JPH037906B2 - - Google Patents
Info
- Publication number
- JPH037906B2 JPH037906B2 JP5456784A JP5456784A JPH037906B2 JP H037906 B2 JPH037906 B2 JP H037906B2 JP 5456784 A JP5456784 A JP 5456784A JP 5456784 A JP5456784 A JP 5456784A JP H037906 B2 JPH037906 B2 JP H037906B2
- Authority
- JP
- Japan
- Prior art keywords
- test solution
- ligand
- reagent system
- test
- change
- 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
Links
- 239000012085 test solution Substances 0.000 claims description 134
- 239000003153 chemical reaction reagent Substances 0.000 claims description 129
- 239000003446 ligand Substances 0.000 claims description 120
- 230000008859 change Effects 0.000 claims description 48
- 238000000034 method Methods 0.000 claims description 43
- 230000005284 excitation Effects 0.000 claims description 25
- 238000005259 measurement Methods 0.000 claims description 19
- 238000004040 coloring Methods 0.000 claims description 16
- 238000010521 absorption reaction Methods 0.000 claims description 8
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 238000005558 fluorometry Methods 0.000 claims description 3
- 238000012360 testing method Methods 0.000 description 45
- 238000006243 chemical reaction Methods 0.000 description 38
- 239000000523 sample Substances 0.000 description 35
- BAWFJGJZGIEFAR-NNYOXOHSSA-N NAD zwitterion 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](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-N 0.000 description 27
- 229950006238 nadide Drugs 0.000 description 27
- 239000000243 solution Substances 0.000 description 25
- NMWCVZCSJHJYFW-UHFFFAOYSA-M sodium;3,5-dichloro-2-hydroxybenzenesulfonate Chemical compound [Na+].OC1=C(Cl)C=C(Cl)C=C1S([O-])(=O)=O NMWCVZCSJHJYFW-UHFFFAOYSA-M 0.000 description 24
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 22
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 21
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 20
- 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 19
- 239000012086 standard solution Substances 0.000 description 18
- 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 17
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 17
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 16
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 15
- 239000000126 substance Substances 0.000 description 15
- 239000008103 glucose Substances 0.000 description 14
- 235000018102 proteins Nutrition 0.000 description 14
- 102000004169 proteins and genes Human genes 0.000 description 14
- 108090000623 proteins and genes Proteins 0.000 description 14
- JORABGDXCIBAFL-UHFFFAOYSA-M iodonitrotetrazolium chloride Chemical compound [Cl-].C1=CC([N+](=O)[O-])=CC=C1N1[N+](C=2C=CC(I)=CC=2)=NC(C=2C=CC=CC=2)=N1 JORABGDXCIBAFL-UHFFFAOYSA-M 0.000 description 13
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 12
- DDRJAANPRJIHGJ-UHFFFAOYSA-N creatinine Chemical compound CN1CC(=O)NC1=N DDRJAANPRJIHGJ-UHFFFAOYSA-N 0.000 description 12
- 235000012000 cholesterol Nutrition 0.000 description 11
- 230000003993 interaction Effects 0.000 description 11
- 239000002245 particle Substances 0.000 description 11
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 10
- CVSVTCORWBXHQV-UHFFFAOYSA-N creatine Chemical compound NC(=[NH2+])N(C)CC([O-])=O CVSVTCORWBXHQV-UHFFFAOYSA-N 0.000 description 10
- 239000011777 magnesium Substances 0.000 description 10
- 229910052749 magnesium Inorganic materials 0.000 description 10
- 239000000203 mixture Substances 0.000 description 10
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 9
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 9
- 102000003855 L-lactate dehydrogenase Human genes 0.000 description 9
- 108700023483 L-lactate dehydrogenases Proteins 0.000 description 9
- LEHOTFFKMJEONL-UHFFFAOYSA-N Uric Acid Chemical compound N1C(=O)NC(=O)C2=C1NC(=O)N2 LEHOTFFKMJEONL-UHFFFAOYSA-N 0.000 description 9
- TVWHNULVHGKJHS-UHFFFAOYSA-N Uric acid Natural products N1C(=O)NC(=O)C2NC(=O)NC21 TVWHNULVHGKJHS-UHFFFAOYSA-N 0.000 description 9
- 229930195712 glutamate Natural products 0.000 description 9
- 229940116269 uric acid Drugs 0.000 description 9
- KPGXRSRHYNQIFN-UHFFFAOYSA-N 2-oxoglutaric acid Chemical compound OC(=O)CCC(=O)C(O)=O KPGXRSRHYNQIFN-UHFFFAOYSA-N 0.000 description 8
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 8
- DRBBFCLWYRJSJZ-UHFFFAOYSA-N N-phosphocreatine Chemical compound OC(=O)CN(C)C(=N)NP(O)(O)=O DRBBFCLWYRJSJZ-UHFFFAOYSA-N 0.000 description 8
- 239000011575 calcium Substances 0.000 description 8
- 229910052791 calcium Inorganic materials 0.000 description 8
- 238000004737 colorimetric analysis Methods 0.000 description 8
- FSYKKLYZXJSNPZ-UHFFFAOYSA-N sarcosine Chemical compound C[NH2+]CC([O-])=O FSYKKLYZXJSNPZ-UHFFFAOYSA-N 0.000 description 8
- 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 7
- 108010088751 Albumins Proteins 0.000 description 7
- 102000009027 Albumins Human genes 0.000 description 7
- 108060003951 Immunoglobulin Proteins 0.000 description 7
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 description 7
- 238000002835 absorbance Methods 0.000 description 7
- 102000018358 immunoglobulin Human genes 0.000 description 7
- 239000008363 phosphate buffer Substances 0.000 description 7
- 238000002834 transmittance Methods 0.000 description 7
- 241000283690 Bos taurus Species 0.000 description 6
- NBSCHQHZLSJFNQ-GASJEMHNSA-N D-Glucose 6-phosphate Chemical compound OC1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H](O)[C@H]1O NBSCHQHZLSJFNQ-GASJEMHNSA-N 0.000 description 6
- VFRROHXSMXFLSN-UHFFFAOYSA-N Glc6P Natural products OP(=O)(O)OCC(O)C(O)C(O)C(O)C=O VFRROHXSMXFLSN-UHFFFAOYSA-N 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 230000005540 biological transmission Effects 0.000 description 6
- 239000004202 carbamide Substances 0.000 description 6
- 229940109239 creatinine Drugs 0.000 description 6
- 229940027941 immunoglobulin g Drugs 0.000 description 6
- 239000000725 suspension Substances 0.000 description 6
- XTWYTFMLZFPYCI-KQYNXXCUSA-N 5'-adenylphosphoric acid Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O XTWYTFMLZFPYCI-KQYNXXCUSA-N 0.000 description 5
- XTWYTFMLZFPYCI-UHFFFAOYSA-N Adenosine diphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(O)=O)C(O)C1O XTWYTFMLZFPYCI-UHFFFAOYSA-N 0.000 description 5
- -1 Copper tartrate Sodium tartrate Trilithium acetate Chemical compound 0.000 description 5
- 102000004420 Creatine Kinase Human genes 0.000 description 5
- 108010042126 Creatine kinase Proteins 0.000 description 5
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 5
- 229910019142 PO4 Inorganic materials 0.000 description 5
- 108010042687 Pyruvate Oxidase Proteins 0.000 description 5
- 239000002253 acid Substances 0.000 description 5
- DZBUGLKDJFMEHC-UHFFFAOYSA-N acridine Chemical compound C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 description 5
- 229960003624 creatine Drugs 0.000 description 5
- 239000006046 creatine Substances 0.000 description 5
- 108010074605 gamma-Globulins Proteins 0.000 description 5
- 230000035699 permeability Effects 0.000 description 5
- 210000002966 serum Anatomy 0.000 description 5
- RLFWWDJHLFCNIJ-UHFFFAOYSA-N 4-aminoantipyrine Chemical compound CN1C(C)=C(N)C(=O)N1C1=CC=CC=C1 RLFWWDJHLFCNIJ-UHFFFAOYSA-N 0.000 description 4
- 102000005548 Hexokinase Human genes 0.000 description 4
- 108700040460 Hexokinases Proteins 0.000 description 4
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 4
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 4
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 4
- 108010077895 Sarcosine Proteins 0.000 description 4
- 108010060059 Sarcosine Oxidase Proteins 0.000 description 4
- 102000008118 Sarcosine oxidase Human genes 0.000 description 4
- 229960003767 alanine Drugs 0.000 description 4
- 229940009098 aspartate Drugs 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 230000003287 optical effect Effects 0.000 description 4
- 239000010452 phosphate Substances 0.000 description 4
- 229940043230 sarcosine Drugs 0.000 description 4
- 108010029731 6-phosphogluconolactonase Proteins 0.000 description 3
- 102100031126 6-phosphogluconolactonase Human genes 0.000 description 3
- 108010089254 Cholesterol oxidase Proteins 0.000 description 3
- 108010077078 Creatinase Proteins 0.000 description 3
- 108010015776 Glucose oxidase Proteins 0.000 description 3
- 239000004366 Glucose oxidase Substances 0.000 description 3
- 108010018962 Glucosephosphate Dehydrogenase Proteins 0.000 description 3
- JVTAAEKCZFNVCJ-REOHCLBHSA-N L-lactic acid Chemical compound C[C@H](O)C(O)=O JVTAAEKCZFNVCJ-REOHCLBHSA-N 0.000 description 3
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 3
- WMFOQBRAJBCJND-UHFFFAOYSA-M Lithium hydroxide Chemical compound [Li+].[OH-] WMFOQBRAJBCJND-UHFFFAOYSA-M 0.000 description 3
- AUNGANRZJHBGPY-SCRDCRAPSA-N Riboflavin Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-SCRDCRAPSA-N 0.000 description 3
- 108010055297 Sterol Esterase Proteins 0.000 description 3
- 102000000019 Sterol Esterase Human genes 0.000 description 3
- 239000013504 Triton X-100 Substances 0.000 description 3
- 229920004890 Triton X-100 Polymers 0.000 description 3
- 108010092464 Urate Oxidase Proteins 0.000 description 3
- 108010046334 Urease Proteins 0.000 description 3
- LIPOUNRJVLNBCD-UHFFFAOYSA-N acetyl dihydrogen phosphate Chemical compound CC(=O)OP(O)(O)=O LIPOUNRJVLNBCD-UHFFFAOYSA-N 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 239000012916 chromogenic reagent Substances 0.000 description 3
- 239000000975 dye Substances 0.000 description 3
- 229940116332 glucose oxidase Drugs 0.000 description 3
- 235000019420 glucose oxidase Nutrition 0.000 description 3
- 229940116871 l-lactate Drugs 0.000 description 3
- CPBJMKMKNCRKQB-UHFFFAOYSA-N 3,3-bis(4-hydroxy-3-methylphenyl)-2-benzofuran-1-one Chemical compound C1=C(O)C(C)=CC(C2(C3=CC=CC=C3C(=O)O2)C=2C=C(C)C(O)=CC=2)=C1 CPBJMKMKNCRKQB-UHFFFAOYSA-N 0.000 description 2
- GOLORTLGFDVFDW-UHFFFAOYSA-N 3-(1h-benzimidazol-2-yl)-7-(diethylamino)chromen-2-one Chemical compound C1=CC=C2NC(C3=CC4=CC=C(C=C4OC3=O)N(CC)CC)=NC2=C1 GOLORTLGFDVFDW-UHFFFAOYSA-N 0.000 description 2
- 108010017192 4-hydroxy-4-methyl-2-oxoglutarate aldolase Proteins 0.000 description 2
- IKHGUXGNUITLKF-UHFFFAOYSA-N Acetaldehyde Chemical compound CC=O IKHGUXGNUITLKF-UHFFFAOYSA-N 0.000 description 2
- 102100029589 Acylpyruvase FAHD1, mitochondrial Human genes 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 108010000659 Choline oxidase Proteins 0.000 description 2
- QNAYBMKLOCPYGJ-UHFFFAOYSA-N D-alpha-Ala Natural products CC([NH3+])C([O-])=O QNAYBMKLOCPYGJ-UHFFFAOYSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- QNAYBMKLOCPYGJ-UWTATZPHSA-N L-Alanine Natural products C[C@@H](N)C(O)=O QNAYBMKLOCPYGJ-UWTATZPHSA-N 0.000 description 2
- 108090001060 Lipase Proteins 0.000 description 2
- 102000004882 Lipase Human genes 0.000 description 2
- 239000004367 Lipase Substances 0.000 description 2
- UFWIBTONFRDIAS-UHFFFAOYSA-N Naphthalene Chemical compound C1=CC=CC2=CC=CC=C21 UFWIBTONFRDIAS-UHFFFAOYSA-N 0.000 description 2
- 108010069823 Oxaloacetate decarboxylase Proteins 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- LOUPRKONTZGTKE-WZBLMQSHSA-N Quinine Chemical compound C([C@H]([C@H](C1)C=C)C2)C[N@@]1[C@@H]2[C@H](O)C1=CC=NC2=CC=C(OC)C=C21 LOUPRKONTZGTKE-WZBLMQSHSA-N 0.000 description 2
- SMWDFEZZVXVKRB-UHFFFAOYSA-N Quinoline Chemical compound N1=CC=CC2=CC=CC=C21 SMWDFEZZVXVKRB-UHFFFAOYSA-N 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 108090000340 Transaminases Proteins 0.000 description 2
- PNNCWTXUWKENPE-UHFFFAOYSA-N [N].NC(N)=O Chemical compound [N].NC(N)=O PNNCWTXUWKENPE-UHFFFAOYSA-N 0.000 description 2
- 235000004279 alanine Nutrition 0.000 description 2
- POJWUDADGALRAB-UHFFFAOYSA-N allantoin Chemical compound NC(=O)NC1NC(=O)NC1=O POJWUDADGALRAB-UHFFFAOYSA-N 0.000 description 2
- MWPLVEDNUUSJAV-UHFFFAOYSA-N anthracene Chemical compound C1=CC=CC2=CC3=CC=CC=C3C=C21 MWPLVEDNUUSJAV-UHFFFAOYSA-N 0.000 description 2
- RWZYAGGXGHYGMB-UHFFFAOYSA-N anthranilic acid Chemical compound NC1=CC=CC=C1C(O)=O RWZYAGGXGHYGMB-UHFFFAOYSA-N 0.000 description 2
- VBRNLOQCBCPPHL-UHFFFAOYSA-N calmagite Chemical compound CC1=CC=C(O)C(N=NC=2C3=CC=CC=C3C(=CC=2O)S(O)(=O)=O)=C1 VBRNLOQCBCPPHL-UHFFFAOYSA-N 0.000 description 2
- 150000001840 cholesterol esters Chemical class 0.000 description 2
- 239000002131 composite material Substances 0.000 description 2
- 239000012954 diazonium Substances 0.000 description 2
- 150000001989 diazonium salts Chemical class 0.000 description 2
- GNGACRATGGDKBX-UHFFFAOYSA-N dihydroxyacetone phosphate Chemical compound OCC(=O)COP(O)(O)=O GNGACRATGGDKBX-UHFFFAOYSA-N 0.000 description 2
- 238000000295 emission spectrum Methods 0.000 description 2
- DEFVIWRASFVYLL-UHFFFAOYSA-N ethylene glycol bis(2-aminoethyl)tetraacetic acid Chemical compound OC(=O)CN(CC(O)=O)CCOCCOCCN(CC(O)=O)CC(O)=O DEFVIWRASFVYLL-UHFFFAOYSA-N 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 229940072221 immunoglobulins Drugs 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
- 235000019421 lipase Nutrition 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- VLAPMBHFAWRUQP-UHFFFAOYSA-L molybdic acid Chemical compound O[Mo](O)(=O)=O VLAPMBHFAWRUQP-UHFFFAOYSA-L 0.000 description 2
- 238000004848 nephelometry Methods 0.000 description 2
- 230000009871 nonspecific binding Effects 0.000 description 2
- 238000006395 oxidase reaction Methods 0.000 description 2
- 235000021317 phosphate Nutrition 0.000 description 2
- 239000001103 potassium chloride Substances 0.000 description 2
- 235000011164 potassium chloride Nutrition 0.000 description 2
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 2
- YGSDEFSMJLZEOE-UHFFFAOYSA-N salicylic acid Chemical compound OC(=O)C1=CC=CC=C1O YGSDEFSMJLZEOE-UHFFFAOYSA-N 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 239000012488 sample solution Substances 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 229940083618 sodium nitroprusside Drugs 0.000 description 2
- 238000010998 test method Methods 0.000 description 2
- 102000014898 transaminase activity proteins Human genes 0.000 description 2
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- YHDUQRGEHNIMJY-DFWYDOINSA-N (2s)-2-aminopentanedioic acid;2-oxopropanoic acid Chemical compound CC(=O)C(O)=O.OC(=O)[C@@H](N)CCC(O)=O YHDUQRGEHNIMJY-DFWYDOINSA-N 0.000 description 1
- GOCCREQJUBABAL-UHFFFAOYSA-N 2,2-dihydroxyacetic acid Chemical compound OC(O)C(O)=O GOCCREQJUBABAL-UHFFFAOYSA-N 0.000 description 1
- KQCMTOWTPBNWDB-UHFFFAOYSA-N 2,4-dichloroaniline Chemical compound NC1=CC=C(Cl)C=C1Cl KQCMTOWTPBNWDB-UHFFFAOYSA-N 0.000 description 1
- WXTMDXOMEHJXQO-UHFFFAOYSA-N 2,5-dihydroxybenzoic acid Chemical compound OC(=O)C1=CC(O)=CC=C1O WXTMDXOMEHJXQO-UHFFFAOYSA-N 0.000 description 1
- VXARDGMDMUMINC-UHFFFAOYSA-N 2-(2-phenylethenyl)pyridin-3-amine Chemical compound NC1=CC=CN=C1C=CC1=CC=CC=C1 VXARDGMDMUMINC-UHFFFAOYSA-N 0.000 description 1
- XNCSCQSQSGDGES-UHFFFAOYSA-N 2-[2-[bis(carboxymethyl)amino]propyl-(carboxymethyl)amino]acetic acid Chemical compound OC(=O)CN(CC(O)=O)C(C)CN(CC(O)=O)CC(O)=O XNCSCQSQSGDGES-UHFFFAOYSA-N 0.000 description 1
- QLKGUVGAXDXFFW-UHFFFAOYSA-M 2-hydroxyethyl(trimethyl)azanium;acetate Chemical compound CC([O-])=O.C[N+](C)(C)CCO QLKGUVGAXDXFFW-UHFFFAOYSA-M 0.000 description 1
- MAFGWKUWMUNBRW-UHFFFAOYSA-N 2-oxobutanedioic acid Chemical compound C(=O)(C(=O)O)CC(=O)O.C(=O)(C(=O)O)CC(=O)O.C(=O)(C(=O)O)CC(=O)O MAFGWKUWMUNBRW-UHFFFAOYSA-N 0.000 description 1
- YLYRAZVVSJOILG-UHFFFAOYSA-N 2-oxobutanedioic acid;2-oxopentanedioic acid Chemical compound OC(=O)CC(=O)C(O)=O.OC(=O)CCC(=O)C(O)=O YLYRAZVVSJOILG-UHFFFAOYSA-N 0.000 description 1
- FZJGROBMVACKNO-UHFFFAOYSA-N 2-oxopentanedioic acid;2-oxopropanoic acid Chemical compound CC(=O)C(O)=O.OC(=O)CCC(=O)C(O)=O FZJGROBMVACKNO-UHFFFAOYSA-N 0.000 description 1
- BCHZICNRHXRCHY-UHFFFAOYSA-N 2h-oxazine Chemical compound N1OC=CC=C1 BCHZICNRHXRCHY-UHFFFAOYSA-N 0.000 description 1
- KKAJSJJFBSOMGS-UHFFFAOYSA-N 3,6-diamino-10-methylacridinium chloride Chemical compound [Cl-].C1=C(N)C=C2[N+](C)=C(C=C(N)C=C3)C3=CC2=C1 KKAJSJJFBSOMGS-UHFFFAOYSA-N 0.000 description 1
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- LOUPRKONTZGTKE-UHFFFAOYSA-N cinchonine Natural products C1C(C(C2)C=C)CCN2C1C(O)C1=CC=NC2=CC=C(OC)C=C21 LOUPRKONTZGTKE-UHFFFAOYSA-N 0.000 description 1
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- 238000000502 dialysis Methods 0.000 description 1
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- KBYOBAICCHNMNJ-UHFFFAOYSA-L diperchloryloxymercury Chemical compound [Hg+2].[O-]Cl(=O)(=O)=O.[O-]Cl(=O)(=O)=O KBYOBAICCHNMNJ-UHFFFAOYSA-L 0.000 description 1
- KRTVZTOXNMQDSQ-UHFFFAOYSA-M dipotassium cyanide hydroxide Chemical compound [C-]#N.[K+].[OH-].[K+] KRTVZTOXNMQDSQ-UHFFFAOYSA-M 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
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- 150000002430 hydrocarbons Chemical class 0.000 description 1
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- 238000010561 standard procedure Methods 0.000 description 1
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- 235000021286 stilbenes Nutrition 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
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Description
本発明は改良螢光測定法に関する。
臨床医学診断分野における普通の非同位元素分
析法には以後配位子とよばれる臨床的に重要物質
の分光又は螢光測定法がある。この方法は非常に
敏感で特殊であり、光学的性質、即ち試験溶液中
の特定配位子の存在で生ずる試験溶液の透光性又
は螢光性変化の測定に基づく。
分光試験における試験溶液内の測定される配位
子と配位子に特定の試薬系間の相互作用は試験溶
液の透光性に測定できる変化を生ずる。透過性の
変化とは既知強さの光線が試験溶液をとおつた時
の特定波長帯内の試験溶液によつて吸収された又
は散乱された光量をいう。試験溶液の透過性の変
化は既知強さをもつ単色光を試験溶液にとおし透
過又は散乱光強さの入射光強さに対する比率を検
査して測定する。試験溶液の透過性の検出できる
変化を生成するため殆んど全配位子が特定波長の
エネルギーを吸収するか又は試験溶液中特定試薬
系と相互作用するかいづれかである事実は多数の
特殊分光測定法の開発となつている。試験溶液中
の配位子測定として試験溶液の透光性の変化測定
に基づく分光測定には例えば試験溶液の色変化に
よる比色試験および試験溶液内の濁度変化による
濁度又は比濁試験がある。比色試験では試験溶液
の透光性の変化は一般に試験溶液の吸光度といい
測定する配位子と配位子に特定の試薬系の相互作
用による試験溶液の色の変化によるのである。試
験溶液の吸光度はその中の配位子濃度に比例す
る。比色試験は試験溶液の透光性、特に色の検出
できる変化を生成するため試験溶液中で問題の特
定配位子と相互作用しうる発色試薬系を利用す
る。特定配位子測定に便利な発色試薬系が多数開
発されており市販されている。比濁試験の原理は
試験溶液を光が通過する際特定物質により散乱さ
れる又は阻止される光量の測定にある。比濁試験
において問題の配位子は試験溶液中に粒子の懸濁
物をつくるため配位子に特定の試薬系と相互に作
用する。既知強さをもつ光線が試験溶液をとおる
際配位子と試薬系の相互作用で生じた粒子懸濁液
は入射光を阻止又は散乱して試験溶液をとおる光
強さを減少する。濁度測定における透光性変化は
試験溶液をとおる光強さの減少をいい、粒子懸濁
物によつて散乱又は阻止された入射光量に比例し
存在する粒子数とこの粒子の断面積による。比濁
試験は問題の配位子が配位子に特定の試薬系と相
互作用して試験溶液中粒子懸濁物を生成する点で
濁度試験と同じである。比濁試験では試験溶液の
透光性変化も粒子懸濁物によつて散乱され又は阻
止される入射光量に比例にするが、試験溶液をと
おる光強さを測定する濁度試験とちがつて試験溶
液への光入射角における散乱又は阻止された光を
測定する。したがつて比濁試験では透光性の変化
は試験溶液に入る光と入射光に対する角で散乱さ
れた光との強さの差をいう。濁度試験と比濁試験
は血液、尿、脊髄液等の分析で有効な発色試薬系
のないため比較する比色試験のない蛋白質の様な
配位子測定に使われる。ヨーエとクリムマンはニ
ユーヨーク市ウイレイ アンド サンズ社編
Photoelectric Chemical Analysis巻：
Nephelometry（1929）に種々の比濁試験につい
て記載している。
一般に螢光試験においては試験溶液中の配位子
は化学的に又は免疫学的に螢光性複合物又は共役
物に変えられて試験溶液の螢光性に検知できる変
化を生ずる。試験溶液の螢光性変化は生成した螢
光性複合物又は共役物を螢光体の励起波長帯内の
波長をもつ単色光で励起し螢光体の放射波長帯内
の波長をもつ放射光強さを測定してえられる。放
射光の螢光強さは配位子濃度に比例する。しかし
試験溶液によつて放出された螢光強さは測定する
配位子が試料中にある蛋白質又はりん酸塩の様な
非螢光性妨害物と複合する場合又は測定する配位
子を含む試料がフイルターとして働らくに十分な
色をもつて放射螢光強さを減少する場合抑制され
る。螢光測定試験の感度と特異性を最大とするた
めこれらの抑制要素があるならば分析前に非螢光
性妨害物又は色生成物質を除去するか又は試料の
第２部分に加えた内部標準を用いてこの要素の存
在を補償しまた内部標準をもつ試料を用いて全試
験法を行なうかいづれかでこれら抑制要素をなく
す必要があることがよくわかる。
本発明の目的は試験溶液によつて放射された螢
光強さが測定する配位子と配位子の存在で試験溶
液の透光性の変化を生じうる試薬系との相互作用
により生ずる透光性変化に比例する様な試験溶液
中の配位子の螢光測定法を提供するものである。
また本発明の目的は試験溶液中の配位子濃度の分
光測定か螢光測定のいづれかを利用できる新規の
試薬組成物の提供にある。
本発明は配位子を含むと思われる試料中の配位
子測定法に関するもので、その方法は上記試料、
螢光体の有効量および測定する配位子の存在で螢
光体の励起および（又は）放射波長帯となる波長
帯内で試験溶液の透光性を変えうる試薬系の有効
量を混合して試験溶液を生成し、試験溶液を螢光
体の励起波長帯内の波長をもつ光で照射した後試
験溶液によつて放射された螢光強さを試料中の配
位子濃度の尺度として測定することより成る。
本発明は更に配位子を含む溶液の透光性変化を
生じうる試薬系の有効量および試薬と配位子を含
む溶液の透光性変化と関聯した波長帯と重複する
励起および（又は）放射波長帯をもつ螢光体の有
効量より成る上記配位子を含むと思われる料中の
配位子螢光測定用新規試薬組成物に関する。
本発明の方法により生物学的試料中の配位子は
試験溶液中に上記試料、試薬系有効量および螢光
体有効量を混合して測定される。試験溶液を螢光
体の励起波長帯内の波長をもつ光で照射し試験溶
液によつて放出された螢光強さを試料中の配位子
濃度の指示として測定するのである。試験溶液に
よつて放出された螢光強さは配位子と試薬系の相
互作用から生ずる試験溶液の透光性変化に比例す
る。
本明細書で使う“試験溶液の透光性変化”とは
既知強さの光線が試験溶液をとおる時特定波長帯
内で試験溶液により吸収又は散乱された光量をい
い、一般に試験溶液の色又は濁度の変化による。
特に透過性の変化は配位子と配位子に特定な試薬
系の相互作用から実質的に生ずる様な特定波長帯
内の試験溶液により吸収された又は散乱された光
量変化をいう。透過性の変化は一般に既知強さを
もつ単色光を試験溶液にとおし入射光強さに対す
る透過又は散乱光強さの比を測定して求められ
る。透過性変化、即ち特定波長帯内の試験溶液に
より吸収又は散乱された光量変化は試験溶液中の
配位子濃度に比例する。今や配位子、試薬系およ
び螢光体を含む試験溶液において螢光体の励起お
よび（又は）放射波長帯と重なる波長帯内で配位
子と試薬系の相互作用から生ずる透過性の変化も
試験溶液から放出される螢光強さの比例的変化と
なることがわかつている。故に本発明の方法によ
り試験溶液により放出される螢光強さの変化は試
験溶液の配位子濃度に比例する。重要なことは本
発明の方法による測定する配位子、試薬系および
螢光体を含む試験溶液により放出された螢光強さ
の変化は試薬系と螢光体のみを含む試験溶液から
放出される螢光強さと比べると全く配位子と試薬
系の相互作用によつて生じた透過性変化によるも
のである。試験溶液中螢光体と他のどの成分、即
ち測定する配位子又は試薬系との間には化学的又
は免疫学的のいづれの反応もない。故に試験溶液
により放出された螢光強さは螢光体と試験溶液内
にあるかもしれない発色物質又は懸濁粒子との間
の分子間距離によるものではない。
本発明の方法によつて測定できる配位子には比
色的、濁度的又は比濁的に測定できる臨床的に重
要な物質がある。即ち配位子は試験溶液中の配位
子濃度に比例して透過性の検出できる変化を生ず
る様試薬系と相互作用しうるものでなければなら
ない。本発明の方法により分析できる代表的配位
子には例えばグルコース、ウル酸、コレステロー
ル、クレアチニン、ラクテイト、ラクテイト デ
ヒドロジエナーゼ（LDH）、トリグリセリド、イ
ムノグロブリン、ユリネステラーゼ、血清グルタ
メイト オキサラクテイトトランスアミナーゼ、
（SGOT）、血清グルタメイト ピルヴエイトトラ
ンスアミナーゼ（SGPT）、クレアチンホスホキ
ナーゼ（CPK）、エタノール、全蛋白質、アルブ
ミン、カルシウム、ビリルビン、血液尿素窒素
（BUN）、アンモニア、マグネシウム、りん、塩
化物等がある。
“螢光体”とは試薬系と配位子を含む溶液の透
光特性に関連した螢光特性をもつ化合物又はは組
成物をいう。特に螢光体と関連した励起又は放射
波長帯は配位子と試薬系の相互作用から生ずる試
験溶液の透光性変化と関連した波長帯と重複する
必要がある。また上記のとおり螢光体と測定する
配位子又は試薬系の間の化学的又は免疫学的結合
はない。他の考えは試薬系のPHに関する。螢光体
は試薬系に対し測定する配位子と相互反応するに
有効なPH範囲内で螢光を発しなければならない。
また本発明の方法に有効な螢光体は非結合および
非複合状態において螢光性である。比色試験にお
いて螢光体と関連する励起波長帯又は放射波長帯
は少なくともある程度配位子と呈色試薬系の相互
作用に関連する吸収波長帯と重複する必要があ
る。最大試験感度のためには配位子と呈色試薬系
の相互作用から生ずる吸収波長帯が螢光体に関連
する励起波長帯と放射帯の両方と重複することが
好ましい。濁度試験又は比濁試験では螢光体に関
連した励起又は放射波長帯は少なくもある程度配
位子と濁度測定又は比濁測定試薬系を含む試験溶
液の濁度が測定される様な波長帯と重複する必要
がある。本明細書で透光性変化と関連した波長帯
および螢光体の励起および（又は）放射波長帯の
“重複”はそれぞれの波長帯の一部又は全部の重
複のいずれかをいう。
本発明の方法には広く種々の螢光体が使用でき
る。すでに述べたとおり螢光体の選択は測定する
特定配位子と使用する試薬系によるだろう。本発
明の方法に使用できる螢光体の代表的なものは例
えばフルオレシイン、ローダミン、フラビン、ク
ーマリン、ナフタレン、アクリジン、アンスラセ
ン、多核溶融炭化水素、スチルベン、アントラニ
ル酸、アミノスチリルピリジン、キノリン、サリ
チル酸、シアニン、オクソノール、フエナンチジ
ン、フルオレスカミン並びにその誘導体と塩があ
る。使用できる螢光体の特例には例えばエオシ
ン、ローダミン、アミノナフタレンスルホネイ
ト、アクリフラビン、フルオレシイン、ジヒドロ
キシ安息香酸、ヒドロキシキノリン、NADH、
リボフラビン、ブリリアントサルフアフラビン、
キニン、ナフトールスルホン酸、チオフラビン、
クーマリン、アクリジンオレンジ、８−キノリン
−１−ナフタレンスルホン酸、オキサジン、ウン
ベリフエロン、アクリジン、レゾルフインおよび
これらの誘導体と塩がある。本発明の方法に有効
な螢光体の選択はこの分野の技術をもつ者には容
易に確認できるだろう。本明細書で使う“螢光体
の有効量”とは試験溶液に配位子とそれに特定の
試薬系を加えた場合試験溶液によつて放出される
螢光強さの認められる変化を生ずるに十分な試験
溶液中の螢光体濃度をいう。この有効量は一般に
この技術分野の知識ある者によつて確かめられ、
例えば特定試薬系、測定する配位子、特定螢光体
又は螢光強さ測定器械の様な１又は２以上の要素
による。
本明細書で使う“試薬系”とは問題の配位子の
存在で螢光体の励起および（又は）放射波長帯と
重なる波長帯内の試験溶液の透過性変化を生ずる
１又は２以上の試薬を含む化学系をいう。本発明
の方法に有効な試薬系は測定する特定配位子に依
りまた測定する透過性変化が試験溶液の色又は濁
度の変化によるかどうかである。試験溶液の色変
化が試験溶液の透光性変化と関連する比色試験で
は呈色試薬系が試薬系として使われる。濁度、即
ち懸濁粒子によつて阻止又は散乱される光量が試
験溶液の透過性変化と関連する濁度又は比濁試験
では濁度測定試薬系又は比濁測定試薬系がそれぞ
れ使われる。
本明細書で使用する“呈色試薬系”とは透過
性、特に螢光体の励起および（又は）放射波長帯
と重なる波長帯内で試験溶液の比色性の認められ
る変化を生ずる様測定する配位子と特定反応順序
によつて反応する１又は２以上の試薬を含む化学
系をいう。本発明の目的のためこの呈色試薬系を
含む種々の試薬はその添加順序が特定反応順序に
よつて制限されない限り単独で又は混合して試験
溶液に添加できる。配位子の比色測定に利用でき
る呈色試薬系はこの分野ではよく知られており、
例えばニユーヨーク、ヘーバーメデイカルデイビ
ジヨン、ハーパー アンド ロー（1964）のヘン
リーらのClivical Chemistry、Principles and
Techniques；W.B.サウンダース カンパニー
（1970）のテイーツのFundamentals of Clinical
Chemistryの文献がある。種々の試験道具箱と試
薬系が市販されており標準法と試薬を用いる。一
般にこの比色法は配位子が発色試薬を含む呈色試
薬系と反応して試験溶液中に認めうる変化を生じ
るという原理に基づく。代表的呈色試薬系には例
えば終点と運動測定を含むオキシダーゼ反応系と
NADH／NAD反応系がある。例えばオキシダー
ゼ反応系は配位子と反応して過酸化水素を放出し
それが次いでペルオキシダーゼの存在で染料と反
応して試料中の配位子量の指示として試験溶液の
比色性変化を生ずる酸化性酵素を利用する。
NADH／NAD反応系はNADのNADHへの還元
又はNADHのNADへの酸化とつづく染料系との
反応による試料中の配位子濃度尺度としての試験
溶液の比色性変化生成に基づく。本明細書で使う
“試薬系の有効量”とは配位子の存在において試
験溶液の比色性の認めうる変化を生ずるに十分な
試薬系の量をいう。この有効量はこの技術知識を
もつものには容易に確かめられるだろう。
次は本発明の方法により使用できる種々の呈色
試薬系とその反応順序を示すに役立つだろう。次
の記号を本明細書で使用している：
DHBS ３，５−ジクロロ−２−ヒドロキシベ
ンゼンスルホン酸ナトリウム、
AAP ４−アミノアンチピリジン、
HRPO 西洋わさび ペルオキシダーゼ、
NAD 酸化されたニコチンアミド−アデニンジ
ヌクレオチド、
NADH 還元されたニコチンアミド−アデニン
ジヌクレオチド、
LDH ラクテイト デヒドロジエナーゼ、
SGOT 血清グルタミツク オキザルアセチツク
トランスアミナーゼ、
SGPT 血清グルタミツク ピルヴイツク トラ
ンスアミナーゼ、
CPK クレアチン ホスホキナーゼ、
INT ２−（ｐ−アイオドフエニル）−３−（ｐ−
ニトロフエニル）−５−フエニルテトラゾリウ
ム塩化物、
ATP アデノシン トリホスフエイト
ADP アデノシン ジホスフエイト
EGTA エチレングリコール−ビス（β−アミ
ノエチレンエーテル）−Ｎ，N′−テトラ酢酸
また次の反応順序のある生成物の特定構造が不
確実なため試験溶液の色を生じ分光測定試験で測
定される特定反応順序の生成物は詳しく固定しな
い限り一般に本明細書では“発色体”という。
NADH／NAD系を示している反応順序11−16に
おいて試験溶液の色を生ずる生成物はフオルマジ
ンである。血液尿素窒素の試験を示す反応順序21
において試験溶液の色を生ずる生成物はインドフ
エノールである。塩化物の試験を示す反応順序24
においては試験溶液の色を生ずる生成物はチオシ
アン酸第２鉄である。
１ 配位子：グルコース
発色試薬系：グルコースオキシダーゼ
DHBS
AAP
HRPO
反応順序：
Ｄ−グルコース＋O2＋H2Oグルコースオキシダーゼ
―――――――――――――→
Ｄ−グルコン酸＋H2O2
2H2O＋DHBS＋AAPHRPO
―――――→
発色体
２ 配位子：ウル酸
発色試薬系：ウリカーゼ
DHBS
AAP
HRPO
反応順序：
ウル酸＋O2＋2H2Oウリカーズ
――――――→
アラントイン＋CO2＋H2O2
2H2O＋DHBS＋AAPHRPO
―――――→
発色体
３ 配位子：コレステロール（コレステロールと
コレステロールエステル）
発色試薬系：コレステロールエステラーゼ
コレステロールオキシダーゼ
DHBS
AAP
HRPO
反応順序：
コレステロールエステルコレステロールエステラー
ゼ
――――――――――――――――→
コレステロール
コレステロール＋O2コレステロールオキシダーゼ
――――――――――――――――→
△4コレステロン＋H2O2
2H2O2＋DHBS＋AAPHRPO
―――――→
発色体
４ 配位子：クレアチニン
発色試薬系：クレアチニナーゼ
クレアチナーゼ
サルコシンオキシダーゼ
DHBS
AAP
HRPO
反応順序：
クレアチニン＋H2Oクレアチニナーゼ
――――――――――→
クレアチン
クレアチン＋H2Oクレアチナーゼ
―――――――――→
サルコシン＋尿素
サルコシン＋H2O＋O2サルコシンオキシダーゼ
――――――――――――――→
2H2O2＋DHBS＋AAPHRPO
―――――→
発色体
５ 配位子：ラクテイト
発色試薬系：NAD
LDH
ピルヴエイトオキシダーゼ
DHBS
AAP
HRPO
反応順序
ラクテイト＋NADLDH
――――→
ピルヴエイト＋NADH
ピルヴエイト＋O2ピルヴエイトオキシダーゼ
―――――――――――――――→
アセチルホスフエイト＋CO2＋H2O2
2H2O2＋DHBS＋AAPHRPO
―――――→
発色体
６ 配位子：トリグリセリド
発色試薬系：リパーゼ
グリセロールキナーゼ
グリセロールホスフエイトオキシダーゼ
DHBS
AAP
HRPO
反応順序：
トリグリセリド＋3H2Oリパーゼ
―――――→
グリセロール＋脂肪酸
グリセロール＋ATPグリセロールキナーゼ
――――――――――――→
グリセロール−３−ホスフエイト＋ADP
グリセロール＋３−ホスフエイト
グリセロールホスフエイトオキシダーゼ
――――――――――――――――――――――→
ジヒドロキシアセトンホスフエイト＋H2O2
2H2O2＋DHBS＋AAPHRPO
―――――→
発色体
７ 配位子：コリネステラーゼ
発色試薬系：アセチルコリネステラーゼ
コリンオキシダーゼ
DHBS
AAP
HRPO
反応順序：
アセチルコリンアセチルコリネステラーゼ
―――――――――――――――→
コリン＋アセテイト
コリン＋O2コリンオキシダーゼ
―――――――――――→
2H2O2
2H2O2＋DHBS＋AAPHRPO
―――――→
発色体
８ 配位子：SGOT
発色試薬系：アスパレイト
α−ケトグルタレイト
オキサロアセテイトデカルボキシラーゼ
ピルヴエイトオキシダーゼ
DHBS
AAP
HRPO
反応順序：
アスパレイト＋α−ケトグルタレイトSGOT
―――――→
グルタメイト＋オキサロアセテイト
オキサロアセテイトオキサロアセテイトデカルボキ
シラーゼ
――――――――――――――――――――――→
ピルヴエイト＋CO2
ピルヴエイト＋O2ピルヴエイトオキシダーゼ
―――――――――――――――→
H2O2＋アセチルホスフエイト＋CO2
2H2O2＋DHBS＋AAPHRPO
―――――→
発色体
９ 配位子：SGPT
発色試薬系：Ｌ−アラニン
α−ケトグルタレイト
ピルヴエイトオキシダーゼ
DHBS
AAP
HRPO
反応順序：
アラニン＋α−ケトグルタレイトSGPT
―――――→
グルタメイト＋ピルヴエイト
ピルヴエイト＋O2ピルヴエイトオキシダーゼ
―――――――――――――――→
アセチルホスフエイト＋H2O2＋CO2
2H2O2＋DHBS＋AAPHRPO
―――――→
発色体
10 配位子：CPK
配色系：クレアチンホスフエイト
クレアチナーゼ
サルコシンオキシダーゼ
DHBS
AAP
HRPO
反応順序：
クレアチンホスフエイト＋ADPCPX
――――→
クレアチン＋ATP
クレアチン＋H2Oクレアチナーゼ
――――――――→
サルコシン＋尿素
サルコシン＋H2O＋O2サルコシンオキシダーゼ
――――――――――――――→
Ｏ
‖
グリシン＋HCH＋H2O2
2H2O2＋DHBS＋AAPHRPO
―――――→
発色体
11 配位子：エタノール
発色試薬系：NAD
アルコールデヒドロジエナーゼ
INT
デイアフオラーゼ
反応順序：
エタノール＋NADアルコールデヒドロジエナーゼ
―――――――――――――――――→
NADH＋アセタルデヒド
NADH＋INTデイアフオラーゼ
―――――――――→
NAD＋ホルマジン
12 配位子：SGOT
発色試薬系：アスパラテイト
α−ケトグルタレイト
NAD
グルタメイトデヒドロジエナーゼ
デイアフオラーゼ
INT
反応順序：
アスパレイト＋α−ケトグルタレイトSGOT
―――――→
グルタメイト＋オキサロアセテイト
NAD＋グルタメイト＋H2Oグルタメイトデヒドロジエ
ナーゼ
――――――――――――――――→
NADH＋２
−オクソグルタレイト＋NH3
NADA＋INTデイアフオラーゼ
―――――――――→
NAD＋ホルマジン
13 配位子：SGPT
発色試薬系：Ｌ−アラニン
α−ケトグルタレイト
NAD
グルタレイトデヒドロジエナーゼ
デイアフオラーゼ
INT
反応順序
アラニン＋α−ケトグルタレイトSGPT
―――――→
グルタメイト＋ピルヴエイト
グルタメイト＋NAD＋H2Oグルタメイトデヒドロジエ
ナーゼ
――――――――――――――――――→
NADH
＋２−オクソグルタレイト＋NH3
NADH＋INTデイアフオラーゼ
――――――――――→
NAD＋ホルマジン
14 配位子：グルコース
発色試薬系：ATP
ヘクソキナーゼ
NAD
グルコース−６−ホスフエイトデヒドロジエナ
ーゼ
INT
デイアフオラーゼ
反応順序：
グルコース＋ATPヘクソキナーゼ
――――――――→
グルコース−６−ホスフエイト＋ADP
グルコース−６−ホスフエイト
＋NADグルコース‐６‐ホスフエイトデヒドロジエ
ナーゼ
＋NADグルコース‐６‐ホスフエイトデヒドロジエ
ナーゼ
―――――――――――――――――――――――――
――――→
NAD＋グルコネイト−６−ホスフエイ
ト
NADH＋INHデイアフオラーゼ
―――――――――→
NAD＋ホルマジン
15 配位子 CPK
発色試薬系 クレアチンホスフエイト
ADP
グルコース
ヘクソキナーゼ
NAD
グルコース−６−ホスフエイトデヒドロジエナ
ーゼ
INT
デイアホラーゼ
反応順序：
クレアチンホスフエイト＋ADPCPK
――――→
クレアチン＋ATP
グルコース＋ATPヘクソキナーゼ
――――――――→
グルコース−６−ホスフエイト＋ADP
グルコース−６−ホスフエイト＋NAD
グルコース‐６‐ホスフエイト
―――――――――――――――――――→
NADH＋グルコネイト−６−ホスフエイト
NADH＋INTデイアホラーゼ
――――――――→
NAD＋ホルマジン
16 配位子：LDH
発色試薬系：Ｌ−ラクテイト
NAD
INT
デイアホラーゼ
反応順序：
Ｌ−ラクテイト＋NADLAD
NADH＋ピルヴエイト
NADH＋INTデイアフオラーゼ
―――――――――→
NAD＋ホルマジン
17 配位子：全蛋白質
発色試薬系：銅タータレイト
ナトリウムタータレイト
リチウムアセテイト
反応順序：
蛋白質＋銅タータレイト＋ナトリウムタータレイト
＋リチウム水酸化物→発色体
18 配位子：アルブミン
発色試薬系：ブロムクレオゾルグリーン
反応順序：
アルブミン＋ブロムクレオゾルグリーン→発色
体
19 配位子：カルシウム
発色試薬系：Ｏ−クレゾールフタレインコンプ
レクソン
８−キノリノ硫酸塩
反応順序：
カルシウム＋Ｏ−クレゾールフタレイン＋８−キノ
リノル硫酸塩→発色体
20 配位子：ビリルビン
発色試薬系：２，４−ジクロロアニリンのジア
ゾニウム塩
メタノール
スルフアミン酸
反応順序：
ビリルビン＋２，４−ジクロロアニリンのジアゾニ
ウム塩メタノール
――――――――→
スルフアミン酸発色体
21 配位子：血液尿素窒素（尿素）
発色試薬系：ウレアーゼ
次亜塩素酸ナトリウム
フエノール
水酸化ナトリウム
ナトリウムニトロプルシド
反応順序：
尿素ウレアーゼ
――――――→
2NH3＋CO2
2NH3＋2NaClO→2NH2Cl＋2NaOH
2NH2Cl＋２フエノール＋2NaOH→2p−アミノフエノ
ール＋2NaCl＋2H2O
２ｐ−アミノフエノール＋２フエノール＋O2→２イ
ンドフエノール＋2H2O
22 配位子：マグネシウム
発色試薬系：塩化カリウム
カルマガイト
シアン化カリウム
水酸化カリウム
EGTA
反応順序：
マグネシウム＋KCl＋カルマガイト＋KCNKOH
――――→
発色体
23 配位子：りん
発色試薬系：モリブデン酸
硫 酸
触 媒
反応順序：
ホスフエイト＋モリブデン酸
＋硫酸触媒
―――→
発色体
24 配位子：塩化物
発色試薬系：尿素
チオシアン酸カリウム
塩化第２水銀
過塩素酸
過塩素酸第２水銀
過塩素酸第２鉄
反応順序：
2Cl-＋Hg（SCN）2→HgCl2＋2SCN-
3SCN-＋Fe++→Fe（SCN）3
本明細書で使つている“濁度測定試薬系”とは
特定方法により測定する配位子と相互作用して螢
光体の励起および（又は）放射波長帯と重なる波
長帯内で試験溶液の透光性、特に濁度の認めうる
変化を生ずる様な１又は２以上の試薬を含む化学
系をいう。本発明の目的のため濁度測定試薬系を
含む種々の試薬は特定反応順序によつてその添加
順序が制限されなければ単独で又は他と混合して
試験溶液に添加できる。種々の濁度測定試薬系は
この技術分野でよく知られている。濁度測定試薬
系を用いる重要な１試験は人のイムノグロブリン
の濁度測定試験である。この試験の基本原理は測
定するイムノグロブリンに特定の抗血清より成る
濁度測定試薬系と問題のイムノグロブリンの反応
による懸濁粒子より成る特殊複合物の生成に基づ
く。抗血清−イムノグロブリン複合物の生成によ
る懸濁粒子は試験溶液の濁度変化を生ずる。故に
試験溶液に人のイムノグロブリン以上に過剰の抗
血清を使用すると試料中のイムノグロブリン濃度
が増すにつれて懸濁液をとおる光は減少する。本
明細書で使う“比濁測定試薬系”とは特定方法に
より測定する配位子と相互作用し螢光体の励起お
よび（又は）放射波長帯と重なる波長帯内で試験
溶液の透光性、特に濁度の認めうる変化を生ずる
様な１又は２以上の試薬を含む化学系をいう。比
濁測定試験は濁度測定試験と同じ原理に基づく
が、但し試験溶液の比濁測定は濁度測定とちがつ
て入射光に対する角度における散乱光を測定する
のである。多くの濁度および比濁測定法がこの分
野で知られているが、この試験に使う試薬系はこ
の分野の知識ある者には容易に確かめられるであ
ろう。
本発明の方法を行なうには試験溶液を螢光計セ
ル中に入れる。励起波長の選択は使う螢光体、配
位子および試薬系による。放射スペクトルを測定
する特定波長又は帯波長は一般に放射最大によ
る。一定強さの光源を使つて放射スペクトルを検
べ特定波長又は波長の特定帯における放射強さを
測定してこの結果を既知標準と比較できる。本発
明の試験方法を測定する配位子の既知量を含む標
準と実質的に同じ方法で未知試料について行なつ
て未知試料中にある配位子量を定性的に又は定量
的に測定できるのである。
本発明の方法により測定できる配位子濃度が殆
んど使う特定螢光計と使う特定試薬系によるが、
0.01乃至0.1ｍＭの濃度の配位子を含む試料が測
定されている。
試験溶液のPHは一般に使う特定試薬系による。
試薬系のPH範囲内で螢光体が螢光を出すことが必
要である点で試薬系のPHは螢光体選択の条件であ
ろう。
ある配位子と螢光体においては配位子と螢光体
の蛋白質への非特定結合が少量であるが問題にな
らぬ量あつてもよい。蛋白質妨害が問題であれば
試料の超過、ゲル過、沈澱、透析等による予
備処理によつて試験溶液の蛋白質濃度を最少とす
ればよい。また配位子又は螢光体の蛋白質への非
特定結合は試験溶液にトリトンＸ−100などの様
な表面活性剤の添加によつて最少にできる。
本発明の方法は一般に約15−40℃、好ましくは
約25乃至40℃の温度範囲で行なわれる。試験を一
定温度で行なうことも好ましい。
次の実施例は本発明の方法を示すに役立つであ
ろう。試薬濃度と他の種々の助変数は本発明の方
法を示すのみで本発明を限定するものではない。
市販箱、即ちアボツトＡ−Gent臨床化学試薬を
用いる試験は一般に箱に用意された内容物により
試薬系として使われる。唯一の追加試薬は使用す
る特定螢光体である。また次の実施例における各
試験溶液の螢光強さ（FI）はアボツトTDX分析器
を用いて励起用485nｍと放射用525nｍの波長に
おいて測定している。次の実施例の標準曲線は各
標準液について測定した螢光強さ対標準溶液濃度
をプロツトしてつくることができる。また直線標
準曲線を望むならば各標準溶液について測定した
螢光強さ対標準液濃度の対数の負数をプロツトす
ればよい。
実施例 １
グルコース試験
未知又はグルコース標準を含む試料5μにグ
ルコースオキシダーゼ2200単位／mlを含む液25μ
、0.2MDHBSおよび10-5Mフルオレシインを
含む液25μ、および0.1％４−アミノアンチピレ
ンと西洋わさびペルオキシダーゼ400単位／mlを
含む液25μを含む牛ガンマグロブリンとのりん
酸塩緩衝液（PH7.5）205mlを加えた。えた試験溶
液を35℃において最大色生成をさせた。吸光度と
螢光強さを測定し０−500mg／dlグルコースを含
む標準液に対してえた結果を次表に示している。
The present invention relates to an improved fluorometry method. Common non-isotopic analytical methods in the field of clinical medical diagnostics include spectroscopic or fluorometric methods of clinically important substances, hereinafter referred to as ligands. This method is very sensitive and specialized and is based on the measurement of optical properties, ie, changes in the translucency or fluorescence of the test solution caused by the presence of specific ligands in the test solution. Interactions between the ligand to be measured and the ligand-specific reagent system within the test solution in a spectroscopic test result in a measurable change in the light transmission properties of the test solution. Transmission change refers to the amount of light absorbed or scattered by a test solution within a particular wavelength band when a beam of known intensity passes through the test solution. Changes in the transmittance of the test solution are measured by passing monochromatic light of known intensity through the test solution and examining the ratio of the transmitted or scattered light intensity to the incident light intensity. The fact that almost all the ligands either absorb energy at specific wavelengths or interact with specific reagent systems in the test solution to produce a detectable change in the permeability of the test solution Measurement methods are currently being developed. Spectroscopic measurements based on the measurement of changes in the translucency of the test solution for measuring ligands in the test solution include, for example, colorimetric tests based on color changes in the test solution, and turbidity or nephelometric tests based on turbidity changes in the test solution. be. In a colorimetric test, the change in the light transmittance of the test solution is generally referred to as the absorbance of the test solution, and is due to the change in color of the test solution due to the interaction between the ligand to be measured and the ligand-specific reagent system. The absorbance of the test solution is proportional to the concentration of ligand therein. Colorimetric tests utilize a chromogenic reagent system that can interact with a particular ligand of interest in a test solution to produce a detectable change in the translucency of the test solution, particularly in color. Many coloring reagent systems convenient for specific ligand measurements have been developed and are commercially available. The principle of the turbidimetric test is to measure the amount of light that is scattered or blocked by a particular substance as it passes through the test solution. In turbidimetric tests, the ligand in question interacts with a reagent system specific to the ligand to create a suspension of particles in the test solution. When a light beam of known intensity passes through the test solution, the particle suspension created by the interaction of the ligand and reagent system blocks or scatters the incident light, reducing the light intensity passing through the test solution. Transmission change in turbidity measurements refers to the decrease in light intensity passing through the test solution, which is proportional to the amount of incident light scattered or blocked by the particle suspension and depends on the number of particles present and the cross-sectional area of these particles. Nephelometric tests are similar to turbidity tests in that the ligand in question interacts with a ligand-specific reagent system to produce a suspension of particles in the test solution. In nephelometric tests, the change in light transmission of the test solution is also proportional to the amount of incident light that is scattered or blocked by the particle suspension, unlike turbidity tests, which measure the intensity of light passing through the test solution. Measure the scattered or blocked light at the angle of light incidence on the test solution. Therefore, in a turbidimetric test, the change in translucency refers to the difference in intensity between the light entering the test solution and the light scattered at an angle relative to the incident light. Turbidity tests and nephelometric tests are used to measure ligands such as proteins for which there is no colorimetric test for comparison, as there is no effective coloring reagent system for analyzing blood, urine, spinal fluid, etc. Yohe and Krimman, edited by Wiley and Sons, New York.
Photoelectric Chemical Analysis Volume:
Nephelometry (1929) describes various turbidimetric tests. Generally, in a fluorescence test, a ligand in a test solution is chemically or immunologically converted into a fluorescent compound or conjugate, resulting in a detectable change in the fluorescence of the test solution. To change the fluorescence of the test solution, the generated fluorescent composite or conjugate is excited with monochromatic light having a wavelength within the excitation wavelength range of the fluorophore, and emitted light having a wavelength within the emission wavelength range of the fluorophore is produced. It can be obtained by measuring strength. The fluorescence intensity of synchrotron radiation is proportional to the ligand concentration. However, the fluorescence intensity emitted by the test solution may be reduced if the ligand to be measured is complexed with non-fluorescent interfering substances such as proteins or phosphates present in the sample, or if the ligand to be measured is present in the sample. Suppression occurs when the sample has sufficient color to act as a filter, reducing the emitted fluorescence intensity. To maximize the sensitivity and specificity of the fluorometric test, remove non-fluorescent interferences or color-producing substances, if any, before analysis or add an internal standard to the second portion of the sample. It is clear that it is necessary to eliminate these suppressing factors either by compensating for the presence of this factor using a standard or by performing the entire test method using a sample with an internal standard. It is an object of the present invention that the intensity of fluorescence emitted by a test solution is determined by the interaction of the ligand with the reagent system, which in the presence of the ligand can cause a change in the translucency of the test solution. It provides a method for fluorometric measurement of ligands in test solutions that is proportional to the change in optical properties.
It is also an object of the present invention to provide a new reagent composition that can utilize either spectroscopic or fluorometric measurements of the concentration of ligands in test solutions. The present invention relates to a method for measuring a ligand in a sample that is thought to contain a ligand.
Mixing an effective amount of a fluorophore and an effective amount of a reagent system that, in the presence of the ligand to be measured, can alter the translucency of the test solution within a wavelength range that is the excitation and/or emission wavelength range of the fluorophore. After irradiating the test solution with light having a wavelength within the excitation wavelength range of the fluorophore, the intensity of the fluorescence emitted by the test solution is used as a measure of the concentration of the ligand in the sample. It consists of measuring. The present invention further provides an effective amount of a reagent system capable of producing a change in the optical transparency of a solution containing the ligand, and an excitation and/or excitation that overlaps with the wavelength band associated with the change in optical transmission of the solution containing the reagent and the ligand. The present invention relates to novel reagent compositions for the determination of ligand fluorescence in materials believed to contain the above-mentioned ligands, comprising an effective amount of a fluorophore having an emission wavelength band. According to the method of the present invention, the ligand in a biological sample is determined by mixing the sample, an effective amount of the reagent system, and an effective amount of the fluorophore in a test solution. The test solution is irradiated with light having a wavelength within the excitation wavelength range of the fluorophore and the intensity of the fluorescence emitted by the test solution is measured as an indication of the concentration of the ligand in the sample. The intensity of fluorescence emitted by the test solution is proportional to the change in translucency of the test solution resulting from the interaction of the ligand and reagent system. As used herein, "translucency change of a test solution" refers to the amount of light absorbed or scattered by the test solution within a specific wavelength band when a light beam of known intensity passes through the test solution, and generally refers to the color or color of the test solution. Due to changes in turbidity.
In particular, a change in transmittance refers to a change in the amount of light absorbed or scattered by a test solution within a particular wavelength band that substantially results from the interaction of the ligand and the ligand-specific reagent system. Changes in transmittance are generally determined by passing monochromatic light of known intensity through the test solution and measuring the ratio of the transmitted or scattered light intensity to the incident light intensity. The change in transmission, ie, the amount of light absorbed or scattered by the test solution within a particular wavelength band, is proportional to the concentration of the ligand in the test solution. Changes in permeability resulting from interaction of the ligand and reagent system within the wavelength range that overlaps the excitation and/or emission wavelength range of the fluorophore in a test solution that now contains the ligand, reagent system, and fluorophore are also included. It has been found that there is a proportional change in the fluorescence intensity emitted from the test solution. Therefore, the change in fluorescence intensity emitted by the test solution according to the method of the invention is proportional to the ligand concentration of the test solution. Importantly, the changes in fluorescence intensity emitted by the test solution containing the ligand, reagent system, and fluorophore measured by the method of the present invention are the same as those emitted from the test solution containing only the reagent system and fluorophore. This is entirely due to the change in transmittance caused by the interaction between the ligand and the reagent system. There is no reaction, either chemical or immunological, between the fluorophore and any other components in the test solution, ie, the ligand or reagent system to be measured. Therefore, the fluorescence intensity emitted by the test solution is not due to the intermolecular distance between the fluorophore and any colored substances or suspended particles that may be within the test solution. Ligands that can be measured by the method of the invention include clinically important substances that can be measured colorimetrically, turbiditically, or nephelometrically. That is, the ligand must be capable of interacting with the reagent system in a manner that produces a detectable change in permeability that is proportional to the concentration of the ligand in the test solution. Representative ligands that can be analyzed by the method of the invention include, for example, glucose, uric acid, cholesterol, creatinine, lactate, lactate dehydrogenase (LDH), triglycerides, immunoglobulins, urinesterase, serum glutamate oxalactate transaminase,
(SGOT), serum glutamate pyruvate transaminase (SGPT), creatine phosphokinase (CPK), ethanol, total protein, albumin, calcium, bilirubin, blood urea nitrogen (BUN), ammonia, magnesium, phosphorus, chloride, etc. . "Fluorophore" refers to a compound or composition that has fluorescent properties that are related to the light-transmitting properties of a solution containing the reagent system and the ligand. In particular, the excitation or emission wavelength range associated with the fluorophore should overlap with the wavelength range associated with the change in translucency of the test solution resulting from interaction of the ligand and reagent system. Also, as mentioned above, there is no chemical or immunological bond between the fluorophore and the measuring ligand or reagent system. Another consideration concerns the pH of the reagent system. The fluorophore must fluoresce within an effective pH range for the reagent system to interact with the ligand to be measured.
Fluorophores useful in the methods of the invention are also fluorescent in the unbound and unconjugated state. In a colorimetric test, the excitation or emission wavelength band associated with the fluorophore must overlap, at least to some extent, with the absorption wavelength band associated with the interaction of the ligand and color reagent system. For maximum test sensitivity, it is preferred that the absorption wavelength band resulting from the interaction of the ligand and color reagent system overlap with both the excitation and emission bands associated with the fluorophore. In a turbidity or nephelometric test, the excitation or emission wavelength range associated with the fluorophore is such that the turbidity of the test solution containing at least some ligand and the turbidimetric or turbidimetric reagent system is measured. It must overlap with the obi. As used herein, "overlap" of wavelength bands associated with translucency changes and excitation and/or emission wavelength bands of fluorophores refers to either partial or complete overlap of the respective wavelength bands. A wide variety of fluorescers can be used in the method of the invention. As mentioned above, the choice of fluorophore will depend on the particular ligand being measured and the reagent system used. Typical fluorophores that can be used in the method of the present invention include, for example, fluorescein, rhodamine, flavin, coumarin, naphthalene, acridine, anthracene, polynuclear fused hydrocarbon, stilbene, anthranilic acid, aminostyrylpyridine, quinoline, salicylic acid, and cyanine. , oxonol, phenanthidine, fluorescamine and their derivatives and salts. Specific examples of fluorophores that can be used include eosin, rhodamine, aminonaphthalene sulfonate, acriflavine, fluorescein, dihydroxybenzoic acid, hydroxyquinoline, NADH,
riboflavin, brilliant sulfaflavin,
quinine, naphtholsulfonic acid, thioflavin,
Examples include coumarin, acridine orange, 8-quinoline-1-naphthalenesulfonic acid, oxazine, umbelliferone, acridine, resorufin and their derivatives and salts. The selection of fluorophores useful in the method of the invention will be readily apparent to those skilled in the art. As used herein, an "effective amount of fluorophore" means an amount that, when added to a test solution, of a ligand and a particular reagent system produces an appreciable change in the intensity of the fluorescein emitted by the test solution. Refers to the concentration of fluorophore in sufficient test solution. This effective amount is generally ascertained by one knowledgeable in the art;
For example, it depends on one or more factors such as a specific reagent system, a ligand to be measured, a specific fluorophore, or a fluorescence intensity measuring instrument. As used herein, "reagent system" refers to one or more systems that, in the presence of the ligand of interest, cause a change in the transmittance of a test solution within a wavelength range that overlaps the excitation and/or emission wavelength range of the fluorophore. A chemical system that includes reagents. The reagent systems useful in the method of the invention depend on the particular ligand being measured and whether the change in permeability being measured is due to a change in color or turbidity of the test solution. A color reagent system is used as the reagent system in colorimetric tests where a change in color of the test solution is related to a change in translucency of the test solution. In turbidity or nephelometric tests in which turbidity, ie the amount of light blocked or scattered by suspended particles, is related to changes in the permeability of the test solution, turbidity or nephelometric reagent systems are used, respectively. As used herein, a "color reagent system" refers to a transmittance measurement that produces an appreciable change in the colorimetric properties of a test solution, particularly within a wavelength range that overlaps the excitation and/or emission wavelength range of the fluorophore. A chemical system that includes a ligand and one or more reagents that react in a specific reaction order. For purposes of the present invention, various reagents, including this color-forming reagent system, can be added to the test solution alone or in mixtures, so long as the order of addition is not limited by the particular reaction order. Color reagent systems available for colorimetric determination of ligands are well known in the field;
For example, Henry et al.'s Clinical Chemistry, Principles and
Techniques; Teates's Fundamentals of Clinical by WB Saunders Company (1970)
There is a literature on Chemistry. A variety of test kits and reagent systems are commercially available and use standard methods and reagents. Generally, this colorimetric method is based on the principle that a ligand reacts with a color-forming reagent system that includes a color-forming reagent to produce an observable change in the test solution. Typical color reagent systems include, for example, oxidase reaction systems that include endpoint and kinetic measurements;
There is a NADH/NAD reaction system. For example, oxidase reaction systems react with the ligand to release hydrogen peroxide, which then reacts with the dye in the presence of the peroxidase to produce a colorimetric change in the test solution as an indication of the amount of ligand in the sample. Use sex enzymes.
The NADH/NAD reaction system is based on the reduction of NAD to NADH or the oxidation of NADH to NAD and subsequent reaction with a dye system to produce a colorimetric change in the test solution as a measure of the concentration of the ligand in the sample. As used herein, an "effective amount of a reagent system" refers to an amount of a reagent system sufficient to cause an appreciable change in the colorimetric properties of the test solution in the presence of the ligand. This effective amount will be readily ascertainable to those skilled in the art. The following will serve to illustrate the various color-forming reagent systems and their reaction sequences that can be used according to the method of the invention. The following symbols are used herein: DHBS sodium 3,5-dichloro-2-hydroxybenzenesulfonate, AAP 4-aminoantipyridine, HRPO horseradish peroxidase, NAD oxidized nicotinamide-adenine dinucleotide. , NADH reduced nicotinamide-adenine dinucleotide, LDH lactate dehydrogenase, SGOT serum glutamic oxalacetic transaminase, SGPT serum glutamic pyruvic transaminase, CPK creatine phosphokinase, INT 2-(p-iodophenyl)-3-( p-
Nitrophenyl)-5-phenyltetrazolium chloride, ATP Adenosine triphosphate ADP Adenosine diphosphate EGTA Ethylene glycol-bis(β-aminoethylene ether)-N,N'-tetraacetic acid Identification of products with the following reaction sequence The products of a specific reaction sequence that give rise to the color of the test solution and are measured in spectrometric tests due to structural uncertainty are generally referred to herein as "chromophores" unless specifically fixed.
The product that gives rise to the color of the test solution in reaction sequences 11-16 showing the NADH/NAD system is formazine. Reaction sequence 21 showing blood urea nitrogen test
The product that gives rise to the color of the test solution in is indophenol. Reaction sequence 24 showing chloride test
The product that gives rise to the color of the test solution is ferric thiocyanate. 1 Ligand: Glucose Color reagent system: Glucose oxidase DHBS AAP HRPO Reaction order: D-glucose + O 2 + H 2 O glucose oxidase――――――――――――→ D-gluconic acid + H 2 O 2 2H 2 O + DHBS + AAPHRPO ――――――→ Chromogenic body 2 Ligand: Uric acid Coloring reagent system: Uricase DHBS AAP HRPO Reaction order: Uric acid + O 2 +2H 2 O uricase ――――――→ Allantoin + CO 2 + H 2 O 2 2H 2 O＋DHBS＋AAPHRPO ――――――→ Chromogenic body 3 Ligand: Cholesterol (cholesterol and cholesterol ester) Coloring reagent system: Cholesterol esterase Cholesterol oxidase DHBS AAP HRPO Reaction order: Cholesterol ester Cholesterol esterase ―――――――― ――――――――→ Cholesterol Cholesterol + O 2 cholesterol oxidase ――――――――――――――――→ △ 4 cholesterol + H 2 O 2 2H 2 O 2 + DHBS + AAPHRPO ――――――→ Chromogen 4 Ligand: Creatinine Color reagent system: Creatinase Creatinase Sarcosine oxidase DHBS AAP HRPO Reaction order: Creatinine + H 2 O creatininase――――――――――→ Creatine Creatine + H 2 O creatinase― ――――――――→ Sarcosine + Urea Sarcosine + H 2 O + O 2 Sarcosine Oxidase ――――――――――――――→ 2H 2 O 2 +DHBS+AAPHRPO ――――→ Chromogen 5 Ligand: Lactate Color reagent system: NAD LDH Pyruvate oxidase DHBS AAP HRPO Reaction order Lactate + NADLDH ――――→ Pyruvate + NADH Pyruvate + O 2 Pyruvate oxidase ――――――――――――――→ Acetyl phosphate + CO 2 + H 2 O 2 2H 2 O 2 + DHBS + AAPHRPO ――――――→ Color former 6 Ligand: Triglyceride Color reagent system: Lipase glycerol Kinase Glycerol Phosphate Oxidase DHBS AAP HRPO Reaction order: Triglyceride + 3H 2 O lipase――――――→ Glycerol + Fatty acid Glycerol + ATP Glycerol Kinase――――――――――→ Glycerol-3-phosphate + ADP Glycerol + 3 -Phosphate Glycerol phosphate oxidase――――――――――――――――――――――→ Dihydroxyacetone phosphate + H 2 O 2 2H 2 O 2 +DHBS + AAPHRPO ――――――→ Chromophore 7 Ligand: Cholinesterase Color reagent system: Acetylcholinesterase Choline oxidase DHBS AAP HRPO Reaction order: Acetylcholine acetylcholinesterase――――――――――――――→ Choline + Acetate Choline + O 2 Choline oxidase ――――――――――――→ 2H 2 O 2 2H 2 O 2 +DHBS+AAPHRPO ――――――→ Chromophore 8 Ligand: SGOT Color reagent system: Aspartate α-ketoglutarate oxaloacetate decarboxylase Pyruvate oxidase DHBS AAP HRPO Reaction order: Aspartate + α-ketoglutarate SGOT ――――――→ Glutamate + Oxaloacetate Oxaloacetate Oxaloacetate decarboxylase ―――――――――――――――― ――――――→ Pyruvate + CO 2 Pyruvate + O 2 Pyruvate oxidase ――――――――――――――→ H 2 O 2 + Acetyl phosphate + CO 2 2H 2 O 2 + DHBS + AAPHRPO ―― ---→ Chromogen 9 Ligand: SGPT Color reagent system: L-alanine α-ketoglutarate pyruvate oxidase DHBS AAP HRPO Reaction order: Alanine + α-ketoglutarate SGPT ---→ Glutamate + Pyruvate Piruvate + O 2 Pyruvate oxidase――――――――――――――→ Acetyl phosphate + H 2 O 2 + CO 2 2H 2 O 2 + DHBS + AAPHRPO ――――――→ Chromogen 10 Ligand: CPK Color scheme System: Creatine Phosphate Creatinase Sarcosine Oxidase DHBS AAP HRPO Reaction order: Creatine Phosphate + ADPCPX ――――→ Creatine + ATP Creatine + H 2 O Creatinase ――――――――→ Sarcosine + Urea Sarcosine + H 2 O + O 2 Sarcosine Oxidase ――――――――――――――→ O ‖ Glycine + HCH + H 2 O 2 2H 2 O 2 + DHBS + AAPHRPO ――――――→ Color former 11 Ligand: Ethanol Color reagent system: NAD Alcohol dehydrodienase INT diafluorase reaction order: Ethanol + NAD Alcohol dehydrogenase――――――――――――――――→ NADH + Acetaldehyde NADH + INT diafluorase ――――――――――→ NAD + Formazin 12 Ligand: SGOT Coloring reagent system: Aspartate α-ketoglutarate NAD Glutamate dehydrodienase deafuorase INT Reaction order: Aspartate + α-ketoglutarate SGOT ------→ Glutamate + Oxaloacetate NAD + Glutamate + H 2 O glutamate dehydrodienase ――――――――――――――――→ NADH+2 -Oxoglutarate+NH 3 NADA+INT diafluorase――――――――――→ NAD+Formazin 13 Ligand: SGPT Color reagent system: L-alanine α-ketoglutarate NAD Glutarate dehydrodienase dehydrogenase INT Reaction order Alanine + α-ketoglutarate SGPT ――――――→ Glutamate + Pyruvate Glutamate + NAD + H 2 O glutamate dehydrodienase ―――――――――――― ――――――→ NADH + 2-Oxoglutarate + NH 3 NADH + INT Deafluorase ――――――――――→ NAD + Formazine 14 Ligand: Glucose Coloring Reagent System: ATP Hexokinase NAD Glucose-6-Phosphate Dehydro Dienase INT Deafluorase reaction order: Glucose + ATP hexokinase ---> Glucose-6-phosphate + ADP Glucose-6-phosphate +NAD Glucose-6-phosphate dehydrogenase +NAD Glucose-6-phosphate dehydrogenase ――――――――――――――――――――――――
――――→ NAD + gluconate-6-phosphate NADH + INH diafluorase ――――――――――→ NAD + formazine 15 Ligand CPK Coloring reagent system Creatine phosphate ADP Glucose hexokinase NAD Glucose-6-phosphate dehydrogenase Nase INT Diaphorase reaction order: Creatine phosphate + ADPCPK ---→ Creatine + ATP Glucose + ATP hexokinase ---> Glucose-6-phosphate + ADP Glucose-6-phosphate + NAD Glucose-6-phosphate --- ――――――――――――――――→ NADH + gluconate-6-phosphate NADH + INT diaphorase ――――――――→ NAD + formazin 16 Ligand: LDH Coloring reagent system: L-lactate NAD INT Diaphorase reaction order: L-lactate + NADLAD NADH + Pyruvate NADH + INT Diaphorase ――――――――――→ NAD + Formazine 17 Ligand: Total protein Coloring reagent system: Copper tartrate Sodium tartrate Trilithium acetate Reaction order: Protein + Copper tartrate + sodium tartrate + lithium hydroxide → chromophore 18 Ligand: albumin Color reagent system: Bromucreosol green Reaction order: Albumin + Bromucreosol green → chromophore 19 Ligand: Calcium chromophore system: O -Cresolphthalein complexon 8-quinolinosulfate Reaction sequence: Calcium + O-cresolphthalein + 8-quinolinol sulfate → Color former 20 Ligand: Bilirubin Color reagent system: Diazonium salt of 2,4-dichloroaniline Methanol sulfate Amic acid reaction sequence: Bilirubin + diazonium salt of 2,4-dichloroaniline methanol――――――――→ Sulfamic acid color former 21 Ligand: Blood urea nitrogen (urea) Color reagent system: Urease sodium hypochlorite Sodium phenol hydroxide Sodium nitroprusside Reaction order: Urea urease ------→ 2NH 3 +CO 2 2NH 3 +2NaClO→2NH 2 Cl+2NaOH 2NH 2 Cl+2phenol+2NaOH→2p-aminophenol+2NaCl+2H 2 O 2p -aminophenol+2phenol+O 2 → 2 indophenol + 2H 2 O 22 Ligand: Magnesium Coloring reagent system: Potassium chloride Calmagite Potassium cyanide Potassium hydroxide EGTA Reaction order: Magnesium + KCl + Calmagite + KCNKOH ――――→ Coloring body 23 Ligand: Phosphorous coloring reagent system :Molybdic acid sulfur Acid catalyst reaction order: Phosphate + molybdic acid + sulfuric acid catalyst---→ Color former 24 Ligand: Chloride Color reagent system: Urea Potassium thiocyanate Mercury chloride Perchloric acid Perchloric acid 2 Mercury ferric perchlorate reaction order: 2Cl - +Hg (SCN) 2 →HgCl 2 +2SCN - 3SCN - +Fe ++ →Fe (SCN) 3The "turbidity measurement reagent system" used in this specification is not specified. 1 or 2 which interacts with the ligand to be measured by the method to produce an appreciable change in the translucency, especially the turbidity, of the test solution within a wavelength range that overlaps with the excitation and/or emission wavelength range of the fluorophore. A chemical system containing two or more reagents. For purposes of the present invention, various reagents, including the turbidity measurement reagent system, can be added to the test solution alone or mixed with others, provided that the order of addition is not limited by the particular reaction order. Various turbidity measurement reagent systems are well known in the art. One important test using a turbidity measurement reagent system is the turbidity measurement test for human immunoglobulins. The basic principle of this test is based on the reaction of a turbidity measuring reagent system consisting of an antiserum specific to the immunoglobulin to be measured and the immunoglobulin in question to form a special composite consisting of suspended particles. Suspended particles due to the formation of antiserum-immunoglobulin complexes cause a change in the turbidity of the test solution. Therefore, if an excess of antiserum over human immunoglobulin is used in the test solution, the light passing through the suspension will decrease as the immunoglobulin concentration in the sample increases. As used herein, "nephelometric reagent system" means the ability of a test solution to interact with the ligand to be measured by a specified method and to transmit light in a test solution within a wavelength range that overlaps with the excitation and/or emission wavelength range of the fluorophore. , especially a chemical system containing one or more reagents that produces an appreciable change in turbidity. Nephelometric tests are based on the same principles as turbidimetric tests, except that nephelometry of test solutions, unlike turbidity measurements, measures the scattered light at an angle to the incident light. Although many turbidity and nephelometric methods are known in the art, the reagent systems used for this test will be readily ascertained by those skilled in the art. To carry out the method of the invention, a test solution is placed in a fluorometer cell. The choice of excitation wavelength depends on the fluorophore, ligand, and reagent system used. The particular wavelength or band of wavelengths at which the emission spectrum is measured generally depends on the emission maximum. Using a light source of constant intensity, one can examine the emission spectrum, measure the intensity of radiation at a particular wavelength or band of wavelengths, and compare the results with known standards. The test method of the present invention can be performed on an unknown sample in substantially the same manner as a standard containing a known amount of the ligand to be measured, and the amount of ligand in the unknown sample can be qualitatively or quantitatively determined. be. The concentration of ligand that can be measured by the method of the present invention depends mostly on the specific fluorometer used and the specific reagent system used.
Samples containing concentrations of the ligand between 0.01 and 0.1 mM have been measured. The pH of the test solution depends on the specific reagent system commonly used.
The pH of the reagent system is likely to be a condition for selecting a phosphor in that it is necessary for the phosphor to emit fluorescence within the pH range of the reagent system. For certain ligands and fluorophores, there may be a small but not problematic amount of non-specific binding of the ligand and fluorophore to the protein. If protein interference is a problem, the protein concentration of the test solution can be minimized by pretreatment such as excess sample, gel filtration, precipitation, dialysis, etc. Also, non-specific binding of the ligand or fluorophore to the protein can be minimized by the addition of a surfactant, such as Triton X-100, to the test solution. The process of the invention is generally carried out at a temperature range of about 15-40°C, preferably about 25-40°C. It is also preferred to conduct the test at constant temperature. The following examples will serve to illustrate the method of the invention. The reagent concentrations and various other parameters are merely illustrative of the method of the invention and are not intended to limit the invention.
Tests using commercially available boxes, ie, Abbott A-Gent Clinical Chemistry Reagents, are generally used as reagent systems depending on the contents provided in the box. The only additional reagent is the specific fluorophore used. In addition, the fluorescence intensity (F I ) of each test solution in the following examples was measured using an Abbott TDX analyzer at wavelengths of 485 nm for excitation and 525 nm for emission. The standard curves in the following examples can be constructed by plotting the measured fluorescence intensity versus standard solution concentration for each standard solution. If a linear standard curve is desired, the negative number of the logarithm of the fluorescence intensity versus the concentration of the standard solution measured for each standard solution may be plotted. Example 1 Glucose test 5μ of sample containing unknown or glucose standard and 25μ of solution containing 2200 units/ml of glucose oxidase
, 25 μ of a solution containing 0.2 MDHBS and 10 -5 M fluorescein, and 25 μ of a solution containing 0.1% 4-aminoantipyrene and 400 units/ml of horseradish peroxidase in a phosphate buffer (PH 7.5) with bovine gamma globulin. ) 205ml was added. The test solutions obtained were allowed to reach maximum color production at 35°C. The absorbance and fluorescence intensity were measured for standard solutions containing 0-500 mg/dl glucose, and the results are shown in the table below.
【表】
これらの結果から未知試料中のグルコース濃度
が決定できる様な標準曲線をえることができる。
実施例 ２
ウル酸試験
未知又はウル酸標準を含む試料20μにウリカ
ーゼ10単位／mlを含む液25μ、0.2M DHBSと
10-5Mフルオレシインを含む液25μおよび0.1％
４−アミノアンチピレンと西洋わさびペルオキシ
ダーゼ400単位／mlを含む液25μを含む牛ガン
マグロブリンとのりん酸塩緩衝液（PH7.5）2.05
mlを加えた。えた試験溶液を35℃で５分間培養し
試験溶液の螢光強さを測定した。０−９mg／dlの
ウル酸を含む試料からえられた結果を次表に示
す。
ウル酸濃度（mg／mg） FI
０ 57588
２ 54440
４ 52431
５ 51426
６ 50774
９ 47767
この結果から未知試料中のウル酸濃度を決定で
きる様な標準曲線をつくることができる。
次表は上記表の結果からつくつた標準曲線を用
いてDade Moni−Trolおよび化学調整試料
について測定したウル酸濃度を表わす。[Table] From these results, a standard curve can be obtained from which the glucose concentration in unknown samples can be determined. Example 2 Uric acid test 20μ of sample containing unknown or uric acid standard, 25μ of solution containing 10 units/ml of uricase, 0.2M DHBS and
25 μ of solution containing 10 -5 M fluorescein and 0.1%
Phosphate buffer (PH7.5) 2.05 with bovine gamma globulin containing 25μ of a solution containing 4-aminoantipyrene and horseradish peroxidase 400 units/ml
Added ml. The resulting test solution was incubated at 35°C for 5 minutes, and the fluorescence intensity of the test solution was measured. The results obtained from samples containing 0-9 mg/dl of uric acid are shown in the following table. Uric acid concentration (mg/mg) F I 0 57588 2 54440 4 52431 5 51426 6 50774 9 47767 From these results, a standard curve can be created from which the uric acid concentration in the unknown sample can be determined. The following table represents the uric acid concentrations determined for Dade Moni-Trol and chemical preparation samples using the standard curve constructed from the results of the above table.
【表】
実施例 ３
コレステロール試験
未知又はコレステロール標準を含む試料10μ
をコレステロールエステラーゼ110単位／ml、コ
レステロールオキシダーゼ100単位／ml、20％ト
リトンＸ−100および0.2Mコレイトを含む液25μ
；約１×10-5Mフルオレシインと0.2M DHBS
を含む液25μ；0.1％４−アミノアンチピレンお
よび西洋わさびペルオキシダーゼ400μ／mlを含
む液25μを含む牛ガンマグロブリンとのりん酸
塩緩衝液（PH7.5）2.05mlを加えた。
えた試験溶液を35℃で５分間培養した。コレス
テロール０−400mg／dl含む標準液からえた結果
は次表のとおりである：
コレステロール酸濃度（mg／dl） FI
０ 65175
50 56909
100 49998
200 38404
300 29149
400 22273
この結果から未知試料中のコレステロール濃度
が決定できる様な標準曲線をえられる。
次表は上記表中の結果からつくつた標準曲線を
用いてDade Moni−Trol とについて決定
したコレステロール濃度を示す。[Table] Example 3 Cholesterol test 10 μ sample containing unknown or cholesterol standard
25μ of a solution containing cholesterol esterase 110 units/ml, cholesterol oxidase 100 units/ml, 20% Triton X-100 and 0.2M cholate.
approximately 1×10 -5 M fluorescein and 0.2 M DHBS
25μ of a solution containing 0.1% 4-aminoantipyrene and 400μ/ml of horseradish peroxidase and 2.05ml of a phosphate buffer with bovine gamma globulin (PH7.5) were added. The test solution obtained was incubated at 35°C for 5 minutes. The results obtained from standard solutions containing 0-400 mg/dl of cholesterol are shown in the following table: Cholesterol acid concentration (mg/dl) F I 0 65175 50 56909 100 49998 200 38404 300 29149 400 22273 From this result, cholesterol in the unknown sample can be determined. A standard curve is obtained from which concentrations can be determined. The following table shows the cholesterol concentrations determined for Dade Moni-Trol using the standard curve constructed from the results in the above table.
【表】
実施例 ４
全蛋白質試験
1.5×10-7Mフルオレシインを含む様調節され
た再構成されたアボツトＡ−Gent全蛋白質試薬
３mlに未知又は標準を含む試料50μを加えた。
試薬と試料を混合し少なくも10分間20℃で培養し
た。試験溶液の螢光強さを測定し蛋白質０−８
ｇ／dlを含む標準液についてえた結果を次表に示
している。
蛋白質濃度（mg／dl） FI
０ 54068
４ 37383
６ 32760
８ 27792
この結果から未知試料中の全蛋白質濃度を決定
できる様な標準曲線をえられる。
実施例 ５
LDH試験
未知又は標準液を含む試料20μにリチウム−
Ｌ−ラクテイト250mg／mlとNAD125mg／mlを含
む液25μ、INT25mg／mlと１×10-5Mフルオレ
シインを含む50％エタノール溶液25μおよびジ
アホラーゼ100単位／mlを含む50％グリセロール
液25μを含む牛ガンマグロブリンとのりん酸塩
緩衝液2.05mlを加えた。試料と試験溶液を混合し
えた試験溶液を35℃で14分間培養し試験溶液の螢
光強さを測定した。LDH0−500単位／を含む
標準液でえた結果を次表に示している。
LDH活性（単位／ml） FI
０ 45797
50 43170
100 41470
200 38484
300 35909
500 28021
この結果から未知試料中のLDH濃度を決定で
きる様な標準曲線がえられる。
実施例 ６
クレアチニン試験
ピクリン酸5.5mlと2.5N水酸化ナトリウム1.1ml
を含む水溶液20mlに10-5Mフルオレシイン200μ
を加えて10-7Mフルオレシインを含む試薬液をつ
くつた。上記試薬液２mlに未知又は標準を含む試
料100μを加えた。えた試験溶液を35℃で約15
分間培養し試験溶液の螢光強さを測定した。クレ
アチニン０−10mg／dlを含む標準液についてえた
結果を次表に示す：
クレアチニン濃度（mg／dl） FI
０ 8282
２ 7665
６ 6159
10 4972
この結果から未知試料中のクレアチニン濃度を
決定できる様な標準曲線がえられる。
実施例 ７
BUN試験
未知又は標準を含む試料20μに0.1Mりん酸カ
リウム（PH7.5）緩衝液；牛ガンマグロブリン；
0.1％アジ化ナトリウム；ウレアーゼ130単位／ml
を含む５％グリセロール液25μ；１ml水にナト
リウムニトロプルシツド300mgの液をフエノール
５ml、グリセロール１mlおよびエタノール１mlお
よび十分なフルオレシインに加えてつくつた
10-5Mフルオレシイン液25μ；および5.7％次亜
塩素酸ナトリウムを含む液50mlに水酸化ナトリウ
ム25ｇを加えてつくつた液25μより成る試験溶
液2.050mlを加えた。試料と試験溶液を混合しえ
た試験溶液を35℃で約５分間培養した。試験溶液
の螢光強さを測定し尿素０−100mg／dlを含む標
準液の螢光強さを次表に示している：尿素濃度（mg／dl）
FI
０ 31847
6.25 28713
12.5 25745
25 20962
50 15538
100 8798
この結果から未知試料中の尿素濃度を決定でき
る様な標準曲線がえられる。
実施例 ８
ビリルビン試験
10-7Mフルオレシインを含む様調整された再構
成されたアボツトＡ−Gentビリルビン試薬２ml
に未知又は標準を含む試料50μを加えた。試薬
と試料を混合し37℃で約10分間培養した。えた試
験溶液の螢光強さを測定しビリルビン０−20.2
mg／dlを含む標準液についてえた結果を次表に示
している：ビリルビン濃度（mg／dl）
FI
０ 65256
４ 59371
10.1 50353
20.2 32740
この結果から未知試料中のビリルビン濃度を決
定できる様な標準曲線がえられる。
次表は上記表の結果からつくつた標準曲線を用
いてDade Moni−Trolと化学調整試料につ
いて決定したビリルビン濃度を表わす。[Table] Example 4 Total Protein Test 50 μ of sample containing unknown or standard was added to 3 ml of reconstituted Abbott A-Gent total protein reagent adjusted to contain 1.5×10 −7 M fluorescein.
Reagents and samples were mixed and incubated at 20°C for at least 10 minutes. The fluorescence intensity of the test solution was measured and the protein 0-8
The results obtained for standard solutions containing g/dl are shown in the following table. Protein concentration (mg/dl) F I 0 54068 4 37383 6 32760 8 27792 From this result, a standard curve can be obtained from which the total protein concentration in unknown samples can be determined. Example 5 LDH test Lithium-
Bovine gamma containing 25μ of a solution containing 250mg/ml of L-lactate and 125mg/ml of NAD, 25μ of a 50% ethanol solution containing 25mg/ml of INT and 1×10 -5 M fluorescein, and 25μ of a 50% glycerol solution containing 100 units/ml of diaphorase. 2.05 ml of phosphate buffer with globulin was added. The test solution obtained by mixing the sample and test solution was incubated at 35°C for 14 minutes, and the fluorescence intensity of the test solution was measured. The results obtained with the standard solution containing 0-500 units of LDH are shown in the table below. LDH activity (units/ml) F I 0 45797 50 43170 100 41470 200 38484 300 35909 500 28021 From this result, a standard curve from which the LDH concentration in unknown samples can be determined can be obtained. Example 6 Creatinine test Picric acid 5.5ml and 2.5N sodium hydroxide 1.1ml
200μ of 10 -5 M fluorescein in 20ml of an aqueous solution containing
was added to prepare a reagent solution containing 10 -7 M fluorescein. 100μ of a sample containing unknown or standard was added to 2ml of the above reagent solution. The test solution was heated at 35℃ for about 15 minutes.
After incubation for a minute, the fluorescence intensity of the test solution was measured. The results obtained for standard solutions containing 0-10 mg/ dl of creatinine are shown in the following table: Creatinine concentration (mg/dl) A standard curve is obtained. Example 7 BUN test 20μ of sample containing unknown or standard 0.1M potassium phosphate (PH7.5) buffer; bovine gamma globulin;
0.1% sodium azide; urease 130 units/ml
25 μl of a 5% glycerol solution containing 300 mg of sodium nitroprusside in 1 ml of water was prepared by adding 5 ml of phenol, 1 ml of glycerol and 1 ml of ethanol and enough fluorescein.
2.050 ml of a test solution consisting of 25 μl of a 10 −5 M fluorescein solution; and 25 μl of a solution prepared by adding 25 g of sodium hydroxide to 50 ml of a solution containing 5.7% sodium hypochlorite was added. The test solution obtained by mixing the sample and test solution was incubated at 35° C. for about 5 minutes. The fluorescence intensity of the test solution was measured and the fluorescence intensity of the standard solution containing urea 0-100mg/dl is shown in the following table: Urea concentration (mg/dl) F I 0 31847 6.25 28713 12.5 25745 25 20962 50 15538 100 8798 This result provides a standard curve from which the urea concentration in unknown samples can be determined. Example 8 Bilirubin Test 2 ml reconstituted Abbott A-Gent bilirubin reagent adjusted to contain 10 -7 M fluorescein.
50 μ of sample containing unknown or standard was added to the sample. The reagent and sample were mixed and incubated at 37°C for about 10 minutes. The fluorescence intensity of the test solution was measured and bilirubin was 0-20.2.
The results obtained for standard solutions containing mg/dl are shown in the following table: Bilirubin concentration (mg/dl) F I 0 65256 4 59371 10.1 50353 20.2 32740 A standard from which the bilirubin concentration in unknown samples can be determined from the results. A curve is obtained. The following table presents the bilirubin concentrations determined for Dade Moni-Trol and chemically prepared samples using the standard curve constructed from the results of the above table.
【表】
実施例 ９
カルシウム試験
1.5×10-7Mフルオレシインを含む様調整され
た再構成アボツトＡ−Gentカルシウム試薬３ml
に未知又はカルシウム標準を含む試料100μを
加えた。試薬と試料を混合し20℃で約10分間培養
した。試験溶液の螢光強さを測定しカルシウム０
−12mg／dlを含む標準液についてえた結果を次表
に示している：カルシウム酸濃度（mg／dl）
FI
０ 41881
８ 30762
10 29029
12 26416
この結果から未知試料中のカルシウム濃度を決
定できる様な標準曲線がえられる。
実施例 10
アルブミン試験
５×10-5Mブリリアントサルフアフラヴインを
含む様調整された再構成アボツトＡ−Gentアル
ブミン試薬２mlに未知又は標準を含む試料10μ
を加えた。試薬と試料を混合し20℃で５分間培養
した。えた試験溶液の螢光強さを測定しアルブミ
ン０−８ｇ／dlを含む標準液についてえた結果は
次表の通りである：アルブミン濃度（mg／dl）
FI
０ 2888
３ 2430
８ 2056
この結果から未知試料中のアルブミン濃度を決
定できる様な標準曲線がえることができる。
実施例 11
塩化物試験
５×10-5Mブリリアントサルフアフラヴインを
含む様調整されたハーレコ塩化物デペローピング
試薬２mlに未知又は標準を含む試料20μを加え
た。えた混合物に0.2M過塩素酸第２水銀溶液１
滴を加ええた試験溶液の螢光強さを測定した。塩
化物０−200meq／を含む標準液についてえた
結果を次表に示す：塩化物濃度（meq／）
FI
０ 54047
50 48959
100 23900
200 10368
この結果から未知試料中の塩化物濃度を決定で
きる様な標準曲線がえられる。
次表は上記表の結果からつくつた標準曲線を用
いてDade Moni−Trolと化学調整試料につ
いてえた塩化物酸濃度を示している：[Table] Example 9 Calcium test 3 ml of reconstituted Abbot A-Gent calcium reagent adjusted to contain 1.5×10 -7 M fluorescein
100 μ of sample containing unknown or calcium standard was added to the sample. The reagent and sample were mixed and incubated at 20°C for about 10 minutes. Measure the fluorescence intensity of the test solution and find that calcium is 0.
The results obtained for the standard solution containing -12 mg/ dl are shown in the following table: Calcium acid concentration (mg/dl) A standard curve is obtained. Example 10 Albumin Test 10μ of sample containing unknown or standard in 2ml of reconstituted Abbott A-Gent albumin reagent adjusted to contain 5 x 10 -5 M brilliant sulfaflavin.
added. The reagent and sample were mixed and incubated at 20°C for 5 minutes. The fluorescence intensity of the test solution obtained was measured and the results obtained for the standard solution containing 0-8 g/dl of albumin are shown in the following table: Albumin concentration (mg/dl) F I 0 2888 3 2430 8 2056 From this result A standard curve can be generated from which the albumin concentration in unknown samples can be determined. Example 11 Chloride Test 20μ of sample containing unknown or standard was added to 2ml of Harleco Chloride Deperoping Reagent prepared to contain 5 x 10 -5 M brilliant sulfaflavin. Add 0.2M mercuric perchlorate solution to the mixture
The fluorescence intensity of the test solution to which the drops were added was measured. The results obtained for standard solutions containing 0-200 meq/chloride are shown in the following table: Chloride concentration (meq/) F I 0 54047 50 48959 100 23900 200 10368 From this result, the chloride concentration in the unknown sample can be determined. A standard curve is obtained. The following table shows the chloride acid concentrations obtained for Dade Moni-Trol and chemically prepared samples using the standard curve created from the results in the table above:
【表】
実施例 12
マグネシウム試験
５×10-7Mフルオレシインを含む様調整された
マグネシウム染料試薬（P.L.バイオケミカルズ
社）1.0mlにマグネシウム主体試薬（P.L.バイオ
ケミカルズ社）1.0mlと未知又は標準を含む試料
20μを加えた。えた試験溶液を20℃で少なくも
１分間放置し試験溶液の螢光強さを測定した。マ
グネシウム０−6.0meq／を含む標準液につい
てえた結果を次表に示している：マグネシウム濃度（meq／）
FI
０ 31612
2.0 22510
4.0 17330
6.0 12802
この結果から未知試料中のマグネシウム濃度を
決定できる標準曲線がえられる。
次表は上記の結果からつくつた標準曲線を用い
てDade Moni−Trolおよび化学調整試料に
ついて測定したマグネシウム濃度を示している：[Table] Example 12 Magnesium test 1.0ml of magnesium dye reagent (PL Biochemicals) adjusted to contain 5×10 -7 M fluorescein contains 1.0ml of magnesium-based reagent (PL Biochemicals) and unknown or standard. sample
Added 20μ. The test solution was allowed to stand at 20°C for at least 1 minute and the fluorescence intensity of the test solution was measured. The results obtained for standard solutions containing 0-6.0 meq/magnesium are shown in the following table: Magnesium concentration (meq/) F I 0 31612 2.0 22510 4.0 17330 6.0 12802 A standard from which the magnesium concentration in unknown samples can be determined. A curve is obtained. The following table shows the magnesium concentrations determined for Dade Moni-Trol and chemically prepared samples using the standard curve generated from the above results:
【表】
実施例 13
グルコース試験
未知又はグルコース標準を含む試料5μにグ
ルコースオキシダーゼ2200単位／ml、チメルゾル
0.025mg／mlおよび1.5M硫酸アンモニウムを含む
液25μ；0.2MDHBS、0.1％フエリシアン化カ
リウム、0.1％４−アミノアンチピレン、0.1％ト
リトンＸ−100、チメルゾル0.025mg／mlおよびリ
ボフラヴイン5.633×10-4モル／を含む0.1Mり
ん酸塩緩衝液25μ；および西洋わさびペルオキ
シダーゼ400単位／ml、10％子牛血清、0.6ｍ
MEDTA、0.1％ANS、１％トリトンＸ−100、
リパーゼ2000単位／ml、チメルゾル0.025ｇ／ml
を含む0.1Mりん酸塩緩衝液25試料を含み牛ガン
マグロブリンを含むりん酸塩緩衝液（PH7.0）
2.05mlを加えた。えた試験溶液を35℃で最大色生
成をえる迄培養した。吸光度と螢光強さを測定し
グルコース０−500mg／dlを含む標準液について
えた結果は次表のとおりである：グルコース濃度（mg／dl）
FI
０ 37432
50 28731
100 22019
200 12547
300 7291
500 2269
実施例 14
イムノグロブリンＧ（IgG）試験
アボツトＡ−GentイムノグロブリンＧ臨床化
学診断箱で供給された試薬を使い山羊抗−人間
IgG抗血清400μを４％ポリエチレングリコール
を含むりん酸塩緩衝塩溶液11.6mlと混合してつく
つた働らく抗血清試薬2.5mlに箱で供給された標
準を用いてアボツトＡ−Gentイムノグロブリン
Ｇ臨床化学診断箱の指示によりつくつた標準液
100μを加えた。混合物に10-5Mフルオレシイン
液25μを加え試験溶液の螢光強さを測定しイム
ノグロブリンG382−6093mg／dlを含む標準液か
らえた結果は次表のとおりである：IgG濃度（mg／dl）
FI
382 10556
761 10262
1526 10132
3046 9397
6093 8849
前記した実施例で明白なとおり、本発明の方法
は広範な試験に応用できるのである。既知発色試
薬系を用いて未知配位子の螢光測定を可能にする
他に本発明の方法は発色試薬系を用いて試験の直
線性を増す。特に本発明の方法は高吸光度値にお
ける試験の直線性範囲を増す。器械の制限によつ
て比色試験の直線性は2.0以上の吸光度をもつ発
色体濃度において実質的に減少することはよく知
られている。本発明の方法を用いて2.0以上の吸
光度をもつ発色体濃度をつくる試薬系と配位子の
濃度を用いて試験の直線性を拡張することは可能
である。
上述のとおり本発明は試験溶液中の配位子濃度
の分光測定又は螢光測定のいづれかに利用できる
新規の試薬組成物に関する。この様な試薬組成物
は配位子に特定の試薬系、即ち測定する配位子を
含む溶液の透光性に変化を与えうる試薬系の有効
量および試薬系と測定する配位子を含む溶液の透
光性変化と関連する波長帯と重なる励起および
（又は）放射波長帯をもつ螢光体の有効量より成
る。本発明の方法により配位子を螢光測定するに
便利な試薬組成物生成に要する螢光体有効量は試
験溶液の透光性測定を妨害しないことはわかつて
いる。故に測定する配位子とその配位子に特定の
本発明の試薬組成物を含む試験溶液はその中の配
位子濃度決定のため分光測定又は螢光測定でき
る。
本発明を特定実施態様について記述したが、本
発明の真意や範囲から逸脱しない限り種々の同等
法、変更法および修正法もできることは明らかで
あろうから、実施態様が本発明を限定するものと
考えるべきでないしまた同等の実施態様も本発明
に包含されるものと考えるものである。[Table] Example 13 Glucose Test Glucose oxidase 2200 units/ml, thymersol to 5μ of sample containing unknown or glucose standard
25μ of a solution containing 0.025mg/ml and 1.5M ammonium sulfate; 0.2MDHBS, 0.1% potassium ferricyanide, 0.1% 4-aminoantipyrene, 0.1 % Triton 25μ of 0.1M phosphate buffer containing; and 400 units/ml of horseradish peroxidase, 10% calf serum, 0.6M
MEDTA, 0.1% ANS, 1% Triton X-100,
Lipase 2000 units/ml, Thimersol 0.025g/ml
0.1M phosphate buffer containing 25 samples containing phosphate buffer containing bovine gamma globulin (PH7.0)
Added 2.05ml. The resulting test solution was incubated at 35°C until maximum color production was achieved. The results obtained by measuring the absorbance and fluorescence intensity for standard solutions containing glucose 0-500 mg/dl are shown in the following table: Glucose concentration (mg/dl) F I 0 37432 50 28731 100 22019 200 12547 300 7291 500 2269 Example 14 Immunoglobulin G (IgG) Test Goat anti-human using reagents supplied in the Abbott A-Gent Immunoglobulin G Clinical Chemistry Box.
A working antiserum reagent made by mixing 400 µl of IgG antiserum with 11.6 ml of phosphate buffered saline containing 4% polyethylene glycol was prepared using the standard supplied in the box with Abbott A-Gent Immunoglobulin G Clinical. Standard solution prepared according to the instructions of the chemical diagnostic box
Added 100μ. Adding 25μ of 10 -5 M fluorescein solution to the mixture and measuring the fluorescence intensity of the test solution, the results obtained from the standard solution containing immunoglobulin G382-6093mg/dl are shown in the following table: IgG concentration (mg/dl) F I 382 10556 761 10262 1526 10132 3046 9397 6093 8849 As is clear from the examples described above, the method of the invention can be applied to a wide range of tests. In addition to allowing fluorescence measurements of unknown ligands using a known chromogenic reagent system, the method of the present invention uses a chromogenic reagent system to increase the linearity of the test. In particular, the method of the invention increases the linearity range of the test at high absorbance values. It is well known that due to instrumental limitations, the linearity of colorimetric tests is substantially reduced at chromophore concentrations with absorbance greater than 2.0. Using the method of the present invention, it is possible to extend the linearity of the test using reagent systems and ligand concentrations that create chromophore concentrations with absorbances greater than 2.0. As stated above, the present invention relates to novel reagent compositions that can be used for either spectroscopic or fluorometric measurements of ligand concentrations in test solutions. Such reagent compositions include a specific reagent system for the ligand, i.e., an effective amount of the reagent system capable of altering the translucency of a solution containing the ligand to be measured, and the reagent system and the ligand to be measured. It comprises an effective amount of a fluorophore having excitation and/or emission wavelength bands that overlap with the wavelength band associated with the change in translucency of the solution. It has been found that the effective amount of fluorophore required to produce a reagent composition convenient for fluorometry of ligands by the method of the present invention does not interfere with the measurement of translucency of the test solution. Thus, a test solution containing the ligand to be measured and a reagent composition of the invention specific to that ligand can be spectrophotometrically or fluorometrically measured to determine the concentration of the ligand therein. Although the invention has been described with respect to particular embodiments, it will be obvious that various equivalents, alterations and modifications may be made without departing from the spirit or scope of the invention, so the embodiments should not be construed as limitations on the invention. It is not to be considered and equivalent embodiments are considered to be encompassed by this invention.
Claims (1)
び（又は）放射波長帯と重複する波長帯内で
試験溶液の透光性に変化を与えうる様な試薬
系の有効量 を混合し、 (b) 試験溶液を螢光体の励起波長帯内の波長をも
つ光で照射した後、 (c) 試験溶液により放射された螢光強さを試料中
の配位子濃度の尺度として測定する ことを特徴とする配位子を含むと思われる試料中
の配位子測定法。 ２ 試薬系が発色試薬系又は濁度測定試薬系であ
る特許請求の範囲第１項に記載の方法。 ３ 試薬系が発色試薬系である特許請求の範囲第
２項に記載の方法。 ４ 試験溶液の透光性変化と関連する吸光波長帯
が螢光体の励起波長帯と重複する特許請求の範囲
第３項に記載の方法。 ５ 試験溶液の透光性変化と関連する吸光波長帯
が螢光体の放射波長帯と重複する特許請求の範囲
第３項に記載の方法。 ６ 試験溶液の透光性変化と関連した吸光波長帯
が螢光体の励起および放射波長帯と重なる特許請
求の範囲第３項に記載の方法。 ７ 配位子を含んでいると思われる試料と配位子
の存在で波長帯内で試験溶液の透光性変化を与え
うる試薬系の有効量を含む試験溶液中の配位子測
定用の改良螢光測定法において、試験溶液にその
透光性変化と関連した波長帯と重複する励起およ
び（又は）放射波長帯をもつ螢光体の有効量を加
え、試験溶液を螢光体の励起波長帯内の波長をも
つ光で照射した後試験溶液によつて放射された螢
光を試料中の配位子濃度の尺度として測定するこ
とを特徴とする改良法。 ８ 試薬系が発色試薬系又は濁度測定試薬系であ
る特許請求の範囲第７項に記載の方法。 ９ 試薬系が発色試薬系である特許請求の範囲第
８項に記載の方法。 １０ 螢光体が試験溶液の透光性変化と関連した
吸光波長帯と重複する励起波長帯をもつ特許請求
の範囲第９項に記載の方法。 １１ 螢光体が試験溶液の透光性変化と関連した
吸光波長帯と重複する放射波長帯をもつ特許請求
の範囲第９項に記載の方法。 １２ 螢光体が試験溶液の透光性変化と関連した
吸光波長帯と重複する励起波長帯と放射波長帯を
もつ特許請求の範囲第９項に記載の方法。[Claims] 1. (a) to produce a test solution: (i) a sample suspected of containing the ligand; (ii) an effective amount of fluorophore; (iii) in the presence of the ligand to be measured. (b) mixing an effective amount of a reagent system capable of producing a change in the translucency of the test solution within a wavelength range that overlaps the excitation and/or emission wavelength range of the fluorophore; (c) measuring the fluorescence intensity emitted by the test solution as a measure of the concentration of the ligand in the sample; A method for measuring ligands in samples that are likely to contain them. 2. The method according to claim 1, wherein the reagent system is a coloring reagent system or a turbidity measuring reagent system. 3. The method according to claim 2, wherein the reagent system is a coloring reagent system. 4. The method according to claim 3, wherein the absorption wavelength band associated with the change in translucency of the test solution overlaps with the excitation wavelength band of the phosphor. 5. The method according to claim 3, wherein the absorption wavelength band associated with the change in translucency of the test solution overlaps with the emission wavelength band of the phosphor. 6. The method of claim 3, wherein the absorption wavelength band associated with the change in translucency of the test solution overlaps with the excitation and emission wavelength bands of the fluorophore. 7 For the measurement of ligands in test solutions containing a sample suspected of containing a ligand and an effective amount of a reagent system capable of altering the translucency of the test solution within the wavelength range in the presence of the ligand. In a modified fluorometry method, an effective amount of a fluorophore with excitation and/or emission wavelength bands that overlap the wavelength band associated with the change in translucency is added to the test solution; An improved method characterized in that the fluorescence emitted by the test solution after irradiation with light having a wavelength within the wavelength range is measured as a measure of the concentration of the ligand in the sample. 8. The method according to claim 7, wherein the reagent system is a coloring reagent system or a turbidity measuring reagent system. 9. The method according to claim 8, wherein the reagent system is a coloring reagent system. 10. The method of claim 9, wherein the fluorophore has an excitation wavelength band that overlaps with the absorption wavelength band associated with the translucency change of the test solution. 11. The method of claim 9, wherein the phosphor has an emission wavelength band that overlaps with the absorption wavelength band associated with the change in translucency of the test solution. 12. The method of claim 9, wherein the fluorophore has excitation and emission wavelength bands that overlap with the absorption wavelength band associated with the translucency change of the test solution.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US06/469,323 US4495293A (en) | 1983-02-24 | 1983-02-24 | Fluorometric assay |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS60202361A JPS60202361A (en) | 1985-10-12 |
| JPH037906B2 true JPH037906B2 (en) | 1991-02-04 |
Family
ID=23863331
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP5456784A Granted JPS60202361A (en) | 1983-02-24 | 1984-03-23 | Improved fluorescence measuring method |
Country Status (3)
| Country | Link |
|---|---|
| JP (1) | JPS60202361A (en) |
| AU (1) | AU564398B2 (en) |
| ZA (1) | ZA841911B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3376685D1 (en) * | 1982-12-21 | 1988-06-23 | Ares Serono Nv | Assay technique |
| US5370114A (en) * | 1992-03-12 | 1994-12-06 | Wong; Jacob Y. | Non-invasive blood chemistry measurement by stimulated infrared relaxation emission |
-
1984
- 1984-03-13 AU AU25556/84A patent/AU564398B2/en not_active Ceased
- 1984-03-14 ZA ZA841911A patent/ZA841911B/xx unknown
- 1984-03-23 JP JP5456784A patent/JPS60202361A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| AU2555684A (en) | 1985-09-19 |
| AU564398B2 (en) | 1987-08-13 |
| JPS60202361A (en) | 1985-10-12 |
| ZA841911B (en) | 1984-09-07 |
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