CA1144859A - Diacetyldichlorofluorescin with a source of hydrogen peroxide for assay of peroxidase - Google Patents
Diacetyldichlorofluorescin with a source of hydrogen peroxide for assay of peroxidaseInfo
- Publication number
- CA1144859A CA1144859A CA000346350A CA346350A CA1144859A CA 1144859 A CA1144859 A CA 1144859A CA 000346350 A CA000346350 A CA 000346350A CA 346350 A CA346350 A CA 346350A CA 1144859 A CA1144859 A CA 1144859A
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- CA
- Canada
- Prior art keywords
- hydrogen peroxide
- enzyme
- source
- substrate
- composition
- 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.)
- Expired
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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
-
- 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
- C12Q2326/40—Triphenylmethane dye chromogens, e.g. fluorescein derivatives
-
- 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
- Y10S435/97—Test strip or test slide
-
- 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
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T436/00—Chemistry: analytical and immunological testing
- Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
- Y10T436/142222—Hetero-O [e.g., ascorbic acid, etc.]
- Y10T436/143333—Saccharide [e.g., DNA, etc.]
- Y10T436/144444—Glucose
-
- 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
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T436/00—Chemistry: analytical and immunological testing
- Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
- Y10T436/145555—Hetero-N
- Y10T436/147777—Plural nitrogen in the same ring [e.g., barbituates, creatinine, etc.]
- Y10T436/148888—Uric acid
Abstract
Abstract An assay for substances demonstrating peroxidative activity is based on the peroxidase catalyzed oxidation of dichlorofluoresein to dichlorofluorescein by peroxide. The dichloro-fluoresein is formed in situ from diacetyldichloro-fluoresein either by the direct addition of peroxide or by the inclusion of a peroxide generating system in the reaction mixture.
Description
4~
--1~
DIACET~LDICHLOROFLUORESCIN WITH A SOU~CE OF
HYDROGEN PEROXIDE FOR ASSAY OF PEROXIDASE
The oxidation of dichlorofluorescin by hydro~en peroxide to dichlorofluorescein, catalyzed by the enzyme peroxidase, has been used as an assay technique for hydrogen peroxide (Black, M.J. & Brandt, R.B.~ Anal, Biochem, 58:241 ~1974}).
A drawback of this technique is that since the dichlorofluores-Cill .iS unstable to air oxidation, it must be generated as neededby hydrolysis of diacetyldichlorofluorescin at alkaline pH
followed by neutralization and dilution in the assay tube.
The assay of peroxidase, particularly at relatively low levels~ is useful in certain immunoassay techniques wherein a peroxidase is used as the label for a ligand in a competitive binding or displacement immunological determination. Such assay techniques are described in U.S. Patent 3,817,837; U.S.
Patent 3,154,090 and in Van Weeman and Schuurs, BIOCHIMIE, 54:842 (1972).
There is a need for a peroxidase assay which is capable of detecting low levels of the enzyme, i.e., on the order of _10 7M, as are used in imrnunoassays. Such an assay should be rapid and eliminate the requirement for the dichlorofluores-cin being prepared as needed in a separate step. Furthermore, it is most desirable thak all o~ the reagents can be stored in a stable t dry condition until immediately prior to use.
A new peroxidase assay ~or substances demonstrating peroxidative activity is described which comprises the steps of 1) contacting an - ~ . .
- . : :',. ':~, ' : . ' assay composition comprising diacetyldichloro-fluorescin and a source of hydrogen peroxide with a sample to be assayed and 2~ detecting the resulting dichlorofluorescein which is formed from the peroxidase catalyzed oxidation of the reaction product of diacetyldichlorofluorescin with hydrogen peroxide. In a preferred embodi-ment, the dichlorofluorescein is detected by rate of fluorescence increase. As long as all reactants other than the peroxidase are present in excess, the rate of fluorescence increase is linearly related to the amount of peroxidase in the sample under assay.
ln accordance with the present invention, a composition is provided which is useful for the determinat~on of a substance having peroxidative activity. The composition consists essentially of diacetyldichlorofluorescin and a source of hydrogen peroxide in a concentration of from about 10 6 to 10 2M with the ratio of diacetyldi-chlorofluorescin to hydrogen peroxide or generated hydrogen peroxide ranging from 1:5 to 1:100.
The present invention also provides dry reagent compositions that include all of the materials required to perform a peroxidase de-termination. Such compositions can be stored dry and provide stable and adaptable compositions for a multitude of applications wherein peroxidase detection and/or quantification may be useful or desirable.
The assay compositions of the present invention comprise diacetyldichlorofluorescin, hydrogen peroxide or a composition which generates hydrogen peroxide ~i.e., a source of hydrogen per-oxide~, e.g., glucose and glucose oxidase or uric acid and uricase, and, generally, a buffer to maintain the pH of the assay composition at be-tween 7 and 9.
.~
-2a- -In accordance with a further embodiment, an element is provided for the detection of per-oxidase which comprises a layer which comprises a spreading agent and a reagent layer in fluid contact under conditions of use, the reagent layer comprises a reagent consisting essentially of diacetyldichlorofluorescin and a dry source of hydrogen peroxide at a concentration of from about 10 6 to 10 2M, the ratio of diacetyldichloro-fluorescin to generated hydrogen peroxide beingin the range of from 1:5 to 1:100.
In accordance with a further embodiment, a method is provided for assaying peroxidase which comprises the steps of:
a) contacting a sample for analysis with a composition consisting essentially of di-acetyldichlorofluorescin and a source of hydrogen peroxide in a concentration of from about 10 6 to 10 2M to produce dichlorofluorescein, the ratio of .
: .
85~
diacetyldichlorofluorescin to hydrogen peroxide or generated hydrogen peroxide ranging from 1:5 to 1:100; and b) detecting the dichlorofluorescin.
In yet a further aspect of the present teachings, there is provided a method of assaying for antigen or antibody present in a sample to be tested which comprises:
a) labeling a known amount of antigen or antibody with peroxidase and adding the la-beled antigen or antibody with the sample to be tested to a ]cnown amount of corresponding antigen or antibody; and b) determining the amount of unknown anti-gen or antibody by (i) contacting in an aqueous medium the mixture of diacetyldichlorofluorescin and a source of hydrogen peroxide at a concentration of from about 10 6 to 10 2M to produce dichloro-fluorescein, the ratio of diacetyldichlorofluore-scin to hydrogen peroxide or generated hydrogen peroxide being in the range of from 1:5 to 1:100;
(ii) detecting the dichlorofluorescein.
Particular advantages of the peroxidase assay method described herein are:
(1) Simplicity: dichlorofluorescin is generated ln situ during the assay reaction, thus eliminating costly preparation steps;
--1~
DIACET~LDICHLOROFLUORESCIN WITH A SOU~CE OF
HYDROGEN PEROXIDE FOR ASSAY OF PEROXIDASE
The oxidation of dichlorofluorescin by hydro~en peroxide to dichlorofluorescein, catalyzed by the enzyme peroxidase, has been used as an assay technique for hydrogen peroxide (Black, M.J. & Brandt, R.B.~ Anal, Biochem, 58:241 ~1974}).
A drawback of this technique is that since the dichlorofluores-Cill .iS unstable to air oxidation, it must be generated as neededby hydrolysis of diacetyldichlorofluorescin at alkaline pH
followed by neutralization and dilution in the assay tube.
The assay of peroxidase, particularly at relatively low levels~ is useful in certain immunoassay techniques wherein a peroxidase is used as the label for a ligand in a competitive binding or displacement immunological determination. Such assay techniques are described in U.S. Patent 3,817,837; U.S.
Patent 3,154,090 and in Van Weeman and Schuurs, BIOCHIMIE, 54:842 (1972).
There is a need for a peroxidase assay which is capable of detecting low levels of the enzyme, i.e., on the order of _10 7M, as are used in imrnunoassays. Such an assay should be rapid and eliminate the requirement for the dichlorofluores-cin being prepared as needed in a separate step. Furthermore, it is most desirable thak all o~ the reagents can be stored in a stable t dry condition until immediately prior to use.
A new peroxidase assay ~or substances demonstrating peroxidative activity is described which comprises the steps of 1) contacting an - ~ . .
- . : :',. ':~, ' : . ' assay composition comprising diacetyldichloro-fluorescin and a source of hydrogen peroxide with a sample to be assayed and 2~ detecting the resulting dichlorofluorescein which is formed from the peroxidase catalyzed oxidation of the reaction product of diacetyldichlorofluorescin with hydrogen peroxide. In a preferred embodi-ment, the dichlorofluorescein is detected by rate of fluorescence increase. As long as all reactants other than the peroxidase are present in excess, the rate of fluorescence increase is linearly related to the amount of peroxidase in the sample under assay.
ln accordance with the present invention, a composition is provided which is useful for the determinat~on of a substance having peroxidative activity. The composition consists essentially of diacetyldichlorofluorescin and a source of hydrogen peroxide in a concentration of from about 10 6 to 10 2M with the ratio of diacetyldi-chlorofluorescin to hydrogen peroxide or generated hydrogen peroxide ranging from 1:5 to 1:100.
The present invention also provides dry reagent compositions that include all of the materials required to perform a peroxidase de-termination. Such compositions can be stored dry and provide stable and adaptable compositions for a multitude of applications wherein peroxidase detection and/or quantification may be useful or desirable.
The assay compositions of the present invention comprise diacetyldichlorofluorescin, hydrogen peroxide or a composition which generates hydrogen peroxide ~i.e., a source of hydrogen per-oxide~, e.g., glucose and glucose oxidase or uric acid and uricase, and, generally, a buffer to maintain the pH of the assay composition at be-tween 7 and 9.
.~
-2a- -In accordance with a further embodiment, an element is provided for the detection of per-oxidase which comprises a layer which comprises a spreading agent and a reagent layer in fluid contact under conditions of use, the reagent layer comprises a reagent consisting essentially of diacetyldichlorofluorescin and a dry source of hydrogen peroxide at a concentration of from about 10 6 to 10 2M, the ratio of diacetyldichloro-fluorescin to generated hydrogen peroxide beingin the range of from 1:5 to 1:100.
In accordance with a further embodiment, a method is provided for assaying peroxidase which comprises the steps of:
a) contacting a sample for analysis with a composition consisting essentially of di-acetyldichlorofluorescin and a source of hydrogen peroxide in a concentration of from about 10 6 to 10 2M to produce dichlorofluorescein, the ratio of .
: .
85~
diacetyldichlorofluorescin to hydrogen peroxide or generated hydrogen peroxide ranging from 1:5 to 1:100; and b) detecting the dichlorofluorescin.
In yet a further aspect of the present teachings, there is provided a method of assaying for antigen or antibody present in a sample to be tested which comprises:
a) labeling a known amount of antigen or antibody with peroxidase and adding the la-beled antigen or antibody with the sample to be tested to a ]cnown amount of corresponding antigen or antibody; and b) determining the amount of unknown anti-gen or antibody by (i) contacting in an aqueous medium the mixture of diacetyldichlorofluorescin and a source of hydrogen peroxide at a concentration of from about 10 6 to 10 2M to produce dichloro-fluorescein, the ratio of diacetyldichlorofluore-scin to hydrogen peroxide or generated hydrogen peroxide being in the range of from 1:5 to 1:100;
(ii) detecting the dichlorofluorescein.
Particular advantages of the peroxidase assay method described herein are:
(1) Simplicity: dichlorofluorescin is generated ln situ during the assay reaction, thus eliminating costly preparation steps;
(2) Reagent Stability: since all re-quired reagents are capable of dry storage, the assay composition exhibits extended shelf life and can be adapted to numerous assay applications;
~3) Speed: the assay compositions are capable of assaying for peroxidase activity in one minute or less.
Peroxidative activity is well known in the art and includes, for example, peroxidative r~
:
, ~4~5~3 enzymes, cytochromes, hemoglobin, etc. Thus, the assay procedure discussed herein is useful not only in the assay of what are technlcally termed peroxidase enzymes, but also in the determination of other materials demonstrating "peroxidative activity."
The reaction which occurs in one of the preferred assay compositions described herein is as follows:
Glucose Oxidase Glucose + 2 ~ Gluconic Acid + H2O2 H202+ Diacetyldichlorofluorescin-~Dichlorofluorescin + 2 HOAC
Peroxidase Dichlorofluorescin + H202 ~ ~ Dichlorofluorescein (Fluorescent) As described hereinabove, the assay compositions of the present invention comprise diacetyldichlorofluorescin and a source of hydrogen peroxide.
Diacetyldichlorofluorescin is readily and commercially available from ~astman Organic Chemicals, Rochester, New York.
Any source of hydrogen peroxide can be useful in the successful practice of the instant invention. Of course, hydrogen peroxide itself may be used as the source of the material. This is preferred when the assay reaction is carried out in solution or when the hydrogen peroxide can be added to the reagent system at about the same time as the sample under assay.
In many circ~mstances, it may, however, be desirable to supply a reagent composition of the type described herein in dry powder form ready for reconstltution with water or to provide ~4~
the assay composition in the form Or a paper or other fibrous or strip material which has been impregnated with reagent which is reconstituted upon application of a liquid assay sample thereto. Yet a further desirable embodiment can be the incorporation of the reagent into one or more layers of a multilayer element of the type described in U.S. Patent 3,992,158.
Under such circumstances, a source of hydrogen peroxide that is dry until contacted with water should be used. Preferred sources are mixtures of an enzyme that demonstrates oxidative activity and substrates for such an enzyme~ for example, glucose and glucose oxidase, uric acid and uricase, and cholesterol and cholesterol oxldase. Any of these and numerous other similar mixtures Or oxidase and substrate when contacted in water and in the presence of oxy~en result in decomposition of the substrate with tne concomitant production of hydrogen peroxide. Such materials are particularly useful because they can be freeze-dried or lyophilized to provide dried powders that can be reconstituted with water. Of course, the enzyme and the substrate must be kept separated until the production of the hydrogen peroxide is desired.
Furthermore, since dichlorofluorescin is quite unstable, its formation by the reaction Or peroxide and diacetyldichlorofluorescin should not be initiated until the substance under assay having per~xidative activity has been added to the assa~y composition. Other useful hydrogen peroxide sources include chemically bound hydrogen peroxldes, such as urea peroxide (Robeco Chemicals, Inc.).
, ' s~
The concentration of the reagents in the assay composition will depend, to a large extent, on the concentration of the material under assay - higher concentrations of reagent being necessary for higher concentrations of material under assay. For concentrations of substance having peroxidative activity between about 10 5 and about 10 lOM, the following composition has been found particularly useful:
diacetyldichlorofluorescin 10 7-10-4M
oxidative enzyme 1-50 units/ml substrate 1-100 mg/dl ~uffer to maintain pH at 7-9.
To be useful, a buffer should not interfere with the assay reaction. ~arbital or phosphate buffers at a concentration of between about 0.01 and 0.1ll have been found useful.
When hydrogen peroxide is used as the peroxide source, a concentration Or between about 10 5 and 5 x 10 4M has been found useful in the above-described assay composition.
In a further embodiment of the present invention, the assaying composition comprising diacetyldichlorofluorescin and a source of hydrogen peroxide can be used to determine the amount of peroxidative activity present in a patient's hemoglobin. Thus, a sample of hemoglobin can be added to the assaying composition and the measurement of the fluorescence rate of the resulting dichlorofluorescin will determine the amount of peroxidative activity in the hemoglobin.
The method for, detecting a substance having peroxidative act~ivity comprises contacting, in an aqueous medium, the sample under assay with a composition comprising dlacetyldichloro-fluorescin and a source of hydrogen peroxide and . . ~ .
.
5~
then detecting the resulting dichlorofluorescein.
Since the resulting dichlorofluorescein is fluorescent, it is easily detected by measuring the fluorescence over a short period of time.
5 The amount of substance having peroxidative activity in the sample is directly proportional to the rate of fluorescence increase per minute of the resulting dichlorofluorescin.
The assay composition can be employed in dry chemistry techniques. It can, for example~ be incorporated by imbibition impregnation or by coating techniques into a reagent zone of a dry test element, e.g., a reagent layer of a dip-and-read fibrous test strip or a reagent layer o~ a non-fibrous multilayer element, as described in U.S. Patents 3,992,158 and 4,042,335.
The element can comprise zones of reagent, which zones may be in the form of layers, and, optionally, spreading agent.
Elements containing these zones are described in U.S. Patents 3,992,158 and 4,069,016.
The substantially dry element can be contacted with a sample for analysis and the resulting dichlorofluorescein can be detected.
25 If the sample itself does not contain water, it could be added with the sample to the dry element.
A particularly preferred test element for the detection Or a substance havlng peroxidative activity, e.g. peroxidase, in a liquid comprises 30 a spreading layer, a reagent layer and a registration layer, all in ~luid contact under conditions of use, and a support~ the,reagent layer intervenlng the spreading layer an~ the re~istration layer, and the registration layer intervening the 35 reagent layer and the support, the reagent layer comprising diacetyldichlorofluorescin and a ' source of hydrogen peroxlde. The registration layer is described in detail in U.S. Patent 4,042,335, columns 11 and 12.
A particularly preferred embodiment of the invention comprises a method for assaying for antigen or antibody present in a sample.
This method comprises labeling a known antigen (or antibody) with a substance having peroxidative activity, e.g. peroxidase, and carrying out the immunoassay in the conventional manner. Thus, the unknown sample can be added along with the labeled sample to a liquid or web comprising the corresponding antibody (or antigen).
The labeled and unlabeled antigen (or antibody) will combine with the corresponding antibody (or antigen) to form an antigen-antibody precipitate and ~ree labeled antigen (or antibody) as well as free unlabeled antigen (or antibody). The determination of the unXnown antibody (or antigen) is made by measuring the free labeled antigen (or antibody) or the labeled antigen-antibody precipitate.
The measurement of labeled antigen or antibody can be accomplished by assaying for the substance having peroxidat~ve activlty by adding the resulting solution to an assaying composition comprising diacetyldichlorfluorescin and a source of hydrogen peroxide and determinlng the amount Or substance having peroxldative activity by measuring the rate of fluorescence of the resulting dichlorofluorescein.
The followin~,examples demons~rate the successful practice of~the present lnvention.
Example 1 -- Fluor scence Assay for Peroxidase To a 1 cm2 cuvette was added 1.4 ml phosphate buffer solution (PBS) containing O.OlM
-. - .
.. , - .
.. ~
, ~4~8S~
sodium phosphate, 0.1 N NaCl, pH 7.5, 0.200 ml diacetyldichlorofluorescin (1 x 10-5M in PBS) and 200 ~1 hydrogen peroxide (1 x 10 3M in water). Reaction was initiated by the addltion 5 o~ peroxidase (horseradish) of the indicated concentration. The rate of fluorescence increase was measured in a Farrand Mk I fluorometer with excitation mo~ochrometer at 490 nm and emission - monochrometer at 520 nm. The rate was linear for 5 minutes. In Figure 1, the rates of fluorescence increase per minute are plotted as a function of peroxidase concentration.
Peroxidase concentrations of up to 1.32 x 10 5M were measurable when the hydrogen 1~ peroxide concentration was increased fifty fold and the dichlorofluorescin concentration increased twofold.
Example 2 -- Multilayered Element ~or the Fluorometric Determination of Peroxidase .
A multilayered element was prepared according to the following:
A polycarbonate support was coated with a reagent layer comprised of deionized gelatin (Type V) (4.5 g/m2), 5,5-dimethyl-1,3-cyclohexanedione (0.4 g/m2), glucose oxidase (538 Units/m2), bis(vinyl sulfonyl methyl)ether (0.05 g/m2), Surfactant lOG, a nonylphenoxy-polyglycidol from Olin Corp., (0.01 g/m2) 5 zinc acetate (0.01 g/m2), dichlorofluorescin diacetate (0.01 g/m2) in 0.02 M phosphate buffer at pH
8.0; an enzyme layer comprising deionized gelatin (2.9 g/m2), colloldal silver (1.02 g/m2~, catalase (86,000 U/m2)~'bis(vinyl sulfonyl methyl)ether (0.04 g/m2), Surfactant lOG (0.007 g/m );
a subbing layer comprised of poly-N-isopropylacryl-amide (0.4 g/m2); and a spreading layer comprised ~' :
s~
of TiO2 (70.0 g/m2), cellulose acetate (10.0 g/m2), glucose (0.5 g/m2), and polyacrylamide particles (L60 mesh) (0.01 g/m2).
The element was tested by applylng thereto a 10 ~1 drop of solutlon containing dinitrophenyl peroxidase con~ugate* (DNP.POD) and measuring the rate at which the fluorescent signal increased, when excited at 460 nm and emitted at 520 nm, as in Example 1. Results are shown in Table I.
Table Conc. DNP'POD
(M) Relative Rate 5.0 x 10- 7.5 2.5 x 10-6 4.8 1.25 x 10-6 1.92 6.25 x 10-7 0.79
~3) Speed: the assay compositions are capable of assaying for peroxidase activity in one minute or less.
Peroxidative activity is well known in the art and includes, for example, peroxidative r~
:
, ~4~5~3 enzymes, cytochromes, hemoglobin, etc. Thus, the assay procedure discussed herein is useful not only in the assay of what are technlcally termed peroxidase enzymes, but also in the determination of other materials demonstrating "peroxidative activity."
The reaction which occurs in one of the preferred assay compositions described herein is as follows:
Glucose Oxidase Glucose + 2 ~ Gluconic Acid + H2O2 H202+ Diacetyldichlorofluorescin-~Dichlorofluorescin + 2 HOAC
Peroxidase Dichlorofluorescin + H202 ~ ~ Dichlorofluorescein (Fluorescent) As described hereinabove, the assay compositions of the present invention comprise diacetyldichlorofluorescin and a source of hydrogen peroxide.
Diacetyldichlorofluorescin is readily and commercially available from ~astman Organic Chemicals, Rochester, New York.
Any source of hydrogen peroxide can be useful in the successful practice of the instant invention. Of course, hydrogen peroxide itself may be used as the source of the material. This is preferred when the assay reaction is carried out in solution or when the hydrogen peroxide can be added to the reagent system at about the same time as the sample under assay.
In many circ~mstances, it may, however, be desirable to supply a reagent composition of the type described herein in dry powder form ready for reconstltution with water or to provide ~4~
the assay composition in the form Or a paper or other fibrous or strip material which has been impregnated with reagent which is reconstituted upon application of a liquid assay sample thereto. Yet a further desirable embodiment can be the incorporation of the reagent into one or more layers of a multilayer element of the type described in U.S. Patent 3,992,158.
Under such circumstances, a source of hydrogen peroxide that is dry until contacted with water should be used. Preferred sources are mixtures of an enzyme that demonstrates oxidative activity and substrates for such an enzyme~ for example, glucose and glucose oxidase, uric acid and uricase, and cholesterol and cholesterol oxldase. Any of these and numerous other similar mixtures Or oxidase and substrate when contacted in water and in the presence of oxy~en result in decomposition of the substrate with tne concomitant production of hydrogen peroxide. Such materials are particularly useful because they can be freeze-dried or lyophilized to provide dried powders that can be reconstituted with water. Of course, the enzyme and the substrate must be kept separated until the production of the hydrogen peroxide is desired.
Furthermore, since dichlorofluorescin is quite unstable, its formation by the reaction Or peroxide and diacetyldichlorofluorescin should not be initiated until the substance under assay having per~xidative activity has been added to the assa~y composition. Other useful hydrogen peroxide sources include chemically bound hydrogen peroxldes, such as urea peroxide (Robeco Chemicals, Inc.).
, ' s~
The concentration of the reagents in the assay composition will depend, to a large extent, on the concentration of the material under assay - higher concentrations of reagent being necessary for higher concentrations of material under assay. For concentrations of substance having peroxidative activity between about 10 5 and about 10 lOM, the following composition has been found particularly useful:
diacetyldichlorofluorescin 10 7-10-4M
oxidative enzyme 1-50 units/ml substrate 1-100 mg/dl ~uffer to maintain pH at 7-9.
To be useful, a buffer should not interfere with the assay reaction. ~arbital or phosphate buffers at a concentration of between about 0.01 and 0.1ll have been found useful.
When hydrogen peroxide is used as the peroxide source, a concentration Or between about 10 5 and 5 x 10 4M has been found useful in the above-described assay composition.
In a further embodiment of the present invention, the assaying composition comprising diacetyldichlorofluorescin and a source of hydrogen peroxide can be used to determine the amount of peroxidative activity present in a patient's hemoglobin. Thus, a sample of hemoglobin can be added to the assaying composition and the measurement of the fluorescence rate of the resulting dichlorofluorescin will determine the amount of peroxidative activity in the hemoglobin.
The method for, detecting a substance having peroxidative act~ivity comprises contacting, in an aqueous medium, the sample under assay with a composition comprising dlacetyldichloro-fluorescin and a source of hydrogen peroxide and . . ~ .
.
5~
then detecting the resulting dichlorofluorescein.
Since the resulting dichlorofluorescein is fluorescent, it is easily detected by measuring the fluorescence over a short period of time.
5 The amount of substance having peroxidative activity in the sample is directly proportional to the rate of fluorescence increase per minute of the resulting dichlorofluorescin.
The assay composition can be employed in dry chemistry techniques. It can, for example~ be incorporated by imbibition impregnation or by coating techniques into a reagent zone of a dry test element, e.g., a reagent layer of a dip-and-read fibrous test strip or a reagent layer o~ a non-fibrous multilayer element, as described in U.S. Patents 3,992,158 and 4,042,335.
The element can comprise zones of reagent, which zones may be in the form of layers, and, optionally, spreading agent.
Elements containing these zones are described in U.S. Patents 3,992,158 and 4,069,016.
The substantially dry element can be contacted with a sample for analysis and the resulting dichlorofluorescein can be detected.
25 If the sample itself does not contain water, it could be added with the sample to the dry element.
A particularly preferred test element for the detection Or a substance havlng peroxidative activity, e.g. peroxidase, in a liquid comprises 30 a spreading layer, a reagent layer and a registration layer, all in ~luid contact under conditions of use, and a support~ the,reagent layer intervenlng the spreading layer an~ the re~istration layer, and the registration layer intervening the 35 reagent layer and the support, the reagent layer comprising diacetyldichlorofluorescin and a ' source of hydrogen peroxlde. The registration layer is described in detail in U.S. Patent 4,042,335, columns 11 and 12.
A particularly preferred embodiment of the invention comprises a method for assaying for antigen or antibody present in a sample.
This method comprises labeling a known antigen (or antibody) with a substance having peroxidative activity, e.g. peroxidase, and carrying out the immunoassay in the conventional manner. Thus, the unknown sample can be added along with the labeled sample to a liquid or web comprising the corresponding antibody (or antigen).
The labeled and unlabeled antigen (or antibody) will combine with the corresponding antibody (or antigen) to form an antigen-antibody precipitate and ~ree labeled antigen (or antibody) as well as free unlabeled antigen (or antibody). The determination of the unXnown antibody (or antigen) is made by measuring the free labeled antigen (or antibody) or the labeled antigen-antibody precipitate.
The measurement of labeled antigen or antibody can be accomplished by assaying for the substance having peroxidat~ve activlty by adding the resulting solution to an assaying composition comprising diacetyldichlorfluorescin and a source of hydrogen peroxide and determinlng the amount Or substance having peroxldative activity by measuring the rate of fluorescence of the resulting dichlorofluorescein.
The followin~,examples demons~rate the successful practice of~the present lnvention.
Example 1 -- Fluor scence Assay for Peroxidase To a 1 cm2 cuvette was added 1.4 ml phosphate buffer solution (PBS) containing O.OlM
-. - .
.. , - .
.. ~
, ~4~8S~
sodium phosphate, 0.1 N NaCl, pH 7.5, 0.200 ml diacetyldichlorofluorescin (1 x 10-5M in PBS) and 200 ~1 hydrogen peroxide (1 x 10 3M in water). Reaction was initiated by the addltion 5 o~ peroxidase (horseradish) of the indicated concentration. The rate of fluorescence increase was measured in a Farrand Mk I fluorometer with excitation mo~ochrometer at 490 nm and emission - monochrometer at 520 nm. The rate was linear for 5 minutes. In Figure 1, the rates of fluorescence increase per minute are plotted as a function of peroxidase concentration.
Peroxidase concentrations of up to 1.32 x 10 5M were measurable when the hydrogen 1~ peroxide concentration was increased fifty fold and the dichlorofluorescin concentration increased twofold.
Example 2 -- Multilayered Element ~or the Fluorometric Determination of Peroxidase .
A multilayered element was prepared according to the following:
A polycarbonate support was coated with a reagent layer comprised of deionized gelatin (Type V) (4.5 g/m2), 5,5-dimethyl-1,3-cyclohexanedione (0.4 g/m2), glucose oxidase (538 Units/m2), bis(vinyl sulfonyl methyl)ether (0.05 g/m2), Surfactant lOG, a nonylphenoxy-polyglycidol from Olin Corp., (0.01 g/m2) 5 zinc acetate (0.01 g/m2), dichlorofluorescin diacetate (0.01 g/m2) in 0.02 M phosphate buffer at pH
8.0; an enzyme layer comprising deionized gelatin (2.9 g/m2), colloldal silver (1.02 g/m2~, catalase (86,000 U/m2)~'bis(vinyl sulfonyl methyl)ether (0.04 g/m2), Surfactant lOG (0.007 g/m );
a subbing layer comprised of poly-N-isopropylacryl-amide (0.4 g/m2); and a spreading layer comprised ~' :
s~
of TiO2 (70.0 g/m2), cellulose acetate (10.0 g/m2), glucose (0.5 g/m2), and polyacrylamide particles (L60 mesh) (0.01 g/m2).
The element was tested by applylng thereto a 10 ~1 drop of solutlon containing dinitrophenyl peroxidase con~ugate* (DNP.POD) and measuring the rate at which the fluorescent signal increased, when excited at 460 nm and emitted at 520 nm, as in Example 1. Results are shown in Table I.
Table Conc. DNP'POD
(M) Relative Rate 5.0 x 10- 7.5 2.5 x 10-6 4.8 1.25 x 10-6 1.92 6.25 x 10-7 0.79
3.13 x 10-7 0.27 1.57 x 10-7 0,09 7.85 x 10-8 0.05 3.93 x 10-8 0.028 O O
_ *Prepared by coupling dinitrobenzene sulfonic -- acid to peroxi~ase obtained ~rom horseradish
_ *Prepared by coupling dinitrobenzene sulfonic -- acid to peroxi~ase obtained ~rom horseradish
Claims (41)
1. A composition useful for the determination of a substance having peroxidative activity, said composition consisting essentially of diacetyldichlorofluorescin and a source of hydrogen peroxide in a concentration of from about 10- 6 to 10-2M, the ratio of diacetyldichlorofluorescin to hydrogen peroxide or generated hydrogen peroxide ranging from 1:5 to 1:100.
2. The composition of Claim 1 wherein said source of hydrogen peroxide comprises an enzyme and an enzyme substrate which, when contacted in an aqueous medium in the presence of oxygen, release hydrogen peroxide.
3. The composition of Claim 1 wherein said source of hydrogen peroxide is hydrogen peroxide.
4. The composition of Claim 1 wherein said source of hydrogen peroxide is urea peroxide.
5. The composition of Claim 2 wherein said enzyme is an oxidative enzyme and said substrate is the corresponding substrate for said oxidative enzyme.
6. The composition of Claim 2 wherein said enzyme is glucose oxidase and said substrate is glucose.
7. The composition of Claim 2 wherein said enzyme is uricase and said substrate is uric acid.
8. The composition of Claim 2 wherein said enzyme is cholesterol oxidase and said substrate is cholesterol.
9. The composition of Claim 1 further including a buffer which maintains the pH of the composition at a pH of between about 7 and 9.
10. The composition of Claim 1 wherein the concentration of diacetyldichlorofluorescin is from about 10-7 to 10-3M.
11. A method for assaying peroxidase com-prising the steps of (a) contacting a sample for analysis and a com-position consisting essentially of diacetyl-dichlorofluorescin and a source of hydrogen peroxide in a concentration from about 10- 6 to 10-2M to produce dichlorofluorescein, the ratio of diacetyldichlorofluorescin to hydrogen peroxide or generated hydrogen peroxide ranging from 1:5 to 1:100; and (b) detecting the dichlorofluorescein.
12. The method of Claim 11 wherein the com-position comprises an aqueous medium.
13. The method of Claim 11 wherein the di-chlorofluorescein is detected by fluorescence rate measurements.
14. The method of Claim 11 wherein said source of hydrogen peroxide comprises an enzyme and an enzyme substrate which, when contacted in an aqueous medium in the presence of oxygen, release hydrogen peroxide.
15. The method of Claim 11 wherein said source of hydrogen peroxide is hydrogen peroxide.
16. The method of Claim 14 wherein said en-zyme is an oxidative enzyme and said substrate is the corresponding substrate for said oxidative enzyme.
17. The method of Claim 11 wherein the con-centration of diacetyldichlorofluorescin in said com-position is from about 10- 7 to about 10-3M.
18. The method of Claim 11 wherein said com-position further includes a buffer which maintains the pH of the composition at a pH of between about 7 and 9.
19. A method for assaying peroxidase com-prising the steps of (a) contacting a substantially dry element com-prising a reagent having a composition con-sisting essentially of diacetyldichloro-fluorescin and a dry source of hydrogen per-oxide at a concentration of from about 10-6 to 10-2M with a sample for analysis, the ratio of diacetyldichlorofluorescin to gener-ated hydrogen peroxide ranging from 1:5 to 1:100; and (b) detecting the dichlorofluorescein resulting from said contact.
20. The method of Claim 19 wherein the di-chlorofluorescein is detected by fluorescence rate measurements.
21. The method of Claim 19 wherein said source of hydrogen peroxide comprises an enzyme and an enzyme substrate which, when contacted in an aqueous medium in the presence of oxygen, release hydrogen peroxide.
22. The method of Claim 19 wherein said source of hydrogen peroxide is urea peroxide.
23. A dry test element for the assay of peroxidase in an aqueous liquid, said element having a reagent zone containing an assay composition for peroxidase, said assay composition consisting es-sentially of diacetyldichlorofluorescin and a dry source of hydrogen peroxide at a concentration of from about 10-6 to 10-2M, the ratio of diacetyldi-chlorofluorescin to generated hydrogen peroxide ranging from 1:5 to 1:100.
24. The element of Claim 23 wherein said source of hydrogen peroxide comprises an enzyme and an enzyme substrate which, when contacted in an aqueous medium in the presence of oxygen, release hydrogen peroxide.
25. The element of Claim 23 wherein said source of hydrogen peroxide is urea peroxide.
26. The element of Claim 23 wherein said enzyme is an oxidative enzyme and said substrate is the corresponding substrate for said oxidative enzyme.
27. An element for the detection of peroxidase comprising fibrous material which is im-pregnated with a reagent consisting essentially of diacetyldichlorofluorescin and a source of hydrogen peroxide at a concentration of from about 10-6 to 10-2M, the ratio of diacetyldichlorofluorescin to hydrogen peroxide or generated hydrogen peroxide ranging from 1:5 to 1:100.
28. The element of Claim 27 wherein said fibrous material is paper.
29. The element of Claim 27 wherein said source of hydrogen peroxide is hydrogen peroxide.
30. The element of Claim 27 wherein said source of hydrogen peroxide is urea peroxide.
31. The element of Claim 27 wherein said source of hydrogen peroxide comprises an enzyme and an enzyme substrate which, when contacted in an aqueous medium in the presence of oxygen, release hydrogen peroxide.
32. An element for the detection of peroxidase comprising a layer which comprises a spreading agent and a reagent layer in fluid contact under conditions of use, said reagent layer comprising a reagent consisting essentially of diacetyldichloro-fluorescin and a dry source of hydrogen peroxide at a concentration of from about 10-6 to 10-2M, the ratio of diacetyldichlorofluorescin to generated hydrogen peroxide ranging from 1:5 to 1:100.
33. The element of Claim 32 wherein said source of hydrogen peroxide comprises an enzyme and an enzyme substrate which, when contacted in an aqueous medium in the presence of oxygen, release hydrogen peroxide.
34. The element of Claim 32 wherein said source of hydrogen peroxide is urea peroxide,
35. The element of Claim 33 wherein said enzyme is an oxidative enzyme and said substrate is the corresponding substrate for said oxidative enzyme.
36. An element for the detection of peroxidase in a liquid, the element comprising a layer containing a spreading agent, a reagent layer and a registration layer all in fluid contact under conditions of use and a support, the reagent layer intervening the layer which contains a spreading agent and the registration layer intervening the reagent layer and the support, the reagent layer comprising a reagent consisting essentially of diacetyldichloro-fluorescin and a dry source of hydrogen peroxide at a concentration of from about 10-6 to 10-2M, the ratio of diacetyldichlorofluorescin to generated hydrogen peroxide ranging from 1:5 to 1:100.
37. The element of Claim 36 wherein said source of hydrogen peroxide comprises an enzyme and an enzyme substrate which, when contacted in an aqueous medium in the presence of oxygen, release hydrogen peroxide.
38. The element of Claim 36 wherein said source of hydrogen peroxide is urea peroxide.
39. The element of Claim 37 wherein said enzyme is An oxidative enzyme and said substrate is the corresponding substrate for said oxidative enzyme.
40. The element of Claim 36 wherein said reagent layer further comprises a buffer which main-tains the pH of the reagent at a pH of between about 7 and 9.
41. A method of assaying for antigen or antibody present in a sample to be tested comprising:
(a) labeling a known amount of antigen or anti-body with peroxidase and adding said labeled antigen or antibody with said sample to be tested to a known amount of corresponding antigen or antibody; and (b) determining the amount of unknown antigen or antibody by (i) contacting in an aqueous medium the mixture of diacetyldichlorofluorescin and a source of hydrogen peroxide at a concentration of from about 10-6 to 10-2M to produce dichlorofluorescein, the ratio of diacetyldichlorofluorescin to hydrogen peroxide or generated hydrogen peroxide ranging from 1:5 to 1:100;
(ii) detecting the dichlorofluorescein.
(a) labeling a known amount of antigen or anti-body with peroxidase and adding said labeled antigen or antibody with said sample to be tested to a known amount of corresponding antigen or antibody; and (b) determining the amount of unknown antigen or antibody by (i) contacting in an aqueous medium the mixture of diacetyldichlorofluorescin and a source of hydrogen peroxide at a concentration of from about 10-6 to 10-2M to produce dichlorofluorescein, the ratio of diacetyldichlorofluorescin to hydrogen peroxide or generated hydrogen peroxide ranging from 1:5 to 1:100;
(ii) detecting the dichlorofluorescein.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US06/018,532 US4269938A (en) | 1979-03-08 | 1979-03-08 | Assay of peroxidatively active materials |
| US18,532 | 1979-03-08 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1144859A true CA1144859A (en) | 1983-04-19 |
Family
ID=21788413
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000346350A Expired CA1144859A (en) | 1979-03-08 | 1980-02-25 | Diacetyldichlorofluorescin with a source of hydrogen peroxide for assay of peroxidase |
Country Status (5)
| Country | Link |
|---|---|
| US (1) | US4269938A (en) |
| EP (1) | EP0015767B1 (en) |
| JP (1) | JPS55124499A (en) |
| CA (1) | CA1144859A (en) |
| DE (1) | DE3063025D1 (en) |
Families Citing this family (28)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS57144996A (en) * | 1981-02-27 | 1982-09-07 | Fuji Photo Film Co Ltd | Film for quantitative analysis |
| JPS57150399A (en) * | 1981-03-13 | 1982-09-17 | Fuji Photo Film Co Ltd | Reagent for determining hydrogen peroxide and its determination method |
| US4956300A (en) * | 1982-01-05 | 1990-09-11 | Helena Laboratories Corporation | Aid for determining the presence of occult blood, method of making the aid, and method of using the aid |
| US4857455A (en) * | 1982-11-26 | 1989-08-15 | Syntex (U.S.A) Inc. | Method for the determination of peroxidase using fluorogenic substrates |
| US4495293A (en) * | 1983-02-24 | 1985-01-22 | Abbott Laboratories | Fluorometric assay |
| US4615972A (en) * | 1983-11-04 | 1986-10-07 | Immuno Concepts, Inc. | Stabilization of indicators for detecting enzyme activity |
| US5702913A (en) * | 1983-12-21 | 1997-12-30 | Helena Laboratories Corporation | Chromgen-reagent test system |
| US5273888A (en) * | 1984-01-16 | 1993-12-28 | Helena Laboratories Corporation | Chemical test kit and method for determining the presence of blood in a specimen and for verifying the effectiveness of the chemicals |
| JPS60200167A (en) * | 1984-03-24 | 1985-10-09 | Toyobo Co Ltd | Quantitative analysis of hydrogen peroxide by chemiluminescent method |
| DE3679690D1 (en) * | 1985-03-19 | 1991-07-18 | Abbott Lab | DIAGNOSTIC SAMPLE USING MICROPEROXIDASE. |
| GB8514288D0 (en) * | 1985-06-06 | 1985-07-10 | Amersham Int Plc | Enzyme assay of body fluids |
| JPS63501983A (en) * | 1985-12-12 | 1988-08-04 | デニソン マニユフアクチユアリング カンパニ− | Measurement of test substances |
| US4935346A (en) | 1986-08-13 | 1990-06-19 | Lifescan, Inc. | Minimum procedure system for the determination of analytes |
| US5081040A (en) * | 1987-06-29 | 1992-01-14 | Helena Laboratories Corporation | Composition and kit for testing for occult blood in human and animal excretions, fluids, or tissue matrixes |
| US4906896A (en) * | 1988-10-03 | 1990-03-06 | Science Applications International Corporation | Disk and washer linac and method of manufacture |
| US5217874A (en) * | 1989-04-04 | 1993-06-08 | Helena Laboratories Corporation | Fecal occult blood test product with positive and negative controls |
| US5196167A (en) * | 1989-04-04 | 1993-03-23 | Helena Laboratories Corporation | Fecal occult blood test product with positive and negative controls |
| AU6602890A (en) * | 1989-10-05 | 1991-04-28 | Exoxemis, Inc. | Haloperoxidase acid optimum chemiluminescence assay system |
| US5108899A (en) * | 1989-10-31 | 1992-04-28 | Exoxemis, Inc. | Chemiluminescence assay of in vivo inflammation |
| US5605152A (en) * | 1994-07-18 | 1997-02-25 | Minimed Inc. | Optical glucose sensor |
| US6114177A (en) * | 1997-11-12 | 2000-09-05 | Phytochem Technologies, Inc. | Fluorometric assay for measurement of antioxidant activity |
| US6458326B1 (en) | 1999-11-24 | 2002-10-01 | Home Diagnostics, Inc. | Protective test strip platform |
| US6525330B2 (en) | 2001-02-28 | 2003-02-25 | Home Diagnostics, Inc. | Method of strip insertion detection |
| US6562625B2 (en) | 2001-02-28 | 2003-05-13 | Home Diagnostics, Inc. | Distinguishing test types through spectral analysis |
| US6541266B2 (en) | 2001-02-28 | 2003-04-01 | Home Diagnostics, Inc. | Method for determining concentration of an analyte in a test strip |
| US8426213B2 (en) * | 2007-03-05 | 2013-04-23 | Advanced Liquid Logic Inc | Hydrogen peroxide droplet-based assays |
| US20100081159A1 (en) * | 2008-09-26 | 2010-04-01 | Lebedeva Irina V | Profiling reactive oxygen, nitrogen and halogen species |
| CN106574929B (en) | 2014-07-25 | 2019-08-09 | 贝克顿·迪金森公司 | Analyte testing item test and for its implement test-strips and kit |
Family Cites Families (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| NL154600B (en) * | 1971-02-10 | 1977-09-15 | Organon Nv | METHOD FOR THE DETERMINATION AND DETERMINATION OF SPECIFIC BINDING PROTEINS AND THEIR CORRESPONDING BINDABLE SUBSTANCES. |
| US3654090A (en) * | 1968-09-24 | 1972-04-04 | Organon | Method for the determination of antigens and antibodies |
| NL154598B (en) * | 1970-11-10 | 1977-09-15 | Organon Nv | PROCEDURE FOR DETERMINING AND DETERMINING LOW MOLECULAR COMPOUNDS AND PROTEINS THAT CAN SPECIFICALLY BIND THESE COMPOUNDS AND TEST PACKAGING. |
| US3992158A (en) * | 1973-08-16 | 1976-11-16 | Eastman Kodak Company | Integral analytical element |
| US3964870A (en) * | 1974-03-29 | 1976-06-22 | Boehringer Mannheim G.M.B.H. | Diagnostic composition for the determination of glucose |
| US4042335A (en) * | 1975-07-23 | 1977-08-16 | Eastman Kodak Company | Integral element for analysis of liquids |
| US4089747A (en) * | 1976-08-09 | 1978-05-16 | Eastman Kodak Company | Compositions for the detection of hydrogen peroxide |
| US4069016A (en) * | 1977-01-14 | 1978-01-17 | Eastman Kodak Company | Assay for bilirubin |
| IL51668A (en) * | 1977-03-16 | 1981-12-31 | Israel State | Analytical method for the quantitative determination of immunogens and antibodies and a kit therefor |
| JPS5443791A (en) * | 1977-09-13 | 1979-04-06 | Seishin Seiyaku Kk | Method and reagent for measuring hydrogen peroxide |
-
1979
- 1979-03-08 US US06/018,532 patent/US4269938A/en not_active Expired - Lifetime
-
1980
- 1980-02-25 CA CA000346350A patent/CA1144859A/en not_active Expired
- 1980-03-06 JP JP2744580A patent/JPS55124499A/en active Pending
- 1980-03-07 DE DE8080300714T patent/DE3063025D1/en not_active Expired
- 1980-03-07 EP EP80300714A patent/EP0015767B1/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| US4269938A (en) | 1981-05-26 |
| DE3063025D1 (en) | 1983-06-16 |
| EP0015767A1 (en) | 1980-09-17 |
| JPS55124499A (en) | 1980-09-25 |
| EP0015767B1 (en) | 1983-05-11 |
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