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
  • C12Q1/54—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving glucose or galactose
• • 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
  • C12Q2326/30—2,2'-Azinobis (3-ethylbenzothiazoline-6-sulfonic acid), i.e. ABTS

Definitions

• ABSTRACT Compositions comprising glucose oxidase, peroxidase, a chromogen, a buffer, an azide, and 2,2'-azino-di-(3- ethylbenzothiazoline-6-sulfonic acid) provide remarkably stable test reagents for the enzymatic determination of glucose.
• the present invention is concerned with a reagent composition and process for the enzymatic determination of glucose by use of the enzymes glucose oxidase and peroxidase.
• test reagents In the case of test reagents, it isgenerally desired that these contain all the necessary reagents in a pre-mixed state in order, on the one hand, to reduce as much as possible the expenditure of labor in carrying out large numbers of routine investigations and, on the other hand, to prevent errors arising in the mixing of several components.
• these reagent mixtures should be in solid form but should be easily dissolved in water at the time of use and then remain stable in aqueous solution for as long as possible in order to avoid having to prepare new solutions continuously.
• the present invention provides a reagent composition for the determination of glucose, which comprises glucose oxidase, peroxidase, a chromogen and a buffer, together with an azide and 2,2-azino-di- (3-ethyl-benzothiazoline--sulphonic acid), hereinafter abbreviated as ABTS.
• ABTS can be used simultaneously as stabilizer and as the chromogen.
• it is expediently present in the reagent in an amount of 0.25 to 1.5 grams/liter, preferably of about 0.5 grams/liter, of aqueous solution or, in the case of a solid mixture intended for the preparation of one liter of solution, is present therein in an equivalent amount.
• the enzymes GOD and POD are expediently present in the reagent according to the present invention in amounts such that the mutarotation of the glucose and not the enzymes determines the rate of the reaction. This requirement is fulfilled by GOD concentrations of 2.5 to 15 X- 10 U/liter (i.e., units per liter) and of POD concentrations of 30 to I000 U/liter.
• the buffer substance used should produce, in aqueous solution, a pH value of between 6.2 and 7.5 and preferably of between 6.5 and 7.2. All buffer substances can be used which do not inhibit the reaction, a phosphate buffer (pH 7.0) being preferably used. However, tris buffer is unsuitable.
• a preferred reagent according to the present invention for the determination of blood sugar comprises:
• An especially preferred reagent according to the present invention comprises:
• a further preferred reagent according to the present invention comprises:
• the reagent according to the present invention can be in the form of an aqueous solution of. the components.
• This aqueous solution has a good stability and can, depending upon the composition and especially upon the chromogen used, be stored ready for use for several weeks.
• chromogen and enzymes had to be prepared in different solutions and, on the other hand, these solutions only had a limited period of use.
• the reagents according to the present in vention have several advantages, especially an outstanding stability. Their storage at refrigerator temperature is more than one year. Furthermore, solid mixtures according to the invention, e.g., in the form of powders or possibly of tablets, have the advantage of being very easily soluble in water.
• a further surprising advantage of the reagent according to the present invention is that the amount of POD can be substantially reduced from the previously used amount of about 250 U/liter to about 30 U/liter. Nevertheless, POD does not become the velocitydetermining reaction component. This enables a substantial saving of POD.
• a reagent solution was prepared by dissolving 1.5 grams of the above solid powder mixture in 100 ml. water. This solution contained all reagents necessary for the determination of the glucose and, when stored at +4C. can be used for at least 8 weeks.
• the reagent solution were mixed with 0.2 ml. of the glucose-containing solution (obtained, for example, by the deproteinization of blood or serum with a known deproteinisation agent, such as perchloric acid or uranyl acetate).
• a known deproteinisation agent such as perchloric acid or uranyl acetate.
• the mixture was left to stand for 25 minutes at ambient temperature and then the intensity of the colored material formed was measured. This was proportional to the glucose concentration present in the test solution.
• a standard sample of known glucose concentration which was determined at the same time, the desired glucose concentration of the test solution can easily be calculated.
• EXAMPLE 2 Stability of the peroxidase
• the reagent solution described in Example 1 was compared with a reagent solution prepared in the same way but which did not contain ABTS, by measuring the activity of the peroxidase at different times.
• the following Table shows the results obtained.
• Reagent composition for the enzymatic determination of glucose which composition comprises glucose oxidase, peroxidase, a chromogen, a buffer, an azide, and 2,2'-azino-di-(3-ethyl-benzothiazoline-o-sulfonic acid).
• composition as claimed in claim 1 which comprises:
• composition as claimed in claim 5 wherein said composition is in the form of an aqueous solution.
• composition as claimed in claim 1 which comprises:
• composition as claimed in claim 1 which comprises:

Landscapes

• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Life Sciences & Earth Sciences (AREA)
• Health & Medical Sciences (AREA)
• Zoology (AREA)
• Wood Science & Technology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Engineering & Computer Science (AREA)
• Microbiology (AREA)
• Biochemistry (AREA)
• Physics & Mathematics (AREA)
• Molecular Biology (AREA)
• Biotechnology (AREA)
• Biophysics (AREA)
• Analytical Chemistry (AREA)
• Immunology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• General Engineering & Computer Science (AREA)
• General Health & Medical Sciences (AREA)
• Genetics & Genomics (AREA)
• Emergency Medicine (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
• Investigating Or Analysing Biological Materials (AREA)

Applications Claiming Priority (1)

Publications (1)

Family

ID=5781013

Family Applications (1)

Country Status (15)

Cited By (18)

• 1971
  • 1971-06-29 AT AT563271A patent/AT308049B/de not_active IP Right Cessation
  • 1971-07-16 IL IL37324A patent/IL37324A/xx unknown
  • 1971-07-21 DK DK359271A patent/DK134796C/da active
  • 1971-07-29 CA CA119,471A patent/CA952416A/en not_active Expired
  • 1971-08-11 IT IT27438/71A patent/IT941678B/it active
  • 1971-08-11 US US00170949A patent/US3721607A/en not_active Expired - Lifetime
  • 1971-08-23 SU SU1696900A patent/SU513645A3/ru active
  • 1971-08-24 JP JP6467371A patent/JPS5335479B1/ja active Pending
  • 1971-08-24 GB GB39579/71A patent/GB1300338A/en not_active Expired
  • 1971-08-25 SE SE10768/71A patent/SE368872B/xx unknown
  • 1971-08-25 CH CH1242871A patent/CH560901A5/xx not_active IP Right Cessation
  • 1971-08-25 CH CH1242771A patent/CH560900A5/xx not_active IP Right Cessation
  • 1971-08-25 NL NL717111668A patent/NL154832B/xx not_active IP Right Cessation
  • 1971-08-27 FR FR7131226A patent/FR2131198A5/fr not_active Expired
  • 1971-08-27 FI FI712405A patent/FI51638C/fi active
  • 1971-08-27 HU HUBO1316A patent/HU163669B/hu unknown

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