CA1068586A - .alpha.-AMYLASE ASSAY UTILIZING MODIFIED STARCH AS THE SUBSTRATE - Google Patents
.alpha.-AMYLASE ASSAY UTILIZING MODIFIED STARCH AS THE SUBSTRATEInfo
- Publication number
- CA1068586A CA1068586A CA281,158A CA281158A CA1068586A CA 1068586 A CA1068586 A CA 1068586A CA 281158 A CA281158 A CA 281158A CA 1068586 A CA1068586 A CA 1068586A
- Authority
- CA
- Canada
- Prior art keywords
- phosphate
- amylase
- glucose
- alpha
- sample
- 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
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/40—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving amylase
Abstract
Abstract of the Disclosure A specific and sensitive method for the determination of .alpha.-amylase activities in a sample is disclosed. The method utilizes a partially modified starch substrate which contains blockages to the action of exo-carbohydrases but not to .alpha.-amylase, an endo-carbohydrase.
Cleavage of the modified substrate by .alpha.-amylase leads to free nonreducing ends which can then be attacked by a suitable exo-carbohydrase serving as the coupling enzyme.
The reaction sequence is as follows: Blocked starch .alpha.-amylase unblocked starch phosphoglucomutase inorganic phosphate glucose-1-phosphate phosphoglucomutase glucose-6-phosphate glucose-6-phosphate dehydrogenase, NAD
NADH. The rate of increase of absorbance due to NADH
formation can be measured to determine the level of .alpha.-amylase activity in a sample, particularly one of serum or urine origin. The values obtained are useful in clinical diagnosis.
Cleavage of the modified substrate by .alpha.-amylase leads to free nonreducing ends which can then be attacked by a suitable exo-carbohydrase serving as the coupling enzyme.
The reaction sequence is as follows: Blocked starch .alpha.-amylase unblocked starch phosphoglucomutase inorganic phosphate glucose-1-phosphate phosphoglucomutase glucose-6-phosphate glucose-6-phosphate dehydrogenase, NAD
NADH. The rate of increase of absorbance due to NADH
formation can be measured to determine the level of .alpha.-amylase activity in a sample, particularly one of serum or urine origin. The values obtained are useful in clinical diagnosis.
Description
~068586 Back~round Of The Invention a-Amylase levels in samples can be of considerable importance, particularly in biological fluids such as serum or urine. High levels of serum a-amylase usually signify disorder of the pancreas. a-amylase hydrolyzes the ~ 4 ;~ glucan linkages of polysaccharides and oligosaccharides in a random fashion. The final produc~s are mainly maltose and some glucose. Limit dextrins are also produced when branch-chained starch is the substrate. Currently, a-amylase activity is measured by one of the following methods: 1) Decrease in viscosity of the starch solution; 2) Decrease in the ~-turbidity of starch suspension; 3) Decrease in the intensity of the starch-iodine complex; 4) Increase in the amount of reducing ends (saccharogenic assay); 5) Formation of glucose in a-glucosidase-coupled reaction; or 6) Intensity of chromagen ; in solution after cleavage of stained insoluble starch.
All of these current methods have one or several of ; the following disadvantages: Poor sensitivity; Long lag time;
Insoluble substrate; High background reading, especially when glucose is present; Multi-step procedure; or Short linearity.
.'' .
I Summary Of The Invention . .
-1 The method described herein concerns an assay for the enzyme a-amylase (a-1,4-glucan-4-glucanohydrolase, EC
3.2.1.1) especially that of serum or urine origin, and has .~, ,~ ~
`' ~068586 particular application in clinical diagnosis. The method -involves 1) the use of a modified starch which contains ~lockages to the action of the exo-acting enzymes (Marshall, J.J.
and Whelan, W.J., 1971, Anal. Biochem., 43, 316-321; Marshall, J.J., 1970, Anal. Biochem., 2, 132-145) such as phosphorylase and 2) the coupling of the reaction to a polysaccharide phosphorylase so that glucose-l-phosphate is formed when ~-.. ~
a-amylase is present. Phosphorylase attacks the starch from ;~
the nonreducing ends. Thus it cannot utilize the modified 10 starch (with blocked nonreducing ends) as a substrate. ~-a-Amylase, on the other hand, is an endo-acting enzyme which hydrolyzes the internal a-l--~ 4 bonds of the starch. Therefore, the modified starch can serve as a substrate for a-amylase. The hydrolytic action of a-amylase results in the revealing of nonreducing ends which can then be utilized by phosphorylase to form glucose-l-phosphate. The overall reaction sequence is as follows:
Modified (blocked) starch a-amylase ~ Nonreducing ends (unblocked starch) Nonreducing ends inorganic phosphate (Pi) phosphorylase b plus adenosine 5'-monophosphate (AMP) ~ or phosphorylase ~ -= ~ or maltodextrin phosphorylase ~ glucose-l-phosphate Glucose-l-phosphate phospho~lucomutase > glucose-6-phosphate Glucose-6-phosphate nicotinamide adeninedinucleotide (NAD~, glucose-6-phosphate dehydro~enase, ~r reduced nicotinamide adeninedinucleotide plus 6-phosphogluconate. `
... .
;' .
. .
; The rate of increase of absorbance at 340 nm. due to NADH
formation can be followed by a photometer to monitor the enzymatic activity of a-amylase. With the addition of a tetrazolium dye, 2-p-iodophenyl-3-nitrophenyl-5-phenyl tetrazolium chloride (INT) and an electron carrier, the assay ~ can be converted into a colorimetric one: ~ -; INT plus NADH diaphorase or phenazine methosulfate INTH
(Absorbance peak = 500 nm.) plus NAD. --Compared with current methods, the method described herein has the following advantages:
1) Since the reaction is coupled through the formation of glucose-l-phosphate and glucose-6-phosphate, which are not present at appreciable levels in body flulds, the assay is free from lnterference by glucose and other reducing sugars. Consequently, a sample blank -l, need not be performed.q 2) The coupling enzymes used in the reaction, , namely, phosphorylase, phosphoglucomutase, and glucose-6-phosphate dehydrogenase, all have high activity at pH 7, the optimal pH for a-amylase. Hence the assay can be performed ~') under the optimal reaction conditions. In ... .
~ other methods where the reaction is coupled j to a-glucosidase or glucoamylase, either a ;
lower pH or a large amount of the coupling enzyme is required.
.
. .
~ . :
~.
3) Each bond hydrolyzed by a-amylase immediately results in the formation of glucose-l-phosphate.
Therefore, this reaction has little or no lag -time. The a-glucosidase-coupled reaction has a long lag time since a build-up of maltose is `
necessary prior to detection.
4) The activity of a-amylase can be measured in a slngle operation in a continuous manner.
Other tests which utilize iodometric, visco-metric, saccharogenis principles or dyed substrates require separate steps. ` `
5) The assay offers high sensitivity and long linearity. The viscometric, turbidimetric, ~-and dyed-substrate approaches suffer from --; either low sensitivity or short linearity ~ -~
~with respect to both the amount of a-amylase and time).
In one particular a3pect the present invention provides . .
a method of assaying for a-amylase in a sample which comprises ``
incubating said sample with a modified starch substrate con-taining blocked nonreducing ends in the presence of a suitable - phosphorylase, phosphoglucomutase, and glucose-6-phosphate dehydrogenase as the coupling enzymes for the measurement of ~he liberated nonreducing ends due to the action of a-amylase.
In another particular aspect the present invention provides a reagent for use in assaying for a-amylase in a sample com-prising, in association:
phosphate buffer, i pH range of 6-8 20-100 mmoles/liter Mg salts 1-5 mmoles/liter adenosine 5'-mono-phosphate 0.1-0.5 mmoles/liter '' , ~ `' jl/ ~ -4-R'' : ' . ''`~
~068586 ~ nicotinamide adeninedinu-cleotide 1-5 mmoles/liter glucose-l, 6-diphosphate 0.1-5 U/mmoles/liter NaCl 2-100 mmoles/liter 4% periodate .
oxidized, NaBHb reduced starch 2-5 g/liter reagent Phosphorylase b (rabbit muscle) 8-30 U/ml reagent Phosphoglucomutase 1-10 Utml reagent Glucose-6-phosphate dehydrogenase 5-50 U/ml reagent Detailed Description of the Invention .
The preferred .substrate is a modified branch-chained : ;
polysaccharide, preferably modified to the extent of 2-10%.
The substrate used in the following description is a modified amylopectin which is prepared by limited oxidation with NaI04, followed by reduction by NaBH4, then by treatment with - an exo-acting enzyme such as gluco-amylase, ~-amylase, or phosphorylase to remove the unreacted nonreducing ends.
. .
' :
., , 20 . . .
.: - .
,':
`' .
~ ` -4a-- ` 10685~6 ~ ~ ~
: However, other methods of modification can be -used. Starch glycollate and modified glycogen, amylose or soluble starch, for instance, can serve as substrates in ; this reaction, though they are not as desirable. :.
The extent of oxidation also affects the efficiency of the substrate. The substrate employed herein is 4%
oxidized amylopectin, but starches modified by lower or higher percentage of oxidation (2-8%) have been shown to be good substrates.
A single reagent, containing all of the ingredients .
for the determination of a-amylase, is represented by the following formula:
- .
In~redient Concentration .
::~ Phosphate buffer, :: -:~ 15 pH 7.0 20-100 mmoles/liter :
MgC12 1-5 mmoles/liter :
. AMP 0.1-0.5 mmoles/liter .
.; NAD 1-5 mmoles/liter -Glucose-l, 6-.~ 20 diphosphate 0.1-5 umoles/liter NaCl 2-100 mmoles/liter 4% oxidized, reduced starch 2-5 g/liter reagent Phosphosylase b 8-30 U/ml reagent --. 25(rabbit muscle) Phosphoglucomatase 1-10 U/ml reagent Glucose-6-phosphate . dehydrogenase 5-50 U/ml reagent : Assay temperature = 37C
-:
.
:
~ ~ `
~68586 It should be emphasized that phosphorylase a -or phosphorylase from other mammalian organs or from plant or microbial sources can be used to replace phosphosylase b.
Maltodextrin phosphosylase (Schwartz, M. and Hofnung, M., 1967, European J. Biochem; ~ 132-145) can also be used. In addition, a mixture of several phosphorylases can be employed to achieve higher efficiency. An analog may be used in place of AMP. In colorimetric assay the following ingredients are added:
INT 0.4g/liter reagent Diaphorase 0.5U/ml reagent . .
Any electron acceptor such as PMS can be used to replace diaphorase.
As described, the method involves the use of a modified starch in conjunction with a phosphorylase as the ~- , key coupling enzyme to produce an ultraviolet (UV) or a ` colorimetric test. Changes in the type of the modified starch or the source of phosphosylase used are considered to be within the scope of the invention. The glucose-6-phosphate dehydrogenase can be obtained from yeast, leuconostoc or other sources. The electron transfer agent can be an enzyme such as diaphorase, phenazine methosulfate or other suitable electron carrier.
The body fluid to be assay for ~-amylase can be a serum or a urine sample. When human serum is the sample, :' 20 ul. of serum is added to 1.0 ml of the above described assay mixture and the reaction is followed at 340 nanometers (nm) (or 500 nm in the colorimetric test) for 5 minutes at 37 with a suitable spectrophotometer. The procedure has been adapted to the Abbott Bichrometic Analyzer 100, using the following settings:
~' . .
37 C.
340/380 filter (500/600 filter for -colorimetric test) 5 minute cycle FRR (First revolution reading) mode Rate-up 1:51 syringe plate (1:101 plate for lS color test) :
When the previously described assay mixture and procedure are used, the test is linear to 0.3 absorbance unit per minute at 340 nm, equivalent to 6000U a-amylase/
liter serum. This method correlates very well with the established iodometric method of Street and Close (Street, H.V., and Close, J.R., 1956, Clin. Chim. Acta., 1, 256).
One Street and Close unit is equivalent to about five international units.
'~ ' :'' -:
..
All of these current methods have one or several of ; the following disadvantages: Poor sensitivity; Long lag time;
Insoluble substrate; High background reading, especially when glucose is present; Multi-step procedure; or Short linearity.
.'' .
I Summary Of The Invention . .
-1 The method described herein concerns an assay for the enzyme a-amylase (a-1,4-glucan-4-glucanohydrolase, EC
3.2.1.1) especially that of serum or urine origin, and has .~, ,~ ~
`' ~068586 particular application in clinical diagnosis. The method -involves 1) the use of a modified starch which contains ~lockages to the action of the exo-acting enzymes (Marshall, J.J.
and Whelan, W.J., 1971, Anal. Biochem., 43, 316-321; Marshall, J.J., 1970, Anal. Biochem., 2, 132-145) such as phosphorylase and 2) the coupling of the reaction to a polysaccharide phosphorylase so that glucose-l-phosphate is formed when ~-.. ~
a-amylase is present. Phosphorylase attacks the starch from ;~
the nonreducing ends. Thus it cannot utilize the modified 10 starch (with blocked nonreducing ends) as a substrate. ~-a-Amylase, on the other hand, is an endo-acting enzyme which hydrolyzes the internal a-l--~ 4 bonds of the starch. Therefore, the modified starch can serve as a substrate for a-amylase. The hydrolytic action of a-amylase results in the revealing of nonreducing ends which can then be utilized by phosphorylase to form glucose-l-phosphate. The overall reaction sequence is as follows:
Modified (blocked) starch a-amylase ~ Nonreducing ends (unblocked starch) Nonreducing ends inorganic phosphate (Pi) phosphorylase b plus adenosine 5'-monophosphate (AMP) ~ or phosphorylase ~ -= ~ or maltodextrin phosphorylase ~ glucose-l-phosphate Glucose-l-phosphate phospho~lucomutase > glucose-6-phosphate Glucose-6-phosphate nicotinamide adeninedinucleotide (NAD~, glucose-6-phosphate dehydro~enase, ~r reduced nicotinamide adeninedinucleotide plus 6-phosphogluconate. `
... .
;' .
. .
; The rate of increase of absorbance at 340 nm. due to NADH
formation can be followed by a photometer to monitor the enzymatic activity of a-amylase. With the addition of a tetrazolium dye, 2-p-iodophenyl-3-nitrophenyl-5-phenyl tetrazolium chloride (INT) and an electron carrier, the assay ~ can be converted into a colorimetric one: ~ -; INT plus NADH diaphorase or phenazine methosulfate INTH
(Absorbance peak = 500 nm.) plus NAD. --Compared with current methods, the method described herein has the following advantages:
1) Since the reaction is coupled through the formation of glucose-l-phosphate and glucose-6-phosphate, which are not present at appreciable levels in body flulds, the assay is free from lnterference by glucose and other reducing sugars. Consequently, a sample blank -l, need not be performed.q 2) The coupling enzymes used in the reaction, , namely, phosphorylase, phosphoglucomutase, and glucose-6-phosphate dehydrogenase, all have high activity at pH 7, the optimal pH for a-amylase. Hence the assay can be performed ~') under the optimal reaction conditions. In ... .
~ other methods where the reaction is coupled j to a-glucosidase or glucoamylase, either a ;
lower pH or a large amount of the coupling enzyme is required.
.
. .
~ . :
~.
3) Each bond hydrolyzed by a-amylase immediately results in the formation of glucose-l-phosphate.
Therefore, this reaction has little or no lag -time. The a-glucosidase-coupled reaction has a long lag time since a build-up of maltose is `
necessary prior to detection.
4) The activity of a-amylase can be measured in a slngle operation in a continuous manner.
Other tests which utilize iodometric, visco-metric, saccharogenis principles or dyed substrates require separate steps. ` `
5) The assay offers high sensitivity and long linearity. The viscometric, turbidimetric, ~-and dyed-substrate approaches suffer from --; either low sensitivity or short linearity ~ -~
~with respect to both the amount of a-amylase and time).
In one particular a3pect the present invention provides . .
a method of assaying for a-amylase in a sample which comprises ``
incubating said sample with a modified starch substrate con-taining blocked nonreducing ends in the presence of a suitable - phosphorylase, phosphoglucomutase, and glucose-6-phosphate dehydrogenase as the coupling enzymes for the measurement of ~he liberated nonreducing ends due to the action of a-amylase.
In another particular aspect the present invention provides a reagent for use in assaying for a-amylase in a sample com-prising, in association:
phosphate buffer, i pH range of 6-8 20-100 mmoles/liter Mg salts 1-5 mmoles/liter adenosine 5'-mono-phosphate 0.1-0.5 mmoles/liter '' , ~ `' jl/ ~ -4-R'' : ' . ''`~
~068586 ~ nicotinamide adeninedinu-cleotide 1-5 mmoles/liter glucose-l, 6-diphosphate 0.1-5 U/mmoles/liter NaCl 2-100 mmoles/liter 4% periodate .
oxidized, NaBHb reduced starch 2-5 g/liter reagent Phosphorylase b (rabbit muscle) 8-30 U/ml reagent Phosphoglucomutase 1-10 Utml reagent Glucose-6-phosphate dehydrogenase 5-50 U/ml reagent Detailed Description of the Invention .
The preferred .substrate is a modified branch-chained : ;
polysaccharide, preferably modified to the extent of 2-10%.
The substrate used in the following description is a modified amylopectin which is prepared by limited oxidation with NaI04, followed by reduction by NaBH4, then by treatment with - an exo-acting enzyme such as gluco-amylase, ~-amylase, or phosphorylase to remove the unreacted nonreducing ends.
. .
' :
., , 20 . . .
.: - .
,':
`' .
~ ` -4a-- ` 10685~6 ~ ~ ~
: However, other methods of modification can be -used. Starch glycollate and modified glycogen, amylose or soluble starch, for instance, can serve as substrates in ; this reaction, though they are not as desirable. :.
The extent of oxidation also affects the efficiency of the substrate. The substrate employed herein is 4%
oxidized amylopectin, but starches modified by lower or higher percentage of oxidation (2-8%) have been shown to be good substrates.
A single reagent, containing all of the ingredients .
for the determination of a-amylase, is represented by the following formula:
- .
In~redient Concentration .
::~ Phosphate buffer, :: -:~ 15 pH 7.0 20-100 mmoles/liter :
MgC12 1-5 mmoles/liter :
. AMP 0.1-0.5 mmoles/liter .
.; NAD 1-5 mmoles/liter -Glucose-l, 6-.~ 20 diphosphate 0.1-5 umoles/liter NaCl 2-100 mmoles/liter 4% oxidized, reduced starch 2-5 g/liter reagent Phosphosylase b 8-30 U/ml reagent --. 25(rabbit muscle) Phosphoglucomatase 1-10 U/ml reagent Glucose-6-phosphate . dehydrogenase 5-50 U/ml reagent : Assay temperature = 37C
-:
.
:
~ ~ `
~68586 It should be emphasized that phosphorylase a -or phosphorylase from other mammalian organs or from plant or microbial sources can be used to replace phosphosylase b.
Maltodextrin phosphosylase (Schwartz, M. and Hofnung, M., 1967, European J. Biochem; ~ 132-145) can also be used. In addition, a mixture of several phosphorylases can be employed to achieve higher efficiency. An analog may be used in place of AMP. In colorimetric assay the following ingredients are added:
INT 0.4g/liter reagent Diaphorase 0.5U/ml reagent . .
Any electron acceptor such as PMS can be used to replace diaphorase.
As described, the method involves the use of a modified starch in conjunction with a phosphorylase as the ~- , key coupling enzyme to produce an ultraviolet (UV) or a ` colorimetric test. Changes in the type of the modified starch or the source of phosphosylase used are considered to be within the scope of the invention. The glucose-6-phosphate dehydrogenase can be obtained from yeast, leuconostoc or other sources. The electron transfer agent can be an enzyme such as diaphorase, phenazine methosulfate or other suitable electron carrier.
The body fluid to be assay for ~-amylase can be a serum or a urine sample. When human serum is the sample, :' 20 ul. of serum is added to 1.0 ml of the above described assay mixture and the reaction is followed at 340 nanometers (nm) (or 500 nm in the colorimetric test) for 5 minutes at 37 with a suitable spectrophotometer. The procedure has been adapted to the Abbott Bichrometic Analyzer 100, using the following settings:
~' . .
37 C.
340/380 filter (500/600 filter for -colorimetric test) 5 minute cycle FRR (First revolution reading) mode Rate-up 1:51 syringe plate (1:101 plate for lS color test) :
When the previously described assay mixture and procedure are used, the test is linear to 0.3 absorbance unit per minute at 340 nm, equivalent to 6000U a-amylase/
liter serum. This method correlates very well with the established iodometric method of Street and Close (Street, H.V., and Close, J.R., 1956, Clin. Chim. Acta., 1, 256).
One Street and Close unit is equivalent to about five international units.
'~ ' :'' -:
..
Claims (19)
1. A method of assaying for .alpha.-amylase in a sample which comprises incubating said sample with a modified starch substrate containing blocked nonreducing ends in the presence of a suitable phosphorylase, phosphoglucomutase, and glucose-6-phosphate dehydrogenase as the coupling enzymes for the measurement of the liberated nonreducing ends due to the action of .alpha.-amylase.
2. A method according to Claim 1 in which said sample is a biological fluid.
3. A method according to Claim 1 wherein said phosphorylase is selected from animal, plant or microbial sources.
4. A method according to Claim 3 in which the phosphorylase is in the a or b sub-form.
5. A method according to Claim 1 in which the liberated nonreducing ends in the presence of phosphate are converted to glucose-1-phosphate.
6. A method according to Claim 1 in which said phosphoglucomutase is selected from animal, plant or microbial sources.
7, A method according to Claim 1 in which the glucose-6-phosphate which is formed is reacted in the presence of nicotinamide adeninedinucleotide (NAD) or nicotinamide adeninedinucleotide phosphate (NADP) and glucose-6-phosphate dehydrogenase which are thereby converted to 6-phosphogluconate and reduced nicotinamide adeninedinucleotide (NADH) or reduced nicotinamide adeninedinucleotide phosphate (NADPH), respectively.
8. A method according to Claim 7 in which said glucose-6-phosphate dehydrogenase is selected from yeast or leuconostoc sources,
9. A method according to Claim 7 in which the formed reduced nicotinamide adeninedinucleotide or reduced nicotinamide adeninedinucleotide is coupled to a suitable dye in the presence of a suitable electron transfer agent.
10. A method according to Claim 9 in which said dye is 2-p-iodophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride.
11. A method according to Claim 9 in which the electron transfer agent is an enzyme selected from the group consisting of diaphorase or phenazine methosulfate.
12. A method according to Claim 1 in which the blocked substrate is selected from the group consisting of branched or nonbranched polysaccharide.
13. A method according to Claim 12 in which the substrate is a modified branched chain polysaccharide.
14. A reagent for use in assaying for .alpha.-amylase in a sample comprising, in association:
15. A reagent according to Claim 14 in which the activator is adenosine 5'-monophosphate or an analog thereof.
16. A reagent according to Claim 14 in the form of a dry, single solid.
17. A reagent according to Claim 16 in which the dry reagent is made ready for use by the addition of water or other suitable liquid.
18. A method according to Claim 13 in which the polysaccharide may be modified to the extent of 2-10%,
19. A method according to Claim 2 wherein said sample is serum.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US70177876A | 1976-07-01 | 1976-07-01 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1068586A true CA1068586A (en) | 1979-12-25 |
Family
ID=24818638
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA281,158A Expired CA1068586A (en) | 1976-07-01 | 1977-06-22 | .alpha.-AMYLASE ASSAY UTILIZING MODIFIED STARCH AS THE SUBSTRATE |
Country Status (5)
| Country | Link |
|---|---|
| JP (1) | JPS533891A (en) |
| CA (1) | CA1068586A (en) |
| DE (1) | DE2729636B2 (en) |
| FR (1) | FR2356939A1 (en) |
| GB (1) | GB1543130A (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0005867B2 (en) * | 1978-06-06 | 1986-07-09 | Cooperbiomedical, Inc. | An alpha-amylase assay, a reagent composition and a reagent system therefor |
| US4304854A (en) | 1978-06-06 | 1981-12-08 | Worthington Biochemical Corporation | Alpha-amylase assay and substrates for use therein |
| JPS5768798A (en) * | 1980-10-14 | 1982-04-27 | Toyo Jozo Co Ltd | Novel measurement of amylase activity |
| US4649108A (en) * | 1984-07-26 | 1987-03-10 | Genzyme Corporation | Alpha amylase assay |
| US5043436A (en) * | 1986-11-20 | 1991-08-27 | Kurita Water Ind., Ltd. | Substrate for measurement of alpha-amylase activity |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA973499A (en) * | 1970-07-28 | 1975-08-26 | Hayashibara, Ken | Process for the preparation of amylose as the substrate for the quantitative analysis of amylose |
| US3879263A (en) * | 1973-09-06 | 1975-04-22 | Du Pont | Method for the determination of amylase |
| US4097336A (en) * | 1976-02-13 | 1978-06-27 | Beckman Instruments, Inc. | Reagent system for beta-amylase assay |
-
1977
- 1977-06-22 CA CA281,158A patent/CA1068586A/en not_active Expired
- 1977-06-29 JP JP7668477A patent/JPS533891A/en active Granted
- 1977-06-30 FR FR7720191A patent/FR2356939A1/en active Granted
- 1977-06-30 GB GB2751577A patent/GB1543130A/en not_active Expired
- 1977-06-30 DE DE19772729636 patent/DE2729636B2/en not_active Ceased
Also Published As
| Publication number | Publication date |
|---|---|
| DE2729636B2 (en) | 1979-03-15 |
| FR2356939A1 (en) | 1978-01-27 |
| JPS533891A (en) | 1978-01-13 |
| DE2729636A1 (en) | 1978-02-02 |
| GB1543130A (en) | 1979-03-28 |
| JPS5639637B2 (en) | 1981-09-14 |
| FR2356939B1 (en) | 1983-11-25 |
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