NO842258L - STABILIZED PRODUCTS FOR ISOENOMY REGULATION. - Google Patents
STABILIZED PRODUCTS FOR ISOENOMY REGULATION.Info
- Publication number
- NO842258L NO842258L NO842258A NO842258A NO842258L NO 842258 L NO842258 L NO 842258L NO 842258 A NO842258 A NO 842258A NO 842258 A NO842258 A NO 842258A NO 842258 L NO842258 L NO 842258L
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- Prior art keywords
- reagent according
- lactose
- net
- present
- reagent
- Prior art date
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- 239000003153 chemical reaction reagent Substances 0.000 claims description 55
- 108010044467 Isoenzymes Proteins 0.000 claims description 49
- 239000003381 stabilizer Substances 0.000 claims description 45
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 29
- 239000008101 lactose Substances 0.000 claims description 24
- 102000004190 Enzymes Human genes 0.000 claims description 23
- 108090000790 Enzymes Proteins 0.000 claims description 23
- 230000000694 effects Effects 0.000 claims description 21
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 claims description 12
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 claims description 12
- GUBGYTABKSRVRQ-CUHNMECISA-N D-Cellobiose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-CUHNMECISA-N 0.000 claims description 12
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 12
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 claims description 12
- 229930195725 Mannitol Natural products 0.000 claims description 12
- 239000000594 mannitol Substances 0.000 claims description 12
- 235000010355 mannitol Nutrition 0.000 claims description 12
- 235000000346 sugar Nutrition 0.000 claims description 11
- 108090000623 proteins and genes Proteins 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 9
- 102000004169 proteins and genes Human genes 0.000 claims description 9
- 239000003795 chemical substances by application Substances 0.000 claims description 8
- 230000000087 stabilizing effect Effects 0.000 claims description 7
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 claims description 6
- 125000003071 maltose group Chemical group 0.000 claims description 6
- 229910021645 metal ion Inorganic materials 0.000 claims description 6
- 239000000872 buffer Substances 0.000 claims description 5
- -1 monosaccharide sugars Chemical class 0.000 claims 10
- 239000007864 aqueous solution Substances 0.000 claims 4
- 239000000243 solution Substances 0.000 claims 4
- 229910019142 PO4 Inorganic materials 0.000 claims 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims 3
- 239000010452 phosphate Substances 0.000 claims 3
- 239000004599 antimicrobial Substances 0.000 claims 1
- 238000001879 gelation Methods 0.000 claims 1
- 230000005764 inhibitory process Effects 0.000 claims 1
- 230000000813 microbial effect Effects 0.000 claims 1
- 238000004321 preservation Methods 0.000 claims 1
- 239000000047 product Substances 0.000 description 8
- 102000003855 L-lactate dehydrogenase Human genes 0.000 description 7
- 108700023483 L-lactate dehydrogenases Proteins 0.000 description 7
- 238000007710 freezing Methods 0.000 description 7
- 230000008014 freezing Effects 0.000 description 7
- 239000000463 material Substances 0.000 description 6
- 210000001519 tissue Anatomy 0.000 description 6
- DDRJAANPRJIHGJ-UHFFFAOYSA-N creatinine Chemical compound CN1CC(=O)NC1=N DDRJAANPRJIHGJ-UHFFFAOYSA-N 0.000 description 4
- 102100036475 Alanine aminotransferase 1 Human genes 0.000 description 3
- 108010082126 Alanine transaminase Proteins 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 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 description 2
- 108700016171 Aspartate ammonia-lyases Proteins 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical group OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical class O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 108090000340 Transaminases Proteins 0.000 description 2
- 229960001456 adenosine triphosphate Drugs 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 229940109239 creatinine Drugs 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 239000003349 gelling agent Substances 0.000 description 2
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 210000003205 muscle Anatomy 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 102000014898 transaminase activity proteins Human genes 0.000 description 2
- 229910002012 Aerosil® Inorganic materials 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- PWKSKIMOESPYIA-BYPYZUCNSA-N L-N-acetyl-Cysteine Chemical compound CC(=O)N[C@@H](CS)C(O)=O PWKSKIMOESPYIA-BYPYZUCNSA-N 0.000 description 1
- 108090000279 Peptidyltransferases Proteins 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- 102000001253 Protein Kinase Human genes 0.000 description 1
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 description 1
- 229960004308 acetylcysteine Drugs 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 210000005013 brain tissue Anatomy 0.000 description 1
- 230000000747 cardiac effect Effects 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 230000001627 detrimental effect Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 150000002016 disaccharides Chemical class 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000005496 eutectics Effects 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 230000008571 general function Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 229960003180 glutathione Drugs 0.000 description 1
- 230000034659 glycolysis Effects 0.000 description 1
- 210000005003 heart tissue Anatomy 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 150000002772 monosaccharides Chemical group 0.000 description 1
- 230000002107 myocardial effect Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 108060006633 protein kinase Proteins 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
Landscapes
- Enzymes And Modification Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Fats And Perfumes (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Description
Foreliggende oppfinnelse vedrører stabiliserte kontroll-produkter som er nyttige i det kliniske miljø og angår spesielt stabiliserte isoenzym-kontrollreagenser innbefattende CK- og LDH-isoenzymer. The present invention relates to stabilized control products which are useful in the clinical environment and relates in particular to stabilized isoenzyme control reagents including CK and LDH isoenzymes.
Isoenzymer eller isozymer som de alternativt kalles, er enzymer i flere former som kan utføre den samme generelle funksjon, men ved forskjellige hastigheter. De er til-strekkelig forskjellige i kjemisk sammensetning slik at de vanligvis kan separeres elektroforetisk. Et slikt isoenzym, laktat-dehydrogenase (LDH), finnes i fem elektroforetisk adskilte fraksjoner. Hver av disse elektroforetiske typer av LDH er en tetramer bestående av Isoenzymes, or isozymes as they are alternatively called, are enzymes in multiple forms that can perform the same general function but at different rates. They are sufficiently different in chemical composition that they can usually be separated electrophoretically. One such isoenzyme, lactate dehydrogenase (LDH), is found in five electrophoretically separated fractions. Each of these electrophoretic types of LDH is a tetramer consisting of
- to polypeptidkjede-enheter, H og M, som er til stede i forskjellige proporsjoner: H4, MH3,<M>2<H>2, HM3og M4. Disse fem isoenzymer erforskjellige hva angår katalytisk aktivitet (affinitet for substratet, pyruvat målt ved hjelp av Michaelis-konstanten), aminosyresammensetning, varmelabilitet og immunologisk respons. De to peptidene H og M er kodet av forskjellige gener. Den type enzym som er til stede, er således under genetisk kontroll og reguleres av betingelsene i det miljø som utøves på cellen. Likeledes er kreatinin-kinase (CK) et annet isoenzym som inneholder underenheter av enten M-deler eller B-deler og.kan således være til stede som MM, BB eller MB. MB-formen er klinisk signifikant som en indikator for myokardial informasjon, men denne form er ustabil og er tilbøyelig til å dissosiere og gjendannes til MM- eller BB-typer. Det er et formål å stabilisere en kontrollreagens som har MB-formen. - two polypeptide chain units, H and M, which are present in different proportions: H4, MH3,<M>2<H>2, HM3 and M4. These five isoenzymes are different in terms of catalytic activity (affinity for the substrate, pyruvate measured using the Michaelis constant), amino acid composition, heat-lability and immunological response. The two peptides H and M are encoded by different genes. The type of enzyme that is present is thus under genetic control and is regulated by the conditions in the environment exerted on the cell. Likewise, creatinine kinase (CK) is another isoenzyme that contains subunits of either M parts or B parts and can thus be present as MM, BB or MB. The MB form is clinically significant as an indicator of myocardial information, but this form is unstable and prone to dissociate and revert to MM or BB types. It is a purpose to stabilize a control reagent that has the MB form.
De forskjellige proporsjoner eller kombinasjoner av isoenzymer som er til stede i vevet, kan relateres til de spesifikke behov i den aktuelle celle, og påvirkes således av slike faktorer som grad av differensiering og utvikling av cellen, samt av nivå og type av metabolisme som fore- kommer i cellen. Følgelig gir fordelingen mellom de forskjellige former for isoenzymer diagnostisk signifikante data. F.eks. utviser LDH signifikant kontroll over cellulær glykolyse. Spesielt er MH3- og H^-isoenzymtyper dominerende i vev med ren aerob eller "respiratorisk metabolisme. De kan følgelig anvendes som diagnostiske verktøy ved bestemmelse av tilstanden hos muskler slik som hjertet, spesielt med CK-isoenzymer, hjernen,leveren og andre organer i tilfellet for alanin-aminotransferase (ALT) eller aspartase-aminotransferanse (AST). Nok et annet isoenzym av betydning er alkalisk fosfatase-gammaglutamyl-transpeptidase. The different proportions or combinations of isoenzymes that are present in the tissue can be related to the specific needs of the cell in question, and are thus affected by such factors as the degree of differentiation and development of the cell, as well as by the level and type of metabolism that occurs enters the cell. Consequently, the distribution between the different forms of isoenzymes provides diagnostically significant data. E.g. LDH exhibits significant control over cellular glycolysis. In particular, MH3 and H^ isoenzyme types are predominant in tissues with purely aerobic or "respiratory metabolism. They can therefore be used as diagnostic tools in determining the condition of muscles such as the heart, especially with CK isoenzymes, the brain, the liver and other organs in the case for alanine aminotransferase (ALT) or aspartase aminotransferase (AST).Yet another important isoenzyme is alkaline phosphatase-gammaglutamyl transpeptidase.
Med en slik oppmerksomhet rettet mot bestemmelse av isoenzym-nivåer, hvilket er spesielt viktig i tilfelle for pasienter ''med kritisk hjertetilstand, er det aksiomatisk at tilstrekkelige kontroller må være tilgjengelige for å sikre riktig operasjon av manuelle automatiske metoder som er beregnet for bestemmelse av disse nivåer. Hittil har slike kontroller som har vært tilgjengelige, typisk vært ustabile p.g.a. enzymenes egen høye ustabile natur. With such attention directed to the determination of isozyme levels, which is particularly important in the case of patients with "critical cardiac condition", it is axiomatic that adequate controls must be available to ensure the correct operation of manual automated methods intended for the determination of these levels. Until now, such controls that have been available have typically been unstable due to the enzymes own highly unstable nature.
Det er et formål med foreliggende oppfinnelse å tilveiebringe kontrollreagenser som har de nødvendige nivåer av isoenzymer til stede deri i et stabilisert format. It is an object of the present invention to provide control reagents which have the required levels of isoenzymes present therein in a stabilized format.
Et annet formål med oppfinnelsen er å tilveiebringe metoder hvorved isoenzym-kontrollreagenser kan stabiliseres. Another object of the invention is to provide methods by which isoenzyme control reagents can be stabilized.
Ifølge formålene og prinsippene ved foreliggende oppfinnelse er det tilveiebragt isoenzym-kontrollreagenser for kreatinin-kinase, laktat-dehydrogenase, alanin-aminotransferanse og aspartase-aminotransferase, hvilke er vesentlig stabilisert ved tilsetning av nettformede ("plexiform") stabiliseringsmidler. Det foretrukne nettformede stabiliseringsmiddel velges fra gruppen bestående av maltose, manitol, cellobiose og laktose, idet det sistnevnte er det som er mest foretrukket. Det nettformede stabiliseringsmiddel tilveiebringes fordelaktig i en sluttkonsentrasjon i området på ca. 2-8%, idet den idelle konsentrasjon foreligger ved ca. 6%. Den ideelle isoenzym-kontrollreagens vil ha vesentlig alt vann fjernet, slik som ved lyofilisering, for å forbedre langvarig lagring og stabilitet. According to the purposes and principles of the present invention, isoenzyme control reagents for creatinine kinase, lactate dehydrogenase, alanine aminotransferase and aspartase aminotransferase are provided, which are significantly stabilized by the addition of net-shaped ("plexiform") stabilizing agents. The preferred net stabilizer is selected from the group consisting of maltose, mannitol, cellobiose and lactose, the latter being the most preferred. The net-shaped stabilizer is advantageously provided in a final concentration in the range of approx. 2-8%, the ideal concentration being at approx. 6%. The ideal isozyme control reagent will have substantially all water removed, such as by lyophilization, to improve long-term storage and stability.
Som et resultat kan kontrollreagensproduktene ifølge foreliggende oppfinnelse fremstilles slik at de virker og oppfører seg på den måte som er mer lik dem hos en pasient, og de foreliggende reagenser er virkelig i stand , til å benyttes i hvilken som helst av de tre separeringssystemer som i dag er i bruk: Kolonne-kromatografi og de immunobaserte separasjonssystemene. As a result, the control reagent products of the present invention can be prepared to act and behave in a manner more similar to that in a patient, and the present reagents are indeed capable of being used in any of the three separation systems as in are currently in use: Column chromatography and the immuno-based separation systems.
Som antydet ovenfor har det hittil vært store vanskeligheter med å stabilisere en slik isoenzym-kontrollreagens og spesielt stabilisering av de forskjellige isoenzym-underenhetene slik som CK-isoenzym-enhet MB uten skadelig innvirkning på andre underenhets-konponenter eller tester for disse. Ved tilsetning av det nettformede stabiliseringsmiddel ifølge foreliggende oppfinnelse har disse isoenzymer og deres underenhets-bestanddeler nå blitt stabilisert og kan holdes i oppløsning i betydelig lengre tidsrom enn det som tidligere var mulig. I tørr form, oppnådd når vesentlig alt vann er fjernet,slik som ved lyofilisering, blir stabilitetsperioden forøket i enda større grad. As indicated above, there has hitherto been great difficulty in stabilizing such an isoenzyme control reagent and in particular stabilizing the various isoenzyme subunits such as CK isoenzyme unit MB without detrimental effects on other subunit components or tests for these. By adding the net-shaped stabilizer according to the present invention, these isoenzymes and their subunit components have now been stabilized and can be kept in solution for a significantly longer period of time than was previously possible. In dry form, obtained when substantially all water has been removed, such as by lyophilization, the stability period is increased to an even greater extent.
Tilsetningen av det nettformede stabiliseringsmiddel ifølge oppfinnelsen resulterer i nedsettelse avdet faktiske frysepunkt. Frysepunktsnedsettelse er vanligvis forbundet med langsommere frysehastigheter, men hurtigere frysing av produktet ifølge oppfinnelsen har imidlertid blitt observert. Det synes imidlertid som om det nettformede stabiliseringsmiddel når det er tilsatt til foreliggende produkt, resulterter i tap av platået for det eutektiske punkt, og således faktisk øker fryse-hastigheten. Forbundet med dette fenomen er den observasjon at de kaker som dannes under frysing, er ensartet krystallinske i motsetning til de ofte forekommende pulverformer. Dette kan tyde på at det nettformede stabiliseringsmiddel holder bestanddelene i en stabil, tredimensjonal "krystallinsk" struktur og derved hjelper fjerningen av vann, stabiliseringen av selve bestanddelene,samt gjør rekonstitueringen av det lyofiliserte materiale hurtigere. Disse og andre komplekse virkninger er mer fullstendig beskrevet i norsk The addition of the net-shaped stabilizer according to the invention results in a reduction of the actual freezing point. Freezing point depression is usually associated with slower freezing rates, but faster freezing of the product according to the invention has, however, been observed. However, it appears that the net stabilizer when added to the present product results in a loss of the eutectic point plateau, and thus actually increases the freezing rate. Associated with this phenomenon is the observation that the cakes formed during freezing are uniformly crystalline in contrast to the often occurring powder forms. This may indicate that the net-shaped stabilizer keeps the components in a stable, three-dimensional "crystalline" structure and thereby helps the removal of water, the stabilization of the components themselves, and makes the reconstitution of the lyophilized material faster. These and other complex effects are more fully described in Norwegian
.patentsøknad nr.patent application no
For a fremstxlle isoenzym-kontrollreagensene ifølge foreliggende oppfinnelse ble det valgt et spesielt humant vev i overensstemmelse med den type av enzymholdige produkt som skulle fremstilles. For CK MB-underenheten velges f.eks. hjertevev, for CK MM enzym-underenheten anvendes muskelvev, mens for CK BB-underenheten anvendes hjernevev. Likeledes kan disse og andre vev innbefattende ikke-humane vev anvendes som kildemateriale for isoenzymene eller deres underenheter ifølge velkjent teknikk. In order to produce the isoenzyme control reagents according to the present invention, a special human tissue was chosen in accordance with the type of enzyme-containing product to be produced. For the CK MB subunit, e.g. heart tissue, for the CK MM enzyme subunit muscle tissue is used, while for the CK BB subunit brain tissue is used. Likewise, these and other tissues including non-human tissues can be used as source material for the isoenzymes or their subunits according to well-known techniques.
Vevet behandles deretter ved maling osv. for dannelse av en egnet isoenzym-isolasjon ved enten kromatografi, elektroforese, eller et annet immunologisk basert system ifølge velkjente teknikker. De således isolerte enzymkomponentene kan tilsettes i diagnostisk signifikante mengdeforhold til en fortrinnsvis på forhånd klaret, humansera-basis. Det humane sera blir fordelaktig klaret for å fjerne lipider enten ved filtrering, frysing, rekonstituering og filtrering eller ved tilsetning av siliciumdioksydforbindelser slik som aerosil ifølge velkjente metoder. The tissue is then processed by staining etc. to form a suitable isozyme isolation by either chromatography, electrophoresis, or another immunologically based system according to well known techniques. The thus isolated enzyme components can be added in diagnostically significant proportions to a preferably pre-clarified, human sera base. The human sera is advantageously clarified to remove lipids either by filtration, freezing, reconstitution and filtration or by the addition of silicon dioxide compounds such as aerosil according to well-known methods.
Det er deretter fordelaktig at det til dette materialet tilsettes en stabiliserende kofaktor etter behov for å hjelpe opprettholdelsen av bindingssted-aktivitet. F.eks., for CK-isoenzymene er sulfhydrylholdige forbindelser slik som glutation eller ditiotreotol eller n-acetyl-cystein nyttig. Videre, det ideelle produkt innbefatter også gelateringsmidler for fjerning av metallioner, dersom slike er til stede, hvilke kan forstyrre enzymaktivitet. Dersom enzymets aktivitet ikke påvirkes av tilstede-værelsen av metallioner, er det ikke nødvendig å inkludere dette . middel. Likeledes, dersom ingen metallioner er til stede, kan dette middel også elimineres. Et eksempel på et slikt gelateringsmiddel er etylendiamintetraeddiksyre It is then advantageous that a stabilizing cofactor is added to this material as needed to help maintain binding site activity. For example, for the CK isoenzymes, sulfhydryl-containing compounds such as glutathione or dithiothreotol or n-acetyl-cysteine are useful. Furthermore, the ideal product also includes gelling agents to remove metal ions, if present, which may interfere with enzyme activity. If the enzyme's activity is not affected by the presence of metal ions, it is not necessary to include this. medium. Likewise, if no metal ions are present, this agent can also be eliminated. An example of such a gelling agent is ethylenediaminetetraacetic acid
.(EDTA)..(EDTA).
Til slutt vil isoenzymproduktene ifølge foreliggende oppfinnelse ytterligere omfatte en svak, ikke-fosfatbuffer. Det er foretrukket at det anvendes en ikke-fosfatbuffer for å unngå fosforholdige forbindelser som ellers kan forstyrre noen av reaksjonene, spesielt de som innebærer adenintrifosfat (ATP)-cykler. Bufferen bør være "svak", dvs. i området på ca. 10-200 millimolar konsentrasjon. Bufferens pH-verdi bør ideelt velges eller justeres for fortrinnsvis å optimalisere aktiviteten og maksimere stabiliteten. Med f.eks. CK- og LDH-isoenzymene er en pH-verdi på omtrent 7 forelaktig, mens den ideelle pH-verdi for de alkaliske fosfatase-isoenzymene er i området fra ca. 7,6 til 7,8. Finally, the isoenzyme products according to the present invention will further comprise a weak, non-phosphate buffer. It is preferred that a non-phosphate buffer is used to avoid phosphorus-containing compounds that may otherwise interfere with some of the reactions, especially those involving adenine triphosphate (ATP) cycles. The buffer should be "weak", i.e. in the range of approx. 10-200 millimolar concentration. The pH of the buffer should ideally be chosen or adjusted to preferably optimize activity and maximize stability. With e.g. For the CK and LDH isoenzymes, a pH value of about 7 is favorable, while the ideal pH value for the alkaline phosphatase isoenzymes is in the range from about 7.6 to 7.8.
Sluttlig tilsettes nettformede stabiliseringsmidler for å tilveiebringe stabiliteten og andre ovenfor nevnte fordeler. Et slikt nettformet stabiliseringsmiddel velges fra gruppen bestående av monosakkarid- og disakkarid-reduserende sukkere og vil fortrinnsvis velges fra gruppen bestående av manitol, maltose, cellobiose og laktose. Som beskrevet i det ovenfor nevnte norske patent, er det mest foretrukne, hva angår stabilitetsfordeler og økonomiske betraktninger, laktose. Man har funnet det ønskelig å ha det nettformede stabiliseringsmiddel i et slutt-konsentrasjonsområde på ca. 2-8%, idet den mest foretrukne utførelse omfatter ca. 6%. Finally, net-shaped stabilizers are added to provide the stability and other advantages mentioned above. Such a net-shaped stabilizer is selected from the group consisting of monosaccharide and disaccharide reducing sugars and will preferably be selected from the group consisting of mannitol, maltose, cellobiose and lactose. As described in the above-mentioned Norwegian patent, the most preferred, as far as stability advantages and economic considerations are concerned, is lactose. It has been found desirable to have the net-shaped stabilizer in a final concentration range of approx. 2-8%, with the most preferred embodiment comprising approx. 6%.
Det nettformede stabiliseringsmiddel forårsaker ikke bare retardering av dissosiasjonen av isoenzymer hvis underenheter har tendens til å dissosiere temmelig lett, men stabiliserer også isoenzymets elektroforetiske mønstere. Konsentrasjonen av det nettformede stabiliseringsmiddel velges slik at enzymets aktivitet bibeholdes selv om produktet er frysetørket, og kan fortrinnsvis optimaliseres for maksimum enzymaktivitet. Dersom for lite nettformet stabiliseringsmiddel inkorporeres i sluttproduktet, så oppnås utilstrekkelig beskyttelse, men det er uønsket å tilsette for meget nettformet stabiliseringsmiddel som i høye konsentrasjoner ikke bare kan utfelles fra oppløsningen, men også kan blokkere proteinets aktive sete. Således har høye konsentrasjoner av det nettformede stabiliseringsmiddel faktisk tilbøyelighet til, å redusere enzymaktivitet og blir følgelig unngått. The net-shaped stabilizer not only causes retardation of the dissociation of isoenzymes whose subunits tend to dissociate rather easily, but also stabilizes the electrophoretic patterns of the isoenzymes. The concentration of the net-shaped stabilizer is chosen so that the activity of the enzyme is maintained even if the product is freeze-dried, and can preferably be optimized for maximum enzyme activity. If too little network-shaped stabilizer is incorporated into the final product, insufficient protection is achieved, but it is undesirable to add too much network-shaped stabilizer, which in high concentrations can not only precipitate from the solution, but can also block the protein's active site. Thus, high concentrations of the reticulated stabilizing agent actually tend to reduce enzyme activity and are therefore avoided.
Et eksempel på forøket stabilitet er angitt i tabell 1. De deri angitte data representerer aktiviteten for et CK-isoenzym-kontrollmateriale lagret i vandig form ved 37°C. Tabellen sammenligner de data som observeres,med et ustabilisert isoenzym-kontrollmateriale med det stabiliserte isoenzym-kontrollmateriale fra dag 0 til dag 30. Enda større økninger i stabilitet kan forventes ved lavere temperaturer, dvs. lagring 5°C samt i lyofilisert lagringsformat. An example of increased stability is shown in Table 1. The data shown therein represent the activity of a CK isozyme control material stored in aqueous form at 37°C. The table compares the data observed with an unstabilized isoenzyme control material with the stabilized isoenzyme control material from day 0 to day 30. Even greater increases in stability can be expected at lower temperatures, i.e. storage at 5°C and in lyophilized storage format.
Claims (47)
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US50121383A | 1983-06-06 | 1983-06-06 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| NO842258L true NO842258L (en) | 1984-12-07 |
Family
ID=23992572
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| NO842258A NO842258L (en) | 1983-06-06 | 1984-06-05 | STABILIZED PRODUCTS FOR ISOENOMY REGULATION. |
Country Status (6)
| Country | Link |
|---|---|
| JP (1) | JP2596911B2 (en) |
| AU (1) | AU583474B2 (en) |
| CA (1) | CA1230300A (en) |
| DK (1) | DK275184A (en) |
| IL (1) | IL72030A0 (en) |
| NO (1) | NO842258L (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA1226794A (en) * | 1983-06-06 | 1987-09-15 | Michael K. Hoskins | Stabilized multiparameter control product |
| US4663295A (en) * | 1983-06-29 | 1987-05-05 | Ciba Corning Diagnostics Corp. | Estrogen-progesterone control reagents and methods for making same |
| CA1226795A (en) * | 1983-06-06 | 1987-09-15 | Michael K. Hoskins | Stabilized coagulation control products |
| JP2575648B2 (en) * | 1986-04-24 | 1997-01-29 | 国際試薬株式会社 | Method for stabilizing creatine kinase |
| IT1394539B1 (en) * | 2009-05-19 | 2012-07-05 | Sentinel Ch S P A | USE OF A POLYMERASE STABILIZER FOR THE LIOFILIZATION AND PREPARATION OF READY-TO-USE KITS |
Family Cites Families (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS4834912A (en) * | 1971-09-09 | 1973-05-23 | ||
| JPS589688A (en) * | 1981-07-06 | 1983-01-20 | Toyobo Co Ltd | Stable enzymic composition |
| JPS5865218A (en) * | 1981-10-13 | 1983-04-18 | Kowa Co | Preparation of tissual plasminogen activator |
| JPS58107178A (en) * | 1981-12-22 | 1983-06-25 | Mitsui Toatsu Chem Inc | Stable freeze-dried product of beta-galactosidase or its composite |
| JPS58134991A (en) * | 1981-12-28 | 1983-08-11 | Takeda Chem Ind Ltd | Stabilization of serratiopeptidase |
| CA1226794A (en) * | 1983-06-06 | 1987-09-15 | Michael K. Hoskins | Stabilized multiparameter control product |
| US4663295A (en) * | 1983-06-29 | 1987-05-05 | Ciba Corning Diagnostics Corp. | Estrogen-progesterone control reagents and methods for making same |
| CA1226795A (en) * | 1983-06-06 | 1987-09-15 | Michael K. Hoskins | Stabilized coagulation control products |
-
1984
- 1984-05-02 CA CA000453361A patent/CA1230300A/en not_active Expired
- 1984-06-04 DK DK275184A patent/DK275184A/en not_active Application Discontinuation
- 1984-06-05 NO NO842258A patent/NO842258L/en unknown
- 1984-06-05 IL IL72030A patent/IL72030A0/en unknown
- 1984-06-05 AU AU29085/84A patent/AU583474B2/en not_active Ceased
- 1984-06-06 JP JP59114690A patent/JP2596911B2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| JPS606192A (en) | 1985-01-12 |
| CA1230300A (en) | 1987-12-15 |
| DK275184D0 (en) | 1984-06-04 |
| DK275184A (en) | 1984-12-07 |
| AU583474B2 (en) | 1989-05-04 |
| IL72030A0 (en) | 1984-10-31 |
| AU2908584A (en) | 1984-12-13 |
| JP2596911B2 (en) | 1997-04-02 |
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