JP3536191B2 - Human lactoferrin analysis method, infectious disease screening method and screening kit - Google Patents
Human lactoferrin analysis method, infectious disease screening method and screening kitInfo
- Publication number
- JP3536191B2 JP3536191B2 JP22174496A JP22174496A JP3536191B2 JP 3536191 B2 JP3536191 B2 JP 3536191B2 JP 22174496 A JP22174496 A JP 22174496A JP 22174496 A JP22174496 A JP 22174496A JP 3536191 B2 JP3536191 B2 JP 3536191B2
- Authority
- JP
- Japan
- Prior art keywords
- human lactoferrin
- antibody
- sample
- solution
- urine
- 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.)
- Expired - Lifetime
Links
- 101000798114 Homo sapiens Lactotransferrin Proteins 0.000 title claims description 115
- 102000050459 human LTF Human genes 0.000 title claims description 115
- 238000000034 method Methods 0.000 title claims description 47
- 238000012216 screening Methods 0.000 title claims description 16
- 208000035473 Communicable disease Diseases 0.000 title claims description 15
- 238000004458 analytical method Methods 0.000 title claims description 12
- 208000015181 infectious disease Diseases 0.000 title claims description 7
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims description 50
- 210000002700 urine Anatomy 0.000 claims description 46
- 235000021242 lactoferrin Nutrition 0.000 claims description 31
- 102000010445 Lactoferrin Human genes 0.000 claims description 30
- 108010063045 Lactoferrin Proteins 0.000 claims description 30
- CSSYQJWUGATIHM-IKGCZBKSSA-N l-phenylalanyl-l-lysyl-l-cysteinyl-l-arginyl-l-arginyl-l-tryptophyl-l-glutaminyl-l-tryptophyl-l-arginyl-l-methionyl-l-lysyl-l-lysyl-l-leucylglycyl-l-alanyl-l-prolyl-l-seryl-l-isoleucyl-l-threonyl-l-cysteinyl-l-valyl-l-arginyl-l-arginyl-l-alanyl-l-phenylal Chemical compound C([C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 CSSYQJWUGATIHM-IKGCZBKSSA-N 0.000 claims description 30
- 229940078795 lactoferrin Drugs 0.000 claims description 30
- 229910052742 iron Inorganic materials 0.000 claims description 25
- 239000003085 diluting agent Substances 0.000 claims description 16
- 238000003317 immunochromatography Methods 0.000 claims description 16
- 230000009257 reactivity Effects 0.000 claims description 16
- 239000011324 bead Substances 0.000 claims description 15
- 230000001580 bacterial effect Effects 0.000 claims description 12
- 210000003608 fece Anatomy 0.000 claims description 12
- 208000019206 urinary tract infection Diseases 0.000 claims description 12
- 206010012742 Diarrhoea infectious Diseases 0.000 claims description 11
- 208000001848 dysentery Diseases 0.000 claims description 11
- 230000004520 agglutination Effects 0.000 claims description 10
- 239000004094 surface-active agent Substances 0.000 claims description 10
- 102000004190 Enzymes Human genes 0.000 claims description 9
- 108090000790 Enzymes Proteins 0.000 claims description 9
- 238000003018 immunoassay Methods 0.000 claims description 8
- 239000004816 latex Substances 0.000 claims description 8
- 229920000126 latex Polymers 0.000 claims description 8
- 230000001900 immune effect Effects 0.000 claims description 6
- 239000002202 Polyethylene glycol Substances 0.000 claims description 5
- 229920001223 polyethylene glycol Polymers 0.000 claims description 5
- ORLFVWPPBMVPNZ-UHFFFAOYSA-N 1-(6-methylheptyl)-4-[4-(6-methylheptyl)phenoxy]benzene Chemical compound C1=CC(CCCCCC(C)C)=CC=C1OC1=CC=C(CCCCCC(C)C)C=C1 ORLFVWPPBMVPNZ-UHFFFAOYSA-N 0.000 claims description 2
- YGYXNQFLPOCERC-UHFFFAOYSA-N CCCCCCCCCCCCCCC(S([O-])(=O)=O)CC[NH+](C)C Chemical compound CCCCCCCCCCCCCCC(S([O-])(=O)=O)CC[NH+](C)C YGYXNQFLPOCERC-UHFFFAOYSA-N 0.000 claims 1
- 239000000523 sample Substances 0.000 description 66
- 239000000243 solution Substances 0.000 description 52
- 239000000872 buffer Substances 0.000 description 28
- 210000004027 cell Anatomy 0.000 description 23
- 239000012898 sample dilution Substances 0.000 description 23
- 238000005259 measurement Methods 0.000 description 17
- 238000002965 ELISA Methods 0.000 description 16
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 15
- 239000012528 membrane Substances 0.000 description 15
- 230000000694 effects Effects 0.000 description 14
- 210000004408 hybridoma Anatomy 0.000 description 14
- 210000000265 leukocyte Anatomy 0.000 description 14
- 241000699666 Mus <mouse, genus> Species 0.000 description 13
- 241000283973 Oryctolagus cuniculus Species 0.000 description 13
- 239000013504 Triton X-100 Substances 0.000 description 12
- 229920004890 Triton X-100 Polymers 0.000 description 12
- 239000007853 buffer solution Substances 0.000 description 12
- 239000002953 phosphate buffered saline Substances 0.000 description 12
- 230000003053 immunization Effects 0.000 description 11
- IZWSFJTYBVKZNK-UHFFFAOYSA-N lauryl sulfobetaine Chemical compound CCCCCCCCCCCC[N+](C)(C)CCCS([O-])(=O)=O IZWSFJTYBVKZNK-UHFFFAOYSA-N 0.000 description 11
- 229920001213 Polysorbate 20 Polymers 0.000 description 10
- 239000002609 medium Substances 0.000 description 10
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 10
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 10
- 239000000427 antigen Substances 0.000 description 9
- 102000036639 antigens Human genes 0.000 description 9
- 108091007433 antigens Proteins 0.000 description 9
- 238000001514 detection method Methods 0.000 description 9
- 230000007910 cell fusion Effects 0.000 description 8
- 238000002649 immunization Methods 0.000 description 8
- 239000000203 mixture Substances 0.000 description 8
- 229920006395 saturated elastomer Polymers 0.000 description 8
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 7
- 108010010803 Gelatin Proteins 0.000 description 7
- 102000008133 Iron-Binding Proteins Human genes 0.000 description 7
- 108010035210 Iron-Binding Proteins Proteins 0.000 description 7
- 206010035226 Plasma cell myeloma Diseases 0.000 description 7
- 239000002671 adjuvant Substances 0.000 description 7
- 239000008273 gelatin Substances 0.000 description 7
- 229920000159 gelatin Polymers 0.000 description 7
- 235000019322 gelatine Nutrition 0.000 description 7
- 235000011852 gelatine desserts Nutrition 0.000 description 7
- 201000000050 myeloid neoplasm Diseases 0.000 description 7
- 239000013641 positive control Substances 0.000 description 7
- 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 6
- ZWEVPYNPHSPIFU-AUGHYPCGSA-N (2r,3s,4r,5r)-2,3,4,5,6-pentahydroxy-n-[3-[3-[[(2r,3s,4r,5r)-2,3,4,5,6-pentahydroxyhexanoyl]amino]propyl-[(4r)-4-[(3r,5s,7r,8r,9s,10s,12s,13r,14s,17r)-3,7,12-trihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1h-cyclopenta[a]phenan Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)N(CCCNC(=O)[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO)CCCNC(=O)[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO)C)[C@@]2(C)[C@@H](O)C1 ZWEVPYNPHSPIFU-AUGHYPCGSA-N 0.000 description 6
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 238000007865 diluting Methods 0.000 description 6
- 238000011084 recovery Methods 0.000 description 6
- 230000035945 sensitivity Effects 0.000 description 6
- 239000008279 sol Substances 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- UMCMPZBLKLEWAF-BCTGSCMUSA-N 3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulfonate Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCC[N+](C)(C)CCCS([O-])(=O)=O)C)[C@@]2(C)[C@@H](O)C1 UMCMPZBLKLEWAF-BCTGSCMUSA-N 0.000 description 5
- 239000007760 Iscove's Modified Dulbecco's Medium Substances 0.000 description 5
- 239000007983 Tris buffer Substances 0.000 description 5
- 238000002835 absorbance Methods 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 239000002504 physiological saline solution Substances 0.000 description 5
- 235000018102 proteins Nutrition 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 210000004989 spleen cell Anatomy 0.000 description 5
- 108090001008 Avidin Proteins 0.000 description 4
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 4
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 4
- 229940098773 bovine serum albumin Drugs 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000006911 enzymatic reaction Methods 0.000 description 4
- 230000002550 fecal effect Effects 0.000 description 4
- 239000008187 granular material Substances 0.000 description 4
- 230000002163 immunogen Effects 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 3
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 3
- 108090000371 Esterases Proteins 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- 241000699670 Mus sp. Species 0.000 description 3
- 239000004677 Nylon Substances 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 238000004220 aggregation Methods 0.000 description 3
- 230000002776 aggregation Effects 0.000 description 3
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 3
- 229960002685 biotin Drugs 0.000 description 3
- 235000020958 biotin Nutrition 0.000 description 3
- 239000011616 biotin Substances 0.000 description 3
- 230000000903 blocking effect Effects 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 239000012228 culture supernatant Substances 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012091 fetal bovine serum Substances 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 229920001778 nylon Polymers 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000012488 sample solution Substances 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- BPYKTIZUTYGOLE-IFADSCNNSA-N Bilirubin Chemical compound N1C(=O)C(C)=C(C=C)\C1=C\C1=C(C)C(CCC(O)=O)=C(CC2=C(C(C)=C(\C=C/3C(=C(C=C)C(=O)N\3)C)N2)CCC(O)=O)N1 BPYKTIZUTYGOLE-IFADSCNNSA-N 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- 229920000742 Cotton Polymers 0.000 description 2
- 229920002271 DEAE-Sepharose Polymers 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 239000004793 Polystyrene Substances 0.000 description 2
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 2
- 210000000683 abdominal cavity Anatomy 0.000 description 2
- 229910052782 aluminium Inorganic materials 0.000 description 2
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
- 210000000628 antibody-producing cell Anatomy 0.000 description 2
- 229910021538 borax Inorganic materials 0.000 description 2
- 239000001569 carbon dioxide Substances 0.000 description 2
- 229910002092 carbon dioxide Inorganic materials 0.000 description 2
- 239000011545 carbonate/bicarbonate buffer Substances 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 239000000356 contaminant Substances 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- ZBCBWPMODOFKDW-UHFFFAOYSA-N diethanolamine Chemical compound OCCNCCO ZBCBWPMODOFKDW-UHFFFAOYSA-N 0.000 description 2
- KAKKHKRHCKCAGH-UHFFFAOYSA-L disodium;(4-nitrophenyl) phosphate;hexahydrate Chemical compound O.O.O.O.O.O.[Na+].[Na+].[O-][N+](=O)C1=CC=C(OP([O-])([O-])=O)C=C1 KAKKHKRHCKCAGH-UHFFFAOYSA-L 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 235000020256 human milk Nutrition 0.000 description 2
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 238000012417 linear regression Methods 0.000 description 2
- 238000000691 measurement method Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 229940126619 mouse monoclonal antibody Drugs 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 229920003023 plastic Polymers 0.000 description 2
- 239000004033 plastic Substances 0.000 description 2
- 239000003755 preservative agent Substances 0.000 description 2
- 230000002335 preservative effect Effects 0.000 description 2
- 238000005185 salting out Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 239000004328 sodium tetraborate Substances 0.000 description 2
- 235000010339 sodium tetraborate Nutrition 0.000 description 2
- 210000000952 spleen Anatomy 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 239000012085 test solution Substances 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 description 1
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- 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 1
- TVZGACDUOSZQKY-LBPRGKRZSA-N 4-aminofolic acid Chemical compound C1=NC2=NC(N)=NC(N)=C2N=C1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 TVZGACDUOSZQKY-LBPRGKRZSA-N 0.000 description 1
- IRLPACMLTUPBCL-KQYNXXCUSA-N 5'-adenylyl sulfate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OS(O)(=O)=O)[C@@H](O)[C@H]1O IRLPACMLTUPBCL-KQYNXXCUSA-N 0.000 description 1
- 239000004925 Acrylic resin Substances 0.000 description 1
- 229920000178 Acrylic resin Polymers 0.000 description 1
- 206010003445 Ascites Diseases 0.000 description 1
- 241000271566 Aves Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 1
- QXNVGIXVLWOKEQ-UHFFFAOYSA-N Disodium Chemical compound [Na][Na] QXNVGIXVLWOKEQ-UHFFFAOYSA-N 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 1
- 229930182566 Gentamicin Natural products 0.000 description 1
- 239000006173 Good's buffer Substances 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 229920002302 Nylon 6,6 Polymers 0.000 description 1
- 241000283977 Oryctolagus Species 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- OFOBLEOULBTSOW-UHFFFAOYSA-N Propanedioic acid Natural products OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- BHATUINFZWUDIX-UHFFFAOYSA-N Zwittergent 3-14 Chemical compound CCCCCCCCCCCCCC[N+](C)(C)CCCS([O-])(=O)=O BHATUINFZWUDIX-UHFFFAOYSA-N 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 229960003896 aminopterin Drugs 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 239000005441 aurora Substances 0.000 description 1
- 238000006149 azo coupling reaction Methods 0.000 description 1
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 1
- 239000002981 blocking agent Substances 0.000 description 1
- 238000004820 blood count Methods 0.000 description 1
- 239000003618 borate buffered saline Substances 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- 229960002645 boric acid Drugs 0.000 description 1
- 230000003139 buffering effect Effects 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 238000010370 cell cloning Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 229940106164 cephalexin Drugs 0.000 description 1
- ZAIPMKNFIOOWCQ-UEKVPHQBSA-N cephalexin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@@H]3N(C2=O)C(=C(CS3)C)C(O)=O)=CC=CC=C1 ZAIPMKNFIOOWCQ-UEKVPHQBSA-N 0.000 description 1
- 235000013330 chicken meat Nutrition 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 229940127089 cytotoxic agent Drugs 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 239000012470 diluted sample Substances 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 229960002518 gentamicin Drugs 0.000 description 1
- 210000004251 human milk Anatomy 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000016784 immunoglobulin production Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 description 1
- 239000011976 maleic acid Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- 229960000564 nitrofurantoin Drugs 0.000 description 1
- NXFQHRVNIOXGAQ-YCRREMRBSA-N nitrofurantoin Chemical compound O1C([N+](=O)[O-])=CC=C1\C=N\N1C(=O)NC(=O)C1 NXFQHRVNIOXGAQ-YCRREMRBSA-N 0.000 description 1
- 239000002736 nonionic surfactant Substances 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 238000013102 re-test Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 210000001635 urinary tract Anatomy 0.000 description 1
- 239000002888 zwitterionic surfactant Substances 0.000 description 1
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
Description
【発明の詳細な説明】Detailed Description of the Invention
【０００１】[0001]
【産業上の利用分野】本発明は、便又は尿中のヒトラク
トフェリンを分析する方法に関する。また、ヒトラクト
フェリンを分析することからなる感染症（例えば、尿路
感染症又は細菌感染性下痢症）のスクリーニング方法及
びスクリーニング用キットにも関する。TECHNICAL FIELD The present invention relates to a method for analyzing human lactoferrin in feces or urine. It also relates to a screening method and a screening kit for an infectious disease (for example, urinary tract infection or bacterial infectious diarrhea), which comprises analyzing human lactoferrin.
【０００２】[0002]
【従来の技術】尿路感染症や細菌感染性下痢症等におい
ては、その感染（炎症）部位から、便中や尿中に多量の
白血球が浸潤する。従って、尿路感染症や細菌感染性下
痢症の診断には、便又は尿検体の細菌培養検査と並行し
て、白血球の鏡検法が用いられている。しかし、白血球
は便又は尿中では非常に不安定で破砕し易いため、鏡検
法では操作に迅速性と熟練度を要求されるだけでなく、
正確性に欠け、更には保存便又は保存尿検体には適用困
難である。そのため、鏡検法に代わる尿中の白血球検出
法として、白血球の顆粒中に存在するエステラーゼ活性
を、酵素反応を組み入れたアゾ・カップリング反応を用
いて測定し、対象とする尿路感染症を検出する方法［例
えば、Ｊ．Ｃｌｉｎ．Ｍｉｃｒｏｂｉｏｌ．，ｐ．８５
２〜ｐ．８５４（１９８２）］が開発されている。2. Description of the Related Art In urinary tract infections, bacterial infectious diarrhea, etc., a large amount of leukocytes infiltrate in feces and urine from the site of infection (inflammation). Therefore, in order to diagnose urinary tract infection and bacterial infectious diarrhea, leukocyte microscopic examination is used in parallel with bacterial culture examination of a stool or urine sample. However, white blood cells are extremely unstable in stool or urine and are easily crushed. Therefore, the microscopic method not only requires quickness and skill in operation, but also
It lacks accuracy and is difficult to apply to stored feces or stored urine samples. Therefore, as a method for detecting leukocytes in urine as an alternative to microscopy, the esterase activity present in the leukocyte granules was measured using an azo coupling reaction incorporating an enzymatic reaction, and the target urinary tract infection was detected. Method of detection [eg. Clin. Microbiol. , P. 85
2 to p. 854 (1982)] has been developed.
【０００３】[0003]
【発明が解決しようとする課題】しかしながら、エステ
ラーゼ活性測定法は、尿保存剤のホルムアルデヒドによ
る偽陽性や、尿中のタンパク質、セファレキシンやゲン
タマイシン等の抗生物質、尿保存剤のホウ酸、アスコル
ビン酸、ビリルビン及び化学療法剤ニトロフラントイン
等の影響による偽陰性が起こるうえに、便検体には適用
不可能である。However, the method for measuring esterase activity is false positive with formaldehyde as a urine preservative, protein in urine, antibiotics such as cephalexin and gentamicin, boric acid as a urine preservative, ascorbic acid, False negatives occur due to the effects of bilirubin and the chemotherapeutic agent nitrofurantoin, and they are not applicable to fecal samples.
【０００４】一方、白血球の顆粒内には、鉄結合性タン
パク質であるラクトフェリンも多量（４．９μｇヒトラ
クトフェリン／１０6 個白血球）に存在しており、便又
は尿中の白血球のマーカーとなり得ることが知られてい
る［Ｊ．Ｃｌｉｎ．Ｍｉｃｒｏｂｉｏｌ．，３０
（５），ｐ．１２３８〜ｐ．１２４２（１９９２）］。
従って、本発明の目的は、ヒトラクトフェリンを測定対
象とし、便又は尿中の白血球の顆粒内の鉄結合性タンパ
ク質であるヒトラクトフェリンを検出することにより、
鏡検法やエステラーゼ活性測定法に代わる尿路感染症や
細菌感染性下痢症等の感染症の確実な検出方法を提供す
ることにある。On the other hand, lactoferrin, which is an iron-binding protein, is also present in a large amount in the granules of leukocytes (4.9 μg human lactoferrin / 10 6 leukocytes) and can be a marker for leukocytes in stool or urine. Is known [J. Clin. Microbiol. , 30
(5), p. 1238-p. 1242 (1992)].
Therefore, an object of the present invention is to measure human lactoferrin, by detecting human lactoferrin, which is an iron-binding protein in the granules of leukocytes in stool or urine,
An object of the present invention is to provide a reliable method for detecting infectious diseases such as urinary tract infection and bacterial infectious diarrhea, which is an alternative to the microscopic method and the esterase activity measuring method.
【０００５】[0005]
【課題を解決するための手段】本発明は、ヒトラクトフ
ェリンの鉄飽和度に反応性が影響されない抗ヒトラクト
フェリン抗体を用いることを特徴とする、便又は尿中の
ヒトラクトフェリンを免疫学的に分析する方法に関す
る。また、本発明は、前記の分析方法によって便又は尿
中のヒトラクトフェリンを分析することを特徴とする、
感染症のスクリーニング方法にも関する。更に、本発明
は、ヒトラクトフェリンの鉄飽和度に反応性が影響され
ない抗ヒトラクトフェリン抗体、ヒトラクトフェリンの
鉄飽和度に反応性が影響されない標識化抗ヒトラクトフ
ェリン抗体、及び界面活性剤含有検体希釈液を含む、感
染症のスクリーニング用キットにも関する。本明細書に
おいて「分析」とは、分析対象であるヒトラクトフェリ
ンの存在を検出すること、及びヒトラクトフェリンを定
量的又は半定量的に測定することのいずれをも含む。従
って、本明細書において「分析方法」とは、分析対象で
あるヒトラクトフェリンの存在を検出する方法、及びヒ
トラクトフェリンを定量的又は半定量的に測定する方法
のいずれをも含む。The present invention is an immunological analysis of human lactoferrin in stool or urine characterized by using an anti-human lactoferrin antibody whose reactivity is not affected by the iron saturation of human lactoferrin. On how to do. Further, the present invention is characterized by analyzing human lactoferrin in feces or urine by the above-mentioned analysis method,
It also relates to a method of screening for infectious diseases. Furthermore, the present invention is an anti-human lactoferrin antibody whose reactivity is not affected by the iron saturation of human lactoferrin, a labeled anti-human lactoferrin antibody whose reactivity is not affected by the iron saturation of human lactoferrin, and a surfactant-containing sample diluent. The present invention also relates to a kit for screening an infectious disease. As used herein, the term “analysis” includes both detecting the presence of human lactoferrin, which is an analysis target, and quantitatively or semiquantitatively measuring human lactoferrin. Therefore, in the present specification, the “analytical method” includes both a method for detecting the presence of human lactoferrin to be analyzed and a method for quantitatively or semi-quantitatively measuring human lactoferrin.
【０００６】[0006]
【発明の実施の形態】以下、本発明を詳細に説明する。
ラクトフェリンは鉄結合性タンパク質であるが、鉄結合
量は必ずしも一定でなく、便又は尿中におけるヒトラク
トフェリンの鉄飽和度も現在のところ明らかではない。
従って、ヒトラクトフェリンを免疫学的に分析する場合
に、ポリクローナル抗体及びモノクローナル抗体とし
て、その反応性がヒトラクトフェリンの鉄飽和度に影響
されない抗体を選択して用いれば、正確に感染症に起因
するヒトラクトフェリンを検出することができる。この
ように分析したヒトラクトフェリンの値から、例えば尿
路感染症や細菌感染性下痢症等を正確にスクリーニング
することができる。BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below.
Although lactoferrin is an iron-binding protein, the amount of iron binding is not always constant, and the iron saturation of human lactoferrin in stool or urine is not clear at present.
Therefore, when immunologically analyzing human lactoferrin, if antibodies that are not affected by the iron saturation of human lactoferrin are selected and used as polyclonal antibodies and monoclonal antibodies, humans accurately caused by infectious diseases can be selected. Lactoferrin can be detected. From the value of human lactoferrin thus analyzed, for example, urinary tract infection, bacterial infectious diarrhea, etc. can be accurately screened.
【０００７】本発明で用いられる抗ヒトラクトフェリン
抗体は、ポリクローナル抗体あるいはモノクローナル抗
体のいずれであることもできる。ポリクローナル抗体の
調製又はモノクローナル抗体を産生するハイブリドーマ
の分離並びにモノクローナル抗体の調製は、従来公知の
手段に従って実施することができる。例えば、ラクトフ
ェリン（例えば、哺乳動物、特にヒトの母乳、又は白血
球由来のヒトラクトフェリン）を免疫原として用い、哺
乳動物や鳥類（例えば、ウサギ、マウス、又は鶏等）を
通常の方法により免疫することにより行う。免疫には、
例えば、免疫原溶液を等量のフロイントの完全アジュバ
ント又は不完全アジュバントと乳化混合し、ウサギやマ
ウス等に接種（初回免疫）し、以後、２〜４週間の間隔
で同様の操作を行い、数回免疫する方法等がある。ポリ
クローナル抗体は、このように免疫した動物等の血液を
採取し、抗血清として調製する。The anti-human lactoferrin antibody used in the present invention may be either a polyclonal antibody or a monoclonal antibody. Preparation of a polyclonal antibody or isolation of a hybridoma producing a monoclonal antibody and preparation of a monoclonal antibody can be carried out according to conventionally known means. For example, using lactoferrin (eg, human lactoferrin derived from breast milk of mammals, particularly humans, or leukocytes) as an immunogen, and immunizing mammals or birds (eg, rabbits, mice, chickens, etc.) by a usual method. By. For immunity,
For example, an immunogen solution is emulsified and mixed with an equal amount of Freund's complete adjuvant or incomplete adjuvant, and inoculated into rabbits or mice (primary immunization), and then the same operation is performed at intervals of 2 to 4 weeks. There are methods such as immunization. The polyclonal antibody is prepared as an antiserum by collecting blood from the animal thus immunized.
【０００８】またモノクローナル抗体を作製する場合
は、例えば、最終免疫して数日後のマウス等から得た脾
臓細胞等を、細胞融合工程に用いることができる。細胞
融合のもう一方の親細胞であるミエローマ細胞（骨髄腫
細胞）としては、各種の公知の細胞株、例えば、ｐ３・
ＮＳ−１／１・Ａｇ４．１［Ｅｕｒ．Ｊ．Ｉｍｍｕｎｏ
ｌ．，５；５１１−５１７（１９７５）］、ＳＰ２／０
−Ａｇ１４［Ｎａｔｕｒｅ，２７６；２６９−２７０
（１９７８）］、Ｐ３−Ｘ６３−Ａｇ８．６５３［Ｊ．
Ｉｍｍｕｎｏｌ．，１２３；１５４８−１５５０（１９
７９）］等を使用することができる。When producing a monoclonal antibody, for example, spleen cells obtained from a mouse or the like several days after the final immunization can be used in the cell fusion step. As the other parent cell of the cell fusion, myeloma cells (myeloma cells), various known cell lines such as p3.
NS-1 / 1.Ag4.1 [Eur. J. Immuno
l. , 5; 511-517 (1975)], SP2 / 0.
-Ag14 [Nature, 276; 269-270.
(1978)], P3-X63-Ag8.653 [J.
Immunol. , 123; 1548-1550 (19
79)] and the like can be used.
【０００９】細胞融合は通常の方法、例えば、公知の融
合促進剤（ポリエチレングリコールなど）を用いて行う
ことができ、細胞の混合比率も、例えば、マウスの脾臓
細胞に対してミエローマ細胞を約１／５〜１／１０程度
の割合で行うことができる。細胞融合剤としては、例え
ば、市販の細胞融合用ポリエチレングリコール［分子量
１５００（Ｂｏｅｈｒｉｎｇｅｒ Ｍａｎｎｈｅｉｍ
製）］を用いることができる。細胞融合は、前記の細胞
融合剤中で脾臓細胞とミエローマ細胞とをよく混合する
ことによって行い、選別用培地（例えば、ＨＡＴ培地）
中で培養することによりハイブリドーマのみを増殖した
後、その培養上清中の抗体産生の有無を、例えばＥＬＩ
ＳＡ法によって測定し、目的とするハイブリドーマを分
離することができる。このハイブリドーマを、細胞クロ
ーニング法を用いてクローン化し、モノクローナル抗体
産生細胞を得ることができる。また、ヒトラクトフェリ
ンの鉄飽和度に影響されないモノクローナル抗体を産生
するハイブリドーマの選択は、そのハイブリドーマの培
養上清のヒトラクトフェリンとの反応性を、鉄飽和度の
異なる種々のヒトラクトフェリンとの反応性に関して
（例えば、ＥＬＩＳＡ法で）確認することによって、行
うことができる。分離したハイブリドーマは、通常の培
地で培養することができ、液体窒素等の中で容易に長期
間保存することができる。The cell fusion can be carried out by a conventional method, for example, using a known fusion promoter (polyethylene glycol etc.), and the mixing ratio of cells is, for example, about 1 myeloma cell to about 1 mouse spleen cell. It can be performed at a ratio of about / 5 to 1/10. Examples of the cell fusion agent include commercially available polyethylene glycol for cell fusion [molecular weight 1500 (Boehringer Mannheim
Manufactured)] can be used. Cell fusion is carried out by thoroughly mixing spleen cells and myeloma cells in the above-mentioned cell fusion agent, and selecting medium (for example, HAT medium)
After proliferating only the hybridoma by culturing in the medium, the presence or absence of antibody production in the culture supernatant is determined by, for example, ELI.
The hybridoma of interest can be separated by measurement by the SA method. This hybridoma can be cloned using a cell cloning method to obtain a monoclonal antibody-producing cell. In addition, the selection of hybridomas that produce a monoclonal antibody that is not affected by the iron saturation of human lactoferrin was performed by comparing the reactivity of the culture supernatant of the hybridoma with human lactoferrin with respect to the reactivity with various human lactoferrins having different iron saturations. This can be done by checking (for example, by the ELISA method). The isolated hybridoma can be cultured in an ordinary medium and can be easily stored in liquid nitrogen or the like for a long period of time.
【００１０】また、本発明で用いるモノクローナル抗体
は、目的のモノクローナル抗体を産生するハイブリドー
マを培養することにより、容易に調製することができ
る。ハイブリドーマの培養は、例えば、約５％二酸化炭
素、約３７℃及び加湿の条件下で、培養に適した任意の
培地を用いて行うことができるが、好適にはイスコフ改
変ダルベッコ培地に１０％ウシ胎児血清（以下ＦＢＳと
いう）を含む培地を用いる。またイン・ビボの場合に
は、例えばマウスの腹腔中で培養するのが好ましい。The monoclonal antibody used in the present invention can be easily prepared by culturing a hybridoma that produces the desired monoclonal antibody. Cultivation of the hybridoma can be carried out, for example, under conditions of about 5% carbon dioxide, about 37 ° C. and humidification, using any medium suitable for culture, but preferably 10% bovine in Iscove's modified Dulbecco's medium. A medium containing fetal serum (hereinafter referred to as FBS) is used. In the case of in vivo, it is preferable to culture in the abdominal cavity of a mouse, for example.
【００１１】抗体溶液としては、ポリクローナル抗体の
場合には抗血清を、モノクローナル抗体の場合にはイン
・ビトロで培養した培養液又はマウスの腹腔中で培養し
て得た腹水を用いることができるが、これらを出発材料
として用いて、更に分離・精製して得た抗体を用いるこ
ともできる。抗体の分離、精製には、タンパク質の分
離、精製に一般的な方法、例えば硫安塩析、イオン交換
クロマトグラフィー、分子篩カラムクロマトグラフィ
ー、アフィニテイークロマトグラフィー、又は透析等の
方法を用いることができる。As the antibody solution, an antiserum can be used in the case of a polyclonal antibody, and a culture solution cultured in vitro or an ascites obtained by culturing in the abdominal cavity of a mouse can be used in the case of a monoclonal antibody. Alternatively, an antibody obtained by further separating and purifying using these as starting materials can also be used. For separating and purifying the antibody, a method generally used for separating and purifying proteins, such as salting out with ammonium sulfate, ion exchange chromatography, molecular sieve column chromatography, affinity chromatography, or dialysis can be used. .
【００１２】こうして得られた抗体（すなわち、反応性
がヒトラクトフェリンの鉄飽和度に影響されない抗体）
１種又は２種以上を用いて、後述するヒトラクトフェリ
ンの免疫学的分析方法、及び感染症のスクリーニング方
法を実施することができ、更には感染症のスクリーニン
グ用キットを構成することができる。なお、ヒトラクト
フェリンは１分子当り最大２分子の鉄を結合するので、
本明細書において「ヒトラクトフェリンの鉄飽和度」と
は、ヒトラクトフェリン１分子当りの結合鉄分子数が２
（飽和型）、１（部分飽和型）又は０（非結合型）のい
ずれかであることを意味する。また、ヒトラクトフェリ
ンは鉄を結合するか否かで（特に鉄結合部位の）立体
（高次）構造がわずかに変化する。従って、「反応性が
ヒトラクトフェリンの鉄飽和度に影響されない抗体」と
は、鉄結合の有無（度合い）により生ずるヒトラクトフ
ェリン立体（高次）構造の変化に無関係に一定の結合能
を有する抗体をいう。The antibody thus obtained (that is, the antibody whose reactivity is not affected by the iron saturation of human lactoferrin)
One or more of them can be used to carry out the immunological analysis method for human lactoferrin and the screening method for infectious diseases, which will be described later, and can also constitute a screening kit for infectious diseases. Since human lactoferrin binds up to 2 molecules of iron per molecule,
In the present specification, "iron saturation of human lactoferrin" means that the number of bound iron molecules per molecule of human lactoferrin is 2
(Saturated type), 1 (partially saturated type) or 0 (non-bonded type) is meant. In addition, human lactoferrin slightly changes its steric (higher order) structure (particularly at the iron binding site) depending on whether or not it binds iron. Therefore, "an antibody whose reactivity is not affected by the iron saturation of human lactoferrin" means an antibody having a certain binding ability regardless of the change in the human lactoferrin steric (higher order) structure caused by the presence (degree) of iron binding. Say.
【００１３】前記の方法に従い、例えば、ヒトラクトフ
ェリンで免疫したウサギの抗血清から、常法により、ウ
サギポリクローナル抗ヒトラクトフェリン抗体を得るこ
とができる。また、例えば、ヒトラクトフェリンで免疫
したマウスの脾細胞と、マウスの骨髄腫細胞とを融合さ
せてモノクローナル抗体ヒトラクトフェリン抗体産生細
胞を作製し、培養上清からマウスモノクローナル抗ヒト
ラクトフェリン抗体を得ることができる。本発明では、
これらのウサギポリクローナル抗ヒトラクトフェリン抗
体及びマウスモノクローナル抗ヒトラクトフェリン抗体
のうち、ヒトラクトフェリンの鉄飽和度に反応性が影響
されない抗体を選択して用いる。本発明者は、後記実施
例で示すように、ヒトラクトフェリンの鉄飽和度に反応
性が影響されない抗体として、実際に、ウサギポリクロ
ーナル抗体（以下、Ｈ１５抗体と称す）、及びマウスモ
ノクローナル抗体（以下、１５Ｇ１１−０４抗体、１７
Ｂ０４−０８抗体、１７Ｇ０５−１０抗体及び３２Ｄ０
１−１０抗体と称す）を得ることができた。According to the above-mentioned method, for example, a rabbit polyclonal anti-human lactoferrin antibody can be obtained from the antiserum of a rabbit immunized with human lactoferrin by a conventional method. In addition, for example, a mouse spleen cell immunized with human lactoferrin and a mouse myeloma cell are fused to prepare a monoclonal antibody human lactoferrin antibody-producing cell, and a mouse monoclonal anti-human lactoferrin antibody can be obtained from the culture supernatant. it can. In the present invention,
Among these rabbit polyclonal anti-human lactoferrin antibody and mouse monoclonal anti-human lactoferrin antibody, an antibody whose reactivity is not affected by the iron saturation of human lactoferrin is selected and used. The present inventor, as will be shown in Examples described later, is a rabbit polyclonal antibody (hereinafter referred to as H15 antibody), and a mouse monoclonal antibody (hereinafter, referred to as the following, as an antibody whose reactivity is not affected by the iron saturation of human lactoferrin). 15G11-04 antibody, 17
B04-08 antibody, 17G05-10 antibody and 32D0
1-10 antibody) was obtained.
【００１４】本発明においては、検体として便又は尿を
使用する。この便又は尿をそのまま用いることもできる
が、正常便又は正常尿中に存在するヒトラクトフェリン
量では、ヒトラクトフェリンを検出することができない
ように便又は尿試料を希釈することにより、検出感度を
調整して用いるのが好ましい。検体希釈液としては、反
応系に影響を与えない水性液体であれば特に限定され
ず、例えば、精製水、生理食塩水又は各種緩衝液を用い
ることができる。緩衝液としては、ｐＨ６〜９に緩衝能
を有する従来公知の緩衝液、例えば、リン酸緩衝液、ト
リス緩衝液、ＨＥＰＥＳ緩衝液等のグッド緩衝液、ホウ
酸塩緩衝液等を用いることができる。希釈液として好ま
しくは緩衝液を使用し、特に好ましくはホウ酸塩緩衝液
を用いる。In the present invention, stool or urine is used as the sample. Although this stool or urine can be used as it is, the detection sensitivity is adjusted by diluting the stool or urine sample so that human lactoferrin cannot be detected with the amount of human lactoferrin present in normal stool or normal urine. It is preferable to use it. The sample diluent is not particularly limited as long as it is an aqueous liquid that does not affect the reaction system, and for example, purified water, physiological saline, or various buffer solutions can be used. As the buffer solution, a conventionally known buffer solution having a buffering capacity at pH 6 to 9, for example, phosphate buffer solution, Tris buffer solution, Good buffer solution such as HEPES buffer solution, borate buffer solution, etc. can be used. . A buffer solution is preferably used as the diluent, and a borate buffer solution is particularly preferably used.
【００１５】この際、検体中に含まれるタンパク質及び
／又は脂質等（非イオン性及びイオン性夾雑物）が、抗
体及び金コロイド、メンブレン、ラテックス粒子、磁気
ビーズ粒子、又はポリスチレン製プレート等に対して非
特異的に吸着し、測定に影響を与えることがある。検体
希釈液に界面活性剤を添加することにより、こうした非
特異的な吸着を回避することができる。界面活性剤とし
ては、例えば、非イオン系界面活性剤及び両性イオン系
界面活性剤を使用することが好ましい。特に、Ｎ−テト
ラデシル−Ｎ，Ｎ−ジメチル−３−アンモニオ−１−プ
ロパンスルホン酸（以下、Ｚｗｉｔｔｅｒｇｅｎｔとい
う）、３−［（３−コラシドプロピル）ジメチルアンモ
ニオ］−プロパンスルホン酸（以下、ＣＨＡＰＳとい
う）、Ｎ，Ｎ−ビス（３−Ｄ−グルコンアミドプロピ
ル）コラミド（以下、ＢＩＧＣＨＡＰという）、及びポ
リエチレングルコールモノ−ｐ−イソオクチルフェニル
エーテル（商品名「Ｔｒｉｔｏｎ Ｘ−１００」；以
下、Ｔｒｉｔｏｎ Ｘ−１００という）からなる４種の
界面活性剤を添加した検体希釈液を用いることにより、
非イオン性及びイオン性夾雑物による影響を効果的に排
除することができる。更に、牛血清アルブミン（ＢＳ
Ａ）を共存させるとより効果的である。At this time, proteins and / or lipids (nonionic and ionic contaminants) contained in the sample are against the antibodies and colloidal gold, membranes, latex particles, magnetic bead particles, polystyrene plates, etc. Non-specifically, it may affect the measurement. By adding a surfactant to the sample diluent, such non-specific adsorption can be avoided. As the surfactant, it is preferable to use, for example, a nonionic surfactant and a zwitterionic surfactant. In particular, N-tetradecyl-N, N-dimethyl-3-ammonio-1-propanesulfonic acid (hereinafter, referred to as Zwittergent), 3-[(3-colasidepropyl) dimethylammonio] -propanesulfonic acid (hereinafter, CHAPS). , N, N-bis (3-D-gluconamidopropyl) colamide (hereinafter referred to as BIGCHAP), and polyethylene glycol mono-p-isooctyl phenyl ether (trade name "Triton X-100"; hereinafter, Triton X-100) and the use of a sample diluent containing 4 kinds of surfactants
The effect of nonionic and ionic contaminants can be effectively eliminated. Furthermore, bovine serum albumin (BS
It is more effective to coexist with A).
【００１６】これら添加剤の検体希釈液中濃度は、Ｚｗ
ｉｔｔｅｒｇｅｎｔは好ましくは０．０１〜０．１５ｗ
／ｖ％、より好ましくは０．０５〜０．１ｗ／ｖ％、Ｃ
ＨＡＰＳは好ましくは０．０１〜０．５ｗ／ｖ％、より
好ましくは０．０５〜０．１ｗ／ｖ％、、ＢＩＧＣＨＡ
Ｐは好ましくは０．０１〜０．３ｗ／ｖ％、より好まし
くは０．０５〜０．１ｗ／ｖ％、Ｔｒｉｔｏｎ Ｘ−１
００は、好ましくは０．０１〜０．５ｗ／ｖ％、より好
ましくは０．１〜０．３ｗ／ｖ％の範囲内で用いる。更
に、必要に応じてＢＳＡを添加する場合には、その検体
希釈液中濃度は、好ましくは０．１〜１．０ｗ／ｖ％、
より好ましくは０．３〜０．７ｗ／ｖ％の範囲内で用い
る。The concentration of these additives in the sample diluent is Zw.
Ittergent is preferably 0.01 to 0.15 w
/ V%, more preferably 0.05 to 0.1 w / v%, C
HAPS is preferably 0.01 to 0.5 w / v%, more preferably 0.05 to 0.1 w / v%, BIGCHA
P is preferably 0.01 to 0.3 w / v%, more preferably 0.05 to 0.1 w / v%, Triton X-1.
00 is preferably used in the range of 0.01 to 0.5 w / v%, more preferably 0.1 to 0.3 w / v%. Furthermore, when BSA is added as necessary, the concentration in the sample diluent is preferably 0.1 to 1.0 w / v%,
More preferably, it is used within the range of 0.3 to 0.7 w / v%.
【００１７】上記で得られた抗ヒトラクトフェリン抗体
を用いて、便又は尿中のヒトラクトフェリンを免疫学的
に分析するには、従来公知の任意の免疫学的分析手段を
適宜選択して適用することが可能である。例えば、酵素
免疫法、又は磁気ビーズ酵素免疫法を利用してラクトフ
ェリンを測定するには、例えば、上記ポリクローナル抗
体（例えば、Ｈ１５抗体）をマイクロプレート又は磁気
ビーズに固相化する。そして、適当なブロッキング剤
（例えば、１．０％ゼラチン溶液）でブロッキングした
後、例えば、便試料（例えば、５０ｍｇ程度）を上記検
体希釈液で約１００〜４０００倍に希釈する。また、尿
試料（例えば、１００μｌ程度）を前記検体希釈液で約
５〜１００倍に希釈する。それぞれの希釈液を試料溶液
として上記固相化抗体と反応させる。続いて、市販の標
識化キット（例えば、Ａｍｅｒｓｈａｍ製ビオチン標識
キット；商品名ＲＰＮ２２０２等）を用いて標識した上
記ポリクローナル抗体（例えば、Ｈ１５抗体）と反応さ
せる。更に、標識アビジン（例えば、アルカリホスファ
ターゼ標識アビジン）を反応させた後、適当な基質（例
えば、ｐ−ニトロフェニルリン酸二ナトリウム等）を加
えて発色させ、分光学的にその吸光度を検出する。一
方、コントロール試験として、試料の代わりにヒトラク
トフェリン標準試薬シリーズを用いること以外は前記と
同様の操作を実施して、ヒトラクトフェリン標準曲線を
作成し、前記の試料から得られた吸光度と比較すること
によって、前記試料中のヒトラクトフェリンの存在・不
在を検出し、又はヒトラクトフェリン量を定量的に測定
することができる。前記の酵素免疫法又は磁気ビーズ酵
素免疫法は、２種のモノクローナル抗体の組合せ、又は
ポリクローナル抗体とモノクローナル抗体との組合せに
よって実施することもできる。To immunologically analyze human lactoferrin in feces or urine using the anti-human lactoferrin antibody obtained above, any conventionally known immunological analysis means is appropriately selected and applied. It is possible. For example, in order to measure lactoferrin using the enzyme immunoassay or the magnetic bead enzyme immunoassay, for example, the above polyclonal antibody (eg, H15 antibody) is immobilized on a microplate or magnetic beads. Then, after blocking with an appropriate blocking agent (for example, 1.0% gelatin solution), for example, a stool sample (for example, about 50 mg) is diluted about 100 to 4000 times with the above-mentioned sample diluent. Also, a urine sample (for example, about 100 μl) is diluted about 5 to 100 times with the sample diluent. Each diluted solution is used as a sample solution and reacted with the above-mentioned immobilized antibody. Then, it is reacted with the above-mentioned polyclonal antibody (for example, H15 antibody) labeled with a commercially available labeling kit (for example, biotin labeling kit manufactured by Amersham; trade name RPN2202 and the like). Furthermore, after reacting with labeled avidin (for example, alkaline phosphatase-labeled avidin), an appropriate substrate (for example, disodium p-nitrophenylphosphate or the like) is added to develop color, and the absorbance is spectroscopically detected. On the other hand, as a control test, perform the same operation as above except that the human lactoferrin standard reagent series is used instead of the sample to prepare a human lactoferrin standard curve, and compare it with the absorbance obtained from the sample. Thus, the presence / absence of human lactoferrin in the sample can be detected, or the amount of human lactoferrin can be quantitatively measured. The enzyme immunoassay or the magnetic bead enzyme immunoassay described above can also be carried out by a combination of two kinds of monoclonal antibodies or a combination of a polyclonal antibody and a monoclonal antibody.
【００１８】また、ラテックス凝集法、又は磁気ビーズ
凝集法を利用してヒトラクトフェリンを免疫学的に分析
する場合には、例えば、上記ポリクローナル抗体（例え
ば、Ｈ１５抗体）をラテックスビーズ又は磁気ビーズに
固相化する。便試料（例えば、５０ｍｇ程度）を上記検
体希釈液で２０〜１００倍に希釈する。また、尿試料
（例えば、１００μｌ程度）を上記検体希釈液で２０〜
１０倍に希釈する。それぞれの希釈液を試料溶液として
上記固相化抗体と反応させる。室温で数分間（例えば、
３〜１０分間）反応させた後、ラテックス又は磁気ビー
ズの凝集の有無を目視あるいは機械的に検出する。前記
のラテックス凝集法又は磁気ビーズ凝集法は、１種又は
２種以上のモノクローナル抗体の組合せ、又はポリクロ
ーナル抗体と１種以上のモノクローナル抗体との組合せ
を用いることによって実施することもできる。When human lactoferrin is immunologically analyzed by using the latex agglutination method or magnetic bead agglutination method, for example, the above polyclonal antibody (eg, H15 antibody) is immobilized on latex beads or magnetic beads. To be compatible. A stool sample (for example, about 50 mg) is diluted 20 to 100 times with the sample diluent. In addition, a urine sample (for example, about 100 μl) is diluted with the sample diluent 20
Dilute 10 times. Each diluted solution is used as a sample solution and reacted with the above-mentioned immobilized antibody. For a few minutes at room temperature (for example,
After reacting (for 3 to 10 minutes), the presence or absence of aggregation of latex or magnetic beads is visually or mechanically detected. The above-mentioned latex agglutination method or magnetic bead agglutination method can also be carried out by using a combination of one or more monoclonal antibodies, or a combination of a polyclonal antibody and one or more monoclonal antibodies.
【００１９】更に、イムノクロマトグラフ法を利用して
ヒトラクトフェリンを免疫学的に分析する場合には、例
えば、上記ポリクローナル抗体（例えば、Ｈ１５抗
体）、あるいは上記モノクローナル抗体（例えば、１５
Ｇ１１−０４抗体、１７Ｂ０４−０８抗体、１７Ｇ０４
−０８抗体、又は３２Ｄ０１−１０抗体）をヒトラクト
フェリン検出用抗体として用い、そしてヤギ抗ウサギＩ
ｇＧ抗体（Ｋａｐｐｅｌ製等）又はウサギ抗マウスＩｇ
抗体（ＤＡＫＯ製等）を陽性コントロール抗体として用
いて、それらの抗体を支持体（例えばナイロンメンブレ
ン）上に固相化する。一方、上記ポリクローナル抗体
（例えば、Ｈ１５抗体）、及び上記モノクローナル抗体
（例えば、１５Ｇ１１−０４抗体、１７Ｂ０４−０８抗
体、１７Ｇ０４−０８抗体、又は３２Ｄ０１−１０抗
体）を金コロイド（例えば、Ａｕｒｏ Ｂｅａｄｓ Ｇ
５，Ｇ１０，Ｇ３０；Ｐｈａｒｍａｃｉａ製等）で標識
し、これらの標識抗体を吸水パット（例えば、Ｌｏｐｒ
ｏｓｏｒｂ；Ｐｏｌｅ製等）に塗布して乾燥させた後、
上記の抗体固相化ナイロンメンブレンの一端に貼付す
る。そして、便試料（例えば、５０ｍｇ程度）を上記検
体希釈液で１００〜４０００倍に、そして尿試料（例え
ば、１００μｌ程度）を上記検体希釈液で２〜１００倍
に希釈して調製した各希釈試料液を、試料液として吸水
パット上に塗布する。室温で５〜１０分間放置した後、
ナイロンメンブレン上に固相化されたヒトラクトフェリ
ン検出用抗体及び陽性コントロール抗体による金コロイ
ドの捕獲に基づく着色（例えば赤紫−紫色）の有無を目
視あるいは機械的に判定する。このような操作法によ
り、試料中のヒトラクトフェリンを正確に分析すること
ができる。Further, when immunologically analyzing human lactoferrin using an immunochromatographic method, for example, the above polyclonal antibody (for example, H15 antibody) or the above monoclonal antibody (for example, 15 antibody) is used.
G11-04 antibody, 17B04-08 antibody, 17G04
-08 antibody, or 32D01-10 antibody) as an antibody for detecting human lactoferrin, and goat anti-rabbit I
gG antibody (Kappel etc.) or rabbit anti-mouse Ig
Using an antibody (manufactured by DAKO, etc.) as a positive control antibody, those antibodies are immobilized on a support (for example, a nylon membrane). On the other hand, the above polyclonal antibody (for example, H15 antibody) and the above monoclonal antibody (for example, 15G11-04 antibody, 17B04-08 antibody, 17G04-08 antibody, or 32D01-10 antibody) are colloidal gold (for example, Aurora Beads G).
5, G10, G30; manufactured by Pharmacia, etc., and these labeled antibodies are labeled with a water-absorbing pad (for example, Lopr).
osorb; made by Pole, etc.) and dried,
Attach it to one end of the antibody-immobilized nylon membrane. Each diluted sample prepared by diluting a stool sample (for example, about 50 mg) 100 to 4000 times with the sample diluent and a urine sample (for example, about 100 μl) 2 to 100 times with the sample diluent The solution is applied as a sample solution onto a water absorbing pad. After leaving at room temperature for 5-10 minutes,
The presence or absence of coloration (for example, red purple-purple) based on the capture of the gold colloid by the human lactoferrin detection antibody and the positive control antibody immobilized on the nylon membrane is visually or mechanically determined. By such an operation method, human lactoferrin in the sample can be accurately analyzed.
【００２０】なお、本発明において標識化されたポリク
ローナル抗体及び／又はモノクローナル抗体を用いる場
合には、従来公知の任意の方法によって、従来公知の任
意の標識（例えば、酵素タンパク質や金属コロイド）を
付けることができる。また、標識化されたポリクローナ
ル抗体及び／又はモノクローナル抗体として用いるラテ
ックス感作抗体も、従来公知の任意の方法によって調製
することができる。When the labeled polyclonal antibody and / or monoclonal antibody is used in the present invention, any conventionally known label (for example, enzyme protein or metal colloid) is attached by any conventionally known method. be able to. A latex-sensitized antibody used as a labeled polyclonal antibody and / or monoclonal antibody can also be prepared by any conventionally known method.
【００２１】図１に、酵素免疫法、イムノクロマトグラ
フ法、ラテックス凝集法、磁気ビーズ凝集法、磁気ビー
ズ酵素免疫法によるヒトラクトフェリンの測定可能濃度
範囲を示す。こうして、得られた結果は、感染症、例え
ば尿路感染症や細菌感染性下痢症等の診断指標として適
用することができる。酵素免疫法、又は磁気ビーズ酵素
免疫法により、ヒトラクトフェリンが便中に４０μｇ／
ｇ又はｍｌ便又はそれ以上、尿中に２００ｎｇ／ｍｌ尿
又はそれ以上の量で検出された場合、またラテックス凝
集法、磁気ビーズ凝集法、又はイムノクロマトグラフ法
でヒトラクトフェリンが陽性の場合には、消化管及び尿
路中の細菌が陽性であるものと判断することができ、こ
うして尿路感染症及び細菌感染性下痢症等のスクリーニ
ング法が提供される。FIG. 1 shows the measurable concentration range of human lactoferrin by enzyme immunoassay, immunochromatography, latex agglutination, magnetic bead agglutination, and magnetic bead enzyme immunoassay. Thus, the obtained results can be applied as a diagnostic index for infectious diseases such as urinary tract infection and bacterial infectious diarrhea. Human lactoferrin in the stool was 40 μg /
g or ml stool or more, when 200 ng / ml urine or more is detected in urine, and when human lactoferrin is positive by latex agglutination, magnetic bead agglutination, or immunochromatography, Bacteria in the digestive tract and urinary tract can be determined to be positive, thus providing a screening method for urinary tract infection, bacterial infectious diarrhea and the like.
【００２２】本発明によるスクリーニング用キットは、
少なくともヒトラクトフェリンの鉄飽和度に反応性が影
響されない抗ヒトラクトフェリン抗体、ヒトラクトフェ
リンの鉄飽和度に反応性が影響されない標識化抗ヒトラ
クトフェリン抗体、及び界面活性剤含有検体希釈液から
構成することができる。それらの具体的な構成は、選択
する免疫学的手段によって適宜変更可能である。以下
に、イムノクロマトグラフ法によるスクリーニング用キ
ットの一例を示す。The screening kit according to the present invention comprises:
At least an anti-human lactoferrin antibody whose reactivity is not affected by the iron saturation of human lactoferrin, a labeled anti-human lactoferrin antibody whose reactivity is not affected by the iron saturation of human lactoferrin, and a surfactant-containing sample diluent it can. Specific configurations thereof can be appropriately changed depending on the selected immunological means. An example of a screening kit for immunochromatography is shown below.
【００２３】便中ヒトラクトフェリン測定用のイムノク
ロマトグラフィー用キットの１態様
（１）ヒトラクトフェリン測定用イムノクロマトグラフ
小片（抗ヒトラクトフェリン抗体、標識化抗ヒトラクト
フェリン抗体、及び陽性コントロール抗体を含む）
（２）検体希釈用緩衝液
（３）試料採取用具（例えば、滅菌アルミニウム軸綿
棒、又は滅菌定量キャピラリー）
（４）試料希釈用容器（例えば、スプーン付キャップ、
及び検体希釈用緩衝液一定量入りの採便容器） Immunos for measuring human lactoferrin in stool
One aspect of kit for chromatography (1) Immunochromatographic piece for measuring human lactoferrin (including anti-human lactoferrin antibody, labeled anti-human lactoferrin antibody, and positive control antibody) (2) Sample dilution buffer (3) Sampling Tools (for example, sterilized aluminum swabs or sterilized quantitative capillaries) (4) Sample dilution containers (for example, caps with spoons,
And a stool collection container containing a fixed amount of sample dilution buffer)
【００２４】試験工程
（１）便検体を滅菌アルミニウム軸綿棒に十分に含ませ
る。
（２）綿棒を採便容器内の検体希釈用緩衝液中に浸し、
十分に洗い込み、便検体の希釈液を調製する。なお、粘
性便、軟様便、又は固形便等のように、綿棒で採取困難
な検体は、添付の定量キャピラリー又は採便容器のキャ
ップに付属しているスプーンで採取した後、採便容器内
の検体希釈用緩衝液中に浸し、均一に懸濁するまでよく
混和する。
（３）上記便検体の希釈液を添付の検体希釈用緩衝液を
用いて更に希釈し、被検液とする。
（４）被検液（例えば、約２００μｌ）をとり、イムノ
クロマトグラフ小片先端の吸水パッドを浸し、所定時間
（例えば、１０分間）静置した後、イムノクロマトグラ
フ小片の「ヒトラクトフェリン検出ゾーン」及び「陽性
コントロールゾーン」の着色の有無により判定する。例
えば、金コロイドを標識物として用いた場合には、以下
の表１の基準に従い判定する。 Test Step (1) Sterile aluminum swabs are thoroughly included in the stool specimen. (2) Dip a swab into the sample dilution buffer in the stool collection container,
Thoroughly wash and prepare a diluted solution of the stool sample. Samples that are difficult to collect with a cotton swab, such as viscous stool, soft stool, or solid stool, should be collected in the attached fixed-volume capillary or the spoon attached to the cap of the stool collection container, and then stored in the stool collection container. Immerse the sample in the sample dilution buffer and mix well until uniformly suspended. (3) The diluted solution of the above-mentioned fecal sample is further diluted with the attached sample dilution buffer to obtain a test solution. (4) Take a test liquid (for example, about 200 μl), immerse the water absorption pad at the tip of the immunochromatographic piece, and let it stand for a predetermined time (for example, 10 minutes). Then, the “human lactoferrin detection zone” and Judge by the presence or absence of coloration in the "positive control zone". For example, when gold colloid is used as a label, the judgment is made according to the criteria shown in Table 1 below.
【００２５】[0025]
【表１】 ヒトラクトフェリン検出ゾーン 陽性コントロールゾーン 判 定
赤紫 〜 紫色 赤紫 〜 紫色 陽 性
無 色 赤紫 〜 紫色 陰 性
赤紫 〜 紫色 無 色 再試験 無 色 無 色 再試験
尿検体についても、希釈率を適宜変更して、前記と同様
の工程によって、ラクトフェリンを分析することができ
る。[Table 1] For even human lactoferrin detection zone positive control zone determination Priority determination reddish purple to purplish red purple to purplish-positive non-color magenta-violet-negative magenta-violet-free color retested No color No color retest urine samples, the dilution rate appropriate Alternatively, lactoferrin can be analyzed by steps similar to those described above.
【００２６】[0026]
【実施例】以下、実施例によって本発明を具体的に説明
するが、これらは本発明の範囲を限定するものではな
い。以下の実施例において、ヒトラクトフェリンをｈＬ
Ｆと称することがある。実施例１：ウサギポリクローナル抗ヒトラクトフェリン
抗体の作製
免疫原として、ヒトラクトフェリン（Ｓｉｇｍａ製；Ｌ
−０５２０）（４ｍｇ／ｍｌ）の生理食塩水溶液５００
μｌを等量のフロイント完全アジュバント（ヤトロン
製）と十分に混合し、日本白色種ウサギ（１４週齢；
雌）に対して皮下に初回免疫を行った。４週間後、ヒト
ラクトフェリン（１ｍｇ／ｍｌ）の生理食塩水溶液５０
０μｌを等量のフロインド不完全アジュバント（和光純
薬工業製）と十分に混合し、上記ウサギに対して皮下に
追加免疫を行った。更に４週間後、追加免疫と同様の方
法で最終免疫を行った。最終免疫から２週間後に採血を
行い、抗血清を回収した。この抗血清に対して３３％飽
和硫安塩析を行って、ＩｇＧ粗画分を得た後、このＩｇ
Ｇ粗画分を、２０ｍＭリン酸緩衝液（ｐＨ７．５）に再
溶解し、ＤＥＡＥセファロースＣＬ−６Ｂカラムに負荷
した。ＤＥＡＥセファロースＣＬ−６Ｂカラムからの素
通り溶出画分を回収した後、限外濾過式タンパク質濃縮
装置（分子量カットオフ＝３００００）で濃縮し、ウサ
ギポリクローナル抗ｈＬＦ抗体とし、−４０℃下で保存
した。The present invention will be described in detail below with reference to examples, but these do not limit the scope of the present invention. In the examples below, human lactoferrin was added to hL
Sometimes referred to as F. Example 1: Rabbit polyclonal anti-human lactoferrin
Preparation of antibody As an immunogen, human lactoferrin (manufactured by Sigma; L
-0520) (4 mg / ml) of saline solution 500
μl was thoroughly mixed with an equal amount of Freund's complete adjuvant (manufactured by Yatron), and a Japanese white rabbit (14 weeks old;
Female) was subcutaneously immunized. 4 weeks later, a physiological saline solution of human lactoferrin (1 mg / ml) 50
0 μl was thoroughly mixed with an equal amount of Freund's incomplete adjuvant (manufactured by Wako Pure Chemical Industries, Ltd.), and the rabbit was boosted subcutaneously. After a further 4 weeks, the final immunization was performed in the same manner as the booster immunization. Two weeks after the final immunization, blood was collected to collect antisera. The antiserum was subjected to salting out with 33% saturated ammonium sulfate to obtain a crude IgG fraction.
The G crude fraction was redissolved in 20 mM phosphate buffer (pH 7.5) and loaded onto a DEAE Sepharose CL-6B column. The flow-through elution fraction from the DEAE Sepharose CL-6B column was collected and then concentrated with an ultrafiltration protein concentrator (molecular weight cutoff = 30000) to obtain a rabbit polyclonal anti-hLF antibody, which was stored at -40 ° C.
【００２７】便検体約２０ｍｇを精密に量り取り、検体
希釈用緩衝液〔０．０７％Ｚｗｉｔｔｅｒｇｅｎｔ（Ｃ
ａｌｂｉｏｃｈｅｍ製）、０．０７％ＣＨＡＰＳ（同仁
化学製）、０．０７％ＢＩＧＣＨＡＰ（同仁化学製）、
０．２％Ｔｒｉｔｏｎ Ｘ−１００（Ｂｉｏ−Ｒａｄ
Ｌａｂ．製）、及び０．５％牛血清アルブミン（Ｓｉｇ
ｍａ製）を含むホウ酸塩緩衝液；ｐＨ８．２〕を便の希
釈倍率が１００倍となるように正確に加えた。便検体が
均一に分散するまで超音波処理をした後、綿栓濾過し
た。濾液を１４０００ｒｐｍで１分間遠心分離して得た
上清をＥＬＩＳＡ測定用被検液とした。便中ｈＬＦ量が
多い検体は、検体希釈用緩衝液で更に適宜希釈し、測定
に用いた。About 20 mg of a stool sample was precisely weighed and a sample dilution buffer [0.07% Zwittergent (C
albiochem), 0.07% CHAPS (made by Dojindo), 0.07% BIGCHAP (made by Dojindo),
0.2% Triton X-100 (Bio-Rad
Lab. Made) and 0.5% bovine serum albumin (Sig
borate buffer solution (pH 8.2) containing ma) was accurately added so that the stool dilution ratio was 100 times. After ultrasonic treatment until the stool sample was uniformly dispersed, it was filtered with a cotton plug. The supernatant obtained by centrifuging the filtrate at 14000 rpm for 1 minute was used as a test solution for ELISA measurement. A sample having a large amount of hLF in stool was further diluted with a sample dilution buffer and used for the measurement.
【００２８】実施例２：モノクローナル抗体の作製法
（１）ヒトラクトフェリン（Ｓｅｒｖａ Ｆｅｉｎｂｉ
ｏｃｈｅｍｉｃａ製；Ｎｏ．２７４２２）を２ｍｇ／ｍ
ｌの濃度で生理食塩水に溶かし、免疫用抗原溶液とし
た。また、ヒトラクトフェリン（Ｓｅｒｖａ Ｆｅｉｎ
ｂｉｏｃｈｅｍｉｃａ製；Ｎｏ．２７４２２；鉄非結合
型）、ヒトラクトフェリン（Ｓｉｇｍａ製；Ｌ−０５２
０；鉄部分飽和型）、及びヒトラクトフェリン（Ｓｉｇ
ｍａ製；Ｌ−３７７０；鉄飽和型）を各々１０μｇ／ｍ
ｌの濃度で０．０５Ｍ炭酸塩／重炭酸塩緩衝液（ｐＨ
９．６）に溶かし、分析用抗原溶液とした。免疫用抗原
溶液１００μｌに同量のフロインド完全アジュバントを
加え、充分に混合して均質のゾルを調製した。このゾル
１００μｌを雌マウス（５週齢；ＢａＬＢ／ｃ）の皮下
に投与した。４週間後、免疫用抗原溶液を生理食塩水で
４倍希釈した希釈液１００μｌに同量のフロインド不完
全アジュバントを加え、充分に混合して均質のゾルを調
製した。このゾル１００μｌを上記雌マウスの皮下に投
与した。更に４週間後、免疫用抗原溶液を生理食塩水で
４倍希釈した希釈液１００μｌに同量のフロインド不完
全アジュバントを加え、充分に混合して均質のゾルを調
製した。このゾル１００μｌを上記雌マウスに腹腔内投
与した。 Example 2: Method for producing monoclonal antibody (1) Human lactoferrin (Serva Feinbi)
ochemica; No. 27422) at 2 mg / m
It was dissolved in physiological saline at a concentration of 1 to prepare an immunizing antigen solution. In addition, human lactoferrin (Serva Fein
manufactured by biochemica; 27422; iron non-binding type, human lactoferrin (manufactured by Sigma; L-052)
0; partially iron-saturated type), and human lactoferrin (Sig
ma; L-3770; iron saturated type) 10 μg / m each
0.05M carbonate / bicarbonate buffer (pH)
It was dissolved in 9.6) to give an antigen solution for analysis. The same amount of Freund's complete adjuvant was added to 100 μl of the immunizing antigen solution and mixed well to prepare a homogeneous sol. 100 μl of this sol was subcutaneously administered to female mice (5 weeks old; BaLB / c). After 4 weeks, the same amount of Freund's incomplete adjuvant was added to 100 µl of a diluted solution of the antigen solution for immunization which was 4-fold diluted with physiological saline, and they were mixed sufficiently to prepare a homogeneous sol. 100 μl of this sol was subcutaneously administered to the female mouse. After a further 4 weeks, the same amount of Freund's incomplete adjuvant was added to 100 μl of a diluted solution of the antigen solution for immunization which was diluted 4-fold with physiological saline, and they were mixed sufficiently to prepare a homogeneous sol. 100 μl of this sol was intraperitoneally administered to the female mouse.
【００２９】血清中の抗ヒトラクトフェリン抗体価の上
昇したマウスの脾臓を摘出し、イスコフ改変ダルベッコ
培地により、シャーレ内で摘出脾臓を３回洗浄した後、
ハサミで切れ目を入れてから、絞り出すように単細胞を
取り出して懸濁液を調製した。単細胞懸濁液をメッシュ
で濾過して大きな固形物を除いた。得られた濾液に、マ
ウスのミエローマ細胞Ｐ３−Ｘ６３−Ａｇ８−６．５．
３を細胞数の比で５：１（脾細胞：ミエローマ細胞）に
なるように混ぜ、遠心処理（１０００ｒｐｍ、５分間）
して細胞を集めた。次に、遠心管を指で弾いて沈渣を攪
拌してから３７℃に暖めておいたポリエチレングリコー
ル（分子量１，５００）溶液を、遠心管を回転させなが
ら１分間かけて徐々に加えた。室温下で１分間静置した
後、前記のイスコフ改変ダルベッコ培地５ｍｌを５分間
かけて徐々に加えた後、ウシ胎児血清（ＦＢＳ）１ｍｌ
をすばやく加え、細胞融合を停止した。そして前記のイ
スコフ改変ダルベッコ培地４０ｍｌを追加して、室温下
で１０分間静置した後、遠心処理した。得られた細胞を
細胞数が２×１０6 個／ｍｌとなるように１０％ＦＢ
Ｓ、ヒポキサンチン、及びチミジンを含むイスコフ改変
ダルベッコ培地（ＨＴ培地）に懸濁した。この細胞懸濁
液を９６ウェルプラスチックプレートに１００μｌ／ウ
ェルずつ分注して、３７℃にて、５％二酸化炭素及び９
５％加湿空気の条件下で培養を開始した。２４時間後
に、アミノプテリン加ＨＴ培地（ＨＡＴ培地）を１５０
μｌ／ウェルずつ分注し、更に１０〜１４日間同条件下
で継続培養した。培養液中の抗ヒトラクトフェリン抗体
活性を調べ、目的としている抗体を産生しているウェル
の細胞について９６ウェルプラスチックプレートでＨＴ
培地を用い、限界希釈法によりハイブリドーマのクロー
ニングを行った。クローニングの結果、抗ヒトラクトフ
ェリン抗体を産生しているハイブリドーマ９株を得た。After removing the spleen of the mouse in which the anti-human lactoferrin antibody titer in the serum was increased and washing the extracted spleen three times in a Petri dish with Iscove's modified Dulbecco's medium,
A suspension was prepared by cutting out with single scissors and then squeezing out single cells. The single cell suspension was filtered through a mesh to remove large solids. The resulting filtrate contains mouse myeloma cells P3-X63-Ag8-6.5.
3 was mixed at a cell number ratio of 5: 1 (spleen cells: myeloma cells) and centrifuged (1000 rpm, 5 minutes).
Then the cells were collected. Next, the centrifuge tube was flipped with a finger to stir the precipitate, and then a polyethylene glycol (molecular weight 1,500) solution warmed to 37 ° C. was gradually added over 1 minute while rotating the centrifuge tube. After standing at room temperature for 1 minute, 5 ml of the above Iscove's modified Dulbecco's medium was gradually added over 5 minutes, and then 1 ml of fetal bovine serum (FBS) was added.
Was rapidly added to stop cell fusion. Then, 40 ml of the above Iscove's modified Dulbecco's medium was added, and the mixture was allowed to stand at room temperature for 10 minutes and then centrifuged. The obtained cells were mixed with 10% FB so that the number of cells was 2 × 10 6 cells / ml.
The cells were suspended in Iscove's modified Dulbecco's medium (HT medium) containing S, hypoxanthine, and thymidine. 100 μl / well of this cell suspension was dispensed into a 96-well plastic plate, and at 37 ° C., 5% carbon dioxide and 9% were added.
Culture was started under the condition of 5% humidified air. After 24 hours, the HT medium containing aminopterin (HAT medium) was added to 150
Each μl / well was dispensed, and the cells were continuously cultured under the same conditions for 10 to 14 days. The anti-human lactoferrin antibody activity in the culture solution was examined, and the cells in the well producing the desired antibody were HT-treated with a 96-well plastic plate.
Hybridomas were cloned using the medium by the limiting dilution method. As a result of cloning, 9 hybridoma strains producing anti-human lactoferrin antibody were obtained.
【００３０】ハイブリドーマ９株の各々をペニシリン、
ストレプトマイシン及びファンギソンを各々２．５μｇ
／ｍｌずつ含む組織培養用無血清培地セルグロッサーＨ
（ハイブリドーマ用；住友製薬製）で培養し、培養液を
得た。培養液を３３％硫安分画し、得られたモノクロー
ナル抗体を５０ｍＭリン酸塩緩衝生理食塩液（ｐＨ７．
４）（以下、ＰＢＳという）に溶かして透析し、抗体を
得た。Each of the 9 hybridoma strains was treated with penicillin,
2.5 μg each of streptomycin and fungison
/ Ml Serum-free medium for tissue culture Cell Grosser H
(For hybridoma; manufactured by Sumitomo Pharmaceutical Co., Ltd.) to obtain a culture solution. The culture solution was fractionated with 33% ammonium sulfate, and the obtained monoclonal antibody was added to 50 mM phosphate buffered saline (pH 7.
4) It was dissolved in (hereinafter referred to as PBS) and dialyzed to obtain an antibody.
【００３１】（２）抗体の選定
上記３種類の分析用抗原溶液をマイクロプレート（Ｃｏ
ｓｔａｒ製；Ｎｏ．３５９０）に５０μｌ／ウェルずつ
分注し、４℃下で一夜静置して抗原を固相化した。各ウ
ェルをホウ酸塩緩衝生理食塩液（ｐＨ８．２）（以下、
ＢＢＳという）で２回洗浄した後、ＢＢＳに溶かしたゼ
ラチン溶液（１％）を２５０μｌ／ウェルの量で各ウェ
ルに分注し、３７℃下で１時間保温してブロッキング
し、ＢＢＳで５回洗浄した。前記実施例１で調製したウ
サギポリクローナル抗ヒトラクトフェリン抗体及び前記
実施例２（１）で調製したマウスモノクローナル抗ヒト
ラクトフェリン抗体を、０．１％ゼラチン及び０．１％
ポリオキシエチレンソルビタンモノラウレート（商品名
＝Ｔｗｅｅｎ２０）を含むＰＢＳに濃度１．０ｍｇ〜６
８６ｐｇ／ｍｌの範囲で順次希釈した希釈液を５０μｌ
／ウェルの量で各ウェルに分注し、３７℃下で１時間反
応させ、０．１％Ｔｗｅｅｎ２０を含むＰＢＳで５回洗
浄した。ビオチン標識プロテインＡ（Ａｍｅｒｓｈａｍ
製；ＲＰＮ１０１２）又は、ビオチン標識抗マウスＩｇ
Ｇ（Ａｍｅｒｓｈａｍ製；ＲＰＮ１１７７）を、０．１
％ゼラチン及び０．１％Ｔｗｅｅｎ２０を含むＰＢＳで
１０００倍希釈した希釈液を５０μｌ／ウェルの量で各
ウェルに分注し、３７℃下で１時間反応させた後、０．
１％のＴｗｅｅｎ２０を含むＰＢＳで５回洗浄した。(2) Selection of antibody The above-mentioned three types of analysis antigen solutions were applied to a microplate (Co
made by star; 50 μl / well in 3590) and allowed to stand overnight at 4 ° C. to immobilize the antigen. Each well was loaded with borate buffered saline (pH 8.2) (hereinafter,
After washing twice with BBS), a gelatin solution (1%) dissolved in BBS was dispensed into each well in an amount of 250 μl / well, kept warm at 37 ° C. for 1 hour to block, and 5 times with BBS. Washed. The rabbit polyclonal anti-human lactoferrin antibody prepared in Example 1 and the mouse monoclonal anti-human lactoferrin antibody prepared in Example 2 (1) were mixed with 0.1% gelatin and 0.1%.
A concentration of 1.0 mg to 6 in PBS containing polyoxyethylene sorbitan monolaurate (trade name = Tween 20)
50 μl of serially diluted dilution in the range of 86 pg / ml
/ Well was dispensed into each well, reacted at 37 ° C for 1 hour, and washed 5 times with PBS containing 0.1% Tween20. Biotin labeled protein A (Amersham
Manufactured by RPN1012) or biotin-labeled anti-mouse Ig
G (manufactured by Amersham; RPN1177) is 0.1
Diluted solution 1000-fold diluted with PBS containing 0.1% gelatin and 0.1% Tween 20 was dispensed into each well in an amount of 50 μl / well, reacted at 37 ° C. for 1 hour, and
The cells were washed 5 times with PBS containing 1% Tween20.
【００３２】アルカリホスファターゼ標識アビジン（Ｄ
ＡＫＯ製；Ｄ３６５）を、０．１％Ｔｗｅｅｎ２０を含
むＢＢＳで１０００倍希釈した希釈液を５０μｌ／ウェ
ルの量で各ウェルに分注し、３７℃下で３０分間反応さ
せた後、ＢＢＳで５回洗浄した。次いで、０．００５％
塩化マグネシウムを含む９．７％ジエタノールアミン緩
衝液（ｐＨ９．８）に、ｐ−ニトロフェニルリン酸二ナ
トリウム六水和物を１ｍｇ／ｍｌの濃度で溶かした溶液
を、基質溶液として、５０μｌ／ウェルの量で各ウェル
に分注し、室温下で１０分間静置し、酵素反応させた
後、４Ｍ水酸化ナトリウム溶液を５０μｌ／ウェルの量
で各ウェルに分注し、反応を止めた。反応液の吸光度を
波長４０５ｎｍにて分光光度計（Ｂｉｏ−Ｔｅｋ Ｉｎ
ｓｔｒｕｍｅｎｔｓ製；ＥＬ３１２ｅ型）で測定し、シ
グモイド結合曲線を作製した。結果を図２及び図３に示
す。その結果、３種類の分析用抗原に対するシグモイド
結合曲線がほぼ重なる（即ち、鉄飽和度の異なる３種類
のヒトラクトフェリンに対する結合能が同等）抗体とし
て、ウサギポリクローナル抗体（Ｈ１５抗体）、及びマ
ウスモノクローナル抗体（１５Ｇ１１−０４、１７Ｂ０
４−０８、１７Ｇ０５−１０、及び３２Ｄ０１−１０抗
体）、ならびにそれらのモノクローナル抗体を分泌する
ハイブリドーマ４株（１５Ｇ１１−０４、１７Ｂ０４−
０８、１７Ｇ０５−１０、及び３２Ｄ０１−１０）を得
た。Alkaline phosphatase labeled avidin (D
AKO; D365) was diluted 1000 times with BBS containing 0.1% Tween 20, and the diluted solution was dispensed into each well at a volume of 50 μl / well, reacted at 37 ° C. for 30 minutes, and then reacted with BBS for 5 minutes. Washed twice. Then 0.005%
A solution prepared by dissolving p-nitrophenyl phosphate disodium hexahydrate at a concentration of 1 mg / ml in a 9.7% diethanolamine buffer solution (pH 9.8) containing magnesium chloride was used as a substrate solution at 50 μl / well. The amount was dispensed into each well, the mixture was allowed to stand at room temperature for 10 minutes to cause an enzyme reaction, and then 4 M sodium hydroxide solution was dispensed into each well in an amount of 50 μl / well to stop the reaction. The absorbance of the reaction solution was measured with a spectrophotometer (Bio-Tek In) at a wavelength of 405 nm.
sigmoid binding curve was prepared by measuring with an EL312e model manufactured by Instruments). The results are shown in FIGS. 2 and 3. As a result, rabbit polyclonal antibody (H15 antibody) and mouse monoclonal antibody were used as the antibodies having almost the same sigmoid binding curves for the three types of analytical antigens (that is, the same binding ability for three types of human lactoferrin having different iron saturation). (15G11-04, 17B0
4-08, 17G05-10, and 32D01-10 antibodies), and four hybridomas secreting these monoclonal antibodies (15G11-04, 17B04-).
08, 17G05-10, and 32D01-10) were obtained.
【００３３】図２において、○は鉄非結合型ヒトラクト
フェリン（ａｐｏ−ｈＬＦ；Ｓｅｒｖａ Ｆｅｉｎｂｉ
ｏｃｈｅｍｉｃａ製；Ｎｏ．２７４２２）に対する反応
性、●は鉄部分飽和型ヒトラクトフェリン（ｐｓａｔ−
ｈＬＦ；Ｓｉｇｍａ製；Ｌ−０５２０）に対する反応
性、そして□は鉄飽和型ヒトラクトフェリン（ｈｏｌｏ
−ｈＬＦ；Ｓｉｇｍａ製；Ｌ−３７７０）に対する反応
性を示す。図３において、○は鉄非結合型ヒトラクトフ
ェリン（ａｐｏ−ｈＬＦ；Ｓｅｒｖａ Ｆｅｉｎｂｉｏ
ｃｈｅｍｉｃａ製；Ｎｏ．２７４２２）に対する反応
性、□は鉄部分飽和型ヒトラクトフェリン（ｐｓａｔ−
ｈＬＦ；Ｓｉｇｍａ製；Ｌ−０５２０）に対する反応
性、そして△は鉄飽和型ヒトラクトフェリン（ｈｏｌｏ
−ｈＬＦ；Ｓｉｇｍａ製；Ｌ−３７７０）に対する反応
性を示す。In FIG. 2, ∘ indicates non-iron-binding human lactoferrin (apo-hLF; Serva Feinbi).
ochemica; No. 27422), and ● represents iron partially saturated human lactoferrin (psat-
hLF; manufactured by Sigma; L-0520), and □ are iron-saturated human lactoferrin (holo).
-HLF; manufactured by Sigma; L-3770). In FIG. 3, ◯ indicates non-iron-binding human lactoferrin (apo-hLF; Serva Feinbio).
manufactured by Chemica; No. 27422), □ indicates iron partially saturated human lactoferrin (psat-
hLF; manufactured by Sigma; L-0520), and Δ are iron-saturated human lactoferrin (holo).
-HLF; manufactured by Sigma; L-3770).
【００３４】実施例３：ヒトラクトフェリンＥＬＩＳＡ
測定法
マイクロプレート（Ｃｏｓｔａｒ製；Ｎｏ．３５９０）
の各ウェルに、Ｈ１５抗体溶液（１０μｇ／ｍｌ；０．
０５Ｍ炭酸塩／重炭酸塩緩衝液；ｐＨ９．６）を５０μ
ｌずつ分注し、４℃下で一夜保存した後、ＢＢＳで３回
洗浄した。１％ゼラチン（Ｂｉｏ−Ｒａｄ Ｌａｂ．
製；ＥＩＡ ｐｕｒｉｔｙ）を含むＰＢＳを各ウェルに
２５０μｌずつ分注し、３７℃下で１時間保温してブロ
ッキングした。０．１％Ｔｗｅｅｎ２０を含むＰＢＳで
５回洗浄した。検体希釈用緩衝液（０．０７％Ｚｗｉｔ
ｔｅｒｇｅｎｔ、０．０７％ＣＨＡＰＳ、０．０７％Ｂ
ＩＧＣＨＡＰ、０．２％Ｔｒｉｔｏｎ Ｘ−１００、検
体及び０．５％牛血清アルブミンを含むホウ酸塩緩衝
液；ｐＨ８．２）で希釈したヒトラクトフェリン標準
品、便検体又は尿検体を各ウェルに５０μｌずつ加え、
３７℃下で１時間反応させた。０．１％Ｔｗｅｅｎ２０
を含むＰＢＳで５回洗浄した。 Example 3: Human lactoferrin ELISA
Measurement method Microplate (manufactured by Costar; No. 3590)
H15 antibody solution (10 μg / ml;
50M of 05M carbonate / bicarbonate buffer; pH 9.6)
The solution was dispensed in liters, stored overnight at 4 ° C., and washed 3 times with BBS. 1% gelatin (Bio-Rad Lab.
PBS (manufactured by EIA purity) was dispensed into each well in an amount of 250 μl and incubated at 37 ° C. for 1 hour for blocking. The plate was washed 5 times with PBS containing 0.1% Tween20. Sample dilution buffer (0.07% Zwit
tergent, 0.07% CHAPS, 0.07% B
50 μl of human lactoferrin standard, stool sample or urine sample diluted with borate buffer containing IGCHAP, 0.2% Triton X-100, sample and 0.5% bovine serum albumin; pH 8.2) to each well Add one by one,
The reaction was carried out at 37 ° C for 1 hour. 0.1% Tween 20
It was washed 5 times with PBS containing.
【００３５】予め、市販の試薬（Ａｍｅｒｓｈａｍ製；
ＲＰＮ２２０３）を用いてビオチン標識したＨ１５抗体
溶液（２μｇ／ｍｌ；０．１％Ｔｗｅｅｎ２０及び０．
１％ゼラチンを含むＰＢＳ）を各ウェルに５０μｌずつ
分注し、３７℃で１時間反応させた後、０．１％Ｔｗｅ
ｅｎ２０を含むＰＢＳで５回洗浄した。アルカリホスフ
ァターゼ標識アビジン（ＤＡＫＯ製；Ｄ３６５）を、
０．１％Ｔｗｅｅｎ２０及び０．１％ゼラチンを含むＰ
ＢＳで１０００倍に希釈した希釈液を各ウェルに５０μ
ｌずつ分注し、３７℃下で３０分間反応させた後、ＢＢ
Ｓで５回洗浄した。ｐ−ニトロフェニルリン酸二ナトリ
ウム六水和物溶液（１０ｍｇ／ｍｌ；０．００５％Ｍｇ
Ｃｌ2 を含む９．７ｖ／ｖ％ジエタノールアミン緩衝
液；ｐＨ９．８）を各ウェルに５０μｌずつ分注した。
室温下で１０分間酵素反応させ、４ＭＮａＯＨ溶液を各
ウェルに５０μｌずつ分注して酵素反応を停止させた。
最後に、各ウェルの黄色の呈色をマイクロプレート用分
光光度計（Ｂｉｏ−Ｔｅｋ Ｉｎｓｔｒｕｍｅｎｔｓ
製；ＥＬ３１２ｅ型）を用いて、４０５ｎｍの波長光で
吸光度を測定した。検量線から便又は尿中のヒトラクト
フェリン濃度を算出した。Commercially available reagents (manufactured by Amersham;
HPN antibody solution (2 μg / ml; 0.1% Tween 20 and 0.
50 μl of PBS containing 1% gelatin) was dispensed into each well and reacted at 37 ° C. for 1 hour, and then 0.1% Twe.
The cells were washed 5 times with PBS containing en20. Alkaline phosphatase labeled avidin (DAKO; D365),
P containing 0.1% Tween 20 and 0.1% gelatin
Dilute the solution 1000 times with BS to each well 50μ
Dispense 1 liter each and react at 37 ° C for 30 minutes, then BB
Washed 5 times with S. Disodium p-nitrophenyl phosphate hexahydrate solution (10 mg / ml; 0.005% Mg
50 μl of 9.7 v / v% diethanolamine buffer solution containing Cl 2 ; pH 9.8) was dispensed into each well.
The enzyme reaction was carried out at room temperature for 10 minutes, and 50 μl of 4M NaOH solution was dispensed into each well to stop the enzyme reaction.
Finally, the yellow color of each well was analyzed by a spectrophotometer for microplates (Bio-Tek Instruments).
Manufactured by EL312e) was used to measure the absorbance with light having a wavelength of 405 nm. The human lactoferrin concentration in feces or urine was calculated from the calibration curve.
【００３６】図４は、ＥＬＩＳＡによるヒトラクトフェ
リンの標準曲線を示したものである。図５〜図１０は、
便中ヒトラクトフェリンのＥＬＩＳＡ測定の際に用いた
検体希釈用緩衝液に添加した界面活性剤の効果を検討し
た結果を示したものである。すなわち、界面活性剤の組
成の異なる検体希釈用緩衝液で便検体を２０〜１０００
倍希釈した状態でヒトラクトフェリンの標準添加実験を
行った。使用した界面活性剤の種類と濃度〔ＢＢＳ（ｐ
Ｈ８．２）中〕を以下の表２に示す。FIG. 4 shows a standard curve of human lactoferrin by ELISA. 5 to FIG.
2 shows the results of examining the effect of a surfactant added to a sample diluting buffer used in ELISA measurement of human lactoferrin in stool. That is, a stool sample is diluted with a sample-diluting buffer solution having a different composition of the surfactant from 20 to 1,000.
A standard addition experiment of human lactoferrin was carried out in a state of being diluted twice. Type and concentration of surfactant used [BBS (p
H8.2)] is shown in Table 2 below.
【００３７】[0037]
【表２】 図 ５及び６ ７及び８ ９及び１０ Ｚｗｉｔｔｅｒｇｅｎｔ ０．１％ ０．１％ ０．０７％ ＣＨＡＰＳ なし ０．１％ ０．０７％ ＢＩＧＣＨＡＰ ０．１％ なし ０．０７％ Ｔｒｉｔｏｎ Ｘ−１００ ０．２％ ０．２％ ０．２％ＢＳＡ ０．５％ ０．５％ ０．５％ [Table 2] Figures 5 and 6 7 and 8 9 and 10 Zwittergent 0.1% 0.1% 0.07% CHAPS none 0.1% 0.07% BIGCHAP 0.1% none 0.07% Triton X-100 0.2 % 0.2% 0.2% BSA 0.5% 0.5% 0.5%
【００３８】なお、図５〜図１０において、●は検体希
釈用緩衝液のみの場合を示す。また、図５、図７及び図
９において、○は２０倍希釈便検体、□は５０倍希釈便
検体、そして△は１００倍希釈便検体の場合を示す。更
に、図６、図８及び図１０において、○は２００倍希釈
便検体、□は５００倍希釈便検体、そして△は１０００
倍希釈便検体の場合を示す。図５〜図１０の結果から明
らかなように、０．０７％Ｚｗｉｔｔｅｒｇｅｎｔ、
０．０７％ＣＨＡＰＳ、０．０７％ＢＩＧＣＨＡＰ、
０．２％ＴｒｉｔｏｎＸ−１００、及び０．５％牛血清
アルブミンを含むＢＢＳを検体希釈用緩衝液として用い
た場合には、便検体中の夾雑物の影響が最も少なくな
り、ヒトラクトフェリンを定量的に測定することができ
た。In FIGS. 5 to 10, the solid circles indicate the case where only the sample diluting buffer solution is used. In addition, in FIGS. 5, 7, and 9, ∘ indicates a 20-fold diluted stool sample, □ indicates a 50-fold diluted stool sample, and Δ indicates a 100-fold diluted stool sample. Further, in FIGS. 6, 8 and 10, ∘ indicates a 200-fold diluted stool sample, □ indicates a 500-fold diluted stool sample, and Δ indicates 1000.
The case of double-diluted stool sample is shown. As is clear from the results of FIGS. 5 to 10, 0.07% Zwittergent,
0.07% CHAPS, 0.07% BIGCHAP,
When BBS containing 0.2% Triton X-100 and 0.5% bovine serum albumin was used as a sample dilution buffer, the influence of impurities in the stool sample was minimized, and human lactoferrin was quantitatively determined. Could be measured.
【００３９】前記検体希釈用緩衝液を用いて、ヒトラク
トフェリンの便又は尿への添加回収実験を行った結果を
図１１、図１２及び図１３に示す。すなわち、成人健常
人から採取して前記検体希釈用緩衝液で２００倍に希釈
した便検体１（図１１内の○）、成人健常人から採取し
て前記検体希釈用緩衝液で１００倍に希釈した便検体２
（図１１内の□）、及び成人健常人から採取して前記検
体希釈用緩衝液で１００倍に希釈した便検体３（図１１
内の△）のそれぞれについて種々濃度のａｐｏ−ｈＬＦ
標準品を添加し、前記のＥＬＩＳＡ法により各検体につ
き３回測定した。図１１には、平均値（○、□及び△）
と標準偏差The results of a recovery experiment of human lactoferrin added to feces or urine using the above-mentioned sample dilution buffer are shown in FIGS. 11, 12 and 13. That is, the stool sample 1 (○ in FIG. 11) that was collected from a healthy adult person and diluted 200 times with the sample dilution buffer, and 100 times diluted with the sample dilution buffer solution from an adult healthy person. Stool sample 2
(□ in FIG. 11), and fecal sample 3 (FIG. 11) that was collected from a healthy adult person and diluted 100-fold with the sample dilution buffer.
Of each of Δ) in various concentrations of apo-hLF
The standard product was added, and measurement was performed 3 times for each sample by the above-mentioned ELISA method. In Fig. 11, the average value (○, □ and △)
And standard deviation
【外１】
とを示す。なお、検体１、検体２及び検体３の直線回帰
式は以下のとおりであった。
検体１：ｙ＝０．９３６ｘ＋５．１５
検体２：ｙ＝０．８７２ｘ＋１．２８６
検体３：ｙ＝０．９７６ｘ＋０．８９０
すなわち、３種類の便検体を１００倍又は２００倍希釈
した状態での添加回収率は、８７．２％〜９７．６％で
あった。[Outer 1] And indicates. The linear regression equations for Sample 1, Sample 2 and Sample 3 were as follows. Specimen 1: y = 0.936x + 5.15 Specimen 2: y = 0.872x + 1.286 Specimen 3: y = 0.976x + 0.890 That is, addition recovery of three types of fecal specimens diluted 100-fold or 200-fold The rate was 87.2% to 97.6%.
【００４０】また、成人健常人から採取した２種類の尿
について、前記検体希釈用緩衝液で５倍に希釈した尿検
体１（図１２内の●）、１０倍に希釈した尿検体２（図
１２内の○）、同様にもう一方の尿を前記検体希釈用緩
衝液で５倍に希釈した尿検体３（図１３内の●）、１０
倍に希釈した尿検体４（図１３内の○）のそれぞれにつ
いて上記と同様に測定を行った。検体１、検体２、検体
３及び検体４の直線回帰式は以下のとおりであった。
検体１：ｙ＝０．９１６ｘ＋７．６５０
検体２：ｙ＝０．９６５ｘ＋１．８６１
検体３：ｙ＝１．１３０ｘ＋３．２２７
検体４：ｙ＝０．９２５ｘ＋３．５１４
すなわち、添加回収率は、９１．６％〜１１３％であっ
た。In addition, two kinds of urine collected from healthy adult persons were diluted 5 times with the sample dilution buffer (1 in FIG. 12) and 10 times diluted with urine sample 2 (see FIG. 12). 12), similarly, the other urine is a urine sample 3 (● in FIG. 13) obtained by diluting the other urine five-fold with the sample dilution buffer.
The same measurement as above was performed for each of the urine specimens 4 (O in FIG. 13) that were diluted twice. The linear regression equations for Sample 1, Sample 2, Sample 3, and Sample 4 were as follows. Specimen 1: y = 0.916x + 7.650 Specimen 2: y = 0.965x + 1.861 Specimen 3: y = 1.130x + 3.227 Specimen 4: y = 0.925x + 3.514 That is, the addition recovery rate is 91.6. % To 113%.
【００４１】実施例４：イムノクロマトグラフ法による
ヒトラクトフェリンの測定
本例では、Ｈ１５抗体及び金コロイド標識Ｈ１５抗体、
又は金コロイド標識１７Ｂ０４−０８抗体を用いてヒト
ラクトフェリンイムノクロマトグラフ法を実施した。ナ
イロン６６製メンブレン（Ｐｏｌｅ製；Ｂｉｏｄｙｎｅ
Ｃ、又はＩｍｍｕｎｏｄｙｎｅ ＡＢＣ；孔径＝３μ
ｍ又は５μｍ）を６ｍｍ×３５ｍｍのサイズに切断し
た。Ｂｉｏｄｙｎｅ Ｃは、０．５％ジシクロヘキシル
カルボジイミドの塩化メチレン溶液に浸し、室温下で３
０分間反応させ、塩化メチレンで洗浄し、風乾した後に
用いた。また、Ｉｍｍｕｎｏｄｙｎｅ ＡＢＣは、その
ままイムノクロマトグラフ用メンブレンとして用いた。
各々のメンブレンの一端から、１５ｍｍ及び２３ｍｍの
位置にＨ１５抗体溶液（１０ｍｇ〜０．１ｍｇ／ｍｌ；
ＢＢＳ）、及び陽性コントロール抗体としてヤギ抗ウサ
ギＩｇＧ抗体溶液（Ｋａｐｐｅｌ製）又はウサギ抗マウ
スＩｇ抗体溶液（ＤＡＫＯ製）（各３ｍｇ／ｍｌ；ＢＢ
Ｓ）を０．５μｌずつ塗布し、室温下で１時間静置し固
相化した。メンブレンを０．５％モノエタノールアミン
のＢＢＳ溶液に浸し、室温下で３０分間反応させブロッ
キングした。メンブレンを０．５％カゼイン（ハマース
テングレード）溶液（０．１Ｍマレイン酸緩衝液；ｐＨ
７．５；０．４５μｍ濾過）に浸し、室温下で３０分間
静置し更にブロッキングした。メンブレンをＰＢＳで２
回、蒸留水（０．４５μｍで濾過したもの）で３回洗浄
し、風乾した後、シリカゲルデシケーター内に保存し、
ヒトラクトフェリンイムノクロマトグラフ法用メンブレ
ンとした。 Example 4: By immunochromatographic method
Measurement of human lactoferrin In this example, H15 antibody and colloidal gold-labeled H15 antibody,
Alternatively, human lactoferrin immunochromatography was carried out using gold colloid-labeled 17B04-08 antibody. Nylon 66 Membrane (Pole; Biodyne
C, or Immunodyne ABC; Pore size = 3μ
m or 5 μm) was cut into a size of 6 mm × 35 mm. Biodyne C was immersed in a 0.5% solution of dicyclohexylcarbodiimide in methylene chloride, and allowed to stand at room temperature for 3 days.
The mixture was reacted for 0 minutes, washed with methylene chloride and air-dried before use. In addition, Immunodyne ABC was used as it was as a membrane for immunochromatography.
H15 antibody solution (10 mg-0.1 mg / ml; 15 mm and 23 mm) from one end of each membrane.
BBS) and a goat anti-rabbit IgG antibody solution (manufactured by Kappel) or a rabbit anti-mouse Ig antibody solution (manufactured by DAKO) as a positive control antibody (each 3 mg / ml; BB).
0.5 μl each of S) was applied, and allowed to stand at room temperature for 1 hour to solidify. The membrane was immersed in a BBS solution of 0.5% monoethanolamine, reacted at room temperature for 30 minutes, and blocked. Use a 0.5% casein (Hamersten grade) solution (0.1M maleic acid buffer; pH) for the membrane.
7.5; 0.45 μm filtration) and allowed to stand at room temperature for 30 minutes for further blocking. Membrane with PBS 2
Once, washed 3 times with distilled water (0.45 μm filtered), air dried and stored in a silica gel desiccator,
The membrane was used for human lactoferrin immunochromatography.
【００４２】一方、金コロイド液（Ａｍｅｒｓｈａｍ
製；粒径１０ｎｍ；ＲＰＮ４７６）８０ｍｌ、Ｈ１５抗
体溶液（１ｍｇ／ｍｌ）、及び１７Ｂ０４−０８抗体溶
液（１ｍｇ／ｍｌ）各々１０ｍｌを、２ｍＭＮａ2 Ｂ4
Ｏ7 緩衝液（ｐＨ９．０）（以下、Ｂｏｒａｘ緩衝液と
称す）３０００ｍｌで４℃下にて一夜透析した。Ｈ１５
抗体溶液及び１７Ｂ０４−０８抗体溶液は１００，００
０×ｇで１時間遠心分離した後、その上清を１００，０
００×ｇで１時間遠心分離したＢｏｒａｘ緩衝液で、各
々１５０μｇ／ｍｌ、及び２４０μｇ／ｍｌとなるよう
に希釈した。金コロイド溶液各々４０ｍｌに希釈したＨ
１５抗体溶液又は１７Ｂ０４−０８抗体溶液４．０ｍｌ
を加え、室温下にて２分間攪拌した。１０％ＢＳＡ溶液
（ｐＨ９．０；０．４５μｍ濾過）４．９ｍｌを加えて
攪拌した後、２〜８℃下にて４５，０００×ｇで３０分
間遠心分離し、上清を除去した。１％ＢＳＡを含む２０
ｍＭトリス、１５０ｍＭＮａＣｌ緩衝液（ｐＨ８．０；
０．４５μｍ濾過）（以下、ＢＳＡ−Ｔｒｉｓと称す）
４０ｍｌを加えて攪拌した後、２〜８℃下にて４５，０
００×ｇで３０分間遠心分離し、上清を除去した。更
に、ＢＳＡ−Ｔｒｉｓ４０ｍｌを加えて攪拌した後、２
〜８℃下にて４５，０００×ｇで３０分間遠心分離し、
上清を除去した。On the other hand, a gold colloid solution (Amersham)
Made; particle size 10 nm; RPN476) 80 ml, H15 antibody solution (1 mg / ml), and 17B04-08 antibody solution (1 mg / ml) 10 ml each, 2 mM Na 2 B 4
The mixture was dialyzed against 3000 ml of O 7 buffer (pH 9.0) (hereinafter referred to as Borax buffer) at 4 ° C. overnight. H15
The antibody solution and 17B04-08 antibody solution are 100,000.
After centrifuging at 0 xg for 1 hour, the supernatant was washed with 100,0
Diluted with Borax buffer, which was centrifuged at 00 × g for 1 hour, to 150 μg / ml and 240 μg / ml, respectively. Gold colloid solution diluted to 40 ml each H
15 antibody solution or 17B04-08 antibody solution 4.0 ml
Was added, and the mixture was stirred at room temperature for 2 minutes. After adding 4.9 ml of 10% BSA solution (pH 9.0; 0.45 μm filtration) and stirring, the mixture was centrifuged at 45,000 × g for 30 minutes at 2-8 ° C., and the supernatant was removed. 20 including 1% BSA
mM Tris, 150 mM NaCl buffer (pH 8.0;
0.45 μm filtration) (hereinafter referred to as BSA-Tris)
After adding 40 ml and stirring, 45,0 under 2-8 degreeC.
The supernatant was removed by centrifugation at 00 × g for 30 minutes. Furthermore, after adding 40 ml of BSA-Tris and stirring, 2
Centrifuge at 45,000 xg for 30 minutes at ~ 8 ° C,
The supernatant was removed.
【００４３】沈殿した金コロイドを波長５２０ｎｍの吸
光度が２．５となるようＢＳＡ−Ｔｒｉｓで希釈した
後、４℃下に保存し、ヒトラクトフェリンイムノクロマ
トグラフ法用金コロイド標識抗体懸濁液とした。金コロ
イド標識抗体懸濁液は、その５０μｌを吸水パット（Ｐ
ｏｌｅ製；Ｌｏｐｒｏｓｏｒｂ；６ｍｍ×１０ｍｍ）に
塗布した後、風乾し、イムノクロマトグラフ法用吸水パ
ッドとして用いた。上記イムノクロマトグラフ法用メン
ブレンをアクリル樹脂製支持体（１０×５０ｍｍ）に貼
付した後、その一端に上記イムノクロマトグラフ法用吸
水パッドを貼付して、ヒトラクトフェリン測定用イムノ
クロマトグラフキットとした。前記検体希釈用緩衝液で
希釈したヒトラクトフェリン標準品、便又は尿検体各２
００μｌにイムノクロマトグラフキット上の吸水パッド
を浸し、室温下で５〜１０分間静置した。メンブレンの
Ｈ１５抗体を塗布した部分、及び陽性コントロール抗体
を塗布した部分の金コロイドの捕獲に基づく、赤紫−紫
色の着色により、ヒトラクトフェリンの有無を判定し
た。The precipitated gold colloid was diluted with BSA-Tris so that the absorbance at a wavelength of 520 nm was 2.5 and then stored at 4 ° C. to obtain a gold colloid-labeled antibody suspension for human lactoferrin immunochromatography. 50 μl of the gold colloid-labeled antibody suspension is used as a water-absorbing pad (P
Ole; Loprosorb; 6 mm x 10 mm), and then air-dried to be used as a water absorption pad for immunochromatography. After sticking the membrane for immunochromatography to an acrylic resin support (10 × 50 mm), the water absorption pad for immunochromatography was stuck to one end of the support to obtain an immunochromatographic kit for measuring human lactoferrin. Human lactoferrin standard product diluted with the sample dilution buffer, feces or urine samples 2 each
The water-absorbing pad on the immunochromatographic kit was immersed in 00 μl, and allowed to stand at room temperature for 5 to 10 minutes. The presence or absence of human lactoferrin was determined by the red-purple-purple coloration based on the capture of the gold colloid on the H15 antibody-coated portion of the membrane and the positive control antibody-coated portion.
【００４４】表３は、イムノクロマトグラフ法によるヒ
トラクトフェリン標準溶液測定結果を示したものであ
る。表３において、＋は陽性、−は陰性、±は偽陽性で
あることを示す（以下の表４及び表５も同様）。Ｈ１５
抗体はポリクローナル抗体であるため、抗体過剰領域で
は金コロイドの抗原濃度依存性凝集が起こり、Ｂｉｏｄ
ｙｎｅ Ｃを用いた場合には、その影響で５００μｇ／
ｍｌ以上の検出が不可能であったが、Ｉｍｍｕｎｏｄｙ
ｎｅ ＡＢＣでは、凝集による影響が軽減し、更に、２
０ｎｇ／ｍｌの検出感度が得られた。１７Ｂ０４−０８
抗体を用いた場合は１０ｎｇ／ｍｌの検出感度が得られ
た。Table 3 shows the results of human lactoferrin standard solution measurement by immunochromatography. In Table 3, + indicates positive, − indicates negative, and ± indicates false positive (the same applies to Tables 4 and 5 below). H15
Since the antibody is a polyclonal antibody, antigen concentration-dependent aggregation of gold colloid occurs in the antibody excess region, and Biod
When yne C is used, 500 μg /
It was impossible to detect more than ml, but Immunody
ne ABC reduces the effect of aggregation, and
A detection sensitivity of 0 ng / ml was obtained. 17B04-08
A detection sensitivity of 10 ng / ml was obtained when the antibody was used.
【００４５】表４は、イムノクロマトグラフ法によるヒ
トラクトフェリン標準品の検出感度の調整結果を示した
ものである。金コロイド標識抗体量は一定であっても、
メンブレン上に固相化する抗体濃度を変化させることに
より、ヒトラクトフェリン検出感度を３０〜３００ｎｇ
／ｍｌの範囲で調整することができた。Table 4 shows the adjustment result of the detection sensitivity of the human lactoferrin standard product by the immunochromatography method. Even if the amount of gold colloid-labeled antibody is constant,
By changing the concentration of the antibody immobilized on the membrane, the detection sensitivity of human lactoferrin is 30 to 300 ng.
It could be adjusted in the range of / ml.
【００４６】表５は成人健常人血液より分離した白血球
（多核球）の破砕上清中のヒトラクトフェリンのイムノ
クロマトグラフ法による測定結果を示したものである。
白血球数１０6 個がヒトラクトフェリン量４．９μｇに
相当する（Ｊ．Ｉｍｍｕｎｏｌ．Ｍｅｔｈｏｄｓ、６
５、ｐ１８３〜ｐ１９０、１９８３）ことから、ヒト母
乳由来のラクトフェリンを免疫原として作製した抗ヒト
ラクトフェリン抗体を用いた本イムノクロマトグラフ法
により白血球中のヒトラクトフェリンを効率よく測定す
ることができる。Table 5 shows the measurement results of human lactoferrin in the crushed supernatant of white blood cells (polynuclear cells) separated from the blood of healthy adults by immunochromatography.
A white blood cell count of 10 6 corresponds to a human lactoferrin amount of 4.9 μg (J. Immunol. Methods, 6).
5, p183 to p190, 1983), human lactoferrin in leukocytes can be efficiently measured by the present immunochromatographic method using an anti-human lactoferrin antibody prepared using human breast milk-derived lactoferrin as an immunogen.
【００４７】表６は、成人健常便及び成人健常起床時初
尿中のヒトラクトフェリン量をＥＬＩＳＡ法により測定
した結果を示したものである。各々、平均値Table 6 shows the results of measuring the amount of human lactoferrin in the adult normal stool and the initial urine of the adult healthy wake-up by the ELISA method. Average value
【外２】 及び標準偏差（ＳＤ）よりカットオフ値[Outside 2] And cutoff value from standard deviation (SD)
【外３】
を算出すると３９．３μｇ／ｇ便、１６７ｎｇ／ｍｌ尿
となった。従って、例えば感度１０ｎｇ／ｍｌ（メンブ
レン；Ｉｍｍｕｎｏｄｙｎｅ ＡＢＣ、固相化Ｈ１５抗
体濃度３ｍｇ／ｍｌ、金コロイド標識抗体；１７Ｂ０４
−０８）のイムノクロマトグラフキットで測定する場
合、便検体は４０００倍、尿検体は１０〜２０倍に希釈
可能である。[Outside 3] Was calculated to be 39.3 μg / g stool and 167 ng / ml urine. Therefore, for example, the sensitivity is 10 ng / ml (membrane: Immunodyne ABC, immobilized H15 antibody concentration 3 mg / ml, colloidal gold-labeled antibody; 17B04).
When measured with the immunochromatographic kit of (08), a stool sample can be diluted 4000 times and a urine sample can be diluted 10 to 20 times.
【００４８】表７は、細菌感染性下痢患者由来便及び成
人健常便中のヒトラクトフェリンをＥＬＩＳＡ法及びイ
ムノクロマトグラフ法で測定した結果を示したものであ
る。その結果、前者中にはヒトラクトフェリンが多量に
含まれており、イムノクロマトグラフ法では３０検体中
２７検体を検出することができた。一方、成人健常便中
のヒトラクトフェリン量は少量であり、偽陽性結果を生
じた検体は２８検体中２検体であった。Table 7 shows the results of measuring human lactoferrin in feces derived from patients with bacterial infectious diarrhea and healthy adult feces by the ELISA method and the immunochromatographic method. As a result, the former contained a large amount of human lactoferrin, and 27 out of 30 samples could be detected by immunochromatography. On the other hand, the amount of human lactoferrin in healthy adult stool was small, and the number of samples that gave false positive results was 2 out of 28 samples.
【００４９】表８は、尿路感染症患者由来の尿及び成人
健常起床時初尿中のヒトラクトフェリンをＥＬＩＳＡ法
及びイムノクロマトグラフ法で測定した結果を示したも
のである。その結果、イムノクロマトグラフ法により、
前者全１８検体中１７検体を検出することができた。ま
た、成人健常起床時初尿中で偽陽性結果を生じた検体は
全２３検体中わずか１検体であった。Table 8 shows the results of measurement of human lactoferrin in urine derived from patients with urinary tract infection and in the first urine of adult healthy wake-up by ELISA method and immunochromatographic method. As a result, by immunochromatography,
It was possible to detect 17 of the former 18 samples. In addition, only 1 sample out of the 23 samples gave a false positive result in the first urine of an adult healthy wake-up.
【００５０】[0050]
【表３】 [Table 3]
【００５１】[0051]
【表４】 [Table 4]
【００５２】[0052]
【表５】 [Table 5]
【００５３】[0053]
【表６】 [Table 6]
【００５４】[0054]
【表７】 [Table 7]
【００５５】[0055]
【表８】 [Table 8]
【００５６】[0056]
【発明の効果】本発明によれば、便又は尿中の白血球の
顆粒内の鉄結合性タンパク質であるヒトラクトフェリン
を免疫学的に分析することができるので、尿路感染症や
細菌感染性下痢症等の感染症を確実に検出することがで
きる。INDUSTRIAL APPLICABILITY According to the present invention, human lactoferrin, which is an iron-binding protein in leukocyte granules in stool or urine, can be immunologically analyzed. Therefore, urinary tract infection and bacterial infectious diarrhea It is possible to reliably detect infectious diseases such as infectious diseases.
【図１】各種の免疫学的測定方法によるヒトラクトフェ
リンの測定可能濃度範囲を示した説明図である。FIG. 1 is an explanatory diagram showing a measurable concentration range of human lactoferrin by various immunological measurement methods.
【図２】飽和度の異なる３種類のヒトラクトフェリンに
対するポリクローナル抗体Ｈ１５の反応性を示すグラフ
である。FIG. 2 is a graph showing the reactivity of polyclonal antibody H15 against three types of human lactoferrin having different degrees of saturation.
【図３】飽和度の異なる３種類のヒトラクトフェリンに
対する４種のモノクローナル抗体の反応性を示すグラフ
である。FIG. 3 is a graph showing the reactivity of four types of monoclonal antibodies against three types of human lactoferrin having different degrees of saturation.
【図４】ＥＬＩＳＡによるヒトラクトフェリンの標準曲
線を示すグラフである。FIG. 4 is a graph showing a standard curve of human lactoferrin by ELISA.
【図５】２０〜１００倍希釈便検体中のヒトラクトフェ
リンに対して、ＥＬＩＳＡ測定の際に用いた検体希釈用
緩衝液に添加したＺｗｉｔｔｅｒｇｅｎｔ、ＢＩＧＣＨ
ＡＰ、Ｔｒｉｔｏｎ Ｘ−１００及びＢＳＡの効果を示
すグラフである。FIG. 5: Zwittergent and BIGCH added to the sample dilution buffer used in the ELISA measurement for human lactoferrin in a 20-100-fold diluted stool sample.
It is a graph which shows the effect of AP, Triton X-100, and BSA.
【図６】２００〜１０００倍希釈便検体中のヒトラクト
フェリンに対して、ＥＬＩＳＡ測定の際に用いた検体希
釈用緩衝液に添加したＺｗｉｔｔｅｒｇｅｎｔ、ＢＩＧ
ＣＨＡＰ、Ｔｒｉｔｏｎ Ｘ−１００及びＢＳＡの効果
を示すグラフである。FIG. 6: Zwittergent and BIG added to the sample dilution buffer used in the ELISA measurement for human lactoferrin in 200 to 1000-fold diluted stool samples
It is a graph which shows the effect of CHAP, Triton X-100, and BSA.
【図７】２０〜１００倍希釈便検体中のヒトラクトフェ
リンに対して、ＥＬＩＳＡ測定の際に用いた検体希釈用
緩衝液に添加したＺｗｉｔｔｅｒｇｅｎｔ、ＣＨＡＰ
Ｓ、Ｔｒｉｔｏｎ Ｘ−１００及びＢＳＡの効果を示す
グラフである。FIG. 7: Zwittergent and CHAP added to the sample dilution buffer used in the ELISA measurement for human lactoferrin in a stool sample diluted 20 to 100 times.
It is a graph which shows the effect of S, Triton X-100, and BSA.
【図８】２００〜１０００倍希釈便検体中のヒトラクト
フェリンに対して、ＥＬＩＳＡ測定の際に用いた検体希
釈用緩衝液に添加したＺｗｉｔｔｅｒｇｅｎｔ、ＣＨＡ
ＰＳ、Ｔｒｉｔｏｎ Ｘ−１００及びＢＳＡの効果を示
すグラフである。FIG. 8: Zwittergent, CHA added to the sample dilution buffer used in the ELISA measurement for human lactoferrin in 200 to 1000-fold diluted stool samples
It is a graph which shows the effect of PS, Triton X-100, and BSA.
【図９】２０〜１００倍希釈便検体中のヒトラクトフェ
リンに対して、ＥＬＩＳＡ測定の際に用いた検体希釈用
緩衝液に添加したＺｗｉｔｔｅｒｇｅｎｔ、ＣＨＡＰ
Ｓ、ＢＩＧＣＨＡＰ、Ｔｒｉｔｏｎ Ｘ−１００及びＢ
ＳＡの効果を示すグラフである。FIG. 9: Zwittergent and CHAP added to the sample dilution buffer used in the ELISA measurement for human lactoferrin in a 20-100-fold diluted stool sample.
S, BIGCHAP, Triton X-100 and B
It is a graph which shows the effect of SA.
【図１０】２００〜１０００倍希釈便検体中のヒトラク
トフェリンに対して、ＥＬＩＳＡ測定の際に用いた検体
希釈用緩衝液に添加したＺｗｉｔｔｅｒｇｅｎｔ、ＣＨ
ＡＰＳ、ＢＩＧＣＨＡＰ、Ｔｒｉｔｏｎ Ｘ−１００及
びＢＳＡの効果を示すグラフである。FIG. 10: Zwittergent, CH added to the sample dilution buffer used in ELISA measurement for human lactoferrin in 200-1000-fold diluted stool samples
It is a graph which shows the effect of APS, BIGCHAP, Triton X-100, and BSA.
【図１１】成人健常便検体に対してヒトラクトフェリン
の添加回収実験を行った結果を示すグラフである。FIG. 11 is a graph showing the results of a human lactoferrin addition and recovery experiment conducted on adult healthy stool specimens.
【図１２】成人健常尿検体に対してヒトラクトフェリン
の添加回収実験を行った結果を示すグラフである。FIG. 12 is a graph showing the results of a human lactoferrin addition and recovery experiment conducted on adult healthy urine samples.
【図１３】成人健常尿検体に対してヒトラクトフェリン
の添加回収実験を行った結果を示すグラフである。FIG. 13 is a graph showing the results of a human lactoferrin addition and recovery experiment conducted on an adult healthy urine sample.
フロントページの続き (72)発明者 大田 美佐子 東京都千代田区東神田１丁目11番４号 株式会社ヤトロン内 (58)調査した分野(Int.Cl.7，ＤＢ名) G01N 33/53 Front page continuation (72) Inventor Misako Ota 1-11-4 Higashi-Kanda, Chiyoda-ku, Tokyo Inside Yatron Co., Ltd. (58) Fields investigated (Int.Cl. 7 , DB name) G01N 33/53
Claims (7)
が影響されない抗ヒトラクトフェリン抗体を用いること
を特徴とする、便又は尿中のヒトラクトフェリンを免疫
学的に分析する方法。1. A method for immunologically analyzing human lactoferrin in feces or urine, which comprises using an anti-human lactoferrin antibody whose reactivity is not affected by the iron saturation of human lactoferrin.
ロマトグラフ法、ラテックス凝集法、磁気ビーズ凝集
法、又は磁気ビーズ酵素免疫法である請求項１に記載の
分析方法。2. The analysis method according to claim 1, wherein the immunological method is an enzyme immunoassay, an immunochromatography method, a latex agglutination method, a magnetic bead agglutination method, or a magnetic bead enzyme immunoassay method.
は尿中のヒトラクトフェリンを分析することを特徴とす
る、感染症のスクリーニング方法。3. A method for screening an infectious disease, which comprises analyzing human lactoferrin in feces or urine by the analysis method according to claim 1.
症である請求項３に記載のスクリーニング方法。4. The screening method according to claim 3, wherein the infectious disease is urinary tract infection or bacterial infectious diarrhea.
が影響されない抗ヒトラクトフェリン抗体、ヒトラクト
フェリンの鉄飽和度に反応性が影響されない標識化抗ヒ
トラクトフェリン抗体、及び界面活性剤含有検体希釈液
を含む、感染症のスクリーニング用キット。5. An anti-human lactoferrin antibody whose reactivity is not affected by the iron saturation of human lactoferrin, a labeled anti-human lactoferrin antibody whose reactivity is not affected by the iron saturation of human lactoferrin, and a sample diluent containing a surfactant. A kit for screening for infectious diseases, comprising:
テトラデシル−Ｎ，Ｎ−ジメチル−３−アンモニオ−１
−プロパンスルホン酸、３−［（３−コラシドプロピ
ル）ジメチルアンモニオ］−プロパンスルホン酸、Ｎ，
Ｎ−ビス（３−Ｄ−グルコンアミドプロピル）コラミ
ド、及びポリエチレングルコール モノ−ｐ−イソオク
チルフェニルエーテルを含む請求項５に記載のスクリー
ニング用キット。6. The sample diluent is N-as a surfactant.
Tetradecyl-N, N-dimethyl-3-ammonio-1
-Propanesulfonic acid, 3-[(3-Colasidepropyl) dimethylammonio] -propanesulfonic acid, N,
The screening kit according to claim 5, comprising N-bis (3-D-gluconamidopropyl) colamide, and polyethylene glycol mono-p-isooctylphenyl ether.
症である請求項５に記載のスクリーニング用キット。7. The screening kit according to claim 5, wherein the infectious disease is urinary tract infection or bacterial infectious diarrhea.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP22174496A JP3536191B2 (en) | 1996-08-05 | 1996-08-05 | Human lactoferrin analysis method, infectious disease screening method and screening kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP22174496A JP3536191B2 (en) | 1996-08-05 | 1996-08-05 | Human lactoferrin analysis method, infectious disease screening method and screening kit |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH1048209A JPH1048209A (en) | 1998-02-20 |
| JP3536191B2 true JP3536191B2 (en) | 2004-06-07 |
Family
ID=16771553
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP22174496A Expired - Lifetime JP3536191B2 (en) | 1996-08-05 | 1996-08-05 | Human lactoferrin analysis method, infectious disease screening method and screening kit |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP3536191B2 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2356867A1 (en) * | 1998-12-28 | 2000-07-06 | Kabushiki Kaisya Advance | System for identifying microorganism |
-
1996
- 1996-08-05 JP JP22174496A patent/JP3536191B2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| JPH1048209A (en) | 1998-02-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP5807300B2 (en) | Method and reagent for measuring C-reactive protein in sample | |
| US8404479B2 (en) | Simple membrane assay method and kit | |
| WO2022193980A1 (en) | Antibody or antigen-binding fragment thereof for novel coronavirus nucleocapsid protein, and application thereof | |
| JP4268358B2 (en) | Antibody and immunological assay | |
| JP3536191B2 (en) | Human lactoferrin analysis method, infectious disease screening method and screening kit | |
| US20030092086A1 (en) | Method for detecting streptococcus sobrinus and antibody therefor | |
| US5525476A (en) | Immunoassay, monoclonal antibody, and hybridoma | |
| JP2024003083A (en) | Detection reagent or kit for detecting RS virus and method for detecting RS virus | |
| WO2024166923A1 (en) | SARS-CoV-2 DETECTION METHOD AND KIT THEREFOR | |
| JP2006118936A (en) | Membrane enzyme immunoassay | |
| EP0233048B1 (en) | Method of detecting urinary tract infection or inflammation | |
| JP2004069672A (en) | Method and kit for measuring oxide appolipoprotein ai and oxide lipoprotein containing it | |
| WO1990007114A1 (en) | Solid phase immuno-assay with labelled conjugate | |
| CN116990507B (en) | A method for early screening of monkeypox virus infection | |
| JP4491572B2 (en) | Reagents and methods for measuring substances to be measured in samples by enzyme immunoassay | |
| JP4588053B2 (en) | Method and kit for measuring oxidized apolipoprotein AI and oxidized lipoprotein containing the same | |
| JP7274904B2 (en) | Reagent for detecting human metapneumovirus | |
| JP2001302697A (en) | Polyclonal antibody and method for producing the same | |
| WO1997010508A1 (en) | Method for the diagnosis of coeliac disease | |
| WO2024166922A1 (en) | SARS-CoV-2 DETECTION METHOD AND KIT THEREFOR | |
| JP2002017397A (en) | Method for immunologically assaying norwalk virus, method for screening infectious disease and screening kit | |
| JPS63250567A (en) | Enzyme immunoassay | |
| JPWO2001081927A1 (en) | Method for detecting Streptococcus sobrinus and antibody therefor | |
| JPH07501396A (en) | HLA (human leukocyte antigen) type classification method | |
| JP2004361206A (en) | Membrane assay |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20040225 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20040302 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20040303 |
|
| R150 | Certificate of patent or registration of utility model |
Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
| S531 | Written request for registration of change of domicile |
Free format text: JAPANESE INTERMEDIATE CODE: R313531 |
|
| R350 | Written notification of registration of transfer |
Free format text: JAPANESE INTERMEDIATE CODE: R350 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20090326 Year of fee payment: 5 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20100326 Year of fee payment: 6 |
|
| S111 | Request for change of ownership or part of ownership |
Free format text: JAPANESE INTERMEDIATE CODE: R313111 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20100326 Year of fee payment: 6 |
|
| R371 | Transfer withdrawn |
Free format text: JAPANESE INTERMEDIATE CODE: R371 |
|
| S111 | Request for change of ownership or part of ownership |
Free format text: JAPANESE INTERMEDIATE CODE: R313111 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20100326 Year of fee payment: 6 |
|
| R350 | Written notification of registration of transfer |
Free format text: JAPANESE INTERMEDIATE CODE: R350 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20100326 Year of fee payment: 6 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20110326 Year of fee payment: 7 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20130326 Year of fee payment: 9 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20140326 Year of fee payment: 10 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| S531 | Written request for registration of change of domicile |
Free format text: JAPANESE INTERMEDIATE CODE: R313531 |
|
| S533 | Written request for registration of change of name |
Free format text: JAPANESE INTERMEDIATE CODE: R313533 |
|
| R350 | Written notification of registration of transfer |
Free format text: JAPANESE INTERMEDIATE CODE: R350 |
|
| EXPY | Cancellation because of completion of term |