JPH03133396A - Reagent composition for ammonia analysis and one-body type multilayer analyzing element containing thereof - Google Patents
Reagent composition for ammonia analysis and one-body type multilayer analyzing element containing thereofInfo
- Publication number
- JPH03133396A JPH03133396A JP27163589A JP27163589A JPH03133396A JP H03133396 A JPH03133396 A JP H03133396A JP 27163589 A JP27163589 A JP 27163589A JP 27163589 A JP27163589 A JP 27163589A JP H03133396 A JPH03133396 A JP H03133396A
- Authority
- JP
- Japan
- Prior art keywords
- layer
- ammonia
- reagent
- pyruvate
- composition
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 title claims abstract description 89
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 65
- 229910021529 ammonia Inorganic materials 0.000 title claims abstract description 44
- 239000000203 mixture Substances 0.000 title claims abstract description 39
- 238000004458 analytical method Methods 0.000 title claims abstract description 19
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 claims abstract description 35
- 102000013009 Pyruvate Kinase Human genes 0.000 claims abstract description 20
- 101710088194 Dehydrogenase Proteins 0.000 claims abstract description 16
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 claims abstract description 10
- 229930182816 L-glutamine Natural products 0.000 claims abstract description 9
- 239000000758 substrate Substances 0.000 claims description 17
- 238000011161 development Methods 0.000 claims description 14
- 102000003960 Ligases Human genes 0.000 claims description 5
- 108090000364 Ligases Proteins 0.000 claims description 5
- 239000002253 acid Substances 0.000 claims description 4
- 108010031852 Pyruvate Synthase Proteins 0.000 claims description 2
- 108060006633 protein kinase Proteins 0.000 claims description 2
- 102000053067 Pyruvate Dehydrogenase Acetyl-Transferring Kinase Human genes 0.000 claims 1
- 108700009582 Pyruvate Dehydrogenase Acetyl-Transferring Kinase Proteins 0.000 claims 1
- 102000004190 Enzymes Human genes 0.000 abstract description 24
- 108090000790 Enzymes Proteins 0.000 abstract description 24
- 108020005115 Pyruvate Kinase Proteins 0.000 abstract description 18
- -1 polyethylene terephthalate Polymers 0.000 abstract description 12
- 229920000139 polyethylene terephthalate Polymers 0.000 abstract description 5
- 239000005020 polyethylene terephthalate Substances 0.000 abstract description 5
- 238000001035 drying Methods 0.000 abstract description 4
- 239000012528 membrane Substances 0.000 abstract description 4
- 125000003831 tetrazolyl group Chemical group 0.000 abstract description 3
- 239000000463 material Substances 0.000 abstract 2
- 239000010410 layer Substances 0.000 description 124
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 description 38
- 229940076788 pyruvate Drugs 0.000 description 34
- 229940088598 enzyme Drugs 0.000 description 21
- DDRJAANPRJIHGJ-UHFFFAOYSA-N creatinine Chemical compound CN1CC(=O)NC1=N DDRJAANPRJIHGJ-UHFFFAOYSA-N 0.000 description 20
- 229940107700 pyruvic acid Drugs 0.000 description 19
- 238000006243 chemical reaction Methods 0.000 description 18
- 238000000034 method Methods 0.000 description 17
- 108010010803 Gelatin Proteins 0.000 description 14
- 229920000159 gelatin Polymers 0.000 description 14
- 239000008273 gelatin Substances 0.000 description 14
- 235000019322 gelatine Nutrition 0.000 description 14
- 235000011852 gelatine desserts Nutrition 0.000 description 14
- 229920000642 polymer Polymers 0.000 description 14
- 238000003892 spreading Methods 0.000 description 13
- ZKHQWZAMYRWXGA-KQYNXXCUSA-J ATP(4-) Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-J 0.000 description 12
- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 12
- 229940109239 creatinine Drugs 0.000 description 10
- 239000012992 electron transfer agent Substances 0.000 description 10
- 238000006911 enzymatic reaction Methods 0.000 description 10
- 238000005259 measurement Methods 0.000 description 10
- 239000004744 fabric Substances 0.000 description 9
- 102000005396 glutamine synthetase Human genes 0.000 description 9
- 108020002326 glutamine synthetase Proteins 0.000 description 9
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 9
- 210000002381 plasma Anatomy 0.000 description 9
- 229960002363 thiamine pyrophosphate Drugs 0.000 description 9
- 235000008170 thiamine pyrophosphate Nutrition 0.000 description 9
- 239000011678 thiamine pyrophosphate Substances 0.000 description 9
- YXVCLPJQTZXJLH-UHFFFAOYSA-N thiamine(1+) diphosphate chloride Chemical compound [Cl-].CC1=C(CCOP(O)(=O)OP(O)(O)=O)SC=[N+]1CC1=CN=C(C)N=C1N YXVCLPJQTZXJLH-UHFFFAOYSA-N 0.000 description 9
- 239000012790 adhesive layer Substances 0.000 description 8
- 230000000694 effects Effects 0.000 description 8
- 239000007788 liquid Substances 0.000 description 8
- 238000010521 absorption reaction Methods 0.000 description 7
- 239000012491 analyte Substances 0.000 description 7
- 238000001514 detection method Methods 0.000 description 7
- 229920001477 hydrophilic polymer Polymers 0.000 description 7
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 6
- 108010046334 Urease Proteins 0.000 description 6
- PNNCWTXUWKENPE-UHFFFAOYSA-N [N].NC(N)=O Chemical compound [N].NC(N)=O PNNCWTXUWKENPE-UHFFFAOYSA-N 0.000 description 6
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 5
- 150000001768 cations Chemical class 0.000 description 5
- 239000011325 microbead Substances 0.000 description 5
- 239000002736 nonionic surfactant Substances 0.000 description 5
- 239000002491 polymer binding agent Substances 0.000 description 5
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 4
- 229910019142 PO4 Inorganic materials 0.000 description 4
- 108010042687 Pyruvate Oxidase Proteins 0.000 description 4
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 4
- 210000001124 body fluid Anatomy 0.000 description 4
- 239000010839 body fluid Substances 0.000 description 4
- 229920002678 cellulose Polymers 0.000 description 4
- 239000011248 coating agent Substances 0.000 description 4
- 238000000576 coating method Methods 0.000 description 4
- 230000003287 optical effect Effects 0.000 description 4
- 239000010452 phosphate Substances 0.000 description 4
- 238000004445 quantitative analysis Methods 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 241000193830 Bacillus <bacterium> Species 0.000 description 3
- UDMBCSSLTHHNCD-UHFFFAOYSA-N Coenzym Q(11) Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(O)=O)C(O)C1O UDMBCSSLTHHNCD-UHFFFAOYSA-N 0.000 description 3
- GWEVSGVZZGPLCZ-UHFFFAOYSA-N Titan oxide Chemical compound O=[Ti]=O GWEVSGVZZGPLCZ-UHFFFAOYSA-N 0.000 description 3
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000001913 cellulose Substances 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 239000000835 fiber Substances 0.000 description 3
- 239000010419 fine particle Substances 0.000 description 3
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 3
- 239000000543 intermediate Substances 0.000 description 3
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 3
- 229920000620 organic polymer Polymers 0.000 description 3
- 239000008363 phosphate buffer Substances 0.000 description 3
- 238000005375 photometry Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 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 2
- JEOLGCWRLRXABW-UHFFFAOYSA-N 2,5-diphenyl-2h-tetrazol-2-ium;chloride Chemical compound [Cl-].C1=CC=CC=C1C1=[NH+]N(C=2C=CC=CC=2)N=N1 JEOLGCWRLRXABW-UHFFFAOYSA-N 0.000 description 2
- MWMOPIVLTLEUJO-UHFFFAOYSA-N 2-oxopropanoic acid;phosphoric acid Chemical compound OP(O)(O)=O.CC(=O)C(O)=O MWMOPIVLTLEUJO-UHFFFAOYSA-N 0.000 description 2
- RLFWWDJHLFCNIJ-UHFFFAOYSA-N 4-aminoantipyrine Chemical compound CN1C(C)=C(N)C(=O)N1C1=CC=CC=C1 RLFWWDJHLFCNIJ-UHFFFAOYSA-N 0.000 description 2
- IKHGUXGNUITLKF-UHFFFAOYSA-N Acetaldehyde Chemical compound CC=O IKHGUXGNUITLKF-UHFFFAOYSA-N 0.000 description 2
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 2
- 241000193385 Geobacillus stearothermophilus Species 0.000 description 2
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 2
- 241000186660 Lactobacillus Species 0.000 description 2
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 2
- 239000004372 Polyvinyl alcohol Substances 0.000 description 2
- 239000004373 Pullulan Substances 0.000 description 2
- 229920001218 Pullulan Polymers 0.000 description 2
- 108010053763 Pyruvate Carboxylase Proteins 0.000 description 2
- 102100039895 Pyruvate carboxylase, mitochondrial Human genes 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 2
- UDMBCSSLTHHNCD-KQYNXXCUSA-N adenosine 5'-monophosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O UDMBCSSLTHHNCD-KQYNXXCUSA-N 0.000 description 2
- LNQVTSROQXJCDD-UHFFFAOYSA-N adenosine monophosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(CO)C(OP(O)(O)=O)C1O LNQVTSROQXJCDD-UHFFFAOYSA-N 0.000 description 2
- 239000000853 adhesive Substances 0.000 description 2
- 230000001070 adhesive effect Effects 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 230000004888 barrier function Effects 0.000 description 2
- IISBACLAFKSPIT-UHFFFAOYSA-N bisphenol A Chemical compound C=1C=C(O)C=CC=1C(C)(C)C1=CC=C(O)C=C1 IISBACLAFKSPIT-UHFFFAOYSA-N 0.000 description 2
- 210000004556 brain Anatomy 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 208000028659 discharge Diseases 0.000 description 2
- 239000006185 dispersion Substances 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 229960002989 glutamic acid Drugs 0.000 description 2
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- RSAZYXZUJROYKR-UHFFFAOYSA-N indophenol Chemical compound C1=CC(O)=CC=C1N=C1C=CC(=O)C=C1 RSAZYXZUJROYKR-UHFFFAOYSA-N 0.000 description 2
- 229940039696 lactobacillus Drugs 0.000 description 2
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 2
- KHPXUQMNIQBQEV-UHFFFAOYSA-N oxaloacetic acid Chemical compound OC(=O)CC(=O)C(O)=O KHPXUQMNIQBQEV-UHFFFAOYSA-N 0.000 description 2
- 125000006353 oxyethylene group Chemical group 0.000 description 2
- 239000006174 pH buffer Substances 0.000 description 2
- 229940085991 phosphate ion Drugs 0.000 description 2
- 229920002451 polyvinyl alcohol Polymers 0.000 description 2
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 2
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 2
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 2
- 235000019423 pullulan Nutrition 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- FBWNMEQMRUMQSO-UHFFFAOYSA-N tergitol NP-9 Chemical compound CCCCCCCCCC1=CC=C(OCCOCCOCCOCCOCCOCCOCCOCCOCCO)C=C1 FBWNMEQMRUMQSO-UHFFFAOYSA-N 0.000 description 2
- FFJCNSLCJOQHKM-CLFAGFIQSA-N (z)-1-[(z)-octadec-9-enoxy]octadec-9-ene Chemical compound CCCCCCCC\C=C/CCCCCCCCOCCCCCCCC\C=C/CCCCCCCC FFJCNSLCJOQHKM-CLFAGFIQSA-N 0.000 description 1
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 description 1
- PKDBCJSWQUOKDO-UHFFFAOYSA-M 2,3,5-triphenyltetrazolium chloride Chemical compound [Cl-].C1=CC=CC=C1C(N=[N+]1C=2C=CC=CC=2)=NN1C1=CC=CC=C1 PKDBCJSWQUOKDO-UHFFFAOYSA-M 0.000 description 1
- JHCINPODUYPSLQ-UHFFFAOYSA-N 2-bromo-3-(bromomethyl)-6-propan-2-ylphenol Chemical compound CC(C)C1=CC=C(CBr)C(Br)=C1O JHCINPODUYPSLQ-UHFFFAOYSA-N 0.000 description 1
- 101710169336 5'-deoxyadenosine deaminase Proteins 0.000 description 1
- 229930024421 Adenine Natural products 0.000 description 1
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 1
- 108010052875 Adenine deaminase Proteins 0.000 description 1
- 102000055025 Adenosine deaminases Human genes 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 108090000531 Amidohydrolases Proteins 0.000 description 1
- 102000004092 Amidohydrolases Human genes 0.000 description 1
- 108010024976 Asparaginase Proteins 0.000 description 1
- 102000015790 Asparaginase Human genes 0.000 description 1
- 108700016171 Aspartate ammonia-lyases Proteins 0.000 description 1
- 101000950981 Bacillus subtilis (strain 168) Catabolic NAD-specific glutamate dehydrogenase RocG Proteins 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- FRPHFZCDPYBUAU-UHFFFAOYSA-N Bromocresolgreen Chemical compound CC1=C(Br)C(O)=C(Br)C=C1C1(C=2C(=C(Br)C(O)=C(Br)C=2)C)C2=CC=CC=C2S(=O)(=O)O1 FRPHFZCDPYBUAU-UHFFFAOYSA-N 0.000 description 1
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 229920001747 Cellulose diacetate Polymers 0.000 description 1
- 229920002284 Cellulose triacetate Polymers 0.000 description 1
- CKLJMWTZIZZHCS-UHFFFAOYSA-N D-OH-Asp Natural products OC(=O)C(N)CC(O)=O CKLJMWTZIZZHCS-UHFFFAOYSA-N 0.000 description 1
- 241000588722 Escherichia Species 0.000 description 1
- 239000001856 Ethyl cellulose Substances 0.000 description 1
- ZZSNKZQZMQGXPY-UHFFFAOYSA-N Ethyl cellulose Chemical compound CCOCC1OC(OC)C(OCC)C(OCC)C1OC1C(O)C(O)C(OC)C(CO)O1 ZZSNKZQZMQGXPY-UHFFFAOYSA-N 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 102000016901 Glutamate dehydrogenase Human genes 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 108010073324 Glutaminase Proteins 0.000 description 1
- 102000009127 Glutaminase Human genes 0.000 description 1
- 108030001320 Glutamine kinases Proteins 0.000 description 1
- 108010012029 Guanine Deaminase Proteins 0.000 description 1
- 102000013587 Guanine deaminase Human genes 0.000 description 1
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 1
- 108010020056 Hydrogenase Proteins 0.000 description 1
- 239000005909 Kieselgur Substances 0.000 description 1
- CKLJMWTZIZZHCS-UWTATZPHSA-N L-Aspartic acid Natural products OC(=O)[C@H](N)CC(O)=O CKLJMWTZIZZHCS-UWTATZPHSA-N 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- 229930195714 L-glutamate Natural products 0.000 description 1
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 description 1
- PWHULOQIROXLJO-UHFFFAOYSA-N Manganese Chemical compound [Mn] PWHULOQIROXLJO-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 108010011939 Pyruvate Decarboxylase Proteins 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- PLXBWHJQWKZRKG-UHFFFAOYSA-N Resazurin Chemical class C1=CC(=O)C=C2OC3=CC(O)=CC=C3[N+]([O-])=C21 PLXBWHJQWKZRKG-UHFFFAOYSA-N 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 235000010724 Wisteria floribunda Nutrition 0.000 description 1
- NNLVGZFZQQXQNW-ADJNRHBOSA-N [(2r,3r,4s,5r,6s)-4,5-diacetyloxy-3-[(2s,3r,4s,5r,6r)-3,4,5-triacetyloxy-6-(acetyloxymethyl)oxan-2-yl]oxy-6-[(2r,3r,4s,5r,6s)-4,5,6-triacetyloxy-2-(acetyloxymethyl)oxan-3-yl]oxyoxan-2-yl]methyl acetate Chemical compound O([C@@H]1O[C@@H]([C@H]([C@H](OC(C)=O)[C@H]1OC(C)=O)O[C@H]1[C@@H]([C@@H](OC(C)=O)[C@H](OC(C)=O)[C@@H](COC(C)=O)O1)OC(C)=O)COC(=O)C)[C@@H]1[C@@H](COC(C)=O)O[C@@H](OC(C)=O)[C@H](OC(C)=O)[C@H]1OC(C)=O NNLVGZFZQQXQNW-ADJNRHBOSA-N 0.000 description 1
- XJLXINKUBYWONI-DQQFMEOOSA-N [[(2r,3r,4r,5r)-5-(6-aminopurin-9-yl)-3-hydroxy-4-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2s,3r,4s,5s)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate Chemical compound NC(=O)C1=CC=C[N+]([C@@H]2[C@H]([C@@H](O)[C@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-DQQFMEOOSA-N 0.000 description 1
- LIPOUNRJVLNBCD-UHFFFAOYSA-N acetyl dihydrogen phosphate Chemical compound CC(=O)OP(O)(O)=O LIPOUNRJVLNBCD-UHFFFAOYSA-N 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 229960000643 adenine Drugs 0.000 description 1
- 229960005305 adenosine Drugs 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- 210000001557 animal structure Anatomy 0.000 description 1
- 229960003272 asparaginase Drugs 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-M asparaginate Chemical compound [O-]C(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-M 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 229960005261 aspartic acid Drugs 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000000035 biogenic effect Effects 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 125000002529 biphenylenyl group Chemical group C1(=CC=CC=2C3=CC=CC=C3C12)* 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-N carbonic acid Chemical compound OC(O)=O BVKZGUZCCUSVTD-UHFFFAOYSA-N 0.000 description 1
- 229920006217 cellulose acetate butyrate Polymers 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 235000013330 chicken meat Nutrition 0.000 description 1
- 239000005515 coenzyme Substances 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 108010029444 creatinine deiminase Proteins 0.000 description 1
- 229940071120 dehydroacetate Drugs 0.000 description 1
- 238000007357 dehydrogenase reaction Methods 0.000 description 1
- 238000006356 dehydrogenation reaction Methods 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 238000002845 discoloration Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000003248 enzyme activator Substances 0.000 description 1
- 235000019325 ethyl cellulose Nutrition 0.000 description 1
- 229920001249 ethyl cellulose Polymers 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 229930182478 glucoside Natural products 0.000 description 1
- 150000008131 glucosides Chemical class 0.000 description 1
- 229930195712 glutamate Natural products 0.000 description 1
- 229940049906 glutamate Drugs 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 229920001600 hydrophobic polymer Polymers 0.000 description 1
- 238000005470 impregnation Methods 0.000 description 1
- 208000017169 kidney disease Diseases 0.000 description 1
- 238000010030 laminating Methods 0.000 description 1
- 229910001425 magnesium ion Inorganic materials 0.000 description 1
- 229910052748 manganese Inorganic materials 0.000 description 1
- 239000011572 manganese Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- JZMJDSHXVKJFKW-UHFFFAOYSA-M methyl sulfate(1-) Chemical compound COS([O-])(=O)=O JZMJDSHXVKJFKW-UHFFFAOYSA-M 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 229930029653 phosphoenolpyruvate Natural products 0.000 description 1
- DTBNBXWJWCWCIK-UHFFFAOYSA-N phosphoenolpyruvic acid Chemical compound OC(=O)C(=C)OP(O)(O)=O DTBNBXWJWCWCIK-UHFFFAOYSA-N 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 229920000515 polycarbonate Polymers 0.000 description 1
- 239000004417 polycarbonate Substances 0.000 description 1
- 229920005596 polymer binder Polymers 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 229910001414 potassium ion Inorganic materials 0.000 description 1
- JKVUQLWTIZFTMF-UHFFFAOYSA-M potassium;2-oxopropanoate Chemical compound [K+].CC(=O)C([O-])=O JKVUQLWTIZFTMF-UHFFFAOYSA-M 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- AZIQALWHRUQPHV-UHFFFAOYSA-N prop-2-eneperoxoic acid Chemical compound OOC(=O)C=C AZIQALWHRUQPHV-UHFFFAOYSA-N 0.000 description 1
- 230000022558 protein metabolic process Effects 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 150000004728 pyruvic acid derivatives Chemical class 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000000661 sodium alginate Substances 0.000 description 1
- 235000010413 sodium alginate Nutrition 0.000 description 1
- 229940005550 sodium alginate Drugs 0.000 description 1
- 239000008279 sol Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 239000004408 titanium dioxide Substances 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は尿素窒素、アンモニア、クレアチニン等のアン
モニウムイオンを生成しうる被分析成分（アナライト）
を測定するための試薬系及びそれを含む一体型多層分析
要素に関するものである。[Detailed Description of the Invention] [Industrial Application Field] The present invention is directed to an analyte that can produce ammonium ions such as urea nitrogen, ammonia, and creatinine.
The present invention relates to a reagent system for measuring and an integrated multilayer analytical element containing the same.
蛋白質代謝の中間体及び最終生成物である尿素窒素（Ｂ
ＵＮ）、アンモニア、クレアチニン等の測定は腎疾患の
診断、治療経過の判断等のために必要である。従来、こ
れらは湿式法で分析され、例えば尿素窒素は強酸性条件
下でテレフタルアルデヒド及びフェニレンジアミン誘導
体を直接反応させて発色を比色定量する方法（Ｄ、Ｊ、
Ｊｕｎｇ、 Ｃｌ１ｎ。Urea nitrogen (B
Measurement of UN), ammonia, creatinine, etc. is necessary for diagnosing renal disease and determining the course of treatment. Conventionally, these have been analyzed using a wet method. For example, urea nitrogen has been analyzed using a colorimetric method (D, J,
Jung, Cl1n.
Ｃｈｅｍ、、２Ｌ １１３６（１９７５）） 、尿素に
ウレアーゼを作用させて生成したアンモニウムイオンを
ネスラー試薬を用いるかあるいはインドフェノール法で
比色定量する方法で定量されていた。インドフェノール
法はその後多くの研究者によって改良がなされている（
例えば、Ｈ，０ｋｕｄａ ｅｔ ａｌ、Ｔｏｋｕｓｈｉ
ｍａＪ、Ｅｘｐｔｌ、Ｍｅｄ、、 １２．（１）、１ｌ
−２３（１９６５）） 。この方法は強酸性条件下で反
応させるので装置が腐蝕しやすいという問題があり、感
度が低いこと及び測定に時間を要することも問題であっ
た。Chem, 2L 1136 (1975)), ammonium ions produced by the action of urease on urea were quantified using Nessler's reagent or colorimetrically using the indophenol method. The indophenol method has since been improved by many researchers (
For example, H.Kuda et al., Tokushi
maJ, Exptl, Med, 12. (1), 1l
-23 (1965)). This method has the problem that the reaction is carried out under strongly acidic conditions, so the equipment is easily corroded, and there are also problems that the sensitivity is low and the measurement takes time.
酵素法についても、グルタミン酸脱水素酵素を利用して
ＮＡＤＰＨの減少量をＵ■吸収の測定して求める方法が
開発されている（Ａ、Ｍｏｎｄｚａｃ ｅｔａｌ、 Ｊ
、Ｌａｂ、ＣＩｉＣｌｌｎｏ、、６６、５２６（１９６
５））。この方法も感度及び精度に問題があった。グル
タミン酸及びＡＴＰの存在下でグルタミン合成酵素を作
用させ、さらにピルビン酸キナーゼ及びピルビン酸オキ
シダーゼを作用させて生成した過酸化水素をペルオキシ
ダーゼと４−アミノアンチピリンを利用して比色定置す
る方法も開発されている（特開昭６２−３８００号公報
）。Regarding the enzymatic method, a method has been developed that uses glutamate dehydrogenase to determine the amount of decrease in NADPH by measuring U absorption (A, Mondzac et al., J.
, Lab, CIiCllno, , 66, 526 (196
5)). This method also had problems with sensitivity and accuracy. A method has also been developed in which hydrogen peroxide produced by the action of glutamine synthetase in the presence of glutamate and ATP and further action of pyruvate kinase and pyruvate oxidase is colorimetrically fixed using peroxidase and 4-aminoantipyrine. (Japanese Unexamined Patent Publication No. 62-3800).
一方、簡便な操作で迅速に定量できる方法として乾式法
も知られている。乾式法に用いる分析器具の例として、
日本分析化学会編分析ライブリー３「臨床化学分析■含
窒素成分」第２版（東京化学同人、１９７９年発行）１
３〜１４頁、「臨床検査」第５巻（第６号）３８７〜３
９１頁（１９６１年発行）米国特許３０１１８７４号等
には、濾紙片に細帯状の試薬層（ウレアーゼとアルカリ
剤を含み、尿素からアンモニアガスを生成させる）、細
帯状の障壁物層、細帯状の指示下層（ｐｌ（指示薬ブロ
ムクレゾールグリーンを含む）がこの層に設けられたＢ
ＵＮ定量分析用試薬片が開示されている。特開昭５０−
９３４９４号公報には、濾紙片にｐＨ支支持ジブロムチ
モールブルー含浸し、ついでウレアーゼと親水性ポリマ
ーからなる障壁形成体との混合物をさらに含浸し、最後
にエチルセルロース半透膜を設けたＢＵＮ定量分析用試
験片が開示されている。また、特開昭５２−３４８８号
公報（特公昭５８−１９０６２号）には水平浸透性の透
明支持体の上に支持薬層、アンモニア薄層が均一に浸透
可能で液体状水およびアルカリ性妨害物が浸透不可能な
セルロースアセテートブチレート等の疎水性ポリマーの
均質（非孔性）薄層からなる障壁物層、ウレアーゼおよ
びアルカリ剤を含む試薬層、および多孔性展ＩＩｎ層を
この順に一体に積層してなるＢＵＮ定量定量分析体型多
層分析要素等が提案されている。On the other hand, a dry method is also known as a method that allows rapid quantitative determination with simple operations. Examples of analytical instruments used in the dry method include:
Analysis Library 3 “Clinical Chemistry Analysis ■Nitrogen-containing Components” edited by the Japanese Society of Analytical Chemistry, 2nd edition (Tokyo Kagaku Doujin, published in 1979) 1
Pages 3-14, "Clinical Examination" Volume 5 (No. 6) 387-3
91 pages (published in 1961), US Pat. An indicator lower layer (PL (containing the indicator bromcresol green) is provided in this layer B)
A reagent strip for UN quantitative analysis is disclosed. Japanese Patent Application Publication 1973-
Publication No. 93494 discloses a BUN quantitative analysis method in which a piece of filter paper is impregnated with pH-supporting dibromthymol blue, then further impregnated with a mixture of urease and a barrier former made of a hydrophilic polymer, and finally an ethyl cellulose semipermeable membrane is provided. Disclosed is a test piece for In addition, Japanese Patent Application Laid-Open No. 52-3488 (Japanese Patent Publication No. 58-19062) discloses that a support drug layer and a thin ammonia layer are formed on a horizontally permeable transparent support to allow for uniform permeation of liquid water and alkaline interference. A barrier layer consisting of a homogeneous (non-porous) thin layer of a hydrophobic polymer such as cellulose acetate butyrate that is impenetrable to cellulose, a reagent layer containing urease and an alkaline agent, and a porous expanded IIn layer are laminated together in this order. A BUN quantitative quantitative analysis type multilayer analysis element has been proposed.
従来の乾式法においてはいずれもアンモニアガスを試験
片ないし分析要素内を拡散させており、二の拡散が定量
性に欠けるという問題があった。In all conventional dry methods, ammonia gas is diffused within the test piece or analysis element, and the second problem is that the diffusion lacks quantitative properties.
本発明の目的は蛋白質代謝の中間体及び最終生成物であ
る尿素窒素（ＢＵＮ） 、タレアチニン、アンモニア等
を簡便な方法で高精度で定量できる分析用試薬組成物と
分析要素を提供することにある。An object of the present invention is to provide an analytical reagent composition and an analytical element that can quantify intermediates and final products of protein metabolism such as urea nitrogen (BUN), taleatinin, ammonia, etc. with a simple method and with high precision. .
本発明者らは上記目的を達成するべく鋭意検討を行った
結果、基本的には蛋白質代謝中間体や最終生成物の測定
に共通的に利用できるアンモニウムイオン検出法であっ
て発色反応に結びつけられる反応原理として、前述のグ
ルタミン合成酵素にピルビン酸キナーゼ及びピルビン酸
オキシダーゼを組合せ、生成した過酸化水素を比色定置
する方法を開発した（特願平１− 号、同
号）。しかしながら、公知のピル
ビン酸オキシダーゼは熱安定性に劣るという欠点が判明
した。The present inventors conducted intensive studies to achieve the above objective, and found that the present inventors have developed an ammonium ion detection method that can basically be used commonly for measuring protein metabolic intermediates and final products, and is linked to color reactions. As a reaction principle, a method was developed in which the above-mentioned glutamine synthetase was combined with pyruvate kinase and pyruvate oxidase, and the produced hydrogen peroxide was colorimetrically fixed (Japanese Patent Application No. 1999, same issue). However, it has been found that known pyruvate oxidases have a drawback of poor thermostability.
そこで、本発明者らはさらに検討を進め、熱安定性の優
れたピルビン酸デヒドロゲナーゼ反応系をピルビン酸オ
キシダーゼの代わりに用いる手段を案出するに至り、こ
れによって前記目的を達成することができた。Therefore, the present inventors further investigated and came up with a means to use a pyruvate dehydrogenase reaction system with excellent thermostability in place of pyruvate oxidase, thereby achieving the above object. .
かかる本発明は、少なくともＬ〜グルタミン合成酵素、
ＡＴＰ、ピルビン酸キナーゼ、フォスフオニノールピル
ビン酸、ピルビン酸脱水素酵素、ＦＡＤ、ＴＰＰ、２価
陽イオン、燐酸イオン、電子伝達剤及び電子受容性指示
薬からなる、体液中のアンモニアを又はアンモニア生成
基質を分析するためのアンモニア又はアンモニア生成基
質分析用試薬組成物及びそれを含む一体型多層分近要素
に関するものである。The present invention provides at least L~glutamine synthetase,
Ammonia in body fluids or ammonia production, consisting of ATP, pyruvate kinase, phosphooninol pyruvate, pyruvate dehydrogenase, FAD, TPP, divalent cations, phosphate ions, electron transfer agents and electron accepting indicators. The present invention relates to a reagent composition for analyzing ammonia or ammonia-producing substrates for analyzing a substrate, and an integrated multilayer separation element containing the same.
本発明の試薬組成物及び分析要素においてアンモニウム
イオンを検出する反応系は下記のように進行する。The reaction system for detecting ammonium ions in the reagent composition and analytical element of the present invention proceeds as follows.
Ｓ
グルタミン酸−１−ＡＴＰ ＋Ｎｌ＋４°＝グルタミン
＋ＡＤＰ±燐酸
Ｋ
ＡＤＰ＋フォスフオニノールピルビン酸→Ａ Ｔ Ｐ−
←ピルビン酸
十ＣＯ２
還元された電子受容体
ＧＳ：グルタミン合成酵素
ＰＫ：ピルビン酸キナーゼ
ＰＤＨｎピルビン酸脱水酸酢水
素酵素ルタミン合成酵素（Ｅ Ｃ６，３，１，２）はＢ
ｒｅｖｉｂａｃｔｅｒｉｕｍ ｆｌａｖｕｍ 、大腸菌
（Ｅｓｃｈｅｒｉｃｈｉａ ｃ。S Glutamic acid-1-ATP +Nl+4°=Glutamine+ADP±K phosphate ADP+Phosphoninolpyruvate→A T P-
←Pyruvate decaCO2 Reduced electron acceptor GS: Glutamine synthase PK: Pyruvate kinase PDHn Pyruvate dehydroacetate hydrogenase Lutamine synthase (E C6, 3, 1, 2) is B
revibacterium flavum, Escherichia c.
１ｉ ＡＴＣＣ９６３７等）、Ｂａｃｉｌｌｕｓ ｓｔ
ｅａｒｏｔｈｅｒｍｏｐｈｉｌｕｓ等が産生じ、羊脳、
牛脳等からも分離しうる。Ｂ。1i ATCC9637 etc.), Bacillus st.
earothermophilus etc. are produced, sheep brain,
It can also be isolated from bovine brain, etc. B.
ｓｔｅａｒｏｔｈｅｒｍｏｐｈｉｌｕｓの産生ずる酵素
は耐熱性酵素であって市販されており、本発明の試薬組
成物及び分析要素に特に好ましい。The enzyme produced by P. stearothermophilus is a thermostable enzyme that is commercially available and is particularly preferred for the reagent compositions and analytical elements of the present invention.
ピルビン酸キナーゼ［Ｅ Ｃ２，７，１，４０）はＢａ
ｃｉｌｌｕｓ ｓｔｅａｒｏｔｈｅｒｍｏｐｈｉｌｕｓ
、 Ｂａｃｉｌｌｕｓ ｓｔｅａｒｏｔｈｅｒｍｏｐ
ｈｉｌｕｓ Ｂａｃｉｌｌｕｓ Ｉｉｃｈｅｎｉｆｏｒ
ｍｉｓ等が産生しうるほか、ラット、ウサギ、イヌ、ニ
ワトリ等の動物の筋肉、ブタの心臓などから取得しうる
。特に好ましいピルビン酸脱水素酵素として非ＮＡＤＨ
依存性のものを挙げることができる。Pyruvate kinase [E C2,7,1,40) is Ba
cillus stearothermophilus
, Bacillus stearothermop
hilus Bacillus Iichenifor
In addition to being produced by M.mis, it can also be obtained from the muscles of animals such as rats, rabbits, dogs, and chickens, and the heart of pigs. As a particularly preferred pyruvate dehydrogenase, non-NADH
Dependencies can be mentioned.
ピルビン酸脱水素酵素はＬａｃｔｏｂａｃｉｌｌｕｓ
ｄｅｌｂｒｕｅｃｋｉｉなどの諸種の細菌から抽出され
た酵素や動物臓器から抽出された酵素を用いることがで
きる。Pyruvate dehydrogenase is Lactobacillus
Enzymes extracted from various types of bacteria such as Delbrueckii and enzymes extracted from animal organs can be used.
好ましいＰＤＨはＮＡＤＨ非依存性のものである。Preferred PDHs are NADH independent.
諸種の生物起源のＰＤＨのうちでり、 ｄｅｌｂｒｕｅ
ｃｋｉｉ起源の酵素（例えば東洋紡績■から市販されて
いる。）は好ましい酵素である。この酵素は分子量約１
６万、等電点ｐＨ４，４±０．１で、ＦＡＤ、ＴＰＰ等
の補酵素を必要とするが、燐酸をも基質とする点で従来
のピルビン酸デヒドロゲナーゼと異なる酵素である。Ｍ
ｉｃｈａｅｌ ｉｓ定数はピルビン酸に対して４．４
Ｘｌ０−’Ｍ 、燐酸に対して１．４ Ｘｌ０−２Ｍで
ある。また、４５°Ｃの５０ｍＭ ｐＨ７，０燐酸緩衝
液中での活性値減少は極めて少なく、少なくとも３時間
にわたって実質的に活性値の減少は見られなかった。Out of various biogenic PDHs, delbrue
Enzymes of C. ckii origin (commercially available, for example, from Toyobo ■) are preferred enzymes. This enzyme has a molecular weight of approximately 1
60,000, has an isoelectric point of pH 4.4±0.1, and requires coenzymes such as FAD and TPP, but is different from conventional pyruvate dehydrogenase in that it also uses phosphoric acid as a substrate. M
The is constant is 4.4 for pyruvate
Xl0-'M, 1.4 Xl0-2M for phosphoric acid. Furthermore, the decrease in activity value in 50 mM pH 7.0 phosphate buffer at 45°C was extremely small, and virtually no decrease in activity value was observed for at least 3 hours.
この酵素に限らず一般にＰＤＨは熱安定性が良く、約５
０°Ｃの周囲温度（緩衝液中など）で活性値の減少は僅
少なものが多い。またいずれも市販されており、購入し
て用いることができる。一方、本発明の分析要素用とし
てはこれらの酵素は純品でなくともよ（、例えば微生物
の発酵液あるいはそれから沈澱等の手段で得た粗製酵素
を利用することもできる。Not limited to this enzyme, PDH in general has good thermostability, about 5
At an ambient temperature of 0°C (in a buffer solution, etc.), the decrease in activity value is often slight. All of them are commercially available and can be purchased and used. On the other hand, for use in the analytical elements of the present invention, these enzymes do not need to be pure products (for example, it is also possible to use a fermentation solution of a microorganism or a crude enzyme obtained from it by means such as precipitation).
２価陽イオンは前記の共役酵素を活性化させるものであ
り、Ｍｇｇ＋、Ｍｎ２＋等を含む。ホスホエノールピル
ベートはピルビン酸キナーゼの基質であり、チアミンピ
ロ燐酸はピルビン酸デヒドロゲナーゼの活性化に必要な
試薬成分である。アデノシン三燐酸（ＡＴＰ）はＬ−グ
ルタミン合成酵素の基質であり、燐酸イオンはピルビン
酸脱水素反応において燐酸源として使用されるものであ
る。電子伝達剤と電子受容性指示薬からなっている。Divalent cations activate the above-mentioned coupled enzymes, and include Mgg+, Mn2+, and the like. Phosphoenolpyruvate is a substrate for pyruvate kinase, and thiamine pyrophosphate is a necessary reagent component for activation of pyruvate dehydrogenase. Adenosine triphosphate (ATP) is a substrate for L-glutamine synthetase, and phosphate ions are used as a phosphate source in the pyruvate dehydrogenation reaction. It consists of an electron transfer agent and an electron-accepting indicator.
電子伝達剤としてはＪ、 Ｆ、Ｂｕｒｄ、ｅｔ ａ！、
Ｃｌ１ｎ、 Ｃｈｉｍ Ａｃｔａ、 ４６ ．２３
（１９７３） 、 Ｓ、 Ｎａｋａｍｕｒａ、
ｅｔ ａｌＣｌｉｎ、 Ｃｈｉｍ、Ａｃｔａ、 １０
１，３２１ （１９８０）等に記載の電子伝達剤やジア
ホラーゼ（ＥＣ１，６，４，３３等の酵素が使用できる
。例えば、■−メトキシー５−メチルフェナジニウムメ
チルサルフエート（１−メトキシＰＭＳ） 、メクロラ
スブルー（９−ジメチルアミノベンゾ−α−フェナゾニ
ウムクロリド）、５−メチルツェナジニウムメチルサル
フェート（ＰＭＳ）等である。Ｂａｃｉｌｌｕｓ ｓｔ
ｅａｒｏｔｈｅｒｍｏｐｈｉＩｕｓの産生ずるジアホラ
ーゼは５０’Ｃでも活性が低下しない程熱安定性が高く
、特に好ましい。Examples of electron transfer agents include J, F, Burd, et a! ,
Cl1n, Chim Acta, 46. 23
(1973), Nakamura, S.
et alClin, Chim, Acta, 10
1,321 (1980) and enzymes such as diaphorase (EC1,6,4,33) can be used.For example, ■-Methoxy5-methylphenazinium methylsulfate (1-methoxyPMS) , Meclorus blue (9-dimethylaminobenzo-α-phenazonium chloride), 5-methylzenazinium methyl sulfate (PMS), etc. Bacillus st.
The diaphorase produced by P. aerothermophilus is particularly preferred because it has such high thermostability that its activity does not decrease even at 50'C.
電子受容性Ｉ旨示薬としては還元性発色剤として公知の
テトラゾリウム塩類やレサズリン誘導体を使用できる。As the electron-accepting I indicator, tetrazolium salts and resazurin derivatives, which are known as reducing coloring agents, can be used.
テトラゾリウム塩類の例としては、３−（ｐ−インドフ
ェニル）−２−（ｐ−ニトロフェニル−５−フェニル−
２Ｈ−テトラゾリウムクロリド（ＩＮＴ）、３− （４
，５−ジメチル−２チアジル）−２，５−ジフェニル−
２Ｈ−テトラゾリウムプロミド（ＭＴＴ）、３．３’
−（４，４“−ビフェニレン）−ビス（２，５−ジフェ
ニル−２Ｈ−テトラゾリウムクロリド）（ネオ−ＴＢ）
、３．３’−（３，３°−ジメトキシ−４，４′ビフエ
ニレン）ビス（２−（ｐ−ニトロフェニル）５−フェニ
ル−２Ｈテトラヅリウムクロリド〕にドローＴＢ）、３
．３’ −（３，３’ −ジメトキシ−４，４゛−ビ
フェニレン）ビス（２，５ジフェニル−２Ｈテトラゾリ
ウムクロリド）（ＴＢ）、３．３’ −（３，３’−ジ
メトキシ４．４”−ビフェニレン）ビスＣ２，５−ビス
（ｐ−ニトロフェニル）−２Ｈテトラゾリウムクロリド
）（ＴＮＴＢ）等々を挙げることができる。Examples of tetrazolium salts include 3-(p-indophenyl)-2-(p-nitrophenyl-5-phenyl-
2H-tetrazolium chloride (INT), 3- (4
,5-dimethyl-2thiazyl)-2,5-diphenyl-
2H-tetrazolium bromide (MTT), 3.3'
-(4,4"-biphenylene)-bis(2,5-diphenyl-2H-tetrazolium chloride) (Neo-TB)
, 3.3'-(3,3°-dimethoxy-4,4'biphenylene)bis(2-(p-nitrophenyl)5-phenyl-2H tetradurium chloride), 3
．． 3'-(3,3'-dimethoxy-4,4'-biphenylene)bis(2,5diphenyl-2H tetrazolium chloride) (TB), 3.3'-(3,3'-dimethoxy4.4"- biphenylene)bisC2,5-bis(p-nitrophenyl)-2H tetrazolium chloride) (TNTB), and the like.
アンモニアを生成する基質とアンモニアを生成させる酵
素としては、
Ｌ−アスパラギン：アスパラギナーゼ（ＥＣ３，５，１
，１）Ｌ−アスパラギン酸：アスパルターゼ（ＥＣ４，
３，１，１）アデニン：アデニンデアミナーゼ（ＥＣ３
，５，４，２）アデノシン：アデノシンデアミナーゼ（
ＥＣ３，５，４，４）アデノシンモノ燐酸：アデノシン
モノ燐酸デアミナーゼ（ＥＣ３，５，４，１７）
尿素：ウレアーゼ（ＥＣ３，５，１，３）グアニン：グ
アニンデアミナーゼ（ＥＣ３，５，４，３）グルタミン
：グルタミナーゼ（ＥＣ３，５，１、２）クレアチニン
：クレアチニンデイミナーゼ（ＣＣ３，５，４，２１）
笠の組合せが挙げられる。The substrate that generates ammonia and the enzyme that generates ammonia include L-asparagine:asparaginase (EC3, 5, 1
, 1) L-aspartic acid: aspartase (EC4,
3,1,1) Adenine: Adenine deaminase (EC3
, 5, 4, 2) Adenosine: Adenosine deaminase (
EC3,5,4,4) Adenosine monophosphate: adenosine monophosphate deaminase (EC3,5,4,17) Urea: urease (EC3,5,1,3) Guanine: guanine deaminase (EC3,5,4,3) Examples include combinations of glutamine: glutaminase (EC3, 5, 1, 2) and creatinine: creatinine deiminase (CC 3, 5, 4, 21).
本発明の一体型多層分析要素は少なくとも光透過性水平
透過性支持体、試薬層及び展開層がこの順に積層されて
いる。The integrated multilayer analytical element of the present invention has at least a horizontally transparent support, a reagent layer, and a developing layer laminated in this order.
光透過性水平透過性支持体は一般の多層分析要素に使用
されている公知のものを利用すればよい。As the light-transmitting horizontally transparent support, any known support used in general multilayer analytical elements may be used.
その具体例としては、ポリエチレンテレフタレート、ビ
スフェノールＡのポリカルボネート、ポリスチレン、セ
ルロースエステル（セルロースジアセテート、セルロー
ストリアセテート、セルロースアセテ−１−プロピオネ
ート等）などのポリマーからなる厚さ約５０ｐｍから約
１ｍｍ、好ましくは約８０μｍから約３００μｍの範囲
の透明支持体を用いることができる。支持体の表面には
必要により前記の諸特許明細書等により公知の下塗層ま
たは接着層を設けて支持体の上に設けられる親水性層等
との接着を強固にすることができる。Specific examples thereof include polymers such as polyethylene terephthalate, polycarbonate of bisphenol A, polystyrene, and cellulose esters (cellulose diacetate, cellulose triacetate, cellulose acetate-1-propionate, etc.), with a thickness of about 50 pm to about 1 mm, preferably. A transparent support in the range of about 80 μm to about 300 μm can be used. If necessary, a known undercoat layer or adhesive layer may be provided on the surface of the support as described in the above-mentioned patent specifications to strengthen the adhesion with a hydrophilic layer or the like provided on the support.
試薬層は前記の酵素及び試薬組成物の全部又は一部を含
有する層であり、非孔性で吸水性の層又は微多孔性で水
浸透性の層である。The reagent layer is a layer containing all or part of the enzyme and reagent composition described above, and is a non-porous, water-absorbing layer or a microporous, water-permeable layer.
実質的に非孔性で吸水性の試薬層に用いられる親水性ポ
リマーバインダーは水吸収時の膨潤率が３０°Ｃで約１
５０％から約２０００％、好ましくは約２５０％から約
１５００％の範囲の天然または合成親水性ポリマーであ
る。その例として、ゼラチン（酸処理ゼラチン、脱イオ
ンゼラチン等）、ゼラチン誘導体（フタル化ゼラチン、
ヒドロキシアクリレートグラフトゼラチン等）、アガロ
ース、プルラン、プルラン誘導体、ポリアクリルアミド
、ポリビニルアルコール、ポリビニルピロリドン等をあ
げることができる。実質的に非孔性の試薬層の乾燥厚さ
は約３μｍから約５０１！ｍ、好ましくは約５μ印から
約３０鐸の範囲であり、被覆量で約３ｇ／イから約５０
ｇ／ポ、好ましくは約５ｇ／ｒｒｆから約３０ｇ／ｒｒ
ｆの範囲である。試薬層は実質的に透明であることが好
ましい。The hydrophilic polymer binder used in the substantially non-porous, water-absorbing reagent layer has a swelling rate of approximately 1 at 30°C upon water absorption.
Natural or synthetic hydrophilic polymers ranging from 50% to about 2000%, preferably from about 250% to about 1500%. Examples include gelatin (acid-treated gelatin, deionized gelatin, etc.), gelatin derivatives (phthalated gelatin,
(hydroxyacrylate grafted gelatin, etc.), agarose, pullulan, pullulan derivatives, polyacrylamide, polyvinyl alcohol, polyvinylpyrrolidone, and the like. The dry thickness of the substantially non-porous reagent layer ranges from about 3 μm to about 501 μm! m, preferably in the range of about 5 μm to about 30 m, and the coating amount is about 3 g/m to about 50 m
g/po, preferably about 5 g/rrf to about 30 g/rr
This is the range of f. Preferably, the reagent layer is substantially transparent.
微多孔性で水浸透性の試薬層は後述する布地展開層と同
様の布地、特開昭５７−１４８２５０に記載の有機ポリ
マー繊維バルブ含有抄造紙、特開昭５７−１２５８４７
等に記載の繊維と親水性ポリマーの分散液を塗布して形
成した多孔性層等の繊維質多孔性層；特公昭５３−２１
６７７、米国特許３９９２１５８等に記載のメンブラン
フィルタ−層（プラッシュポリマー層）、ポリマーミク
ロビーズ等の微粒子が親水性ポリマーバインダーで点接
触状に接着されてなる連続微空隙含有等方的多孔性層、
特開昭５５−９０８５９に記載のポリマーミクロビーズ
が水で膨潤しないポリマー接着剤で点接触状に接触され
てなる連続空隙含有等方的多孔性層（三次元格子状粒状
構造物層）等の非繊維等方的多孔性層等の微多孔性層に
試薬組成物を含有させた層又は固体微粒子と親水性ポリ
マーバインダーから構成される微多孔性構造体に試薬組
成物を含有させた層である。微多孔性試薬層の乾燥時の
層厚は約７μｍから約２５０μｍ、好ましくは約１０μ
ｍから約２５０ μｍの範囲である。微多孔性試薬層の
上に展開層を設ける場合には、両層の間は特開昭６１−
４９５９ 、特開昭６２−１３８７５６等に記載の多孔
性接着によるのが好ましい。The microporous and water-permeable reagent layer is made of the same fabric as the fabric spreading layer described later, a paper made from organic polymer fiber valves as described in JP-A-57-148250, and JP-A-57-125847.
A fibrous porous layer such as a porous layer formed by applying a dispersion of fibers and a hydrophilic polymer described in Japanese Patent Publication No. 53-21
677, a membrane filter layer (plush polymer layer) described in US Pat.
An isotropic porous layer containing continuous voids (three-dimensional lattice-like granular structure layer), etc., in which polymer microbeads are contacted in a point-contact manner with a polymer adhesive that does not swell with water, as described in JP-A-55-90859. A layer in which a reagent composition is contained in a microporous layer such as a non-fibrous isotropic porous layer, or a layer in which a reagent composition is contained in a microporous structure composed of solid fine particles and a hydrophilic polymer binder. be. The dry layer thickness of the microporous reagent layer is about 7 μm to about 250 μm, preferably about 10 μm.
m to approximately 250 μm. When a developing layer is provided on the microporous reagent layer, the space between the two layers is
4959, JP-A No. 62-138756, etc., porous adhesion is preferably used.
本発明の多層分析要素の１態様として、展開層に前述の
試薬組成物を含有させ、展開層の下に（必要におうじて
接着層を介して）吸水層又は検出層を設けた構成の多層
分析要素がある。別の１態様として、試薬層のポリマー
バインダーとして、水性液体試料の溶媒に溶解又はゾル
化するポリマー、例えばポリビニルアルコール、ポリビ
ニルピロリドン、カルホキシミチルセルロース、アルギ
ン酸ナトリウム、非硬化ゼラチン、寒天等を用いて試薬
層を形成し、その上に前記の微多孔性試薬層として用い
る微多孔性の薄シート状部材を多孔性検出層として積層
接着した構成の多層分析要素がある。One embodiment of the multilayer analysis element of the present invention is a multilayer structure in which the development layer contains the above-mentioned reagent composition, and a water absorption layer or a detection layer is provided below the development layer (through an adhesive layer if necessary). There is an analytical element. In another embodiment, the polymer binder of the reagent layer is a polymer that dissolves or becomes a sol in the solvent of the aqueous liquid sample, such as polyvinyl alcohol, polyvinylpyrrolidone, carboximityl cellulose, sodium alginate, unhardened gelatin, agar, etc. There is a multilayer analytical element having a structure in which a reagent layer is formed, and a microporous thin sheet-like member used as the microporous reagent layer is laminated and adhered thereon as a porous detection layer.
試薬層には公知のｐＨ緩衝剤組成物、高分子ｐＨ緩衝剤
、塩基性ポリマー、酸性ポリマー、高分子媒染剤等を含
有させることができる。The reagent layer can contain a known pH buffer composition, a polymer pH buffer, a basic polymer, an acidic polymer, a polymer mordant, and the like.
展開層としては特公昭５３−２１６７７号公報等に開示
のメンブランフィルタ（プラッシュポリマー層）、ポリ
マーミクロビーズ、ガラスミクロビーズ、珪藻土が親水
性ポリマーバインダーに保持されてなる連続微空隙含有
多孔性層、特開昭５５−９０８５９号公報に開示のポリ
マーミクロビーズ、ガラスミクロビーズ笠で水が膨潤し
ないポリマー接着剤で点接触状に接着されてなる連続微
空隙含有多孔性層（三次元格子状粒状構造物Ｎ）等の非
繊維質等方的多孔性展開層、特開昭５５−１６４３５６
号公報、特開昭５７−６６３５９号公報等に開示の織物
展開層、特願昭５９−７９１５８号明細書に開示の織物
展開層、特願昭５７−１４８２５０号公報に開示の有機
ポリマー繊維バルブを含む抄造紙からなる展開層等の繊
維質多孔性展開層等があげられる。As the developing layer, a membrane filter (plush polymer layer) disclosed in Japanese Patent Publication No. 53-21677 etc., a continuous microporous porous layer formed by polymer microbeads, glass microbeads, diatomaceous earth held in a hydrophilic polymer binder, A continuous microvoid-containing porous layer (three-dimensional lattice-like granular structure) in which polymer microbeads and glass microbeads disclosed in JP-A-55-90859 are bonded in point contact with a polymer adhesive that does not swell with water. Non-fibrous isotropic porous spread layer such as product N), JP-A-55-164356
The fabric spreading layer disclosed in Japanese Patent Application No. 57-66359, the fabric spreading layer disclosed in Japanese Patent Application No. 59-79158, the organic polymer fiber valve disclosed in Japanese Patent Application No. 57-148250, etc. Examples include a fibrous porous spread layer such as a spread layer made of paper containing.
展開層は直接あるいは必要により接着層を介して積層さ
れる。The spreading layer is laminated directly or via an adhesive layer if necessary.
多層分析要素には前述の諸層のほかに必要に応じて特開
昭５１−４０１９１、特開昭５３−１３１０８９等に開
示の検出層または媒染層、特開昭５４−２９７００等に
開示のミグレーション阻止層、特開昭５１−４０１９１
等に開示の中間層、特開昭５６−８５４９に開示の試薬
含有微粒子分散層等を組み込んで設けることができる。In addition to the above-mentioned layers, the multilayer analysis element may include a detection layer or a mordant layer disclosed in JP-A-51-40191, JP-A-53-131089, etc., and a MIG disclosed in JP-A-54-29700, etc., as necessary. ration prevention layer, JP-A-51-40191
It is possible to incorporate an intermediate layer disclosed in et al., a reagent-containing fine particle dispersed layer disclosed in JP-A-56-8549, and the like.
展開層、試薬層、吸水層、検出層、接着層等には界面活
性剤、好ましくはノニオン性界面活性剤を含有させるこ
とができる。ノニオン性界面活性剤の例として、ｐ−オ
クチルフェノキシポリエトキシエタノール、ｐ−ノニル
フェノキシポリグリシドール、ポリオキシエチレンオレ
イルエーテル、ポリオキシエチレンソルビクンモノラウ
レート、オキチルグルコシド等がある。ノニオン性界面
活性剤を試薬層、吸水層又は検出層に含有させることに
より分析操作時に水性液体試料中の水が試薬層、吸水層
又は検出層に実質的に一様に吸収されやすくなり、また
展開層との液体接触が迅速かつ実質的に一様になる。ノ
ニオン性界面活性剤を展開層に含有させることにより水
性液体試料の展開作用（メータリング作用）がより良好
になる。ノニオン性界面活性剤の展開層における含有量
は展開層１ｒ′Ｉ′ｒ当り約１０■〜約３．０ｇ、好ま
しくは約１０■〜約２．０ｇの範囲である。The spreading layer, reagent layer, water absorption layer, detection layer, adhesive layer, etc. can contain a surfactant, preferably a nonionic surfactant. Examples of nonionic surfactants include p-octylphenoxypolyethoxyethanol, p-nonylphenoxypolyglycidol, polyoxyethylene oleyl ether, polyoxyethylene sorbicun monolaurate, oxytyl glucoside, and the like. By including a nonionic surfactant in the reagent layer, water absorption layer, or detection layer, water in an aqueous liquid sample is easily absorbed substantially uniformly into the reagent layer, water absorption layer, or detection layer during analysis operations, and Liquid contact with the spreading layer is rapid and substantially uniform. By containing a nonionic surfactant in the spreading layer, the spreading action (metering action) of the aqueous liquid sample becomes better. The content of the nonionic surfactant in the spreading layer ranges from about 10 to about 3.0 g, preferably from about 10 to about 2.0 g per 1r'I'r of the spreading layer.
本発明の分析要素を尿素窒素分析用とする場合には例え
ばウレアーゼを、クレアチニン分析用とする場合にはク
レアチニンディミナーゼあるいはタレアチニンアミドヒ
ドロラーゼ、タレアチンアミジノヒドロラーゼ及びウレ
アーゼを試薬層、展開層あるいはその他の適当な層へ含
有せしめればよい。When the analytical element of the present invention is used for urea nitrogen analysis, for example, urease is used, and when it is used for creatinine analysis, creatinine diminase, taleatinin amidohydrolase, taleatinamidinohydrolase, and urease are used in the reagent layer, developing layer, or other layers. It may be contained in an appropriate layer.
本発明の一体型多層分析要素は」二連のような分析要素
において、ピルビン酸を基質とするが電子伝達剤と電子
受容性指示薬反応系に作用する反応生成物を与えない酵
素反応試薬組成物を含有するピルビン酸除去層を試薬層
の支持体と反対側に設けることができる。The integrated multilayer analytical element of the present invention is an enzyme reaction reagent composition that uses pyruvate as a substrate but does not give a reaction product that acts on the electron transfer agent and electron-accepting indicator reaction system. A pyruvic acid removal layer containing the reagent layer can be provided on the opposite side of the reagent layer to the support.
このような酵素反応試薬組成物としては、フォスフオニ
ノールピルビン酸合成酵素〔Ｅ Ｃ２，７，９゜２〕と
ＡＴＰの組合せ、ピルビン酸燐酸ジキナーゼ（ＥＣ２，
７，９，１）とＡＴＰと燐酸塩の組合せ、ピルビン酸カ
ルボキシラーゼ（ＥＣ６，４，１，１）とＡＴＰと炭酸
塩の組合せ等を挙げることができる。Such enzyme reaction reagent compositions include a combination of phosphoninolpyruvate synthase [EC2,7,9゜2] and ATP, pyruvate phosphate dikinase (EC2,
Examples include a combination of ATP and phosphate, and a combination of pyruvate carboxylase (EC6, 4, 1, 1), ATP and carbonate.
フォスフオニノールピルビン酸合成酵素とＡＴＰの組合
せを用いることによって２価のマンガン・ｆオンの存在
下で試料中のピルビン酸をフォスフオニノールピルビン
酸に変えることができ、ピルビン酸燐酸ジキナーゼとＡ
ＴＰと燐酸塩の組合せを用いることによってマグふシウ
ムイオンとアンモニウムイオン又はカリウムイオンの存
在下でピルビン酸をフォスフオニノールピルビン酸に変
えることができる。ピルビン酸カルボキシラーゼとＡＴ
Ｐと炭酸塩の組合せを用いることによってビオチンとマ
グネシウムイオンの存在下でピルビン酸をオキサロ酢酸
に変えることができる。ピルビン酸を除去する反応系は
逆反応の起こらないものあるいは起こりにくいものが好
ましい。By using a combination of phosphooninolpyruvate synthase and ATP, pyruvate in a sample can be converted to phosphooninolpyruvate in the presence of divalent manganese f-one, and pyruvate phosphate dikinase and A
By using a combination of TP and phosphate, pyruvate can be converted to phosphoninolpyruvate in the presence of magfusium and ammonium or potassium ions. Pyruvate carboxylase and AT
By using a combination of P and carbonate, pyruvate can be converted to oxaloacetate in the presence of biotin and magnesium ions. The reaction system for removing pyruvic acid is preferably one in which reverse reaction does not occur or is difficult to occur.
また、上記の酵素反応と共役した反応系をさらに導入し
て生成物をさらに別生成物に変えあるいは副生成物を反
応前の化合物に戻してやることも好ましい。例えば、ピ
ルビン酸デカルボキシレートでアセトアルデヒドと炭酸
に分解する反応は逆反応が起らない点で好ましい。前記
の共役反応系は数多く存在し、それらのなかから本発明
に適当なものを選択することができる。It is also preferable to further introduce a reaction system coupled to the above enzyme reaction to convert the product into another product or to return the by-product to the compound before the reaction. For example, a reaction in which pyruvate decarboxylate is decomposed into acetaldehyde and carbonic acid is preferable because no reverse reaction occurs. There are many conjugate reaction systems mentioned above, and one suitable for the present invention can be selected from them.
酵素反応試薬組成物にはｐｌ＋緩衝剤、酵素活性化剤等
をさらに組合せることができる。The enzyme reaction reagent composition can further include pl+buffer, enzyme activator, etc.
上記の酵素反応試薬組成物を含むピルビン酸除去層は試
薬層の支持体と反対側に設けて試料中のあるいは目的成
分から生じたアンモニアから生成されたピルビン酸を酵
素反応させる前に試料に予め含まれていたピルビン酸を
他の物質に変えるようにする。このピルビン酸除去層は
新たに独立して設けてもよく、展開層、光遮蔽層等に酵
素反応試薬組成物を含有させて兼用させることもできる
。The pyruvic acid removal layer containing the above enzymatic reaction reagent composition is provided on the opposite side of the reagent layer to the support, and is applied to the sample before enzymatically reacting the pyruvic acid generated from ammonia in the sample or from the target component. Converts the pyruvic acid contained in it into other substances. This pyruvic acid removal layer may be newly provided independently, or the enzymatic reaction reagent composition may be contained in the development layer, light shielding layer, etc., so that they can also be used.
新たに設ける場合には例えば前述の試薬層で説明した親
水性ポリマーバインダー層又は微多孔性層を利用するこ
とができる。また、ピルビン酸除去層は１層に限られる
ものではなく、酵素反応試薬組成物の各成分を２層以上
に分けて含有せしめてもよい。When newly provided, for example, the hydrophilic polymer binder layer or microporous layer described in the above-mentioned reagent layer can be used. Further, the number of layers for removing pyruvic acid is not limited to one layer, and each component of the enzyme reaction reagent composition may be divided into two or more layers and contained therein.
一方、グルタミン合成酵素、ピルビン酸キナーゼ、ピル
ビン酸脱水素酵素、電子伝達剤、電子受容性指示薬等も
成分の一部は試薬層以外の層に含有させてもよいが、そ
の場合であっても試料中のあるいは目的成分から生じた
アンモニアをピルビン酸に変える反応がピルビン酸除去
層によって試料中に予め含まれていたピルビン酸が除去
された後に起こるように配置する必要がある。On the other hand, some of the components such as glutamine synthetase, pyruvate kinase, pyruvate dehydrogenase, electron transfer agents, and electron-accepting indicators may be contained in layers other than the reagent layer; The arrangement must be such that the reaction of converting ammonia in the sample or generated from the target component into pyruvic acid occurs after the pyruvic acid previously contained in the sample is removed by the pyruvic acid removal layer.
本発明の一体型多層分析要素における含有量はグルタミ
ン合成酵素が２００〜３万Ｉｌｌ／ｒ７ｆ程度、好まし
くは５００〜２万ＴＯ／ｒｎ程度、ピルビン酸キナーゼ
が２０００〜６万ＩＵ／Ｍ程度、好ましくは３０００〜
４万／ｎ（程度、ピルビン酸脱水素酵素が３０００〜１
０万■口／ｒＴｆ程度、好ましくは５０００〜５万ＩＵ
／ ｍ程度がｊ適当である。The content of the integrated multilayer analytical element of the present invention is about 200 to 30,000 Ill/r7f of glutamine synthase, preferably about 500 to 20,000 TO/rn, and about 2000 to 60,000 IU/M of pyruvate kinase, preferably about 200 to 30,000 Ill/r7f. is 3000～
40,000/n (approximately, pyruvate dehydrogenase is 3000-1
About 00,000 mouths/rTf, preferably 5,000 to 50,000 IU
/m is appropriate.
本発明の分析要素においてはまたグルタミン合成酵素、
ピルビン酸キナーゼ又はアンモニア生成基質をアンモニ
アに変換する酵素の含有量の少なくとも一つを速度限定
量（ｒａｔｅ ｌｉｍｉｔｉｎｇ ａｍｏｕｎｔ）とす
る。これはピルビン酸脱水素酵素の量（活性値）に対し
てグルタミン合成酵素、ピルビン酸キナーゼ又はアンモ
ニア生成基質をアンモニアに変換する酵素の量（活性値
）が前記の反応の進行する速度を支配するような量であ
ることを意味する。The analytical element of the present invention also includes glutamine synthetase,
The content of at least one of pyruvate kinase or an enzyme that converts an ammonia-producing substrate to ammonia is a rate limiting amount. This is because the amount (activity value) of glutamine synthetase, pyruvate kinase, or an enzyme that converts an ammonia-producing substrate into ammonia with respect to the amount (activity value) of pyruvate dehydrogenase controls the rate at which the above reaction proceeds. It means such an amount.
速度限定量は試料が血漿または血清の場合には一般に、
グルタミン合成酵素は２００〜２万ＩＵ／ｒｄ程度、ピ
ルビン酸キナーゼは２００〜２万ＩＩ／ｒＴＩ程度そし
てアンモニア生成基質をアンモニアに変換する酵素は２
００〜２００００／ ｒｒｆ程度である。ピルビン酸脱
水素酵素は充分量あればよく、通常３０００〜１０万１
０／イ程度、好ましくは５０００〜５万ＩＩ／耐程度で
ある。The rate-limiting amount is generally determined when the sample is plasma or serum.
Glutamine synthetase is about 200 to 20,000 IU/rd, pyruvate kinase is about 200 to 20,000 II/rTI, and the enzyme that converts ammonia-producing substrate to ammonia is about 2
It is about 00-20000/rrf. A sufficient amount of pyruvate dehydrogenase is sufficient, usually 3,000 to 100,000.
It is about 0/I, preferably about 5,000 to 50,000 II/I.
本発明の多層分析要素は前述の諸特許明細書に記載の公
知の方法により調製することができる。The multilayer analytical element of the present invention can be prepared by known methods as described in the aforementioned patent specifications.
本発明の多層分析要素は一辺約１５ｍｍから約３０ｍｍ
の正方形またはほぼ同サイズの円形等の小片に裁断し、
特公昭５７−２８３３１、実公昭５６−１４２４５４、
特開昭５７−６３４５２、実開昭５８−３２３５０、特
表昭５８−５０１１４４等に記載のスライド枠に収めて
化学分析スライドとして用いることが、製造、包装、輸
送、保存、測定操作等諸種の観点で好ましい。使用目的
によっては長いテープ状でカセットまたはマガジンに収
めて用いること、または小片を開口のあるカードに貼付
または収めて用いることなどもできる。The multilayer analytical element of the present invention has a side of about 15 mm to about 30 mm.
Cut into small pieces such as squares or circles of approximately the same size,
Special Publication No. 57-28331, Real Publication No. 56-142454,
JP-A-57-63452, Utility Model Publication 58-32350, Japanese Patent Application Publication No. 58-501144, etc., it is possible to use it as a chemical analysis slide by placing it in the slide frame described in Japanese Patent Publication No. 57-63452, Utility Model Application Publication No. 58-32350, Japanese Patent Application Publication No. 58-501144, etc. Preferred from this point of view. Depending on the purpose of use, it can be used in the form of a long tape and stored in a cassette or magazine, or small pieces can be attached or stored in a card with an opening.
本発明の多層分析要素は前述の諸特許明細３等に記載の
操作により液体試料中のアナライトの分析を実施できる
。例えば約５μｅから約３０μｅ、好ましくは８μｒか
ら１５μｅの範囲の全血、血漿、血清、尿等に水性液体
試料摘を展開層に点着し、１分から１０分の範囲で、約
２０’Ｃから約４０’Ｃの範囲の実質的に一定の温度で
、好ましくは３７°Ｃ近傍の実質的に一定の温度でイン
クベーションし、要素内の発色または変色を可視光又は
紫外光の吸収極大波長またはその近傍の波長の光を用い
て光透過性支持体側から反射測光し、予め作成した検量
線を用（Ａで比色測定法の原理により液体試料中のアナ
ライトの含有量を求めることができる。The multilayer analysis element of the present invention can analyze an analyte in a liquid sample by the operations described in the above-mentioned patent specifications 3 and the like. For example, an aqueous liquid sample of whole blood, plasma, serum, urine, etc. in the range of about 5μe to about 30μe, preferably 8μr to 15μe is spotted on the developing layer, and the temperature is about 20°C for 1 to 10 minutes. The incubation is carried out at a substantially constant temperature in the range of about 40'C, preferably around 37'C, to induce color development or discoloration within the element at wavelengths of maximum absorption of visible or ultraviolet light or Reflection photometry is performed from the light-transmitting support side using light with a wavelength in the vicinity of that wavelength, and a previously prepared calibration curve is used to determine the content of the analyte in the liquid sample using the principle of colorimetric measurement in .
測定操作は特開昭６０−１２５５４３、特開昭６０−２
２０８６２、特開昭６１−２９４３６７、特開昭５８−
１６１８６７、特開昭６３−３１３０６３等に記載の化
学分析装置により極めて容易な操作で高精度の定量分析
を実施できる。Measurement operations are performed using JP-A-60-125543 and JP-A-60-2.
20862, JP-A-61-294367, JP-A-58-
161867, JP-A No. 63-313063, etc., highly accurate quantitative analysis can be carried out with extremely easy operation.
本発明の分析要素で採用されているアンモニア測定用の
反応試薬系はアンモニウムイオンをグルタミン合成酵素
がグルタミン酸と反応させてグルタミンに変換し、その
際生成したＡＤＰをピルビン酸キナーゼがフォスフオニ
ノールピルビン酸ト反応させてピルビン酸を生成させる
。このピルビン酸をピルビン酸脱水素酵素がアセチル燐
酸に変えその際に電子受容性化合物が還元される。この
電子受容性化合物に電子伝達剤と電子受容性指示薬を用
いることによって比色定量するのである。In the reaction reagent system for ammonia measurement employed in the analytical element of the present invention, glutamine synthetase reacts ammonium ions with glutamic acid to convert them into glutamine, and pyruvate kinase converts the ADP produced at this time into glutamine. Acid reaction produces pyruvic acid. Pyruvate dehydrogenase converts this pyruvate into acetyl phosphate, at which time the electron-accepting compound is reduced. Colorimetric determination is performed by using this electron-accepting compound with an electron transfer agent and an electron-accepting indicator.
このような反応系では試料中に予め含まれているピルビ
ン酸も検出されて誤差の原因となる。そこで、このピル
ビン酸を試料中のあるいは目的成分から生じたアンモニ
アからピルビン酸が生成される前にピルビン酸除去用の
酵素反応試薬組成物で他の物質に変えて誤差を除いてい
る。例えば、この酵素反応試薬組成物にピルビン酸デカ
ルホキシラーゼ（ＥＣ４，１，１，１）を用いた場合に
はピルビン酸をオキサロ酢酸に変えてアンモニア測定分
析用の反応系で反応しないようにしている。また、グル
タミン合成酵素、ピルビン酸キナーゼあるいはアンモニ
ア生成基質をアンモニアに変換する酵素の含有量を規制
することによって被分析物の発色速度を規制し、この発
色速度を求めることによって試料中に含まれているピル
ビン酸による発色の影響を排除している。In such a reaction system, pyruvic acid previously contained in the sample is also detected, causing errors. Therefore, the error is removed by replacing this pyruvic acid with another substance using an enzyme reaction reagent composition for removing pyruvic acid before pyruvic acid is generated from ammonia in the sample or from the target component. For example, if pyruvate decarboxylase (EC4,1,1,1) is used in this enzyme reaction reagent composition, pyruvate should be changed to oxaloacetate to prevent it from reacting in the reaction system for ammonia measurement and analysis. ing. In addition, the rate of color development of the analyte can be regulated by regulating the content of glutamine synthetase, pyruvate kinase, or an enzyme that converts an ammonia-producing substrate into ammonia, and by determining the rate of color development, the amount of analyte contained in the sample can be determined. This eliminates the influence of color development caused by pyruvic acid.
本発明の好ましい実施態様は次の通りである。Preferred embodiments of the invention are as follows.
実施態様１
（１）少なくともＬ−グルタミン合成酵素、ＡＴＰ、ピ
ルビン酸キナーゼ、フォスフオニノールピルビン酸、ピ
ルビン酸脱水素酵素、ＦＡＤ、ＴＰＰ。Embodiment 1 (1) At least L-glutamine synthetase, ATP, pyruvate kinase, phosphooninolpyruvate, pyruvate dehydrogenase, FAD, and TPP.
２価陽イオン、燐酸イオン、電子伝達剤及び電子受容性
指示薬からなる電子受容性指示薬組成物、体液中のアン
モニアを又はアンモニア生成基質を分析するためのアン
モニア又はアンモニア生成基質分析用試薬組成物。An electron-accepting indicator composition comprising a divalent cation, a phosphate ion, an electron transfer agent, and an electron-accepting indicator, and a reagent composition for analyzing ammonia or an ammonia-producing substrate for analyzing ammonia in body fluids or an ammonia-producing substrate.
実施態様２
（２）体液中のアンモニア生成基質を分析するための一
体型多層分析要素であって水平透過性支持体、Ｌ−グル
タミン合成酵素、ＡＴＰ、ピルビン酸キナーゼ、フォス
フオニノールピルビン酸、ピルビン酸脱水素酵素、ＦＡ
Ｄ、、ＴＰＰ、２価陽イオン、燐酸イオン、電子伝達剤
及び電子受容性指示薬からなる電子受容性指示薬組成物
とを含む１以上の試５層、並びに多孔性展開層から構成
され、かつ検体中のアンモニア生成基質をアンモニアに
変換する酵素を試薬層又は展開層に律速量含有する一体
型多層分析要素。Embodiment 2 (2) An integrated multilayer analytical element for analyzing ammonia-producing substrates in body fluids, comprising a horizontally permeable support, L-glutamine synthetase, ATP, pyruvate kinase, phosphooninol pyruvate, pyruvate dehydrogenase, FA
D. Consisting of one or more test layers containing TPP, a divalent cation, a phosphate ion, an electron-accepting indicator composition consisting of an electron transfer agent, and an electron-accepting indicator, and a porous spreading layer; An integrated multilayer analytical element whose reagent layer or development layer contains a rate-limiting amount of an enzyme that converts the ammonia-producing substrate contained therein into ammonia.
（３）体液中のアンモニア又はアンモニア生成基質を分
析するための一体型多層分析要素であって水平透過性支
持体、Ｌ−グルタミン合成酵素、ＡＴＰ、ピルビン酸キ
ナーゼ、フォスフオニノールピルビン酸、ピルビン酸脱
水素酵素、ＦＡＤ、ＴＰＰ、２価陽イオン、燐酸イオン
、電子伝達剤及び電子受容性指示薬からなる電子受容性
指示薬組成物とを含む１以上の試薬層、検体が試薬層へ
到達する以前にアナライトを含んだ検体が一様に通過し
得る検体中の内因性ピルビン酸除去層並びに多孔性展開
層を有する一体型多層分析要素。(3) An integrated multilayer analytical element for analyzing ammonia or ammonia-generating substrates in body fluids, comprising a horizontally permeable support, L-glutamine synthetase, ATP, pyruvate kinase, phosphooninol pyruvate, pyruvate. one or more reagent layers containing acid dehydrogenase, FAD, TPP, divalent cations, phosphate ions, an electron transfer agent, and an electron-accepting indicator composition consisting of an electron-accepting indicator, before the analyte reaches the reagent layer; An integrated multilayer analytical element having a layer for removing endogenous pyruvate in a sample and a porous spreading layer through which a sample containing an analyte can uniformly pass.
（４） Ｌ −グルタミン合成酵素、ピルビン酸キナー
ゼ及び該電子伝達剤がジアホラーゼであり、ジアホラー
ゼがいずれもバチルス・ステアロサーモフィルス由来の
耐熱性酵素であって、かつピルビン酸脱水素酵素がラク
トバチルス・デルブルエキイ由来の５０°Ｃ以下で熱的
に安定な酵素であることを（１）、（２）又は（３）に
記載の試薬組成物又は分析要素。(4) L-glutamine synthetase, pyruvate kinase, and the electron transfer agent are diaphorases, all diaphorases are thermostable enzymes derived from Bacillus stearothermophilus, and pyruvate dehydrogenase is derived from Lactobacillus stearothermophilus. - The reagent composition or analytical element according to (1), (2) or (3), which is an enzyme derived from A. delbruekii and is thermally stable at 50°C or lower.
実施例１ 〔アンモニア定量用一体型多層分析要素〕ゼ
ラチン下塗層を有する厚さ１８５μｍの無色透明ポリエ
チレンテレフタレート（ＰＥＴ）フィルム支持体の上に
下記組成のアンモニア測定用試薬層を乾燥後の層厚が約
１５μｍになるようにして塗布し乾燥した。Example 1 [Integrated multilayer analytical element for quantifying ammonia] Layer thickness after drying a reagent layer for measuring ammonia having the following composition on a colorless transparent polyethylene terephthalate (PET) film support having a gelatin undercoat layer and a thickness of 185 μm It was coated to a thickness of about 15 μm and dried.
アンモニア測定用試薬組成（１ボ当りの被覆量）Ｌ−グ
ルタミン酸 ０．０４４ ｇフォスフオ
ニノールピルビンＭＯ，７５ｇＡＴＰ
２ ｇ燐酸緩衝剤（ｐｌ＋７．０）
０．２ ｍｏｌこの試薬層の上に０．２ｇの
ノニルフェノキシポリエトキシエタノール（平均１０オ
キシ工チレン単位含有）、８ｇの二酸化チタン微粒子（
ルチル型、粒子径０．２５〜０，４０μｌ）および１ｇ
の脱イオンゼラチンの割合の水分散液を塗布し乾燥して
乾燥層厚が約５μｍの光遮蔽層を設けた。Reagent composition for ammonia measurement (coating amount per bottle) L-glutamic acid 0.044 g Phosphoninolpyruvin MO, 75 g ATP
2 g phosphate buffer (pl+7.0)
On top of this 0.2 mol reagent layer, 0.2 g of nonylphenoxy polyethoxyethanol (containing an average of 10 oxyethylene units) and 8 g of titanium dioxide fine particles (
Rutile type, particle size 0.25-0.40μl) and 1g
An aqueous dispersion of deionized gelatin in a proportion of .
次いで、この光遮蔽層の上に下記組成の内因性ピルビン
酸除去層を乾燥後の層厚が１０μｍになるようにして塗
布し乾燥した。Next, on this light shielding layer, an endogenous pyruvic acid removing layer having the following composition was applied and dried so that the layer thickness after drying was 10 μm.
ピルビン酸除去層用試薬組成（１ポ当りの被覆量）チア
ミンピロ燐酸（ＴＰＰ） ６００ ｍｇＦＡＤ
４００ ｍｇＭｇ”’
１００ ｍｇＮＴＢ
８００ ｍｇ脱イオン
ゼラチン １０ ｇ燐酸（ｐＨ７，０燐
酸緩衝液） ０．２ ｍｏｌ脱イオンゼ
ラチン ７ｇ更にこの内因性ピルビン
酸除去層上には、４ｇ（２，７ｇ／ｎｉ）の脱イオンゼ
ラチンと０ 、２ ｇ （１３５ｍｇ／ ｒｒｆ ）の
ノニルフェノキシポリエトキシエタノール（平均１０オ
キシ工チレン単位含有）を１００ｍｊ！の水に熔解させ
た溶液を塗布し乾燥して乾燥層厚約２μｍの接着層を設
けた。Reagent composition for pyruvic acid removal layer (coating amount per pot) Thiamine pyrophosphate (TPP) 600 mgFAD
400 mgMg"'
100mgNTB
800 mg deionized gelatin 10 g phosphoric acid (pH 7.0 phosphate buffer) 0.2 mol deionized gelatin 7 g Furthermore, on this endogenous pyruvate removal layer, 4 g (2.7 g/ni) of deionized gelatin and , 2 g (135 mg/rrf) of nonylphenoxy polyethoxyethanol (containing an average of 10 oxyethylene units) to 100 mj! A solution dissolved in water was applied and dried to form an adhesive layer with a dry thickness of about 2 μm.
一方、太さ約５００相当のＰＥＴ紡績糸からなる厚さ約
２５０−の水洗脱脂済トリコット編生地で、その片面を
６０秒グロー放電処理（１，６ｋＷ／ｒｒｆ ；酸素濃
度は減圧調整）した編物生地を用意した。On the other hand, it is a washed and degreased tricot knitted fabric with a thickness of about 250 mm made of PET spun yarn with a thickness equivalent to about 500 mm, and one side of which has been treated with glow discharge for 60 seconds (1.6 kW/rrf; oxygen concentration adjusted under reduced pressure). I prepared the dough.
次いで、接着層を水でほぼ・均一に湿潤させ、その上に
前述のトリコント編物生地をグロー放電処理時の電極側
表面を接着層に向きあわせてかさね全体を加圧ローラー
間を通過させ接着層に均一にラミネートして編物展開層
を設けた。Next, the adhesive layer is almost uniformly wetted with water, and the above-mentioned tricontact knitted fabric is placed on top of it, with the electrode side surface facing the adhesive layer at the time of glow discharge treatment, and the entire wrap is passed between pressure rollers to form the adhesive layer. A knitted fabric development layer was provided by uniformly laminating the fabric.
次いで、劇物展開層の上から下記組成の馬物展開層含浸
処理用水溶液を１ｒｒｆ当り １５０ｍｆｆ１の８１１
合で塗布し乾燥してアンモニア濃度定量用一体型多層分
析要素を調製した。Next, an aqueous solution for impregnating the horse animal development layer with the following composition was added to the top of the deleterious substance development layer at a rate of 150mff1 per 1rrf.
An integrated multilayer analytical element for ammonia concentration determination was prepared by coating and drying.
織物展開層含浸処理用水溶液の組成
水
１０００ ｇ
この分析要素を１５ｍｍ Ｘ １５ｍ＋ｎの正方形のチ
ップに裁断し、特開昭５７−６３４５２号公報に記載の
有機ポリマー製スライド枠に収めて性能評価のための測
定を行った。検体として、健康で正常な成人の血液をヘ
パリン採血遠心分離して得た血漿を用いた。Composition of aqueous solution for fabric spreading layer impregnation treatment 1000 g of water This analysis element was cut into square chips of 15 mm x 15 m+n and placed in an organic polymer slide frame as described in JP-A-57-63452 for performance evaluation. Measurements were made. Plasma obtained by centrifuging the blood of a healthy normal adult was used as a specimen.
この血漿に塩化アンモニウム及びピルビン酸カリウムを
各々添加し、各々のアンモニア及びピルビン酸濃度を溶
液法による標準測定法で測定しておいた。反射光学濃度
値の測定は、血漿検体を１０μＬ点着し、３７“Ｃで６
分インクベーションし、透明支持体側から要素内の発色
を波長５４０ ｎｍで反射測光したところ、下記の結果
を得た。Ammonium chloride and potassium pyruvate were each added to this plasma, and the respective ammonia and pyruvate concentrations were measured using a standard measurement method using a solution method. To measure the reflected optical density value, place 10μL of plasma sample and heat at 37"C for 6 hours.
After incubation for several minutes, the color development within the element was measured by reflection photometry at a wavelength of 540 nm from the transparent support side, and the following results were obtained.
実施例２〔タレアチニン定量用一体型多層分析要素］実
施例１と同様にして、透明支持体上に、クレアチニンデ
イミダーゼを速度限定量にした下記組成の第１試薬層、
第２試薬層の順に塗布し、さらに光遮蔽層、展開層を塗
布してタレアチニン分析用一体型多層分析要素を作製し
た。Example 2 [Integrated multilayer analytical element for quantifying taleatinin] In the same manner as in Example 1, a first reagent layer having the following composition containing a rate-limiting amount of creatinine deimidase was placed on a transparent support,
The second reagent layer was coated in this order, and then a light shielding layer and a developing layer were coated to produce an integrated multilayer analytical element for taleatinin analysis.
第２試薬層
タレアチニンデイミダーゼ １５００ Ｕ／ｎ（
Ｌ−グルタミン酸
フォスフオニノールピルビン酸
ＴＰ
脱イオンゼラチン
第１試薬層
チアミンピロ燐酸
ＡＤ
Ｍｇ”
ＴＢ
イミダゾール緩衝剤（ｐＨ７，０）
０．２１
０．０８
０．０３
ｍｏｌｅ／ボ
ｍｏｌｅ／ボ
ｍｏｌｅ／ボ
ｇ／ボ
０．００１ｍｏｌｅ／ ｒｄ
Ｏ，０９ｍｍｏｌｅ／ ｒｄ
Ｏ，０５ｕ １Ｗｏｌｅ／ ｎｊ
ｏ、０８ ｍｏｌｅ／ｍ
０、１ ｍｏｌｅ／ ｒｒｆ
脱イオンゼラチン ７ ｇ／ボこの
ようにして調整されたタレアチニン定量用一体型多層分
析要素は、実施例１と同様にして打機ポリマー製スライ
ド枠に収めて性能評価のための測定を行った。検体とし
て、健康で正常な成人の血液をヘパリン採血し、遠心分
離して得た血漿を用いた。得られた血漿にクレアチニン
を添加し、下表のクレアチニン濃度の血漿を調製した。Second reagent layer Taleatinine deimidase 1500 U/n (
L-glutamate phosphooninol pyruvate TP Deionized gelatin 1st reagent layer Thiamine pyrophosphate AD Mg” TB Imidazole buffer (pH 7,0) 0.21 0.08 0.03 mole/bo mole/bo mole/bog /Bo 0.001mole/rd O,09mmole/rdO,05u 1Wole/njo,08 mole/m 0,1 mole/rrf Deionized gelatin 7 g/Bo Integrated multilayer for the determination of taleatinin prepared in this manner The analysis element was placed in a perforated polymer slide frame in the same manner as in Example 1, and measurements for performance evaluation were performed.As a sample, blood from a healthy, normal adult was collected with heparin and then centrifuged. Creatinine was added to the obtained plasma to prepare plasma having the creatinine concentration shown in the table below.
血漿のクレアチニン濃度とピルビン酸濃度を溶液法によ
る標準測定法で測定しておいた。反射光学濃度値の測定
は、血漿検体を１０μＬ点着し、３７°Ｃでインクヘー
ションしながら、点着後２分後と５分後の時点で透明支
持体側から要素内の発色を波長５４０ ｎｎ＋で反射測
光した。得られた５分後と２分後の反射光学濃度値の差
（下表ではΔＯＤ、（５分−２分）と記す）を求めた。Plasma creatinine and pyruvate concentrations were measured using a standard solution method. To measure the reflected optical density value, 10 μL of plasma sample was spotted, and while inking at 37°C, the color development inside the element was detected from the transparent support side at a wavelength of 540 at 2 and 5 minutes after spotting. Reflection photometry was performed using nn+. The difference between the obtained reflected optical density values after 5 minutes and after 2 minutes (denoted as ΔOD, (5 minutes - 2 minutes) in the table below) was determined.
なお、反射光学濃度値の測定は富士写真フィルム■製Ｆ
ＤＣ５０００アナライザーを用いて行った。The reflective optical density value was measured using Fuji Photo Film F.
This was done using a DC5000 analyzer.
評価結果を下記に示す。The evaluation results are shown below.
〔発明の効果］
本発明の分析要素は熱安定性が良好であり、試料に含ま
れているピルビン酸の影古を排除してアンモニア及びそ
の測定試薬系を利用した尿素窒素、クレアチニン等を正
確に定量することができる。[Effect of the invention] The analytical element of the present invention has good thermal stability, eliminates the influence of pyruvic acid contained in the sample, and accurately measures urea nitrogen, creatinine, etc. using ammonia and its measurement reagent system. can be quantified.
Claims (2)
ピルビン酸デヒドロゲナーゼ、及び電子受容性指示薬組
成物を含むアンモニア分析試薬組成物(1) L-glutamine synthetase, pyruvate kinase,
Ammonia analysis reagent composition comprising pyruvate dehydrogenase and an electron-accepting indicator composition
の試薬層及び展開層がこの順に積層されている一体型多
層分析要素であって、前記試薬層にＬ−グルタミン合成
酵素、ピルビン酸キナーゼ、ピルビン酸デヒドロゲナー
ゼ、及び電子受容性指示組成物を含むアンモニア分析試
薬組成物が含有されていることを特徴とするアンモニア
又はアンモニア生成基質分析用一体型多層分析要素(2) An integrated multilayer analytical element in which at least one reagent layer and a development layer are laminated in this order on a horizontally transparent light-transparent support, the reagent layer containing L-glutamine synthetase, pyruvin An integrated multilayer analytical element for analyzing ammonia or ammonia-producing substrates, characterized in that it contains an ammonia analytical reagent composition comprising acid kinase, pyruvate dehydrogenase, and an electron-accepting indicator composition.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP27163589A JPH03133396A (en) | 1989-10-20 | 1989-10-20 | Reagent composition for ammonia analysis and one-body type multilayer analyzing element containing thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP27163589A JPH03133396A (en) | 1989-10-20 | 1989-10-20 | Reagent composition for ammonia analysis and one-body type multilayer analyzing element containing thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH03133396A true JPH03133396A (en) | 1991-06-06 |
Family
ID=17502814
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP27163589A Pending JPH03133396A (en) | 1989-10-20 | 1989-10-20 | Reagent composition for ammonia analysis and one-body type multilayer analyzing element containing thereof |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH03133396A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006095757A1 (en) * | 2005-03-09 | 2006-09-14 | Fujifilm Corporation | Multilayer analytical element |
| WO2009136497A1 (en) * | 2008-05-09 | 2009-11-12 | パナソニック株式会社 | Method, device and apparatus for measuring creatinine concentration, and method, device and apparatus for measuring salt content in urine using same |
-
1989
- 1989-10-20 JP JP27163589A patent/JPH03133396A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006095757A1 (en) * | 2005-03-09 | 2006-09-14 | Fujifilm Corporation | Multilayer analytical element |
| JP4874953B2 (en) * | 2005-03-09 | 2012-02-15 | 富士フイルム株式会社 | Multi-layer analysis element |
| WO2009136497A1 (en) * | 2008-05-09 | 2009-11-12 | パナソニック株式会社 | Method, device and apparatus for measuring creatinine concentration, and method, device and apparatus for measuring salt content in urine using same |
| JP4430134B2 (en) * | 2008-05-09 | 2010-03-10 | パナソニック株式会社 | Creatinine concentration measuring method, measuring device and measuring apparatus, and urinary salt content measuring method, measuring device and measuring apparatus using them |
| CN101883982A (en) * | 2008-05-09 | 2010-11-10 | 松下电器产业株式会社 | Method, device and apparatus for measuring the concentration of creatinine, and method, device and apparatus for measuring the amount of salt in urine using the same |
| US7879617B2 (en) | 2008-05-09 | 2011-02-01 | Panasonic Corporation | Method, device and apparatus for measuring the concentration of creatinine, and method, device and apparatus for measuring the amount of salt in urine using the same |
| JPWO2009136497A1 (en) * | 2008-05-09 | 2011-09-08 | パナソニック株式会社 | Creatinine concentration measuring method, measuring device and measuring apparatus, and urinary salt content measuring method, measuring device and measuring apparatus using them |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP2509429B2 (en) | One-step test of aspartate aminotransferase | |
| EP0200540B1 (en) | Analytical element and method for determination of creatine kinase isoenzyme | |
| US4503145A (en) | Quantitative analysis film | |
| JPH0550275B2 (en) | ||
| US4812399A (en) | Analytical element and method for the determination of creatinine or creatine | |
| US4897347A (en) | Multilayer analysis film for analyzing transaminase | |
| CA1227113A (en) | Enzymatic atp/fmn assay | |
| JPH0332360B2 (en) | ||
| JPH0526865A (en) | Measuring method using dry process analysis element and dry process analysis element | |
| US6130054A (en) | Test strip for creatine kinase activity measurement | |
| US4176008A (en) | Method, composition and element for the detection of nitrogen-containing compounds | |
| US4927752A (en) | Support used in bioluminescent dosing of enzymes, substrates or enzymatic inhibitors | |
| CA2306554A1 (en) | Uric acid assay device with stabilized uricase reagent composition | |
| US4788140A (en) | Analytical element containing photosensitive compound and filter layer and method of use | |
| JP2513509B2 (en) | Urea analysis reagent composition and integrated multilayer analytical element containing the same | |
| JPH03133396A (en) | Reagent composition for ammonia analysis and one-body type multilayer analyzing element containing thereof | |
| US4939085A (en) | Oxidized coenzyme-containing dry analytical element | |
| EP0200541B1 (en) | Immunochemical method and analytical composition and element for determination of creatine kinase-mb | |
| JPH0394698A (en) | Integrated multi-layer analytic element containing ammonia determination reagent composition | |
| JPH0391497A (en) | Integrated multi-layer analytic element containing reagent composition for determination of ammonium ion | |
| JP3493411B2 (en) | Reagent composition for potassium ion measurement and test piece | |
| EP0989190B1 (en) | Dry analytical element for quantitative analysis of creatine kinase MB isozyme | |
| JPH0481438B2 (en) | ||
| US5998158A (en) | Glucose free, stable dry analytical element for quantitative analysis of creatine kinase MB isozyme | |
| JPH0595798A (en) | Determination of pyruvic acid and determination of bio-component using the method |