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Description  |
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BACKGROUND
This invention relates to a process and diagnostic device for the
determination of ammonia and of substrates which react with the formation
of ammonia.
The determination of urea in body fluids is of great importance for the
diagnosis and monitoring of diseases of the kidney. For some time now, a
number of wet chemical processes have been available for this purpose
which depend upon the following principles: In the case of a direct
method, urea is reacted with diacetylmonoxime and the adsorption of the
resulting compound is measured photometrically. Other more modern methods
have, prior to the color reaction, an enzymatic step in which the urea is
decomposed to ammonium carbonate by means of the enzyme urease. The
ammonia is subsequently reacted, for example in the known Nessler color
reaction or is converted according to berthelot's method, with
phenol/hypochlorite, into a colored indophenol. The extinction of the
colored reaction products formed by these reactions is finally a measure
of the amount of urea initially present.
These processes are themselves of sufficient exactitude but, nevertheless,
require the use of expensive photometers and trained personnel with
sufficient experience in precise pipetting and in handling reagents, some
of which are of limited stability and are also corrosive. Since these
processes cannot be applied to whole blood, additional laboratory devices
are necessary in order to obtain serum or plasma.
In emergency cases, for example in cases of uraemic coma, it is essential
for the therapeutic measures which have to be taken that an accurate
analytical result be available in the shortest possible time. Therefore,
extensive efforts have also been made to develop methods for the
determination of urea in body fluids which enable analysis results to be
obtained in the shortest possible time which are of good diagnostic value.
Rapid tests in the form of test strips have recently achieved importance
because of simplicity of handling and, in some cases, also the shorter
reaction time, as well as the usability at the sick bed and in
emergencies, and also use by untrained personnel or even by the patients
themselves.
A rapid test for the determination of urea in body fluids, such as blood,
serum and plasma, is described in German Pat. No. 1,240,306. In this case,
urease, pH indicator, buffer and adjuvants are impregnated together onto a
paper. Upon dropping a body fluid thereon, neutral-reacting urea contained
therein is converted into ammonium carbonate, which has an alkaline
reaction. The change in pH is indicated by a change in color of the pH
indicator and the urea content of the sample can be estimated by
comparison with comparative colors. In spite of certain advantages, such
as ease of handling and a short reaction time, these test strips can only
be used for rough estimations because of the difficulty of differentiating
their reaction colors and because of the marked influence of the acid-base
content of the body fluid.
The problem of dependence upon the acid-base content of body fluids is
solved by strips described in German Pat. No. 1,245,619 in which an
absorbent paper strip is impregnated side-by-side with three different
solutions. in the case of these test strips, the urea-containing solution
is first absorbed into a urease-containing test zone in which the urea
reacts to give ammonium carbonate. The solution is then transferred by
capillary forces to a neighboring alkaline test zone which liberates
gaseous ammonia from the ammonium carbonate which passes over the gas
space surrounding the test strips into a third test zone and colors a pH
indicator present therein corresponding to the concentration of the urea.
Transmission of the alkaline solution is prevented by means of an
approximately 2 mm. wide hydrophobic zone between the second and third
reaction zones. Since, in this manner, only gaseous ammonia passes over to
the third reaction zone, other bases and acids present do not disturb the
reaction. Furthermore, by means of buffering, the first test zone can be
adjusted to an optimum pH value for urease so long as the degree of
alkalinity of the second test zone is sufficient to liberate the ammonia
quantitatively.
In spite of results which are of good diagnostic value, these test strips
suffer from a number of disadvantages. The determination can only be
carried out with serum or plasma but not with blood because the blood
corpuscles disturb the chromatography. This necessitates centrifuging the
blood, i.e., a large sample is needed and can only be obtained by a
physician or appropriately well-trained personnel. In order to make the
transmission of the ammonia through the gas space reproducible, the test
strips must, in addition, be suspended in a special reaction chamber and
precisely fixed relative to the sample during the reaction period. For the
chromatography in the first and second reaction zones and especially for
the diffusion of the ammonia through the gas space, a reaction time of 30
minutes is necessary, so that the test strips can scarcely be called rapid
tests.
In German Pat. No. 2,249,647, there is described a similarly constructed
test strip which possesses some constructional improvements. In this case,
determinations can also be carried out on whole blood. However, in normal
cases, these test strips also require a reaction time of 30 minutes and
also require the use of a reaction chamber.
A further disadvantage of both of the above-mentioned test strips is the
fact that the concentration of the urea takes place with a visual
longitudinal measurement of the colorchanging zone of the indicator paper
with an unclear boundary zone which is difficult to assess and cannot be
carried out by the visual or precise measurement of the color depth with a
remission photometer.
In German Pat. No. 2,626,367, there is described a device with several
layers lying one on top of the other. For the detection of urea, these
consist of a urease-containing layer, an alkaline buffer-containing layer
and an indicator layer for detecting gaseous ammonia. In order to prevent
the undesired diffusion of sample fluid from the urease layer to the
indicator zone in addition to the gaseous ammonia, the urease layer and
the indicator layer lying thereunder are separated by a hydrophobic,
gas-permeable film or the indicator layer is made in the form of a
hydrophobic film. The indicator layer, the intensity of the color of which
differs according to the urea content of the sample, is measured through a
carrier film and the concentration of the urea is thus determined.
In order to achieve reaction times of about 10 minutes, this test device
must be incubated at 40.degree.-50.degree. C. because of the relatively
high impermeability of the separating layer. Because of the cost of
apparatus which this involves, it is not possible to use this test device
as a rapid test.
Thus, all the previously known test strips for the determination of urea
suffer from substantial disadvantages.
The present invention provides a device for the detection of urea in body
fluids which: is simple to handle and does not require the additional use
of a reaction chamber; enable a urea determination to be carried out not
only in serum or plasma but also in small amounts of whole blood; at
ambient temperature requires a reaction time of at most 10 minutes and
preferably of about 5 minutes; gives semiquantitative results in the case
of visual assessment; and in the case of remission photometric evaluation,
enables a quantitative determination of urea to be carried out.
SUMMARY
According to the present invention, there is provided a diagnostic device
for the determination of ammonia and of substrates reacting with the
formation of ammonia, comprising a handle, an indicator layer for gaseous
ammonia fixed thereon and an alkaline buffer-containing reaction layer
which optionally contains additional reagents reacting with the substrate
with the formation of ammonia, wherein the indicator layer is securely
attached to the handle and above it is arranged the reaction layer at a
distance of 10 to 200.mu. by means of a distance piece, the distance piece
and the reaction layer being easily removable from the indicator layer.
DESCRIPTION OF THE DRAWING
The present invention will be more fully understood from the following
description taken in conjunction with the accompanying drawing which is an
exploded side view of a device according to the invention.
DESCRIPTION
The device of the invention can be used not only for the detection and
quantitative determination of ammonia itself but also of ammonium salts
and of substrates which react with the liberation of ammonia when, instead
of urease, there are used the appropriate reagents which react with the
substrate in question with the formation of ammonia. The following
substrate/reagent combinations are mentioned by way of example:
urea/urease;
creatinine/creatinine deiminase;
amino acid/amino acid dehydrogenase;
amino acid/amino acid oxidase;
amino acid/amino acid dehydratase;
amino acid/ammonia lyase;
amine/amine oxidase;
diamine/amine oxidase;
glucose/phosphoramidate-hexose-phosphoric transferase and phosphoramidate;
adenosine diphosphate/carbamate kinase and carbamoyl phosphate;
acid amide/amidohydrolase;
nucleobase/deaminase;
nucleoside/deaminase; and
nucleotide/deaminase.
The present invention is characterized by an ammonia-forming and
simultaneously ammonia-expelling reaction layer fixed at a small but
exactly reproducible distance above an indicator layer without the free
diffusion of the ammonia through films, layers and the like being
hindered. Free diffusion and a small distance are prerequisites for a
short reaction time. Only with an exactly reproducible distance of both
layers, even in the case of large scale production, can such a test strip
satisfy the quantitative requirements.
One embodiment of the device of the invention is shown in the drawing. The
device includes an indicator layer 1 which preferably consists of an
absorbent carrier, such as paper, a porous synthetic resin or the like,
and contains a reagent mixture, the remission photometric properties of
which are changed gradually by the action of gaseous ammonia, depending
upon the amount of ammonia. The indicator layer preferably contains an
appropriate pH indicator, which is preferably a
tetranitrodiphenylmethylpyridine indicator (as described in commonly-owned
copending application Ser. No. 34,720 filed Apr. 30, 1979) and a buffer,
for example a citrate, tartrate or malonate buffer or also a polymeric
buffer, for example a polyacrylate or polymethacrylate or copolymer
thereof or some other carboxyl group-containing polymer.
Over the indicator layer 1 there is arranged a spacer 2 which has a
constant thickness of about 10 to 200.mu. and also a uniform hold surface,
the open area of which accounts for at least 25% of the total surface
area. The spacer can consist of a hydrophobed woven or knitted mesh or of
a perforated film which satisfies the above requirements, it being
preferable to employ a hydrophobed woven fabric. Such meshes are known,
for example, as screen printing cloth or as bolting cloth.
In the case of a perforated film, there can be used a film with many
relatively small holes which then corresponds practically to a woven mesh
but a film can also be used which only has one large hole exactly over the
middle of the test region.
Above the spacer 2, there is a reaction layer 3 which preferably consists
of an absorbent carrier, for example paper. This reaction layer 3 contains
the ammonia-forming reagents, an alkaline buffer, for example an
ethylenediaminetetraacetate (EDTA), tris-(hydroxy-methyl)-aminomethane
(TRIS) or a phosphate buffer, with a pH of 7 to 9.5, a TRIS buffer with a
pH of 8.5 being preferred, and adjuvants, such as wetting agents and/or
stabilizers.
The reaction layer 3 is preferably held firmly with a mesh 4, for example a
woven or knitted material or the like, in the manner described in German
Pat. No. 2,118,455 but it can also be securely stuck or sealed onto the
distance piece in some other manner. As shown in the drawing, one end of
the spacer 2 extends beyond layer 3 and mesh 4 so that all three members
can be readily stripped off or removed together at the end of a test to
reveal indicator lever 1.
For carrying out a substrate determination, a drop of test fluid is applied
to the reaction layer 3. In order to prevent the upward escape of ammonia,
the upper side of the reaction layer is preferably covered with an
appropriate layer, for example with an adhesive label or the like. After a
reaction time of 2 to 10 minutes and normally of 5 to 7 minutes, the
reaction layer and the spacer are pulled off. The coloration of the thus
uncovered indicator layer can be measured quantitatively with a remission
photometer or semiquantitatively by visual comparison with standard
colors.
The following examples are intended to illustrate the present invention:
EXAMPLE 1
Quantitative Determination of Urea in Serum
(a) Urease paper (3)
Filter paper is impregnated with a solution with the following composition,
dried and then cut up into 6 mm. wide strips:
______________________________________
urease (5U/g.) 6 g.
dithioerythritol 0.1 g.
0.3M TRIS . HCl buffer (pH 8.5)
100 ml.
______________________________________
(b) Indicator
Filter paper is impregnated with a solution with the following composition,
dried and also cut up into strips with a width of 6 mm.
______________________________________
N-[bis-(2,4-dinitrophenyl)-
0.39 g.
methyl]-4-tert.-butyl-
pyridinium chloride
ethylene glycol monomethyl
42 ml.
ether
0.25 M sodium malonate buffer
48 ml.
(pH 2.8)
______________________________________
(c) Spacer (2)
Screen printing cloth with a filament thickness of about 100.mu. and with
an open surface area of about 35% of the whole surface area is rendered
hydrophobic with silicone resin and cut up into 25 to 40 mm. wide strips.
(d) Covering mesh (4)
Hydrophilic nylon mesh of about 60.mu. thickness and 40.mu. filament
thickness and with a free holed surface area of about 65% of the whole
surface area is cut up into 15 mm. wide bands.
(e) Handle (5)
As carrier film and handle, there is used a 6 to 10 cm. wide, approximately
0.2 to 0.3 mm. thick band of melt adhesive-coated polyester film.
Production of the test strips
Urease paper (3), indicator paper (1) and spacer (2) are sealed together
with a mesh (4) covering the urease paper, as shown in the drawing, onto
the end of the 6 to 10 cm. wide handle (5) coated with melt adhesive and
the resultant band is cut up into 6 mm. wide strips so that 6.times.6 mm.
test zones result on a 6 to 10 cm. long handle.
10 .mu.l. of serum is dropped onto the covering mesh of such a strip and
covered with an adhesive label. After a reaction time of 7 minutes, the
urease paper (3) and covering mesh (4) are removed, together with the
spacer (2). The coloration of the indicator layer (1) is measured from
above with a remission photometer. Depending upon the urea concentration,
the following measurement values are obtained:
______________________________________
measurement signal (scale
mg. urea/100 ml.
divisions) .+-. 1 s; average
serum value from 10 values
______________________________________
20 12.9 .+-. 0.75
40 27.5 .+-. 1.5
60 46.1 .+-. 1.9
80 61.5 .+-. 1.4
100 69.0 .+-. 0.4
150 77.2 .+-. 0.5
200 79.4 .+-. 0.5
______________________________________
EXAMPLE 2
Quantitative Detection of Urea in Blood
(a) Urease paper (3)
Same as Example 1.
(b) Indicator paper (1)
Filter paper is impregnated with a solution of the following composition
and dried:
______________________________________
N-[bis-(2,4-dinitrophenyl)-methyl]-
0.44 g.
4-tert.-butyl-pyridinium chloride
ethylene glycol monomethyl ether
40 ml.
polyacrylate ("Acrytex" SL 865 of
6 g.
the firm Rohm)
water 60 ml.
("Acrytex" is a Registered Trade Mark).
______________________________________
(c) Spacer (2)
20.mu. thick polypropylene film with a perforated surface of 35 to 45% of
the whole surface area.
(d) Covering mesh (4)
Same as Example 1.
(e) Handle (5)
Same as Example 1.
The production of the test strips and the urea determination are carried
out as described in Example 1 but with the use of EDTA blood plasma, the
following results being obtained:
______________________________________
measurement signals (scale
mg. urea/100 m.
divisions) .+-. 1 s; average
plasma values from 10 values
______________________________________
20 11.6 .+-. 0.6
40 23.0 .+-. 1.3
60 41.2 .+-. 1.9
80 56.7 .+-. 1.5
100 66.4 .+-. 0.5
150 75.3 .+-. 0.5
200 78.2 .+-. 0.4
______________________________________
EXAMPLE 3
Semiquantitative Detection of Urea in Blood
(a) Urease paper (3)
Same as Example 1.
(b) Indicator paper (1)
Filter paper is impregnated with a solution of the following composition
and dried at 70.degree. C.:
______________________________________
bromophenol blue 0.1 g.
ethylene glycol monomethyl ether
9 ml.
tartaric acid 0.4 g.
water 21 m.
______________________________________
(c) Spacer (2)
Polyamide fleece with a thickness of about 80.mu. and hydrophobed with
silicone resin.
(d) Covering mesh (4) and Handles (5)
Same as Example 1.
Preparation is the same as Example 1.
For urea determination in whole blood, a drop of blood is applied to the
test strip. After a reaction time of 7 minutes, reaction colors which can
be visually easily differentiated are obtained according to the urea
content:
______________________________________
mg. urea/100 ml.
blood color
______________________________________
20 yellow
40 greenish-yellow
60 yellow-green
80 green
100 blue-green
150 greenish-blue
200 blue
______________________________________
EXAMPLE 4
Quantitative Detection of Creatinine in Serum or Blood
(a) Creatinine deiminase paper
Filter paper is impregnated with a solution of the following composition,
dried and cut up into 6 mm. wide bands:
______________________________________
creatinine deiminase 2000 U
dithioerythritol 0.1 g.
0.3M TRIS . HCl buffer (pH 8.5)
100 ml.
______________________________________
(b) Indicator layer
A mass of the following composition is coated with a thickness of 0.1 mm.
onto a polycarbonate film, dried and cut up into 6 mm. bands:
______________________________________
N-[bis-(2,4-dinitrophenyl)-
0.14 g.
methyl]-4-tert.-butyl-
pyridinium chloride
hydroxypropylcellulose 0.18 g.
("Culminal" PK 82 (Henkel))
water 30 ml.
0.01N hydrochloric acid 1.5 ml.
("Culminal" is a Registered Trade Mark).
______________________________________
(c) Spacer
Screen printing cloth of about 100.mu. filament thickness and with an open
surface area of about 35% of the whole surface area is hydrophobed with
silicone resin and cut up into 25 to 40 mm. wide bands.
(d) Covering mesh
Hydrophilic nylon mesh of about 60.mu. thickness and 40.mu. filament
strength and with approximately 65% free hole surface area of the whole
surface area is cut up into 15 mm. wide bands.
(e) Handle
As carrier foil and handle, there is used a 6 to 10 cm. wide and
approximately 0.2 to 0.3 mm. thick band of melt adhesive-coated polyester
film.
Production of the test strips
Creatinine deiminase paper (3), indicator paper (1) and spacer (2) are
sealed, together with a mesh (4) covering the creatinine deiminase paper
as shown in the FIGURE of the accompanying drawing, onto the end of the 6
to 10 cm. wide film coated with melt adhesive and the resultant band is
cut up into 6 to 10 mm. wide strips, so that 6.times.6 mm. test zones
result on a 6 to 10 cm. long handle.
10 .mu.l. of serum are applied to the covering mesh of such a strip and
closed with an adhesive label. After a reaction time of 7 minutes, the
creatinine deiminase paper and the covering mesh are removed, together
with the spacer. The coloration of the indicator layer is measured from
above with a remission photometer. Depending upon the creatinine
concentration, the following measurement values are obtained:
______________________________________
mg. creatinine/ measurement signal
100 ml. (scale divisions)
______________________________________
0 28
0.5 37
1 47
2 60
3 68
4 73
5 76
6 78
7 80
8 82
9 83
10 84
______________________________________
The creatinine concentrations determined in this manner are, of course,
still falsified by the ammonia which is also present in the serum. The
true creatinine values are obtained when the ammonia concentration values
obtained according to the following Example 5 are substracted or the
ammonia is removed from the serum before the measurement by suitable
means, for example with an ion exchanger.
The falsification due to ammonia is only of importance in the case of
creatinine determinations but not in the case of the urea determination
according to Examples 1 and 2 because of the more favorable ratios of the
normal values occurring in the serum:
______________________________________
ammonia 0.016-0.038 mMol/l.
creatinine 0.044-0.097 mMol/l.
urea 3.5-8.3 mMol/l.
______________________________________
EXAMPLE 5
Quantitative Determination of Ammonia in Serum or Blood
(a) Ammonia-expelling paper
Filter paper is impregnated with an aqueous solution of 0.3 M TRIS.HCl
buffer (pH 9), dried and cut up into 6 mm. side bands.
(b)-(e) Analogous to Example 4
The production and use of the test strips is also analogous to Example 4.
Depending upon the ammonia concentrations, the following measurement values
are obtained:
______________________________________
.mu.g. ammonia/ measurement signal
100 ml. (scale divisions)
______________________________________
0 27
50 41
100 50
150 54
200 59
300 65
600 75
1500 85
______________________________________
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