U.S. patent application number 12/906170 was filed with the patent office on 2011-08-11 for devices and systems for biomarker detection.
Invention is credited to Richard P. Gill, Richard Selinfreund, Rakesh Vig.
Application Number | 20110195872 12/906170 |
Document ID | / |
Family ID | 44352629 |
Filed Date | 2011-08-11 |
United States Patent
Application |
20110195872 |
Kind Code |
A1 |
Selinfreund; Richard ; et
al. |
August 11, 2011 |
DEVICES AND SYSTEMS FOR BIOMARKER DETECTION
Abstract
Devices and systems for biomarker detection . In at least one
embodiment of a diagnostic testing device of the present
disclosure, the device comprises a fluid collector to collect a
body fluid, and a housing, to test and retain the body fluid. The
housing, in at least one embodiment, comprises a collection
chamber, having an open end, to receive the fluid collector and
contain a stabilization reagent, at least one membrane test strip,
to indicate the presence or absence of at least one diagnostic
marker, and an immunoassay-based fingerprint acquisition pad in
fluid communication with the collection chamber.
Inventors: |
Selinfreund; Richard; (Terre
Haute, IN) ; Vig; Rakesh; (Durham, CT) ; Gill;
Richard P.; (Essex, CT) |
Family ID: |
44352629 |
Appl. No.: |
12/906170 |
Filed: |
October 18, 2010 |
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Current U.S.
Class: |
506/39 |
Current CPC
Class: |
Y10T 436/105831
20150115; Y10T 436/10 20150115; A61B 10/0045 20130101; Y10T
436/2525 20150115; G01N 33/54393 20130101; G01N 33/50 20130101;
C12Q 1/40 20130101 |
Class at
Publication: |
506/39 |
International
Class: |
C40B 60/12 20060101
C40B060/12 |
Claims
1. A diagnostic testing device, comprising: a fluid collector to
collect a body fluid; and a housing, to test and retain the body
fluid, the housing comprising: a collection chamber, having an open
end, to receive the fluid collector and contain a stabilization
reagent to completely or substantially prevent the degradation or
inactivation of a diagnostic marker in the body fluid; at least one
membrane test strip, in fluid communication with the collection
chamber, to indicate the presence or absence of at least one
diagnostic marker; and an immunoassay-based fingerprint acquisition
pad in fluid communication with the collection chamber, the
acquisition pad having at least one control zone including a
control reagent to identify the body fluid donor, the reagent
including a member of a predetermined ligand/receptor binding pair;
and a plurality of reaction zones, each of which includes a
reaction reagent to determine the presence of a specific diagnostic
marker in the fluid sample, the reaction reagent including a member
of a predetermined ligand/receptor binding pair.
2. The device of claim 1, wherein the stabilizing agent is useful
to completely or substantially prevent degradation or inactivation
of a diagnostic marker selected from the group consisting of a
protein, a glycoprotein, a nucleic acid, an enzyme, an enzyme
inhibitor, and a metabolite.
3. The device of claim 1, wherein the stabilizing agent is useful
to completely or substantially prevent degradation or inactivation
of a diagnostic marker indicative of a disease.
4. The device of claim 3, wherein the disease state is selected
from the group consisting of a cancer, a metabolic disease, a
cardiac disease, diabetes, a bacterial infection, a viral
infection, a fungal infection, and a parasitic infection.
5. The device of claim 1, wherein the stabilizing agent is selected
from the group consisting of Fixanal.RTM. Buffer 6.0, acetic acid,
aluminum hydroxide bentonite, aluminum sulfate hydrate, benzoic
acid, aluminum potassium sulfate dodecahydrate, caffeine, and
3-tert-butyl-hydroxyanisole, or a combination thereof.
6. The device of claim 1, wherein the stabilizing agent is capable
of inhibiting degradation of the diagnostic marker to an inhibitory
degree, wherein the inhibitory degree is selected from the group
consisting of at least about 40%, at least about 45%, at least
about 50%, at least about 55%, at least about 60%, at least about
65%, at least about 70%, at least about 75%, at least about 80%, at
least about 85%, at least about 90%, at least about 95%, and at
least about 99%.
7. The device of claim 1, wherein the stabilizing agent has a
concentration selected from the group consisting of about 200 parts
per million (ppm) to about 2000 ppm, about 400 ppm to about 1600
ppm, about 600 ppm to about 1400 ppm, about 800 ppm to about 1200
ppm, and about 400 ppm to about 600 ppm.
8. The device of claim 1, wherein the stabilizing agent is able to
inhibit the degradation or inactivation of the diagnostic marker
for an inhibitory period selected from the group consisting of at
least one minute, at least about five minutes, at least about ten
minutes, at least about fifteen minutes, at least about thirty
minutes, at least about one hour, at least about two hours, at
least about four hours, and at least about eight hours.
9. The device of claim 1, further comprising an interface in
communication with processor, wherein the processor is capable of
analyzing the results from the diagnostic testing device, and
transmitting the analyzed results to a recipient.
10. A diagnostic testing device, comprising: a fluid collector to
collect a body fluid; and a housing, to test and retain the body
fluid, the housing comprising: a collection chamber, having an open
end, to receive the fluid collector and containing a stabilization
reagent to completely or substantially prevent the degradation or
inactivation of a diagnostic marker in the body fluid; at least one
membrane test strip in fluid communication with the collection
chamber, to indicate the presence or absence of at least one
diagnostic marker; and an immunoassay-based fingerprint acquisition
pad in fluid communication with the collection chamber, the
acquisition pad having at least one control zone including a
control reagent to identify the fluid sample donor, the reagent
including a member of a predetermined ligand/receptor binding pair;
and a plurality of reaction zones, each of which includes a
reaction reagent to determine the presence of a specific diagnostic
marker in the fluid sample, the reaction reagent including a member
of a predetermined ligand/receptor binding pair wherein the
stabilizing agent is selected from the group consisting of
Fixanal.RTM. Buffer 6.0, acetic acid, aluminum hydroxide bentonite,
aluminum sulfate hydrate, benzoic acid, aluminum potassium sulfate
dodecahydrate, caffeine, and 3-tert-butyl-hydroxyanisole, or a
combination thereof; and wherein the body fluid is selected from
the group consisting of saliva, a mucous secretion, tears, sweat,
semen, urine, a vaginal secretion, exhalate, blood, serum, and an
anal secretion.
11. The device of claim 1, further comprising an interface in
communication with processor, wherein the processor is capable of
analyzing the results from the diagnostic testing device, and
transmitting the analyzed results to a recipient.
12. The device of claim 10, wherein the stabilizing agent is
capable of inhibiting degradation of the diagnostic marker to an
inhibitory degree, wherein the inhibitory degree is selected from
the group consisting of at least about 40%, at least about 45%, at
least about 50%, at least about 55%, at least about 60%, at least
about 65%, at least about 70%, at least about 75%, at least about
80%, at least about 85%, at least about 90%, at least about 95%,
and at least about 99%.
13. The device of claim 10, wherein the stabilizing agent has a
concentration selected from the group consisting of about 200 parts
per million (ppm) to about 2000 ppm, about 400 ppm to about 1600
ppm, about 600 ppm to about 1400 ppm, about 800 ppm to about 1200
ppm, and about 400 ppm to about 600 ppm.
14. The device of claim 10, wherein the stabilizing agent is able
to inhibit the degradation or inactivation of the diagnostic marker
for an inhibitory period selected from the group consisting of at
least one minute, at least about five minutes, at least about ten
minutes, at least about fifteen minutes, at least about thirty
minutes, at least about one hour, at least about two hours, at
least about four hours, and at least about eight hours.
Description
PRIORITY
[0001] This U.S. utility patent application claims the priority
benefit of U.S. Provisional Patent Application Ser. No. 61/390,002,
filed Oct. 5, 2010; U.S. Provisional Patent Application Ser. No.
61/385,347, filed Sep. 22, 2010; U.S. Provisional Patent
Application Ser. No. 61/383,401, filed Sep. 16, 2010; U.S.
Provisional Patent Application Ser. No. 61/379,598, filed Sep. 2,
2010; U.S. Provisional Patent Application Ser. No. 61/378,960,
filed Sep. 1, 2010; U.S. Provisional Patent Application Ser. No.
61/373,619, filed Aug. 13, 2010; U.S. Provisional Patent
Application Ser. No. 61/364,964, filed Jul. 16, 2010; U.S.
Provisional Patent Application Ser. No. 61/364,969, filed Jul. 16,
2010; U.S. Provisional Patent Application Ser. No. 61/364,975,
filed Jul. 16, 2010; U.S. Provisional Patent Application Ser. No.
61/364,978, filed Jul. 16, 2010; U.S. Provisional Patent
Application Ser. No. 61/364,982, filed Jul. 16, 2010; U.S.
Provisional Patent Application Ser. No. 61/365,179, filed Jul. 16,
2010; U.S. Provisional Patent Application Ser. No. 61/322,768,
filed Apr. 9, 2010; and U.S. Provisional Patent Application Ser.
No. 61/303,165, filed Feb. 10, 2010. The contents of each of these
applications are hereby incorporated by reference in their entirety
into this disclosure.
BACKGROUND
[0002] Biomarkers are molecular indicators of a specific biological
property, a biochemical feature or facet that can be used to
measure the progress of a disease or the effects of a treatment.
For example, serum low-density lipoprotein (LDL) is a biomarker of
cholesterol and blood pressure, while the P53 gene is a biomarker
for cancer. For chronic diseases and conditions, such as diabetes
and allergies, accurate diagnosis is particularly important,
especially where the side effects of a treatment are severe.
[0003] Diagnostic tests using biomarkers as molecular indicators
not only detect the presence or absence of the biomarker, but often
must measure the exact concentration of a biomarker to determine
whether an abnormal condition exists. Because of the requirement
for accuracy, the process of sample collection, preparation, and
analysis are often complicated and time consuming. Currently,
blood-based assays for biomarker presence or activity are
considered to be the "gold standard" for biomarker-type assays.
[0004] Despite the desire for accurate results, rapid, point of
care analysis of biological samples using biomarkers is becoming
increasingly important in the present medical environment due to
the need for quick results.
SUMMARY
[0005] The disclosure of the present application provides various
diagnostic testing devices.
[0006] In at least one embodiment of a diagnostic testing device of
the present disclosure, the device comprises a fluid collector to
collect a body fluid, and a housing, to test and retain the body
fluid. The housing, in at least one embodiment, comprises a
collection chamber, having an open end, to receive the fluid
collector and contain a stabilization reagent to completely or
substantially prevent the degradation or inactivation of a
diagnostic marker in the body fluid, at least one membrane test
strip, in fluid communication with the collection chamber, to
indicate the presence or absence of at least one diagnostic marker,
and an immunoassay-based fingerprint acquisition pad in fluid
communication with the collection chamber, the acquisition pad
having at least one control zone including a control reagent to
identify the body fluid donor, the reagent including a member of a
predetermined ligand/receptor binding pair, and a plurality of
reaction zones, each of which includes a reaction reagent to
determine the presence of a specific diagnostic marker in the fluid
sample, the reaction reagent including a member of a predetermined
ligand/receptor binding pair.
[0007] In at least one embodiment of a diagnostic testing device of
the present disclosure, the stabilizing agent is useful to
completely or substantially prevent degradation or inactivation of
a diagnostic marker selected from the group consisting of a
protein, a glycoprotein, a nucleic acid, an enzyme, an enzyme
inhibitor, and a metabolite.
[0008] In at least one embodiment of a diagnostic testing device of
the present disclosure, the stabilizing agent is useful to
completely or substantially prevent degradation or inactivation of
a diagnostic marker indicative of a disease.
[0009] In at least one embodiment of a diagnostic testing device of
the present disclosure, the disease state is selected from the
group consisting of a cancer, a metabolic disease, a cardiac
disease, diabetes, a bacterial infection, a viral infection, a
fungal infection, and a parasitic infection.
[0010] In at least one embodiment of a diagnostic testing device of
the present disclosure, the stabilizing agent is selected from the
group consisting of Fixanal.RTM. Buffer 6.0, acetic acid, aluminum
hydroxide bentonite, aluminum sulfate hydrate, benzoic acid,
aluminum potassium sulfate dodecahydrate, caffeine, and
3-tert-butyl-hydroxyanisole, or a combination thereof.
[0011] In at least one embodiment of a diagnostic testing device of
the present disclosure, the stabilizing agent is capable of
inhibiting degradation of the diagnostic marker to an inhibitory
degree, wherein the inhibitory degree is selected from the group
consisting of at least about 40%, at least about 45%, at least
about 50%, at least about 55%, at least about 60%, at least about
65%, at least about 70%, at least about 75%, at least about 80%, at
least about 85%, at least about 90%, at least about 95%, and at
least about 99%.
[0012] In at least one embodiment of a diagnostic testing device of
the present disclosure, the stabilizing agent has a concentration
selected from the group consisting of about 200 parts per million
(ppm) to about 2000 ppm, about 400 ppm to about 1600 ppm, about 600
ppm to about 1400 ppm, about 800 ppm to about 1200 ppm, and about
400 ppm to about 600 ppm.
[0013] In at least one embodiment of a diagnostic testing device of
the present disclosure, the stabilizing agent is able to inhibit
the degradation or inactivation of the diagnostic marker for an
inhibitory period selected from the group consisting of at least
one minute, at least about five minutes, at least about ten
minutes, at least about fifteen minutes, at least about thirty
minutes, at least about one hour, at least about two hours, at
least about four hours, and at least about eight hours.
[0014] In at least one embodiment of a diagnostic testing device of
the present disclosure, the device further comprising an interface
in communication with processor, wherein the processor is capable
of analyzing the results from the diagnostic testing device, and
transmitting the analyzed results to a recipient.
DESCRIPTION OF THE DRAWINGS
[0015] The features and advantages of the present disclosure, and
the manner of attaining them, will be more apparent and better
understood by reference to the following descriptions taken in
conjunction with the accompanying figures, wherein:
[0016] FIGS. 1A and B show a diagram of a detection system,
according to at least one embodiment of the present disclosure;
[0017] FIGS. 2-4 shows a diagram of a method of producing a bead
system, according to at least one embodiment of the present
disclosure;
[0018] FIG. 5 shows a flowchart of a method for creating a bead
system, according to at least one embodiment of the present
disclosure;
[0019] FIGS. 6A and B show a diagram of a test strip, according to
at least one embodiment of the present disclosure;
[0020] FIG. 6C show a diagram of a microarray, according to at
least one embodiment of the present disclosure;
[0021] FIG. 6D shows a diagram of a microfluidic system, according
to at least one embodiment of the present disclosure;
[0022] FIG. 7 shows a flowchart of a method of stabilizing
diagnostic markers, according to at least one embodiment of the
present disclosure;
[0023] FIG. 8 shows a flowchart of a method analyzing a diagnostic
markers, according to at least one embodiment of the present
disclosure;
[0024] FIG. 9 shows a an exemplary system framework, according to
at least one embodiment of the present disclosure;
[0025] FIG. 10 shows a graphical depiction of .alpha.-amylase
inhibition according to at least one embodiment of the present
disclosure;
[0026] FIG. 11 shows a graphical depiction of .alpha.-amylase
inhibition according to at least one embodiment of the present
disclosure;
[0027] FIG. 12 shows a graphical depiction of lysozyme inhibition
according to at least one embodiment of the present disclosure;
[0028] FIG. 13 shows a graphical depiction of a galactose oxidase
screen according to at least one embodiment of the present
disclosure;
[0029] FIG. 14A shows a three dimensional plot of glycated
Hemoglobin A1c (HbA1c) analyzed by high pressure liquid
chromatography (HPLC) according to at least one embodiment of the
present disclosure;
[0030] FIG. 14B shows a ultraviolet (UV) image of HbA1c analyzed by
HPLC according to at least one embodiment of the present
disclosure;
[0031] FIG. 14C shows a chromatogram of HbA1c analyzed by HPLC
according to at least one embodiment of the present disclosure;
[0032] FIG. 15A shows a three dimensional plot of HbA1c analyzed by
HPLC following exposure to saliva according to at least one
embodiment of the present disclosure;
[0033] FIG. 15B shows a UV image of HbA1c analyzed by HPLC
following exposure to saliva according to at least one embodiment
of the present disclosure;
[0034] FIG. 15C shows a chromatogram of HbA1c analyzed by HPLC
following exposure to saliva according to at least one embodiment
of the present disclosure;
[0035] FIG. 16A shows a three dimensional plot of HbA1c analyzed by
HPLC following exposure to saliva and a stabilizing mixture
according to at least one embodiment of the present disclosure;
[0036] FIG. 16B shows a UV image of HbA1c analyzed by HPLC
following exposure to saliva and a stabilizing mixture according to
at least one embodiment of the present disclosure;
[0037] FIG. 16C shows a chromatogram of HbA1c analyzed by HPLC
following exposure to saliva and a stabilizing mixture according to
at least one embodiment of the present disclosure;
[0038] FIG. 17 shows a comparison of chromatograms from HPLC
analyzed HbA1c (FIG. 17A), HbA1c+Saliva (FIG. 17B), and
HbA1c+Saliva+Stabilizing mixture (FIG. 17C) according to at least
one embodiment of the present disclosure;
[0039] FIG. 18 shows a comparison of UV images from HPLC analyzed
HbA1c (FIG. 18A), HbA1c+Saliva (FIG. 18B), and
HbA1c+Saliva+Stabilizing mixture (FIG. 18C) according to at least
one embodiment of the present disclosure;
[0040] FIG. 19 shows a comparison of three dimensional plots from
HPLC analyzed HbA1c (FIG. 19A), HbA1c+Saliva (FIG. 19B), and
HbA1c+Saliva+Stabilizing mixture (FIG. 19C) according to at least
one embodiment of the present disclosure;
[0041] FIG. 20A shows a three dimensional plot of Cancer Antigen
19-9 (CA 19-9) analyzed by high pressure liquid chromatography
(HPLC) according to at least one embodiment of the present
disclosure;
[0042] FIG. 20B shows a ultraviolet (UV) image of CA 19-9 analyzed
by HPLC according to at least one embodiment of the present
disclosure;
[0043] FIG. 21A shows a three dimensional plot of CA 19-9 analyzed
by HPLC following exposure to saliva according to at least one
embodiment of the present disclosure;
[0044] FIG. 21B shows a UV image of CA 19-9 analyzed by HPLC
following exposure to saliva according to at least one embodiment
of the present disclosure;
[0045] FIG. 22A shows a three dimensional plot of CA 19-9 analyzed
by HPLC following exposure to saliva and a stabilizing mixture
according to at least one embodiment of the present disclosure;
[0046] FIG. 22B shows a UV image of CA 19-9 analyzed by HPLC
following exposure to saliva and a stabilizing mixture according to
at least one embodiment of the present disclosure;
[0047] FIG. 23 shows a comparison of three dimensional plots from
HPLC analyzed CA 19-9 (FIG. 23A), CA 19-9+Saliva (FIG. 23B), and CA
19-9+Saliva+Stabilizing mixture (FIG. 23C) according to at least
one embodiment of the present disclosure;
[0048] FIG. 24 shows a comparison of UV images from HPLC analyzed
CA 19-9 (FIG. 24A), CA 19-9+Saliva (FIG. 24B), and CA
19-9+Saliva+Stabilizing mixture (FIG. 24C) according to at least
one embodiment of the present disclosure;
[0049] FIG. 25 shows a graphical depiction of Peroxidase activity
inhibition by an inhibitor cocktail according to at least one
embodiment of the present disclosure;
[0050] FIG. 26 shows a graphical depiction of Peroxidase activity
inhibition by caffeine according to at least one embodiment of the
present disclosure;
[0051] FIG. 27 shows a graphical depiction of Peroxidase activity
inhibition by 3-tert-butyl-hydroxyanisole according to at least one
embodiment of the present disclosure;
[0052] FIG. 28 shows a graphical depiction of Peroxidase activity
inhibition by benzoic acid according to at least one embodiment of
the present disclosure;
[0053] FIG. 29 shows a graphical depiction of Peroxidase activity
inhibition by aluminum potassium sulfate dodecahydrate according to
at least one embodiment of the present disclosure;
[0054] FIG. 30 shows a schematic of a diagnostic system, according
to at least one embodiment of the present disclosure;
[0055] FIG. 31 shows a graphical representation of an assay
component, according to at least one embodiment of the present
disclosure; and
[0056] FIG. 32 shows a graphical depiction of a fluid collection
device, according to at least one embodiment of the present
disclosure.
DETAILED DESCRIPTION
[0057] For the purposes of promoting an understanding of the
principles of the present disclosure, reference will now be made to
the embodiments illustrated in the drawings, and specific language
will be used to describe the same. It will nevertheless be
understood that no limitation of the scope of this disclosure is
thereby intended.
[0058] The disclosure of the present application provides various
compositions, systems, and methods for biomarker stabilization and
analysis. Specifically, formulations are disclosed herein, which
function to preserve the level of diagnostic markers, such as
biomarkers, in body fluids, from enzymatic alteration, or
degradation. Additionally, methods for the collection and analysis
of body fluids are disclosed. The methods and formulations
disclosed herein can be used to improve the sampling and testing of
a body fluid by conditioning the body fluids for the stabilization
of specific biomolecules and/or drug metabolites. As used herein,
the term "body fluids" includes fluids produced by the body, such
as saliva, or fractions thereof, mucous secretions, tears, sweat,
bile, semen, urine, vaginal secretions, exhalations, anal
secretions, blood, plasma, serum and mixtures of thereof. Body
fluids may also comprise cancer cells, peripheral blood mononuclear
cells, lymphocytes, lymph fluid, and other tissue secretions or
fluid.
Saliva
[0059] Saliva is clear, viscous fluid with a slightly alkaline pH
and a pI range from 11.5-3.0. It is hypotonic, composed of about
99.5% water, and also contains ions (e.g., K.sup.+, Na.sup.+,
Ca.sup.2+, Mg.sup.2+, H.sup.+, Cl.sup.-, HCO.sub.3.sup.-, I.sup.-,
F.sup.-, HPO.sub.4.sup.2-), and small organic molecules (e.g.,
ureas, hormones, lipids, DNA, and RNA). There are multiple
contributors to the composition of saliva. Saliva has a complex
"proteome"-10.sup.6 D glycoproteins to 1000D peptides. It contains
secretory products of salivary glands, products of B cells, PMNs,
epithelial cells, and bacteria. Major (e.g., parotid,
submandibular, and sublingual) and minor (e.g., palatine and
retromolar) glands contribute to the composition of saliva, along
with extraneous contributors such as gingival crevicular fluid,
serum proteins, white blood cells and their byproducts, oral
epithelial cells, oral bacteria, food debris and dissolved food
components.
[0060] Saliva from different glands may differ in composition. For
example, saliva from the parotid gland is dominated by serous
secretory cells, whereas saliva from the submandibular and
sublingual glands and minor glands are mixed serous or mostly
mucous. There can also be qualitative and quantitative differences
in saliva output that affect its composition. Glandular
contribution to saliva is affected by level of gland activity. The
amount of saliva secreted per minute or the salivary flow rate
influences the concentration of the constituents as well as the
proportion of the constituents from each of the three pairs of
major salivary glands and the minor salivary glands.
I. Stabilizing Agents
[0061] According to at least one embodiment of the present
disclosure, conditioning of body fluids for the preservation of
diagnostic markers may be accomplished through the use of
stabilizing agents, which may be incorporated into one or more
carrier vehicles, such as rinses, gums, beverages, and
confectionaries. For example, specific rinses and/or pre-rinses
specially formulated according to the specific molecule or
molecules (the diagnostic marker) to be detected in the body fluid,
may contain a stabilization agent. The use of rinses and pre-rinses
of the present disclosure to condition the body fluid may enhance
the sensitivity for detection of specific diagnostic markers for
clinical diagnosis. Additionally, this process may improve the
signal-to-noise ratio for a better diagnostic yield. Further, each
rinse/pre-rinse can be specifically formulated to reduce or prevent
false positive and false negative results.
[0062] In at least one embodiment of a stabilizing agent of the
present disclosure, the stabilizing agent may act to
prevent/decrease the degradation, or reduction of activity, of
diagnostic markers. In at least one embodiment, the stabilizing
agent may be comprised of one or more Generally Regarded as Safe
(GRAS) compound, as determined by the Food and Drug Administration
of the United States of America, such as those listed in Table I.
An exemplary embodiment of a stabilization agent may act to
stabilize a diagnostic marker, and/or to bind to a destructive
component to prevent the destructive component from degrading or
decreasing the activity of the diagnostic marker.
[0063] In additional exemplary embodiments of the stabilization
agent, the stabilizing agent may comprise at least 2, at least 3,
at least 4, at least 5, at least 6, at least 7, at least 8, at
least 9, at least 10, at least 15, at least 20, or at least 25
compounds which acts to stabilize the diagnostic marker, and/or to
bind to a destructive component to prevent the destructive
component from degrading or decreasing the activity of the
diagnostic marker. Further, in various embodiments the stabilizing
agent of the present disclosure may be present in at a level of
between about 200 ppm to about 2,000 ppm, about 400 ppm to about
1600 ppm, about 600 ppm to about 1400 ppm, about 800 ppm to about
1200 ppm. Moreover, in at least one embodiment, the stabilizing
agent may be present at a level of approximately 500 ppm.
TABLE-US-00001 TABLE I GRAS Compounds as Stabilizing Agents 1 Agar
Sigma-Aldrich 2 Alginic acid sodium salt Sigma-Aldrich 3 L-Ascorbic
acid Sigma-Aldrich 4 (+)-Sodium ascorbate Sigma-Aldrich 5 Buffer
solution Fluka 6 Sodium citrate Sigma-Aldrich 7 Tragacanth
Sigma-Aldrich 8 Gum arabic, from acacia tree Sigma-Aldrich 9 Sodium
tripolyphosphate Sigma-Aldrich 10 Potassium citrate monobasic
Sigma-Aldrich 11 Sodium pyrophosphate tetrabasic Sigma-Aldrich 12
Calcium citrate Sigma-Aldrich 13 Citric acid Sigma-Aldrich 14
Potassium sodium tartrate tetrahydrate Sigma-Aldrich 15 Sodium
benzoate Sigma-Aldrich 16 Benzoic acid Sigma-Aldrich 17 D-aspartic
acid Sigma-Aldrich 18 Sodium phosphate dibasic Sigma-Aldrich 19
Malic acid Sigma-Aldrich 20 Sodium propionate Sigma-Aldrich 21
Sodium L-ascorbate Sigma-Aldrich 22 DL-Phenylalanine Sigma-Aldrich
23 Calcium L-ascorbate dihydrate Sigma-Aldrich 24 Choline chloride
Sigma-Aldrich 25 L-Ascorbic acid Sigma-Aldrich 26 Sodium diacetate
SAFC 27 PH buffer 4.01 Orion 28 PH Buffer 7.00 Orion 29 PH buffer
10.0 Orion 30 Fixanal Buffer 6.0 Sigma-Aldrich 31 Ammonium Chloride
Sigma-Aldrich 32 Ammonium Citrate tribasic, anhydrous Sigma-Aldrich
33 Caffeine Sigma-Aldrich 34 Aluminum sulfate hydrate Sigma-Aldrich
35 Beeswax refined Sigma-Aldrich 36 Aluminum Hydroxide
Sigma-Aldrich 37 Bentonite Sigma-Aldrich 38 Ammonium bicarbonate
Sigma-Aldrich 39 3-tert--butyl-hydroxyanisole Sigma-Aldrich 40
Benzoic acid Sigma-Aldrich 41 trans-aconitic acid Sigma-Aldrich 42
Aluminum potassium sulfate Sigma-Aldrich dodecahydrate 43 Adpic
acid Sigma-Aldrich 44 Ammonium hydrogen phosphate Sigma-Aldrich 45
Ammonium dihydrogen phosphate Sigma-Aldrich 46 Ammonium carbonate
Sigma-Aldrich 47 Acetic acid Sigma-Aldrich
[0064] According to at least one exemplary embodiment, the
stabilizing agent may block at least one enzymatic activity of a
destructive component. The destructive component may be one or more
component of a bodily fluid which acts to degrade, or decrease the
activity of a diagnostic marker. Further, the destructive component
may be one or more Amylases, Lysozymes, Peroxidases, Glycosidases,
Esterases, Proteases, and/or Peptidases. In an exemplary
embodiment, the stabilizing agent may be a naturally occurring or
artificial protease inhibitor, DNase inhibitor, or RNase inhibitor.
Specifically, a natural stabilizing agent may include any component
found from food products (including, but not limited to, the
Solanaceae family), herbs, or spices that acts to decrease the
effect of a destructive component. For example, exemplary
embodiments of the natural stabilizing agents may be any of the
inhibitors found in lima beans, soybeans, or avian eggs (such as
ovomucoid glycoprotein protease inhibitors) (See Table II).
Moreover, an embodiment of the stabilizing agent may also comprise
a molecule which specifically inhibits the activity of a molecule
which targets the diagnostic marker for degradation or
inactivation. Further, the stabilizing agent may act to alter the
composition of the body fluid (such as pH, or ion concentration) to
decrease the degradation or inactivation of the diagnostic
marker.
TABLE-US-00002 TABLE II Molecular Inhibitory Material weight power
Details Lima beans 8-10 kDa 2.2 times There are six different lima
bean inhibitors. weight Ovomucoid 8-10 kDa 1.2 times Ovomucoids are
the glycoprotein protease inhibitors weight found in raw avian egg
white. There are other protease inhibitors in ovomucoids as well.
Soybeans 20.7-22.3 kDa 1.2 times Soybeans contain several
inhibitors; the one in the weight chart is considered the primary
one. All of them bind chymotrypsin to a lesser degree.
[0065] As used herein, "diagnostic marker" may be any biological
molecule whose presence, specific concentration, or specific
activity may be indicative of a disease state or heath/lifestyle
characteristic. As used herein, "indicative of a disease state"
refers to the presence, progression, prevention, remission,
amelioration, or prognosis of a disease. This also includes, but is
not limited to, the effects of a therapeutic on a disease. The
disease state analyzed through use of the diagnostic marker may in
an exemplary embodiment be directed towards any cancer, metabolic
disease (such as diabetes I or II), cardiovascular disease, or the
predisposition of any of the same. Additionally, a disease state
may also include the infection of the patient by a bacterial,
virus, yeast, fungus, or parasite.
[0066] A. Disease States
[0067] Bacterial infections, as referenced herein, may include a
mammalian infection by any bacterial species. Embodiments of the
bacterial species, as used herein, may include, but are not limited
to, Bacillus anthracis, Bacillus cereus, Staphylococcus aureus,
Listeria monocytogenes, Streptococcus pneumoniae, Streptococcus
pyogenes, Clostridium botulinum, Clostridium difficile, Clostridium
perfringens, Clostridium tetani, Borrelia burgdorferi, Treponema
pallidum, Chlamydia trachomatis, Chlamydophila psittaci,
Corynebacterium diphtherias, Mycobacterium tuberculosis, and
Mycobacterium avium, Rickettsia prowazekii, Rickettsia rickettsii,
Rickettsia typhi, Anaplasma phagocytophilum, Ehrlichia chaffeensis,
Brucella melitensis, Bordetella pertussis, Burkholderia mallei, B.
pseudomallei, Neisseria gonorrhoeae, Neisseria meningitides,
Campylobacter jejuni, Helicobacter pylori, Legionella pneumophila,
Acinetobacter baumannii, Moraxella catarrhalis, Pseudomonas
aeruginosa, Aeromonas sp., Vibrio cholerae, Vibrio
parahaemolyticus, Thiotrichales sp., Haemophilus influenzae,
Klebsiella pneumoniae, Proteus mirabilis, Yersinia pestis, Yersinia
enterocolitica, Shigella flexneri, Salmonella enterica, and
Escherichia coli.
[0068] Viral infections, as referenced herein, may include a
mammalian infection by any viral species. Embodiments of the viral
species, as used herein, may include, but are not limited to,
single-stranded DNA viruses, including the genus Parvoviridae,
double-stranded DNA viruses, including the genus Papillomaviridae,
Polyomaviridae, Poxyiridae, Herpesviridae (ex. Herpes Simplex Virus
1, 2, 6, Varicella-zoster virus, Epstein-Barr virus EBV, Human
cytomegalovirus, and human herpesvirus 7), single-stranded RNA
viruses, Reo- and Retroviruses (ex. Hepatitis B virus, Human
immunodeficiency virus 1 and 2, and Human T-lymphotropic virus
1).
[0069] Fungal infections, as referenced herein, may include a
mammalian infection by any bacterial species. Embodiments of the
fungal species, as used herein, may include, but are not limited
to, Fusarium oxysporum, Pneumocystis jirovecii, Aspergillus spp.,
Coccidioides immitis/posadasii, Candida albicans, Filobasidiella
neoformans, Trichosporon, Encephalitozoon cuniculi, Enterocytozoon
bieneusi, Mucor circinelloides, Rhizopus oryzae, and Lichtheimia
corymbifera.
[0070] Parasite infections, as referenced herein, may include a
mammalian infection by any parasite. Embodiments of the parasite
may be, but are not limited to, Entamoeba histolytica, Babesia
microti, Babesia sp. WA1, Trypanosoma cruzi, Taenia solium,
Echinococcus granulosus, Leishmania braziliensis, L. donovani, L.
tropica, Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale
and Plasmodium malariae, Plasmodium knowlesi, Paragonimus
westermani, chistosoma sp., S. mansoni, S. haematobium, S.
japonicum, Strongyloides stercoralis, Toxocara canis, Toxoplasma
gondii, Trichinella spiralis African trypanosomiasis, Ancylostoma,
Angiostrongylus, Anisakis, Baylisascaris procyonis, Clonorchis
(Opisthorchis) sinensis, Echinococcus multilocularis, Fasciola
hepatica, Filariasis, Gnathostoma
[0071] Exemplary embodiments of diagnostic markers include those
listed in Table III. Further exemplary embodiments for the
diagnosis of type-II diabetes include, but are not limited to,
Albumin, Alpha-1 antitrypsin (A1AT)--G Protein, Cystatin C,
Cystatin A, alpha-2-macroglobulin (A2MG), Uteroglobin,
Transthyretin (TTR), Annexin A1, Annexin A2, Annexin A3 and
Calnexin.
[0072] Diagnostic markers directed towards cardiovascular disease,
in an exemplary embodiment, may include, but are not limited to,
any isoform of creatine kinase, troponin I and T, LD, Myoglobin,
ALT/AST, H-FABP, and Glycogen phosphorylase B.
[0073] Diagnostic markers directed towards cancer, in addition to
those described in Table III, may include, but are not limited to,
Aldose reductase, Angiogenin, Annexin A1, B-cell activating factor
(BAFF), B-cell lymphoma 2 (BCL2)-like 2, Beta Human chorionic
gonadotropin, Ca15-3, Calcyclin, Calvasculin, Cathepsin D,
Caveolin-1, Chromogranin A, Alpha-crystallin B chain (CRYAB),
Endostatin, Eotaxin-2, Epithelial cell adhesion molecule (EpCAM),
Ezrin, fatty acid binding protein 4 (FABP4), Galectin-3,
.gamma.-glutamylcysteine ligase regulatory chain (GCLR), Gelsolin,
Glucose 6-phosphate (G6P), Glycoprotein 130 (gp130), Glutathione
S-transferase Mu 1 (GSTM1), Hepsin, High-mobility group protein B1
(HMGB-1), Insulin-like growth factor binding protein 1 (IGFBP-1),
Insulin-like growth factor binding protein 4 (IGFBP-4),
Insulin-like growth factor binding protein 5 (IGFBP-5),
Insulin-like growth factor binding protein 6 (IGFBP-6), LGL,
latency associated peptide (LAP), macrophage stimulating protein
(MSP), MHC class I polypeptide-related sequence A (MICA),
Nucleoside diphosphate kinase B (NME2), Neuron-specific Enolase
(NSE), Osteopontin, Osteoprotegerin, Pepsinogen, Peroxiredoxin,
Phosphoserine aminotransferase (PSAT1), Prostate Specific Antigen,
Receptor tyrosine-protein kinase erbB-3 (ErbB3), Serpin B3,
Vascular smooth muscle cell growth factor R2 (VSGF R2/KDR),
Vascular endothelial growth factor R3 (VEGF R3/Flt-4),
Thyroglobulin, Tyrosine kinase with immunoglobulin-like and
EGF-like domains 2 (TIE-2), Tissue plasminogen activator (tPA),
Transforming growth factor beta (TGF-.beta.1), Tumor necrosis
factor receptor 1 (TNF-R1), urokinase-type Plasminogen Activator
(uPA), urokinase-type Plasminogen Activator Receptor (uPAR), BrcaI,
BrcaII, kallikreins, e-cadherin, Hox peptide, and Engrailed-2.
[0074] Diagnostic markers directed towards a bacterial or virus may
include any surface or secreted antigen from the bacteria or virus,
or a conserved nucleotide sequence.
TABLE-US-00003 TABLE III Present in Relevant Serum/ Present Class
of Tumor Marker Cancer Plasma in Saliva Molecules
.alpha.-fetoprotein Heptacellular Yes Yes Protein carcinoma Cancer
Antigen Breast cancer Yes Yes Protein (CA15-3) Cancer Antigen
Ovarian cancer Yes Yes Protein (CA 125) Carcino- Many epithelial
Yes Yes embryonic cancers antigen Prostate Prostate cancer Yes Yes
Protein specific antigen c-erb-2 Breast cancer Yes Nucleic acid
P16, p53 Oral SCC BRACA1, Breast cancer Yes Yes Nucleic acid BRACA2
Hemoglobin-A1c Yes Protein
[0075] B. Diagnostic Markers
[0076] Further, diagnostic markers according to an embodiment of
the present disclosure may include one or more of: 1,25
dihydroxy-vitamin D, 17-Hydroxyprogesterone, 25-hydroxy-vitamin D,
Antineutrophil Cytoplasmic Antibodies, 5-Hydroxy Tryptamine,
5-hydroxyindoleacetic acid, Acetoacetate, Activated Partial
Thromboplastin Time, Adrenocorticotropic Hormone, Alanine
aminotransferase, Alanine transaminase, Albumin,
Albumin-to-Creatinine ratio, Albumin/Globulin ratio, Alcohol,
Aldolase, Aldosterone, Aldosterone and plasma renin activity,
Aldosterone and Renin, Alkaline Phosphatase, Allergen-specific IgE,
Alpha tryptase, Alpha-1 Antitrypsin, Alpha-fetoprotein,
Alpha1-antitrypsin, Alzheimer biomarkers (including Tau protein and
Amyloid Beta 42 peptides), Amylase, ANCA Antibodies, ANCA/MPO/PR3
Antibodies, Angiotensin-Converting Enzyme, Anti-citrulline
antibody, anti-cyclic citrullinated peptide antibody,
Anti-retroviral drug resistance testing, anti-ribonucleoprotein,
anti-Sjogren's Syndrome A, anti-Sjogren's Syndrome B, Anti-Smooth
Muscle Antibody, anti-topoisomerase, Anticardiolipin Antibodies,
Antidiuretic Hormone, Antifactor Xa heparin, Antiglobulin,
Antihistidyl Transfer RNA, Synthase Antibodies, Antimicrosomal
antibody, Antimitochondrial Antibody and Antimitochondrial M2
Antibody, Antineutrophil Cytoplasmic Antibodies, Antinuclear
Antibody test, Antiphospholipid antibodies, Antiphospholipids,
Antistreptolysin O titer, Antithrombin, Antithyroglobulin antibody,
Antithyroid antibodies, Apolipoprotein A-I, Apolipoprotein B-100,
Arginine Vasopressin, Aspartate aminotransferase, Aspartate
transaminase, B-type natriuretic peptide, Beta hCG, Beta tryptase,
Beta-2 Microglobulin, Beta-hydroxybutyrate, Beta-hydroxybutyric
acid, Beta2 Microglobulin, Bicarbonate, Bilirubin, cholesterol,
Blood clotting factors, Bordetella pertussis Antibodies, Borrelia
burgdorferi antibodies, IgM/IgG, Brain natriuretic peptide, Breast
Cancer Gene 1 and Breast Cancer Gene 2, c-ANCA, c-erbB-2,
C-peptide, C-Reactive Protein, Caffeine, Calcidiol, Calcifidiol,
Calcitonin, Calcitriol, Calcium, Calcofluor white stain, Cancer
Antigen 125, Cancer Antigen 15-3, Cancer Antigen 19-9, Cancer
antigen-breast, Cancer antigen-GI, Carbamazepine, Carcinoembryonic
Antigen, Cardiac-specific Troponin I and Troponin T, Cardiolipin
Antibodies, Catecholamines, Celiac Disease Tests, Ceruloplasmin,
Chickenpox, Chickenpox and Shingles Tests, Chlamydia, Chloride,
Cholesterol, Chromogranin A, Chymotrypsin, Citrulline antibody,
Coagulation Factors, Cobalamin, Complement Component C3, Complement
Component C4, Complexed PSA, Conjugated bilirubin, Copper,
Corticotropin, Cortisol, Cotinine, Creatine Kinase, Creatine
Kinase-MB, Creatinine, Cryoglobulin, Cryoprotein, Cyclic
Citrullinated Peptide Antibody, Cyclosporine, Cystatin C, Cystic
Fibrosis Gene Mutation Panel, Cystic fibrosis genotyping,
Cytomegalovirus, Cytotoxic T-cells, Dehydroepiandrosterone Sulfate,
Delta-aminolevulinic acid, Depakene, Depakote, Des-gamma-carboxy
prothrombin, DHEA Sulfate, Diabetes mellitus autoantibody panel,
Digoxin, Dilantin, Direct Anti-human Globulin test, Direct
Antiglobulin Test, Direct bilirubin, Direct Low-density lipoprotein
cholesterol, Dopamine, Drug screen, eGFR, Epidermal Growth Factor
Receptor, Epinephrine, Epstein-Barr Virus Antibodies,
Erythropoietin, Estradiol, Estriol, Estrogens, Estrone, Ethanol,
F-Actin Antibody, Factor I, Factor V Leiden, Factor V Leiden
Mutation and PT 20210 Mutation, Factor V Leiden mutation: Activated
protein C resistance, Factor V R506Q, Ferritin, Fetal fibronectin,
Fibrin degradation fragment, Fibrinogen, Fluorescent Antinuclear
Antibody, Fluorescent treponemal antibody absorption, Folic Acid,
Follicle-stimulating hormone, Fragment D-dimer, Fructosamine,
Gamma-glutamyl transferase, Gamma-glutamyl transpeptidase, Gastrin,
Genital Human Papillomavirus, Glucose-6-Phosphate Dehydrogenase,
Glutamic Acid Decarboxylase Autoantibodies, Gluten-Sensitive
Enteropathy Tests, Glycated Albumin, Glycated hemoglobin, Heavy
Metals (such as lead, mercury, iron, copper, and zinc), Hemoglobin,
Hemoglobin-binding Protein, Hemogram, Heparin Anti-Xa, Hepatitis A,
Hepatitis B, Hepatitis C, Herpes Simplex Virus, Type1 and Type 2,
Herpes Zoster, Heterophile Antibodies, High-density lipoprotein
cholesterol, High-sensitivity C-reactive protein, Homocysteine,
Human calcitonin, Human chorionic gonadotropin, Human epidermal
growth factor receptor, Human Growth Hormone, Human
immunodeficiency virus antibody test, Human Immunodeficiency Virus
Genotypic Resistance Testing, Human Leukocyte Antigen,
Immunoreactive trypsinogen, Insulin Autoantibodies, Insulin
C-peptide, Insulin-like Growth Factor-1, Insulinoma-Associated-2
Autoantibodies, Intact PTH, Interstitial Cell Stimulating Hormone,
Iron, Ischemia-Modified Albumin, Islet autoantibodies Islet Cell
Cytoplasmic Autoantibodies, Janus Kinase 2, Ketone bodies Ketones,
blood, Lactate, Lactate dehydrogenase, Lactic Acid, Lactic
dehydrogenase, Lead, Lipoprotein, Lithium, Low-density lipoprotein
cholesterol, Lupus Antibody, Luteinizing hormone, Lyme antibodies
IgM/IgG by Western blot, Magnesium, Mast cell tryptase, Measles or
Mumps IgM and IgG Antibodies, Mercury, Metanephrine and
Normetanephrine, Methotrexate, Methylmalonic Acid, Microalbumin,
Mitochondrial Antibody, Mononuclear heterophile test, Mycophenolic
acid, Mycoplasma, Mycoplasma pneumoniae IgG and IgM antibodies,
Myeloperoxidase Antibodies, Myoglobin, N-terminal pro b-type
natriuretic peptide, Neonatal bilirubin, Nicotine, Norepinephrine,
p24 antigen, Parathyroid Hormone, Parvovirus, Pertussis,
Phenobarbital, Phenyloin, Phosphate, Phosphorus,
Platelet-activating factor acetylhydrolase, porphobilinogen,
Potassium, Prealbumin, Presenilin 1 gene, Procalcitonin,
Progesterone, Proinsulin C-peptide, Prolactin, Prostate Specific
Antigen, Protein C, Protein C and Protein S, Protein Tyrosine
Phosphatase-like Autoantibodies, Proteinase 3 Antibodies,
Prothrombin 20210 mutation, Prothrombin 20210 mutation: PT G20210A,
factor II 20210, Red Blood Cell Antibody Identification, Renin,
Respiratory Syncytial Virus, Rheumatoid Factor, Ristocetin
Cofactor, Rubella, Scleroderma antibodies, Serine Protease 3,
Serotonin, Siderophilin, Sirolimus, Smith antibody, Smooth Muscle
Antibody, Sodium, Soluble Mesothelin-Related Peptides, Somatomedin
C, Somatotropin, Syphilis, Tacrolimus, Tegretol.RTM., Testosterone,
Testosterone-estrogen Binding Globulin, Theophylline, Theophylline
and Caffeine, Thiopurine methyltransferase, Thiopurine
S-methyltransferase, Thymotaxin, Thyrocalcitonin, Thyroglobulin,
Thyroid peroxidase antibody, Thyroid stimulating hormone receptor
antibody, Thyroid stimulating immunoglobulin, Thyroid-stimulating
hormone, Thyroperoxidase antibody, Thyrotropin, Thyroxine,
Thyroxine-binding prealbumin, Transferrin, Transthyretin test,
Treponema pallidum particle agglutination assay, Trichomonas,
Trichomoniasis, Triglycerides, Triiodothyronine, Troponins,
Trypsin, Trypsin and Chymotrypsin, Trypsinogen, Tryptase, Valproic
acid, Vancomycin, Vanillylmandelic acid, Varicella Zoster Virus,
Vasopressin, Very Low-Density Lipoprotein Cholesterol, Viral
Hepatitis A Antibody, Viral Hepatitis C. Viral Load (HIV), Vitamin
B12, Vitamin B12 & Folate, Vitamin D, Vitamin D2, Vitamin D3,
Vitamin K, von Willebrand Factor, West Nile Virus, Westergren
sedimentation rate, and Zinc Protoporphyrin.
[0077] C. Exemplary Targets for Stabilizing Agent(s)
[0078] 1. Amylase
[0079] Amylase is an enzyme that breaks down starch into sugar, and
is found in such places as human saliva, where it begins the
chemical process of digestion. The .alpha.-amylases are
calciummetalloenzymes that are completely unable to function in the
absence of calcium. Through acting at random locations on the
starch chain, .alpha.-amylase breaks down carbohydrates into
maltotriose and maltose from amylase. In animals, .alpha.-amylase
is a major digestive enzyme that has an optimum pH of 6.7 to
7.0.
[0080] In at least one embodiment of a stabilizing agent of the
present disclosure, the stabilizing agent may be an inhibitor of
.alpha.-amylase. Further, the stabilizing agent may be active in a
biological fluid, such as in saliva. In an exemplary embodiment, a
stabilizing agent may comprise one or more of Fixanal.RTM. Buffer
6.0 (Sigma-Aldrich Co.), aluminum sulfate hydrate, aluminum
hydroxide, bentonite, and aluminum potassium sulfate
dodecahydrate.
[0081] 2. Lysozyme
[0082] Lysozyme, also known as muramidase or N-acetylmuramide
glycanhydrolase, is a glycoside hydrolase, that damage bacterial
cell walls by catalyzing hydrolysis of 1,4-beta-linkages between
N-acetylmuramic acid and N-acetyl-D-glucosamine residues in a
peptidoglycan and between N-acetyl-D-glucosamine residues in
chitodextrins. Lysozyme is abundant in a number of secretions, such
as tears, saliva, human milk, and mucus. It is also present in
cytoplasmic granules of the polymorphonuclear neutrophils
(PMN).
[0083] In at least one embodiment, a stabilizing agent of the
present disclosure may be an inhibitor of lysozyme. Further, the
stabilizing agent may be active in a biological fluid, such as
saliva. In an exemplary embodiment, a stabilizing agent may
comprise one or more of Fixanal.RTM. Buffer 6.0 (Sigma-Aldrich
Co.), bentonite, benzoic acid, and acetic acid.
[0084] 3. Peroxidase
[0085] Peroxidase are a class of oxidoreductase enzymes that
catalyze the oxidation of a compound by the decomposition of
hydrogen peroxide or an organic peroxide. These peroxidases may be
found in many different bodily fluids. For example, saliva contains
both salivary peroxidase (SPX) and myeloperoxidase (MPO). These
peroxides may act on a number of different diagnostic markers found
in bodily fluids to mask the detection or analysis of the
markers.
[0086] In at least one embodiment of a stabilizing agent of the
present disclosure, the stabilizing agent may be an inhibitor
peroxidase activity. Further, the stabilizing agent may be active
in a biological fluid, such as saliva, blood, serum, or cancer
cells. In an exemplary embodiment, a stabilizing agent may comprise
one or more of aluminum sulfate hydrate, benzoic acid, and aluminum
potassium sulfate dodecahydrate.
[0087] 4. Insulin Resistance
[0088] In at least one embodiment of a stabilizing agent of the
present disclosure, the stabilizing agent may preserve a diagnostic
marker for insulin resistance and/or glucose intolerance. Further,
the stabilizing agent may be active in a biological fluid, such as
saliva. In an exemplary embodiment, a diagnostic marker may be one
or more of .alpha.-hydroxybutyrate, 1-linoleoyl-GPC, palmitate,
Glycine, 3-methyl-2-oxybutyrate. Moreover, a diagnostic marker may
be any marker of early stage insulin resistance and glucose
intolerance in a non-diabetic individual.
[0089] 5. Galactose Oxidase
[0090] Galactose oxidase is a member of the family of
oxidoreductaases, and has been shown to participate in galactose
metabolism. Because of this activity, galactose oxidase may be used
to detect mucin-like glycoproteins. These glycoproteins are a major
component of mucus secreted by epithelial and glandular cells and
are primarily responsible for the protective properties of the
viscoeleastic mucous barrier. Mucins have been implicated in the
process of cholesterol gallstone formation, and has been identified
as having abnormal expression in some cancers.
[0091] In at least one embodiment of a stabilizing agent of the
present disclosure, the stabilizing agent may be an inhibitor of
galactose oxidase. Further, the stabilizing agent may be active in
a biological fluid, such as saliva. In an exemplary embodiment, a
stabilizing agent may comprise one or more of aluminum potassium
sulfate dodecahydrate, 3-tert-butyl-hydroxyanisole, benzoic acid,
and acetic acid.
[0092] 6. Glycated Hemoglobin
[0093] Glycated hemoglobin (HbA1c) is a form of hemoglobin that is
indicative of the average plasma glucose concentration over a
period of time. Due to the transport of components of the plasma
into bodily fluids, such as saliva, the level of HbA1c may be
determined in bodily fluids in addition to plasma.
[0094] In at least one embodiment of a stabilizing agent of the
present disclosure, the stabilizing agent may be an inhibitor of
HbA1c degradation. Further, the stabilizing agent may be active in
a biological fluid, such as saliva. In an exemplary embodiment, a
stabilizing agent may comprise one or more of acetic acid, aluminum
hydroxide bentonite, aluminum sulfate hydrate, aluminum potassium
sulfate dodecahydrate, benzoic acid, caffeine, and
3-tert-butyl-hydroxyanisole, or a combination thereof.
[0095] 7. Cancer Antigen 19-9 (CA 19-9)
[0096] Cancer Antigen 19-9 (CA 19-9), which may in some instances
be referred to as Cancer Angigen 19.9, Cancer antigen-GI or CA-GI,
is an antigen associated with various cancers, such as prostate and
colon cancer. At least one use of CA 19-9 may be see whether a
pancreatic tumor is secreting antigen. If that is the case, then
the levels may decrease when the tumor is treated, and they may
rise again if the disease recurs.
[0097] In at least one embodiment of a stabilizing agent of the
present disclosure, the stabilizing agent may be an inhibitor of CA
19-9 degradation. Further, the stabilizing agent may be active in a
biological fluid, such as saliva, blood, serum, or cancer cells. In
an exemplary embodiment, a stabilizing agent may comprise one or
more of aluminum sulfate hydrate, benzoic acid, and aluminum
potassium sulfate dodecahydrate.
[0098] Further, in an additional exemplary embodiment, the
components of a stabilizing agent may be present at or about equal
amounts, such as for example 1:1:1 in a stabilizing agent with
three active components. Additionally, the stabilizing agents may
in at least one embodiment inhibit degradation or inactivation of a
diagnostic marker by at least about 40%, at least about 45%, at
least about 50%, at least about 55%, at least about 60%, at least
about 65%, at least about 70%, at least about 75%, at least about
80%, at least about 85%, at least about 90%, at least about 95%, or
at least about 99%. Moreover, the stabilizing agent may, in at
least one embodiment, inhibit degradation of a diagnostic marker
for at least about 1 minute, at least about 5 minutes, at least
about 10 minutes, at least about 15 minutes, at least about 30
minutes, at least about 1 hour, at least about 2 hours, at least
about 4 hours, or at least about 8 hours.
Additional Components for Stabilizing Agent Carriers:
[0099] According to at least one embodiment of the present
disclosure, stabilizing agents may be incorporated into various
carriers for their use with mammals. Such carriers may include
various rinses, gums, lozenge, mouthwash, beverages, confectionary,
washes, or other applicable vehicles to deliver a stabilizing agent
to the site of a diagnostic marker.
[0100] Embodiments of a carrier, such as a rinse, according to the
present disclosure may also comprise one or more additional
component which may include an anti-caking agent, a chemical
preservative, an emulsifying agent, a nutrient and dietary
supplement, a sequestrant, a stabilizer, an additive, a synthetic
and flavoring substance. Exemplary embodiments of these components
are listed herein (See section entitled "Additional Components for
Rinse"). Further, the one or more additional component may be a
substance which has been labeled as Generally Recognized As Safe
(GRAS) by the Food and Drug Administration of the United States of
America.
[0101] A exemplary embodiment of a carrier of the present
disclosure may be a mouthwash composition which may comprise water
and pharmaceutically acceptable excipients or additives such as:
one or more oils, such as an oil selected from the group comprising
anethole, anisole, camphor, methyl salicylate, vanillin, eugenol,
furaneol, linalool, menthol, thymol, cinnamaldehyde, citral, methyl
butanoate, pentylbutanoate, pentylpentanoate, tea tree oil,
peppermint oil, spearmint oil, pineapplemint oil and eucalyptus
oil; sweetening agents, for example sorbitol; thickening agents,
such as xanthan gum, carrageenan, carbomer, or HPMC (hydroxypropyl
methyl cellulose); preservative agents, such as sodium benzoate,
methyl paraben, or propyl paraben; emulsifiers, such as polysorbate
80 (or Tween.TM. 80); and/or at least one antacid such as aluminium
or magnesium hydroxide.
[0102] Gum compositions may be provided in a variety of different
forms, such as, for example, slab, pellet, sticks, balls, cubes,
center-fill gums, candy gum, multi-region gum, multi-layer gum,
bubble gum, deposited gums and compressed gums. The gum
compositions also may include at least one flavor and a variety of
optional additives. In some embodiments, the gum compositions
described herein may be formed in a rope or sheet form and then
processed into individual forms as described above.
[0103] A gum composition also may include a gum base. The gum base
may include any component known in the chewing gum art. Such
components may be water soluble, water-insoluble or a combination
thereof. For example, the gum base may include elastomers, bulking
agents, waxes, elastomer solvents, emulsifiers, plasticizers,
fillers and mixtures thereof.
[0104] A ingestible beverage composition of the present disclosure
may comprise any formulation commonly known in the art.
[0105] At least one embodiment of a carrier of the present
application may comprise a liquid. The liquid, in at least one
embodiment, is a non-toxic liquid. Further, the liquid may comprise
water, a beverage containing water, or glycerin. Moreover, in at
least one embodiment, a stabilizing agent may be at least partially
dissolved in the liquid.
[0106] It will be appreciated that the above list of excipients
and/or additives is provided merely by way of example and that
various other such components may be used in the formulation of the
present invention.
[0107] The following components, in at least one embodiment of the
rinse of the present disclosure, may include:
[0108] 1. ANTI-CAKING AGENTS: aluminum calcium silicate, calcium
silicate, magnesium silicate, sodium calcium aluminosilicate, and
tricalcium silicate.
[0109] 2. CHEMICAL PRESERVATIVES: ascorbic acid, ascorbyl
palmitate, benzoic acid, butylated hydroxyanisole, butylated
hydroxytoluene, calcium ascorbate, calcium propionate, calcium
sorbate, caprylic acid, dilauryl thiodipropionate, erythorbic acid,
gum guaiac, methylparaben, potassium bisulfite, potassium
metabisulfite, potassium sorbate, propionic acid, propyl gallate,
propylparaben, sodium ascorbate, sodium benzoate, sodium bisulfite,
sodium metahisulfite, sodium propionate, sodium sorbate, sodium
sulfite, sorbic acid, stannous chloride, sulfur dioxide,
thiodipropionic acid, tocopherols.
[0110] 3. EMULSIFYING AGENTS: cholic acid, desoxycholic acid,
diacetyl tartaric acid esters of (M)mono- and diglycerides,
glycocholic acid, mono- and diglycerides, monosodium phosphate
derivatives of above, propylene glycol, ox bile extract,
taurocholic acid.
[0111] 4. NUTRIENTS AND DIETARY SUPPLEMENTS: alanine, arginine,
ascorbic acid, aspartic acid, biotin, calcium carbonate, calcium
citrate, calcium glycerophosphate, calcium oxide, calcium
pantothenate, calcium phosphate, calcium pyrophosphate, calcium
sulfate, carotene, choline bitartrate, choline chloride, copper
gluconate, cuprous iodide, cysteine, cystine, ferric phosphate,
ferric pyrophosphate, ferric sodium pyrophosphate, ferrous
gluconate, ferrous lactate, ferrous sulfate, glycine, histidine,
inositol, iron (reduced), isoleucine, leucine, linoleic acid,
lysine, magnesium oxide, magnesium phosphate, magnesium sulfate,
manganese chloride, manganese citrate, manganese gluconate,
manganese glycerophosphate, manganese hypophosphite, manganese
sulfate, manganous oxide, mannitol, methionine, methionine hydroxy
analogue, niacin, niacinamide D-pantothenyl alcohol, phenylalanine,
potassium chloride, potassium glycerophosphate, potassium iodide,
proline, pyridoxine hydrochloride, riboflavin,
riboflavin-5-phosphate, serine, sodium pantothenate, sodium
phosphate, sorbitol, thiamine hydrochloride, thiamine mononitrate,
threonine, tocopherols, tocopherol acetate, tyrosine, valine,
vitamin A, vitamin A acetate, vitamin A palmitate, vitamin B12,
vitamin D2, vitamin D3, zinc sulfate, zinc gluconate, zinc
chloride, zinc oxide, zinc stearate.
[0112] 5. SEQUESTRANTS: calcium acetate, calcium chloride, calcium
citrate, calcium diacetate, calcium gluconate, calcium
hexametaphosphate, calcium phosphate. monobasic, calcium phytate,
citric acid, dipotassium phosphate, disodium phosphate, isopropyl
citrate, monoisopropyl citrate, potassium citrate, sodium acid
phosphate, sodium citrate, sodium diacetate, sodium gluconate,
sodium hexametaphosphate, sodium metaphosphate, sodium phosphate,
sodium potassium tartrate, sodium pyrophosphate, sodium
pyrophosphate, tetra, sodium tartrate, sodium thiosulfate, sodium
tripolyphosphate, stecaryl citrate, tartaric acid.
[0113] 6. STABILIZERS: acacia (gum arabic), agar-agar, ammonium
alginate, calcium alginate, carob bean gum, chondrus extract,
ghatti gum, guar gum, potassium alginate, sodium alginate,
sterculia (or karava) gum, tragacanth.
[0114] 7. ADDITIVES: acetic acid, adipic acid, aluminum ammonium
sulfate, aluminum potassium sulfate aluminum sodium sulfate,
aluminum sulfate, ammonium bicarbonate, ammonium carbonate,
ammonium hydroxide, ammonium phosphate, ammonium sulfate, bees wax,
bentonite, butane, caffeine, calcium carbonate, calcium chloride,
calcium citrate, calcium gluconate, calcium hydroxide, calcium
lactate, calcium oxide, calcium phosphate, caramel, carbon dioxide,
carnauba wax, citric acid, dextrans, ethyl formate, glutamic acid,
glutamic acid hydrochloride, glycerin, glyceryl monostearate,
helium, hydrochloric acid, hydrogen peroxide, lactic acid,
lecithin, magnesium carbonate, magnesium hydroxide, magnesium
oxide, magnesium stearate, malic acid, methylcellulose,
monoammonium glutamate, monopotassium glutamate, nitrogen, nitrous
oxide, papain, phosphoric acid, potassium acid tartrate, potassium
bicarbonate, potassium carbonate, potassium citrate, potassium
hydroxide, potassium sulfate, propane, propylene glycol, rennet,
silica aerogel, sodium acetate, sodium acid pyrophosphate, sodium
aluminum phosphate, sodium bicarbonate, sodium carbonate, sodium
citrate, sodium carboxy-methylcellulose, sodium caseinate, sodium
citrate, sodium hydroxide, sodium pectinate, sodium phosphate,
sodium potassium tartrate, sodium sesquicarbonate, sodium
tripolyphosphate, succinic acid, sulfuric acid, tartaric acid,
triacetin, triethyl citrate.
[0115] 8. SYNTHETIC FLAVORING SUBSTANCES: acetaldehyde, acetoin,
aconitic acid, anethole, benzaldehyde, N-butyric acid, d- or
l-carvone cinnamaldehyde, citral, decanal, diacetyl, ethyl acetate,
ethyl butyrate, ethyl vanillin, eugenol, geraniol, geranyl acetate,
glycerol tributyrate limonene, linalool, linalyl acetate, l-malic
acid, methyl anthranilate, 3-methyl-3-phenyl glycidic acid, ethyl
ester, piperonal, vanillin.
II. Indicator Carriers
[0116] In at least one additional embodiment of a carrier, of the
present disclosure, the carrier comprises an indicator compound
capable of binding to and/or reacting with a target to produce an
indicator signal. The target in an exemplary embodiment may be a
diagnostic marker, such as the presence of glucose or glucose in
excess of a defined threshold, the presence of one or more
glycosylated protein related to diabetes or pre-diabetes, the
presence of cancer (or pre-cancer) markers, and the presence of
cardiac (or pre-cardiac) markers.
[0117] A. Indicators
[0118] The indicator compound in an exemplary embodiment of a
carrier according to the present disclosure may be any compound,
chemical, or biological component which may interact with a target
or byproduct of a target. For example, an indicator compound may
comprise an antibody, a reactive chemical compound, a labeled
molecule, or any combination thereof. Antibodies used in an
embodiment of the present disclosure may be monoclonal or
polyclonal and derived from any species (e.g. human, rat, mouse,
rabbit, pig). Further, indicator molecules may be aptamers,
proteins, peptides, small organic molecules, natural compounds
(e.g. steroids), non-peptide polymers, MHC multimers (including
MHC-dextramers, MHC-tetramers, MHC-pentamers and other
MHC-multimers), or any other molecules that specifically and
efficiently bind to other molecules are also marker molecules.
[0119] Labeled molecules, for use as indicator compounds, may be
any molecule that absorbs, excites, or modifies radiation, such as
the absorption of light (e.g. dyes and chromophores) and the
emission of light after excitation (fluorescence from
fluorochromes). Additionally, labeled molecules may have an
enzymatic activity, by which it catalyzes a reaction between
chemicals in the near environment of the labeling molecules,
producing a signal which include production of light
(chemi-luminescence) or precipitation of chromophors, dyes, or a
precipitate that can be detected by an additional layer of
detection molecules.
[0120] Fluorescence labels may produce the presence of light at a
single wavelength, or a shift in wavelengths. Exemplary fluorescent
labels may include: [0121] Fluor dyes, Pacific Blue.TM., Pacific
Orange.TM., Cascade Yellow.TM.; [0122] AlexaFluor.RTM. (AF); o
AF405, AF488.AF500, AF514, AF532, AF546, AF555, AF568, AF594,
AF610, AF633, AF635, AF647, AF680, AF700, AF710, AF750, AF800;
[0123] Fluorescein (Flu) or any derivate of that, ex. FITC
(fluorescein isothiocyanate) Cy-Dyes o Cy2, Cy3, Cy3.5, Cy5, Cy5.5,
Cy7 Fluorescent Proteins; o RPE(R-phycoerythrin), PerCp,
APC(Allophycocyanin); other of phycobillin containing proteins,
e.g. phycobiliprotein o Green fluorescent proteins (GFP); [0124]
GFP and GFP derivated mutant proteins; BFP1CFP, YFP, DsRed, T1,
Dimer2, mRFP1, MBanana, mOrange, dTomato, tdTomato, mTangerine,
mStrawberry, mCherry Tandem dyes: o RPE-Cy5, RPE-Cy5.5, RPE-Cy7,
RPE-AlexaFluor.RTM. tandem conjugates; [0125] RPE-Alexa610,
RPE-TxRed o APC-Aleca600, APC-Alexa610, APC-Alexa750, APC-Cy5,
APC-Cy5.5 Multi fluorochrome assemblies o Multiple fluorochromes
attached to a polymer molecule, such as a peptide/protein, Dex, or
poly-sacceride. o Any combination of the fluorescent dyes involving
in generation of FRET; [0126] (Fluorescence resonance energy
transfer) based techniques, Multiple fluorochromes associated or
coupled to a polymeric molecule, or consisting of polymeric
residues, lonophors; ion chelating fluorescent probes or Probes
that change wavelength when binding a specific ion, such as
calcium; [0127] Probes that change intensity when binding to a
specific ion, such as calcium; and [0128] Combinations of
fluorochromes on the same marker. The marker is not identified by a
single fluorochrome but by a code of identification being a
specific combination of fluorochromes, as well as inter related
ratio's of intensities.
[0129] B. Target
[0130] The target of an exemplary indicator compound of the present
disclosure may be any diagnostic marker or diagnostic condition for
a disease state (or pre-disease state) of an individual. For
example, the disease state may be diabetes, pre-diabetes, cardiac
disease, pre-cardiac conditions, cancers of any stage and type, or
pre-cancerous conditions. Specifically, the target may be the
presence, or level of, glucose found in saliva for diabetes.
Alternately, the target may be the presence, or level of,
glycosylated proteins, such as advanced glycosylation end products.
Further, the target may be the presence or level of cardiac
markers, such as Creatine kinase-MB (CK-MB), myoglobin,
homocysteine, C-reactive protein (CRP), troponin T (cTnT), and
troponin I (cTnI). Moreover, the target may be the presence of
cancer markers, such as Cancer Antigen 125, Cancer Antigen 15.3,
Cancer Antigen 19.9, Prostate specific antigen, Carcinoembryonic
Antigen, Alpha Feto-Protein, Epidermal growth factor receptor,
Kallikrein 3, Vascular endothelial growth factor A (VEGF),
Calcitonin, Chromogranin A, Gastrin, S100 alpha chain,
Somatostatin, Thyroglobulin, V-erb-b2, cyckub-dependent kinase
inhibitor 1 (p21), Breast Cancer Antigen 1 and 2 (BRCA1 and 2),
MutL homolog 1 (MLH1), MutS homolog 2 (MSH2), MutS homolog 6
(MSH6), and postmeiotic segregation increased 1 and 2 (PMS1 and 2).
Moreover, the target may be the presence or level of any diagnostic
marker included herein.
[0131] C. Indicator Signal
[0132] The indictor signal in at least one embodiment of the
present disclosure may comprise any detectable signal, including
but not limited to, color change, fluorescence, and
chemical/structural change of the target (and/or indicator
compound) so as to be amenable to reacting with a secondary
detection marker. Further, the detection of a signal may involve a
secondary reactive molecule.
[0133] D. Glucose
[0134] In an embodiment of the present disclosure, glucose may be
detected through an indicator rinse by a chemical or enzymatic
method. Exemplary embodiments of this method may include alkaline
copper reduction, alkaline ferricyanide reduction, glucose oxidase,
and/or hexokinase.
[0135] In an exemplary embodiment of an indicator rinse of the
present disclosure, the rinse may further comprise one or more
additional component as described herein. These additional
components may include stabilizing agents, as well as GRAS
components (such as those listed herein).
[0136] E. Glycosylated Hemoglobin
[0137] In an embodiment of the present disclosure, glycated
Hemoglobin A1c (HbA1c) may be detected through any known method for
the detection of HbA1c or fragments thereof, such as high pressure
liquid chromatography, immunoassays, and an indicator rinse by a
chemical or enzymatic method. In an exemplary embodiment of an
indicator rinse of the present disclosure, the rinse may further
comprise one or more additional component as described herein.
These additional components may include stabilizing agents, as well
as GRAS components (such as those listed herein).
[0138] F. Cancer Antigen 19-9
[0139] In an embodiment of the present disclosure, cancer antigen
19-9 (CA 19-9) may be detected through any known method for the
detection of CA 19-9 or fragments thereof, such as high pressure
liquid chromatography, immunoassays, and an indicator rinse by a
chemical or enzymatic method. In an exemplary embodiment of an
indicator rinse of the present disclosure, the rinse may further
comprise one or more additional component as described herein.
These additional components may include stabilizing agents, as well
as GRAS components (such as those listed herein).
III. Diagnostic Marker Detection
[0140] In an embodiment of the present disclosure, a diagnostic
marker susceptible to degradation or alteration by a destructive
component may be detected through any known method, including, but
not limited to, high pressure liquid chromatography, immunoassays,
and an indicator rinse by a chemical or enzymatic method. Further,
the detection may be conducted in a micro well, deep well,
microarray, microfluidic device, high throughput screen, or any
applicable diagnostic device.
[0141] A. Rapid Diagnostic
[0142] Detection of a diagnostic marker may be conducted according
to a rapid detection system of the present disclosure. Turning to
FIG. 1A, a detection platform 100 may comprise a platform 102
having a plurality of test wells 104, where each well is capable of
containing a plurality of test spots 106. The plurality of test
wells 104 may include at least 2, at least 4, at least 8, at least
16, at least 32, at least 64, or at least 256 wells. The plurality
of tests spots 106 may include at least 2, at least 4, at least 8,
at least 16, at least 32, or at least 64 test spots 106. Further,
each of the plurality of test spots 106 may comprise one or more
diagnostic marker or control marker.
[0143] An embodiment of detection platform 100 may further be sized
and shaped to allow for automated detection and analysis of a
diagnostic or control marker. In at least one embodiment, the
detection system 100 may comprise an identifier 108, such as a
barcode or Radio-frequency identification (RFID) tag, to allow for
the identification of the detection system 100 and data collected
from the analysis of test spots 106.
[0144] Further, detection platform 100 may be comprised of any
applicable material sufficient to house or define test wells 104.
Exemplary embodiments of applicable materials for detection
platform 100 may be a polymer, glass, metal, quartz, nylon,
silica-based material, resin, or combination thereof. Further,
embodiments of test wells 104 may include not only a depression
defined or housed by platform 102, but may also include a defined
area or region of the platform 102 (such as may be found on a
microarray based platform).
[0145] Turning to FIG. 1B, at least one embodiment of detection
system 150 of the present disclosure is shown. An exemplary
embodiment of detection system 150 comprises an embodiment of
detection platform 100, a detection device 120 capable of
determining a binding characteristic between a detection agent and
a diagnostic marker on the detection platform 100, and a computer
processor 130 coupled to a computer database 135 and to the
detection device 120. In at least one embodiment, the computer
processor 130 controlling the detection device 120 is able to (1)
determine the binding characteristic of a detection agent to a
diagnostic agent, (2) compare the binding characteristic among each
of a plurality of samples tested, (3) generate a binding report
using the compared binding characteristics, and deliver the binding
report to a recipient or external computer (not shown) in
communication with the computer processor 130.
[0146] A binding characteristic as used herein may include any
measurement of the attraction (or repulsion) of two molecules (such
as a detection agent and a diagnostic marker).
[0147] Detection system 100 may also comprise an embodiment of a
bead-based system of the present disclosure having at least one
bead type. For example, a dual bead system may be used wherein at
least one bead type is magnetic or paramagnetic. These beads may
also be bound to a carrier protein with a conjugated hapten.
Further, the beads may have an analyte capture monoclonal antibody
reagent bound to it. Additionally, the beads, in at least one
embodiment, do not retain their magnetic properties when removed
from a magnetic field. In at least one embodiment, the beads may be
Europium micro-particles. The sizes of the Europium micro-particles
may be varied as needed to accommodate the method of detection
used.
[0148] At least one embodiment of a bead based system may comprise
a latex particle (See FIGS. 2-4). The latex particle according to
at least one embodiment may be a Carboxylate modified latex
(CML)bead. Further, an exemplary embodiment of a bead may include
bovine serum albumin (BSA) and/or human serum albumin (HSA).
Additionally, an embodiment of a bead based system of the present
disclosure may include a linker coupled to HSA and/or BAS. The
linker according to an embodiment, may comprise a maleimide
compound. At least one exemplary embodiment of a maleimide compound
may be Succinimidyl 4-[N-Maleimidomethyl] Cyclohexane-1-Carboxylate
(SMCC) or N-Hydroxysuccinimide-activated hexa(ethylene glycol)
undecane Thiol (NHS). Moreover, at least one exemplary embodiment
of a bead based system may further comprise an antibody linked to
maleimide on the bead. Accordingly, at least one embodiment of the
bead based system comprises a latex particle coupled to BAS or HSA,
maleimide coupled to BAS or HSA, and an antibody coupled to
maleimide.
[0149] The latex particle, according to at least one embodiment may
be in a size range from about 0.02 .mu.m to about 7.0 .mu.m.
[0150] Turning to FIG. 5, at least one at least one method 500 of
coupling an antibody to a bead comprises the step 502 of modifying
an embodiment of a bead (such as a latex bead/particle) with BSA or
HAS, wherein the BSA or HSA may further comprise a maleimide
containing molecule, such as SMCC or NHS. Additionally, an
embodiment of the method 500 may further comprise the step 504 of
attaching a diagnostic marker binding agent (such as an antibody)
to the bead. Step 504 of attaching the diagnostic marker binding
agent to the bead may be accomplished through binding the
diagnostic marker binding agent to maleimide. Further, method 500
may also comprise the step 506 of attaching one or more stabilizing
agent to an embodiment of a bead. Additionally, while an embodiment
of method 500 may use latex beads for attachment of a maleimide
containing molecule and/or a stabilizing agent, alternate
embodiments of the bead may comprise silicon, a polymer, or other
applicable materials.
[0151] According to at least one embodiment of the present
disclosure, one or more stabilizing agent may be attached or
adsorbed to any one of an embodiment of a detection platform 100,
test strip, microchip, or microfluidic device.
[0152] B. Test Strip
[0153] Turning to FIGS. 6A and B, exemplary embodiments of a
diagnostic testing device 600 are described. An exemplary
embodiment of diagnostic testing device 600 comprises housing
structure 602 which comprises a collection chamber 604 capable of
receiving body fluids, at least one membrane test strip 606 in
fluid communication with collection chamber 604, an
immunoassay-based fingerprint acquisition pad 608 in fluid
communication with the collection chamber, and a plurality of
reaction zones 610 which may allow the visual display of the
presence of a predetermined diagnostic marker. For example,
reaction zone 610 may in an exemplary embodiment be capable of
displaying a characteristic of the predetermined diagnostic marker,
such as the presence of, the concentration of, or a concentration
above or below a predetermined value for the predetermined
diagnostic agent.
[0154] In an exemplary embodiment of diagnostic testing device 600,
diagnostic testing device 600 further comprises a fluid collector
612 which is operable to collect body fluids from a subject, and
wherein the fluid collector is capable of supply collected body
fluid to collection chamber 604.
[0155] Exemplary embodiments of test strip 606 may comprise any
material capable of adsorbing or attaching a stabilizing agent and
may bind a diagnostic marker. Further, exemplary embodiments of
test strip 606 may comprise compositions comprising a polyvinyl
chloride-silica combination, nitrocellulose, or any suitable
synthetic, resinous material. At least one embodiment, housing 602
is capable of reducing the exposure of the test strip 606 at sites
other than the collection chamber 604 to foreign material. Test
strip 606 in at least one embodiment may be capable of interfacing
with an analysis device 615. The analysis device 615 in an
exemplary embodiment may be capable of measuring the level of the
predetermined diagnostic marker in the sample.
[0156] Turning to FIG. 6C, an exemplary embodiment of a microarray
system 620 is described. An exemplary embodiment of microarray
system 620 may comprise a microarray product 622 having a
microarray identifier 624, and a plurality of microarray sites 626,
a control microarray product 628 located on at least one microarray
site 626 and bound to microarray product 622, and operably coupled
to a computer processor 130 for providing information regarding the
identification and concentration of markers on the microarray
product 622 based on the microarray identifier 624. Additionally,
microarray product 622 may further comprise, in at least one
embodiment, an embodiment of a stabilization agent 630 located on
at least one microarray site 626 and bound to microarray product
622. Further, microarray product 622 may comprise one or more
diagnostic marker 632 located on at least one microarray site 626
and bound to microarray product 622. Moreover, microarray system
620, in at least one embodiment, may further comprise a microarray
detector 634 operable to detect a signal from at least one of the
control microarray product 628 and/or diagnostic marker 632.
[0157] A microchip or microarrays may refer to an array of distinct
polynucleotides affixed to a substrate, such as glass, plastic,
paper, nylon or other type of membrane, filter, chip, or any other
suitable solid support. The polynucleotides can be synthesized
directly on the substrate, or synthesized separate from the
substrate and then affixed to the substrate. In an embodiment, the
microarray may be prepared and used at least according to the
methods described in U.S. Pat. No. 5,837,832, Chee et al., PCT
application WO95/11995 (Chee et al.), Lockhart, D. J. et al. (1996;
Nat. Biotech. 14: 1675-1680) and Schena, M. et al. (1996; Proc.
Natl. Acad. Sci. 93: 10614-10619), all of which are incorporated
herein in their entirety by reference.
[0158] Turning to FIG. 6D, an exemplary embodiment of microfluidic
device 640 is described. An embodiment of microfluidic device 640
comprises a sample reservoir 642 for receiving a body fluid (or
other fluid sample) through a sample input 644, where the sample
reservoir 642 is fluidly connected to a detector array 646.
Additionally, in at least one embodiment of microfluidic device
640, detector array 646 comprises a reaction site 647 and a results
display 648. Moreover, an exemplary embodiment of microfluidic
device 640 may further comprise a filter device 650 capable of
separating at least one unwanted component from a fluid sample and
fluidly coupled between sample reservoir 642 and detector array
646. Detector array 646 may also, in at least one embodiment
comprise a control reagent display 652, where at least one control
reagent may be visually detected.
[0159] Further, microfluidic device 640 may also, in at least one
embodiment, be able to couple to a processor 655. Processor 655 may
be able to compare at least one reading, such as from results
display 648 or control reagent display 652 from an embodiment of
microfluidic device 640 to at least one additional stored reading
on a computer database 657 of the processor 655. An embodiment of
microfluidic system 660 of the present disclosure, may comprise one
or more microfluidic device 640 operationally coupled to a
processor 655.
[0160] Embodiments of microfluidic devices 640, which may also be
referred to as "lab-on-a-chip" systems, biomedical
micro-electro-mechanical systems (bioMEMs), or multicomponent
integrated systems, are exemplary kits/systems for analyzing
polynucleotide regions. Such systems may miniaturize and
compartmentalize processes such as probe/target hybridization,
nucleic acid amplification, and capillary electrophoresis reactions
in a single functional device. Such microfluidic devices 640
typically utilize detection reagents in at least one aspect of the
system, and such detection reagents may be used to detect one or
more polynucleotide region of the present disclosure. Exemplary
microfluidic devices 640 may comprise a pattern of microchannels
designed onto a glass, silicon, quartz, or plastic wafer included
on a microchip. The movements of the samples may be controlled by
electric, electroosmotic or hydrostatic forces applied across
different areas of the microchip to create functional microscopic
valves and pumps with no moving parts. Varying the voltage can be
used as a means to control the liquid flow at intersections between
the micro-machined channels and to change the liquid flow rate for
pumping across different sections of the microchip.
[0161] In at least one embodiment of a microarray or microfludic
device 640, the microarray or microfluidic device may comprise one
or more stabilizing agent capable of reducing the degradation of a
predefined diagnostic marker. For example, a stabilizing agent may
be incorporated onto the surface of the microarray system 620,
and/or on at least one part of the microfluidic device 640.
IV. Methods of Biomarker Stabilization
[0162] In at least one embodiment of a method for stabilizing
biomarkers, as described in FIG. 7, the method 700 comprises the
step 702 of mixing a body fluid with an embodiment of a stabilizing
agent. Optionally, the step of mixing a body fluid with a
stabilizing agent may comprise the step 704 of introducing the
stabilizing agent into the patient, such as through the oral
cavity, so that the stabilizing agent mixes with the bodily fluid,
and the step 706 of retrieving a body fluid comprising a stabilized
diagnostic marker. In an additional exemplary embodiment of a
method of stabilizing a diagnostic marker, the method comprises the
step 708 of isolating a body fluid having a diagnostic marker, the
step 710 of treating the body fluid with an embodiment of a
carrier, such as a rinse, gum, or beverage, as described herein,
and the step 712 of analyzing the diagnostic marker. Isolation of
the body fluid may occur through any customary mechanism,
including, but not limited to, rinsing, swabbing, suction,
collection, and lavage. Further, the rinse in an exemplary
embodiment of a method 700 of the present disclosure, may be
ingested to stabilize a diagnostic marker present in the bodily
fluid, such as in urine, semen, anal secretions, and vaginal
secretions.
[0163] The step 702 of mixing a body fluid with a carrier may occur
prior to, or after isolation step 708 of the body fluid. As an
exemplary embodiment, a rinse may be used to rinse the mouth.
Afterwards, the rinse containing the treated body fluid may be
collected by spitting, suction, or other means. Alternately,
expirated saliva may be mixed with a rinse to treat the saliva ex
vivo. Similar treatment of body fluids may be performed with any
type of body fluid.
[0164] In analyzing the treated body fluid, the diagnostic marker
may be analyzed through any known means. Optionally, the analysis
of the treated body fluid may include the separation of solid
materials from soluble materials through means such as filtration,
or by centrifugation. Analysis of the treated body fluid may use
any appropriate technique, such as western blot analysis,
Enzyme-Linked Immunosorbent Assay (ELISA), protein activity assays,
reverse transcription polymerase chain reaction (RT-PCR),
microarray, high pressure liquid chromatography, or any comparable
assay to determine a characteristic of the diagnostic marker. Such
an analysis, in an exemplary embodiment, may be of a modified
product, a cleavage product, or cleavage pattern, of a diagnostic
marker.
[0165] In an exemplary embodiment of the analyzing step 712, the
treated body fluid may be placed in contact with an embodiment of a
test strip, such as a nitrocellulose strip, prior to detection with
an appropriate probe specific for the diagnostic marker. The
nitrocellulose strip may be designed to trap or filter particulates
in the body fluid. This may reduce or eliminate potential
contaminants or other substances in the oral fluid that would
otherwise reduce the signal to noise ration during the detection
step. A probe (such as an antibody) with affinity to the diagnostic
marker may be affixed or immobilized to a specific location on the
nitrocellulose membrane for detection of the analyte. Utilizing
standard principles of immunoassay detection (or nucleotide
detection), the signal generated may be visible to the eye or
detectable by an instrument.
[0166] In an exemplary embodiment of a method of analyzing a
stabilized diagnostic marker of the present disclosure as depicted
in FIG. 8, the method 800 comprises the step 802 of operating a
system for the detection of a diagnostic marker in a body fluid,
where the system comprises a diagnostic device having a plurality
of test wells each capable of containing at least one diagnostic
marker binding agent, and a detection device capable of interacting
with diagnostic device, wherein the detection device is capable of
detecting an interaction between the at least one diagnostic marker
binding agent and a diagnostic marker. Following step 802, the
sample of bodily fluid may be diluted in step 803 with a reagent
which may contain an embodiment of stabilizing agent as described
herein. At least one embodiment of method 800 further comprises the
steps of: contacting 804 a sample of a bodily fluid with at least
one diagnostic marker binding agent, incubating 806 the contacted
sample in the test well at a first temperature for a pre-determined
period of time. The step 804 of contacting of the sample of bodily
fluid with at least one diagnostic marker binding agent may be
accomplished through a magnetic field applied to the test well.
[0167] In at least one embodiment of method 800, the first
temperature may be at room or at body temperature (such as about
22.degree. C. or about 37.degree. C.). The pre-determined period of
time may, for example, be between five and fifteen minutes. The
incubated and contacted sample may then be removed in step 808 from
the plurality of test wells, and the test wells analyzed in step
810 to detect a binding event with the detection device.
Alternately, a stabilizing agent may be incubated with the
diagnostic marker binding agent (also referred to as a detection
agent) prior to introduction of a bodily fluid. In at least one
embodiment of the method, each diagnostic marker binding agent may
have a different monoclonal antibody designed to bind a specific
hapten molecule.
[0168] Following the detection of a characteristic of a treated
diagnostic marker (the results of which may be termed a "profile"
such as a "cancer profile"), the characteristic may in step 812 be
compared to a standard value to determine the presence or absence
of a disease state. The characteristic determined may be an
activity level, the concentration of the diagnostic marker, or a
particular modification, such as glycosylation or methylation.
[0169] Optionally, an embodiment of the method 800 may further
comprise the step 814 of customizing a detection method through
screening of applicable pre-clinical samples through a database of
stabilizing agents/cocktails. Further, an embodiment of method 800
of diagnostic marker detection may further comprise the step 816 of
comparing a level or characteristic (such as a predictable
degradation products) of a diagnostic agent with a library of known
characteristics. Moreover, an exemplary embodiment of method 800
may additionally comprise the step 818 of determining a diagnosis
of a disease state using the compared characteristic. In at least
one embodiment, a method of diagnostic marker detection may be
automated, and capable of detecting at least about ten thousand
samples in a twenty-four hour period. Method 800 in at least one
embodiment, may be at least partially completed by a computer
processor. Additionally, the processor may be in communication with
at least one more processor and/or a computer database. For
instance, in at least one embodiment of method 800, most of the
steps of the method may be completed with or monitored by a
computer processor. Moreover, method 800, in at least one
embodiment, may use a computer processor to perform additional step
820 of generating a report by using a comparison of determined
characteristics (such as a binding report where a comparison is
performed of binding characteristics), and step 822 of delivering
the report to a recipient (such as a user, or secondary
processor).
[0170] A method of biomarker stabilization may, in an exemplary
embodiment, may be used to diagnose a particular disease state or
condition of a patient. For example, a disease state or condition
as used with this method may include one or more of Acid-Base
Disorders, Acidosis and Alkalosis, Acidosis/Alkalosis, Acute
inflammatory demyelinating polyneuropathy, Acute myocardial
infarct, Addison's Disease, Adrenal Insufficiency, Adrenal
Insufficiency & Addison's Disease, Alcohol dependence,
Alcoholism, Allergies, Alzheimer's Disease, Anemia, Angina
Pectoris, Anthrax, Arthritis, Asthma, Atypical Pneumonia,
Autoimmune Disorders, Autoimmune thyroiditis, Avian flu, Benign
Prostatic Hyperplasia, Benign Prostatic Hypertrophy, Bioterrorism
Agents, Bleeding Disorders, Bone Marrow Disorders, Breast Cancer,
Cardiovascular Disease, Celiac Disease, Cervical Cancer, Chlamydia,
Chronic Fatigue and Immune Dysfunction Syndrome, Chronic Fatigue
Syndrome, Chronic thyroiditis, Colon Cancer, Community-Acquired
Pneumonia, Congestive Heart Failure, Conn's Syndrome, Cushing's
Syndrome, Cystic Fibrosis, Degenerative Joint Disease, Diabetes,
Diarrhea, Diffuse thyrotoxic goiter, Diseases of the Pancreas,
Disseminated lupus erythematosus, Double pneumonia, Down Syndrome,
Encephalitis, Endocrine Syndromes, Endocrine System and Syndromes,
Epilepsy, Fibromyalgia, Flu, Folate Deficiency, Fungal Infections,
Genital Herpes, Gonorrhea, Gout, Gouty arthritis, Graves' Disease,
Guillain-Barre Syndrome, Influenza H1N1, Hashimoto's Thyroiditis,
Healthcare-Associated Pneumonia, Heart Attack, Heart Attack and
Acute Coronary Syndrome, Heart Disease, Hemochromatosis, Hepatitis,
Herpes, Herpes Zoster, High blood pressure, Hospital-Acquired
Pneumonia, Human Immunodeficiency Virus, Human Papillomavirus,
Hypercoagulable Disorders, Hypersensitivity, Hypertension,
Hyperthyroidism, Hypothyroidism, Infectious Arthritis, Infectious
polyneuritis, Infertility, Inflammatory Bowel Disease, Influenza,
Influenza A, Influenza B, Insulin Resistance, Jaundice, Juvenile
Rheumatoid Arthritis, Kidney and Urinary Tract Function, Disorders,
and Diseases, Kidney Disease, Landry's ascending paralysis, Lead
Poisoning, Leukemia, Liver Disease, Lobar pneumonia, Lower
Respiratory Tract Infection, Lung Diseases, Lupus, Lupus
erythematosus, Lyme Disease, Lymphoma, Malnutrition, Meningitis,
Meningitis and Encephalitis, Menopause, Metabolic Syndrome,
Metabolic Syndrome/Syndrome X, Multiple Myeloma, Multiple
Sclerosis, Myeloproliferative Disorders, Myocardial infarct, Neural
Tube Defects, Nontuberculous Mycobacteria, Osteoarthritis,
Osteoporosis, Ovarian Cancer, Pancreatic Cancer, Pancreatic
Diseases, Pancreatic Insufficiency, Pancreatitis, Pelvic
Inflammatory Disease, Peptic Ulcer, Pituitary Disorders, Pneumonia,
Polycystic ovarian syndrome, Pregnancy, Primary hyperaldosteronism,
Prostate Cancer, Proteinuria, Rheumatoid Arthritis, Septic
Arthritis, Sexually Transmitted Diseases, Sexually transmitted
infections, Shingles, Sickle Cell Anemia, Sickle Cell Disease,
Sjogren's Syndrome, Staph Wound Infections, Staph Wound Infections
and Methicillin Resistant Staphylococcus aureus, Stein-Leventhal
syndrome, Stroke, Swine flu, Syphilis, Systemic Lupus
Erythematosus, Testicular Cancer, Thalassemia, Thyroid Diseases,
Travelers' Diseases, Trichomonas, Tuberculosis, Types of Liver
Disease, Urinary Tract Infection, Venereal diseases, Vitamin B12
and Folate Deficiency, Vitamin B12 Deficiency, Vitamin K
Deficiency, Walking pneumonia, West Nile Virus, Wilson's Disease,
Wound and Skin Infections.
V. Computational Analysis
[0171] In at least one embodiment of the systems, methods, and
devices of the present disclosure, a computer processor and a
database may be used in or during the system, method, or device. An
exemplary embodiment of a system framework comprising at least one
computer processor and at least one database, as may be used in the
embodiments of the present disclosure is shown in FIG. 9.
[0172] In at least one embodiment, a result from a diagnostic
method may be compared using a computer processor to at least one
additional result from the diagnostic test, and/or to a result
stored on a database. Such a comparison, in at least one embodiment
of the present disclosure, may reveal the stabilizing agent with a
greater effect on the binding characteristic, may allow for the
diagnosis of a disease state or health/lifestyle characteristic.
Further, such a comparison, in at least one embodiment, may be used
to develop, confirm, or modify a therapeutic treatment of a patient
having a disease state or health/lifestyle characteristic.
[0173] As shown in exemplary system framework 900 shown in FIG. 9,
one or more user computers 902 may be operably connected to a
system server 904. A user computer 902 may be a computer, computing
device, or system of a type known in the art, such as a personal
computer, mainframe computer, workstation, notebook computer,
laptop computer, hand-held computer, wireless mobile telephone,
personal digital assistant device, and the like.
[0174] One or more administrator computers 906 may also be operably
connected to system server 904 including through a network 908 such
as the Internet. Administrator computers 906, similar to user
computers, may be computers, computing devices, or systems of a
type known in the art, such as personal computers, mainframe
computers, workstations, notebook computers, laptop computers,
hand-held computers, wireless mobile telephones, personal digital
assistant devices, and the like. In addition, user computers 902
and administrator computers 906 may each comprise such software
(operational and application), hardware, and componentry as would
occur to one of skill of the art, such as, for example, one or more
microprocessors, memory, input/output devices, device controllers,
and the like. User computers and administrator computers 906 may
also comprise one or more data entry means (not shown in FIG. 9)
operable by a user of client computer and/or an administrator
computer, such as, for example, a keyboard, keypad, pointing
device, mouse, touchpad, touchscreen, microphone, and/or other data
entry means known in the art. User computers 902 and administrator
computers 906 also may comprise an audio display means (not shown
in FIG. 9) such as one or more loudspeakers and/or other means
known in the art for emitting an audibly perceptible output. The
configuration of user computers and administrator computers 906 in
a particular implementation of one or more systems of the present
disclosure is left to the discretion of the practitioner.
[0175] System server 904 may comprise one or more server computers,
computing devices, or systems of a type known in the art. System
server 904 may comprise server memory. System server 904 may
comprise one or more components of solid-state electronic memory,
such as random access memory. System server 904 may also comprise
an electromagnetic memory such as one or more hard disk drives
and/or one or more floppy disk drives or magnetic tape drives, and
may comprise an optical memory such as a Compact Disk Read Only
Memory (CD-ROM) drive. System server 904 may further comprise such
software (operational and application), hardware, and componentry,
as would occur to one of skill of the art, such as, for example,
microprocessors, input/output devices, device controllers, video
display means, and the like.
[0176] System server 904 may comprise one or more host servers,
computing devices, or computing systems configured and programmed
to carry out the functions allocated to system server 904. System
server 904 may be operated by, or under the control of, a "system
operator," which may be an individual or a business entity. For
purposes of clarity, System server 904 is shown in FIG. 9 and
referred to herein as a single server. System server 904 need not,
however, be a single server. System server 904 may comprise a
plurality of servers or other computing devices or systems
connected by hardware and software that collectively are operable
to perform the functions allocated to the various systems of
present disclosure. Specifically, system server 904 may be operable
to be a web server, configured and programmed to carry out the
functions allocated to a system server according to the present
disclosure. Further, although user computers 902 and administrator
computers 906 may be connected directly to system server 904, these
computers may be connected to system server 904 through any
suitable network, such as network 908. Further, in one embodiment,
the users need not be provided access to system server 904, but
instead the content posts from users are made by the user(s) and
saved to one or more particular locations and the posts are
accessed or harvested by the administrator or system
automatically.
[0177] System server 904 may be operably connected to the various
user computers 902 and/or an administrator computers 906 by network
908, which in an embodiment of the present disclosure comprises the
Internet, a global computer network. However, network 908 need not
comprise the Internet. Network 908 may comprise any means for
electronically interconnecting system server 904 and a user
computer 902 and/or an administrator computer 906. Thus, it will be
appreciated by those of ordinary skill in the art that the network
908 may comprise the Internet, the commercial telephone network,
one or more local area networks, one or more wide area networks,
one or more wireless communications networks, coaxial cable, fiber
optic cable, twisted-pair cable, the equivalents of any of the
foregoing, or the combination of any two or more of the foregoing.
In an embodiment where system server 904 and user computer 902
and/or an administrator computer 906 comprise a single computing
device operable to perform the functions delegated to both system
server 904 and user computer 902 and/or an administrator computer
906 according to the present disclosure, network 908 comprises the
hardware and software means interconnecting system server 904 and
user computer 902 and/or an administrator computer 906 within the
single computing device. Network 908 may comprise packet switched
facilities, such as the Internet, circuit switched facilities, such
as the public switched telephone network, radio based facilities,
such as a wireless network, etc.
[0178] The various systems, methods, schema, ontologies, and
architectures of the present disclosure may be used for purposes
outside of the medical transcription field as referenced in the
various examples cited herein. For example, the system for
analyzing verbal records may comprise various components and
relationships suitable for use in any number of areas where various
experiences are utilized and processed, with feedback being fed
back into system componentry to improve overall system outcomes. In
addition, various components described herein may share a name (or
a portion thereof) but have duplicative reference numbers, and
therefore the descriptions for the various components should read
in view of one another.
[0179] In addition, and regarding the various systems of the
present disclosure, such systems may be operable, as desired by a
user of such systems, to generate visual, electronic (video, audio,
database, transcript, etc.), and/or printed reports, outputs,
outcomes, and the like. Such exemplary outputs may be used for any
number of purposes, and may be useful generally to "report"
results, data, and/or knowledge contained within and generated from
such systems. Furthermore, the disclosure of the present
application further encompasses uses of the various methods,
systems, architectures, etc., to perform various tasks in
connection therewith.
EXAMPLES
Examples
1. .alpha.-Amylase Inhibition Saliva Screening Method
[0180] At least one assay used for the detection of
.alpha.-amylases activity, as used herein includes the use of a
chromagenic substrate, 2-chloro-p-nitrophenol linked to
maltotriose. Enzymatic activity of .alpha.-amylase on the substrate
yields 2-chloro-p-nitrophenol, which can be measured
spectrophotometrically at 405 nm. The amount of .alpha.-amylase
activity present in the experimental sample is directly
proportional to the increase in absorbance at 405 nm.
[0181] For the assay, test compounds are mixed with saliva, prior
to the addition of .alpha.-amylase substrate. The test compounds,
such as GRAS materials, are prepared at a concentration of 5,000
ppm in distilled water. Following this preparation, 300 .mu.l of
.alpha.-amylase substrate (Salimetrics .alpha.-amylase Assay Kit)
that has been pre-heated to 37.degree. C. is added to 20 .mu.l of
the test compound. To initiate the assay, 10 .mu.l of a dilute
(3.5%) or full strength (100%) saliva solution is added to the
mixture. Following the initiation, each mixture is measured at 1
minute intervals using a spectrophotometer (such as Molecular
Devices M5 reader) at 405 nm. Temperatures of the mixtures are kept
constant at 37.degree. C. during the assay.
2. .alpha.-Amylase Inhibitor Assays
[0182] A series of GRAS compounds were tested using the
.alpha.-amylase assay protocol described here. The tested compounds
are included in Table I. From the screen of GRAS compounds, five
compounds (CDI-030, 034, 036, 037 and 042) were shown to have
.alpha.-Amylase Inhibitor Activity in dilute (3.5%) saliva (See
FIG. 10). Further details of these five compounds may also be seen
in Table 4.
TABLE-US-00004 TABLE 4 0% 100% Saliva Amylase Time CDI-30 CDI-34
CDI-36 CDI-37 CDI-42 Control Control 0 min 1.60 0.40 3.63 4.00 0.80
4.00 0.09 2.03 0.36 3.20 3.85 1.13 4.00 0.09 2.02 0.40 3.46 4.00
0.90 4.00 0.09 1.87 0.45 4.00 4.00 0.84 4.00 0.10 % CV 10.67 9.13
9.42 1.85 16.35 0.00 1.84 Ave 1.88 0.40 3.57 3.96 0.92 4.00 0.09 %
Std 46.96 10.04 89.29 99.08 22.93 100.00 2.34 % Inhibition 53.04
89.96 10.71 0.92 77.07 0.00 97.66 5 min 4.00 0.49 4.00 4.00 1.65
4.00 0.09 4.00 0.50 4.00 4.00 2.15 4.00 0.09 4.00 0.50 4.00 4.00
1.80 4.00 0.09 4.00 0.52 4.00 4.00 1.81 4.00 0.10 % CV 0.00 2.32
0.00 0.00 11.31 0.00 2.79 Ave 4.00 0.50 4.00 4.00 1.85 4.00 0.09 %
Std 100.00 12.61 100.00 100.00 46.24 100.00 2.36 % Inhibition 0.00
87.39 0.00 0.00 53.76 0.00 97.64 15 min 4.00 0.53 4.00 4.00 2.37
4.00 0.09 4.00 0.61 4.00 4.00 2.38 4.00 0.10 4.00 0.54 4.00 4.00
2.25 4.00 0.09 4.00 0.56 4.00 4.00 2.17 4.00 0.10 % CV 0.00 6.67
0.00 0.00 4.33 0.00 1.88 Ave 4.00 0.56 4.00 4.00 2.29 4.00 0.09 %
Std 100.00 14.02 100.00 100.00 57.35 100.00 2.37 % Inhibition 0.00
85.98 0.00 0.00 42.65 0.00 97.64
Among these five compounds, CDI-034 and CDI-42 were shown to
inhibit .alpha.-Amylase in 100% saliva samples (See FIG. 11).
3. Lysozyme Inhibition Saliva Screening Method
[0183] At least one assay used for the detection of
.alpha.-amylases activity, as used herein includes the use of a
Micrococcus lysodeikticus labeled with fluorescein. The assay
measures lysozyme activity on Micrococcus lysodeikticus cell walls,
which are labeled to such a degree that the fluorescence is
quenched. Lysozyme activity can relieve this quenching; yielding
increased fluorescence that is proportional to lysozyme
activity.
[0184] For the assay, test compounds are mixed with saliva, prior
to the addition of lysozyme substrate. The test compounds, such as
GRAS materials, are prepared at a concentration of 5,000 ppm in
distilled water. Following this preparation, 50 .mu.l of lysozyme
substrate at 1 mg/ml (Molecular Probes EnzChek Lysozyme Assay Kit)
that has been pre-heated to 37.degree. C. is added to 50 .mu.l of
the test compound. To initiate the assay, 50 .mu.l of a dilute
(3.5%) or full strength (100%) saliva solution is added to the
mixture. Following the initiation, each mixture is measured at 1
minute intervals using a spectrophotometer (such as Molecular
Devices M5 reader) for absorption at 494 nm and fluorescence
emission at 518 nm. Temperatures of the mixtures are kept constant
at 37.degree. C. during the assay.
4. Lysozyme Inhibitor Assays
[0185] A series of GRAS compounds were tested using the Lysozyme
assay protocol described above. The tested compounds are included
in Table I. From the screen of GRAS compounds, four compounds
including Fixanal.RTM. Buffer 6.0 (Sigma-Aldrich Co., CDI-030),
bentonite (CDI-37), benzoic acid (CDI-40), and acetic acid
(CDI-047) were shown to have Lysozyme Inhibitor Activity in dilute
(3.5%) saliva (See FIG. 12). Further details of these five
compounds may also be seen in Table 5.
TABLE-US-00005 TABLE 5 Cell Location RFU Reading (60 mins) CDI
Candidate # 30 F 4 51.13 37 E 5 92.06 40 H 5 35.82 47 G 6 33.53
CONTROLS Pooled Saliva E 12 127.33 Fresh Saliva F 12 182.02
Lysozyme G 12 233.43
Among these four compounds, benzoic acid (CDI-40), and acetic acid
(CDI-047) were shown to inhibit Lysozyme in 100% saliva samples
(data not shown).
5. Galactose Oxidase Inhibition Saliva Screening Method
[0186] At least one assay used for the detection of galactose
oxidase activity, as used herein, includes the use of a chromagenic
or fluorogenic substrate, such as with Amplex.RTM. Red (Molecular
Probes). In an embodiment of the assay used herein, galactose
oxidase catalyzes the oxidation of galactose at the C6 position and
generates hydrogen peroxide (H2O2). The H2O2 then, in the presence
of horseradish peroxidase (HRP), reacts with 1:1 stoichiometry with
Amplex.RTM. Red reagent to generate the red-fluorescent oxidation
product, resorufin. Resorufin has absorption and fluorescence
emission maxima of approximately 571 nm and 585 nm, respectively,
and because the extinction coefficient is high (54,000 cm-1M-1),
the assay can be performed either fluorometrically or
spectrophotometrically.
[0187] For the assay, test compounds are mixed with Amplex Red,
reaction buffer, horseradish peroxidase (HRP) at 100 U/ml, and
galactose stock solution (according to manufacturers recommended
protocol, Molecular Probes A22179), prior to the addition of
saliva. The test compounds, such as GRAS materials, are prepared
under various concentration for testing. Following this
preparation, 50 .mu.l of the test compound solution (with Amplex
Red, reaction buffer, and HRP) is added to 50 .mu.l of saliva.
Following the initiation, each mixture is measured at 1 minute
intervals using a spectrophotometer (such as Molecular Devices M5
reader) using excitation in the range of 530-560 nm and emission
detection at about 590 nm or absorbance at 560 nm. Temperatures of
the mixtures are kept constant at 37.degree. C. during the
assay.
6. Galactose Oxidase Inhibitor Assays
[0188] A series of GRAS compounds were tested using the Galactose
Oxidase assay protocol described herein. The tested compounds are
included in Table I. From the screen of GRAS compounds, four
compounds (CDI-039, 040, 042, and 047) were shown to have Galactose
Oxidase Inhibitor Activity in dilute (3.5%) saliva (See FIG. 13).
FIG. 13 includes the following symbols which are: open
circle=Aluminum potassium sulfate dodecahydrate, open
square=3-tert-butyl-hydroxyanisole, open triangle=benzoic acid,
open diamond=acetic acid, filled circle=fresh saliva, and filled
square=water (control blank).
7. Stabilization of Glycosylated Hemoglobin
[0189] At least one procedure used for the stabilization and
detection of glycosylated hemoglobin (HbA1c), as described herein,
includes the incubation of HbA1c with a stabilizing agent prior to,
or concurrently with, detection. Analysis of samples was conducted
on a HPLC 1100 series (Agilent Technologies) with a 2.1.times.150
(3.50.mu.) C18 reverse phase column. The mobile phase used was
acetonitrile with 6.5 mM Ammonium Carbonate. A HbA1c standard was
prepared by dissolving HbA1c (Sigma Aldrich, 405 RM) with 1.0 ml of
deionized water. The samples analyzed by HPLC included pure HbA1c
(FIG. 14), 100 ml HbA1c mixed with 100 ml of fresh saliva (FIG.
15), and 200 ml HbA1c mixed with 100 ml of fresh saliva and 100 ml
of a stabilizing compound comprising aluminum sulfate hydrate,
benzoic acid, and aluminum potassium sulfate dodecahydrate (each
present in a 1:1:1 ratio) (FIG. 16). While the pure HbA1c sample
was analyzed fresh, samples of HbA1c with saliva were incubated
overnight at room temperature prior to analysis. For each sample
analyzed, the data was displayed through a three dimensional plot
(FIGS. 14A, 15A, and 16A), a UV image (FIGS. 14B, 15B, and 16B),
and a chromatogram (FIGS. 14C, 15C, and 16C). Additionally, a
comparison of the analysis HbA1c in the presence of saliva with and
without stabilizing agent is shown in FIGS. 17 (chromatography), 18
(UV image), and 19 (three dimensional plot).
8. HbA1c Protection Assays
[0190] A series of GRAS compounds were tested, as shown in FIGS.
14-16, for the ability to protect HbA1c from degradation by
components in saliva. The tested compounds are included in Table I.
As described above, a stabilizing compound comprising aluminum
sulfate hydrate, benzoic acid, and aluminum potassium sulfate
dodecahydrate (each present in a 1:1:1 ratio) minimized the
degradation of HbA1c, and yielded a defined pattern for HbA1c.
9. Stabilization of Cancer Antigen 19-9
[0191] At least one procedure used for the stabilization and
detection of Cancer Antigen 19-9 (CA 19-9) as described herein,
includes the incubation of CA 19-9 with a stabilizing agent prior
to, or concurrently with, detection. Analysis of samples was
conducted on a HPLC 1100 series (Agilent Technologies) with a
2.1.times.150 (3.50.mu.) C18 reverse phase column. The mobile phase
used was acetonitrile with 6.5 mM Ammonium Carbonate. A CA 19-9
standard was prepared by dissolving CA 19-9 with 1.0 ml of
deionized water. The samples analyzed by HPLC included pure CA 19-9
(FIG. 20), 100 ml CA 19-9 mixed with 100 ml of fresh saliva (FIG.
21), and 200 ml CA 19-9 mixed with 100 ml of fresh saliva and 100
ml of a stabilizing compound comprising aluminum sulfate hydrate,
benzoic acid, and aluminum potassium sulfate dodecahydrate (each
present in a 1:1:1 ratio) (FIG. 22). While the pure CA 19-9 sample
was analyzed fresh, samples of CA 19-9 with saliva were incubated
overnight at room temperature prior to analysis. For each sample
analyzed, the data was displayed through a three dimensional plot
(FIGS. 20A, 21A, and 22A) and a UV image (FIGS. 20B, 21B, and 22B).
Additionally, a comparison of the analysis CA 19-9 in the presence
of saliva with and without stabilizing agent is shown in FIGS. 23
(three dimensional plot) and 24 (UV image).
10. CA 19-9 Protection Assays
[0192] A series of GRAS compounds were tested, as shown in FIGS.
20-22, for the ability to protect CA 19-9 from degradation by
components in saliva. The tested compounds are included in Table I.
As described above, a stabilizing compound comprising aluminum
sulfate hydrate, benzoic acid, and aluminum potassium sulfate
dodecahydrate (each present in a 1:1:1 ratio) minimized the
degradation of CA 19-9 in at least one embodiment.
11. Peroxidase Assay
[0193] To determine the effect of various GRAS compounds as
inhibitors of peroxidase activity, a standard assay using
Amplex.RTM. Red (Life Technologies), which releases a fluorescent
oxidation product. This oxidation product, named resorufin, has an
excitation and emission maxima of approximately 571 nm and 585 nm,
respectively, and because the extinction coefficient is high
(58,000.+-.5,000 cm-1M-1), it allows for the use with fluorometric
or spectrophotometric assays.
[0194] The test compounds, such as GRAS materials or cocktails of
same, were prepared at a concentration of 500 ppm in distilled
water. For the assay, 50 .mu.l samples of test compounds were mixed
with 20-30 .mu.l of saliva (either "pooled" or "unpooled") prior to
the addition of Amplex.RTM. Red. Following the mixture of the test
compounds with saliva, 500 of Amplex.RTM. Red/HRP working solution
(See Protocol for Invitrogen Assay #A22188, which is incorporated
herein in its entirety), which has a concentration of 100 .mu.M of
Amplex.RTM. Red, is added to the test compound/saliva mixture.
Following the initiation of the reaction, each mixture is measured
at 1 minute intervals using a spectrophotmoter (such as a Molecular
Devices M5 reader) for excitation in the range of 530-560 nm and
fluorescence emission detection at approximately 590 nl, or for
absorption at approximately 560 nm. Reactions were allowed to run
for 30 minutes during these assays.
12. Peroxidase Protection Assays
[0195] A series of GRAS compounds and cocktails of GRAS compounds
were tested, as shown in FIGS. 25-29, for the ability to inhibit
degradation of a peroxidase-sensitive marker by components in
saliva. Various embodiments of the tested compounds are included in
Table I. As described above, a stabilizing compound/cocktail
comprising aluminum sulfate hydrate, benzoic acid, and aluminum
potassium sulfate dodecahydrate (each present in a 1:1:1 ratio)
minimized the degradation of a peroxidase sensitive marker in at
least one embodiment (FIG. 25, open circle=blank control, open
square=stabilizing cocktail). Further, inhibition assays were also
conducted with caffeine (FIG. 26, CDI #33; Key: open circle=pooled
saliva, open square=unpooled saliva, open triangle=caffeine, open
triangle=caffeine (repeat)), 3-tert-butyl-hydroxyanisole (FIG. 27,
CDI #39; Key: open circle=pooled saliva, open square=unpooled
saliva, open triangle=3-tert-butyl-hydroxyanisole, open
triangle=3-tert-butyl-hydroxyanisole (repeat)), benzoic acid (FIG.
28, CDI #16; Key: open circle=pooled saliva, open square=unpooled
saliva, open triangle=benzoic acid, open triangle=benzoic acid
(repeat)), and aluminum potassium sulfate dodecahydrate (FIG. 29,
CDI #42; Key: open circle=pooled saliva, open square=unpooled
saliva, open triangle=aluminum potassium sulfate dodecahydrate,
open triangle=aluminum potassium sulfate dodecahydrate (repeat)),
each of which showed significant peroxidase inhibition
capabilities.
13. Diagnostic Devices
[0196] In an example of a diagnostic device, as shown in FIG. 30, a
test slide with 28 test wells are spotted with 12 to 18 sets of
diagnostic markers. These reagents may include diagnostic markers,
or controls for the diagnosis. The diagnostic device, in this
example, has four diagnostic markers spotted in duplicate, along
with two positive and two negative control marker. Each marker is
spotted with a competitor, such as anti-haptin, which binds the
capture antigen particle to the marker. The device may have four
different hatpin/anti-haptin systems to serve as the generic
capture system. A unique haptin may be conjugated to a fully
paramagnetic particle (PmMp). The PmMp is also coated with a
capture monoclonal antibody (MAb) specific for the marker. A second
particle (polystyrene microparticle with Europium) is conjugated
with the second or detection MAb. In the diagnostic device shown in
FIG. 30, the hash marks on the side of the diagnostic device
(slide) are indentations molded into the plastic, which may be used
as cog grooves. Additionally the slide is designed to fit into a
processor that may have more than one processing station, as shown
in FIG. 32.
[0197] In performing a method of detecting a diagnostic marker
using an embodiment of the diagnostic device, such as that in FIGS.
30 and 32, the user adds a pre-determined amount of specimen (such
as 120 .mu.l) to a specimen diluent (such as 40 to 120 .mu.l), and
adds the mixture to each test well. Following addition of the
diluted specimen, the test slide is incubated at 37.degree. C. for
about 5 to about 15 minutes with agitation. Following the
incubation period, the test wells move over a magnetic source that
brings the PmMp to the bottom of the well, so that there is maximal
binding of the PmMp with the anti-haptin bound in the test well.
Next, specimen and assay reagents are removed, wash buffer is added
and aspirated off. Lastly, each well is scanned using a
visualization device, such as a CCD camera, and then the
fluorescent signal (or other signal) of each marker is
determined.
14. Fluid Collection Device
[0198] An example of a collection device for the collection of an
bodily fluids is depicted in FIG. 31. In this example, the
collection device is capable of collecting at least 500 .mu.l of
bodily fluid, such as saliva. The cap is first removed from the
tube. Squeezing the bulb provides suction adequate to pull an
amount of saliva into the bulb. In some cases, a filter may be
included in the tube to remove unwanted materials (such as
particulate and mucous). The filter may be of any known filter
material, such as rayon, that is appropriate for the purposes
described herein. Following the obtaining of the bodily fluid in
the collection device, a cap is placed on the tube, and a second
cap is removed from a dispensing tube. The dispensing tube is in
fluid communication with the bulb and is sized and shaped to allow
for drops of a predetermined size to be formed when the bulb is
squeezed. For example, the dispensing tube may allow drops of 30
.mu.l to be formed when the bulb is squeezed.
[0199] While various embodiments of the systems, methods, and
compositions of the present disclosure have been described in
considerable detail herein, the embodiments are merely offered by
way of non-limiting examples of the disclosure described herein. It
will therefore be understood that various changes and modifications
may be made, and equivalents may be substituted for elements
thereof, without departing from the scope of the disclosure.
Indeed, this disclosure is not intended to be exhaustive or to
limit the scope of the present disclosure.
[0200] Further, in describing representative embodiments, the
disclosure may have presented a method and/or process as a
particular sequence of steps. However, to the extent that the
method or process does not rely on the particular order of steps
set forth herein, the method or process should not be limited to
the particular sequence of steps described. Other sequences of
steps may be possible. Therefore, the particular order of the steps
disclosed herein should not be construed as limitations of the
present disclosure. In addition, disclosure directed to a method
and/or process should not be limited to the performance of their
steps in the order written. Such sequences may be varied and still
remain within the scope of the present disclosure.
* * * * *