U.S. patent application number 12/645387 was filed with the patent office on 2010-06-17 for rapid test for glycated albumin.
This patent application is currently assigned to EPINEX DIAGNOSTICS, INC.. Invention is credited to Henry J. Smith, Asad Zaidi.
Application Number | 20100151488 12/645387 |
Document ID | / |
Family ID | 34393012 |
Filed Date | 2010-06-17 |
United States Patent
Application |
20100151488 |
Kind Code |
A1 |
Smith; Henry J. ; et
al. |
June 17, 2010 |
Rapid Test for Glycated Albumin
Abstract
A rapid immunochromatographic assay system is provided for
measuring the amount of glycated albumin in a blood sample relative
to the total level of albumin in the sample. The assay system is
comprised of a disposable cassette that contains the test strips
and testing reagents, and a measurement device that automatically
reads, calculates and displays the test results over a period of
time. The test cassette contains two test strips that are used to
measure glycated albumin and total albumin respectively. The strips
are contiguous beneath the single sample application well so that
the same sample is tested simultaneously by both test strips. Part
of the sample will migrate thru the glycated albumin test strip
where it will react with the glycated albumin test reagents to
yield a glycated albumin result, while part of the sample will
migrate thru the total albumin test strip where it will react with
the total albumin test reagents to yield a total albumin result.
The test cassette is placed within a measuring device such as a
reflectance spectrometer or fluorometer, that reads, calculates and
expresses the result as the percentage of glycated albumin relative
to total albumin in the sample. The results of successive testing
that are performed over a period of time are stored in the
instrument's memory and displayed in a numerical or graphical
format so that the individual's glycated albumin levels can be
monitored over time.
Inventors: |
Smith; Henry J.; (Temecula,
CA) ; Zaidi; Asad; (Irvine, CA) |
Correspondence
Address: |
K&L Gates LLP
1900 MAIN STREET, SUITE 600
IRVINE
CA
92614-7319
US
|
Assignee: |
EPINEX DIAGNOSTICS, INC.
Tustin
CA
|
Family ID: |
34393012 |
Appl. No.: |
12/645387 |
Filed: |
December 22, 2009 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10538392 |
Apr 27, 2006 |
7659107 |
|
|
PCT/US04/31202 |
Sep 22, 2004 |
|
|
|
12645387 |
|
|
|
|
60505392 |
Sep 23, 2003 |
|
|
|
Current U.S.
Class: |
435/7.1 ;
435/287.2 |
Current CPC
Class: |
Y10S 436/81 20130101;
Y10S 435/805 20130101; Y10S 435/81 20130101; Y10S 435/973 20130101;
Y10S 436/807 20130101; Y10S 436/815 20130101; G01N 2333/765
20130101; G01N 33/6842 20130101; Y10S 435/975 20130101; Y10S
436/808 20130101; G01N 33/558 20130101; G01N 33/68 20130101; Y10S
435/97 20130101 |
Class at
Publication: |
435/7.1 ;
435/287.2 |
International
Class: |
G01N 33/53 20060101
G01N033/53; C12M 1/34 20060101 C12M001/34 |
Claims
1-26. (canceled)
27. A system for detecting glycated albumin in a sample and
determining the percent glycated albumin comprising: a first assay
measuring glycated albumin in said sample; a second assay measuring
total albumin in the same sample as said first assay; means for
calculating percent glycated albumin.
28. The system of claim 27 wherein said first assay comprises:
contacting a drop of blood with an anti-glycated albumin antibody
such that glycated albumin present in said blood binds to said
anti-glycated albumin antibody; and detecting said bound glycated
albumin.
29. The system of claim 27 wherein said second assay comprises:
contacting a drop of blood with an anti-albumin antibody such that
albumin present in said blood binds to said anti-albumin antibody;
and detecting said bound albumin.
30. The system of claim 27 wherein said system is an
immunochromatographic system.
31. The system of claim 30 wherein said immunochromatographic
system for determining the percent glycated albumin in a blood
sample comprises: a first test strip that measures glycated albumin
and a second test strip that measures total albumin; and a
measurement device that reads, calculates and displays the result
as the percentage of glycated albumin compared to total albumin in
the sample.
32. The system of claim 31, wherein said first test strip is
comprised of microparticles coated with a first antibody to
glycated albumin and an immobilization agent covalently bound to
said test strip.
33. The system of claim 32 wherein said immobilization agent is an
antibody to glycated albumin.
34. The system of claim 32 wherein said microparticles are selected
from the group consisting of colloidal gold particles, latex
particles, polystyrene particles, acrylic particles or other solid
phase microparticles.
35. The system of claim 31 wherein said second test strip is
comprised of microparticles coated with a first antibody to albumin
and a second antibody to albumin covalently bound to said test
strip.
36. The system of claim 35 wherein said microparticles are selected
from the group consisting of colloidal gold particles, latex
particles, polystyrene particles, acrylic particles or other solid
phase microparticles.
37. The system of either of claim 34 or 36, wherein said
microparticles may be colored or tagged with a fluorescent
compound.
38. The system of claim 31 wherein the first test strip and the
second test strip may be arranged in parallel; or opposite to each
other; or at an angle to each other.
39. The system of claim 31 wherein the first test strip and the
second test strip are enclosed in a rigid cassette.
40. The system of claim 31 wherein said measurement device is a
reflectance spectrometer or a fluorometer comprising: a detector
for measuring the glycated albumin test result; a detector for
measuring the glycated albumin control band; a detector for
measuring the total albumin test result; a detector for measuring
the total albumin control band; an internal computer chip for
measurement and calculation; a liquid crystal display; an external
port to transfer data to an external computer and/or printer; a
battery and/or an external power source; and a rigid external case
with an aperture for inserting the test cassette.
41. The system of claim 40 whereby the one or more than one test
result can by displayed on said measurement device's liquid crystal
display in numerical format or in graphical format.
42. The system of claim 40 further comprising an internal memory
chip capable of storing one or more than one test result.
43. The system of claim 42 whereby the one or more than one test
result can be transferred to an external computer or printer.
44. A method of monitoring glycated albumin using a point-of-care
assay and determining a percent glycated albumin level comprising:
depositing a drop of blood into a sample well of an
immunochromatography system test cassette; transferring said blood
into the sample application pad thereby allowing blood plasma to
pass into a first conjugate pad of a first test strip and into a
second conjugate pad of a second test strip; binding said blood
plasma to anti-glycated albumin antibody-coated microparticles in
said first conjugate pad; binding said blood plasma to anti-total
albumin antibody-coated microparticles in said second first
conjugate pad; allowing blood plasma-bound antibody-coated
microparticles to migrate across said conjugate pad to a fixed band
of membrane-bound antibody; binding said blood plasma-bound
antibody-coated microparticles to said membrane bound antibody to
form a visible band; inserting said immunochromatography system
test cassette into a measurement device; providing numerical
results of glycated albumin levels from said first test strip and
total albumin levels from said second test strip; and calculating
said percent glycated albumin.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 10/538,392 filed Apr. 27, 2006, which is an
application under Section 371 of International patent application
number PCT/US2004/031202 filed Sep. 22, 2004 which in turn claims
the benefit under 35 USC .sctn.119(e) to U.S. provisional patent
application Ser. No. 60/505,392 filed Sep. 23, 2003, the entire
contents of all of which are incorporated by reference herein.
FIELD OF THE INVENTION
[0002] This invention relates generally to medical devices for
measuring levels of glycated albumin in blood from patients with
diabetes. More specifically, the present invention uses lateral
flow immunochromatography to measure both glycated albumin and
total albumin in a single sample. Additionally the present
invention provides methods for monitoring levels of glycated
albumin in the blood of diabetes patients using a point-of-care
assay and medical device.
BACKGROUND OF THE INVENTION
[0003] Diabetes mellitus, or diabetes, is a disease characterized
by elevated levels of plasma glucose. Uncontrolled hyperglycemia is
associated with increased risk of vascular disease including,
nephropathy, neuropathy, retinopathy, hypertension, and death.
There are two major forms of diabetes: Type 1 diabetes (or
insulin-dependent diabetes) and Type 2 diabetes (or non
insulin-dependent diabetes). The American Diabetes Association has
estimated that approximately 6% of the world population has
diabetes.
[0004] The goal of diabetic therapy is to maintain a normal level
of glucose in the blood. The American Diabetic Association has
recommended that diabetics monitor their blood glucose level at
least three times a day in order to adjust their insulin dosages
and/or their eating habits and exercise regimen. However, glucose
tests can only measure a point in time result and do not provide an
overall assessment of glycemic control over a period of time. The
measurement of glycated albumin has proven to be valuable measure
of the effectiveness of glycemic control over the preceding 2-3
weeks. The basis for measuring glycated albumin depends on the
nonenzymatic glycosylation of albumin and is directly proportional
to the level of glucose in plasma over a period of time. The
half-life of albumin in plasma is 2-3 weeks and as glycosylation
occurs at a constant rate over time the level of glycated albumin
provides a measure of the average blood glucose level over the
preceding two to three weeks.
[0005] Frequent monitoring of the individual's glycated albumin
would provide an accurate assessment of overall effectiveness of
glycemic control in the individual.
[0006] Current methodology for performing tests for glycated
albumin are complex to perform or require expensive instrumentation
and are generally performed in laboratories. It would be
advantageous to develop a simplified point-of-care assay that could
be utilized in a doctor's office or by the patient and there is
intensive research to develop such a test.
[0007] The present invention describes a simplified point-of-care
assay that utilizes disposable test strips and a reusable measuring
instrument.
SUMMARY OF THE INVENTION
[0008] The present invention is directed to medical devices and
methods for monitoring levels of glycated albumin in the blood of
diabetes patients using a point-of-care assay and medical device.
Specifically, the present invention uses lateral flow
immunochromatography to measure both glycated albumin and total
albumin in a single sample.
[0009] In an embodiment of the present invention, an
immunochromatographic system is provided for measuring glycated
albumin in a blood sample comprising a first test strip that
measures glycated albumin and a second test strip that measures
total albumin; and a measurement device that reads, calculates and
displays the result as the percentage of glycated albumin compared
to total albumin in the sample.
[0010] In another embodiment of the present invention, the first
test strip is comprised of microparticles coated with a first
antibody to glycated albumin and an immobilization agent covalently
bound to the membrane strip. The immobilization agent is a second
antibody to glycated albumin or phenyl boronic acid.
[0011] In alternative embodiments of the present invention the
first and second antibodies to glycated albumin are individually
monoclonal or polyclonal antibodies. The polyclonal antibodies may
be the whole antiserum, the IgG fraction or the purified
antibody.
[0012] In an embodiment of the present invention, the
microparticles of the first test strip are selected from the group
consisting of colloidal gold particles, latex particles,
polystyrene particles, acrylic particles or other solid phase
microparticles. Additionally the size of the microparticles can
vary from approximately 5 nm to approximately 50 nm in
diameter.
[0013] In another embodiment of the present invention, the second
test strip is comprised of microparticles coated with a first
antibody to albumin and an second antibody to albumin covalently
bound to the membrane strip.
[0014] In an embodiment of the present invention, the first and
second antibodies to albumin are individually monoclonal or
polyclonal antibodies. The polyclonal anti-albumin antibodies may
be the whole antiserum, the IgG fraction or the purified
antibody.
[0015] In an embodiment of the present invention, the
microparticles of the second test strip are selected from the group
consisting of colloidal gold particles, latex particles,
polystyrene particles, acrylic particles or other solid phase
microparticles. Additionally the size of the microparticles can
vary from approximately 5 nm to approximately 50 nm in
diameter.
[0016] In another embodiment of the present invention the
microparticles of either of the first or second test strips can
have particle size diameters of 10 nm, 20 nm, 30 nm and 40 nm.
[0017] In yet another embodiment of the present invention, the
microparticles of either the first or second test strip can either
colored or tagged with a fluorescent compound.
[0018] In an embodiment of the present invention, the first test
strip and the second test strip may be arranged in parallel; or
opposite to each other; or at an angle to each other. Additionally
the first test strip and the second test strip are enclosed in a
rigid cassette.
[0019] In an embodiment of the present invention the measurement
device is a reflectance spectrometer comprising: a reflectance
detector for measuring the glycated albumin test result; a
reflectance detector for measuring the glycated albumin control
band; a reflectance detector for measuring the total albumin test
result; a reflectance detector for measuring the total albumin
control band; an internal computer chip for measurement and
calculation; a liquid crystal display; an external port to transfer
data to an external computer and/or printer; a battery and/or an
external power source; and a rigid external case with an aperture
for inserting the test cassette.
[0020] In an embodiment of the present invention the measurement
device is a fluorometer composed comprising: a fluorescence
detector for measuring the glycated albumin test result; a
fluorescence detector for measuring the glycated albumin control
band; a fluorescence detector for measuring the total albumin test
result; a fluorescence detector for measuring the total albumin
control band; an internal computer chip for measurement and
calculation; a liquid crystal display; an external port to transfer
data to an external computer and/or printer; a battery and/or an
external power source; and a rigid external case with an aperture
for inserting the test cassette.
[0021] In another embodiment of the present invention the
measurement device further comprises an internal memory chip
capable of storing one or more than one test result.
[0022] In yet another embodiment of the present invention, the
measurement device can display one or more than one test result on
the measurement device's liquid crystal display in numerical format
or in graphical format. Additionally the test results can be
transferred to an external computer or printer.
[0023] In an embodiment of the present invention, a method of
monitoring glycated albumin using a point-of-care assay is provided
comprising: depositing a drop of blood into a sample well of an
immunochromatography system test cassette; transferring said blood
into the sample application pad thereby allowing blood plasma to
pass into a first conjugate pad of a first test strip; binding said
blood plasma to anti-glycated albumin antibody-coated
microparticles in said first conjugate pad; allowing blood
plasma-bound anti-glycated albumin antibody-coated microparticles
to migrate across said first conjugate pad to a fixed band of
membrane-bound anti-glycated albumin antibody; binding said blood
plasma-bound anti-glycated albumin antibody-coated microparticles
to said membrane bound anti-glycated albumin antibody to form a
visible band; inserting said immunochromatography system test
casette into a measurement device; and providing numerical results
of glycated albumin levels.
[0024] In an embodiment of the present invention, the method of
monitoring glycated albumin using a point-of-care assay further
comprises: depositing a drop of blood into a sample well of an
immunochromatography system cassette; transferring said blood into
the sample application pad thereby allowing blood plasma to pass
into a second conjugate pad of a second test strip; binding said
blood plasma to anti-total albumin antibody-coated microparticles
in said second first conjugate pad; allowing blood plasma-bound
anti-total albumin antibody-coated microparticles to migrate across
said second conjugate pad to a fixed band of membrane-bound
anti-total albumin antibody; binding said blood plasma-bound
anti-total albumin antibody-coated microparticles to said membrane
bound anti-total albumin antibody to form a visible band; inserting
said immunochromatography system test cassette into a measurement
device; and providing numerical results of total albumin
levels.
[0025] In another embodiment of the present invention, a method of
monitoring glycated albumin using a point-of-care assay is provided
wherein glycated albumin levels and said total albumin levels are
used to determine percent glycated albumin.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIG. 1 depicts a first view of the test strips made in
accordance with the teachings of the present invention.
[0027] FIG. 2 depicts a second view of the test strips made in
accordance with the teachings of the present invention.
[0028] FIG. 3 depicts a side view of the test strips made in
accordance with the teachings of the present invention.
[0029] FIG. 4 depicts a reflectance spectrometer as used with the
test strips made in accordance with the methods of the present
invention.
[0030] FIG. 4b depicts a fluorometer as used with the test strips
made in accordance with the methods of the present invention.
[0031] FIG. 5 depicts a first view of a test strip cassette made in
accordance with the teachings of the present invention.
[0032] FIG. 6 depicts a second view of a test strip cassette made
in accordance with the teachings of the present invention.
[0033] FIG. 7 depicts a reflectance spectrometer and test strip as
used in accordance with the methods of the present invention.
DESCRIPTION OF THE INVENTION
[0034] This invention utilizes the principle of lateral flow
immunochromatography to measure both glycated albumin and total
albumin. The patient's blood sample is placed in a test cassette
that contains reagents to separate the plasma from the red blood
cells and to perform the test. The test cassette is then inserted
into a measuring instrument that reads, calculates and reports the
result.
[0035] The rapid assay for glycated albumin is an
immunochromatographic method that utilizes antibodies to glycated
albumin and antibodies to total albumin on test strips. In order to
measure the percent of glycated albumin to total albumin, two
procedures are involved. The first procedure utilizes an
immunochromatographic test strip to measure glycated albumin. The
second procedure utilizes an immunochromatographic test strip to
measure total albumin. Both strips are contained within a single
exterior cassette (FIG. 1) that is inserted into a measuring
instrument (FIG. 7) that automatically reads, calculates and
displays the result.
[0036] Glycated Albumin Test
[0037] The test strip for measuring glycated albumin is shown in
FIGS. 1 and 2. The test strip consists of a solid phase support
(1), including but not limited to a cellulose nitrate membrane, to
which antibody to glycated albumin has been fixed to the
solid-phase substrate as a band (2). A sample application pad (3)
contacts a conjugate pad (4) containing microparticles coated with
anti-glycated albumin antibody. A control band is provided to bind
excess unreacted microparticles (5) and a reservoir pad (6) is
provided at the distal end of the membrane to absorb excess sample
fluid. The test strip is enclosed within a rigid cassette
containing a sample well and window segments to allow for
visualization and measurement of the test result.
[0038] To perform the test a small volume of blood is placed into
the sample well. The blood migrates into the sample application pad
which filters and binds the red blood cells allowing the plasma to
pass through into the conjugate pad where it reacts with the
antibody coated microparticles. Any glycated albumin present binds
to the anti-glycated albumin antibody-coated microparticles. The
microparticles continue to migrate across the cellulose membrane
until they come into contact with the fixed band of anti-glycated
albumin antibody. Any glycated albumin bound to microparticles
becomes bound to the membrane and causes the bound microparticles
to form a visible band. The intensity of the band is proportional
to the amount of glycated albumin bound to the microparticles. The
intensity of the visible band is estimated visually by comparison
to a visual standard or measured in an instrument developed for
this purpose.
[0039] Total Albumin Test
[0040] The test strip for measuring total albumin is shown in FIGS.
1 and 2. It consists of a solid phase substrate (1), including but
not limited to a cellulose nitrate membrane (1) to which antibody
to albumin has been fixed as a band (7). A sample application pad
(3) contacts a conjugate pad (8) containing microparticles coated
with anti-albumin antibody. A control band (9) is provided to bind
excess unreacted microparticles and a reservoir pad (6) is provided
at the distal end of the membrane to absorb excess sample fluid.
The test strip is enclosed within a rigid cassette containing a
sample well and window segments to allow for visualization and
measurement of the test result.
[0041] To perform the test a small volume of blood is placed into
the sample well. The blood migrates from the sample application pad
which filters and binds the red blood cells allowing the plasma to
pass into the conjugate pad where it reacts with the antibody
coated microparticles. Any albumin present binds to the
anti-albumin antibody coated microparticles. The microparticles
continue to migrate across the cellulose membrane until they come
into contact with the fixed band of anti-albumin antibody. Any
albumin bound to microparticles becomes bound to the membrane and
causes the bound microparticles to form a visible band. The
intensity of the band is proportional to the amount of albumin
bound to the microparticles. The intensity of the visible band is
estimated visually by comparison to a visual standard or measured
in an instrument developed for this purpose.
[0042] Measuring Instrument
[0043] In one embodiment of this invention, the measuring
instrument is a reflectance spectrophotometer that is specifically
designed to measure the intensity of the glycated albumin test band
on the glycated albumin test strip, the total albumin test band on
the total albumin test strip, and to calculate a result from these
readings. The instrument has two sets of detectors: one detector
set is for measuring glycated albumin and the other detector set is
for measuring total albumin. The result is then calculated
according to a mathematical algorithm derived from data obtained
from measurement of standards of glycated and total albumin. The
result is expressed as the percent of glycated albumin compared to
total albumin present.
[0044] Alternatively, other methods for measuring the density of
the aggregated microparticles may be employed. For example, in
another embodiment of the present invention, the measuring
instrument may be a fluorometer that measures the fluorescence that
is emitted from microroparticles that have been tagged with a
fluorescent dye including, but not limited to, fluorescein or
rhodamine red. In this embodiment there will be an excitatory beam
of light projected onto the test bands and onto the control bands,
and the emitted light from each band will be individually read by
the corresponding detectors sensitive to the wavelength of the
emitted light. The data reduction and reporting of the result will
be as described above for the reflectance spectrophotometer.
EXAMPLE 1
Glycated Albumin Test
[0045] A blood sample, such as that obtained from a finger stick,
is placed in the sample well and allowed to absorb into the sample
application pad. The sample application pad is composed of porous
cellulose material but other woven or porous materials including
but not limited to glass fibers may be used. The sample application
pad has a porosity that does not allow the passage of red blood
cells but allows the passage of the plasma. Alternatively, the
application pad can be treated with binding agents such as lectins
that bind the red blood cells and prevent them from passage through
the application membrane.
[0046] The filtered plasma sample then flows into a conjugate pad
containing microparticles. The conjugate pad is composed of porous
cellulose material but other woven or porous materials such as
glass fibers may be used. The microparticles are composed of
materials including, but not limited to, colloidal gold, latex
particles, acrylic particles or polystyrene particles with
diameters that may range from approximately 5 nm to 50 nm.
Microparticles composed of other materials may also be employed and
are within the scope of this invention. In alternative embodiment
of the present invention, colored or fluorescent tagged
microparticles can be employed to increase the sensitivity of the
system.
[0047] In embodiments of the present invention, the microparticles
are coated with either polyclonal or monoclonal antibodies to
glycated albumin. The polyclonal anti-glycated albumin antibodies
are prepared in immunized animals, including but not limited to
rabbits, sheep, goats, or other immunized species of animals, or by
monoclonal antibody techniques. Either the whole antiserum, or the
IgG purified fraction, or the affinity purified antibody to
glycated albumin may be employed. The methods for immunization of
animals and the preparation and purification of antibody is
performed according to standard laboratory procedures and are known
to those skilled in the art.
[0048] Similarly, the methods of developing monoclonal antibodies
is performed according to standard laboratory procedures and are
known to those skilled in the art. The microparticles may be coated
with the antibody by passive adsorption, by chemical conjugation
such as covalent binding, or through binding to an intermediate
agent such as to Protein A-coated microparticles. The methods for
coating microparticles are performed according to standard
laboratory procedures and are familiar to those skilled in the
art.
[0049] When the test sample comes into contact with the antibody
coated microparticles, the antibody will bind any glycated albumin
present. The microparticles will continue to migrate across the
membrane until they reach the band of anti-glycated albumin
antibody fixed to the membrane. Any microparticles containing bound
glycated albumin will become bound to the fixed band of
anti-glycated albumin antibody to form a visible band.
[0050] Alternatively the membrane may be treated with chemicals
known to bind glycated proteins such as phenyl boronic acids which
are applied as a band to the membrane strip. Any microparticles
containing bound glycated albumin will become bound to the fixed
band of phenyl boronic acid to form a visible band. Independent of
the method by which the glycated albumin becomes bound to the test
strip, the density of the band formed will be directly proportional
to the amount of glycated albumin present in the blood sample. The
density of the band can be measured using a reflectance
spectrometer for colored microparticles or a fluorometer for
microparticles tagged with a fluorescent compound. The measurements
are used to calculate the percentage of glycated albumin compared
to total albumin in the blood sample.
[0051] In order to verify that the test strips are functioning
correctly each test strip can additionally have a control band
located distal to the test band. For the glycated albumin test
strip this control band is composed of antibody directed against
the species antibody that was used to coat the microparticles. For
example, if rabbit anti-human glycated albumin antibody used to
coat the microparticles, then the control band would use another
species such as goat or sheep antibodies directed against rabbit
IgG immunoglobulin. The antibodies in the control band bind to the
excess unreacted antibody-coated microparticles that were not bound
to the test band but continued to migrate across the membrane until
bound by the control reagent. The intensity of the control band is
measured using a reflectance spectrometer or fluorometer and the
data is used to determine if the test is performing correctly.
EXAMPLE 2
Total Albumin Test
[0052] A blood sample, such as that obtained from a finger stick,
is placed in the sample well and allowed to absorb into the sample
application pad. The sample application pad is composed of porous
cellulose material but other woven or porous materials, including
but not limited to glass fibers may be used. The sample application
has a porosity that does not allow the passage of red blood cells
but allows the passage of the plasma. Alternatively, the
application pad can be treated with binding agents such as lectins
that bind the red blood cells and prevent them from passage through
the application membrane.
[0053] The filtered plasma sample then flows into a conjugate pad
containing microparticles. The conjugate pad is composed of porous
cellulose material but other woven or porous materials, including
but not limited to glass fibers may be used. The microparticles are
composed of materials including, but not limited to, colloidal
gold, latex particles, acrylic particles or polystyrene particles
with diameters that may range from approximately 5 nm to 50 nm.
Microparticles composed of other materials may also be employed and
are within the scope of this invention. Colored or fluorescence
tagged microparticles may be employed to increase the sensitivity
of measurement of the result.
[0054] In embodiments of the present invention, the microparticles
are coated with either polyclonal or monoclonal antibodies to
glycated albumin. The polyclonal anti-albumin antibodies are
prepared in immunized animals including but not limited to rabbits,
sheep, goats, or other immunized species of animals, or by
monoclonal antibody techniques. Either the whole antiserum, or the
IgG purified fraction, or the affinity purified antibody to albumin
may be employed. The methods for immunization of animals and the
preparation and purification of antibody is performed according to
standard laboratory procedures and are known to those skilled in
the art. Similarly, the methods of developing monoclonal antibodies
are performed according to standard laboratory procedures and are
known to those skilled in the art.
[0055] The microparticles may be coated with the antibody by
passive adsorption, by chemical conjugation such as covalent
binding, or through binding to an intermediate agent such as to
Protein A-coated microparticles. The methods for coating
microparticles are performed according to standard laboratory
procedures and are familiar to those skilled in the art.
[0056] When the test sample comes into contact with the antibody
coated microparticles the antibody binds any albumin present. The
microparticles continue to migrate across the membrane until they
reach the band of anti-albumin antibody fixed to the membrane. Any
microparticles containing bound albumin become bound to the fixed
band of anti-albumin antibody to form a visible band. The density
of the band formed is directly proportional to the amount of
albumin present in the blood sample. The density of the band is
measured using a reflectance spectrometer for colored
microparticles or a fluorometer for microparticles tagged with a
fluorescent compound. The measurements are used to calculate the
percentage of glycated albumin compared to total albumin in the
blood sample.
[0057] In order to verify that the test strips are functioning
correctly each test strip has an additional band of fixed reagent
located distal to the test band. For the test strip, this control
band is composed of antibody directed against the species antibody
that was used to coat the microparticles. For example, if rabbit
anti-human albumin antibody was used to coat the microparticles
then the control band uses another species such as goat or sheep
antibodies directed against rabbit IgG immunoglobulin. The
antibodies in the control band bind to the excess unreacted
antibody coated microparticles that were not bound to the test band
but continued to migrate across the membrane until bound by the
control reagent. The intensity of the control band is measured
using a reflectance spectrometer or fluorometer and the data is
used to determine if the test is performing correctly.
EXAMPLE 3
The Measuring Instrument
[0058] The measuring instrument shown in FIG. 4a is a reflectance
spectrometer and is composed of the following components: A
detector (10) calibrated to read the reflectance of the
microparticles fixed to the glycated albumin band on the glycated
albumin test strip; a detector (11) calibrated to read the
reflectance of the microparticles fixed to the control band on the
glycated albumin test strip; a detector (12) calibrated to read the
reflectance of the microparticles fixed to the total albumin band
on the total albumin test strip; a detector (13) calibrated to read
the reflectance of the microparticles fixed to the control band on
the total albumin test strip; a computing chip and electronic
circuitry (14) to collect the data from the detectors and to
calculate the result.
[0059] The calculations are based on a mathematical algorithm and a
reference standard curve. The standard curve is derived from value
assigned standards and the instrument is precalibrated at the
manufacturing facility before it is distributed. The result is
expressed as the percent of glycated albumin compared to total
albumin and displayed on a liquid crystal display (15). Successive
results obtained over a period of time are stored in the instrument
and can be retrieved on demand and displayed in numerical format or
in graphical format. Typically, the result will be displayed along
with the date of the test. The user may then select to have all the
previous stored test results and their date displayed, or have all
the results presented as a graph so that any trends can be
identified. In order to enter commands to the internal computer the
instrument may contain either buttons or a keyboard on its exterior
case.
[0060] The results can also be downloaded via an external port to
an external computer and/or printed on an external printer (16).
The instrument's electronics are powered by an internal battery
(17) and/or external power source (18). The components are housed
in a rigid exterior case (19) with a window (20) for the display
monitor and an aperture (21) for inserting the test cassette.
[0061] Alternatively, the measuring instrument may be a fluorometer
(FIG. 4b) that measures the density of aggregated microparticles
that have been tagged with a fluorescent dye such fluorescein or
rhodamine. The fluorometer is composed of the following components:
A detector (22) calibrated to read the fluorescence of the
microparticles fixed to the glycated albumin band on the glycated
albumin test strip; a detector (23) calibrated to read the
fluorescence of the microparticles fixed to the control band on the
glycated albumin test strip; a detector (24) calibrated to read the
fluorescence of the microparticles fixed to the total albumin band
on the total albumin test strip; a detector (25) calibrated to read
the fluorescence of the microparticles fixed to the control band on
the total albumin test strip; a computing chip and electronic
circuitry (26) to collect the data from the detectors and to
calculate the result. Using fluorescein tagged microparticles as an
example, the excitatory beam of light (492 nm wavelength) is
projected onto the test bands and onto the control bands, and the
emitted light from each band is individually read by the
corresponding detectors sensitive to the wavelength (518 nm) of the
emitted light. Alternatively, other fluorescent compounds may be
used and the wavelength of the exciting beam and the wavelength of
the resulting fluorescence to be measured is adjusted
accordingly.
[0062] The calculations are based on a mathematical algorithm and a
reference standard curve. The standard curve is derived from value
assigned standards and the instrument is precalibrated at the
manufacturing facility before distribution. The result is expressed
as the percent of glycated albumin compared to total albumin and is
displayed on a liquid crystal display (27). Successive results
obtained over a period of time are stored in the instrument and can
be retrieved on demand and displayed in numerical format or in
graphical format. Typically, the result is displayed along with the
date of the test. The user may then select to have all the previous
stored test results and their date displayed, or have all the
results presented as a graph so that any trends can be identified.
In order to enter commands to the internal computer the instrument
may contain either buttons or a keyboard on its exterior case.
[0063] The results can also be downloaded via an external port to
an external computer and/or printed on an external printer (28).
The instrument's electronics are powered by an internal battery
(29) and/or external power source (30). The components are housed
in a rigid exterior case (31) with a window (32) for the display
monitor and an aperture (33) for inserting the test cassette.
[0064] In one embodiment of this invention, the test cassette is
designed to enclose two test strips arranged in a parallel fashion
(FIG. 1) and the sample application well is constructed so that the
test sample fluid can migrate across both test strips
simultaneously. However, other test cassette configurations may be
employed using the same principles described in this invention and
are considered to be within the scope of this invention. For
example, the sample application well may be centrally located with
the glycated albumin test strip and the total albumin test strip
pointing outward in a radial direction. FIG. 5 shows the test
strips arrangement as diametrically opposite to each other and FIG.
6 shows the test strips to be at an angle to each other. In these
examples the test cassette is in the shape of a rectangular or
square configuration. The aperture in the measuring instrument for
inserting these cassettes is adjusted to accommodate the shape of
these cassettes.
[0065] In an embodiment of the present invention, the measuring
instrument is a reflectance spectrometer which measures a
particular wavelength of the light reflected from the colored
microparticles. The amount of reflected light measured at the test
band and control band sites is directly proportional to the density
of the aggregated microparticles at each site
[0066] Alternatively, a fluorometer may be used as the measuring
instrument. In this example, the microparticles are tagged with an
internal fluorescent dye such as fluorescein or rhodamine red. The
fluorescence-tagged microparticles are excited at one wavelength of
light which causes them to fluoresce at a different wavelength of
light. The amount of fluorescence measured at the test band and
control band sites is directly proportional to the density of the
aggregated microparticles at each site.
[0067] In another embodiment of the present invention, the
measuring instrument is of small size, compact and lightweight. In
general, it is similar in appearance and design to the various
handheld glucometers in common usage. Such variations are cosmetic
in nature and are considered to be within the scope of this
invention.
* * * * *