JPH04252954A - Measuring method, reagent and kit for protein - Google Patents
Measuring method, reagent and kit for proteinInfo
- Publication number
- JPH04252954A JPH04252954A JP939791A JP939791A JPH04252954A JP H04252954 A JPH04252954 A JP H04252954A JP 939791 A JP939791 A JP 939791A JP 939791 A JP939791 A JP 939791A JP H04252954 A JPH04252954 A JP H04252954A
- Authority
- JP
- Japan
- Prior art keywords
- antibody
- sequence
- kpi
- app
- app751
- 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.)
- Withdrawn
Links
- 238000000034 method Methods 0.000 title claims abstract description 48
- 108090000623 proteins and genes Proteins 0.000 title claims description 29
- 102000004169 proteins and genes Human genes 0.000 title claims description 27
- 239000003153 chemical reaction reagent Substances 0.000 title claims description 19
- 208000024827 Alzheimer disease Diseases 0.000 claims abstract description 19
- 101710137189 Amyloid-beta A4 protein Proteins 0.000 claims abstract description 19
- 101710151993 Amyloid-beta precursor protein Proteins 0.000 claims abstract description 19
- 102100022704 Amyloid-beta precursor protein Human genes 0.000 claims abstract description 19
- DZHSAHHDTRWUTF-SIQRNXPUSA-N amyloid-beta polypeptide 42 Chemical compound C([C@@H](C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](C)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(O)=O)[C@@H](C)CC)C(C)C)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@@H](NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC(O)=O)C(C)C)C(C)C)C1=CC=CC=C1 DZHSAHHDTRWUTF-SIQRNXPUSA-N 0.000 claims abstract description 11
- 239000012634 fragment Substances 0.000 claims description 19
- 239000002243 precursor Substances 0.000 claims description 4
- 239000000427 antigen Substances 0.000 abstract description 30
- 102000036639 antigens Human genes 0.000 abstract description 30
- 108091007433 antigens Proteins 0.000 abstract description 30
- 238000001514 detection method Methods 0.000 abstract description 23
- 238000002965 ELISA Methods 0.000 abstract description 15
- 238000003118 sandwich ELISA Methods 0.000 abstract description 12
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 108091005804 Peptidases Proteins 0.000 abstract description 2
- 239000004365 Protease Substances 0.000 abstract description 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 32
- 239000002953 phosphate buffered saline Substances 0.000 description 31
- 210000004027 cell Anatomy 0.000 description 29
- 208000018910 keratinopathic ichthyosis Diseases 0.000 description 27
- 239000000872 buffer Substances 0.000 description 22
- 102000004190 Enzymes Human genes 0.000 description 19
- 108090000790 Enzymes Proteins 0.000 description 19
- 108020001507 fusion proteins Proteins 0.000 description 19
- 102000037865 fusion proteins Human genes 0.000 description 19
- 239000000523 sample Substances 0.000 description 19
- 239000000758 substrate Substances 0.000 description 19
- 108010005774 beta-Galactosidase Proteins 0.000 description 18
- 238000002360 preparation method Methods 0.000 description 18
- 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 17
- 102000005936 beta-Galactosidase Human genes 0.000 description 17
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 15
- 239000004202 carbamide Substances 0.000 description 15
- 239000013612 plasmid Substances 0.000 description 15
- 150000001413 amino acids Chemical class 0.000 description 14
- 241000588724 Escherichia coli Species 0.000 description 13
- 102000004142 Trypsin Human genes 0.000 description 13
- 108090000631 Trypsin Proteins 0.000 description 13
- 230000003053 immunization Effects 0.000 description 13
- 238000002649 immunization Methods 0.000 description 13
- 239000002244 precipitate Substances 0.000 description 13
- 239000012588 trypsin Substances 0.000 description 13
- 230000002401 inhibitory effect Effects 0.000 description 12
- 108020004707 nucleic acids Proteins 0.000 description 12
- 102000039446 nucleic acids Human genes 0.000 description 12
- 150000007523 nucleic acids Chemical class 0.000 description 12
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 12
- 239000000126 substance Substances 0.000 description 12
- 238000012360 testing method Methods 0.000 description 12
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 11
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 10
- 229940098773 bovine serum albumin Drugs 0.000 description 10
- 210000004408 hybridoma Anatomy 0.000 description 10
- 239000002609 medium Substances 0.000 description 10
- 210000002966 serum Anatomy 0.000 description 10
- 239000007790 solid phase Substances 0.000 description 10
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 9
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 9
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 9
- 208000037259 Amyloid Plaque Diseases 0.000 description 9
- 230000001580 bacterial effect Effects 0.000 description 9
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 9
- 241000283973 Oryctolagus cuniculus Species 0.000 description 8
- 239000002671 adjuvant Substances 0.000 description 8
- 229960002685 biotin Drugs 0.000 description 8
- 235000020958 biotin Nutrition 0.000 description 8
- 239000011616 biotin Substances 0.000 description 8
- 238000005119 centrifugation Methods 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 8
- 238000000746 purification Methods 0.000 description 8
- 108020004414 DNA Proteins 0.000 description 7
- 241000699670 Mus sp. Species 0.000 description 7
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 7
- 239000012228 culture supernatant Substances 0.000 description 7
- 201000010099 disease Diseases 0.000 description 7
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 7
- 238000002372 labelling Methods 0.000 description 7
- 238000003756 stirring Methods 0.000 description 7
- 238000011282 treatment Methods 0.000 description 7
- 238000005406 washing Methods 0.000 description 7
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 6
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 6
- 229960000723 ampicillin Drugs 0.000 description 6
- 238000010828 elution Methods 0.000 description 6
- 238000003127 radioimmunoassay Methods 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- 108090001008 Avidin Proteins 0.000 description 5
- 239000011324 bead Substances 0.000 description 5
- 210000004369 blood Anatomy 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 210000004556 brain Anatomy 0.000 description 5
- 210000004899 c-terminal region Anatomy 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- 238000005259 measurement Methods 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 239000011780 sodium chloride Substances 0.000 description 5
- YUDPTGPSBJVHCN-DZQJYWQESA-N 4-methylumbelliferyl beta-D-galactoside Chemical compound C1=CC=2C(C)=CC(=O)OC=2C=C1O[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O YUDPTGPSBJVHCN-DZQJYWQESA-N 0.000 description 4
- 229920000936 Agarose Polymers 0.000 description 4
- 101100007328 Cocos nucifera COS-1 gene Proteins 0.000 description 4
- 241000701959 Escherichia virus Lambda Species 0.000 description 4
- 108091028043 Nucleic acid sequence Proteins 0.000 description 4
- 229920001213 Polysorbate 20 Polymers 0.000 description 4
- 108010076504 Protein Sorting Signals Proteins 0.000 description 4
- 108010090804 Streptavidin Proteins 0.000 description 4
- 238000003556 assay Methods 0.000 description 4
- 230000027455 binding Effects 0.000 description 4
- 238000010367 cloning Methods 0.000 description 4
- 239000011248 coating agent Substances 0.000 description 4
- 238000004925 denaturation Methods 0.000 description 4
- 230000036425 denaturation Effects 0.000 description 4
- 239000000499 gel Substances 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- -1 polyethylene Polymers 0.000 description 4
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 4
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 4
- 239000012723 sample buffer Substances 0.000 description 4
- 239000012086 standard solution Substances 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 206010003445 Ascites Diseases 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 3
- 241000699666 Mus <mouse, genus> Species 0.000 description 3
- 239000004743 Polypropylene Substances 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 229940122618 Trypsin inhibitor Drugs 0.000 description 3
- 101710162629 Trypsin inhibitor Proteins 0.000 description 3
- 125000000539 amino acid group Chemical group 0.000 description 3
- 230000000890 antigenic effect Effects 0.000 description 3
- 238000009835 boiling Methods 0.000 description 3
- 230000007910 cell fusion Effects 0.000 description 3
- 238000000576 coating method Methods 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 239000003599 detergent Substances 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 238000000502 dialysis Methods 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 239000013613 expression plasmid Substances 0.000 description 3
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 3
- 230000014509 gene expression Effects 0.000 description 3
- 230000001900 immune effect Effects 0.000 description 3
- 108020004999 messenger RNA Proteins 0.000 description 3
- 230000002285 radioactive effect Effects 0.000 description 3
- 230000009257 reactivity Effects 0.000 description 3
- 239000012064 sodium phosphate buffer Substances 0.000 description 3
- 239000002753 trypsin inhibitor Substances 0.000 description 3
- 239000013598 vector Substances 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- KUWPCJHYPSUOFW-YBXAARCKSA-N 2-nitrophenyl beta-D-galactoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=CC=CC=C1[N+]([O-])=O KUWPCJHYPSUOFW-YBXAARCKSA-N 0.000 description 2
- XZKIHKMTEMTJQX-UHFFFAOYSA-N 4-Nitrophenyl Phosphate Chemical compound OP(O)(=O)OC1=CC=C([N+]([O-])=O)C=C1 XZKIHKMTEMTJQX-UHFFFAOYSA-N 0.000 description 2
- SPBDXSGPUHCETR-JFUDTMANSA-N 8883yp2r6d Chemical compound O1[C@@H](C)[C@H](O)[C@@H](OC)C[C@@H]1O[C@@H]1[C@@H](OC)C[C@H](O[C@@H]2C(=C/C[C@@H]3C[C@@H](C[C@@]4(O[C@@H]([C@@H](C)CC4)C(C)C)O3)OC(=O)[C@@H]3C=C(C)[C@@H](O)[C@H]4OC\C([C@@]34O)=C/C=C/[C@@H]2C)/C)O[C@H]1C.C1C[C@H](C)[C@@H]([C@@H](C)CC)O[C@@]21O[C@H](C\C=C(C)\[C@@H](O[C@@H]1O[C@@H](C)[C@H](O[C@@H]3O[C@@H](C)[C@H](O)[C@@H](OC)C3)[C@@H](OC)C1)[C@@H](C)\C=C\C=C/1[C@]3([C@H](C(=O)O4)C=C(C)[C@@H](O)[C@H]3OC\1)O)C[C@H]4C2 SPBDXSGPUHCETR-JFUDTMANSA-N 0.000 description 2
- 102000013455 Amyloid beta-Peptides Human genes 0.000 description 2
- 108010090849 Amyloid beta-Peptides Proteins 0.000 description 2
- 108060003951 Immunoglobulin Proteins 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 239000004793 Polystyrene Substances 0.000 description 2
- 206010039966 Senile dementia Diseases 0.000 description 2
- 206010070834 Sensitisation Diseases 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- 230000003187 abdominal effect Effects 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical class N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 239000012752 auxiliary agent Substances 0.000 description 2
- WQZGKKKJIJFFOK-FPRJBGLDSA-N beta-D-galactose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-FPRJBGLDSA-N 0.000 description 2
- 238000011088 calibration curve Methods 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 210000004748 cultured cell Anatomy 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000002405 diagnostic procedure Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000003113 dilution method Methods 0.000 description 2
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 102000018358 immunoglobulin Human genes 0.000 description 2
- 230000016784 immunoglobulin production Effects 0.000 description 2
- 238000010324 immunological assay Methods 0.000 description 2
- 239000002198 insoluble material Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 238000000691 measurement method Methods 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 229920002401 polyacrylamide Polymers 0.000 description 2
- 229920002223 polystyrene Polymers 0.000 description 2
- XOJVVFBFDXDTEG-UHFFFAOYSA-N pristane Chemical compound CC(C)CCCC(C)CCCC(C)CCCC(C)C XOJVVFBFDXDTEG-UHFFFAOYSA-N 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 239000008213 purified water Substances 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 230000008313 sensitization Effects 0.000 description 2
- 239000001488 sodium phosphate Substances 0.000 description 2
- 229910000162 sodium phosphate Inorganic materials 0.000 description 2
- 238000005063 solubilization Methods 0.000 description 2
- 230000007928 solubilization Effects 0.000 description 2
- 238000000527 sonication Methods 0.000 description 2
- 210000004988 splenocyte Anatomy 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 2
- 210000002700 urine Anatomy 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- YMXHPSHLTSZXKH-RVBZMBCESA-N (2,5-dioxopyrrolidin-1-yl) 5-[(3as,4s,6ar)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoate Chemical compound C([C@H]1[C@H]2NC(=O)N[C@H]2CS1)CCCC(=O)ON1C(=O)CCC1=O YMXHPSHLTSZXKH-RVBZMBCESA-N 0.000 description 1
- KHLLRHIUKOJXLL-UHFFFAOYSA-N (4-carbamimidoylphenyl)methanesulfonyl fluoride;hydron;chloride Chemical compound Cl.NC(=N)C1=CC=C(CS(F)(=O)=O)C=C1 KHLLRHIUKOJXLL-UHFFFAOYSA-N 0.000 description 1
- 230000006269 (delayed) early viral mRNA transcription Effects 0.000 description 1
- GEYOCULIXLDCMW-UHFFFAOYSA-N 1,2-phenylenediamine Chemical compound NC1=CC=CC=C1N GEYOCULIXLDCMW-UHFFFAOYSA-N 0.000 description 1
- CWRWJDAEKWYUJT-CGKXPTHNSA-N 1-(9S,13S,12-oxophytodienoyl)-2-(7Z,10Z,13Z)-hexadecatrienoyl-3-(beta-D-galactosyl)-sn-glycerol Chemical compound C([C@H](OC(=O)CCCCC\C=C/C\C=C/C\C=C/CC)COC(=O)CCCCCCC[C@@H]1[C@@H](C(=O)C=C1)C\C=C/CC)O[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O CWRWJDAEKWYUJT-CGKXPTHNSA-N 0.000 description 1
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 description 1
- AYXZIZMZXAORLO-UFLZEWODSA-N 5-[(3as,4s,6ar)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoic acid;1-hydroxypyrrolidine-2,5-dione Chemical compound ON1C(=O)CCC1=O.N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 AYXZIZMZXAORLO-UFLZEWODSA-N 0.000 description 1
- LPXQRXLUHJKZIE-UHFFFAOYSA-N 8-azaguanine Chemical compound NC1=NC(O)=C2NN=NC2=N1 LPXQRXLUHJKZIE-UHFFFAOYSA-N 0.000 description 1
- 229960005508 8-azaguanine Drugs 0.000 description 1
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonium chloride Substances [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 101710168454 Beta-galactosidase A Proteins 0.000 description 1
- 101000984722 Bos taurus Pancreatic trypsin inhibitor Proteins 0.000 description 1
- 101100322583 Caenorhabditis elegans add-2 gene Proteins 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 229920002271 DEAE-Sepharose Polymers 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 238000009007 Diagnostic Kit Methods 0.000 description 1
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 description 1
- 102000002464 Galactosidases Human genes 0.000 description 1
- 108010093031 Galactosidases Proteins 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 1
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 1
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- MQUQNUAYKLCRME-INIZCTEOSA-N N-tosyl-L-phenylalanyl chloromethyl ketone Chemical group C1=CC(C)=CC=C1S(=O)(=O)N[C@H](C(=O)CCl)CC1=CC=CC=C1 MQUQNUAYKLCRME-INIZCTEOSA-N 0.000 description 1
- 241000283977 Oryctolagus Species 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 229920002535 Polyethylene Glycol 1500 Polymers 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 239000007640 basal medium Substances 0.000 description 1
- 230000006287 biotinylation Effects 0.000 description 1
- 238000007413 biotinylation Methods 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 210000000133 brain stem Anatomy 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 235000011148 calcium chloride Nutrition 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 239000003990 capacitor Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 208000013677 cerebrovascular dementia Diseases 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000008021 deposition Effects 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
- HDFXRQJQZBPDLF-UHFFFAOYSA-L disodium hydrogen carbonate Chemical compound [Na+].[Na+].OC([O-])=O.OC([O-])=O HDFXRQJQZBPDLF-UHFFFAOYSA-L 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 239000007789 gas 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
- 239000011521 glass Substances 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 239000003547 immunosorbent Substances 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 210000003000 inclusion body Anatomy 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000005304 joining Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- HOVAGTYPODGVJG-UHFFFAOYSA-N methyl beta-galactoside Natural products COC1OC(CO)C(O)C(O)C1O HOVAGTYPODGVJG-UHFFFAOYSA-N 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 210000000066 myeloid cell Anatomy 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 101150006061 neur gene Proteins 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 230000009871 nonspecific binding Effects 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 210000003200 peritoneal cavity Anatomy 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 229920000728 polyester Polymers 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000002331 protein detection Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000011535 reaction buffer Substances 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000003001 serine protease inhibitor Substances 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical group OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- WCTAGTRAWPDFQO-UHFFFAOYSA-K trisodium;hydrogen carbonate;carbonate Chemical compound [Na+].[Na+].[Na+].OC([O-])=O.[O-]C([O-])=O WCTAGTRAWPDFQO-UHFFFAOYSA-K 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
- 108010088854 urinastatin Proteins 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
【０００１】0001
【産業上の利用分野】本発明はタンパクの測定方法、試
薬及びキットに関するものである。更に詳しくは、アル
ツハイマー病アミロイド前駆体蛋白ＡＰＰ７５１、及び
ＡＰＰ７７０に含まれるＫＰＩ配列を認識する抗体を少
なくともその組み合わせの一方に用いることを特徴とす
るサンドイッチＥＬＩＳＡ法による、ＫＰＩ配列を含む
アルツハイマー病アミロイド前駆体蛋白又はその断片の
免疫学的測定方法、試薬及びキットに関するものである
。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for measuring proteins, a reagent, and a kit. More specifically, the production of Alzheimer's disease amyloid precursor containing a KPI sequence by a sandwich ELISA method characterized in that at least one of the combinations uses an antibody that recognizes the KPI sequence contained in Alzheimer's disease amyloid precursor proteins APP751 and APP770. This invention relates to immunological assay methods, reagents, and kits for proteins or fragments thereof.
【０００２】0002
【従来の技術】近年、人口構成の高齢化に伴い、老人性
痴呆症をはじめとする、脳神経系の疾病が社会問題化し
つつある。これらの疾病の中には発症の原因や簡便な診
断法、治療法の確立されていない疾病も多く、その研究
開発が強く望まれている。このような病気には、アルツ
ハイマー型老人性痴呆症及びアルツハイマー病（以下こ
の２つを併せて、ＡＤと略す）が含まれる。ＡＤ患者の
脳には、老人斑アミロイドβ蛋白（以下老人斑アミロイ
ドと略す）を主要構成成分とする老人斑と呼ばれる構造
体が、正常人に比し、多く沈着することから老人斑アミ
ロイドとＡＤ発症の関係が注目されてきた。Ｋａｎｇら
により、老人斑アミロイドには前駆体（以下ＡＰＰと略
す）が存在すること（以下Ｋａｎｇらにより発見された
ＡＰＰをＡＰＰ６９５と略す）が報告され（Ｋａｎｇら
、Ｎａｔｕｒｅ ３２５ ７３３−７３６ １９
８７、特開昭６３−２２２６９３号公報参照）、さらに
本発明者らの研究等により、ＡＰＰには少なくとも３種
類（ＡＰＰ６９５に加えてＡＰＰ７５１及びＡＰＰ７７
０）存在することが明らかになった（Ｐｏｎｔｅら、Ｎ
ａｔｕｒｅ ３３１ ５２５−５２７ １９８８
、Ｔａｎｚｉら、Ｎａｔｕｒｅ ３３１ ５２８−
５３０ １９８８、Ｋｉｔａｇｕｃｈｉら、Ｎａｔｕ
ｒｅ ３３１ ５３０−５３２ １９８８、欧州
特許公開番号０３０４０１３号公報）。更に、７１４ア
ミノ酸からなるアルツハイマー病アミロイド前駆体蛋白
ＡＰＰ７１４（Ｇｏｌｄｅら、Ｎｅｕｒｏｎ ４
２５３−２６７ １９９０）、及び老人斑アミロイド
部分はもたない、５６３アミノ酸からなるＡＰＰ５６３
（ｄｅ Ｓａｕｖａｇｅら、Ｓｃｉｅｎｃｅ ２４
５ ６５１−６５３ １９８９）もマイナー成分な
がらその存在が知られている（図１）。ＡＰＰ７５１及
びＡＰＰ７７０にはＫｕｎｉｔｚ型プロテアーゼインヒ
ビターと高い相同性を有する領域が存在すること、及び
実際にプロテアーゼインヒビター活性が存在することが
明らかになった（図１）。ＡＤ患者における老人斑アミ
ロイドの脳内沈着を、プロテアーゼインヒビターによる
脳内の代謝阻害、ＡＰＰの不完全分解の結果と考えるな
らば、プロテアーゼインヒビター活性を有するＡＰＰの
過剰発現がＡＤの発症に何らかの役割をはたしている可
能性が考えられる。この見地から、本発明者らは、３種
類のＡＰＰメッセンジャーＲＮＡの発現量をＡＤ患者と
正常人の脳について検討したところ、ＡＤ患者において
ＡＰＰ７７０メッセンジャーＲＮＡの発現量が２倍程高
いという結果を得た。またＡＰＰ７５１メッセンジャー
ＲＮＡもＡＤ患者で対照に比べ１．１倍から１．３倍高
いという結果を得ている（Ｔａｎａｋａら、Ｂ．Ｂ．Ｒ
．Ｃ．１５７ ４７２−４７９ １９８８及びＢ．
Ｂ．Ｒ．Ｃ．１６５ １４０６−１４１４ １９９
０）。またヒト髄液を被検体としてプロテアーゼインヒ
ビター（ＫＰＩ）領域を含むＡＰＰタンパクまたはその
断片量をトリプシンＥＬＩＳＡ法（Ｋｉｔａｇｕｃｈｉ
ら、Ｂ．Ｂ．Ｒ．Ｃ．１６６１４５３−１４５９ １
９９０及び特願平１−２１４１１８、特願平２−２２０
８８９）により測定した結果、ＡＤ群は脳血管性痴呆群
に比し、有意に高値を示すという結果がえられている（
Ｋｉｔａｇｕｃｈｉら、Ｂ．Ｂ．Ｒ．Ｃ．１６６１４５
３−１４５９ １９９０、及び、第３１回日本神経学
会総会抄録集２４１ページＩＩ−Ｊ−０５ １９９０
年）。この結果は、ＡＰＰ７７０及びＡＰＰ７５１中に
共通に存在するプロテアーゼインヒビター（ＫＰＩ；こ
こでＫＰＩとはＡＰＰ７７０およびＡＰＰ７５１のＮ末
端側から数えて２８９番目のＧｌｕから３４４番目のＡ
ｌａまでの５６残基を指し、種々のプロテアーゼに対し
て阻害活性を有する）領域を認識するウサギ抗血清と、
トリプシン（このプロテアーゼインヒビター領域と強く
結合する）とを用いたトリプシンＥＬＩＳＡ法により得
られたものである。（但しＥＬＩＳＡとは、Ｅｎｚｙｍ
ｅ Ｌｉｎｋｅｄ Ｉｍｍｕｎｏｓｏｒｂｅｎｔ
Ａｓｓａｙの略である）。しかしながら、例えば、血
清や血漿を被検体とし、且つトリプシンを固相に結合し
て第１抗体の代わりに用いる場合には、血液中のトリプ
シンインヒビターとトリプシンが結合してしまうため、
アッセイプレート固相への被検体タンパク（即ちＡＰＰ
）の結合量が充分でない場合がある。そこで、トリプシ
ンＥＬＩＳＡ法に代わるＡＰＰ７５１及びＡＰＰ７７０
タンパクの高感度検出方法が望まれている。BACKGROUND OF THE INVENTION In recent years, with the aging of the population, diseases of the brain and nervous system, including senile dementia, are becoming a social problem. Among these diseases, there are many diseases for which the causes, simple diagnostic methods, and treatments have not been established, and research and development of these diseases is strongly desired. Such diseases include Alzheimer's type senile dementia and Alzheimer's disease (hereinafter these two will be collectively abbreviated as AD). In the brains of AD patients, structures called senile plaques, whose main component is senile plaque amyloid-β protein (hereinafter abbreviated as senile plaque amyloid), are deposited in larger amounts than in normal people, so it is possible to distinguish between senile plaque amyloid and AD. The relationship between disease onset and onset has been attracting attention. Kang et al. reported that senile plaque amyloid has a precursor (hereinafter abbreviated as APP) (hereinafter the APP discovered by Kang et al. is abbreviated as APP695) (Kang et al., Nature 325 733-736 19
87, see Japanese Patent Application Laid-Open No. 63-222693), and research by the present inventors has revealed that there are at least three types of APP (in addition to APP695, APP751 and APP77).
0) was found to exist (Ponte et al., N
ature 331 525-527 1988
, Tanzi et al., Nature 331 528-
530 1988, Kitaguchi et al., Natu
re 331 530-532 1988, European Patent Publication No. 0304013). Furthermore, Alzheimer's disease amyloid precursor protein APP714 (Golde et al., Neuron 4
253-267 1990), and APP563, which consists of 563 amino acids and does not have the senile plaque amyloid part.
(de Sauvage et al., Science 24
5 651-653 1989) is also known to exist, although it is a minor component (Figure 1). It has been revealed that APP751 and APP770 have regions with high homology to Kunitz-type protease inhibitors, and that they actually have protease inhibitor activity (FIG. 1). If we consider that the intracerebral deposition of senile plaque amyloid in AD patients is a result of intracerebral metabolic inhibition by protease inhibitors and incomplete degradation of APP, then overexpression of APP, which has protease inhibitor activity, may play a role in the development of AD. It is possible that this is the case. From this point of view, the present inventors investigated the expression levels of three types of APP messenger RNA in the brains of AD patients and normal people, and found that the expression level of APP770 messenger RNA was about twice as high in AD patients. Ta. APP751 messenger RNA was also found to be 1.1 to 1.3 times higher in AD patients than in controls (Tanaka et al., B.B.R.
．． C. 157 472-479 1988 and B.
B. R. C. 165 1406-1414 199
0). In addition, using human cerebrospinal fluid as a sample, the amount of APP protein or its fragments containing the protease inhibitor (KPI) region was measured using the trypsin ELISA method (Kitaguchi).
et al., B. B. R. C. 1661453-1459 1
990 and Japanese Patent Application No. 1-214118, Japanese Patent Application No. 2-220
889), the AD group showed significantly higher values than the cerebrovascular dementia group (
Kitaguchi et al., B. B. R. C. 166145
3-1459 1990 and the 31st Annual Meeting of the Japanese Society of Neurology Abstracts 241 pages II-J-05 1990
Year). This result indicates that the protease inhibitor (KPI; here KPI refers to the Glu at position 289 to the A at position 344 counting from the N-terminus of APP770 and APP751.
A rabbit antiserum that recognizes a region (representing 56 residues up to la and having inhibitory activity against various proteases);
This was obtained by trypsin ELISA using trypsin (which strongly binds to this protease inhibitor region). (However, ELISA is Enzyme
e Linked Immunosorbent
It is an abbreviation of Assay). However, for example, when serum or plasma is used as the sample and trypsin is bound to a solid phase and used instead of the first antibody, the trypsin inhibitor in the blood binds to the trypsin.
Transferring the analyte protein (i.e. APP) to the assay plate solid phase
) binding amount may not be sufficient. Therefore, APP751 and APP770 were used instead of the trypsin ELISA method.
A highly sensitive protein detection method is desired.
【０００３】ＫＰＩ配列を認識する抗体に関しては、合
成ペプチドを抗原として作製したものが報告されている
が（Ａｎｄｅｒｓｏｎら、ＥＭＢＯ Ｊｏｕｒｎａｌ
８３６２７−３６３２、１９８９、Ｐａｌｍｅｒｔ
ら、Ｎｅｕｒｏｌｏｇｙ ４０１０２８−１０３４
１９９０）、これらはいずれもウェスタンブロティン
グにしか使用されておらずＥＬＩＳＡには使用されてい
ない。ウェスタンブロティング法に於いては、被検体を
濃縮、脱塩、さらに界面活性剤や還元剤による変性処理
等の操作を行った後、電気泳動によりタンパク質の分離
を行い、これに対して抗原抗体反応を行わなければなら
ない。そのため操作が煩雑で多数の検体について測定が
困難であること、被検試料に含まれるＡＰＰ以外の來雑
タンパクの量によって検出感度が著しく左右されること
等の点で問題がある。また、ＡＰＰのＣ末端側（ＡＰＰ
６９５、７５１、７７０に共通な配列。ＡＰＰ６９５で
いえば第６６７−６７６残基）に対する抗体でラジオイ
ムノアッセイ法（以下ＲＩＡと略す）によって、血清中
のＡＰＰ濃度を検討したところ、ＡＤ／対照に有為差は
なかったとする報告もある（Ｒｕｍｂｌｅら、Ｎｅｗ
Ｅｎｇｌａｎｄ Ｊ．Ｍｅｄ．３２０、１４４６−
１４５２、１９８９）。ただし髄液や血液等体液中に存
在するＡＰＰの殆どは、膜貫通領域よりＣ末端側の配列
を有していない（例えばＰａｌｍｅｒｔら、Ｐｒｏｃ．
Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ ８６、６３３
８−６３４２、１９８９、Ｐａｌｍｅｒｔら、Ｎｅｕｒ
ｏｌｏｇｙ ４０、１０２８−１０３４、１９９０、
ｖａｎ Ｎｏｓｔｒａｎｄら、Ｓｃｉｅｎｃｅ ２
４８、７４５−７４８、１９９０、Ｃｏｌｅら、Ｂｉｏ
ｃｈｅｍ．Ｂｉｏｐｈｙｓ．Ｒｅｓ．Ｃｏｍｍ．１７０
、２８８−２９５、１９９０など）。これらの現状に於
いて、臨床現場でのＫＰＩ配列を有するＡＰＰの特異的
かつ迅速な定量方法の確立が求められていた。Regarding antibodies that recognize the KPI sequence, antibodies prepared using synthetic peptides as antigens have been reported (Anderson et al., EMBO Journal
83627-3632, 1989, Palmer
et al., Neurology 401028-1034
(1990), both of which have been used only for Western blotting and not for ELISA. In the Western blotting method, the specimen is concentrated, desalted, and denatured with a surfactant or reducing agent, and then the proteins are separated by electrophoresis, and the antigen and antibody are separated. A reaction must take place. Therefore, there are problems in that the operation is complicated and it is difficult to measure a large number of specimens, and the detection sensitivity is significantly affected by the amount of contaminant proteins other than APP contained in the test sample. In addition, the C-terminal side of APP (APP
Sequence common to 695, 751, and 770. There is also a report that when the serum APP concentration was examined by radioimmunoassay (hereinafter abbreviated as RIA) using an antibody against APP695 (residues 667-676), there was no significant difference between AD and control ( Rumble et al., New
England J. Med. 320, 1446-
1452, 1989). However, most of the APPs present in body fluids such as cerebrospinal fluid and blood do not have a sequence on the C-terminal side of the transmembrane region (for example, Palmer et al., Proc.
Natl. Acad. Sci. USA 86, 633
8-6342, 1989, Palmer et al., Neur.
ology 40, 1028-1034, 1990,
van Nostrand et al., Science 2
48, 745-748, 1990, Cole et al., Bio
chem. Biophys. Res. Comm. 170
, 288-295, 1990, etc.). Under these circumstances, there has been a need to establish a specific and rapid method for quantifying APP having a KPI sequence in clinical practice.
【０００４】0004
【発明が解決しようとする課題】従って、本発明は、測
定操作及び試料の調製が簡便であるとともに、高感度に
ＫＰＩ配列を有するＡＰＰ（ＡＰＰ７５１及びＡＰＰ７
７０）及びその断片を測定することのできる、測定方法
、試薬及びキットを提供することを目的とする。SUMMARY OF THE INVENTION Therefore, the present invention provides simple measurement operations and sample preparation, as well as highly sensitive APPs (APP751 and APP7) having a KPI sequence.
The present invention aims to provide a measurement method, reagent, and kit that can measure 70) and its fragments.
【０００５】[0005]
【課題を解決するための手段】上記目的達成のため、本
発明者が鋭意研究を行った結果、ＫＰＩ配列を有するＡ
ＰＰ（ＡＰＰ７５１及びＡＰＰ７７０）及びその断片の
定量方法として、ＫＰＩ配列を認識する抗体を少なくと
も１種類用いた２抗体サンドイッチＥＬＩＳＡ法によっ
て可能となることを見いだし、さらに当該方法により、
測定操作及び試料の調製が簡便であるとともに、高感度
にこれらを定量できる試薬及びキットを作成し、本発明
を完成するに至った。[Means for Solving the Problems] In order to achieve the above object, the present inventor conducted intensive research and found that A
We have discovered that a two-antibody sandwich ELISA method using at least one type of antibody that recognizes the KPI sequence can be used as a method for quantifying PP (APP751 and APP770) and its fragments, and furthermore, by this method,
The inventors have completed the present invention by creating reagents and kits that are easy to perform measurement operations and sample preparation, and can quantify them with high sensitivity.
【０００６】以下、本発明について詳細に説明する。サ
ンドイッチＥＬＩＳＡ法による免疫学的測定方法とは、
抗原上の２つの異なった抗体認識部位（これを抗原決定
基という）にそれぞれ結合する２種類の抗体を用いて抗
原の有無又はその量を測定する方法である。本発明にお
いて用いられる、ＫＰＩ配列を有するＡＰＰの検出のた
めの抗体としては、一方がＫＰＩ配列を特異的に認識す
る抗体であることが要件であり、他方に用いられる抗体
としてはＡＰＰ６９５、ＡＰＰ７５１、又はＡＰＰ７７
０等各種ＡＰＰを認識する抗体であれば特に限定はない
。ＫＰＩ配列を認識する抗体として例えば本発明実施例
１に記載した抗ＺｔＰＩポリクローナル抗体が挙げられ
る。The present invention will be explained in detail below. What is the immunoassay method using sandwich ELISA method?
This is a method of measuring the presence or absence of an antigen or its amount using two types of antibodies that each bind to two different antibody recognition sites (referred to as antigenic determinants) on an antigen. One of the antibodies used in the present invention for detecting APP having a KPI sequence must be an antibody that specifically recognizes the KPI sequence, and the antibodies used for the other are APP695, APP751, or APP77
There is no particular limitation as long as the antibody recognizes various APPs such as APP0. Examples of antibodies that recognize the KPI sequence include the anti-ZtPI polyclonal antibody described in Example 1 of the present invention.
【０００７】他方に用いられる抗体としては、ＡＰＰ６
９５、ＡＰＰ７５１又はＡＰＰ７７０等各種ＡＰＰを認
識する抗体やＡＰＰ６９５、ＡＰＰ７５１又はＡＰＰ７
７０の膜貫通領域からカルボキシル末端までを除去した
各種ＡＰＰ誘導体を認識するポリクローナル抗体やモノ
クローナル抗体等を挙げることができる。例えば、本発
明者らによって作成された、ＡＰＰ６９５のＣ末端部分
１０３アミノ酸（膜貫通領域及び細胞内部分）が欠失し
たＡＰＰ６９５誘導体であるＡＰＰ５９２（実施例参照
）を免疫して得られた、ウサギ抗ＡＰＰ５９２ポリクロ
ーナル抗体等及びマウス抗ＡＰＰ５９２モノクローナル
抗体等が挙げられる。モノクローナル抗体の例としては
、微工研菌寄第１１９４２号（ＦＥＲＭ Ｐ−１１９
４２，モノクローナル抗体１Ｂ２を産生する）、微工研
菌寄第１１９４３号（ＦＥＲＭ Ｐ−１１９４３，モ
ノクローナル抗体３Ｂ２を産生する）、及び微工研菌寄
第１１９４４号（ＦＥＲＭ Ｐ−１１９４４，モノク
ローナル抗体６Ａ３を産生する）のハイブリドーマが産
生する抗体等が挙げられる。或いは、ＫＰＩ配列上の異
なる抗原決定基を認識する抗体との組み合わせであって
も良い。このような例としては本発明者らによって作成
された抗ＺｔＰＩポリクローナル抗体が挙げられる。[0007] The other antibody used is APP6.
Antibodies that recognize various APPs such as 95, APP751 or APP770, APP695, APP751 or APP7
Examples include polyclonal antibodies and monoclonal antibodies that recognize various APP derivatives in which the transmembrane region of 70 to the carboxyl terminus has been removed. For example, rabbits obtained by immunization with APP592 (see Examples), which is an APP695 derivative created by the present inventors and in which the 103 amino acids of the C-terminal portion of APP695 (transmembrane region and intracellular portion) are deleted. Examples include anti-APP592 polyclonal antibody, mouse anti-APP592 monoclonal antibody, and the like. As an example of a monoclonal antibody, FERM P-119
42, producing monoclonal antibody 1B2), FERM P-11943, producing monoclonal antibody 3B2), and FERM P-11944, producing monoclonal antibody 6A3 Antibodies produced by hybridomas that produce Alternatively, it may be a combination with antibodies that recognize different antigenic determinants on the KPI sequence. Such an example includes the anti-ZtPI polyclonal antibody generated by the present inventors.
【０００８】これらの抗体は完全な形の抗体のままで用
い得ることは勿論のこと、その本質的結合能が維持され
る抗体断片、例えば、Ｆａｂ、Ｆａｂ′、（Ｆａｂ′）
２等として用いることもできる。本発明における被検試
料としては特に限定はないが、具体的には、髄液、血液
（血漿及び血清）、尿等が挙げられる。また、脳等の組
織や細胞の抽出物を用いることもできる。[0008] These antibodies can of course be used as intact antibodies, but also as antibody fragments that maintain their essential binding ability, such as Fab, Fab', (Fab').
It can also be used as a second grade. Test samples in the present invention are not particularly limited, but specific examples include cerebrospinal fluid, blood (plasma and serum), urine, and the like. Furthermore, extracts of tissues and cells such as the brain can also be used.
【０００９】本発明において用いられる不溶性担体とし
ては、例えはポリスチレン、ポリエチレン、ポリプロピ
レン、ポリエステル、フッ素樹脂、ガラス、金属及びこ
れらの組み合わせ等を例示することができる。また不溶
性担体の形状としては、例えばトレー状、球状、繊維状
、棒状、盤状、容器状、セル、試験管等の種々の形状で
本発明を実施することができる。多数の被検試料を同時
に測定できる点では、９６穴マイクロプレート等が好ま
しい。Examples of the insoluble carrier used in the present invention include polystyrene, polyethylene, polypropylene, polyester, fluororesin, glass, metal, and combinations thereof. Furthermore, the present invention can be carried out with various shapes of the insoluble carrier, such as a tray shape, a spherical shape, a fiber shape, a rod shape, a disk shape, a container shape, a cell, a test tube, and the like. A 96-well microplate or the like is preferable in that a large number of test samples can be measured simultaneously.
【００１０】本発明に於いては第２抗体として用いられ
る抗体を直接、酵素や放射性同位元素或いはＦＩＴＣ等
の蛍光物質で標識してもよい。或いは、かかる第２抗体
に対する抗体を標識したものを第３抗体として用いても
よい。或いは第２抗体を、ビオチン等のマーカー化合物
で標識し、このマーカー化合物に特異的に結合する物質
（ビオチンの場合、ストレプトアビジンやアビジン）を
、酵素や蛍光色素、放射性同位元素等で標識したものを
接合させるという方法も用いられる。[0010] In the present invention, the antibody used as the second antibody may be directly labeled with an enzyme, a radioactive isotope, or a fluorescent substance such as FITC. Alternatively, a labeled antibody against such second antibody may be used as the third antibody. Alternatively, the second antibody is labeled with a marker compound such as biotin, and a substance that specifically binds to this marker compound (in the case of biotin, streptavidin or avidin) is labeled with an enzyme, fluorescent dye, radioisotope, etc. A method of joining is also used.
【００１１】本発明に於いては最終的な検出手段は特に
限定せず、ＡＰＰ７５１、ＡＰＰ７７０に共通な配列を
認識する１組の２種類の抗体の一方として、これらのＡ
ＰＰに含まれるＫＰＩ配列を認識する抗体を用いること
が要件であり、かかる組み合わせにより認識固定された
後の検出手段は如何なるものでも構わない。しかしなが
ら、本発明に用いることができる代表的検出法を例示す
ると、第２抗体を酵素、放射性同位元素、蛍光物質或い
は他の結合物質で標識したものを用いる検出方法となる
。[0011] In the present invention, the final detection means is not particularly limited, and these A
It is necessary to use an antibody that recognizes the KPI sequence contained in PP, and any detection means may be used after recognition and immobilization by such a combination. However, a typical detection method that can be used in the present invention is a detection method using a second antibody labeled with an enzyme, a radioisotope, a fluorescent substance, or other binding substance.
【００１２】酵素を用いる検出方法では、酵素としては
、β−Ｄ−ガラクトシダーゼ（β−Ｇａｌ）、ホースラ
ディッシュパーオキシダーゼ（ＨＲＰ）、アルカリ性ホ
スファターゼ（ＡＰ）等があり、それぞれの酵素に対応
する比色基質、或いは蛍光基質を加え、比色計、または
蛍光光度計を用いて生成物を定量する。上記酵素に対す
る代表的基質としては、β−Ｇａｌの基質として、ｏ−
ニトロフェニル−β−Ｄ−ガラクトシド（比色基質）、
４−メチルウンベリフェリル−β−Ｄ−ガラクトシド（
蛍光基質）等、ＨＲＰの基質としてｏ−フェニレンジア
ミン（比色基質）等、ＡＰの基質として、ｐ−ニトロフ
ェニルリン酸（比色基質）等を挙げることができる。放
射性同位元素を用いる検出方法としては、１２５−Ｉ等
で標識された第２抗体を用い、γ−ウェルカウンター等
を用いて放射活性を測定する。また、第２抗体をビオチ
ン標識し、更にアビジンで標識した酵素と反応させ、以
下酵素を用いる検出の場合と同様の反応を行って、検出
することも可能である。また蛍光物質を用いる方法では
、蛍光物質として、フルオレッセインイソチオシアネー
ト（ＦＩＴＣ）等を例示することができる。これらは例
示したものに限らず、免疫学的測定方法に使用されてい
るものであれば、他のものでも使用できる。[0012] In the detection method using an enzyme, enzymes include β-D-galactosidase (β-Gal), horseradish peroxidase (HRP), alkaline phosphatase (AP), etc., and the colorimetric method corresponding to each enzyme is used. A substrate or fluorescent substrate is added and the product is quantified using a colorimeter or fluorometer. Typical substrates for the above enzymes include o-
nitrophenyl-β-D-galactoside (colorimetric substrate),
4-Methylumbelliferyl-β-D-galactoside (
Examples of the substrate for AP include p-nitrophenyl phosphate (colorimetric substrate) and the like. As a detection method using a radioisotope, a second antibody labeled with 125-I or the like is used, and radioactivity is measured using a γ-well counter or the like. It is also possible to perform detection by biotin-labeling the second antibody and reacting it with an avidin-labeled enzyme, and then performing the same reaction as in the case of detection using an enzyme. In the method using a fluorescent substance, examples of the fluorescent substance include fluorescein isothiocyanate (FITC). These are not limited to those exemplified, but other types can be used as long as they are used in immunological assay methods.
【００１３】本発明で用いる抗体を使用して、ＲＩＡを
組むことも可能である。ＲＩＡの１つの方法としてＡＰ
Ｐ７５１やＡＰＰ７７０又はその断片を、放射性同位元
素で標識して標識抗原とし、これと試料中の抗原（非標
識抗原）とが、本発明で用いる抗体を不溶性担体に結合
したものに、競争的に結合させ、標識抗原の結合の度合
いで、試料中の抗原量を測定する方法が挙げられる。即
ち、試料中の抗原量が多ければ、不溶性担体に結合した
標識抗原は少なくなり、遊離の標識抗原が多くなる。試
料中の抗原量が少なければ、その逆となる。また、ＲＩ
Ａの他の方法として、先述した様に、本発明で用いる第
２抗体を放射性同位元素で標識し、試料中の抗原と結合
した標識抗体量を測定する方法も挙げられる。[0013] It is also possible to construct RIA using the antibodies used in the present invention. AP as one method of RIA
P751, APP770, or a fragment thereof is labeled with a radioactive isotope to make a labeled antigen, and this and the antigen in the sample (unlabeled antigen) are competitively attached to the antibody used in the present invention bound to an insoluble carrier. An example of this method is to bind the labeled antigen and measure the amount of antigen in the sample based on the degree of binding of the labeled antigen. That is, if the amount of antigen in the sample is large, the amount of labeled antigen bound to the insoluble carrier will decrease, and the amount of free labeled antigen will increase. The opposite is true if the amount of antigen in the sample is low. Also, R.I.
As another method of A, as mentioned above, there is also a method of labeling the second antibody used in the present invention with a radioactive isotope and measuring the amount of the labeled antibody bound to the antigen in the sample.
【００１４】次に本発明による、ＫＰＩ配列を含むアル
ツハイマー病アミロイド前駆体蛋白の測定方法について
具体的に説明する。ＫＰＩ配列を認識する抗体（第１抗
体）を適当な不溶性担体、例えば９６穴ポリスチレン製
マイクロプレートに固定化する（以下これを“固定化抗
体”という）。固定用バッファーとしては、炭酸ナトリ
ウム−炭酸水素ナトリウムバッファー（ｐＨ９．６）、
或いはリン酸緩衝塩類溶液（以下ＰＢＳ（−）と略す）
等が用いられる。固定は、４℃で一晩放置するのが一般
的であるが、例えば、室温で２時間程度静置することも
可能である。このようにして第１抗体を固定したマイク
ロプレートをＰＢＳ（−）等によって洗浄する。次いで
不溶性担体と、測定しようとする試薬又は被検試料との
非特異的結合を避けるために、適当な物質、例えば１〜
３％ウシ血清アルブミンやスキムミルク／ＰＢＳ（−）
溶液で、不溶性担体の表面を被覆する。被覆は、３７℃
や室温で２時間程度、静置又はプレートミキサー等で攪
拌しながら行うのが一般的である。被覆後、再びマイク
ロプレートをＰＢＳ（−）等で洗浄する。このようにし
て得られた、第１抗体が固定化された不溶性担体を被検
試料と一定時間及び一定温度で接触させ反応させる。反
応は４℃で一晩プレートミキサー等で攪拌しながら行う
のが一般的であるが、室温や３７℃で１〜２時間程度行
うことも可能である。この間に固定化抗体（第１抗体）
と被検試料中のＫＰＩ配列を含むＡＰＰ又はその断片が
結合する。次いでＰＢＳ（−）等で洗った後、適当な標
識物質、例えば、β−ガラクトシダーゼ、ホースラディ
ッシュパーオキシダーゼ、アルカリ性ホスファターゼ等
の酵素や、１２５−Ｉ等の放射性同位元素で標識化した
ＡＰＰ６９５、ＡＰＰ７５１、及びＡＰＰ７７０を認識
する抗体（第２抗体）の溶液を、不溶性担体上の固定化
抗体に結合したＡＰＰ又はその断片と一定時間及び一定
温度例えば、４℃で一晩、或いは室温で１〜２時間接触
させ第２抗体と反応させる。これを再び、ＰＢＳ（−）
等で洗い、次いで不溶性担体上に存在する第２抗体に標
識された標識物質の量を測定する。酵素を用いる検出方
法では、酵素としては、β−Ｄ−ガラクトシダーゼ（β
−Ｇａｌ）、ホースラディッシュパーオキシダーゼ、（
ＨＲＰ）、アルカリ性ホスファターゼ（ＡＰ）等があり
、それぞれの酵素に対応する比色基質、或いは蛍光基質
を加え、比色計、または蛍光光度計を用いて生成物を定
量する。上記酵素に対する代表的基質としては、β−Ｇ
ａｌの基質として、ｏ−ニトロフェニル−β−Ｄ−ガラ
クトシド（比色基質）、４−メチルウンベリフェニル−
β−Ｄ−ガラクトシド（蛍光基質）等、ＨＲＰの基質と
して、ｏ−フェニレンジアミン（比色基質）等、ＡＰの
基質としてｐ−ニトロフェニルリン酸（比色基質）等を
挙げることができる。放射性同位元素を用いる検出方法
としては、１２５−Ｉ等で標識された第２抗体を用い、
γ−ウェルカウンター等を用いて放射活性を測定する。
またアビジンのビオチンに対する高親和性を利用して、
第２抗体をビオチン標識し、更にアビジンやストレプト
アビジンで標識した酵素をと反応させ、以下酵素を用い
る検出の場合と同様の反応を行って、検出することも可
能である。Next, a method for measuring Alzheimer's disease amyloid precursor protein containing a KPI sequence according to the present invention will be explained in detail. An antibody that recognizes the KPI sequence (first antibody) is immobilized on a suitable insoluble carrier, such as a 96-well polystyrene microplate (hereinafter referred to as "immobilized antibody"). As the fixation buffer, sodium carbonate-sodium hydrogen carbonate buffer (pH 9.6),
Or phosphate buffered saline solution (hereinafter abbreviated as PBS(-))
etc. are used. For fixation, it is common to leave it at 4°C overnight, but it is also possible to leave it at room temperature for about 2 hours, for example. The microplate on which the first antibody is immobilized in this way is washed with PBS(-) or the like. Next, in order to avoid non-specific binding between the insoluble carrier and the reagent to be measured or the test sample, a suitable substance, e.g.
3% bovine serum albumin or skim milk/PBS (-)
The solution coats the surface of the insoluble carrier. Coating is at 37℃
It is generally carried out at room temperature for about 2 hours while standing or stirring with a plate mixer or the like. After coating, the microplate is washed again with PBS(-) or the like. The thus obtained insoluble carrier on which the first antibody is immobilized is brought into contact with the test sample for a certain period of time and at a certain temperature to cause a reaction. The reaction is generally carried out overnight at 4°C while stirring with a plate mixer, but it is also possible to carry out the reaction at room temperature or 37°C for about 1 to 2 hours. During this time, the immobilized antibody (first antibody)
APP or a fragment thereof containing the KPI sequence in the test sample binds to the test sample. Next, after washing with PBS(-) etc., APP695, APP751, labeled with an appropriate labeling substance, for example, an enzyme such as β-galactosidase, horseradish peroxidase, alkaline phosphatase, or a radioisotope such as 125-I, A solution of an antibody (second antibody) that recognizes APP770 is mixed with APP or a fragment thereof bound to an immobilized antibody on an insoluble carrier for a certain period of time and at a certain temperature, for example, overnight at 4°C, or for 1 to 2 hours at room temperature. and react with the second antibody. This again, PBS (-)
Then, the amount of the labeling substance labeled with the second antibody present on the insoluble carrier is measured. In the detection method using an enzyme, β-D-galactosidase (β-D-galactosidase) is used as the enzyme.
-Gal), horseradish peroxidase, (
HRP), alkaline phosphatase (AP), etc., and the product is quantified using a colorimeter or fluorometer by adding a colorimetric or fluorescent substrate corresponding to each enzyme. Typical substrates for the above enzymes include β-G
As substrates for al, o-nitrophenyl-β-D-galactoside (colorimetric substrate), 4-methylumbelliphenyl-
Substrates for HRP such as β-D-galactoside (fluorescent substrate) include o-phenylenediamine (colorimetric substrate), and substrates for AP include p-nitrophenyl phosphate (colorimetric substrate). As a detection method using a radioisotope, a second antibody labeled with 125-I etc. is used,
Radioactivity is measured using a γ-well counter or the like. In addition, by utilizing the high affinity of avidin for biotin,
Detection can also be carried out by labeling the second antibody with biotin, reacting it with an enzyme labeled with avidin or streptavidin, and performing the same reaction as in the case of detection using an enzyme.
【００１５】一方、被検試料の代わりに種々の濃度の標
準品、例えば組換えＤＮＡ法により産生した、精製アル
ツハイマー病アミロイド前駆体蛋白又はその断片、或い
は標準となる髄液や血液、尿などを用いて標準検量線を
作製し、これと、被検試料の測定値を対比させ、被検試
料中のアルツハイマー病アミロイド前駆体蛋白の量を算
出し、定量することができる。第１抗体としてＫＰＩ配
列を認識する抗体、第２抗体としてＡＰＰを認識する抗
体について述べたが、第１抗体と第２抗体の組み合わせ
は逆であっても構わないし、第１抗体、第２抗体がＫＰ
Ｉ配列上の異なる抗原決定基を認識する抗体との組み合
わせであってもよい。On the other hand, instead of the test sample, standard products of various concentrations, such as purified Alzheimer's disease amyloid precursor protein or its fragment produced by recombinant DNA method, or standard cerebrospinal fluid, blood, urine, etc. A standard calibration curve is prepared using the method, and by comparing this with the measured values of the test sample, the amount of Alzheimer's disease amyloid precursor protein in the test sample can be calculated and quantified. Although we have described an antibody that recognizes the KPI sequence as the first antibody and an antibody that recognizes APP as the second antibody, the combination of the first antibody and the second antibody may be reversed, or the combination of the first antibody and the second antibody is KP
It may also be a combination with antibodies that recognize different antigenic determinants on the I sequence.
【００１６】本発明に於ける測定試薬は、一方を不溶性
担体に結合した第１抗体と他方の第２抗体とにより構成
される。また、この試薬を能率よく、且つ簡便に利用す
るために、これら抗体以外に種々の補助剤を含めてキッ
トを形成することができる。係る補助剤としては、例え
ば、検量線作製用標準品、試薬を溶解させるための溶解
剤、不溶化担体を洗浄するために使用される洗浄剤、抗
体の標識物質として酵素を使用した場合、酵素活性を測
定するための基質、その反応停止剤等の免疫学的測定試
薬のキットとして通常使用されるものが挙げられる。The measurement reagent in the present invention is composed of a first antibody, one of which is bound to an insoluble carrier, and the other of which is a second antibody. Furthermore, in order to utilize this reagent efficiently and easily, a kit can be formed containing various auxiliary agents in addition to these antibodies. Such auxiliary agents include, for example, standards for preparing calibration curves, lysing agents for dissolving reagents, detergents used for washing insolubilized carriers, enzymes used as labeling substances for antibodies, enzyme activity Examples include those commonly used as kits for immunological measurement reagents, such as substrates and reaction terminators for measuring .
【００１７】[0017]
【実施例】以下に実施例により本発明を詳述するが、本
発明は該実施例によって限定されるものではない。EXAMPLES The present invention will be explained in detail with reference to Examples below, but the present invention is not limited by these Examples.
【００１８】[0018]
【実施例１】 抗原、アッセイ用タンパク及び抗体の
作成
［工程１］ 抗ＺｔＰＩ（抗ＫＰＩ）ポリクローナル
抗体の作成及び精製
［工程１−１］ β−ガラクトシダーゼ・ＫＰＩ融合
蛋白の大腸菌による発現
（ｉ） 融合タンパク発現プラスミドの構築大腸菌β−
ガラクトシダーゼのＮ末端にｓＰＩ（ＡＰＰ７７０の２
８８番目のＡｒｇ残基から３５９番目のＡｒｇ残基まで
を含む。従ってＫＰＩを含む。）を結合した融合タンパ
ク（ＺＰＩ）を発現しうるプラスミドを構築した。具体
的には、発現用ベクターｐＥＸ１（ベーリンガー・マン
ハイム山之内社）を制限酵素ＢａｍＨＩ及びＰｓｔＩで
切断し、直鎖状ベクターＤＮＡ断片を得た。
一方、欧州特許出願公開番号０３０４０１３号の実施例
４に記載のプラスミドｐＰＩｔｒｐ７５−１をＢａｍＨ
Ｉ及びＰｓｔＩで切断し、アガロースゲル電気泳動後０
．３ｋｂｐの断片を、フナコシ社製ジーンクリーン・キ
ットを用いて単離した。得られた２つの断片をＴ４ＤＮ
Ａリガーゼを用いて連結せしめた。[Example 1] Preparation of antigen, assay protein, and antibody [Step 1] Preparation and purification of anti-ZtPI (anti-KPI) polyclonal antibody [Step 1-1] Expression of β-galactosidase/KPI fusion protein in Escherichia coli (i) Construction of fusion protein expression plasmid E. coli β-
sPI (APP770 2) at the N-terminus of galactosidase
It includes the 88th Arg residue to the 359th Arg residue. Therefore, it includes KPIs. ) was constructed. A plasmid capable of expressing a fusion protein (ZPI) was constructed. Specifically, expression vector pEX1 (Boehringer Mannheim Yamanouchi) was cut with restriction enzymes BamHI and PstI to obtain a linear vector DNA fragment. On the other hand, plasmid pPItrp75-1 described in Example 4 of European Patent Application Publication No. 0304013 was transferred to BamH
After cutting with I and PstI and agarose gel electrophoresis,
．． A 3 kbp fragment was isolated using the Funakoshi GeneClean kit. The two obtained fragments were transformed into T4DN.
Ligation was performed using A ligase.
【００１９】該反応物を、マニアティスらの実験書（Ｔ
．Ｍａｎｉａｔｉｓら、Ｍｏｌｅｃｕｌａｒ Ｃｌｏ
ｎｉｎｇ．Ａ Ｌａｂｏｒａｔｏｒｙ Ｍａｎｕａ
ｌ、Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌ
ａｂｏｒａｔｏｒｙ（１９８２））に記載されている方
法に従って調製した大腸菌ＰＯＰ２１３６株（ベーリン
ガー・マンハイム山之内社）コンピテントセルに加え、
形質転換せしめた。アンピシリン耐性形質転換コロニー
６個を選び、各プラスミドを抽出し、ＢａｍＨＩ及びＰ
ｓｔＩで切断し解析したところ、すべて目的とするプラ
スミドを有してした。得られたプラスミドをｐＥＸ−Ｚ
ＰＩと命名した。本プラスミドは、ラムダ・ファージ由
来のＰＲプロモーターの制御により、β−ガラクトシダ
ーゼ・ＫＰＩの融合タンパクを発現しうる。The reaction product was prepared according to Maniatis et al.'s experimental book (T
．． Maniatis et al., Molecular Clo
ning. A Laboratory Manual
l, Cold Spring Harbor L
In addition to E. coli strain POP2136 (Boehringer Mannheim Yamanouchi) competent cells prepared according to the method described in the laboratory (1982),
Transformed. Six ampicillin-resistant transformed colonies were selected, each plasmid was extracted, and BamHI and P
When cut with stI and analyzed, all of them contained the desired plasmid. The obtained plasmid was pEX-Z
It was named PI. This plasmid can express a β-galactosidase/KPI fusion protein under the control of the PR promoter derived from lambda phage.
【００２０】（ｉｉ） 融合タンパクの生産本実施例工
程１−１の（ｉ） で得たプラスミドｐＥＸ−ＺＰＩを
保持する大腸菌ＰＯＰ２１３６／ｐＥＸ−ＺＰＩを、５
０μｇ／ｍｌのアンピシリン含有Ｌ培地で、３０℃一晩
培養せしめた。本培養液１ｍｌを、５０μｇ／ｍｌアン
ピシリン含有Ｌ培地５０ｍｌに添加し、３０℃にて３時
間培養した後、４２℃の恒温培養槽に移し、さらに３時
間培養した。４２℃において、ラムダ・ファージＰＲプ
ロモーターのリプレッサーは不活化され、ＰＲプロモー
ターが作動することによって、目的とする融合タンパク
が生産される。(ii) Production of fusion protein Escherichia coli POP2136/pEX-ZPI carrying the plasmid pEX-ZPI obtained in step 1-1 (i) of this example was transformed into 5
The cells were cultured overnight at 30°C in L medium containing 0 μg/ml ampicillin. 1 ml of the main culture solution was added to 50 ml of L medium containing 50 μg/ml ampicillin and cultured at 30° C. for 3 hours, then transferred to a constant temperature culture tank at 42° C. and cultured for an additional 3 hours. At 42°C, the repressor of the lambda phage PR promoter is inactivated, and the desired fusion protein is produced by operating the PR promoter.
【００２１】該培養液を遠心分離し、菌体を集め、菌体
の一部を、レムリーの方法〔Ｌａｅｍｍｌｉ、Ｕ．Ｋ．
，Ｎａｔｕｒｅ、２２７．６８０−６８５（１９７０）
〕に従って、ＳＤＳ・サンプルバッファー中で煮沸後、
１０％ポリアクリルアミドゲルを用いてＳＤＳ−ポリア
クリルアミドゲル電気泳動に供した。泳動後、ゲルをク
マシー染色した結果、分子量約１２０Ｋｄの目的とする
融合タンパクが存在することが確認された。以下このβ
ガラクトシダーゼ・ＫＰＩ融合タンパクをＺＰＩと略す
。[0021] The culture solution is centrifuged, the bacterial cells are collected, and a portion of the bacterial cells are collected by the method of Laemmli [Laemmli, U.S.; K.
, Nature, 227.680-685 (1970)
] After boiling in SDS/sample buffer,
SDS-polyacrylamide gel electrophoresis was performed using a 10% polyacrylamide gel. After electrophoresis, the gel was stained with Coomassie, and it was confirmed that the desired fusion protein with a molecular weight of about 120 Kd was present. Below this β
Galactosidase/KPI fusion protein is abbreviated as ZPI.
【００２２】（ｉｉｉ）ＺＰＩの可溶化本実施例工程１
−１の（ｉｉ）で得た培養菌体の一部を、尿素を終濃度
０Ｍ，２Ｍ，４Ｍ，６Ｍ，８Ｍをそれぞれ含む２０ｍＭ
トリス塩酸（ｐＨ８．０）に懸濁し、オータケ・ソニケ
ーター（大岳製作所）を用いて菌体を超音波破壊し、遠
心分離により不溶物を集めた。得られた不溶物を本実施
例工程１−１の（ｉｉ）と同様に処理して、ＳＤＳポリ
アクリルアミドゲル電気泳動法により解析した。(iii) ZPI solubilization Step 1 of this example
A part of the cultured bacterial cells obtained in (ii) of -1 was added to 20mM containing urea at final concentrations of 0M, 2M, 4M, 6M, and 8M, respectively.
The cells were suspended in Tris-HCl (pH 8.0), the cells were disrupted by ultrasonic waves using an Otake sonicator (Otake Seisakusho), and insoluble materials were collected by centrifugation. The obtained insoluble matter was treated in the same manner as in step 1-1 (ii) of this example, and analyzed by SDS polyacrylamide gel electrophoresis.
【００２３】クマシー染色の結果、目的物ＺＰＩのバン
ドは、尿素０Ｍ，２Ｍおよび４Ｍ含有バッファーで抽出
した不溶画分では検出されたが、尿素６Ｍおよび８Ｍ含
有バッファー抽出後の不溶画分では認められなかった。
以上の結果から、目的融合タンパクは産生後大腸菌体内
で不溶物としてタンパク封入体（Ｉｎｃｌｕｓｉｏｎ
ｂｏｄｙ）を形成し、この不溶物は６Ｍ以上の尿素で
可溶化されることが判明した。As a result of Coomassie staining, a band of the target product ZPI was detected in the insoluble fractions extracted with buffers containing 0M, 2M, and 4M urea, but was not observed in the insoluble fractions extracted with buffers containing 6M and 8M urea. There wasn't. From the above results, the target fusion protein forms protein inclusion bodies as insoluble matter in E. coli after production.
It was found that this insoluble material was solubilized by urea of 6M or more.
【００２４】（ｉｖ） ＺＰＩの部分精製本実施例工程
１−１の（ｉｉ）と同様にして、大腸菌ＰＯＰ２１３６
／ｐＥＸ−ＺＰＩを培養し、２００ｍｌの培養液から菌
体を回収した。該培養菌体を、４０ｍｌの４Ｍ尿素を含
む２０ｍＭトリス塩酸（ｐＨ８．０）バッファーに懸濁
し、菌体を超音波破壊した。菌体破壊物を遠心分離（１
００００×ｇ，１０分間）し、沈澱物を回収した。次い
でこの沈澱物を２０ｍｌの８Ｍ尿素を含む２０ｍＭトリ
ス塩酸（ｐＨ８．０）バッファーに再懸濁し、超音波処
理により沈澱を分散せしめた。遠心分離（１００００×
ｇ，１０分間）後上清を回収した。以上の操作により、
４Ｍ尿素で可溶な大腸菌タンパク、および８Ｍ尿素に不
溶な大腸菌タンパクが除去され、ＺＰＩの部分精製液が
得られた。この部分精製液の一部をＳＤＳ−ポリアクリ
ルアミドゲル電気泳動法で解析したところ、全タンパク
質のうち５０％以上が目的融合タンパクＺＰＩであった
。(iv) Partial purification of ZPI E. coli POP2136 was purified in the same manner as (ii) of Step 1-1 of this example.
/pEX-ZPI was cultured, and bacterial cells were collected from 200 ml of the culture solution. The cultured cells were suspended in 40 ml of 20 mM Tris-HCl (pH 8.0) buffer containing 4M urea, and the cells were disrupted by ultrasonication. Centrifuging the destroyed bacterial cells (1
0000×g for 10 minutes) and the precipitate was collected. Next, this precipitate was resuspended in 20 ml of 20 mM Tris-HCl (pH 8.0) buffer containing 8 M urea, and the precipitate was dispersed by sonication. Centrifugation (10000x
g, 10 minutes) and then the supernatant was collected. By the above operations,
E. coli proteins soluble in 4 M urea and E. coli proteins insoluble in 8 M urea were removed to obtain a partially purified solution of ZPI. When a part of this partially purified solution was analyzed by SDS-polyacrylamide gel electrophoresis, more than 50% of the total protein was the target fusion protein ZPI.
【００２５】（ｖ）プロテアーゼインヒビター領域の抽
出本実施例工程１−１の（ｉｖ）で得たＺＰＩ部分精製
液２０ｍｌを、２０ｍＭトリス塩酸（ｐＨ８．０）２Ｌ
に対して２回透析し、尿素を除去せしめると同時にタン
パクの立体構造を回復せしめた。得られた透析液を３７
℃にて保温した後、ＴＰＣＫ処理済みトリプシン固定化
アガロースビーズ（シグマ社）１００単位を加え、３７
℃で１時間振とうすることにより、ＺＰＩ中のβガラク
トシダーゼ領域を切断すると同時にプロテアーゼインヒ
ビター領域を吸着させた。かかる懸濁液を、５′プライ
ム→３′プライム社のセレクトＳＴＭ空カラムに充填し
、アガロースビーズを回収した。アガロースビーズとバ
ッファーとの分離は、１，０００ｒｐｍ、１分間の遠心
操作により行った（以下の記述中のアガロースビーズの
回収も同様にして行った）。回収したビーズを、０．３
Ｍ ＮａＣｌ、１０ｍＭ ＣａＣｌ２ 、１０ｍＭ
ＨＣｌ（ｐＨ２）からなる溶出液６ｍｌで溶出した
。溶出は計３回行い、各溶出液を５ＮＮａＯＨを用いて
中和した後、トリプシン阻害活性を測定した。阻害活性
の測定は、欧州特許公開番号０３０４０１３号実施例４
に記載のｓＰＩのトリプシン阻害活性測定と同様の方法
によった。(v) Extraction of protease inhibitor region 20 ml of the partially purified ZPI solution obtained in step 1-1 (iv) of this example was added to 2 L of 20 mM Tris-HCl (pH 8.0).
The protein was dialyzed twice to remove urea and at the same time restore the three-dimensional structure of the protein. The obtained dialysate was
After incubating at ℃, 100 units of TPCK-treated trypsin-immobilized agarose beads (Sigma) were added,
By shaking at ℃ for 1 hour, the β-galactosidase region in ZPI was cleaved and at the same time the protease inhibitor region was adsorbed. This suspension was packed into a Select STM empty column manufactured by 5'Prime→3' Prime Co., Ltd., and the agarose beads were collected. Separation of the agarose beads and buffer was performed by centrifugation at 1,000 rpm for 1 minute (collection of agarose beads in the following description was performed in the same manner). The collected beads were 0.3
M NaCl, 10mM CaCl2, 10mM
Elution was performed with 6 ml of an eluent consisting of HCl (pH 2). Elution was performed three times in total, and after neutralizing each eluate with 5N NaOH, trypsin inhibitory activity was measured. The inhibitory activity was measured according to European Patent Publication No. 0304013, Example 4.
The trypsin inhibitory activity of sPI was measured in the same manner as described in .
【００２６】各溶出液に含まれるトリプシン・インヒビ
ター活性濃度は、溶出分画の順に、ＢＰＴＩに換算して
１６．４μｇ／ｍｌ、１．５μｇ／ｍｌ、０．２７μｇ
／ｍｌであった。これらの溶出分画を、ＺｔＰＩ画分と
する。
（ｖｉ） プロテアーゼ・インヒビターの解析本実施例
工程１−１の（ｖ） で得た溶出液に、あらかじめ−２
０℃に冷却したアセトンを４倍容量加え、−２０℃にて
１時間冷却した。さらに、冷却遠心分離（１０，０００
×ｇ１５分間）し、沈澱を回収し、得られた沈澱を１ｍ
ｌの２０ｍＭトリス塩酸（ｐＨ７．５）に再溶解した。
本実施例工程１−１の（ｖ） と同様にしてトリプシン
阻害活性量を測定した。その結果、欧州特許公開番号０
３０４０１３号実施例４に記載のｓＰＩに換算して６２
μｇ相当のトリプシン・インヒビターが含まれているこ
とが判明した。[0026] The concentration of trypsin inhibitor activity contained in each eluate is 16.4 μg/ml, 1.5 μg/ml, and 0.27 μg in terms of BPTI in the order of elution fractions.
/ml. These elution fractions are designated as ZtPI fractions. (vi) Analysis of protease inhibitors Add -2
Four times the volume of acetone cooled to 0°C was added, and the mixture was cooled at -20°C for 1 hour. Furthermore, refrigerated centrifugation (10,000
x g for 15 minutes), collect the precipitate, and transfer the obtained precipitate to 1 m
1 of 20mM Tris-HCl (pH 7.5). Trypsin inhibitory activity was measured in the same manner as in step 1-1 (v) of this example. As a result, European patent publication number 0
62 in terms of sPI described in Example 4 of No. 304013
It was found that it contained trypsin inhibitor equivalent to μg.
【００２７】得られたインヒビター溶液の一部を、ＳＤ
Ｓ−ポリアクリルアミドゲル電気泳動法により解析した
ところ、分子量約６Ｋｄのメジャーな蛋白質が検出され
た。
［工程１−２］ 抗ＺｔＰＩ抗血清の作成本実施例工
程１−１の（ｉｖ）で得られたＺＰＩ３００μｇを、前
述したレムリーの方法によるＳＤＳ−ポリアクリルアミ
ドゲル電気泳動で用いるサンプルバッファー（２％ＳＤ
Ｓ、５％β−ＭＥを含む、ただし色素は加えなかった）
中で、１００℃で５分間処理したのち、４倍容のアセト
ンを加え、−２０℃で１時間冷却を行い、９０００回転
２０分間の遠心操作を行い、沈澱を回収した。その沈澱
をＰＢＳ（−）に懸濁し、これとフロイント完全アジュ
バント（ＦＣＡ）とをよく混合してウサギに皮下注射し
た。３１日後にＦＣＡのかわりにフロイント不完全アジ
ュバントを用いた以外は同じ処理（免疫）を行った。さ
らに１４日後、３００μｇの本実施例工程１−１の（ｖ
） で得られたＺｔＰＩを同様の処理を行い免疫した。
さらに１６日後、ＺｔＰＩを用いて同じ処理を行った。
さらに１５日後、欧州特許公開番号０３０４０１３号実
施例４に記載のｓＰＩ１５０μｇを同じ処理をして免疫
した。このウサギから５０ｍｌ採血し、抗血清を得た。
以後この抗血清を抗ＺｔＰＩ抗血清と呼ぶ。血清中の抗
体価は、非変性及びＳＤＳ変性のｓＰＩ（２５ｎｇ／ウ
ェル）を抗原とする固相ＥＬＩＳＡによって行った。最
終的に得られた抗血清の力価は、対照（抗原なしのウェ
ル）の３倍以上の発色を示す抗血清の希釈度で示すと、
非変性ｓＰＩに対して６４０００倍、ＳＤＳ変性ｓＰＩ
に対して１２８０００倍であった。また尿中トリプシン
インヒビター（日本ケミカルリサーチ社）とは全く反応
しなかった。即ち、本抗ＺｔＰＩ抗血清はＫＰＩ配列に
対する高い特異性と抗体価を有することがわかった。
［工程１−３］ 抗ＺｔＰＩ ＩｇＧの精製本実施
例工程１−２で得た抗ＺｔＰＩ血清２０ｍｌを５６℃に
て３０分インキュベートして非働化させた後、等量のＰ
ＢＳ（−）を加えて希釈し、４℃で１００％飽和硫安溶
液（予め０．１Ｎアンモニア溶液にてｐＨ６．５に調整
したもの）を最終濃度３３％飽和になる様攪拌しながら
徐々に加えた。加え終わってから更に１２時間攪拌を続
けた後、３０００×ｇで３０分間冷却遠心した。
得られた沈澱を５０％飽和硫安溶液に懸濁し、再び冷却
遠心して沈澱を得た。沈澱を１０ｍｌの１０ｍＭリン酸
ナトリウムバッファー（ｐＨ８．０）に溶解し、透析チ
ューブにつめて５Ｌの同バッファーに対して３回透析を
行った。得られた透析試料を予め１０ｍＭリン酸ナトリ
ウムバッファー（ｐＨ８．０）で平衡化させたＤＥＡＥ
−セルロースカラム（ＤＥ５２、ワットマン社）にアプ
ライし、同バッファーで溶出させた。素通り画分を集め
、セントリプレップＴＭ３０（アミコン社）で濃縮し、
１５．７ｍｇの部分精製抗ＺｔＰＩ抗体を得た。部分精
製抗体をアフィプレップＴＭプロテインＡカートリッジ
（バイオラッド社）を用い、バイオラッド社発行添付プ
ロトコールに従って、更に精製を行い、精製抗ＺｔＰＩ
ＩｇＧ１２．７ｍｇを得た。
［工程２］ ＡＰＰの調製
（ｉ） ＡＰＰ５９２の調製
ＡＰＰ６９５のＣ末端側１０３アミノ酸を欠失させた蛋
白である５９２アミノ酸（シグナルペプチドを含む）か
ら成るＡＰＰ５９２（図２）は、欧州特許公開番号０３
０４０１３号実施例１に記載の方法で得たヒト老人斑ア
ミロイド前駆体ＡＰＰ６９５ｃＤＮＡから出発して、上
述欧州特許公開番号０３０４０１３号実施例５に記載の
方法でサル腎臓由来のＣＯＳ−１細胞で産生させ培養上
清中へ分泌させ、精製した。以下詳述する。A part of the obtained inhibitor solution was added to SD
When analyzed by S-polyacrylamide gel electrophoresis, a major protein with a molecular weight of about 6 Kd was detected. [Step 1-2] Preparation of anti-ZtPI antiserum 300 μg of ZPI obtained in step 1-1 (iv) of this example was added to the sample buffer (2% SD
S, with 5% β-ME, but no dye added)
After processing at 100°C for 5 minutes, 4 times the volume of acetone was added, cooled at -20°C for 1 hour, centrifuged at 9000 rpm for 20 minutes, and the precipitate was collected. The precipitate was suspended in PBS(-), mixed well with complete Freund's adjuvant (FCA), and subcutaneously injected into rabbits. After 31 days, the same treatment (immunization) was performed except that incomplete Freund's adjuvant was used instead of FCA. After another 14 days, 300 μg of (v
) was subjected to the same treatment and immunized. After another 16 days, the same treatment was performed using ZtPI. After another 15 days, the mice were immunized with 150 μg of sPI described in Example 4 of European Patent Publication No. 0304013 using the same treatment. 50 ml of blood was collected from this rabbit to obtain antiserum. This antiserum is hereinafter referred to as anti-ZtPI antiserum. Antibody titer in serum was determined by solid-phase ELISA using non-denatured and SDS-denatured sPI (25 ng/well) as antigen. The final titer of the antiserum is expressed as the dilution of the antiserum that shows 3 times more color than the control (well without antigen).
64,000 times greater than undenatured sPI, SDS denatured sPI
It was 128,000 times that of the previous year. Furthermore, it did not react at all with urinary trypsin inhibitor (Nippon Chemical Research Co., Ltd.). That is, the present anti-ZtPI antiserum was found to have high specificity and antibody titer for the KPI sequence. [Step 1-3] Purification of anti-ZtPI IgG After inactivating 20 ml of the anti-ZtPI serum obtained in step 1-2 of this example at 56°C for 30 minutes, an equal amount of P
Dilute with BS(-), and gradually add 100% saturated ammonium sulfate solution (previously adjusted to pH 6.5 with 0.1N ammonia solution) at 4°C while stirring to reach a final concentration of 33% saturation. Ta. After the addition was completed, stirring was continued for an additional 12 hours, and then refrigerated centrifugation was performed at 3000 xg for 30 minutes. The obtained precipitate was suspended in a 50% saturated ammonium sulfate solution and centrifuged again under cooling to obtain a precipitate. The precipitate was dissolved in 10 ml of 10 mM sodium phosphate buffer (pH 8.0), packed into a dialysis tube, and dialyzed three times against 5 L of the same buffer. The obtained dialyzed sample was pre-equilibrated with 10mM sodium phosphate buffer (pH 8.0) using DEAE.
- It was applied to a cellulose column (DE52, Whatman) and eluted with the same buffer. Collect the flow-through fractions, concentrate with Centriprep TM30 (Amicon),
15.7 mg of partially purified anti-ZtPI antibody was obtained. The partially purified antibody was further purified using Affiprep™ Protein A cartridge (Bio-Rad) according to the attached protocol published by Bio-Rad, and purified anti-ZtPI was obtained.
12.7 mg of IgG was obtained. [Step 2] Preparation of APP (i) Preparation of APP592 APP592 (Figure 2), which consists of 592 amino acids (including a signal peptide) and is a protein in which the C-terminal 103 amino acids of APP695 are deleted, is disclosed in European Patent Publication No. 03.
Starting from the human senile plaque amyloid precursor APP695 cDNA obtained by the method described in Example 1 of No. 04013, it was produced in monkey kidney-derived COS-1 cells by the method described in Example 5 of the above-mentioned European Patent Publication No. 0304013. It was secreted into the culture supernatant and purified. The details will be explained below.
【００２８】欧州特許公開番号０３０４０１３号実施例
５工程２記載のプラスミドｐＳＶＭＴ５９２をＣＯＳ−
１細胞に導入した。即ち、ｐＳＶＭＴ５９２ ２０μ
ｇを、１×１０６ 個のＣＯＳ−１細胞に対し、ショ糖
含有ＰＢＳ（２７２ｍＭショ糖、７ｍＭリン酸ナトリウ
ム（ｐＨ７．４）１ｍＭＭｇＣｌ２）中で、バイオラッ
ド社のジーンパルサーＴＭを用い、電圧４００Ｖ、キャ
パシター３μＦ，１．０〜１．３ｍ秒で、３０秒の間隔
をおいて２回電気パルスを与えることにより導入を行っ
た。導入後の細胞を、１０％ウシ胎児血清（以下ＦＣＳ
と略す）を含むダルベッコの変法最小基本培地中で３７
℃、５％ＣＯ２ の条件下で２４時間培養後、ダルベッ
コのリン酸緩衝塩類溶液（Ｃａ２＋、Ｍｇ２＋不含）（
以下ＰＢＳ（−）と略す）で洗浄し、ＦＣＳを除いた後
、ＦＣＳを含まない培地に交換し、以後４８時間毎に培
地を交換し、計３回培養上清を回収した。更に導入を３
０回行い、計９００ｍｌの培養上清を得た。Plasmid pSVMT592 described in Example 5 Step 2 of European Patent Publication No. 0304013 was transformed into COS-
1 cell. That is, pSVMT592 20μ
g was applied to 1 x 106 COS-1 cells in sucrose-containing PBS (272 mM sucrose, 7 mM sodium phosphate (pH 7.4), 1 mM MgCl2) at a voltage of 400 V using Bio-Rad's Gene PulsarTM. The introduction was carried out by applying two electrical pulses with a 30 second interval between 1.0 and 1.3 msec using a 3 μF capacitor. After introduction, cells were treated with 10% fetal calf serum (hereinafter referred to as FCS).
37 in Dulbecco's modified minimal basal medium containing
After culturing for 24 hours at ℃ and 5% CO2, Dulbecco's phosphate buffered saline solution (Ca2+, Mg2+ free) (
After washing with PBS (hereinafter abbreviated as "-") to remove FCS, the medium was replaced with an FCS-free medium, and thereafter, the medium was replaced every 48 hours, and the culture supernatant was collected three times in total. Further introduction 3
This was repeated 0 times to obtain a total of 900 ml of culture supernatant.
【００２９】ついで、プラスミド導入ＣＯＳ細胞の培養
上清に対し終濃度２ｍＭになるよう（ｐ−アミジノフェ
ニル）メタンスルホニルフルオリド塩酸塩（和光純薬工
業株式会社）を添加し、ＣｅｎｔｒｉｐｒｅｐＴＭ３０
（アミコン社）を用いて濃縮を行いさらに同装置中で２
０ｍＭＴｒｉｓ−塩酸（ｐＨ８．０）にバッファー交換
を行った。次に、ＤＥＡＥ−Ｓｅｐｈａｒｏｓｅカラム
（ファルマシア社）を用い、上記試料をアプライし、２
０ｍＭＴｒｉｓ−塩酸（ｐＨ８．０）、ＮａＣｌ勾配０
−１Ｍの条件でカラムクロマトグラフィーを行った。ク
ロマトグラフィーの各画分について、後述する参考例２
において大腸菌で発現させたＡＰＰ１９−３７９ （Ｚ
ＫＸ）に対して作成したウサギ抗ＡＰＰ１９−３７９
（ＺＫＸ）抗血清を用いた固相ＥＬＩＳＡを行い、ＡＰ
Ｐ溶出位置を同定した。（ＺＫＸは、ＡＰＰ７７０中の
Ｎ末端から１９番目のアミノ酸残基から３７９番目のア
ミノ酸残基までを含むポリペプチドとβ−ガラクトシダ
ーゼとの融合タンパクであり、大腸菌中に発現させ、６
Ｍ尿素による可溶化を行い精製した。（精製法は参考例
２で詳述した。）ＺＫＸは、ＡＰＰ７７０、ＡＰＰ７５
１、ＡＰＰ６９５に共通な配列のほかに、プロテアーゼ
インヒビター部分及びＡＰＰＰ７７０に特異的な配列を
含むが、ＡＰＰの膜貫通領域からＣ末端までの配列は含
まない）。
ＡＰＰ５９２は、０．３−０．４ＭＮａＣｌで溶出され
た。ＥＬＩＳＡ陽性画分をセントリコンＴＭ３０（アミ
コン社）を用いて濃縮し、さらに同装置中で５０ｍＭリ
ン酸ナトリウム（ｐＨ７．２）にバッファー交換した。Next, (p-amidinophenyl)methanesulfonyl fluoride hydrochloride (Wako Pure Chemical Industries, Ltd.) was added to the culture supernatant of the plasmid-introduced COS cells to a final concentration of 2 mM, and CentriprepTM30
(Amicon) for concentration and further 2
Buffer exchange was performed to 0mM Tris-HCl (pH 8.0). Next, the above sample was applied to a DEAE-Sepharose column (Pharmacia), and 2
0mM Tris-HCl (pH 8.0), NaCl gradient 0
Column chromatography was performed under -1M conditions. Regarding each fraction of chromatography, refer to Reference Example 2 described below.
APP19-379 (Z
Rabbit anti-APP19-379 created against KX)
(ZKX) Solid-phase ELISA using antiserum was performed, and AP
The P elution position was identified. (ZKX is a fusion protein of β-galactosidase and a polypeptide containing the 19th amino acid residue to the 379th amino acid residue from the N-terminus of APP770.
It was purified by solubilization with M urea. (The purification method was detailed in Reference Example 2.) ZKX is APP770, APP75
1. In addition to the sequences common to APP695, it contains a protease inhibitor portion and a sequence specific to APP770, but does not contain the sequence from the transmembrane region to the C-terminus of APP). APP592 was eluted at 0.3-0.4M NaCl. The ELISA-positive fraction was concentrated using Centricon TM30 (Amicon), and buffer exchanged into 50 mM sodium phosphate (pH 7.2) in the same device.
【００３０】次にヘパリンカラムを用いたアフィニティ
ークロマトグラフィーを行った。即ち、ＤＥＡＥ−Ｓｅ
ｐｈａｒｏｓｅカラム（ファルマシア社）による部分精
製画分をヘパリン−５ＰＷカラム（東ソー株式会社）に
アプライし、ＮａＣｌ勾配０−２Ｍの条件でクロマトグ
ラフィーを行った。クロマトグラフィーの各画分につい
て同様にウサギ抗ＡＰＰ１９−３７９ （ＺＫＸ）抗血
清を用いた固相ＥＬＩＳＡを行い、ＡＰＰ５９２溶出位
置を同定した。ＡＰＰ５９２は１．０−１．２ＭＮａＣ
ｌで溶出した。ＥＬＩＳＡ陽性画分をセントリコンＴＭ
３０（アミコン社）を用いて濃縮後、２０ｍＭＴｒｉｓ
−塩酸（ｐＨ８．０）にバッファー交換した。これらを
７．５％ＳＤＳ−ＰＡＧＥにかけ、Ｃｏｏｍａｓｓｉｅ
染色を行い、純度及び分子量の確認を行った。ｐＳＶＭ
Ｔ５９２を導入したＣＯＳ−１細胞培養上清から、上記
操作を行うことにより得られたタンパクは、ＳＤＳ−Ｐ
ＡＧＥ上でややブロードな単一バンドであり、見かけの
分子量は９４．５ｋＤａと決定された。更に、この蛋白
のＮ末端のアミノ酸配列を、アプライド社気相シーケン
サーで解析したところ、ＡＰＰ６９５のうちシグナルペ
プチド（Ｎ末端から１７アミノ酸）が除去された、ＡＰ
Ｐ６９５のＮ末端から１８番目のＬｅｕから３３番目の
Ｇｌｎまでのアミノ酸配列（ＡＰＰ７５１、ＡＰＰ７７
０にも共通）が確認された。こうして、精製ＡＰＰ５９
２が得られた。Next, affinity chromatography using a heparin column was performed. That is, DEAE-Se
The partially purified fraction by a Pharose column (Pharmacia) was applied to a heparin-5PW column (Tosoh Corporation), and chromatography was performed under the conditions of a 0-2M NaCl gradient. Solid-phase ELISA using rabbit anti-APP19-379 (ZKX) antiserum was similarly performed on each chromatographic fraction to identify the APP592 elution position. APP592 is 1.0-1.2M NaC
It eluted at 1. ELISA positive fraction to Centricon™
After concentration using 30 (Amicon), 20mM Tris
- Buffer exchanged to hydrochloric acid (pH 8.0). These were subjected to 7.5% SDS-PAGE and Coomassie
Staining was performed to confirm purity and molecular weight. pSVM
The protein obtained by performing the above procedure from the culture supernatant of COS-1 cells introduced with T592 was subjected to SDS-P.
It was a rather broad single band on AGE, and the apparent molecular weight was determined to be 94.5 kDa. Furthermore, when the amino acid sequence of the N-terminus of this protein was analyzed using an Applied gas phase sequencer, it was found that the signal peptide (17 amino acids from the N-terminus) of APP695 was removed.
Amino acid sequence from the 18th Leu to the 33rd Gln from the N-terminus of P695 (APP751, APP77
0) was confirmed. Thus, purified APP59
2 was obtained.
【００３１】（ｉｉ） ＡＰＰ６４８及びＡＰＰ６６７
の調製
ＡＰＰ７５１、ＡＰＰ７７０各々のＣ末端側１０３アミ
ノ酸を欠失させた蛋白である、６４８アミノ酸（シグナ
ルペプチドを含む）から成るＡＰＰ６４８（図２）、及
び、６６７アミノ酸（シグナルペプチドを含む）から成
るＡＰＰ６６７（図２）は、欧州特許公開番号０３０４
０１３号実施例５工程２記載のプラスミドｐＳＶＭＴ６
６７、及びｐＳＶＭＴ６４８を、本実施例工程２（ｉ）
と同様にして、ＣＯＳ−１細胞への導入、培養上清の
回収、精製を行った。精製に際しては、クロマトグラフ
ィーの各フラクションの確認に、ウサギ抗ＡＰＰ１９−
３７９ 抗血清の他に、ＡＰＰ６６７については、特開
平２−１３８９９５号公報の実施例９で得たモノクロー
ナル抗体ＡＤＪ５−３−７（微工研菌寄第１０３８８号
）も用いた。さらに、ＡＰＰ６６７及びＡＰＰ６４８に
ついては、そのトリプシン阻害活性も確認手段の一つと
し、その両者とも強い阻害活性を示すことを確認した（
阻害活性測定は、欧州特許公開番号０３０４０１３号実
施例に記載のｓＰＩのトリプシン阻害活性測定の方法に
準じて行った）。精製したＡＰＰ６４８、ＡＰＰ６６７
の分子量は、還元条件下のＳＤＳ−ＰＡＧＥで、それぞ
れ１００．５ｋＤａ、１０５．５ｋＤａであった。
［工程３］ 抗ＡＰＰ抗体の作製
［工程３−１］ 抗ＡＰＰモノクローナル抗体産生ハ
イブリドーマ３Ｂ２及び６Ａ３の作製
（ｉ）マウスの感作
ハイブリドーマ作成のための抗原は、以下のようにして
調製した。本実施例工程２（ｉ） で得た精製ＡＰＰ５
９２をＰＢＳ（−）に溶かし、ＬａｅｍｍｌｉのＳＤＳ
−ＰＡＧＥサンプルバッファー（Ｌａｅｍｍｌｉ Ｕ
．Ｋ．Ｎａｔｕｒｅ２２７ ６８０−６８５（１９７
０））中で１００℃、３分間煮沸後、４倍容のアセトン
を加え、−２０℃で１時間冷却を行い、９０００回転２
０分間の遠心操作を行った。こうして過剰量のＳＤＳを
除去し、沈査よりＳＤＳ変性ＡＰＰ５９２を回収し、こ
れをハイブリドーマ作成のための抗原として用いた。免
疫は２０μｇのＳＤＳ変性ＡＰＰ５９２を５００μｌの
ＰＢＳ（−）に溶かしたものをそれぞれ３匹の６週齢の
Ｂａｌｂ／ｃ雌マウスの皮下にフロイントの完全アジュ
バントと共に注射し、以後２週間の間隔で合計３回の免
疫を行った。さらに、１週間の間隔で２回皮下注射を行
った。(ii) APP648 and APP667
Preparation of APP751 and APP770, which are proteins with the C-terminal 103 amino acids deleted, APP648 (Figure 2) consisting of 648 amino acids (including the signal peptide), and APP667 consisting of 667 amino acids (including the signal peptide). (Figure 2) is European Patent Publication No. 0304
Plasmid pSVMT6 described in No. 013, Example 5, Step 2
67, and pSVMT648 in step 2(i) of this example.
In the same manner as above, introduction into COS-1 cells, collection of culture supernatant, and purification were performed. During purification, rabbit anti-APP19-
In addition to the 379 antiserum, for APP667, the monoclonal antibody ADJ5-3-7 obtained in Example 9 of JP-A-2-138995 (Feikoken Bibori No. 10388) was also used. Furthermore, for APP667 and APP648, their trypsin inhibitory activity was also confirmed as a means of confirmation, and it was confirmed that both of them exhibited strong inhibitory activity (
The inhibitory activity was measured according to the method for measuring the trypsin inhibitory activity of sPI described in the Examples of European Patent Publication No. 0304013). Purified APP648, APP667
The molecular weights were 100.5 kDa and 105.5 kDa, respectively, by SDS-PAGE under reducing conditions. [Step 3] Preparation of anti-APP antibody [Step 3-1] Preparation of anti-APP monoclonal antibody-producing hybridomas 3B2 and 6A3 (i) Sensitization of mice Antigens for the preparation of hybridomas were prepared as follows. Purified APP5 obtained in step 2(i) of this example
92 in PBS(-) and Laemmli's SDS
-PAGE sample buffer (Laemmli U
．． K. Nature227 680-685 (197
After boiling at 100℃ for 3 minutes in 0)), add 4 times the volume of acetone, cool at -20℃ for 1 hour, and boil at 9000 rpm for 2 minutes.
Centrifugation was performed for 0 minutes. In this manner, excess SDS was removed, and SDS-denatured APP592 was recovered from the sediment, which was used as an antigen for hybridoma production. For immunization, 20 μg of SDS-denatured APP592 dissolved in 500 μl of PBS (-) was subcutaneously injected into three 6-week-old Balb/c female mice together with complete Freund's adjuvant, and the total injection was performed at intervals of 2 weeks thereafter. Three immunizations were performed. Furthermore, two subcutaneous injections were performed at one week intervals.
【００３２】免疫の過程で抗原に対する血清の抗体価の
上昇を単クローン抗体実験マニュアル（講談社サイエン
ティフィク富山朔二著）に従って、固相ＥＬＩＳＡ法に
より確認した。
（ｉｉ） 細胞融合
単クローン抗体実験マニュアル（講談社サイエンティフ
ィク富山朔二著）に従って細胞融合を行った。即ち、血
清の抗体価の最も高かったマウスに２００μｌのＰＢＳ
（−）に溶かした２５μｇのＳＤＳ変性ＡＰＰ５９２を
尾静脈より注射し、ブーストとした。その３日後に、脾
細胞を無菌的に取り出し、ステンレスメッシュで単細胞
にほぐし、脾細胞の１／５量の８−アザグアニン耐性骨
髄細胞腫株Ｐ３Ｘ６３−Ａｇ８−６５３細胞（株式会社
免疫生物研究所より入手）と、ＭｅｄｉａＩ培地（
株式会社 免疫生物研究所）を混合し、遠沈後、細胞
のペレットに５０％ＰＥＧ１５００（ベーリンガー社製
）を加え、融合操作を行った。[0032] During the course of immunization, an increase in serum antibody titer against the antigen was confirmed by solid-phase ELISA according to the Monoclonal Antibody Experimental Manual (written by Sakuji Toyama, Kodansha Scientific). (ii) Cell fusion Cell fusion was performed according to the Monoclonal Antibody Experiment Manual (written by Sakuji Toyama, Kodansha Scientific). That is, 200 μl of PBS was added to the mouse whose serum antibody titer was highest.
25 μg of SDS-denatured APP592 dissolved in (-) was injected through the tail vein to serve as a boost. Three days later, the splenocytes were removed aseptically and loosened into single cells using a stainless steel mesh, and 8-azaguanine-resistant myeloid cell line P3X63-Ag8-653 (1/5 amount of splenocytes) (from Immuno-Biological Laboratories Co., Ltd.) obtained) and Media I medium (
After mixing and centrifugation, 50% PEG1500 (manufactured by Boehringer) was added to the cell pellet to perform a fusion operation.
【００３３】その後、融合細胞を遠心して上清を吸引除
去した後、３０％ＦＣＳ入りＨＡＴ培地を加え、９６穴
マイクロプレート１１枚にまいた。融合後、約２週間で
約９５０ウェルからコロニーが生育し、これらの培養上
清についてＳＤＳ変性ＡＰＰ５９２をスクリーニング用
抗原とした固相ＥＬＩＳＡを行い、抗原と強く反応する
クローンを得、限界希釈法によるクローニングを３回繰
り返した。３回のクローニングにより安定した抗体産生
を示す２つのクローンを３Ｂ２及び６Ａ３と各々命名し
、これを平成３年１月１０日付けで微生物工業技術研究
所に寄託した。（３Ｂ２の受託番号は、微工研菌寄第１
１９４３号、および、６Ａ３のそれは微工研菌寄第１１
９４４号）。これらのハイブリドーマが産生するモノク
ローナル抗体３Ｂ２及び６Ａ３の反応性を本実施例工程
２（ｉ） 及び（ｉｉ）で作成した、ＡＰＰ６６７、Ａ
ＰＰ６４８、ＡＰＰ５９２に対して検討したところ、後
述するように、ＳＤＳ変性、非変性何れの条件でもこれ
ら３種のＡＰＰ誘導体と強く反応した。即ち、これらの
モノクローナル抗体は、ＡＰＰ７７０、ＡＰＰ７５１、
ＡＰＰ６９５の何れをも認識できることがわかった。
［工程３−２］ 抗ＡＰＰモノクローナル抗体産生ハ
イブリドーマ１Ｂ２の作製
（ｉ） マウスの感作
後述するように参考例２で得たＺＫＸを、上述の本実施
例工程３−１（ｉ） 記載と同様の方法で変性させた。
このＳＤＳ変性ＺＫＸ２０μｇを５００μｌのＰＢＳ（
−）に溶かしたものをそれぞれ４匹の６週齢のＢａｌｂ
／ｃ雌マウスの皮下にフロイントの完全アジュバントと
共に注射し、２週間後に同様の免疫を行った。さらに、
２週間の間隔で、ＳＤＳ変性ＺＫＸの免疫を、フロイン
トの不完全アジュバントを用いて６回行った。その２週
間ののちＳＤＳ変性ＺＫＸ２０μｇを静注した。約１カ
月後、今度は２０μｇのＳＤＳ変性ＡＰＰ５９２をフロ
イントの完全アジュバントと共に皮下に注射し、さらに
１週間後同様にしてＡＰＰ５９２による免疫を行った。[0033] Thereafter, the fused cells were centrifuged and the supernatant was removed by suction, and then HAT medium containing 30% FCS was added and plated on 11 96-well microplates. Colonies grew from about 950 wells in about two weeks after fusion, and solid-phase ELISA was performed on these culture supernatants using SDS-denatured APP592 as a screening antigen.Clones that strongly reacted with the antigen were obtained, and clones were isolated by limiting dilution method. Cloning was repeated three times. Two clones showing stable antibody production after cloning three times were named 3B2 and 6A3, respectively, and were deposited with the Microbial Technology Research Institute on January 10, 1991. (The accession number of 3B2 is
No. 1943 and that of 6A3 are published in the 11th issue of
No. 944). The reactivity of monoclonal antibodies 3B2 and 6A3 produced by these hybridomas was determined using APP667 and A, which were prepared in steps 2(i) and (ii) of this example.
When PP648 and APP592 were examined, as described below, they strongly reacted with these three APP derivatives under both SDS denaturation and non-denaturation conditions. That is, these monoclonal antibodies are APP770, APP751,
It was found that any of APP695 could be recognized. [Step 3-2] Preparation of anti-APP monoclonal antibody-producing hybridoma 1B2 (i) After sensitization of mice, ZKX obtained in Reference Example 2 was subjected to the same procedure as described in Step 3-1 (i) of the present example described above. It was denatured using the method. 20 μg of this SDS-denatured ZKX was added to 500 μl of PBS (
-) of 4 6-week-old Bals.
/c female mice were subcutaneously injected together with complete Freund's adjuvant, and 2 weeks later a similar immunization was performed. moreover,
Six immunizations of SDS-modified ZKX were performed using incomplete Freund's adjuvant at two-week intervals. Two weeks later, 20 μg of SDS-denatured ZKX was intravenously injected. Approximately one month later, 20 μg of SDS-modified APP592 was injected subcutaneously together with Freund's complete adjuvant, and one week later, immunization with APP592 was performed in the same manner.
【００３４】免疫の過程で抗原に対する血清の抗体価の
上昇を本実施例工程３−１と同様にして、固相ＥＬＩＳ
Ａ法により確認した。
（ｉｉ） 細胞融合
血清の抗体価の最も高かったマウスに２００μｌのＰＢ
Ｓ（−）に溶かした２５μｇのＳＤＳ変性ＡＰＰ５９２
を尾静脈より注射し、ブーストとした。これ以降の操作
は、本実施例工程３−１（ｉｉ）と同様に行い、ＡＰＰ
５９２と強く反応するクローンを得、限界希釈法による
クローニングを３回繰り返した。３回のクローニングに
より安定した抗体産生を示すクローンを１Ｂ２と命名し
、これを平成３年１月１０日付けで微生物工業技術研究
所に寄託した（微工研菌寄第１１９４２号）。このハイ
ブリドーマが産生するモノクローナル抗体１Ｂ２の反応
性を本実施例工程２（ｉ） 及び（ｉｉ）で作成した、
ＡＰＰ６６７、ＡＰＰ６４８、ＡＰＰ５９２に対して検
討したところ、後述するように、ＳＤＳ変性、非変性何
れの条件でもこれら３種のＡＰＰ誘導体と強く反応した
。即ち、モノクローナル抗体１Ｂ２は、ＡＰＰ７７０、
ＡＰＰ７５１、ＡＰＰ６９５の何れをも認識できること
がわかった。
［工程４］ モノクローナル抗体の特徴づけ（ｉ）免
疫グロブリンのクラス及びサブクラスの決定モノクロー
ナル抗体１Ｂ２，３Ｂ２，６Ａ３の免疫グロブリンのク
ラス及びサブクラスをアイソタイピングキット（アマー
シャム社）を使用して決定した。その結果、１Ｂ２につ
いてはＩｇＧ２ｂ、Ｌ鎖はｋ、３Ｂ２についてはＩｇＧ
２ｂ、Ｌ鎖はｋ、６Ａ３についてはＩｇＧ２ａ、Ｌ鎖は
ｋと決定された。
（ｉｉ）特異性の決定
上記モノクローナル抗体の特異性を以下に示す試験によ
り決定した。[0034] During the immunization process, the increase in serum antibody titer against the antigen was measured using solid-phase ELIS in the same manner as in step 3-1 of this example.
Confirmed by method A. (ii) 200 μl of PB to the mouse with the highest antibody titer in the cell fusion serum.
25 μg of SDS-denatured APP592 dissolved in S(-)
was injected into the tail vein as a boost. The subsequent operations are performed in the same manner as in step 3-1(ii) of this example, and the APP
A clone that strongly reacted with 592 was obtained, and cloning by limiting dilution method was repeated three times. A clone showing stable antibody production after three clonings was named 1B2, and was deposited with the Microbial Technology Research Institute on January 10, 1991 (Feikoken Bacterium Deposit No. 11942). The reactivity of monoclonal antibody 1B2 produced by this hybridoma was determined by the steps 2(i) and (ii) of this example.
When APP667, APP648, and APP592 were investigated, as described below, they strongly reacted with these three APP derivatives under both SDS denaturation and non-denaturation conditions. That is, monoclonal antibody 1B2 contains APP770,
It was found that both APP751 and APP695 could be recognized. [Step 4] Characterization of monoclonal antibodies (i) Determination of immunoglobulin class and subclass The immunoglobulin class and subclass of monoclonal antibodies 1B2, 3B2, and 6A3 were determined using an isotyping kit (Amersham). As a result, IgG2b for 1B2, k for the L chain, and IgG2b for 3B2.
2b, the L chain was determined to be k, and for 6A3, IgG2a, the L chain was determined to be k. (ii) Determination of specificity The specificity of the above monoclonal antibody was determined by the test shown below.
【００３５】各種ＡＰＰ（ＡＰＰ６６７、ＡＰＰ６４８
、ＡＰＰ５９２）を用い、非変性及びＳＤＳ−変性処理
を行い、通常の固相ＥＬＩＳＡ法を用いて判定を行った
。一次抗体として上記モノクローナル抗体を２μｇ／ｍ
ｌのＩｇＧ濃度で加え、二次抗体はβ−ガラクトシダー
ゼ標識ヒツジＦ（ａｂ′）２ 抗マウスＩｇ（アマーシ
ャム社）を用い、基質は４−メチルウンベリフェリル−
β−Ｄ−ガラクトピラノシドを用い、３０分間酵素反応
を行った後、蛍光リーダーＦＣＡ（ＰＡＮＤＥＸ社）を
用いて蛍光強度を測定した。その結果を表１に示す。[0035] Various APPs (APP667, APP648
, APP592), non-denaturing and SDS-denaturing treatments were performed, and determination was made using a conventional solid-phase ELISA method. 2 μg/m of the above monoclonal antibody as the primary antibody
The secondary antibody was β-galactosidase-labeled sheep F(ab')2 anti-mouse Ig (Amersham), and the substrate was 4-methylumbelliferyl-
After performing an enzyme reaction for 30 minutes using β-D-galactopyranoside, the fluorescence intensity was measured using a fluorescence reader FCA (PANDEX). The results are shown in Table 1.
【００３６】本発明によるモノクローナル抗体、１Ｂ２
、３Ｂ２、６Ａ３は、いずれも、３種のＡＰＰ（ＡＰＰ
６６７、ＡＰＰ６４８及びＡＰＰ５９２）のすべてを、
強く認識した。ネガティブコントロールとしてウシ血清
アルブミン（ＢＳＡ）との反応性も検討したが、これら
３つのモノクローナル抗体はＢＳＡとは全く反応しなか
った。
［工程５］ 腹水化モノクローナル抗体からのＩｇＧ
の精製
大量のモノクローナル抗体を得るため工程３で得た１Ｂ
２、３Ｂ２、６Ａ３の各ハイブリドーマ１×１０７ 個
を、ＭｅｄｉａＩ培地（株式会社 免疫生物研究所）
０．５ｍｌに浮遊させ、予め１週間前に腹腔内に０．５
ｍｌのプリスタン（アルドリッチ社）投与しておいたＢ
ａｌｂ／ｃマウスの腹腔に注射した。約２週間で腹部の
肥大が認められ、腹腔切開により腹水を採取した。各ハ
イブリドーマにつき７匹のマウスを処理し、各々約１０
ｍｌずつの腹水を得た。得られた各ハイブリドーマ由来
腹水５ｍｌずつを、アフィプレップＴＭプロテインＡカ
ートリッジ（バイオラッド社）を用いて精製を行った。
精製はバイオラッド社発行の添付プロトコールに従った
。
１Ｂ２クローンから７．９ｍｇ、３Ｂ２クローンから４
．３ｍｇ、６Ａ３クローンから３．３ｍｇの精製ＩｇＧ
が得られた。
〔工程６〕抗体のビオチン化
本実施例工程１−３及び工程５で得た抗ＺｔＰＩ抗体及
び１Ｂ２、３Ｂ２及び６Ａ３精製モノクローナル抗体各
２ｍｇを用い、免疫実験操作法（第９巻、３５３９−３
５４９ １９８２ 日本免疫学会編）に従って抗体
のビオチン化を行った。予め０．１ＭＮａＨＣＯ３ 溶
液に対して透析、バッファー交換を行い、濃度を１ｍｇ
／ｍｌに調整しておいた精製１Ｂ２、３Ｂ２、６Ａ３、
及び抗ＺｔＰＩ抗体に、１ｍｇ／ｍｌの濃度に調整した
ＮＨＳ−ビオチン（Ｎ−ヒドロキシサクシニミド−ビオ
チン、ピアス社）のジメチルスルホキシド溶液２８０μ
ｌを加え、室温で４時間静かに攪拌した。次に、４°Ｃ
で３ＬのＰＢＳ（−）に対して４回の透析を行い、ビオ
チン標識１Ｂ２、３Ｂ２、６Ａ３及び抗ＺｔＰＩ抗体を
得た。Monoclonal antibody according to the invention, 1B2
, 3B2, and 6A3 are all three types of APP (APP
667, APP648 and APP592),
I strongly recognized it. Reactivity with bovine serum albumin (BSA) was also examined as a negative control, but these three monoclonal antibodies did not react with BSA at all. [Step 5] IgG from ascitic monoclonal antibody
1B obtained in step 3 to obtain a large amount of purified monoclonal antibody.
1 x 107 hybridomas of 2, 3B2, and 6A3 were cultured in MediaI medium (Immuno-Biological Laboratories Co., Ltd.).
Suspend in 0.5ml and inject 0.5ml intraperitoneally one week beforehand.
ml of pristane (Aldrich) was administered to B.
injected into the peritoneal cavity of alb/c mice. After about 2 weeks, abdominal enlargement was observed, and ascites was collected through abdominal incision. Seven mice were treated for each hybridoma, approximately 10
ml of ascites was obtained. 5 ml of the obtained ascites derived from each hybridoma was purified using an Affiprep™ Protein A cartridge (Bio-Rad). Purification was performed according to the attached protocol published by Bio-Rad. 7.9 mg from 1B2 clone, 4 from 3B2 clone
．． 3 mg, 3.3 mg purified IgG from 6A3 clone
was gotten. [Step 6] Biotinylation of Antibodies Using 2 mg each of the anti-ZtPI antibody obtained in Steps 1-3 and 5 of this Example and the purified monoclonal antibodies 1B2, 3B2, and 6A3, the Immunization Experiment Procedures (Vol. 9, 3539-3)
549 1982 edited by the Japanese Society of Immunology), antibodies were biotinylated. Dialysis and buffer exchange were performed on 0.1M NaHCO3 solution in advance, and the concentration was adjusted to 1mg.
/ml purified 1B2, 3B2, 6A3,
and anti-ZtPI antibody, 280 μl of a dimethyl sulfoxide solution of NHS-biotin (N-hydroxysuccinimide-biotin, Pierce) adjusted to a concentration of 1 mg/ml.
1 was added thereto, and the mixture was gently stirred at room temperature for 4 hours. Next, 4°C
Dialysis was performed four times against 3 L of PBS(-) to obtain biotin-labeled 1B2, 3B2, 6A3 and anti-ZtPI antibody.
【００３７】[0037]
【実施例２】 抗ＺｔＰＩ抗体と抗ＺｔＰＩ抗体の組
み合わせによる２抗体サンドイッチＥＬＩＳＡ実施例１
工程１−３で得た精製抗ＺｔＰＩ抗体を５μｇ／ｍｌの
濃度で０．１Ｍ炭酸ナトリウム−炭酸水素ナトリウムバ
ッファー（ｐＨ９．６）に溶解し、９６穴マイクロプレ
ートに１ウェルあたり５０μｌずつ加え、室温で２時間
静置した。プレートをＰＢＳ（−）で３回洗浄の後、１
％ウシ血清アルブミン（シグマ社）を含むＰＢＳ（−）
を加え、３７°Ｃで２時間被覆した。次に０．１％ウシ
血清アルブミンを含むＰＢＳ（−）中で希釈した精製Ａ
ＰＰ６６７標準液（２〜２０００ｎｇ／ｍｌ）を加え、
攪拌しながら室温で１時間処理を行った。次に、０．０
５％のＴｗｅｅｎ−２０を含むＰＢＳ（−）でプレート
を３回洗浄した後、実施例１の工程６で得たビオチン標
識した精製抗ＺｔＰＩ ＩｇＧを１μｇ／ｍｌの濃度
で５０μｌずつ各ウェルに加え、攪拌しながら室温で１
時間反応を行った。０．０５％のＴｗｅｅｎ−２０を含
むＰＢＳ（−）で３回洗浄した後、β−ガラクトシダー
ゼ標識アビジンＤ（ベクター社）を加え、更に攪拌しな
がら室温で１時間反応させた。０．０５％のＴｗｅｅｎ
−２０を含むＰＢＳ（−）で５回洗浄の後、２ｍＭ
ＭｇＣｌ２ を含むＰＢＳ（−）中に溶かした４−メチ
ルウンベリフェリル−β−Ｄ−ガラクトピラノシド（１
ｍｇ／ｍｌ）を加え、室温で３０分反応させた後、ＦＣ
Ａ（ＰＡＮＤＥＸ社）で蛍光強度を測定した。結果は図
３に示す様に検出限界が約１ｎｇ／ウェルであった。[Example 2] Two-antibody sandwich ELISA Example 1 using a combination of anti-ZtPI antibody and anti-ZtPI antibody
The purified anti-ZtPI antibody obtained in step 1-3 was dissolved in 0.1 M sodium carbonate-sodium bicarbonate buffer (pH 9.6) at a concentration of 5 μg/ml, added to a 96-well microplate in an amount of 50 μl per well, and incubated at room temperature. It was left undisturbed for 2 hours. After washing the plate 3 times with PBS(-),
PBS (-) containing % bovine serum albumin (Sigma)
was added and coated for 2 hours at 37°C. Purified A was then diluted in PBS(-) containing 0.1% bovine serum albumin.
Add PP667 standard solution (2-2000ng/ml),
The treatment was carried out at room temperature for 1 hour while stirring. Next, 0.0
After washing the plate three times with PBS (-) containing 5% Tween-20, 50 μl of the biotin-labeled purified anti-ZtPI IgG obtained in Step 6 of Example 1 was added to each well at a concentration of 1 μg/ml. , 1 at room temperature with stirring.
A time reaction was performed. After washing three times with PBS (-) containing 0.05% Tween-20, β-galactosidase-labeled avidin D (Vector) was added, and the mixture was reacted for 1 hour at room temperature with further stirring. 0.05% Tween
After washing 5 times with PBS(-) containing -20, 2mM
4-Methylumbelliferyl-β-D-galactopyranoside (1
mg/ml) and reacted at room temperature for 30 minutes, then FC
Fluorescence intensity was measured using A (PANDEX). As shown in FIG. 3, the detection limit was approximately 1 ng/well.
【００３８】[0038]
【実施例３】 抗ＺｔＰＩ抗体と１Ｂ２モノクローナ
ル抗体の組み合わせによる２抗体サンドイッチＥＬＩＳ
Ａ実施例２と同様の方法を用いて抗ＺｔＰＩ抗体と１Ｂ
２モノクローナル抗体との組み合わせによる２抗体サン
ドイッチＥＬＩＳＡの検討を行った。結果は図４に示す
様に検出限界が約３ｎｇ／ウェルであった。[Example 3] Two-antibody sandwich ELIS using a combination of anti-ZtPI antibody and 1B2 monoclonal antibody
A Using the same method as in Example 2, anti-ZtPI antibody and 1B
A two-antibody sandwich ELISA in combination with two monoclonal antibodies was investigated. As shown in FIG. 4, the detection limit was about 3 ng/well.
【００３９】[0039]
【実施例４】 抗ＺｔＰＩ抗体と３Ｂ２モノクローナ
ル抗体の組み合わせによる２抗体サンドイッチＥＬＩＳ
Ａ実施例２と同様の方法を用いて抗ＺｔＰＩ抗体と３Ｂ
２モノクローナル抗体の組み合わせによる２抗体サンド
イッチＥＬＩＳＡの検討を行った。結果は図５に示す様
に検出限界が約５ｎｇ／ウェルであった。[Example 4] Two-antibody sandwich ELIS using a combination of anti-ZtPI antibody and 3B2 monoclonal antibody
A Using the same method as in Example 2, anti-ZtPI antibody and 3B
A two-antibody sandwich ELISA using a combination of two monoclonal antibodies was investigated. As shown in FIG. 5, the detection limit was about 5 ng/well.
【００４０】[0040]
【実施例５】 抗ＺｔＰＩ抗体と６Ａ３モノクローナ
ル抗体の組み合わせによる２抗体サンドイッチＥＬＩＳ
Ａ実施例２と同様の方法を用いて抗ＺｔＰＩ抗体と抗６
Ａ３モノクローナル抗体の組み合わせによる２抗体サン
ドイッチＥＬＩＳＡの検討を行った。結果は図６に示す
様に検出限界が約２ｎｇ／ウェルであった。[Example 5] Two-antibody sandwich ELIS using a combination of anti-ZtPI antibody and 6A3 monoclonal antibody
A Using the same method as in Example 2, anti-ZtPI antibody and anti-6
A two-antibody sandwich ELISA using a combination of A3 monoclonal antibodies was investigated. As shown in FIG. 6, the detection limit was approximately 2 ng/well.
【００４１】[0041]
【参考例１】 抗ＺｔＰＩ抗体を用いたＲＩＡ（ラジ
オイムノアッセイ）法によるＡＰＰ６６７の検出（ｉ）
バッファー
１５０ｍＭ ＮａＣｌ、０．１％ゼラチン（バイオラ
ッド社）及び０．０２％ＮａＮ３ を含む５０ｍＭリン
酸ナトリウムバッファー（ｐＨ７．０）を調製し、バッ
ファーＡとした。[Reference Example 1] Detection of APP667 by RIA (radioimmunoassay) using anti-ZtPI antibody (i)
Buffer A 50 mM sodium phosphate buffer (pH 7.0) containing 150 mM NaCl, 0.1% gelatin (Bio-Rad) and 0.02% NaN3 was prepared and designated as buffer A.
【００４２】（ｉｉ） １２５Ｉ−ＡＰＰ６６７の調
製実施例１工程２（ｉｉ）で得た精製ＡＰＰ６６７をボ
ルトン−ハンター法（Ｂｏｌｔｏｎら、Ｂｉｏｃｈｅｍ
．Ｊ．１３３、５２９−５３９、１９７３）によって１
２５ Ｉ標識し、 １２５Ｉ−ＡＰＰ６６７を得た。
（ｉｉｉ） ＡＰＰ６６７標準液の調製実施例１工程
２（ｉｉ）で得た精製ＡＰＰ６６７を２０ｍＭ Ｔｒ
ｉｓ−ＨＣｌ（ｐＨ８．０）に溶解し、バッファーＡで
希釈し、種々の濃度のＡＰＰ６６７標準液を作成した。(ii) Preparation of 125I-APP667 The purified APP667 obtained in Example 1 step 2(ii) was purified by the Bolton-Hunter method (Bolton et al., Biochem).
．． J. 133, 529-539, 1973) by 1
25I labeling was performed to obtain 125I-APP667. (iii) Preparation of APP667 standard solution The purified APP667 obtained in Example 1 step 2(ii) was diluted with 20mM Tr.
It was dissolved in is-HCl (pH 8.0) and diluted with buffer A to prepare APP667 standard solutions at various concentrations.
【００４３】（ｉｖ） ＡＰＰ６６７のＲＩＡＡＰ
Ｐ６６７標準液１００μｌを、バッファーＡで１μｇ／
ｍｌの濃度に調製した精製抗ＺｔＰＩ抗体（実施例１工
程１−１（ｖｉｉｉ））溶液２００μｌ、 １２５Ｉ−
標識ＡＰＰ６６７のバッファーＡ溶液１００μｌ（１０
０００ｃｐｍ）、及びバッファーＡ２００μｌを加えて
混和後、４°Ｃ、１８時間インキュベートした。デキス
トラン−炭素液（デキストランＴ−７０（フナコシ薬品
株式会社）、０．００２５％溶液と活性炭Ｎｏｒｉｔ
Ａ（シグマ社）０．２５％液で等量混合した液）２０
０μｌを添加して、４°Ｃで１８時間静置後、２．５０
０×ｇで２０分間遠心分離し、上清の放射能をガンマウ
ェルカウンターにて測定した。測定限界は約１０ｎｇ／
アッセイであった。(iv) RIAAP of APP667
100 μl of P667 standard solution was diluted with buffer A to 1 μg/
200 μl of purified anti-ZtPI antibody (Example 1 step 1-1 (viii)) solution prepared to a concentration of 125I-
100 μl of a buffer A solution of labeled APP667 (10
000 cpm) and 200 μl of buffer A were added and mixed, followed by incubation at 4°C for 18 hours. Dextran-carbon liquid (Dextran T-70 (Funakoshi Pharmaceutical Co., Ltd.), 0.0025% solution and activated carbon Norit
A (Sigma) 0.25% liquid mixed in equal amounts) 20
After adding 0μl and leaving it at 4°C for 18 hours, 2.50
The mixture was centrifuged at 0xg for 20 minutes, and the radioactivity of the supernatant was measured using a gamma well counter. The measurement limit is approximately 10ng/
It was an assay.
【００４４】[0044]
【実施例６】 ＡＰＰ検出用キットの作製第１抗体と
して抗ＺｔＰＩ抗体、第２抗体としてビオチン標識−抗
ＺｔＰＩ抗体、標準ＡＰＰとして精製ＡＰＰ６６７、標
識検出試薬としてβ−ガラクトシダーゼ標識−ストレプ
トアビジン、４−メチルウンベリフェリル−β−Ｄ−ガ
ラクトピラノシド、基質溶解剤としてジメチルスルホキ
シド、バッファーとしてＰＢＳ（−）、洗浄剤としてＴ
ｗｅｅｎ−２０、酸素反応用バッファーとして１ｍＭＭ
ｇＣｌ２ 含有ＰＢＳ（−）、被覆剤としてウシ血清ア
ルブミンを用い、ＡＰＰ検出キットを作製した（表２）
。このキットを用いてＫＰＩ配列を含むＡＰＰの検出を
行った。[Example 6] Preparation of APP detection kit Anti-ZtPI antibody as the first antibody, biotin-labeled anti-ZtPI antibody as the second antibody, purified APP667 as the standard APP, β-galactosidase-labeled streptavidin as the label detection reagent, 4- Methylumbelliferyl-β-D-galactopyranoside, dimethyl sulfoxide as a substrate solubilizer, PBS (-) as a buffer, T as a detergent.
ween-20, 1mM as oxygen reaction buffer
An APP detection kit was prepared using gCl2-containing PBS (-) and bovine serum albumin as a coating agent (Table 2).
. Using this kit, APP containing the KPI sequence was detected.
【００４５】粉末ＰＢＳ（−）（日水製薬株式会社）９
．６ｇを精製水に溶解し、１ｌのＰＢＳ（−）溶液とし
た。洗浄剤としてＴｗｅｅｎ−２０を用いて０．０５％
Ｔｗｅｅｎ−２０を含有するＰＢＳ（−）溶液を調製し
た。被覆剤としてウシ血清アルブミン１ｇを１００ｍｌ
のＰＢＳ（−）に溶解し、１％ウシ血清アルブミン溶液
とした。Powder PBS (-) (Nissui Pharmaceutical Co., Ltd.) 9
．． 6 g was dissolved in purified water to make 1 liter of PBS(-) solution. 0.05% using Tween-20 as detergent
A PBS(-) solution containing Tween-20 was prepared. 100ml of 1g of bovine serum albumin as a coating material
was dissolved in PBS(-) to obtain a 1% bovine serum albumin solution.
【００４６】第１抗体は抗ＺｔＰＩ抗体１ｍｇを１ｍｌ
のＰＢＳ（−）に溶解したものを２００倍濃縮液として
調製した。第２抗体はビオチン標識−抗ＺｔＰＩ抗体１
ｍｇを１ｍｌのＰＢＳ（−）に溶解したものを１０００
倍濃縮液として調製した。β−ガラクトシダーゼ標識−
ストレプトアビジン１ｍｇを１ｍｌのＰＢＳ（−）に溶
解したものを１０００倍濃縮液として調製した。[0046] The first antibody is 1 ml of 1 mg of anti-ZtPI antibody.
A 200-fold concentrated solution was prepared by dissolving this in PBS(-). Second antibody is biotin labeled-anti-ZtPI antibody 1
1000mg dissolved in 1ml of PBS(-)
It was prepared as a double concentrated solution. β-galactosidase label-
A 1000-fold concentrated solution was prepared by dissolving 1 mg of streptavidin in 1 ml of PBS(-).
【００４７】β−ガラクトシダーゼの基質として４−メ
チルウンベリフェリル−β−Ｄ−ガラクトピラノシド１
０ｍｇを１００μｌのジメチルスルホキシドに溶解した
ものを５００倍濃縮液として用時調製した。標準ＡＰＰ
（精製ＡＰＰ６６７１０μｇ及び１０ｍｇのウシ血清ア
ルブミンを含むＰＢＳ（−）溶液１ｍｌの凍結乾燥品）
は１ｍｌの精製水に溶解した。4-Methylumbelliferyl-β-D-galactopyranoside 1 as a substrate for β-galactosidase
A 500-fold concentrated solution was prepared by dissolving 0 mg in 100 μl of dimethyl sulfoxide before use. Standard APP
(Lyophilized product of 1 ml of PBS(-) solution containing 10 μg of purified APP667 and 10 mg of bovine serum albumin)
was dissolved in 1 ml of purified water.
【００４８】各調製した試薬で、実施例２に準じてＫＰ
Ｉ配列を含むＡＰＰの検出を実施し、同様の結果を得た
。[0048] With each prepared reagent, KP was prepared according to Example 2.
Detection of APP containing the I sequence was performed and similar results were obtained.
【００４９】[0049]
【実施例７】 本キットを用いたヒト髄液中のＫＰＩ
配列を含むＡＰＰの定量
２名のアルツハイマー病患者から得られた髄液を検体と
して、本発明実施例６記載のキットを用いて、髄液中の
全ＡＰＰを定量した。方法は実施例２に準じた。結果は
、５４９ｎｇ／ｍｌ及び４０３ｎｇ／ｍｌであった。[Example 7] KPI in human cerebrospinal fluid using this kit
Quantification of APP containing sequences Using cerebrospinal fluid obtained from two Alzheimer's disease patients as samples, total APP in the cerebrospinal fluid was quantified using the kit described in Example 6 of the present invention. The method was the same as in Example 2. The results were 549ng/ml and 403ng/ml.
【００５０】[0050]
【参考例２】 免疫用抗原ＺＫＸタンパク及び抗ＺＫ
Ｘ抗体の作成
［工程１］ 免疫用抗原ＺＫＸの調製まず、βーガラ
クトシダーゼの一部分と、ＡＰＰ７７０のＮ末端から数
えて１９番目残基から３７９番目残基までの断片との融
合蛋白であるＺＫＸの産生について述べる。[Reference Example 2] Immunization antigen ZKX protein and anti-ZK
Preparation of X antibody [Step 1] Preparation of immunization antigen ZKX First, we prepared ZKX, which is a fusion protein of a part of β-galactosidase and a fragment from the 19th residue to the 379th residue counting from the N-terminus of APP770. Let's talk about production.
【００５１】［工程１−１］発現プラスミド ｐＥＸ
−ＺＫＸの構築
発現用ベクターｐＥＸ２（ベーリンガー・マンハイム山
之内社）を 制限酵素Ｂｓｕ３６Ｉ（ＭｓｔＩＩ）及び
ＳａｌＩで切断し、直鎖状ベクターＤＮＡ断片を得た。
一方、欧州特許出願８８１１３２８３．１号（公開番号
０３０４０１３号、対応の日本出願は特願昭６３−２０
１９９８号）の実施例１に記載のプラスミドｐＧＢＰ２
（ＡＴＣＣ−６７５０２）をＫｐｎＩ及びＸｈｏＩで切
断し、アガロースゲル電気泳動によりＡＰＰ７７０のＮ
末端側約３６０残基をコードするＤＮＡ断片を、フナコ
シ社製ジーン・クリーン・キットを用いて単離した。さ
らに配列表記載の６つの合成リンカー、１、２，３，４
、５、６（これらのリンカーをアニールさせると図７に
示したようにＭｓｔＩＩ切断末端とＫｐｎＩ切断末端を
有し、ｐＥＸ２のＭｓｔＩＩサイトに結合してもフレー
ムはずれずに連続するアミノ酸をコードする）をアプラ
イドバイオシステムズ社ＤＮＡ合成機３８０Ａを用いて
合成し、さきに得られた２つのＤＮＡ断片とともにＴ４
ＤＮＡリガーゼを用いて連結せしめた。図７にこの概要
を示す。[Step 1-1] Expression plasmid pEX
- Construction of ZKX The expression vector pEX2 (Boehringer Mannheim Yamanouchi) was digested with restriction enzymes Bsu36I (MstII) and SalI to obtain a linear vector DNA fragment. On the other hand, European Patent Application No. 88113283.1 (Publication No. 0304013, the corresponding Japanese application is Japanese Patent Application No. 88113283.1 (Publication No. 0304013)
1998), plasmid pGBP2 described in Example 1 of
(ATCC-67502) was cut with KpnI and XhoI, and the N of APP770 was analyzed by agarose gel electrophoresis.
A DNA fragment encoding about 360 residues on the terminal side was isolated using a Gene Clean kit manufactured by Funakoshi. Furthermore, six synthetic linkers listed in the sequence listing, 1, 2, 3, 4
, 5, 6 (When these linkers are annealed, it has an MstII cut end and a KpnI cut end as shown in Figure 7, and encodes consecutive amino acids without moving out of frame even when bound to the MstII site of pEX2) was synthesized using Applied Biosystems DNA Synthesizer 380A, and T4 was synthesized with the two previously obtained DNA fragments.
They were ligated using DNA ligase. Figure 7 shows an overview of this.
【００５２】該反応物を、マニアティスらの実験書に記
載されている方法に従って調製した大腸菌ＰＯＰ２１３
６株（ベーリンガー・マンハイム山之内社）コンピテン
トセルに加え、形質転換せしめた。アンピシリン耐性形
質転換コロニー６個を選び、各プラスミドを抽出し、Ｂ
ａｍＨＩで切断し解析したところ、すべて目的とするプ
ラスミドを有していた。得られたプラスミドをｐＥＸ−
ＺＫＸと命名した。本プラスミドは、ラムダ・ファージ
由来のＰＲ プロモーターの制御により、βガラクトシ
ダーゼのＮ末端約１５０アミノ酸とＡＰＰ７７０のＮ末
端から数えて１９番目残基から３７９番目残基までの配
列を含む融合タンパクＺＫＸを発現しうる。ＺＫＸは、
ＡＰＰ７７０、ＡＰＰ７５１、ＡＰＰ６９５に共通な配
列の一部のほかに、プロテアーゼインヒビター部分及び
ＡＰＰＰ７７０特異的な配列を含むが、ＡＰＰの膜貫通
領域からＣ末端までの配列は含まない。The reaction product was prepared using Escherichia coli POP213 prepared according to the method described in the experimental paper of Maniatis et al.
6 strains (Boehringer Mannheim Yamanouchi) were added to competent cells and transformed. Six ampicillin-resistant transformed colonies were selected, each plasmid was extracted, and B
When cut with amHI and analyzed, all of them contained the desired plasmid. The obtained plasmid was pEX-
It was named ZKX. This plasmid expresses the fusion protein ZKX, which contains approximately 150 amino acids at the N-terminus of β-galactosidase and the sequence from residue 19 to residue 379 counting from the N-terminus of APP770, under the control of the PR promoter derived from lambda phage. I can do it. ZKX is
In addition to part of the sequence common to APP770, APP751, and APP695, it contains a protease inhibitor portion and a sequence specific to APP770, but does not contain the sequence from the transmembrane region to the C-terminus of APP.
【００５３】［工程１−２］ 融合タンパクの生産参
考例２工程１−１で得たプラスミドｐＥＸ−ＺＫＸを保
持する大腸菌ＰＯＰ２１３６／ｐＥＸ−ＺＫＸを、５０
μｇ／ｍｌのアンピシリン含有Ｌ培地で、３０℃一晩培
養せしめた。本培養液１ｍｌを、５０μｇ／ｍｌアンピ
シリン含有Ｌ培地５０ｍｌに添加し、３０℃にて３時間
培養した後、４２℃の恒温培養槽に移し、さらに３時間
培養した。４２℃において、ラムダ・ファージＰＲ プ
ロモーターのリプレッサーは不活化され、ＰＲ プロモ
ーターが作動することによって、目的とする融合タンパ
クが生産される。[Step 1-2] Fusion protein production reference example 2 Escherichia coli POP2136/pEX-ZKX carrying the plasmid pEX-ZKX obtained in step 1-1 was incubated at 50%
The cells were cultured overnight at 30°C in L medium containing μg/ml ampicillin. 1 ml of the main culture solution was added to 50 ml of L medium containing 50 μg/ml ampicillin and cultured at 30° C. for 3 hours, then transferred to a constant temperature culture tank at 42° C. and cultured for an additional 3 hours. At 42°C, the repressor of the lambda phage PR promoter is inactivated, and the desired fusion protein is produced by operating the PR promoter.
【００５４】該培養液を遠心分離し菌体を集め、菌体の
一部を、レムリーの方法［Ｌａｅｍｍｌｉ Ｕ．Ｋ．
、Ｎａｔｕｒｅ ２２７ ６８０−６８５ （１
９７０）］に従って、ＳＤＳ−ＰＡＧＥサンプルバッフ
ァー中で煮沸後、１０％ポリアクリルアミドゲルを用い
てＳＤＳ−ポリアクリルアミドゲル電気泳動に供した。
泳動後、ゲルをクマシー染色した結果、分子量約５８ｋ
Ｄａの目的とする融合タンパクが存在することが確認さ
れた。以下、この部分βガラクトシダーゼ・部分ＡＰＰ
７７０融合タンパクをＺＫＸと略す。
［工程１−３］ＺＫＸの部分精製
参考例２工程１−２と同様にして、大腸菌ＰＯＰ２１３
６／ｐＥＸ−ＺＫＸを培養し、２００ｍｌの培養液から
菌体を回収した。該培養菌体を、４０ｍｌの４Ｍ尿素を
含む２０ｍＭトリス塩酸（ｐＨ８．０）バッファーに懸
濁し、菌体を超音波破壊した。菌体破壊物を遠心分離（
１００００×ｇ、１０分間）し、沈澱物を回収した。
次いでこの沈澱物を２０ｍｌの８Ｍ尿素を含む２０ｍＭ
トリス塩酸（ｐＨ８．０）バッファーに再懸濁し、超音
波処理により沈澱を分散せしめた。遠心分離（１０００
０×ｇ、１０分間）後上清を回収した。以上の操作によ
り、４Ｍ尿素で可溶な大腸菌タンパク、および８Ｍ尿素
に不溶な大腸菌タンパクが除去され、ＺＫＸの部分精製
液が得られた。この部分精製液の一部をＳＤＳ−ポリア
クリルアミドゲル電気泳動法で解析したところ、全タン
パク質のうち５０％以上が目的融合タンパクＺＫＸであ
った。ＺＫＸのトリプシン阻害活性を、欧州特許公開番
号０３０４０１３号実施例に記載のｓＰＩのトリプシン
阻害活性測定方法に準じて測定したところ強い阻害活性
を示した。また、本発明実施例１工程６記載の抗ＺｔＰ
Ｉ抗血清を用いたウエスタンブロットでも、このＺＫＸ
は強く反応した。[0054] The culture solution was centrifuged to collect the bacterial cells, and a portion of the bacterial cells were collected using the Laemmli method [Laemmli U. et al. K.
, Nature 227 680-685 (1
970)] and then subjected to SDS-polyacrylamide gel electrophoresis using a 10% polyacrylamide gel after boiling in SDS-PAGE sample buffer. After electrophoresis, the gel was stained with Coomassie, and the molecular weight was approximately 58k.
It was confirmed that the desired fusion protein of Da existed. Below, this partial β-galactosidase/partial APP
770 fusion protein is abbreviated as ZKX. [Step 1-3] ZKX Partial Purification Reference Example 2 In the same manner as Step 1-2, Escherichia coli POP213
6/pEX-ZKX was cultured, and bacterial cells were collected from 200 ml of the culture solution. The cultured cells were suspended in 40 ml of 20 mM Tris-HCl (pH 8.0) buffer containing 4M urea, and the cells were disrupted by ultrasonic waves. Centrifuging the destroyed bacterial cells (
10,000×g for 10 minutes), and the precipitate was collected. This precipitate was then diluted with 20 ml of 20 mM urea containing 8M urea.
It was resuspended in Tris-HCl (pH 8.0) buffer, and the precipitate was dispersed by sonication. Centrifugation (1000
0×g for 10 min), the supernatant was collected. Through the above operations, E. coli proteins soluble in 4 M urea and E. coli proteins insoluble in 8 M urea were removed, and a partially purified solution of ZKX was obtained. When a part of this partially purified solution was analyzed by SDS-polyacrylamide gel electrophoresis, more than 50% of the total protein was the target fusion protein ZKX. When the trypsin inhibitory activity of ZKX was measured according to the method for measuring the trypsin inhibitory activity of sPI described in the Examples of European Patent Publication No. 0304013, it was found to have a strong inhibitory activity. In addition, the anti-ZtP described in Step 6 of Example 1 of the present invention
Western blotting using I antiserum also showed that this ZKX
reacted strongly.
【００５５】［工程１−４］抗ＺＫＸ抗血清の作成参考
例２工程１−３で得られたＺＫＸを兎に免疫して抗血清
を得た。即ち、実施例１工程３記載のＳＤＳ変性ＡＰＰ
５９２作成と同様の方法でＳＤＳ変性ＺＫＸを作成し、
２００μｇのＳＤＳ変性ＺＫＸを１ｍｌのＰＢＳ（−）
に溶かしたものを、それぞれ２匹の家兎の背中の皮下に
フロイントの完全アジュバントと共に注射し、以後２週
間の間隔でフロイントの不完全アジュバントと共に合計
６回の免疫を行った。血清中の抗体価は、非変性及びＳ
ＤＳ変性のＺＫＸ（２５ｎｇ／ウェル）を抗原とする固
相ＥＬＩＳＡによって行った。最終的に得られた抗血清
の力価は、対照（抗原無しのウェル）の２倍以上の発色
を示す抗血清の希釈度で示すと、非変性ＺＫＸに対して
１６０００倍、ＳＤＳ変性ＺＫＸに対して３２０００倍
であり、実施例１工程２記載のＡＰＰ６６７、ＡＰＰ５
９２、さらに本実施例１工程（ｖｉ）記載のＺｔＰＩに
対しても強く反応した。[Step 1-4] Preparation of anti-ZKX antiserum Reference Example 2 A rabbit was immunized with ZKX obtained in step 1-3 to obtain an antiserum. That is, the SDS-modified APP described in Step 3 of Example 1
Create SDS-denatured ZKX in the same manner as 592 creation,
200 μg of SDS-denatured ZKX in 1 ml of PBS(-)
The solution was injected subcutaneously into the backs of two domestic rabbits together with complete Freund's adjuvant, and then immunizations with incomplete Freund's adjuvant were performed at two-week intervals for a total of six times. Antibody titers in serum are non-denatured and S
This was performed by solid-phase ELISA using DS-denatured ZKX (25 ng/well) as an antigen. The final titer of the antiserum was 16,000 times that of non-denatured ZKX and 16,000 times that of SDS-denatured ZKX, as shown by the dilution of the antiserum that showed more than twice the color development of the control (well without antigen). APP667 and APP5 described in Example 1 Step 2
92, and also strongly reacted with ZtPI described in step (vi) of Example 1.
【００５６】[0056]
【表１】[Table 1]
【００５７】[0057]
【表２】[Table 2]
【００５８】[0058]
【発明の効果】本発明の測定方法、試薬及びキットを用
いることにより、ＫＰＩ配列を含むアルツハイマー病ア
ミロイド前駆体蛋白及びその断片が簡便に且つ、高感度
に検出、定量することが可能となり、アルツハイマー病
の診断方法、試薬及び診断キットとして有用である。ま
た、アルツハイマー病アミロイド前駆体蛋白を用いる研
究の場に於ける研究試薬としても有用である。Effects of the Invention By using the measurement method, reagent, and kit of the present invention, Alzheimer's disease amyloid precursor protein containing the KPI sequence and its fragments can be easily and highly sensitively detected and quantified, and Alzheimer's disease It is useful as a disease diagnostic method, reagent, and diagnostic kit. It is also useful as a research reagent in research using Alzheimer's disease amyloid precursor protein.
【００５９】[0059]
【００６０】
配列番号：１
配列の長さ：３９
配列の型：核酸
鎖の数：一本鎖
トポロジー：直線状
配列の種類：他の核酸 合成ＤＮＡ
配列の特徴
特徴を表す記号：リンカー１
配列
ＴＧＡＧＧＣＣＧＡＴ ＡＣＴＧＣＴＧＴＣＧ ＴＣＣ
ＣＣＴＣＡＡＡ ＣＴＧＧＣＡＧＡＴ
３９SEQ ID NO: 1 Sequence length: 39 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence characteristics Characteristic symbols: Linker 1 Sequence TGAGGCCGAT ACTGCTGTCG TCC
CCTCAA CTGGCAGAT
39
【００６１
】
配列番号：２
配列の長さ：４２
配列の型：核酸
鎖の数：一本鎖
トポロジー：直線状
配列の種類：他の核酸 合成ＤＮＡ
配列の特徴
特徴を表す記号：リンカー２
配列
ＧＣＡＣＧＧＴＴＡＣ ＧＡＴＧＣＧＣＣＣＡ ＴＧＴ
ＡＣＡＣＣＡＡ ＣＧＴＡＡＣＣＴＡＴ ＣＣ
４２0061
] Sequence number: 2 Sequence length: 42 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence characteristics Symbols representing characteristics: Linker 2 Sequence GCACGGTTAC GATGCGCCCA TGT
ACACCAACGTAACCTAT CC
42
【００６２
】
配列番号：３
配列の長さ：５０
配列の型：核酸
鎖の数：一本鎖
トポロジー：直線状
配列の種類：他の核酸 合成ＤＮＡ
配列の特徴
特徴を表す記号：リンカー３
配列
ＣＡＴＴＡＣＧＧＴＣ ＡＡＴＣＣＧＣＣＧＴ ＴＴＧ
ＴＴＣＣＣＡＣ ＧＧＧＡＴＣＣＡＴＧ ＡＧＣＴＣＧ
ＧＴＡＣ ５０0062
] Sequence number: 3 Sequence length: 50 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Characteristics of the sequence Characteristic symbols: Linker 3 Sequence CATTACGGTC AATCCGCCGT TTG
TTCCCAC GGGATCCATG AGCTCG
GTAC 50
【００６３
】
配列番号：４
配列の長さ：４２
配列の型：核酸
鎖の数：一本鎖
トポロジー：直線状
配列の種類：他の核酸 合成ＤＮＡ
配列の特徴
特徴を表す記号：リンカー４
配列
ＣＧＡＧＣＴＣＡＴＧ ＧＡＴＣＣＣＧＴＧＧ ＧＡＡ
ＣＡＡＡＣＧＧ ＣＧＧＡＴＴＧＡＣＣ ＧＴ
４２0063
] Sequence number: 4 Sequence length: 42 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence characteristics Symbols representing characteristics: Linker 4 Sequence CGAGCTCATG GATCCCGTGG GAA
CAAACGG CGGATTGACC GT
42
【００６４
】
配列番号：５
配列の長さ：４２
配列の型：核酸
鎖の数：一本鎖
トポロジー：直線状
配列の種類：他の核酸 合成ＤＮＡ
配列の特徴
特徴を表す記号：リンカー５
配列
ＡＡＴＧＧＧＡＴＡＧ ＧＴＴＡＣＧＴＴＧＧ ＴＧＴ
ＡＣＡＴＧＧＧ ＣＧＣＡＴＣＧＴＡＡ ＣＣ
４２0064
] Sequence number: 5 Sequence length: 42 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence characteristics Symbols representing characteristics: Linker 5 Sequence AATGGGATAG GTTACGTTGG TGT
ACATGGG CGCATCGTAA CC
42
【００６５
】
配列番号：６
配列の長さ：４０
配列の型：核酸
鎖の数：一本鎖
トポロジー：直線状
配列の種類：他の核酸 合成ＤＮＡ
配列の特徴
特徴を表す記号：リンカー６
配列
ＧＴＧＣＡＴＣＴＧＣ ＣＡＧＴＴＴＧＡＧＧ ＧＧＡ
ＣＧＡＣＧＡＣ ＡＧＴＡＴＣＧＧＣＣ
４０0065
] Sequence number: 6 Sequence length: 40 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Characteristics of the sequence Characteristic symbols: Linker 6 Sequence GTGCATCTGC CAGTTTGAGG GGA
CGACGAC AGTATCGGCC
40
【図１】アルツハイマー病アミロイド前駆体蛋白ＡＰＰ
６９５、ＡＰＰ７５１、ＡＰＰ７７０、ＡＰＰ７１４、
ＡＰＰ５６３の模式図である。ｂからｆの黒く塗りつぶ
した部分はプロテアーゼインヒビター活性を有する５６
アミノ酸から成る領域、点々部分はＡＰＰ７７０に固有
の１９アミノ酸から成る領域、斜線部は脳に沈着する老
人斑アミロイド部分を示す。ａはＡＰＰのうち脳に沈着
するアミロイドβ蛋白部分（影をつけた部分）と膜貫通
領域を示す。ＡＰＰ５６３はβ蛋白部分をもたない。[Figure 1] Alzheimer's disease amyloid precursor protein APP
695, APP751, APP770, APP714,
It is a schematic diagram of APP563. Blacked areas from b to f have protease inhibitor activity56
A region consisting of amino acids, the dotted portions indicate a region consisting of 19 amino acids unique to APP770, and the shaded area indicates a senile plaque amyloid portion deposited in the brain. a shows the amyloid β protein portion of APP that is deposited in the brain (shaded portion) and the transmembrane region. APP563 does not have a β protein portion.
【図２】分泌型ＡＰＰ（ＡＰＰ６６７、ＡＰＰ６４８、
ＡＰＰ５９２）の構造を示す模式図である。図中の数字
は各ＡＰＰのＮ末端から数えたアミノ酸残基の番号を示
す。[Figure 2] Secretory APP (APP667, APP648,
APP592) is a schematic diagram showing the structure of APP592). The numbers in the figure indicate the amino acid residue numbers counted from the N-terminus of each APP.
【図３】抗ＺｔＰＩ抗体と抗ＺｔＰＩ抗体の組み合わせ
による２抗体サンドイッチＥＬＩＳＡ[Figure 3] Two-antibody sandwich ELISA using a combination of anti-ZtPI and anti-ZtPI antibodies
【図４】抗ＺｔＰＩ抗体と１Ｂ２モノクローナル抗体の
組み合わせによる２抗体サンドイッチＥＬＩＳＡFigure 4: Two-antibody sandwich ELISA using a combination of anti-ZtPI antibody and 1B2 monoclonal antibody
【図５
】抗ＺｔＰＩ抗体と３Ｂ２モノクローナル抗体の組み合
わせによる２抗体サンドイッチＥＬＩＳＡ[Figure 5
] Two-antibody sandwich ELISA using a combination of anti-ZtPI antibody and 3B2 monoclonal antibody
【図６】抗Ｚ
ｔＰＩ抗体と６Ａ３モノクローナル抗体の組み合わせに
よる２抗体サンドイッチＥＬＩＳＡ[Figure 6] Anti-Z
Two-antibody sandwich ELISA using a combination of tPI antibody and 6A3 monoclonal antibody
【図７】β−ガラク
トシダーゼの一部分とＡＰＰ７７０の部分タンパクとの
融合タンパクであるＺＫＸの発現プラスミド作成の模式
図である。FIG. 7 is a schematic diagram of the construction of an expression plasmid for ZKX, which is a fusion protein of a portion of β-galactosidase and a partial protein of APP770.
Claims (3)
白ＡＰＰ７５１、及びＡＰＰ７７０に含まれるＫＰＩ配
列を認識する抗体を、少なくともその組み合わせの一方
に用いることを特徴とする２抗体サンドイッチＥＬＩＳ
Ａ法による、ＫＰＩ配列を含むアルツハイマー病アミロ
イド前駆体蛋白又はその断片の免疫学的測定方法。[Claim 1] Two-antibody sandwich ELIS characterized in that an antibody that recognizes the KPI sequence contained in Alzheimer's disease amyloid precursor protein APP751 and APP770 is used in at least one of the combinations.
A method for immunologically measuring Alzheimer's disease amyloid precursor protein or a fragment thereof containing a KPI sequence using Method A.
方を不溶性担体に結合した第１次試薬と、もう一方の抗
体である第２次試薬とからなる、ＫＰＩ配列を含むアル
ツハイマー病アミロイド前駆体蛋白又はその断片の測定
試薬。2. Alzheimer's disease amyloid containing a KPI sequence, comprising a primary reagent in which one of the two antibodies according to claim 1 is bound to an insoluble carrier, and a secondary reagent that is the other antibody. A reagent for measuring precursor proteins or fragments thereof.
一部とする、ＫＰＩ配列を含むアルツハイマー病アミロ
イド前駆体蛋白又はその断片の測定キット。3. A kit for measuring Alzheimer's disease amyloid precursor protein or a fragment thereof containing the KPI sequence, which comprises the measuring reagent according to claim 2 as a component.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP939791A JPH04252954A (en) | 1991-01-29 | 1991-01-29 | Measuring method, reagent and kit for protein |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP939791A JPH04252954A (en) | 1991-01-29 | 1991-01-29 | Measuring method, reagent and kit for protein |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH04252954A true JPH04252954A (en) | 1992-09-08 |
Family
ID=11719292
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP939791A Withdrawn JPH04252954A (en) | 1991-01-29 | 1991-01-29 | Measuring method, reagent and kit for protein |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH04252954A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5705401A (en) * | 1991-11-12 | 1998-01-06 | The University Of Melbourne | Method of assaying for alzheimer's disease |
| US6114133A (en) * | 1994-11-14 | 2000-09-05 | Elan Pharmaceuticals, Inc. | Methods for aiding in the diagnosis of Alzheimer's disease by measuring amyloid-β peptide (x-≧41) |
| US7700309B2 (en) | 1994-11-14 | 2010-04-20 | Elan Pharmaceuticals, Inc. | Methods for aiding in the diagnosis of alzheimer's disease by measuring amyloid-β peptide (X->41) and tau |
| US8450275B2 (en) | 2010-03-19 | 2013-05-28 | Baxter International Inc. | TFPI inhibitors and methods of use |
| US8466108B2 (en) | 2008-12-19 | 2013-06-18 | Baxter International Inc. | TFPI inhibitors and methods of use |
| US8962563B2 (en) | 2009-12-21 | 2015-02-24 | Baxter International, Inc. | TFPI inhibitors and methods of use |
-
1991
- 1991-01-29 JP JP939791A patent/JPH04252954A/en not_active Withdrawn
Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5705401A (en) * | 1991-11-12 | 1998-01-06 | The University Of Melbourne | Method of assaying for alzheimer's disease |
| US6114133A (en) * | 1994-11-14 | 2000-09-05 | Elan Pharmaceuticals, Inc. | Methods for aiding in the diagnosis of Alzheimer's disease by measuring amyloid-β peptide (x-≧41) |
| US7700309B2 (en) | 1994-11-14 | 2010-04-20 | Elan Pharmaceuticals, Inc. | Methods for aiding in the diagnosis of alzheimer's disease by measuring amyloid-β peptide (X->41) and tau |
| US7811769B2 (en) | 1994-11-14 | 2010-10-12 | Elan Pharmaceuticals, Inc. | Methods for aiding in the diagnosis of Alzheimer's disease by measuring amyloid-β peptide (x-≧41) and tau |
| US9777051B2 (en) | 2008-12-19 | 2017-10-03 | Baxalta GmbH | TFPI inhibitors and methods of use |
| US8466108B2 (en) | 2008-12-19 | 2013-06-18 | Baxter International Inc. | TFPI inhibitors and methods of use |
| US9873720B2 (en) | 2008-12-19 | 2018-01-23 | Baxalta GmbH | TFPI inhibitors and methods of use |
| US11001613B2 (en) | 2008-12-19 | 2021-05-11 | Takeda Pharmaceutical Company Limited | TFPI inhibitors and methods of use |
| US8962563B2 (en) | 2009-12-21 | 2015-02-24 | Baxter International, Inc. | TFPI inhibitors and methods of use |
| US9018167B2 (en) | 2010-03-19 | 2015-04-28 | Baxter International Inc. | TFPI inhibitors and methods of use |
| US9556230B2 (en) | 2010-03-19 | 2017-01-31 | Baxalta GmbH | TFPI inhibitors and methods of use |
| US8450275B2 (en) | 2010-03-19 | 2013-05-28 | Baxter International Inc. | TFPI inhibitors and methods of use |
| US10201586B2 (en) | 2010-03-19 | 2019-02-12 | Baxalta GmbH | TFPI inhibitors and methods of use |
| US11793855B2 (en) | 2010-03-19 | 2023-10-24 | Takeda Pharmaceutical Company Limited | TFPI inhibitors and methods of use |
| US10800816B2 (en) | 2012-03-21 | 2020-10-13 | Baxalta GmbH | TFPI inhibitors and methods of use |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP3544544B2 (en) | Use of human neutrophil lipocalin (HNL) as a diagnostic marker and anti-HNL-antibody preparation | |
| WO2011034128A1 (en) | Collagen neoepitope antibody | |
| US10907141B2 (en) | Rep protein for use in a diagnostic assay | |
| US5506111A (en) | Method of immunological assaying of human osteocalcin, reagent and kit therefor, antibody to human osteocalcin, hybridoma producing said antibody, and method of producing it | |
| JP2012082210A (en) | Antibody for assaying adamts13 activity and method for assaying the activity | |
| JPH04252954A (en) | Measuring method, reagent and kit for protein | |
| WO1990009587A1 (en) | Immunoassay of human osteocalcin, reagent and kit therefor, antihuman osteocalcin antibody, hybridoma producing said antibody, and method of producing said antibody | |
| JPH04252955A (en) | Measuring method, reagent and kit for protein | |
| JPH04252956A (en) | Measuring method, reagent and kit for protein | |
| JPH04252195A (en) | Monoclonal antibody and hybridoma | |
| JPH10226700A (en) | Immunoassay for detecting mia | |
| EP0339302B1 (en) | Reagent system for immunologically assaying complex of human plasminogen activator inhibitor and human tissue plasminogen activator, and assay kit therefor | |
| KR20070042994A (en) | Anti-synoviolin antibody | |
| JP2003130880A (en) | Reagent and kit for measuring immunity of abnormal prion, and method of measuring immunity of abnormal prion by using the same | |
| JP4533995B2 (en) | Anti-ADAMTS13 monoclonal antibody | |
| US5196307A (en) | Cloned human centromere autoantigen | |
| US20060057134A1 (en) | Antibody against antibacterial peptide and utiliazation thereof | |
| JP2558956B2 (en) | Immunological assay method for human osteocalcin, reagent and kit therefor, antibody against human osteocalcin, hybridoma producing the same, and method for producing the same | |
| JPH05252987A (en) | Anti-igf-ii monoclonal antibody | |
| JP2000028607A (en) | NOVEL MONOCLONAL ANTIBODY AND IMMUNOLOGICAL ANALYSIS OF NICK beta2 GLYCOPROTEIN I | |
| JP4608570B2 (en) | Novel monoclonal antibody and immunological analysis method for nick β2 glycoprotein I | |
| JP2000060551A (en) | Anti-prion antibody | |
| JPH07196698A (en) | Anti-human phospholipase a2-activated protein antibody and hybridoma producing the same and reagent for assaying human phospholipase a2-activated protein using the same and assay | |
| JP2007254428A (en) | Antibody against natural unmodified cdt, method for producing antibody, hybridoma and immunoassay method | |
| WO2001013111A2 (en) | Allele detection of pgm1, gc, and aat |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A300 | Application deemed to be withdrawn because no request for examination was validly filed |
Free format text: JAPANESE INTERMEDIATE CODE: A300 Effective date: 19980514 |