U.S. patent application number 12/852312 was filed with the patent office on 2011-03-17 for devices for detecting renal disorders.
This patent application is currently assigned to Rules-Based Medicine, Inc.. Invention is credited to Karri L. Ballard, Dominic P. Eisinger, Samuel T. LaBrie, James P. Mapes, Ralph L. McDade, Michael D. Spain.
Application Number | 20110065608 12/852312 |
Document ID | / |
Family ID | 43544701 |
Filed Date | 2011-03-17 |
United States Patent
Application |
20110065608 |
Kind Code |
A1 |
LaBrie; Samuel T. ; et
al. |
March 17, 2011 |
Devices for Detecting Renal Disorders
Abstract
Devices for diagnosing, monitoring, or determining a renal
disorder in a mammal are described. In particular, devices for
diagnosing, monitoring, or determining a renal disorder using
measured concentrations of a combination of three or more analytes
in a test sample taken from the mammal are described.
Inventors: |
LaBrie; Samuel T.; (Austin,
TX) ; Mapes; James P.; (Lakeway, TX) ; McDade;
Ralph L.; (Austin, TX) ; Eisinger; Dominic P.;
(Keene, NY) ; Ballard; Karri L.; (Austin, TX)
; Spain; Michael D.; (Austin, TX) |
Assignee: |
Rules-Based Medicine, Inc.
Austin
TX
|
Family ID: |
43544701 |
Appl. No.: |
12/852312 |
Filed: |
August 6, 2010 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
61327389 |
Apr 23, 2010 |
|
|
|
61232091 |
Aug 7, 2009 |
|
|
|
Current U.S.
Class: |
506/18 |
Current CPC
Class: |
G01N 2333/765 20130101;
G01N 2333/4725 20130101; G01N 2333/475 20130101; G01N 2333/70539
20130101; Y10T 436/147777 20150115; G01N 2333/91177 20130101; G01N
2800/347 20130101; G01N 33/5302 20130101; G01N 2333/4703 20130101;
G01N 33/566 20130101; G01N 2333/8146 20130101; G01N 33/6893
20130101; G01N 2333/8139 20130101; G01N 2333/47 20130101; G01N
2333/82 20130101; G01N 2800/56 20130101; G01N 2333/70503 20130101;
G01N 2333/52 20130101; G01N 2333/4706 20130101; G01N 2800/52
20130101; G01N 2333/4727 20130101; G01N 2800/34 20130101; G01N
2333/775 20130101; G01N 2800/60 20130101 |
Class at
Publication: |
506/18 |
International
Class: |
C40B 40/10 20060101
C40B040/10 |
Claims
1. An assay device for diagnosing, monitoring, or determining a
renal disorder in a mammal, the device comprising a panel of
biomarkers for diagnosing, monitoring, or determining a renal
disorder comprising six antibodies immobilized on a contact
surface, wherein the antigenic determinants of the antibodies are
analytes associated with renal disorder, wherein the analytes are
selected from the group consisting of alpha-1 microglobulin, beta-2
microglobulin, cystatin C, KIM-1, THP, and TIMP-1.
2. An assay device for diagnosing, monitoring, or determining a
renal disorder in a mammal, the device comprising a panel of
biomarkers for diagnosing, monitoring, or determining a renal
disorder comprising three or more antibodies immobilized on a
contact surface, wherein the antigenic determinants of the
antibodies are analytes associated with renal disorder, wherein the
analytes are selected from the group consisting of alpha-1
microglobulin, beta-2 microglobulin, calbindin, clusterin, CTGF,
creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL,
osteopontin, THP, TIMP-1, TFF-3, VEGF, BLC, CD40, IGF BP2, MMP3,
peptide YY, stem cell factor, TNF RII, AXL, Eotaxin 3, FABP, FGF
basic, myoglobin, resistin, TRAIL R3, endothelin 1, NrCAM, Tenascin
C, VCAM1, and cortisol.
3. The assay device of claim 2, wherein the three or more
antibodies have antigenic determinants for analytes selected from
the group consisting of alpha-1 microglobulin, beta-2
microglobulin, cystatin C, KIM-1, THP, and TIMP-1.
4. The assay device of claim 2, wherein the renal disorder
comprises obstructive uropathy, and wherein the three or more
antibodies have antigenic determinants for analytes selected from
the group consisting of creatinine, THP, A1M, clusterin, NGAL, and
osteopontin.
5. The assay device of claim 2, wherein the renal disorder
comprises obstructive uropathy, wherein the panel of biomarkers has
six antibodies having antigenic determinants for analytes selected
from the group consisting of creatinine, THP, alpha-1
microglobulin, clusterin, NGAL, and osteopontin.
6. The assay device of claim 2, wherein the renal disorder
comprises glomerulonephritis, and wherein the three or more
antibodies have antigenic determinants for analytes selected from
the group consisting of creatinine, KIM-1, TIMP-1, alpha-1
microglobulin, THP, and osteopontin.
7. The assay device of claim 2, wherein the renal disorder
comprises glomerulonephritis, and wherein the panel of biomarkers
has six antibodies having antigenic determinants for analytes
selected from the group consisting of creatinine, KIM-1, TIMP-1,
alpha-1 microglobulin, THP, and osteopontin.
8. The assay device of claim 2, wherein the renal disorder
comprises kidney toxicity, and wherein the three or more antibodies
have antigenic determinants for analytes selected from the group
consisting of creatinine, KIM-1, THP, osteopontin, NGAL, and
TIMP-1.
9. The assay device of claim 2, wherein the renal disorder
comprises kidney toxicity, and wherein the panel of biomarkers has
six antibodies having antigenic determinants for analytes selected
from the group consisting of creatinine, KIM-1, THP, osteopontin,
NGAL, and TIMP-1.
10. The assay device of claim 2, wherein the renal disorder
comprises diabetic nephropathy, and wherein the three or more
antibodies have antigenic determinants for analytes selected from
the group consisting of microalbumin, alpha-1 microglobulin, NGAL,
KIM-1, THP, and clusterin.
11. The assay device of claim 2, wherein the renal disorder
comprises diabetic nephropathy, and wherein the panel of biomarkers
has six antibodies having antigenic determinants for analytes
selected from the group consisting of microalbumin, alpha-1
microglobulin, NGAL, KIM-1, THP, and clusterin.
12. The assay device of claim 2, wherein the renal disorder
comprises kidney transplant rejection and chronic allograft
nephropathy, and wherein the panel comprises three or more
antibodies having antigenic determinants for analytes selected from
the group consisting of BLC, CD40, IGF BP2, MMP3, peptide YY, stem
cell factor, TNF RII, AXL, Eotaxin 3, FABP, FGF basic, myoglobin,
resistin, TRAIL R3, endothelin 1, NrCAM, Tenascin C, VCAM1, and
cortisol.
13. The assay device of claim 2, wherein the panel of biomarkers
comprises ten or more antibodies having antigenic determinants for
analytes selected from the group consisting of alpha-1
microglobulin, beta-2 microglobulin, calbindin, clusterin, CTGF,
creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL,
osteopontin, THP, TIMP-1, TFF-3, and VEGF.
14. The assay device of claim 2, wherein the panel of biomarkers
has sixteen antibodies having antigenic determinants for the
analytes comprising alpha-1 microglobulin, beta-2 microglobulin,
calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha,
KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, and
VEGF.
15. The assay device of claim 2, wherein the contact surface
comprises a substrate capable of immobilizing analytes captured by
the antibodies.
16. The assay device of claim 15, wherein the substrate comprises a
porous material selected from the group consisting of paper,
nitrocellulose, cellulose, glass, glass fiber mesh, silica gel,
synthetic resins, plastic strips, beads, the inner surface of a
well, the surface of a microtitration tray, and combinations
thereof.
17. The assay device of claim 2, further comprising a plurality of
indicators, wherein one of the plurality of indicators is attached
to one of the three or more antibodies
18. The assay device of claim 2, wherein the plurality of
indicators comprises visual indicators and electrochemical
indicators.
19. The assay device of claim 18, wherein the visual indicators are
selected from the group consisting of nanoparticulate gold,
polyurethane microspheres loaded with dye compounds, latex
microspheres loaded with dye compounds, carbon black, fluorophores,
phycoerythrin, radioactive isotopes, nanoparticles, and enzymes
such as horseradish peroxidase or alkaline phosphatase that react
with a chemical substrate to form a colored product.
20. The assay device of claim 18, wherein the electrochemical
indicators are selected from the group consisting of ascorbate,
vitamin E, glutathione, polyphenols, catechols, quercetin,
phytoestrogens, penicillin, carbazole, murranes, phenols,
carbonyls, benzoates, and trace metal ions such as nickel, copper,
cadmium, iron, and mercury.
21. The assay device of claim 2, wherein the assay method comprises
electrophoresis, mass spectrometry, protein microarrays, western
blot, immunohistochemical staining, enzyme-linked immunosorbent
assay methods, and particle-based capture-sandwich
immunoassays.
22. The assay device of claim 2, wherein the renal disorder
comprises glomerulonephritis, interstitial nephritis, tubular
damage, vasculitis, glomerulosclerosis, acute renal failure,
chronic renal failure, nephrosis, nephropathy, polycystic kidney
disease, Bright's disease, renal transplant, chronic unilateral
obstructive uropathy, chronic bilateral obstructive uropathy, acute
unilateral obstructive uropathy, and acute bilateral obstructive
uropathy.
23. The assay device of claim 2, wherein the renal disorder
comprises renal damage caused by exposure to secondary agents and
conditions including therapeutic drugs, recreational drugs,
contrast agents, toxins, nephrolithiasis, ischemia, liver
transplantation, heart transplantation, lung transplantation, and
hypovolemia.
24. The assay device of claim 2, wherein the renal disorder
comprises renal damage secondary to a primary disease state
including diabetes, hypertension, autoimmune diseases including
lupus, Wegener's granulomatosis, and Goodpasture syndrome, primary
hyperoxaluria, kidney transplant rejection, sepsis, nephritis
secondary to infection of the kidney, rhabdomyolysis, multiple
myeloma, and prostate diseases.
25. The assay device of claim 2, wherein the mammal is selected
from the group consisting of humans, apes, monkeys, rats, mice,
dogs, cats, pigs, and livestock including cattle and oxen.
26. An assay device for diagnosing, monitoring, or determining a
renal disorder in a mammal, the device comprising: a. three or more
capture antibodies, wherein the antigenic determinants of the
capture antibodies are analytes associated with a renal disorder,
wherein the analytes are selected from the group consisting of
alpha-1 microglobulin, beta-2 microglobulin, calbindin, clusterin,
CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL,
osteopontin, THP, TIMP-1, TFF-3, VEGF, BLC, CD40, IGF BP2, MMP3,
peptide YY, stem cell factor, TNF RII, AXL, Eotaxin 3, FABP, FGF
basic, myoglobin, resistin, TRAIL R3, endothelin 1, NrCAM, Tenascin
C, VCAM1, and cortisol; b. three or more capture agents comprising
an antigenic moiety, wherein one of the capture agents is attached
to each of the capture antibodies; c. three or more detection
antibodies, wherein the antigenic determinant of the detection
antibodies is the antigenic moiety; and d. three or more
indicators, wherein each of the indicators is attached to one of
the detection antibodies.
27. The assay device of claim 26, wherein the three or more capture
antibodies have antigenic determinants for the analytes selected
from the group consisting of alpha-1 microglobulin, beta-2
microglobulin, cystatin C, KIM-1, THP, and TIMP-1.
28. The assay device of claim 26, wherein the panel of biomarkers
comprises six or more antibodies having antigenic determinants for
the analytes comprising alpha-1 microglobulin, beta-2
microglobulin, cystatin C, KIM-1, THP, and TIMP-1.
29. The assay device of claim 26, wherein the panel of biomarkers
comprises ten or more antibodies having antigenic determinants for
the analytes comprising alpha-1 microglobulin, beta-2
microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C,
GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1,
TFF-3, and VEGF.
30. The assay device of claim 26, wherein the panel of biomarkers
comprises sixteen or more antibodies having antigenic determinants
for the analytes comprising alpha-1 microglobulin, beta-2
microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C,
GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1,
TFF-3, and VEGF.
31. A kit for diagnosing, monitoring, or determining a renal
disorder in a mammal, the kit comprising: a. the assay device of
claim 2; and b. a collection apparatus suitable for collecting a
sample of bodily fluid from the mammal.
32. The kit of claim 31, wherein the collection apparatus comprises
urine sample cups, urethral catheters, swabs, hypodermic needles,
thin needles, hollow needles, metabolic cages, and aspiration
needles.
33. A kit for diagnosing, monitoring, or determining a renal
disorder in a mammal, the kit comprising: a. the assay device of
claim 26; and b. a collection apparatus suitable for collecting a
sample of bodily fluid from the mammal.
34. The kit of claim 33, wherein the collection apparatus comprises
urine sample cups, urethral catheters, swabs, hypodermic needles,
thin needles, hollow needles, metabolic cages, and aspiration
needles.
35. An assay device for diagnosing, monitoring, or determining a
renal disorder in a mammal, the device comprising a panel of
biomarkers having sixteen antibodies immobilized on a contact
surface, wherein the antigenic determinants of the antibodies are
analytes associated with renal disorder, wherein the analytes are
selected from the group consisting of alpha-1-microglobulin,
beta-2-microglobulin, calbindin, clusterin, CTGF, creatinine,
cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP,
TIMP-1, TFF-3, and VEGF.
36. A platform for diagnosing, monitoring, or determining a renal
disorder in a mammal, the platform comprising at least 6 antibodies
selected from the group consisting of alpha-1-microglobulin,
beta-2-microglobulin, calbindin, clusterin, CTGF, creatinine,
cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP,
TIMP-1, TFF-3, and VEGF.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the priority of U.S. provisional
application Ser. No. 61/327,389, filed Apr. 23, 2010, and U.S.
provisional application Ser. No. 61/232,091, filed Aug. 7, 2009,
each of which is hereby incorporated by reference in its entirety,
and is related to U.S. patent application Ser. Nos. [Not Yet
Assigned], entitled Methods and Devices for Detecting Obstructive
Uropathy and Associated Disorders, Computer Methods and Devices for
Detecting Kidney Damage, Methods and Devices for Detecting Kidney
Damage, Methods and Devices for Detecting Kidney Transplant
Rejection, Methods and Devices for Detecting Diabetic Nephropathy
and Associated Disorders, and Methods and Devices for Detecting
Glomerulonephritis and Associated Disorders, Attorney Docket Nos.
060075-, filed on the same date as this application, the entire
contents of which are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The invention encompasses devices for diagnosing,
monitoring, or determining a renal disorder in a mammal. In
particular, the present invention provides methods and devices for
diagnosing, monitoring, or determining renal disorders in a mammal
using measured concentrations of a combination of three or more
analytes in a test sample taken from the mammal.
BACKGROUND OF THE INVENTION
[0003] The urinary system, in particular the kidneys, perform
several critical functions such as maintaining electrolyte balance
and eliminating toxins from the bloodstream. In the human body, the
pair of kidneys together process roughly 20% of the total cardiac
output, amounting to about 1 L/min in a 70-kg adult male. Because
compounds in circulation are concentrated in the kidney up to
1000-fold relative to the plasma concentration, the kidney is
especially vulnerable to injury due to exposure to toxic
compounds.
[0004] Renal disorders and disease are serious conditions that
generally affect the function of the kidney. The disorders
discussed herein may arise from a variety of causes, including
intrinsic disease processes, such as inflammation and necrosis of
the kidney. In addition, renal disorders may also arise from
secondary sources including drugs that are toxic to the kidneys and
alternative disease states that cause secondary adverse effects on
the kidney, such as diabetes and hypertension. Prevention of renal
disorders is largely dependent on early diagnosis of the condition.
Existing diagnostic tests such as BUN and serum creatine tests
typically detect only advanced stages of kidney damage. Other
diagnostic tests such as kidney tissue biopsies or CAT scans have
the advantage of enhanced sensitivity to earlier stages of kidney
damage, but these tests are also generally costly, slow, and/or
invasive.
[0005] A need exists in the art for a fast, simple, reliable, and
sensitive method of detecting obstructive uropathy or an associated
disorder. In a clinical setting, the early detection of kidney
damage would help medical practitioners to diagnose and treat
kidney damage more quickly and effectively.
SUMMARY OF THE INVENTION
[0006] The present invention provides methods and devices for
diagnosing, monitoring, or determining a renal disorder in a
mammal. In particular, the present invention provides methods and
devices for diagnosing, monitoring, or determining a renal disorder
using measured concentrations of a combination of three or more
analytes in a test sample taken from the mammal.
[0007] In one aspect, the present invention encompasses an assay
device for diagnosing, monitoring, or determining a renal disorder
in a mammal, the device comprising a panel of biomarkers for
diagnosing, monitoring, or determining a renal disorder comprising
six antibodies immobilized on a contact surface, wherein the
antigenic determinants of the antibodies are analytes associated
with renal disorder, wherein the analytes are selected from the
group consisting of alpha-1 microglobulin, beta-2 microglobulin,
cystatin C, KIM-1, THP, and TIMP-1.
[0008] In another aspect, the invention encompasses an assay device
for diagnosing, monitoring, or determining a renal disorder in a
mammal, the device comprising a panel of biomarkers for diagnosing,
monitoring, or determining a renal disorder comprising three or
more antibodies immobilized on the contact surface, wherein the
antigenic determinants of the antibodies are analytes associated
with renal disorder, wherein the analytes are selected from the
group consisting of alpha-1 microglobulin, beta-2 microglobulin,
calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha,
KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, VEGF,
BLC, CD40, IGF BP2, MMP3, peptide YY, stem cell factor, TNF RII,
AXL, Eotaxin 3, FABP, FGF basic, myoglobin, resistin, TRAIL R3,
endothelin 1, NrCAM, Tenascin C, VCAM1, and cortisol. It is also
recognized that the assay device may include combinations of 6, 10,
or 16 antibodies with antigenic determinants corresponding to the
analytes disclosed herein.
[0009] In another aspect, the invention encompasses an assay device
for diagnosing, monitoring, or determining a renal disorder in a
mammal, the device comprising: (a) three or more capture
antibodies, wherein the antigenic determinants of the capture
antibodies are analytes associated with a renal disorder, wherein
the analytes are selected from the group consisting of alpha-1
microglobulin, beta-2 microglobulin, calbindin, clusterin, CTGF,
creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL,
osteopontin, THP, TIMP-1, TFF-3, VEGF, BLC, CD40, IGF BP2, MMP3,
peptide YY, stem cell factor, TNF RII, AXL, Eotaxin 3, FABP, FGF
basic, myoglobin, resistin, TRAIL R3, endothelin 1, NrCAM, Tenascin
C, VCAM1, and cortisol; (b) three or more capture agents comprising
an antigenic moiety, wherein one of the capture agents is attached
to each of the capture antibodies; (c) three or more detection
antibodies, wherein the antigenic determinant of the detection
antibodies is the antigenic moiety; and (d) three or more
indicators, wherein each of the indicators is attached to one of
the detection antibodies.
[0010] In a further aspect, the invention encompasses a kit for
diagnosing, monitoring, or determining a renal disorder in a
mammal, where the kit includes: (a) an assay device having a panel
of biomarkers for diagnosing, monitoring, or determining a renal
disorder comprising three or more antibodies immobilized on the
contact surface, wherein the antigenic determinants of the
antibodies are analytes associated with renal disorder, wherein the
analytes are selected from the group consisting of alpha-1
microglobulin, beta-2 microglobulin, calbindin, clusterin, CTGF,
creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL,
osteopontin, THP, TIMP-1, TFF-3, and VEGF; and (b) a collection
apparatus suitable for collecting a sample of bodily fluid from the
mammal.
[0011] In yet another aspect, the invention encompasses a kit for
diagnosing, monitoring, or determining a renal disorder in a
mammal, where the kit includes: (a) an assay device having (i)
three or more capture antibodies, wherein the antigenic
determinants of the capture antibodies are analytes associated with
a renal disorder, wherein the analytes are selected from the group
consisting of alpha-1 microglobulin, beta-2 microglobulin,
calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha,
KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, and
VEGF; (ii) three or more capture agents comprising an antigenic
moiety, wherein one of the capture agents is attached to each of
the capture antibodies; (iii) three or more detection antibodies,
wherein the antigenic determinant of the detection antibodies is
the antigenic moiety; and (iv) three or more indicators, wherein
each of the indicators is attached to one of the detection
antibodies; and (b) a collection apparatus suitable for collecting
a sample bodily fluid from the mammal.
[0012] In still another aspect, the invention encompasses an assay
device for diagnosing, monitoring, or determining a renal disorder
in a mammal, the device comprising a panel of biomarkers having
sixteen antibodies immobilized on a contact surface, wherein the
antigenic determinants of the antibodies are analytes associated
with renal disorder, wherein the analytes are selected from the
group consisting of alpha-1-microglobulin, beta-2-microglobulin,
calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha,
KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, and
VEGF.
[0013] In a further aspect, the invention encompasses a platform
for diagnosing, monitoring, or determining a renal disorder in a
mammal, the platform comprising at least 6 antibodies selected from
the group consisting of alpha-1-microglobulin,
beta-2-microglobulin, calbindin, clusterin, CTGF, creatinine,
cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP,
TIMP-1, TFF-3, and VEGF.
[0014] Other aspects and iterations of the invention are described
in more detail below.
DESCRIPTION OF FIGURES
[0015] FIG. 1 depicts four graphs comparing (A) the concentrations
of alpha-1 microglobulin in the urine of normal controls, kidney
cancer patients, and patients with other cancer types; (B) the
concentrations of beta-2 microglobulin in the urine of normal
controls, kidney cancer patients, and patients with other cancer
types; (C) the concentrations of NGAL in the urine of normal
controls, kidney cancer patients, and patients with other cancer
types; and (D) the concentrations of THP in the urine of normal
controls, kidney cancer patients, and patients with other cancer
types.
[0016] FIG. 2 shows the four different disease groups from which
samples were analyzed, and a plot of two different estimations on
eGFR outlining the distribution within each group.
[0017] FIG. 3 is a number of scatter plots of results on selected
proteins in urine and plasma. The various groups are indicated as
follows--control: blue, AA: red, DN: green, GN: yellow, OU: orange.
(A) A1M in plasma, (B) cystatin C in plasma, (C) B2M in urine, (D)
cystatin C in urine.
[0018] FIG. 4 depicts the multivariate analysis of the disease
groups and their respective matched controls using plasma results.
Relative importance shown using the random forest model.
[0019] FIG. 5 depicts three graphs showing the mean AUROC and its
standard deviation (A) for plasma samples, and mean error rates (B)
and mean AUROC (C) from urine samples for each classification
method used to distinguish disease samples vs. normal samples.
Disease encompasses analgesic abuse (AA), glomerulonephritis (GN),
obstructive uropathy (OU), and diabetic nephropathy (DN).
Normal=NL.
[0020] FIG. 6 depicts three graphs showing the average importance
of analytes and clinical variables from 100 bootstrap runs measured
by random forest (A and B) or boosting (C) to distinguish disease
(AA+GN+ON+DN) samples vs. normal samples from plasma (A) and urine
(B and C).
[0021] FIG. 7 depicts three graphs showing the mean AUROC and its
standard deviation (A) for plasma samples, and mean error rates (B)
and mean AUROC (C) from urine samples for each classification
method used to distinguish analgesic abuse samples vs. normal
samples. Abbreviations as in FIG. 4.
[0022] FIG. 8 depicts three graphs showing the average importance
of analytes and clinical variables from 100 bootstrap runs measured
by random forest (A and B) or boosting (C) to distinguish analgesic
abuse samples vs. normal samples from plasma (A) and urine (B and
C).
[0023] FIG. 9 depicts three graphs showing the mean AUROC and its
standard deviation (A) for plasma samples, and mean error rates (B)
and mean AUROC (C) from urine samples for each classification
method used to distinguish analgesic abuse samples vs. diabetic
nephropathy samples. Abbreviations as in FIG. 4.
[0024] FIG. 10 depicts three graphs showing the average importance
of analytes and clinical variables from 100 bootstrap runs measured
by random forest (A and B) or boosting (C) to distinguish analgesic
abuse samples vs. diabetic nephropathy samples from plasma (A) and
urine (B and C).
[0025] FIG. 11 depicts three graphs showing the mean AUROC and its
standard deviation (A) for plasma samples, and mean error rates (B)
and mean AUROC (C) from urine samples for each classification
method used to distinguish glomerulonephritis samples vs. analgesic
abuse samples. Abbreviations as in FIG. 4.
[0026] FIG. 12 depicts three graphs showing the average importance
of analytes and clinical variables from 100 bootstrap runs measured
by random forest (A and B) or boosting (C) to distinguish
glomerulonephritis samples vs. analgesic abuse samples from plasma
(A) and urine (B and C).
[0027] FIG. 13 depicts three graphs showing the mean AUROC and its
standard deviation (A) for plasma samples, and mean error rates (B)
and mean AUROC (C) from urine samples for each classification
method used to distinguish obstructive uropathy samples vs.
analgesic abuse samples. Abbreviations as in FIG. 4.
[0028] FIG. 14 depicts three graphs showing the average importance
of analytes and clinical variables from 100 bootstrap runs measured
by random forest (A and B) or boosting (C) to distinguish
obstructive uropathy samples vs. analgesic abuse samples from
plasma (A) and urine (B and C).
DETAILED DESCRIPTION OF THE INVENTION
[0029] It has been discovered that a multiplexed panel of three or
more, six or more, and preferably sixteen, biomarkers may be used
to detect renal disorders. As used herein, the term "renal
disorder" includes, but is not limited to glomerulonephritis,
interstitial nephritis, tubular damage, vasculitis,
glomerulosclerosis, diabetic nephropathy, analgesic nephropathy,
and acute tubular necrosis. As used herein, the term
"glomerulonephritis" refers to a disorder characterized by
inflammation of the glomeruli. The term may encompass chronic
glomerulonephritis, acute glomerulonephritis, primary
glomerulonephritis, or secondary glomerulonephritis. As used
herein, the term "diabetic nephropathy" refers to a disorder
characterized by angiopathy of capillaries in the kidney glomeruli.
The term encompasses Kimmelstiel-Wilson syndrome, or nodular
diabetic glomerulosclerosis and intercapillary glomerulonephritis.
Additionally, the present invention encompasses biomarkers that may
be used to detect a disorder associated with diabetic nephropathy.
As used herein, the phrase "a disorder associated with diabetic
nephropathy" refers to a disorder that stems from angiopathy of
capillaries in the kidney glomeruli. For instance, non-limiting
examples of associated disorders may include nephritic syndrome,
chronic kidney failure, and end-stage kidney disease. The devices
of the present invention may also be used to detect secondary
kidney damage or toxicity caused by exposure to a toxic compound
including but not limited to therapeutic drugs, recreational drugs,
medical imaging contrast agents, and toxins. Non-limiting examples
of therapeutic drugs may include an analgesic (e.g. aspirin,
acetaminophen, ibuprofen, naproxen sodium), an antibiotic (e.g. an
aminoglycoside, a beta lactam (cephalosporins, penicillins,
penems), rifampin, vancomycin, a sulfonamide, a fluoroquinolone,
and a tetracycline), or a chemotherapy agent (e.g. Cisplatin
(Platinol.RTM.), Carboplatin (Paraplatin.RTM.), Cytarabine
(Cytosar-U.RTM.), Gemtuzumab ozogamicin (Mylotarg.RTM.),
Gemcitabine (Gemzar.RTM.), Melphalan (Alkeran.RTM.), Ifosfamide
(Ifex.RTM.), Methotrexate (Rheumatrex.RTM.), Interleukin-2
(Proleukin.RTM.), Oxaliplatin (Eloxatin.RTM.), Streptozocin
(Zanosar.RTM.), Pemetrexed (Alimta.RTM.), Plicamycin
(Mithracin.RTM.), and Trimetrexate (Neutrexin.RTM.). Further, the
term renal disorder may include kidney damage due to kidney stones,
ischemia, liver transplantation, heart transplantation, lung
transplantation, or hypovolemia. Moreover, the devices of the
current invention may be used to detect renal disorders including
kidney damage cause by other disease states including but not
limited to diabetes, hypertension, autoimmune diseases including
lupus, Wegener's granulomatosis, Goodpasture syndrome, primary
hyperoxaluria, kidney transplant rejection, sepsis, nephritis
secondary to any infection of the kidney, rhabdomyolysis, multiple
myeloma, and prostate disease.
[0030] In addition, the devices and systems of the current
invention may be used to detect renal disorders including acute
kidney transplant rejection or chronic allograft nephropathy.
Importantly, the devices of the invention may be used to
distinguish between an acute rejection reaction and a chronic
allograft nephropathy. Alternatively, the devices of the present
invention may be used to distinguish between a successful
transplant and rejection. As used herein, the term "rejection"
refers to a recipient response to a foreign antigen derived from
the transplanted kidney. The phrase "acute rejection" refers to an
immune related response to the foreign kidney. The response is
primarily T-cell driven and originates from an HLC mismatch between
the donor and recipient. The phrase "chronic allograft nephropathy"
refers to a chronic inflammatory and immune response mediated
reaction to a foreign kidney. Chronic allograft nephropathy may
result in damage to the kidney manifested by diffuse interstitial
fibrosis glomerular changes, typically membranous and sclerotic in
nature, as well as intimal fibrosis of the blood vessels with
tubular atrophy and loss of tubular structures.
[0031] Additionally, the present invention encompasses devices
comprising biomarkers that may be used to detect a renal disorder
associated with kidney transplant rejection. As used herein, the
phrase "a disorder associated with kidney transplant rejection"
refers to a disorder that stems from a host response to a foreign
antigen derived from the transplated kidney. For instance,
non-limiting examples of associated disorders may include chronic
kidney failure and end-stage kidney disease.
[0032] The devices of the present invention may also be utilized to
detect a renal disorder including obstructive uropathy or an
associated disorder in a mammal that includes determining the
presence or concentration of a combination of three or more sample
analytes in a test sample containing the bodily fluid of the
mammal. As used herein, the term "obstructive uropathy" refers to a
structural or functional hindrance of normal urine flow. The term
may encompass chronic unilateral obstructive uropathy, chronic
bilateral obstructive uropathy, acute unilateral obstructive
uropathy, or acute bilateral obstructive uropathy. Additionally,
the present invention encompasses biomarkers that may be used to
detect a disorder associated with obstructive uropathy. As used
herein, the phrase "a disorder associated with obstructive
uropathy" refers to a disorder that stems from a structural or
functional hindrance of normal urine flow. For instance,
non-limiting examples of associated disorders may include
hydronephrosis and obstructive nephropathy. The measured
concentrations of the combination of sample analytes is compared to
the entries of a dataset in which each entry contains the minimum
diagnostic concentrations of a combination of three of more
analytes reflective of obstructive uropathy or an associated
disorder. Other embodiments provide computer-readable media encoded
with applications containing executable modules, systems that
include databases and processing devices containing executable
modules configured to diagnose, monitor, or determine a renal
disorder in a mammal. Still other embodiments provide
antibody-based devices for diagnosing, monitoring, or determining
obstructive uropathy or an associated disorder in a mammal.
[0033] The biomarkers included in a multiplexed panel of the
invention are analytes known in the art that may be detected in the
urine, serum, plasma and other bodily fluids of mammals. As such,
the analytes of the multiplexed panel may be readily extracted from
the mammal in a test sample of bodily fluid. The concentrations of
the analytes within the test sample may be measured using known
analytical techniques such as a multiplexed antibody-based
immunological assay. The combination of concentrations of the
analytes in the test sample may be compared to empirically
determined combinations of minimum diagnostic concentrations and
combinations of diagnostic concentration ranges associated with
healthy kidney function to determine whether a renal disorder is
indicated in the mammal.
[0034] The analytes used as biomarkers in the multiplexed assay,
methods of diagnosing, monitoring, or determining a renal disorder
using measurements of the analytes, systems and applications used
to analyze the multiplexed assay measurements, and antibody-based
devices used to measure the analytes are described in detail
below.
I. Analytes in Multiplexed Assay
[0035] One embodiment of the invention measures the concentrations
of three or more, six or more, ten or more, and preferably sixteen,
biomarker analytes within a test sample taken from a mammal and
compares the measured analyte concentrations to minimum diagnostic
concentrations to diagnose, monitor, or determine obstructive
uropathy or an associated renal disorder in a mammal. In this
aspect, the biomarker analytes are known in the art to occur in the
urine, plasma, serum and other bodily fluids of mammals. The
biomarker analytes are proteins that have known and documented
associations with early renal damage in humans. As defined herein,
the biomarker analytes include but are not limited to alpha-1
microglobulin, beta-2 microglobulin, calbindin, clusterin, CTGF,
creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL,
osteopontin, THP, TIMP-1, TFF-3, and VEGF. A description of each
biomarker analyte is given below. In one embodiment, the biomarker
analytes include alpha-1-microglobulin, beta-2-microglobulin,
cystatin-C, KIM-1, THP, and TIMP-1.
(a) Alpha-1 Microglobulin (A1M)
[0036] Alpha-1 microglobulin (A1M, Swiss-Prot Accession Number
P02760) is a 26 kDa glycoprotein synthesized by the liver and
reabsorbed in the proximal tubules. Elevated levels of A1M in human
urine are indicative of glomerulotubular dysfunction. A1M is a
member of the lipocalin super family and is found in all tissues.
Alpha-1-microglobulin exists in blood in both a free form and
complexed with immunoglobulin A (IgA) and heme. Half of plasma A1M
exists in a free form, and the remainder exists in complexes with
other molecules including prothrombin, albumin, immunoglobulin A
and heme. Nearly all of the free A1M in human urine is reabsorbed
by the megalin receptor in proximal tubular cells, where it is then
catabolized. Small amounts of A1M are excreted in the urine of
healthy humans. Increased A1M concentrations in human urine may be
an early indicator of renal damage, primarily in the proximal
tubule.
(b) Beta-2 Microglobulin (B2M)
[0037] Beta-2 microglobulin (B2M, Swiss-Prot Accession Number
P61769) is a protein found on the surfaces of all nucleated cells
and is shed into the blood, particularly by tumor cells and
lymphocytes. Due to its small size, B2M passes through the
glomerular membrane, but normally less than 1% is excreted due to
reabsorption of B2M in the proximal tubules of the kidney.
Therefore, high plasma levels of B2M occur as a result of renal
failure, inflammation, and neoplasms, especially those associated
with B-lymphocytes.
(c) Calbindin
[0038] Calbindin (Calbindin D-28K, Swiss-Prot Accession Number
P05937) is a Ca-binding protein belonging to the troponin C
superfamily. It is expressed in the kidney, pancreatic islets, and
brain. Calbindin is found predominantly in subpopulations of
central and peripheral nervous system neurons, in certain
epithelial cells involved in Ca2+ transport such as distal tubular
cells and cortical collecting tubules of the kidney, and in enteric
neuroendocrine cells.
(d) Clusterin
[0039] Clusterin (Swiss-Prot Accession Number P10909) is a highly
conserved protein that has been identified independently by many
different laboratories and named SGP2, S35-S45, apolipoprotein J,
SP-40, 40, ADHC-9, gp80, GPIII, and testosterone-repressed prostate
message (TRPM-2). An increase in clusterin levels has been
consistently detected in apoptotic heart, brain, lung, liver,
kidney, pancreas, and retinal tissue both in vivo and in vitro,
establishing clusterin as a ubiquitous marker of apoptotic cell
loss. However, clusterin protein has also been implicated in
physiological processes that do not involve apoptosis, including
the control of complement-mediated cell lysis, transport of
beta-amyloid precursor protein, shuttling of aberrant beta-amyloid
across the blood-brain barrier, lipid scavenging, membrane
remodeling, cell aggregation, and protection from immune detection
and tumor necrosis factor induced cell death.
(e) Connective Tissue Growth Factor (CTGF)
[0040] Connective tissue growth factor (CTGF, Swiss-Prot Accession
Number P29279) is a 349-amino acid cysteine-rich polypeptide
belonging to the CCN family. In vitro studies have shown that CTGF
is mainly involved in extracellular matrix synthesis and fibrosis.
Up-regulation of CTGF mRNA and increased CTGF levels have been
observed in various diseases, including diabetic nephropathy and
cardiomyopathy, fibrotic skin disorders, systemic sclerosis,
biliary atresia, liver fibrosis and idiopathic pulmonary fibrosis,
and nondiabetic acute and progressive glomerular and
tubulointerstitial lesions of the kidney. A recent cross-sectional
study found that urinary CTGF may act as a progression promoter in
diabetic nephropathy.
(f) Creatinine
[0041] Creatinine is a metabolite of creatine phosphate in muscle
tissue, and is typically produced at a relatively constant rate by
the body. Creatinine is chiefly filtered out of the blood by the
kidneys, though a small amount is actively secreted by the kidneys
into the urine. Creatinine levels in blood and urine may be used to
estimate the creatinine clearance, which is representative of the
overall glomerular filtration rate (GFR), a standard measure of
renal function. Variations in creatinine concentrations in the
blood and urine, as well as variations in the ratio of urea to
creatinine concentration in the blood, are common diagnostic
measurements used to assess renal function.
(g) Cystatin C (Cyst C)
[0042] Cystatin C (Cyst C, Swiss-Prot Accession Number P01034) is a
13 kDa protein that is a potent inhibitor of the C1 family of
cysteine proteases. It is the most abundant extracellular inhibitor
of cysteine proteases in testis, epididymis, prostate, seminal
vesicles and many other tissues. Cystatin C, which is normally
expressed in vascular wall smooth muscle cells, is severely reduced
in both atherosclerotic and aneurismal aortic lesions.
(h) Epidermal Growth Factor (EGF)
[0043] Epidermal growth factor (EGF, Swiss-Prot Accession Number
P07522) is a small protein that functions as a potent mitogen. EGF
promotes cell growth and differentiation, is essential in
embryogenesis, and is important in wound healing. It is produced by
many normal cell types and is made in large amounts by certain
types of tumors.
(i) Glutathione S-Transferase alpha (GST-alpha)
[0044] Glutathione S-transferase alpha (GST-alpha, Swiss-Prot
Accession Number P08263) belongs to a family of enzymes that
utilize glutathione in reactions contributing to the transformation
of a wide range of compounds, including carcinogens, therapeutic
drugs, and products of oxidative stress. These enzymes play a key
role in the detoxification of such substances.
(j) Glutathione S-Transferase mu (GST-mu)
[0045] Glutathione S-transferase mu (GST-mu, Swiss-Prot Accession
Number PO4905) functions in the detoxification of electrophilic
compounds, including carcinogens, therapeutic drugs, environmental
toxins and products of oxidative stress, by conjugation with
glutathione. The genes encoding the mu class of enzymes are
organized in a gene cluster on chromosome 1 p13.3 and are known to
be highly polymorphic. Genetic variations in GST-mu can change a
mammal's susceptibility to carcinogens and toxins as well as affect
the toxicity and efficacy of certain drugs. Null mutations of this
class mu gene have been linked with an increase in a number of
cancers.
(k) Kidney Injury Molecule-1 (KIM-1)
[0046] Kidney injury molecule-1 (KIM-1, Swiss-Prot Accession Number
Q96D42) is an immunoglobulin superfamily cell-surface protein
highly upregulated on the surface of injured kidney epithelial
cells. It is also known as TIM-1 (T-cell immunoglobulin mucin
domain-1), as it is expressed at low levels by subpopulations of
activated T-cells and hepatitis A virus cellular receptor-1
(HAVCR-1). KIM-1 is increased in expression more than any other
protein in the injured kidney and is localized predominantly to the
apical membrane of the surviving proximal epithelial cells.
(l) Microalbumin
[0047] Albumin is the most abundant plasma protein in humans and
other mammals. Albumin is essential for maintaining the osmotic
pressure needed for proper distribution of body fluids between
intravascular compartments and body tissues. Healthy, normal
kidneys typically filter out albumin from the urine. The presence
of albumin in the urine may indicate damage to the kidneys. Albumin
in the urine may also occur in patients with long-standing
diabetes, especially type 1 diabetes. The amount of albumin
eliminated in the urine has been used to differentially diagnose
various renal disorders. For example, nephrotic syndrome usually
results in the excretion of about 3.0 to 3.5 grams of albumin in
human urine every 24 hours. Microalbuminuria, in which less than
300 mg of albumin is eliminated in the urine every 24 hours, may
indicate the early stages of diabetic nephropathy.
(m) Neutrophil Gelatinase-Associated Lipocalin (NGAL)
[0048] Neutrophil gelatinase-associated lipocalin (NGAL, Swiss-Prot
Accession Number P80188) forms a disulfide bond-linked heterodimer
with MMP-9. It mediates an innate immune response to bacterial
infection by sequestrating iron. Lipocalins interact with many
different molecules such as cell surface receptors and proteases,
and play a role in a variety of processes such as the progression
of cancer and allergic reactions.
(n) Osteopontin (OPN)
[0049] Osteopontin (OPN, Swiss-Prot Accession Number P10451) is a
cytokine involved in enhancing production of interferon-gamma and
IL-12, and inhibiting the production of IL-10. OPN is essential in
the pathway that leads to type I immunity. OPN appears to form an
integral part of the mineralized matrix. OPN is synthesized within
the kidney and has been detected in human urine at levels that may
effectively inhibit calcium oxalate crystallization. Decreased
concentrations of OPN have been documented in urine from patients
with renal stone disease compared with normal individuals.
(o) Tamm-Horsfall Protein (THP)
[0050] Tamm-Horsfall protein (THP, Swiss-Prot Accession Number
P07911), also known as uromodulin, is the most abundant protein
present in the urine of healthy subjects and has been shown to
decrease in individuals with kidney stones. THP is secreted by the
thick ascending limb of the loop of Henley. THP is a monomeric
glycoprotein of .about.85 kDa with .about.30% carbohydrate moiety
that is heavily glycosylated. THP may act as a constitutive
inhibitor of calcium crystallization in renal fluids.
(p) Tissue Inhibitor of Metalloproteinase-1 (TIMP-1)
[0051] Tissue inhibitor of metalloproteinase-1 (TIMP-1, Swiss-Prot
Accession Number P01033) is a major regulator of extracellular
matrix synthesis and degradation. A certain balance of MMPs and
TIMPs is essential for tumor growth and health. Fibrosis results
from an imbalance of fibrogenesis and fibrolysis, highlighting the
importance of the role of the inhibition of matrix degradation role
in renal disease.
(q) Trefoil Factor 3 (TFF3)
[0052] Trefoil factor 3 (TFF3, Swiss-Prot Accession Number Q07654),
also known as intestinal trefoil factor, belongs to a small family
of mucin-associated peptides that include TFF1, TFF2, and TFF3.
TFF3 exists in a 60-amino acid monomeric form and a 118-amino acid
dimeric form. Under normal conditions TFF3 is expressed by goblet
cells of the intestine and the colon. TFF3 expression has also been
observed in the human respiratory tract, in human goblet cells and
in the human salivary gland. In addition, TFF3 has been detected in
the human hypothalamus.
(r) Vascular Endothelial Growth Factor (VEGF)
[0053] Vascular endothelial growth factor (VEGF, Swiss-Prot
Accession Number P15692) is an important factor in the
pathophysiology of neuronal and other tumors, most likely
functioning as a potent promoter of angiogenesis. VEGF may also be
involved in regulating blood-brain-barrier functions under normal
and pathological conditions. VEGF secreted from the stromal cells
may be responsible for the endothelial cell proliferation observed
in capillary hemangioblastomas, which are typically composed of
abundant microvasculature and primitive angiogenic elements
represented by stromal cells.
(s) Vascular Endothelial Growth Factor A (VEGF A)
[0054] Vascular endothelial growth factor A (VEGF A, Swiss-Prot
Accession Number Q00731) is a growth factor active in angiogenesis,
vasculogenesis and endothelial cell growth. It induces endothelial
cell proliferation, promotes cell migration, inhibits apoptosis,
and induces permeabilization of blood vessles. It is important in
the pathophysiology of neuronal and other tumors, likely
functioning as a potent promoter of angiogenesis. Due to its
influences on vascular permeability, VEGF A may be involved in
altering blood-brain-barrier functions under normal and
pathological conditions. The production and secretion of VEGF by
mammalian retinal pigment epithelial cells may be important in the
pathogenesis of ocular neovascularization.
(t) B-lymphocyte Chemoattractant (BLC)
[0055] B-lymphocyte chemoattractant (BLC, Swiss-Prot Accession
Number 043927) is also referred to as C-X-C motif chemokine 13,
Small-inducible cytokine B13, B lymphocyte chemoattractant, CXC
chemokine BLC, and B cell-attracting chemokine 1. BLC functions as
a potent chemoattractant for B lymphocytes, but not T lymphocytes,
monocytes, or neutrophils. Its specific receptor BLR1 is a G
protein-coupled receptor originally isolated from Burkitt's
lymphoma cells. Among cells of the hematopoietic lineages, the
expression of BRL1, now designated CXCR5, is restricted to B
lymphocytes and a subpopulation of T helper memory cells.
(u) Cluster of Differentiation Surface Receptors 40 (CD40)
[0056] Cluster of Differentiation Surface Receptors 40 (CD40, Swiss
Prot Accession Number P25942) is also referred to TNFRSF5 (Tumor
necrosis factor receptor superfamily member 5. CD40 is a member of
the tumor necrosis factor-receptor superfamily of proteins. CD40
has been found to be essential in mediating a broad variety of
immune and inflammatory responses including T cell-dependent
immunoglobulin class switching, memory B cell development, and
germinal center formation.
(v) Insulin-Like Growth Factor Binding Protein 2 (IGF BP2)
[0057] Insulin-like Growth Factor Binding Protein 2 (IGF BP2, Swiss
Prot Accession Number P18065) functions to prolong the half-life of
the insulin growth factors and have been shown to either inhibit or
stimulate the growth promoting effects of the insulin growth
factors on cell culture. Specifically, during development,
insulin-like growth factor binding protein-2 is expressed in a
number of tissues with the highest expression level found in the
central nervous system. IGFBP-2 exhibits a 2-10 fold higher
affinity for IGF II than for IGF I.
(w) Matrix Metalloproteinase-3 (MMP3)
[0058] Matrix Metalloproteinase-3 (MMP3, Swiss Prot Accession
Number P08254) is also known as stromelysin-1 and Transin-1. MMP3
is involved in the breakdown of extracellular matrix in normal
physiological processes, such as embryonic development,
reproduction, and tissue remodeling, as well as in disease
processes, such as arthritis and metastasis. Most MMP's are
secreted as inactive proproteins which are activated when cleaved
by extracellular proteinases. MMP3 encodes an enzyme which degrades
fibronectin, laminin, collagens III, IV, IX, and X, and cartilage
proteoglycans. The enzyme is thought to be involved in wound
repair, progression of atherosclerosis, and tumor initiation. MMP3
is part of a cluster of MMP genes which localize to chromosome
11q22.3.
(x) Peptide YY (PYY)
[0059] Peptide YY (PYY, Swiss-Prot Accession Number P10082) is also
known as peptide tyrosine tyrosine and pancreatic peptide
YY.sub.3-36. Peptide YY exerts its action through neuropeptide Y
receptors, inhibits gastric motility and increases water and
electrolyte absorption in the colon. PYY may also suppress
pancreatic secretion. It is secreted by the neuroendocrine cells in
the ileum and colon in response to a meal, and has been shown to
reduce appetite. PYY works by slowing the gastric emptying; hence,
it increases efficiency of digestion and nutrient absorption after
meal. Research has also indicated that PYY may be useful in
removing aluminum accumulated in the brain.
(y) Stem Cell Factor (SCF)
[0060] Stem Cell Factor (SCF, UniProtKB/TrEMBL Q13528) is also
known as kit-ligand, KL, and steel factor. SCF functions SCF plays
an important role in the hematopoiesis during embryonic
development. Sites where hematopoiesis takes place, such as the
fetal liver and bone marrow, all express SCF. SCF may serve as
guidance cues that direct hematopoietic stem cells (HSCs) to their
stem cell niche (the microenvironment in which a stem cell
resides), and it plays an important role in HSC maintenance.
Non-lethal point mutants on the c-Kit receptor can cause anemia,
decreased fertility, and decreased pigmentation. During
development, the presence of the SCF also plays an important role
in the localization of melanocytes, cells that produce melanin and
control pigmentation. In melanogenisis, melanoblasts migrate from
the neural crest to their appropriate locations in the epidermis.
Melanoblasts express the Kit receptor, and it is believed that SCF
guides these cells to their terminal locations. SCF also regulates
survival and proliferation of fully differentiated melanocytes in
adults. In spermatogenesis, c-Kit is expressed in primordial germ
cells, spermatogonia, and in primordial oocytes. It is also
expressed in the primordial germ cells of females. SCF is expressed
along the pathways that the germ cells use to reach their terminal
destination in the body. It is also expressed in the final
destinations for these cells. Like for melanoblasts, this helps
guide the cells to their appropriate locations in the body
(z) Tumor Necrosis Factor Receptor Type II (TNF RII)
[0061] Tumor Necrosis Factor Receptor Type II (TNF RII, Swiss-Prot
Accession Number P20333) is also known as p75, p80 TNF alpha
receptor, and TNFRSF1B. TNF RII is a protein that in humans is
encoded by the TNFRSF1B gene. The protein encoded by this gene is a
member of the Tumor necrosis factor receptor superfamily, which
also contains TNFRSF1A. The protein encoded by this gene is a
member of the TNF-receptor superfamily. This protein and
TNF-receptor 1 form a heterocomplex that mediates the recruitment
of two anti-apoptotic proteins, c-IAP1 and c-IAP2, which possess E3
ubiquitin ligase activity. The function of IAPs in TNF-receptor
signaling is unknown; however, c-IAP1 is thought to potentiate
TNF-induced apoptosis by the ubiquitination and degradation of
TNF-receptor-associated factor 2, which mediates anti-apoptotic
signals. Knockout studies in mice also suggest a role of this
protein in protecting neurons from apoptosis by stimulating
antioxidative pathways.
(aa) AXL Oncogene
[0062] AXL (Swiss-Prot Accession Number P30530) is also known as
UFO, ARK, and tyrosine-protein kinase receptor UFO. The protein
encoded by AXL is a member of the receptor tyrosine kinase
subfamily. Although it is similar to other receptor tyrosine
kinases, the AXL protein represents a unique structure of the
extracellular region that juxtaposes IgL and FNIII repeats. AXL
transduces signals from the extracellular matrix into the cytoplasm
by binding growth factors like vitamin K-dependent protein
growth-arrest-specific gene 6. It is involved in the stimulation of
cell proliferation. This receptor can also mediate cell aggregation
by homophilic binding. AXL is a chronic myelogenous
leukemia-associated oncogene and also associated with colon cancer
and melanoma.
(bb) Eotaxin 3
[0063] Eotaxin 3 (Swiss-Prot Accession Number P51671) is also known
as C-C motif chemokine 11 (CCL11), small inducible cytokine A11,
and eosinophil chemotactic protein. Eotaxin 3 is a small cytokine
belonging to the CC chemokine family that is also called Eotaxin-3,
Macrophage inflammatory protein 4-alpha (MIP-4-alpha), Thymic
stroma chemokine-1 (TSC-1), and IMAC. It is expressed by several
tissues including heart, lung and ovary, and in endothelial cells
that have been stimulated with the cytokine interleukin 4.[1][2]
CCL26 is chemotactic for eosinophils and basophils and elicits its
effects by binding to the cell surface chemokine receptor CCR3.
(cc) Fatty Acid Binding Protein (FABP)
[0064] Fatty Acid Binding Protein (FABP, Swiss-Prot Accession
Number Q01469) is also known as epidermal-type fatty acid binding
protein, and fatty-acid binding protein 5. This gene encodes the
fatty acid binding protein found in epidermal cells, and was first
identified as being upregulated in psoriasis tissue. Fatty acid
binding proteins are a family of small, highly conserved,
cytoplasmic proteins that bind long-chain fatty acids and other
hydrophobic ligands. It is thought that FABPs roles include fatty
acid uptake, transport, and metabolism.
(dd) Basic Fibroblast Growth Factor (FGF basic)
[0065] Basic Fibroblast Growth Factor (FGF basic, Swiss-Prot
Accession NumberP09038) is also known as heparin-binding growth
factor. In normal tissue, basic fibroblast growth factor is present
in basement membranes and in the subendothelial extracellular
matrix of blood vessels. It stays membrane-bound as long as there
is no signal peptide. It has been hypothesized that, during both
wound healing of normal tissues and tumor development, the action
of heparan sulfate-degrading enzymes activates FGF basic, thus
mediating the formation of new blood vessels. Additionally, FGF
basic is a critical component of human embryonic stem cell culture
medium; the growth factor is necessary for the cells to remain in
an undifferentiated state, although the mechanisms by which it does
this are poorly defined. It has been demonstrated to induce gremlin
expression which in turn is known to inhibit the induction of
differentiation by bone morphogenetic proteins. It is necessary in
mouse-feeder cell dependent culture systems, as well as in feeder
and serum-free culture systems.
(ee) Myoglobin
[0066] Myoglobin (Swiss-Prot Accession Number P02144) is released
from damaged muscle tissue (rhabdomyolysis), which has very high
concentrations of myoglobin. The released myoglobin is filtered by
the kidneys but is toxic to the renal tubular epithelium and so may
cause acute renal failure. Myoglobin is a sensitive marker for
muscle injury, making it a potential marker for heart attack in
patients with chest pain.
(ff) Resistin (RETN)
[0067] Resistin (RETN, UniProtKB/TrEMBL Q76B53) is theorized to
participate in the inflammatory response. Resistin has also been
shown to increase transcriptional events leading to an increased
expression of several pro-inflammatory cytokines including (but not
limited to) interleukin-1 (IL-1), interleukin-6 (IL-6),
interleukin-12 (IL-12), and tumor necrosis factor-.alpha.
(TNF-.alpha.) in an NF-KB-mediated fashion. It has also been
demonstrated that resistin upregulates intracellular adhesion
molecule-1 (ICAM1) vascular cell-adhesion molecule-1 (VCAM1) and
CCL2, all of which are occupied in chemotactic pathways involved in
leukocyte recruitment to sites of infection. Resistin itself can be
upregulated by interleukins and also by microbial antigens such as
lipopolysaccharide, which are recognized by leukocytes. Taken
together, because resistin is reputed to contribute to insulin
resistance, results such as those mentioned suggest that resistin
may be a link in the well-known association between inflammation
and insulin resistance. In fact, recent data have shown positive
correlations between obesity, insulin resistance, and chronic
inflammation which is believed to be directed in part by resistin
signaling.
(gg) Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand
Receptor 3 (TRAIL R3)
[0068] TRAIL R3 (Swiss-Prot Accession Number P83626 (mouse)) is
also known as tumor necrosis factor-related apoptosis-inducing
ligand receptor 3, and tumor necrosis factor receptor mouse
homolog. TRAIL R3 is a decoy receptor for TRAIL, a member of the
tumor necrosis factor family. In several cell types decoy receptors
inhibit TRAIL-induced apoptosis by binding TRAIL and thus
preventing its binding to proapoptotic TRAIL receptors.
(hh) Endothelin 1 (ET1)
[0069] Endothelin 1 (ET1, UniProtKB/TrEMBL Q6FH53) is also known as
EDN1 and EDN1 protein. Endothelin 1 is a protein that constricts
blood vessels and raises blood pressure. It is normally kept in
balance by other mechanisms, but when over-expressed, it
contributes to high blood pressure (hypertension) and heart
disease. Endothelin 1 peptides and receptors are implicated in the
pathogenesis of a number of disease states, including cancer and
heart disease.
(ii) Neuronal Cell Adhesion Molecule (NrCAM)
[0070] Neuronal Cell Adhesion Molecule (NrCAM, UniProtKB/TrEMBL
Q14CA1) encodes a neuronal cell adhesion molecule with multiple
immunoglobulin-like C2-type domains and fibronectin type-III
domains. This ankyrin-binding protein is involved in neuron-neuron
adhesion and promotes directional signaling during axonal cone
growth. This gene is also expressed in non-neural tissues and may
play a general role in cell-cell communication via signaling from
its intracellular domain to the actin cytoskeleton during
directional cell migration. Allelic variants of this gene have been
associated with autism and addiction vulnerability.
(jj) Tenascin C (TN-C)
[0071] Tenascin C (TN-C, UniProt/TrEMBL Q99857) has anti-adhesive
properties, causing cells in tissue culture to become rounded after
it is added to the medium. One mechanism to explain this may come
from its ability to bind to the extracellular matrix glycoprotein
fibronectin and block fibronectin's interactions with specific
syndecans. The expression of tenascin-C in the stroma of certain
tumors is associated with a poor prognosis.
(kk) Vascular Cell Adhesion Molecule 1 (VCAM1)
[0072] Vascular Cell Adhesion Molecule 1 (VCAM1, Swiss-Prot
Accession Number P19320) is also known as vascular cell adhesion
protein 1. VCAM1 mediates the adhesion of lymphocytes, monocytes,
eosinophils, and basophils to vascular endothelium. It also
functions in leukocyte-endothelial cell signal transduction, and it
may play a role in the development of atherosclerosis and
rheumatoid arthritis. Upregulation of VCAM-1 in endothelial cells
by cytokines occurs as a result of increased gene transcription
(e.g., in response to Tumor necrosis factor-alpha (TNF-.alpha.) and
Interleukin-1 (IL-1)) and through stabilization of Messenger RNA
(mRNA) (e.g., Interleukin-4 (IL-4)). The promoter region of the
VCAM-1 gene contains functional tandem NF-.kappa.B (nuclear
factor-kappa B) sites. The sustained expression of VCAM-1 lasts
over 24 hours. Primarily, the VCAM-1 protein is an endothelial
ligand for VLA-4 (Very Late Antigen-4 or .alpha.4.beta.1) of the
.beta.1 subfamily of integrins, and for integrin .alpha.4.beta.7.
VCAM-1 expression has also been observed in other cell types (e.g.,
smooth muscle cells). It has also been shown to interact with EZR
and Moesin. Certain melanoma cells can use VCAM-1 to adhere to the
endothelium, and VCAM-1 may participate in monocyte recruitment to
atherosclerotic sites.
(ll) Cortisol
[0073] Cortisol (Swiss-Prot Accession Number P08185) is also known
as corticosteroid-binding globulin, transcortin, and Serpin A6.
Cortisol is a steroid hormone or glucocorticoid produced by the
adrenal gland. It is released in response to stress, and to a low
level of blood glucocorticoids. Its primary functions are to
increase blood sugar through gluconeogenesis, suppress the immune
system, and aid in fat, protein and carbohydrate metabolism. It
also decreases bone formation. In addition, cortisol can weaken the
activity of the immune system. Cortisol prevents proliferation of
T-cells by rendering the interleukin-2 producer T-cells
unresponsive to interleukin-1 (IL-1), and unable to produce the
T-cell growth factor. Cortisol also has a negative feedback effect
on interleukin-1. IL-1 must be especially useful in combating some
diseases; however, endotoxin bacteria have gained an advantage by
forcing the hypothalamus to increase cortisol levels via forcing
secretion of CRH hormone, thus antagonizing IL-1 in this case. The
suppressor cells are not affected by GRMF, so that the effective
set point for the immune cells may be even higher than the set
point for physiological processes. It reflects leukocyte
redistribution to lymph nodes, bone marrow, and skin.
II. Combinations of Analytes Measured by Multiplexed Assay
[0074] The device for diagnosing, monitoring, or determining a
renal disorder involves determining the presence or concentrations
of a combination of sample analytes in a test sample. The
combinations of sample analytes, as defined herein, are any group
of three or more analytes selected from the biomarker analytes,
including but not limited to alpha-1 microglobulin, beta-2
microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C,
GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1,
TFF-3, VEGF, BLC, CD40, IGF BP2, MMP3, peptide YY, stem cell
factor, TNF RII, AXL, Eotaxin 3, FABP, FGF basic, myoglobin,
resistin, TRAIL R3, endothelin 1, NrCAM, Tenascin C, VCAM1, and
cortisol. In one embodiment, the combination of analytes may be
selected to provide a group of analytes associated with renal
disorder in a mammal.
[0075] In one embodiment, the devices and systems of the current
invention detect the combination of sample analytes, and may
include any three of the biomarker analytes. In other embodiments,
the combination of sample analytes may be any four, any five, any
six, any seven, any eight, any nine, any ten, any eleven, any
twelve, any thirteen, any fourteen, any fifteen, or all sixteen of
the sixteen biomarker analytes. In another embodiment, the
combination of sample analytes may comprise a combination listed in
Table A.
TABLE-US-00001 TABLE A alpha-1 microglobulin beta-2 microglobulin
calbindin alpha-1 microglobulin beta-2 microglobulin clusterin
alpha-1 microglobulin beta-2 microglobulin CTGF alpha-1
microglobulin beta-2 microglobulin creatinine alpha-1 microglobulin
beta-2 microglobulin cystatin C alpha-1 microglobulin beta-2
microglobulin GST-alpha alpha-1 microglobulin beta-2 microglobulin
KIM-1 alpha-1 microglobulin beta-2 microglobulin microalbumin
alpha-1 microglobulin beta-2 microglobulin NGAL alpha-1
microglobulin beta-2 microglobulin osteopontin alpha-1
microglobulin beta-2 microglobulin THP alpha-1 microglobulin beta-2
microglobulin TIMP-1 alpha-1 microglobulin beta-2 microglobulin
TFF-3 alpha-1 microglobulin beta-2 microglobulin VEGF alpha-1
microglobulin calbindin clusterin alpha-1 microglobulin calbindin
CTGF alpha-1 microglobulin calbindin creatinine alpha-1
microglobulin calbindin cystatin C alpha-1 microglobulin calbindin
GST-alpha alpha-1 microglobulin calbindin KIM-1 alpha-1
microglobulin calbindin microalbumin alpha-1 microglobulin
calbindin NGAL alpha-1 microglobulin calbindin osteopontin alpha-1
microglobulin calbindin THP alpha-1 microglobulin calbindin TIMP-1
alpha-1 microglobulin calbindin TFF-3 alpha-1 microglobulin
calbindin VEGF alpha-1 microglobulin clusterin CTGF alpha-1
microglobulin clusterin creatinine alpha-1 microglobulin clusterin
cystatin C alpha-1 microglobulin clusterin GST-alpha alpha-1
microglobulin clusterin KIM-1 alpha-1 microglobulin clusterin
microalbumin alpha-1 microglobulin clusterin NGAL alpha-1
microglobulin clusterin osteopontin alpha-1 microglobulin clusterin
THP alpha-1 microglobulin clusterin TIMP-1 alpha-1 microglobulin
clusterin TFF-3 alpha-1 microglobulin clusterin VEGF alpha-1
microglobulin CTGF creatinine alpha-1 microglobulin CTGF cystatin C
alpha-1 microglobulin CTGF GST-alpha alpha-1 microglobulin CTGF
KIM-1 alpha-1 microglobulin CTGF microalbumin alpha-1 microglobulin
CTGF NGAL alpha-1 microglobulin CTGF osteopontin alpha-1
microglobulin CTGF THP alpha-1 microglobulin CTGF TIMP-1 alpha-1
microglobulin CTGF TFF-3 alpha-1 microglobulin CTGF VEGF alpha-1
microglobulin creatinine cystatin C alpha-1 microglobulin
creatinine GST-alpha alpha-1 microglobulin creatinine KIM-1 alpha-1
microglobulin creatinine microalbumin alpha-1 microglobulin
creatinine NGAL alpha-1 microglobulin creatinine osteopontin
alpha-1 microglobulin creatinine THP alpha-1 microglobulin
creatinine TIMP-1 alpha-1 microglobulin creatinine TFF-3 alpha-1
microglobulin creatinine VEGF alpha-1 microglobulin cystatin C
GST-alpha alpha-1 microglobulin cystatin C KIM-1 alpha-1
microglobulin cystatin C microalbumin alpha-1 microglobulin
cystatin C NGAL alpha-1 microglobulin cystatin C osteopontin
alpha-1 microglobulin cystatin C THP alpha-1 microglobulin cystatin
C TIMP-1 alpha-1 microglobulin cystatin C TFF-3 alpha-1
microglobulin cystatin C VEGF alpha-1 microglobulin GST-alpha KIM-1
alpha-1 microglobulin GST-alpha microalbumin alpha-1 microglobulin
GST-alpha NGAL alpha-1 microglobulin GST-alpha osteopontin alpha-1
microglobulin GST-alpha THP alpha-1 microglobulin GST-alpha TIMP-1
alpha-1 microglobulin GST-alpha TFF-3 alpha-1 microglobulin
GST-alpha VEGF alpha-1 microglobulin KIM-1 microalbumin alpha-1
microglobulin KIM-1 NGAL alpha-1 microglobulin KIM-1 osteopontin
alpha-1 microglobulin KIM-1 THP alpha-1 microglobulin KIM-1 TIMP-1
alpha-1 microglobulin KIM-1 TFF-3 alpha-1 microglobulin KIM-1 VEGF
alpha-1 microglobulin microalbumin NGAL alpha-1 microglobulin
microalbumin osteopontin alpha-1 microglobulin microalbumin THP
alpha-1 microglobulin microalbumin TIMP-1 alpha-1 microglobulin
microalbumin TFF-3 alpha-1 microglobulin microalbumin VEGF alpha-1
microglobulin NGAL osteopontin alpha-1 microglobulin NGAL THP
alpha-1 microglobulin NGAL TIMP-1 alpha-1 microglobulin NGAL TFF-3
alpha-1 microglobulin NGAL VEGF alpha-1 microglobulin osteopontin
THP alpha-1 microglobulin osteopontin TIMP-1 alpha-1 microglobulin
osteopontin TFF-3 alpha-1 microglobulin osteopontin VEGF alpha-1
microglobulin THP TIMP-1 alpha-1 microglobulin THP TFF-3 alpha-1
microglobulin THP VEGF alpha-1 microglobulin TIMP-1 TFF-3 alpha-1
microglobulin TIMP-1 VEGF alpha-1 microglobulin TFF-3 VEGF beta-2
microglobulin calbindin clusterin beta-2 microglobulin calbindin
CTGF beta-2 microglobulin calbindin creatinine beta-2 microglobulin
calbindin cystatin C beta-2 microglobulin calbindin GST-alpha
beta-2 microglobulin calbindin KIM-1 beta-2 microglobulin calbindin
microalbumin beta-2 microglobulin calbindin NGAL beta-2
microglobulin calbindin osteopontin beta-2 microglobulin calbindin
THP beta-2 microglobulin calbindin TIMP-1 beta-2 microglobulin
calbindin TFF-3 beta-2 microglobulin calbindin VEGF beta-2
microglobulin clusterin CTGF beta-2 microglobulin clusterin
creatinine beta-2 microglobulin clusterin cystatin C beta-2
microglobulin clusterin GST-alpha beta-2 microglobulin clusterin
KIM-1 beta-2 microglobulin clusterin microalbumin beta-2
microglobulin clusterin NGAL beta-2 microglobulin clusterin
osteopontin beta-2 microglobulin clusterin THP beta-2 microglobulin
clusterin TIMP-1 beta-2 microglobulin clusterin TFF-3 beta-2
microglobulin clusterin VEGF beta-2 microglobulin CTGF creatinine
beta-2 microglobulin CTGF cystatin C beta-2 microglobulin CTGF
GST-alpha beta-2 microglobulin CTGF KIM-1 beta-2 microglobulin CTGF
microalbumin beta-2 microglobulin CTGF NGAL beta-2 microglobulin
CTGF osteopontin beta-2 microglobulin CTGF THP beta-2 microglobulin
CTGF TIMP-1 beta-2 microglobulin CTGF TFF-3 beta-2 microglobulin
CTGF VEGF beta-2 microglobulin creatinine cystatin C beta-2
microglobulin creatinine GST-alpha beta-2 microglobulin creatinine
KIM-1 beta-2 microglobulin creatinine microalbumin beta-2
microglobulin creatinine NGAL beta-2 microglobulin creatinine
osteopontin beta-2 microglobulin creatinine THP beta-2
microglobulin creatinine TIMP-1 beta-2 microglobulin creatinine
TFF-3 beta-2 microglobulin creatinine VEGF beta-2 microglobulin
cystatin C GST-alpha beta-2 microglobulin cystatin C KIM-1 beta-2
microglobulin cystatin C microalbumin beta-2 microglobulin cystatin
C NGAL beta-2 microglobulin cystatin C osteopontin beta-2
microglobulin cystatin C THP beta-2 microglobulin cystatin C TIMP-1
beta-2 microglobulin cystatin C TFF-3 beta-2 microglobulin cystatin
C VEGF beta-2 microglobulin GST-alpha KIM-1 beta-2 microglobulin
GST-alpha microalbumin beta-2 microglobulin GST-alpha NGAL beta-2
microglobulin GST-alpha osteopontin beta-2 microglobulin GST-alpha
THP beta-2 microglobulin GST-alpha TIMP-1 beta-2 microglobulin
GST-alpha TFF-3 beta-2 microglobulin GST-alpha VEGF beta-2
microglobulin KIM-1 microalbumin beta-2 microglobulin KIM-1 NGAL
beta-2 microglobulin KIM-1 osteopontin beta-2 microglobulin KIM-1
THP beta-2 microglobulin KIM-1 TIMP-1 beta-2 microglobulin KIM-1
TFF-3 beta-2 microglobulin KIM-1 VEGF beta-2 microglobulin
microalbumin NGAL beta-2 microglobulin microalbumin osteopontin
beta-2 microglobulin microalbumin THP beta-2 microglobulin
microalbumin TIMP-1 beta-2 microglobulin microalbumin TFF-3 beta-2
microglobulin microalbumin VEGF beta-2 microglobulin NGAL
osteopontin beta-2 microglobulin NGAL THP beta-2 microglobulin NGAL
TIMP-1 beta-2 microglobulin NGAL TFF-3 beta-2 microglobulin NGAL
VEGF beta-2 microglobulin osteopontin THP beta-2 microglobulin
osteopontin TIMP-1 beta-2 microglobulin osteopontin TFF-3 beta-2
microglobulin osteopontin VEGF beta-2 microglobulin THP TIMP-1
beta-2 microglobulin THP TFF-3 beta-2 microglobulin THP VEGF beta-2
microglobulin TIMP-1 TFF-3 beta-2 microglobulin TIMP-2 VEGF beta-2
microglobulin TFF-3 VEGF calbindin clusterin CTGF calbindin
clusterin creatinine calbindin clusterin cystatin C calbindin
clusterin GST-alpha calbindin clusterin KIM-1 calbindin clusterin
microalbumin calbindin clusterin NGAL calbindin clusterin
osteopontin calbindin clusterin THP calbindin clusterin TIMP-1
calbindin clusterin TFF-3 calbindin clusterin VEGF calbindin CTGF
creatinine calbindin CTGF cystatin C calbindin CTGF GST-alpha
calbindin CTGF KIM-1 calbindin CTGF microalbumin calbindin CTGF
NGAL calbindin CTGF osteopontin calbindin CTGF THP calbindin CTGF
TIMP-1 calbindin CTGF TFF-3 calbindin CTGF VEGF calbindin
creatinine cystatin C calbindin creatinine GST-alpha calbindin
creatinine KIM-1 calbindin creatinine microalbumin calbindin
creatinine NGAL calbindin creatinine osteopontin calbindin
creatinine THP calbindin creatinine TIMP-1 calbindin creatinine
TFF-3 calbindin creatinine VEGF calbindin cystatin C GST-alpha
calbindin cystatin C KIM-1 calbindin cystatin C microalbumin
calbindin cystatin C NGAL calbindin cystatin C osteopontin
calbindin cystatin C THP calbindin cystatin C TIMP-1 calbindin
cystatin C TFF-3 calbindin cystatin C VEGF calbindin GST-alpha
KIM-1 calbindin GST-alpha microalbumin calbindin GST-alpha NGAL
calbindin GST-alpha osteopontin calbindin GST-alpha THP calbindin
GST-alpha TIMP-1 calbindin GST-alpha TFF-3 calbindin GST-alpha VEGF
calbindin KIM-1 microalbumin calbindin KIM-1 NGAL
calbindin KIM-1 osteopontin calbindin KIM-1 THP calbindin KIM-1
TIMP-1 calbindin KIM-1 TFF-3 calbindin KIM-1 VEGF calbindin
microalbumin NGAL calbindin microalbumin osteopontin calbindin
microalbumin THP calbindin microalbumin TIMP-1 calbindin
microalbumin TFF-3 calbindin microalbumin VEGF calbindin NGAL
osteopontin calbindin NGAL THP calbindin NGAL TIMP-1 calbindin NGAL
TFF-3 calbindin NGAL VEGF calbindin osteopontin THP calbindin
osteopontin TIMP-1 calbindin osteopontin TFF-3 calbindin
osteopontin VEGF calbindin THP TIMP-1 calbindin THP TFF-3 calbindin
THP VEGF calbindin TIMP-1 TFF-3 calbindin TIMP-1 VEGF calbindin
TFF-3 VEGF clusterin CTGF creatinine clusterin CTGF cystatin C
clusterin CTGF GST-alpha clusterin CTGF KIM-1 clusterin CTGF
microalbumin clusterin CTGF NGAL clusterin CTGF osteopontin
clusterin CTGF THP clusterin CTGF TIMP-1 clusterin CTGF TFF-3
clusterin CTGF VEGF clusterin creatinine cystatin C clusterin
creatinine GST-alpha clusterin creatinine KIM-1 clusterin
creatinine microalbumin clusterin creatinine NGAL clusterin
creatinine osteopontin clusterin creatinine THP clusterin
creatinine TIMP-1 clusterin creatinine TFF-3 clusterin creatinine
VEGF clusterin cystatin C GST-alpha clusterin cystatin C KIM-1
clusterin cystatin C microalbumin clusterin cystatin C NGAL
clusterin cystatin C osteopontin clusterin cystatin C THP clusterin
cystatin C TIMP-1 clusterin cystatin C TFF-3 clusterin cystatin C
VEGF clusterin GST-alpha KIM-1 clusterin GST-alpha microalbumin
clusterin GST-alpha NGAL clusterin GST-alpha osteopontin clusterin
GST-alpha THP clusterin GST-alpha TIMP-1 clusterin GST-alpha TFF-3
clusterin GST-alpha VEGF clusterin KIM-1 microalbumin clusterin
KIM-1 NGAL clusterin KIM-1 osteopontin clusterin KIM-1 THP
clusterin KIM-1 TIMP-1 clusterin KIM-1 TFF-3 clusterin KIM-1 VEGF
clusterin microalbumin NGAL clusterin microalbumin osteopontin
clusterin microalbumin THP clusterin microalbumin TIMP-1 clusterin
microalbumin TFF-3 clusterin microalbumin VEGF clusterin NGAL
osteopontin clusterin NGAL THP clusterin NGAL TIMP-1 clusterin NGAL
TFF-3 clusterin NGAL VEGF clusterin osteopontin THP clusterin
osteopontin TIMP-1 clusterin osteopontin TFF-3 clusterin
osteopontin VEGF clusterin THP TIMP-1 clusterin THP TFF-3 clusterin
THP VEGF clusterin TIMP-1 TFF-3 clusterin TIMP-1 VEGF clusterin
TFF-3 VEGF CTGF creatinine cystatin C CTGF creatinine GST-alpha
CTGF creatinine KIM-1 CTGF creatinine microalbumin CTGF creatinine
NGAL CTGF creatinine osteopontin CTGF creatinine THP CTGF
creatinine TIMP-1 CTGF creatinine TFF-3 CTGF creatinine VEGF CTGF
cystatin C GST-alpha CTGF cystatin C KIM-1 CTGF cystatin C
microalbumin CTGF cystatin C NGAL CTGF cystatin C osteopontin CTGF
cystatin C THP CTGF cystatin C TIMP-1 CTGF cystatin C TFF-3 CTGF
cystatin C VEGF CTGF GST-alpha KIM-1 CTGF GST-alpha microalbumin
CTGF GST-alpha NGAL CTGF GST-alpha osteopontin CTGF GST-alpha THP
CTGF GST-alpha TIMP-1 CTGF GST-alpha TFF-3 CTGF GST-alpha VEGF CTGF
KIM-1 microalbumin CTGF KIM-1 NGAL CTGF KIM-1 osteopontin CTGF
KIM-1 THP CTGF KIM-1 TIMP-1 CTGF KIM-1 TFF-3 CTGF KIM-1 VEGF CTGF
microalbumin NGAL CTGF microalbumin osteopontin CTGF microalbumin
THP CTGF microalbumin TIMP-1 CTGF microalbumin TFF-3 CTGF
microalbumin VEGF CTGF NGAL osteopontin CTGF NGAL THP CTGF NGAL
TIMP-1 CTGF NGAL TFF-3 CTGF NGAL VEGF CTGF osteopontin THP CTGF
osteopontin TIMP-1 CTGF osteopontin TFF-3 CTGF osteopontin VEGF
CTGF THP TIMP-1 CTGF THP TFF-3 CTGF THP VEGF CTGF TIMP-1 TFF-3 CTGF
TIMP-1 VEGF CTGF TFF-3 VEGF creatinine cystatin C GST-alpha
creatinine cystatin C KIM-1 creatinine cystatin C microalbumin
creatinine cystatin C NGAL creatinine cystatin C osteopontin
creatinine cystatin C THP creatinine cystatin C TIMP-1 creatinine
cystatin C TFF-3 creatinine cystatin C VEGF creatinine GST-alpha
KIM-1 creatinine GST-alpha microalbumin creatinine GST-alpha NGAL
creatinine GST-alpha osteopontin creatinine GST-alpha THP
creatinine GST-alpha TIMP-1 creatinine GST-alpha TFF-3 creatinine
GST-alpha VEGF creatinine KIM-1 microalbumin creatinine KIM-1 NGAL
creatinine KIM-1 osteopontin creatinine KIM-1 THP creatinine KIM-1
TIMP-1 creatinine KIM-1 TFF-3 creatinine KIM-1 VEGF creatinine
microalbumin NGAL creatinine microalbumin osteopontin creatinine
microalbumin THP creatinine microalbumin TIMP-1 creatinine
microalbumin TFF-3 creatinine microalbumin VEGF creatinine NGAL
osteopontin creatinine NGAL THP creatinine NGAL TIMP-1 creatinine
NGAL TFF-3 creatinine NGAL VEGF creatinine osteopontin THP
creatinine osteopontin TIMP-1 creatinine osteopontin TFF-3
creatinine osteopontin VEGF creatinine THP TIMP-1 creatinine THP
TFF-3 creatinine THP VEGF creatinine TIMP-1 TFF-3 creatinine TIMP-1
VEGF creatinine TFF-3 VEGF cystatin C GST-alpha KIM-1 cystatin C
GST-alpha microalbumin cystatin C GST-alpha NGAL cystatin C
GST-alpha osteopontin cystatin C GST-alpha THP cystatin C GST-alpha
TIMP-1 cystatin C GST-alpha TFF-3 cystatin C GST-alpha VEGF
cystatin C KIM-1 microalbumin cystatin C KIM-1 NGAL cystatin C
KIM-1 osteopontin cystatin C KIM-1 THP cystatin C KIM-1 TIMP-1
cystatin C KIM-1 TFF-3 cystatin C KIM-1 VEGF cystatin C
microalbumin NGAL cystatin C microalbumin osteopontin cystatin C
microalbumin THP cystatin C microalbumin TIMP-1 cystatin C
microalbumin TFF-3 cystatin C microalbumin VEGF cystatin C NGAL
osteopontin cystatin C NGAL THP cystatin C NGAL TIMP-1 cystatin C
NGAL TFF-3 cystatin C NGAL VEGF cystatin C osteopontin THP cystatin
C osteopontin TIMP-1 cystatin C osteopontin TFF-3 cystatin C
osteopontin VEGF cystatin C THP TIMP-1 cystatin C THP TFF-3
cystatin C THP VEGF cystatin C TIMP-1 TFF-3 cystatin C TIMP-1 VEGF
cystatin C TFF-3 VEGF GST-alpha KIM-1 microalbumin GST-alpha KIM-1
NGAL GST-alpha KIM-1 osteopontin GST-alpha KIM-1 THP GST-alpha
KIM-1 TIMP-1 GST-alpha KIM-1 TFF-3 GST-alpha KIM-1 VEGF GST-alpha
microalbumin NGAL GST-alpha microalbumin osteopontin GST-alpha
microalbumin THP GST-alpha microalbumin TIMP-1 GST-alpha
microalbumin TFF-3 GST-alpha microalbumin VEGF GST-alpha NGAL
osteopontin GST-alpha NGAL THP GST-alpha NGAL TIMP-1 GST-alpha NGAL
TFF-3 GST-alpha NGAL VEGF GST-alpha osteopontin THP GST-alpha
osteopontin TIMP-1 GST-alpha osteopontin TFF-3 GST-alpha
osteopontin VEGF GST-alpha THP TIMP-1
GST-alpha THP TFF-3 GST-alpha THP VEGF GST-alpha TIMP-1 TFF-3
GST-alpha TIMP-1 VEGF GST-alpha TFF-3 VEGF KIM-1 microalbumin NGAL
KIM-1 microalbumin osteopontin KIM-1 microalbumin THP KIM-1
microalbumin TIMP-1 KIM-1 microalbumin TFF-3 KIM-1 microalbumin
VEGF KIM-1 NGAL osteopontin KIM-1 NGAL THP KIM-1 NGAL TIMP-1 KIM-1
NGAL TFF-3 KIM-1 NGAL VEGF KIM-1 osteopontin THP KIM-1 osteopontin
TIMP-1 KIM-1 osteopontin TFF-3 KIM-1 osteopontin VEGF KIM-1 THP
TIMP-1 KIM-1 THP TFF-3 KIM-1 THP VEGF KIM-1 TIMP-1 TFF-3 KIM-1
TIMP-1 VEGF KIM-1 TFF-3 VEGF microalbumin NGAL osteopontin
microalbumin NGAL THP microalbumin NGAL TIMP-1 microalbumin NGAL
TFF-3 microalbumin NGAL VEGF microalbumin osteopontin THP
microalbumin osteopontin TIMP-1 microalbumin osteopontin TFF-3
microalbumin osteopontin VEGF microalbumin THP TIMP-1 microalbumin
THP TFF-3 microalbumin THP VEGF microalbumin TIMP-1 TFF-3
microalbumin TIMP-1 VEGF microalbumin TFF-3 VEGF NGAL osteopontin
THP NGAL osteopontin TIMP-1 NGAL osteopontin TFF-3 NGAL osteopontin
VEGF NGAL THP TIMP-1 NGAL THP TFF-3 NGAL THP VEGF NGAL TIMP-1 TFF-3
NGAL TIMP-1 VEGF NGAL TFF-3 VEGF osteopontin THP TIMP-1 osteopontin
THP TFF-3 osteopontin THP VEGF osteopontin TIMP-1 TFF-3 osteopontin
TIMP-1 VEGF osteopontin TFF-3 VEGF THP TIMP-1 TFF-3 THP TIMP-1 VEGF
THP TFF-3 VEGF TIMP-1 TFF-3 VEGF
[0076] In one exemplary embodiment, the combination of sample
analytes may include creatinine, KIM-1, and THP. In another
exemplary embodiment, the combination of sample analytes may
include microalbumin, creatinine, and KIM-1. In yet another
exemplary embodiment, the combination of sample analytes may
include creatinine, THP, and A1M. In still another exemplary
embodiment, the combination of sample analytes may include
microalbumin, TIMP-1, and osteopontin.
[0077] In still another embodiment, the devices and systems of the
current invention may be used to diagnose, monitor or determine the
presence of obstructive uropathy. The combination of sample
analytes may include any three of the biomarker analytes previously
discussed. In an additional embodiment, the devices and systems to
diagnose, monitor or determine the presence of obstructive uropathy
include three or more biomarker analytes, including creatinine,
THP, A1M, clusterin, NGAL, and osteopontin. In a further
embodiment, the devices and systems to diagnose, monitor or
determine the presence of obstructive uropathy includes six
biomarker analytes, including creatinine, THP, A1M, clusterin,
NGAL, and osteopontin.
[0078] In yet another embodiment, the devices and systems of the
current invention may be used to diagnose, monitor or determine the
presence of glomerulonephritis. The combination of sample analytes
may include any three of the biomarker analytes previously
discussed. In an additional embodiment, the devices and systems to
diagnose, monitor or determine the presence of glomerulonephritis
include three or more biomarker analytes, including creatinine,
KIM-1, TIMP-1, alpha-1 microglobulin, THP, and osteopontin. In a
further embodiment, the devices and systems to diagnose, monitor or
determine the presence of glomerulonephropathy includes six
biomarker analytes, including creatinine, KIM-1, TIMP-1, alpha-1
microglobulin, THP, and osteopontin.
[0079] In an additional embodiment, the devices and systems of the
current invention may be used to diagnose, monitor or determine the
presence of kidney damage or toxicity. The combination of sample
analytes may include any three of the biomarker analytes previously
discussed. In anotherembodiment, the devices and systems to
diagnose, monitor or determine the presence of kidney damage or
toxicity include three or more biomarker analytes, including
creatinine, KIM-1, THP, osteopontin, NGAL, and TIMP-1. In a further
embodiment, the devices and systems to diagnose, monitor or
determine the presence of kidney damage or toxicity include six
biomarker analytes, including creatinine, KIM-1, THP, osteopontin,
NGAL, and TIMP-1.
[0080] In a further embodiment, the devices and systems of the
current invention may be used to diagnose, monitor or determine the
presence of diabetic nephropathy. The combination of sample
analytes may include any three of the biomarker analytes previously
discussed. In another embodiment, the devices and systems to
diagnose, monitor or determine the presence of diabetic nephropathy
include three or more biomarker analytes, including microalbumin,
alpha-1 microglobulin, NGAL, KIM-1, THP, and clusterin. In a
further embodiment, the devices and systems to diagnose, monitor or
determine the presence of diabetic nephropathy include six
biomarker analytes, including microalbumin, alpha-1 microglobulin,
NGAL, KIM-1, THP, and clusterin.
[0081] In another embodiment, the devices and systems of the
current invention detect the combination of sample analytes, and
may include any three of the biomarker analytes discussed
previously to diagnose kidney transplant rejection or other
associated disease as discussed previously. In other embodiments,
the combination of sample analytes may be any four, any five, any
six, any seven, any eight, any nine, any ten, any eleven, any
twelve, any thirteen, any fourteen, any fifteen, any sixteen, any
seventeen, any eighteen, or any nineteen biomarker analytes. In
another embodiment, the combination of sample analytes may comprise
a combination listed in Table B.
TABLE-US-00002 TABLE B BLC CD40 IGF BP2 BLC CD40 MMP3 BLC CD40
peptide YY BLC CD40 stem cell factor BLC CD40 TNF RII BLC CD40 AXL
BLC CD40 Eotaxin 3 BLC CD40 FABP BLC CD40 FGF basic BLC CD40
myoglobin BLC CD40 resistin BLC CD40 TRAIL R3 BLC CD40 endothilin 1
BLC CD40 NrCAM BLC CD40 Tenascin C BLC CD40 VCAM1 BLC CD40 cortisol
BLC IGF BP2 MMP3 BLC IGF BP2 peptide YY BLC IGF BP2 stem cell
factor BLC IGF BP2 TNF RII BLC IGF BP2 AXL BLC IGF BP2 Eotaxin 3
BLC IGF BP2 FABP BLC IGF BP2 FGF basic BLC IGF BP2 myoglobin BLC
IGF BP2 resistin BLC IGF BP2 TRAIL R3 BLC IGF BP2 endothilin 1 BLC
IGF BP2 NrCAM BLC IGF BP2 Tenascin C BLC IGF BP2 VCAM1 BLC IGF BP2
cortisol BLC MMP3 peptide YY BLC MMP3 stem cell factor BLC MMP3 TNF
RII BLC MMP3 AXL BLC MMP3 Eotaxin 3 BLC MMP3 FABP BLC MMP3 FGF
basic BLC MMP3 myoglobin BLC MMP3 resistin BLC MMP3 TRAIL R3 BLC
MMP3 endothilin 1 BLC MMP3 NrCAM BLC MMP3 Tenascin C BLC MMP3 VCAM1
BLC MMP3 cortisol BLC peptide YY stem cell factor BLC peptide YY
TNF RII BLC peptide YY AXL BLC peptide YY Eotaxin 3 BLC peptide YY
FABP BLC peptide YY FGF basic BLC peptide YY myoglobin BLC peptide
YY resistin BLC peptide YY TRAIL R3 BLC peptide YY endothilin 1 BLC
peptide YY NrCAM BLC peptide YY Tenascin C BLC peptide YY VCAM1 BLC
peptide YY cortisol BLC stem cell factor TNF RII BLC stem cell
factor AXL BLC stem cell factor Eotaxin 3 BLC stem cell factor FABP
BLC stem cell factor FGF basic BLC stem cell factor myoglobin BLC
stem cell factor resistin BLC stem cell factor TRAIL R3 BLC stem
cell factor endothilin 1 BLC stem cell factor NrCAM BLC stem cell
factor Tenascin C BLC stem cell factor VCAM1 BLC stem cell factor
cortisol BLC TNF RII AXL BLC TNF RII Eotaxin 3 BLC TNF RII FABP BLC
TNF RII FGF basic BLC TNF RII myoglobin BLC TNF RII resistin BLC
TNF RII TRAIL R3 BLC TNF RII endothilin 1 BLC TNF RII NrCAM BLC TNF
RII Tenascin C BLC TNF RII VCAM1 BLC TNF RII cortisol BLC AXL
Eotaxin 3 BLC AXL FABP BLC AXL FGF basic BLC AXL myoglobin BLC AXL
resistin BLC AXL TRAIL R3 BLC AXL endothilin 1 BLC AXL NrCAM BLC
AXL Tenascin C BLC AXL VCAM1 BLC AXL cortisol BLC Eotaxin 3 FABP
BLC Eotaxin 3 FGF basic BLC Eotaxin 3 myoglobin BLC Eotaxin 3
resistin BLC Eotaxin 3 TRAIL R3 BLC Eotaxin 3 endothilin 1 BLC
Eotaxin 3 NrCAM BLC Eotaxin 3 Tenascin C BLC Eotaxin 3 VCAM1 BLC
Eotaxin 3 cortisol BLC FABP FGF basic BLC FABP myoglobin BLC FABP
resistin BLC FABP TRAIL R3 BLC FABP endothilin 1 BLC FABP NrCAM BLC
FABP Tenascin C BLC FABP VCAM1 BLC FABP cortisol BLC FGF basic
myoglobin BLC FGF basic resistin BLC FGF basic TRAIL R3 BLC FGF
basic endothilin 1 BLC FGF basic NrCAM BLC FGF basic Tenascin C BLC
FGF basic VCAM1 BLC FGF basic cortisol BLC myoglobin resistin BLC
myoglobin TRAIL R3 BLC myoglobin endothilin 1 BLC myoglobin NrCAM
BLC myoglobin Tenascin C BLC myoglobin VCAM1 BLC myoglobin cortisol
BLC resistin TRAIL R3 BLC resistin endothilin 1 BLC resistin NrCAM
BLC resistin Tenascin C BLC resistin VCAM1 BLC resistin cortisol
BLC TRAIL R3 endothilin 1 BLC TRAIL R3 NrCAM BLC TRAIL R3 Tenascin
C BLC TRAIL R3 VCAM1 BLC TRAIL R3 cortisol BLC endothilin 1 NrCAM
BLC endothilin 1 Tenascin C BLC endothilin 1 VCAM1 BLC endothilin 1
cortisol BLC NrCAM Tenascin C BLC NrCAM VCAM1 BLC NrCAM cortisol
BLC Tenascin C VCAM1 BLC Tenascin C cortisol BLC VCAM1 cortisol
CD40 IGF BP2 MMP3 CD40 IGF BP2 peptide YY CD40 IGF BP2 stem cell
factor CD40 IGF BP2 TNF RII CD40 IGF BP2 AXL CD40 IGF BP2 Eotaxin 3
CD40 IGF BP2 FABP CD40 IGF BP2 FGF basic CD40 IGF BP2 myoglobin
CD40 IGF BP2 resistin CD40 IGF BP2 TRAIL R3 CD40 IGF BP2 endothilin
1 CD40 IGF BP2 NrCAM CD40 IGF BP2 Tenascin C CD40 IGF BP2 VCAM1
CD40 IGF BP2 cortisol CD40 MMP3 peptide YY CD40 MMP3 stem cell
factor CD40 MMP3 TNF RII CD40 MMP3 AXL CD40 MMP3 Eotaxin 3 CD40
MMP3 FABP CD40 MMP3 FGF basic CD40 MMP3 myoglobin CD40 MMP3
resistin CD40 MMP3 TRAIL R3 CD40 MMP3 endothilin 1 CD40 MMP3 NrCAM
CD40 MMP3 Tenascin C CD40 MMP3 VCAM1 CD40 MMP3 cortisol CD40
peptide YY stem cell factor CD40 peptide YY TNF RII CD40 peptide YY
AXL CD40 peptide YY Eotaxin 3 CD40 peptide YY FABP CD40 peptide YY
FGF basic CD40 peptide YY myoglobin CD40 peptide YY resistin CD40
peptide YY TRAIL R3 CD40 peptide YY endothilin 1 CD40 peptide YY
NrCAM CD40 peptide YY Tenascin C CD40 peptide YY VCAM1 CD40 peptide
YY cortisol CD40 stem cell factor TNF RII CD40 stem cell factor AXL
CD40 stem cell factor Eotaxin 3 CD40 stem cell factor FABP CD40
stem cell factor FGF basic CD40 stem cell factor myoglobin CD40
stem cell factor resistin CD40 stem cell factor TRAIL R3 CD40 stem
cell factor endothilin 1 CD40 stem cell factor NrCAM CD40 stem cell
factor Tenascin C CD40 stem cell factor VCAM1 CD40 stem cell factor
cortisol CD40 TNF RII AXL CD40 TNF RII Eotaxin 3 CD40 TNF RII FABP
CD40 TNF RII FGF basic CD40 TNF RII myoglobin CD40 TNF RII resistin
CD40 TNF RII TRAIL R3 CD40 TNF RII endothilin 1 CD40 TNF RII NrCAM
CD40 TNF RII Tenascin C CD40 TNF RII VCAM1 CD40 TNF RII cortisol
CD40 AXL Eotaxin 3 CD40 AXL FABP CD40 AXL FGF basic CD40 AXL
myoglobin CD40 AXL resistin CD40 AXL TRAIL R3 CD40 AXL endothilin 1
CD40 AXL NrCAM CD40 AXL Tenascin C CD40 AXL VCAM1 CD40 AXL cortisol
CD40 Eotaxin 3 FABP CD40 Eotaxin 3 FGF basic CD40 Eotaxin 3
myoglobin CD40 Eotaxin 3 resistin CD40 Eotaxin 3 TRAIL R3 CD40
Eotaxin 3 endothilin 1 CD40 Eotaxin 3 NrCAM CD40 Eotaxin 3 Tenascin
C CD40 Eotaxin 3 VCAM1 CD40 Eotaxin 3 cortisol CD40 FABP FGF basic
CD40 FABP myoglobin CD40 FABP resistin CD40 FABP TRAIL R3
CD40 FABP endothilin 1 CD40 FABP NrCAM CD40 FABP Tenascin C CD40
FABP VCAM1 CD40 FABP cortisol CD40 FGF basic myoglobin CD40 FGF
basic resistin CD40 FGF basic TRAIL R3 CD40 FGF basic endothilin 1
CD40 FGF basic NrCAM CD40 FGF basic Tenascin C CD40 FGF basic VCAM1
CD40 FGF basic cortisol CD40 myoglobin resistin CD40 myoglobin
TRAIL R3 CD40 myoglobin endothilin 1 CD40 myoglobin NrCAM CD40
myoglobin Tenascin C CD40 myoglobin VCAM1 CD40 myoglobin cortisol
CD40 resistin TRAIL R3 CD40 resistin endothilin 1 CD40 resistin
NrCAM CD40 resistin Tenascin C CD40 resistin VCAM1 CD40 resistin
cortisol CD40 TRAIL R3 endothilin 1 CD40 TRAIL R3 NrCAM CD40 TRAIL
R3 Tenascin C CD40 TRAIL R3 VCAM1 CD40 TRAIL R3 cortisol CD40
endothilin 1 NrCAM CD40 endothilin 1 Tenascin C CD40 endothilin 1
VCAM1 CD40 endothilin 1 cortisol CD40 NrCAM Tenascin C CD40 NrCAM
VCAM1 CD40 NrCAM cortisol CD40 Tenascin C VCAM1 CD40 Tenascin C
cortisol CD40 VCAM1 cortisol IGF BP2 MMP3 peptide YY IGF BP2 MMP3
stem cell factor IGF BP2 MMP3 TNF RII IGF BP2 MMP3 AXL IGF BP2 MMP3
Eotaxin 3 IGF BP2 MMP3 FABP IGF BP2 MMP3 FGF basic IGF BP2 MMP3
myoglobin IGF BP2 MMP3 resistin IGF BP2 MMP3 TRAIL R3 IGF BP2 MMP3
endothilin 1 IGF BP2 MMP3 NrCAM IGF BP2 MMP3 Tenascin C IGF BP2
MMP3 VCAM1 IGF BP2 MMP3 cortisol IGF BP2 peptide YY stem cell
factor IGF BP2 peptide YY TNF RII IGF BP2 peptide YY AXL IGF BP2
peptide YY Eotaxin 3 IGF BP2 peptide YY FABP IGF BP2 peptide YY FGF
basic IGF BP2 peptide YY myoglobin IGF BP2 peptide YY resistin IGF
BP2 peptide YY TRAIL R3 IGF BP2 peptide YY endothilin 1 IGF BP2
peptide YY NrCAM IGF BP2 peptide YY Tenascin C IGF BP2 peptide YY
VCAM1 IGF BP2 peptide YY cortisol IGF BP2 stem cell factor TNF RII
IGF BP2 stem cell factor AXL IGF BP2 stem cell factor Eotaxin 3 IGF
BP2 stem cell factor FABP IGF BP2 stem cell factor FGF basic IGF
BP2 stem cell factor myoglobin IGF BP2 stem cell factor resistin
IGF BP2 stem cell factor TRAIL R3 IGF BP2 stem cell factor
endothilin 1 IGF BP2 stem cell factor NrCAM IGF BP2 stem cell
factor Tenascin C IGF BP2 stem cell factor VCAM1 IGF BP2 stem cell
factor cortisol IGF BP2 TNF RII AXL IGF BP2 TNF RII Eotaxin 3 IGF
BP2 TNF RII FABP IGF BP2 TNF RII FGF basic IGF BP2 TNF RII
myoglobin IGF BP2 TNF RII resistin IGF BP2 TNF RII TRAIL R3 IGF BP2
TNF RII endothilin 1 IGF BP2 TNF RII NrCAM IGF BP2 TNF RII Tenascin
C IGF BP2 TNF RII VCAM1 IGF BP2 TNF RII cortisol IGF BP2 AXL
Eotaxin 3 IGF BP2 AXL FABP IGF BP2 AXL FGF basic IGF BP2 AXL
myoglobin IGF BP2 AXL resistin IGF BP2 AXL TRAIL R3 IGF BP2 AXL
endothilin 1 IGF BP2 AXL NrCAM IGF BP2 AXL Tenascin C IGF BP2 AXL
VCAM1 IGF BP2 AXL cortisol IGF BP2 Eotaxin 3 FABP IGF BP2 Eotaxin 3
FGF basic IGF BP2 Eotaxin 3 myoglobin IGF BP2 Eotaxin 3 resistin
IGF BP2 Eotaxin 3 TRAIL R3 IGF BP2 Eotaxin 3 endothilin 1 IGF BP2
Eotaxin 3 NrCAM IGF BP2 Eotaxin 3 Tenascin C IGF BP2 Eotaxin 3
VCAM1 IGF BP2 Eotaxin 3 cortisol IGF BP2 FABP FGF basic IGF BP2
FABP myoglobin IGF BP2 FABP resistin IGF BP2 FABP TRAIL R3 IGF BP2
FABP endothilin 1 IGF BP2 FABP NrCAM IGF BP2 FABP Tenascin C IGF
BP2 FABP VCAM1 IGF BP2 FABP cortisol IGF BP2 FGF basic myoglobin
IGF BP2 FGF basic resistin IGF BP2 FGF basic TRAIL R3 IGF BP2 FGF
basic endothilin 1 IGF BP2 FGF basic NrCAM IGF BP2 FGF basic
Tenascin C IGF BP2 FGF basic VCAM1 IGF BP2 FGF basic cortisol IGF
BP2 myoglobin resistin IGF BP2 myoglobin TRAIL R3 IGF BP2 myoglobin
endothilin 1 IGF BP2 myoglobin NrCAM IGF BP2 myoglobin Tenascin C
IGF BP2 myoglobin VCAM1 IGF BP2 myoglobin cortisol IGF BP2 resistin
TRAIL R3 IGF BP2 resistin endothilin 1 IGF BP2 resistin NrCAM IGF
BP2 resistin Tenascin C IGF BP2 resistin VCAM1 IGF BP2 resistin
cortisol IGF BP2 TRAIL R3 endothilin 1 IGF BP2 TRAIL R3 NrCAM IGF
BP2 TRAIL R3 Tenascin C IGF BP2 TRAIL R3 VCAM1 IGF BP2 TRAIL R3
cortisol IGF BP2 endothilin 1 NrCAM IGF BP2 endothilin 1 Tenascin C
IGF BP2 endothilin 1 VCAM1 IGF BP2 endothilin 1 cortisol IGF BP2
NrCAM Tenascin C IGF BP2 NrCAM VCAM1 IGF BP2 NrCAM cortisol IGF BP2
Tenascin C VCAM1 IGF BP2 Tenascin C cortisol IGF BP2 VCAM1 cortisol
MMP3 peptide YY stem cell factor MMP3 peptide YY TNF RII MMP3
peptide YY AXL MMP3 peptide YY Eotaxin 3 MMP3 peptide YY FABP MMP3
peptide YY FGF basic MMP3 peptide YY myoglobin MMP3 peptide YY
resistin MMP3 peptide YY TRAIL R3 MMP3 peptide YY endothilin 1 MMP3
peptide YY NrCAM MMP3 peptide YY Tenascin C MMP3 peptide YY VCAM1
MMP3 peptide YY cortisol MMP3 stem cell factor TNF RII MMP3 stem
cell factor AXL MMP3 stem cell factor Eotaxin 3 MMP3 stem cell
factor FABP MMP3 stem cell factor FGF basic MMP3 stem cell factor
myoglobin MMP3 stem cell factor resistin MMP3 stem cell factor
TRAIL R3 MMP3 stem cell factor endothilin 1 MMP3 stem cell factor
NrCAM MMP3 stem cell factor Tenascin C MMP3 stem cell factor VCAM1
MMP3 stem cell factor cortisol MMP3 TNF RII AXL MMP3 TNF RII
Eotaxin 3 MMP3 TNF RII FABP MMP3 TNF RII FGF basic MMP3 TNF RII
myoglobin MMP3 TNF RII resistin MMP3 TNF RII TRAIL R3 MMP3 TNF RII
endothilin 1 MMP3 TNF RII NrCAM MMP3 TNF RII Tenascin C MMP3 TNF
RII VCAM1 MMP3 TNF RII cortisol MMP3 AXL Eotaxin 3 MMP3 AXL FABP
MMP3 AXL FGF basic MMP3 AXL myoglobin MMP3 AXL resistin MMP3 AXL
TRAIL R3 MMP3 AXL endothilin 1 MMP3 AXL NrCAM MMP3 AXL Tenascin C
MMP3 AXL VCAM1 MMP3 AXL cortisol MMP3 Eotaxin 3 FABP MMP3 Eotaxin 3
FGF basic MMP3 Eotaxin 3 myoglobin MMP3 Eotaxin 3 resistin MMP3
Eotaxin 3 TRAIL R3 MMP3 Eotaxin 3 endothilin 1 MMP3 Eotaxin 3 NrCAM
MMP3 Eotaxin 3 Tenascin C MMP3 Eotaxin 3 VCAM1 MMP3 Eotaxin 3
cortisol MMP3 FABP FGF basic MMP3 FABP myoglobin MMP3 FABP resistin
MMP3 FABP TRAIL R3 MMP3 FABP endothilin 1 MMP3 FABP NrCAM MMP3 FABP
Tenascin C MMP3 FABP VCAM1 MMP3 FABP cortisol MMP3 FGF basic
myoglobin MMP3 FGF basic resistin MMP3 FGF basic TRAIL R3 MMP3 FGF
basic endothilin 1 MMP3 FGF basic NrCAM MMP3 FGF basic Tenascin C
MMP3 FGF basic VCAM1 MMP3 FGF basic cortisol MMP3 myoglobin
resistin MMP3 myoglobin TRAIL R3 MMP3 myoglobin endothilin 1 MMP3
myoglobin NrCAM MMP3 myoglobin Tenascin C MMP3 myoglobin VCAM1 MMP3
myoglobin cortisol MMP3 resistin TRAIL R3 MMP3 resistin endothilin
1 MMP3 resistin NrCAM MMP3 resistin Tenascin C MMP3 resistin VCAM1
MMP3 resistin cortisol
MMP3 TRAIL R3 endothilin 1 MMP3 TRAIL R3 NrCAM MMP3 TRAIL R3
Tenascin C MMP3 TRAIL R3 VCAM1 MMP3 TRAIL R3 cortisol MMP3
endothilin 1 NrCAM MMP3 endothilin 1 Tenascin C MMP3 endothilin 1
VCAM1 MMP3 endothilin 1 cortisol MMP3 NrCAM Tenascin C MMP3 NrCAM
VCAM1 MMP3 NrCAM cortisol MMP3 Tenascin C VCAM1 MMP3 Tenascin C
cortisol MMP3 VCAM1 cortisol peptide YY stem cell factor TNF RII
peptide YY stem cell factor AXL peptide YY stem cell factor Eotaxin
3 peptide YY stem cell factor FABP peptide YY stem cell factor FGF
basic peptide YY stem cell factor myoglobin peptide YY stem cell
factor resistin peptide YY stem cell factor TRAIL R3 peptide YY
stem cell factor endothilin 1 peptide YY stem cell factor NrCAM
peptide YY stem cell factor Tenascin C peptide YY stem cell factor
VCAM1 peptide YY stem cell factor cortisol peptide YY TNF RII AXL
peptide YY TNF RII Eotaxin 3 peptide YY TNF RII FABP peptide YY TNF
RII FGF basic peptide YY TNF RII myoglobin peptide YY TNF RII
resistin peptide YY TNF RII TRAIL R3 peptide YY TNF RII endothilin
1 peptide YY TNF RII NrCAM peptide YY TNF RII Tenascin C peptide YY
TNF RII VCAM1 peptide YY TNF RII cortisol peptide YY AXL Eotaxin 3
peptide YY AXL FABP peptide YY AXL FGF basic peptide YY AXL
myoglobin peptide YY AXL resistin peptide YY AXL TRAIL R3 peptide
YY AXL endothilin 1 peptide YY AXL NrCAM peptide YY AXL Tenascin C
peptide YY AXL VCAM1 peptide YY AXL cortisol peptide YY Eotaxin 3
FABP peptide YY Eotaxin 3 FGF basic peptide YY Eotaxin 3 myoglobin
peptide YY Eotaxin 3 resistin peptide YY Eotaxin 3 TRAIL R3 peptide
YY Eotaxin 3 endothilin 1 peptide YY Eotaxin 3 NrCAM peptide YY
Eotaxin 3 Tenascin C peptide YY Eotaxin 3 VCAM1 peptide YY Eotaxin
3 cortisol peptide YY FABP FGF basic peptide YY FABP myoglobin
peptide YY FABP resistin peptide YY FABP TRAIL R3 peptide YY FABP
endothilin 1 peptide YY FABP NrCAM peptide YY FABP Tenascin C
peptide YY FABP VCAM1 peptide YY FABP cortisol peptide YY FGF basic
myoglobin peptide YY FGF basic resistin peptide YY FGF basic TRAIL
R3 peptide YY FGF basic endothilin 1 peptide YY FGF basic NrCAM
peptide YY FGF basic Tenascin C peptide YY FGF basic VCAM1 peptide
YY FGF basic cortisol peptide YY myoglobin resistin peptide YY
myoglobin TRAIL R3 peptide YY myoglobin endothilin 1 peptide YY
myoglobin NrCAM peptide YY myoglobin Tenascin C peptide YY
myoglobin VCAM1 peptide YY myoglobin cortisol peptide YY resistin
TRAIL R3 peptide YY resistin endothilin 1 peptide YY resistin NrCAM
peptide YY resistin Tenascin C peptide YY resistin VCAM1 peptide YY
resistin cortisol peptide YY TRAIL R3 endothilin 1 peptide YY TRAIL
R3 NrCAM peptide YY TRAIL R3 Tenascin C peptide YY TRAIL R3 VCAM1
peptide YY TRAIL R3 cortisol peptide YY endothilin 1 NrCAM peptide
YY endothilin 1 Tenascin C peptide YY endothilin 1 VCAM1 peptide YY
endothilin 1 cortisol peptide YY NrCAM Tenascin C peptide YY NrCAM
VCAM1 peptide YY NrCAM cortisol peptide YY Tenascin C VCAM1 peptide
YY Tenascin C cortisol peptide YY VCAM1 cortisol stem cell factor
TNF RII AXL stem cell factor TNF RII Eotaxin 3 stem cell factor TNF
RII FABP stem cell factor TNF RII FGF basic stem cell factor TNF
RII myoglobin stem cell factor TNF RII resistin stem cell factor
TNF RII TRAIL R3 stem cell factor TNF RII endothilin 1 stem cell
factor TNF RII NrCAM stem cell factor TNF RII Tenascin C stem cell
factor TNF RII VCAM1 stem cell factor TNF RII cortisol stem cell
factor AXL Eotaxin 3 stem cell factor AXL FABP stem cell factor AXL
FGF basic stem cell factor AXL myoglobin stem cell factor AXL
resistin stem cell factor AXL TRAIL R3 stem cell factor AXL
endothilin 1 stem cell factor AXL NrCAM stem cell factor AXL
Tenascin C stem cell factor AXL VCAM1 stem cell factor AXL cortisol
stem cell factor Eotaxin 3 FABP stem cell factor Eotaxin 3 FGF
basic stem cell factor Eotaxin 3 myoglobin stem cell factor Eotaxin
3 resistin stem cell factor Eotaxin 3 TRAIL R3 stem cell factor
Eotaxin 3 endothilin 1 stem cell factor Eotaxin 3 NrCAM stem cell
factor Eotaxin 3 Tenascin C stem cell factor Eotaxin 3 VCAM1 stem
cell factor Eotaxin 3 cortisol stem cell factor FABP FGF basic stem
cell factor FABP myoglobin stem cell factor FABP resistin stem cell
factor FABP TRAIL R3 stem cell factor FABP endothilin 1 stem cell
factor FABP NrCAM stem cell factor FABP Tenascin C stem cell factor
FABP VCAM1 stem cell factor FABP cortisol stem cell factor FGF
basic myoglobin stem cell factor FGF basic resistin stem cell
factor FGF basic TRAIL R3 stem cell factor FGF basic endothilin 1
stem cell factor FGF basic NrCAM stem cell factor FGF basic
Tenascin C stem cell factor FGF basic VCAM1 stem cell factor FGF
basic cortisol stem cell factor myoglobin resistin stem cell factor
myoglobin TRAIL R3 stem cell factor myoglobin endothilin 1 stem
cell factor myoglobin NrCAM stem cell factor myoglobin Tenascin C
stem cell factor myoglobin VCAM1 stem cell factor myoglobin
cortisol stem cell factor resistin TRAIL R3 stem cell factor
resistin endothilin 1 stem cell factor resistin NrCAM stem cell
factor resistin Tenascin C stem cell factor resistin VCAM1 stem
cell factor resistin cortisol stem cell factor TRAIL R3 endothilin
1 stem cell factor TRAIL R3 NrCAM stem cell factor TRAIL R3
Tenascin C stem cell factor TRAIL R3 VCAM1 stem cell factor TRAIL
R3 cortisol stem cell factor endothilin 1 NrCAM stem cell factor
endothilin 1 Tenascin C stem cell factor endothilin 1 VCAM1 stem
cell factor endothilin 1 cortisol stem cell factor NrCAM Tenascin C
stem cell factor NrCAM VCAM1 stem cell factor NrCAM cortisol stem
cell factor Tenascin C VCAM1 stem cell factor Tenascin C cortisol
stem cell factor VCAM1 cortisol TNF RII AXL Eotaxin 3 TNF RII AXL
FABP TNF RII AXL FGF basic TNF RII AXL myoglobin TNF RII AXL
resistin TNF RII AXL TRAIL R3 TNF RII AXL endothilin 1 TNF RII AXL
NrCAM TNF RII AXL Tenascin C TNF RII AXL VCAM1 TNF RII AXL cortisol
TNF RII Eotaxin 3 FABP TNF RII Eotaxin 3 FGF basic TNF RII Eotaxin
3 myoglobin TNF RII Eotaxin 3 resistin TNF RII Eotaxin 3 TRAIL R3
TNF RII Eotaxin 3 endothilin 1 TNF RII Eotaxin 3 NrCAM TNF RII
Eotaxin 3 Tenascin C TNF RII Eotaxin 3 VCAM1 TNF RII Eotaxin 3
cortisol TNF RII FABP FGF basic TNF RII FABP myoglobin TNF RII FABP
resistin TNF RII FABP TRAIL R3 TNF RII FABP endothilin 1 TNF RII
FABP NrCAM TNF RII FABP Tenascin C TNF RII FABP VCAM1 TNF RII FABP
cortisol TNF RII FGF basic myoglobin TNF RII FGF basic resistin TNF
RII FGF basic TRAIL R3 TNF RII FGF basic endothilin 1 TNF RII FGF
basic NrCAM TNF RII FGF basic Tenascin C TNF RII FGF basic VCAM1
TNF RII FGF basic cortisol TNF RII myoglobin resistin TNF RII
myoglobin TRAIL R3 TNF RII myoglobin endothilin 1 TNF RII myoglobin
NrCAM TNF RII myoglobin Tenascin C TNF RII myoglobin VCAM1 TNF RII
myoglobin cortisol TNF RII resistin TRAIL R3 TNF RII resistin
endothilin 1 TNF RII resistin NrCAM TNF RII resistin Tenascin C TNF
RII resistin VCAM1 TNF RII resistin cortisol TNF RII TRAIL R3
endothilin 1 TNF RII TRAIL R3 NrCAM TNF RII TRAIL R3 Tenascin C TNF
RII TRAIL R3 VCAM1 TNF RII TRAIL R3 cortisol TNF RII endothilin 1
NrCAM TNF RII endothilin 1 Tenascin C TNF RII endothilin 1 VCAM1
TNF RII endothilin 1 cortisol TNF RII NrCAM Tenascin C TNF RII
NrCAM VCAM1 TNF RII NrCAM cortisol TNF RII Tenascin C VCAM1 TNF RII
Tenascin C cortisol TNF RII VCAM1 cortisol AXL Eotaxin 3 FABP
AXL Eotaxin 3 FGF basic AXL Eotaxin 3 myoglobin AXL Eotaxin 3
resistin AXL Eotaxin 3 TRAIL R3 AXL Eotaxin 3 endothilin 1 AXL
Eotaxin 3 NrCAM AXL Eotaxin 3 Tenascin C AXL Eotaxin 3 VCAM1 AXL
Eotaxin 3 cortisol AXL FABP FGF basic AXL FABP myoglobin AXL FABP
resistin AXL FABP TRAIL R3 AXL FABP endothilin 1 AXL FABP NrCAM AXL
FABP Tenascin C AXL FABP VCAM1 AXL FABP cortisol AXL FGF basic
myoglobin AXL FGF basic resistin AXL FGF basic TRAIL R3 AXL FGF
basic endothilin 1 AXL FGF basic NrCAM AXL FGF basic Tenascin C AXL
FGF basic VCAM1 AXL FGF basic cortisol AXL myoglobin resistin AXL
myoglobin TRAIL R3 AXL myoglobin endothilin 1 AXL myoglobin NrCAM
AXL myoglobin Tenascin C AXL myoglobin VCAM1 AXL myoglobin cortisol
AXL resistin TRAIL R3 AXL resistin endothilin 1 AXL resistin NrCAM
AXL resistin Tenascin C AXL resistin VCAM1 AXL resistin cortisol
AXL TRAIL R3 endothilin 1 AXL TRAIL R3 NrCAM AXL TRAIL R3 Tenascin
C AXL TRAIL R3 VCAM1 AXL TRAIL R3 cortisol AXL endothilin 1 NrCAM
AXL endothilin 1 Tenascin C AXL endothilin 1 VCAM1 AXL endothilin 1
cortisol AXL NrCAM Tenascin C AXL NrCAM VCAM1 AXL NrCAM cortisol
AXL Tenascin C VCAM1 AXL Tenascin C cortisol AXL VCAM1 cortisol
Eotaxin 3 FABP FGF basic Eotaxin 3 FABP myoglobin Eotaxin 3 FABP
resistin Eotaxin 3 FABP TRAIL R3 Eotaxin 3 FABP endothilin 1
Eotaxin 3 FABP NrCAM Eotaxin 3 FABP Tenascin C Eotaxin 3 FABP VCAM1
Eotaxin 3 FABP cortisol Eotaxin 3 FGF basic myoglobin Eotaxin 3 FGF
basic resistin Eotaxin 3 FGF basic TRAIL R3 Eotaxin 3 FGF basic
endothilin 1 Eotaxin 3 FGF basic NrCAM Eotaxin 3 FGF basic Tenascin
C Eotaxin 3 FGF basic VCAM1 Eotaxin 3 FGF basic cortisol Eotaxin 3
myoglobin resistin Eotaxin 3 myoglobin TRAIL R3 Eotaxin 3 myoglobin
endothilin 1 Eotaxin 3 myoglobin NrCAM Eotaxin 3 myoglobin Tenascin
C Eotaxin 3 myoglobin VCAM1 Eotaxin 3 myoglobin cortisol Eotaxin 3
resistin TRAIL R3 Eotaxin 3 resistin endothilin 1 Eotaxin 3
resistin NrCAM Eotaxin 3 resistin Tenascin C Eotaxin 3 resistin
VCAM1 Eotaxin 3 resistin cortisol Eotaxin 3 TRAIL R3 endothilin 1
Eotaxin 3 TRAIL R3 NrCAM Eotaxin 3 TRAIL R3 Tenascin C Eotaxin 3
TRAIL R3 VCAM1 Eotaxin 3 TRAIL R3 cortisol Eotaxin 3 endothilin 1
NrCAM Eotaxin 3 endothilin 1 Tenascin C Eotaxin 3 endothilin 1
VCAM1 Eotaxin 3 endothilin 1 cortisol Eotaxin 3 NrCAM Tenascin C
Eotaxin 3 NrCAM VCAM1 Eotaxin 3 NrCAM cortisol Eotaxin 3 Tenascin C
VCAM1 Eotaxin 3 Tenascin C cortisol Eotaxin 3 VCAM1 cortisol FABP
FGF basic myoglobin FABP FGF basic resistin FABP FGF basic TRAIL R3
FABP FGF basic endothilin 1 FABP FGF basic NrCAM FABP FGF basic
Tenascin C FABP FGF basic VCAM1 FABP FGF basic cortisol FABP
myoglobin resistin FABP myoglobin TRAIL R3 FABP myoglobin
endothilin 1 FABP myoglobin NrCAM FABP myoglobin Tenascin C FABP
myoglobin VCAM1 FABP myoglobin cortisol FABP resistin TRAIL R3 FABP
resistin endothilin 1 FABP resistin NrCAM FABP resistin Tenascin C
FABP resistin VCAM1 FABP resistin cortisol FABP TRAIL R3 endothilin
1 FABP TRAIL R3 NrCAM FABP TRAIL R3 Tenascin C FABP TRAIL R3 VCAM1
FABP TRAIL R3 cortisol FABP endothilin 1 NrCAM FABP endothilin 1
Tenascin C FABP endothilin 1 VCAM1 FABP endothilin 1 cortisol FABP
NrCAM Tenascin C FABP NrCAM VCAM1 FABP NrCAM cortisol FABP Tenascin
C VCAM1 FABP Tenascin C cortisol FABP VCAM1 cortisol FGF basic
myoglobin resistin FGF basic myoglobin TRAIL R3 FGF basic myoglobin
endothilin 1 FGF basic myoglobin NrCAM FGF basic myoglobin Tenascin
C FGF basic myoglobin VCAM1 FGF basic myoglobin cortisol FGF basic
resistin TRAIL R3 FGF basic resistin endothilin 1 FGF basic
resistin NrCAM FGF basic resistin Tenascin C FGF basic resistin
VCAM1 FGF basic resistin cortisol FGF basic TRAIL R3 endothilin 1
FGF basic TRAIL R3 NrCAM FGF basic TRAIL R3 Tenascin C FGF basic
TRAIL R3 VCAM1 FGF basic TRAIL R3 cortisol FGF basic endothilin 1
NrCAM FGF basic endothilin 1 Tenascin C FGF basic endothilin 1
VCAM1 FGF basic endothilin 1 cortisol FGF basic NrCAM Tenascin C
FGF basic NrCAM VCAM1 FGF basic NrCAM cortisol FGF basic Tenascin C
VCAM1 FGF basic Tenascin C cortisol FGF basic VCAM1 cortisol
myoglobin resistin TRAIL R3 myoglobin resistin endothilin 1
myoglobin resistin NrCAM myoglobin resistin Tenascin C myoglobin
resistin VCAM1 myoglobin resistin cortisol myoglobin TRAIL R3
endothilin 1 myoglobin TRAIL R3 NrCAM myoglobin TRAIL R3 Tenascin C
myoglobin TRAIL R3 VCAM1 myoglobin TRAIL R3 cortisol myoglobin
endothilin 1 NrCAM myoglobin endothilin 1 Tenascin C myoglobin
endothilin 1 VCAM1 myoglobin endothilin 1 cortisol myoglobin NrCAM
Tenascin C myoglobin NrCAM VCAM1 myoglobin NrCAM cortisol myoglobin
Tenascin C VCAM1 myoglobin Tenascin C cortisol myoglobin VCAM1
cortisol resistin TRAIL R3 endothilin 1 resistin TRAIL R3 NrCAM
resistin TRAIL R3 Tenascin C resistin TRAIL R3 VCAM1 resistin TRAIL
R3 cortisol resistin endothilin 1 NrCAM resistin endothilin 1
Tenascin C resistin endothilin 1 VCAM1 resistin endothilin 1
cortisol resistin NrCAM Tenascin C resistin NrCAM VCAM1 resistin
NrCAM cortisol resistin Tenascin C VCAM1 resistin Tenascin C
cortisol resistin VCAM1 cortisol TRAIL R3 endothilin 1 NrCAM TRAIL
R3 endothilin 1 Tenascin C TRAIL R3 endothilin 1 VCAM1 TRAIL R3
endothilin 1 cortisol TRAIL R3 NrCAM Tenascin C TRAIL R3 NrCAM
VCAM1 TRAIL R3 NrCAM cortisol TRAIL R3 Tenascin C VCAM1 TRAIL R3
Tenascin C cortisol TRAIL R3 VCAM1 cortisol endothilin 1 NrCAM
Tenascin C endothilin 1 NrCAM VCAM1 endothilin 1 NrCAM cortisol
endothilin 1 Tenascin C VCAM1 endothilin 1 Tenascin C cortisol
endothilin 1 VCAM1 cortisol NrCAM Tenascin C VCAM1 NrCAM Tenascin C
cortisol NrCAM VCAM1 cortisol Tenascin C VCAM1 cortisol
III. Test Sample
[0082] The method for diagnosing, monitoring, or determining a
renal disorder involves determining the presence of sample analytes
in a test sample. A test sample, as defined herein, is an amount of
bodily fluid taken from a mammal. Non-limiting examples of bodily
fluids include urine, blood, plasma, serum, saliva, semen,
perspiration, tears, mucus, and tissue lysates. In an exemplary
embodiment, the bodily fluid contained in the test sample is urine,
plasma, or serum.
(a) Mammals
[0083] A mammal, as defined herein, is any organism that is a
member of the class Mammalia. Non-limiting examples of mammals
appropriate for the various embodiments may include humans, apes,
monkeys, rats, mice, dogs, cats, pigs, and livestock including
cattle and oxen. In an exemplary embodiment, the mammal is a
human.
(b) Devices and Methods of Taking Bodily Fluids from Mammals
[0084] The bodily fluids of the test sample may be taken from the
mammal using any known device or method so long as the analytes to
be measured by the multiplexed assay are not rendered undetectable
by the multiplexed assay. Non-limiting examples of devices or
methods suitable for taking bodily fluid from a mammal include
urine sample cups, urethral catheters, swabs, hypodermic needles,
thin needle biopsies, hollow needle biopsies, punch biopsies,
metabolic cages, and aspiration.
[0085] In order to adjust the expected concentrations of the sample
analytes in the test sample to fall within the dynamic range of the
multiplexed assay, the test sample may be diluted to reduce the
concentration of the sample analytes prior to analysis. The degree
of dilution may depend on a variety of factors including but not
limited to the type of multiplexed assay used to measure the
analytes, the reagents utilized in the multiplexed assay, and the
type of bodily fluid contained in the test sample. In one
embodiment, the test sample is diluted by adding a volume of
diluent ranging from about 1/2 of the original test sample volume
to about 50,000 times the original test sample volume.
[0086] In one exemplary embodiment, if the test sample is human
urine and the multiplexed assay is an antibody-based
capture-sandwich assay, the test sample is diluted by adding a
volume of diluent that is about 100 times the original test sample
volume prior to analysis. In another exemplary embodiment, if the
test sample is human serum and the multiplexed assay is an
antibody-based capture-sandwich assay, the test sample is diluted
by adding a volume of diluent that is about 5 times the original
test sample volume prior to analysis. In yet another exemplary
embodiment, if the test sample is human plasma and the multiplexed
assay is an antibody-based capture-sandwich assay, the test sample
is diluted by adding a volume of diluent that is about 2,000 times
the original test sample volume prior to analysis.
[0087] The diluent may be any fluid that does not interfere with
the function of the multiplexed assay used to measure the
concentration of the analytes in the test sample. Non-limiting
examples of suitable diluents include deionized water, distilled
water, saline solution, Ringer's solution, phosphate buffered
saline solution, TRIS-buffered saline solution, standard saline
citrate, and HEPES-buffered saline.
IV. Multiplexed Assay Device
[0088] In one embodiment, the concentration of a combination of
sample analytes is measured using a multiplexed assay device
capable of measuring up to 189 of the biomarker analytes. A
multiplexed assay device, as defined herein, is an assay capable of
simultaneously determining the concentration of three or more, four
or more, five or more, six or more, seven or more, eight or more,
nine or more, ten or more, eleven or more, twelve or more, thirteen
or more, fourteen or more, fifteen or more, sixteen or more,
seventeen or more, eighteen or more, nineteen or more, or twenty or
more of the biomarker analytes using a single device and/or method.
Any known method of measuring the concentration of the biomarker
analytes may be used for the multiplexed assay device. Non-limiting
examples of measurement methods suitable for the multiplexed assay
device include electrophoresis, mass spectrometry, protein
microarrays, surface plasmon resonance, and immunoassays including,
but not limited to western blot, immunohistochemical staining,
enzyme-linked immunosorbent assay (ELISA) methods, vibrational
detection using MicroElectroMagnetic Devices (MEMS), and
particle-based capture-sandwich immunoassays.
(a) Multiplexed Immunoassay Device
[0089] In one embodiment, the concentrations of the analytes in the
test sample are measured using a multiplexed immunoassay device
that utilizes capture antibodies marked with indicators to
determine the concentration of the sample analytes.
(i) Capture Antibodies
[0090] In the same embodiment, the multiplexed immunoassay device
includes three or more capture antibodies. Capture antibodies, as
defined herein, are antibodies in which the antigenic determinant
is one of the Biomarker Analytes known in the art to have a
documented association with early renal damage in humans. The
biomarker analytes include, but are note limited to
alpha-1-microglobulin, beta-2-microglobulin, calbindin, clusterin,
CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL,
osteopontin, THP, TIMP-1, TFF-3, and VEGF. Each of the at least
three capture antibodies has a unique antigenic determinant that is
one of the biomarker analytes. When contacted with the test sample,
the capture antibodies form antigen-antibody complexes in which the
analytes serve as antigens.
[0091] The term "antibody," as used herein, encompasses a
monoclonal ab, an antibody fragment, a chimeric antibody, and a
single-chain antibody.
[0092] In some embodiments, the capture antibodies may be attached
to a platform or other substrate having a contact surface in order
to immobilize any analytes captured by the capture antibodies. The
platform generally incorporates a porous material for immobilizing
the analytes. Non-limiting examples of suitable substrates include
paper, nitrocellulose, cellulose, glass, glass fiber mesh, silica
gel, synthetic resins, or plastic strips, beads, or surfaces, such
as the inner surface of the well of a microtitration tray. Suitable
beads may include polystyrene or latex microspheres.
(ii) indicators
[0093] In one embodiment of the multiplexed immunoassay device, an
indicator is attached to each of the three or more capture
antibodies. The indicator, as defined herein, is any compound that
registers a measurable change to indicate the presence of one of
the sample analytes when bound to one of the capture antibodies.
Non-limiting examples of indicators include visual indicators and
electrochemical indicators.
[0094] Visual indicators, as defined herein, are compounds that
register a change by reflecting a limited subset of the wavelengths
of light illuminating the indicator, by fluorescing light after
being illuminated, or by emitting light via chemiluminescence. The
change registered by visual indicators may be in the visible light
spectrum, in the infrared spectrum, or in the ultraviolet spectrum.
Non-limiting examples of visual indicators suitable for the
multiplexed immunoassay device include nanoparticulate gold,
organic particles such as polyurethane or latex microspheres loaded
with dye compounds, carbon black, fluorophores, phycoerythrin,
radioactive isotopes, nanoparticles, quantum dots, and enzymes such
as horseradish peroxidase or alkaline phosphatase that react with a
chemical substrate to form a colored or chemiluminescent
product.
[0095] Electrochemical indicators, as defined herein, are compounds
that register a change by altering an electrical property. The
changes registered by electrochemical indicators may be an
alteration in conductivity, resistance, capacitance, current
conducted in response to an applied voltage, or voltage required to
achieve a desired current. Non-limiting examples of electrochemical
indicators include redox species such as ascorbate (vitamin C),
vitamin E, glutathione, polyphenols, catechols, quercetin,
phytoestrogens, penicillin, carbazole, murranes, phenols,
carbonyls, benzoates, and trace metal ions such as nickel, copper,
cadmium, iron and mercury.
[0096] In this same embodiment, the test sample containing a
combination of three or more sample analytes is contacted with the
capture antibodies and allowed to form antigen-antibody complexes
in which the sample analytes serve as the antigens. After removing
any uncomplexed capture antibodies, the concentrations of the three
or more analytes are determined by measuring the change registered
by the indicators attached to the capture antibodies.
[0097] In one exemplary embodiment, the indicators are polyurethane
or latex microspheres loaded with dye compounds and
phycoerythrin.
(b) Multiplexed Sandwich Immunoassay Device
[0098] In another embodiment, the multiplexed immunoassay device
has a sandwich assay format. In this embodiment, the multiplexed
sandwich immunoassay device includes three or more capture
antibodies as previously described. However, in this embodiment,
each of the capture antibodies is attached to a capture agent that
includes an antigenic moiety. The antigenic moiety serves as the
antigenic determinant of a detection antibody, also included in the
multiplexed immunoassay device of this embodiment. In addition, an
indicator is attached to the detection antibody.
[0099] In this same embodiment, the test sample is contacted with
the capture antibodies and allowed to form antigen-antibody
complexes in which the sample analytes serve as antigens. The
detection antibodies are then contacted with the test sample and
allowed to form antigen-antibody complexes in which the capture
agent serves as the antigen for the detection antibody. After
removing any uncomplexed detection antibodies the concentration of
the analytes are determined by measuring the changes registered by
the indicators attached to the detection antibodies.
(c) Multiplexing Approaches
[0100] In the various embodiments of the multiplexed immunoassay
devices, the concentrations of each of the sample analytes may be
determined using any approach known in the art. In one embodiment,
a single indicator compound is attached to each of the three or
more antibodies. In addition, each of the capture antibodies having
one of the sample analytes as an antigenic determinant is
physically separated into a distinct region so that the
concentration of each of the sample analytes may be determined by
measuring the changes registered by the indicators in each
physically separate region corresponding to each of the sample
analytes.
[0101] In another embodiment, each antibody having one of the
sample analytes as an antigenic determinant is marked with a unique
indicator. In this manner, a unique indicator is attached to each
antibody having a single sample analyte as its antigenic
determinant. In this embodiment, all antibodies may occupy the same
physical space. The concentration of each sample analyte is
determined by measuring the change registered by the unique
indicator attached to the antibody having the sample analyte as an
antigenic determinant.
(d) Microsphere-Based Capture-Sandwich Immunoassay Device
[0102] In an exemplary embodiment, the multiplexed immunoassay
device is a microsphere-based capture-sandwich immunoassay device.
In this embodiment, the device includes a mixture of three or more
capture-antibody microspheres, in which each capture-antibody
microsphere corresponds to one of the biomarker analytes. Each
capture-antibody microsphere includes a plurality of capture
antibodies attached to the outer surface of the microsphere. In
this same embodiment, the antigenic determinant of all of the
capture antibodies attached to one microsphere is the same
biomarker analyte.
[0103] In this embodiment of the device, the microsphere is a small
polystyrene or latex sphere that is loaded with an indicator that
is a dye compound. The microsphere may be between about 3 .mu.m and
about 5 .mu.m in diameter. Each capture-antibody microsphere
corresponding to one of the biomarker analytes is loaded with the
same indicator. In this manner, each capture-antibody microsphere
corresponding to a biomarker analyte is uniquely color-coded.
[0104] In this same exemplary embodiment, the multiplexed
immunoassay device further includes three or more biotinylated
detection antibodies in which the antigenic determinant of each
biotinylated detection antibody is one of the biomarker analytes.
The device further includes a plurality of streptaviden proteins
complexed with a reporter compound. A reporter compound, as defined
herein, is an indicator selected to register a change that is
distinguishable from the indicators used to mark the
capture-antibody microspheres.
[0105] The concentrations of the sample analytes may be determined
by contacting the test sample with a mixture of capture-antigen
microspheres corresponding to each sample analyte to be measured.
The sample analytes are allowed to form antigen-antibody complexes
in which a sample analyte serves as an antigen and a capture
antibody attached to the microsphere serves as an antibody. In this
manner, the sample analytes are immobilized onto the
capture-antigen microspheres. The biotinylated detection antibodies
are then added to the test sample and allowed to form
antigen-antibody complexes in which the analyte serves as the
antigen and the biotinylated detection antibody serves as the
antibody. The streptaviden-reporter complex is then added to the
test sample and allowed to bind to the biotin moieties of the
biotinylated detection antibodies. The antigen-capture microspheres
may then be rinsed and filtered.
[0106] In this embodiment, the concentration of each analyte is
determined by first measuring the change registered by the
indicator compound embedded in the capture-antigen microsphere in
order to identify the particular analyte. For each microsphere
corresponding to one of the biomarker analytes, the quantity of
analyte immobilized on the microsphere is determined by measuring
the change registered by the reporter compound attached to the
microsphere.
[0107] For example, the indicator embedded in the microspheres
associated with one sample analyte may register an emission of
orange light, and the reporter may register an emission of green
light. In this example, a detector device may measure the intensity
of orange light and green light separately. The measured intensity
of the green light would determine the concentration of the analyte
captured on the microsphere, and the intensity of the orange light
would determine the specific analyte captured on the
microsphere.
[0108] Any sensor device may be used to detect the changes
registered by the indicators embedded in the microspheres and the
changes registered by the reporter compound, so long as the sensor
device is sufficiently sensitive to the changes registered by both
indicator and reporter compound. Non-limiting examples of suitable
sensor devices include spectrophotometers, photosensors,
colorimeters, cyclic coulometry devices, and flow cytometers. In an
exemplary embodiment, the sensor device is a flow cytometer.
(e) Vibrational Detection Device
[0109] In another exemplary embodiment, the multiplexed immunoassay
device has a vibrational detection format using a MEMS. In this
embodiment, the immunoassay device uses capture antibodies as
previously described. However, in this embodiment, the capture
antibodies are attached to a microscopic silicon microcantilever
beam structure. The microcantilevers are micromechanical beams that
are anchored at one end, such as diving spring boards that can be
readily fabricated on silicon wafers and other materials. The
microcantilever sensors are physical sensors that respond to
surface stress changes due to chemical or biological processes.
When fabricated with very small force constants, they can measure
forces and stresses with extremely high sensitivity. The very small
force constant of a cantilever allows detection not surface stress
variation due to the binding of an analyte to the capture antibody
on the microcantilever. Binding of the analyte results in a
differential surface stress due to adsorption-induced forces, which
manifests as a deflection which can be measured. The vibrational
detection may be multiplexed. For more details, see Datar et al.,
MRS Bulletin (2009) 34:449-459 and Gaster et al., Nature Medicine
(2009) 15:1327-1332, both of which are hereby incorporated by
reference in their entireties.
[0110] It will be understood by one skilled in the art that the
devices described herein, as well as all those embodiments within
the scope of the current invention may be incorporated into a kit.
Generally, the kit may include any of the devices described herein
in addition to a collection apparatus suitable for collecting a
sample of bodily fluid from the mammal. The collection apparatus
may include, but it not limited to urine sample cups, urethral
catheters, swabs, hypodermic needles, thin needles, hollow needles,
metabolic cages, aspiration needles, and combinations thereof.
EXAMPLES
[0111] The following examples illustrate various iterations of the
invention.
Example 1
Least Detectable Dose and Lower Limit of Quantitation of Assay for
Analytes Associated with Renal Disorders
[0112] To assess the least detectable doses (LDD) and lower limits
of quantitation (LLOQ) of a variety of analytes associated with
renal disorders, the following experiment was conducted. The
analytes measured were alpha-1 microglobulin (A1M), beta-2
microglobulin (B2M), calbindin, clusterin, CTGF, cystatin C,
GST-alpha, KIM-1, NGAL, osteopontin (OPN), THP, TIMP-1, TFF-3, and
VEGF.
[0113] The concentrations of the analytes were measured using a
capture-sandwich assay using antigen-specific antibodies. For each
analyte, a range of standard sample dilutions ranging over about
four orders of magnitude of analyte concentration were measured
using the assay in order to obtain data used to construct a
standard dose response curve. The dynamic range for each of the
analytes, defined herein as the range of analyte concentrations
measured to determine its dose response curve, is presented
below.
[0114] To perform the assay, 5 .mu.L of a diluted mixture of
capture-antibody microspheres were mixed with 5 .mu.L of blocker
and 10 .mu.L of pre-diluted standard sample in each of the wells of
a hard-bottom microtiter plate. After incubating the hard-bottom
plate for 1 hour, 10 .mu.L of biotinylated detection antibody was
added to each well, and then the hard-bottom plate was incubated
for an additional hour. 10 .mu.L of diluted
streptavidin-phycoerythrin was added to each well and then the
hard-bottom plate was incubated for another 60 minutes.
[0115] A filter-membrane microtiter plate was pre-wetted by adding
100 .mu.L wash buffer, and then aspirated using a vacuum manifold
device. The contents of the wells of the hard-bottom plate were
then transferred to the corresponding wells of the filter-membrane
plate. All wells of the hard-bottom plate were vacuum-aspirated and
the contents were washed twice with 100 .mu.L of wash buffer. After
the second wash, 100 .mu.L of wash buffer was added to each well,
and then the washed microspheres were resuspended with thorough
mixing. The plate was then analyzed using a Luminex 100 Analyzer
(Luminex Corporation, Austin, Tex., USA). Dose response curves were
constructed for each analyte by curve-fitting the median
fluorescence intensity (MFI) measured from the assays of diluted
standard samples containing a range of analyte concentrations.
[0116] The least detectable dose (LDD) was determined by adding
three standard deviations to the average of the MFI signal measured
for 20 replicate samples of blank standard solution (i.e. standard
solution containing no analyte). The MFI signal was converted to an
LDD concentration using the dose response curve and multiplied by a
dilution factor of 2.
[0117] The lower limit of quantification (LLOQ), defined herein as
the point at which the coefficient of variation (CV) for the
analyte measured in the standard samples was 30%, was determined by
the analysis of the measurements of increasingly diluted standard
samples. For each analyte, the standard solution was diluted by 2
fold for 8 dilutions. At each stage of dilution, samples were
assayed in triplicate, and the CV of the analyte concentration at
each dilution was calculated and plotted as a function of analyte
concentration. The LLOQ was interpolated from this plot and
multiplied by a dilution factor of 2.
[0118] The LDD and LLOQ results for each analyte are summarized in
Table 2:
TABLE-US-00003 TABLE 2 LDD, LLOQ, and Dynamic Range of Analyte
Assay Dynamic Range Analyte Units LDD LLOQ minimum maximum
Calbindin ng/mL 1.1 3.1 0.516 2580 Clusterin ng/mL 2.4 2.3 0.676
3378 CTGF ng/mL 1.3 3.8 0.0794 400 GST-alpha ng/mL 1.4 3.6 0.24
1,200 KIM-1 ng/mL 0.016 0.028 0.00478 24 VEGF pg/mL 4.4 20 8.76
44,000 .beta.-2M .mu.g/mL 0.012 0.018 0.0030 15 Cystatin C ng/mL
2.8 3.7 0.60 3,000 NGAL ng/mL 4.1 7.8 1.2 6,000 Osteopontin ng/mL
29 52 3.9 19,500 TIMP-1 ng/mL 0.71 1.1 0.073 365 A-1M .mu.g/mL
0.059 0.29 0.042 210 THP .mu.g/mL 0.46 0.30 0.16 800 TFF-3 .mu.g/mL
0.06 0.097 0.060 300
[0119] The results of this experiment characterized the least
detectible dose and the lower limit of quantification for fourteen
analytes associated with various renal disorders using a
capture-sandwich assay.
Example 2
Precision of Assay for Analytes Associated with Renal Disorders
[0120] To assess the precision of an assay used to measure the
concentration of analytes associated with renal disorders, the
following experiment was conducted. The analytes measured were
alpha-1 microglobulin (A1M), beta-2 microglobulin (B2M), calbindin,
clusterin, CTGF, cystatin C, GST-alpha, KIM-1, NGAL, osteopontin
(OPN), THP, TIMP-1, TFF-3, and VEGF. For each analyte, three
concentration levels of standard solution were measured in
triplicate during three runs using the methods described in Example
1. The percent errors for each run at each concentration are
presented in Table 3 for all of the analytes tested:
TABLE-US-00004 TABLE 3 Precision of Analyte Assay Average Run 2
Interrun concentration Run 1 Error Run 2 Error Analyte (ng/mL)
Error (%) (%) Error (%) (%) Calbindin 4.0 6 2 6 13 36 5 3 2 7 281 1
6 0 3 Clusterin 4.4 4 9 2 6 39 5 1 6 8 229 1 3 0 2 CTGF 1.2 10 17 4
14 2.5 19 19 14 14 18 7 5 13 9 GST-alpha 3.9 14 7 5 10 16 13 7 10
11 42 1 16 6 8 KIM-1 0.035 2 0 5 13 0.32 4 5 2 8 2.9 0 5 7 4 VEGF
65 10 1 6 14 534 9 2 12 7 5,397 1 13 14 9 .beta.-2M 0.040 6 1 8 5
0.43 2 2 0 10 6.7 6 5 11 6 Cystatin C 10.5 4 1 7 13 49 0 0 3 9 424
2 6 2 5 NGAL 18.1 11 3 6 13 147 0 0 6 5 1,070 5 1 2 5 Osteopontin
44 1 10 2 11 523 9 9 9 7 8,930 4 10 1 10 TIMP-1 2.2 13 6 3 13 26 1
1 4 14 130 1 3 1 4 A-1M 1.7 11 7 7 14 19 4 1 8 9 45 3 5 2 4 THP 9.4
3 10 11 11 15 3 7 8 6 37 4 5 0 5 TFF-3 0.3 13 3 11 12 4.2 5 8 5 7
1.2 3 7 0 13
[0121] The results of this experiment characterized the precision
of a capture-sandwich assay for fourteen analytes associated with
various renal disorders over a wide range of analyte
concentrations. The precision of the assay varied between about 1%
and about 15% error within a given run, and between about 5% and
about 15% error between different runs. The percent errors
summarized in Table 2 provide information concerning random error
to be expected in an assay measurement caused by variations in
technicians, measuring instruments, and times of measurement.
Example 3
Linearity of Assay for Analytes Associated with Renal Disorders
[0122] To assess the linearity of an assay used to measure the
concentration of analytes associated with renal disorders, the
following experiment was conducted. The analytes measured were
alpha-1 microglobulin (A1M), beta-2 microglobulin (B2M), calbindin,
clusterin, CTGF, cystatin C, GST-alpha, KIM-1, NGAL, osteopontin
(OPN), THP, TIMP-1, TFF-3, and VEGF. For each analyte, three
concentration levels of standard solution were measured in
triplicate during three runs using the methods described in Example
1. Linearity of the assay used to measure each analyte was
determined by measuring the concentrations of standard samples that
were serially-diluted throughout the assay range. The % recovery
was calculated as observed vs. expected concentration based on the
dose-response curve. The results of the linearity analysis are
summarized in Table 4.
TABLE-US-00005 TABLE 4 Linearity of Analyte Assay Expected Observed
Recovery Analyte Dilution concentration concentration (%) Calbindin
1:2 61 61 100 (ng/mL) 1:4 30 32 106 1:8 15 17 110 Clusterin 1:2 41
41 100 (ng/mL) 1:4 21 24 116 1:8 10 11 111 CTGF 1:2 1.7 1.7 100
(ng/mL) 1:4 0.84 1.0 124 1:8 0.42 0.51 122 GST-alpha 1:2 25 25 100
(ng/mL) 1:4 12 14 115 1:8 6.2 8.0 129 KIM-1 1:2 0.87 0.87 100
(ng/mL) 1:4 0.41 0.41 101 1:8 0.21 0.19 93 VEGF 1:2 2,525 2,525 100
(pg/mL) 1:4 1,263 1,340 106 1:8 631 686 109 .beta.-2M 1:100 0.63
0.63 100 (.mu.g/mL) 1:200 0.31 0.34 106 1:400 0.16 0.17 107
Cystatin C 1:100 249 249 100 (ng/mL) 1:200 125 122 102 1:400 62 56
110 NGAL 1:100 1,435 1,435 100 (ng/mL) 1:200 718 775 108 1:400 359
369 103 Osteopontin 1:100 6,415 6,415 100 (ng/mL) 1:200 3,208 3,275
102 1:400 1,604 1,525 95 TIMP-1 1:100 35 35 100 (ng/mL) 1:200 18 18
100 1:400 8.8 8.8 100 A-1M 1:2000 37 37 100 (.mu.g/mL) 1:4000 18 18
99 1:8000 9.1 9.2 99 THP 1:2000 28 28 100 (.mu.g/mL) 1:4000 14 14
96 1:8000 6.7 7.1 94 TFF-3 1:2000 8.8 8.8 100 (.mu.g/mL) 1:4000 3.8
4.4 86 1:8000 1.9 2.2 86
[0123] The results of this experiment demonstrated reasonably
linear responses of the sandwich-capture assay to variations in the
concentrations of the analytes in the tested samples.
Example 4
Spike Recovery of Analytes Associated with Renal Disorders
[0124] To assess the recovery of analytes spiked into urine, serum,
and plasma samples by an assay used to measure the concentration of
analytes associated with renal disorders, the following experiment
was conducted. The analytes measured were alpha-1 microglobulin
(A1M), beta-2 microglobulin (B2M), calbindin, clusterin, CTGF,
cystatin C, GST-alpha, KIM-1, NGAL, osteopontin (OPN), THP, TIMP-1,
TFF-3, and VEGF. For each analyte, three concentration levels of
standard solution were spiked into known urine, serum, and plasma
samples. Prior to analysis, all urine samples were diluted 1:2000
(sample: diluent), all plasma samples were diluted 1:5 (sample:
diluent), and all serum samples were diluted 1:2000 (sample:
diluent).
[0125] The concentrations of the analytes in the samples were
measured using the methods described in Example 1. The average %
recovery was calculated as the proportion of the measurement of
analyte spiked into the urine, serum, or plasma sample (observed)
to the measurement of analyte spiked into the standard solution
(expected). The results of the spike recovery analysis are
summarized in Table 5.
TABLE-US-00006 TABLE 5 Spike Recovery of Analyte Assay in Urine,
Serum, and Plasma Samples Recovery in Recovery in Recovery in Spike
Urine Serum Plasma Analyte Concentration Sample (%) Sample (%)
Sample (%) Calbindin 66 76 82 83 (ng/mL) 35 91 77 71 18 80 82 73
average 82 80 76 Clusterin 80 72 73 75 (ng/mL) 37 70 66 72 20 90 73
70 average 77 70 72 CTGF 8.4 91 80 79 (ng/mL) 4.6 114 69 78 2.4 76
80 69 average 94 77 75 GST-alpha 27 75 84 80 (ng/mL) 15 90 75 81
7.1 82 84 72 average 83 81 78 KIM-1 0.63 87 80 83 (ng/mL) .029 119
74 80 0.14 117 80 78 average 107 78 80 VEGF 584 88 84 82 (pg/mL)
287 101 77 86 123 107 84 77 average 99 82 82 .beta.-2M 0.97 117 98
98 (.mu.g/mL) 0.50 124 119 119 0.24 104 107 107 average 115 108 105
Cystatin C 183 138 80 103 (ng/mL) 90 136 97 103 40 120 97 118
average 131 91 108 NGAL 426 120 105 111 (ng/mL) 213 124 114 112 103
90 99 113 average 111 106 112 Osteopontin 1,245 204 124 68 (ng/mL)
636 153 112 69 302 66 103 67 average 108 113 68 TIMP-1 25 98 97 113
(ng/mL) 12 114 89 103 5.7 94 99 113 average 102 95 110 A-1M 0.0028
100 101 79 (.mu.g/mL) 0.0012 125 80 81 0.00060 118 101 82 average
114 94 81 THP 0.0096 126 108 90 (.mu.g/mL) 0.0047 131 93 91 0.0026
112 114 83 average 123 105 88 TFF-3 0.0038 105 114 97 (.mu.g/mL)
0.0019 109 104 95 0.0010 102 118 93 average 105 112 95
[0126] The results of this experiment demonstrated that the
sandwich-type assay is reasonably sensitive to the presence of all
analytes measured, whether the analytes were measured in standard
samples, urine samples, plasma samples, or serum samples.
Example 5
Matrix Interferences of Analytes Associated with Renal
Disorders
[0127] To assess the matrix interference of hemoglobin, bilirubin,
and triglycerides spiked into standard samples, the following
experiment was conducted. The analytes measured were alpha-1
microglobulin (A1M), beta-2 microglobulin (B2M), calbindin,
clusterin, CTGF, cystatin C, GST-alpha, KIM-1, NGAL, osteopontin
(OPN), THP, TIMP-1, TFF-3, and VEGF. For each analyte, three
concentration levels of standard solution were spiked into known
urine, serum, and plasma samples. Matrix interference was assessed
by spiking hemoglobin, bilirubin, and triglyceride into standard
analyte samples and measuring analyte concentrations using the
methods described in Example 1. A % recovery was determined by
calculating the ratio of the analyte concentration measured from
the spiked sample (observed) divided by the analyte concentration
measured form the standard sample (expected). The results of the
matrix interference analysis are summarized in Table 6.
TABLE-US-00007 TABLE 6 Matrix Interference of Hemoglobin,
Bilirubin, and Triglyceride on the Measurement of Analytes Matrix
Compound Maximum Overall Spiked into Spike Recovery Analyte Sample
Concentration (%) Calbindin Hemoglobin 500 110 (mg/mL) Bilirubin 20
98 Triglyceride 500 117 Clusterin Hemoglobin 500 125 (mg/mL)
Bilirubin 20 110 Triglyceride 500 85 CTGF Hemoglobin 500 91 (mg/mL)
Bilirubin 20 88 Triglyceride 500 84 GST-alpha Hemoglobin 500 100
(mg/mL) Bilirubin 20 96 Triglyceride 500 96 KIM-1 Hemoglobin 500
108 (mg/mL) Bilirubin 20 117 Triglyceride 500 84 VEGF Hemoglobin
500 112 (mg/mL) Bilirubin 20 85 Triglyceride 500 114 .beta.-2M
Hemoglobin 500 84 (.mu.g/mL) Bilirubin 20 75 Triglyceride 500 104
Cystatin C Hemoglobin 500 91 (ng/mL) Bilirubin 20 102 Triglyceride
500 124 NGAL Hemoglobin 500 99 (ng/mL) Bilirubin 20 92 Triglyceride
500 106 Osteopontin Hemoglobin 500 83 (ng/mL) Bilirubin 20 86
Triglyceride 500 106 TIMP-1 Hemoglobin 500 87 (ng/mL) Bilirubin 20
86 Triglyceride 500 93 A-1M Hemoglobin 500 103 (.mu.g/mL) Bilirubin
20 110 Triglyceride 500 112 THP Hemoglobin 500 108 (.mu.g/mL)
Bilirubin 20 101 Triglyceride 500 121 TFF-3 Hemoglobin 500 101
(.mu.g/mL) Bilirubin 20 101 Triglyceride 500 110
[0128] The results of this experiment demonstrated that hemoglobin,
bilirubin, and triglycerides, three common compounds found in
urine, plasma, and serum samples, did not significantly degrade the
ability of the sandwich-capture assay to detect any of the analytes
tested.
Example 6
Sample Stability of Analytes Associated with Renal Disorders
[0129] To assess the ability of analytes spiked into urine, serum,
and plasma samples to tolerate freeze-thaw cycles, the following
experiment was conducted. The analytes measured were alpha-1
microglobulin (A1M), beta-2 microglobulin (B2M), calbindin,
clusterin, CTGF, cystatin C, GST-alpha, KIM-1, NGAL, osteopontin
(OPN), THP, TIMP-1, TFF-3, and VEGF. Each analyte was spiked into
known urine, serum, and plasma samples at a known analyte
concentration. The concentrations of the analytes in the samples
were measured using the methods described in Example 1 after the
initial addition of the analyte, and after one, two and three
cycles of freezing and thawing. In addition, analyte concentrations
in urine, serum and plasma samples were measured immediately after
the addition of the analyte to the samples as well as after storage
at room temperature for two hours and four hours, and after storage
at 4.degree. C. for 2 hours, four hours, and 24 hours.
[0130] The results of the freeze-thaw stability analysis are
summarized in Table 7. The % recovery of each analyte was
calculated as a percentage of the analyte measured in the sample
prior to any freeze-thaw cycles.
TABLE-US-00008 TABLE 7 Freeze-Thaw Stability of the Analytes in
Urine, Serum, and Plasma Period Urine Sample Serum Sample Plasma
Sample and Recovery Recovery Recovery Analyte Temp Concentration
(%) Concentration (%) Concentration (%) Calbindin Control 212 100
31 100 43 100 (ng/mL) 1X 221 104 30 96 41 94 2X 203 96 30 99 39 92
3X 234 110 30 97 40 93 Clusterin 0 315 100 232 100 187 100 (ng/mL)
1X 329 104 227 98 177 95 2X 341 108 240 103 175 94 3X 379 120 248
107 183 98 CTGF 0 6.7 100 1.5 100 1.2 100 (ng/mL) 1X 7.5 112 1.3 82
1.2 94 2X 6.8 101 1.4 90 1.2 100 3X 7.7 115 1.2 73 1.3 107 GST- 0
12 100 23 100 11 100 alpha 1X 13 104 24 105 11 101 (ng/mL) 2X 14
116 21 92 11 97 3X 14 111 23 100 12 108 KIM-1 0 1.7 100 0.24 100
0.24 100 (ng/mL) 1X 1.7 99 0.24 102 0.22 91 2X 1.7 99 0.22 94 0.19
78 3X 1.8 107 0.23 97 0.22 93 VEGF 0 1,530 100 1,245 100 674 100
(pg/mL) 1X 1,575 103 1,205 97 652 97 2X 1,570 103 1,140 92 612 91
3X 1,700 111 1,185 95 670 99 .beta.-2M 0 0.0070 100 1.2 100 15 100
(.mu.g/mL) 1X 0.0073 104 1.1 93 14 109 2X 0.0076 108 1.2 103 15 104
3X 0.0076 108 1.1 97 13 116 Cystatin C 0 1,240 100 1,330 100 519
100 (ng/mL) 1X 1,280 103 1,470 111 584 113 2X 1,410 114 1,370 103
730 141 3X 1,420 115 1,380 104 589 113 NGAL 0 45 100 245 100 84 100
(ng/mL) 1X 46 102 179 114 94 112 2X 47 104 276 113 91 108 3X 47 104
278 113 91 109 Osteopontin 0 38 100 1.7 100 5.0 100 (ng/mL) 1X 42
110 1.8 102 5.5 110 2X 42 108 1.5 87 5.5 109 3X 42 110 1.3 77 5.4
107 TIMP-1 0 266 100 220 100 70 100 (ng/mL) 1X 265 100 220 10 75
108 2X 255 96 215 98 77 110 3X 295 111 228 104 76 109 A-1M 0 14 100
26 100 4.5 100 (.mu.g/mL) 1X 13 92 25 96 4.2 94 2X 15 107 25 96 4.3
97 3X 16 116 23 88 4.0 90 THP 0 4.6 100 31 100 9.2 100 (.mu.g/mL)
1X 4.4 96 31 98 8.8 95 2X 5.0 110 31 100 9.2 100 3X 5.2 114 27 85
9.1 99 TFF-3 0 4.6 100 24 100 22 100 (.mu.g/mL) 1X 4.4 96 23 98 22
103 2X 5.0 110 24 103 22 101 3X 5.2 114 19 82 22 102
[0131] The results of the short-term stability assessment are
summarized in Table 8. The % recovery of each analyte was
calculated as a percentage of the analyte measured in the sample
prior to any short-term storage.
TABLE-US-00009 TABLE 8 Short-Term Stability of Analytes in Urine,
Serum, and Plasma Storage Urine Sample Serum Sample Plasma Sample
Time/ Sample Recovery Sample Recovery Sample Recovery Analyte Temp
Conc. (%) Conc. (%) Conc. (%) Calbindin Control 226 100 33 100 7
100 (ng/mL) 2 hr/ 242 107 30 90 6.3 90 room temp 2 hr. @ 228 101 29
89 6.5 93 4.degree. C. 4 hr @ 240 106 28 84 5.6 79 room temp 4 hr.
@ 202 89 29 86 5.5 79 4.degree. C. 24 hr. @ 199 88 26 78 7.1 101
4.degree. C. Clusterin Control 185 100 224 100 171 100 (ng/mL) 2 hr
@ 173 94 237 106 180 105 room temp 2 hr. @ 146 79 225 100 171 100
4.degree. C. 4 hr @ 166 89 214 96 160 94 room temp 4 hr. @ 157 85
198 88 143 84 4.degree. C. 24 hr. @ 185 100 207 92 162 94 4.degree.
C. CTGF Control 1.9 100 8.8 100 1.2 100 (ng/mL) 2 hr @ 1.9 99 6.7
76 1 83 room temp 2 hr. @ 1.8 96 8.1 92 1.1 89 4.degree. C. 4 hr @
2.1 113 5.6 64 1 84 room temp 4 hr. @ 1.7 91 6.4 74 0.9 78
4.degree. C. 24 hr. @ 2.2 116 5.9 68 1.1 89 4.degree. C. GST-
Control 14 100 21 100 11 100 alpha 2 hr @ 11 75 23 107 11 103
(ng/mL) room temp 2 hr. @ 13 93 22 104 9.4 90 4.degree. C. 4 hr @
11 79 21 100 11 109 room temp 4 hr. @ 12 89 21 98 11 100 4.degree.
C. 24 hr. @ 13 90 22 103 14 129 4.degree. C. KIM-1 Control 1.5 100
0.23 100 0.24 100 (ng/mL) 2 hr @ 1.2 78 0.2 86 0.22 90 room temp 2
hr. @ 1.6 106 0.23 98 0.21 85 4.degree. C. 4 hr @ 1.3 84 0.19 82
0.2 81 room temp 4 hr. @ 1.4 90 0.22 93 0.19 80 4.degree. C. 24 hr.
@ 1.1 76 0.18 76 0.23 94 4.degree. C. VEGF Control 851 100 1215 100
670 100 (pg/mL) 2 hr @ 793 93 1055 87 622 93 room temp 2 hr. @ 700
82 1065 88 629 94 4.degree. C. 4 hr @ 704 83 1007 83 566 84 room
temp 4 hr. @ 618 73 1135 93 544 81 4.degree. C. 24 hr. @ 653 77
1130 93 589 88 4.degree. C. .beta.-2M Control 0.064 100 2.6 100 1.2
100 (.mu.g/mL) 2 hr @ 0.062 97 2.4 92 1.1 93 room temp 2 hr. @
0.058 91 2.2 85 1.2 94 4.degree. C. 4 hr @ 0.064 101 2.2 83 1.2 94
room temp 4 hr. @ 0.057 90 2.2 85 1.2 98 4.degree. C. 24 hr. @ 0.06
94 2.5 97 1.3 103 4.degree. C. Cystatin C Control 52 100 819 100
476 100 (ng/mL) 2 hr @ 50 96 837 102 466 98 room temp 2 hr. @ 44 84
884 108 547 115 4.degree. C. 4 hr @ 49 93 829 101 498 105 room temp
4 hr. @ 46 88 883 108 513 108 4.degree. C. 24 hr. @ 51 97 767 94
471 99 4.degree. C. NGAL Control 857 100 302 100 93 100 (ng/mL) 2
hr @ 888 104 287 95 96 104 room temp 2 hr. @ 923 108 275 91 92 100
4.degree. C. 4 hr @ 861 101 269 89 88 95 room temp 4 hr. @ 842 98
283 94 94 101 4.degree. C. 24 hr. @ 960 112 245 81 88 95 4.degree.
C. Osteopontin Control 2243 100 6.4 100 5.2 100 (ng/mL) 2 hr @ 2240
100 6.8 107 5.9 114 room temp 2 hr. @ 2140 95 6.4 101 6.2 120
4.degree. C. 4 hr @ 2227 99 6.9 108 5.8 111 room temp 4 hr. @ 2120
95 7.7 120 5.2 101 4.degree. C. 24 hr. @ 2253 100 6.5 101 6 116
4.degree. C. TIMP-1 Control 17 100 349 100 72 100 (ng/mL) 2 hr @ 17
98 311 89 70 98 room temp 2 hr. @ 16 94 311 89 68 95 4.degree. C. 4
hr @ 17 97 306 88 68 95 room temp 4 hr. @ 16 93 329 94 74 103
4.degree. C. 24 hr. @ 18 105 349 100 72 100 4.degree. C. A-1M
Control 3.6 100 2.2 100 1 100 (.mu.g/mL) 2 hr @ 3.5 95 2 92 1 105
room temp 2 hr. @ 3.4 92 2.1 97 0.99 99 4.degree. C. 4 hr @ 3.2 88
2.2 101 0.99 96 room temp 4 hr. @ 3 82 2.2 99 0.97 98 4.degree. C.
24 hr. @ 3 83 2.2 100 1 101 4.degree. C. THP Control 1.2 100 34 100
2.1 100 (.mu.g/mL) 2 hr @ 1.2 99 34 99 2 99 room temp 2 hr. @ 1.1
90 34 100 2 98 4.degree. C. 4 hr @ 1.1 88 27 80 2 99 room temp 4
hr. @ 0.95 79 33 97 2 95 4.degree. C. 24 hr. @ 0.91 76 33 98 2.4
116 4.degree. C. TFF-3 Control 1230 100 188 100 2240 100 (.mu.g/mL)
2 hr @ 1215 99 179 95 2200 98 room temp 2 hr. @ 1200 98 195 104
2263 101 4.degree. C. 4 hr @ 1160 94 224 119 2097 94 room temp 4
hr. @ 1020 83 199 106 2317 103 4.degree. C. 24 hr. @ 1030 84 229
122 1940 87 4.degree. C.
[0132] The results of this experiment demonstrated that the
analytes associated with renal disorders tested were suitably
stable over several freeze/thaw cycles, and up to 24 hrs. of
storage at a temperature of 4.degree. C.
Example 8
Diagnosis of Renal Damage Using Detection of Analytes in Human
Urine Samples
[0133] To assess the effectiveness of a human kidney toxicity panel
to detect renal damage due to disease states, the following
experiment was conducted. Urine samples were obtained from healthy
control patients (n=5), renal cancer patients (n=4) and "other"
cancer patients (n=8) afflicted with lung cancer, pancreatic
cancer, liver cancer, or colon cancer. All urine samples were
diluted as described in Example 4 and subjected to a
sandwich-capture assay as described in Example 1. Urine
concentrations of analytes included in a human kidney toxicity
panel were measured by the assay, including alpha-1 microglobulin
(A1M), beta-2 microglobulin (B2M), calbindin, clusterin, CTGF,
cystatin C, GST-alpha, KIM-1, NGAL, osteopontin (OPN), THP, TIMP-1,
TFF-3, and VEGF.
[0134] FIG. 1 summarizes the urine concentrations of those analytes
that differed significantly from control urine concentrations. The
urine concentrations of A1M, NGAL, and THP were slightly elevated
for the renal cancer patient group and more significantly elevated
for the "other" cancer patient group. Urine B2M concentrations
appeared to be elevated for both the renal cancer and "other"
cancer patient groups, although the BRM concentrations exhibited
more variability than the other analyte concentrations shown in
FIG. 1.
[0135] The results of this experiment demonstrated that panels of
analytes detected in urine samples were capable of identifying
patients having renal damage resulting from renal cancer and other
cancers.
Example 9
Analysis of Kidney Biomarkers in Plasma and Urine from Patients
with Renal Injury
[0136] A screen for potential protein biomarkers in relation to
kidney toxicity/damage was performed using a panel of biomarkers,
in a set of urine and plasma samples from patients with documented
renal damage. The investigated patient groups included diabetic
nephropathy (DN), obstructive uropathy (OU), analgesic abuse (AA)
and glomerulonephritis (GN) along with age, gender and BMI matched
control groups. Multiplexed immunoassays were applied in order to
quantify the following protein analytes: Alpha-1 Microglobulin
(.alpha.1M), KIM-1, Microalbumin, Beta-2-Microglobulin (.beta.32M),
Calbindin, Clusterin, CystatinC, TreFoilFactor-3 (TFF-3), CTGF,
GST-alpha, VEGF, Calbindin, Osteopontin, Tamm-HorsfallProtein
(THP), TIMP-1 and NGAL.
[0137] Li-Heparin plasma and mid-stream spot urine samples were
collected from four different patient groups. Samples were also
collected from age, gender and BMI matched control subjects. 20
subjects were included in each group resulting in a total number of
160 urine and plasma samples. All samples were stored at
-80.degree. C. before use. Glomerular filtration rate for all
samples was estimated using two different estimations (Modification
of Diet in Renal Disease or MDRD, and the Chronic Kidney Disease
Epidemiology Collaboration or CKD-EPI) to outline the eGFR
(estimated glomerular filtration rate) distribution within each
patient group (FIG. 2). Protein analytes were quantified in human
plasma and urine using multiplexed immunoassays in the Luminex
xMAP.TM. platform. The microsphere-based multiplex immunoassays
consist of antigen-specific antibodies and optimized reagents in a
capture-sandwich format. Output data was given as g/ml calculated
from internal standard curves. Because urine creatinine (uCr)
correlates with renal filtration rate, data analysis was performed
without correction for uCr. Univariate and multivariate data
analysis was performed comparing all case vs. control samples as
well as cases vs. control samples for the various disease
groups.
[0138] The majority of the measured proteins showed a correlation
to eGFR. Measured variables were correlated to eGFR using Pearson's
correlations coefficient, and samples from healthy controls and all
disease groups were included in the analysis. 11 and 7 proteins
displayed P-values below 0.05 for plasma and urine (Table 9)
respectively.
TABLE-US-00010 TABLE 9 Correlation analysis of eGFR and variables
for all case samples URINE PLASMA Variable Pearson's r P-Value
Variable Pearson's r P-Value Alpha-1- -0.08 0.3 Alpha-1- -0.33
Microglobulin Microglobulin Beta-2- -0.23 0.003 Beta-2- -0.39
Microglobulin Microglobulin Calbindin -0.16 0.04 Calbindin -0.18
<0.02 Clusterin -0.07 0.4 Clusterin -0.51 CTGF -0.08 0.3 CTGF
-0.05 0.5 Creatinine -0.32 Cystatin-C -0.42 <0.0001 Cystatin-C
-0.24 0.002 GST-alpha -0.12 0.1 GST-alpha -0.11 0.2 KIM-1 -0.17
0.03 KIM-1 -0.08 0.3 NGAL -0.28 <0.001 Microalbumin_UR -0.17
0.03 Osteopontin -0.33 NGAL -0.15 0.07 THP -0.31 Osteopontin -0.19
0.02 TIMP-1 -0.28 <0.001 THP -0.05 0.6 TFF3 -0.38 TIMP-1 -0.19
0.01 VEGF -0.14 0.08 TFF2 -0.09 0.3 VEGF -0.07 0.4 P values
<0.0001 are shown in bold italics P values <0.005 are shown
in bold P values <0.05 are shown in italics
[0139] For the various disease groups, univariate statistical
analysis revealed that in a direct comparison (T-test) between
cases and controls, a number of proteins were differentially
expressed in both urine and plasma (Table 10 and FIG. 3). In
particular, clusterin showed a marked differential pattern in
plasma.
TABLE-US-00011 TABLE 10 Differentially regulated proteins by
univariate statistical analysis Group Matrix Protein p-value AA
Urine Calbindin 0.016 AA Urine NGAL 0.04 AA Urine Osteopontin 0.005
AA Urine Creatinine 0.001 AA Plasma Calbindin 0.05 AA Plasma
Clusterin 0.003 AA Plasma KIM-1 0.03 AA Plasma THP 0.001 AA Plasma
TIMP-1 0.02 DN Urine Creatinine 0.04 DN Plasma Clusterin 0.006 DN
Plasma KIM-1 0.01 GN Urine Creatinine 0.004 GN Urine Microalbumin
0.0003 GN Urine NGAL 0.05 GN Urine Osteopontin 0.05 GN Urine TFF3
0.03 GN Plasma Alpha 1 Microglobulin 0.002 GN Plasma Beta 2
Microglobulin 0.03 GN Plasma Clusterin 0.00 GN Plasma Cystatin C
0.01 GN Plasma KIM-1 0.003 GN Plasma NGAL 0.03 GN Plasma THP 0.001
GN Plasma TIMP-1 0.003 GN Plasma TFF3 0.01 GN Plasma VEGF 0.02 OU
Urine Clusterin 0.02 OU Urine Microalbumin 0.007 OU Plasma
Clusterin 0.00
[0140] Application of multivariate analysis yielded statistical
models that predicted disease from control samples (plasma results
are shown in FIG. 4).
[0141] In conclusion, these results form a valuable base for
further studies on these biomarkers in urine and plasma both
regarding baseline levels in normal populations and regarding the
differential expression of the analytes in various disease groups.
Using this panel of analytes, error rates from adaboosting and/or
random forest were low enough (<10%) to allow a prediction model
to differentiate between control and disease patient samples.
Several of the analytes showed a greater correlation to eGFR in
plasma than in urine.
Example 10
Statistical Analysis of Kidney Biomarkers in Plasma and Urine from
Patients with Renal Injury
[0142] Urine and plasma samples were taken from 80 normal control
group subjects and 20 subjects from each of four disorders:
analgesic abuse, diabetic nephropathy, glomerulonephritis, and
obstructive uropathy. The samples were analyzed for the quantity
and presence of 16 different proteins (alpha-1 microglobulin
(.alpha.1M), beta-2 microglobulin (.beta.2M), calbindin, clusterin,
CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL,
osteopontin, THP, TIMP-1, TFF-3, and VEGF) as described in Example
1 above. The goal was to determine the analytes that distinguish
between a normal sample and a diseased sample, a normal sample and
an obstructive uropathy (OU) sample, and finally, an
glomerulonephritis sample from the other disease samples (diabetic
nephropathy (DN), analgesic abuse (AA), and glomerulonephritis
(GN)).
[0143] From the above protein analysis data, bootstrap analysis was
used to estimate the future performance of several classification
algorithms. For each bootstrap run, training data and testing data
was randomly generated. Then, the following algorithms were applied
on the training data to generate models and then apply the models
to the testing data to make predictions: automated Matthew's
classification algorithm, classification and regression tree
(CART), conditional inference tree, bagging, random forest,
boosting, logistic regression, SVM, and Lasso. The accuracy rate
and ROC areas were recorded for each method on the prediction of
the testing data. The above was repeated 100 times. The mean and
the standard deviation of the accuracy rates and of the ROC areas
were calculated.
[0144] The mean error rates and AUROC were calculated from urine
and AUROC was calculated from plasma for 100 runs of the above
method for each of the following comparisons: disease (AA+GN+OU+DN)
vs. normal (FIG. 5, Table 11), AA vs. normal (FIG. 7, Table 13), DN
vs. AA (FIG. 9, Table 15, AA vs. GN (FIG. 11, Table 17), and AA vs.
OU (FIG. 13, Table 19).
[0145] The average relative importance of 16 different analytes
(alpha-1 microglobulin, beta-2 microglobulin, calbindin, clusterin,
CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL,
osteopontin, THP, TIMP-1, TFF-3, and VEGF) and 4 different clinical
variables (weight, BMI, age, and gender) from 100 runs were
analyzed with two different statistical methods--random forest
(plasma and urine samples) and boosting (urine samples)--for each
of the following comparisons: disease (AA+GN+OU+DN) vs. normal
(FIG. 6, Table 12), AA vs. normal (FIG. 8, Table 14), DN vs. AA
(FIG. 10, Table 16), AA vs. GN (FIG. 12, Table 18), and AA vs. OU
(FIG. 14, Table 20).
TABLE-US-00012 TABLE 11 Disease v. Normal Standard Mean deviation
method AUROC AUROC random 0.931 0.039 forest bagging 0.919 0.045
svm 0.915 0.032 boosting 0.911 0.06 lasso 0.897 0.044 logistic
0.891 0.041 regression ctree 0.847 0.046 cart 0.842 0.032 matt 0.83
0.023
TABLE-US-00013 TABLE 12 Disease v. Normal relative analyte
importance Creatinine 11.606 Kidney_Injury_M 8.486 Tamm_Horsfall_P
8.191 Total_Protein 6.928 Osteopontin 6.798 Neutrophil_Gela 6.784
Tissue_Inhibito 6.765 Vascular_Endoth 6.716 Trefoil_Factor.sub.--
6.703 Cystatin_C 6.482 Alpha_1_Microgl 6.418 Beta_2_Microglo 6.228
Glutathione_S_T 6.053 clusterin 5.842
TABLE-US-00014 TABLE 13 AA v. NL Standard deviation Mean of method
AUROC AUROC cart 1 0 bagging 1 0 boosting 1 0 lasso 0.998 0.008
ctree 0.998 0.015 random 0.997 0.012 forest svm 0.977 0.033
logistic 0.933 0.092 regression matt 0.873 0.112
TABLE-US-00015 TABLE 14 AA v. NL Relative analyte importance
Creatinine 17.800 Tissue_Inhibito 9.953 Total_Protein 8.837
Tamm_Horsfall_P 7.379 Cystatin_C 6.237 Kidney_Injury_M 6.174
Beta_2_Microglo 5.915 Neutrophil_Gela 5.761 Alpha_1_Microgl 5.742
Trefoil_Factor.sub.-- 5.736 Osteopontin 5.561 Vascular_Endoth 5.338
clusterin 4.892 Glutathione_S_T 4.675
TABLE-US-00016 TABLE 15 AA v. DN Standard Mean deviation method
AUROC AUROC lasso 0.999 0.008 random 0.989 0.021 forest svm 0.988
0.039 boosting 0.988 0.022 bagging 0.972 0.036 logistic 0.969 0.057
regression cart 0.93 0.055 ctree 0.929 0.063 matt 0.862 0.12
TABLE-US-00017 TABLE 16 AA v. DN Relative analyte importance
Creatinine 17.57 Total_Protein 10.90 Tissue_Inhibito 8.77 clusterin
6.89 Glutathione_S_T 6.24 Alpha_1_Microgl 6.15 Beta_2_Microglo 6.06
Cystatin_C 5.99 Trefoil_Factor.sub.-- 5.88 Kidney_Injury_M 5.49
Vascular_Endoth 5.38 Tamm_Horsfall_P 5.33 Osteopontin 4.86
Neutrophil_Gela 4.47
TABLE-US-00018 TABLE 17 AA v. GN Standard deviation Mean of method
AUROC AUROC svm 0.689 0.11 boosting 0.675 0.102 bagging 0.674 0.106
random 0.66 0.096 forest matt 0.631 0.085 cart 0.626 0.089 logistic
0.614 0.091 regression lasso 0.606 0.102 ctree 0.53 0.061
TABLE-US-00019 TABLE 18 AA v. GN Relative analyte importance
Creatinine 10.780 Alpha_1_Microgl 8.847 Kidney_Injury_M 8.604
clusterin 8.109 Total_Protein 7.679 Glutathione_S_T 7.493
Neutrophil_Gela 6.721 Vascular_Endoth 6.461 Cystatin_C 6.444
Beta_2_Microglo 6.261 Trefoil_Factor.sub.-- 6.184 Tamm_Horsfall_P
5.872 Tissue_Inhibito 5.690 Osteopontin 4.855
TABLE-US-00020 TABLE 19 AA v. OU Standard deviation Mean of method
AUROC AUROC random 0.814 0.11 forest bagging 0.792 0.115 svm 0.788
0.112 lasso 0.786 0.118 boosting 0.757 0.117 matt 0.687 0.111
logistic 0.683 0.116 regression cart 0.665 0.097 ctree 0.659
0.118
TABLE-US-00021 TABLE 20 AA v. OU Relative analyte importance
Total_Protein 11.502 Tissue_Inhibito 9.736 Cystatin_C 9.161
Alpha_1_Microgl 8.637 Trefoil_Factor.sub.-- 7.329 Osteopontin 7.326
Beta_2_Microglo 6.978 Neutrophil_Gela 6.577 Glutathione_S_T 6.100
Tamm_Horsfall_P 6.066 Kidney_Injury_M 6.038 Vascular_Endoth 5.946
clusterin 4.751 Creatinine 3.854
[0146] It should be appreciated by those of skill in the art that
the techniques disclosed in the examples above represent techniques
discovered by the inventors to function well in the practice of the
invention. Those of skill in the art should, however, in light of
the present disclosure, appreciate that many changes can be made in
the specific embodiments that are disclosed and still obtain a like
or similar result without departing from the spirit and scope of
the invention, therefore all matter set forth or shown in the
accompanying drawings is to be interpreted as illustrative and not
in a limiting sense.
* * * * *