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Claims  |
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We claim;
1. A method for the simultaneous determination of glucose and urea in a
sample with a single reagent system in a reaction mixture comprising:
adding in a single step a reagent system containing enzymatic reagents and
reactants for the simultaneous determination of glucose and urea, each
enzymatic reagent and reactant being selected such that it capable of
contributing to a discrete electromagnetic radiation signal for either
glucose or urea, wherein the determination of glucose does not interfere
with the determination of urea, and the determination of urea does not
interfere with the determination of glucose;
simultaneously reacting a sample containing glucose and urea with the
reagent system; and
monitoring changes in absorbance or fluorescence of the resulting reaction
mixture at two or more wavelengths, thereby separately but simultaneously
determining the concentration of glucose and urea in the sample.
2. A method for the simultaneous determination of glucose and urea in a
sample with a single reagent system in a reaction mixture comprising:
adding in a single step a reagent system containing enzymatic reagents and
chromophores for the simultaneous determination of glucose and urea, each
enzymatic reagent and chromophore being selected such that it is capable
of contributing to a discrete electromagnetic radiation absorbance signal
for either glucose or urea, wherein the determination of glucose does not
interfere with determination of urea, and the determination of urea does
not interfere with the determination of glucose;
simultaneously reacting a sample containing glucose and urea with the
reactant system; and
monitoring changes in absorbance or fluorescence of the resulting reaction
mixture at two or more wavelengths, thereby separately but simultaneously
determining the concentration of glucose and urea, in the sample.
3. The method of claim 2 wherein said chromophore used in the glucose
determination is selected from the group consisting of: NAD(H), NADP(H),
thio-NAD(H), thio-NADP(H), hypoxanthine-NAD(H), hypoxanthine-NADP(H),
pyrroloquinone and chromogenic oxygen acceptors.
4. The method of claim 2 wherein said chromophore sued in the area
determination is selected from the group consisting of: NAD(H), NADP(H),
thio-NAD(H), thio-NADP(H), hypoxanthine-NAD(H), hypoxanthine-NADP(H) and
pH indicator dyes.
5. The method of claim 2 wherein said reagent comprises an NADP(H)-specific
enzyme system for the determination of glucose with thio-NADP(H) as a
chromophore; and an NAD(H)-specific enzyme system for the determination of
urea with NAD(H) as a chromophore.
6. The method of claim 2 wherein said reagent comprises an NAD(H)-specific
enzyme system for the determination of glucose with NAD(H) as a
chromophore; and an NAD(H)-specific enzyme system for the determination of
urea with thio-NADP(H) as a chromophore.
7. The method of claim 2 wherein said reagent comprises an oxidized or
reduced pyrroloquinone-specific enzyme system for the determination of
glucose with 2,6-dichlorophenolindophenol as a chromophore; and an
NAD(P)(H) enzyme system for the determination of urea with NAD(P)(H) as a
chromophore.
8. The method of claim 5 wherein said reagent comprises glucose
dehydrogenase and thio-NADP for the determination of glucose; and urease,
alpha-ketoglutarate, GLDH, and NADH for the determination of urea.
9. The method of claim 8 which additionally includes mutarotase for the
determination of glucose.
10. The method of claim 5 wherein said reagent comprises NADP specific
glucose dehydrogenase an thio-NADP for the determination of glucose; and
urease, pyruvate, NADH specific ADH, an LDH inhibitor, and NADH for the
determination of urea.
11. The method of claim 10 which additionally includes mutarotase for the
determination of glucose.
12. The method of claim 5 wherein said reagent comprises NADP specific
glucose-6-phosphate dehydrogenase, hexokinase, ATP, Mg and thio-NADP for
the determination of glucose; and urease, pyruvate, NADH specific ADH, an
LDH inhibitor, and NADH for the determination of urea.
13. The method of claim 5 wherein said reagent comprises NADP specific
glucose-6-phosphate dehydrogenase, hexokinase, ATP, Mg and thio-NADP for
the determination of glucose; and urease, alpha-ketoglutarate, NADH
specific GLDH, and NADH for the determination of urea.
14. The method of claim 5 wherein said reagent comprises NADP specific
glucose dehydrogenase and thio-NADP for the determination of glucose; and
urease, 2-oxoisocapnoate, NADH specific leucine dehydrogenase, an LDH
inhibitor and NADH for the determination of urea.
15. The method of claim 14 which additionally includes mutarotase for the
determination of glucose.
16. The method of claim 5 wherein said reagent comprises NADP specific
glucose-6-phosphate dehydrogenase, hexokinase, ATP, Mg and thio-NADP for
the determination of glucose; and urease, 2-oxoisocapnoate, NADH specific
leucine dehydrogenase, an LDH inhibitor and NADH for the determination of
urea.
17. The method of claim 5 wherein said reagent comprises NADP specific
glucose dehydrogenase and thio-NADP for the determination of glucose; and
urease, glutamine synthetase, ATP, phosphoenolpyruvate, pyruvate kinase,
NADH specific lactate dehydrogenase, and NADH for the determination of
urea.
18. The method of claim 17 which additionally includes mutarotase for the
determination of glucose.
19. The method of claim 2 wherein said reagent comprises NAD(P) glucose
dehydrogenase and NAD(P) for the determination of glucose; and urease, and
a pH indicator dye for the determination of urea.
20. The method of claim 19 which additionally includes mutarotase for the
determination of glucose.
21. The method of claim 2 wherein said reagent comprises NAD(P)
glucose-6-phosphate dehydrogenase, hexokinase, ATP, Mg and NAD(P) for the
determination of glucose; and urease, and a pH indicator dye for the
determination of urea.
22. The method of claim 7 wherein said reagent comprises glucose
dehydrogenase (E.C.1.1.99.10) and 2,6-dichloroindophenol for the
determination of glucose; and urease, alpha-ketoglutarate, GLDH, and NADH
for the determination of urea.
23. The method of claim 22 which additionally includes mutarotase for the
determination of glucose.
24. The method of claim 7 wherein said reagent comprises glucose
dehydrogenase (E.C.1.1.99.10) and 2,6-dichloroindophenol for the
determination of glucose; and urease, pyruvate, ADH, an LDH inhibitor, and
NAD(P)(H) for the determination of urea.
25. The method of claim 24 which additionally includes mutarotase for the
determination of glucose.
26. The method of claim 7 wherein said reagent comprises glucose
dehydrogenase (E.C.1.1.99.10) and 2, 6 dichloroindophenol for the
determination of glucose; and urease, 2-oxoisocapnoate, leucine
dehydrogenase, an LDH inhibitor and NAD(P)(H) for the determination of
urea.
27. The method of claim 26 which additionally includes mutarotase for the
determination of glucose.
28. The method of claim 7 wherein said reagent comprises glucose
dehydrogenase (E.C.1.1.99.10) and 2,6 -dichloroindophenol for the
determination of glucose; and urease, glutamine synthetase, ATP,
phosphoenol-pyruvate, pyruvate kinase, lactate dehydrogenase, NADH for the
determination of urea.
29. The method of claim 28 which additionally includes mutarotase for the
determination of glucose.
30. The method of claims 1 or 2 wherein the changes in absorbance or
fluorescence are monitored by measuring changes in a reaction rate or an
endpoint reaction.
31. The method of claim 2 wherein said reagent comprises glucose oxidase,
hydrogen peroxide and a chromogenic oxygen acceptor for the determination
of glucose; and urease, GLDH, alpha-ketoglutarate and NAD(P)(H) for the
determination of urea.
32. The method of claim 2 wherein said reagent comprises glucose oxidase,
hydrogen peroxide and a chromogenic oxygen acceptor for the determination
of glucose; and urease, ADH, pyruvate, an LDH inhibitor and NAD(P)(H) for
the determination of urea.
33. The method of claim 2 wherein said reagent comprises glucose oxidase,
hydrogen peroxide and a chromogenic oxygen acceptor for the determination
of glucose; and urease, leucine dehydrogenase, 2-oxoisocaproate, an LDH
inhibitor and NAD(P)(H) for the determination of urea.
34. The method of claim 2 wherein said reagent comprises glucose oxidase,
hydrogen peroxide and a chromogenic oxygen acceptor for the determination
of glucose; and urease, glutamine synthetase, ATP, phosphoenol-pyruvate,
pyruvate kinase, lactate dehydrogenase and NAD(P)(H) for the determination
of urea. |
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Claims |
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Description |
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BACKGROUND OF THE INVENTION
The present invention relates to a simultaneous assay for glucose and urea
with a single reagent by monitoring concurrent reactions which produce
changes in the electromagnetic radiation absorbance characteristics of the
sample. In one aspect, the invention relates to the simultaneous
measurement of glucose and urea in blood serum by monitoring two
concurrent reactions at two or more different wavelengths.
In the field of diagnostics, various assays are designed to identify or
quantify an analyte, such as glucose or urea, which may be present in a
sample material. Unfortunately the assay is usually only specific to one
analyte even though it may be desirable to diagnose more than one analyte
for any given sample. This leads to multiple testing on the same sample
which increases diagnosis cost and decreases efficiency. It is therefore
desirable to develop diagnostic testing which can identify or quantify
multiple analytes in an efficient manner.
For example, glucose and urea are two of the more common tests performed in
the clinical chemistry laboratory. Analysis of glucose is typically done
using either a hexokinase or glucose oxidase method (Tietz, N. W.,
Textbook of Clinical Chemistry, 1986, p. 785). In the hexokinase method
glucose is converted to glucose- 6 -phosphate hexokinase and adenosine
triphosphate. Glucose-6 -phosphate then reacts with glucose- 6 -phosphate
dehydrogenase (G-6-PDH) to produce 6 -phosphogluconate, with the
concomitant reduction of nicotinamide-adenine dinucleotide (NAD) producing
an increase in absorbance at 340 nm. The glucose oxidase method involves
oxidation of glucose to gluconic acid and hydrogen peroxide by glucose
oxidase. The hydrogen peroxide then reacts with peroxidase and a
chromogenic oxygen acceptor to produce a color change in the 400-550 nm
range. A third method for identifying glucose uses glucose dehydrogenase
(Tietz, N. W., Textbook of Clinical Chemistry, 1986, p. 790). Glucose
dehydrogenase converts glucose to gluconolactone with the concomitant
reduction of NAD.
Analysis of urea is typically done using the urease/glutamate dehydrogenase
method (Tietz, N. W., Textbook of Clinical Chemistry, 1986, p. 1268).
Urease converts urea to ammonia and carbon dioxide. The ammonia produced
reacts with glutamate dehydrogenase and alpha ketoglutarate to produce
glutamate, with the concomitant oxidation of NADH producing a decrease in
absorbance at 340 nm.
The assays mentioned above are performed with separate reagents in separate
cuvettes. This costs the clinical chemistry lab time and money. By
combining the two tests into one test the lab would be able to realize an
increase in productivity and also a cost savings.
Combining the two tests is not a straightforward task. Reagents must be
selected that allow precise measurement of one analyte (i.e., glucose),
without interfering in the measurement of the second analyte (i.e., urea).
For example, the combination of the glucose hexokinase and urea urease
methods is eliminated by the fact that both use the NAD/NADH reaction. The
combination of the glucose oxidase and urea urease methods is eliminated
by the fact that peroxidase in the glucose reaction would oxidize the NADH
in the urea reaction.
One way of combining the two assays in a single reaction vessel is to do a
sequential assay such as disclosed in U.S. Pat. No. 4,425,427 to Luderer.
European Application No. 133064 to Cam discloses another sequential assay
where reagent for a first component is added to the vessel and at some
later time a concentration is determined for the first component. Then a
second reagent, which either quenches the first reaction or is added after
the first reaction is complete, is added to the vessel to trigger a
reaction with the second component. At some later time the concentration
of the second component is determined. These reactions can either be
monitored at the same wavelength or at different wavelengths (either
through the use of filter wheels or diode arrays).
U.S. Pat. No. 3,925,162 describes the simultaneous measurement of enzyme
activity in body fluids. In this approach the substrate for each of the
enzymes to be identified are added to a reaction medium with other
reagents and changes in the absorbance or fluorescence of the resulting
reaction system are measured. The present invention is an approach where a
single reagent system is used to simultaneously identify or quantify at
least two analytes by monitoring the electromagnetic signal of the
reaction mixture.
SUMMARY OF THE INVENTION
The present invention is directed toward a method for the simultaneous
determination of glucose and urea with a single reagent system. The method
comprises adding a reagent system containing a reactant for each of the
analytes to be determined, each reactant being selected such that it is
capable of giving a unique electromagnetic radiation absorbance for the
particular analyte which does not interfere with the determination of the
other analyte. The analytes are reacted with their respective reactant
under conditions such that the reaction takes place simultaneously. The
concentration of the analyte is determined by measuring changes in
absorbance or fluorescence of the resulting reaction mixture at a
plurality of wavelengths which are characteristic for each of the analytes
to be determined. The analyte concentration is measured by either
monitoring the reaction rates or the reaction endpoint.
In another aspect the present invention is a method for the simultaneous
determination of glucose and urea with a single reagent system by adding a
reagent system containing a chromophore for each of the analytes to be
determined, each chromophore being selected such that it is capable of
giving a unique absorbance band for the particular analyte which does not
interfere with the determination of the other analyte. The analytes are
reacted with their respective chromophore under conditions such that the
reaction takes place simultaneously. The concentration of the analytes is
determined by monitoring changes in absorbance or fluorescence of the
resulting reaction mixture at a plurality of wavelengths which are
characteristic for each of the analytes to be determined.
In a preferred embodiment the reagent system is an enzyme system where
chromophores are chosen for glucose and urea which have distinguishable
energy spectra such that they can be simultaneously determined. Preferred
chromophores for the determination of glucose are NAD(H), NADP(H),
thio-NAD(H), thio-NADP(H), hypoxanthine-NAP(H), hypoxanthine-NADP(H),
pyrroloquinone, peroxide/chromogenic oxygen acceptor or analogs thereof.
Preferred chromophores for the determination of urea are NAD(H), NADP(H),
thio-NAD(H), thio-NADP(H), hypoxanthine-NAD(H), hypoxanthine-NADP(H),
indicator dyes or analogs thereof. The NAD or NAP chromophores can be used
in either a reduced or oxidised state as is indicated by the (H). This is
because either form can be used to monitor a change in absorbance or
fluorescence.
A reagent system useful in performing the present simultaneous assay
comprises hexokinase, ATP, NADP specific glucose-6-phosphate dehydrogenase
(G-6-PDH), magnesium and thio-NADP for the determination of glucose; and
urease, pyruvate, lactate dehydrogenase (LDH) inhibitor, NADH specific
alanine dehydrogenase (ADH), and NADH for the determination of urea.
Preferably, the LDH inhibitor is oxalate.
The simultaneous assay can also be performed using a reagent comprising
hexokinase, ATP, NADP specific G-6-PDH, magnesium and thio NADP for the
determination of glucose; and urease, alpha ketoglutarate, NADH specific
GLDH, and NADH for the determination of urea.
Another reagent system for performing the simultaneous assay can comprise
NADP specific glucose dehydrogenase (with or without mutarotase) and
thio-NADP for the determination of glucose; and urease, 2-oxoisocaproate,
NADH specific leucine dehydrogenase, an LDH inhibitor and NADH for the
determination of urea.
The simultaneous assay can also be performed with a reagent system
comprising glucose dehydrogenase (with or without mutarotase) and thio
NADP for the determination of glucose; and urease, alpha ketoglutarate,
GLDH, and NADH for the determination of urea
The glucose dehydrogenase reaction can also be coupled to the following
urea reaction. Urease is used to produce ammonia from urea. The ammonia is
reacted with L-glutamate and ATP in the presence of glutamine synthetase
to produce ADP. The ADP is reacted with phosphoenolpyruvate in the
presence of pyruvate kinase to produce pyruvate. The pyruvate is reacted
with NADH in the presence of LDH to produce lactate and oxidized NAD.
Both the hexokinase/G-6-PDH and glucose dehydrogenase assays can be coupled
to a urea reaction comprising urease, 2-oxoisocapnoate, oxalate, leucine
dehydrogenase, and NADH for the determination of urea.
In yet another reagent system for performing the simultaneous assay can
comprise glucose dehydrogenase and NAD in concentrations sufficient to
allow a rate determination of glucose and urease and an indicator dye in
concentrations sufficient to allow an endpoint determination of urea.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a method for simultaneously measuring a
plurality of analytes in a biological fluid. The method utilizes a single
reagent for measurement of each of the analytes by monitoring several
electromagnetic signals simultaneously.
The electromagnetic signals can be monitored simultaneously by a
spectrophotometer, or spectrofluorometer. The measurement of changes in
the reaction mixture can be carried out on any of the instruments by
conventional procedures. The particular change in the system ,i.e.,
wavelength, is not critical, but it is preferable that the changes or
differences in wavelength be as great as possible provided they can be
monitored simultaneously. Preferably, the reaction mixture spectra is
simultaneously monitored to observe reaction rate changes as well as
endpoint determinations.
In a simultaneous assay a reagent containing all the components for
reaction with the analytes to be measured are added to the sample and the
reactions are monitored by the instrument. Typically, a simultaneous assay
is done in a single cuvette with a single reagent, eliminating the need
for a second reagent dispense or other optional steps generally associated
with multiple analyte assays.
A key to the design of a simultaneous assay is the selection of reagents
that will allow the reactions to proceed simultaneously, but without
interfering with each other in determination of results in the clinically
relevant range. A reactant is chosen for each of the analytes to be
determined, each reactant being selected such that it is capable of giving
a unique electromagnetic radiation absorbance for the particular analyte
which permits determination of other analytes. A reactant can be a
chromophore or indicator dye where the reaction will be monitored by
spectra wavelength. For example, by choosing appropriate chromophores an
assay can be developed that will measure glucose and urea simultaneously
as described below.
Preferred chromophores for the determination of glucose are NAD(H),
NADP(H), thio-NAD(H), thio-NADP(H), hypoxanthine-NAP(H),
hypoxanthine-NADP(H), pyrroloquinone, peroxide/chromogenic oxygen acceptor
or analogs thereof. Preferred chromophores for the determination of urea
are NAD(H), NADP(H), thio-NAD(H), thio-NADP(H), hypoxanthine-NAD(H),
hypoxanthine-NADP(H), indicator dyes or analogs thereof. The NAD or NAP
chromophores can be used in either a reduced or oxidised state as is
indicated by the (H). This is because either form can be used to monitor a
change in absorbance or fluorescence.
The reagent and sample are mixed such that each of the analytes is
contacted with their respective reactant under conditions such that the
reaction takes place simultaneously. The addition and mixing of the sample
and reagent is monitored by instrumentation appropriate for the reaction
taking place such as measuring changes in absorbance or fluorescence of
the resulting reaction mixture at a plurality of wavelengths which are
characteristic for each of the analytes to be determined.
Preferably the monitoring of the reaction mixture is begun as soon as the
reagent and sample are intermixed. This allows for monitoring of changes
in either the reaction rate or endpoint reaction for the particular
electromagnetic signal being monitored.
In one example the subject method allows for the simultaneous measurement
of glucose and urea in blood serum using a single reagent. The glucose and
urea reactions proceed at the same time, with measurement of the two
different reactions monitored at two separate wavelengths by a
spectrophotometer. The spectrophotometer employs a diode array detector
having the capability of simultaneously monitoring many wavelengths.
In one aspect, the measurement of glucose is through the use of
nicotinamide-adenine dinucleotide phosphate (NADP) specific
glucose-6-phosphate dehydrogenase (G-6-PDH) coupled with thio-NADP. The
absorbance maximum of thio-NADP is at 404 nm, with relatively little
absorbance change at 340 nm. This allows coupling of the glucose reagent
with a urea reagent using reduced nicotinamide-adenine dinucleotide (NADH)
and alanine dehydrogenase (ADH) since the NADH can be monitored at 340 nm
with little or no absorbance change at 404 nm.
In another approach the G-6-PDH/thio-NADP reaction can be coupled with a
urea reagent using urease, alpha-ketoglutarate, NADH, and glutamate
dehydrogenase. In yet another approach, the G-6-PDH/thio-NADP reaction is
coupled with a urea reagent using urease, NADH, leucine dehydrogenase, and
2-oxoisocaproate or analogs thereof. It should be noted that in the
preceding approaches the GDH/thio-NADP reaction may replace the
G-6-PDH/thio-NADP reaction.
In another aspect the GDH/thio-NADP reaction can be coupled with a urea
reagent using urease, L-glutamate, ATP, glutamine synthetase,
phosphoenolpyruvate, pyruvate kinase, NADH, and LDH.
Alternatively, the G-6-PDH/thio-NADP or GDH/thio-NADP reaction can be
coupled with an indicator reaction similar to that described by Chang in
U.S. Pat. No. 3,950,226. Here non-specific glucose dehydrogenase
(E.C.1.1.1.47) is used and the glucose reaction is monitored by following
the change in absorbance at 340 nm as NAD is reduced to NADH. Tie urea
reaction is monitored by following the change in absorbance of an
indicator dye as ammonia is produced by the action of urease on urea.
Since the urea reaction is dependent on a change in pH, sensitivity can be
increased by using a weakly buffered system and by adjusting the pH away
from the pK of the buffer.
Another approach is to use glucose dehydrogenase (E.C.1.1.99.10) specific
for pyrroloquinone dyes such as 2,6-dichlorophenol indophenol (DCIP). The
glucose reaction is monitored at 600 nm as DCIP is reduced. The urea part
of the assay uses the ADH/NADH (alternatively, GLDH/NADH, leucine
dehydrogenase/NADH or glutamine synthetase/NADH) method and the urea
reaction is monitored at 340 nm as NADH is oxided to NAD.
In a preferred approach the glucose part of the assay consists of
hexokinase, ATP, magnesium, G-6-PDH, and thio-NADP. Glucose in the sample
is phosphorylated by hexokinase in the presence of ATP. The
glucose-6-phosphate produced is subsequently oxidized by an NDP specific
G-6-PDH with the concomitant reduction of thio-NADP to thio-NADPH. The
reduction of thio-NADP is monitored at 404 nm.
The urea art of the assay consists of urease, pyruvate, an LDH inhibitor
such as oxalate, ADH, and NADH. Urea in the sample is hydrolyzed to carbon
dioxide and ammonia by urease. Ammonia subsequently reacts with ADH in the
presence of pyruvate with the concomitant oxidation of NADH to NAD. The
oxidation of NADH is monitored at 340 nm. The preferred LDH inhibitors are
oxalate and oxamate which inhibit LDH in the sample.
ADH preferentially uses NADH over thio-NADPH and so production of
thio-NADPH in the glucose part of the assay will not interfere with the
determination of urea. Also, since the G-6-PDH is specific for NADP,
production of NAD in the urea part of the assay will not interfere with
the determination of glucose.
Thio-NADPH has a maximum absorbance at 404 nm, but also has an absorbance
at 340 nm which does not change in going from oxidized to reduced form.
Thus the combined absorbance at 340 nm of thio-NADP and NADH limits the
amount of either which can be in solution. The optimum concentrations will
be in the range of 0.1 to 0.8 mM. Varying the ratio of NADH to thio-NADP
in the reagent will affect the linear ranges of the chromophores.
To increase linearity for urea, an option is to increase the NADH
concentration and read the delta absorbance at 364 nm. However, since
there is an absorbance change from thio-NADP at 364 nm, a correction
factor will need to be applied to the 364 nm wavelength reading.
A number of ADH substrates were successfully tried, with pyruvate proving
optimal. Likewise, a number of LDH inhibitors were successfully tried,
with oxalate proving optimal.
A number of buffer systems were tried at various pH values. Triethanolamine
(TEA) buffer (pH 8.0-8.8) performed optimally.
To further describe the instant invention the following examples are
provided.
EXAMPLE 1
Glucose/Urea Simultaneous Assay
The following procedure describes a method for performing a simultaneous
assay for glucose and urea by monitoring the endpoints of both the glucose
and the urea reactions. A reagent system was prepared by mixing the
following (U/L is units per liter and mM is millimoles per liter):
1000 U/L hexokinase
2000 U/L G-6-PDH
2.0 mM ATP
1.0 mM magnesium aspartate
0.25 mM thio-NADP
100000 U/L urease
40000 U/L ADH
10 mM pyruvate
40 mM oxalate
0.6 mM NADH
50 mM TEA pH 8.0
Sample is added to the reagent at a ratio of 1:201 and the reaction is
allowed to proceed. After 3 minutes the absorbance is read at 340 nm and
at 404 nm. Concentrations are calculated by comparison with standard
curves.
EXAMPLE 2
Glucose/Urea Simultaneous Assay
The following procedure describes a method for performing a simultaneous
assay for glucose and urea by monitoring the glucose rate of reaction and
the urea reaction endpoint. A reagent system was prepared by mixing the
following (U/L is units per liter and mM is millimoles per liter):
1000 U/L hexokinase
200 U/L G-6-PDH
2.0 mM ATP
1.0 mM magnesium aspartate
0.25 mM thio-NADP
100000 U/L urease
40000 U/L ADH
10 mM pyruvate
40 mM oxalate
0.6 mM NADH
50 mM TEA pH 8.0
Sample is added to the reagent at a ratio of 1:201 and the reaction is
allowed to proceed. The glucose rate of reaction is monitored at 404 nm by
taking a read every 60 sec for three minutes, starting at 60 seconds.
After 3 minute the absorbance is read at 340 nm. Concentrations are
calculated by comparison with standard curves.
EXAMPLE 3
Glucose/Urea Simultaneous Assay
The following procedure describes a method for performing a simultaneous
assay for glucose and urea by monitoring the glucose reaction endpoint and
the urea reaction rate. A reagent system was prepared by mixing the
following (U/L is units per liter and mM is millimoles per liter):
1000 U/L hexokinase
2000 U/L G-6-PDH
2.0 mM ATP
1.0 mM magnesium aspartate
0.25 mM thio-NADP
100000 U/L urease
3000 U/L ADH
10 mM pyruvate
40 mM oxalate
0.25 mM NADH
50 mM TEA pH 8.0
Sample is added to the reagent at a ratio of 1:201 and the reaction is
allowed to proceed. The urea is followed at 340 nm by reading every 60
seconds for three minutes. After 3 minutes the absorbance is read at 404
nm. Concentrations are calculated by comparison with standard curves.
EXAMPLE 4
Glucose/Urea Simultaneous Assay
The following procedure describes a method for performing a simultaneous
assay for glucose and urea by monitoring the rates of both the glucose and
the urea reactions. A reagent system was prepared by mixing the following
(U/L is units per liter and mM is millimoles per liter):
1000 U/L hexokinase
200 U/L G-6-PDH
2.0 mM ATP
1.0 mM magnesium aspartate
0.25 mM thio-NADP
100000 U/L urease
3000 U/L ADH
10 mM pyruvate
40 mM oxalate
0.25 mM NADH
50 mM TEA pH 8.0
Sample is added to the reagent at a ratio of 1:201 and the reaction is
allowed to proceed. Every 60 seconds for three minute the absorbance is
read at 340 nm and at 404 nm. Concentrations are calculated by comparison
with standard curves.
EXAMPLE 5
Glucose/Urea Simultaneous Assay
The following procedure describes a method for performing a simultaneous
assay for glucose and urea by monitoring the endpoints of both the glucose
and the urea reactions. A reagent system was prepared by mixing the
following (U/L is units per liter and mM is millimoles per liter):
10,000 U/L glucose dehydrogenase
2000 U/L mutarotasg
0.25 mM thio-NADP
100000 U/L urease
40000 U/L ADH
10 mM pyruvate
40 mM oxalate
0.6 mM NADH
50 mM TEA pH 8.0
Sample is added to the reagent at a ratio of 1:201 and the reaction is
allowed to proceed. After 3 minutes the absorbance is read at 340 nm and
404 nm. Concentrations are calculated by comparison with standard curves.
EXAMPLE 6
Glucose/Urea Simultaneous Assay
The following procedure describes a method for performing a simultaneous
assay for glucose and urea by monitoring the glucose rate of reaction and
the urea reaction endpoint. A reagent system was prepared by mixing the
following (U/L is units per liter and mM is millimoles per liter):
1000 U/L glucose dehydrogenase
200 U/L mutarotase
0.25 mM thio-NADP
100000 U/L urease
40000 U/L ADH
10 mM pyruvate
40 mM oxalate
0.6 mM NADH
50 mM TEA pH 8.0
Sample is added to the reagent at a ratio of 1:201 and the reaction is
allowed to proceed. The glucose rate of reaction is monitored at 404 nm by
taking a read every 60 seconds for three minutes, starting at 60 seconds.
After 3 minutes the absorbance is read at 340 nm. Concentrations are
calculated by comparison with standard curves.
EXAMPLE 7
Glucose/Urea Simultaneous Assay
The following procedure describes a method for performing a simultaneous
assay for glucose and urea by monitoring the glucose reaction endpoint and
the urea reaction rate. A reagent system was prepared by mixing the
following (U/L is units per liter and mM is millimoles per liter):
10,000 U/L glucose dehydrogenase
2000 U/L mutarotase
0.25 mM thio-NADP
100000 U/L urease
3000 U/L ADH
10 mM pyruvate
40 mM oxalate
0.6 mM NADH
50 mM TEA pH 8.0
Sample is added to the reagent at a ratio of 1:201 and the reaction is
allowed to proceed. The urea is followed at 340 nm by reading every 60
seconds for three minutes. After 3 minutes the absorbance is read at 404
nm. Concentrations are calculated by comparison with standard curves.
EXAMPLE 8
Glucose/Urea Simultaneous Assay
The following procedure describes a method for performing a simultaneous
assay for glucose and urea by monitoring the rates of both the glucose and
the urea reactions. A reagent system was prepared by mixing the following
(U/L is units per liter and mM is millimoles per liter):
1000 U/L glucose dehydrogenase
200 U/L mutarotase
0.25 mM thio-NADP
100000 U/L urease
3000 U/L ADH
10 mM pyruvate
40 mM oxalate
0.6 mM NADH
50 mM TEA pH 8.0
Sample is added to the reagent at a ratio of 1:201 and the reaction is
allowed to proceed. Every 60 seconds for three minutes the absorbance is
read at 340 nm and at 404 nm. Concentrations are calculated by comparison
with standard curves.
EXAMPLE 9
Glucose/Urea Simultaneous Assay
The following procedure describes a method for performing a simultaneous
assay for glucose and urea by monitoring the endpoints of both the glucose
and the urea reactions. A reagent system was prepared by mixing the
following (U/L is units per liter and mM is millimoles per liter):
1000 U/L hexokinase
2000 U/L G-6-PDH
2.0 mM ATP
1.0 mM magnesium aspartate
0.25 mM thio-NADP
100000 U/L urease
8000 U/L GLDH
10 mM alpha ketoglutarate
0.6 mM NADH
50 mM TEA pH 8.0
Sample is added to the reagent at a ratio of 1:201 and the reaction is
allowed to proceed. After 3 minutes the absorbance is read at 340 nm and
at 404 nm. Concentrations are calculated by comparison with standard
curves.
EXAMPLE 10
Glucose/Urea Simultaneous Assay
The following procedure describes a method for performing a simultaneous
assay for glucose and urea by monitoring the glucose rate of reaction and
the urea reaction endpoint. A reagent system was prepared by mixing the
following (U/L is units per liter and mM is millimoles per liter):
1000 U/L hexokinase
200 U/L G-6-PDH
2.0 mM ATP
1.0 mM magnesium apartate
100000 U/L urease
8000 U/L GLDH
10 mM alpha ketoglutarate
0.6 mM NADH
50 mM TEA pH 8.0
Sample is added to the reagent at a ratio of 1:201 and the reaction is
allowed to proceed. The glucose rate of reaction is monitored at 404 nm by
taking a read every 60 seconds for three minutes, starting at 60 seconds.
After 3 minutes the absorbance is read at 340 nm. Concentrations are
calculated by comparison with standard curves.
EXAMPLE 11
Glucose/Urea Simultaneous Assay
The following procedure describes a method for performing a simultaneous
assay for glucose and urea by monitoring the glucose reaction endpoint and
the urea reaction rate. A reagent system was prepared by mixing the
following (U/L is units per liter and mM is millimoles per liter):
1000 U/L hexokinase
2000 U/L G-6-PDH
2.0 mM ATP
1.0 mM magnesium aspartate
0.25 mM thio-NADP
100000 U/L urease
800 U/L GLDH
10 mM alpha-ketoglutarate
0.25 mM NADH
50 mM TEA pH 8.0
Sample is added to the reagent at a ratio of 1:201 and the reaction is
allowed to proceed. The urea is followed at 340 nm by reading every 60
seconds for three minutes. After 3 minutes the absorbance is read at 404
nm. Concentrations are calculated by comparison with standard curves.
EXAMPLE 12
Glucose/Urea Simultaneous Assay
The following procedure describes a method for performing a simultaneous
assay for glucose and urea by monitoring the rates of both the glucose and
the urea reactions. A reagent system was prepared by mixing the following
(U/L is unites per liter and mM is millimoles per liter):
1000 U/L hexokinase
200 U/L G-6-PDH
2.0 mM ATP
1.0 mM magnesium aspartate
0.25 mM thio-NADP
100000 U/L urease
800 U/L GLDH
10 mM alpha ketoglutarate
0.25 mM NADH
50 mM TEA pH 8.0
Sample is added to the reagent at a ratio of 1:201 and the reaction is
allowed to proceed. Every 60 seconds for three minutes the absorbance is
read at 340 nm and at 404 nm. Concentration are calculated by comparison
with standard curves.
EXAMPLE 13
Glucose/Urea Simultaneous Assay
The following procedure describes a method for performing a simultaneous
assay for glucose and urea by monitoring the endpoints of both the glucose
and the urea reactions. A reagent system was prepared by mixing the
following (U/L is units per liter and mM is millimoles per liter):
10,000 U/L glucose dehydrogenase
2,000 U/L mutarotase
0.25 mM thio-NADP
25000 U/L GLDH
10 mM alpha ketoglutarate
0.6 mM NADH
50 mM triethanolamine (TEA) pH 8.0
Sample was added to the reagent at a ratio of 1:201 and the reaction was
allowed to proceed. After 3 minutes the absorbance was read at 364 nm and
at 404 nm. Concentrations were calculated by comparison with standard
curves.
EXAMPLE 14
Glucose/Urea Simultaneous Assay
The following procedure describes a method for performing a simultaneous
assay for glucose and urea by monitoring the glucose rate of reaction and
the urea reaction endpoint. A reagent system was prepared by mixing the
following:
1000 U/L glucose dehydrogenase
200 U/L Mutarotase
0.25 mM thio-NADP
85000 U/L urease
25000 U/L GLDH
10 mM alpha ketoglutarate
0.6 mM NADH
50 mM TEA pH 8.0
Sample was added to the reagent at a ratio of 1:201 and the reaction was
allowed to proceed. The glucose rate of reaction was monitored at 404 nm
by taking a reading every 60 seconds for three minutes, starting at 60
seconds. After 3 minutes the absorbance is read at 364 nm. Concentrations
are calculated by comparison with standard curves.
EXAMPLE 15
Glucose/Urea Simultaneous Assay
The following procedure describes a method for performing a simultaneous
assay for glucose and urea by monitoring the glucose reaction endpoint and
the urea reaction rate. A reagent system was prepared by mixing the
following:
10,000 U/L glucose dehydrogenase
2,000 U/L Mutarotase
0.25 mM thio-NADP
50000 U/L urease
800 U/L GLDH
10 mM alpha ketoglutarate
0.6 mM NADH
50 mM TEA pH 8.0
Sample was added to the reagent at a ratio of 1:201 and the reaction was
allowed to proceed. The urea was followed at 364 nm by reading every 60
seconds for three minutes. After 3 minutes the absorbance is read at 404
nm. Concentrations were calculated by comparison with standard curves.
EXAMPLE 16
Glucose/Urea Simultaneous Assay
The following procedure describes a method for performing a simultaneous
assay for glucose and urea by monitoring the glucose reaction rate and the
urea reaction rate. A reagent system was prepared by mixing the following:
1000 U/L glucose dehydrogenase
0.25 mM thio-NADP
200 U/L Mutarotase
50000 U/L urease
800 U/L GLDH
10 mM alpha ketoglutarate
0.6 mM NADH
50 mM Tris pH 8.0
Sample was added to the reagent at a ratio of 1:201 and the reaction was
allowed to proceed. Every 60 seconds for three minutes the absorbance is
read at 364 nm and at 404 nm. Concentrations were calculated by comparison
with standard curves
EXAMPLE 17
Glucose/Urea Simultaneous with Leucine Dehydrogenase
This method would essentially be the same as in Examples 1-8, except the
ADH would be replaced with leucine dehydrogenase and the pyruvate would be
replaced with 2-oxoisocapnoate (in concentrations suitable for endpoint or
rate determination).
EXAMPLE 18
Glucose/Urea Simultaneous with glutamine synthetase
The glucose reaction for this method would be the same as in Examples 5-8.
The urea part of the reaction would consist of the following (in
concentrations suitable for endpoint or rate determination): urease,
glutamine synthetase, ATP, phosphoenolpyruvate, pyruvate kinase, NADH, and
LDH.
EXAMPLE 19
Glucose/Urea Simultaneous Assay
The following procedure describes a method for performing a simultaneous
assay for glucose and urea which employs a different method than employed
in Examples 1-18. The glucose part of the assay consists of hexokinase,
G-6-PDH, magnesium, ATP, and NAD(P). The reduction of NAD to NADH is
monitored at 340 nm.
The urea part of the assay consists of urease and an indicator dye. The
production of ammonia by the action of urease on urea is monitored by a
change in color of the indicator dye at 564 nm.
A reagent system was prepared by mixing the following:
1000 U/L hexokinase
200 U/L G-6-PDH
2.0 mM ATP
1.0 mM magnesium aspartate
0.6 mM NAD
100,000 U/L urease
0.075 mM Phenol Red
50 mM Tartrate (pH 6.8)
The glucose reaction was monitored at 340 nm every 60 seconds for three
minutes starting after three minutes. The urea delta absorbance was read
at 564 nm after 3 minutes.
EXAMPLE 20
Glucose/Urea Simultaneous Assay
This procedure involves the same urea method as in Example 19. The glucose
part of the assay consists of glucose dehydrogenase with NAD(P) as
coenzyme for the glucose dehydrogenase. The reduction of NAD to NADH is
monitored at 340 nm.
A reagent system was prepared by mixing the following:
50 mM tartrate pH 6.8
0.075 mM Phenol Red
1000 U/L glucose dehydrogenase
200 U/L Mutarotase
0.6 mM NAD
20000 U/L urease
The glucose reaction was monitored at 340 nm every 60 seconds for three
minutes starting after three minutes. The urea delta absorbance was read
at 564 nm after 3 minutes.
EXAMPLE 21
Glucose/Urea Simultaneous Assay
The following procedure describes a method for performing a simultaneous
assay for glucose and urea which employs a different method than employed
in Examples 1-20. The glucose part of the assay consists of glucose
dehydrogenase with DCIP as a coenzyme for the glucose dehydrogenase. The
reduction of DCIP is monitored at 600 nm.
The urea part of the assay consists of urease, ADH, pyruvate, oxalate, and
NADH as the coenzyme for ADH. The oxidation of NADH to NAD is monitored at
340 nm.
EXAMPLE 22
Glucose/Urea Simultaneous Assay
This procedure uses the same glucose method as in Example 21. The urea part
of the assay consists of urease, GLDH, alpha ketoglutarate, and NADH as
the coenzyme for GLDH.
EXAMPLE 23
Glucose/Urea Simultaneous Fluorescent Assay
The following procedure describes a method for performing a simultaneous
assay for glucose and urea which employs fluorescence to determine the
substrate concentration. In this method a spectrofluorometer is used to
monitor the simultaneous reactions. The components of the assay are
essentially the same as in Example 1. The glucose part of the assay is
measured by following the fluorescence emission at 550 with excitation at
400 nm as thio-NADP is reduced to thio-NADPH. The urea part of the assay
is measured by following the fluorescence emission at 440 nm with
excitation at 340 nm as NADH is oxidized to NAD.
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