Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
W095/13536 ~ 7 ~ ~ ~ 7 PCT~S94/1344
GLUCOSE CONTROL MATERIAL FOR TEST STRIPS
BACKGROUND
The present invention relates to control material
useful in validating testing devices such as test strips
and dipsticks. More particularly, the present invention
relates to a non-serum based, aqueous glucose control
material and to a method for making said control
material.
The field of clinical chemistry and clinical
analysis is concerned, inter alia, with the determination
and quantification of various substances in body fluids.
Many examples of the substances which are determined can
be given, and these include cholesterol, urea, cations,
and glucose. These examples of analytes, as well as
others, are assayed in diverse body fluids such as urine
and blood.
The monitoring of the level of glucose in blood is
important to the management of diabetes. The level of
glucose in the blood is controlled by the amount of
carbohydrate ingested and by insulin. Too much insulin
lowers the glucose level, and too little will result in
an abnormally high level of glucose. Both circumstances
lead to serious health problems for the diabetic.
Most of the glucose testing done outside of the
hospital laboratory is done in non-laboratory settings
such as nurses' stations, physicians' offices and at
WO95/13536 2 1 7 5 9 0 7 PCT~S94/1344~
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home. Testing is frequently done by measuring the amount
of glucose in urine. As the level of glucose rises in
the blood, it exceeds the ability of the kidney to
reabsorb it, and glucose is excreted into the urine.
Although measurement of glucose in urine is useful,
measurement of glucose in blood provides a more accurate
reflection of the condition of the subject. Urine
glucose does not accurately reflect the level of glucose
in the blood since the level of glucose in urine is
determined by the level of glucose in the blood and the
ability of the kidney to reabsorb the glucose.
Therefore, the urine sample cannot tell the diabetic how
low his glucose level is.
Dry reagent test strips, sometimes referred to as
dipsticks, are widely used for detecting glucose in urine
and blood. These devices are characterized by their
simplicity of use. In general, such test strips comprise
plastic strips provided at one end thereof with an
absorbent paper portion which has been impregnated with
reagents such as an enzyme system and a color indicator
compound which produces or changes color to form a
detectable signal when the test strip is contacted with
the analyte being determined. This change in color can
be measured by comparing the color formed on the strip
with a standard color chart calibrated to various glucose
concentrations. More recently, however, to more
accurately control the level of glucose in blood,
Wo95/13536 ~ 2 1 75 9 0 7 PCT~S94/1344~
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instruments have been developed which measure the color
change in a reflectance photometer and thereby produce
quantitative results. Examples of reaction systems which
measure glucose using reflectance measurements include
oxidative reactions, such as the glucose
oxidase/peroxidase method, and reductive reactions, such
as the glucose oxidase/ferricyanide method. The latter
method is described in detail in Freitag, U.S. Pat. No.
4,929,545, the content of which is herein incorporated by
reference. Instruments have also been developed which
determine glucose by means of electrochemical methods in
which a change in current is measured.
It will be understood that clinical analysis of the
type described herein requires that any testing system be
extremely accurate. In particular, when automated
systems and instruments are used, it is essential to
ensure that the elements of the analysis are reliable and
that the measurement taken is valid. It is for this
purpose that control reagents are used.
Westgard and Klee, in Textbook of Clinical
ChemistrY, N.W. Tietz, Ed., 1986, p. 430, define "control
material" as "a specimen, or solution, which is analyzed
solely for quality control purposes and is not used for
calibration purposes." This standard reference work goes
on to describe some of the requisites of control
materials as follows: "They need to be stable materials,
available in aliquots or vials, that can be analyzed
W 0 95/13536 ~ 1 7~9~7 PCT~US94/1344~
periodically over a long time. There should be little
vial-to-vial variation so that differences between
repeated measurements can be attributed to the analytical
method alone.".
The above-cited reference, at page 433, discusses
how the matrix of the control material should be the same
as the material being analyzed. To that end, modified
human serum is discussed as one type of control material.
Indeed, the art now recognizes the term "control serum"
as referring to control material based upon serum. This
terminology will be used herein and is different from the
term "control reagent," which, as used hereafter, refers
to a control material which is not based upon, and which
does not contain, serum of any type.
As has been pointed out above, one of the criteria
which control materials have to satisfy is stability.
Control materials based upon serum, however, are
inherently unstable due to the various components
contained therein. Further, sera will vary from source
to source, so uniformity from lot to lot cannot be
guaranteed. Thus, it is sometimes desirable to have a
control material based upon a non-serum or serum-free
medium.
An example of a serum-free control medium, or
"control reagent" as used herein, is described in U.S.
Pat. No. 4,729,959, issued to Ryan, which is directed to
"a stable glucose reference control." This control
WO95/13536 ~ 2 1 7 5 q 0 7 PCT~S9411344~
contains glucose in a range of from about 40 to 500
mg/dl, together with fixed red blood cells, in an aqueous
suspension. The range of glucose concentrations given
are sufficient to cover just about all ranges of glucose
found in, e.g., blood.
The Ryan '959 patent points to a problem with
aqueous control reagents at column 1, lines 50-55.
Briefly, erythrocytes impart a degree of viscosity to
blood which is absent in water based systems. This
problem was also recognized in U.S. Pat. No. 3,920,580
issued to Mast. This patent teaches that aqueous
solutions had not been consistent, and that a lack of
reproducibility was observed when dry reagent strips were
used with such materials. Mast teaches that suitable
reagents could be prepared by using an antidiffusing
agent in combination with glucose and water. The
antidiffusing agents taught by Mast include
polyvinylpyrrolidone, polyvinyl alcohol, polyethylene
glycol, dextran, and bovine serum albumin. Beneficial
amounts are taught to be between about 3 and 35 percent
of antidiffusing agent. The control solution may also
include adjuvants to obtain a particular color or
physical appearance, which include colored latex
particles and water-insoluble lake dyes.
Terashima, in European Appl. No. 266,216, discloses
control or calibration solutions containing a water-
insolu~ie dispersed phase, which can be a solid polymer
WO95/13536 2 1 75 90 7 PCT~S94/1344~
or copolymer having a molecular weight of 105 to 106, a
liquid phase, or an emulsion of natural polymers such as
sodium alginate. Particle sizes taught are about 0.01 ~m
to about 10 ~m, and amounts taught are 1 to 50 percent by
weight, preferably 10 to 50 percent by weight.
Louderback, in U.S. Pat. No. 3,977,995, teaches a
calibrating fluid for automated instruments for blood
cell counting and hemoglobin determination comprising a
solution of hemoglobin which contains latex particles.
The latex particles have a particle size of from about 5
to 20 microns, approximately the size of leukocytes, and
are employed in the calibrating fluid at a concentration
of 8,000 to 22,000 particles per microliter.
Kennamer et al., in U.S. Pat. No. 5,028,542, the
content of which is herein incorporated by reference,
describe a non-serum based, glucose measurement control
reagent in which the viscosity agent polystyrene
sulfonate is used.
It has now been found that a suitable glucose
control reagent can be formed without using any of the
organic, polymeric materials referred to in Mast and
others in the art as required ingredients. Rather, by
combining an inorganic, non-polymeric clay mineral with a
predetermined amount of glucose and water, along with
additional optional materials, a suitable glucose control
reagent can be made.
W0 95/13536 - ` : 2 1 7 5 9 0 7 PCT/US94/1344~
SUMMARY OF THE INVENTION
The present invention is a non-serum based glucose
control reagent which comprises a predetermined, known
amount of glucose, water, and an inorganic clay mineral.
Preferred clay minerals are selected from the smectite
group of clays, and an especially preferred clay is
hectorite. The preferred concentration for the clay
mineral is between about 0.1 and 1 percent by weight. It
was found, quite unexpectedly, that the composition of
the present invention is useful in validating testing
devices such as test strips for the measurement of
glucose. Further, it was surprisingly discovered that
the control reagent of the invention is useful with a
variety of types of glucose testing devices, including
those devices employing oxidative glucose measurement
methods, devices employing reductive glucose measurement
methods, and also with devices utilizing electrochemical
methods for determining glucose. Additional materials
such as a buffer, a preservative, or a surfactant, either
alone or in various additive combinations, may be mixed
with the three required components. Another aspect of
the present invention is a method for making the control
reagent by mixing the glucose, water, and the clay
mineral together.
A preferred clay mineral used in the invention is
selected from the smectite, or montmorillonite, group of
clays, which includes montmorillonite, beidellite,
WO95tl3536 - 2 ~ PCT~S94/13445
nontronite, hectorite, saponite and sauconite. Less
common smectite clays include volkhonskoite, medmonite
and pimelite. An especially preferred clay mineral is
hectorite. Smectites are crystalline clay minerals that
carry a lattice charge and characteristically expand when
solvated with water and alcohols. Hectorite is
preferably and conveniently used in the form of a
rheological additive, a specially processed hectorite
clay gellant with a fine particle size which makes it
readily dispersible in aqueous systems using conventional
high speed dispersers. Wet particles should be fine
enough to pass through a No. 200 sieve and thus less than
about 75 ~m in size. Clay minerals from other groups
such as kaolinite and attapulgite clays are also within
the scope and spirit of the present invention. In
general, the clay chosen must be in a purified form free
from grit, very fine-grained, and dispersible in liquid.
Essential to the invention are a predetermined
amount of glucose, water, and the recited clay mineral.
The water is used, of course, to create a reagent
solution in which the clay particles are suspended. By
"predetermined" is meant that, prior to formulation of
the actual reagent, a concentration of glucose has been
selected. This concentration may vary, as those skilled
in the art will recognize. As has been mentioned above,
the art recognized, e.g., a range of from 40 to 500
mg/dl, but one may envision lower ranges to, e.g., about
WO95/13536 ~ 2 ~ 7 5 9 ~ 7 PCT~S9~/1344~
20 mg/dl. Some typical ranges would be from about 60 to
about 240 mg/dl, or from about 60 to about 300 mg/dl.
The essential features of the invention, when the
reagent is in the form of a dispersion or solution, are
the solvent (water), the predetermined amount of glucose,
and the clay mineral. The clay mineral may be present
in, e.g., a range of about O.l to about l percent by
weight of the reagent. The weight percent of the clay
mineral will be determined by the final reagent viscosity
desired and the desired diffusion or permeability
characteristics of the control material with the
particular testing device with which it is to be used.
Such characteristics will vary according to the
particular clay chosen and its specific properties, which
include the predominant content of the clay mineral,
which is typically a hydrated silicate of aluminum, iron,
or magnesium, the fineness of individual clay particles,
which may be of colloidal size in at least one dimension,
rheological properties, and the property of thixotropy in
various degrees of complexity. Of course, the particular
clay selected should also be one whose reactivity does
not adversely interfere with the determination of
glucose. It is not necessary that the control material
have the same viscosity as whole blood: however, it is
desirable that the permeability of the material, i.e.,
the diffusion rate of the analyte, through the reagent
matrix of the test strip approximate that of whole blood.
woss/13536 2 1 ~ 5 9 0 7 pcT~ss~ll344s
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Optional additional components of the control
material include typical additives such as buffers,
preservatives, and surfactants. The art is replete with
specific examples of suitable and useful additives for
control material, and the skilled artisan will be able to
determine useful amounts from a review of the art.
It may also be desirable to include a colored or
colorable substance in the reagent mixture. This can be
desirable because body fluid samples frequently possess a
particular color as one of their properties. As the
control reagent is being used to calibrate per a body
fluid sample, it can be useful to calibrate against
conditions as similar to the tested fluid as possible,
including color.
BRIEF DESCRIPTION OF THE DRAWING
The present invention will be better understood by
reference to the following detailed description of the
invention when considered in combination with the drawing
that forms part of the specification, wherein:
Fig. 1 is a graph showing the percent reflectance of
the control material of the present invention at varying
levels of glucose.
WO95/13~36 ~ 2 1 7 5 9 0 7 PCT~S9411344~
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DESCRIPTION OF PREFERRED EMBODIMENTS
EXAMPLE 1
Pre~aration of Control Reagent
A preferred formulation of the control reagent of
the present invention was prepared as follows: A 1
percent by weight aqueous dispersion of BENTONE~ EW
(Rheox, Inc., Hightstown, NJ) hectorite clay particles
was prepared. It has been found that best results are
obtained if the dispersion is made under high shear
(minimum 3000 rpm) for at least 15 minutes using tepid
water between pH 7 and 8. This is necessary to break up
the hectorite platelets and thus ensure complete
hydration of the particles and stabilization of
viscosity. Although not an essential feature in the
composition of the present invention, several biocides
were also added, 0.30% by weight 2-phenoxyethanol, 0.30%
by weight imidazolidinyl urea (available as GERMALL~ 115,
GAF Chemicals Corp.), and 0.15% by weight methylparaben.
The dispersion then had glucose added to it in a
predetermined amount, which was found to be 23 mg/dl as
measured using a hexokinase reference method.
EXAMPLE 2
Efficacy of Control Reaqent with Reductive Method
The control reagent described in Example 1 was then
tested for its efficacy. As explained above, one of the
most important features of a control reagent is its
W095/13~36 ~1 75~7 PCT~Sg~tl344~
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consistency, meaning that values obtained using it should
be fairly uniform from run to run.
With this in mind, the control reagent of Example l
was applied to 5 different lots of test strips containing
the glucose determination system described in U.S. Pat.
No. 4~929~545. Briefly, this publication describes the
determination of glucose using a reagent containing a
glucose oxidase/ferricyanide/ferric compound system.
Ten replicates of each strip lot were measured using
10 different ACCU-CHEKX EASY instruments (Boehringer
Mannheim Corp., Indianapolis, IN), and the mean glucose
values and standard deviations were calculated. This
procedure was then repeated using a commercially-
available, non-serum based glucose control reagent,
referred to herein as "Reference E". The results are set
forth in Table l below.
Table l
Strip
Lot No.Control Mean Std. Dev.
n5c Bentone 2 .0 .9
A o Bentone _ . ~. 2
~0~ Bentone .; ~.8
~:o_ Bentone 7. ~-
~0 Bentone ~7. ;.6
~ cRe_erence :
4~5Re~erence _ 72.2 8.8
406Re_erence _ 69. 6. 8
410Reference E 57.~ 7.3
420Reference E 52. 11.0
The hexokinase-measured glucose level was 23 mg/dl
for the 1% Bentone control and l9 mg/dl for the Reference
E control material. These results show a level of
wossll3536 ~ 2 1 7 5 ~ 0 7 PCT~S94/1344~
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consistency well within that required of a control
reagent, as is indicated by the comparative standard
deviation values reported for each set of tests.
EXAMPLE 3
Efficacy of Control Reagent with Oxidative Method
A control reagent comprising a 1 percent aqueous
dispersion of VAN GEL~ ES (R.T. Vanderbilt Company, Inc.,
Norwalk, CT) clay particles was prepared using the
following: 2.0 g Van Gel ES, 200.0 g deionized water,
and 0.20 g PLURONIC~ L-35 (polyoxyalkylene ether from
BASF Corp.). To this was added 0.5 M MES/CAPS buffer to
make 50 mM. The mixture was homogenized for 15 minutes.
Eleven aliquots of this dispersion then had glucose added
to them in predetermined amounts ranging from 26.0 to
429.0 mg/dl as measured using a hexokinase reference
method.
Seven replicates of each control were then measured
using bG~ Test Strips (Boehringer Mannheim Corp.,
Indianapolis, IN), which utilize an oxidative glucose
oxidase/peroxidase method, and seven different ACCU-CHEK
II instruments. The percent reflectance readings (two
reaction pads per strip) were recorded, and standard
deviations and coefficients of variation were calculated.
The results obtained are shown in Table 2 below.
WO95/13536 2 1 7 5 9 0 7 PCT~Sg4/13445
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Table 2
Glucose Mean Mean Std. ~td. Coeff. Coeff.
(mg/dl) (%R) (%R) Dev. Dev. Var. Var.
bG b~ bG3 bG~ bG3 bG6
:6 7 .~ .1 0.~ 0.~ 0.00~ 0.0:
7 7 .~~ .7 0.~ 0.~ 0.00~ 0.0
~1 7~.~~.7 0. 0.~ 0.00 0.0_~
,2 7~ .0 0.~0 0. ~J 0.00 0.0_.
99 ~ ._2,.3 ,.~7 0. 2 0.0_ 0.0_~
1~2 ~.~4.0 .~ 0. 7 0.0 ' 0Ø
.. 9.4 _.0~ 0._ 0.0~ 0.0_
4 ~4.'~5.0 0.9; 0. 0.0~8 0.0~1
.~o _7._3.0 0.29 0._ 0.0 6 0.0~4
.,9 9.19.5 0.25 0.2 0.027 0.030
429 7.19.1 0.21 0.6 0.030 0.069
A dose response curve was also plotted, and this has
been reproduced as Figure 1. These results obtained
using an oxidative glucose measurement method also show a
level of performance well within that required of a
control reagent.
EXAMPLE 4
EfficacY of Control Reaqent with Electrochemical Method
A 0.55 percent by weight aqueous dispersion of
Bentone EW clay particles was prepared as described in
Example 1. Several biocides were also added, 0.30% by
weight 2-phenoxyethanol, 0.30% by weight Germall 115, and
0.15% by weight methylparaben. Thirteen aliquots of this
dispersion then had glucose added to them in
predetermined amounts ranging from 21.0 to 661.0 mg/dl as
measured using a hexokinase reference method. One
aliquot had no glucose added.
WO95/13536 2 1 7 ~ 9 0 7 PCT~S94/1344~
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Using the electrochemical, amperometric biosensor
method described in PCT Application No. PCT/US90/07374,
ten replicates of each control were then measured, and
the current readings at 10 seconds were recorded.
Standard deviations and coefficients of variation were
calculated, and these were compared with values obtained
using capillary blood samples having glucose levels
ranging from 1.3 to 787.8 mg/dl. The results obtained
are shown in Table 3 below.
Table 3
Glucose Mean Coeff. of
(mg/dl) Control (~am~s) Std. Dev. Var.
0.0 . 5 4entone. 1 0.08 5.0
21.0 . 5 Bentone~.~3 0.15 3.4
30.0 . Bentone~.~2 0.1 .99
'9.0 . 4entone~.44 0.2 ;.3
'.0 . Bentone,.~ 0.~, ;.,
, .0 . Bentone9.2 0.~
.0 . Bentone9.~2 0.,~ 3.77
.0 . Bentone2. ~ 0., 4 2.74
~ .0 . ~ Bentone_4.0~ 0.2 2.04
1. .0 . ~ Bentone .. ,.6~ 0.~, :.. ;9
1;~.0 . Ben~one 9.7. o.
:. .0 . Ben one .2.7
.0 . Ben one .70 ~ . 4
... 0 . Bentone ~ . 8 7. 10.~
_.3 b_ood sample .~o 0.. ~ 7. ,
2_.0 b_oo sample _.:4 0.......... 2.
4'. b:oo~ samp.e~. 6 0._ 2.
'., b:.ooc samp.. e .. ~ 0.2; _. ;
1; . b_oo~ samp.e_ .~ 0. 2 ... .
3 _.0 b_oo samp_e~ .:. . 0 -.2
.0 b_oo~ samp:.e~7.44 .54 2.28
.0 b:.ooc samp e8.79 .90 2.14
. ,.8 ~_oo~ samp_e1 1.91 .14 7.27
These results show a level of consistency well
within that required of a control reagent, as is
indicated by the comparative standard deviation values
W095/13536 2 1 7 5 9 0 7 PCT~S94/13445
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and coefficients of variation reported for each set of
tests.
It will be understood that the specification and
examples are illustrative but not limitative of the
present invention, and that other embodiments within the
spirit and scope of the invention will suggest themselves
to those skilled in the art.
