GB2055200A - Enhancement of the sensitivity of bioluminescent and chemiluminescent assays with enzymatic cycling - Google Patents
Enhancement of the sensitivity of bioluminescent and chemiluminescent assays with enzymatic cycling Download PDFInfo
- Publication number
- GB2055200A GB2055200A GB8023995A GB8023995A GB2055200A GB 2055200 A GB2055200 A GB 2055200A GB 8023995 A GB8023995 A GB 8023995A GB 8023995 A GB8023995 A GB 8023995A GB 2055200 A GB2055200 A GB 2055200A
- Authority
- GB
- United Kingdom
- Prior art keywords
- atp
- cycling
- enzymatic
- concentration
- bioluminescent
- 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.)
- Granted
Links
- 230000001351 cycling effect Effects 0.000 title claims abstract description 69
- 230000002255 enzymatic effect Effects 0.000 title claims abstract description 46
- 238000003556 assay Methods 0.000 title claims abstract description 27
- 230000035945 sensitivity Effects 0.000 title abstract description 15
- 239000000758 substrate Substances 0.000 claims abstract description 36
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 claims abstract description 25
- 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 claims abstract description 17
- 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 claims abstract description 12
- 238000006243 chemical reaction Methods 0.000 claims description 20
- 102000004190 Enzymes Human genes 0.000 claims description 17
- 108090000790 Enzymes Proteins 0.000 claims description 17
- 239000003153 chemical reaction reagent Substances 0.000 claims description 12
- 241000254158 Lampyridae Species 0.000 claims description 11
- 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 claims description 11
- 230000003321 amplification Effects 0.000 claims description 11
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 11
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 claims description 7
- 230000007306 turnover Effects 0.000 claims description 7
- 108020005115 Pyruvate Kinase Proteins 0.000 claims description 5
- 102000013009 Pyruvate Kinase Human genes 0.000 claims description 5
- 102000002281 Adenylate kinase Human genes 0.000 claims description 4
- 108020000543 Adenylate kinase Proteins 0.000 claims description 4
- 230000001580 bacterial effect Effects 0.000 claims description 4
- 239000000126 substance Substances 0.000 claims description 4
- ACFIXJIJDZMPPO-NNYOXOHSSA-N NADPH 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](OP(O)(O)=O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 ACFIXJIJDZMPPO-NNYOXOHSSA-N 0.000 claims description 3
- 101710204616 Pyruvate kinase 2 Proteins 0.000 claims description 2
- 238000011534 incubation Methods 0.000 claims description 2
- 239000002773 nucleotide Substances 0.000 claims description 2
- 150000002978 peroxides Chemical class 0.000 claims description 2
- 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 claims 14
- 102000003829 Adenylate kinase 2 Human genes 0.000 claims 1
- 108090000115 Adenylate kinase 2 Proteins 0.000 claims 1
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 claims 1
- 230000000694 effects Effects 0.000 claims 1
- 238000006911 enzymatic reaction Methods 0.000 claims 1
- 125000003729 nucleotide group Chemical group 0.000 claims 1
- 238000005259 measurement Methods 0.000 abstract description 19
- 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 abstract description 16
- 150000003222 pyridines Chemical class 0.000 abstract description 15
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 abstract description 6
- 238000005558 fluorometry Methods 0.000 abstract description 6
- 238000002798 spectrophotometry method Methods 0.000 abstract description 4
- ZKHQWZAMYRWXGA-KQYNXXCUSA-N Adenosine triphosphate 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-N 0.000 abstract description 3
- GFFGJBXGBJISGV-UHFFFAOYSA-N adenyl group Chemical class N1=CN=C2N=CNC2=C1N GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 abstract description 3
- 238000001952 enzyme assay Methods 0.000 abstract description 2
- DRBBFCLWYRJSJZ-UHFFFAOYSA-N N-phosphocreatine Chemical compound OC(=O)CN(C)C(=N)NP(O)(O)=O DRBBFCLWYRJSJZ-UHFFFAOYSA-N 0.000 description 14
- 238000000034 method Methods 0.000 description 14
- 239000000523 sample Substances 0.000 description 14
- 229950006238 nadide Drugs 0.000 description 9
- 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 8
- 230000029918 bioluminescence Effects 0.000 description 8
- 238000005415 bioluminescence Methods 0.000 description 8
- 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 6
- 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 6
- CVSVTCORWBXHQV-UHFFFAOYSA-N creatine Chemical compound NC(=[NH2+])N(C)CC([O-])=O CVSVTCORWBXHQV-UHFFFAOYSA-N 0.000 description 6
- 229940049920 malate Drugs 0.000 description 5
- BJEPYKJPYRNKOW-UHFFFAOYSA-N malic acid Chemical compound OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 description 5
- KHPXUQMNIQBQEV-UHFFFAOYSA-L oxaloacetate(2-) Chemical compound [O-]C(=O)CC(=O)C([O-])=O KHPXUQMNIQBQEV-UHFFFAOYSA-L 0.000 description 5
- 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 4
- BIRSGZKFKXLSJQ-SQOUGZDYSA-N 6-Phospho-D-gluconate Chemical compound OP(=O)(O)OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O BIRSGZKFKXLSJQ-SQOUGZDYSA-N 0.000 description 4
- IKHGUXGNUITLKF-UHFFFAOYSA-N Acetaldehyde Chemical compound CC=O IKHGUXGNUITLKF-UHFFFAOYSA-N 0.000 description 4
- 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 4
- 238000010438 heat treatment Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- DTBNBXWJWCWCIK-UHFFFAOYSA-K phosphonatoenolpyruvate Chemical compound [O-]C(=O)C(=C)OP([O-])([O-])=O DTBNBXWJWCWCIK-UHFFFAOYSA-K 0.000 description 4
- 108010021809 Alcohol dehydrogenase Proteins 0.000 description 3
- 102000007698 Alcohol dehydrogenase Human genes 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 239000005089 Luciferase Substances 0.000 description 3
- 108010026217 Malate Dehydrogenase Proteins 0.000 description 3
- 102000013460 Malate Dehydrogenase Human genes 0.000 description 3
- BAWFJGJZGIEFAR-NNYOXOHSSA-O NAD(+) 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-O 0.000 description 3
- 102000004316 Oxidoreductases Human genes 0.000 description 3
- 108090000854 Oxidoreductases Proteins 0.000 description 3
- 229960003624 creatine Drugs 0.000 description 3
- 239000006046 creatine Substances 0.000 description 3
- 229930029653 phosphoenolpyruvate Natural products 0.000 description 3
- AWUCVROLDVIAJX-GSVOUGTGSA-N sn-glycerol 3-phosphate Chemical compound OC[C@@H](O)COP(O)(O)=O AWUCVROLDVIAJX-GSVOUGTGSA-N 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 102000004420 Creatine Kinase Human genes 0.000 description 2
- 108010042126 Creatine kinase Proteins 0.000 description 2
- 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 2
- 101710088194 Dehydrogenase Proteins 0.000 description 2
- YTNIXZGTHTVJBW-SCRDCRAPSA-L FMNH2(2-) Chemical compound [O-]P(=O)([O-])OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2NC2=C1NC(=O)NC2=O YTNIXZGTHTVJBW-SCRDCRAPSA-L 0.000 description 2
- 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 2
- 102100035172 Glucose-6-phosphate 1-dehydrogenase Human genes 0.000 description 2
- 102100023903 Glycerol kinase Human genes 0.000 description 2
- 108010093096 Immobilized Enzymes Proteins 0.000 description 2
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 2
- 108060001084 Luciferase Proteins 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- 238000010521 absorption reaction Methods 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
- 150000001299 aldehydes Chemical class 0.000 description 2
- 239000012491 analyte Substances 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- FVTCRASFADXXNN-SCRDCRAPSA-N flavin mononucleotide Chemical compound OP(=O)(O)OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O FVTCRASFADXXNN-SCRDCRAPSA-N 0.000 description 2
- 229940013640 flavin mononucleotide Drugs 0.000 description 2
- FVTCRASFADXXNN-UHFFFAOYSA-N flavin mononucleotide Natural products OP(=O)(O)OCC(O)C(O)C(O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O FVTCRASFADXXNN-UHFFFAOYSA-N 0.000 description 2
- 239000011768 flavin mononucleotide Substances 0.000 description 2
- 229960002163 hydrogen peroxide Drugs 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 description 2
- 239000011777 magnesium Substances 0.000 description 2
- 235000021317 phosphate Nutrition 0.000 description 2
- 235000019231 riboflavin-5'-phosphate Nutrition 0.000 description 2
- 238000007086 side reaction Methods 0.000 description 2
- JVTAAEKCZFNVCJ-UWTATZPHSA-M (R)-lactate Chemical compound C[C@@H](O)C([O-])=O JVTAAEKCZFNVCJ-UWTATZPHSA-M 0.000 description 1
- CIEYFQRZEULHJV-UHFFFAOYSA-N 1,1-dihydroxypropan-2-one;phosphoric acid Chemical compound OP(O)(O)=O.CC(=O)C(O)O CIEYFQRZEULHJV-UHFFFAOYSA-N 0.000 description 1
- WGLQHUKCXBXUDV-UHFFFAOYSA-N 3-aminophthalic acid Chemical compound NC1=CC=CC(C(O)=O)=C1C(O)=O WGLQHUKCXBXUDV-UHFFFAOYSA-N 0.000 description 1
- 102000004567 6-phosphogluconate dehydrogenase Human genes 0.000 description 1
- 108020001657 6-phosphogluconate dehydrogenase Proteins 0.000 description 1
- 108010029731 6-phosphogluconolactonase Proteins 0.000 description 1
- 102000006267 AMP Deaminase Human genes 0.000 description 1
- 108700016228 AMP deaminases Proteins 0.000 description 1
- 229930024421 Adenine Natural products 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
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 108010066906 Creatininase Proteins 0.000 description 1
- IGXWBGJHJZYPQS-SSDOTTSWSA-N D-Luciferin Chemical compound OC(=O)[C@H]1CSC(C=2SC3=CC=C(O)C=C3N=2)=N1 IGXWBGJHJZYPQS-SSDOTTSWSA-N 0.000 description 1
- 108010001539 D-lactate dehydrogenase Proteins 0.000 description 1
- FNZLKVNUWIIPSJ-UHNVWZDZSA-N D-ribulose 5-phosphate Chemical compound OCC(=O)[C@H](O)[C@H](O)COP(O)(O)=O FNZLKVNUWIIPSJ-UHNVWZDZSA-N 0.000 description 1
- CYCGRDQQIOGCKX-UHFFFAOYSA-N Dehydro-luciferin Natural products OC(=O)C1=CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 CYCGRDQQIOGCKX-UHFFFAOYSA-N 0.000 description 1
- 108700033376 EC 1.1.1.49 Proteins 0.000 description 1
- 108700035269 EC 1.1.1.8 Proteins 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 108010014531 FMN Reductase Proteins 0.000 description 1
- BJGNCJDXODQBOB-UHFFFAOYSA-N Fivefly Luciferin Natural products OC(=O)C1CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 BJGNCJDXODQBOB-UHFFFAOYSA-N 0.000 description 1
- 108010018962 Glucosephosphate Dehydrogenase Proteins 0.000 description 1
- 102000016901 Glutamate dehydrogenase Human genes 0.000 description 1
- 108700023156 Glutamate dehydrogenases Proteins 0.000 description 1
- 229930195714 L-glutamate Natural products 0.000 description 1
- 102000003855 L-lactate dehydrogenase Human genes 0.000 description 1
- 108700023483 L-lactate dehydrogenases Proteins 0.000 description 1
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 1
- DDWFXDSYGUXRAY-UHFFFAOYSA-N Luciferin Natural products CCc1c(C)c(CC2NC(=O)C(=C2C=C)C)[nH]c1Cc3[nH]c4C(=C5/NC(CC(=O)O)C(C)C5CC(=O)O)CC(=O)c4c3C DDWFXDSYGUXRAY-UHFFFAOYSA-N 0.000 description 1
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 description 1
- 102000002247 NADPH Dehydrogenase Human genes 0.000 description 1
- 108010014870 NADPH Dehydrogenase Proteins 0.000 description 1
- 101710163410 Probable glycerol kinase Proteins 0.000 description 1
- FNZLKVNUWIIPSJ-UHFFFAOYSA-N Rbl5P Natural products OCC(=O)C(O)C(O)COP(O)(O)=O FNZLKVNUWIIPSJ-UHFFFAOYSA-N 0.000 description 1
- 229960000643 adenine Drugs 0.000 description 1
- 229960005305 adenosine Drugs 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- DNJIEGIFACGWOD-UHFFFAOYSA-N ethyl mercaptane Natural products CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 description 1
- 229930195712 glutamate Natural products 0.000 description 1
- AWUCVROLDVIAJX-UHFFFAOYSA-N glycerol 1-phosphate Chemical compound OCC(O)COP(O)(O)=O AWUCVROLDVIAJX-UHFFFAOYSA-N 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 229910001425 magnesium ion Inorganic materials 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- -1 nucleotide phosphates Chemical class 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Bioluminescent and chemiluminescent assays coupled with enzymatic cycling systems greatly increase the versatility and sensitivity of substrate and enzyme assays based on enzymatic cycling principle. Besides the normal pyridine nucleotide cycling system it is possible to use the adenine nucleotide system when the produced substrates are measured by the bioluminescent assay of ATP. Utilizing the bioluminescent assays of NADH, NADPH or ATP, or the chemiluminescent assay of H2O2 or O2 it is possible to measure more substrates produced in the enzymatic cycling systems than has been possible by spectrophotometry or fluorometry. The sensitivity of the measurement with the luminescent assay systems are 2 to 3 orders of magnitude higher than with fluorometry.
Description
SPECIFICATION
Enhancement of the sensitivity of bioluminescent and chemiluminescent assays with enzymatic cycling
Enzymatic cycling is a method of a chemical amplification of an analyte (W. J. Blaedel and R. G.
Boguslaski; Anal. Chem. 50:1026-1032, 1 978). Enzymatic cycling applies alternate oxidating and reduction of pyridine nucleotides by two specific enzymes in a cycling fashion (T. Kato, S. J. Berger, J. A.
Cater and D. H. Lowry; Anal. Biochem. 53: 86-97, 1973). During this enzymatic cycling one molecule is accumulated during each cycle of the pyridine nucleoxide. This type of system can turn over from 2,000 to 30,000 times per hour multiplying the quantity the analyte by this high factor. Enzymatic cycling has been applied to increase the sensitivity of spectrophotometric and fluorometric substrate and enzyme assays.
Fig. 1. schematically shows the enzymatic cycling applying nicotinamide-adenine dinucleotide in oxidated form (NAD+) and reduced form (NADH) and alcohol dehydrogenase enzyme (EC 1.1.1.1) and malic dehydrogenase enzyme (EC 1.1.1.37) to produce aldehyde and malate substrates. The turnover is over 30.000 times per hour and the number of actual molecules produced is proportional to the concentration of NAD+ or NADH in the sample.
-If the enzymes are pure, there are no side reactions utilizing the pyridine nucleotides (NAD and
NADH), the concentration of the pyridine nucleotides in the sample stays constant throughout the cycling procedure. Amplification factor is determined by the turnover rates of the enzymes and the final concentration of accumulated substrate is proportional to the stoichiometric amount of pyridine nucleotide in the sample.
Useful indicator reactions for measuring the produced substrates in the system, shown in
Figure 1., are: 1. Malate + NADPH + malic
enzyme pyruvate + CO2 + NADP + H+ or 2. Malate + NAD + malic
dehydrogenase oxalacetate + NADH +
The measurement of malate is made either spectrophotometrically or fluorometrically on the basis of absorption or fluorescence of pyridine nucleotides, respectively.
In theory it should also be possible to measure oxalacetate produced in the cycling reaction in
Figure 1., but when the cycling is stopped by heating samples to 100 C, oxalacetate breaks down.
The procedure of enzymatic cycling with pyridine nucleotides of the system in Figure 1. is as following:
For the cycling reagent there is prepared a mixture of controlled quantities of alcohol dehydrogenase (20--200 Mg/ml) and malic dehydrogenase (0.5-10 Iss/ml) and non-limiting concentrations of ethanol (200-500 mM) and oxalacetate (1-2 mM) in 100 mM Tris-HCI (pH 8.0) buffer containing 2 mM mercapto-ethanol and 0.02% bovine serum albumin.
1. 10 yI sample containing NAD is added to 100,us of cycling reagent at OOC. Blanks and standards are prepared similarly. Everything should be pipetted as fast as possible to avoid differences in the reaction times.
2. Samples are incubated for 60 minutes at +250C.
3. Samples are heated for 3 minutes at 1000C.
4. Indicator reagent is added and incubated 3-20 minutes to convert malate to pyruvate (reaction 1) or to oxalacetate (reaction 2) and the fluorescence of produced NADP or NADH, respectively, is measured.
The disadvantages of the spectrophotometric and fluorometric measurements of the concentration of substrates produced in the enzymatic cycling are that the lower limit of fluorometric measurement is with one cycling in the order of 1 0-14 moles and with two cyclings 1 0-15 moles. These limits are caused by the sensitivity of fluorescence principle itself and the blank values of the reagent system. Sensitivity of spectrophotometry is even less. Both these methods also suffer from non-specific absorption (spectrophotometry) and fluorescence of other substrates than the measured product (fluorometry).
Bioluminescence assays are of two to three orders magnitude more sensitive and also more specific to the measured substance than either spectrophotometry or fluorometry. The practical sensitivity limit of firefly bioluminescence is at 10-16 moles and photobacterial bioluminescence 10-'5 moles (Wettermark, G., H. Stymne, S. E. Brolin and B. Petterson. Anal. Biochem. 63 :293-307, 1975; Haggerty, C., E. Jablonski, L. Star and DeLuca. Anal. Biochem. 88:162-173, 1978). Thus the direct measurement is already more sensitive than the enzymatic cycling method using fluorometry.
However, when enzymatic cycling is applied, it is possible to get down to 1 0-1S1 0-20 moles of a substrate.
Bioluminescent assay coupled with the enzymatic cycling offers great versatility as it is possible to apply both adenine and pyridine nucleotides for the cycling process as compared to conventional
methods of applying the pyridine nucleotides only.
Bioluminescent assays of ATP with firefly system and pyridine nucleotides (NADH, NADPH) with
bacterial system are known per se (M. DeLuca: Bioluminescence and Chemiluminescence, Methods-in
Enzymology, Vol. 48, Academic Press, New York, N.Y., 1978). The utilization of enzymatic cycling in
connection with bioluminescent assays is not known before. The need to further improve the sensitivity
of bioluminescent assays with enzymatic cycling applies to measurement of substrates and enzymes with low cost instrumentation that do not offer the high photon sensitivity, or with sensitive instruments to allow the detection of single bacteria or measurement of enzyme and substrates in single cellular
level as well as for immuno-assays.
Exampies of enzymatic cycling for bioluminescent assays are as following:
A. Amplification of ATP (adenosine triphosphatej concentration. ATP concentration in the sample
can be amplified several orders of magnitude by an enzymatic cycling system illustrated in Figure 2.
The amplification of ATP is accomplished through an enzymatic cycling with adenosine
monophosphate (AMP) and phosphoenol pyruvate (PEP) as substrates and adenylate kinase (ATP: AMP
phosphotransferase EC 2.7.4.3) and pyruvate kinase (ATP: pyruvate 2-O-ph;osphotransferase EC
2.7.1.40) as enzymes. With each turnover one molecule of ATP is produced in excess by pyruvate
kinase 2 ADP + 2 PEP
PK
pyruvate + 2 ATP.
-In 60 minutes the original ATP concentration can be amplified 1.000 to 10.000 times. The reaction produces one extra molecule of ATP for each cycle and the produced ATP is directly proportional to the original concentration of ATP in the sample and the number of enzyme cycles. Produced ATP is measured with the firefly bioluminescent system.
The inhibition of high concentration of AMP in the firefly reaction can be eliminated by converting
AMP to other metabolites with enzymes, e.g. with AMP deaminase.
Procedure for the amplification of ATP concentration in the sample: - A 10-100 yI sample containing 1 0-1a1 0-12 moles of ATP is pipetted to -100-1,000 yI, cycling reagent containing 50-500 units/ml of adenylate kinase and 5-50 units/ml pyruvate kinase, and 1-5 mM AMP (adenosine monophosphate) and 1-5 mM PEP-(phosphonol pyruvate) in 0.1 M
Tris-HCI or phosphate buffer, pH 7.0-8.0 containing 5-20 mM Mg+±ion and 5-20 mM K±ion and 4 mM EDTA. Samples, blanks and ATP standards-are pipetted within 5 minutes and reagents and samples kept cool at +OOC during the pipetting to lower the reaction rate during pipetting.
- Samples are incubated at 25-3O0C for 30 or 60 minutes.
-The cyc!ing reaction is stopped by heating the samples to 1 000C for 3 minutes.
-Accumulated ATP is measured with the firefly bioluminescent assay as following: An aliquote of sample solution 10-1,000 yI, but preferably 100 pl is taken and added to 10-100 yI firefly luciferin-luciferase reagent, but preferably 100 ,al containing 0.01-2 y9, but preferably 0.1--0.5 ug luciferase and 0.01-1 mM, but preferably 0.1-0.5 mM luciferin in a biochemical buffer having 5-50 mM magnesium ion, preferably 7-10 mM and pH between 7-8.5, but preferably between 7.4-7.8, and the emitted light intensity measured as a peak or as an integrated value over a preset time. The value of light intensity is converted to ATP by comparing it to the light intensity produced by a known ATP standard, measured similarly.
-The original ATP concentration in the samples is calculated by comparing the value after cycling to those of standards treated similarly and after substrating blank values from both. Enzymes have to be purified and free from contaminating enzymes that could cause side reactions. Substrates (AMP and PEP) should not have ADP (adenosine diphosphate) or AMP contamination.
B. Amplification of a substrate through ATP-ADP conversion is enzymatic cycling: An analogous method to conventional enzymatic cycling (Figure 1.) is illustrated in Figure 3. by the amplification of creatine phosphate (CP) with creatine and PEP as substrate and ATP in sample with creatine kinase (ATP: creatine N-phosphotransferase EC 2.7.3.2) and pyruvate kinase. One molecule of creatine phosphate (CP) and pyruvate are produced during each turnover of the ATP-ADP system. After stopping the cycling by heating, creatine phosphate can be converted to creatine and ATP in the indicator reaction during a 5-1 5 minute incubation.
Creatininase is used in the indicator reagent to pull the
CP + ADF
CK
ATP + creatine reaction to completion. The quantity of CP produced is proportional to the quantity of ATP in the sample. ATP produced in the indicator reaction is measured with the firefly bioluminescent assay.
C. Amplification of a substrate through pyridine nucleotide oxido-reduction in enzymatic cycling: It is possible to produce substrates that can be measured with the bioluminescent assay of ATP using a NADH-NAD or NADPH-NADP cycling system. Figure 4. shows an example for production of glycerol3-phosphate (G-3-P) using NAD±NADH cycling, dihydroxyaceton phosphate (DAP) and lactate as substrates and glycerin-3-phosphate dehydrogenase (SM-glycerol-3 phosphate: NAD 2-oxidereductase
EC 1.1.1.8) and lactate dehydrogenase (D-lactate: NAD oxidoreductase EC 1.1.1.28) as cycling enzymes. Produced glycerol-3-phosphate is converted with glycerokinase (ATP: glycerol-3 phosphotransferase EC 2.7.1.30) and ADP to ATP to be measured with the firefly bioluminescent assay.
D. Amplification of a substrate with pyridine nucleotide oxidoreduction for measurement
with bacterial bioluminescence: Figure 5. gives an example of an enzymatic cycling system where 6-phospho-gluconate and glutamate are produced with NADP-NADPH oxidoreduction system.
6-P-gluconate is converted to ribulose-5-phosphate and NADPH. NADP concentration in the sample is amplified with glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP l-oxidoreductase (EC 1.1.1.49) and glutamate dehydrogenase (L-glutamate: NAD(P) oxidoreductase EC 1.4.1.3) using glucose-6-phosphate, a-ketoglutarate and ammonia as substrates. Produced 6-phosphoglucomate (6-P-gluconate) is converted to a stoechiometric quantity of NADPH with NADP and 6-phosphogluconate dehydrogenase (6-phospho-D-gluconate: NADP 2-oxidoreductase EC 1.1.1.44).
Produced NADPH is measured with bacterial bioluminescence using the system given in Figure 6.
Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is used to reduce flavin mononucleotide (FMN) to FMNH2 by NADPH: FMN oxidoreductase (EC 1.6.99.-) FMNH2 serves a role of the luceferin and reacts with oxygen and a long-chain aldehyde (8-1 6 carbon chain) when catalyzed by luciferase, producing photons in the blue regions of visible light.
Bioluminescence assays of ATP, NADH and NADPH greatly increase the sensitivity and versatility of enzymatic cycling methods for measurements of substrates. The application of ATP-ADP conversion systems allows the use of many of the about 220 ATP-specific enzymes for enzymatic cycling, thus the possibility of measurement of adenylate and other nucleotide phosphates as well as
ATP-specific enzymes.
E. Utilization of chemiluminescent assays for measurement of substrates produced in enzymatic cycling: High specificity and sensitivity of certain chemiluminescent assays of hydrogen-peroxide and suporoxide increase the range of substrates measurements in conjunction with enzymatic cycling. This is performed by applying oxidase enzymes which react with a product of an enzymatic cycling system.
In Figure 1. acetaldehyde is one product of the cycling reaction. After stopping the reaction by heating, the following indicator reaction involving an oxidase enzyme can be used to produce hydrogen peroxide: acetaldehyde + H20 - aldehyde
oxidase H2O2 + 1 Produced peroxide is then measured with luminol (5-amino-2.3-dihydrophtalazine-1 .4-dione) in alkaline medium using a first transition series metal as a catalyst:: luminol + 2H2O2 + OH - Fe++
pH 10-12 aminophthalic acid + 3H2O + N2 + photon (R max. 425 nm)
With this it is possible to measure down to 10-'0 moles of H202, thus concentration of NAD+ down to 1 0-14 moles can be assayed with chemiiuminescence using enzymatic cycling procedure.
Utilization of both adenylate phosphate and pyridine nucleotides in enzymatic cycling systems increase the versatility of chemical amplification with enzymatic cycling. The sensitivity and versatility of enzymatic cycling technique is further increased by applying bioluminescent and chemiluminescent assays for the measurement of the produced substrates. With fluorometry and enzymatic cycling it is possible to measure pyridine nucleotides down to 1 0-111 0-12 moles with single cycling and down to 10-'5 moles with double cycling. With bioluminescence it is possible to measure pyridine nucleotides down to 10-'4 and adenine nucleotides down to 10-'5 moles with a single cycle.
Chemiluminescence allows the measurement of substrates down to 10-14 moles with single cycle.
With double cycling and utilizing luminescent assays for measurement, it is possible to go down another four orders of magnitude, thus to 10-'8--1 0-'9 moles, if the cycling reagents are highly purified.
Luminescent measurement increases the sensitivity of the enzymatic cycling method up to 2-3 orders of magnitude and increases the number as compared with the conventional spectrophotometric and fluorometric methods.
In practice the enzymatic cycling for measurement of a substrate can be performed by adding to a sample a prepared mixture of other substrates and the enzymes required in the particular cycling system. This means that only one reagent mixture is needed. It is also possible to apply immobilized enzymes for the cycling, in which case only the additional substrates have to be dispensed to the sample after the sample is in contact with the immobilized enzymes.
Enzymatic cycling for amplifying concentration of a substrate for measurement of it with bioluminescent or chemiluminescent assay is simple to perform with ready-made reagent mixtures, and offers a sensitivity limit of more than two orders of magnitude over fluorometric method for the same assays. Application of bioluminescent and chemiluminescent measurement widely increase the choice of enzymatic cycling system because ATP, NADH, NADPH and H202 based reactions can be measured.
Claims (11)
1. A system of utilizing bioluminescent and chemiluminescent assays for measuring substrate concentrations and-enzyme activities in conjunction with different enzymatic cycling systems.
2. A system of claim 1 wherein the enzymatic cycling system is based on the chemical amplification of adenosine triphosphate (ATP) by an ADP-ATP cycling using AMP and PEP as substrates, and adenylate kinase and pyruvate kinase as cycling reagents to produce one extra molecule of ATP with each turnover of the enzyme systems,
AMP +ATP adenylate kinase
2 ADP
ZADP + PEP pyruvate kinase
2 ATP + pyruvate
3. A system of claim 2 wherein the enzyme system is let to turn over 100--1000 times to increase the original ATP concentration by the same factor during a 0.5-2 hour incubation time.
4. A system of claim 3 wherein the produced ATP is measured with the firefly bioluminescent system.
5. A system of claim 1 wherein the NAD-NADH of NADP-NADPH enzymatic cycling system produces a substrate that can enzymatically converted to ATP.
6. A system of claim 1 wherein an ADP-ATP enzymatic cycling system produces a substrate that enzymatically can be converted to ATP.
7. A system of claim 5, wherein the concentration of produced ATP is measured with the firefly bioluminescent system, and the obtained ATP concentration converted to the concentration of the original concentration of the pyrudine nucleotide of interest.
8. A system of claim 7 wherein the concentration of ATP after enzymatic cycling and enzymatic conversion of the accumulated substrate is measured as ATP with the firefly bioluminescent assay, and that result converted to the original concentration of ATP or ADP in the sample.
9. A system of claim 1 wherein the enzymatic cycling is used to accumulate a substrate that in an enzymatic reaction produces superoxide or peroxide to be measured with chemiluminescence assay.
10. A system of claim 1 wherein an NAD-NADH or NADP-NADPH system produces a substratethat through an enzymatic indicator reaction is converted to NADH or NADPH.
11. A system of claim 10 wherein the concentration of NADH or NADPH is measured with the bacterial bioluminescent reaction.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB8023995A GB2055200B (en) | 1979-07-24 | 1980-07-22 | Luminescent assays with enzymatic cycling enhancement of the sensitivity of bioluminescent and chemi |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB7925712 | 1979-07-24 | ||
| GB8023995A GB2055200B (en) | 1979-07-24 | 1980-07-22 | Luminescent assays with enzymatic cycling enhancement of the sensitivity of bioluminescent and chemi |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| GB2055200A true GB2055200A (en) | 1981-02-25 |
| GB2055200B GB2055200B (en) | 1984-05-02 |
Family
ID=26272292
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| GB8023995A Expired GB2055200B (en) | 1979-07-24 | 1980-07-22 | Luminescent assays with enzymatic cycling enhancement of the sensitivity of bioluminescent and chemi |
Country Status (1)
| Country | Link |
|---|---|
| GB (1) | GB2055200B (en) |
Cited By (20)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0049606A1 (en) * | 1980-10-02 | 1982-04-14 | Colin Henry Self | Detection method, use and diagnostic aid |
| FR2545504A1 (en) * | 1983-04-25 | 1984-11-09 | Toyo Jozo Kk | METHOD OF TITRATION USING NAD-SYNTHETASE AND PROCESS FOR PRODUCTION OF ENZYME |
| FR2547926A1 (en) * | 1983-06-27 | 1984-12-28 | Biomerieux Sa | Process for assaying oestrogens and androgens by amplification and bioluminescence |
| DE4011297A1 (en) * | 1989-04-06 | 1990-10-11 | Japan Spectroscopic Co | MEASURING METHOD AND MEASURING DEVICE FOR ALDEHYDE MEASUREMENT |
| FR2699930A1 (en) * | 1992-12-24 | 1994-07-01 | Kabore Paul | Bioluminescent determn of adenosine phosphates |
| WO1994025619A1 (en) * | 1993-04-23 | 1994-11-10 | Celsis Limited | Detection of biological material |
| US6159693A (en) * | 1998-03-13 | 2000-12-12 | Promega Corporation | Nucleic acid detection |
| US6235480B1 (en) | 1998-03-13 | 2001-05-22 | Promega Corporation | Detection of nucleic acid hybrids |
| US6268146B1 (en) | 1998-03-13 | 2001-07-31 | Promega Corporation | Analytical methods and materials for nucleic acid detection |
| US6270973B1 (en) | 1998-03-13 | 2001-08-07 | Promega Corporation | Multiplex method for nucleic acid detection |
| US6270974B1 (en) | 1998-03-13 | 2001-08-07 | Promega Corporation | Exogenous nucleic acid detection |
| US6277578B1 (en) | 1998-03-13 | 2001-08-21 | Promega Corporation | Deploymerization method for nucleic acid detection of an amplified nucleic acid target |
| US6312902B1 (en) | 1998-03-13 | 2001-11-06 | Promega Corporation | Nucleic acid detection |
| US6335162B1 (en) | 1998-03-13 | 2002-01-01 | Promega Corporation | Nucleic acid detection |
| US6391551B1 (en) | 1998-03-13 | 2002-05-21 | Promega Corporation | Detection of nucleic acid hybrids |
| GB2375171A (en) * | 2000-12-15 | 2002-11-06 | Lumitech | Method for detecting protein kinase activity using a bioluminescence reaction |
| US6703211B1 (en) | 1998-03-13 | 2004-03-09 | Promega Corporation | Cellular detection by providing high energy phosphate donor other than ADP to produce ATP |
| US7090975B2 (en) | 1998-03-13 | 2006-08-15 | Promega Corporation | Pyrophosphorolysis and incorporation of nucleotide method for nucleic acid detection |
| CN108872205A (en) * | 2018-06-19 | 2018-11-23 | 深圳上泰生物工程有限公司 | The application of object detection method of content and detection reagent in the detection method |
| EP4159755A4 (en) * | 2020-05-25 | 2024-07-31 | Yokogawa Electric Corporation | METHOD FOR DETECTING A TARGET MOLECULE IN A SAMPLE, AND TARGET MOLECULE DETECTION KIT |
-
1980
- 1980-07-22 GB GB8023995A patent/GB2055200B/en not_active Expired
Cited By (29)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0049606A1 (en) * | 1980-10-02 | 1982-04-14 | Colin Henry Self | Detection method, use and diagnostic aid |
| FR2545504A1 (en) * | 1983-04-25 | 1984-11-09 | Toyo Jozo Kk | METHOD OF TITRATION USING NAD-SYNTHETASE AND PROCESS FOR PRODUCTION OF ENZYME |
| DE3415436A1 (en) * | 1983-04-25 | 1985-05-15 | Toyo Jozo K.K., Tagata, Shizuoka | ASSAY METHOD USING NAD SYNTHETASE AND A METHOD FOR PRODUCING THE ENZYME |
| FR2547926A1 (en) * | 1983-06-27 | 1984-12-28 | Biomerieux Sa | Process for assaying oestrogens and androgens by amplification and bioluminescence |
| DE4011297A1 (en) * | 1989-04-06 | 1990-10-11 | Japan Spectroscopic Co | MEASURING METHOD AND MEASURING DEVICE FOR ALDEHYDE MEASUREMENT |
| DE4011297C2 (en) * | 1989-04-06 | 1998-08-06 | Japan Spectroscopic Co | Device for collecting and measuring gaseous aldehydes from a gaseous sample |
| FR2699930A1 (en) * | 1992-12-24 | 1994-07-01 | Kabore Paul | Bioluminescent determn of adenosine phosphates |
| WO1994025619A1 (en) * | 1993-04-23 | 1994-11-10 | Celsis Limited | Detection of biological material |
| US6043047A (en) * | 1993-04-23 | 2000-03-28 | Celsis International, Plc | Sample-collecting and assay device for use in the detection of biological material |
| US6277578B1 (en) | 1998-03-13 | 2001-08-21 | Promega Corporation | Deploymerization method for nucleic acid detection of an amplified nucleic acid target |
| US6391551B1 (en) | 1998-03-13 | 2002-05-21 | Promega Corporation | Detection of nucleic acid hybrids |
| US6268146B1 (en) | 1998-03-13 | 2001-07-31 | Promega Corporation | Analytical methods and materials for nucleic acid detection |
| US6270973B1 (en) | 1998-03-13 | 2001-08-07 | Promega Corporation | Multiplex method for nucleic acid detection |
| US6270974B1 (en) | 1998-03-13 | 2001-08-07 | Promega Corporation | Exogenous nucleic acid detection |
| US6159693A (en) * | 1998-03-13 | 2000-12-12 | Promega Corporation | Nucleic acid detection |
| US6312902B1 (en) | 1998-03-13 | 2001-11-06 | Promega Corporation | Nucleic acid detection |
| US6335162B1 (en) | 1998-03-13 | 2002-01-01 | Promega Corporation | Nucleic acid detection |
| US6379898B2 (en) | 1998-03-13 | 2002-04-30 | John W. Shultz | Nucleic acid detection |
| US6235480B1 (en) | 1998-03-13 | 2001-05-22 | Promega Corporation | Detection of nucleic acid hybrids |
| US7090975B2 (en) | 1998-03-13 | 2006-08-15 | Promega Corporation | Pyrophosphorolysis and incorporation of nucleotide method for nucleic acid detection |
| US6730479B2 (en) | 1998-03-13 | 2004-05-04 | Promega Corporation | Detection of nucleic acid hybrids |
| US6703211B1 (en) | 1998-03-13 | 2004-03-09 | Promega Corporation | Cellular detection by providing high energy phosphate donor other than ADP to produce ATP |
| US6653078B2 (en) | 1998-03-13 | 2003-11-25 | Promega Corporation | Multiplex method for nucleic acid detection |
| US6599711B2 (en) | 2000-12-15 | 2003-07-29 | Lumitech (Uk) Limited | Methods and kits for detecting protein kinases |
| GB2375171B (en) * | 2000-12-15 | 2003-03-12 | Lumitech | Methods and kits for detecting protein kinases |
| US6911319B2 (en) | 2000-12-15 | 2005-06-28 | Lumitech (Uk) Limited | Methods and kits for detecting protein kinases |
| GB2375171A (en) * | 2000-12-15 | 2002-11-06 | Lumitech | Method for detecting protein kinase activity using a bioluminescence reaction |
| CN108872205A (en) * | 2018-06-19 | 2018-11-23 | 深圳上泰生物工程有限公司 | The application of object detection method of content and detection reagent in the detection method |
| EP4159755A4 (en) * | 2020-05-25 | 2024-07-31 | Yokogawa Electric Corporation | METHOD FOR DETECTING A TARGET MOLECULE IN A SAMPLE, AND TARGET MOLECULE DETECTION KIT |
Also Published As
| Publication number | Publication date |
|---|---|
| GB2055200B (en) | 1984-05-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| GB2055200A (en) | Enhancement of the sensitivity of bioluminescent and chemiluminescent assays with enzymatic cycling | |
| EP0794260B1 (en) | Bioluminescence reagent and method for quantitative determination of adenosine phosphate ester using the reagent | |
| Wienhausen et al. | Bioluminescent assays of picomole levels of various metabolites using immobilized enzymes | |
| US4806415A (en) | Method and system for determining the presence of adenosine triphosphate or flavin mononucleotide | |
| JPH0472520B2 (en) | ||
| US7122333B2 (en) | Method and reagent for visually measuring ATP | |
| EP0199363B1 (en) | Method of terminating isocitrate dehydrogenase reaction | |
| US4927752A (en) | Support used in bioluminescent dosing of enzymes, substrates or enzymatic inhibitors | |
| EP0207493B1 (en) | Method of terminating isocitrate dehydrogenase reaction | |
| JPH0220239B2 (en) | ||
| Ugarova et al. | Bioluminescent assay of creatine kinase activity using immobilized firefly extract | |
| EP0270291A1 (en) | Riboflavin-linked assay procedures and materials | |
| US20020068310A1 (en) | Method and reagant for quantitative determination of 1,5-anhydroglucitol | |
| US5250416A (en) | Method for highly sensitive determination of NADH using kinase | |
| WO2012050536A1 (en) | Method of the adenosine diphosphate quantitative determination | |
| US3838010A (en) | Photometric method for determining lactic acid dehydrogenase or glucose-6-phosphate dehydrogenase | |
| Ferrier | An enzymatic cycling method for 3-acetylpyridine adenine dinucleotide to increase the sensitivity of enzymatic methods which employ this NAD analog | |
| US20040219622A1 (en) | Phosphine-containing formulations for chemiluminescent luciferase assays | |
| US20060286618A1 (en) | AMP Assay | |
| JPH047200B2 (en) | ||
| JP2001252095A (en) | Method for measuring trace components and composition used therefor | |
| Coulet et al. | Immobilized biological compounds in bio-and chemiluminescence assays | |
| EP0392021B1 (en) | Method for analyzing components | |
| JPS6066993A (en) | How to measure biological fluid components | |
| JPH07121233B2 (en) | Reagent for quantitative determination of inorganic phosphorus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 732 | Registration of transactions, instruments or events in the register (sect. 32/1977) | ||
| PCNP | Patent ceased through non-payment of renewal fee |