DK159124B - PROCEDURE FOR KINETIC ANALYSIS OF SUR PHOSPHATASE AND REAGENT FOR USE IN EXERCISING THE PROCEDURE - Google Patents
PROCEDURE FOR KINETIC ANALYSIS OF SUR PHOSPHATASE AND REAGENT FOR USE IN EXERCISING THE PROCEDURE Download PDFInfo
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Opfindelsen angår en fremgangsmåde ved kinetisk analyse for sur phosphatase og reagens til anvendelse ved udøvelse af fremgangsmåden.The invention relates to a method of kinetic analysis for acid phosphatase and reagents for use in the practice of the process.
5 Fra Bergmeyer, H.U., "Methoden der enzymatischen Analyse", 3. Aufl.,5 From Bergmeyer, H.U., "Methods of Enzymatic Analysis", 3rd ed.,
Bd. 1, s. 532-543, 826-829 og 923-927, Verlag Chemie, Weinheim/Berg-strasse, 1974, kendes metoder til aktivitetsbestemmelse af phospho-glucomutase, 6-phosphogluconatdehydrogenase, phosphoglucoseisomerase og 3-phosphoglyceratkinase. PhosphogTucomutaseaktivitet bestemmes 10 under anvendelse af glucose-6-phosphatdehydrogenase som indikatorenzym. Endvidere kan α-amylaseaktivitet bestemmes ved at måle maltose og glucose, der hidrører fra nedbrydningen af stivelse med a-amylase. Fra "J. Biol. Chem.", 199, 153-163 (1952) og "J. Biol. Chem.", 236, 2186-2189 (1961) er det kendt, at α-maltose med enzymet 15 maltosephosphorylase under tilstedeværelse af phosphationer kan omdannes til glucose og /J-D-glucose-l-phosphat ved, at /J-D-glucose- 1-phosphat danner et substrat for phosphoglucomutase til dannelse af glucose-6-phosphat. I "Analytical Biochemistry", 57, 303-305 (1974) beskrives en fremgangsmåde til kvantitativ bestemmelse af maltose 20 under anvendelse af maltosephosphorylase koblet med et glucoseoxi-dasesystem. Fra "Analytical Biochemistry", 53, 108-114 (1973) kendes en fremgangsmåde til spektrofotometrisk bestemmelse af /J-glucose-1-phosphat i nærvær af a-glucose-l-phosphat og glucose-6-phosphat og under anvendelse af /?-mutase. I "J. Gen. Appl. Microbiol.", 14, 25 359-371 (1968) beskrives oprensning af og 6-phosphogluconatdehydro- genases egenskab til oxidativ decarboxylering af 6-phosphogluconat under dannelse af ribulose-5-phosphat og COg. Endvidere beskrives der i "Årztl. Lab.", 17, 340-343, 1971, bestemmelse af a-amylaseak-tiviteten sammenkoblet med α-glucosidase, hexokinase og glucose-6-30 phosphatdehydrogenase som indikatorenzymer. I "Agr. Biol. Chem.", 37, 2813-2819, 1973, beskrives oprensning af maltosephosphorylase og undersøgelse af dette enzyms specificitet med henblik på anvendelse af samme til kvantitativ bestemmelse af maltose i nærvær af stivel-seshydrolysater. Fra "Methods of Enzymatic Analyses", vol. 1, 35 Academic Press, New York, s. 109-110 og 123-127, 1974, kendes endvidere bestemmelse af glucose ved en "hjælpereaktion" under anvendelse af et "hjælpeenzym", som omdanner glucose til glucose-6-phosphat, der kan indgå som substrat i en NADP-afhængig indikatorreaktion. Reaktionerne er således koblede. Der kan desuden udføres 2Bd. 1, pp. 532-543, 826-829 and 923-927, Verlag Chemie, Weinheim / Berg-strasse, 1974, methods for the determination of phospho-glucomutase, 6-phosphogluconate dehydrogenase, phosphoglucose isomerase and 3-phosphoglycerate kinase are known. PhosphogTucomutase activity is determined using glucose-6-phosphate dehydrogenase as indicator enzyme. Furthermore, α-amylase activity can be determined by measuring maltose and glucose resulting from the breakdown of starch with α-amylase. From "J. Biol. Chem.", 199, 153-163 (1952) and "J. Biol. Chem.", 236, 2186-2189 (1961), it is known that α-maltose with the enzyme maltose phosphorylase in the presence of of phosphate ions can be converted to glucose and / JD-glucose-1-phosphate by forming / JD-glucose-1-phosphate as a substrate for phosphoglucomutase to form glucose-6-phosphate. Analytical Biochemistry, 57, 303-305 (1974) discloses a method for quantitative determination of maltose 20 using maltose phosphorylase coupled with a glucose oxidase system. From "Analytical Biochemistry", 53, 108-114 (1973), a method for spectrophotometric determination of β-glucose-1-phosphate is known in the presence of α-glucose-1-phosphate and glucose-6-phosphate and using ? -mutase. J. Gen. Appl. Microbiol., 14, 25 359-371 (1968) describes the purification of and 6-phosphogluconate dehydrogenase property for oxidative decarboxylation of 6-phosphogluconate to form ribulose-5-phosphate and CO Further, in "Årztl. Lab.", 17, 340-343, 1971, determination of α-amylase activity coupled with α-glucosidase, hexokinase and glucose-6-30 phosphate dehydrogenase are described as indicator enzymes. Agr. Biol. Chem., 37, 2813-2819, 1973 discloses purification of maltose phosphorylase and study of the specificity of this enzyme for use in the same for quantitative determination of maltose in the presence of starch hydrolysates. Further, from Methods of Enzymatic Assays, Vol. 1, 35 Academic Press, New York, pp. 109-110 and 123-127, 1974, determination of glucose by a "helper reaction" using a "auxiliary enzyme" known as converts glucose into glucose-6-phosphate, which can act as a substrate in a NADP-dependent indicator reaction. Thus, the reactions are coupled. In addition, 2 can be performed
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flere "hjælpeaktioner" efter hinanden. I beskrivelsen til US patent nr. 3.823.071 beskrives en fremgangsmåde til bestemmelse af surt prostataphosphataseenzym i en biologisk væske, ved hvilken en vandig opløsning af den biologiske væske, der som eneste substrat indehol-5 der et alkalimetal- eller alkalisk jordartsmetalsalt af thymol-phthaleinmonophosphat, inkuberes, og at den ved inkubationen omdannede substratmængde bestemmes. Der beskrives endvidere i US patent nr. 3.799.843 en fremgangsmåde til bestemmelse af sur phosphatase under anvendelse af en substratsammensætning omfattende et thymol-10 phthaleinsalt som farvedannende reagens.several "auxiliary actions" in succession. U.S. Patent No. 3,823,071 discloses a process for determining acidic prostate phosphatase enzyme in a biological fluid in which an aqueous solution of the biological fluid containing the sole substrate contains an alkali metal or alkaline earth metal salt of thymol. phthalein monophosphate is incubated and the amount of substrate converted during incubation is determined. Further, U.S. Patent No. 3,799,843 discloses a process for determining acid phosphatase using a substrate composition comprising a thymol-phthalein salt as a color-forming reagent.
Formålet med den foreliggende opfindelse er at tilvejebringe en fremgangsmåde til analyse for sur phosphatase, med hvilken fremgangsmåde der tilvejebringes en metode til hurtig, enkel, pålidelig 15 og reproducerbar bestemmelse af sur phosphatase i en forelagt prøve.The object of the present invention is to provide a method for assaying for acid phosphatase, which method provides a method for rapid, simple, reliable and reproducible determination of acid phosphatase in a submitted sample.
Dette formål opnås med fremgangsmåden ifølge opfindelsen, som er ejendommelig ved, (a) at der ved en pH på fra ca. 4,5 til ca. 6 udføres samtidige 20 reaktioner, som indbefatter: (I) omsætning af et organisk phosphat udvalgt fra gruppen bestående af /J-glycerophosphat, phenylphosphat, p-nitro-phenylphosphat, a-naphthylphosphat, adenosin-3'-monophos-phat, thymolphthaleinmonophosphat og phenolphthaleinmono- 25 phosphat i nærværelse af den sure phosphatase og under frigørelse af uorganisk phosphat, (II) omsætning af maltose med phosphationer i nærværelse af maltosephosphorylase til dannelse af glucose og β-D-glu-cose-l-phosphat, 30 (Ill)omsætning af /l-D-glucose-l-phosphat i nærværelse af /J-D-phosphoglucomutase til dannelse af glucose-6-phosphat, og (IV) omsætning af glucose-6-phosphat i nærværelse af glucose-6-phosphatdehydrogenase og et co-enzym udvalgt fra gruppen 35 bestående af ^-nicotinamid-adenindinucleotid, Ø-nicotin- amid-adenindinucleotidphosphat og blandinger heraf til dannelse af den reducerede form af co-enzymet og 6-phos-phogluconatet, (b) at dannelseshastigheden for det reducerede co-enzym måles,This object is achieved by the process according to the invention, which is characterized in that (a) that at a pH of from ca. 4.5 to approx. 6, simultaneous reactions are carried out which include: (I) reaction of an organic phosphate selected from the group consisting of β-glycerophosphate, phenylphosphate, p-nitro-phenylphosphate, α-naphthyl phosphate, adenosine 3'-monophosphate, thymolphthalein monophosphate and phenolphthalein monophosphate in the presence of the acidic phosphatase and during the release of inorganic phosphate, (II) reaction of maltose with phosphate ions in the presence of maltose phosphorylase to form glucose and β-D-glucose-1-phosphate, 30 reaction of / lD-glucose-1-phosphate in the presence of / JD-phosphoglucomutase to form glucose-6-phosphate, and (IV) reaction of glucose-6-phosphate in the presence of glucose-6-phosphate dehydrogenase and a co-enzyme selected from the group 35 consisting of β-nicotinamide adenine dinucleotide, β-nicotinamide adenine dinucleotide phosphate and mixtures thereof to form the reduced form of the coenzyme and the 6-phosphogluconate, (b) reducing the rate of formation thereof. a co-enzyme is measured,
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3 hvorhos den sure phosphatase, som måles, er hastighedsbegrænsende, og hvorhos der benyttes en ikke-phosphatpuffer til regulering af pH-værdien.3 wherein the acid phosphatase being measured is rate limiting and where a non-phosphate buffer is used to adjust the pH.
5 Den kinetiske analyse for sur phosphatase omfatter således følgende samtidige reaktioner: (I) organisk phosphat (R-P04 ) sur PhosPhataser po4'"" + r ___ MpThus, the kinetic analysis for acid phosphatase includes the following simultaneous reactions: (I) organic phosphate (R-PO 4) acidic PhosPhatases po4 + + r ___ Mp
10 (II) maltose + P04 _^ glucose + Ø-D-G-l-P(II) maltose + PO 4 _ glucose + Ø-D-G-1-P
(III) Ø-D-G-l-P ^~PGM^ G-6-P(III) Ø-D-G-1-P ^ ~ PGM ^ G-6-P
(IV) G-6-P + NAD G~6PDH ^ 6-P-G + NADH(IV) G-6-P + NAD G ~ 6PDH ^ 6-P-G + NADH
15 og i en foretrukken udførelsesform også følgende reaktion: (V) 6-P-G + NAD 6'PDH ^ ribulose-5-P + NADH + 00£ 20 Nedenstående forkortelser er anvendt i ovenstående reaktionsskemaer og i det følgende:15 and, in a preferred embodiment, also the following reaction: (V) 6-P-G + NAD 6'PDH ^ ribulose-5-P + NADH + 00 £ 20 The following abbreviations are used in the above reaction schemes and in the following:
Forkortelser: P04”’ : phosphation 25 MP : maltosephosphorylase β-D-G1P : /?-D-glucose-l-phosphat /3-PGM : /5-D-phosphoglucomutase G-1,6-diP : D-glucose-l,6-diphosphat G-6-P : glucose-6-phosphat 30 6-PG : 6-phosphogluconat G6PDH : glucose-6-phosphatdehydrogenase 6PDH : 6-phosphogluconatdehydrogenase NAD : Ø-nicotinamid-adenindinucleotid NADH : reduceret form af Ø-nicotinamid-adenindinukleotid 35Abbreviations: PO4 ": phosphate 25 MP: maltose phosphorylase β-D-G1P: β-D-glucose-1-phosphate / 3-PGM: / 5-D-phosphoglucomutase G-1,6-diP: D-glucose 1,6-diphosphate G-6-P: glucose-6-phosphate 30 6-PG: 6-phosphogluconate G6PDH: glucose-6-phosphate dehydrogenase 6PDH: 6-phosphogluconate dehydrogenase NAD: β-nicotinamide adenine dinucleotide NADH: reduced form of nicotinamide adenine dinucleotide 35
Skønt en hvilken som helst organisk phosphatforbindelse kan benyttes ved den første reaktion:Although any organic phosphate compound can be used in the first reaction:
(I) R-P04~- sur Phosphatase^ + R(I) R-PO 4 ~ - Acid Phosphatase + R
44
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vælges den organiske phosphatforbindelse fortrinsvis blandt Ø-gly-cerophosphat, phenylphosphat, p-nitrophenylphosphat, a-naphthylphosphat, adenosin-3'-monophosphat, thymolphthaleinmonophosphat og phenolphthaleinmonophosphat og er fortrinsvis a-naphthylphosphat.preferably, the organic phosphate compound is selected from β-glycerophosphate, phenyl phosphate, p-nitrophenyl phosphate, α-naphthyl phosphate, adenosine 3'-monophosphate, thymolphthalein monophosphate and phenolphthalein monophosphate and is preferably α-naphthyl phosphate.
55
Ved ovenstående kinetiske analyse for sur phosphatase er det nødvendigt, at mængden af sur phosphatase er hastighedsbegrænsende. Det organiske phosphat hydrolyseres af sur phosphatase til phosphat-ioner.In the above kinetic analysis for acid phosphatase, it is necessary that the amount of acid phosphatase be rate limiting. The organic phosphate is hydrolyzed by acid phosphatase to phosphate ions.
1010
Med hensyn til den anden ovenfor nævnte reaktion:Regarding the second reaction mentioned above:
MPMP
(II) maltose + phosphat glucose + /J-D-G-l-P(II) maltose + phosphate glucose + / J-D-G-l-P
15 omsættes tilsat maltose med de phosphationer, som dannes ved den første reaktion, under anvendelse af maltosephosphorylase som enzymatisk katalysator til dannelse af glucose og ^-D-glucose-1-phosphat.15, added maltose is reacted with the phosphate ions formed in the first reaction using maltose phosphorylase as enzymatic catalyst to form glucose and β-D-glucose-1-phosphate.
20 Maltosephosphorylase er et enzym, som katalyserer reaktionen mellem α-maltose og uorganisk phosphat.Maltose phosphorylase is an enzyme that catalyzes the reaction between α-maltose and inorganic phosphate.
Den foretrukne kilde for maltosephosphorylase er en stamme af mikroorganismen Lactobacillus brevis (ATCC 8287), som er blevet 25 dyrket af Beckman Instruments, Inc., Microbics Operations, Carlsbad, Californien, og enzymet er blevet ekstraheret og renset ved sædvanlige fremgangsmåder derfra. Andre kilder for dette enzym er stammer af Neisseria meninqitides, Neisseria perflava og andre Lactobacil-li-stammer.The preferred source of maltose phosphorylase is a strain of the microorganism Lactobacillus brevis (ATCC 8287) which has been grown by Beckman Instruments, Inc., Microbics Operations, Carlsbad, California, and the enzyme has been extracted and purified by conventional methods therefrom. Other sources of this enzyme are strains of Neisseria meninqitides, Neisseria perflava and other Lactobacil-li strains.
3030
Med hensyn til den tredie af ovennævnte reaktioner:With regard to the third of the above reactions:
(III) β-ϋ-G-l-P G-6-P(III) β-ϋ-G-l-P G-6-P
35 katalyserer enzymet Ø-phosphoglucomutase (/J-PGM) omdannelsen af Ø-D-glucose-l-phosphat til glucose-6-phosphat. Den foretrukne kilde for J3-PGM er Lactobacillus brevis (ATCC 8287). Den dyrkes og renses ved sædvanlige enzymrensningsmetoder. Andre kilder er stammer af Neisseria meninqitides, Neisseria perflava og Euqlena gracilis.35, the enzyme catalyzes β-phosphoglucomutase (/ J-PGM) conversion of β-D-glucose-1-phosphate to glucose-6-phosphate. The preferred source for J3-PGM is Lactobacillus brevis (ATCC 8287). It is grown and purified by conventional enzyme purification methods. Other sources are strains of Neisseria meninqitides, Neisseria perflava and Euqlena gracilis.
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Fortrinsvis er glucose-l,6-diphosphat (G-l,6-diP) til stede i enzymsystemet for at virke som cofaktor for Ø-PGM. /J-PGM kræver /J-formen af G~1,6-diP for aktivitet, men det antages, at α-formen af denne cofaktor også kan virke.Preferably, glucose-1,6-diphosphate (G-1,6-diP) is present in the enzyme system to act as cofactor for β-PGM. The / J PGM requires the / J form of G ~ 1,6-diP for activity, but it is believed that the α form of this cofactor may also work.
5 +2 +25 + 2 + 2
Fortrinsvis er der også divalente kationer i form af Mn , Mg , ^2 +7 i oPreferably, there are also divalent cations in the form of Mn, Mg, 2 +7 in o
Co , Zn eller Ni til stede i enzymsystemet for at virke som cofaktor for Ø-PGM. Kationerne Mn+2, Mg+2 eller Co+2 foretrækkes fremfor Zn+2 eller Ni+2.Co, Zn or Ni present in the enzyme system to act as cofactor for β-PGM. The cations Mn + 2, Mg + 2 or Co + 2 are preferred over Zn + 2 or Ni + 2.
1010
Hvad angår den fjerde af ovennævnte reaktioner:Regarding the fourth of the above reactions:
(IV) G-6-P + NAD G"6PPH ^ 6-P-G + NADH(IV) G-6-P + NAD G "6PPH ^ 6-P-G + NADH
15 omsættes glucose-6-phosphat med /l-nicotinamid-adenindinucleotid og G6PDH under dannelse af 6-phosphogluconat og NADH.15, glucose 6-phosphate is reacted with / 1-nicotinamide adenine dinucleotide and G6PDH to form 6-phosphogluconate and NADH.
Den foretrukne kilde for G-6-PDH er Leuconostoc mesenteroides (ATCC 12291), men det kan fås fra andre kilder.The preferred source for G-6-PDH is Leuconostoc mesenteroides (ATCC 12291), but it can be obtained from other sources.
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Formålet med den eventuelle femte reaktion er at forøge sensitiviteten og nøjagtigheden af analysen ved at forøge den dannede mængde NADH: 25 (V) 6-P-G + NAD 6~PDH ^ ribulose-5-P + NADH + C02The purpose of the optional fifth reaction is to increase the sensitivity and accuracy of the assay by increasing the amount of NADH formed: 25 (V) 6-P-G + NAD 6 ~ PDH ^ ribulose-5-P + NADH + CO 2
Den foretrukne kilde til enzymet 6-PDH er Leuconostoc mesenteroides (ATCC 12291), hvorfra enzymet er blevet dyrket og renset ved sædvan-30 lige kendte fremgangsmåder, men det kan fås fra andre kilder.The preferred source of the enzyme 6-PDH is Leuconostoc mesenteroides (ATCC 12291), from which the enzyme has been grown and purified by conventional methods, but it can be obtained from other sources.
Hastigheden for phosphationfrigørelse bestemmes således ved måling af hastigheden for dannet NADH, NADPH eller blandinger heraf under anvendelse af de koblede enzymatiske reaktioner ifølge den forelig-35 gende opfindelse.Thus, the rate of phosphate release is determined by measuring the rate of NADH, NADPH or mixtures thereof formed using the coupled enzymatic reactions of the present invention.
NADH-Dannelseshastigheden og omregningen af denne hastighed til sur phosphatasekoncentrationen gennemføres ved velkendte metoder. En af disse metoder benytter spektrofotometrisk udstyr til måling af 6The NADH formation rate and conversion of this rate to the acid phosphatase concentration is accomplished by well known methods. One of these methods uses spectrophotometric equipment to measure 6
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ændringen i lysabsorbans som følge af dannelsen af NADH ved bølgelængder beliggende mellem 300 og 370 millimicron (nm) i et temperaturområde på fra ca. 15eC til ca. 50°C. En bølgelængde på ca. 340 nm ved ca. 37°C foretrækkes.the change in light absorbance due to the formation of NADH at wavelengths located between 300 and 370 millimicrons (nm) in a temperature range of from ca. 15 ° C to approx. 50 ° C. A wavelength of approx. 340 nm at approx. 37 ° C is preferred.
5 Når hastigheden for absorbansforandringen måles, kan koncentrationen af sur phosphatase beregnes efter følgende ligning, hvori absorbansforandringen måles ved en bølgelængde på 340 nm og ved en temperatur på 37*C.When the rate of absorbance change is measured, the concentration of acid phosphatase can be calculated by the following equation in which the absorbance change is measured at a wavelength of 340 nm and at a temperature of 37 ° C.
10 IU/liter = ΔΑ x Vt x 1000 Vs x 6,22 15 ΔΑ = forandring i absorbans/minut10 IU / liter = ΔΑ x Vt x 1000 Vs x 6.22 15 ΔΑ = change in absorbance / minute
Vj. = totalt reaktionsrumfangVj. = total reaction volume
Vs = rumfang prøve indeholdende a-amylase 6,22 = millimolært absorptivitetstal for NADH ved 340 nm.Vs = volume of sample containing α-amylase 6.22 = millimolar absorbance number of NADH at 340 nm.
20 pH-Værdien for den sure phosphataseanalyse holdes i området fra ca.The pH value of the acid phosphatase assay is kept in the range of approx.
4 til under 7, fortrinsvis fra ca. 4,5 til ca. 6, og navnlig fra ca.4 to less than 7, preferably from ca. 4.5 to approx. 6, and in particular from ca.
5 til ca. 6. Reagenssysfemet Ran pufres ved hjæl~p af”en hvilken som helst ikke-phosphatpuffer med en pH-værdi på fra ca. 4 til under 7, og som er forligelig med de reagenser, som anvendes. Eksempler på 25 sådanne ikke-phosphatpuffere er natriumcitrat, natriumhydrogenmaleat og natriumkakodylat. (Natriumcitrat er den foretrukne puffer til brug i forbindelse med det kinetiske reagenssystem for sur phosphatase).5 to approx. 6. The reagent system Ran is buffered using any non-phosphate buffer having a pH of about 4 to less than 7 and which are compatible with the reagents used. Examples of 25 such non-phosphate buffers are sodium citrate, sodium hydrogen maleate and sodium cocodylate. (Sodium citrate is the preferred buffer for use in the acidic phosphatase kinetic reagent system).
30 Opfindelsen omhandler også et reagens til anvendelse ved udøvelse af fremgangsmåden ifølge opfindelsen, hvilket reagens er ejendommeligt ved, at det omfatter: (a) maltose, 35 (b) et organisk phosphat udvalgt fra gruppen bestående af /J-glycerophosphat, phenolphosphat, p-nitrophenolphosphat, α-naphthylphosphat, adenosin-3'-monophosphat, thymolphtha-leinmonophosphat og phenolphthaleinmonophosphat, (c) maltosephosphorylase, 7The invention also provides a reagent for use in the practice of the invention, which reagent is characterized in that it comprises: (a) maltose, (b) an organic phosphate selected from the group consisting of / J-glycerophosphate, phenol phosphate, p -nitrophenol phosphate, α-naphthyl phosphate, adenosine 3'-monophosphate, thymolphthalein monophosphate and phenolphthalein monophosphate, (c) maltose phosphorylase, 7
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(d) et co-enzym udvalgt fra gruppen bestående af /1-nicotin-amid-adenindinucleotid, Ø-nicotinamid-adenindinucleotid-phosphat og blandinger heraf, (e) glucose-6-phosphatdehydrogenase, 5 (f) /J-D-phosphoglucomutase, og eventuelt (g) 6-phosphogluconatdehydrogenase, hvorhos ovenstående stoffer er til stede i en sådan mængde, at den sure phosphatase, som skal bestemmes, er hastighedsbegrænsende, og 10 (h) en ikke-phosphatholdig puffer med en pH-værdi på fra ca.(d) a co-enzyme selected from the group consisting of / 1-nicotinamide-adenine dinucleotide, β-nicotinamide-adenine dinucleotide phosphate and mixtures thereof, (e) glucose-6-phosphate dehydrogenase, 5 (f) / JD-phosphoglucomutase, and optionally (g) 6-phosphogluconate dehydrogenase, wherein the above substances are present in an amount such that the acidic phosphatase to be determined is rate limiting, and 10 (h) a non-phosphate-containing buffer having a pH of about .
4,5 til ca. 6,0.4.5 to approx. 6.0.
Reagenssystemet kan opbevares og benyttes i form af en vandig opløs-15 ning, eller opløsningen kan frysetørres ved sædvanlige foranstaltninger og rekonstitueres med vand, når den er klar til brug. Reagenssystemet kan også fremstilles under anvendelse af dets bestanddele i pulveriseret form, som opløses med vand" umiddelbart inden brugen.The reagent system can be stored and used in the form of an aqueous solution, or the solution can be freeze-dried by usual means and reconstituted with water when ready for use. The reagent system can also be prepared using its constituents in powdered form which is dissolved with water immediately prior to use.
2020
Eksempler på analysereagenssammensætninger for sur phosphatase er angivet i nedenstående skema.Examples of assay reagent compositions for acid phosphatase are given in the table below.
EKSEMPELEXAMPLE
2525
Bestanddele i analyseblanding for sur phosphatase Nødvendig mindsteIngredients in assay mixture for acid phosphatase Minimum required
Bestanddel Foretrukket område mængde_Ingredient Preferred range quantity_
30 Organisk phosphat 1-5 mM 0,5 mMOrganic phosphate 1-5 mM 0.5 mM
Maltose 5-20 mM 2 mMMaltose 5-20 mM 2 mM
Maltosephosphorylase 1-5 IU/ml 0,5 IU/ml jff-Phosphoglucomutase 0,3-2 IU/ml 0,1 IU/mlMaltose Phosphorylase 1-5 IU / ml 0.5 IU / ml jff-Phosphoglucomutase 0.3-2 IU / ml 0.1 IU / ml
Co-enzym (NAD, NADP) 0,2-4 mM 0,1 mMCoenzyme (NAD, NADP) 0.2-4 mM 0.1 mM
35 Glucose-6-phosphat DH 2-10 IU/ml 1 IU/mlGlucose-6-phosphate DH 2-10 IU / ml 1 IU / ml
Di valent kation 1-5 mM 0The valent cation 1-5 mM 0
Glucose-l,6-diphosphat 0,02-0,2 mM 0Glucose-1,6-diphosphate 0.02-0.2 mM 0
Ikke-phosphatpuffer 0,02-0,05 M 0,01 MNon-phosphate buffer 0.02-0.05 M 0.01 M
Claims (11)
Applications Claiming Priority (6)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US65797676 | 1976-02-13 | ||
| US05/657,976 US4036697A (en) | 1976-02-13 | 1976-02-13 | Kinetic assay for alpha-amylase |
| US75851877 | 1977-01-11 | ||
| US05/758,518 US4097336A (en) | 1976-02-13 | 1977-01-11 | Reagent system for beta-amylase assay |
| DK061077A DK160844C (en) | 1976-02-13 | 1977-02-11 | PROCEDURE FOR KINETIC DETERMINATION OF ALFA AMYLASE IN Aqueous SOLUTIONS AND REAGENT SYSTEM FOR USE THEREOF |
| DK61077 | 1977-02-11 |
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| Publication Number | Publication Date |
|---|---|
| DK268380A DK268380A (en) | 1980-06-23 |
| DK159124B true DK159124B (en) | 1990-09-03 |
| DK159124C DK159124C (en) | 1991-02-11 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| DK268280A DK158956C (en) | 1976-02-13 | 1980-06-23 | PROCEDURE FOR KINETIC OR END-POINT ANALYSIS OF INORGANIC PHOSPHATE AND REAGENT USE IN EXERCISE OF THE PROCEDURE |
| DK268380A DK159124C (en) | 1976-02-13 | 1980-06-23 | PROCEDURE FOR KINETIC ANALYSIS OF SUR PHOSPHATASE AND REAGENT FOR USE IN EXERCISING THE PROCEDURE |
| DK268180A DK159123C (en) | 1976-02-13 | 1980-06-23 | PROCEDURE FOR KINETIC ANALYSIS OF BETA AMYLASE AND REAGENT USE IN EXERCISE OF THE PROCEDURE |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| DK268280A DK158956C (en) | 1976-02-13 | 1980-06-23 | PROCEDURE FOR KINETIC OR END-POINT ANALYSIS OF INORGANIC PHOSPHATE AND REAGENT USE IN EXERCISE OF THE PROCEDURE |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| DK268180A DK159123C (en) | 1976-02-13 | 1980-06-23 | PROCEDURE FOR KINETIC ANALYSIS OF BETA AMYLASE AND REAGENT USE IN EXERCISE OF THE PROCEDURE |
Country Status (1)
| Country | Link |
|---|---|
| DK (3) | DK158956C (en) |
-
1980
- 1980-06-23 DK DK268280A patent/DK158956C/en not_active IP Right Cessation
- 1980-06-23 DK DK268380A patent/DK159124C/en not_active IP Right Cessation
- 1980-06-23 DK DK268180A patent/DK159123C/en not_active IP Right Cessation
Also Published As
| Publication number | Publication date |
|---|---|
| DK158956C (en) | 1990-12-31 |
| DK268280A (en) | 1980-06-23 |
| DK159123B (en) | 1990-09-03 |
| DK159123C (en) | 1991-02-11 |
| DK268180A (en) | 1980-06-23 |
| DK159124C (en) | 1991-02-11 |
| DK268380A (en) | 1980-06-23 |
| DK158956B (en) | 1990-08-06 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PBP | Patent lapsed |