U.S. patent application number 12/689102 was filed with the patent office on 2010-05-13 for biomarkers for differentiating between type 1 and type 2 diabetes.
Invention is credited to Mark A. Atkinson, Tamir M. Ellis, Alba Esther Morales, Clive H. Wasserfall.
Application Number | 20100120629 12/689102 |
Document ID | / |
Family ID | 35064202 |
Filed Date | 2010-05-13 |
United States Patent
Application |
20100120629 |
Kind Code |
A1 |
Ellis; Tamir M. ; et
al. |
May 13, 2010 |
Biomarkers for Differentiating Between Type 1 and Type 2
Diabetes
Abstract
Biomarkers that are diagnostic of type 1 diabetes, type 2
diabetes and/or diabetic disorder are identified. Detection of
different biomarkers of the invention are also diagnostic of the
degree of severity of type 1 diabetes, type 2 diabetes and/or
diabetic disorder. An analysis includes the parameters of matching
for BMI and Tanner stage. Receiver-operator characteristic (ROC)
curves were established to assess association of the biomarkers
with a disease.
Inventors: |
Ellis; Tamir M.;
(Gainesville, FL) ; Morales; Alba Esther;
(Maumelle, AR) ; Atkinson; Mark A.; (Gainesville,
FL) ; Wasserfall; Clive H.; (Gainesville,
FL) |
Correspondence
Address: |
SALIWANCHIK LLOYD & SALIWANCHIK;A PROFESSIONAL ASSOCIATION
PO Box 142950
GAINESVILLE
FL
32614
US
|
Family ID: |
35064202 |
Appl. No.: |
12/689102 |
Filed: |
January 18, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10873101 |
Jun 21, 2004 |
7648825 |
|
|
12689102 |
|
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|
60480041 |
Jun 20, 2003 |
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Current U.S.
Class: |
506/12 ;
435/29 |
Current CPC
Class: |
G01N 33/74 20130101;
Y10T 436/24 20150115; G01N 33/564 20130101; Y10T 436/145555
20150115; G01N 33/6863 20130101; G01N 33/6893 20130101; G01N 33/92
20130101; Y10S 436/811 20130101; G01N 2800/042 20130101 |
Class at
Publication: |
506/12 ;
435/29 |
International
Class: |
C12Q 1/02 20060101
C12Q001/02; C40B 30/10 20060101 C40B030/10 |
Claims
1. A method for differentiating between type 1 and type 2 diabetes
in a subject, the method comprising the steps of: (a) obtaining a
sample from the subject; (b) determining the amount of at least one
biomarker in the sample; and (c) correlating the amount of said
biomarker in the sample with the presence of either type 1 and type
2 diabetes in the subject.
2. The method of claim 1, wherein the levels of at least two
biomarkers in the sample are determined and extrapolated into a
ratio and the ratio is correlated with the presence of either type
1 or type 2 diabetes in the subject.
3. A method for differentiating between type 1 and type 2 diabetes
in a subject, the method comprising the steps of: (a) obtaining a
sample from the subject; (b) determining the amount of adiponectin
or leptin in the sample; and (c) correlating the amount of
adiponectin or leptin in the sample with the presence of either
type 1 or type 2 diabetes in the subject.
4. The method of claim 3, wherein the levels of both adiponectin
and leptin in the sample are determined and extrapolated into a
ratio of adiponectin:leptin and/or leptin:adiponectin, and the
ratio is correlated with the presence of either type 1 or type 2
diabetes in the subject.
5. A method for detection and diagnosis of type 1 diabetes, type 2
diabetes and/or diabetic disorder comprising: detecting at least
one or more protein biomarkers in a subject sample, and;
correlating the detection of one or more protein biomarkers with a
diagnosis of type 1 diabetes, type 2 diabetes and/or diabetic
disorder, wherein the correlation takes into account the detection
of one or more biomarker in each diagnosis, as compared to normal
subjects wherein the one or more protein markers are selected from:
adiponectin, leptin, ghrelin, resistin, autoantibodies to insulin,
autoantibodies to glutamic acid decarboxylase, autoantibodies to
IL-2, autoantibodies to IA-2, incretins, TNF-.alpha., IL-6, and;
correlating the detection of one or more protein biomarkers with a
diagnosis of type 1 diabetes, type 2 diabetes and/or diabetic
disorder, wherein the correlation takes into account the detection
of one or more protein biomarkers in each diagnosis, as compared to
normal subjects.
6. The method of claim 5, wherein one or more protein biomarkers
are used to diagnose type 1 diabetes, type 2 diabetes and/or
diabetic disorder.
7. The method of claim 5, comprising: generating data on
immobilized subject samples on a biochip array, by subjecting the
biochip array to laser ionization and detecting intensity of signal
for mass/charge ratio; and, transforming the data into computer
readable form; and executing an algorithm that classifies the data
according to user input parameters, for detecting signals that
represent markers present in patients suffering from type 1
diabetes, type 2 diabetes and/or diabetic disorder and are lacking
in formal subject controls.
8. A method for detection and diagnosis of type 1 diabetes, type 2
diabetes and/or diabetic disorder comprising: detecting at least
one or more protein biomarkers in a subject sample, and;
correlating the detection of one or more protein biomarkers with a
diagnosis of type 1 diabetes, type 2 diabetes and/or diabetic
disorder, wherein the correlation takes into account the detection
of one or more biomarker in each diagnosis, as compared to normal
subjects wherein the one or more protein markers are selected from:
adiponectin, leptin, ghrelin, resistin, autoantibodies to insulin,
autoantibodies to glutamic acid decarboxylase, autoantibodies to
IL-2, autoantibodies to IA-2, incretins, TNF-.alpha., and IL-6,
fragments, or variants thereof, and; correlating the detection of
one or more protein biomarkers with a diagnosis of type 1 diabetes,
type 2 diabetes and/or diabetic disorder, wherein the correlation
takes into account the detection of one or more protein biomarkers
in each diagnosis, as compared to normal subjects.
9. The method claim 8, wherein one or more protein biomarkers are
used to diagnose type 1 diabetes, type 2 diabetes and/or diabetic
disorder.
10. A kit for diagnosing type 1 diabetes, type 2 diabetes and/or
diabetic disorder in a subject, the kit comprising: (a) a substrate
for holding a biological sample isolated from a human subject
suspected of having type 1 diabetes, type 2 diabetes and/or
diabetic disorder, (b) an agent that specifically binds at least
one or more of the diabetic proteins; and, (c) printed instructions
for reacting the agent with the biological sample or a portion of
the biological sample to detect the presence or amount of at least
one marker in the biological sample.
Description
[0001] This application is a divisional application of co-pending
U.S. application Ser. No. 10/873,101, filed Jun. 21, 2004; which
claims the benefit of U.S. Provisional Application No. 60/480,041,
filed Jun. 20, 2003; which are hereby incorporated by reference in
their entirety.
FIELD OF THE INVENTION
[0002] The invention provides for the reliable detection and
identification of biomarkers, important for the diagnosis and
prognosis of type 1 diabetes, type 2 diabetes and/or diabetic
disorders. More particularly, the invention relates to compositions
and methods for differentiating between type 1 and type 2 diabetes
by measuring levels of adiponectin and leptin.
BACKGROUND
[0003] A worldwide epidemic exists with respect to diabetes; a fact
in large part due to increased rates of obesity. Recent studies
have established adipose tissue as an endocrine organ capable of
hormone and cytokine secretion. Adiponectin, is an
anti-inflammatory and anti-atherogenic hormone exclusively
synthesized in adipose tissue. Serum adiponectin levels are
decreased in obese adults including those with Type 2 diabetes
(T2D), and increase during weight loss or treatment with
thiazolidinediones. Indeed, adiponectin has been proposed to
independently protect against T2D. Adiponection appears to increase
insulin sensitivity by regulating glucose and lipid metabolism.
Indeed, a major effect of adiponectin involves the enhancement of
insulin action in liver and hence, hepatic glucose output.
[0004] Leptin, another obesity related hormone, is critical in the
regulation of energy balance and body weigh. Like adiponectin, it
also is secreted mainly by adipocytes. However, in contrast to
adiponectin, leptin concentration in serum is decreased in obese
adults including those with T2D. Leptin levels are directly
correlated with total body fat.
[0005] Detection and diagnosis of diabetes has proved to be
difficult to assess in children and adolescents. There is
therefore, an urgent need in the art to identify markers that
discriminate between type 1 and type 2 diabetes and/or metabolic
disorders.
SUMMARY
[0006] The present invention identifies protein markers that are
differentially present in the samples of patients suffering from
type 1 diabetes, type 2 diabetes and/or diabetic disorders as
compared to samples of control subjects. The present invention also
provides sensitive and quick methods and kits that can be used as
an aid for diagnosis of type 1 diabetes, type 2 diabetes and/or
diabetic disorders by detecting these markers. The measurement of
these markers, alone or in combination, in patient samples provides
information that a diagnostician can correlate with a probable
diagnosis of the extent of type 1 diabetes, type 2 diabetes and/or
diabetic disorder.
[0007] In a preferred embodiment, the invention provides a method
for differentiating between type 1 and type 2 diabetes in a
pediatric subject. Preferably, the method comprises the steps of
obtaining a serum sample from the subject; determining the amount
of biomarker in the sample; and correlating the amount of at least
one biomarker in the sample with the presence of either type 1 and
type 2 diabetes in the subject.
[0008] In another preferred embodiment, the levels of at least two
biomarkers in the sample are determined and extrapolated into a
ratio and the ratio is correlated with the presence of either type
1 and type 2 diabetes in the subject. Preferably, the ratio is
calculated by a multivariant analysis associating biomarker levels
with anthropometrical parameters and disease state.
[0009] In another preferred embodiment, the multivariant analysis
further includes matching for BMI and Tanner stage. Preferably, the
ratio of biomarkers is determined by differences in
receiver-operator characteristic (ROC) curves. In accordance with
the invention, the calculation of area under the receiver-operator
characteristic (ROC) curves determines the ratio of biomarkers.
[0010] In another preferred embodiment, calculation of the ratio of
biomarkers is determines specificity for type 1 or type 2 diabetes.
Preferably, specific detection of biomarker ratios is about 70% as
compared to a healthy subject, more preferable, specific detection
of biomarker ratios is about 90% as compared to a healthy subject,
more preferable, specific detection of biomarker ratios is up to
100% as compared to a healthy subject.
[0011] In another preferred embodiment, calculation of the ratio of
biomarkers is determines sensitivity for type 1 or type 2 diabetes
biomarkers. Preferably, the sensitivity of detection of biomarker
ratios is about 70% as compared to a healthy subject, more
preferable, sensitivity of detection of biomarker ratios is about
90% as compared to a healthy subject, more preferable, sensitivity
of detection of biomarker ratios is up to 100% as compared to a
healthy subject.
[0012] In another preferred embodiment, the ratio of at least two
biomarkers differentiates between type 1 and type 2 diabetes in a
subject.
[0013] In a preferred embodiment, the invention provides a method
for differentiating between type 1 and type 2 diabetes in a
pediatric subject. Preferably, the method comprises the steps of
obtaining a serum sample from the subject; determining the amount
of adiponectin or leptin in the sample; and correlating the amount
of adiponectin or leptin in the sample with the presence of either
type 1 and type 2 diabetes in the subject. Preferably, other
biomarkers include, but not limited to, for example, ghrelin,
resistin, autoantibodies to insulin, autoantibodies to glutamic
acid decarboxylase, autoantibodies to IL-2, autoantibodies to IA-2,
incretins, TNF-.alpha., and IL-6.
[0014] In another preferred embodiment, the levels of both
adiponectin and leptin in the sample are determined and
extrapolated into a ratio (adiponectin:leptin or
leptin:adiponectin), or any combination thereof, including, but not
limited to ghrelin, resistin, autoantibodies to insulin,
autoantibodies to glutamic acid decarboxylase, autoantibodies to
IL-2, autoantibodies to IA-2, incretins, TNF-.alpha., and IL-6, and
the ratio is correlated with the presence of either type 1 and type
2 diabetes in the subject.
[0015] In another preferred embodiment, the ratio is calculated by
a multivariant analysis associating serum adiponectin and leptin
levels with anthropometrical parameters and disease state.
Preferably, the multivariant analysis further includes matching for
BMI and Tanner stage.
[0016] In another preferred embodiment, the ratio of adiponectin
and leptin biomarkers is determined by differences in
receiver-operator characteristic (ROC) curves. In accordance with
the invention, calculation of area under the receiver-operator
characteristic (ROC) curves determines the ratio of adiponectin and
lectin biomarkers.
[0017] In another preferred embodiment, calculation of the ratio of
adiponectin and leptin determines specificity for type 1 or type 2
diabetes. Preferably, wherein specific detection of adiponectin and
lectin biomarker ratios is about 70% as compared to a healthy
subject, more preferable, specific detection of adiponectin and
lectin biomarker ratios is about 90% as compared to a healthy
subject, more preferable, specific detection of adiponectin and
lectin biomarker ratios is up to 100% as compared to a healthy
subject.
[0018] In another preferred embodiment, calculation of the ratio of
adiponectin and leptin determines sensitivity for type 1 or type 2
diabetes. Preferably, sensitivity of detection of adiponectin and
lectin biomarker ratios is about 70% as compared to a healthy
subject, more preferable, sensitivity of detection of adiponectin
and lectin biomarker ratios is about 90% as compared to a healthy
subject, more preferable, sensitivity of detection of adiponectin
and lectin biomarker ratios is up to 100% as compared to a healthy
subject.
[0019] In another aspect, preferably a single biomarker is used in
combination with one or more biomarkers from normal, healthy
individuals for diagnosing type 1 diabetes, type 2 diabetes and/or
diabetic disorder and progression of disease, more preferably a
plurality of the markers are used in combination with one or more
biomarkers from normal, healthy individuals for diagnosing type 1
diabetes, type 2 diabetes and/or diabetic disorder and progression
of disease. It is preferred that one or more protein biomarkers are
used in comparing protein profiles from patients susceptible to, or
suffering from type 1 diabetes, type 2 diabetes and/or diabetic
disorder diagnosis, with normal subjects. For example, adiponectin,
leptin, ghrelin, resistin, autoantibodies to insulin,
autoantibodies to glutamic acid decarboxylase, autoantibodies to
IL-2, autoantibodies to IA-2, incretins, TNF-.alpha., and IL-6,
fragments, variants or any combination thereof.
[0020] In another preferred embodiment, detection methods include
use of a biochip array. Biochip arrays useful in the invention
include protein and nucleic acid arrays. One or more markers are
immobilized on the biochip array and subjected to laser ionization
to detect the molecular weight of the markers. Analysis of the
markers is, for example, by molecular weight of the one or more
markers against a threshold intensity that is normalized against
total ion current. Preferably, logarithmic transformation is used
for reducing peak intensity ranges to limit the number of markers
detected.
[0021] In another preferred method, data is generated on
immobilized subject samples on a biochip array, by subjecting said
biochip array to laser ionization and detecting intensity of signal
for mass/charge ratio; and, transforming the data into computer
readable form; and executing an algorithm that classifies the data
according to user input parameters, for detecting signals that
represent markers present in type 1 diabetes, type 2 diabetes
and/or diabetic disorder patients and are lacking in non-diabetic
and/or non-diseased subject controls.
[0022] Preferably the biochip surfaces are, for example, ionic,
anionic, comprising immobilized nickel ions, a mixture of positive
and negative ions, one or more antibodies, single or double
stranded nucleic acids, proteins, peptides or fragments thereof,
amino acid probes, phage display libraries.
[0023] In other preferred methods one or more of the markers are
detected using laser desorption/ionization mass spectrometry,
comprising, providing a probe adapted for use with a mass
spectrometer comprising an adsorbent attached thereto, and;
contacting the subject sample with the adsorbent, and; desorbing
and ionizing the marker or markers from the probe and detecting the
deionized/ionized markers with the mass spectrometer.
[0024] Preferably, the laser desorption/ionization mass
spectrometry comprises, providing a substrate comprising an
adsorbent attached thereto; contacting the subject sample with the
adsorbent; placing the substrate on a probe adapted for use with a
mass spectrometer comprising an adsorbent attached thereto; and,
desorbing and ionizing the marker or markers from the probe and
detecting the desorbed/ionized marker or markers with the mass
spectrometer.
[0025] The adsorbent can for example be, hydrophobic, hydrophilic,
ionic or metal chelate adsorbent, such as, nickel or an antibody,
single- or double stranded oligonucleotide, amino acid, protein,
peptide or fragments thereof.
[0026] In another embodiment, a process for purification of a
biomarker, comprising fractioning a sample comprising one or more
protein biomarkers by size-exclusion chromatography and collecting
a fraction that includes the one or more biomarker; and/or
fractionating a sample comprising the one or more biomarkers by
anion exchange chromatography and collecting a fraction that
includes the one or more biomarkers. Fractionation is monitored for
purity on normal phase and immobilized nickel arrays. Generating
data on immobilized marker fractions on an array, is accomplished
by subjecting said array to laser ionization and detecting
intensity of signal for mass/charge ratio; and, transforming the
data into computer readable form; and executing an algorithm that
classifies the data according to user input parameters, for
detecting signals that represent markers present in type 1
diabetes, type 2 diabetes and/or diabetic disorder patients and are
lacking in non-diabetic and/or non-diseased subject controls.
Preferably fractions are subjected to gel electrophoresis and
correlated with data generated by mass spectrometry. In one aspect,
gel bands representative of potential markers are excised and
subjected to enzymatic treatment and are applied to biochip arrays
for peptide mapping.
[0027] In another preferred embodiment, the presence of certain
biomarkers is indicative of the extent of type 1 diabetes, type 2
diabetes and/or diabetic disorder. For example, detection of one or
more biomarkers would be indicative of type 1 diabetes, type 2
diabetes and/or diabetic disorder and the presence of one or more
would be indicative of the extent of type 1 diabetes, type 2
diabetes and/or diabetic disorder diagnosis. Preferably, the
biomarkers can be compared to a known protein indicative of
diabetes such as the insulin levels.
[0028] Preferred methods for detection and diagnosis of type 1
diabetes, type 2 diabetes and/or diabetic disorder comprise
detecting at least one or more protein biomarkers in a subject
sample, and; correlating the detection of one or more protein
biomarkers with a diagnosis of type 1 diabetes, type 2 diabetes
and/or diabetic disorder, wherein the correlation takes into
account the detection of one or more biomarker in each diagnosis,
as compared to normal subjects, wherein the one or more protein
markers comprise, for example, adiponectin, lectin and insulin.
[0029] In another preferred embodiment, the invention provides a
kit for analyzing type 1 diabetes, type 2 diabetes and/or diabetic
disorder in a subject. The kit, preferably includes: (a) a
substrate for holding a biological sample isolated from a human
subject suspected of having type 1 diabetes, type 2 diabetes and/or
diabetic disorder, (b) an agent that specifically binds at least
one or more of the diabetic proteins; and (c) printed instructions
for reacting the agent with the biological sample or a portion of
the biological sample to detect the presence or amount of at least
one marker in the biological sample.
[0030] Preferably, the biological sample is a fluid, for example,
blood or serum, and the agent can be an antibody, aptamer, or other
molecule that specifically binds at least one or more of the
diabetic proteins. The kit can also include a detectable label such
as one conjugated to the agent, or one conjugated to a substance
that specifically binds to the agent (e.g., a secondary
antibody).
[0031] Other aspects of the invention are described infra.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] FIG. 1 is a series of graphs showing adiponectin levels (A),
leptin (levels), and adiponectin/leptin ratios in the serum of
control (non-diabetic), T1D, and T2D pediatric subjects.
[0033] FIGS. 2A-2F are graphs showing adiponectin and leptin levels
in healthy pediatric subjects and those with diabetes. Serum
adiponectin levels in indicated subject groups with BMI>85
percentile (FIG. 2A), those with Tanner 4 or 5 (FIG. 2B), or all
study participants (FIG. 2C). Serum leptin levels in study subject
groups with BMI>85 percentile (FIG. 2D), those with Tanner 4 or
5 (FIG. 2E), or all study participants (FIG. 2F). Bar represents
mean value. T1D (type 1 diabetes); T2D (type 2 diabetes).
[0034] FIG. 3 is a graph showing adiponectin and leptin levels in
healthy pediatric subjects and those with diabetes. Serum
adiponectin to leptin ratios in indicated subject groups. Bar
represents mean value; y-axis, log.sub.2 scale. T1D (type 1
diabetes); T2D (type 2 diabetes).
[0035] FIG. 4 is a graph showing adiponectin and leptin ratios in
healthy pediatric subjects and those with diabetes as a function of
race. Bar represents mean value; y-axis, log.sub.2 scale. AA
(African American); C (Caucasian); T1D (type 1 diabetes); T2D (type
2 diabetes).
[0036] FIG. 5 is a graph showing the ROC curve for specificity and
sensitivity of adiponectin-to-leptin ratio in those with type 1
diabetes (T1D) versus type 2 diabetes (T2D).
DETAILED DESCRIPTION
[0037] The invention provides the research leading to the present
invention investigated children and adolescents with diabetes for
production of these hormones not only for additional mechanistic
information that could be derived but in addition, to identify any
diagnostic value these markers would provide in discriminating
between these disorders which are sometimes difficult to
distinguish in this age group.
DEFINITIONS
[0038] Prior to setting forth the invention, it may be helpful to
an understanding thereof to set forth definitions of certain terms
that will be used hereinafter.
[0039] "Marker" in the context of the present invention refers to a
polypeptide (of a particular apparent molecular weight) which is
differentially present in a sample taken from patients having
diabetic disorders as compared to a comparable sample taken from
control subjects (e.g., a person with a negative diagnosis, normal
or healthy subject).
[0040] "Complementary" in the context of the present invention
refers to detection of at least two biomarkers, which when detected
together provides increased sensitivity and specificity as compared
to detection of one biomarker alone.
[0041] The phrase "differentially present" refers to differences in
the quantity and/or the frequency of a marker present in a sample
taken from patients having for example, diabetes as compared to a
control subject. For example, a marker can be a polypeptide which
is present at an elevated level or at a decreased level in samples
of patients with diabetes compared to samples of control subjects.
Alternatively, a marker can be a polypeptide which is detected at a
higher frequency or at a lower frequency in samples of patients
compared to samples of control subjects. A marker can be
differentially present in terms of quantity, frequency or both.
[0042] A polypeptide is differentially present between the two
samples if the amount of the polypeptide in one sample is
statistically significantly different from the amount of the
polypeptide in the other sample. For example, a polypeptide is
differentially present between the two samples if it is present at
least about 120%, at least about 130%, at least about 150%, at
least about 180%, at least about 200%, at least about 300%, at
least about 500%, at least about 700%, at least about 900%, or at
least about 1000% greater than it is present in the other sample,
or if it is detectable in one sample and not detectable in the
other.
[0043] Alternatively or additionally, a polypeptide is
differentially present between the two sets of samples if the
frequency of detecting the polypeptide in samples of patients'
suffering from diabetic disorders, is statistically significantly
higher or lower than in the control samples. For example, a
polypeptide is differentially present between the two sets of
samples if it is detected at least about 120%, at least about 130%,
at least about 150%, at least about 180%, at least about 200%, at
least about 300%, at least about 500%, at least about 700%, at
least about 900%, or at least about 1000% more frequently or less
frequently observed in one set of samples than the other set of
samples.
[0044] "Diagnostic" means identifying the presence or nature of a
pathologic condition. Diagnostic methods differ in their
sensitivity and specificity. The "sensitivity" of a diagnostic
assay is the percentage of diseased individuals who test positive
(percent of "true positives"). Diseased individuals not detected by
the assay are "false negatives." Subjects who are not diseased and
who test negative in the assay, are termed "true negatives." The
"specificity" of a diagnostic assay is 1 minus the false positive
rate, where the "false positive" rate is defined as the proportion
of those without the disease who test positive. While a particular
diagnostic method may not provide a definitive diagnosis of a
condition, it suffices if the method provides a positive indication
that aids in diagnosis.
[0045] A "test amount" of a marker refers to an amount of a marker
present in a sample being tested. A test amount can be either in
absolute amount (e.g., .mu.g/ml) or a relative amount (e.g.,
relative intensity of signals).
[0046] A "diagnostic amount" of a marker refers to an amount of a
marker in a subject's sample that is consistent with a diagnosis of
Type 1 or Type 2 diabetes and/or diabetic disorders. A diagnostic
amount can be either in absolute amount (e.g., .mu.g/ml) or a
relative amount (e.g., relative intensity of signals).
[0047] A "control amount" of a marker can be any amount or a range
of amount which is to be compared against a test amount of a
marker. For example, a control amount of a marker can be the amount
of a marker in a person without diabetes. A control amount can be
either in absolute amount (e.g., .mu.g/ml) or a relative amount
(e.g., relative intensity of signals).
[0048] As used herein, "diabetic proteins" refers to any protein
that is detectable in an individual with type 1 or type 2 diabetes,
such as for example, adiponectin, leptin, insulin, ghrelin,
resistin, autoantibodies to insulin, autoantibodies to glutamic
acid decarboxylase, autoantibodies to IL-2, autoantibodies to IA-2,
incretins, TNF-.alpha., and IL-6, fragments, variants or any
combination thereof and the like.
[0049] As used herein, a "pharmaceutically acceptable" component is
one that is suitable for use with humans and/or animals without
undue adverse side effects (such as toxicity, irritation, and
allergic response) commensurate with a reasonable benefit/risk
ratio.
[0050] The terms "patient" or "individual" are used interchangeably
herein, and is meant a mammalian subject to be treated, with human
patients being preferred. In some cases, the methods of the
invention find use in experimental animals, in veterinary
application, and in the development of animal models for disease,
including, but not limited to, rodents including mice, rats, and
hamsters; and primates.
[0051] As used herein, "ameliorated" or "treatment" refers to a
symptom which is approaches a normalized value, e.g., is less than
50% different from a normalized value, preferably is less than
about 25% different from a normalized value, more preferably, is
less than 10% different from a normalized value, and still more
preferably, is not significantly different from a normalized value
as determined using routine statistical tests.
[0052] "Probe" refers to a device that is removably insertable into
a gas phase ion spectrometer and comprises a substrate having a
surface for presenting a marker for detection. A probe can comprise
a single substrate or a plurality of substrates.
[0053] "Substrate" or "probe substrate" refers to a solid phase
onto which an adsorbent can be provided (e.g., by attachment,
deposition, etc.).
[0054] "Adsorbent" refers to any material capable of adsorbing a
marker. The term "adsorbent" is used herein to refer both to a
single material ("monoplex adsorbent") (e.g., a compound or
functional group) to which the marker is exposed, and to a
plurality of different materials ("multiplex adsorbent") to which
the marker is exposed. The adsorbent materials in a multiplex
adsorbent are referred to as "adsorbent species." For example, an
addressable location on a probe substrate can comprise a multiplex
adsorbent characterized by many different adsorbent species (e.g.,
anion exchange materials, metal chelators, or antibodies), having
different binding characteristics. Substrate material itself can
also contribute to adsorbing a marker and may be considered part of
an "adsorbent."
[0055] "Adsorption" or "retention" refers to the detectable binding
between an absorbent and a marker either before or after washing
with an eluant (selectivity threshold modifier) or a washing
solution.
[0056] "Eluant" or "washing solution" refers to an agent that can
be used to mediate adsorption of a marker to an adsorbent. Eluants
and washing solutions are also referred to as "selectivity
threshold modifiers." Eluants and washing solutions can be used to
wash and remove unbound materials from the probe substrate
surface.
[0057] "Resolve," "resolution," or "resolution of marker" refers to
the detection of at least one marker in a sample. Resolution
includes the detection of a plurality of markers in a sample by
separation and subsequent differential detection. Resolution does
not require the complete separation of one or more markers from all
other biomolecules in a mixture. Rather, any separation that allows
the distinction between at least one marker and other biomolecules
suffices.
[0058] "Gas phase ion spectrometer" refers to an apparatus that
measures a parameter which can be translated into mass-to-charge
ratios of ions formed when a sample is volatilized and ionized.
Generally ions of interest bear a single charge, and mass-to-charge
ratios are often simply referred to as mass. Gas phase ion
spectrometers include, for example, mass spectrometers, ion
mobility spectrometers, and total ion current measuring
devices.
[0059] "Mass spectrometer" refers to a gas phase ion spectrometer
that includes an inlet system, an ionization source, an ion optic
assembly, a mass analyzer, and a detector.
[0060] "Laser desorption mass spectrometer" refers to a mass
spectrometer which uses laser as means to desorb, volatilize, and
ionize an analyte.
[0061] "Detect" refers to identifying the presence, absence or
amount of the object to be detected.
[0062] The terms "polypeptide," "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is an analog or mimetic of a corresponding
naturally occurring amino acid, as well as to naturally occurring
amino acid polymers. Polypeptides can be modified, e.g., by the
addition of carbohydrate residues to form glycoproteins. The terms
"polypeptide," "peptide" and "protein" include glycoproteins, as
well as non-glycoproteins.
[0063] "Detectable moiety" or a "label" refers to a composition
detectable by spectroscopic, photochemical, biochemical,
immunochemical, or chemical means. For example, useful labels
include .sup.32P, .sup.35S, fluorescent dyes, electron-dense
reagents, enzymes (e.g., as commonly used in an ELISA),
biotin-streptavidin, dioxigenin, haptens and proteins for which
antisera or monoclonal antibodies are available, or nucleic acid
molecules with a sequence complementary to a target. The detectable
moiety often generates a measurable signal, such as a radioactive,
chromogenic, or fluorescent signal, that can be used to quantify
the amount of bound detectable moiety in a sample. Quantitation of
the signal is achieved by, e.g., scintillation counting,
densitometry, or flow cytometry.
[0064] "Antibody" refers to a polypeptide ligand substantially
encoded by an immunoglobulin gene or immunoglobulin genes, or
fragments thereof, which specifically binds and recognizes an
epitope (e.g., an antigen). The recognized immunoglobulin genes
include the kappa and lambda light chain constant region genes, the
alpha (.alpha.), gamma (.gamma.), delta (.delta.), epsilon
(.epsilon.), and mu (.mu.) heavy chain constant region genes, and
the myriad immunoglobulin variable region genes. Antibodies exist,
e.g., as intact immunoglobulins or as a number of well
characterized fragments produced by digestion with various
peptidases. This includes, e.g., Fab' and F(ab)'.sub.2 fragments.
The term "antibody," as used herein, also includes antibody
fragments either produced by the modification of whole antibodies
or those synthesized de novo using recombinant DNA methodologies.
It also includes polyclonal antibodies, monoclonal antibodies,
chimeric antibodies, humanized antibodies, or single chain
antibodies. "Fc" portion of an antibody refers to that portion of
an immunoglobulin heavy chain that comprises one or more heavy
chain constant region domains, CH.sub.1, CH.sub.2 and CH.sub.3, but
does not include the heavy chain variable region.
[0065] "Immunoassay" is an assay that uses an antibody to
specifically bind an antigen (e.g., a marker). The immunoassay is
characterized by the use of specific binding properties of a
particular antibody to isolate, target, and/or quantify the
antigen.
[0066] The phrase "specifically (or selectively) binds" to an
antibody or "specifically (or selectively) immunoreactive with,"
when referring to a protein or peptide, refers to a binding
reaction that is determinative of the presence of the protein in a
heterogeneous population of proteins and other biologics. Thus,
under designated immunoassay conditions, the specified antibodies
bind to a particular protein at least two times the background and
do not substantially bind in a significant amount to other proteins
present in the sample. Specific binding to an antibody under such
conditions may require an antibody that is selected for its
specificity for a particular protein. For example, polyclonal
antibodies raised to marker "X" from specific species such as rat,
mouse, or human can be selected to obtain only those polyclonal
antibodies that are specifically immunoreactive with marker "X" and
not with other proteins, except for polymorphic variants and
alleles of marker "X". This selection may be achieved by
subtracting out antibodies that cross-react with marker "X"
molecules from other species. A variety of immunoassay formats may
be used to select antibodies specifically immunoreactive with a
particular protein. For example, solid-phase ELISA immunoassays are
routinely used to select antibodies specifically immunoreactive
with a protein (see, e.g., Harlow & Lane, Antibodies, A
Laboratory Manual (1988), for a description of immunoassay formats
and conditions that can be used to determine specific
immunoreactivity). Typically a specific or selective reaction will
be at least twice background signal or noise and more typically
more than 10 to 100 times background.
[0067] "Energy absorbing molecule" or "EAM" refers to a molecule
that absorbs energy from an ionization source in a mass
spectrometer thereby aiding desorption of analyte, such as a
marker, from a probe surface. Depending on the size and nature of
the analyte, the energy absorbing molecule can be optionally used.
Energy absorbing molecules used in MALDI are frequently referred to
as "matrix." Cinnamic acid derivatives, sinapinic acid ("SPA"),
cyano hydroxy cinnamic acid ("CHCA") and dihydroxybenzoic acid are
frequently used as energy absorbing molecules in laser desorption
of bioorganic molecules.
[0068] "Sample" is used herein in its broadest sense. A sample
comprising polynucleotides, polypeptides, peptides, antibodies and
the like may comprise a bodily fluid; a soluble fraction of a cell
preparation, or media in which cells were grown; a chromosome, an
organelle, or membrane isolated or extracted from a cell; genomic
DNA, RNA, or cDNA, polypeptides, or peptides in solution or bound
to a substrate; a cell; a tissue; a tissue print; a fingerprint,
skin or hair; and the like.
[0069] "Substantially purified" refers to nucleic acid molecules or
proteins that are removed from their natural environment and are
isolated or separated, and are at least about 60% free, preferably
about 75% free, and most preferably about 90% free, from other
components with which they are naturally associated.
[0070] "Substrate" refers to any rigid or semi-rigid support to
which nucleic acid molecules or proteins are bound and includes
membranes, filters, chips, slides, wafers, fibers, magnetic or
nonmagnetic heads, gels, capillaries or other tubing, plates,
polymers, and microparticles with a variety of surface forms
including wells, trenches, pins, channels and pores.
[0071] As used herein, "diabetic disorders" refers to complications
due to diabetes. For example, complications such as retinopathy,
nephropathy and neuropathy develop with angiopathy as a prime
factor in diabetic individuals.
[0072] As used herein "diabetes" refers to type I and type II
diabetes. Diabetes is classified according to the types of disease
into insulin dependent diabetes (IDDM; type I diabetes) and
non-insulin dependent diabetes (NIDDM; type II diabetes).
[0073] In a preferred embodiment, detection of one or more
biomarkers is diagnostic for Type 1 and Type 2 diabetes and/or
diabetic disorders. For example, detection of adiponectin and/or
lectin, ghrelin, resistin, autoantibodies to insulin,
autoantibodies to glutamic acid decarboxylase, autoantibodies to
IL-2, autoantibodies to IA-2, incretins, TNF-.alpha., and IL-6,
fragments, peptides or variants thereof. In accordance with the
invention, adjusting for BMI and pubertal stage, adiponectin levels
were elevated in T1D and decreased in T2D. Conversely, elevations
in leptin concentrations were observed in cases of pediatric
T2D.
[0074] In another preferred embodiment, detection of a biomarkers
that are differentially present in an individual are diagnostic of
type 1 diabetes, type 2 diabetes and/or diabetic disorders. For
example, adiponectin/leptin ratios were dramatically different
amongst healthy children (11.8 [95% CI 4.8-18.7]) and those with
T1D (6.1 [3.8-8.3] or T2D (0.4 [0.3-0.5]) (p<0.0001). Other
combinations can include, for example, adiponectin, leptin,
ghrelin, resistin, autoantibodies to insulin, autoantibodies to
glutamic acid decarboxylase, autoantibodies to IL-2, autoantibodies
to IA-2, incretins, TNF-.alpha., and IL-6, fragments, variants or
any combination thereof.
[0075] In another preferred embodiment, the invention provides for
the quantitative detection of biomarkers diagnostic of type 1 and
type 2 diabetes and/or diabetic disorders. Depending on the type
and severity of disease biomarkers are differentially present and
the ratios of these biomarkers are indicative of diabetes and/or
diabetic disorders. For example, the ratio of adiponectin to leptin
as compared to healthy individuals.
[0076] In another preferred embodiment, detection of certain
biomarkers are diagnostic of the specific type of diabetes. For
example, detection of adiponectin and leptin proteins, peptides,
fragments and derivatives thereof is diagnostic of type 1 or type 2
diabetes.
[0077] In another preferred embodiment, type 1, type 2 and/or
diabetic disorders in a subject is analyzed by (a) providing a
biological sample isolated from a subject suspected of having
diabetes; (b) detecting in the sample the presence or amount of at
least one marker selected from one or more biomarker proteins; and
(c) correlating the presence or amount of the marker with the
presence or type of diabetes in the subject. Preferably, a sample
from a diabetic individual, such as serum, adipocytes, pancreatic
cells and the like, in in vitro culture or in situ in an animal
subjects express higher levels of diabetic proteins as compared to
non-diabetic individuals. Preferably, the samples comprise cells,
for example, a biopsy of adipocyte tissue and pancreas are suitable
biological samples for use in the invention. In addition,
adiponectin/lectin are detected in the circulating blood and other
biofluids (e.g. urine, sweat, s saliva, etc.). Thus, other suitable
biological samples include, but not limited to such cells or fluid
secreted from these cells. Obtaining biological fluids such as
blood, plasma, serum, saliva and urine, from a subject is typically
much less invasive and traumatizing than obtaining a solid tissue
biopsy sample. Thus, samples, which are biological fluids, are
preferred for use in the invention.
[0078] A biological sample can be obtained from a subject by
conventional techniques. Blood can be obtained by venipuncture,
while plasma and serum can be obtained by fractionating whole blood
according to known methods. Surgical techniques for obtaining solid
tissue samples are well known in the art.
[0079] Any animal that expresses the diabetic biomarker proteins,
such as for example, adiponectin, leptin, can be used as a subject
from which a biological sample is obtained. Preferably, the subject
is a mammal, such as for example, a human, dog, cat, horse, cow,
pig, sheep, goat, chicken, primate, rat, or mouse. More preferably,
the subject is a human. Particularly preferred are subjects
suspected of having or at risk for developing type 1 type 2
diabetes and related diabetic disorders.
[0080] In a preferred embodiment, samples are obtained from
children and adolescents with type 1 diabetes (n=41), type 2
diabetes (n=17), and nondiabetic individuals of similar age from
the general population (n=43) were investigated. An analysis
included the parameters of matching for BMI and Tanner stage.
Receiver-operator characteristic (ROC) curves were established to
assess these analytes association with a disease.
[0081] The biomarkers of the invention can be detected in a sample
by any means. Methods for detecting the biomarkers are described in
detail in the materials and methods and Examples which follow. For
example, immunoassays, include but are not limited to competitive
and non-competitive assay systems using techniques such as western
blots, radioimmunoassays, ELISA (enzyme linked immunosorbent
assay), "sandwich" immunoassays, immunoprecipitation assays,
precipitin reactions, gel diffusion precipitin reactions,
immunodiffusion assays, fluorescent immunoassays and the like. Such
assays are routine and well known in the art (see, e.g., Ausubel et
al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John
Wiley & Sons, Inc., New York, which is incorporated by
reference herein in its entirety). Exemplary immunoassays are
described briefly below (but are not intended by way of
limitation).
[0082] Immunoprecipitation protocols generally comprise lysing a
population of cells in a lysis buffer such as RITA buffer (1% NP-40
or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl,
0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with
protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF,
aprotinin, sodium vanadate), adding an antibody of interest to the
cell lysate, incubating for a period of time (e.g., 1-4 hours) at
4.degree. C., adding protein A and/or protein G sepharose beads to
the cell lysate, incubating for about an hour or more at 4.degree.
C., washing the beads in lysis buffer and resuspending the beads in
SDS/sample buffer. The ability of the antibody to immunoprecipitate
a particular antigen can be assessed by, e.g., western blot
analysis. One of skill in the art would be knowledgeable as to the
parameters that can be modified to increase the binding of the
antibody to an antigen and decrease the background (e.g.,
pre-clearing the cell lysate with sepharose beads). For further
discussion regarding immunoprecipitation protocols see, e.g.,
Ausubel et al, eds, 1994, Current Protocols in Molecular Biology,
Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.
[0083] Western blot analysis generally comprises preparing protein
samples, electrophoresis of the protein samples in a polyacrylamide
gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the
antigen), transferring the protein sample from the polyacrylamide
gel to a membrane such as nitrocellulose, PVDF or nylon, blocking
the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat
milk), washing the membrane in washing buffer (e.g., PBS-Tween 20),
blocking the membrane with primary antibody (the antibody of
interest) diluted in blocking buffer, washing the membrane in
washing buffer, blocking the membrane with a secondary antibody
(which recognizes the primary antibody, e.g., an anti-human
antibody) conjugated to an enzymatic substrate (e.g., horseradish
peroxidase or alkaline phosphatase) or radioactive molecule (e.g.,
.sup.32P or .sup.125I) diluted in blocking buffer, washing the
membrane in wash buffer, and detecting the presence of the antigen.
One of skill in the art would be knowledgeable as to the parameters
that can be modified to increase the signal detected and to reduce
the background noise. For further discussion regarding western blot
protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in
Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at
10.8.1.
[0084] ELISAs comprise preparing antigen (i.e. diabetic biomarker),
coating the well of a 96 well microtiter plate with the antigen,
adding the antibody of interest conjugated to a detectable compound
such as an enzymatic substrate (e.g., horseradish peroxidase or
alkaline phosphatase) to the well and incubating for a period of
time, and detecting the presence of the antigen. In ELISAs the
antibody of interest does not have to be conjugated to a detectable
compound; instead, a second antibody (which recognizes the antibody
of interest) conjugated to a detectable compound may be added to
the well. Further, instead of coating the well with the antigen,
the antibody may be coated to the well, in this case, a second
antibody conjugated to a detectable compound may be added following
the addition of the antigen of interest to the coated well. One of
skill in the art would be knowledgeable as to the parameters that
can be modified to increase the signal detected as well as other
variations of ELISAs known in the art. For further discussion
regarding ELISAs see, e.g., Ausubel et al, eds, 1994, Current
Protocols in Molecular Biology, Vol. 1, John Wiley & Sons,
Inc., New York at 11.2.1.
Identification of New Markers
[0085] In a preferred embodiment, a biological sample is obtained
from a patient suffering from or susceptible to diabetes.
Biological samples comprising biomarkers from other patients and
control subjects (i.e. normal healthy individuals of similar age,
sex, physical condition) are used as comparisons. Biological
samples are extracted as discussed above. Preferably, the sample is
prepared prior to detection of biomarkers. Typically, preparation
involves fractionation of the sample and collection of fractions
determined to contain the biomarkers. Methods of pre-fractionation
include, for example, size exclusion chromatography, ion exchange
chromatography, heparin chromatography, affinity chromatography,
sequential extraction, gel electrophoresis and liquid
chromatography. The analytes also may be modified prior to
detection. These methods are useful to simplify the sample for
further analysis. For example, it can be useful to remove high
abundance proteins, such as albumin, from blood before
analysis.
[0086] In one embodiment, a sample can be pre-fractionated
according to size of proteins in a sample using size exclusion
chromatography. For a biological sample wherein the amount of
sample available is small, preferably a size selection spin column
is used. In general, the first fraction that is eluted from the
column ("fraction 1") has the highest percentage of high molecular
weight proteins; fraction 2 has a lower percentage of high
molecular weight proteins; fraction 3 has even a lower percentage
of high molecular weight proteins; fraction 4 has the lowest amount
of large proteins; and so on. Each fraction can then be analyzed by
immunoassays, gas phase ion spectrometry, and the like, for the
detection of markers.
[0087] In another embodiment, a sample can be pre-fractionated by
anion exchange chromatography. Anion exchange chromatography allows
pre-fractionation of the proteins in a sample roughly according to
their charge characteristics. For example, a Q anion-exchange resin
can be used (Q HYPER D F, Biosepra), and a sample can be
sequentially eluted with eluants having different pH's. Anion
exchange chromatography allows separation of biomarkers in a sample
that are more negatively charged from other types of biomarkers.
Proteins that are eluted with an eluant having a high pH is likely
to be weakly negatively charged, and a fraction that is eluted with
an eluant having a low pH is likely to be strongly negatively
charged. Thus, in addition to reducing complexity of a sample,
anion exchange chromatography separates proteins according to their
binding characteristics.
[0088] In yet another embodiment, a sample can be pre-fractionated
by heparin chromatography. Heparin chromatography allows
pre-fractionation of the markers in a sample also on the basis of
affinity interaction with heparin and charge characteristics.
Heparin, a sulfated mucopolysaccharide, will bind markers with
positively charged moieties and a sample can be sequentially eluted
with eluants having different pH's or salt concentrations. Markers
eluted with an eluant having a low pH are more likely to be weakly
positively charged. Markers eluted with an eluant having a high pH
are more likely to be strongly positively charged. Thus, heparin
chromatography also reduces the complexity of a sample and
separates markers according to their binding characteristics.
[0089] In yet another embodiment, a sample can be pre-fractionated
by isolating proteins that have a specific characteristic, e.g. are
glycosylated. For example, a blood, or serum sample can be
fractionated by passing the sample over a lectin chromatography
column (which has a high affinity for sugars). Glycosylated
proteins will bind to the lectin column and non-glycosylated
proteins will pass through the flow through. Glycosylated proteins
are then eluted from the lectin column with an eluant containing a
sugar, e.g., N-acetyl-glucosamine and are available for further
analysis.
[0090] Thus there are many ways to reduce the complexity of a
sample based on the binding properties of the proteins in the
sample, or the characteristics of the proteins in the sample.
[0091] In yet another embodiment, a sample can be fractionated
using a sequential extraction protocol. In sequential extraction, a
sample is exposed to a series of adsorbents to extract different
types of biomarkers from a sample. For example, a sample is applied
to a first adsorbent to extract certain proteins, and an eluant
containing non-adsorbent proteins (i.e., proteins that did not bind
to the first adsorbent) is collected. Then, the fraction is exposed
to a second adsorbent. This further extracts various proteins from
the fraction. This second fraction is then exposed to a third
adsorbent, and so on.
[0092] Any suitable materials and methods can be used to perform
sequential extraction of a sample. For example, a series of spin
columns comprising different adsorbents can be used. In another
example, a multi-well comprising different adsorbents at its bottom
can be used. In another example, sequential extraction can be
performed on a probe adapted for use in a gas phase ion
spectrometer, wherein the probe surface comprises adsorbents for
binding biomarkers. In this embodiment, the sample is applied to a
first adsorbent on the probe, which is subsequently washed with an
eluant. Markers that do not bind to the first adsorbent are removed
with an eluant. The markers that are in the fraction can be applied
to a second adsorbent on the probe, and so forth. The advantage of
performing sequential extraction on a gas phase ion spectrometer
probe is that markers that bind to various adsorbents at every
stage of the sequential extraction protocol can be analyzed
directly using a gas phase ion spectrometer.
[0093] In yet another embodiment, biomarkers in a sample can be
separated by high-resolution electrophoresis, e.g., one or
two-dimensional gel electrophoresis. A fraction containing a marker
can be isolated and further analyzed by gas phase ion spectrometry.
Preferably, two-dimensional gel electrophoresis is used to generate
two-dimensional array of spots of biomarkers, including one or more
markers. See, e.g., Jungblut and Thiede, Mass Spectr. Rev.
16:145-162 (1997).
[0094] The two-dimensional gel electrophoresis can be performed
using methods known in the art. See, e.g., Deutscher ed., Methods
In Enzymology vol. 182. Typically, biomarkers in a sample are
separated by, e.g., isoelectric focusing, during which biomarkers
in a sample are separated in a pH gradient until they reach a spot
where their net charge is zero (i.e., isoelectric point). This
first separation step results in one-dimensional array of
biomarkers. The biomarkers in one dimensional array is further
separated using a technique generally distinct from that used in
the first separation step. For example, in the second dimension,
biomarkers separated by isoelectric focusing are further separated
using a polyacrylamide gel, such as polyacrylamide gel
electrophoresis in the presence of sodium dodecyl sulfate
(SDS-PAGE). SDS-PAGE gel allows further separation based on
molecular mass of biomarkers. Typically, two-dimensional gel
electrophoresis can separate chemically different biomarkers in the
molecular mass range from 1000-200,000 Da within complex
mixtures.
[0095] Biomarkers in the two-dimensional array can be detected
using any suitable methods known in the art. For example,
biomarkers in a gel can be labeled or stained (e.g., Coomassie Blue
or silver staining). If gel electrophoresis generates spots that
correspond to the molecular weight of one or more markers of the
invention, the spot can be further analyzed by densitometric
analysis or gas phase ion spectrometry. For example, spots can be
excised from the gel and analyzed by gas phase ion spectrometry.
Alternatively, the gel containing biomarkers can be transferred to
an inert membrane by applying an electric field. Then a spot on the
membrane that approximately corresponds to the molecular weight of
a marker can be analyzed by gas phase ion spectrometry. In gas
phase ion spectrometry, the spots can be analyzed using any
suitable techniques, such as MALDI or SELDI.
[0096] Prior to gas phase ion spectrometry analysis, it may be
desirable to cleave biomarkers in the spot into smaller fragments
using cleaving reagents, such as proteases (e.g., trypsin). The
digestion of biomarkers into small fragments provides a mass
fingerprint of the biomarkers in the spot, which can be used to
determine the identity of markers if desired.
[0097] In yet another embodiment, high performance liquid
chromatography (HPLC) can be used to separate a mixture of
biomarkers in a sample based on their different physical
properties, such as polarity, charge and size. HPLC instruments
typically consist of a reservoir of mobile phase, a pump, an
injector, a separation column, and a detector. Biomarkers in a
sample are separated by injecting an aliquot of the sample onto the
column. Different biomarkers in the mixture pass through the column
at different rates due to differences in their partitioning
behavior between the mobile liquid phase and the stationary phase.
A fraction that corresponds to the molecular weight and/or physical
properties of one or more markers can be collected. The fraction
can then be analyzed by gas phase ion spectrometry to detect
markers.
[0098] Optionally, a marker can be modified before analysis to
improve its resolution or to determine its identity. For example,
the markers may be subject to proteolytic digestion before
analysis. Any protease can be used. Proteases, such as trypsin,
that are likely to cleave the markers into a discrete number of
fragments are particularly useful. The fragments that result from
digestion function as a fingerprint for the markers, thereby
enabling their detection indirectly. This is particularly useful
where there are markers with similar molecular masses that might be
confused for the marker in question. Also, proteolytic
fragmentation is useful for high molecular weight markers because
smaller markers are more easily resolved by mass spectrometry. In
another example, biomarkers can be modified to improve detection
resolution. For instance, neuraminidase can be used to remove
terminal sialic acid residues from glycoproteins to improve binding
to an anionic adsorbent and to improve detection resolution. In
another example, the markers can be modified by the attachment of a
tag of particular molecular weight that specifically bind to
molecular markers, further distinguishing them. Optionally, after
detecting such modified markers, the identity of the markers can be
further determined by matching the physical and chemical
characteristics of the modified markers in a protein database
(e.g., SwissProt).
[0099] After preparation, biomarkers in a sample are typically
captured on a substrate for detection. Traditional substrates
include antibody-coated 96-well plates or nitrocellulose membranes
that are subsequently probed for the presence of proteins.
Preferably, the biomarkers are identified using immunoassays as
described above. However, preferred methods also include the use of
biochips. Preferably the biochips are protein biochips for capture
and detection of proteins. Many protein biochips are described in
the art. These include, for example, protein biochips produced by
Packard BioScience Company (Meriden Conn.), Zyomyx (Hayward,
Calif.) and Phylos (Lexington, Mass.). In general, protein biochips
comprise a substrate having a surface. A capture reagent or
adsorbent is attached to the surface of the substrate. Frequently,
the surface comprises a plurality of addressable locations, each of
which location has the capture reagent bound there. The capture
reagent can be a biological molecule, such as a polypeptide or a
nucleic acid, which captures other biomarkers in a specific manner.
Alternatively, the capture reagent can be a chromatographic
material, such as an anion exchange material or a hydrophilic
material. Examples of such protein biochips are described in the
following patents or patent applications: U.S. Pat. No. 6,225,047
(Hutchens and Yip, "Use of retentate chromatography to generate
difference maps," May 1, 2001), International publication WO
99/51773 (Kuimelis and Wagner, "Addressable protein arrays," Oct.
14, 1999), International publication WO 00/04389 (Wagner et al.,
"Arrays of protein-capture agents and methods of use thereof," Jul.
27, 2000), International publication WO 00/56934 (Englert et al.,
"Continuous porous matrix arrays," Sep. 28, 2000).
[0100] In general, a sample containing the biomarkers is placed on
the active surface of a biochip for a sufficient time to allow
binding. Then, unbound molecules are washed from the surface using
a suitable eluant. In general, the more stringent the eluant, the
more tightly the proteins must be bound to be retained after the
wash. The retained protein biomarkers now can be detected by
appropriate means.
[0101] Analytes captured on the surface of a protein biochip can be
detected by any method known in the art. This includes, for
example, mass spectrometry, fluorescence, surface plasmon
resonance, ellipsometry and atomic force microscopy. Mass
spectrometry, and particularly SELDI mass spectrometry, is a
particularly useful method for detection of the biomarkers of this
invention.
[0102] Preferably, a laser desorption time-of-flight mass
spectrometer is used in embodiments of the invention. In laser
desorption mass spectrometry, a substrate or a probe comprising
markers is introduced into an inlet system. The markers are
desorbed and ionized into the gas phase by laser from the
ionization source. The ions generated are collected by an ion optic
assembly, and then in a time-of-flight mass analyzer, ions are
accelerated through a short high voltage field and let drift into a
high vacuum chamber. At the far end of the high vacuum chamber, the
accelerated ions strike a sensitive detector surface at a different
time. Since the time-of-flight is a function of the mass of the
ions, the elapsed time between ion formation and ion detector
impact can be used to identify the presence or absence of markers
of specific mass to charge ratio.
[0103] Matrix-assisted laser desorption/ionization mass
spectrometry, or MALDI-MS, is a method of mass spectrometry that
involves the use of an energy absorbing molecule, frequently called
a matrix, for desorbing proteins intact from a probe surface. MALDI
is described, for example, in U.S. Pat. No. 5,118,937 (Hillenkamp
et al.) and U.S. Pat. No. 5,045,694 (Beavis and Chait). In MALDI-MS
the sample is typically mixed with a matrix material and placed on
the surface of an inert probe. Exemplary energy absorbing molecules
include cinnamic acid derivatives, sinapinic acid ("SPA"), cyano
hydroxy cinnamic acid ("CHCA") and dihydroxybenzoic acid. Other
suitable energy absorbing molecules are known to those skilled in
this art. The matrix dries, forming crystals that encapsulate the
analyte molecules. Then the analyte molecules are detected by laser
desorption/ionization mass spectrometry. MALDI-MS is useful for
detecting the biomarkers of this invention if the complexity of a
sample has been substantially reduced using the preparation methods
described above.
[0104] Surface-enhanced laser desorption/ionization mass
spectrometry, or SELDI-MS represents an improvement over MALDI for
the fractionation and detection of biomolecules, such as proteins,
in complex mixtures. SELDI is a method of mass spectrometry in
which biomolecules, such as proteins, are captured on the surface
of a protein biochip using capture reagents that are bound there.
Typically, non-bound molecules are washed from the probe surface
before interrogation. SELDI is described, for example, in: U.S.
Pat. No. 5,719,060 ("Method and Apparatus for Desorption and
Ionization of Analytes," Hutchens and Yip, Feb. 17, 1998) U.S. Pat.
No. 6,225,047 ("Use of Retentate Chromatography to Generate
Difference Maps," Hutchens and Yip, May 1, 2001) and Weinberger et
al., "Time-of-flight mass spectrometry," in Encyclopedia of
Analytical Chemistry, R. A. Meyers, ed., pp 11915-11918 John Wiley
& Sons Chichesher, 2000.
[0105] Markers on the substrate surface can be desorbed and ionized
using gas phase ion spectrometry. Any suitable gas phase ion
spectrometers can be used as long as it allows markers on the
substrate to be resolved. Preferably, gas phase ion spectrometers
allow quantitation of markers.
[0106] In one embodiment, a gas phase ion spectrometer is a mass
spectrometer. In a typical mass spectrometer, a substrate or a
probe comprising markers on its surface is introduced into an inlet
system of the mass spectrometer. The markers are then desorbed by a
desorption source such as a laser, fast atom bombardment, high
energy plasma, electrospray ionization, thermospray ionization,
liquid secondary ion MS, field desorption, etc. The generated
desorbed, volatilized species consist of preformed ions or neutrals
which are ionized as a direct consequence of the desorption event.
Generated ions are collected by an ion optic assembly, and then a
mass analyzer disperses and analyzes the passing ions. The ions
exiting the mass analyzer are detected by a detector. The detector
then translates information of the detected ions into
mass-to-charge ratios. Detection of the presence of markers or
other substances will typically involve detection of signal
intensity. This, in turn, can reflect the quantity and character of
markers bound to the substrate. Any of the components of a mass
spectrometer (e.g., a desorption source, a mass analyzer, a
detector, etc.) can be combined with other suitable components
described herein or others known in the art in embodiments of the
invention.
[0107] In another embodiment, an immunoassay can be used to detect
and analyze markers in a sample. This method comprises: (a)
providing an antibody that specifically binds to a marker; (b)
contacting a sample with the antibody; and (c) detecting the
presence of a complex of the antibody bound to the marker in the
sample.
[0108] To prepare an antibody that specifically binds to a marker,
purified markers or their nucleic acid sequences can be used.
Nucleic acid and amino acid sequences for markers can be obtained
by further characterization of these markers. For example, each
marker can be peptide mapped with a number of enzymes (e.g.,
trypsin, V8 protease, etc.). The molecular weights of digestion
fragments from each marker can be used to search the databases,
such as SwissProt database, for sequences that will match the
molecular weights of digestion fragments generated by various
enzymes. Using this method, the nucleic acid and amino acid
sequences of other markers can be identified if these markers are
known proteins in the databases.
[0109] Alternatively, the proteins can be sequenced using protein
ladder sequencing. Protein ladders can be generated by, for
example, fragmenting the molecules and subjecting fragments to
enzymatic digestion or other methods that sequentially remove a
single amino acid from the end of the fragment. Methods of
preparing protein ladders are described, for example, in
International Publication WO 93/24834 (Chait et al.) and U.S. Pat.
No. 5,792,664 (Chait et al.). The ladder is then analyzed by mass
spectrometry. The difference in the masses of the ladder fragments
identify the amino acid removed from the end of the molecule.
[0110] If the markers are not known proteins in the databases,
nucleic acid and amino acid sequences can be determined with
knowledge of even a portion of the amino acid sequence of the
marker. For example, degenerate probes can be made based on the
N-terminal amino acid sequence of the marker. These probes can then
be used to screen a genomic or cDNA library created from a sample
from which a marker was initially detected. The positive clones can
be identified, amplified, and their recombinant DNA sequences can
be subcloned using techniques which are well known. See, e.g.,
Current Protocols for Molecular Biology (Ausubel et al., Green
Publishing Assoc. and Wiley-Interscience 1989) and Molecular
Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Cold Spring
Harbor Laboratory, NY 2001).
[0111] Using the purified markers or their nucleic acid sequences,
antibodies that specifically bind to a marker can be prepared using
any suitable methods known in the art. See, e.g., Coligan, Current
Protocols in Immunology (1991); Harlow & Lane, Antibodies: A
Laboratory Manual (1988); Coding, Monoclonal Antibodies: Principles
and Practice (2d ed. 1986); and Kohler & Milstein, Nature
256:495-497 (1975). Such techniques include, but are not limited
to, antibody preparation by selection of antibodies from libraries
of recombinant antibodies in phage or similar vectors, as well as
preparation of polyclonal and monoclonal antibodies by immunizing
rabbits or mice (see, e.g., Huse et al., Science 246:1275-1281
(1989); Ward et al., Nature 341:544-546 (1989)).
[0112] After the antibody is provided, a marker can be detected
and/or quantified using any of suitable immunological binding
assays known in the art (see, e.g., U.S. Pat. Nos. 4,366,241;
4,376,110; 4,517,288; and 4,837,168). Useful assays include, for
example, an enzyme immune assay (EIA) such as enzyme-linked
immunosorbent assay (ELISA), a radioimmune assay (RIA), a Western
blot assay, or a slot blot assay. These methods are also described
in, e.g., Methods in Cell Biology: Antibodies in Cell Biology,
volume 37 (Asai, ed. 1993); Basic and Clinical Immunology (Stites
& Ten, eds., 7th ed. 1991); and Harlow & Lane, supra.
[0113] Generally, a sample obtained from a subject can be contacted
with the antibody that specifically binds the marker. Optionally,
the antibody can be fixed to a solid support to facilitate washing
and subsequent isolation of the complex, prior to contacting the
antibody with a sample. Examples of solid supports include glass or
plastic in the form of, e.g., a microtiter plate, a stick, a bead,
or a microbead. Antibodies can also be attached to a probe
substrate or PROTEINCHIP.RTM. array described above. The sample is
preferably a biological fluid sample taken from a subject. Examples
of biological fluid samples include cerebrospinal fluid, blood,
serum, plasma, urine, tears, saliva etc. In a preferred embodiment,
the biological fluid comprises serum. The sample can be diluted
with a suitable eluant before contacting the sample to the
antibody.
[0114] After incubating the sample with antibodies, the mixture is
washed and the antibody-marker complex formed can be detected. This
can be accomplished by incubating the washed mixture with a
detection reagent. This detection reagent may be, e.g., a second
antibody which is labeled with a detectable label. Exemplary
detectable labels include magnetic beads (e.g., DYNABEADS.TM.),
fluorescent dyes, radiolabels, enzymes (e.g., horse radish
peroxide, alkaline phosphatase and others commonly used in an
ELISA), and colorimetric labels such as colloidal gold or colored
glass or plastic beads. Alternatively, the marker in the sample can
be detected using an indirect assay, wherein, for example, a
second, labeled antibody is used to detect bound marker-specific
antibody, and/or in a competition or inhibition assay wherein, for
example, a monoclonal antibody which binds to a distinct epitope of
the marker is incubated simultaneously with the mixture.
[0115] Throughout the assays, incubation and/or washing steps may
be required after each combination of reagents. Incubation steps
can vary from about 5 seconds to several hours, preferably from
about 5 minutes to about 24 hours. However, the incubation time
will depend upon the assay format, marker, volume of solution,
concentrations and the like. Usually the assays will be carried out
at ambient temperature, although they can be conducted over a range
of temperatures, such as 10.degree. C. to 40.degree. C.
[0116] Immunoassays can be used to determine presence or absence of
a marker in a sample as well as the quantity of a marker in a
sample. First, a test amount of a marker in a sample can be
detected using the immunoassay methods described above. If a marker
is present in the sample, it will form an antibody-marker complex
with an antibody that specifically binds the marker under suitable
incubation conditions described above. The amount of an
antibody-marker complex can be determined by comparing to a
standard. A standard can be, e.g., a known compound or another
protein known to be present in a sample. As noted above, the test
amount of marker need not be measured in absolute units, as long as
the unit of measurement can be compared to a control.
[0117] The methods for detecting these markers in a sample have
many applications. For example, one or more markers can be measured
to aid in the diagnosis of diabetes and/or diabetic disorders. In
another example, the methods for detection of the markers can be
used to monitor responses in a subject to treatment. In another
example, the methods for detecting markers can be used to assay for
and to identify compounds that modulate expression of these markers
in vivo or in vitro.
[0118] Data generated by desorption and detection of markers can be
analyzed using any suitable means. In one embodiment, data is
analyzed with the use of a programmable digital computer. The
computer program generally contains a readable medium that stores
codes. Certain code can be devoted to memory that includes the
location of each feature on a probe, the identity of the adsorbent
at that feature and the elution conditions used to wash the
adsorbent. The computer also contains code that receives as input,
data on the strength of the signal at various molecular masses
received from a particular addressable location on the probe. This
data can indicate the number of markers detected, including the
strength of the signal generated by each marker.
[0119] Data analysis can include the steps of determining signal
strength (e.g., height of peaks) of a marker detected and removing
"outliers" (data deviating from a predetermined statistical
distribution). The observed peaks can be normalized, a process
whereby the height of each peak relative to some reference is
calculated. For example, a reference can be background noise
generated by instrument and chemicals (e.g., energy absorbing
molecule) which is set as zero in the scale. Then the signal
strength detected for each marker or other biomolecules can be
displayed in the form of relative intensities in the scale desired
(e.g., 100). Alternatively, a standard (e.g., a serum protein) may
be admitted with the sample so that a peak from the standard can be
used as a reference to calculate relative intensities of the
signals observed for each marker or other markers detected.
[0120] The computer can transform the resulting data into various
formats for displaying. In one format, referred to as "spectrum
view or retentate map," a standard spectral view can be displayed,
wherein the view depicts the quantity of marker reaching the
detector at each particular molecular weight. In another format,
referred to as "peak map," only the peak height and mass
information are retained from the spectrum view, yielding a cleaner
image and enabling markers with nearly identical molecular weights
to be more easily seen. In yet another format, referred to as "gel
view," each mass from the peak view can be converted into a
grayscale image based on the height of each peak, resulting in an
appearance similar to bands on electrophoretic gels. In yet another
format, referred to as "3-D overlays," several spectra can be
overlaid to study subtle changes in relative peak heights. In yet
another format, referred to as "difference map view," two or more
spectra can be compared, conveniently highlighting unique markers
and markers which are up- or down-regulated between samples. Marker
profiles (spectra) from any two samples may be compared visually.
In yet another format, Spotfire Scatter Plot can be used, wherein
markers that are detected are plotted as a dot in a plot, wherein
one axis of the plot represents the apparent molecular mass of the
markers detected and another axis represents the signal intensity
of markers detected. For each sample, markers that are detected and
the amount of markers present in the sample can be saved in a
computer readable medium. This data can then be compared to a
control (e.g., a profile or quantity of markers detected in
control, e.g., normal, healthy subjects in whom diabetes injury is
undetectable).
Diagnosis and Differentiation between Type 1 and Type 2
Diabetes
[0121] In another aspect, the invention provides methods for aiding
a type 1 diabetes, type 2 diabetes and/or diabetic disorder
diagnosis using one or more markers. For example, proteins
identified from patients in Table 1, peptides, fragments or
derivatives thereof. These markers can be used singularly or in
combination with other markers in any set. The markers are
differentially present in samples of a human patient, for example a
type 1 patient, and a normal subject in whom diabetes is
undetectable. For example, some of the markers are expressed at an
elevated level and/or are present at a higher frequency in human
patients with type 1 diabetes, type 2 diabetes and/or diabetic
disorders than in normal subjects. Therefore, detection of one or
more of these markers in a person would provide useful information
regarding the probability that the person may have type 1 versus
type 2 diabetes and/or diabetic disorder. Examples of diabetic
biomarkers include, but not limited to adiponectin, leptin,
ghrelin, resistin, autoantibodies to insulin, autoantibodies to
glutamic acid decarboxylase, autoantibodies to IL-2, autoantibodies
to IA-2, incretins, TNF-.alpha., and IL-6, fragments, variants or
any combination thereof.
[0122] In a preferred embodiment, a multivariant analysis is
performed associating serum adiponectin and leptin levels with
anthropometrical parameters and disease state. See for example,
Table 1. Specifically type 1 diabetes was diagnosed through a
clinical evaluation of a number of factors including a symptomatic
history (e.g., polydipsia, polyphagia, polyuria), weight loss, BMI,
ketoacidosis, and the presence of a type 1 diabetes-associated
autoantibody (described below). For cases of pediatric type 2
diabetes, a diagnosis was established by historical (e.g., family
history of type 2 diabetes), symptomatic history, physical (e.g.,
BMI, race, acanthosis nigricans), and laboratory data including the
absence of type 1 diabetes-associated autoantibodies (Kaufman F.
Rev Endocr. Metab. Disord. 4:33 42, 2003). All healthy control
subjects were also autoantibody negative.
[0123] In a preferred embodiment, statistical analyses were
undertaken with GraphPad Prizm 4.0 (GraphPad, San Diego, Calif.)
using Fisher's exact test, receiver-operator characteristic (ROC)
analysis, linear regression, t testing, or ANOVA (Kruskal-Wallis)
with Dunn's post-testing. P<0.05 was deemed significant. ROC
plots were constructed comparing type 1 with type 2 diabetic
subjects (i.e., area under the ROC curve of 0.969 [95% CI
0.93-1.00]; P<0.0001) to determine an appropriate cutoff value
for the adiponectin-to-leptin ratio (FIG. 5). At a ratio cutoff of
<0.9, the sensitivity was 100% (range 80-100%) with specificity
of 80% (65-91%) for type 2 as opposed to type 1 diabetes. At a
ratio cutoff of <0.7, sensitivity was 88% (64-99%) with
specificity of 90% (77-97%).
[0124] Accordingly, embodiments of the invention include methods
for aiding a type 1 diabetes, type 2 diabetes and/or diabetic
disorder diagnosis using one or more markers, wherein the method
comprises: (a) detecting at least one marker in a sample, wherein
the marker is adiponectin, leptin peptides, fragments and
derivatives thereof; and (b) correlating the detection of the
marker or markers with a probable diagnosis of type 1 diabetes,
type 2 diabetes and/or diabetic disorder. The correlation may take
into account the amount of the marker or markers in the sample
compared to a control amount of the marker or markers (up or down
regulation of the marker or markers) (e.g., in normal subjects in
whom diabetes is undetectable). The correlation may take into
account the presence or absence of the markers in a test sample and
the frequency of detection of the same markers in a control. The
correlation may take into account both of such factors to
facilitate determination of whether a subject has type 1 diabetes,
type 2 diabetes and/or diabetic disorder and the degree of severity
of the disease, or not.
[0125] Any suitable samples can be obtained from a subject to
detect markers. Preferably, a sample is a serum sample from the
subject. If desired, the sample can be prepared as described above
to enhance detectability of the markers. For example, to increase
the detectability of markers, a blood serum sample from the subject
can be preferably fractionated by, e.g., Cibacron blue agarose
chromatography and single stranded DNA affinity chromatography,
anion exchange chromatography and the like. Sample preparations,
such as pre-fractionation protocols, is optional and may not be
necessary to enhance detectability of markers depending on the
methods of detection used. For example, sample preparation may be
unnecessary if antibodies that specifically bind markers are used
to detect the presence of markers in a sample.
[0126] Any suitable method can be used to detect a marker or
markers in a sample. For example, an immunoassay or gas phase ion
spectrometry can be used as described above. Using these methods,
one or more markers can be detected. Preferably, a sample is tested
for the presence of a plurality of markers. Detecting the presence
of a plurality of markers, rather than a single marker alone, would
provide more information for the diagnostician. Specifically, the
detection of a plurality of markers in a sample would increase the
percentage of true positive and true negative diagnoses and would
decrease the percentage of false positive or false negative
diagnoses.
[0127] The detection of the marker or markers is then correlated
with a probable diagnosis of type 1 diabetes, type 2 diabetes
and/or diabetic disorder. In some embodiments, the detection of the
mere presence or absence of a marker, without quantifying the
amount of marker, is useful and can be correlated with a probable
diagnosis of type 1 diabetes, type 2 diabetes and/or diabetic
disorder. For example, adiponectin, leptin, proteins, fragments or
derivatives thereof, can be more frequently detected in patients
with type 1 diabetes, type 2 diabetes and/or diabetic disorder than
in normal subjects.
[0128] In other embodiments, the detection of markers can involve
quantifying the markers to correlate the detection of markers with
a probable diagnosis of type 1 diabetes, type 2 diabetes and/or
diabetic disorder, degree of severity of type 1 diabetes, type 2
diabetes and/or diabetic disorder and the like. Thus, if the amount
of the markers detected in a subject being tested is higher
compared to a control amount, then the subject being tested has a
higher probability of having type 1 diabetes, type 2 diabetes
and/or diabetic disorder.
[0129] Similarly, in another embodiment, the detection of markers
can further involve quantifying the markers to correlate the
detection of markers with a probable diagnosis of type 1 diabetes,
type 2 diabetes and/or diabetic disorder, degree of severity of
type 1 diabetes, type 2 diabetes and/or diabetic disorder and the
like, wherein the markers are present in lower quantities in blood
serum samples from patients than in blood serum samples of normal
subjects. Thus, if the amount of the markers detected in a subject
being tested is lower compared to a control amount, then the
subject being tested has a higher probability of having type 1
diabetes, type 2 diabetes and/or diabetic disorder.
[0130] When the markers are quantified, it can be compared to a
control. A control can be, e.g., the average or median amount of
marker present in comparable samples of normal subjects in whom
type 1 diabetes, type 2 diabetes and/or diabetic disorder, is
undetectable. The control amount is measured under the same or
substantially similar experimental conditions as in measuring the
test amount. For example, if a test sample is obtained from a
subject's blood serum sample and a marker is detected using a
particular probe, then a control amount of the marker is preferably
determined from a serum sample of a patient using the same probe.
It is preferred that the control amount of marker is determined
based upon a significant number of samples from normal subjects who
do not have type 1 diabetes, type 2 diabetes and/or diabetic
disorder so that it reflects variations of the marker amounts in
that population.
[0131] Data generated by mass spectrometry can then be analyzed by
a computer software. The software can comprise code that converts
signal from the mass spectrometer into computer readable form. The
software also can include code that applies an algorithm to the
analysis of the signal to determine whether the signal represents a
"peak" in the signal corresponding to a marker of this invention,
or other useful markers. The software also can include code that
executes an algorithm that compares signal from a test sample to a
typical signal characteristic of "normal" and human type 1
diabetes, type 2 diabetes and/or diabetic disorder and determines
the closeness of fit between the two signals. The software also can
include code indicating which the test sample is closest to,
thereby providing a probable diagnosis.
Production of Antibodies to Detect Type 1 Diabetes and Type 2
Diabetes Biomarkers
[0132] Biomarkers obtained from samples in patients suffering from
type 1 diabetes, type 2 diabetes and/or diabetic disorder, degrees
of severity of type 1 diabetes, type 2 diabetes and/or diabetic
disorder and the like, can be prepared as described above.
Furthermore, diabetic biomarkers can be subjected to enzymatic
digestion to obtain fragments or peptides of the biomarkers for the
production of antibodies to different antigenic epitopes that can
be present in a peptide versus the whole protein. Antigenic
epitopes are useful, for example, to raise antibodies, including
monoclonal antibodies, that specifically bind the epitope.
Antigenic epitopes can be used as the target molecules in
immunoassays. (See, for instance, Wilson et al., Cell 37:767-778
(1984); Sutcliffe et al., Science 219:660-666 (1983)).
[0133] Diabetic biomarker epitopes can be used, for example, to
induce antibodies according to methods well known in the art. (See,
for instance, Sutcliffe et al., supra; Wilson et al., supra; Chow
et al., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al.,
J. Gen. Virol. 66:2347-2354 (1985). Diabetic polypeptides
comprising one or more immunogenic epitopes may be presented for
eliciting an antibody response together with a carrier protein,
such as an albumin, to an animal system (such as rabbit or mouse),
or, if the polypeptide is of sufficient length (at least about 25
amino acids), the polypeptide may be presented without a carrier.
However, immunogenic epitopes comprising as few as 8 to 10 amino
acids have been shown to be sufficient to raise antibodies capable
of binding to, at the very least, linear epitopes in a denatured
polypeptide (e.g., in Western blotting).
[0134] Epitope-bearing polypeptides of the present invention may be
used to induce antibodies according to methods well known in the
art including, but not limited to, in vivo immunization, in vitro
immunization, and phage display methods. See, e.g., Sutcliffe et
al., supra; Wilson et al., supra, and Bittle et al., J. Gen.
Virol., 66:2347-2354 (1985). If in vivo immunization is used,
animals may be immunized with free peptide; however, anti-peptide
antibody titer may be boosted by coupling the peptide to a
macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or
tetanus toxoid. For instance, peptides containing cysteine residues
may be coupled to a carrier using a linker such as
maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other
peptides may be coupled to carriers using a more general linking
agent such as glutaraldehyde. Animals such as rabbits, rats and
mice are immunized with either free or carrier-coupled peptides,
for instance, by intraperitoneal and/or intradermal injection of
emulsions containing about 100 .mu.g of peptide or carrier protein
and Freund's adjuvant or any other adjuvant known for stimulating
an immune response. Several booster injections may be needed, for
instance, at intervals of about two weeks, to provide a useful
titer of anti-peptide antibody which can be detected, for example,
by ELISA assay using free peptide adsorbed to a solid surface. The
titer of anti-peptide antibodies in serum from an immunized animal
may be increased by selection of anti-peptide antibodies, for
instance, by adsorption to the peptide on a solid support and
elution of the selected antibodies according to methods well known
in the art.
[0135] Nucleic acids diabetic biomarker epitopes can also be
recombined with a gene of interest as an epitope tag (e.g., the
hemagglutinin ("HA") tag or flag tag) to aid in detection and
purification of the expressed polypeptide. For example, a system
described by Janknecht et al. allows for the ready purification of
non-denatured fusion proteins expressed in human cell lines
(Janknecht et al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-897).
In this system, the gene of interest is subcloned into a vaccinia
recombination plasmid such that the open reading frame of the gene
is translationally fused to an amino-terminal tag consisting of six
histidine residues. The tag serves as a matrix binding domain for
the fusion protein. Extracts from cells infected with the
recombinant vaccinia virus are loaded onto Ni.sup.2+ nitriloacetic
acid-agarose column and histidine-tagged proteins can be
selectively eluted with imidazole-containing buffers.
[0136] The antibodies of the present invention may be generated by
any suitable method known in the art. The antibodies of the present
invention can comprise polyclonal antibodies. Methods of preparing
polyclonal antibodies are known to the skilled artisan (Harlow, et
al., Antibodies: A Laboratory Manual, (Cold spring Harbor
Laboratory Press, 2nd ed. (1988), which is hereby incorporated
herein by reference in its entirety). For example, a polypeptide of
the invention can be administered to various host animals
including, but not limited to, rabbits, mice, rats, etc. to induce
the production of sera containing polyclonal antibodies specific
for the antigen. The administration of the polypeptides of the
present invention may entail one or more injections of an
immunizing agent and, if desired, an adjuvant. Various adjuvants
may be used to increase the immunological response, depending on
the host species, and include but are not limited to, Freund's
(complete and incomplete), mineral gels such as aluminum hydroxide,
surface active substances such as lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, keyhole limpet hemocyanins,
dinitrophenol, and potentially useful human adjuvants such as BCG
(bacille Calmette-Guerin) and Corynebacterium parvum. Such
adjuvants are also well known in the art. For the purposes of the
invention, "immunizing agent" may be defined as a polypeptide of
the invention, including fragments, variants, and/or derivatives
thereof, in addition to fusions with heterologous polypeptides and
other forms of the polypeptides as may be described herein.
[0137] Typically, the immunizing agent and/or adjuvant will be
injected in the mammal by multiple subcutaneous or intraperitoneal
injections, though they may also be given intramuscularly, and/or
through IV. The immunizing agent may include polypeptides of the
present invention or a fusion protein or variants thereof.
Depending upon the nature of the polypeptides (i.e., percent
hydrophobicity, percent hydrophilicity, stability, net charge,
isoelectric point etc.), it may be useful to conjugate the
immunizing agent to a protein known to be immunogenic in the mammal
being immunized. Such conjugation includes either chemical
conjugation by derivatizing active chemical functional groups to
both the polypeptide of the present invention and the immunogenic
protein such that a covalent bond is formed, or through
fusion-protein based methodology, or other methods known to the
skilled artisan. Examples of such immunogenic proteins include, but
are not limited to keyhole limpet hemocyanin, serum albumin, bovine
thyroglobulin, and soybean trypsin inhibitor. Various adjuvants may
be used to increase the immunological response, depending on the
host species, including but not limited to Freund's (complete and
incomplete), mineral gels such as aluminum hydroxide, surface
active substances such as lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,
dinitrophenol, and potentially useful human adjuvants such as BCG
(bacille Calmette-Guerin) and Corynebacterium parvum. Additional
examples of adjuvants which may be employed includes the MPL-TDM
adjuvant (monophosphoryl lipid A, synthetic trehalose
dicorynomycolate). The immunization protocol may be selected by one
skilled in the art without undue experimentation.
[0138] The antibodies of the present invention can also comprise
monoclonal antibodies. Monoclonal antibodies may be prepared using
hybridoma methods, such as those described by Kohler and Milstein,
Nature, 256:495 (1975) and U.S. Pat. No. 4,376,110, by Harlow, et
al., Antibodies: A Laboratory Manual, (Cold spring Harbor
Laboratory Press, 2nd ed, (1988), by Hammerling, et al., Monoclonal
Antibodies and T-Cell Hybridomas (Elsevier, N.Y., (1981)), or other
methods known to the artisan. Other examples of methods which may
be employed for producing monoclonal antibodies includes, but are
not limited to, the human B-cell hybridoma technique (Kosbor et
al., 1983, Immunology Today 4:72; Cole et al., 1983, Proc. Natl.
Acad. Sci. USA 80:2026-2030), and the EBV-hybridoma technique (Cole
et al., 1985, Monoclonal Antibodies And Cancer Therapy, Alan R.
Liss, Inc., pp. 77-96). Such antibodies may be of any
immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any
subclass thereof. The hybridoma producing the mAb of this invention
may be cultivated in vitro or in vivo. Production of high titers of
mAbs in vivo makes this the presently preferred method of
production.
[0139] In a hybridoma method, a mouse, a humanized mouse, a mouse
with a human immune system, hamster, or other appropriate host
animal, is typically immunized with an immunizing agent to elicit
lymphocytes that produce or are capable of producing antibodies
that will specifically bind to the immunizing agent. Alternatively,
the lymphocytes may be immunized in vitro.
[0140] The immunizing agent will typically include polypeptides
identified in diabetes patients, fragments or a fusion protein
thereof. Generally, either peripheral blood lymphocytes ("PBLs")
are used if cells of human origin are desired, or spleen cells or
lymph node cells are used if non-human mammalian sources are
desired. The lymphocytes are then fused with an immortalized cell
line using a suitable fusing agent, such as polyethylene glycol, to
form a hybridoma cell (Goding, Monoclonal Antibodies: Principles
and Practice, Academic Press, (1986), pp. 59-103). Immortalized
cell lines are usually transformed mammalian cells, particularly
myeloma cells of rodent, bovine and human origin. Usually, rat or
mouse myeloma cell lines are employed. The hybridoma cells may be
cultured in a suitable culture medium that preferably contains one
or more substances that inhibit the growth or survival of the
unfused, immortalized cells. For example, if the parental cells
lack the enzyme hypoxanthine guanine phosphoribosyl transferase
(HGPRT or HPRT), the culture medium for the hybridomas typically
will include hypoxanthine, aminopterin, and thymidine ("HAT
medium"), which substances prevent the growth of HGPRT-deficient
cells.
[0141] Preferred immortalized cell lines are those that fuse
efficiently, support stable high level expression of antibody by
the selected antibody-producing cells, and are sensitive to a
medium such as HAT medium. More preferred immortalized cell lines
are murine myeloma lines, which can be obtained, for instance, from
the Salk Institute Cell Distribution Center, San Diego, Calif. and
the American Type Culture Collection, Manassas, Va. As inferred
throughout the specification, human myeloma and mouse-human
heteromyeloma cell lines also have been described for the
production of human monoclonal antibodies (Kozbor, J. Immunol.,
133:3001 (1984); Brodeur et al., Monoclonal Antibody Production
Techniques and Applications, Marcel Dekker, Inc., New York, (1987)
pp. 51-63).
[0142] The culture medium in which the hybridoma cells are cultured
can then be assayed for the presence of monoclonal antibodies
directed against the adiponectin and/or lectin polypeptides of the
present invention. Preferably, the binding specificity of
monoclonal antibodies produced by the hybridoma cells is determined
by immunoprecipitation or by an in vitro binding assay, such as
radioimmunoassay (RIA) or enzyme-linked immunoadsorbant assay
(ELISA). Such techniques are known in the art and within the skill
of the artisan. The binding affinity of the monoclonal antibody
can, for example, be determined by the Scatchard analysis of Munson
and Pollart, Anal. Biochem., 107:220 (1980).
[0143] After the desired hybridoma cells are identified, the clones
may be subcloned by limiting dilution procedures and grown by
standard methods (Goding, supra). Suitable culture media for this
purpose include, for example, Dulbecco's Modified Eagle's Medium
and RPMI-1640. Alternatively, the hybridoma cells may be grown in
vivo as ascites in a mammal.
[0144] The monoclonal antibodies secreted by the subclones may be
isolated or purified from the culture medium or ascites fluid by
conventional immunoglobulin purification procedures such as, for
example, protein A-sepharose, hydroxyapatite chromatography, gel
exclusion chromatography, gel electrophoresis, dialysis, or
affinity chromatography.
[0145] The skilled artisan would acknowledge that a variety of
methods exist in the art for the production of monoclonal
antibodies and thus, the invention is not limited to their sole
production in hybridomas. For example, the monoclonal antibodies
may be made by recombinant DNA methods, such as those described in
U.S. Pat. No. 4,816,567. In this context, the term "monoclonal
antibody" refers to an antibody derived from a single eukaryotic,
phage, or prokaryotic clone. The DNA encoding the monoclonal
antibodies of the invention can be readily isolated and sequenced
using conventional procedures (e.g., by using oligonucleotide
probes that are capable of binding specifically to genes encoding
the heavy and light chains of murine antibodies, or such chains
from human, humanized, or other sources). The hybridoma cells of
the invention serve as a preferred source of such DNA. Once
isolated, the DNA may be placed into expression vectors, which are
then transformed into host cells such as Simian COS cells, Chinese
hamster ovary (CHO) cells, or myeloma cells that do not otherwise
produce immunoglobulin protein, to obtain the synthesis of
monoclonal antibodies in the recombinant host cells.
[0146] Methods for producing and screening for specific antibodies
using hybridoma technology are routine and well known in the art.
In a non-limiting example, mice can be immunized with a biomarker
polypeptide or a cell expressing such peptide. Once an immune
response is detected, e.g., antibodies specific for the antigen are
detected in the mouse serum, the mouse spleen is harvested and
splenocytes isolated. The splenocytes are then fused by well-known
techniques to any suitable myeloma cells, for example cells from
cell line SP20 available from the ATCC. Hybridomas are selected and
cloned by limited dilution. The hybridoma clones are then assayed
by methods known in the art for cells that secrete antibodies
capable of binding a polypeptide of the invention. Ascites fluid,
which generally contains high levels of antibodies, can be
generated by immunizing mice with positive hybridoma clones.
[0147] Accordingly, the present invention provides methods of
generating monoclonal antibodies as well as antibodies produced by
the method comprising culturing a hybridoma cell secreting an
antibody of the invention wherein, preferably, the hybridoma is
generated by fusing splenocytes isolated from a mouse immunized
with an antigen of the invention with myeloma cells and then
screening the hybridomas resulting from the fusion for hybridoma
clones that secrete an antibody able to bind a polypeptide of the
invention. The antibodies detecting diabetic biomarkers, peptides
and derivatives thereof, can be used in immunoassays and other
methods to identify new diabetic biomarkers and for use in the
diagnosis of type 1 diabetes, type 2 diabetes and/or diabetic
disorder.
[0148] Other methods can also be used for the large scale
production of diabetic biomarker specific antibodies. For example,
antibodies can also be generated using various phage display
methods known in the art. In phage display methods, functional
antibody domains are displayed on the surface of phage particles
which carry the polynucleotide sequences encoding them. In a
particular embodiment, such phage can be utilized to display
antigen binding domains expressed from a repertoire or
combinatorial antibody library (e.g., human or murine). Phage
expressing an antigen binding domain that binds the antigen of
interest can be selected or identified with antigen, e.g., using
labeled antigen or antigen bound or captured to a solid surface or
bead. Phage used in these methods are typically filamentous phage
including fd and M13 binding domains expressed from phage with Fab,
Fv or disulfide stabilized Fv antibody domains recombinantly fused
to either the phage gene III or gene VIII protein. Examples of
phage display methods that can be used to make the antibodies of
the present invention include those disclosed in Brinkman et al.,
J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol.
Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol.
24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et
al., Advances in Immunology 57:191-280 (1994); PCT application No.
PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO
92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and
U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717;
5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637;
5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which is
incorporated in pertinent part by reference herein for the reasons
cited in the above text.
[0149] The antibodies of the present invention have various
utilities. For example, such antibodies may be used in diagnostic
assays to detect the presence or quantification of the polypeptides
of the invention in a sample. Such a diagnostic assay can comprise
at least two steps. The first, subjecting a sample with the
antibody, wherein the sample is a tissue (e.g., human, animal,
etc.), biological fluid (e.g., blood, urine, sputum, semen,
amniotic fluid, saliva, etc.), biological extract (e.g., tissue or
cellular homogenate, etc.), a protein microchip (e.g., See Arenkov
P, et al., Anal Biochem., 278(2):123-131 (2000)), or a
chromatography column, etc. And a second step involving the
quantification of antibody bound to the substrate. Alternatively,
the method may additionally involve a first step of attaching the
antibody, either covalently, electrostatically, or reversibly, to a
solid support, and a second step of subjecting the bound antibody
to the sample, as defined above and elsewhere herein.
[0150] Various diagnostic assay techniques are known in the art,
such as competitive binding assays, direct or indirect sandwich
assays and immunoprecipitation assays conducted in either
heterogeneous or homogenous phases (Zola, Monoclonal Antibodies: A
Manual of Techniques, CRC Press, Inc., (1987), pp 147-158). The
antibodies used in the diagnostic assays can be labeled with a
detectable moiety. The detectable moiety should be capable of
producing, either directly or indirectly, a detectable signal. For
example, the detectable moiety may be a radioisotope, such as
.sup.2H, .sup.14C, .sup.32P, or .sup.125I a florescent or
chemiluminescent compound, such as fluorescein isothiocyanate,
rhodamine, or luciferin, or an enzyme, such as alkaline
phosphatase, beta-galactosidase, green fluorescent protein, or
horseradish peroxidase. Any method known in the art for conjugating
the antibody to the detectable moiety may be employed, including
those methods described by Hunter et al., Nature, 144:945 (1962);
David et al., Biochem., 13:1014 (1974); Pain et al., J. Immunol.
Methods, 40:219 (1981); and Nygren, J. Histochem. and Cytochem.,
30:407 (1982).
Kits
[0151] In yet another aspect, the invention provides kits for
aiding a diagnosis of type 1 diabetes, type 2 diabetes and/or
diabetic disorder and degree of severity of type 1 diabetes, type 2
diabetes and/or diabetic disorder, wherein the kits can be used to
detect the markers of the present invention. For example, the kits
can be used to detect any one or more of the markers described
herein, which markers are differentially present in samples of a
patient and normal subjects. For example, adiponectin, leptin,
ghrelin, resistin, autoantibodies to insulin, autoantibodies to
glutamic acid decarboxylase, autoantibodies to IL-2, autoantibodies
to IA-2, incretins, TNF-.alpha., and IL-6, fragments, variants or
any combination thereof. The kits of the invention have many
applications. For example, the kits can be used to differentiate if
a subject has type 1 versus type 2 diabetes, or has a negative
diagnosis, thus aiding type 1 diabetes, type 2 diabetes and/or
diabetic disorder diagnosis. In another example, the kits can be
used to identify compounds that modulate expression of one or more
of the markers in in vitro or in vivo animal models to determine
the effects of treatment.
[0152] In one embodiment, a kit comprises (a) an antibody that
specifically binds to a marker; and (b) a detection reagent. Such
kits can be prepared from the materials described above, and the
previous discussion regarding the materials (e.g., antibodies,
detection reagents, immobilized supports, etc.) is fully applicable
to this section and will not be repeated. Optionally, the kit may
further comprise pre-fractionation spin columns. In some
embodiments, the kit may further comprise instructions for suitable
operation parameters in the form of a label or a separate
insert.
[0153] In an additional embodiment, the invention includes a
diagnostic kit for use in screening serum containing antigens of
the polypeptide of the invention. The diagnostic kit includes a
substantially isolated antibody specifically immunoreactive with
polypeptide or polynucleotide antigens, and means for detecting the
binding of the polynucleotide or polypeptide antigen to the
antibody. In one embodiment, the antibody is attached to a solid
support. In a specific embodiment, the antibody may be a monoclonal
antibody. The detecting means of the kit may include a second,
labeled monoclonal antibody. Alternatively, or in addition, the
detecting means may include a labeled, competing antigen.
[0154] In one diagnostic configuration, test serum is reacted with
a solid phase reagent having a surface-bound antigen obtained by
the methods of the present invention. After binding with specific
antigen antibody to the reagent and removing unbound serum
components by washing, the reagent is reacted with reporter-labeled
anti-human antibody to bind reporter to the reagent in proportion
to the amount of bound anti-antigen antibody on the solid support.
The reagent is again washed to remove unbound labeled antibody, and
the amount of reporter associated with the reagent is determined.
Typically, the reporter is an enzyme which is detected by
incubating the solid phase in the presence of a suitable
fluorometric, luminescent or colorimetric substrate (Sigma, St.
Louis, Mo.).
[0155] The solid surface reagent in the above assay is prepared by
known techniques for attaching protein material to solid support
material, such as polymeric beads, dip sticks, 96-well plate or
filter material. These attachment methods generally include
non-specific adsorption of the protein to the support or covalent
attachment of the protein, typically through a free amine group, to
a chemically reactive group on the solid support, such as an
activated carboxyl, hydroxyl, or aldehyde group. Alternatively,
streptavidin coated plates can be used in conjunction with
biotinylated antigen(s).
[0156] Optionally, the kit may further comprise a standard or
control information so that the test sample can be compared with
the control information standard to determine if the test amount of
a marker detected in a sample is a diagnostic amount consistent
with a diagnosis of type 1 diabetes, type 2 diabetes and/or
diabetic disorder, degree of severity of type 1 diabetes, type 2
diabetes and/or diabetic disorder, and/or effect of treatment on
the patient.
[0157] In another embodiment, a kit comprises: (a) a substrate
comprising an adsorbent thereon, wherein the adsorbent is suitable
for binding a marker, and (b) instructions to detect the marker or
markers by contacting a sample with the adsorbent and detecting the
marker or markers retained by the adsorbent. In some embodiments,
the kit may comprise an eluant (as an alternative or in combination
with instructions) or instructions for making an eluant, wherein
the combination of the adsorbent and the eluant allows detection of
the markers using gas phase ion spectrometry. Such kits can be
prepared from the materials described above, and the previous
discussion of these materials (e.g., probe substrates, adsorbents,
washing solutions, etc.) is fully applicable to this section and
will not be repeated.
[0158] In another embodiment, the kit may comprise a first
substrate comprising an adsorbent thereon (e.g., a particle
functionalized with an adsorbent) and a second substrate onto which
the first substrate can be positioned to form a probe which is
removably insertable into a gas phase ion spectrometer. In other
embodiments, the kit may comprise a single substrate which is in
the form of a removably insertable probe with adsorbents on the
substrate. In yet another embodiment, the kit may further comprise
a pre-fractionation spin column (e.g., Cibacron blue agarose
column, anti-HSA agarose column, size exclusion column, Q-anion
exchange spin column, single stranded DNA column, lectin column,
etc.).
[0159] Optionally, the kit can further comprise instructions for
suitable operational parameters in the form of a label or a
separate insert. For example, the kit may have standard
instructions informing a consumer how to wash the probe after a
sample is contacted on the probe. In another example, the kit may
have instructions for pre-fractionating a sample to reduce
complexity of proteins in the sample. In another example, the kit
may have instructions for automating the fractionation or other
processes.
[0160] The following examples are offered by way of illustration,
not by way of limitation. While specific examples have been
provided, the above description is illustrative and not
restrictive. Any one or more of the features of the previously
described embodiments can be combined in any manner with one or
more features of any other embodiments in the present invention.
Furthermore, many variations of the invention will become apparent
to those skilled in the art upon review of the specification. The
scope of the invention should, therefore, be determined not with
reference to the above description, but instead should be
determined with reference to the appended claims along with their
full scope of equivalents.
[0161] All publications and patent documents cited in this
application are incorporated by reference in their entirety for all
purposes to the same extent as if each individual publication or
patent document were so individually denoted. By their citation of
various references in this document, Applicants do not admit any
particular reference is "prior art" to their invention.
EXAMPLES
Materials and Methods
[0162] Samples from children and adolescents with type 1 diabetes
(n=41), type 2 diabetes (n=17), and nondiabetic individuals of
similar age from the general population (n=43) were investigated.
An analysis included the parameters of matching for BMI and Tanner
stage. Receiver-operator characteristic (ROC) curves were
established to assess these analytes association with a
disease.
[0163] Adiponectin and leptin levels were measured in a single
serum sample (nonfasting, stored at -80.degree. C.) from children
and adolescents with type 1 diabetes, type 2 diabetes, and
nondiabetic individuals of similar age from the general population
(demographics in legend to Table 1). Type 1 and type 2 diabetes
were diagnosed according to American Diabetes Association criteria
(Expert Committee on the Diagnosis and Classification of Diabetes
Mellitus: Report of the Expert Committee on the Diagnosis and
Classification of Diabetes Mellitus. Diabetes Care 26 (Suppl.
1):S5S20, 2003).
[0164] Specifically type 1 diabetes was diagnosed through a
clinical evaluation of a number of factors including a symptomatic
history (e.g., polydipsia, polyphagia, polyuria), weight loss, BMI,
ketoacidosis, and the presence of a type 1 diabetes associated
autoantibody (described below). For cases of pediatric type 2
diabetes, a diagnosis was established by historical (e.g., family
history of type 2 diabetes), symptomatic history, physical (e.g.,
BMI, race, acanthosis nigricans), and laboratory data including the
absence of type 1 diabetes-associated autoantibodies (Kaufman F.
Rev Endocr. Metab. Disord. 4:33-42, 2003). All healthy control
subjects were also autoantibody negative.
Serum Analyte and Autoantibody Detection
[0165] LINCOPLEX.TM. (Linco Research, St. Louis, Mo.) kits were
used for the measurement of human leptin (sensitivity 0.01 ng/ml;
interassay coefficient of variation [CV] 5.0%), while B-Bridge
International (San Jose, Calif.) human adiponectin enzyme-linked
immunosorbent assay kits were used for monitoring serum adiponectin
levels (lower limit 0.02 ng/ml; interassay CV 3.2%). To reduce the
potential for interfering heterophile or natural antibodies, a
serum matrix diluent was provided by the manufacturer of the assay
kits. Tests for autoantibodies against three type 1
diabetes-associated autoantigens performed in all study
participants, including those against insulin autoantibody, GAD
antibody, and insulinoma-associated protein 2 antigen. Assays were
performed as previously described (She J. S., et al. Proc Natl.
Acad, Sci USA 96:8116-8119, 1999).
Statistics
[0166] All statistical analyses were undertaken with GraphPad Prizm
4.0 (GraphPad, San Diego, Calif.) using Fisher's exact test,
receiver-operator characteristic (ROC) analysis, linear regression,
t testing, or ANOVA (Kruskal-Wallis) with Dunn's post-testing.
P<0.05 was deemed significant.
Example 1
Determination of Adiponectin and Leptin Levels In Vivo
[0167] Adiponectin and leptin levels were determined in 18 T2D
children (11M/12F; median age 14 years, range 10-20 years), 20
non-diabetic age matched individuals from the general population
(11M/9F; median age 12.0 years, range 5-21 years), as well as 44
T1D patients (22M/22F; median age 14.0 years, range 6-20 years).
Signed (IRB approved) informed consents and assents were obtained
from the children and their parents. LINCOPLEX.TM. (Linco Research,
St. Louis, Mo., USA) kits were used for measurement of human leptin
(sensitivity, 23.4 pg/mL; assay range, 0.375 ng/mL to 12 ng/mL;
intra-assay CV=4.6-5.8%; inter-assay CV=3.2-7.4%), while B-Bridge
International (San Jose, Calif., USA) human adiponectin ELISA kits
were used for monitoring serum adiponectin levels (sensitivity,
23.4 pg/mL; assay range, 0.375 ng/mL to 12 ng/mL; intra-assay
CV=4.6-5.8%; inter-assay CV=3.2-7.4%). T1D related autoantibody
assays were performed for anti-insulin (IAA), anti-glutamic acid
decarboxylase (GADA), and anti-IA2 autoantibodies (IA-2A). All
radiobinding assays were subjected to utilising an index cut-off
for positivity based on previously investigated control
populations. All statistical analyses were undertaken with GraphPad
Prizm.
[0168] Leptin levels were directly correlated with BMI for the
entire pediatric population studied (r=0.62; p<0.0001). This
association was also observed when analyzed as a function of
disease status. After adjustment to analysis of subjects whose
BMI> or equal to 85.sup.th percentile, children with T2D had
significantly higher leptin levels than healthy children (FIG. 1B;
p<0.003), while those with T1D demonstrated reduced levels in
comparison to healthy children (FIG. 1B; p<0.001). Without
adjustment for BMI, a similar trend was observed in that mean
leptin levels were elevated in T2D subjects (23.1 ng/ml
(15.6-30.6)) versus all healthy controls (7.4 ng/ml (2.8-12);
p<0.0004) or T1D subjects (4.5 ng/ml (3.3-5.7); p<0.001).
Leptin concentrations were gender-dependent, being higher among
females (11.4 ng/ml (7.6-15.1)) than males (6.3 ng/ml (3.2-9.4);
p<0.003) regardless of their diagnosis and BMI.
[0169] Adiponectin to leptin ratios revealed an even more striking
difference between T1D and T2D children. Adiponectin/leptin ratios
were dramatically different amongst healthy children (11.8
(4.8-18.7)) and those with T1D (6.1 (3.8-8.3)) or T2D (0.4
(0.3-0.5)) (FIG. 1C; p<0.0001). As anticipated, when restricting
analysis to include only those with BMI> or equal to 85.sup.th
percentile (FIG. 1D) or Tanner 4-5, the ratios decrease since
increases in BMI positively associate with increasing leptin or
pubertal stage and decreases in Adiponectin. Despite this, the
ratio for T1D was significantly elevated versus T2D subjects
(p<0.0001).
Example 2
Adiponectin Levels in Children and Adolescents
[0170] Adiponectin levels were inversely correlated with BMI for
the entire pediatric population studied (r.sup.2=0.60;
P<0.0001). Analysis of subjects with BMI was >85.sup.th
percentile indicated that control subjects had higher adiponectin
levels than type 2 diabetic subjects (FIG. 2A; control versus type
2 diabetic subjects, P<0.01). Type 1 diabetic subjects were not
significantly different from healthy control subjects (P=NS), yet
type 1 diabetic subjects were higher than those with type 2
diabetes (P<0.01). There was no correlation between adiponectin
levels and sex, but levels were lower in subjects with type 2
diabetes who were Tanner stage 4 or 5 (FIG. 2B; control versus type
1 diabetic subjects, P=NS; control versus type 2 diabetic subjects,
P<0.01; and type 1 diabetic versus type 2 diabetic subjects,
P<0.01). The adiponectin levels in the pediatric type 1 diabetic
subjects (FIG. 2C; 10.2 .mu.g/ml [95% CI 8.6-11.7]) did not differ
from healthy control subjects (10.6 .mu.g/ml [9.2-12.0]; P=NS).
Children with type 2 diabetes (5.5 .mu.g/ml [4.8-6.2]) had
significantly lower adiponectin levels than both of those groups
(control versus type 2 diabetic subjects, P<0.001; type 1
diabetic versus type 2 diabetic subjects, P<0.01).
Example 3
Childhood and Adolescent Leptin Levels
[0171] Leptin levels were directly correlated with BMI for the
entire pediatric population studied (r.sup.2=0.60; P<0.0001).
This association was also observed when analyzed as a function of
disease status. An analysis of subjects whose BMI was >85.sup.th
percentile (FIG. 2D; control versus type 2 diabetic subjects,
P<0.001; type 1 diabetic versus type 2 diabetic subjects,
P<0.01) or those with Tanner stage 4 and 5 (FIG. 2E; control
versus type 2 diabetic subjects, P<0.001; type 1 diabetic versus
type 2 diabetic subjects, P<0.001) indicated children with type
2 diabetes had significantly higher leptin levels than healthy
children and those with type 1 diabetes. However, leptin levels
were not different between type 1 diabetic and control subjects if
evaluating those >85.sup.th percentile (control versus type 1
diabetic subjects; P=NS) or with Tanner stage 4 and 5 (control
versus type 1 diabetic subjects; P=NS). Leptin concentrations were
somewhat higher (albeit not statistically significant) among
females (7.1 ng/ml [95% CI 5.5-8.7]) than males (5.2 ng/ml
[3.8-6.6]; P=0.061), regardless of their diagnosis and BMI. Without
accounting for BMI (i.e., all subjects), a trend was observed in
that mean leptin levels were elevated in type 2 diabetic subjects
(FIG. 2F; 24.3 ng/ml [17.1-31.5]) versus all healthy control (2.7
ng/ml [1.3-4.1]; P<0.001) or type 1 diabetic (5.1 ng/ml
[3.5-6.7]; P<0.001) subjects. Leptin levels were also modestly
elevated in type 1 diabetic subjects compared with control subjects
(P<0.05).
Example 4
Adiponectin-to-Leptin Ratios
[0172] An exploration of adiponectin-to-leptin ratios revealed an
even more striking difference between type 1 and type 2 diabetic
children. Adiponectin-to-leptin ratios were dramatically different
among healthy (20.2 [95% CI 11.3-29.0]) and type 1 diabetic (6.3
[3.8-8.8]) children than those with type 2 diabetes (0.3 [0.2-0.5])
(FIG. 3; control versus type 1 diabetic subjects, P=NS; control
versus type 2 diabetic subjects, P<0.001; type 1 diabetic versus
type 2 diabetic subjects, P<0.001). When restricting analysis to
include only those subjects with BMI>85.sup.th percentile
(control versus type 1 diabetic subjects, P<NS; control versus
type 2 diabetic subjects, P<0.001; type 1 diabetic versus type 2
diabetic subjects, P<0.01) or Tanner stage 4 and 5 (control
versus type 1 diabetic subjects, P<NS; control versus type 2
diabetic subjects, P<0.001; type 1 diabetic versus type 2
diabetic subjects, P<0.001), the ratios decrease because
increases in BMI positively associate with increasing leptin or
pubertal stage and decreases in adiponectin. Despite this, the
ratio for type 1 diabetic and control subjects was significantly
elevated versus type 2 diabetic subjects (P<0.001). To ascertain
potential influences of ethnicity, an analysis was performed that
compared adiponectin-to-leptin ratios as a function of race (FIG.
4). No differences were observed in this ratio when comparing
Caucasian and African American subjects within the same disease
group (all P=NS).
Example 5
Diagnostic Value
[0173] ROC plots were constructed comparing type 1 with type 2
diabetic subjects (i.e., area under the ROC curve of 0.969 [95% CI
0.93-1.00]; P<0.0001) to determine an appropriate cutoff value
for the adiponectin-to-leptin ratio (FIG. 5). At a ratio cutoff of
<0.9, the sensitivity was 100% (range 80-100%) with specificity
of 80% (65-91%) for type 2 as opposed to type 1 diabetes. At a
ratio cutoff of <0.7, sensitivity was 88% (64-99%) with
specificity of 90% (77-97%).
TABLE-US-00001 TABLE 1 Anthropometrical and ethnic variables in the
study population Healthy Type 2 diabetes Type 1 diabetes Control
subjects n 17 4 43 Age (years) 144 (10-20) 13.2 (9-20) 13.7 (6-21)
Men/Women 4/13 21/20 22/21 Tanner 4.4 (2-5) 3.9 (1-5) 3.3 (1-5) BMI
(kg/m.sup.2) 36 (32.6-39.4) 21.7 (20.4-23.1) 20.5 (18.8-22.3)
Disease duration 2.4 (0.1-7) 3.9 (0.1-10) NA (years) Ethnicity (%)
Caucasian 18 7 62 African American 75 2 33 Latino 6 7 5 Treatment
Combination of oral Insulin NA hypoglycemic agents with/without
insulin Data are mean (range) unless otherwise indicated. Data for
BMI are mean (95% CI). No healthy control subject (or any type 2
diabetic subject by their differential diagnosis) was identified
with any of three type 1 diabetes-associated autoantibodies. In
contrast, frequencies of 50-70% positivity were observed for those
autoantibodies in the type 1 diabetic group. NA, not
applicable.
[0174] All publications, patent applications, patents, and other
references mentioned herein are incorporated in pertinent part by
reference herein for the reasons cited in the above text.
* * * * *