EP0213156A1 - Solutions enzymatiques stabilisees comprenant un substrat - Google Patents

Solutions enzymatiques stabilisees comprenant un substrat

Info

Publication number
EP0213156A1
EP0213156A1 EP86901252A EP86901252A EP0213156A1 EP 0213156 A1 EP0213156 A1 EP 0213156A1 EP 86901252 A EP86901252 A EP 86901252A EP 86901252 A EP86901252 A EP 86901252A EP 0213156 A1 EP0213156 A1 EP 0213156A1
Authority
EP
European Patent Office
Prior art keywords
solution
acid
substrate
enzyme
chromogen
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP86901252A
Other languages
German (de)
English (en)
Inventor
David A. Thomas
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Travenol Genentech Diagnostics
Original Assignee
Travenol Genentech Diagnostics
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Travenol Genentech Diagnostics filed Critical Travenol Genentech Diagnostics
Publication of EP0213156A1 publication Critical patent/EP0213156A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
    • 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

Definitions

  • This invention relates to an improved enzyme immunoassay (EIA), enzyme linked immunosorbent assay (ELISA), immunoenzymometric assay or immunoperoxidase assay and pertains most specifically to a substrate solution for determining enzyme activity which contains ethyienediaminetetraacetic acid resulting in increased stability of the substrate solution and decreased substrate drift.
  • EIA enzyme immunoassay
  • ELISA enzyme linked immunosorbent assay
  • immunoenzymometric assay immunoperoxidase assay
  • Enzyme immunoassays, enzyme linked immunosorbent assays and immunoenzymometric assays involve the use of an enzyme, such as a peroxidase, as a label for the unknown in an assay procedure, and measurement of the enzyme activity as an indication of the amount of the unknown in the sample.
  • Immunoperoxidase assays apply the same principles to microscopic examination of cultures or tissues. Such assays typically require the addition of the substrate for the enzyme after the immunoassay in order to determine the amount of "bound" enzyme.
  • Peroxidases, such as horseradish peroxidase require two substrates — a peroxide and a chromogen which affords a colored product upon oxidation. Typically, stock solutions of the peroxide and the chromogen are combined in the appropriate buffer to form a "working" substrate solution.
  • the working solution With the usual chromogen, o-phenylenediamine, the working solution is quickly oxidized nonenzymatically resulting in an increase in color development even in the absence of peroxidase. Thus, the working solution must be used within 1/2 hour of preparation.
  • TMB 3,3',5,5'-tetramethylbenzidine
  • the present invention can be used to determine peroxidase act vity of any enzyme which catalyzes the reaction of the chromogen with peroxide to form a colored compound.
  • peroxidases such as horseradish peroxidase
  • other peroxidases may also be used.
  • “Chelating agent” as used in this invention means any compound in which a dicationic, tricationic or tetracationic metal is bound to two or more atoms or complexes with ligands containing more than one point of attachment.
  • Chelating agents which can be used in the present invention include any chelating agent which binds heavy metals preventing nonenzymatic oxidation of the substrate solution without preventing enzymatic oxidation or the formation or detection of color.
  • Preferred chelating agents are nontoxic and do not present a biohazard.
  • EDTA ethyienediaminetetraacetic acid
  • DTPA diethylenetriaminepentaacetic acid
  • iminodiacetic acid and other derivatives iminodiacetic acid and other derivatives
  • nitrilotriacetic acid and derivatives succinic acid and other diacids
  • citric acid and other hydroxyacids and acetylacetone and other dicarbonyl compounds Preferred chelating agents are EDTA and its derivatives, DTPA and its derivatives, iminodiacetic acid and its derivatives, and nitrilotriacetic acid and its derivatives.
  • EDTA due to its solubility in the buffer of the stock and working solutions and its ability strongly to chelate a variety of metal ions at the pH of the working solution.
  • Chelating agents which are not useful in the present invention are those which actually increase the rate of non-enzymatic oxidation of the chromogen or substrate, such as dip colinic acid. These chelating agents will be obvious to one skilled in the art without undue experimentation.
  • the concentration of chelating agent in the working solutions will be dependent upon effective el mination of drift and minimization of interference with the enzymatic oxidation of the substrate. In general, it is desirable to minimize the concentration of chelating agent.
  • Preferred concentrations are in the range of from about O.OlmM to about lOO M.
  • the most preferred concentrations are in the range of from about 0.05mM to about lOmM.
  • Substrates and chromogens with which the present invention is useful include those which are used in the detection of peroxidases.
  • Chromogens which are useful include those which are well known in the art such as o-phenylenediamine, 2,2'-azinodi (3-ethyl) benzthiazoline-6-sulphonic acid (ABTS), dianisidine, dicarboxidine, TMB, diaminobenzidine.
  • Preferred chromogens include any 3,3',5,5'-tetraalkylbenzidine in which the alkyl groups each contain from 1 to 5 carbon atoms; particularly useful are 3,3',5,5'- tetramethylbenzidine and 3,3',5,5'-tetraet_ylbenzidine.
  • Acid salts such as the hydrochlorides also are useful.
  • the amount of chromogen present in the substrate solution can vary over a considerable range, depending upon the identity and concentration of the peroxidase enzyme whose activity is to be measured; in general, the concentration of chromogen can vary from about 0.1 to about 10 M, preferably from about 1 to about 3mM.
  • the amount of peroxide present also may vary, depending upon the amount of chromogen present, ranging from about 1 to about 20mM, but preferably it is from about 1 to about 6mM. Any of the usual peroxides such as hydrogen peroxide, urea peroxide, or the like can be employed in the substrate solution.
  • the working solution is prepared by mixing a first stock solution containing a chromogen and a chelating agent with a second stock solution comprising an aqueous buffer solution containing the substrate and a chelating agent.
  • the chelating agents of the two solutions may be the same or may differ.
  • the chromogen solution can also comprise stabilizing or solubilizing agents.
  • the working solution is prepared by mixing a first stock solution comprising an aqueous solution containing 5 to 50% N-methyl pyrrolidone by volume, 0.5 to ImM EDTA and 1 to 10 M 3,3',5,5'-tetraalkylbenzidine or an acid salt thereof with a second stock solution comprising an aqueous buffer solution containing 0.5 to ImM EDTA and 4 to 40 mM peroxide, and water if necessary to achieve the desired concentration.
  • a first stock solution comprising an aqueous solution containing 5 to 50% N-methyl pyrrolidone by volume, 0.5 to ImM EDTA and 1 to 10 M 3,3',5,5'-tetraalkylbenzidine or an acid salt thereof
  • a second stock solution comprising an aqueous buffer solution containing 0.5 to ImM EDTA and 4 to 40 mM peroxide, and water if necessary to achieve the desired concentration.
  • Stock solutions can be supplied in the form of a kit which contains in addition a supply of conventional stopping agent solution, antibody- or antigen-coated containers, and antigen or antibody standards, calibrators or controls.
  • the determination of enzyme activity is carried out in the usual manner by incubating the substrate solution with the specimen containing the enzyme to develop a visible color.
  • a conventional stopping agent such as, for example, an aqueous solution of water-soluble flouride, such as sodium fluoride, at pH from about 3 to about 6; or acids such as sulfuric acid, hydrochloric acid, etc.
  • a soluble fluoride is preferred because it provides a stable blue color.
  • the present invention can be employed with any of the usual enzyme immunoassay procedures, either homogeneous or heterogeneous assays, and either single- or double-antibody assays, and including enzyme linked immunosorbent assays, immunoenzymometric assays and immunoperoxidase assays.
  • Aqueous substrate solutions were prepared with the following compositions:
  • the solutions were stored in glass at 4°C.
  • Working substrate solutions with and without EDTA were prepared by combining one part of the respective TMB solution with 4 parts of the respective peroxide/acetate stock solution.
  • the working solutions were stored in clear glass bottles at room temperature or at 4° to 6°C.
  • 200 ⁇ L (microliters) of the solution were acidified with 50 ⁇ l l.ON H ⁇ SO, and the absorbance at 450 nm was measured in a spectrophotometer using water as a blank standard. The results are recorded in Table 1.
  • the working substrate solution which contained EDTA was very stable for several days at 4 to 6°C and at room temperature. Without EDTA the nonenzymatic oxidation was so rapid that after a few hours the solution was no longer usable. The drop in absorbance at 7 days is probably due to the decomposition of the colored product occurring at a faster rate than it is formed.
  • the effect of EDTA on enzyme activity was determined by running a horseradish peroxidase assay and varying the amount of horseradish peroxidase (lng/ml). 400 ⁇ l of the working solution of Example I, with and without EDTA, were added. The reaction was stopped after 10 minutes with 1 ml of 1 M HdonS0. and the absorbances at 450 nm were determined as before.
  • Enzyme immunoassays were performed using the EDTA stabilized TMB/peroxide system for quantitation of carcinoembryonic antigen (CEA).
  • CEA standards were prepared containing 0, 2, 5, 10 and 25 ngm/ml of CEA in serum respectively. 100 ⁇ l of each standard were added along with 100 ⁇ l of 50 nM acetate pH 5.3 buffer to separate IgG (anti-CEA) coated microtiter wells. The assays were incubated for 2.0 hours at 37°C. Each well was washed three times with water. A conjugate was formed by covalently linking A'-CEA IgG to enzyme horseradish peroxidase (HRP) diluted in buffer with protein stabilizer.
  • HRP horseradish peroxidase
  • dipicolinic acid prevented rapid non-enzymatic oxidation of the solution.
  • Dipicolinic acid on the other hand, increased non-enzymatic oxidation relative to the control and would not be suitable for use as a stabilizer.
  • HBsAg Hepatitis B surface antigen
  • TMB/Peroxide substrate system prepared with and without varying chelating agents.
  • Controls were prepared using HBsAg diluted in buffer with 1% bovine serum albumin as a positive and human serum as a negative. 200 ⁇ l of control were added to anti-BHsAg coated microtiter wells. 200 ⁇ l of water was used for the blank. The wells were incubated for 2 hours at 37°C and then washed three times with water.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Urology & Nephrology (AREA)
  • Analytical Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • General Engineering & Computer Science (AREA)
  • Cell Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biophysics (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Une solution comprenant un chromogène ou un substrat s'utilisant dans des immunoanalyses enzymatiques, des analyses d'immunosorption liées à une enzyme, des analyses immunoenzymométriques ou des analyses d'immunopéroxydase est stabilisée pour réduire sensiblement la dérive du substrat. Ceci est obtenu en ajoutant un agent de chélation en une quantité effective pour empêcher sensiblement l'oxydation non enzymatique du substrat ou chromogène.
EP86901252A 1985-02-11 1986-02-04 Solutions enzymatiques stabilisees comprenant un substrat Withdrawn EP0213156A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US70021685A 1985-02-11 1985-02-11
US700216 1985-02-11

Publications (1)

Publication Number Publication Date
EP0213156A1 true EP0213156A1 (fr) 1987-03-11

Family

ID=24812635

Family Applications (1)

Application Number Title Priority Date Filing Date
EP86901252A Withdrawn EP0213156A1 (fr) 1985-02-11 1986-02-04 Solutions enzymatiques stabilisees comprenant un substrat

Country Status (5)

Country Link
EP (1) EP0213156A1 (fr)
JP (1) JPS62502653A (fr)
FI (1) FI863968A (fr)
WO (1) WO1986004610A1 (fr)
ZA (1) ZA86983B (fr)

Families Citing this family (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH073422B2 (ja) * 1985-09-13 1995-01-18 富士レビオ株式会社 酵素免疫測定法用緩衝剤
IL80964A0 (en) * 1986-12-15 1987-03-31 Savyon Diagnostics Ltd Method for preparing aqueous solutions containing chromogenic materials
WO1990002339A1 (fr) * 1988-08-16 1990-03-08 Cetus Corporation Solutions temoins stables pour la detection d'une activite peroxydique
US5206006A (en) * 1989-08-31 1993-04-27 Instrumentation Laboratory Spa Stabilized trinder reagent
IT1231737B (it) * 1989-08-31 1991-12-21 Instrumentation Lab Spa Metodo per la inibizione di colorazione aspecifica e spontanea nei reattivi basati sulla reazione di trinder mediante l'uso di chelanti.
US5532138A (en) * 1990-04-26 1996-07-02 Behringwerke Ag Method and kits for determining peroxidatively active catalysts
US5098830A (en) * 1990-07-16 1992-03-24 Diagnostic Markers, Inc. Very rapid detection of fungal infections
DE4037776A1 (de) * 1990-11-28 1992-06-04 Behringwerke Ag Waschloesung fuer festphasen-immunometrisches verfahren, die einen komplexbildner fuer metallionen enthaelt und ihre verwendung
US5192657A (en) * 1990-12-18 1993-03-09 Ortho Diagnostic Systems, Inc. Stabilized proteolytic solution and reagent kit
US5332662A (en) * 1992-07-31 1994-07-26 Syntex (U.S.A.) Inc. Methods for determining peroxidatively active substances
NZ268301A (en) * 1993-06-21 1997-10-24 Boehringer Mannheim Corp The stabilisation of diagnostic compositions comprising glucose oxidase and/or ferricyanide using succinic acid or a salt thereof
DE19544150C2 (de) * 1995-11-16 2002-06-13 Seramun Diagnostica Gmbh Stabile 3,3',5,5'-Tetramethylbenzidin-Lösungen
JP7232475B2 (ja) * 2020-12-28 2023-03-03 ヤマサ醤油株式会社 ロイコ型色原体の安定化方法

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE2612726C3 (de) * 1976-03-25 1979-03-15 Boehringer Mannheim Gmbh, 6800 Mannheim Stabilisierte Urease
NL7610608A (nl) * 1976-09-24 1978-03-29 Akzo Nv Werkwijze voor het stabiliseren van peroxidase- -bevattende composities.
JPS5534001A (en) * 1978-08-28 1980-03-10 Noda Sangyo Kagaku Kenkyusho Stabilization of sarcosine oxidase
US4378429A (en) * 1979-08-23 1983-03-29 Modrovich Ivan Endre Enzymatic method and stabilized solutions for determining total cholesterol in human serum
US4282316A (en) * 1979-09-11 1981-08-04 Modrovich Ivan Endre Stabilized enzymic solutions for determining urea
DE3025372A1 (de) * 1980-07-04 1982-01-28 Behringwerke Ag, 3550 Marburg Mittel zum nachweis peroxidatisch wirksamer substanzen und verwendung von polyvinylmethylacylamid in einem solchen
JPS57138389A (en) * 1981-02-17 1982-08-26 Toyobo Co Ltd Stable urease composition
DE3124590A1 (de) * 1981-06-23 1983-01-27 Boehringer Mannheim Gmbh, 6800 Mannheim Stabilisiertes reagenz zum nachweis von h(pfeil abwaerts)2(pfeil abwaerts)o(pfeil abwaerts)2(pfeil abwaerts)
JPS58162294A (ja) * 1982-03-18 1983-09-26 Toyobo Co Ltd 固定化複合酵素組成物

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO8604610A1 *

Also Published As

Publication number Publication date
FI863968A0 (fi) 1986-10-01
JPS62502653A (ja) 1987-10-15
ZA86983B (en) 1986-09-24
WO1986004610A1 (fr) 1986-08-14
FI863968A (fi) 1986-10-01

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