fusion, the cells were washed with medium, suspended in HAT medium, and seeded into each well of a 96-well culture plate at 1 x 10 5 cells/well.
（４）抗体産生陽性ウェルのスクリーニング
細胞融合後、10日目の培養上清を回収し、抗体産生陽性ウェルのスクリーニングを後述の実験例２の方法で行った。1712ウェル中126ウェルでELISAにおける吸光度が0.1以上の値を示した。これらのうち、吸光度が高値のもの、またはウェスタンブロッティング法による組換えヒトBDNFに対する反応性を高いもの（５ウェル分）を最終的に選択した。
(4) Screening of wells positive for antibody production After cell fusion, the culture supernatant was collected on the 10th day, and screening of wells positive for antibody production was performed by the method described in Experimental Example 2 below. 126 wells out of 1712 wells showed an absorbance value of 0.1 or more in ELISA. Among these, wells with high absorbance or high reactivity to recombinant human BDNF by Western blotting (5 wells) were finally selected.
（５）クローニング
選択した5ウェル分について、限界希釈法によるクローニングを行った。すなわち、細胞を10%のFCSを含むRPMI培地で5個/mLに調製し、96穴培養プレート2枚分の各ウェルに200 μLずつ添加した。10日後、後述の実験例２の方法で培養上清中のBDNFに対する抗体価を測定し、陽性であることを確認し、それぞれのウェルに由来するクローンを2クローン得た。それぞれの樹立クローンを「2D7」、「3C8」とした。
(5) Cloning The selected 5 wells were cloned by limiting dilution. That is, the cells were adjusted to 5 cells/mL in RPMI medium containing 10% FCS, and 200 μL was added to each well of two 96-well culture plates. After 10 days, the antibody titer against BDNF in the culture supernatant was measured by the method of Experimental Example 2 described below, and it was confirmed to be positive, and two clones derived from each well were obtained. The established clones were named "2D7" and "3C8".
（６）抗体のスクリーニング法
96穴マイクロタイタープレートの各ウェルに、固相化抗原としてリン酸緩衝生理食塩水（PBS（pH7.0））で1μg/mLに調製した組換えヒトBDNFを50μLずつ添加し、25°Cで1時間放置した。次に、0.05% Tween20を含むPBS（pH7.0）（PBST）で3回洗浄後、0.5%ゼラチンを含むPBST（ブロッキング液）を各ウェルに200μLずつ添加し、25°Cで1時間静置した。洗浄後、培養上清を原液のまま各ウェルに50μLずつ添加し、25°Cで1時間静置した。次に、PBSTで3回洗浄後、各ウェルに2500倍希釈したHRP標識抗マウスIgG抗体（KPL社）を50μLずつ添加し、25°Cで1時間静置した。次に、PBSTで3回洗浄後、各ウェルに0.02%の過酸化水素を含む0.1Mクエン酸リン酸緩衝液（pH5.0）で0.5 mg/mLに調製したo-フェニレンジアミン溶液100μLを添加し、25℃で10分間静置した後に、1M硫酸溶液100μLを各ウェルに添加し、呈色反応を停止した。その後490 nmの吸光度をELISAリーダーによって測定した。
(6) Antibody screening method
50μL of recombinant human BDNF, prepared as a solid-phase antigen in phosphate-buffered saline (PBS (pH 7.0)) at 1μg/mL, was added to each well of a 96-well microtiter plate and left at 25°C for 1 hour. Next, after washing three times with PBS (pH 7.0) containing 0.05% Tween 20 (PBST), 200μL of PBST containing 0.5% gelatin (blocking solution) was added to each well and left at 25°C for 1 hour. After washing, 50μL of culture supernatant was added to each well as is and left at 25°C for 1 hour. Next, after washing three times with PBST, 50μL of HRP-labeled anti-mouse IgG antibody (KPL) diluted 2500-fold was added to each well and left at 25°C for 1 hour. Next, after washing three times with PBST, 100 μL of o-phenylenediamine solution adjusted to 0.5 mg/mL in 0.1 M citrate phosphate buffer (pH 5.0) containing 0.02% hydrogen peroxide was added to each well, and after leaving it at 25°C for 10 minutes, 100 μL of 1 M sulfuric acid solution was added to each well to stop the color reaction. The absorbance at 490 nm was then measured using an ELISA reader.
（７）ハイブリドーマの樹立
GANP マウスに対して、免疫抗原（組換えヒトBDNF）を背部皮下に3回免疫した。3回免疫後、抗血清力価の上昇を、組換えヒトBDNFを用いたELISAで確認した。有意な力価上昇がもっとも高値であったマウスから脾臓を摘出し、マウスミエローマP3U1と細胞融合させた。融合脾細胞を96ウェルプレートに播種し、HAT選択培養を行った。播種9日後の培養細胞をサンプルとして、組換えヒトBDNFを用いたELISAを実施した。培養上清評価の結果を元に、組換えヒトBDNFに対する反応性が最も高い2D7および3C8ウェルに関して、2回の限界希釈法によるクローニングおよびスクリーニングを行い2D7および3C8の2クローンのハイブリドーマを樹立した。
(7) Establishment of hybridomas
GANP mice were immunized subcutaneously on the back three times with an immunizing antigen (recombinant human BDNF). After three immunizations, the increase in antiserum titer was confirmed by ELISA using recombinant human BDNF. The spleen was removed from the mouse with the highest significant increase in titer and fused with mouse myeloma P3U1. The fused splenocytes were seeded on a 96-well plate and HAT selection culture was performed. ELISA was performed using recombinant human BDNF using the cultured cells 9 days after seeding as a sample. Based on the results of the culture supernatant evaluation, 2D7 and 3C8 wells, which had the highest reactivity to recombinant human BDNF, were cloned and screened twice by limiting dilution method, and hybridomas of two clones, 2D7 and 3C8, were established.
（８）抗体の精製
ハイブリドーマは、細胞凍結保存液（セルバンカー、ゼノアック）およびプログラムフリーザーを用いて凍結し、液体窒素タンク内で保管した。凍結ハイブリドーマを、37℃恒温槽に浸し、素早く解凍した。5％CO2のインキュベーション条件で、9%ウシ血清含有培地を用いて拡大培養した。拡大培養後、同インキュベーション条件下で培地を無血清培地（Hybridoma-SFM, GIBCO）50mLに置換し培養した。無血清培養開始7日後、培養上清を回収し遠心分離後、フィルターでろ過し、タンパク質精製用培養上清とした。1.5mLのprotein G-sepharose(GE healthcare)カラムを用いて50mLの培養上清中の抗体と結合させ、カラムをPBSで洗浄後、グリシン緩衝液（pH3.0）で溶出し、1 mLずつフラクションを採取した。中和後、A280を測定し、抗体を含むフラクションを回収し、PBSにて透析した。透析後濃縮し、BCA法試薬（Thermo Fisher Scientific）で抗体濃度を測定した。組換えヒトBDNFを用いたELISAにて精製抗体力価を確認し、精製抗体を得た。
(8) Purification of antibodies Hybridomas were frozen using a cell freezing medium (Cellbanker, Zenoac) and a programmable freezer, and stored in a liquid nitrogen tank. The frozen hybridomas were immersed in a 37°C incubator and quickly thawed. Under incubation conditions of 5% CO2 , the hybridomas were expanded using a medium containing 9% bovine serum. After expansion, the medium was replaced with 50 mL of serum-free medium (Hybridoma-SFM, GIBCO) under the same incubation conditions and cultured. Seven days after the start of serum-free culture, the culture supernatant was collected, centrifuged, and filtered to obtain the culture supernatant for protein purification. The antibody in 50 mL of culture supernatant was bound to a 1.5 mL protein G-sepharose (GE healthcare) column, the column was washed with PBS, and eluted with glycine buffer (pH 3.0), and 1 mL fractions were collected. After neutralization, A280 was measured, and the fractions containing the antibody were collected and dialyzed against PBS. After dialysis, the solution was concentrated and the antibody concentration was measured using a BCA reagent (Thermo Fisher Scientific). The titer of the purified antibody was confirmed by ELISA using recombinant human BDNF, and purified antibodies were obtained.
（実施例２：2D7および3C8抗体のBDNF反応性の評価）
目的
マウス由来抗ヒトBDNFモノクローナル抗体2D7および3C8のBDNFに対する結合能を表面プラズモン共鳴分析で検証し、BDNF抗体としての評価を行った。
(Example 2: Evaluation of BDNF reactivity of 2D7 and 3C8 antibodies)
the purpose
The binding ability of mouse-derived anti-human BDNF monoclonal antibodies 2D7 and 3C8 to BDNF was examined by surface plasmon resonance analysis, and they were evaluated as BDNF antibodies.
実験
表面プラズモン共鳴分析に用いるセンサーチップCM5はセンサーチップ表面がカルボキシル基で修飾されている。そのためリガンドの固定化はリガンド表面のアミノ基を利用したアミンカップリング法を行った。具体的には、2D7および3C8の抗体溶液（20μg/ml）を10mM Acetate (pH4.7) で10倍希釈することで正に荷電させ、センサーチップCM5にGE社のAmine Coupling kitを用いたアミンカップリング法により結合させた。
結合実験の準備として、HBS-EP buffer（10mM HEPES, 0.15M NaCl, 3 mM EDTA, 0.005 % Surfactant P 20 ( pH 7.4 )）に溶解した組換えBDNF(15nM)を調整し、以下の測定パラメーターの条件により結合実験を行った。
＜測定パラメータ＞
Temperature: 20℃
Flow: 20 μg/min
Detection volume: 120 μL
Dissociation time: 600 sec
センサーチップ上の2D7または3C8抗体とBDNFとの分子間相互をセンサーグラムという形で観測した。BIACOREでは一定流速でランニングバッファーをセンサーチップ表面に流し、ここにアナライト（本実施例ではBDNF）を15nMの濃度でロードすると、センサーチップ上の2D7または3C8との結合を経時的に計測したセンサーグラムを取得できる。その後、アナライトの添加が終了するとHBS-EP bufferに切り替わり、このときセンサーチップ内のアナライト濃度は瞬時にゼロになり、2D7または3C8からの解離過程が観測される。
The sensor chip CM5 used in the experimental surface plasmon resonance analysis has a carboxyl group modified sensor chip surface. Therefore, the ligand was immobilized by the amine coupling method using the amino group on the ligand surface. Specifically, the 2D7 and 3C8 antibody solutions (20 μg/ml) were diluted 10-fold with 10 mM acetate (pH 4.7) to make them positively charged, and were bound to the sensor chip CM5 by the amine coupling method using GE's Amine Coupling kit.
In preparation for the binding experiment, recombinant BDNF (15 nM) was dissolved in HBS-EP buffer (10 mM HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P 20 (pH 7.4)) and the binding experiment was performed under the following measurement parameters.
<Measurement parameters>
Temperature: 20℃
Flow: 20 μg/min
Detection volume: 120 μL
Dissociation time: 600 seconds
The intermolecular interaction between the 2D7 or 3C8 antibody and BDNF on the sensor chip was observed in the form of a sensorgram. In the BIACORE, a running buffer was flowed over the surface of the sensor chip at a constant flow rate, and an analyte (BDNF in this example) was loaded onto the running buffer at a concentration of 15 nM, whereby a sensorgram was obtained that measured the binding with 2D7 or 3C8 on the sensor chip over time. After that, when the addition of the analyte was completed, the buffer was switched to HBS-EP buffer, at which point the analyte concentration in the sensor chip instantly became zero, and the dissociation process from 2D7 or 3C8 was observed.
2D7抗体の結果を図２に、3C8抗体の結果を図３に示す。2D7および3C8抗体ともに、BDNFとの結合が確認され、また、BDNFの添加終了後もBDNFの結合が維持されていることが確認された。この結果から、2D7および3C8抗体はいずれもBDNFに対して強い結合能を有する抗体であることが示していた。 The results for the 2D7 antibody are shown in Figure 2, and the results for the 3C8 antibody are shown in Figure 3. Both the 2D7 and 3C8 antibodies were confirmed to bind to BDNF, and it was confirmed that the binding to BDNF was maintained even after the addition of BDNF was discontinued. These results indicate that both the 2D7 and 3C8 antibodies have strong binding ability to BDNF.
（実施例３：2D7および3C8抗体のエピトープの決定）
目的
得られた2D7および3C8抗体について、BDNFのアミノ酸配列中のエピトープを同定するために、成熟型BDNF由来のペプチド断片hBD-m1、hBD-m2およびhBD-m3を調製し、各断片と2D7および3C8抗体との結合反応性をウェスタンブロッティング法により確認した。
実験１：hBD-m1, hBD-m2およびhBD-m3発現プラスミドの構築（図４、図５）
ヒトpreproBDNF（配列番号１）の129～176番目のアミノ酸からなる断片（hBDNF-m1 (129-176)断片、図中「hBD-m1」と表記）のN末端にチオレドトキシンタグ（TRX）、Hisタグおよび切断配列（PCS）をN末端に融合させたペプチドをコードするDNA配列をpET32b(+)発現ベクターに組み込んだpET32b(+)-hBDNF-m1 (129-176)を、以下の手順により作製した。
ヒトBDNF断片は、pcDNA-hBDNF-EGFP（Journal of Neuroscience Research, 64: 1-10 (2001)）を鋳型とし、下記の合成PCRプライマーによるPCR反応により調製した。
5’-CACTCTGACCCTGCCCGC-3’ （配列番号５）
5’ TTGGCCTTTTGATACAGGGACC-3’ （配列番号６）
得られた増幅産物をpCR2.1-TOPO TAベクター（Thermo Fisher Scientific）に導入し、プラスミドpCR2.1-TOPO TA- hBDNF -m1(129-176)を構築した。さらにpET-32b(+)ベクター（Novagen社製）に移し替えることにより、pET32b(+)-hBDNF (129-176)を得た。
Example 3: Determination of epitopes of 2D7 and 3C8 antibodies
the purpose
In order to identify the epitopes in the amino acid sequence of BDNF for the obtained 2D7 and 3C8 antibodies, peptide fragments derived from mature BDNF, hBD-m1, hBD-m2, and hBD-m3, were prepared and the binding reactivity of each fragment with the 2D7 and 3C8 antibodies was confirmed by Western blotting.
Experiment 1: Construction of hBD-m1, hBD-m2 and hBD-m3 expression plasmids (Figures 4 and 5)
A DNA sequence encoding a peptide in which a thioredotoxin tag (TRX), a His tag, and a cleavage sequence (PCS) were fused to the N-terminus of a fragment consisting of amino acids 129 to 176 of human preproBDNF (SEQ ID NO: 1) (hBDNF-m1 (129-176) fragment, indicated as "hBD-m1" in the figure) was inserted into the pET32b(+) expression vector to prepare pET32b(+)-hBDNF-m1 (129-176) by the following procedure.
The human BDNF fragment was prepared by PCR using pcDNA-hBDNF-EGFP (Journal of Neuroscience Research, 64: 1-10 (2001)) as a template and the following synthetic PCR primers:
5'-CACTCTGACCCTGCCCGC-3' (SEQ ID NO:5)
5'TTGGCCTTTTGATACAGGGACC-3' (SEQ ID NO: 6)
The resulting amplified product was introduced into the pCR2.1-TOPO TA vector (Thermo Fisher Scientific) to construct the plasmid pCR2.1-TOPO TA-hBDNF-m1(129-176), which was then transferred into the pET-32b(+) vector (Novagen) to obtain pET32b(+)-hBDNF (129-176).
以下の合成プライマーを用いた以外は同様の手順により、hBDNF-m2 (165-213)断片（図中「hBD-m2」と表記）を発現するプラスミド（pET32b(+)-hBDNF (165-213)）を作製した。
5’-ACAGTCCTTGAAAAGGTCCCT-3’ （配列番号７）
5’-GTACGACTGGGTAGTTCGGCA-3’ （配列番号８）
A plasmid (pET32b(+)-hBDNF (165-213)) expressing the hBDNF-m2 (165-213) fragment (represented as "hBD-m2" in the figure) was prepared by the same procedure except that the following synthetic primers were used.
5'-ACAGTCCTTGAAAAGGTCCCT-3' (SEQ ID NO:7)
5'-GTACGACTGGGTAGTTCGGCA-3' (SEQ ID NO: 8)
以下の合成プライマーを用いた以外は同様の手順により、hBDNF-m3 (203-247)断片（図中「hBD-m3」と表記）を発現するプラスミド（pET32b(+)-hBDNF (203-247)）を作製した。
5’-CATTGGAACTC CCAGTGCCG-3’ （配列番号９）
5’-CTATCTTCCCCTTTTAATGGTCAA-3’ （配列番号１０）
A plasmid (pET32b(+)-hBDNF (203-247)) expressing the hBDNF-m3 (203-247) fragment (represented as "hBD-m3" in the figure) was prepared by the same procedure except that the following synthetic primers were used.
5'-CATTGGAACTCCAGTGCCG-3' (SEQ ID NO: 9)
5'-CTATCTTCCCCTTTTAATGGTCAA-3' (SEQ ID NO: 10)
実験２：エピトープの同定実験
１）細胞抽出液の調製
上記実験１で構築した各プラスミドを大腸菌株RosettaTM 2(DE3) (Novagen社)に導入した。得られたシングルコロニーをLB培地で振とう培養し、終濃度0.1M IPTGの添加後4時間追培養した。遠心分離によって回収した大腸菌をサンプルバッファー（2X; 125mM Tris 10% (v/v)メルカプトエタノール 4%(w/v)SDS, 30%(v/v)グルセロール 0.005%(g/v)ブロモフェノールブルー）で懸濁して細胞抽出液を作製した。また、ネガティブコントロールとしてIPTG無添加の細胞抽出液を調整した。
Experiment 2: Identification of epitopes Experiment 1) Preparation of cell extract Each plasmid constructed in Experiment 1 above was introduced into Escherichia coli strain Rosetta TM 2(DE3) (Novagen). The obtained single colonies were cultured with shaking in LB medium, and further cultured for 4 hours after adding IPTG at a final concentration of 0.1M. E. coli collected by centrifugation was suspended in sample buffer (2X; 125mM Tris, 10% (v/v) mercaptoethanol, 4% (w/v) SDS, 30% (v/v) glycerol, 0.005% (g/v) bromophenol blue) to prepare a cell extract. In addition, a cell extract without the addition of IPTG was prepared as a negative control.
２）ウェスタンブロッティング解析
各細胞抽出液をウェスタンブロッティング法で解析した。各細胞抽出液をSDS-PAGE後（ゲル濃度:5-20%）、ブロットしたメンブレンと抗BDNF抗体2D7または3C8それぞれを用いて4℃で一晩3% BSA-TBSTバッファー（3% BSA、20mM Tris、322mM NaCl、0.1% Tween-20、pH7.4）中で振とうし、抗原抗体反応を行った。二次抗体としてHRP標識された抗マウス抗体（Millipore, AB3517）を用いて室温1時間反応後、発光シグナルを検出した。IPTGによる融合タンパク質の発現誘導を確認するために抗マウスHis-tag抗体を用いたウェスタンブロッティングも行った。
2) Western blotting analysis Each cell extract was analyzed by Western blotting. After SDS-PAGE (gel concentration: 5-20%), the blotted membrane and anti-BDNF antibody 2D7 or 3C8 were shaken overnight at 4°C in 3% BSA-TBST buffer (3% BSA, 20 mM Tris, 322 mM NaCl, 0.1% Tween-20, pH 7.4) to perform an antigen-antibody reaction. After 1 hour of reaction at room temperature using HRP-labeled anti-mouse antibody (Millipore, AB3517) as the secondary antibody, luminescence signals were detected. Western blotting was also performed using anti-mouse His-tag antibody to confirm the expression induction of the fusion protein by IPTG.
３）結果（図６）
・2D7のエピトープ解析
2D7抗体は、配列番号１のアミノ酸配列における129から176番目のアミノ酸から構成されるhBD-m1断片（配列番号２）と結合することが確認された。一方、配列番号１のアミノ酸配列における165から213番目のアミノ酸から構成されるhBD-m2断片（配列番号３）および配列番号１のアミノ酸配列における203から247番目のアミノ酸から構成されるhBD-m3断片（配列番号４）との結合は見られなかった。つまり、マウスモノクローナル抗BDNF抗体2D7のエピトープは、BDNFタンパク質のアミノ酸配列の129番目から176番目の領域内に存在することが判明した。
3) Results (Figure 6)
・Epitope analysis of 2D7
It was confirmed that the 2D7 antibody binds to the hBD-m1 fragment (SEQ ID NO: 2) consisting of amino acids 129 to 176 in the amino acid sequence of SEQ ID NO: 1. On the other hand, no binding was observed to the hBD-m2 fragment (SEQ ID NO: 3) consisting of amino acids 165 to 213 in the amino acid sequence of SEQ ID NO: 1 and the hBD-m3 fragment (SEQ ID NO: 4) consisting of amino acids 203 to 247 in the amino acid sequence of SEQ ID NO: 1. In other words, it was found that the epitope of the mouse monoclonal anti-BDNF antibody 2D7 exists within the region from amino acids 129 to 176 in the amino acid sequence of BDNF protein.
・3C8のエピトープ解析
3C8抗体は、配列番号１のアミノ酸配列における129から176番目のアミノ酸から構成されるhBD-m1断片（配列番号２）および配列番号１のアミノ酸配列における165から213番目のアミノ酸から構成されるhBD-m2断片（配列番号３）と結合することが確認された。一方、配列番号１のアミノ酸配列における203から247番目のアミノ酸から構成されるhBD-m3断片（配列番号４）との結合は見られなかった。つまり、マウスモノクローナル抗BDNF抗体3C8のエピトープは、BDNFタンパク質のアミノ酸配列の165番目から176番目の領域内に存在することが判明した。
・Epitope analysis of 3C8
It was confirmed that the 3C8 antibody binds to the hBD-m1 fragment (SEQ ID NO: 2) consisting of amino acids 129 to 176 in the amino acid sequence of SEQ ID NO: 1, and the hBD-m2 fragment (SEQ ID NO: 3) consisting of amino acids 165 to 213 in the amino acid sequence of SEQ ID NO: 1. On the other hand, no binding was observed to the hBD-m3 fragment (SEQ ID NO: 4) consisting of amino acids 203 to 247 in the amino acid sequence of SEQ ID NO: 1. In other words, it was found that the epitope of the mouse monoclonal anti-BDNF antibody 3C8 exists within the region from amino acids 165 to 176 in the amino acid sequence of BDNF protein.
（実施例４：免疫沈降（図７））
目的
生物学的活性やそのための構造を有するタンパク質を認識する抗体は有用である。このような用途への2D7および3C8抗体の適用可能性を検証すべく、免疫沈降を行い、その沈降物についてウェスタンブロッティング解析を行った。
実験
以下の参考文献の手順により、BDNF遺伝子欠損マウスその同腹の野生型マウス（BDNF遺伝子を両アレルに持つ）の各2匹の海馬組織のライセートと、2D7または3C8のモノクローナル抗体を一晩回転混合し、翌日、ProteinGとの回転混合をさらに行うことにより、モノクローナル抗体への結合分子の回収を行った。結合分子の解析のためにはウェスタンブロッティング法を用い、BDNFの検出には抗BDNFマウス由来市販抗体(#3C11, Icosagen)を用いた。
参考文献：
S. Dieni, T. Matsumoto, M. Dekkers, S. Rauskolb, M. Ionescu, R. Deogracias, E. D.Gundelfinger, M. Kojima, S. Nestel, M. Frotscher, Y.-A. Barde, BDNF and its pro-peptide are stored in presynaptic dense core vesicles in brain neurons, Journal of Cell Biology, 2012, vol. 196, no. 6, 775-788.
(Example 4: Immunoprecipitation (Figure 7))
the purpose
Antibodies that recognize proteins with biological activity or structures that support such activity are useful. To verify the applicability of the 2D7 and 3C8 antibodies to such applications, we performed immunoprecipitation and Western blotting analysis of the precipitates.
experiment
According to the procedure described in the following reference, hippocampal tissue lysates from two mice each of a BDNF gene-deficient mouse and its littermate wild-type mouse (which has both alleles of the BDNF gene) were mixed with 2D7 or 3C8 monoclonal antibodies overnight by rotation, and the next day, further mixed with Protein G by rotation to recover the molecules bound to the monoclonal antibodies. Western blotting was used to analyze the bound molecules, and a commercially available anti-BDNF mouse antibody (#3C11, Icosagen) was used to detect BDNF.
References:
S. Dieni, T. Matsumoto, M. Dekkers, S. Rauskolb, M. Ionescu, R. Deogracias, ED Gundelfinger, M. Kojima, S. Nestel, M. Frotscher, Y.-A. Barde, BDNF and its pro-peptide are stored in presynaptic dense core vesicles in brain neurons, Journal of Cell Biology, 2012, vol. 196, no. 6, 775-788.
結果
2D7、3C8ともに、野生型マウスの脳海馬組織ライセートからはBDNFを回収することができ、BDNFを示すバンドが検出された（図７中「Wild」）。一方で、BDNF遺伝子欠損マウスの脳海馬組織ライセートからはBDNFを示すバンドは検出されなかった（図７中「KO」）。この結果から、2D7および3C8抗体はいずれもBDNFに対して高い特異的結合性を有することが示された。ここで、ライセートは低濃度Tween-20を加えた溶解バッファーを用いて調製されたものであり、BDNFは生体内での立体構造を保持しているものと考えられる。つまり、この結果からは、2D7および3C8抗体が脳組織内や体液中で活性型として存在する成熟型BDNFと結合できることが示唆され、2D7および3C8抗体がELISAのような検出系に使用できることが期待された。
result
Both 2D7 and 3C8 were able to recover BDNF from the brain hippocampal tissue lysate of wild-type mice, and a band indicating BDNF was detected ("Wild" in Figure 7). On the other hand, no band indicating BDNF was detected from the brain hippocampal tissue lysate of BDNF gene-deficient mice ("KO" in Figure 7). This result indicates that both 2D7 and 3C8 antibodies have high specific binding affinity to BDNF. Here, the lysate was prepared using a lysis buffer containing low concentration Tween-20, and BDNF is considered to retain its three-dimensional structure in vivo. In other words, this result suggests that 2D7 and 3C8 antibodies can bind to mature BDNF that exists as an active form in brain tissue and body fluids, and it is expected that 2D7 and 3C8 antibodies can be used in detection systems such as ELISA.
（実施例５：2D7および3C8抗体のELISAへの適用）
目的
BDNFが脳だけでなく末梢血にも含まれることや、末梢血でも神経系に類似したメカニズムによりBDNFが産生されることが報告されており（P Chacon-Fernandez et al., Journal of Biological Chemistry (2016) 291: 9872-9881）、血中BDNFの診断系への応用が期待されている。そこで本実施例では、ELISAに2D7および3C8抗体を適用できるかどうかを検証した。
(Example 5: Application of 2D7 and 3C8 antibodies to ELISA)
the purpose
It has been reported that BDNF is found not only in the brain but also in peripheral blood, and that BDNF is produced in peripheral blood by a mechanism similar to that in the nervous system (P Chacon-Fernandez et al., Journal of Biological Chemistry (2016) 291: 9872-9881), and it is expected that BDNF in blood can be applied to diagnostic systems. Therefore, in this example, we examined whether the 2D7 and 3C8 antibodies can be applied to ELISA.
実験
組換えBDNFの希釈系列を作り、一次抗体として2D7抗体（1:1000希釈）、二次抗体としてBiotin Labeling Kit - NH2（DOJINDO, LK03）を用いてビオチン標識した3C8抗体（1:500希釈）を用いた以外はPromega社のBDNF ELISA kit（BDNF Emax ImmunoAssay Systems）に添付されている試薬（抗体は除く）とプロトコールにしたがってサンドイッチELISA解析を行った。
最初にCarbonate coating buffer（0.025 M Sodium bicarbonate, 0.025 M Sodium carbonate, pH 9.7）を用いて1 μg/mLの2D7抗体液を準備し、ELISA用96穴プレートに100 μLずつ添加して4℃で一晩静置した。
翌日、100μL TBSTで各ウェルを1回洗浄後、各ウェルに200μL のBlock & Sample 1X Buffer（Promega社 BDNF Emax（登録商標）ImmunoAssay System）を加え、１時間ブロッキング反応を行った。その後、100μL TBSTを用いて各ウェルを1回洗浄した。ヒトBDNF（Promega社 BDNF Emax（登録商標）ImmunoAssay System, G7610）の希釈系列（250 pg/mL, 125 pg/mL, 62.5 pg/mL, 31.25 pg/mL, 15.625 pg/mL, 7.813 pg/mL）およびBDNFを含まない溶液をBlock & Sample 1X Buffer で調整し、各濃度につき100 μLを２ウェルずつ準備した。このプレートを２時間室温で振盪することにより、当該マウスモノクローナル抗体とBDNFとの結合反応を行った。反応が終わったプレートについて、各ウェルにつき100μL TBSTによる洗浄を５回行った。ELISAの二次抗体反応は、3C8抗体200 ngをBiotin Labeling Kit - NH2（DOJINDO, LK03）の添付プロトコールに従ってビオチン標識したものをBlock & Sample 1X Buffer で1/500倍希釈したものを、ウェルあたり100μL添加し1時間室温で振盪することにより行った。続いて100μL TBSTで各ウェルを 5回洗浄した。続いて、TMB One Solution（プロメガ社）をウェルあたり100μL加え発色反応を行った。発色反応の停止は1N HCl溶液100μLの添加により行い、その後、450 nmの吸光度に基づいて発色を決定した。
A dilution series of the experimental recombinant BDNF was prepared, and sandwich ELISA analysis was performed using the reagents (excluding antibodies) and protocol provided with the Promega BDNF ELISA kit (BDNF Emax ImmunoAssay Systems), except that the 2D7 antibody (1:1000 dilution) was used as the primary antibody, and the 3C8 antibody (1:500 dilution) biotin-labeled with Biotin Labeling Kit - NH2 (DOJINDO, LK03) was used as the secondary antibody.
First, a 1 μg/mL solution of 2D7 antibody was prepared using a carbonate coating buffer (0.025 M sodium bicarbonate, 0.025 M sodium carbonate, pH 9.7), and 100 μL of the solution was added to a 96-well ELISA plate and allowed to stand overnight at 4°C.
The next day, each well was washed once with 100 μL TBST, and then 200 μL of Block & Sample 1X Buffer (Promega BDNF Emax® ImmunoAssay System) was added to each well, and blocking reaction was performed for 1 hour. Then, each well was washed once with 100 μL TBST. A dilution series of human BDNF (Promega BDNF Emax® ImmunoAssay System, G7610) (250 pg/mL, 125 pg/mL, 62.5 pg/mL, 31.25 pg/mL, 15.625 pg/mL, 7.813 pg/mL) and a solution without BDNF were prepared with Block & Sample 1X Buffer, and 100 μL was prepared for each concentration in two wells. The plate was shaken at room temperature for 2 hours to perform a binding reaction between the mouse monoclonal antibody and BDNF. After the reaction, each well of the plate was washed five times with 100 μL TBST. The secondary antibody reaction of the ELISA was performed by adding 100 μL of 1/500-fold diluted 3C8 antibody (200 ng) that was biotinylated according to the protocol attached to the Biotin Labeling Kit - NH2 (DOJINDO, LK03) with Block & Sample 1X Buffer per well and shaking for 1 hour at room temperature. Each well was then washed five times with 100 μL TBST. Then, 100 μL of TMB One Solution (Promega) was added per well to carry out the color reaction. The color reaction was stopped by adding 100 μL of 1N HCl solution, and the color development was determined based on the absorbance at 450 nm.
結果（図８）
上記サンドイッチELISAの結果、ヒトBDNF組換えタンパク質の濃度依存的な検量線が得られた（図８Ａ）。一方、ヒトBDNF組換えタンパク質に代えて、BDNFのファミリータンパク質であるNGF、NT-3およびNT-4（各1ng/mL、ヒト配列を有する）について行った同サンドイッチELISAの結果、結合は検出されなかった（図８Ｂ）。さらに、ヒトBDNF組換えタンパク質に代えて、実施例４と同様の手順により調製されたBDNFノックアウトマウス（同腹１週齢）および野生型マウスの海馬組織ライセートを同サンドイッチELISAにより解析した結果、野生型マウスの脳海馬組織ライセートに含まれるBDNFの濃度を決定できたが、同腹のノックアウトマウスの脳海馬組織ライセートではBDNFは検出感度以下であった（図８Ｂ）。これらの結果から、2D7および3C8抗体がBDNFに対して特異的結合性を有し、BDNFの検出および／または定量に利用可能なものであることが示された。
Results (Figure 8)
As a result of the above sandwich ELISA, a concentration-dependent calibration curve of human BDNF recombinant protein was obtained (FIG. 8A). On the other hand, when the same sandwich ELISA was performed using BDNF family proteins NGF, NT-3 and NT-4 (each 1 ng/mL, having a human sequence) instead of human BDNF recombinant protein, no binding was detected (FIG. 8B). Furthermore, instead of human BDNF recombinant protein, hippocampal tissue lysates of BDNF knockout mice (1 week old littermates) and wild-type mice prepared by the same procedure as in Example 4 were analyzed by the same sandwich ELISA. As a result, the concentration of BDNF in the brain-hippocampal tissue lysates of wild-type mice could be determined, but BDNF was below the detection limit in the brain-hippocampal tissue lysates of the knockout mice of the same littermates (FIG. 8B). These results demonstrated that the 2D7 and 3C8 antibodies have specific binding properties to BDNF and can be used to detect and/or quantify BDNF.
（実施例６：2D7および3C8抗体のproBDNFに対する反応性の評価）
目的
2D7および3C8抗体がBDNF前駆体に対しても特異的結合性を有するかどうかを確認するために、組換えproBDNFの希釈系列を用いてサンドイッチELISA解析を行った。
(Example 6: Evaluation of reactivity of 2D7 and 3C8 antibodies to proBDNF)
the purpose
To confirm whether the 2D7 and 3C8 antibodies also have specific binding to the BDNF precursor, sandwich ELISA analysis was performed using a dilution series of recombinant proBDNF.
実験
ヒトBDNF（Promega社 BDNF Emax（登録商標）ImmunoAssay System, G7610）に代えてヒトproBDNF（Alomone Labs, #B-256）を用いた以外は、実施例５と同様の手順によりサンドイッチELISA解析を行った。
Experimental Sandwich ELISA analysis was carried out in the same manner as in Example 5, except that human proBDNF (Alomone Labs, #B-256) was used instead of human BDNF (Promega BDNF Emax (registered trademark) ImmunoAssay System, G7610).
結果（図９）
上記サンドイッチELISAの結果、ヒトproBDNF組換えタンパク質の濃度依存的な検量線が得られた（図９）。この結果から、2D7および3C8抗体がBDNFと同様にproBDNFに対しても特異的結合性を有し、proBDNFの検出および／または定量に利用可能なものであることが示された。
Results (Figure 9)
As a result of the above sandwich ELISA, a concentration-dependent standard curve of human proBDNF recombinant protein was obtained (Figure 9). This result demonstrated that the 2D7 and 3C8 antibodies have specific binding affinity to proBDNF as well as BDNF and can be used to detect and/or quantify proBDNF.
（実施例７：2D7および3C8抗体の機能性の評価）
目的
2D7および3C8抗体がBDNFの作用機能を阻害または亢進するような機能性を有するかどうかを調べるために、2D7および3C8抗体の存在下または非存在下でBDNF/TrkBシグナル経路を刺激した場合のTrkBのリン酸化状態をウェスタンブロッティングにより解析した。
Example 7: Evaluation of the functionality of 2D7 and 3C8 antibodies
the purpose
To examine whether the 2D7 and 3C8 antibodies have the functionality to inhibit or enhance the action function of BDNF, the phosphorylation state of TrkB when the BDNF/TrkB signaling pathway was stimulated in the presence or absence of the 2D7 and 3C8 antibodies was analyzed by Western blotting.
実験
既報文献（Suzuki S, et al., "Brain-derived neurotrophic factor regulates cholesterol metabolism for synapse development", J. Neurosci., 2007, 27(24):6417-27）に記載の手順に従って、胎生18日齢ラット由来大脳皮質神経細胞の初代培養（5x105細胞/cm2）を調製し、2%（vol/vol）のB27サプリメントを含有する Neurobasal medium（Thermo Fisher Scientific）中で培養した。培養14日目にNeurobasal mediumに培地を交換し、その翌日にヒトBDNF組換えタンパク質（10ng/ml）および／または2D7もしくは3C8抗体（3.6μg/ml）を投与し、30分後に細胞を回収し、ライセートを調製した。なお、BDNFおよび抗体のいずれも投与しなかったものを陰性対照とした。
得られたライセートを、上記鈴木らの報文に記載の手順に従って、ウェスタンブロッティングにより解析した。簡単には、各ライセートから調製したサンプル（5μgタンパク質／レーン）をSDS-PAGEに供し、PVDF膜に転写後、抗リン酸化Trk-B抗体（C35G9, Cell Signaling Technology）または抗TrkB抗体（80E3, Cell Signaling Technology）でブロットした。
Primary cultures of cerebral cortical neurons (5x105 cells/cm2) from 18-day-old rats were prepared according to the procedure described in a previous paper (Suzuki S, et al., "Brain - derived neurotrophic factor regulates cholesterol metabolism for synapse development", J. Neurosci., 2007, 27(24): 6417-27 ) and cultured in Neurobasal medium (Thermo Fisher Scientific) containing 2% (vol/vol) B27 supplement. The medium was replaced with Neurobasal medium on the 14th day of culture, and human BDNF recombinant protein (10ng/ml) and/or 2D7 or 3C8 antibody (3.6μg/ml) were administered on the following day. Cells were harvested 30 minutes later and lysates were prepared. Negative controls were treated with neither BDNF nor antibody.
The obtained lysates were analyzed by Western blotting according to the procedure described in the above paper by Suzuki et al. Briefly, samples (5 μg protein/lane) prepared from each lysate were subjected to SDS-PAGE, transferred to a PVDF membrane, and blotted with anti-phosphorylated Trk-B antibody (C35G9, Cell Signaling Technology) or anti-TrkB antibody (80E3, Cell Signaling Technology).
結果（図１０）
図１０の左パネルはリン酸化Trk-Bの検出結果を示し、BDNFと2D7または3C8抗体とを共投与した場合に、BDNFのみを投与した場合に比べてリン酸化Trk-Bの検出バンドが薄くなっており、リン酸化Trk-Bが減少していることが確認された（図１０左パネル、丸印）。また、図１０の右パネルはTrk-Bの検出結果を示し、BDNFのみを投与した場合には、Trk-Bがリン酸化され、分子量の増加によりTrk-Bの検出バンドが上方向にシフトするが、BDNFと2D7または3C8抗体とを共投与した場合には、上方向へのシフトが若干減少していることが確認された（図１０右パネル、＊印）。以上の結果から、2D7および3C8抗体はいずれもBDNFの機能を抑制できるものである可能性が示された。
Results (Figure 10)
The left panel of Figure 10 shows the results of detecting phosphorylated Trk-B. When BDNF and 2D7 or 3C8 antibodies were co-administered, the detection band of phosphorylated Trk-B was lighter than when BDNF alone was administered, confirming the reduction of phosphorylated Trk-B (Figure 10, left panel, circle). The right panel of Figure 10 shows the results of detecting Trk-B. When BDNF alone was administered, Trk-B was phosphorylated, and the detection band of Trk-B shifted upward due to the increase in molecular weight, but when BDNF and 2D7 or 3C8 antibodies were co-administered, the upward shift was slightly reduced (Figure 10, right panel, *). These results suggest that both 2D7 and 3C8 antibodies may be able to suppress the function of BDNF.
Claims (7)
請求項１または２に記載のモノクローナル抗体またはその抗原結合断片と前記試料とを反応させる工程と
前記モノクローナル抗体またはその抗原結合断片と結合した成熟型BDNFおよびその前駆体を検出する工程と
を含む方法。 1. A method for detecting mature BDNF and its precursors in a sample, comprising:
A method comprising the steps of reacting the monoclonal antibody or its antigen-binding fragment described in claim 1 or 2 with the sample, and detecting mature BDNF and its precursor bound to the monoclonal antibody or its antigen-binding fragment.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2019031623 | 2019-02-25 | ||
| JP2019031623 | 2019-02-25 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2020138963A JP2020138963A (en) | 2020-09-03 |
| JP7481733B2 true JP7481733B2 (en) | 2024-05-13 |
Family
ID=72279894
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2020029569A Active JP7481733B2 (en) | 2019-02-25 | 2020-02-25 | Monoclonal antibody against brain-derived neurotrophic factor and hybridoma producing the antibody |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP7481733B2 (en) |
-
2020
- 2020-02-25 JP JP2020029569A patent/JP7481733B2/en active Active
Non-Patent Citations (3)
| Title |
|---|
| AB1534SP Sigma-Aldrich Anti-Brain Derived Neurotrophic Factor Antibody，MERCK, ホーム > Life Science Research > Antibodies and Assays > Primary Antibodies > Anti-Brain Derived Neurotrophic Factor Antibody，2023年，pp.1-3 |
| RABBIT ANTI-BRAIN DERIVED NEUROTROPHIC FACTOR (IgG Fraction) POLYCLONAL ANTIBODY，CATALOG NO: AB1534SP, MILLIPORE，2011年09月13日，pp.1-3 |
| The Journal of Histochemistry and Cytochemistry，1993年，Vol.41, No.4，pp.521-533 |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2020138963A (en) | 2020-09-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP5439176B2 (en) | Antibodies that specifically bind to TDP-43 aggregates | |
| RU2657517C2 (en) | Adrenomedullin assay and methods for determining mature adrenomedullin | |
| RU2521669C2 (en) | Method of human protein cxcl1 immunoassay | |
| WO1994004563A1 (en) | PEPTIDES CONTAINING RESPECTIVE AMINO ACID SEQUENCES SELECTED FROM AMONG THOSE OF LIPOPROTEIN(a) AND APOLIPOPROTEIN(a), ANTIBODIES RESPECTIVELY RECOGNIZING THESE AMINO ACID SEQUENCES, AND METHOD OF ASSAYING WITH THESE ANTIBODIES | |
| RU2769568C2 (en) | Rep protein as protein antigen for diagnostic analyzes | |
| US9347954B2 (en) | Antibody capable of binding to specific region of periostin, and method of measuring periostin using the same | |
| JP5002001B2 (en) | Method for measuring insulin receptor α-subunit | |
| US20230032690A1 (en) | New tau species | |
| WO2007022015A1 (en) | Methods to evaluate amyloid beta-lowering agents using wild-type mice | |
| KR102245938B1 (en) | Improved REP protein for use in diagnostic assays | |
| KR102628144B1 (en) | Antibodies to human erythroferon and uses thereof | |
| CN105548561B (en) | Diagnostic agent and diagnostic method for alzheimer's disease | |
| JP7481733B2 (en) | Monoclonal antibody against brain-derived neurotrophic factor and hybridoma producing the antibody | |
| CN113508300A (en) | Novel biomarkers for human alzheimer's disease | |
| JP6742590B2 (en) | Antibody or antigen-binding fragment thereof and hybridoma | |
| US11174310B2 (en) | Disulfide-type HMGB1-specific antibody, method for measuring disulfide-type HMGB1 and kit for said measurement, and measurement method capable of quantitating all of HMGB1 molecules including reduced HMGB1, disulfide-type HMGB1 and thrombin-cleavable HMGB1 and kit for said measurement | |
| JP2010536715A (en) | Nesfatin-1 specific antibody and use thereof, and Nesfatin specific antibody and use thereof | |
| JP7051096B2 (en) | Antibodies that specifically recognize bovine procalcitonin, their antigen-binding fragments, and their use. | |
| US9017957B2 (en) | Prostasin partial peptide and anti-prostasin antibody | |
| US20120282633A1 (en) | Antibody specific to activated egfr | |
| WO2018194134A1 (en) | Anti-periostin antibody-immobilized carrier, periostin measurement reagent, and method for stabilizing anti-periostin antibody in carrier having said antibody immobilzed thereon | |
| US7141435B2 (en) | Monoclonal antibodies against human apoptosis inhibitory protein NAIP and method for assaying the NAIP | |
| JP2002171970A (en) | ANTI-Wnt-3a MONOCLONAL ANTIBODY | |
| JP2005314397A (en) | Anti-chondromodulin-1-specific antibody and its use | |
| JP2012141175A (en) | Method for detecting cancer |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| AA64 | Notification of invalidation of claim of internal priority (with term) |
Free format text: JAPANESE INTERMEDIATE CODE: A241764 Effective date: 20200313 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20200313 |
|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20221201 |
|
| A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20231025 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20231027 |
|
| A601 | Written request for extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A601 Effective date: 20231220 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20240219 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20240416 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20240419 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 7481733 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |