U.S. patent application number 11/185239 was filed with the patent office on 2005-11-17 for multilayer reagent test strips and methods for using the same to quantify glycated protein in a physiological sample.
Invention is credited to Guo, Sherry, Leong, Koon-wah, Qian, Suyue.
Application Number | 20050255453 11/185239 |
Document ID | / |
Family ID | 29249861 |
Filed Date | 2005-11-17 |
United States Patent
Application |
20050255453 |
Kind Code |
A1 |
Qian, Suyue ; et
al. |
November 17, 2005 |
Multilayer reagent test strips and methods for using the same to
quantify glycated protein in a physiological sample
Abstract
Multilayer reagent test strips for quantitating glycated protein
in a fluid sample, as well as methods for using the same, are
provided. The subject multilayer test strips include at least a
filter layer, a proteinase layer and a ketoamine oxidase signal
producing and fluid flow control system layer. In using the subject
test strips, a fluid sample is applied to the test strip and a
signal is generated that can be employed to quantitate the glycated
protein level in the sample. The quantitated glycated protein level
can then be employed to determine the amount of glycated protein in
the fluid sample. Also provided are kits and systems that include
the subject test strips and find use in practicing the subject
methods. The subject compositions and methods find use in glycated
protein monitoring applications, among other utilities.
Inventors: |
Qian, Suyue; (Fremont,
CA) ; Guo, Sherry; (San Jose, CA) ; Leong,
Koon-wah; (Sunnyvale, CA) |
Correspondence
Address: |
BOZICEVIC, FIELD & FRANCIS LLP
1900 UNIVERSITY AVENUE
SUITE 200
EAST PALO ALTO
CA
94303
US
|
Family ID: |
29249861 |
Appl. No.: |
11/185239 |
Filed: |
July 19, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11185239 |
Jul 19, 2005 |
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10144562 |
May 10, 2002 |
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6951728 |
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Current U.S.
Class: |
435/4 ; 422/400;
436/170 |
Current CPC
Class: |
G01N 33/526 20130101;
G01N 33/54366 20130101; C12Q 1/37 20130101; C12Q 1/26 20130101 |
Class at
Publication: |
435/004 ;
436/170; 422/056 |
International
Class: |
C12Q 001/00 |
Claims
1-17. (canceled)
18. A method for quantifying the amount of glycated protein in a
physiological sample, said method comprising: providing a
multilayer test strip comprising: a blood separation element for
separating red blood cells from plasma; a protease layer in fluid
communication with said blood separation element; and a ketoamine
oxidase signal producing system layer in fluid communication with
said protease layer; applying said physiological sample to a said
multilayer test strip detecting a signal produced on said test
strip to quantify the amount of glycated protein in said
physiological sample.
19. The method according to claim 18, wherein said physiological
sample is whole blood.
20. The method according to claim 18, wherein said detecting step
is performed by an automated instrument.
21-25. (canceled)
Description
FIELD OF THE INVENTION
[0001] The field of this invention is analyte detection,
particularly glycated protein detection.
BACKGROUND OF THE INVENTION
[0002] Individuals suffering from diabetes mellitus have an
abnormally high blood sugar level generally because the pancreas
does not secrete sufficient amounts of the active hormone insulin
into the bloodstream to regulate carbohydrate metabolism. If an
abnormally high blood sugar level, known as a hyperglycemic
condition, is allowed to continue for prolonged periods, the
individual will suffer from the chronic complications of diabetes,
including retinopathy, nephropathy, neuropathy and cardiovascular
disease. Studies indicate that diabetic patients who are able to
maintain near normal glycemic control greatly reduce the likelihood
of these dire complications. Therefore, several tests have been
developed to measure and control glycemic condition.
[0003] One common medical test to control glycemic condition is the
direct measurement of blood glucose levels by diabetics. Because
blood glucose levels fluctuate significantly throughout a given
day, being influenced by diet, activity, and treatment, depending
on the nature and severity of the individual case, some patients
measure their blood glucose levels up to seven times a day. Based
on the observed pattern in the measured glucose levels, the patient
and physician together make adjustments in diet, exercise and
insulin intake to better manage the disease. Clearly, this
information should be available to the patient immediately.
[0004] However, because of the frequent fluctuation of glucose
levels in a given day, tests which are independent of a patient's
diet, activity, and/or treatment and which provide longer term
indications of blood glucose levels have also been developed. These
tests measure the concentration of glycated proteins or
"protein-bound glucose" (PBG). Proteins, such as those present in
whole blood, serum and other biological fluids react with glucose,
under non-enzymatic conditions, to produce glycated proteins. The
extent of the reaction is directly dependent upon the glucose
concentration of the blood.
[0005] One of the first glycated protein tests developed measures
glycated hemoglobin, namely Hemoglobin A.sub.1c (HbA.sub.1c), which
reflects glycemic control over approximately a 2 to 3 month period.
Other such tests measure serum proteins, such as total glycated
serum protein, or a specific glycated serum. protein, namely
glycated albumin. Glycated albumin reflects an intermediate
glycemic control over approximately a 2 to 3 week period.
[0006] Yet another way to indirectly assess blood sugar
concentration is to analyze glycated protein concentration. The
plasma proteins are glycated in vivo by a non-enzymatic reaction
between glucose and available amino groups of blood proteins,
principally the .gamma.-amino groups of lysine residues and the
.alpha.-amino groups of the protein's terminal amino acid. The
glucose binds to an amino group of the protein to form a Schiff
base, i.e., aldimine, that undergoes molecular rearrangement to
form a stable ketoamine. In the art, such ketoamines are
generically known as "fructosamines." The degree of protein
glycation and fructosamine formation is directly proportional to
blood glucose concentration. Measurement of serum or plasma
glycated protein levels is useful for monitoring diabetic control
because glycated protein concentrations in serum or plasma reflect
an average of blood glucose level over approximately a half month
period.
[0007] One currently employed assay that provides accurate
determinations of blood glycated proteins, levels is the GlyPro.TM.
assay currently marketed by Genzyme Corporation, where this assay
is described in U.S. Pat. No. 6,008,006. While this assay provides
accurate results, it is performed in a clinical lab by a trained
technician with a sophisticated instrument, and is therefore not
suitable for home or physician office use.
[0008] U.S. Pat. Nos. 5,470,752; 5,695,949 and 5,725,774 describe a
multilayer reagent test strip for fructosamine quantification,
where the test strip is designed for home or physician office use.
However, measurements provided by the test strips described herein
tend to be inaccurate, as substances in the fluid sample other than
the fructosamine analyte also react with the signal producing
system and affect the signal generated thereby, leading to
inaccuracies in the ultimate fructosamine quantification achieved
with such test strips.
[0009] Accordingly, there is continued interest in the development
of additional multilayer reagent strips formats that are suitable
for glycated protein quantification, where the test strips are
suitable for use in the home or physician office and provide for
the highly accurate measurements achieved with the currently
employed clinical laboratory based protocols.
[0010] Relevant Literature
[0011] U.S. patents of interest include: U.S. Pat. Nos. 5,470,752;
5,695,949; 5,725,774; 6,008,006. Also of interest are: WO 96/31270;
WO 96/31619; WO 96/34977; EP 821064; and EP 737744.
SUMMARY OF THE INVENTION
[0012] Multilayer reagent test strips for quantitating glycated
protein in a fluid sample, as well as methods for using the same,
are provided. The subject multilayer test strips include at least a
filter layer, a protease layer and a ketoamine oxidase signal
producing and fluid flow control system layer. In using the subject
test strips, a fluid sample is applied to the test strip and a
signal is generated that can be employed to quantitate the glycated
protein level in the sample. Also provided are kits and systems
that include the subject test strips and find use in practicing the
subject methods. The subject compositions and methods find use in
glycated protein monitoring applications, among other
utilities.
BRIEF DESCRIPTION OF THE FIGURES
[0013] FIG. 1 provides an exploded view of a multilayer reagent
test strip according to one embodiment of the subject
invention.
[0014] FIG. 2 provides graphical results of an assay performed with
test strip with a one-layer blood separation layer
configuration.
[0015] FIG. 3 provides graphical results of an assay with a
two-layer blood separation layer configuration.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
[0016] Multilayer reagent test strips for quantitating glycated
protein in a fluid sample, as well as methods for using the same,
are provided. The subject multilayer test strips include at least a
filter layer, a protease layer and a ketoamine oxidase signal
producing fluid flow control system layer. In using the subject
test strips, a fluid sample is applied to the test strip and a
signal is generated that can be employed to quantitate the glycated
protein level in the sample. Also provided are kits and systems
that include the subject test strips and find use in practicing the
subject methods. The subject compositions and methods find use in
glycated protein monitoring applications, among other
utilities.
[0017] Before the subject invention is described further, it is to
be understood that the invention is not limited to the particular
embodiments of the invention described below, as variations of the
particular embodiments may be made and still fall within the scope
of the appended claims. It is also to be understood that the
terminology employed is for the purpose of describing particular
embodiments, and is not intended to be limiting. Instead, the scope
of the present invention will be established by the appended
claims.
[0018] In this specification and the appended claims, the singular
forms "a," "an" and "the" include plural reference unless the
context clearly dictates otherwise. Unless defined otherwise, all
technical and scientific terms used herein have the same meaning as
commonly understood to one of ordinary skill in the art to which
this invention belongs.
[0019] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limit of that range, and any other stated or intervening
value in that stated range, is encompassed within the invention.
The upper and lower limits of these smaller ranges may
independently be included in the smaller ranges, and are also
encompassed within the invention, subject to any specifically
excluded limit in the stated range. Where the stated range includes
one or both of the limits, ranges excluding either or both of those
included limits are also included in the invention.
[0020] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood to one of
ordinary skill in the art to which this invention belongs. Although
any methods, devices and materials similar or equivalent to those
described herein can be used in the practice or testing of the
invention, the preferred methods, devices and materials are now
described.
[0021] All publications mentioned herein are incorporated herein by
reference for the purpose of describing and disclosing the cell
lines, vectors, and methodologies, which are described in the
publications, which might be used in connection with the presently
described invention.
[0022] As summarized above, the subject invention provides
multilayer reagent test strips for quantitating glycated protein in
a sample, as well as systems and kits that include the subject test
strips. In further describing the invention, the test strips are
described first in greater detail, followed by a review of the
methods of using the test strips to quantitate glycated protein
level. Finally, a review of representative systems and kits
according to the subject invention is also provided.
[0023] Multilayer Reagent Test Strips
[0024] As summarized above, the subject invention provides
multilayer reagent test strips, where the subject test strips find
use in quantitating glycated protein in a fluid composition, as
described in greater detail below. The subject reagent test strips
are multilayer reagent test strips, by which is meant that the
subject reagent test strips include a plurality of different
layers, where the layers are in sequential fluid communication,
such that a fluid applied to a first layer of the plurality
sequentially travels through the remaining layers of the plurality
in a sequential manner.
[0025] The number of distinct layers that make up the subject test
strips may vary, typically ranging from about 2 to 10, usually from
about 3 to 7 in many embodiments. In many embodiments, the subject
test strips include a minimum of three different layers, which
layers, in sequential order, are: (a) a blood filter layer for
separating red blood cells from plasma; (b) a protease layer; and
(c) a ketoamine oxidase signal producing and fluid flow control
system layer. As such, many embodiments of the subject test strips
include at least the above layers in sequential order, such that
the second protease layer is in fluid communication with the first
blood filter layer and the third ketoamine oxidase layer is in
fluid communication with the second protease layer. In many
embodiments, one or more additional layers that provide for
additional functionality are also present.
[0026] Blood Separation Layer(s)
[0027] The first element of the subject multilayer test strips is
the blood separation element, which element serves to produce
plasma from whole blood, where the plasma then flows into
subsequent layers for further treatment/analysis. This element may
be present as a two layer separation element, or a single layer
separation element. Each of these distinct embodiments is described
in greater detail below.
[0028] Two Layer Blood Separation/Filter Element
[0029] In certain embodiments, a two layer structure is employed to
produce plasma from whole blood, where the two layer structure
includes a blood separation layer and a filter layer, which layers
work in tandem to provide for red blood cell free plasma which is
subsequently assayed in the subsequent layers. As used herein, the
term "plasma" means the substantially colorless fluid obtained from
a whole blood sample after red blood cells have been removed by the
separation process and device of the present invention. Because
plasma is serum plus the clotting protein fibrinogen, the term
"plasma" is used broadly herein to include both plasma and serum.
The blood separation/filter layers of the subject strips include a
separation matrix and a filter layer.
[0030] The separation matrix of the present invention is a
permeable matrix which does not contain glass fibers and,
therefore, is termed "a permeable non-glass fiber matrix." The term
"permeable" means liquid-permeable, such as permeable to plasma, as
well as permeable or porous to red blood cells when the matrix is
provided in the absence of a polyol. As used herein, the phrase
"matrix being porous to red blood cells in the absence of a polyol"
means that without the polyol contained in or on the matrix the red
blood cells would simply pass through the matrix, virtually
immediately. In the absence of the polyol, red blood cells are not
retained, by filtration or otherwise, in the matrix.
[0031] The polyol contained within or on the matrix chemically
reacts with the whole blood sample so as to clump the red blood
cells. As used herein, "clump" or "clumping" means the collection
into a mass or group, red blood cells distributed in a whole blood
sample. While not wishing to be bound by any theory or mechanism,
the clumping can be the result of agglutination, coagulation, or
the like, or some other chemical interaction between the polyol and
the red blood cells.
[0032] A useful permeable matrix can be a woven or non-woven
material and can be an absorbent or a non-absorbent material which
may or may not be hydrophilic. Especially suitable materials for
the matrix include, for example, woven or non-woven, absorbent or
non-absorbent, nylon, rayon, cotton, acrylic and polyester. In one
embodiment of the invention, the matrix is a non-woven,
non-absorbent polyester. The polyester is preferably a
poly(paraphenylene terephthalate), such as that used in a preferred
polyester sold as Sontara.RTM. (DuPont, Inc., Wilmington, Del.).
Another preferred matrix is the woven, absorbent nylon
Tetex.RTM.3-3710 (Tetko, Inc., Lancaster, N.Y.). Depending upon the
porosity or other properties of the matrix, the clumped red blood
cells either are retained in the matrix or are filtered out by the
filter material as described below. Some of the above-described
matrix materials, such as the non-woven, non-absorbent polyesters,
do not have "pores" in the traditional sense, i.e., that can be
measured, for example, by pore size (microns). In the absence of a
polyol of the present invention such materials essentially have no
limit as the porosity and are porous to red blood cells, which have
an average size of 5 .mu.m. With such macroporous materials, if the
polyol is not present the red blood cells pass through the matrix
almost immediately. For those matrix materials which can be
characterized based on pore size, the matrices used in the present
invention can have a pore size generally of from about 2 .mu.m to
about 10 .mu.m. Such pores sizes can be useful for retaining the
clumped red blood cells. Depending upon the porosity, thickness,
which is generally 200 to 1100 .mu.m, and other properties of the
matrix, such as absorbency, the clumped red blood cells are either
retained in the matrix or captured in a final filter material as
described below.
[0033] The polyol-containing matrix has a first surface for sample
application and a second surface where plasma is received or
becomes available for additional separation. Generally, the first
and second surfaces are presented as opposite sides of the matrix.
The whole blood sample flows in a direction from the first surface
toward the second surface, under conditions which provide such
directional flow, such as, gravitation, vacuum, or external
pressure. To enhance the simplicity of the method, if desired,
separation can be performed by gravity alone. Preferably, the
separation matrix provides for flow in a vertical direction,
preferably by gravitation.
[0034] The separation method and device include a permeable
non-glass fiber matrix containing a polyol. As used herein, the
terms "matrix containing a polyol" and "polyol-containing matrix"
mean that the polyol is separately added to the matrix and is not a
component originally found in the composition or make up of the
matrix, such as cellulose filter paper. Further, "matrix containing
a polyol" means a polyol can be impregnated into the matrix or
coated into or onto the matrix or covalently or non-covalently
bound to the matrix. In a preferred embodiment, the polyol is
impregnated into the matrix.
[0035] As used herein, the term "polyol" means a polyhydroxy
alcohol which is an alkyl or aromatic containing more than one
hydroxyt group. The term "poly" as used in "polyol" does not infer
that the alkyl or aromatic compound is a large polymer made up of
repeating monomeric units, but, instead, means that more than one
hydroxyl group is present in the compound. As discussed more fully
below, with the exception of polysaccharides, the polyols used in
the present invention are simple sugars or sugar alcohols,
oligosaccharides, or other naturally or non-naturally occurring
non-polymeric alkyl or aromatic compounds. Therefore, the term
"polyol" encompasses sugars, alcohol derivatives of sugars, herein
termed "sugar alcohols," and other naturally or
non-naturally.occurring non-polymeric polyols.
[0036] As used herein, "sugar" includes monosaccharides,
oligosaccharides, and polysaccharides. A monosaccharide is a simple
sugar which is as a linear, branched, or cyclic polyhydroxy alcohol
containing either an aldehyde or a ketone group. Exemplary
monosaccharides include, but are not limited to, mannose, glucose,
talose, galactose, xylose, arabinose, lyxose, ribose and fructose.
An oligosaccharide is a linear or branched carbohydrate that
consists from two to ten monosaccharide units joined by means of
glycosidic bonds. Oligosaccharides which can be used in the present
invention include, but are not limited to disaccharides such as
sucrose, trehalose, lactose and maltose. Examples of larger
oligosaccharides which can be used in the invention include the
cyclodextrins, such as alpha-cyclohexylamylose,
beta-cycloheptaamylose, and gamma-cyclooctoamylose, as well as
other oligosaccharides well known in the art. A polysaccharide is
any linear or branched polymer having more than ten monosaccharides
linked together by glycosidic bonds. Exemplary polysaccharides
include, but are not limited to, ficoll, polysucrose, and
hydroxyethyl starch.
[0037] Encompassed within "sugar" are those sugars which are
naturally occurring as well as those which are known but which have
not yet been identified as occurring naturally in plants or
animals. For example, there are five known naturally occurring
aldohexoses, including D-glucose, D-mannose, D-talose, D-galactose,
and L-galactose. However, the aldohexose structure has four chiral
carbons and thus, sixteen possible stereoisomers, all of which are
known, although only the five listed above have been identified as
occurring naturally in plants or animals. Thus, "sugar" encompasses
enantiomers in either the D or L forms of a sugar as well as
racemic mixtures thereof.
[0038] A polyol of the present invention also can be a "sugar
alcohol." A "sugar alcohol" is an alcohol derivative of a mono- or
an oligosaccharide which is generally formed by reduction of the
aldehyde or ketone moiety on the mono- or oligosaccharide.
Exemplary sugar alcohols include, but are not limited to, mannitol,
sorbitol, arabitol, inositol, galactitol, erythritol, and threitol.
Also included within the definition of "sugar alcohol" are the
alcohol derivatives of those mono- and oligosaccharides described
above.
[0039] Where chiral carbons are present in the sugar alcohol, the
sugar alcohol may be in the D or L form, such as D-threitol or
L-threitol, or in a racemic mixture of both the D and L forms. The
sugar alcohol can, but does not have to, be naturally occurring.
That is, the sugar alcohol can be a derivative of a known,
naturally occurring sugar, or, alternatively, it can have a D or L
configuration known to exist but not necessarily identified as
occurring in nature. The sugar alcohol also can be a sugar which is
found naturally in its reduced alcohol form or it can be an alcohol
derivative of a sugar which derivative is not known to exist in
nature.
[0040] In addition to sugar or sugar alcohols, the polyol can be a
non-polymeric naturally occurring or non-naturally occurring
polyol, which includes linear, branched, or cyclic alkyl or
aromatic compounds containing more than one hydroxyl group. As used
herein the term "non-polymeric" means the alkyl or aromatic
compounds are not polymers. Polymers are defined as high molecular
weight compounds consisting of long chains that may be open,
closed, linear, branched, or crosslinked, which chains are composed
of repeating units, called monomers, which may be either identical
or different. As used herein, those polyols which are "naturally
occurring" are ones which occur in nature and those which are
"non-naturally occurring" are not found in nature. Generally, these
naturally occurring or non-naturally occurring alkyl or aromatic
compounds range in size from three to twenty carbons (C.sub.3 to
C.sub.20), and more preferably, from three to ten carbons (C.sub.3
to C.sub.10). Examples of such naturally occurring, non-polymeric
polyols are glycerol, a three-carbon trihydroxy alcohol that occurs
in many lipids, and quinic
acid,1,3,4,5-tetrahydroxycyclo-hexanecarboxylic acid, which acid
can be in the salt form. Examples of non-naturally occurring,
non-polymeric polyols include pentaerythritol and
dipentaerythritol.
[0041] In one embodiment, to apply the polyol to the matrix, the
polyol can simply be dissolved in an aqueous solution generally, at
a concentration of about 20% when used alone, and at about 10%
concentration when combined with a polycationic polymer, which is
generally present in a concentration of about 0.5% to 5% as
discussed more fully below. If desired, multiple layers of matrices
containing polyol at lower concentrations, such as four layers of
matrix containing 5% polyol, also can be used. The polyol and, if
present, the polycationic polymer can alternatively be dissolved in
physiological saline (0.85% NaCl), phosphate buffered saline (PBS),
an organic solvent, or the like.
[0042] In addition to the polyol, a polycationic polymer can, but
does not have to, be added to the matrix. Similar to the addition
of a polyol to the matrix, the polycationic polymer can also be
physically impregnated, coated into or onto, or covalently or
non-covalently bound to the matrix. The polycationic polymer is
also useful for clumping, as well as stabilizing clumped, red blood
cells.
[0043] The polycationic polymer component can be any polymer having
more than one cationic site and are generally based on monomers
which contain an amine group. Suitable polycationic polymers
include, for example, hexadimethrine bromide,
trimethylenehexamethylenediammoniumbromide, polylysine,
polyallylamine, polyarginine, poly(N,N-dimethylaminoethylmeth-
acrylate, copolymers of N,N-dimethylaminoethylmethacrylate and
methylmethacrylate, polyethyleneimine, poly(diallyldimethylammonium
chloride), poly(1,1 -dimethyl-3,5-dimethylenepiperidinium
chloride), and mixtures thereof. The polymerized positively charged
amino acids, such as polylysine, can have the amino acids in either
the D or L forms, such as poly-L-lysine or poly-D-lysine, or a
racemic mixture thereof, such as poly-D,L-lysine.
[0044] As described above, in one embodiment, to apply the cationic
polymer to the matrix, the polymer can be dissolved in an a
solution such as water, physiological saline, PBS, an organic
solvent, or the like, and the matrix then dipped into the polymer
containing solution. Generally, the polymer is in a concentration
of about 0.5% to 5%. Where both polyol and polymer are contained in
the matrix, the order of adding polyol and polymer to the matrix is
irrelevant. For example, polyol and polymer can be simultaneously
or sequentially dissolved in such aqueous solutions or solvents as
those described above and both polyol and polymer simultaneously
applied to the matrix, as described in the Examples below.
Alternatively, polyol and polymer can be applied to the matrix
sequentially in any order.
[0045] Non-hemolytic detergents, such as Pluronic (Pragmatics,
Inc., Elkhart, Ind.), can be added to the aqueous solutions or
solvents described above, generally at a concentration of 0.01% to
0.1%. Such detergents help maximize impregnation of a polyol into
the matrix, thereby improving the flow rate of the whole blood
sample and the plasma. Other optional agents which can further
enhance the flow rate, include, for example, polyvinylpyrrolidone
or similar polymers and other fillers which give the matrix and the
below described filter material stiffness.
[0046] In addition, the matrix may include one or more additional
reagents to remove interference, e.g., KIO.sub.3, KMnO.sub.4,
FeSO.sub.4 for removing ascorbic acid interference, uricase for
removing uric acid interference.
[0047] As indicated above, a filter material can be used in
combination with the matrix of the present invention. Suitable
filter materials include, for example, nylon, cellulose acetate,
polysulfone, synthetic fibers, and polycarbonate. The filter can,
but does not have to, be a membrane. Illustrative filters and
membranes include, for example, BTS polysulfone membrane (Memtek,
Inc., San Diego, Calif.), Ahlstrom synthetic fiber sheets, such as
94-30 A (Ahlstrom Filtration, Inc., Mt. Holly Spring, Pa.), Biodyne
A.RTM.nylon membrane (Pall Corp., East Hills, N.Y.), Ultrabind 450
(Gelman, Ann Arbor, Mich.), and Nucleopore.RTM. polycarbonate
(Costar, Corp., Cambridge, Mass.).
[0048] The need for any additional filter material depends to a
large extent on the porosity, thickness, absorbency or other
properties of the matrix. For example, the clumped red blood cells,
depending upon the above properties of the matrix, can be retained
in the matrix. Alternatively, or in addition thereto, a final
filter material can be used to capture or retain any additional
clumps of red blood cells. Where present, the filter material can
generally have a porosity of up to about 12 .mu.m and preferably
will have a pore size of less than 10 .mu.m, and more preferably 5
.mu.m or less.
[0049] A filter material can be placed underneath the
polyol-containing separation matrix, thereby supporting the
matrix.
[0050] In preferred embodiments of the invention, the blood
separation method and device comprise a non-woven, non-absorbent
polyester matrix impregnated with mannitol and either a nylon or
polysulfone membrane below the matrix. Preferably, the matrix
additionally contains hexadimethrine bromide and KIO.sub.3.
[0051] The above-described two layer filtration element is further
discussed in U.S. Pat. Nos. 5,470,752; 5,695,949 and 5,725774; the
disclosures of which are herein incorporated by reference.
[0052] Single Layer Filtration Layer
[0053] In other embodiments, blood separation is achieve by an
element made up of a single filtration layer. In these embodiments,
the single filtration layer is typically a porous matrix, wherein
the separation takes place as the sample moves through the matrix
from one side to the other. A representative matrix to accomplish
that separation may have pores that trap the red blood cells,
generally pore sizes in the range from about 0.1 .mu.m to about 5
.mu.m. In certain embodiments, the membrane is anisotropic, with a
range of pore sizes; e.g., a broad range of pore sizes. When the
matrix comprises an anisotropic membrane, the first side to which
non-filtered blood is applied may be the large-pore side. For
example, a gradient of pore sizes from about 0.1 .mu.m to about 150
.mu.m may extend through the membrane. On the large-pore side, pore
size is preferably in the range from about 30 .mu.m to about 40
.mu.m. On the side of the membrane where the pores are smallest
(i.e., the side that fluid exits in order to pass on to the next
layer), the void volume is relatively small, and the material of
the membrane is generally quite dense, within a layer that can
typically constitute up to 20% of the membrane's thickness. Within
this layer, pore size is sometimes in the range from about 0.1 to
about 0.8 .mu.m, with a nominal pore size often about 0.3 .mu.m. In
certain embodiments, the matrix is one that not only traps red
blood cells but also minimizes lysing of the cells, so that any
portion of the sample that passes through the matrix to the
downstream layers in the direction of fluid flow does not absorb
light to any appreciable extent at about 700 nm.
[0054] The matrix of the separation layer is generally a
hydrophilic porous membrane. The matrix allows. for the flow of an
aqueous medium through it. Polysulfones and polyamides (nylons) are
examples of suitable matrix materials. Other polymers having
comparable properties may also be used. A preferred method of
preparing the porous material that forms the matrix of the
separation layer is to cast the polymer without a supporting core.
Such a matrix is, for example, the anisotropic polysulfone membrane
available from Memtec, Inc., San Diego, Calif. The terms "matrix"
and "membrane" are used interchangeably herein. Each term is
understood to not be limited to a single layer and may include, for
example, an absorbent layer. A matrix of less than about 500 .mu.m
thickness is usually employed with about 115 to 155 .mu.m being
preferred. A thickness of about 130 to 140 .mu.m is most preferred,
particularly when the matrix is nylon or anisotropic polysulfone.
The matrix generally does not deform on wetting, thus retaining its
original conformation and size, and has sufficient wet strength to
allow for routine manufacture.
[0055] Protease Layer
[0056] Downstream from the blood separation element/layer(s) in the
direction of fluid flow is the protease layer of the subject
multilayer reagent test strips. This layer comprises a matrix or
membrane material and a protease enzyme. The matrix material is one
that is porous and provides for flow of sample fluid through the
material. The matrix that is employed in this layer is typically an
inert porous matrix that provides a support for protease component.
As such, the matrix is one that is permissive of aqueous fluid flow
through it and provides sufficient void space for the protease to
exert its activity on proteins present in fluid that passes through
the matrix. A number of different porous matrices have been
developed for use in various analyte detection assays, which
matrices may differ in terms of materials, pore sizes, dimensions
and the like, where representative matrices include those described
in U.S. Pat. Nos. 55,932,431; 5,874,099; 5,871,767; 5,869,077;
5,866,322; 5,834,001; 5,800,829; 5,800,828; 5,798,113; 5,670,381;
5,663,054; 5,459,080; 5,459,078; 5,441,894 and 5,212,061; the
disclosures of which are herein incorporated by reference. The
dimensions and porosity of the test strip may vary greatly, where
the matrix may or may not have a porosity gradient, e.g., with
larger pores near or at the sample application region and smaller
pores at the detection region. Examples of specific matrix
materials of interest include those prepared from polyamide
(nylon), polysulfone, polyester, polyacrylate, cellulose,
polycarbonate, nitrocellulose, etc.
[0057] The protease layer also includes a protease, i.e., an enzyme
having protease activity. The membrane or matrix of the protease
layer is typically coated with the protease in a manner that
preserves the activity of the protease. The protease of the
protease layer is one that cleaves protein molecules to yield
accessible ketoamine bonds. Any convenient protease may be
employed, where representative proteases include Protease XIV,
Proteinase K, chymotrypsin, substilisin, trypsin, and the like.
[0058] Signal Producing and Fluid Flow Control System Layer
[0059] In fluid communication with, and downstream of, the protease
layer is the ketoamine oxidase signal producing and fluid flow
control system layer. This signal producing system layer includes a
porous matrix or membrane element, such as those described above,
which includes a ketoamine oxidase signal producing system. In the
subject test strips, the one or more members of the signal
producing system are associated, e.g., covalently or non-covalently
attached to, at least a portion of (i.e., the detection region) the
matrix, and in many embodiments to substantially all of the porous
matrix. In many embodiments, the matrix or membrane component is
typically coated with the reagents of the ketoamine oxidase signal
producing system.
[0060] The ketoamine oxidase signal producing system of this
particular layer of the multilayer reagent test strips is an
oxidation signal producing system. By oxidation signal producing
system is meant that in generating the detectable signal from which
the ketoamine concentration in the sample is derived, a ketoamine
bond is oxidized by a ketoamine oxidase to produce an oxidized form
of the substrate and a corresponding or proportional amount of
hydrogen peroxide. The hydrogen peroxide is then employed, in turn,
to generate the detectable product from one or more indicator
compounds (collectively referred to herein as a chromogen system),
where the amount of detectable product produced by the signal
producing system, i.e., the signal, is then related to the amount
of ketoamine in the initial sample. As such, the ketoamine oxidase
oxidation signal producing systems present in the subject test
strips are also correctly characterized as hydrogen peroxide based
signal producing systems or peroxide producing signal producing
systems.
[0061] As indicated above, one member of the subject signal
producing systems is a ketoamine oxidase. Ketoamine oxidases of
interest for use in the subject reagent systems are those that
specifically oxidize the ketoamine bond to produce an oxidized
substrate and a corresponding amount of hydrogen peroxide, where by
corresponding amount is meant that the amount of hydrogen peroxide
that is produced is proportional to the amount of ketoamine present
in the sample. A variety of different suitable ketoamine oxidases
are known to those of skill in the art and may be employed in the
subject invention, where representative ketoamine oxidases of
interest include those described in U.S. Pa. Nos. 5,712,138 and
6,008,006, the disclosures of which are herein incorporated by
reference, as well as in EP 821064 and EP 737744.
[0062] In addition to the ketoamine oxidase activity of the signal
producing system, the signal producing system also includes one or
more indicator compounds, collectively referred to herein as a
chromogen system, where the chromogen system produces a chromogenic
product in the presence of hydrogen peroxide.
[0063] The signal producing systems also include an enzyme that
catalyzes the conversion of a dye substrate into a detectable
product in the presence of hydrogen peroxide, where the amount of
detectable product that is produced by this reaction is
proportional to the amount of hydrogen peroxide that is present.
This second enzyme is generally a peroxidase, where suitable
peroxidases include: horseradish-peroxidase (HRP), soy peroxidase,
recombinantly produced peroxidase and synthetic analogs having
peroxidative activity and the like. See e.g., Ci et al. (1990)
Analytica Chimica Acta, 233:299-302.
[0064] The dye substrates are oxidized by hydrogen peroxide in the
presence of the peroxidase to produce a product that absorbs light
in a predetermined wavelength range, i.e., an indicator dye.
Preferably the indicator dye absorbs strongly at a wavelength
different from that at which the sample or the testing reagent
absorbs strongly. The oxidized form of the indicator may be the
colored, faintly-colored, or colorless final product that evidences
a change in color of the testing side of the membrane. That is to
say, the testing reagent can indicate the presence of an analyte in
a sample by a colored area being bleached or, alternatively, by a
colorless area developing color.
[0065] Dye substrates that are useful in the present invention
include urea derivative dyes. Urea derivative dyes include at least
some of those disclosed in JP 1118768; JP 9019296; EP 38 205, EP
124 287 and EP 251297; the disclosures of which are herein
incorporated by reference. The dye substrate is generally a urea
derivative, having a negative charge, where suitable negatively
charged urea derivatives include those bearing a carboxylate group
or a sulfonate group. Urea derivative dyes of interest are
represented by the following formula:
R.sup.1R.sup.2NCONHR.sup.3,
[0066] wherein
[0067] R.sup.1, R.sup.2 taken together is a N,N-di-substituted
aminoaryl; and
[0068] R.sup.3 is selected from the group consisting of
carboxyalkyl, alkoxycarbonyl, alkylcarbonyl, arylsulfonyl,
sulfoaryl and carboxyaryl.
[0069] The aryl groups of R.sup.1 and R.sup.2 may be bonded via S
to become a phenothiazine derivative type of dye, which is
represented by the following formula: 1
[0070] wherein R.sup.4 and R.sup.5 are independently selected from
NR.sub.2 and OR, where R is hydrogen, (C.sub.1-C.sub.6)-alkyl,
(C.sub.1-C.sub.6)-alkenyl, aryl, substituted aryl, arylalkyl,
substituted arylalkyl; R.sup.3 is defined above; and R.sup.6 and
R.sup.7are independently selected from hydrogen,
(C.sub.1-C.sub.6)-alkyl, (C.sub.1-C.sub.6)-alkenyl, acyl, carboxyl,
sulfonyl, nitro, halogen, hydroxyl, (C.sub.1-C.sub.6)-alkoxyl or
hydroxy-(C.sub.1-C.sub.6)-alkyl.
[0071] Alternatively, the aryl groups of R.sup.1 and R.sup.2 may be
bonded via 0, to form a phenoxazine derivative type of dye, which
is represented by the following formula: 2
[0072] In yet another embodiment, the aryl groups of R.sup.1 and
R.sup.2 is not bonded, which is represented by the following
diphenylamine formula: 3
[0073] Exemplary urea derivative dyes include
10-(carboxymethylaminocarbon-
yl)-3,7-bis(dimethylamino)phenothiazine (leuco methylene blue),
10-(carboxymethylaminocarbonyl)-4,4'-bis(dimethylamino)diphenylamine,
10-propionic acid phenothiazine, and salts thereof. In a preferred
embodiment, the urea derivative dye is
10-(carboxymethylaminocarbonyl)-3,-
7-bis(dimethylamino)phenothiazine, sodium salt.
[0074] A particularly preferred ketoamine oxidase signal producing
system layer is one that includes the layer described in U.S.
application Ser. No. 09/593,827 (the disclosure of which is herein
incorporated by reference) which includes a ketoamine oxidase, as
described above.
[0075] In many embodiments, the ketoamine oxidase signal producing
system layer is a hydrophobic layer which retards entry of aqueous
materials into the layer, such that the plasma fraction of the
sample which is being assayed on the strip resides in the protease
layer for a period of time that is longer than if the signal
producing system layer were not rendered hydrophobic, e.g., by a
time period that is at least about 5 times longer, typically at
least about 10 times longer. This feature of the signal producing
system layer assures that the plasma is present in the protease
layer for a period of time sufficient for the protease to cleave
the proteins present in the plasma sufficiently to yield accessible
ketoamine.
[0076] The signal producing system layer may be rendered
hydrophobic using any convenient protocol. One protocol of
particular interest is that described in U.S. patent application
Ser. No. ______, filed on even date herewith and entitled
"Multilayer Reagent Test Strips That Include at Least One Fluid
Flow Control Layer Methods for Using the Same," (having an attorney
docket no. LIFE-120; LFS 236), the disclosure of which is herein
incorporated by reference. In this protocol, the porous matrix that
includes the signal producing system is coated with an organic
solvent, e.g., by dipping the membrane in an organic solvent.
Organic solvents of interest include, but are not limited to:
chloroform, dichloromethane, halogenated hydrocarbon, hydrocarbon,
ethyl acetate, and the like. Another protocol of interest for
rendering the signal producing system hydrophobic is one that coats
the membrane with a fatty acid or other fluid retention agents,
such as described in U.S. Pat. No. 5,447,689, the disclosure of
which is herein incorporated by reference.
[0077] Additional Layers
[0078] In addition to the above specific filter, protease and
ketoamine oxidase signal producing system layers, the subject
multilayer reagent test strips may also include a number of
additional layers.
[0079] Placed above the above-described layers may be a mesh layer
which serves to hold all of the subsequent or underlying layers
together. The mesh layer may be fabricated from any convenient
material, such as an inert matrix material, as described above. In
addition, a clear polymeric, e.g., polyester or analogous polymeric
material, layer may be present beneath the signal producing system
layer, which layer serves to enhance plasma flow and protect the
plasma sample from drying prior to completion of the assay.
[0080] The above layers are often present in a "stacked"
configuration, as shown in FIG. 1, and may be present in a chamber
bounded by an upper guard piece and a support element.
[0081] Representative Illustrated Embodiment
[0082] FIG. 1 provides an exploded view of a multilayer test strip
according to the subject invention. In FIG. 1, multilayer reagent
test strip 10 includes injection molded guard element 11 on top of
mesh layer 12, which in turn is present above separation layer 13
and filter layer 14, which together make up the blood separation
element. Immediately beneath the separation element is the protease
layer 15. Beneath protease layer 15 is signal producing layer 16,
which is present over polymeric film layer 17. The above layers are
present on support element 18, which includes a manual holding
region 18a and a sample assay region 18b.
[0083] As can be seen in FIG. 1, each of the distinct layers is a
disc shaped layer, where the separated layers are placed one on top
of the other in a stacked configuration. In many embodiments, the
surface area of each of the disparate layers typically ranges from
about 0.1 cm.sup.2 to about 0.25 cm.sup.2, usually from about 0.125
cm.sup.2 to about 0.18 cm.sup.2, such that the disc shaped layers
in the embodiment shown in FIG. 1 generally have a diameter ranging
from about 0.35 cm to about 0.6 cm, usually from about 0.4 cm to
about 0.5 cm.
[0084] The overall dimensions of the support element 18 are
selected to provide for a convenient hand held device, such that
the support element has a width that typically ranges from about
0.25 in to about 0.7 in, usually from about 0.4 5 in to about 0.55
in, and a length that ranges from about 1.5 in to about 3 in,
usually from about 2 in to about 2.5 in.
[0085] The injection molded guard element 11 typically has a width
ranging from about 0.18 in to about 0.5 in, usually from about 0.3
in to about 0.38 in and a length ranging from about 0.35 in to
about 0.68 in, usually from about 0.45 in to about 0.56 in.
[0086] Test Strip Fabrication
[0087] The subject reagent test strips may be fabricated employing
any convenient protocol. Typically, the various layers are
fabricated separately, e.g., by using conventional dipping
protocols in one or more reagent solutions, and then assembled into
a final test strip. A representative fabrication protocol is
provided in the experimental section, infra.
[0088] Methods of Glycated Protien Detection
[0089] The above described multilayer reagent test strips find use
in methods of detecting the presence of, and often the amount of
ketoamine group on protein in a sample. While in principle the
subject methods may be used to determine the presence, and often
concentration, of ketoamine groups in a variety of different
physiological samples, such as urine, tears, saliva, and the like,
they are particularly suited for use in determining the
concentration of ketoamine groups in blood or blood fractions,
e.g., blood derived samples, and more particularly, in whole
blood.
[0090] An important feature of the subject methods is the use of
the subject signal producing systems that include a urea derivative
dye provides for the highly sensitive detection of hydrogen
peroxide. As such, hydrogen peroxide may be detected at
submillimolar concentrations using the subject stable dry reagent
formats, e.g., test strips, where by submillimolar concentration is
typically meant concentrations ranging from 0.010 to 1 mM, usually
from about 0.050 to 0.8 mM. Use of the subject signal producing
systems that include a urea derivative dye provides for more
sensitive detection of hydrogen peroxide as compared to signal
producing systems that include a dye substrate other than a urea
derivative dye, e.g.,
N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline- ,
4-aminoantipyrine.
[0091] In practicing the subject methods, the first step is to
apply a quantity of the physiological sample to the test strip,
where the test strip is described supra. The amount of
physiological sample, e.g., blood, that is applied to the test
strip may vary, but generally ranges from about 2 .mu.L to 40
.mu.L, usually from about 5 .mu.L to 20 .mu.L. Because of the
nature of the subject test strip, the blood sample size that is
applied to the test strip may be relatively small, ranging in size
from about 2 .mu.L to 40 .mu.L, usually from about 5 .mu.L to 20
.mu.L. Where blood is the physiological sample, blood samples of a
variety of different hematocrits may be assayed with the subject
methods, where the hematocrit may range from about 20% to 65%,
usually from about 25% to 60%.
[0092] Following application of the sample to the test strip, the
sample is allowed to flow sequentially through the various layers
of the strip, such that it separates in the separation element, is
digested in the protease layer and reacts with the members of the
signal producing system in the signal producing system layer to
produce a detectable product that is present in an amount
proportional to the initial amount of the ketoamine groups of
interest present in the sample. The amount of detectable product,
i.e., the signal produced by the signal producing system, is then
determined and related to the amount of glycated protein in the
initial sample.
[0093] Utility
[0094] The above-described methods find use in any application
where one wishes to test a fluid sample to determine the
concentration of ketoamine group on protein present therein. Of
particular interest is use of the subject methods to
determine/monitor blood glucose levels. In such applications, the
detected ketoamine group concentration provided by the subject
methods is employed to determine the amount of glycated protein-in
the tested sample, as is known in the art, which is turn is
employed to monitor blood glucose levels, e.g., as is desirable in
the treatment and management of patients suffering from diabetes.
Utilities for the subject test strips are further described in U.S.
Pat. Nos. 5,470,752; 5,695,949; 5,725,774; and 6,008,006; the
disclosures of which are herein incorporated by reference.
[0095] Detection Systems
[0096] Detection systems useful for practicing the subject methods
include a reagent test strip as described above and an signal
detection instrument or reader. In such systems, a physiological
sample is applied to the test strip as described above and the
signal produced by the signal producing system is detected and
related to the presence (and often the amount) of glycated protein
in the sample by the instrument. The above described reaction,
detection and relation steps, and instruments for practicing the
same, are further described in U.S. Pat. Nos. 4,734,360; 4,900,666;
4,935,346; 5,059,394; 5,304,468; 5,306,623; 5,418,142; 5,426,032;
5,515,170; 5,526,120; 5,563,042; 5,620,863; 5,753,429; 5,573,452;
5,780,304; 5,789,255; 5,843,691; 5,846,486; 5,902,731; 5,968,836
and 5,972,294; the disclosures of which are herein incorporated by
reference. In the relation step, the derived glycated protein
concentration takes into account the constant contribution of
competing reactions to the observed signal, e.g., by calibrating
the instrument accordingly.
[0097] Kits
[0098] Also provided by the subject invention are kits for use in
practicing the subject methods. The kits of the subject invention
include a reagent test strip, as described above, and at least one
of a means for obtaining said physiological sample, e.g., a lance
for sticking a finger, a lance actuation means, and the like, and
an standard, e.g., an glycated protein control solution that
contains a standardized concentration of ketoamine or precursor
thereof, e.g., glycated protein. In certain embodiments, the kits
also include a detection instrument, as described above, for
detecting the amount of product produced on the strip following
sample application and relating the detected product to the
presence (and often the amount) of analyte in the sample. Finally,
the kits include instructions for using the subject kit components
in the determination of glycated protein concentration in a
physiological sample. These instructions may be present on one or
more of the packaging, a label insert, containers present in the
kits, and the like.
[0099] The following examples are offered by way of illustration
and not by way of limitation.
EXAMPLES
[0100] I. Reagent Test Strip Preparation
[0101] A: Blood Separation Layer
[0102] Mesh: A Tetko mesh #7-280/44 was placed in detergent
solution of 1% Pluronic (Pragmatics. Inc.) for one minute. Excess
detergent was removed and the mesh was dried by heating at
60.degree. C. for 10 minutes.
[0103] Blood separation matrix: A solution containing 10% mannitol,
1.25% hexadimethrine bromide and 1% KIO.sub.3 in physiological
saline (0.85% NaCl) was impregnated onto Sontara.RTM. #8007
(DuPont. Inc.) on an automated impregnation/drying unit (AFM
Engineering. Santa Anna. Calif.). The drying temperature was
100.degree. C. for approximately 10 minutes.
[0104] Memtec polysulfone membrane was untreated
[0105] B: Digestion Layer
[0106] 200 mg/ml of protease XIV (Sigmna) was dissolved in 100 mM
EPPS, pH 8.0. The nylon mesh 3-200/39(SEFAR) was immersed into the
protease XIV solution. Excess solution was removed and the mesh was
dried by heating at 56.degree. C. for 10 minutes.
[0107] C: Color Formation and Fluid Flow Control Layer
[0108] A dip: It contains 300 U/ml of KAO in 20 mM PBS, pH 7.4, 1
mg/ml HRP, 1% PVP (MW=360K) and 50 mg/ml mannitol. The nylon
membrane (Pall) was dipped into the solution and the excess
solution was removed and the membrane was dried by heating at
56.degree. C. for 5 minutes.
[0109] B dip: After A dipping, the nylon membrane was further
dipped into B dip containing 1 mM DA-60 in 70% methanol. The dipped
membrane was dried by heating at 56.degree. C. for 5 minutes.
[0110] C dip: A and B dipped membrane was quickly pull through into
dichloromethane solvent. Excess solvent was removed and the
membrane was dried under ventilation hood at room temperature for 2
minutes.
[0111] D: Assembly of Test Strip
[0112] Each layer was cut into 0.476 cm ({fraction (3/16)} in)
diameter discs which were assembled in sequence as indicated in
FIG. 1 into final strip.
[0113] II. Glycated Protein Assay Employing the Reagent Test Strip
as Prepared in Example I.
[0114] FIG. 2: One layer blood separation layer strip design was
used. i.e., blood separation layer has polysulfone membrane only.
15 .mu.l of whole patient blood was applied to test strip and the
test strip was incubated at 37.degree. C. for 5 minutes, then the
color intensity was read on the Macbeth reflectance
spectrometer.
[0115] FIG. 3: Comparison of one layer vs. two layer blood
separation strip design. One layer is polysufone membrane only as
blood separation layer. Two layers consist of Sontara.RTM. filter
material and polysulfone membrane both. 15 .mu.l of whole blood
with low, medium and high level glycoprotein was applied to test
strip. The strip was incubated at 40.degree. C. for 5 minutes. The
color intensity was read on a Macbeth reflectance spectrometer.
Significant increase of the slope of color intensity among
different level of glycoprotein in two layer strip design
definitely gives better resolution range. All data points in the
FIG. 3 represent the average of eight replicates and CV is around
10% for both strips.
[0116] It is evident from the above results and discussion that the
subject invention provides a highly sensitive and accurate glycated
protein detection system which is capable of providing results that
are comparable in reliability and accuracy to those currently
achieved only in the clinical laboratory setting, where the system
is one that can be employed by a patient or doctor in a non
laboratory setting, e.g., at home or in the doctor's office. As
such, the subject invention represents a significant contribution
to the art.
[0117] All publications and patents cited in this specification are
herein incorporated by reference as if each individual publication
or patent were specifically and individually indicated to be
incorporated by reference. The citation of any publication is for
its disclosure prior to the filing date and should not be construed
as an admission that the present invention is not entitled to
antedate such publication by virtue of prior invention.
[0118] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it is readily apparent to those of ordinary skill
in the art in light of the teachings of this invention that certain
changes and modifications may be made thereto without departing
from the spirit or scope of the appended claims.
* * * * *