CA1091174A - Stabilized liquid enzyme - Google Patents
Stabilized liquid enzymeInfo
- Publication number
- CA1091174A CA1091174A CA273,920A CA273920A CA1091174A CA 1091174 A CA1091174 A CA 1091174A CA 273920 A CA273920 A CA 273920A CA 1091174 A CA1091174 A CA 1091174A
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- enzyme
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
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- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
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- Biotechnology (AREA)
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- Molecular Biology (AREA)
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- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
Abstract of the Disclosure Labile enzymes are stabilized by treatment with concentrated organic solvent such as 30% aqueous propane diol in the presence of a small amount of polymer such as 0.1%
gelatin and then dilution to 1% solvent with water while main-taining the polymer concentration at at least 0.01%. The diluted composition can be stored for extended periods without loss of significant enzyme catalytic activity. Stability is further enhanced by including from 1 to 18% of salts and 0.1%
bacteriastatic agents with diluted composition. Thus the liquid enzyme can be packaged with substrate and buffered salts.
gelatin and then dilution to 1% solvent with water while main-taining the polymer concentration at at least 0.01%. The diluted composition can be stored for extended periods without loss of significant enzyme catalytic activity. Stability is further enhanced by including from 1 to 18% of salts and 0.1%
bacteriastatic agents with diluted composition. Thus the liquid enzyme can be packaged with substrate and buffered salts.
Description
, lO~iit74 .
Background of the Invention 1. Field of the Invention This invention relates to the stabilization of labile enzymes in liquid media.
Background of the Invention 1. Field of the Invention This invention relates to the stabilization of labile enzymes in liquid media.
2. ~escription of the Prior Art It has recently been estimated that 25% of all in vitro diagn~stic tests conducted annually in this Country are not reliable. Unreliable tests can result in unnecessary medical treàtment, the withho7ding of necessary treatment and lost income.
Because of their high specificity, the use of enzyme determinations has significantly increased during the last few years and indi-cations are that this trend will continue. However, rigorous quality control measures are required to assure the accuracy and consistency of results. This requirement stems from the fact that the exact nature of enzymes, as well as the mechanisms of their action, remain unknown for the most part. At present, the grcatest limitation on the enzyme reagent manufacturer, by far, lies in the unstable characteristics of his products. Current methodologies require the use of numerous labile ingredients, and . .
-` . 1091174 these ingredients are more likely to increase, rather than decrease, in number.
The present commercial state of the art used for ` stabilizing the reactive a~ilitv of enzymes is by locking S them into a solid matrix eithex by freeze drying, dry blending such as used for tableting dried powders, primarily in the pharmaceutical diagnostic and related industries and immobilization by locking the chemical structure of the enzyme into a solid matrix. Contrary to the sophistication these terms imply, these approaches are neither practical nor desirable and are also expensive. The manufacturer is forced to remove the water and supply a partial product, thus relinquishing part of the quality control cycle in the dilution and use of the final product~ ~aboratories are forced to pay the high cost of packaging, reagent waste, freeze drying and dry blending, and usefulness of the product is further - limited by packaging modes and sizes.
Furthermore, good product uniformity is difficult to achieve. This condition is exemplified by the fact that most commercial freeze dried controlled sera reference serum list the acceptable bottle to bottle variation of enzyme constituents at + 10% of the mean.
Summary of the Invention Labile enzymes are chemically modified according to the invention resulting in long term stability without affecting enzymatic reactivity in accordance with the invention.
The invention provides reagents where quality control is assured throughout manufacturing, packaging, storage and use. The inconvenience of rigid package size is eliminated as is the high cost of packaging, freeze drying and reagent waste. Liquid -- 109il~74 enzyme systems provide application flexibility and separation o~ the ingredients is easily accomplished with negligible manufacturing cost providing the flexibility of triggering the desired reaction after all side reactions have been dissipated.
The stabilized enzymes of the invention have been assessed in studies which compared liquid enzyme reagents with fresh reagents. The studies show a 1:1 correlation between liquid and fresh reagents with comparable sensitivity and precision. Providing enzyme reagents in liquid form enhances the colorimetric applicability of present day ~AD/NADH coupled methodologies primarily because the separation of ingredients is easily accomplished. Liquid reagents are especially advantageous where NADH consumption is the basis of measure-ment and the color reagent must be separated from NADH and the reaction main. In the ultraviolet mode, the liquid enzyme system offers better reagent homogeneity and packaging, as well as flexibility in usage, in contrast to the freeze dried or dry media preparations.
In diagnostic enzymology, the stabilization of enzyme reagents in a ready-to-use liquid media is a new and exciting approach to satisfy the needs of the clinical laboratory and the reliability demands of the regulatory authorities. The flexibility of liquid enzyme systems insures their applicability to automated instrumentation, as well as their convenience in manual testings.
Stabilization of labile enzymes is accomplished in accordance with the invention by dissolving lyophilized, dry ~nzymes in an aqueous enzyme base including at least 0.05% of polymer and at least 20~ v/v of organic solvent. The solution is maintained at a temperature below the denaturing point 109~174 suitably below 60C and in most eases below 40C for a`t least 30 minutes, usually 2 to 3 days. The solution is then diluted with water typically at least a 20 times and usually a 30 times dilution while adding further polymer to maintain a level in diluted stage of at least .05 weight ~. The diluted solution suitably at an enzyme eoneentration from 100 to 10,000 I.U. per liter may then be paekaged in separate containers and sealed and is stored refrigerated at temper-atures of 30C or less.
- 10 The diluted solution may also contain substrate buffer and bacteriastatic agent and other eomponents if neeessary. If these other ingredients are added the diluted solution is mixed to obtain a single homogeneous substrate solution before dispensing into individual containers, sealing and storage.
Substrates are organie ehemieals of known strueture whose reaetions or interaetions are catalyzed by enzymes re-sulting in a ehange in the eompound structure, atomic eomposition, or stereo chemieal rotation, fo~ example, laetie acid, L-aspartate or, alphaketoglutarate, L-alanine or the like.
In general, substrates are prone to mierobiological degradation as they serve as food for baeteria, fungi and other microorganisms. Otherwise, these compounds remain stable in aqueous media at or near neutral pH typically from 4-10. Thus if the substrate is added to the enzyme composition the stabilizing media should also contain a buffer to control reaction pll such as an al~ali metal acid phosphate and a bactericidal and/or fungicidal agents which do not chemically react with the substrate or inhibit the enzymatie reaction of
Because of their high specificity, the use of enzyme determinations has significantly increased during the last few years and indi-cations are that this trend will continue. However, rigorous quality control measures are required to assure the accuracy and consistency of results. This requirement stems from the fact that the exact nature of enzymes, as well as the mechanisms of their action, remain unknown for the most part. At present, the grcatest limitation on the enzyme reagent manufacturer, by far, lies in the unstable characteristics of his products. Current methodologies require the use of numerous labile ingredients, and . .
-` . 1091174 these ingredients are more likely to increase, rather than decrease, in number.
The present commercial state of the art used for ` stabilizing the reactive a~ilitv of enzymes is by locking S them into a solid matrix eithex by freeze drying, dry blending such as used for tableting dried powders, primarily in the pharmaceutical diagnostic and related industries and immobilization by locking the chemical structure of the enzyme into a solid matrix. Contrary to the sophistication these terms imply, these approaches are neither practical nor desirable and are also expensive. The manufacturer is forced to remove the water and supply a partial product, thus relinquishing part of the quality control cycle in the dilution and use of the final product~ ~aboratories are forced to pay the high cost of packaging, reagent waste, freeze drying and dry blending, and usefulness of the product is further - limited by packaging modes and sizes.
Furthermore, good product uniformity is difficult to achieve. This condition is exemplified by the fact that most commercial freeze dried controlled sera reference serum list the acceptable bottle to bottle variation of enzyme constituents at + 10% of the mean.
Summary of the Invention Labile enzymes are chemically modified according to the invention resulting in long term stability without affecting enzymatic reactivity in accordance with the invention.
The invention provides reagents where quality control is assured throughout manufacturing, packaging, storage and use. The inconvenience of rigid package size is eliminated as is the high cost of packaging, freeze drying and reagent waste. Liquid -- 109il~74 enzyme systems provide application flexibility and separation o~ the ingredients is easily accomplished with negligible manufacturing cost providing the flexibility of triggering the desired reaction after all side reactions have been dissipated.
The stabilized enzymes of the invention have been assessed in studies which compared liquid enzyme reagents with fresh reagents. The studies show a 1:1 correlation between liquid and fresh reagents with comparable sensitivity and precision. Providing enzyme reagents in liquid form enhances the colorimetric applicability of present day ~AD/NADH coupled methodologies primarily because the separation of ingredients is easily accomplished. Liquid reagents are especially advantageous where NADH consumption is the basis of measure-ment and the color reagent must be separated from NADH and the reaction main. In the ultraviolet mode, the liquid enzyme system offers better reagent homogeneity and packaging, as well as flexibility in usage, in contrast to the freeze dried or dry media preparations.
In diagnostic enzymology, the stabilization of enzyme reagents in a ready-to-use liquid media is a new and exciting approach to satisfy the needs of the clinical laboratory and the reliability demands of the regulatory authorities. The flexibility of liquid enzyme systems insures their applicability to automated instrumentation, as well as their convenience in manual testings.
Stabilization of labile enzymes is accomplished in accordance with the invention by dissolving lyophilized, dry ~nzymes in an aqueous enzyme base including at least 0.05% of polymer and at least 20~ v/v of organic solvent. The solution is maintained at a temperature below the denaturing point 109~174 suitably below 60C and in most eases below 40C for a`t least 30 minutes, usually 2 to 3 days. The solution is then diluted with water typically at least a 20 times and usually a 30 times dilution while adding further polymer to maintain a level in diluted stage of at least .05 weight ~. The diluted solution suitably at an enzyme eoneentration from 100 to 10,000 I.U. per liter may then be paekaged in separate containers and sealed and is stored refrigerated at temper-atures of 30C or less.
- 10 The diluted solution may also contain substrate buffer and bacteriastatic agent and other eomponents if neeessary. If these other ingredients are added the diluted solution is mixed to obtain a single homogeneous substrate solution before dispensing into individual containers, sealing and storage.
Substrates are organie ehemieals of known strueture whose reaetions or interaetions are catalyzed by enzymes re-sulting in a ehange in the eompound structure, atomic eomposition, or stereo chemieal rotation, fo~ example, laetie acid, L-aspartate or, alphaketoglutarate, L-alanine or the like.
In general, substrates are prone to mierobiological degradation as they serve as food for baeteria, fungi and other microorganisms. Otherwise, these compounds remain stable in aqueous media at or near neutral pH typically from 4-10. Thus if the substrate is added to the enzyme composition the stabilizing media should also contain a buffer to control reaction pll such as an al~ali metal acid phosphate and a bactericidal and/or fungicidal agents which do not chemically react with the substrate or inhibit the enzymatie reaction of
3~ the substrate. Typieal examples are 0.1~ sodium azide, benzoie lO9i~74 acid, phenol, thymol, or pentachlorophenol.
It is believed that the selected organic solvent stabilizes the enzyme in liquid media by protecting the functional group site, that is the part of the molecule where S the substrate reaction actua-ly occurs or is catalyzed~and by protecting the enzyme from microbial contamination and thus degradation. There is obviously some physical or chemical reaction occurring in the concentrated solvent media since the enzyme has no catalytic activity for the substrate at this solvent concentration. However on dilution the enzyme is restored to full activity and maintains its full reactivity at high levels over extended storage periods of from a few months to several years. The internal chemical structure of the enzyme molecule need not be preserved. As long as the reactive site is preserved, the catalytic activity of the enzyme remains intact.
Microbial degradation can also be controlled by use of high sale concentrations such as at least 1~ typically 2 to ~ -welght ~ or higher concentration of salts. The salt molecules may also protect the active sites by forming electrostatic bonds protecting the spacial configuration of the enzyme and the active sites.
These and many other objects and attendant advantages of the invention will become apparent as the invention becomes better understood by reference to the follow-ing detailed description.
Description of the Preferred Embodiments Enzymes are large molecular weight, complex protein molecules, usually of unknown chemical structure. They are presently classified by their catalytic activity and extreme 6.
~osll74 substrate specificity. Enzymes may be redefined as biological catalysts, capable of catalyzing a reaction of a single substrate, or a reaction of a similar group of substrates.
.. . ~
Typical enzymes are LDH, MDH, CPK, and the like. The enzyme is present in the diluted, stabilized composition in an amount typically from 100 I.U. to 10,000 I.U.
Substrates are organic chemicals of known structure, I whose reactions or interactions are catalyzed by enzymes resulting in a change in the compound's structure, atomic composition, or stereo-chemical rotation.
In general substrates are prone to microbiological degradation as they serve as food for bacteria, fungi, and other microorganisms. Otherwise, these compounds remain stable in aqueous media at or near neutral p~ (i.e~ pH range of 4-10).
Typical substrates are L-alanine, pyruvate, L-aspartate, alpha-ketoglutarate~ and the like. The substrates are usually in salt form and form part of the salt concentration useful in enhancing stability of the enzyme. The enzymatic stability increases with substrate concentration. However at high substrate concentrations over about 8% enzymatic activity is inhibited. Therefore the substrate concentration should be optimized, generally at about 2 to 4%.
The buffer salt also provides part of the salt con-centration discussed above. The buffer salt is added in an amount necessary to maintain pH between 4-10, typically from 6-8. Generally the buffer is a combination of .1-1% of an alkali metal hydroxide and 0.5 to 3% of an alkali metal acid carbonate or phosphate. The total salt concent also effects thc amount of polymer required. At higher salt content, e.q.
above 4~ by weight, less polymer is required due to the electro-109~17~
static stabilization provided by the salt. However, at highersalt content, the polymer may cloud the solution or precipitate requiring warming the solution to redissolve.
The polymer is preferably provided in the diluted stabilized solution up to an amount that remains in homogenous suspension under refrigeration without precipitation. The polymer is present in an amount from 0.01 to 0.5% preferably from 1 0.05 to 0.25%. Water soluble polymers useful as stabilizing agents in this invention are those that do not inhibit enzymatic activity, and are capable of entrapping the enzyme in the polymer matrix. The polymer may be a synthetic organic material such as polyvinylpyrrolidine or dextran of biologic origin such as gelatin which is denatured collagen.
The solvent must be miscible with water, of neutral or alkaline pH, liquid at room and refrigerator temperatures, and non-degradatively reactive with reactive sites of the enzyme other than formation of electrostatic bondsO Useful solvents are generally polar organic solvents such as ethers, ketones, sulfones, sulfoxides and alcohols such as methanol, ethanol, propanol, butanol, acetone, dioxane, DMS0, dimethylsulfone and THF. However, higher activity at lower solvent concen-tration for the treatment step is found for liquid polyol solvents containing from 2-40H group and containing from 2-10 carbon atmos such as glyceroI~ propanediol, butane diol, ethylene glycol and the like.
The solvent must be present in an amount of at least 20% during the treatment step typically from 25 to 50%. Some solvents require concentrations as high as 70% in order to maintain stabilized activity above 60% enzymatic reactivity.
Specific examples of practice follow:
109117~
EX~5PLE 1 Enzyme Base Material Amount Gelatin ~ 0.1% W/W
1,2 propane diol 30% V/V
Water 70~ V~V
Ammonium sulfate suspension (2.2M) or dry lyophilized LDH enzyme in an amount equivalent to 22,500 IU/l was dissolved in the enzyme base and held at 4-30C for 2-3 10 days.
Substrate Reagent Material Amount L-Alanine 22 g/l Alpha-ketoglutaric acid 1.6 NaO~ 5 Na~2 Gelatin The enzyme base was diluted thirty-fold by addition to the substrate reagent suspension and mixed to obtain a homogenous suspension. The suspension is stored refrigerated.
Projected shelf life under refrigeration is three years with 50-90% activity remaining.
In the clinical diagnostic field the commercial application of these stabilizing methods is represented by, but not limited to, the diagnostic reagents used to determine and quantitate the following constituents in biological fluids:
1. Glutamic-oxalacetic transaminase (SGOT):
2. Glutamic-pyruvic transaminase (SGPT) 3. Lactic dehydrogenase (LD~
-, 109117~
It is believed that the selected organic solvent stabilizes the enzyme in liquid media by protecting the functional group site, that is the part of the molecule where S the substrate reaction actua-ly occurs or is catalyzed~and by protecting the enzyme from microbial contamination and thus degradation. There is obviously some physical or chemical reaction occurring in the concentrated solvent media since the enzyme has no catalytic activity for the substrate at this solvent concentration. However on dilution the enzyme is restored to full activity and maintains its full reactivity at high levels over extended storage periods of from a few months to several years. The internal chemical structure of the enzyme molecule need not be preserved. As long as the reactive site is preserved, the catalytic activity of the enzyme remains intact.
Microbial degradation can also be controlled by use of high sale concentrations such as at least 1~ typically 2 to ~ -welght ~ or higher concentration of salts. The salt molecules may also protect the active sites by forming electrostatic bonds protecting the spacial configuration of the enzyme and the active sites.
These and many other objects and attendant advantages of the invention will become apparent as the invention becomes better understood by reference to the follow-ing detailed description.
Description of the Preferred Embodiments Enzymes are large molecular weight, complex protein molecules, usually of unknown chemical structure. They are presently classified by their catalytic activity and extreme 6.
~osll74 substrate specificity. Enzymes may be redefined as biological catalysts, capable of catalyzing a reaction of a single substrate, or a reaction of a similar group of substrates.
.. . ~
Typical enzymes are LDH, MDH, CPK, and the like. The enzyme is present in the diluted, stabilized composition in an amount typically from 100 I.U. to 10,000 I.U.
Substrates are organic chemicals of known structure, I whose reactions or interactions are catalyzed by enzymes resulting in a change in the compound's structure, atomic composition, or stereo-chemical rotation.
In general substrates are prone to microbiological degradation as they serve as food for bacteria, fungi, and other microorganisms. Otherwise, these compounds remain stable in aqueous media at or near neutral p~ (i.e~ pH range of 4-10).
Typical substrates are L-alanine, pyruvate, L-aspartate, alpha-ketoglutarate~ and the like. The substrates are usually in salt form and form part of the salt concentration useful in enhancing stability of the enzyme. The enzymatic stability increases with substrate concentration. However at high substrate concentrations over about 8% enzymatic activity is inhibited. Therefore the substrate concentration should be optimized, generally at about 2 to 4%.
The buffer salt also provides part of the salt con-centration discussed above. The buffer salt is added in an amount necessary to maintain pH between 4-10, typically from 6-8. Generally the buffer is a combination of .1-1% of an alkali metal hydroxide and 0.5 to 3% of an alkali metal acid carbonate or phosphate. The total salt concent also effects thc amount of polymer required. At higher salt content, e.q.
above 4~ by weight, less polymer is required due to the electro-109~17~
static stabilization provided by the salt. However, at highersalt content, the polymer may cloud the solution or precipitate requiring warming the solution to redissolve.
The polymer is preferably provided in the diluted stabilized solution up to an amount that remains in homogenous suspension under refrigeration without precipitation. The polymer is present in an amount from 0.01 to 0.5% preferably from 1 0.05 to 0.25%. Water soluble polymers useful as stabilizing agents in this invention are those that do not inhibit enzymatic activity, and are capable of entrapping the enzyme in the polymer matrix. The polymer may be a synthetic organic material such as polyvinylpyrrolidine or dextran of biologic origin such as gelatin which is denatured collagen.
The solvent must be miscible with water, of neutral or alkaline pH, liquid at room and refrigerator temperatures, and non-degradatively reactive with reactive sites of the enzyme other than formation of electrostatic bondsO Useful solvents are generally polar organic solvents such as ethers, ketones, sulfones, sulfoxides and alcohols such as methanol, ethanol, propanol, butanol, acetone, dioxane, DMS0, dimethylsulfone and THF. However, higher activity at lower solvent concen-tration for the treatment step is found for liquid polyol solvents containing from 2-40H group and containing from 2-10 carbon atmos such as glyceroI~ propanediol, butane diol, ethylene glycol and the like.
The solvent must be present in an amount of at least 20% during the treatment step typically from 25 to 50%. Some solvents require concentrations as high as 70% in order to maintain stabilized activity above 60% enzymatic reactivity.
Specific examples of practice follow:
109117~
EX~5PLE 1 Enzyme Base Material Amount Gelatin ~ 0.1% W/W
1,2 propane diol 30% V/V
Water 70~ V~V
Ammonium sulfate suspension (2.2M) or dry lyophilized LDH enzyme in an amount equivalent to 22,500 IU/l was dissolved in the enzyme base and held at 4-30C for 2-3 10 days.
Substrate Reagent Material Amount L-Alanine 22 g/l Alpha-ketoglutaric acid 1.6 NaO~ 5 Na~2 Gelatin The enzyme base was diluted thirty-fold by addition to the substrate reagent suspension and mixed to obtain a homogenous suspension. The suspension is stored refrigerated.
Projected shelf life under refrigeration is three years with 50-90% activity remaining.
In the clinical diagnostic field the commercial application of these stabilizing methods is represented by, but not limited to, the diagnostic reagents used to determine and quantitate the following constituents in biological fluids:
1. Glutamic-oxalacetic transaminase (SGOT):
2. Glutamic-pyruvic transaminase (SGPT) 3. Lactic dehydrogenase (LD~
-, 109117~
4. Creatine phosphokinase (CPK):
5. ~-Hydroxybuterid dehydrogenase (~-HBD)
6. Glucose (via Hexokinase-G-6-PDH).
These reagents react similarily, contain some common labile .;, .
ingredients, and some of the chemical reactions involved are common. The following chemical reaction scheme is presented as a model to illustrate the general nature of the reactions involved:
; REACTION SCH~ME 1. --GENERAL MODEL
. 10 Enzyme 1 (1.) SUBSTRATE(S) PRODUCT(S) pH
-~ ~ - Enzyme 2 ¦ (2.) PRODUCT/SUBSTRATE + NAD-NADH -- NADH -NAD+PRODUCT
_ 2 - 2 pH
Catalyst 15 (3.) NADH2 + CHROMOGEN - ~ CHROMOGEN + NAD
(oxidized) - ~ (reduced) All enzymatic reactions listed above will follow this general scheme, where reaction (2.) is usually referred to as the coupling reaction, reactions (2.) or (3.) are the measuring reactions, and reaction (1.) may be characterized as the primary reaction. It is understood however, that not all three reactions are required for measurement in fact, they may ~e limited to two, or one. In the case of the ultraviolet measurement of lactic dehydrogenase (LDH) activity, only reaction (2 ) is involved, 25 as follows:
RE~CTIO~ SCHEME 2. -- LDH
_ LDH
Pyruvate + NADH2 -- ` NAD + Lactate Conversely, more than the three reactions listed may be involved as in the case of Creatine phosphokinase (CPK):
,10 .
)91~`74 REACTION SC~E~E 3. -- CPX
CPK ATP + Creatine HK
S (2,) ATP + Glucose - Glucose-6-Phos. + ADP
; G-6-PDH
(3.) Glucose-6-Phos. + NAD ~ -~ NADH
PMS
~4.) NADH2 + INT ~ INT + NAD
(ox) ~ ~red) SYMBOLS: .
CP 3 Creatine phosphate ADP = Adenosine-5'-diphosphate ATP = Adenosine triphosphate lS HK = ~exokinase NAD = nicotinamide-adenine dinucleotide NAD~2 = nicotinamide-adenine dinucleotide, reduced G-6-PDH = Glucose-6-phosphate dehydrogenase INT = tetrazolium salt PMS = phenazine methosulfate.
In this case, reactions (2.) and (3,) may be considered the coupling reactions, reactions (3.) or (4.) the measuring reactions, and reaction (1.) the primary reaction, Referring to REACTION SCHE~IE 1, --GE~ERAL MODEL, it becomes obvious and is general ~nowledge that the use of the reaction sequence permits the analytical quantitation of either the reacting substrates/products or the catalyzing enzymes.
The quantitation oif these constituents in biological fluids is a well accepted and widely ~sed diagnostic tool in diagnosis and treatment of human and animal disease states.
11 .
~091:1 7~
. ~ . .
It is to be realized that only preferred embodiments of the invention have ~een described and that numerous substitutions, modifications and alterations are permissible without departing from the spirit and scope of S the invention as defined in the following claims.
`
~ 12.
''\
These reagents react similarily, contain some common labile .;, .
ingredients, and some of the chemical reactions involved are common. The following chemical reaction scheme is presented as a model to illustrate the general nature of the reactions involved:
; REACTION SCH~ME 1. --GENERAL MODEL
. 10 Enzyme 1 (1.) SUBSTRATE(S) PRODUCT(S) pH
-~ ~ - Enzyme 2 ¦ (2.) PRODUCT/SUBSTRATE + NAD-NADH -- NADH -NAD+PRODUCT
_ 2 - 2 pH
Catalyst 15 (3.) NADH2 + CHROMOGEN - ~ CHROMOGEN + NAD
(oxidized) - ~ (reduced) All enzymatic reactions listed above will follow this general scheme, where reaction (2.) is usually referred to as the coupling reaction, reactions (2.) or (3.) are the measuring reactions, and reaction (1.) may be characterized as the primary reaction. It is understood however, that not all three reactions are required for measurement in fact, they may ~e limited to two, or one. In the case of the ultraviolet measurement of lactic dehydrogenase (LDH) activity, only reaction (2 ) is involved, 25 as follows:
RE~CTIO~ SCHEME 2. -- LDH
_ LDH
Pyruvate + NADH2 -- ` NAD + Lactate Conversely, more than the three reactions listed may be involved as in the case of Creatine phosphokinase (CPK):
,10 .
)91~`74 REACTION SC~E~E 3. -- CPX
CPK ATP + Creatine HK
S (2,) ATP + Glucose - Glucose-6-Phos. + ADP
; G-6-PDH
(3.) Glucose-6-Phos. + NAD ~ -~ NADH
PMS
~4.) NADH2 + INT ~ INT + NAD
(ox) ~ ~red) SYMBOLS: .
CP 3 Creatine phosphate ADP = Adenosine-5'-diphosphate ATP = Adenosine triphosphate lS HK = ~exokinase NAD = nicotinamide-adenine dinucleotide NAD~2 = nicotinamide-adenine dinucleotide, reduced G-6-PDH = Glucose-6-phosphate dehydrogenase INT = tetrazolium salt PMS = phenazine methosulfate.
In this case, reactions (2.) and (3,) may be considered the coupling reactions, reactions (3.) or (4.) the measuring reactions, and reaction (1.) the primary reaction, Referring to REACTION SCHE~IE 1, --GE~ERAL MODEL, it becomes obvious and is general ~nowledge that the use of the reaction sequence permits the analytical quantitation of either the reacting substrates/products or the catalyzing enzymes.
The quantitation oif these constituents in biological fluids is a well accepted and widely ~sed diagnostic tool in diagnosis and treatment of human and animal disease states.
11 .
~091:1 7~
. ~ . .
It is to be realized that only preferred embodiments of the invention have ~een described and that numerous substitutions, modifications and alterations are permissible without departing from the spirit and scope of S the invention as defined in the following claims.
`
~ 12.
''\
Claims (28)
1. A stabilized liquid enzyme composition used in biological diagnostic determinations and which contains an enzyme, and in which other labile components may be present, said composition comprising an aqueous vehicle containing:
at least 100 I.U. of an enzyme primarily effective in affecting reactivity of one or more specified biological consti-tuents to render a determination of such constituent or consti-tuents in a biological diagnostic determination and which enzyme is normally unstable in an aqueous media;
no more than 5% non-reactive aqueous miscible organic sol-vent which is liquid at least at room temperature and which sol-vent does not materially affect any reaction between the enzyme or the biological constituent or constituents when present in such amount; and at least 0.01% of a water soluble polymer which does not materially affect anv reaction between the enzyme and said one or more biological constituents, and where said composition does not require substantial dilution for use in biological diagnostic determinations.
at least 100 I.U. of an enzyme primarily effective in affecting reactivity of one or more specified biological consti-tuents to render a determination of such constituent or consti-tuents in a biological diagnostic determination and which enzyme is normally unstable in an aqueous media;
no more than 5% non-reactive aqueous miscible organic sol-vent which is liquid at least at room temperature and which sol-vent does not materially affect any reaction between the enzyme or the biological constituent or constituents when present in such amount; and at least 0.01% of a water soluble polymer which does not materially affect anv reaction between the enzyme and said one or more biological constituents, and where said composition does not require substantial dilution for use in biological diagnostic determinations.
2. The composition according to Claim 1 in which the compo-sition further includes at least 0.01% of a bactericical agent to further inhibit deterioration of said enzyme by providing bacteri-ostatic action and by further cooperating in stabilizing the en-zyme degradation.
3. A composition according to Claim 1 in which the solvent is an organic solvent selected from ketones, ethers, sulfones, sulfoxides and alcohols.
4. A composition according to Claim 3 in which the solvent is 1,2-propanediol. 13
5. A composition according to Claim 3 in which the solvent is present in an amount from 0.05 to 5%.
6. A composition according to Claim 1 in which the enzyme has been pretreated with an aqueous media containing at least 20%
of said solvent.
of said solvent.
7. A composition according to Claim 1 in which the polymer is present in an amount from 0.05 to 0.5%.
8. A composition according to Claim 7 in which the polymer is gelatin.
9. A composition according to Claim 7 in which the pH is from 4-10.
10. A composition according to Claim 8 in which the compo-sition further includes from 1% to 8% of salt and from 0.01 to 0.3% of a bacteriacidal agent.
11. A composition according to Claim 10 in which the salt comprises a substrate selected from lactic acid, L-aspartate, alphaketoglutarate, L-alanine or pyruvate and is present in an amount from 2 to 4%.
12. A composition according to Claim 1 in which the compo-sition is used in human medical and body function determinations.
13. The composition according to Claim 12 in that said enzyme is selected from the class consisting of malate dehydrogenase and lactate dehydrogenase.
14. The composition according to Claim 13 in that the bio-logical diagnostic determination is made with biological con-stituents selected from the class consisting of glutamic-oxalacetic transaminase (SGOT) and glutamic-pyruvic transaminase (SGPT).
15. A stabilized liquid enzyme composition used in biological diagnostic determinations of glutamic-oxalacetic transaminase (SGOT) and glutamic-pyruvic transaminase (SGPT), and which compo-sition contains an enzyme effective in determining SGOT and SGPT, and in which other labile components may be present, said compo-sition comprising an aqueous vehicle containing:
at least 100 I.U. of an enzyme selected from the class con-sisting of malate dehydrogenase (MDH) and lactate dehydrogenase (LDH), said enzyme being primarily effective in affecting reac-tivity of the biological constituents SGOT and SGPT to render a determination of such constituents in a biological diagnostic determination and which enzyme is normally unstable in an aqueous media;
no more than 5% non-reactive aqueous miscible organic sol-vent which is liquid at least at room temperature and which sol-vent does not materially affect any reaction between the enzyme and the SGOT or SGPT when present in such amount;
at least 0.01% of a water soluble polymer which does not materially affect any reaction between the enzyme and the SGOT or SGPT; and at least 0.01% of a bactericidal agent to further inhibit deterioration of said enzyme by providing bacteriostatic action and by further cooperating in stabilizing the enzyme against degradation.
at least 100 I.U. of an enzyme selected from the class con-sisting of malate dehydrogenase (MDH) and lactate dehydrogenase (LDH), said enzyme being primarily effective in affecting reac-tivity of the biological constituents SGOT and SGPT to render a determination of such constituents in a biological diagnostic determination and which enzyme is normally unstable in an aqueous media;
no more than 5% non-reactive aqueous miscible organic sol-vent which is liquid at least at room temperature and which sol-vent does not materially affect any reaction between the enzyme and the SGOT or SGPT when present in such amount;
at least 0.01% of a water soluble polymer which does not materially affect any reaction between the enzyme and the SGOT or SGPT; and at least 0.01% of a bactericidal agent to further inhibit deterioration of said enzyme by providing bacteriostatic action and by further cooperating in stabilizing the enzyme against degradation.
16. A composition according to Claim 15 in that said compo-sition does not require substantial dilution for use in said bio-logical diagnostic determinations.
17. A method of stabilizing a labile enzyme used in biological diagnostic determinations and which is unstable in an aqueous media, and which enzyme is primarily effective in affecting the reactivity of one or more biological constituents to render a determination of such constituent or constituents, said method comprising the steps of:
forming a solution of the enzyme molecule in an aqueous media containing at least 20% of non-reactive water-miscible, organic solvent which is liquid at room temperature, and at least 0.05% by weight of water soluble polymer, and a bactericidal agent which inhibits deterioration of said enzyme;
maintaining the enzyme in said solution for a time sufficient to stabilize reactive sites thereof;
diluting the solution with water to an enzyme content of at least 100 I.U., a solvent content of no more than 5% and a water-soluble polymer content of at least 0.01% and which solvent content does not materially affect any reaction between the en-zyme or the biological constituent or constituents when present in such amount, said composition not requiring further substantial dilution for use in biological diagnostic determinations.
forming a solution of the enzyme molecule in an aqueous media containing at least 20% of non-reactive water-miscible, organic solvent which is liquid at room temperature, and at least 0.05% by weight of water soluble polymer, and a bactericidal agent which inhibits deterioration of said enzyme;
maintaining the enzyme in said solution for a time sufficient to stabilize reactive sites thereof;
diluting the solution with water to an enzyme content of at least 100 I.U., a solvent content of no more than 5% and a water-soluble polymer content of at least 0.01% and which solvent content does not materially affect any reaction between the en-zyme or the biological constituent or constituents when present in such amount, said composition not requiring further substantial dilution for use in biological diagnostic determinations.
18. The method of Claim 17 in that a bactericidal agent which inhibits deterioration of the enzyme is added to the solution before dilution thereof.
19. A method according to Claim 17 in which the solvent is an organic solvent selected from ketones, ethers, sulfones, sulfoxides and alcohols.
20. A method according to Claim 18 in which the solvent is 1,2-propanediol present in the treatment step in an amount from 20 to 40%.
21. A method according to Claim 17 in which the polymer is gelatin present in said solution in an amount from 0.05 to 0.5%.
22. A method according to Claim 21 in which the diluted solution contains 1 to 8% of salts including substrate and buffer and 0.01 to 0.3% of a bacteriacidal agent.
23. A method according to Claim 17 in which the solution is used in human medical and body function determinations.
24. The method of Claim 23 in that said enzyme is selected from the class consisting of malate dehydrogenase and lactate dehydrogenase.
25. The method of Claim 24 in that the biological diagnostic determination is made with biological constituents selected from the class consisting of glutamic-oxalacetic transaminase (SGOT) and glutamic-pyruvic transaminase (SGPT).
26. A method of stabilizing a labile enzyme used in biological diagnostic determinations of glutamic oxalacetic transaminase (SGOT) and glutamic-pyruvic transaminase (SGPT), and which enzyme is unstable in an aqueous media, and which enzyme is primarily ef-fective in affecting the reactivity of SGOT or SGPT to render a determination of the SGOT or SGPT, said method comprising the steps of:
forming a solution of an enzyme molecule in an aqueous media containing at least 20% of non-reactive water-miscible, organic solvent which is liquid at room temperature, at least 0.05% by weight of water-soluble polymer, and a bactericidal agent which inhibits deterioration of said enzyme, said enzyme being selected from the class consisting of malate dehydrogenase (MDG) and lactate dehydrogenase (LDH)?
maintaining the enzyme in said solution for a time sufficient to stabilize reactive sites thereof; and diluting the solution with water to an enzyme content of at least 100 I.U., a solvent content of no more than 5% and a water-soluble polymer content of at least 0.01% and which solvent content does not materially affect any reaction between the enzyme and SGOT or SGPT when present in such limited amount.
forming a solution of an enzyme molecule in an aqueous media containing at least 20% of non-reactive water-miscible, organic solvent which is liquid at room temperature, at least 0.05% by weight of water-soluble polymer, and a bactericidal agent which inhibits deterioration of said enzyme, said enzyme being selected from the class consisting of malate dehydrogenase (MDG) and lactate dehydrogenase (LDH)?
maintaining the enzyme in said solution for a time sufficient to stabilize reactive sites thereof; and diluting the solution with water to an enzyme content of at least 100 I.U., a solvent content of no more than 5% and a water-soluble polymer content of at least 0.01% and which solvent content does not materially affect any reaction between the enzyme and SGOT or SGPT when present in such limited amount.
27. The method of Claim 26 in that said composition does not require further substantial dilution for use in biological diag-nostic determinations.
28. A method according to Claim 26 further including the step of storing the diluted composition at refrigerator tempera-ture for a period over one month without significant loss of enzymatic activity.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US66785676A | 1976-03-17 | 1976-03-17 | |
| US667,856 | 1991-03-12 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1091174A true CA1091174A (en) | 1980-12-09 |
Family
ID=24679940
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA273,920A Expired CA1091174A (en) | 1976-03-17 | 1977-03-14 | Stabilized liquid enzyme |
Country Status (7)
| Country | Link |
|---|---|
| JP (1) | JPS52134086A (en) |
| CA (1) | CA1091174A (en) |
| CH (1) | CH630662A5 (en) |
| DE (1) | DE2711754C2 (en) |
| FR (1) | FR2344568A1 (en) |
| GB (1) | GB1550753A (en) |
| SE (1) | SE445045B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA1102225A (en) * | 1976-09-13 | 1981-06-02 | Ivan E. Modrovich | Stabilized liquid enzyme and coenzyme compositions and method of preparing same |
| US4132598A (en) * | 1977-06-03 | 1979-01-02 | Modrovich Ivan Endre | Stabilized liquid phosphate containing diagnostic compositions and method of preparing same |
| US4378430A (en) * | 1979-09-11 | 1983-03-29 | Modrovich Ivan Endre | Method of forming stabilized urease solutions |
| US4652524A (en) * | 1980-10-02 | 1987-03-24 | Ivan E. Modrovich | Soluble stabilized enzymes |
| WO1984000978A1 (en) * | 1982-08-30 | 1984-03-15 | Beckman Instruments Inc | METHOD FOR DETERMINING gamma-GLUTAMYLTRANSFERASE ACTIVITY AND KITS CONTAINING A NOVEL SUBSTRATE SOLUTION FOR USE THEREIN |
| JP3203108B2 (en) * | 1993-08-26 | 2001-08-27 | 協和メデックス株式会社 | Method for stabilizing glucose-6-phosphate dehydrogenase |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3557002A (en) * | 1967-11-15 | 1971-01-19 | Procter & Gamble | Stabilized aqueous enzyme preparation |
-
1977
- 1977-03-14 CA CA273,920A patent/CA1091174A/en not_active Expired
- 1977-03-16 CH CH327077A patent/CH630662A5/en not_active IP Right Cessation
- 1977-03-16 FR FR7707873A patent/FR2344568A1/en active Granted
- 1977-03-17 GB GB1124177A patent/GB1550753A/en not_active Expired
- 1977-03-17 SE SE7703076A patent/SE445045B/en not_active IP Right Cessation
- 1977-03-17 JP JP3101677A patent/JPS52134086A/en active Granted
- 1977-03-17 DE DE19772711754 patent/DE2711754C2/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| DE2711754A1 (en) | 1977-09-22 |
| FR2344568A1 (en) | 1977-10-14 |
| DE2711754C2 (en) | 1985-09-12 |
| FR2344568B1 (en) | 1980-05-09 |
| SE7703076L (en) | 1977-09-18 |
| GB1550753A (en) | 1979-08-22 |
| CH630662A5 (en) | 1982-06-30 |
| JPS52134086A (en) | 1977-11-09 |
| SE445045B (en) | 1986-05-26 |
| JPS6117466B2 (en) | 1986-05-07 |
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