CA1104912A - Method and test kit for serum amylase assay - Google Patents
Method and test kit for serum amylase assayInfo
- Publication number
- CA1104912A CA1104912A CA000282419A CA282419A CA1104912A CA 1104912 A CA1104912 A CA 1104912A CA 000282419 A CA000282419 A CA 000282419A CA 282419 A CA282419 A CA 282419A CA 1104912 A CA1104912 A CA 1104912A
- Authority
- CA
- Canada
- Prior art keywords
- substrate
- sample
- solution
- test kit
- maltase
- 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
-
- 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
-
- 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
- C12Q2334/00—O-linked chromogens for determinations of hydrolase enzymes, e.g. glycosidases, phosphatases, esterases
- C12Q2334/10—O-linked chromogens for determinations of hydrolase enzymes, e.g. glycosidases, phosphatases, esterases p-Nitrophenol derivatives
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Saccharide Compounds (AREA)
Abstract
ABSTRACT OF THE DISCLOSURE
Disclosed herein are a method and a reagent test kit, both using an improved substrate to measure the amylase content of a sample. The substrate used is a glycoside and a substituted aromatic radical attached to the terminal unit of the glycoside. When detached from the polysaccharide, the aglycone exhibits a different spectral absorbance than the substrate.
Disclosed herein are a method and a reagent test kit, both using an improved substrate to measure the amylase content of a sample. The substrate used is a glycoside and a substituted aromatic radical attached to the terminal unit of the glycoside. When detached from the polysaccharide, the aglycone exhibits a different spectral absorbance than the substrate.
Description
BACKGROUND OF THE_INVENTION
l. Field of the Invention: This lnvention relates to amylase assays and to a reagent test kit for use in such assays. More particularly, it relates to amylase assays in which a polysaccharide is used as the amylase : .-substrate.
l. Field of the Invention: This lnvention relates to amylase assays and to a reagent test kit for use in such assays. More particularly, it relates to amylase assays in which a polysaccharide is used as the amylase : .-substrate.
2. Discussion of the Prior Art: a-Amylase is an enzyme which hydrolyzes the ~[l t4] linkages between the glucose units in starch and the lower polymers and oligomers of glucose. This enzyme is produced in the human body, primarily in the pancreas and in the salivary glands, and its concentration in various body ~luids is a useful diagnostic tool ~or physicians. For example, in healthy i.ndi~iduals, serum ~-amylase le~els are relatively constant, 15 but they rise in response to pathological conditions, such as acute pancreatitis.
U.S. Patent 3,879,263, issued ~pril 22, 1975, and U.S. Patent 4,000,042 issued December 28, 1976, disclose a process and reagent tes-t kit for use in deter-mining the ~-amylase content of a sample using the deined oligosaccharides maltotetraose, maltopentaose or malto-hexaose as the amylase substrate. The reaction between ~-amylase and these substrates, preerably in the presence ; of a maltase, produces a specific amount of glucose which .~ 25 can be measured by any conventional glucose detection : system. The additional glucose detection step is an incon~enience. Furthermore, if glucose is present in the ~ :
sample, it must either be removed or compensated for.
Although this can be done by conventional techniques, ;.:
~ ', ' -2- ~ :
~ .
:
'~ : . , .
.
it is an extra step in the process which is a disadvantage.
A. P. Jansen and P. ;. A. B. Wydeveldl Nature, 182, 525 (1958) postulate that ~ nitrophenyl)maltoside could he a substrate for an amylase assay. However, this paper shows that the authors never identified the active agent responsible for their observations. They reported:
(1) Incubation of human urine or saliva samples with a-(p-nitrophenyl)maltoside at 37 for 16 hours produces 4-nitrophenol, identified spectrophotometrically by mixing the hydrolyzate with 0. 02N sodium hydroxide. (2) I'he hydrolysis was inhibited by protein precipitants such as 10% trichloroacetic acid and 0.5N silver nitrate. (3) The hydrolysis was pH-dependent, being most effective at pH
5.~-7Ø They state that this was evidence for "the possible existence of an unidentified carbohydrase". a-(4-Nitrophenyl)maltoside is not believed to be useful for human amylase assay because the cleavage of this compound by ~-amylase is extremely slow.
SUMMARY OF THE INVENTI_N
; 20 Accordlng to the present invention, there is provided a method for determining the amylase content of a sample comprising the steps of:
(a) adding to a solution containing a me~sured amount of the sample a defined pol~saccharide substrate having the folIowing formula:
_ _ .
~i ~ ~C~2oH CH~Oll --r ; ~ ~1 O~ ~ _ o ~ OH ~ _ O ~ OII
IO ~ ~ ~ ~ ~ //OR
; ~C)ll ~01~ ~
: ~ .
~ 3 .
1: .
.
.. : . : : , 9~2 ~ ' : where n is an integer in the range of from 1 to 10, and R is a substituted aromatic radical which, as a detach~d aglycone exhibits a different sp~ctral absorbance than the substrate; and (b~ monitoring the spectral absorbance of the solution.
In the preferred embodiment, n is 2, 3 or 4 and R is a substituted aromatic radical selected from the group consisting of ~} ~J and ~ ~
. ':, : 10 in which X and Y are individually selected rom the group consisting of H, NO2, halogens, alkyls of from 1 to 4 carbon atoms, OR' or CO2R' :
where R' is an alkyl group of from 1 to 6 carbon atoms, , and at least one o~ X and Y is NO2.
In the preferred embodiments, the substrates are ~:
glycosides of maltotetraose, maltopentaose or maltohexaose :
in which the terminal glycoside unit has an aromatic radical attached to it. In the most preferred embodiment, the ter-: 20 minal glycoside unit is an ~-(4-nitrophenyl) glycoside, and a maltase is also added to the solution.
. A reagent test kit is also provided~ This test - :~
kit contains one o the substrates listed above and a maltasc.
:, :
~.
, .
DETAILEO DESCRIPTION OF TH~ INVENTION
The following disclosure, and the invention it describes, is restricted to polymers and oligomers of glucose which are ~[1--~4] linked and which have a sub-5 stituted aromatic radical attached to the terminal (reducing)glucose unit. Such compounds are represented by the general formula CH2CH CH~OII CH2OH
I ~ Q~ H ~ \H
~1 ~ o ~ o ~ 0~1 ,1 . ~
~ OH n ln where n is an integer, R is the substituted aromatie radieal, and the remaining portion of the compound is the glycosyl residue. When detaehed from the glycosyl residue by hydrolysis, the radical R becomes a phenol,-; ROH,or an anion of that phenol, RO ,(depending upon the eondition of the solution) both of which are normallyreferred to as an aglycone.
This eompound, which functions as a substrate for amylase, is a de~ined polysaccharide, or when n<8 a defined oligosaccharide. The term "defined" as applied to polysaccharides has, in the past, been used in a loose sense, often referring to any mixture of polysaccharides in whieh the relative percentages of the various polymers and oligomers is known. As used herein, however, the term "define~ polysaccharide" shall mean a substanee con-taining at least 90~O of a polysaccharide with a givenchain length, i.e., where n is a given inte~er.
:........ .. . .
l~L6?~
~ -Amylase acts as a catalyst in the hydrolysis of polysaccharides into small chain polysaccharides and eventually into maltose. It has been found and reported in the patents listed above that from among all po]y-saccharides, maltotetraose (G4), maltopentasoe (G5) andmaltohexaose (G6) are preferred for use as a substrate in an amylase assay. The G nomenclature is a convenient shorthand for n ~ 4] linked glucose units.
These three substrates are preferred for kinetic and stoichiometric reaso~s. The binding constant of ~-; ; amylase to polysaccharides increases as the number of a[l--~4~ bonds increases, up to about G6 where it levels o~f. For homologs lower than G4, the binding constant is too small to give reasonable reaction rates. For homologs higher than G6, even though the reaction proceeds rapidly, the results are not stoichiometric. Unproductive reactions occur so that n glucose units are not formed when ~-amylase ~-reacts with Gn. Furthermore, the maximum velocity of sub-- strate release from ~-amylase decreases with decreasing n.
.. . .
Where the detection system involves glucose and Gn is used as the substrate, then, within a reasonable time, n glucose units should be produced for every a-amylase interaction with Gn. Otherwise, the percentage of the total glucose ; units released compared to those available must be esti-mated and this leads to error. These factors make G4, G5, and G6 the pre~err~ed substrates.
In the~present system, where the detection ~system depends upon the release o~ a substituted phenol ROH and not glucose formation, stoichiometric considera-~` 30 tions~are not quite as critical and n can be in the range .. .
l ~ -6~
. .
l~q~
of 1 to 10~ However, it is still preferred that the polysaccharide be an oligosaccharide, with n in the range of 1 to 8.
As explained in U.S~ Patent 3,879,2~3, ~he use of a maltase such as ~-glucosidase is not necessary in the measurement of either pancreatic or total N-amylase. This is true when the substrates used are the substrates of the present invention as well as G4, G5 and G6. Furthermore, since the present invention is not dependent upon glucose detection, maltase does not appear to even be necessary in assays ~or salivary a-amylase using the substrates of the present invention. However, the use oE a ~altase does increase the reaction rate in all circumstances. It is particularly use~ul to achieve a truly stoichiometric reaction, because the reaction rate of the maltase with the lower oligosaccharides is greater than the reaction rate of ~-amylase with those substrates. a~Amylase acts to hydrolyze the substrate into smaller fractions, and the maltase acts to complete the hydrolysis to glucose units, 50 that the release of the substituted phenol OCCUl-S stoichiometrically. For this reason, oligo-saccharides of the formula given above, with n = 2, 3 or 4 are the most preferred substrates for the present invention. The discussion which follows, therefore, will be limited to those substrates, particularly those where n is 2 or 3. This limitation, however, is for convenience and is not intended to limit the disclosure.
When a maltase, such as a-glucosidase is used, a side reaction which gives rise to a blank rate occurs because of the reactivity of the maltase with the sub-strate. This means that even ln the absence of ~-amylase, .
: ' there will be release of the phenol. Since the maximum velocity of product release from maltase decreases with increasing n, the growth of a blank rate is slower as n increases. One would expect, then, that the blank r~te 5 for G5 would be less than that for G4. This is verified by experimentation. However, an additional factor is involved in the choice between higher and lower oligo-saccharides (i.e., G4 or G5) as the substrate. In all reactions of the substrate with amylase and reactions of maltase with the substrate, the rate increases, as a function of substrate concentration, to an optimum, at which point it levels off. The substrate concentration at which optimization occurs appears to increase with increasirlg n so that more of the substrate (and the maltase) must be used to optimize (linearize) the standard curve. For expensive chemicals, this is an important consideration.
The substrates of the present invention are the defined polysaccharides covered by the formula given above in which n is an integer between 1 and 10 and R is a sub-stituted aromatic radical which, when detached from the polysaccharide in the form of a phenol or a phenolate anion, exhibits a di.fferent spectral absorbance than the substrate. There are a large number of such radicals.
Chief among them, however, are those radicals selected from the group consisting of y .
~ .
. :
in which X and Y are individually selected from the group consistiny of H, NO2, halogens, alkyls of from 1 to 4 carbon atoms, OR' or CO2R'; where R' is an alkyl of from 1 to 6 carbon atoms, and at least one of X and Y is NO2.
The anions of the phenols formed when these radicals are separated from the glycosyl residue have a maximum absorbance ~max of be-tween about 290 and about 600 nm~
The details of the procedures for preparing these preferred compounds is set forth in Canadian Patent Appli- -cations Serial No. 282,407 (R.C. Burns et al) and Serial No. 282,406 tW.B. Farnham et al), filed on the same day as this application.
Amon~ the preferred embodiments, two compounds are particularly preferred; those in which n is 2 or 3 and R is 4-nitrophenyl. These compounds, ~-(4-nitrophenyl~
maltotetraoside (G4pNp) and ~-(4-nitrophenyl) maltopenta- ~
oside (G5pNp), are used to determine the amylase content o~ ~ -a sample, such as blood serum or urine, accordin~ to the following reaction scheme ~ .
: ': '' ' , " ' . ~, ," ''' :, ' .:
4~
H~e~o~ o~O
OH OH n OH
)l ~Ax 290-305 nm R -. 4 ~ 2 ~ C6H 4 n: 2 n - (4 - NITROPHEN~L ) !~IALTOTETRAOSIDE
n: 3 ~ -t4-NITROPHE~YL) I.~ALTC~ENTAOSIDE
Al~YLASE
~ ' CH20H CH20HCtJ20H
~ H ~ /~ \ H~ ~0\ :
62 'r G3 L Bo~OJ~
R-4-02N~G~J4 n:O a-(4-NITROP~IENYL~ ~.lALTOSIDE
- MAL~ASE
4 ~r5 Gl ~ HO g~NO2 4- NI~ROPHENOL
~,o~
-O~NO2 : ~ .
.
4-NlTROrlJENOlATE ANI()N A~ X 410nm .
The defined oligosaccharide substrate is added to a solution containing a measured amount of the sample to be tested; and the spectral absorbance of the solution is monitored, either as an end point determination or a rate determination using conventional techniques. Usually, as in all enzyme reactions, the reaction solution is main- .
tained at a substantially constant p~ and a substantially constant temperature. When these substances are used, it ~':
: ::
: . , . .
is desirable to perform the assay in a solution which has had its p~I adjus-ted to -the basic range in order to enhance the absorbance at 410 nm. For example~ G4pNp and G5pNp (~max 290-305 nm) and 4-nitrophenol (~max 313 nm~ have a low extinction coefficient at 410 nm compared to 4-nitro-phenolate anion (~max 410 nm).
To best accomplish this, a reagent test kit containing the defined substrate disclosed above and a maltase is used. One exemplary test kit is disclosed in U.S. Patent 3,476,515. This test kit can be used in the analyzer described in U.S. Patent 3,770,382.
A sample o~ ~-(4-nitrophenyl) maltotet~aoside (G~pNp) prepared in accorclance with Example l~I of Canadian Patent Application Serial No. 282,407 (R.C. Burns et al~/
filed on the same da~ as this application, was dissolved in 66.7 mM sodium phosphate buffer, pH 6~5, to provide various ~-substrate concentrations ranging from 2 to 8 mg~3 ml. As described in the aforementioned Canadian Patent Application Serial No. 282,407 (R.C. Burns et al), this substrate sample has been purified using a Sephadex~ LH-20 Chromatographic Column. ~-Glucosidase of various concentrations rang:ing from 2.5 to 12.5 International Units per three millilite~s of solution (IU/3 ml) was then added to the substrate solu-tion and the volume of the solution was hrought up to 3.0 ml. The solution was incubated at 37C. for l to 10 minutes~
After the blank xate was measured at 41Q nm;
using a Gil~ord spectrophotometer, the reaction was initiated by adding 0.1 ml of an Elevated Enzyme Control i Product sold by the E. I. du Pont de Nemours and Company ' ~1~ ~ / ï .
' ., ,, , ,, , ~ ~
(1150 Somogyi Units per deciliter (SU/dl) amylase~ diluted 1:1 with Du Pont Enz~me Diluent. This level of amylase is approximately six times the upper normal serum level.
The total reaction rate was then measured using the Gilford spectrophotometer, and by subtracting the blank rate from the total rate, the net reaction rate was obtained.
A two-variable statistical optimi~ation for the substrate and the ~-glucosidase was run. I`he results of this evaluation are given in Table I in arbitrary Absor-bance units (A) per minute.
TABLE I
,005l .012 .018 12.5 .0912 .113 .llO
,~ .o863 .101 .092 P~ .004.008 .011 7.5 .090.llO .lOg .086.102 .Og8 o ~ .001.008 .002 ~ 2.5 .079.091 .0~0 078.083 .078 2.05.0 8.0 G4pNp mg/3 ml l. Blank rate (A/min) 2. Total rate (A/min)
U.S. Patent 3,879,263, issued ~pril 22, 1975, and U.S. Patent 4,000,042 issued December 28, 1976, disclose a process and reagent tes-t kit for use in deter-mining the ~-amylase content of a sample using the deined oligosaccharides maltotetraose, maltopentaose or malto-hexaose as the amylase substrate. The reaction between ~-amylase and these substrates, preerably in the presence ; of a maltase, produces a specific amount of glucose which .~ 25 can be measured by any conventional glucose detection : system. The additional glucose detection step is an incon~enience. Furthermore, if glucose is present in the ~ :
sample, it must either be removed or compensated for.
Although this can be done by conventional techniques, ;.:
~ ', ' -2- ~ :
~ .
:
'~ : . , .
.
it is an extra step in the process which is a disadvantage.
A. P. Jansen and P. ;. A. B. Wydeveldl Nature, 182, 525 (1958) postulate that ~ nitrophenyl)maltoside could he a substrate for an amylase assay. However, this paper shows that the authors never identified the active agent responsible for their observations. They reported:
(1) Incubation of human urine or saliva samples with a-(p-nitrophenyl)maltoside at 37 for 16 hours produces 4-nitrophenol, identified spectrophotometrically by mixing the hydrolyzate with 0. 02N sodium hydroxide. (2) I'he hydrolysis was inhibited by protein precipitants such as 10% trichloroacetic acid and 0.5N silver nitrate. (3) The hydrolysis was pH-dependent, being most effective at pH
5.~-7Ø They state that this was evidence for "the possible existence of an unidentified carbohydrase". a-(4-Nitrophenyl)maltoside is not believed to be useful for human amylase assay because the cleavage of this compound by ~-amylase is extremely slow.
SUMMARY OF THE INVENTI_N
; 20 Accordlng to the present invention, there is provided a method for determining the amylase content of a sample comprising the steps of:
(a) adding to a solution containing a me~sured amount of the sample a defined pol~saccharide substrate having the folIowing formula:
_ _ .
~i ~ ~C~2oH CH~Oll --r ; ~ ~1 O~ ~ _ o ~ OH ~ _ O ~ OII
IO ~ ~ ~ ~ ~ //OR
; ~C)ll ~01~ ~
: ~ .
~ 3 .
1: .
.
.. : . : : , 9~2 ~ ' : where n is an integer in the range of from 1 to 10, and R is a substituted aromatic radical which, as a detach~d aglycone exhibits a different sp~ctral absorbance than the substrate; and (b~ monitoring the spectral absorbance of the solution.
In the preferred embodiment, n is 2, 3 or 4 and R is a substituted aromatic radical selected from the group consisting of ~} ~J and ~ ~
. ':, : 10 in which X and Y are individually selected rom the group consisting of H, NO2, halogens, alkyls of from 1 to 4 carbon atoms, OR' or CO2R' :
where R' is an alkyl group of from 1 to 6 carbon atoms, , and at least one o~ X and Y is NO2.
In the preferred embodiments, the substrates are ~:
glycosides of maltotetraose, maltopentaose or maltohexaose :
in which the terminal glycoside unit has an aromatic radical attached to it. In the most preferred embodiment, the ter-: 20 minal glycoside unit is an ~-(4-nitrophenyl) glycoside, and a maltase is also added to the solution.
. A reagent test kit is also provided~ This test - :~
kit contains one o the substrates listed above and a maltasc.
:, :
~.
, .
DETAILEO DESCRIPTION OF TH~ INVENTION
The following disclosure, and the invention it describes, is restricted to polymers and oligomers of glucose which are ~[1--~4] linked and which have a sub-5 stituted aromatic radical attached to the terminal (reducing)glucose unit. Such compounds are represented by the general formula CH2CH CH~OII CH2OH
I ~ Q~ H ~ \H
~1 ~ o ~ o ~ 0~1 ,1 . ~
~ OH n ln where n is an integer, R is the substituted aromatie radieal, and the remaining portion of the compound is the glycosyl residue. When detaehed from the glycosyl residue by hydrolysis, the radical R becomes a phenol,-; ROH,or an anion of that phenol, RO ,(depending upon the eondition of the solution) both of which are normallyreferred to as an aglycone.
This eompound, which functions as a substrate for amylase, is a de~ined polysaccharide, or when n<8 a defined oligosaccharide. The term "defined" as applied to polysaccharides has, in the past, been used in a loose sense, often referring to any mixture of polysaccharides in whieh the relative percentages of the various polymers and oligomers is known. As used herein, however, the term "define~ polysaccharide" shall mean a substanee con-taining at least 90~O of a polysaccharide with a givenchain length, i.e., where n is a given inte~er.
:........ .. . .
l~L6?~
~ -Amylase acts as a catalyst in the hydrolysis of polysaccharides into small chain polysaccharides and eventually into maltose. It has been found and reported in the patents listed above that from among all po]y-saccharides, maltotetraose (G4), maltopentasoe (G5) andmaltohexaose (G6) are preferred for use as a substrate in an amylase assay. The G nomenclature is a convenient shorthand for n ~ 4] linked glucose units.
These three substrates are preferred for kinetic and stoichiometric reaso~s. The binding constant of ~-; ; amylase to polysaccharides increases as the number of a[l--~4~ bonds increases, up to about G6 where it levels o~f. For homologs lower than G4, the binding constant is too small to give reasonable reaction rates. For homologs higher than G6, even though the reaction proceeds rapidly, the results are not stoichiometric. Unproductive reactions occur so that n glucose units are not formed when ~-amylase ~-reacts with Gn. Furthermore, the maximum velocity of sub-- strate release from ~-amylase decreases with decreasing n.
.. . .
Where the detection system involves glucose and Gn is used as the substrate, then, within a reasonable time, n glucose units should be produced for every a-amylase interaction with Gn. Otherwise, the percentage of the total glucose ; units released compared to those available must be esti-mated and this leads to error. These factors make G4, G5, and G6 the pre~err~ed substrates.
In the~present system, where the detection ~system depends upon the release o~ a substituted phenol ROH and not glucose formation, stoichiometric considera-~` 30 tions~are not quite as critical and n can be in the range .. .
l ~ -6~
. .
l~q~
of 1 to 10~ However, it is still preferred that the polysaccharide be an oligosaccharide, with n in the range of 1 to 8.
As explained in U.S~ Patent 3,879,2~3, ~he use of a maltase such as ~-glucosidase is not necessary in the measurement of either pancreatic or total N-amylase. This is true when the substrates used are the substrates of the present invention as well as G4, G5 and G6. Furthermore, since the present invention is not dependent upon glucose detection, maltase does not appear to even be necessary in assays ~or salivary a-amylase using the substrates of the present invention. However, the use oE a ~altase does increase the reaction rate in all circumstances. It is particularly use~ul to achieve a truly stoichiometric reaction, because the reaction rate of the maltase with the lower oligosaccharides is greater than the reaction rate of ~-amylase with those substrates. a~Amylase acts to hydrolyze the substrate into smaller fractions, and the maltase acts to complete the hydrolysis to glucose units, 50 that the release of the substituted phenol OCCUl-S stoichiometrically. For this reason, oligo-saccharides of the formula given above, with n = 2, 3 or 4 are the most preferred substrates for the present invention. The discussion which follows, therefore, will be limited to those substrates, particularly those where n is 2 or 3. This limitation, however, is for convenience and is not intended to limit the disclosure.
When a maltase, such as a-glucosidase is used, a side reaction which gives rise to a blank rate occurs because of the reactivity of the maltase with the sub-strate. This means that even ln the absence of ~-amylase, .
: ' there will be release of the phenol. Since the maximum velocity of product release from maltase decreases with increasing n, the growth of a blank rate is slower as n increases. One would expect, then, that the blank r~te 5 for G5 would be less than that for G4. This is verified by experimentation. However, an additional factor is involved in the choice between higher and lower oligo-saccharides (i.e., G4 or G5) as the substrate. In all reactions of the substrate with amylase and reactions of maltase with the substrate, the rate increases, as a function of substrate concentration, to an optimum, at which point it levels off. The substrate concentration at which optimization occurs appears to increase with increasirlg n so that more of the substrate (and the maltase) must be used to optimize (linearize) the standard curve. For expensive chemicals, this is an important consideration.
The substrates of the present invention are the defined polysaccharides covered by the formula given above in which n is an integer between 1 and 10 and R is a sub-stituted aromatic radical which, when detached from the polysaccharide in the form of a phenol or a phenolate anion, exhibits a di.fferent spectral absorbance than the substrate. There are a large number of such radicals.
Chief among them, however, are those radicals selected from the group consisting of y .
~ .
. :
in which X and Y are individually selected from the group consistiny of H, NO2, halogens, alkyls of from 1 to 4 carbon atoms, OR' or CO2R'; where R' is an alkyl of from 1 to 6 carbon atoms, and at least one of X and Y is NO2.
The anions of the phenols formed when these radicals are separated from the glycosyl residue have a maximum absorbance ~max of be-tween about 290 and about 600 nm~
The details of the procedures for preparing these preferred compounds is set forth in Canadian Patent Appli- -cations Serial No. 282,407 (R.C. Burns et al) and Serial No. 282,406 tW.B. Farnham et al), filed on the same day as this application.
Amon~ the preferred embodiments, two compounds are particularly preferred; those in which n is 2 or 3 and R is 4-nitrophenyl. These compounds, ~-(4-nitrophenyl~
maltotetraoside (G4pNp) and ~-(4-nitrophenyl) maltopenta- ~
oside (G5pNp), are used to determine the amylase content o~ ~ -a sample, such as blood serum or urine, accordin~ to the following reaction scheme ~ .
: ': '' ' , " ' . ~, ," ''' :, ' .:
4~
H~e~o~ o~O
OH OH n OH
)l ~Ax 290-305 nm R -. 4 ~ 2 ~ C6H 4 n: 2 n - (4 - NITROPHEN~L ) !~IALTOTETRAOSIDE
n: 3 ~ -t4-NITROPHE~YL) I.~ALTC~ENTAOSIDE
Al~YLASE
~ ' CH20H CH20HCtJ20H
~ H ~ /~ \ H~ ~0\ :
62 'r G3 L Bo~OJ~
R-4-02N~G~J4 n:O a-(4-NITROP~IENYL~ ~.lALTOSIDE
- MAL~ASE
4 ~r5 Gl ~ HO g~NO2 4- NI~ROPHENOL
~,o~
-O~NO2 : ~ .
.
4-NlTROrlJENOlATE ANI()N A~ X 410nm .
The defined oligosaccharide substrate is added to a solution containing a measured amount of the sample to be tested; and the spectral absorbance of the solution is monitored, either as an end point determination or a rate determination using conventional techniques. Usually, as in all enzyme reactions, the reaction solution is main- .
tained at a substantially constant p~ and a substantially constant temperature. When these substances are used, it ~':
: ::
: . , . .
is desirable to perform the assay in a solution which has had its p~I adjus-ted to -the basic range in order to enhance the absorbance at 410 nm. For example~ G4pNp and G5pNp (~max 290-305 nm) and 4-nitrophenol (~max 313 nm~ have a low extinction coefficient at 410 nm compared to 4-nitro-phenolate anion (~max 410 nm).
To best accomplish this, a reagent test kit containing the defined substrate disclosed above and a maltase is used. One exemplary test kit is disclosed in U.S. Patent 3,476,515. This test kit can be used in the analyzer described in U.S. Patent 3,770,382.
A sample o~ ~-(4-nitrophenyl) maltotet~aoside (G~pNp) prepared in accorclance with Example l~I of Canadian Patent Application Serial No. 282,407 (R.C. Burns et al~/
filed on the same da~ as this application, was dissolved in 66.7 mM sodium phosphate buffer, pH 6~5, to provide various ~-substrate concentrations ranging from 2 to 8 mg~3 ml. As described in the aforementioned Canadian Patent Application Serial No. 282,407 (R.C. Burns et al), this substrate sample has been purified using a Sephadex~ LH-20 Chromatographic Column. ~-Glucosidase of various concentrations rang:ing from 2.5 to 12.5 International Units per three millilite~s of solution (IU/3 ml) was then added to the substrate solu-tion and the volume of the solution was hrought up to 3.0 ml. The solution was incubated at 37C. for l to 10 minutes~
After the blank xate was measured at 41Q nm;
using a Gil~ord spectrophotometer, the reaction was initiated by adding 0.1 ml of an Elevated Enzyme Control i Product sold by the E. I. du Pont de Nemours and Company ' ~1~ ~ / ï .
' ., ,, , ,, , ~ ~
(1150 Somogyi Units per deciliter (SU/dl) amylase~ diluted 1:1 with Du Pont Enz~me Diluent. This level of amylase is approximately six times the upper normal serum level.
The total reaction rate was then measured using the Gilford spectrophotometer, and by subtracting the blank rate from the total rate, the net reaction rate was obtained.
A two-variable statistical optimi~ation for the substrate and the ~-glucosidase was run. I`he results of this evaluation are given in Table I in arbitrary Absor-bance units (A) per minute.
TABLE I
,005l .012 .018 12.5 .0912 .113 .llO
,~ .o863 .101 .092 P~ .004.008 .011 7.5 .090.llO .lOg .086.102 .Og8 o ~ .001.008 .002 ~ 2.5 .079.091 .0~0 078.083 .078 2.05.0 8.0 G4pNp mg/3 ml l. Blank rate (A/min) 2. Total rate (A/min)
3. Net rate (A/min) From this evaluation, it can be seen that the blank rate increases as the concentrations of both the G~pNp and the a-glucosidase increase, that the optimum concentration of G4pNp is approximately 4.0 mg/3 ml, and that the optimum concentration of a-glucosidase is approximately 7.5 IU/3 ml.
Using these optimum values of G4pNp and a-glucosi-dase in a reaction solution of 3 ml,'the reaction rates or various amylase sample concentrations were measured and a standard curve was generated. From this curve, the sensitivity in m~/min/SU/dl was measured. The standard curve for this sample is given in Figure l; the blank rate and sensitivity are given for this and other examples ; 10in Table II.
TABLE II
Example Blank Rate Sensitivity (mA/minj (mA/min/SU/dl) 1 5.0 0.115 2 not measured 0.231 3 10.3-13.3 0.110
Using these optimum values of G4pNp and a-glucosi-dase in a reaction solution of 3 ml,'the reaction rates or various amylase sample concentrations were measured and a standard curve was generated. From this curve, the sensitivity in m~/min/SU/dl was measured. The standard curve for this sample is given in Figure l; the blank rate and sensitivity are given for this and other examples ; 10in Table II.
TABLE II
Example Blank Rate Sensitivity (mA/minj (mA/min/SU/dl) 1 5.0 0.115 2 not measured 0.231 3 10.3-13.3 0.110
4 3.0 0.116 -A small amount of the substrate sample used in Example 1 was further purified by High Performance Liquid Chromatography (HPLC) which is a standard puriication techni~ue, well known to those skilled in the art. Using the optimum values for G4pNp and a-glucosidase obtained in Example 1, and the amylase sample of Example 1, a standard ~25 curve was generated using this purified substrate. The sensitivity was also obtained, as described in Example 1.
The standard curve is given in Figure 1, the sensitivity is given in Table II. As can be seen rom Table II, the sensitivity o the assay was increased markedly by the purification, indicating that the substrate sample of Example 1 contained some inhibltor.
-13~
'; ' ~' '' ' ' ' ' :' ' ~ ' "', ' ' '' E~AMPLE 3 The ~-(4-nitrophenyl) maltotetraoside (G~pNp) sample used in this Example was obtained by deacetylation of the HPLC purified acetate of Example lD oE t,he afore-mentioned Canadian Patent Application Serial No. 282,407 (R.C. Burns et al). In particular, to a sample of this acetate, a solution of sodium methoxide and methanol was added and the solution was stirred at room temperature in a closed vessel for 18 hours. The methanol was then removed under reduced pressure.
The G4pNp so formed was dissolved in 66.7 mM
sodium phosphate bu~er, pH 6.5, to provide a substrate concentration of 4 mg/3 ml. Then 7.5 IU/3 ml of ~-glucosi-dase was added to the subskrate solution and the volume o~ ' the solution was brought up to 3.0 ml. The solution was incubated at 37C. for one to ten minutes~
After the blank rate was measured, as described in Example 1 above, the reaction was incubated by adding ',~
0.1 ml of Du Pont Ele~ated Enzym~ Control Product, diluted 1:1 with Du Pont Enzyme Diluent. The reaction rates for various amylase sample concentrations were measured as discussed in Example 1 above, and a standard curve was generated. From this curve, the sensitivity ln m~min~SU/dl was measured. The standard curve ~or this substrate'is given in Figure l; the blank rate and sensitivity are given in Table II.
This is a crude sample; one that has not been purified by chromatographic sepaxation techniques. As a result, the blank rate is very hi,~h,, ran~in~ from 10.3 to 13~3 m~/min, but the sensitivity is equivalent to that o~ the substrate reported in E~ample 1 ~here ini,tial purification was accomplished using a Sephadex~ LH-20 column.
Another series of reactions were run us~ng the conditions descri~ed above, except that five minutes after it was initiated, the reaction was quenched by adding a l.5 ml aliquot of the sample solution in-lo either l.5 ml of 0.2 M Na2CO3 or 5 ml of 0.002 N NaOH. At pH 6.5 r the extinction coefficient o~ the 4-nitrophenol is relatively low because the 4-nitrophenol is no-t all ionized. The increase in pH caused by the quenching is suffIcient to completely ionize the 4-nitrophenol to 4-nitrophenylate anion, thereby increasing the extinction coefficient. This giVes rise to an l'end point" determination for which the standard curves were non-linear, probably because the system was optimiæed ~or a rate and not an end-point approach.
However, a Pive to ten fold lncrease in sensitivity was obseryed.
A sample of a-(4-nitrophenyl) maltotetxaoside~
prepared in accordance with Example lG of the aforementioned Canadian Patent Application Serial No. 282,407 (R.C. Burns et al), was dissolved in 66.7 m~ sodium phosphate buffer, pEI 6.5, to provide a substrate concentration of 4 mg~3 ml.
Then 7.5 IU/3 ml of ~-glucosidase was added to the substrate solution and the volume of the solution was brought up to 3.Q ml. The solution was incubated at 37~C. for one to ten ,: , , ; minutes.
After the blank rate was measured in a Gilford ~ ~`
spectrophotometer at 4l0 n~, the reaction was initiated by addin~ O.l ml of the Du Pont Elevated Enzyme Control Product, diluted l:l with Du Pont Enzy~e Di`luent. The reaction rates ..
., ::
for the various amylase sample concentrations were measured, using -the Gilford spectrophotometer, and a standard curve was generated. From this curve, the sensitivity in m~/min/SU/dl was measured. The standard curve for this sample is given in Figure l; -the blank rate and sensitivity are given in Table II.
This again is a substrate that was purified by using a Sephadex~ LH 20 column. The sensitivity of the assay using the substrate of this Example is equivalent to that o~ the assay reported in Examples 1 and 3. The blank rate, however, is somewhat lower than that of Example 1 and ;~
considerably lower than that o~ Example 3.
EX~MPLE 5 A sample of ~-(4-nitrophenyl) maltopentaoside (G5pNp), prepared in accordance with Example 2E of the aforementioned Canadian Patent Application Serial No. 282,407 (R.C. Burns et al), was dissolved in 6Ç.7 mM sodium phosphate . buf~er, pH 6.5, to provide various substrate concentrations ranging ~rom 4.0 to 12.0 mg/3 ml~ a-Glucosidase of various activity ran~ing from 15 to 45 IU/3 ml was then added to the ;:
substrate solution and the volume o~ the solution was brought up to 3.0 ml. The solution was incubated at 37C~ for one to ten minutes.
Initial tests were conducted using 4.0 m~/3 ml G5pNp and three ~-glucosidase concentratlons, 7.0, 14.0, and 28.0 IU/3 ml. For each of these three: concentrations, as set forth in Example 1~ the standard curves were pro-duced usin~ the amylase sample identi~ied in Example 1~
In each case, the blan.k rate was 3~Q mA~min. The standard curve for the three ~-glucosid~se concentrations a.re given in Figure 2. A11 curves were non-linear which made a --1~--.,~ .
determination o the sensitivity difficulto Sensitivity/
however, is estimated to be greater than .160 mA/min/IU/dl.
Linearit~ increased as a-glucosidase concentration increased indicating that, the ~~~lucosidase concentration was sub-optimal.
A two-variable optimization was performed as described in Example 1. The results of this optimization are given in Table III.
TABLE III
,0031 ~00~ .005 .128 .139 .145 '"""'' ' .1253 .135 .140 H
~ .003 .00~ .012 o~
~ 30 .122 .13S .133 .,.
u~
o .119 .131 .121 , . .
.003 .005 007 .110 .119 .114 4.0 8.0 12.0 G5pNp my/3ml -1, 2, 3 see Table I
From this evaluation, it can be seen that there is little increase in the blank rate as G5pNp or ~-glucosidase concentrations are increased. This is consistent with the situation w1th G5 25 as explained above. This analysis also indicates that the , , , optimum value for either G5pNp or ~-~lucosidase has not been reached at 12.0 m~j3ml or 45 IU/3ml, respectivcly. ~-In the sense that G5pNp has a lower or at least a stable blank rate as a unction o substrate and ~-~luco~idase concentrations, it is a preferred substrate.
However, the large concentrations of G5pNp and ~-glucosidase required for the assay decreases its preferred status.
The disclosure above is intended to instruct those skilled in the art, and is not intended to limit the scope of the invention. Many modiEications well within the skill of the art are intended to he included ~ith the scope of the invention as set forth in the appended claims.
The standard curve is given in Figure 1, the sensitivity is given in Table II. As can be seen rom Table II, the sensitivity o the assay was increased markedly by the purification, indicating that the substrate sample of Example 1 contained some inhibltor.
-13~
'; ' ~' '' ' ' ' ' :' ' ~ ' "', ' ' '' E~AMPLE 3 The ~-(4-nitrophenyl) maltotetraoside (G~pNp) sample used in this Example was obtained by deacetylation of the HPLC purified acetate of Example lD oE t,he afore-mentioned Canadian Patent Application Serial No. 282,407 (R.C. Burns et al). In particular, to a sample of this acetate, a solution of sodium methoxide and methanol was added and the solution was stirred at room temperature in a closed vessel for 18 hours. The methanol was then removed under reduced pressure.
The G4pNp so formed was dissolved in 66.7 mM
sodium phosphate bu~er, pH 6.5, to provide a substrate concentration of 4 mg/3 ml. Then 7.5 IU/3 ml of ~-glucosi-dase was added to the subskrate solution and the volume o~ ' the solution was brought up to 3.0 ml. The solution was incubated at 37C. for one to ten minutes~
After the blank rate was measured, as described in Example 1 above, the reaction was incubated by adding ',~
0.1 ml of Du Pont Ele~ated Enzym~ Control Product, diluted 1:1 with Du Pont Enzyme Diluent. The reaction rates for various amylase sample concentrations were measured as discussed in Example 1 above, and a standard curve was generated. From this curve, the sensitivity ln m~min~SU/dl was measured. The standard curve ~or this substrate'is given in Figure l; the blank rate and sensitivity are given in Table II.
This is a crude sample; one that has not been purified by chromatographic sepaxation techniques. As a result, the blank rate is very hi,~h,, ran~in~ from 10.3 to 13~3 m~/min, but the sensitivity is equivalent to that o~ the substrate reported in E~ample 1 ~here ini,tial purification was accomplished using a Sephadex~ LH-20 column.
Another series of reactions were run us~ng the conditions descri~ed above, except that five minutes after it was initiated, the reaction was quenched by adding a l.5 ml aliquot of the sample solution in-lo either l.5 ml of 0.2 M Na2CO3 or 5 ml of 0.002 N NaOH. At pH 6.5 r the extinction coefficient o~ the 4-nitrophenol is relatively low because the 4-nitrophenol is no-t all ionized. The increase in pH caused by the quenching is suffIcient to completely ionize the 4-nitrophenol to 4-nitrophenylate anion, thereby increasing the extinction coefficient. This giVes rise to an l'end point" determination for which the standard curves were non-linear, probably because the system was optimiæed ~or a rate and not an end-point approach.
However, a Pive to ten fold lncrease in sensitivity was obseryed.
A sample of a-(4-nitrophenyl) maltotetxaoside~
prepared in accordance with Example lG of the aforementioned Canadian Patent Application Serial No. 282,407 (R.C. Burns et al), was dissolved in 66.7 m~ sodium phosphate buffer, pEI 6.5, to provide a substrate concentration of 4 mg~3 ml.
Then 7.5 IU/3 ml of ~-glucosidase was added to the substrate solution and the volume of the solution was brought up to 3.Q ml. The solution was incubated at 37~C. for one to ten ,: , , ; minutes.
After the blank rate was measured in a Gilford ~ ~`
spectrophotometer at 4l0 n~, the reaction was initiated by addin~ O.l ml of the Du Pont Elevated Enzyme Control Product, diluted l:l with Du Pont Enzy~e Di`luent. The reaction rates ..
., ::
for the various amylase sample concentrations were measured, using -the Gilford spectrophotometer, and a standard curve was generated. From this curve, the sensitivity in m~/min/SU/dl was measured. The standard curve for this sample is given in Figure l; -the blank rate and sensitivity are given in Table II.
This again is a substrate that was purified by using a Sephadex~ LH 20 column. The sensitivity of the assay using the substrate of this Example is equivalent to that o~ the assay reported in Examples 1 and 3. The blank rate, however, is somewhat lower than that of Example 1 and ;~
considerably lower than that o~ Example 3.
EX~MPLE 5 A sample of ~-(4-nitrophenyl) maltopentaoside (G5pNp), prepared in accordance with Example 2E of the aforementioned Canadian Patent Application Serial No. 282,407 (R.C. Burns et al), was dissolved in 6Ç.7 mM sodium phosphate . buf~er, pH 6.5, to provide various substrate concentrations ranging ~rom 4.0 to 12.0 mg/3 ml~ a-Glucosidase of various activity ran~ing from 15 to 45 IU/3 ml was then added to the ;:
substrate solution and the volume o~ the solution was brought up to 3.0 ml. The solution was incubated at 37C~ for one to ten minutes.
Initial tests were conducted using 4.0 m~/3 ml G5pNp and three ~-glucosidase concentratlons, 7.0, 14.0, and 28.0 IU/3 ml. For each of these three: concentrations, as set forth in Example 1~ the standard curves were pro-duced usin~ the amylase sample identi~ied in Example 1~
In each case, the blan.k rate was 3~Q mA~min. The standard curve for the three ~-glucosid~se concentrations a.re given in Figure 2. A11 curves were non-linear which made a --1~--.,~ .
determination o the sensitivity difficulto Sensitivity/
however, is estimated to be greater than .160 mA/min/IU/dl.
Linearit~ increased as a-glucosidase concentration increased indicating that, the ~~~lucosidase concentration was sub-optimal.
A two-variable optimization was performed as described in Example 1. The results of this optimization are given in Table III.
TABLE III
,0031 ~00~ .005 .128 .139 .145 '"""'' ' .1253 .135 .140 H
~ .003 .00~ .012 o~
~ 30 .122 .13S .133 .,.
u~
o .119 .131 .121 , . .
.003 .005 007 .110 .119 .114 4.0 8.0 12.0 G5pNp my/3ml -1, 2, 3 see Table I
From this evaluation, it can be seen that there is little increase in the blank rate as G5pNp or ~-glucosidase concentrations are increased. This is consistent with the situation w1th G5 25 as explained above. This analysis also indicates that the , , , optimum value for either G5pNp or ~-~lucosidase has not been reached at 12.0 m~j3ml or 45 IU/3ml, respectivcly. ~-In the sense that G5pNp has a lower or at least a stable blank rate as a unction o substrate and ~-~luco~idase concentrations, it is a preferred substrate.
However, the large concentrations of G5pNp and ~-glucosidase required for the assay decreases its preferred status.
The disclosure above is intended to instruct those skilled in the art, and is not intended to limit the scope of the invention. Many modiEications well within the skill of the art are intended to he included ~ith the scope of the invention as set forth in the appended claims.
Claims (15)
1. A method for the rapid determination of the amylase content of a sample comprising the steps of:
(a) adding to a solution containing a measured amount of the sample a defined polysaccharide substrate having the following formula:
where n is 2, 3 or 4, and R is a substituted aromatic radical which, as a detached aglycone, exhibits a different spectral absorbance than the substrate; and (b) monitoring the spectral absorbance of the solution.
(a) adding to a solution containing a measured amount of the sample a defined polysaccharide substrate having the following formula:
where n is 2, 3 or 4, and R is a substituted aromatic radical which, as a detached aglycone, exhibits a different spectral absorbance than the substrate; and (b) monitoring the spectral absorbance of the solution.
2. The method of Claim 1 wherein R is a substituted aromatic radical selected from the group consisting of in which X and Y are individually selected from the group consisting of H, NO2, halogens, alkyls of from 1 to 4 carbon atoms, OR' or CO2R', where R' is an alkyl of from 1 to 6 carbon atoms, and at least one of X and Y is NO2.
3. The method of Claim 2 wherein R is
4. The method of Claim 3 further comprising the step of adding a maltase to the solution.
5. The method of Claim 4 wherein the maltase is ?-glucosidase.
6. The method of Claim 4 wherein R is 4-nitro-phenyl.
7. The method of Claim 6 wherein the solution is maintained at a substantially constant pH in the basic range and a substantially constant temperature and wherein the spectral absorbance is within one hour of adding the sample to the substrate.
8. The method of Claim 2 wherein the solution is maintained at a substantially constant pH in the basic range and at a substantially constant temperature.
9. In a method for rapidly determining the amylase content of a sample comprising the steps of adding a maltase and a substrate to a solution containing a mea-sured amount of the sample and monitoring the change in spectral absorbance of the solution, the improvement wherein the substrate is ?-(4-nitrophenyl) glycoside of maltotetraose, maltopentaose or maltohexaose.
10. A reagent test kit for rapidly determining the amylase content of a sample comprising:
(a) a defined polysaccharide substrate having the following formula:
wherein n is 2, 3 or 4, and R is a substituted aromatic radical which, when detached from the polysaccharide in the form of a phenolate anion, exhibits a different spectral absorbance than the substrate; and (b) maltase.
(a) a defined polysaccharide substrate having the following formula:
wherein n is 2, 3 or 4, and R is a substituted aromatic radical which, when detached from the polysaccharide in the form of a phenolate anion, exhibits a different spectral absorbance than the substrate; and (b) maltase.
11. The test kit of Claim 10 wherein R is a substituted aromatic radical selected from the group consist-ing of , and in which X and Y are individually selected from the group consisting of H, NO2, halogens, alkyls of from 1 to 4 carbon atoms, OR' or CO2R', where R' is an alkyl of from 1 to 6 carbon atoms, and at least one of X and Y is NO2.
12. The test kit of Claim 11 wherein the maltase is ?-glucsidase.
13. The test kit of Claim 11 wherein R is
14. The test kit of Claim 11 wherein R is 4-nitrophenyl.
15. In a test kit for rapidly determining the amylase content of a sample comprising a maltase and a sub-strate, the improvement wherein the substrate is .alpha. - (4-nitrophenyl) glycoside of maltotetraose, maltopentaose, or maltohexaose.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US70497676A | 1976-07-13 | 1976-07-13 | |
| US704,976 | 1976-07-13 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1104912A true CA1104912A (en) | 1981-07-14 |
Family
ID=24831606
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000282419A Expired CA1104912A (en) | 1976-07-13 | 1977-07-11 | Method and test kit for serum amylase assay |
Country Status (12)
| Country | Link |
|---|---|
| JP (1) | JPS5311092A (en) |
| BE (1) | BE856737A (en) |
| CA (1) | CA1104912A (en) |
| CH (1) | CH633582A5 (en) |
| DE (1) | DE2731421A1 (en) |
| DK (1) | DK155751C (en) |
| FR (1) | FR2358660A1 (en) |
| GB (1) | GB1571642A (en) |
| IE (1) | IE45395B1 (en) |
| IT (1) | IT1114891B (en) |
| LU (1) | LU77751A1 (en) |
| NL (1) | NL179399C (en) |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4102747A (en) * | 1977-07-28 | 1978-07-25 | American Hospital Supply Corporation | Amylase determination |
| DE2755803A1 (en) * | 1977-12-14 | 1979-06-21 | Boehringer Mannheim Gmbh | METHOD AND REAGENT FOR DETERMINING ALPHA-AMYLASE |
| DE2741192C2 (en) * | 1977-09-13 | 1982-07-01 | Boehringer Mannheim Gmbh, 6800 Mannheim | Method for the determination of alpha-amylase |
| AT362526B (en) * | 1977-09-13 | 1981-05-25 | Boehringer Mannheim Gmbh | METHOD AND REAGENT FOR DETERMINING ALPHA AMYLASE |
| DE2752501A1 (en) * | 1977-11-24 | 1979-05-31 | Kurt Prof Dr Wallenfels | Alpha-d-malto:dextrin derivs. as indicators - for colorimetric analysis of amylase(s) |
| JPS5768798A (en) * | 1980-10-14 | 1982-04-27 | Toyo Jozo Co Ltd | Novel measurement of amylase activity |
| DE3328616A1 (en) * | 1983-08-08 | 1985-02-28 | Boehringer Mannheim Gmbh, 6800 Mannheim | OLIGOGLUCOSIDE DERIVATIVES |
| US4649108A (en) * | 1984-07-26 | 1987-03-10 | Genzyme Corporation | Alpha amylase assay |
| US4963479A (en) * | 1986-10-07 | 1990-10-16 | Hoechst Celanese Corporation | Reagent system for an alpha-amylase assay containing aromatic substituted glycoside |
Family Cites Families (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US2094693A (en) * | 1935-06-17 | 1937-10-05 | Trojan Powder Co | Nitration of sugars and their glycosides |
| GB1167083A (en) * | 1966-01-20 | 1969-10-15 | Warner Lambert Pharmaceutical | Assay Method for Amylase |
| FR1508496A (en) * | 1966-01-20 | 1968-01-05 | Warner Lambert Pharmaceutical | Product and method for the assay of amylase |
| SE336915B (en) * | 1968-01-15 | 1971-07-19 | Pharmacia Ab | |
| 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 |
| JPS5185790A (en) * | 1975-01-24 | 1976-07-27 | Ono Pharmaceutical Co | Hitononyooyobitaiekinogurukoamiraazenobiryoteiryoho |
| NL188646C (en) * | 1976-07-13 | 1992-08-17 | Du Pont | PROCESS FOR PREPARING NITROAROMATIC GLYCOSIDES |
-
1977
- 1977-07-11 CA CA000282419A patent/CA1104912A/en not_active Expired
- 1977-07-11 NL NL7707682A patent/NL179399C/en not_active IP Right Cessation
- 1977-07-12 FR FR7721470A patent/FR2358660A1/en active Granted
- 1977-07-12 BE BE179280A patent/BE856737A/en not_active IP Right Cessation
- 1977-07-12 CH CH864077A patent/CH633582A5/en not_active IP Right Cessation
- 1977-07-12 DK DK315777A patent/DK155751C/en not_active IP Right Cessation
- 1977-07-12 IT IT2565377A patent/IT1114891B/en active
- 1977-07-12 DE DE19772731421 patent/DE2731421A1/en active Granted
- 1977-07-13 JP JP8400177A patent/JPS5311092A/en active Granted
- 1977-07-13 LU LU77751A patent/LU77751A1/xx unknown
- 1977-07-13 GB GB2950477A patent/GB1571642A/en not_active Expired
- 1977-07-13 IE IE145577A patent/IE45395B1/en not_active IP Right Cessation
Also Published As
| Publication number | Publication date |
|---|---|
| BE856737A (en) | 1978-01-12 |
| DE2731421C2 (en) | 1988-07-07 |
| IE45395B1 (en) | 1982-08-11 |
| IE45395L (en) | 1978-01-12 |
| GB1571642A (en) | 1980-07-16 |
| DK155751B (en) | 1989-05-08 |
| IT1114891B (en) | 1986-01-27 |
| CH633582A5 (en) | 1982-12-15 |
| FR2358660B1 (en) | 1983-08-12 |
| DE2731421A1 (en) | 1978-02-09 |
| FR2358660A1 (en) | 1978-02-10 |
| DK315777A (en) | 1978-01-14 |
| NL179399B (en) | 1986-04-01 |
| NL7707682A (en) | 1978-01-17 |
| JPS5753079B2 (en) | 1982-11-11 |
| NL179399C (en) | 1986-09-01 |
| DK155751C (en) | 1989-10-23 |
| JPS5311092A (en) | 1978-02-01 |
| LU77751A1 (en) | 1978-02-02 |
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