NO780822L - PEROXYDASE DETERMINATION. - Google Patents
PEROXYDASE DETERMINATION.Info
- Publication number
- NO780822L NO780822L NO780822A NO780822A NO780822L NO 780822 L NO780822 L NO 780822L NO 780822 A NO780822 A NO 780822A NO 780822 A NO780822 A NO 780822A NO 780822 L NO780822 L NO 780822L
- Authority
- NO
- Norway
- Prior art keywords
- peroxidase
- buffer
- mmol per
- sample
- concentration
- Prior art date
Links
- 102000003992 Peroxidases Human genes 0.000 claims description 37
- 108040007629 peroxidase activity proteins Proteins 0.000 claims description 37
- 238000000034 method Methods 0.000 claims description 27
- RLFWWDJHLFCNIJ-UHFFFAOYSA-N 4-aminoantipyrine Chemical compound CN1C(C)=C(N)C(=O)N1C1=CC=CC=C1 RLFWWDJHLFCNIJ-UHFFFAOYSA-N 0.000 claims description 24
- 239000003153 chemical reaction reagent Substances 0.000 claims description 15
- 239000000872 buffer Substances 0.000 claims description 12
- 150000001875 compounds Chemical class 0.000 claims description 10
- 239000001257 hydrogen Substances 0.000 claims description 9
- 229910052739 hydrogen Inorganic materials 0.000 claims description 9
- 239000000758 substrate Substances 0.000 claims description 9
- 239000000203 mixture Substances 0.000 claims description 8
- 239000011149 active material Substances 0.000 claims description 7
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims description 6
- WKBOTKDWSSQWDR-UHFFFAOYSA-N Bromine atom Chemical compound [Br] WKBOTKDWSSQWDR-UHFFFAOYSA-N 0.000 claims description 5
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 claims description 5
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 5
- 125000000217 alkyl group Chemical group 0.000 claims description 5
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Substances BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 claims description 5
- 229910052794 bromium Inorganic materials 0.000 claims description 5
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 5
- 229910052801 chlorine Inorganic materials 0.000 claims description 5
- 239000000460 chlorine Substances 0.000 claims description 5
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 claims description 5
- 239000008363 phosphate buffer Substances 0.000 claims description 5
- 229910052783 alkali metal Inorganic materials 0.000 claims description 4
- 150000001340 alkali metals Chemical class 0.000 claims description 4
- 150000002431 hydrogen Chemical class 0.000 claims description 4
- 238000011534 incubation Methods 0.000 claims description 4
- 239000000523 sample Substances 0.000 description 10
- 102000004190 Enzymes Human genes 0.000 description 9
- 108090000790 Enzymes Proteins 0.000 description 9
- 229940088598 enzyme Drugs 0.000 description 9
- 102100022019 Pregnancy-specific beta-1-glycoprotein 2 Human genes 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 7
- 239000000427 antigen Substances 0.000 description 6
- 102000036639 antigens Human genes 0.000 description 6
- 108091007433 antigens Proteins 0.000 description 6
- 230000000694 effects Effects 0.000 description 5
- 230000001900 immune effect Effects 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 230000008033 biological extinction Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- FAXWFCTVSHEODL-UHFFFAOYSA-N 2,4-dibromophenol Chemical compound OC1=CC=C(Br)C=C1Br FAXWFCTVSHEODL-UHFFFAOYSA-N 0.000 description 2
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- LHGVFZTZFXWLCP-UHFFFAOYSA-N guaiacol Chemical compound COC1=CC=CC=C1O LHGVFZTZFXWLCP-UHFFFAOYSA-N 0.000 description 2
- 230000002779 inactivation Effects 0.000 description 2
- -1 lipoids Proteins 0.000 description 2
- RLSSMJSEOOYNOY-UHFFFAOYSA-N m-cresol Chemical compound CC1=CC=CC(O)=C1 RLSSMJSEOOYNOY-UHFFFAOYSA-N 0.000 description 2
- QWVGKYWNOKOFNN-UHFFFAOYSA-N o-cresol Chemical compound CC1=CC=CC=C1O QWVGKYWNOKOFNN-UHFFFAOYSA-N 0.000 description 2
- IWDCLRJOBJJRNH-UHFFFAOYSA-N p-cresol Chemical compound CC1=CC=C(O)C=C1 IWDCLRJOBJJRNH-UHFFFAOYSA-N 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- HFZWRUODUSTPEG-UHFFFAOYSA-N 2,4-dichlorophenol Chemical compound OC1=CC=C(Cl)C=C1Cl HFZWRUODUSTPEG-UHFFFAOYSA-N 0.000 description 1
- UFBJCMHMOXMLKC-UHFFFAOYSA-N 2,4-dinitrophenol Chemical compound OC1=CC=C([N+]([O-])=O)C=C1[N+]([O-])=O UFBJCMHMOXMLKC-UHFFFAOYSA-N 0.000 description 1
- KUFFULVDNCHOFZ-UHFFFAOYSA-N 2,4-xylenol Chemical compound CC1=CC=C(O)C(C)=C1 KUFFULVDNCHOFZ-UHFFFAOYSA-N 0.000 description 1
- CCBICDLNWJRFPO-UHFFFAOYSA-N 2,6-dichloroindophenol Chemical compound C1=CC(O)=CC=C1N=C1C=C(Cl)C(=O)C(Cl)=C1 CCBICDLNWJRFPO-UHFFFAOYSA-N 0.000 description 1
- ISPYQTSUDJAMAB-UHFFFAOYSA-N 2-chlorophenol Chemical compound OC1=CC=CC=C1Cl ISPYQTSUDJAMAB-UHFFFAOYSA-N 0.000 description 1
- IQUPABOKLQSFBK-UHFFFAOYSA-N 2-nitrophenol Chemical compound OC1=CC=CC=C1[N+]([O-])=O IQUPABOKLQSFBK-UHFFFAOYSA-N 0.000 description 1
- JRBJSXQPQWSCCF-UHFFFAOYSA-N 3,3'-Dimethoxybenzidine Chemical compound C1=C(N)C(OC)=CC(C=2C=C(OC)C(N)=CC=2)=C1 JRBJSXQPQWSCCF-UHFFFAOYSA-N 0.000 description 1
- WDNBURPWRNALGP-UHFFFAOYSA-N 3,4-Dichlorophenol Chemical compound OC1=CC=C(Cl)C(Cl)=C1 WDNBURPWRNALGP-UHFFFAOYSA-N 0.000 description 1
- HORNXRXVQWOLPJ-UHFFFAOYSA-N 3-chlorophenol Chemical compound OC1=CC=CC(Cl)=C1 HORNXRXVQWOLPJ-UHFFFAOYSA-N 0.000 description 1
- RTZZCYNQPHTPPL-UHFFFAOYSA-N 3-nitrophenol Chemical compound OC1=CC=CC([N+]([O-])=O)=C1 RTZZCYNQPHTPPL-UHFFFAOYSA-N 0.000 description 1
- WXNZTHHGJRFXKQ-UHFFFAOYSA-N 4-chlorophenol Chemical compound OC1=CC=C(Cl)C=C1 WXNZTHHGJRFXKQ-UHFFFAOYSA-N 0.000 description 1
- FJKROLUGYXJWQN-UHFFFAOYSA-N 4-hydroxybenzoic acid Chemical compound OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 description 1
- MNVMYTVDDOXZLS-UHFFFAOYSA-N 4-methoxyguaiacol Natural products COC1=CC=C(O)C(OC)=C1 MNVMYTVDDOXZLS-UHFFFAOYSA-N 0.000 description 1
- BTJIUGUIPKRLHP-UHFFFAOYSA-N 4-nitrophenol Chemical compound OC1=CC=C([N+]([O-])=O)C=C1 BTJIUGUIPKRLHP-UHFFFAOYSA-N 0.000 description 1
- 241000222120 Candida <Saccharomycetales> Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 102000011022 Chorionic Gonadotropin Human genes 0.000 description 1
- 108010062540 Chorionic Gonadotropin Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 108010015776 Glucose oxidase Proteins 0.000 description 1
- 239000004366 Glucose oxidase Substances 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 102000004895 Lipoproteins Human genes 0.000 description 1
- 108090001030 Lipoproteins Proteins 0.000 description 1
- 108010062374 Myoglobin Proteins 0.000 description 1
- 102000036675 Myoglobin Human genes 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 229930182558 Sterol Natural products 0.000 description 1
- 241000223996 Toxoplasma Species 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 241000223104 Trypanosoma Species 0.000 description 1
- 108010092464 Urate Oxidase Proteins 0.000 description 1
- LEHOTFFKMJEONL-UHFFFAOYSA-N Uric Acid Chemical compound N1C(=O)NC(=O)C2=C1NC(=O)N2 LEHOTFFKMJEONL-UHFFFAOYSA-N 0.000 description 1
- TVWHNULVHGKJHS-UHFFFAOYSA-N Uric acid Natural products N1C(=O)NC(=O)C2NC(=O)NC21 TVWHNULVHGKJHS-UHFFFAOYSA-N 0.000 description 1
- 239000003513 alkali Chemical group 0.000 description 1
- 229930013930 alkaloid Natural products 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000012928 buffer substance Substances 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 238000012412 chemical coupling Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229940116332 glucose oxidase Drugs 0.000 description 1
- 235000019420 glucose oxidase Nutrition 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 229960001867 guaiacol Drugs 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940084986 human chorionic gonadotropin Drugs 0.000 description 1
- 230000008105 immune reaction Effects 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 150000003141 primary amines Chemical class 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 150000003431 steroids Chemical class 0.000 description 1
- 150000003432 sterols Chemical class 0.000 description 1
- 235000003702 sterols Nutrition 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 229940116269 uric acid Drugs 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
- C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
-
- 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
- C12Q2326/00—Chromogens for determinations of oxidoreductase enzymes
- C12Q2326/50—Phenols; Naphthols; Catechols
-
- 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
- C12Q2326/00—Chromogens for determinations of oxidoreductase enzymes
- C12Q2326/90—Developer
- C12Q2326/96—4-Amino-antipyrine
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
Foreliggende oppfinnelse vedrører en ny fremgangsmåte ved bestemmelse av<p>eroksydase som særlig er av betydning i sammenheng med enzym-immunologiske prøver hvor dette enzymet anvendes for markering av antigener eller antistoffer. The present invention relates to a new method for determining<p>eroxidase which is particularly important in the context of enzyme-immunological tests where this enzyme is used for marking antigens or antibodies.
For enzym-immunologisk bestemmelse av bestanddeler i fysiologiske væsker, i vev eller i celle- og vevsektrakter markeres tilsvarende antigener eller antistoffer med enzymer.. Som markeringsenzym ble det tidligere mest anvendt peroksydase fra pe<p>perrot. Dette enzymet er lett tilgjengelig i høy renhet, har en høy spesifikk aktivitet, er resistent overfor de fleste kjemiske koblingsreagenser, har en god lagringsstabilitet og kan som relativt lite proteinmolekyl (MG = 40 000) lett adskilles fra peroksydase som er bundet til antigenet eller antistoffene. For enzyme-immunological determination of components in physiological fluids, in tissue or in cell and tissue extracts, corresponding antigens or antibodies are marked with enzymes. As a marking enzyme, peroxidase from pe<p>perrot was formerly most commonly used. This enzyme is readily available in high purity, has a high specific activity, is resistant to most chemical coupling reagents, has a good storage stability and, as a relatively small protein molecule (MG = 40,000), can be easily separated from the peroxidase that is bound to the antigen or antibodies .
Til aktivitetsbestemmelse av peroksydase ble tidligere fremfor alt anvendt o-dianisidin, o-tpluidin, guajacol og 2,6-diklorfenolindofenol. Disse kromoforene er generelt dårlig løselige slik at de bare kan anvendes i forsøket i lave konsentrasjoner. Dertil er de ikke i stand til å beskytte peroksydase effektivt mot inaktivering ved H^02. To determine the activity of peroxidase, o-dianisidine, o-tpluidin, guaiacol and 2,6-dichlorophenolindophenol were previously used above all. These chromophores are generally poorly soluble so that they can only be used in the experiment in low concentrations. In addition, they are unable to protect peroxidase effectively against inactivation by H 2 O 2 .
I forskjellige arbeider ble det påvist at det under den katalytiske reaksjonen danner peroksydase med f^O^et stabilt og en enzymatisk inaktiv kompleks, slik åt stadig mindre peroksydase foreligger i aktiv form. Dette resul-terer i at etter en 10 - 20 minutters reaksjonsperiode er peroksydasen ved en inkubasjonstemperatur på 37°C full-stendig inaktivert. Denne inaktiveringen er særlig uheldig ved enzym-immunologiske forsøk, da bestanddeler ofte skal bestemmes i så lave konsentrasjoner at for å oppnå en tilstrekkelig sensibilitet må peroksydasen som er bundet til antigenet eller antistoffet inkuberes opptil 2 timer med den tilsvarende substrat-pufferløsningen. In various works, it was demonstrated that during the catalytic reaction, peroxidase forms a stable and enzymatically inactive complex with f^O^, so that less and less peroxidase exists in active form. This results in the peroxidase being completely inactivated at an incubation temperature of 37°C after a 10-20 minute reaction period. This inactivation is particularly unfortunate in enzyme-immunological experiments, as components often have to be determined in such low concentrations that in order to achieve sufficient sensitivity, the peroxidase that is bound to the antigen or antibody must be incubated for up to 2 hours with the corresponding substrate buffer solution.
Ifølge foreliggende oppfinnelse ble en ny fremgangsmåte utviklet som ikke har de ovennevnte ulemper. Den muliggjør å bestemme de minste mengder peroksydase kvantitativt og dermed oppnå den nødvendige sensibilitet for de ehzym-immunologiske prøver. According to the present invention, a new method was developed which does not have the above-mentioned disadvantages. It makes it possible to determine the smallest amounts of peroxidase quantitatively and thus achieve the necessary sensitivity for the ezzyme immunological samples.
Nærmere definert vedrører foreliggende oppfinnelse en fremgangsmåte ved bestemmelse av<p>eroksydase i en prøvekarakterisert vedat man blander prøven i en puffer under innstilling av en pH-verdi fra 3 til 10,5 med et substrat for peroksydase samt med en blanding av en eller flere forbindelse med formelen More precisely defined, the present invention relates to a method for determining<p>peroxidase in a sample characterized by mixing the sample in a buffer while setting a pH value from 3 to 10.5 with a substrate for peroxidase and with a mixture of one or more connection with the formula
hvor R betyr hydrogen eller alkaliraetall og R-^, R2 r ' , R^og R^hydrogen, brom, klor, nitro, lavere-alkyl eller en karboksylgruppe, og 4-aminoantipyrin, inkuberer den erholdte blanding ved en temperatur mellom 10 - 60°C og bestemmer det i prøven tilstedeværende innhold av peroksydase ved måling av den oppståtte fargeintensitetsøkning pr. tidsenhet. where R means hydrogen or alkali group and R-^, R2 r', R^ and R^hydrogen, bromine, chlorine, nitro, lower alkyl or a carboxyl group, and 4-aminoantipyrine, incubate the resulting mixture at a temperature between 10 - 60°C and determines the content of peroxidase present in the sample by measuring the resulting increase in color intensity per unit of time.
Innenfor rammen av bestemmelséfremgangsmåten ifølge oppfinnelsen kan analyseprøven (f.eks. en fysiologisk-væske eller et celleekstrakt) direkte undersøkes med hensyn til peroksydaseinnhold. Within the framework of the determination method according to the invention, the analysis sample (e.g. a physiological fluid or a cell extract) can be directly examined with regard to peroxidase content.
Fortrinnsvis anvendes dog fremgangsmåten ifølge oppfinnelsen til aktivitetsbestemmelse av peroksydase innenfor rammen av enzym-immunologiske forsøk som beskrevet i Clin. Chem. 22, 733-738 (1976) eller i Clin.Chem. 22, 1243-1255 Preferably, however, the method according to the invention is used to determine the activity of peroxidase within the framework of enzyme immunological experiments as described in Clin. Chem. 22, 733-738 (1976) or in Clin.Chem. 22, 1243-1255
(1976). I dette tilfelle inneholder prøven som undersøkes (1976). In this case, the sample being examined contains
i den enzymatiske delen av den immunologiske fremgangsmåten peroksydasen i en form som er bundet til et immunologisk aktivt materiale (antigen eller antistoff). in the enzymatic part of the immunological method the peroxidase in a form which is bound to an immunologically active material (antigen or antibody).
Uttrykket "immunologisk aktivt materiale" omfatter alle de bestanddeler i fysiologiske væsker, vev, celle- og vevs-ekstrakter, hvor en immunologisk reaksjonspartner er tilstede eller kan dannes. Til disse hører primære aminer, aminosyrer, peptider, . proteiner, lipoproteiner, glykopro-teider, steriner, steroider, lipoider, nukleinsyre, enzymer, hormoner, vitaminer, polysakkarider og alkaloider. The term "immunologically active material" includes all the components in physiological fluids, tissue, cell and tissue extracts, where an immunological reaction partner is present or can be formed. These include primary amines, amino acids, peptides, . proteins, lipoproteins, glycoproteins, sterols, steroids, lipoids, nucleic acid, enzymes, hormones, vitamins, polysaccharides and alkaloids.
Særlig foretrukne immunologiske aktive materialer er humant choriongonadotropin, rheumatoidfaktor, toksoplasma, candida, trypanosoma, CEA-antigen og myoglobin. Particularly preferred immunologically active materials are human chorionic gonadotropin, rheumatoid factor, toxoplasma, candida, trypanosoma, CEA antigen and myoglobin.
For formålet ved foreliggende oppfinnelse er et puffer-system egnet som kan innstilles på en.pH-verdi mellom 3 og 10,5, fortrinnsvis mellom 6 og 9. Særlig foretrukket er en pH-verdi mellom 6,5 og 7,5. For the purpose of the present invention, a buffer system is suitable which can be set to a pH value between 3 and 10.5, preferably between 6 and 9. A pH value between 6.5 and 7.5 is particularly preferred.
Egnede puffersystemer er f.eks. fosfatpuffer, trispuffer og boratpuffer. Pufferkonsentrasjonen er ikke kritisk, men er dog foretrukket 50 til 200 mmol pr. 1. Suitable buffer systems are e.g. phosphate buffer, tris buffer and borate buffer. The buffer concentration is not critical, but is however preferably 50 to 200 mmol per 1.
Som substrat for peroksydasen tjener. J^C^• H2°2kan enten anvendes direkte eller kan dannes ved hjelp av egnede enzymsystemer under reaksjonen. Slike enzymsystemer er f.eks. glukoseoksydase/glykose eller urikase/urinsyre. As a substrate for the peroxidase serves. J^C^• H2°2 can either be used directly or can be formed with the help of suitable enzyme systems during the reaction. Such enzyme systems are e.g. glucose oxidase/glucose or uricase/uric acid.
Ved direkte anvendelse av ^ 2^ 2 velc?es en konsentrasjonBy direct application of ^ 2^ 2 a concentration is selected
fra 0,05 til 10 mmol pr. 1 fortrinnsvis 0,2 til 1 mmol pr. 1. from 0.05 to 10 mmol per 1 preferably 0.2 to 1 mmol per 1.
Forbindelsen med den generelle formel I som anvendes i fremgangsmåten ifølge oppfinnelsen danner sammen med 4-aminoantipyrin under innvirkning av'H00-og<p>eroksydase et fargestoff hvis dannelseshastighet er direkte propors-jonalt med mengden av tilstedeværende peroksydase og kan bestemmes ved måling av den dannede fargeintensitet under definerte forsøksbetingelser. The compound with the general formula I which is used in the method according to the invention forms, together with 4-aminoantipyrine under the influence of H00 and<p>peroxidase, a dye whose rate of formation is directly proportional to the amount of peroxidase present and can be determined by measuring the formed color intensity under defined experimental conditions.
Egnede eksempler på forbindelsen med den generelle formel I er fenol, o-klorfenol, m-klorfenol, p-klorfenol, 2,4-diklorfenol, 2,5-diklorfenyl, 3,4-diklorfenol, 2,4-dibrom-fenol, o-nitrofenol, m-nitrofenol, p-nitrofenol, o-kresol, m-kresol, p-kresol, 2,4-dimetylfenol, 2,4-dinitrofenol og 4-hydroksybenzosyre-. Særlig foretrukket er fenol. Suitable examples of the compound of the general formula I are phenol, o-chlorophenol, m-chlorophenol, p-chlorophenol, 2,4-dichlorophenol, 2,5-dichlorophenyl, 3,4-dichlorophenol, 2,4-dibromophenol, o-nitrophenol, m-nitrophenol, p-nitrophenol, o-cresol, m-cresol, p-cresol, 2,4-dimethylphenol, 2,4-dinitrophenol and 4-hydroxybenzoic acid-. Particularly preferred is phenol.
Konsentrasjonen av forbindelse med formel I ligger med fordel mellom 1 og 200 mmol pr. 1 hvorved 10 til 50 mmol pr. The concentration of compound of formula I is advantageously between 1 and 200 mmol per 1 whereby 10 to 50 mmol per
• 1 er særlig foretrukket.• 1 is particularly preferred.
Konsentrasjonen av 4-aminoantipyrin er fortrinnsvis 0,1 - 10 mmol pr. 1 hvorved 1-3 mmol pr.'1 er særlig foretrukket. The concentration of 4-aminoantipyrine is preferably 0.1 - 10 mmol per 1 whereby 1-3 mmol per 1 is particularly preferred.
Rekkefølgen av tilsetningen av de ovennevnte reagenser velges slik at for kompletering av systemet tilsettes til slutt enten peroksydasen i form av prøven eller substratet for. peroksydasen. The order of the addition of the above-mentioned reagents is chosen so that to complete the system either the peroxidase in the form of the sample or the substrate is finally added. the peroxidase.
Reaksjonstemperaturen er kritisk. Den kan velges mellom 10 og 60°C, fortrinnsvis 25° til 40°C og må holdes konstant under hele reaks jonens varighet. Inkubas jonsvarigheten er ved gitt reaksjonstemperatur avhengig av peroksydaseinn-holdet i prøven. Den ligger ved enzymimmunologiske forsøk mellom 1 minutt og 4 timer, fortrinnsvis mellom 30 og 120 minutter. The reaction temperature is critical. It can be chosen between 10 and 60°C, preferably 25° to 40°C and must be kept constant throughout the duration of the reaction. At a given reaction temperature, the duration of incubation depends on the peroxidase content in the sample. In enzyme immunological tests, it is between 1 minute and 4 hours, preferably between 30 and 120 minutes.
Innholdet av peroksydase som er tilstede i prøven bestemmes ved måling av fargingsintensiteten som oppstår ved den enzymmatiske reaksjonen under definerte forsøksbetingelser (konsentrasjon av reagensene, pH-verdi, temperatur, varighet av inkubasjon). Man måler ekstinksjonsdifferanse som svarer til økningen i fargeintensitet og dermed er direkte pro-<p>orsjonalt med mengden av tilstedeværende peroksydase. The content of peroxidase present in the sample is determined by measuring the staining intensity that occurs in the enzymatic reaction under defined experimental conditions (concentration of the reagents, pH value, temperature, duration of incubation). An extinction difference is measured which corresponds to the increase in color intensity and is thus directly proportional to the amount of peroxidase present.
Målingen av fargeintensiteten skjer med fordel fotometrisk ved en bølgelengde mellom 450 - 580 nm. The measurement of the color intensity is preferably done photometrically at a wavelength between 450 - 580 nm.
Fremgangsmåte ifølge oppfinnelsen egner seg både for manuell og automatiske bestemmelse av peroksydase. The method according to the invention is suitable for both manual and automatic determination of peroxidase.
De nødvendige reagenser for utførelse av fremgangsmåte ifølge oppfinnelsen pakkes fortrinnsvis i et reagenssett. Et reagenssett som er egnet, for formålet ved foreliggende oppfinnelse inneholder med fordel i flere beholdere The necessary reagents for carrying out the method according to the invention are preferably packaged in a reagent set. A reagent set which is suitable for the purpose of the present invention advantageously contains several containers
a) eheller flere forbindelser med formela) or several compounds with formula
hvor R.betyr hydrogen eller alkalimetall og R^, R2, R^ , where R.means hydrogen or alkali metal and R^, R2, R^,
R^og R^_ hydrogen, brom, klor, nitro, lavere-alkyl eller en karboksylgruppe, R^ and R^_ hydrogen, bromine, chlorine, nitro, lower alkyl or a carboxyl group,
og and
b) 4-aminoantipyrin og eventueltb) 4-aminoantipyrine and optionally
c) en puffer for innstilling av en pH-verdi fra 3 til 10,5 c) a buffer for setting a pH value from 3 to 10.5
og eventueltand eventually
d) et substrat for peroksydase.d) a substrate for peroxidase.
Reaksjonssettet kan også inneholde ytterligere en beholder med en kontroll- eller innstillingsløsning og/eller en beholder med en reagens for avbrytelse av reaksjonen og/ eller reagenser for utførelse av en immunologisk bestemmelse. The reaction set can also contain a further container with a control or setting solution and/or a container with a reagent for interrupting the reaction and/or reagents for carrying out an immunological determination.
Det er dog ikke nødvéndig å oppbevare alle reagenser i However, it is not necessary to store all reagents in
separate beholdere. Den foreliggende oppfinnelse omfatter også et reagenssett hvor enkelte av de nødvendige bestanddeler for utførelse av fremgangsmåten iføl<q>e o<p>pfinnelsen oppbevares sammen i. samme beholder. separate containers. The present invention also includes a reagent set where some of the necessary components for carrying out the method according to the invention are stored together in the same container.
Således kan f.eks. puffersubstansen oppbevares sammen med 4-aminoantipyrin i en enkelt beholder. Thus, e.g. the buffer substance is stored together with 4-aminoantipyrine in a single container.
De forskjellige reagenser kan forpakkes såvel i flytende som i fast form. The various reagents can be packaged both in liquid and solid form.
Oppfinnelsen belyses i det følgende ved eksempler nærmere. The invention is explained in more detail in the following examples.
EKSEMPEL 1EXAMPLE 1
En nøyaktig innveiet mengde pepperrot-peroksydase med høye renhetsgrad oppløses i 0,1 mol pr. 1 Na^HPO^-puffer og derav fremstilles forskjellige fortynninger. An accurately weighed amount of horseradish peroxidase with high degrees of purity is dissolved in 0.1 mol per 1 Na^HPO^-buffer and different dilutions are prepared from it.
For aktivitetsbestemmelse av disse peroksydaseløsningene tilblandes 2,0 ml forsøksløsning (0,1 mol pr. 1 fosfatpuffer med pH 7,2 5 med 2 5 mmol pr. 1 fenol, 2 mmol pr. 1 4-aminoantipyrin og 0,8 mmol pr. 1 t^O^) 0,0 5 ml prøve og med 37 C bestemmes ékstinksjonsdifferansen pr. 30 minutter ved bølgelengde 492 nm. To determine the activity of these peroxidase solutions, 2.0 ml test solution (0.1 mol per 1 phosphate buffer with pH 7.2 5 with 25 mmol per 1 phenol, 2 mmol per 1 4-aminoantipyrine and 0.8 mmol per 1 t^O^) 0.0 5 ml sample and at 37 C the extinction difference per 30 minutes at a wavelength of 492 nm.
En standardkurve oppstilles på grunnlag av de ovenfor gitte måleverdier. A standard curve is drawn up on the basis of the measurement values given above.
Den ovenstående fremgangsmåte gjentas med ukjente prøverThe above procedure is repeated with unknown samples
og de derved erholdte måleverdier sammenliknes for bestemmelse av peroksydase-innholdet med peroksydase-standardkurven. and the measurement values thus obtained are compared to determine the peroxidase content with the peroxidase standard curve.
EKSFMPEL 2EXAMPLE 2
For bestemmelse av CEA-innholdet inkuberes prøver med.kanin-CEA-antiserum ved romtemperatur i 2 4 timer i en boratpuffer med pH 8,4. Deretter tilsettes en bestemt mengde pepperrot-peroksydase (POD) markert CEA til denne blandinaen og igjen inkuberes ved romtemperatur i mørke.16 timer. To determine the CEA content, samples are incubated with rabbit CEA antiserum at room temperature for 24 hours in a borate buffer with pH 8.4. A specific amount of horseradish peroxidase (POD) labeled CEA is then added to this mixture and again incubated at room temperature in the dark for 16 hours.
Denne løsningen tilsettes nå gjeit-anti-kanin-immunglobulin som er bundet til et fast stoff. Etter 30 minutter avskilles den faste fasen fra den flytende fasen og peroksydase-aktivi-teten i den ovenstående løsningen bestemmes som følger: Goat anti-rabbit immunoglobulin, which is bound to a solid, is now added to this solution. After 30 minutes, the solid phase is separated from the liquid phase and the peroxidase activity in the above solution is determined as follows:
1,0 ml prøveløsning (0,2 mmol pr. 1 fosfatpuffer med pH1.0 ml sample solution (0.2 mmol per 1 phosphate buffer with pH
7,25 med 50 mmol pr. 1 fenol, 4 mmol pr. 1 4-aminoantipyrin og 1,6 mmol pr. 1 ^C^) blandes med 1,0 ml av ovenstående løsning. Deretter bestemmes ved 37°C ekstinksjonsdifferansen pr. tidsenhet ved -492 nm fotometrisk. 7.25 with 50 mmol per 1 phenol, 4 mmol per 1 4-aminoantipyrine and 1.6 mmol per 1 ^C^) is mixed with 1.0 ml of the above solution. Then, at 37°C, the extinction difference per time unit at -492 nm photometrically.
CEA-innholdet i prøven beregnes ved hjelp av en CEA-standardkurve som ble oppstilt etter utførelse av den beskrevne r fremgangsmåte med standardløsningen av egnet CEA.-innhold. The CEA content in the sample is calculated by means of a CEA standard curve which was drawn up after carrying out the described procedure with the standard solution of suitable CEA content.
Claims (22)
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CH294677 | 1977-03-09 |
Publications (1)
Publication Number | Publication Date |
---|---|
NO780822L true NO780822L (en) | 1978-09-12 |
Family
ID=4245187
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
NO780822A NO780822L (en) | 1977-03-09 | 1978-03-08 | PEROXYDASE DETERMINATION. |
Country Status (12)
Country | Link |
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JP (1) | JPS53110897A (en) |
AU (1) | AU3382778A (en) |
BE (1) | BE864669A (en) |
DE (1) | DE2809755A1 (en) |
DK (1) | DK104578A (en) |
ES (1) | ES467667A1 (en) |
FR (1) | FR2383441A1 (en) |
IL (1) | IL54187A0 (en) |
IT (1) | IT1095456B (en) |
NL (1) | NL7801413A (en) |
NO (1) | NO780822L (en) |
SE (1) | SE7802667L (en) |
Families Citing this family (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4184923A (en) * | 1977-11-03 | 1980-01-22 | Eastman Kodak Company | Reduction of gentisic acid interference in analytical elements |
WO1980000454A1 (en) * | 1978-08-18 | 1980-03-20 | Kyowa Hakko Kogyo Kk | Method of measuring substrate of xanthine oxidase,and composition for the determination |
JPS56117798A (en) * | 1980-02-21 | 1981-09-16 | Toyo Jozo Co Ltd | Kit for measuring lipoid peroxide |
JPS5783287A (en) * | 1980-11-14 | 1982-05-25 | Kyowa Hakko Kogyo Co Ltd | Elimination of hydrogen peroxide |
DE3125667C2 (en) * | 1981-06-30 | 1986-01-23 | Boehringer Mannheim Gmbh, 6800 Mannheim | Method and means for the detection of hydrogen peroxide |
IT1168043B (en) * | 1981-10-22 | 1987-05-20 | Sclavo Inst Sieroterapeut | CHROMOGEN FOR THE COLORIMETRIC DETERMINATION OF PEROXIDES AND METHOD USING THE SAME |
WO1983003104A1 (en) * | 1982-03-03 | 1983-09-15 | Carter, Timothy, Joseph, Nicholas | Enhanced luminescent and luminometric assay |
US4460684A (en) * | 1982-08-02 | 1984-07-17 | Miles Laboratories, Inc. | Ascorbate-resistant broad range glucose test composition, test device and method |
US4521511A (en) * | 1982-09-22 | 1985-06-04 | Enzyme Technology Company | Catalyzed colorimetric and fluorometric substrates for peroxidase enzyme determinations |
EP0108526A3 (en) * | 1982-11-01 | 1984-07-11 | Beckman Instruments, Inc. | Method and reagent for the determination of peroxide and precursors thereof |
DE3463395D1 (en) * | 1983-02-11 | 1987-06-04 | Nat Res Dev | Enhanced luminescent or luminometric assay |
CA1252703A (en) * | 1984-02-27 | 1989-04-18 | Nutan B. Shah | Diagnostic saliva test for detecting periodontal disease |
US4828983A (en) * | 1986-07-10 | 1989-05-09 | Eastman Kodak Company | Use of phenols and anilines to increase the rate of peroxidase catalyzed oxidation of leuco dyes |
GB2450351B (en) | 2007-06-20 | 2012-01-18 | Cozart Bioscience Ltd | Monitoring an Immunoassay |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
IT986838B (en) * | 1972-05-12 | 1975-01-30 | Sclavo Inst Sieroterapeut | COMPLEX OF REAGENTS FOR THE ENZYMATIC DETER MINATION OF GLUCOSE GLUCOSE SYSTEM OXIDASE PEROXIDASE DASES WITH MANUAL AND AUTOMATED METHODS CO WITH READING AT TERM OR IN KINETICS |
-
1978
- 1978-02-07 NL NL7801413A patent/NL7801413A/en not_active Application Discontinuation
- 1978-02-28 IT IT20739/78A patent/IT1095456B/en active
- 1978-03-03 IL IL54187A patent/IL54187A0/en unknown
- 1978-03-03 AU AU33827/78A patent/AU3382778A/en active Pending
- 1978-03-07 JP JP2509578A patent/JPS53110897A/en active Pending
- 1978-03-07 FR FR7806439A patent/FR2383441A1/en not_active Withdrawn
- 1978-03-07 DE DE19782809755 patent/DE2809755A1/en active Pending
- 1978-03-08 DK DK104578A patent/DK104578A/en not_active Application Discontinuation
- 1978-03-08 BE BE185748A patent/BE864669A/en unknown
- 1978-03-08 ES ES467667A patent/ES467667A1/en not_active Expired
- 1978-03-08 NO NO780822A patent/NO780822L/en unknown
- 1978-03-08 SE SE7802667A patent/SE7802667L/en unknown
Also Published As
Publication number | Publication date |
---|---|
DE2809755A1 (en) | 1978-09-14 |
BE864669A (en) | 1978-09-08 |
FR2383441A1 (en) | 1978-10-06 |
AU3382778A (en) | 1979-09-06 |
JPS53110897A (en) | 1978-09-27 |
NL7801413A (en) | 1978-09-12 |
IL54187A0 (en) | 1978-06-15 |
IT1095456B (en) | 1985-08-10 |
IT7820739A0 (en) | 1978-02-28 |
SE7802667L (en) | 1978-09-10 |
ES467667A1 (en) | 1979-09-01 |
DK104578A (en) | 1978-09-10 |
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