SE449005B - REAGENT FOR REMOVAL OF TURBIDITY IN A BIOLOGICAL TEST - Google Patents
REAGENT FOR REMOVAL OF TURBIDITY IN A BIOLOGICAL TESTInfo
- Publication number
- SE449005B SE449005B SE8105737A SE8105737A SE449005B SE 449005 B SE449005 B SE 449005B SE 8105737 A SE8105737 A SE 8105737A SE 8105737 A SE8105737 A SE 8105737A SE 449005 B SE449005 B SE 449005B
- Authority
- SE
- Sweden
- Prior art keywords
- surfactant
- reagent
- acid diethanolamide
- enzyme
- cholesterol
- Prior art date
Links
- 239000003153 chemical reaction reagent Substances 0.000 title claims description 34
- 238000012360 testing method Methods 0.000 title description 3
- 239000004094 surface-active agent Substances 0.000 claims description 29
- 102000004190 Enzymes Human genes 0.000 claims description 22
- 108090000790 Enzymes Proteins 0.000 claims description 22
- 108010055297 Sterol Esterase Proteins 0.000 claims description 16
- 102000000019 Sterol Esterase Human genes 0.000 claims description 16
- 238000002360 preparation method Methods 0.000 claims description 15
- AOMUHOFOVNGZAN-UHFFFAOYSA-N N,N-bis(2-hydroxyethyl)dodecanamide Chemical group CCCCCCCCCCCC(=O)N(CCO)CCO AOMUHOFOVNGZAN-UHFFFAOYSA-N 0.000 claims description 14
- 229940031957 lauric acid diethanolamide Drugs 0.000 claims description 14
- 239000012472 biological sample Substances 0.000 claims description 13
- 239000000523 sample Substances 0.000 claims description 13
- 108090001060 Lipase Proteins 0.000 claims description 11
- 102000004882 Lipase Human genes 0.000 claims description 11
- 125000000217 alkyl group Chemical group 0.000 claims description 11
- 239000000203 mixture Substances 0.000 claims description 11
- 239000004367 Lipase Substances 0.000 claims description 10
- 239000000872 buffer Substances 0.000 claims description 10
- 235000019421 lipase Nutrition 0.000 claims description 10
- 125000003342 alkenyl group Chemical group 0.000 claims description 7
- 239000007864 aqueous solution Substances 0.000 claims description 7
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical group OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 claims description 6
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 claims description 6
- 125000004432 carbon atom Chemical group C* 0.000 claims description 5
- LPMBTLLQQJBUOO-KTKRTIGZSA-N (z)-n,n-bis(2-hydroxyethyl)octadec-9-enamide Chemical group CCCCCCCC\C=C/CCCCCCCC(=O)N(CCO)CCO LPMBTLLQQJBUOO-KTKRTIGZSA-N 0.000 claims description 4
- 244000060011 Cocos nucifera Species 0.000 claims description 3
- 235000013162 Cocos nucifera Nutrition 0.000 claims description 3
- 239000002253 acid Substances 0.000 claims description 3
- BPXGKRUSMCVZAF-UHFFFAOYSA-N n,n-bis(2-hydroxyethyl)decanamide Chemical compound CCCCCCCCCC(=O)N(CCO)CCO BPXGKRUSMCVZAF-UHFFFAOYSA-N 0.000 claims description 3
- SKDZEPBJPGSFHS-UHFFFAOYSA-N n,n-bis(2-hydroxyethyl)tetradecanamide Chemical compound CCCCCCCCCCCCCC(=O)N(CCO)CCO SKDZEPBJPGSFHS-UHFFFAOYSA-N 0.000 claims description 3
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 claims description 2
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 claims description 2
- 239000002202 Polyethylene glycol Substances 0.000 claims description 2
- 239000007983 Tris buffer Substances 0.000 claims description 2
- -1 p-isooctyl phenyl Chemical group 0.000 claims description 2
- 229920001223 polyethylene glycol Polymers 0.000 claims description 2
- 239000012925 reference material Substances 0.000 claims description 2
- KDYFGRWQOYBRFD-UHFFFAOYSA-L succinate(2-) Chemical compound [O-]C(=O)CCC([O-])=O KDYFGRWQOYBRFD-UHFFFAOYSA-L 0.000 claims description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N ether Substances CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims 1
- 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 42
- 235000012000 cholesterol Nutrition 0.000 description 21
- 229940088598 enzyme Drugs 0.000 description 18
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 10
- 210000002966 serum Anatomy 0.000 description 10
- 238000005352 clarification Methods 0.000 description 7
- 230000001000 lipidemic effect Effects 0.000 description 7
- 150000001840 cholesterol esters Chemical class 0.000 description 6
- POULHZVOKOAJMA-UHFFFAOYSA-N dodecanoic acid Chemical compound CCCCCCCCCCCC(O)=O POULHZVOKOAJMA-UHFFFAOYSA-N 0.000 description 6
- 229910000029 sodium carbonate Inorganic materials 0.000 description 5
- 108010089254 Cholesterol oxidase Proteins 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- DRBBFCLWYRJSJZ-UHFFFAOYSA-N N-phosphocreatine Chemical compound OC(=O)CN(C)C(=N)NP(O)(O)=O DRBBFCLWYRJSJZ-UHFFFAOYSA-N 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 230000000813 microbial effect Effects 0.000 description 4
- 150000003626 triacylglycerols Chemical class 0.000 description 4
- 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 3
- 239000005639 Lauric acid Substances 0.000 description 3
- 102000004895 Lipoproteins Human genes 0.000 description 3
- 108090001030 Lipoproteins Proteins 0.000 description 3
- SHPKCSFVQGSAJU-UAIGNFCESA-L dipotassium;(z)-but-2-enedioate Chemical compound [K+].[K+].[O-]C(=O)\C=C/C([O-])=O SHPKCSFVQGSAJU-UAIGNFCESA-L 0.000 description 3
- 239000004615 ingredient Substances 0.000 description 3
- 210000004923 pancreatic tissue Anatomy 0.000 description 3
- OFGDSGVGRWPQJQ-UHFFFAOYSA-N 1h-imidazol-1-ium;acetate Chemical compound CC(O)=O.C1=CNC=N1 OFGDSGVGRWPQJQ-UHFFFAOYSA-N 0.000 description 2
- RLFWWDJHLFCNIJ-UHFFFAOYSA-N 4-aminoantipyrine Chemical compound CN1C(C)=C(N)C(=O)N1C1=CC=CC=C1 RLFWWDJHLFCNIJ-UHFFFAOYSA-N 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 108091000080 Phosphotransferase Proteins 0.000 description 2
- 239000008351 acetate buffer Substances 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- CVSVTCORWBXHQV-UHFFFAOYSA-N creatine Chemical compound NC(=[NH2+])N(C)CC([O-])=O CVSVTCORWBXHQV-UHFFFAOYSA-N 0.000 description 2
- 229940079919 digestives enzyme preparation Drugs 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 210000000496 pancreas Anatomy 0.000 description 2
- 102000020233 phosphotransferase Human genes 0.000 description 2
- 239000008137 solubility enhancer Substances 0.000 description 2
- BJEPYKJPYRNKOW-REOHCLBHSA-N (S)-malic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O BJEPYKJPYRNKOW-REOHCLBHSA-N 0.000 description 1
- ORLFVWPPBMVPNZ-UHFFFAOYSA-N 1-(6-methylheptyl)-4-[4-(6-methylheptyl)phenoxy]benzene Chemical compound C1=CC(CCCCCC(C)C)=CC=C1OC1=CC=C(CCCCCC(C)C)C=C1 ORLFVWPPBMVPNZ-UHFFFAOYSA-N 0.000 description 1
- 108010029731 6-phosphogluconolactonase Proteins 0.000 description 1
- 102100031126 6-phosphogluconolactonase Human genes 0.000 description 1
- 108090000371 Esterases Proteins 0.000 description 1
- 108010018962 Glucosephosphate Dehydrogenase Proteins 0.000 description 1
- 102000005548 Hexokinase Human genes 0.000 description 1
- 108700040460 Hexokinases Proteins 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- OFOBLEOULBTSOW-UHFFFAOYSA-N Propanedioic acid Natural products OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 description 1
- ABBQHOQBGMUPJH-UHFFFAOYSA-M Sodium salicylate Chemical compound [Na+].OC1=CC=CC=C1C([O-])=O ABBQHOQBGMUPJH-UHFFFAOYSA-M 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 241000179532 [Candida] cylindracea Species 0.000 description 1
- XJLXINKUBYWONI-DQQFMEOOSA-N [[(2r,3r,4r,5r)-5-(6-aminopurin-9-yl)-3-hydroxy-4-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2s,3r,4s,5s)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate Chemical compound NC(=O)C1=CC=C[N+]([C@@H]2[C@H]([C@@H](O)[C@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-DQQFMEOOSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- DHKHKXVYLBGOIT-UHFFFAOYSA-N acetaldehyde Diethyl Acetal Natural products CCOC(C)OCC DHKHKXVYLBGOIT-UHFFFAOYSA-N 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- BJEPYKJPYRNKOW-UHFFFAOYSA-N alpha-hydroxysuccinic acid Natural products OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 description 1
- 239000003833 bile salt Substances 0.000 description 1
- 229940093761 bile salts Drugs 0.000 description 1
- 239000013060 biological fluid Substances 0.000 description 1
- 239000008395 clarifying agent Substances 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 229960003624 creatine Drugs 0.000 description 1
- 239000006046 creatine Substances 0.000 description 1
- 229960003964 deoxycholic acid Drugs 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 125000003438 dodecyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 201000005577 familial hyperlipidemia Diseases 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000011976 maleic acid Substances 0.000 description 1
- 239000001630 malic acid Substances 0.000 description 1
- 235000011090 malic acid Nutrition 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- PJUIMOJAAPLTRJ-UHFFFAOYSA-N monothioglycerol Chemical compound OCC(O)CS PJUIMOJAAPLTRJ-UHFFFAOYSA-N 0.000 description 1
- 229920002113 octoxynol Polymers 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 150000002978 peroxides Chemical class 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
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- ZLVSYODPTJZFMK-UHFFFAOYSA-M sodium 4-hydroxybenzoate Chemical compound [Na+].OC1=CC=C(C([O-])=O)C=C1 ZLVSYODPTJZFMK-UHFFFAOYSA-M 0.000 description 1
- NRHMKIHPTBHXPF-TUJRSCDTSA-M sodium cholate Chemical compound [Na+].C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC([O-])=O)C)[C@@]2(C)[C@@H](O)C1 NRHMKIHPTBHXPF-TUJRSCDTSA-M 0.000 description 1
- FHHPUSMSKHSNKW-SMOYURAASA-M sodium deoxycholate Chemical compound [Na+].C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC([O-])=O)C)[C@@]2(C)[C@@H](O)C1 FHHPUSMSKHSNKW-SMOYURAASA-M 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 238000005063 solubilization Methods 0.000 description 1
- 230000007928 solubilization Effects 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 229940035024 thioglycerol Drugs 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
-
- 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
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
-
- 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
- C12Q1/60—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving cholesterol
Description
15 20 25 30 35 449 005 2 Den exakta mekanismen för klarning enligt uppfinningen är ännu inte känd. Man kan emellertid tänka sig, att i fall där patienterna har hyperlipemi den i serumproven uppträdande turbi- diteten beror i första hand på hög triglyceridhalt. The exact mechanism of clarification according to the invention is not yet known. It is conceivable, however, that in cases where patients have hyperlipemia, the turbidity occurring in the serum samples is primarily due to a high triglyceride content.
Triglycerider är vattenolösliga och vanligen dolda i det inre av fettkärnan med kolesterolestrar i lipoprotein- komplex. Klarning av ett lipemiskt prov måste åstadkommas ge- nom spjälkning av lipoprotein med ett ytaktivt medel, såsom laurinsyradietanolamid (DBA),-följd av hydrolys av triglycerid- erna medelst enzymbasen. Det ytaktiva medlet underlättar också upplösningen av däri frigjorda fettsyror. Utan enzym blir det ingen klarning.Triglycerides are water-insoluble and usually hidden inside the fat core with cholesterol esters in lipoprotein complexes. Clarification of a lipemic sample must be accomplished by cleavage of lipoprotein with a surfactant, such as lauric acid diethanolamide (DBA), followed by hydrolysis of the triglycerides by the enzyme base. The surfactant also facilitates the dissolution of fatty acids released therein. Without enzyme there will be no clearance.
Det är föreliggande uppfinnings syfte att erbjuda ett reagens som är effektivt för undanröjande av turbiditet i bio- logiska prov och ett förfarande för åstadkommande därav, sär- skilt i fall när provet skall undersökas fotometriskt eller analyseras på en speciell komponent, såsom kolesterol.It is the object of the present invention to provide a reagent which is effective for eliminating turbidity in biological samples and a method for producing the same, especially in cases where the sample is to be examined photometrically or analyzed on a particular component, such as cholesterol.
Uppfinningen avser ett reagens för bortskaffande av turbiditet från ett biologiskt prov innehållande ett ytaktivt medel av formeln 9 //(CH2CH20)xH R-C-N\\ (CH2CH2O)yH vari R betyder en alkyl- eller alkenylgrupp med 5 till 17 kol- atomer samt x och y är vardera 1, och ett enzym som utgörs av kolesterolesterns eller lipas eller en blandning därav.The invention relates to a reagent for disposing of turbidity from a biological sample containing a surfactant of the formula 9 // (CH 2 CH 2 O) x H RCN \\ (CH 2 CH 2 O) yH wherein R represents an alkyl or alkenyl group having 5 to 17 carbon atoms and x and y is each 1, and an enzyme consisting of the cholesterol ester or lipase or a mixture thereof.
Företrädesvis innehåller det ovannämnda reagenset i form av en buffrad vattenlösning från ca 0,05 g/dl till ca 2,5 g/dl ytaktivt medel, företrädesvis från 0,1 g/dl till ca 0,5 g/dl, och minst ca 0,025 E/ml (enheter per milliliter) enzym (kolesterolesteras) räknat på hela preparatet. Det erhållna preparatet har ett pH inom området från ca 5,5 till ca 7,0.Preferably, the above reagent in the form of a buffered aqueous solution contains from about 0.05 g / dl to about 2.5 g / dl of surfactant, preferably from 0.1 g / dl to about 0.5 g / dl, and at least about 0.025 U / ml (units per milliliter) enzyme (cholesterol esterase) calculated on the whole preparation. The resulting preparation has a pH in the range of from about 5.5 to about 7.0.
När preparatet innehåller ett lipasenzym, utgör det yt- aktiva medlet från ca 0,05 g/dl till ca 2,5 g/dl, före- trädesvis från 0,1 g/dl till ca 0,5 g/dl ca 1,0 E/ml av det bildade preparatet, vilket har ett pH inom området från ca 5,5 till ca 8,0. , och enzymet minst vyn (fl 10 20 25 30 35 3 I båda enzympreparaten kan den använda bufferten vara ett maleat i form av natrium- eller kaliumsaltet, ett fosfat, ett borat, ett citrat, ett succinat, en imidazolacetatbuffcrt, tris etc. Varje lämplig buffert kan emellertid användas. En sådan buffert är vilken som helst som upprätthåller konstant pH i önskat område utan att störa någon av de andra komponent- erna.When the preparation contains a lipase enzyme, the surfactant is from about 0.05 g / dl to about 2.5 g / dl, preferably from 0.1 g / dl to about 0.5 g / dl about 1 0 U / ml of the formed composition, which has a pH in the range from about 5.5 to about 8.0. , and the enzyme at least the view (fl 10 20 25 30 35 3 In both enzyme preparations, the buffer used may be a maleate in the form of the sodium or potassium salt, a phosphate, a borate, a citrate, a succinate, an imidazole acetate buffer, tris, etc. Each however, a suitable buffer may be used, such a buffer being any which maintains a constant pH in the desired range without disturbing any of the other components.
När en maleatbuffert används, t.ex. kalium- eller natriumsaltet, tillsätts den í mängder, som ger från ca 0,05 M till ca 0,5 M, och det erhållna preparatets pH blir från ca 5,0 till ca 7,0.When a maleate buffer is used, e.g. the potassium or sodium salt, it is added in amounts giving from about 0.05 M to about 0.5 M, and the pH of the resulting preparation is from about 5.0 to about 7.0.
Liknande koncentrationer används för de andra buffert- arna.Similar concentrations are used for the other buffers.
Jämte de ovannämnda komponenterna kan en löslighets- förstärkare innefattas i ovanstående enzympreparat. Denna löslig- hetsförstärkare kan utgöras av varje slags material som hjälper det ytaktiva medlet att åstadkomma solubilisering. Exempelvis är gallsalter synnerligen effektiva såsom natriumkolat, natrium- desoxikolat, etc.In addition to the above components, a solubility enhancer may be included in the above enzyme preparations. This solubility enhancer can be any type of material that helps the surfactant to achieve solubilization. For example, bile salts are particularly effective such as sodium carbonate, sodium deoxycholate, etc.
I ett annat föredraget utförande har det ytaktiva med- let den ovanstående formeln, vari R är en alkylgrupp, såsom laurinsyradietanolamid eller oljesyradietanolamid.In another preferred embodiment, the surfactant has the above formula, wherein R is an alkyl group, such as lauric acid diethanolamide or oleic acid diethanolamide.
Enzymet härrör från djurvävnad, t.ex. pankreasvävnad, eller en mikrobiell källa.The enzyme is derived from animal tissue, e.g. pancreatic tissue, or a microbial source.
Lämpligt behandlade biologiska prov är mänskligt serum och plasma.Appropriately treated biological samples are human serum and plasma.
Det ovan beskrivna reagenset resulterar vid kombina- tion med ett biologiskt prov i en effektiv turbiditetsbortskaf- fande verkan. Reagenset erbjuds vanligen som en buffrad vatten- lösning.The reagent described above, when combined with a biological sample, results in an effective turbidity-removing effect. The reagent is usually offered as a buffered aqueous solution.
Inom ramen för uppfinningen ligger också ett reagens för undanröjande av turbiditet i ett biologiskt prov som skall undersökas eller analyseras fotometriskt, innehållande åtminstone ett ytaktivt medel av formeln ~ Û n R_C NZ(CH2CI-*z°)xH *æuzcnznnyn (fl 10 15 20 25 30 35 449 005 H där R betyder en alkyl eller alkenylgrupp innehållande 5 till 17 kolatomer samt x och y är heltal vilkas summa är högst 11, och ett enzym, som utgörs av kolesterolesteras eller lipas eller en annan blandning därav.Also within the scope of the invention is a reagent for eliminating turbidity in a biological sample to be examined or analyzed photometrically, containing at least one surfactant of the formula ~ Û n R_C NZ (CH2Cl- * z °) xH * æuzcnznnyn (fl 10 15 20 449 005 H where R represents an alkyl or alkenyl group containing 5 to 17 carbon atoms and x and y are integers whose sum is not more than 11, and an enzyme consisting of cholesterol esterase or lipase or another mixture thereof.
Ett föredraget rcagcnu är ett med cLL ytakLivt medel av ovanstående formel, vari R är en alkylgrupp,företrädesvis laurylgrupp, summan av x och y är 5 och enzymet är en lipas.A preferred rcagcnu is a cLL surfactant of the above formula, wherein R is an alkyl group, preferably lauryl group, the sum of x and y is 5 and the enzyme is a lipase.
Preparat med detta reagens innehåller lämpligen polyetylenglykol- p-isooktylfenyleter eller andra lämpliga ytaktiva medel.Preparations containing this reagent suitably contain polyethylene glycol p-isooctylphenyl ether or other suitable surfactants.
Ett föredraget reagens, som framkallar effektiv klar- ning av grumliga prov i pH-området 2 till 10 är en blandning av två ytaktiva medel, nämligen laurinsyradietanolamid (x=y=1) 5).A preferred reagent which elicits effective clarification of cloudy samples in the pH range 2 to 10 is a mixture of two surfactants, namely lauric acid diethanolamide (x = y = 1) 5).
Enzympeparat enligt ovan framställs med hjälp av det ovan ytaktiva medlet. och epoxylerad laurinsyra (x + y = Lämpliga ytaktiva medel, som omfattas av ovanstående formel, är laurinsyradietanolamid, myristinsyradietanolamid, kaprinsyradietanolamid, oljesyradietanolamid och kokossyradi- etanolamid.Enzyme preparation according to the above is prepared by means of the above surfactant. and epoxylated lauric acid (x + y = Suitable surfactants included in the above formula are lauric acid diethanolamide, myristic acid diethanolamide, capric acid diethanolamide, oleic acid diethanolamide and coconut acid diethanolamide.
Vid kombination med ett biologiskt prov undanröjer detta reagens effektivt turbiditet i provet och möjliggör noggrann fotometrisk undersökning eller analys. Pâ så sätt behandlar man med fördel ett prov, som skall analyseras fotometriskt på kolesterol. U Föreliggande uppfinning innebär i en aspekt ett reagens, som-effektiwt undanröjer'turbiditet i ett biologiskt prov genom användning av ett speciellt yt- aktivt medel och enzym. I denna aspekt har det ytaktiva medlet formeln 9 ///,(CH2CH2O)xH R-C-N\\\\ (CH2CH20)yH vari R betyder en alkyl- eller alkenylgrupp med 5 till 17 kol- atomer samt x och y betyder vardera 1. Företrädesvis är R en alkylgrupp, såsom exemplifieras av laurinsyradietanolamid. 10 15 20 25 30 449 005 5 Enzymkomponenten är kolesterolesteras eller lipas eller en blandning därav.When combined with a biological sample, this reagent effectively eliminates turbidity in the sample and enables accurate photometric examination or analysis. In this way, a sample is advantageously treated, which is to be analyzed photometrically for cholesterol. In one aspect, the present invention provides a reagent which effectively eliminates turbidity in a biological sample by the use of a particular surfactant and enzyme. In this aspect, the surfactant has the formula 9 ///, (CH 2 CH 2 O) x H RCN \\\\ (CH 2 CH 2 O) y H wherein R represents an alkyl or alkenyl group having 5 to 17 carbon atoms and x and y each represent 1. Preferably R is an alkyl group, as exemplified by lauric acid diethanolamide. The enzyme component is cholesterol esterase or lipase or a mixture thereof.
Det bildade preparatet har helt överraskande befunnits vara högeffektivt för bortskaffande av turbiditet i ett biolo- giskt prov. Det är därför enormt användbart för omvandling av ett biologiskt prov, som är grumligt, till ett klart prov.The formed preparation has surprisingly been found to be highly effective in eliminating turbidity in a biological sample. It is therefore extremely useful for converting a biological sample, which is cloudy, into a clear sample.
Det sålunda behandlade provet kan undersökas eller analyseras kolorimetriskt på någon speciell komponent, så länge de vid undersökningen använda_reagensen inte inhiberar eller stör samverkan mellan det ytaktiva medlet och tvärtom.The sample thus treated can be examined or analyzed colorimetrically on any particular component, as long as the reagents used in the examination do not inhibit or interfere with the interaction between the surfactant and vice versa.
Typiska undersökningar som kan utföras med det ovan angivna reagenset innefattar bestämningar av kolesterol, tri- glycerider och kreatinfosfatkinas.Typical studies that can be performed with the above reagent include determinations of cholesterol, triglycerides and creatine phosphate kinase.
Enzymkomponenten kan härröra från en animalisk källa, såsom pankreasvävnad, eller från en mikrobiell källa.The enzyme component may be derived from an animal source, such as pancreatic tissue, or from a microbial source.
I en andra aspekt av uppfinningen avses ett reagens, som effektivt klarar ett grumligt biologiskt prov, vilket skall undersökas fotometriskt. Reagenset innefattar åtminstone ett ytaktivt medel, definierat vidare än ovan, med formeln 9 /////(CH2CH2O)XH R~C-H \ yH vari R betyder en alkyl- eller alkenylgrupp innehållande från 5 till 17 kolatomer och x och y är heltal, vilkas summa är högst 11, och ett bland kolesterolesteras och lipas och bland- ningar därav valt enzym.In a second aspect of the invention, there is provided a reagent which effectively passes a cloudy biological sample which is to be examined photometrically. The reagent comprises at least one surfactant, defined further than above, of the formula 9 ///// (CH 2 CH 2 O) X H R ~ CH 2 yH wherein R represents an alkyl or alkenyl group containing from 5 to 17 carbon atoms and x and y are integers, whose sum is at most 11, and an enzyme selected from cholesterol esterase and lipase and mixtures thereof.
I en föredragen utföringsform är det ytaktiva medlet såsom ovan, där R betyder en alkylgrupp och x och y är var- dera 1. Exempel på sådana är laurinsyradietanolamid, myristin- syradietanolamid och kaprinsyradietanolamid. Föredragna är även ytaktiva medel, i vilka R är en alkenylgrupp och x och y är 1, såsom oljesyradietanolamid och kokossyradietanolamid.In a preferred embodiment, the surfactant is as above, where R represents an alkyl group and x and y are each 1. Examples of such are lauric acid diethanolamide, myristic acid diethanolamide and capric acid diethanolamide. Also preferred are surfactants in which R is an alkenyl group and x and y are 1, such as oleic acid diethanolamide and coconut acid diethanolamide.
I en annan föredragen utföringsform är det ytaktiva medlet så- är 5. som ovan, vari R betyder en alkylgrupp och summan av X och y . .Lfifl-.waw 449 005 (fl 10 15 20 25 30 35 6 Enzymkomponenten kan härröra från en animalisk källa, såsom pankreasvävnad eller från en mikrobiell källa.In another preferred embodiment, the surfactant is 5. as above, wherein R represents an alkyl group and the sum of X and y. .L fifl-. Waw 449 005 (fl 10 15 20 25 30 35 6 The enzyme component may be derived from an animal source, such as pancreatic tissue or from a microbial source.
När ovannämnda reagens används för undersökningsändamül, kombineras det i form av buffrad vattenlösning med ett bio- logiskt prov. Det grumliga provet klaras och är redo för under- sökningen.When the above reagent is used for testing purposes, it is combined in the form of buffered aqueous solution with a biological sample. The cloudy test is passed and is ready for the examination.
Prov, som effektivt klaras och bereds för undersökning, är exempelvis prov på serum och plasma från människor.Samples that are effectively passed and prepared for examination are, for example, samples of serum and plasma from humans.
Reagansen enligt uppfinningen är unika i åtminstone två hänseenden. De möjliggör bestämning av kompo- nenter i biologiska prov i klart fritt tillstånd utan turbídi- tetsstörningar, och till följd av samverkan mellan det speci- ella ytaktiva medlet och det använda enzymet ger de dessutom möjlighet att använda mindre mängder enzym än man normalt an- vänder, såsom i fallet bestämning av kolesterol. I detta senare avseende kan användningen av mindre mängder utan minskning av reaktionshastigheterna betraktas som en förbättring av den en- zymatiska reaktionens hastighet.The reagents of the invention are unique in at least two respects. They enable the determination of components in biological samples in a clear free state without turbidity disturbances, and as a result of the interaction between the special surfactant and the enzyme used, they also make it possible to use smaller amounts of enzyme than is normally used. , as in the case of determination of cholesterol. In this latter respect, the use of smaller amounts without reducing the reaction rates can be considered as an improvement in the rate of the enzymatic reaction.
Den vanligaste kliniska bestämningen av kolesterol i en biologisk vätska är bestämningen av total kolesterolhalt, innefattande både fri kolesterol och kolesterolestrar. Både kolesterol och dess estrar förekommer i serum tillsammans med andra lipider och diverse proteiner i mikromolekylära komplex, som kallas lipoproteiner, och kolesterolestrar upp- träder normalt som en väsentlig andel (60 - 80 %) av hela halten kolesterol. De är generellt vattenolösliga och är nor- malt inbäddade i komplexet och otillgängliga för enzymer. Vid bestämning av total kolesterol med en helt enzymatisk metod, vare sig automatisk eller manuell, måste både kolesterolen och kolesterolestrarna först frigöras medelst ett lämpligt ytaktivt ämne. Kolesterolestrarna hydrolyseras sedan med kol- esterolesteras till fri kolesterol, vilken i sin tur oxideras med kolesteroloxidas till kolestenon och väteperoxid.The most common clinical determination of cholesterol in a biological fluid is the determination of total cholesterol content, including both free cholesterol and cholesterol esters. Both cholesterol and its esters occur in serum together with other lipids and various proteins in micromolecular complexes, called lipoproteins, and cholesterol esters normally appear as a significant proportion (60-80%) of the entire cholesterol content. They are generally water-insoluble and are normally embedded in the complex and inaccessible to enzymes. When determining total cholesterol by a fully enzymatic method, whether automatic or manual, both the cholesterol and the cholesterol esters must first be released by means of a suitable surfactant. The cholesterol esters are then hydrolyzed with cholesterol esterase to free cholesterol, which in turn is oxidized with cholesterol oxidase to cholesterol and hydrogen peroxide.
Den här angivna metoden kan tillämpas automatiskt genom användning av en automatisk analysator eller kan genom- föras manuellt. 7 Vid iordningsställande av beredningarna för användning vid analys med förfarandet enligt uppfinningen bereder man en . ...___ ...vn 10 15 20 25 30 35 449 005 7 vattenlösning, som jämte det ytaktiva medlet och enzymet inne- häller andra referensmaterial, vilka är förut kända och an- vänds för sådana syften.The method specified here can be applied automatically by using an automatic analyzer or can be performed manually. In preparing the formulations for use in assaying the method of the invention, one is prepared. ...___ ... vn 10 15 20 25 30 35 449 005 7 aqueous solution, which together with the surfactant and the enzyme contains other reference materials, which are previously known and used for such purposes.
Exempelvis använder man vid kolesterolbestämning följ- ande komponenter i nedan angivna halter.For example, the following components are used in cholesterol determination at the levels given below.
Komgonent Kolesterolbestämning Peroxidas 0,8 - 2,0 B/l Kolesteroloxidas ' 0,025 - 0,3 E/ml Kolesterolesteras Ytaktivt medel 0,025 - 0,3 E/mi 0,05 - 0,5 g/dl Natriumkolat 0,05 - 0,5 g/dl Natrium-p-hydroxíbensoat 2,5 - 6 g/dl H-aminoantipyrin 0,5 - 2,0 mM Maleinsyra 0,1 - 0,5 M pH 5,5 - 7,0 Förh. prov/reagens 100 - 400 I ovanstående beredning är det lämpligast att använda n kolesterolesteras från en animalisk källa, t.ex. pankreas; ekvivalenta resultat erhålls emellertid med kolesterolesteras från en mikrobiell källa.Component Cholesterol determination Peroxidase 0.8 - 2.0 B / l Cholesterol oxidase 0.025 - 0.3 U / ml Cholesterol esterase Surfactant 0.025 - 0.3 U / ml 0.05 - 0.5 g / dl Sodium carbonate 0.05 - 0 .5 g / dl Sodium p-hydroxybenzoate 2.5 - 6 g / dl H-aminoantipyrine 0.5 - 2.0 mM Maleic acid 0.1 - 0.5 M pH 5.5 - 7.0 Ref. sample / reagent 100 - 400 In the above preparation it is most convenient to use n cholesterol esterase from an animal source, e.g. pancreas; however, equivalent results are obtained with cholesterol esterase from a microbial source.
Exemgel 1 Klarning som funktion av kolesterolesteras 3 ml klarningsreagens som innehåller 0,1 M kalium- maleat, 0,25 g/dl natriumkolat, 0,2 g/dl laurinsyradietanol- amid, 0,08 till 0,8 E/ml kolesterolesteras, slut-pH 6,0, blandas med 0,025 ml lipemiskt system (triglycerider ca 1H00 ¿ mg/dl). Reaktionsblandningen blir klar inom 10 minuter vid HSOC. Kolesterolesterasen för klaring kanvara från pankreas eller mikroorganismer.Example gel 1 Clarification as a function of cholesterol esterase 3 ml clarifying reagent containing 0.1 M potassium maleate, 0.25 g / dl sodium cholate, 0.2 g / dl lauric acid diethanolamide, 0.08 to 0.8 U / ml cholesterol esterase, final pH 6.0, mixed with 0.025 ml lipemic system (triglycerides approx. 1H00 ¿mg / dl). The reaction mixture is ready within 10 minutes at HSOC. The cholesterol esterase for clearance can be from the pancreas or microorganisms.
Exemgel 2 Användning för kolesterolbestämning För slutpunktskemi, såsom åskâdliggörs av detta exempel, innefattas ingredienserna för kolesterolbestämningen i klar- ningsreagenset. 3 ml beredning, som innehåller 0,125 E/ml kolesterol- esteras, 0,125 B/ml kolesteroloxidas, 1,6 E/ml peroxides, 0,6 mM H-aminoantípyrin, 25 mM natriumhydroxibensoat, 0,25 g/dl natriumkolat, 0,2 g/dl laurinsyradietanolamid och 0,1 M 449 005 8 kaliummaleat, blandas med 0,125 ml prov av lipemiskt serum.' Blandningen inkuberas vid HSOC under 4 - 5 minuter. Sedan be- stäms totala kolesterolhalten genom mätning av färgstyrkan vid 520 nm. Utan klarningseffekten av laurinsyradietanolamiden och kolesterolesterasen ger bestämningen av kolesterol i grumliga prov alltid felaktiga resultat.Example 2 Use for Cholesterol Determination For endpoint chemistry, as illustrated by this example, the ingredients for cholesterol determination are included in the clarifying reagent. 3 ml formulation containing 0.125 U / ml cholesterol esterase, 0.125 B / ml cholesterol oxidase, 1.6 U / ml peroxides, 0.6 mM H-aminoantipyrine, 25 mM sodium hydroxybenzoate, 0.25 g / dl sodium carbonate, 0, 2 g / dl lauric acid diethanolamide and 0.1 M 449 005 8 potassium maleate, mixed with 0.125 ml sample of lipemic serum. The mixture is incubated at HSOC for 4-5 minutes. Then the total cholesterol content is determined by measuring the color strength at 520 nm. Without the clarifying effect of the lauric acid diethanolamide and the cholesterol esterase, the determination of cholesterol in cloudy samples always gives incorrect results.
Exemgel 3 ' Klarning med candida-lipas (Candida cylindracea) 3 ml klarníngsreagens-innehållande 0,2 g/dl laurinsyra- dietanolamid, 0,25 g/dl natriumkolat, 25 E/ml lipas och 0,1 M maleatbuffert, pH 6,0, blandas med 0,025 ml lipemiskt serum.Example gel 3 'Clarida lipase clearance (Candida cylindracea) 3 ml clarification reagent containing 0.2 g / dl lauric acid diethanolamide, 0.25 g / dl sodium carbonate, 25 U / ml lipase and 0.1 M maleate buffer, pH 6, 0, mixed with 0.025 ml of lipemic serum.
Det grumlíga provet blir klart efter 5 minuters ínkubation vid us°c.The cloudy sample becomes clear after 5 minutes of incubation at us ° c.
När en ekvivalent mängd klarningsmedel innehållande en blandning av laurinsyradietanolamid och epoxylerad laurin- syra (x + y = 5) används, erhålls en jämförbar klarning.When an equivalent amount of clarifying agent containing a mixture of lauric acid diethanolamide and epoxylated lauric acid (x + y = 5) is used, a comparable clarification is obtained.
Exemgel H 3 ml av ett klarningsreagens innehållande 0,2 g/dl yLaktivt medel med formeln 9 w/// (CH2CH2O)xH C11H25'C'N (CH2CH2O)yH vari x + y = 5, 0,0 g/dl Triton X~100, 0,2 M kaliummaleat och 25 E/ml lipas, pH 6,0, blandas med 0,05 ml lipemiskt prov och inkuberas vid HSOC. Det grumliga provcL blit klart efter 3 minuter.Example gel H 3 ml of a clarifying reagent containing 0.2 g / dl yL active agent of the formula 9 w /// (CH2CH2O) xH C11H25'C'N (CH2CH2O) yH wherein x + y = 5, 0.0 g / dl Triton X ~ 100, 0.2 M potassium maleate and 25 U / ml lipase, pH 6.0, mixed with 0.05 ml lipemic sample and incubated at HSOC. The cloudy provcL became clear after 3 minutes.
Klarningshastigheten ökas genom höjning av buffert- koncentrationen.The clearance rate is increased by increasing the buffer concentration.
Liknande resultat erhålls genom användning av högre koncentrationer av epoxylerad laurinsyra (x + y = 5) utan bistånd av Triton X-100.Similar results are obtained using higher concentrations of epoxylated lauric acid (x + y = 5) without the assistance of Triton X-100.
Exemgel 5 A. Beredning En diagnostisk reagensberedning framställs som en 1 l vattenlösning under användning av följande ingredienser.Example 5 A. Preparation A diagnostic reagent preparation is prepared as a 1 L aqueous solution using the following ingredients.
M...- 10 15 20 25 30 35 449 005 9 Ingrediens Koncentration Äppelsyra 11,6 g KOH 10,0 g EDTA (K2) 2,7 mM Na-kolat 5,8 mM Na-p-hydroxibensoat 25,0 mM 4-aminoantipyrin 0,6 mM Laurinsyra-dietanolamid 2,0 g Kolesterolesteras ' '125 enheter Kolesteroloxidas 125 enheter Pepparrotsperoxidas 800 enheter pH inställs på 6,0.M ...- 10 15 20 25 30 35 449 005 9 Ingredient Concentration Malic acid 11.6 g KOH 10.0 g EDTA (K2) 2.7 mM Na-carbonate 5.8 mM Na-p-hydroxybenzoate 25.0 mM 4-aminoantipyrine 0.6 mM Lauric acid diethanolamide 2.0 g Cholesterol esterase 125 units Cholesterol oxidase 125 units Horseradish peroxidase 800 units pH is adjusted to 6.0.
Reagenssystemet kan lagras och användas i form av en vattenlösning, eller också kan lösningen frystorkas på kon- ventionellt sätt och rekonstitueras med vatten för att an- vändas.The reagent system can be stored and used in the form of an aqueous solution, or the solution can be lyophilized in a conventional manner and reconstituted with water for use.
B. Bestämning av total kolesterolhalt 3 ml av ovanstående reagens blandas med 0,025 ml serum eller rekonstituerad serumstandard, som innehåller upp till 500 mg/dl kolesterol. Reaktionen utförs med 45°C under H till 5 minuter. genset som blindprov.B. Determination of total cholesterol content 3 ml of the above reagent is mixed with 0.025 ml serum or reconstituted serum standard, which contains up to 500 mg / dl cholesterol. The reaction is carried out at 45 ° C under H for 5 minutes. genset as a blank.
Exemgel 6 Klarning vid bestämning av aktiviteten hos kreatinfos- Provens absorbans vid 525 nm mäts mot rea- fatkinas (CPK) i lipemískt serum 1 ml imidazolacetalbuffert, 0,1 M, pH 6,7, innehållande 0,0 % laurinsyradietanolamíd, 25 E/dl pankreas-kolesterol- 0,25 % natriumkolat och 20 mM tioglycerol, blandas med 0,05 ml lípemiskt serum. Efter 15 minuters inkubation vid s7°c faller rurbidirefen vid sno nm från 2,3 oT (optisk täthet) till 0,02 OT. Det klara provet blandas med 1 ml CPK- reagens, som innehåller 116,7 mM kreatinfosfat, 6,7 mM ADP, 16,7 mM AMP, 6,7 mM EDTA, 6,7 mM NADP, 125 E/dl hexokinas, 100 E/dl glykos-6-fosfatdehydrogenas GBPDH preparat i 0,1 M imidazolacetatbuffert, pH 6,7. Aktíviteten hos CPK övervakas esteras, vid 3H0 nm och 3700 såsom i den konventionella metoden.Example gel 6 Clarification in determining the activity of creatine phosphate- The absorbance of the sample at 525 nm is measured against reapatkin kinase (CPK) in lipemic serum 1 ml imidazole acetal buffer, 0.1 M, pH 6.7, containing 0.0% lauric acid diethanolamide, 25 U / dl pancreatic cholesterol- 0.25% sodium carbonate and 20 mM thioglycerol, mixed with 0.05 ml lipemic serum. After 15 minutes of incubation at s7 ° c, the rurbidirefen drops at sno nm from 2.3 oT (optical density) to 0.02 oT. The clear sample is mixed with 1 ml of CPK reagent containing 116.7 mM creatine phosphate, 6.7 mM ADP, 16.7 mM AMP, 6.7 mM EDTA, 6.7 mM NADP, 125 U / dl hexokinase, 100 E / dl glucose-6-phosphate dehydrogenase GBPDH preparation in 0.1 M imidazole acetate buffer, pH 6.7. The activity of CPK is monitored esterase, at 3H0 nm and 3700 as in the conventional method.
Claims (8)
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| FR2599149B1 (en) * | 1986-05-21 | 1988-08-26 | Univ Nancy | REAGENT FOR TRANSPARIZING BIOLOGICAL MEDIA AND ITS ANALYTICAL APPLICATIONS. |
| DE3620817A1 (en) * | 1986-06-21 | 1987-12-23 | Boehringer Mannheim Gmbh | METHOD FOR THE SPECIFIC DETERMINATION OF THE SERUM FRUCTOSAMINE CONTENT, AND A REAGENT MIXTURE SUITABLE FOR THIS |
| EP1013774B8 (en) | 1998-12-22 | 2006-12-06 | Olympus Life and Material Science Europa GmbH | Liquid reagent for the detection of creatine kinase |
| US7790768B2 (en) | 2003-02-28 | 2010-09-07 | E-L Management Corp. | Method for increasing hair growth |
| JP2006071574A (en) * | 2004-09-06 | 2006-03-16 | Denka Seiken Co Ltd | Immunoturbidimetry and reagent therefor |
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| US2531190A (en) * | 1946-11-12 | 1950-11-21 | Drew & Co Inc E F | Emulsifier consisting of alkylolamine-fatty acid condensation products and esters ofpolyglycols |
| US3260648A (en) * | 1963-08-16 | 1966-07-12 | Warner Lambert Pharmaceutical | Diagnostic aid |
| US3853465A (en) * | 1972-06-09 | 1974-12-10 | Technicon Instr | Turbidity reduction in serum and plasma samples using polyoxyethylated lauric acid compounds |
| US3898130A (en) * | 1974-03-18 | 1975-08-05 | American Hospital Supply Corp | Rapid enzymatic hydrolysis of triglycerides |
| DE2724757C2 (en) * | 1977-06-01 | 1979-08-23 | Boehringer Mannheim Gmbh, 6800 Mannheim | Means for removing cloudiness in serum and process for its preparation |
| DE2816229C2 (en) * | 1978-04-14 | 1983-11-10 | Boehringer Mannheim Gmbh, 6800 Mannheim | Methods and means for removing opacities |
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| AU7287681A (en) | 1982-04-08 |
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| CA1163908A (en) | 1984-03-20 |
| NL8104304A (en) | 1982-05-03 |
| GB2084726B (en) | 1983-11-23 |
| JPH01304898A (en) | 1989-12-08 |
| DE3138602A1 (en) | 1982-06-24 |
| JPS5780560A (en) | 1982-05-20 |
| DE3138602C2 (en) | 1993-02-11 |
| FR2495184A1 (en) | 1982-06-04 |
| IT1144750B (en) | 1986-10-29 |
| AU543239B2 (en) | 1985-04-04 |
| JPH0474000B2 (en) | 1992-11-25 |
| SE8105737L (en) | 1982-04-02 |
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