US3164534A - Diagnostic composition - Google Patents

Diagnostic composition Download PDF

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Publication number
US3164534A
US3164534A US422977A US42297754A US3164534A US 3164534 A US3164534 A US 3164534A US 422977 A US422977 A US 422977A US 42297754 A US42297754 A US 42297754A US 3164534 A US3164534 A US 3164534A
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Prior art keywords
glucose
tablet
urine
enzyme
oxygen
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US422977A
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Alfred H Free
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Bayer Corp
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Miles Laboratories Inc
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    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/805—Test papers

Definitions

  • the invention has for one of its primary objects, the provision of a simple, rapid and convenient method for performing such test with a high degree of simplicity and Without the need for extensive equipment, trained personnel and the like.
  • glucose in such body fluids as urine for example is of signal importance not only in the case of diabetic patients who must control their sugar input, but from another point of view is important in those situations where the detection of disease as a public health measure requires the screening of large numbers of people to determine their physical condition.
  • the aforesaid Compton and Trenneer patent eliminated the need for the extraneous source of heat by providing a built in heat source in a tablet combined with eliminate the possibility of unintentional generation of V for determining the presence or absence of glucose in body fluids, including urine, by a technique which is sim ple, which does not require the use of external, or in fact any, heat source, and which is otherwise free of many of the disadvantages of the prior art compositions and procedures.
  • My invention generally speaking, involves the contacting of the body fluid being tested for reducing sugar content, urine for example, with an enzymatic preparation containing glucose-oxidase and catalase and a subsequent measurement, estimation, or determination of the gluconic acid which is produced when glucose or similar reducing agents are present in the urine.
  • My technique in any one of its numerous embodiments, which will be described in detail hereinafter, is so simple that it can be used as a qualitative, and even quantitative, test, parice ticularly, by any unskilled person who is able only to determine the presence or absence of a color change.
  • Example 1 A diagnostic tablet weighing 5 grains is prepared in.
  • glucose oxidase-catalase enzyme such an enzyme is the product known as Dec 0 or Dee G produced and sold by the Takamine Laboratories, of Clifton, New Jersey.
  • the tablet may contain, conveniently, about 5 mg. of the enzyme together with a suflicient amount of lactose, bentonite, simple inorganic salts, borax, starch and similar fillers and excipients used in tabletting operation.
  • an oxygen-supplying material like strontium peroxide, barium peroxide, sodium pyrophosphate peroxide, sodium perbor silicate, and similar materials may be added.
  • the oxygen needed for the reaction involved might also be supplied by the addition to the tablet of such oxygen-containing compounds as the iodates and related compounds. It is to be understood, of course, that the amount of enzyme used in the tablet is variable, sufficient being present however, so that in use, that there be enough enzyme to catalyze the oxidation of all of the glucose present in the sample of body .fluid being analyzed, to gluconic acid.
  • an indicator which will effect a color change at a pH below the normal pH of body fluids, and particularly which changes color at a pH of approximately 4.5 or below.
  • examples of such indicators are, methyl orange, Congo red, or any other indicator which changes color when the pH of the solution drops below 4.5.
  • a drop or a few drops of the body fluid which is being tested, urine for example, is placed upon the above tablet; if glucose is present in the body fluid, the enzyme will catalyze the oxidation of the glucose to gluconic acid, and a color change on the surface of the tablet indicates the positive presence of glucose in the fluid.
  • a small amount of an effervescent couple to facilitate the contacting of the liquid with the solid materials making up the tablet.
  • a surface-active agent to facilitate the wetting of the tablet by the fluid.
  • Example 2 such asstarch, lactose, borates, such as sodium borate, or other white or preferably colorless compounds.
  • Sufficient effervescent couple may be added so that the tablet readily disintegrates when it is dropped into the urine.
  • the effervescent couple can of course be present in Varying amounts, and may comprise from of 1%, based on the weight of the tablet to as much as 50% or even higher; in any event, only sufficient effervescent couple is used, or need be used to assist in the dissolution of the tablet.
  • gluconic acid has well known chelating properties
  • colored salts of those metals which form chelates with gluconic acid can be incorporated in the tablet or in a test paper used with the tablet to indicate the concentration of gluconic acid obtained when the aforesaid enzyme component of the tablet has a chance to exercise its eifect, in the presence of oxygen, on the glucose present in the urine or other body fluid being tested.
  • metal salts would be the colored salts of such metals as iron, copper, nickel, calcium, barium, magnesium and the like.
  • Another variant of my invention is to use a bibulous mat of paper, asbestos or the like, such as is described for example, in the copending application of Alfred H. Free and Helen Free, Serial No. 178,344, filed August 8, 1950, now abandoned.
  • a bibulous mat of paper, asbestos or the like such as is described for example, in the copending application of Alfred H. Free and Helen Free, Serial No. 178,344, filed August 8, 1950, now abandoned.
  • Such a mat is soaked in a solution of a glucose oXidase-catalase enzyme such as hereinbefore described, dried, and the test performed by applying a drop or two of the urine or other body fluid being tested to the mat in the presence of oxygen or an oxygen supplying compound, as Well as an indicator to indicate the presence or absence of gluconic acid which is formed.
  • a test means for detecting glucose in urine which comprises a bibulous material which has been impregnated with a glucose-free composition comprising glucose-oxidase and an indicator material which undergoes a color reaction induced by a reaction product formed when said enzyme is contacted with glucose in the presence of oxygen.
  • a test means for detecting glucose in urine which comprises a bibulous material which has been impregnated with a glucose-free composition comprising glucose oxidase, catalase, and an indicator material which undergoes a color reaction induced by a reaction product formed when glucose-oxidase is contacted with glucose in the presence of oxygen.

Description

United States Patent O 3,164,534 DIAGNQSTHZ CGWOSITION Alfred E. Free, Elkhart, Ind, assignor to Miles Laboratories, Inc, Ellrlrart, Ind., a corporation of Indiana N Drawing. Filed Apr. 13, 1954, Ser. No. 422,971 3 (Ilaims. (Cl. 195-1935) This invention relates to diagnostic compositions for the detection and estimation of reducing sugars in body fluids.
The invention has for one of its primary objects, the provision of a simple, rapid and convenient method for performing such test with a high degree of simplicity and Without the need for extensive equipment, trained personnel and the like.
The determination of glucose in such body fluids as urine for example, is of signal importance not only in the case of diabetic patients who must control their sugar input, but from another point of view is important in those situations where the detection of disease as a public health measure requires the screening of large numbers of people to determine their physical condition.
There are a number of methods of course, which can be used to measure or estimate the amount of reducing agents in body fluids, particularly urine. The more widely used of the conventional methods are based on the use of alkaline copper solutions which are heated with the material being tested to precipitate cuprous oxide.
The old methods have had the disadvantage that their use has required a certain amount of skill and familiarity with the use of measuring equipment, such as pipettes and the like, the use of liquid reagents some of which,
especially the alkaline ones were dangerous to handle,
and were inconvenient to transport easily.
More recently, a diagnostic tablet described in US. Patent No. 2,387,244 to Compton and Trenneer has found Wide usage because of its relative simplicity, accuracy, economy, and the fact that the test could be executed by unskilled persons, as compared to the older tests.
However, all of these tests, techniques and procedures mentioned above, have one draw-back in common, namely they all require heat to perform the tests. In the older methods this heat was supplied by the use of a Bunsen burner or bysome other extraneous source of heat.
The aforesaid Compton and Trenneer patent eliminated the need for the extraneous source of heat by providing a built in heat source in a tablet combined with eliminate the possibility of unintentional generation of V for determining the presence or absence of glucose in body fluids, including urine, by a technique which is sim ple, which does not require the use of external, or in fact any, heat source, and which is otherwise free of many of the disadvantages of the prior art compositions and procedures.
My invention, generally speaking, involves the contacting of the body fluid being tested for reducing sugar content, urine for example, with an enzymatic preparation containing glucose-oxidase and catalase and a subsequent measurement, estimation, or determination of the gluconic acid which is produced when glucose or similar reducing agents are present in the urine. My technique, in any one of its numerous embodiments, which will be described in detail hereinafter, is so simple that it can be used as a qualitative, and even quantitative, test, parice ticularly, by any unskilled person who is able only to determine the presence or absence of a color change.
The following examples will serve to document a number of specific embodiments of my invention, and illustrate the flexibility of the invention. I have chosen these embodiments hereinafter described, as illustrative of my invention and it will be apparent that various modifications may be made without departing from the spirit and scope of my invention.
Example 1 A diagnostic tablet weighing 5 grains is prepared in.
accordance with conventional blending and tabletting techniques, so that it contains as one essential ingredient a glucose oxidase-catalase enzyme. Such an enzyme is the product known as Dec 0 or Dee G produced and sold by the Takamine Laboratories, of Clifton, New Jersey. The tablet may contain, conveniently, about 5 mg. of the enzyme together with a suflicient amount of lactose, bentonite, simple inorganic salts, borax, starch and similar fillers and excipients used in tabletting operation.
Since a certain amount of oxygen is necessary to carry out the reaction involved, an oxygen-supplying material, like strontium peroxide, barium peroxide, sodium pyrophosphate peroxide, sodium perbor silicate, and similar materials may be added. The oxygen needed for the reaction involved might also be supplied by the addition to the tablet of such oxygen-containing compounds as the iodates and related compounds. It is to be understood, of course, that the amount of enzyme used in the tablet is variable, sufficient being present however, so that in use, that there be enough enzyme to catalyze the oxidation of all of the glucose present in the sample of body .fluid being analyzed, to gluconic acid. In addition to the above components there is added to the composition, in accordance with one form of my invention, a sufficient amount of an indicator which will effect a color change at a pH below the normal pH of body fluids, and particularly which changes color at a pH of approximately 4.5 or below. Examples of such indicators are, methyl orange, Congo red, or any other indicator which changes color when the pH of the solution drops below 4.5.
In use, a drop or a few drops of the body fluid which is being tested, urine for example, is placed upon the above tablet; if glucose is present in the body fluid, the enzyme will catalyze the oxidation of the glucose to gluconic acid, and a color change on the surface of the tablet indicates the positive presence of glucose in the fluid.
' It is desirable to include, as one of the components of such a tablet, a small amount of an effervescent couple to facilitate the contacting of the liquid with the solid materials making up the tablet. Under some circumstances it may be desirable to include a surface-active agent to facilitate the wetting of the tablet by the fluid.
It is also possible if desired, to omit the aforesaid type of indicator from the composition of the tablet and to perform the test by placing an indicator-free tablet on a test paper which has been treated to change color at a pH of about 4.5 or below, add a few drops of the body fluid being tested to the tablet and observe the color reaction if any, in the area which is wetted by the fluid around the periphery of the tablet.
Example 2 such asstarch, lactose, borates, such as sodium borate, or other white or preferably colorless compounds. Sufficient effervescent couple may be added so that the tablet readily disintegrates when it is dropped into the urine. The effervescent couple can of course be present in Varying amounts, and may comprise from of 1%, based on the weight of the tablet to as much as 50% or even higher; in any event, only sufficient effervescent couple is used, or need be used to assist in the dissolution of the tablet.
Besides using a pH indicator in the tablet, or in a test paper, it is of course possible to use as an indicator, compounds which form color salts or addition complexes of one kind or another with the gluconic acid devolved. Since gluconic acid has well known chelating properties, colored salts of those metals which form chelates with gluconic acid can be incorporated in the tablet or in a test paper used with the tablet to indicate the concentration of gluconic acid obtained when the aforesaid enzyme component of the tablet has a chance to exercise its eifect, in the presence of oxygen, on the glucose present in the urine or other body fluid being tested. Examples of such metal salts would be the colored salts of such metals as iron, copper, nickel, calcium, barium, magnesium and the like.
Another variant of my invention is to use a bibulous mat of paper, asbestos or the like, such as is described for example, in the copending application of Alfred H. Free and Helen Free, Serial No. 178,344, filed August 8, 1950, now abandoned. Such a mat is soaked in a solution of a glucose oXidase-catalase enzyme such as hereinbefore described, dried, and the test performed by applying a drop or two of the urine or other body fluid being tested to the mat in the presence of oxygen or an oxygen supplying compound, as Well as an indicator to indicate the presence or absence of gluconic acid which is formed.
Since certain changes in carrying out the above techniques, and certain modifications in the compositions which embodythe invention may be made without departing from its scope, it is intended that all matter contained in the above description shall be interpreted as illustrative and not in a limiting sense. It is also to be understood that the following claims are intended to cover all of the generic and specific features of the invention herein described, and all statements of the scope of the invention which, is a matter of language, might be urine which comprises contacting the urine which is to be tested with a bibulous material which has been impregnated with a glucose-free composition comprising glucose-oxidase and an indicator material which undergoes a color change induced by a reaction product formed when said enzyme is contacted with glucose in the presence of oxygen.
2. A test means for detecting glucose in urine which comprises a bibulous material which has been impregnated with a glucose-free composition comprising glucose-oxidase and an indicator material which undergoes a color reaction induced by a reaction product formed when said enzyme is contacted with glucose in the presence of oxygen.
3. A test means for detecting glucose in urine which comprises a bibulous material which has been impregnated with a glucose-free composition comprising glucose oxidase, catalase, and an indicator material which undergoes a color reaction induced by a reaction product formed when glucose-oxidase is contacted with glucose in the presence of oxygen.
References Cited by the Examiner UNITED STATES PATENTS 1 ,247,522 11/ 17 Fisher 16784.5 2,359,052 9/44 Scharer 23230 2,482,724 9/49 Baker 99186 2,651,592 9/53 Baker -36 2,671,028 3/54 Clark 99192 FOREIGN PATENTS 708,996 1952 Great Britain.
dase, by D. Mueller, Copenhagen, 1936, pp..259271,
p. 261 relied on.
A. LOUIS MONACELL, Primary Examiner.
DONALD J. ARNOLD, ABRAHAM H. VVINKEL- STEIN, TOBIAS E. LEVOW, Examiners.

Claims (1)

1. A METHOD FOR DETECTING THE PRESENCE OF GLUCOSE IN URINE WHICH COMPRISES CONTACTING THE URINE WHICH IS TO BE TESTED WITH A BIBULOUS MATERIAL WHICH HAS BEEN IMPREGNATED WITH A GLUCOSE-FREE COMPOSITION COMPRISING GLUCOSE-OXIDASE AND AN INDICATOR MATERIAL WHICH UNDERGOES A COLOR CHANGE INDUCED BY A REACTION PRODUCT FORMED WHEN SAID ENZYME IS CONTACTED WITH GLUCOSE IN THE PRESENCE OF OXYGEN.
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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4211845A (en) * 1978-11-24 1980-07-08 Miles Laboratories, Inc. Glucose indicator and method
EP0396156A1 (en) 1985-06-28 1990-11-07 Miles Inc. Method of manufacturing a membrane for an electrochemical sensor
US6586195B1 (en) 2001-11-19 2003-07-01 R.E. Davis Chemical Corporation Method of detecting sugars
US20050173245A1 (en) * 2004-02-09 2005-08-11 Benjamin Feldman Membrane suitable for use in an analyte sensor, analyte sensor, and associated method
US20050215871A1 (en) * 2004-02-09 2005-09-29 Feldman Benjamin J Analyte sensor, and associated system and method employing a catalytic agent
US20080081969A1 (en) * 2003-10-31 2008-04-03 Abbott Diabetes Care, Inc. Method of calibrating of an analyte-measurement device, and associated methods, devices and systems
US20080161666A1 (en) * 2006-12-29 2008-07-03 Abbott Diabetes Care, Inc. Analyte devices and methods
US20090054748A1 (en) * 2006-02-28 2009-02-26 Abbott Diabetes Care, Inc. Method and system for providing continuous calibration of implantable analyte sensors
US20090259118A1 (en) * 2008-03-31 2009-10-15 Abbott Diabetes Care Inc. Shallow Implantable Analyte Sensor with Rapid Physiological Response
US11229382B2 (en) 2013-12-31 2022-01-25 Abbott Diabetes Care Inc. Self-powered analyte sensor and devices using the same

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US1247522A (en) * 1914-10-05 1917-11-20 Arthur William Fisher Composition of matter.
US2359052A (en) * 1940-07-01 1944-09-26 Scharer Harry Method for detecting enzyme activity
US2482724A (en) * 1944-07-22 1949-09-20 Ben L Sarett Deoxygenation process
US2651592A (en) * 1950-08-15 1953-09-08 Ben L Sarett Enzymatic process for producing gluconic acid
US2671028A (en) * 1949-10-17 1954-03-02 James D A Clark Method and means for indicating product deterioration
GB708996A (en) * 1954-04-15 1954-05-12 Miles Lab Improvements in or relating to diagnostic compositions

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US1247522A (en) * 1914-10-05 1917-11-20 Arthur William Fisher Composition of matter.
US2359052A (en) * 1940-07-01 1944-09-26 Scharer Harry Method for detecting enzyme activity
US2482724A (en) * 1944-07-22 1949-09-20 Ben L Sarett Deoxygenation process
US2671028A (en) * 1949-10-17 1954-03-02 James D A Clark Method and means for indicating product deterioration
US2651592A (en) * 1950-08-15 1953-09-08 Ben L Sarett Enzymatic process for producing gluconic acid
GB708996A (en) * 1954-04-15 1954-05-12 Miles Lab Improvements in or relating to diagnostic compositions

Cited By (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4211845A (en) * 1978-11-24 1980-07-08 Miles Laboratories, Inc. Glucose indicator and method
EP0396156A1 (en) 1985-06-28 1990-11-07 Miles Inc. Method of manufacturing a membrane for an electrochemical sensor
US6586195B1 (en) 2001-11-19 2003-07-01 R.E. Davis Chemical Corporation Method of detecting sugars
US8219174B2 (en) 2003-10-31 2012-07-10 Abbott Diabetes Care Inc. Method of calibrating an analyte-measurement device, and associated methods, devices and systems
US8219175B2 (en) 2003-10-31 2012-07-10 Abbott Diabetes Care Inc. Method of calibrating an analyte-measurement device, and associated methods, devices and systems
US8684930B2 (en) 2003-10-31 2014-04-01 Abbott Diabetes Care Inc. Method of calibrating an analyte-measurement device, and associated methods, devices and systems
US20080081969A1 (en) * 2003-10-31 2008-04-03 Abbott Diabetes Care, Inc. Method of calibrating of an analyte-measurement device, and associated methods, devices and systems
US8116840B2 (en) 2003-10-31 2012-02-14 Abbott Diabetes Care Inc. Method of calibrating of an analyte-measurement device, and associated methods, devices and systems
US20100282616A1 (en) * 2003-10-31 2010-11-11 Abbott Diabetes Care Inc. Method of Calibrating an Analyte-Measurement Device, and Associated Methods, Devices and Systems
US20090204340A1 (en) * 2003-10-31 2009-08-13 Abbott Diabetes Care Inc. Method Of Calibrating An Analyte-Measurement Device, And Associated Methods, Devices And Systems
US20090275817A1 (en) * 2003-10-31 2009-11-05 Abbott Diabetes Care Inc. Method of Calibrating an Analyte-Measurement Device, and Associated Methods, Devices and Systems
US8165651B2 (en) 2004-02-09 2012-04-24 Abbott Diabetes Care Inc. Analyte sensor, and associated system and method employing a catalytic agent
US7699964B2 (en) 2004-02-09 2010-04-20 Abbott Diabetes Care Inc. Membrane suitable for use in an analyte sensor, analyte sensor, and associated method
US8761857B2 (en) 2004-02-09 2014-06-24 Abbott Diabetes Care Inc. Analyte sensor, and associated system and method employing a catalytic agent
US20070191701A1 (en) * 2004-02-09 2007-08-16 Abbott Diabetes Care, Inc. Analyte Sensor, and Associated System and Method Employing a Catalytic Agent
US20050215871A1 (en) * 2004-02-09 2005-09-29 Feldman Benjamin J Analyte sensor, and associated system and method employing a catalytic agent
US20050173245A1 (en) * 2004-02-09 2005-08-11 Benjamin Feldman Membrane suitable for use in an analyte sensor, analyte sensor, and associated method
US7885698B2 (en) 2006-02-28 2011-02-08 Abbott Diabetes Care Inc. Method and system for providing continuous calibration of implantable analyte sensors
US20110130639A1 (en) * 2006-02-28 2011-06-02 Abbott Diabetes Care Inc. Method and System for Providing Continuous Calibration of Implantable Analyte Sensors
US8506482B2 (en) 2006-02-28 2013-08-13 Abbott Diabetes Care Inc. Method and system for providing continuous calibration of implantable analyte sensors
US20090054748A1 (en) * 2006-02-28 2009-02-26 Abbott Diabetes Care, Inc. Method and system for providing continuous calibration of implantable analyte sensors
US10117614B2 (en) 2006-02-28 2018-11-06 Abbott Diabetes Care Inc. Method and system for providing continuous calibration of implantable analyte sensors
US11872039B2 (en) 2006-02-28 2024-01-16 Abbott Diabetes Care Inc. Method and system for providing continuous calibration of implantable analyte sensors
US20080161666A1 (en) * 2006-12-29 2008-07-03 Abbott Diabetes Care, Inc. Analyte devices and methods
US20090259118A1 (en) * 2008-03-31 2009-10-15 Abbott Diabetes Care Inc. Shallow Implantable Analyte Sensor with Rapid Physiological Response
US11229382B2 (en) 2013-12-31 2022-01-25 Abbott Diabetes Care Inc. Self-powered analyte sensor and devices using the same

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