U.S. patent application number 12/499063 was filed with the patent office on 2010-05-13 for diagnosis and monitoring of diseases.
This patent application is currently assigned to DMI LIFE SCIENCES, INC.. Invention is credited to David Bar-Or, Raphael Bar-Or.
Application Number | 20100120056 12/499063 |
Document ID | / |
Family ID | 32074376 |
Filed Date | 2010-05-13 |
United States Patent
Application |
20100120056 |
Kind Code |
A1 |
Bar-Or; David ; et
al. |
May 13, 2010 |
DIAGNOSIS AND MONITORING OF DISEASES
Abstract
The present invention relates to the diagnosis and monitoring of
diseases and conditions by quantifying markers, including
degradation products of disease-associated proteins, such as
diketopiperazines composed of the two N-terminal amino acids or the
two C-terminal amino acids of such proteins. The methods are useful
for diagnosing or monitoring various diseases, including multiple
sclerosis, Alzheimer's disease and ischemia. The invention further
provides binding partners specific for the markers and compositions
and kits for conducting the methods of the invention.
Inventors: |
Bar-Or; David; (Englewood,
CO) ; Bar-Or; Raphael; (Denver, CO) |
Correspondence
Address: |
SHERIDAN ROSS PC
1560 BROADWAY, SUITE 1200
DENVER
CO
80202
US
|
Assignee: |
DMI LIFE SCIENCES, INC.
Greenwood Village
CO
|
Family ID: |
32074376 |
Appl. No.: |
12/499063 |
Filed: |
July 7, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10679699 |
Oct 2, 2003 |
7575929 |
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12499063 |
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60415908 |
Oct 2, 2002 |
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60489039 |
Jul 21, 2003 |
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60503185 |
Sep 15, 2003 |
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Current U.S.
Class: |
435/7.1 ;
530/350; 530/387.1; 530/388.1 |
Current CPC
Class: |
G01N 33/6842 20130101;
G01N 2800/285 20130101; G01N 33/6848 20130101; G01N 33/6896
20130101 |
Class at
Publication: |
435/7.1 ;
530/350; 530/387.1; 530/388.1 |
International
Class: |
G01N 33/53 20060101
G01N033/53; C07K 16/00 20060101 C07K016/00 |
Claims
1.-46. (canceled)
47. A method for diagnosing or monitoring a disease or condition
comprising the steps of: (a) obtaining a biological sample from a
patient to be diagnosed or monitored; (b) determining the quantity
of a marker in said biological sample, wherein said marker is: (i)
a truncated disease-associated protein lacking its two N-terminal
amino acids, wherein said truncated disease-associated protein is
not human serum albumin; (ii) a truncated disease-associated
protein lacking its two C-terminal amino acids; (iii) a truncated
disease-associated protein lacking its two N-terminal amino acids
and its two C-terminal amino acids; (iv) a diketopiperazine (DKP)
comprising the two N-terminal amino acids of a disease-associated
protein; (v) a DKP comprising the two C-terminal amino acids of a
disease-associated protein; or (vi) two or more markers selected
from those listed in (i) through (v) above; provided that when only
a single DKP is used as the marker, it will not be His-Pro DKP; and
(c) determining if the quantity(ies) of said marker(s) in said
biological sample is(are) indicative of the presence, absence or
status of the disease or condition.
48. The method of claim 47, wherein said disease-associated protein
is myelin basic protein, beta-amyloid, Rh factor, pulmonary
surfactant-associated protein A, B or D, insulin, tau protein,
alpha-synuclein, albumin, C-reactive protein, interleukin 8, S100
proteins, beta-chorionic gonadotropin, fetal erythropoietin,
pregnancy-associated protein A, myoglobin, troponin I, troponin T,
prostate specific antigen, amylase, lipase, alphal-antitrypsyn,
erthyropoietin, activated protein C, tethal chain, zeta chain,
alpha chain, beta chain, delta chain, epsilon chain, gamma AG and
brain natriuretic peptide.
49. The method of claim 47 or 48, wherein said marker is a
truncated disease-associated protein.
50. The method of claim 47 or 48, wherein said marker is X-Y-DKP,
wherein X-Y-DKP is a diketopiperazine composed of amino acids X and
Y, and X and Y are the two N-terminal or the two C-terminal amino
acids of a disease-associated protein.
51. The method of claim 50, wherein X and Y are the two C-terminal
amino acids of a disease-associated protein.
52. The method of claim 50, wherein X and Y are the two N-terminal
amino acids of a disease-associated protein.
53. The method of claim 50, wherein X-Y-DKP is Asp-Ala-DKP,
Met-Ala-DKP, Gln-Asn-DKP, Gly-Leu-DKP or combinations of the
foregoing.
54. The method of claim 50, wherein X-Y-DKP is Gly-Leu-DKP,
Ala-Pro-DKP, Glu-Ala-DKP, Leu-Pro-DKP, Asp-Arg-DKP, His-Gly-DKP or
combinations of the foregoing.
55. The method of claim 50, wherein X-Y-DKP is Arg-His-DKP,
His-Pro-DKP, Ser-Pro-DKP, or combinations of the foregoing.
56. The method of claim 50, wherein X-Y-DKP is Gly-Leu-DKP,
Pro-Glu-DKP, Gln-Gly-DKP, Glu-Ser-DKP, or combinations of the
foregoing.
57. The method of claim 47, wherein the disease or condition is
multiple sclerosis, rheumatoid arthritis, acute respiratory
distress syndrome, cystic fibrosis, diabetes mellitus, Alzheimer's
disease, Parkinson's disease, inflammation, ischemia, cerebral
ischemia, placental ischemia, myocardial infarction, prostate
cancer, pancreatitis, emphysema, renal disease, cancer,
chemotherapy, hemoglobinopathies, anemnias or congestive heart
failure.
58. The method of claim 57 wherein the disease or condition is
Alzheimer's disease.
59. The method of claim 58, wherein the method comprises
determining the quantity in said biological sample of
Asp-Ala-DKP.
60. The method of claim 59 further comprising determining the
quantity of a compound having a mass of about 175 as determined by
liquid chromatography and mass spectrometry.
61. The method of claim 57 wherein the disease or condition is
placental ischemia.
62. The method of claim 61, wherein the method comprises
determining the quantity in said biological sample of: (i)
Gly-Leu-DKP; (ii) Ala-Pro-DKP; or (iii) both Gly-Leu-DKP and
Ala-Pro-DKP.
63. The method of claim 47, wherein step (b) is conducted by mass
spectrometry, chemical assay or immunoassay.
64. The method of claim 63, wherein step (b) is conducted by
immunoassay.
65. The method of claim 64, wherein said immunoassay is conducted
by using one or more binding partners specific for a marker.
66. The method of claim 65, wherein the binding partner is an
antibody or an aptamer.
67. The method of claim 47, wherein said biological sample is a
body fluid.
68. The method of claim 67, wherein said body fluid is serum,
plasma, blood, urine, saliva, cerebrospinal fluid, tears, semen,
vaginal secretion, amniotic fluid or cord blood.
69. The method of claim 68, wherein said body fluid is plasma or
serum.
70. The method of claim 47, wherein said patient is an animal.
71. The method of claims 70, wherein said patient is a human.
72. An isolated binding partner having specificity for a marker
selected from the group consisting of: (a) a truncated
disease-associated protein lacking its two N-terminal amino acids,
wherein said truncated disease-associated protein is not human
serum albumin; (b) a truncated disease-associated protein lacking
its two C-terminal amino acids; (c) a truncated disease-associated
protein lacking its two N-terminal amino acids and its two
C-terminal amino acids; (d) a diketopiperazine (DKP) comprising the
two N-terminal amino acids of a disease-associated protein, wherein
the DKP is not His-Pro DKP; and (e) a DKP comprising the two
C-terminal amino acids of a disease-associated protein, wherein the
DKP is not His-Pro DKP.
73. The isolated binding partner of claim 72, wherein the binding
partner has specificity for a DKP.
74. The isolated binding partner of claim 73, wherein the binding
partner has specificity for Arg-Arg-DKP, Gln-Asn-DKP, Lys-Arg-DKP,
Glu-Phe-DKP, Ser-Met-DKP, Cys-Asn-DKP, Lys-Ala-DKP, Gln-Asn-DKP,
Gly-Leu-DKP, Ala-Ala-DKP, Trp-Pro-DKP, Asn-Ser-DKP, Leu-Pro-DKP,
Asp-Arg-DKP, His-Gly-DKP, Gln-Gly-DKP, Glu-Ser-DKP, Asn-Pro-DKP,
Lys-Leu-DKP, Pro-Cys-DKP, Asn-Lys-DKP, Asp-Arg-DKP, Ala-Pro-DKP or
Arg-His-DKP.
75. The isolated binding partner of claim 73, wherein the binding
partner has specificity for N-acetyl-Ala-Ser-DKP,
N-acetyl-Ala-phosphorylated-Ser-DKP, Asp-Ala-DKP, Glu-Ile-DKP,
Glu-Val-DKP, Phe-Pro-DKP, Ala-Glu-DKP, Phe-Val-DKP, Gly-Ile-DKP,
Met-Ala-DKP, Met-Asp-DKP, Glu-Lys-DKP, Gln-Thr-DKP, Ala-Val-DKP,
Gly-Leu-DKP, Ala-Pro-DKP, Glu-Ala-DKP, Pro-Glu-DKP, Lys-Ser-DKP,
Ile-Val-DKP, Gln-Tyr-DKP, Lys-Glu-DKP, Glu-Asp-DKP, Ala-Pro-DKP,
Ala-Asn-DKP, Ala-Leu-DKP, Ser-Leu-DKP, Val-Leu-DKP, Val-His-DKP,
Gly-His-DKP or Ser-Pro-DKP.
76. The isolated binding partner of any one of claims 72-75,
wherein said binding partner is an antibody.
77. The isolated binding partner of claim 76, wherein said antibody
is a monoclonal antibody.
78. The isolated binding partner of any one of claims 72-75,
wherein said binding partner is an aptamer.
79. A composition comprising the binding partner of any one of
claims 72-75 in a physiologically-acceptable carrier.
80. A kit comprising the binding partner of claim 72-75 and
associated reagents for quantitating the marker.
81. The kit of claim 80, wherein said binding partner is an
antibody.
82. The kit of claim 81, wherein said antibody is a monoclonal
antibody.
83. The kit of claim 80, wherein said binding partner is an
aptamer.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation of U.S. application Ser.
No. 10/679,699, filed Oct. 2, 2003, which claims the benefit of
U.S. Provisional Applications 60/415,908, filed Oct. 2, 2002,
60/489,039, filed Jul. 21, 2003, and 60/503,185, filed Sep. 15,
2003. The complete disclosures of these applications are
incorporated herein by reference for all purposes.
FIELD OF THE INVENTION
[0002] The invention relates to the diagnosis and monitoring of
diseases and medical conditions by quantitating one or more
biochemical markers associated with the diseases or conditions. In
particular, the invention relates to the detection and measurement
of diketopiperazines composed of the two N-terminal amino acids of
disease-associated proteins, diketopiperazines composed of the two
C-terminal amino acids of disease-associated proteins, truncated
disease-associated proteins missing the two N-terminal amino acids
and/or the two C-terminal amino acids, and other biochemical
markers of such diseases and conditions.
BACKGROUND
[0003] Simpler and faster tests for diagnosing and monitoring
diseases and medical conditions are always needed. In addition,
many serious illnesses remain difficult to diagnose and monitor,
and methods of diagnosing and monitoring these diseases and
conditions are critically needed.
[0004] For instance, multiple sclerosis (MS) is difficult to
diagnose because the progress, severity and specific symptoms of MS
are quite variable and unpredictable. There are no laboratory
tests, symptoms or physical findings that can, by themselves,
determine if a person has MS.
[0005] The long established criteria for diagnosing MS are:
[0006] 1. There must be objective evidence of two attacks (i.e.,
two episodes of demyelination in the central nervous system (CNS)).
An attack (also known as an exacerbation, flare or relapse) is
defined clinically as the sudden appearance or worsening of an MS
symptom or symptoms, which lasts at least 24 hours. The objective
evidence comes from findings of a neurological examination and
additional tests.
[0007] 2. The two attacks must be separated in time (at least one
month apart) and space (indicated by evidence of inflammation
and/or damage in different areas of the CNS).
[0008] 3. There must be no other explanation for these attacks or
the symptoms the person is experiencing. Many symptoms that are
common in MS can also be caused by other diseases. Therefore, the
MS diagnosis can only be made by carefully ruling out all
possibilities.
[0009] Over the last twenty years, tests such as magnetic resonance
imaging (MRI), examination of cerebrospinal fluid, and evoked
response testing have played an increasingly important role in the
diagnostic process. In 2001, the International Panel on the
Diagnosis of Multiple Sclerosis issued a revised set of diagnostic
criteria (Annals of Neurology, 50:121-127 (2001)). In addition to
the traditional requirements given above, the revised criteria
provide specific guidelines for using findings of MRI,
cerebrospinal fluid analysis and visual evoked potentials to
provide evidence of the second attack and thereby confirm the
diagnosis more quickly. These guidelines also facilitate the
diagnostic process in those patients who have had steady
progression of disability without distinct attacks. However, even
with these revised criteria, diagnosis of MS is still difficult and
still typically takes several months or even years.
[0010] Due to the possibility of worsening or recurrence of MS,
making a conclusive diagnosis quickly would be of great benefit.
Drugs for the treatment of MS are now available which slow or
prevent progression of the disease in many patients, and a quick
diagnosis would allow early intervention and could significantly
improve the prognosis for many MS patients.
[0011] The diagnosis of Alzheimer's disease is difficult and often
relies on the exclusion of other causes. Various cognitive tests
are employed to possibly identify the disease. However, a
definitive diagnosis is only possible by a brain autopsy after
death. Clearly, a diagnostic test that can provide a diagnosis for
living Alzheimer's disease patients is needed.
[0012] Brain ischemia is currently a clinical diagnosis. Although
certain biochemical markers have been described, such as Enolase,
S-100 family of proteins and others, the imaging techniques
available to the clinician are more reliable and specific. A
reliable and specific biochemical marker for brain ischemia would
be helpful in the diagnosis and monitoring of this disease.
[0013] Early cardiac ischemia is also difficult to diagnose.
Cardiac markers of cellular necrosis, such as creatine kinase
isoenzymes (CK-MB), myoglobin, or troponin, are unreliable markers
of transient myocardial ischemia, particularly when measured in the
first 2 to 6 hours after an ischemic event. Kontos, M. C. and R. L.
Jesse, Am J Cardiol, 2000. 85(5A): p. 32B-39B; Ishikawa, Y., et
al., Clin Chem, 1997. 43(3): p. 467-75; Brogan, G. X., Jr., et al.,
Acad Emerg Med, 1997. 4(1): p. 6-12; Hedges, J. R., et al., Acad
Emerg Med, 1996. 3(1): p. 27-33. Patients who are examined soon
after the onset of ischemic symptoms typically require prolonged
observation to rule out myocardial infarction or myocardial
ischemia. Gomez, M. A., et al., J Am Coll Cardiol, 1996. 28(1): p.
25-33; Zalenski, R. J., et al., Arch Intern Med, 1997. 157(10): p.
1085-91; de Winter, R. J., et al., Ann Emerg Med, 2000. 35(2): p.
113-20; Peacock, W. I., et al. Ann Emerg Med, 2000. 35(3): p.
213-20.
[0014] A novel blood assay method to measure reduced exogenous
cobalt binding to human serum albumin in patients with myocardial
ischemia has been described. Bar-Or et al., J. Emerg. Med., 2000.
19(4): p. 311-5. The albumin-cobalt binding (ACB) assay measures
the binding capacity of exogenous cobalt to the amino terminus
(N-terminus) of human albumin. Under normal conditions, transition
metals, including cobalt, are tightly bound to the exposed
N-terminus of albumin. Kubal, G., P. J. Sadler, and A. Tucker, Eur
J Biochem, 1994. 220(3): p. 781-7. The ACB assay is based on
observations that ischemic conditions may alter the N-terminus of
albumin and rapidly reduce its binding capacity for transition
metals. Berenshtein et al., J. Mol. Cell. Cardiol., 1997. 29(11):
p. 3025-34; Bar-Or et al., Eur. J. Biochem., 2001. 268(1): p.
42-47. Ischemia-induced alterations to albumin would be predicted
to occur minutes or hours before abnormal levels of CK-MB,
myoglobin, or troponin could be detected. However, the ACB assay
has been approved only as a test to rule out cardiac ischemia, and
it would be highly desirable to have an assay that could diagnose
cardiac ischemia, as well as rule it out.
[0015] Low birth weight (LBW) is the leading cause of fetal and
neonatal morbidity and mortality worldwide. LBW is generally
accepted to indicate a weight of less than 2500 grams at delivery,
and may result from a newborn being born at term but small for
gestational age (SGA), being born preterm and appropriate for
gestational age (AGA) or being both preterm and SGA. As such, the
epidemiology of LBW is complex and multifactorial.
[0016] SGA is a statistical definition, indicating that the birth
weight is less than the tenth percentile for gestational age. By
definition then, 10% of newborns are SGA. In practice, some of
these newborns are small and well, fulfilling their genetic growth
potential, and are not at substantial risk. Other SGA newborns on
the other hand are truly growth impaired, failing to meet their
genetic growth potential due to a variety of factors as discussed
below. These newborns are said to suffer from fetal growth
restriction (FGR). In practice, some infants are presumably AGA and
suffer from FGR; that is to say their weight may be at the
20.sup.th percentile for gestational age, but they were genetically
programmed to weigh at the 80.sup.th percentile. These infants are
difficult to identify in a practical sense, as there is no a priori
way of knowing how much an individual "should" weigh.
[0017] FGR leads to LBW both by direct impairment of fetal growth,
and often in addition by necessitating indicated preterm delivery
due to compromised fetal status or associated maternal disease
(e.g., preeclampsia). Morbidity due to LBW and/or prematurity is
varied and substantial and well documented elsewhere. Additionally,
recent data have suggested that a compromised intrauterine
environment can have a profound influence on health in adult life,
the so-called "fetal origins of disease" or Barker hypothesis. Via
these various mechanisms, the disease burden attributable to FGR is
tremendous.
[0018] While the fetus/neonate is often the focus of concern in
pregnancies complicated by FGR, it is important to recall that
these pregnancies are also often complicated by conditions that
directly threaten maternal health. Most notably, preeclampsia,
whose precise pathophysiology remains obscure, has long been felt
to result from placental ischemia. Preeclampsia and its
complications are the leading causes of maternal mortality
worldwide.
[0019] While the differential diagnosis of FGR is diverse,
including chromosomal, toxic, viral and other etiologies, the
majority of cases result from uteroplacental insufficiency (UPI).
UPI may be associated with a variety of maternal diseases
(hypertension, renal disease, systemic lupus erythematosus,
antiphospholipid syndrome, thrombophilia, etc.), pregnancy
complications (placental abruption, preeclampsia), or may be
idiopathic. Regardless of the etiology, the presumed unifying
underlying pathophysiology results from reduced placental blood
flow (ischemia) in either the maternal or the fetal circulation, or
both.
[0020] As a crude measure, it is known that there is a direct
relationship between placental weight and fetal weight, suggesting
that placental resources might control fetal growth to some extent.
There are a large number of placental pathologic lesions associated
with FGR. In general, these are lesions that would be expected to
compromise maternal and/or fetal blood flow. The association
between reduced maternal and/or fetal blood flow (ischemia) and FGR
is also corroborated by a large amount of Doppler flow data in
affected pregnancies. In many cases, these abnormal Doppler flow
waveforms correlate well with abnormal placental pathology.
[0021] While much is known about the pathophysiology of FGR, much
remains to be understood. In the clinical setting, although various
risk factors for FGR are recognized, their positive predictive
values and sensitivities are limited. There can be difficulty
differentiating the FGR fetus from the "SGA but well" fetus.
Recognizing this difference is important to avoid unnecessary
interventions on well pregnancies. Early identification of
pregnancies destined to be affected by FGR might help foster
appropriate follow-up. Timing of delivery is also a matter of
intense interest, balancing the benefits of advancing gestation
against those of continuing in an ischemic environment. Finally, on
a more fundamental level, access to a clinical test to identify
placental ischemia and quantify its severity might ultimately help
foster appropriate treatment or even prevention.
[0022] As noted above, the ACB assay for ruling out ischemia is
based on observations that ischemic conditions may alter the
N-terminus of human serum albumin and rapidly reduce its binding
capacity for transition metals. The nature of the alterations of
the N-terminus of human serum albumin that may account for its
reduced metal binding capacity have not been identified, but
cleavage of 1-4 amino acids has been proposed as one of several
possibilities. See PCT application WO 00/20840. In particular, it
has been hypothesized that cleavage of the N-terminal dipeptide
(Asp-Ala or DA) from human serum albumin and the cyclization of the
dipeptide to form the diketopiperazine (DA-DKP) may partially
explain the observation of reduced metal binding to N-terminus of
human serum albumin in ischemia. Bar-Or et al., Biochem. Biophys.
Res. Commun., 84:856-862 (Jun. 15, 2001). However, this article
does not teach or suggest that DA-DKP can be used as a marker of
ischemia.
[0023] PCT application WO 00/20454 discloses a marker for free
radical damage. The marker is human serum albumin whose N-terminal
metal binding site has been modified by free radical damage.
Reduced metal binding to the altered N-terminus is used to detect
and measure the free radical damage. Several possible modifications
of the N-terminus of human serum albumin that might account for the
reduced metal binding are proposed, including the possibility that
the N-terminal dipeptide (DA) is cleaved by free radicals and that
this dipeptide then cyclizes to form DA-DKP. Although direct
detection of the altered N-terminus of human serum albumin is
suggested as a method of detecting and measuring free radical
damage, measurement of the hypothetical DA-DKP is not taught or
suggested for this purpose.
[0024] Elevated levels of histidine-proline diketopiperazine
(HP-DKP) have been detected in neurological disorders, including
non-medicated schizophrenics and patients suffering from
amyotrophic lateral sclerosis (Prasad, Peptides, 1995 16:1 pp.
151-164), and in patients with renal failure (Takahara et al., J.
Clinical Endocrinol. Metab. 1983 56:2 pp. 312-319). HP-DKP may be
derived from thyrotropin-releasing hormone (TRH) or its precursor
(preproTRH) by unknown mechanisms and/or from other sources
(Prasad, Peptides, 1995 16:1 pp. 151-164).
SUMMARY OF THE INVENTION
[0025] The present invention is based on the discovery of objective
biochemical markers useful for diagnosing and monitoring various
diseases and medical conditions. The markers include
diketopiperazines composed of the two N-terminal amino acids or the
two C-terminal amino acids of disease-associated proteins. The term
"protein" is used herein to mean protein, polypeptide, oligopeptide
or peptide, and the term "disease-associated proteins" is used
herein to mean proteins associated with specific diseases or
conditions, including proteins from organs or tissues
("organ-specific" or "tissue-specific" proteins) affected by a
disease or condition. The markers also include truncated
disease-associated proteins from which the two N-terminal amino
acids and/or the two C-terminal amino acids are missing. These
markers are collectively referred to herein as "target
markers".
[0026] Accordingly, the present invention provides a method of
diagnosing or monitoring a disease or condition comprising
determining the quantity of one or more target markers in a
biological sample and determining if the quantity(ies) of the
marker(s) is(are) indicative of the presence, absence or status of
the disease or condition. The target markers can be measured
rapidly and conveniently, and these measurements provide objective
evidence which will allow a reliable diagnosis to be made easily
and quickly for diseases and conditions, such as, for example,
multiple sclerosis, Alzheimer' s disease and ischemia, particularly
placental ischemia. This method will be of great benefit, since it
will allow treatment of many diseases and conditions to begin much
earlier than is now possible. In addition, the measurement of the
target markers will allow the status of the diseases or conditions
to be monitored, allowing for more effective treatment of many
diseases, conditions and disorders and for the evaluation of new
drugs and other treatments.
[0027] The present invention further provides methods of diagnosing
or monitoring multiple sclerosis (MS) using a MS diagnostic
compound. The methods comprise obtaining a biological sample from a
patient to be tested and measuring the amount of one or more MS
diagnostic compounds in the biological sample. MS diagnostic
compounds include: (i) a compound having a mass of about 175 as
determined by liquid chromatography and mass spectrometry (LC-MS);
(ii) a compound having a mass of about 145 as determined by LC-MS;
(iii) Asp-Ala diketopiperazine (DA-DKP); and (iv)
N-acetyl-alanine-serine diketopiperazine (NAS-DKP). The absence of
compounds (i) and/or (ii) or an elevated level of DA-DKP and/or
NAS-DKP in the biological sample is indicative of MS. Also, an
elevated level of DA-DKP and/or NAS-DKP in the biological sample is
indicative of active MS. Other MS diagnostic compounds are listed
in Tables 1 and 2.
[0028] In a further embodiment, the invention provides methods of
diagnosing or monitoring Alzheimer's disease using various markers
of the disease. In particular, the methods comprise obtaining a
biological sample from a patient to be diagnosed or monitored and
measuring the amount of one or more Alzheimer's diagnostic
compounds in the biological sample. Alzheimer's diagnostic
compounds include: (i) a compound having a mass of about 175 as
determined by liquid chromatography and mass spectrometry; and (ii)
DA-DKP. Both Alzheimer's diagnostic compounds have been found
elevated in the plasma of Alzheimer' s patients. Other Alzheimer's
diagnostic compounds are listed in Tables 1 and 2.
[0029] In yet another embodiment, the invention provides methods of
diagnosing or monitoring placental ischemia in pregnant patients.
The methods comprise obtaining a biological sample from a pregnant
patient and measuring the amount of one or more placental ischemia
diagnostic compounds in the biological sample. Placental ischemia
diagnostic compounds include Gly-Leu diketopiperazine (GL-DKP) and
Ala-Pro diketopiperazine (AP-DKP). Other placental ischemia
diagnostic compounds are listed in Tables 1 and 2.
[0030] The invention also provides novel binding partners having
specificity for the diketopiperazines. The binding partners are
preferably antibodies and/or aptamers that specifically recognize
the diketopiperazines of the present invention. Such binding
partners can be used in the methods of the present invention.
Compositions and kits containing the novel binding partners are
also provided.
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] FIG. 1: Printout from a mass spectrometer. The sample was
recombinant beta-human chorionic gonadotropin processed by liquid
chromatography followed by mass spectrometry.
[0032] FIG. 2: Printout from a mass spectrometer. The sample was a
plasma sample from a pregnant woman (patient 4) processed by liquid
chromatography followed by mass spectrometry.
[0033] FIG. 3: Printout from a mass spectrometer. The sample was
recombinant erythropoietin processed by liquid chromatography
followed by mass spectrometry.
[0034] FIG. 4: Printout from a mass spectrometer. The sample was a
plasma sample from a pregnant woman (patient 4) processed by liquid
chromatography followed by mass spectrometry.
[0035] FIG. 5: A clustering dendogram.
DETAILED DESCRIPTION OF THE PRESENTLY
Preferred Embodiments of the Invention
[0036] The invention generally provides methods of diagnosing and
monitoring diseases, conditions and disorders by quantitating
markers for the diseases and conditions.
[0037] In one embodiment, the present invention provides methods of
diagnosing and monitoring diseases or conditions characterized by
the degradation of disease-associated proteins. The degradation
products include diketopiperazines composed of the two N-terminal
amino acids or the two C-terminal amino acids and the corresponding
truncated disease-associated proteins lacking such terminal amino
acids. Accordingly, the present invention is based on the discovery
that these degradation products are useful markers for diagnosing
and monitoring diseases, conditions and disorders.
[0038] As noted above, the term "disease-associated proteins" is
used herein to mean proteins associated with specific diseases,
conditions or disorders, including proteins from organs or tissues
("organ-specific" or "tissue-specific" proteins) affected by a
disease, condition or disorder. Examples of disease-associated
proteins and their corresponding diseases and conditions are listed
in Tables 1 and 2. Those skilled in the art can readily determine,
without undue experimentation, other disease-associated proteins,
their corresponding diseases or conditions, and useful markers
based on the guidance provided herein.
[0039] The target markers quantified in the methods of this
embodiment are formed by the degradation of the disease-associated
proteins. It is believed that this degradation occurs in diseases
or conditions involving or caused by acidosis, reactive oxygen
species (ROS), inflammation, and/or conditions which cause the
protonation of the N-terminal or C-terminal amino acids of the
disease-associated proteins, such as the binding of certain ligands
to the N-terminal or C-terminal amino acid. Diketopiperazines can
also be formed in vivo due to the action of certain enzymes (e.g.,
dipeptidyl peptidases or carboxypeptidases), and the activity of
these enzymes may be altered in certain diseases, conditions and
disorders. Dipeptidyl peptidases are amino peptidases which cleave
the two amino acids of the N-termini of proteins with some
specificity, while carboxypeptidases cleave amino acids from the
C-termini of proteins. The placenta, for example, is rich in
dipeptidyl peptidase IV. After the cleavage, or under specific
conditions, the enzymes may be responsible for cyclization, as well
as cleavage, of the amino acids. Alternatively, the second step
(cyclization) may be non-enzymatic and may require the protonation
of the N-terminus or C-terminus. Thus, the markers useful in the
present invention include diketopiperazines composed of two amino
acids from either terminal end of a disease-associated protein and
the truncated disease-associated proteins without the two
N-terminal and/or the two C-terminal amino acids.
[0040] As used herein, "X-Y DKP" or "X-Y-DKP" means a
diketopiperazine (cyclic dipeptide) composed of two amino acids, X
and Y, wherein X and Y are the two N-terminal or the two C-terminal
amino acids of a disease-associated protein. X and Y may be the
same or different and each may be any amino acid, including any
post-translationally modified amino acid. Notwithstanding the
foregoing, X-Y DKP may not be His-Pro DKP when a single
diketopiperazine is the only marker measured. Table 3 lists the
conventional three-letter and single-letter abbreviations for each
amino acid. Post-translational modifications of amino acids are
well known and include phosphorylation, acylation, cysteinylation,
nitrosylation, and glycosylation.
[0041] Examples of diketopiperazines useful as markers in the
present invention are listed in Tables 1 and 2 along with their
corresponding diseases and disease-associated proteins. Those
skilled in the art can readily identify, without undue
experimentation, other diketopiperazines derived from the two
N-terminus amino acids or the two C-terminus amino acids of a
disease-associated protein that can be used as target markers of
various diseases and conditions.
TABLE-US-00001 TABLE 1 Disease Protein N-terminal DKP MW Multiple
Sclerosis Myelin basic N-acetyl-Ala- 280 protein (MBP) phospho-Ser
MBP N-acetyl-Ala-Ser 200 Beta-amyloid Asp-Ala 186 Rheumatoid
Arthritis Rh Factor Glu-Ile 242.3 ARDS*, Pulmonary (A) Glu-Val
228.24 Cystic Fibrosis surfactant- (B) Phe-Pro associated
(D)Ala-Glu proteins A, B and D Diabetes Mellitus Insulin Phe-Val
246.34 Gly-Ile 170.24 Alzheimer's disease Beta-amyloid Asp-Ala
186.15 tau protein Met-Ala Parkinson's disease alpha-synuclein
Met-Asp 246.31 Glu-Lys 257.38 Inflammation Albumin Asp-Ala 186.15
(general)** C-reactive Gln-Thr 229.23 protein Interleukin 8 Ala-Val
170.2 Ischemia (general) Albumin Asp-Ala 186.15 Cerebral Ischemia
S100 family of Many Many proteins Placental Ischemia Beta-chorionic
Gly-Leu 170.24 gonadotropin Fetal Ala-Pro 168.18 erythropoietin
Pregnancy- Glu-Ala associated protein A Myocardial Infarction
Myoglobin Gly-Leu 170.24 Troponin I Pro-Glu 226.22 Prostate Cancer
Prostate Lys-Ser 215.28 Specific Antigen (PSA) Ile-Val Pancreatitis
Amylase Gln-Tyr 291.3 Lipase Lys-Glu 257.28 Emphysema alpha1-
Glu-Asp 244.23 antitrypsyn Renal Disease, Cancer, Erythropoietin
Ala-Pro 168.18 Chemotherapy Sepsis Activated Ala-Asn 185.17 protein
C Hemoglobinopathies, Tethal Chain Ala-Leu 199.24 Amemias Zeta
Chain Ser-Leu 215.24 Alpha Chain Val-Leu 227.3 Beta Chain Val-His
236.27 Delta Chain Val-His 236.27 Epsilon Chain Val-His 236.27
Gamma AG Gly-His 194.19 Congestive heart Brain natriuretic His-Pro
234.25 failure peptide Ser-Pro 184.18
TABLE-US-00002 TABLE 2 Disease Protein C-terminal DKP MW Multiple
Sclerosis Myelin basic Arg-Arg 312.36 protein (MBP) Beta-amyloid
Gln-Asn 242.23 Rheumatoid Arthritis Rh Factor Lys-Arg 284.35 ARDS*,
Pulmonary (A) Glu-Phe 276.28 Cystic Fibrosis surfactant- (B)
Ser-Met 218.26 associated (D) Glu-Phe 276.28 proteins A, B and D
Diabetes Mellitus Insulin Cys-Asn 217.24 Lys-Ala 199.24 Alzheimer's
disease Beta-amyloid Gln-Asn 242.23 tau protein Gly-Leu 170.21
Parkinson's disease alpha-synuclein Ala-Ala 142.14 Ala-Ala 142.14
Inflammation Albumin Gly-Leu 170.21 (general)** C-reactive Trp-Pro
283.32 protein Interleukin 8 Asn-Ser 201.17 Ischemia (general)
Albumin Gly-Leu 170.21 Cerebral Ischemia S100 family of Many Many
proteins Placental Ischemia Beta-chorionic Leu-Pro 210.27
gonadotropin Fetal Asp-Arg 271.26 erythropoietin Pregnancy- His-Gly
194.19 associated protein A Myocardial Infarction Myoglobin Gln-Gly
185.18 Troponin I Glu-Ser 216.18 Prostate Cancer Prostate Asn-Pro
211.21 Specific Antigen (PSA) Asn-Pro 211.21 Pancreatitis Amylase
Lys-Leu 241.33 Lipase Pro-Cys 200.25 Emphysema alpha1- Asn-Lys
256.3 antitrypsyn Renal Disease, Cancer, Erythropoietin Asp-Arg
271.26 Chemotherapy Sepsis Activated Ala-Pro 168.18 protein C
Hemoglobinopathies, Tethal Chain Amemias Zeta Chain Alpha Chain
Beta Chain Delta Chain Epsilon Chain Gamma AG Congestive heart
Brain natriuretic Arg-His 293.32 failure peptide *ARDS = acute
respiratory distress syndrome. **Asp-Ala diketopiperazine (DA-DKP)
and/or Gly-Leu diketopiperazine (GL-DKP) derived from albumin, a
circulating protein, will be general markers of inflammation. Other
diketopiperazines derived from disease-associated proteins,
including those found in specific organs or tissues, will be
markers of inflammation in those organs and tissues or associated
with those diseases and conditions.
TABLE-US-00003 TABLE 3 Three-Letter Single-Letter Amino Acid
abbreviation abbreviation Alanine Ala A Arginine Arg R Asparagine
Asn N Aspartic acid Asp D Asparagine or aspartic acid Asx B
Cysteine Cys C Glutamine Gln Q Glutamic acid Glu E Gluatmine or
glutamic acid Glx Z Glycine Gly G Histidine His H Isoleucine Ile I
Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F
Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W
Tyrosine Tyr Y Valine Val V
[0042] Other useful target markers formed by the degradation of the
disease-associated protein are referred to as "truncated
disease-associated proteins." As noted previously, these truncated
disease-associated proteins lack the two N-terminal amino acids
and/or the two C-terminal amino acids and, therefore, can be
utilized as target markers in the present methods. However, the
truncated disease-associated protein may not be human serum albumin
lacking the two N-terminal amino acids. Truncated
disease-associated proteins include, for example, myelin basic
protein missing the amino acids N-acetyl-Ala and Ser from the
N-terminus and beta-amyloid missing the amino acids Asp and Ala
from the N-terminus, both of which are useful target markers of
multiple sclerosis. Truncated beta-amyloid missing the two
N-terminal amino acids Asp and Ala and truncated tau protein
missing the two C-terminal amino acids Gly and Leu are examples of
target markers for Alzheimer' s disease. Those skilled in the art
can readily identify other useful truncated disease-associated
proteins as target markers of various diseases and conditions.
[0043] In the first embodiment of the present invention, the
methods comprise: [0044] (a) obtaining a biological sample from a
patient to be diagnosed or monitored; [0045] (b) determining the
quantity of one or more target markers of the disease or condition;
and [0046] (c) determining if the quantity(ies) of the target
marker(s) is(are) indicative of the presence, absence or status of
the disease or condition.
[0047] In the methods, the target markers can be quantified in any
suitable biological sample derived from the patient to be diagnosed
or monitored. Biological samples include suitable body fluids, such
as serum, plasma, blood, urine, saliva, cerebrospinal fluid, tears,
semen, vaginal secretions, amniotic fluid and cord blood. Also,
lavages, tissue homogenates and cell lysates can be utilized and,
as used herein, biological samples include such preparations.
[0048] The biological samples can be taken from a patient. The term
"patient" includes any animal, preferably mammals, and most
preferably humans. Those skilled in the art can readily determine
appropriate diseases or conditions and their corresponding target
markers for a particular patient.
[0049] The quantity of the target marker can be measured by any
means known to those skilled in the art, including, for example, by
mass spectrometry, immunoassays, chemical assays, sensitive liquid
chromatography without mass spectrometry, and a variety of direct
and indirect photometric techniques. For instance, a variety of
analytical methods can be used to quantitate the target marker by
mass spectrometry. Generally, the marker of interest can be
isolated from the biological sample by a suitable technique, such
as liquid chromatography or two-dimensional gel electrophoresis.
Then the target marker can be quantitated by any mass spectrometry
detection method, such as electrospray ionization mass
spectrometry, liquid chromatography tandem mass spectrometry
(LC-MS), matrix-assisted laser desorption/ionization mass
spectrometry (MALDI-MS), MALDI time-of-flight MS (MALDI-TOF-MS),
etc. See, e.g., Lim et al., Analytical Biochemistry, 295: 45-56
(2001). The target markers can be quantitated using pure marker
standards of known quantity or by comparison to the same target
markers in the same type of biological samples from normal
controls.
[0050] Immunoassays are preferably used to quantitate the target
markers. Immunoassays employ one or more binding partners. A
"binding partner" is any compound or molecule capable of
specifically binding to a target marker. As used herein,
"specifically" means the binding partner binds to the target marker
selectively in the presence of other compounds. Binding partners
are preferably antibodies, aptamers, lectins and other molecules
that can specifically bind to the target marker. Such binding
partners can be used separately or in combination (e.g., antibodies
can be used in combination with aptamers). Suitable binding
partners are described below as a further embodiment of the present
invention.
[0051] Those skilled in the art can readily determine immunoassay
formats suitable for use in the methods of the present invention.
Such immunoassays include homogeneous assays, heterogeneous assays,
enzyme immunoassays (e.g., ELISA), competitive assays, immunometric
(sandwich) assays, turbidimetric assays, nephelometric assays, and
the like. The immunoassays can be performed manually or with an
automated analyzer.
[0052] In a preferred enzyme immunoassay, a binding partner
specific for the target marker is immobilized on a solid substrate.
Suitable solid substrates are well known and include, for example,
glass, polystyrene, polypropylene, polyethylene, nylon,
polyacrylamide and agarose. The biological sample is contacted with
the immobilized binding partner. After washing, the target marker
bound to the solid substrate by the bound binding partner is
reacted with a second binding partner (e.g. a second antibody or a
mixture of antibodies) specific for a known epitope on the target
marker. The second binding partner can be labeled to quantitate the
target marker or a labeled third binding partner or other compound
(e.g., protein A or streptavidin) can be used to quantitate the
marker.
[0053] As an alternative, the target marker can first be separated
from the other constituents of the biological sample by, e.g.,
affinity chromatography. For affinity chromatography, antibodies
specific for the target marker are attached to a solid surface
(e.g., beads in a column) and used to bind the target marker in the
sample. After washing the solid surface, the target markers are
eluted and measured (e.g., by one of the methods described above,
by measuring the absorbance at 280 nm or by any another method
known to those skilled in the art).
[0054] Suitable labels for any of the binding partners (e.g.
primary, secondary or third antibody) are well known in the art.
Such labels include: (i) enzymes (e.g., horseradish peroxidase,
malate dehyrogenase, staphylococcal nuclease, delta-5-steriod
isomerase, yeast alcohol dehydrogenase, alphaglycerophosphate
dehydrogenase, triose phosphate isomerase, alkaline phosphatase,
asparaginase, glucose oxidase, beta-galatosidase, ribonuclease,
urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase
and acetylcholine esterase); (ii) fluorophores (e.g., fluorescein
isothiocyanate, rhodamine, phycoerythrin, phycocyanin,
allophycocyanin, o-phtaldehyde and fluorescamine); (iii)
radionucleotides (e.g., indium-111, technetium-99m, iodine-125,
gallium-67 and gallium-68); (iv) bioluminescent labels (e.g.,
luciferin, luciferase and aequorin); (v) chemiluminescent labels
(e.g., luminol, isoluminol, aromatic acridinium ester, imadazole,
acridinium salt and oxalate ester); (vi) colorimetric labels; (vii)
metal colloid labels; (viii) latex and silica particles with dyes
incorporated into them; (ix) dyes; and (x) affinity labels (e.g.,
biotin). The binding and detection of these labels can be done
using techniques known to those skilled in the art.
[0055] It is then determined if the quantity(ies) of the target
marker(s) in the biological sample is(are) indicative of the
presence, absence or status of a disease or condition. This is
accomplished using any of a variety of well known methods of
statistical analysis. For instance, a clustering technique, such as
the one exemplified in Example 2, can be used. Alternatively, the
determination can be accomplished by comparing the quantity(ies) of
the target marker(s) in the sample to the quantity(ies) of the
target marker(s) in normal patients. "Normal patients" are those
not suffering from the particular disease or condition to be
diagnosed or monitored. For instance, the amount of a target marker
can be compared to a normal range. This normal range is found by
determining the amount of the marker in a large number of samples
from normal individuals by the same method (i.e., same type of
biological sample, same steps, same reagents, same conditions) as
used in assaying the patient sample. If the amount of target marker
is outside the normal range, then the presence of the disease or
condition is indicated. Alternatively, the amount of a target
marker can be compared with a cut-off value that is indicative of
the disease or condition. The cut-off value can be determined by
testing a large number of samples from normal individuals and from
patients known to be suffering from a particular disease or
condition of interest. If the amount of target marker exceeds the
cut-off, then the disease or condition is indicated. Further, the
amount of a target marker and/or the presence of two or more target
markers outside their normal ranges or which exceed their cutoffs
may also be indicative of the status of disease or condition. In
analyzing data, including determining a normal range or cut-off
value, standard statistical methods well known in the art can be
used. Finally, as can be appreciated, the normal ranges and the
cut-off values can be expressed in the units of detection (e.g.,
levels of absorbance or of fluorescence) as a matter of convenience
and ease of making the correlation.
[0056] For example, as noted above, the measurement of DA-DKP can
be used in the diagnosis or monitoring of MS. DA-KDP levels in
normal human patients is in the range of about 50-100 ng/ml. Those
skilled in the art will be able to readily determine when the level
of DA-DKP is significantly elevated to indicate multiple sclerosis
using any of the methods disclosed herein or other known
statistical methods.
[0057] The above methods of the present invention can be used to
diagnose or monitor a number of diseases and conditions. These
diseases and conditions include, but not limited to, those
identified in Tables 1 and 2.
[0058] In a further embodiment, the invention provides objective
biochemical markers useful for the diagnosis and monitoring of
multiple sclerosis (MS) in patients. In particular, the following
markers have been identified in plasma samples by liquid
chromatography followed by mass spectrometry (LC-MS): [0059] 1.
First, a compound of mass 175 (actual mass of 176) has been found
to be missing from the plasma of MS patients as compared to plasma
samples from normal humans. [0060] 2. A compound of mass 145
(actual mass of 146) has also been found to be missing from the
plasma of MS patients as compared to plasma samples from normal
humans. [0061] 3. A compound of mass 185 (actual mass 186) has been
found to be significantly elevated in the plasma of MS patients who
have active disease as compared to plasma samples from normal
humans and from MS patients whose disease is not active. This
compound has been identified as the cyclic dipeptide aspartic
acid-alanine diketopiperazine (DA-DKP). It is interesting to note
that this compound has been shown to inhibit platelet activating
factor and to inhibit the production and/or release of
interleukin-8 (see PCT application WO 02/11676). [0062] 4. A
compound of mass 199 (actual mass 200) has been found to be
significantly elevated in the plasma of MS patients who have active
disease as compared to plasma samples from normal humans and from
MS patients whose disease is not active. This compound has been
identified as N-acetyl-alanine-serine diketopiperazine
(NAS-DKP).
[0063] Thus, the absence of one or both of the compounds of masses
175 and 145 from a plasma sample indicates that the patient has MS.
A significantly elevated level of one or both of the
diketopiperazines of masses 185 and 199 indicates that the patients
are suffering from active MS. MS diagnostic compounds include, but
are not limited to, all of these compounds and the
diketopiperazines and truncated disease-associated proteins of
Tables 1 and 2.
[0064] "Active MS" is used to mean the period when new, additional
or worsening clinical manifestations occur (an attack,
exacerbation, flare or relapse). It is usually associated with
increased myelin/neuron destruction, elevated white blood cells
(>4/hpf) and IgG synthesis rate (>9) in the cerebrospinal
fluid, MRI demyelination plaques, and "black holes" which represent
neuronal loss.
[0065] In yet another embodiment, the invention provides objective
biochemical markers useful for the diagnosis or monitoring of
Alzheimer's disease. The invention provides methods of diagnosing
or monitoring Alzheimer's disease using an Alzheimer's diagnostic
compound. The methods are accomplished by obtaining a biological
sample from a patient to be diagnosed or monitored and determining
the amount of an Alzheimer's diagnostic compound in the biological
sample. Alzheimer's diagnostic compounds include, for example: (i)
a compound having a mass of about 175 as determined by liquid
chromatography and mass spectrometry; and (ii) the diketopiperazine
derived from beta-amyloid which is Asp-Ala DKP (MW 186.15). Both
diagnostic compounds have been found elevated in the plasma of
Alzheimer's patients and are considered diagnostic of the disease.
Other Alzheimer's diagnostic compounds include the
diketopiperazines and truncated disease-associated proteins of
Tables 1 and 2.
[0066] In a further embodiment, the invention provides methods for
the diagnosis or monitoring of placental ischemia in pregnant
patients. These methods comprise obtaining a biological sample from
a pregnant patient and measuring the amount of a placental ischemia
diagnostic compound, including those derived from
pregnancy-associated proteins, in the biological sample. Examples
of placental ischemia diagnostic compounds useful in the methods
include, for example: (i) Gly-Leu diketopiperazine (GL-DKP) derived
from beta-human chlorionic gonadotropin; and (ii) Ala-Pro
diketopiperazine (AP-DKP) derived from fetal erythropoietin. Other
placental ischemia diagnostic compounds include the
diketopiperazines and truncated disease-associated proteins of
Tables 1 and 2.
[0067] Those skilled in the art will be able to readily isolate and
determine the chemical composition of those compounds identified
above only by their masses. Once their chemical compositions are
known, they can be assayed by methods other than mass spectrometry,
including those methods described above, preferably by means of an
immunoassay.
[0068] In yet another embodiment, the invention provides binding
partners useful in the immunoassays described above. Binding
partners include antibodies, antiserum or a purified fraction
thereof, aptamers and other compounds capable of specifically
binding to a target marker. Suitable antibodies include polyclonal
antibodies, monoclonal antibodies, bispecific antibodies, humanized
antibodies, chimeric antibodies, single-chain antibodies, Fab
fragments, F(ab').sub.2 fragments, fragments produced by an Fab
expression library, epitope-binding fragments of any of the
foregoing, and complementarity determining regions (CDRs).
Antibodies suitable for use in the invention can be prepared by
known methods. Particularly suitable antibodies are monoclonal
antibodies having specificity for the diketopiperazines of the
present invention. Since the diketopiperazines are small compounds,
they will preferably be attached to an immunogenic carrier molecule
for use as an immunogen to prepare antibodies specific to them.
Suitable carrier molecules (e.g., KLH) and methods of attaching
molecules to them are well known in the art. The immunogen can be
used to produce monoclonal antibodies using fusion procedures of
Kohler and Milstein, Nature 1975 256 pp. 495-497, with
modifications known to those skilled in the art. The term
"isolated" used in connection with binding partner means the
binding partner is not within the milieu of its natural environment
if found in nature and is not meant to indicate any level of purity
of the binding partner.
[0069] Aptamers can be used in place of, or in combination with,
the antibodies in any of the above described immunoassays. Aptamers
are oligonucleotides that are specific for proteins, peptides,
derivatives of proteins and peptides, inorganic molecules and other
non-nucleotide molecules. See, e.g., PCT applications WO 00/070329,
WO 01/79562 and WO 99/54506, and U.S. Pat. No. 5,756,291, which are
incorporated herein by reference in their entirety. Aptamers
suitable for use in the present invention can be prepared using the
methods described in these references. Briefly, a heterogeneous
population of oligonucleotides of random sequences is synthesized,
and a marker of the invention is mixed with the heterogeneous
population of oligonucleotides. Complexes are formed with some, but
not all, of the sequences present in the oligonucleotide
population. The complexes are isolated, and the oligonucleotides
recovered and amplified (e.g., by PCR). The resulting mixture of
oligonucleotides can be used as the starting material for another
round of complexation, isolation and amplification, and the process
will typically be repeated several times until an aptamer of
satisfactory specificity is obtained and/or until a consensus
aptamer sequence is identified. Suitable labels for aptamers
include dyes, enzymes, radioactive labels, etc.
[0070] The present invention further provides compositions
containing the binding partners described above in a
physiologically-acceptable carrier. Such physiologically-acceptable
carriers are well known in the art and include, for example,
aqueous solutions such as bicarbonate buffers, phosphate buffers,
physiological saline, Ringer's solution and the like.
[0071] The invention also provides kits for quantifying the target
markers. Such kits optionally contain various reagents useful for
conducting the methods of the present invention, including one or
more binding partners specific for a target marker, a labeled
component useful for detecting the target marker, buffers,
diluents, standards, controls, etc. The kits can also contain
bottles, vials, tubes, syringes, microtiter plates or other solid
substrates, instructions and the like.
[0072] The following Examples are intended to illustrate the
embodiments of the invention and are not intended to limit the
invention.
EXAMPLES
Example 1
Diagnosis of Placental Ischemia
[0073] The presence of several diketopiperazines has been detected
in maternal plasma. Of particular interest are the ones derived
from the N-termini of .beta.-human chorionic gonadotropin
(.beta.HCG) and fetal erythropoietin. These are glycine-leucine
diketopiperazine (GL-DKP) and alanine-proline diketopiperazine
(AP-DKP), respectively. AP-DKP, in particular, is elevated in FGR
pregnancy due to elevation of fetal erythropoietin in FGR (Teramo,
et al., Acta Obstet. Gynecol. Scand. 2002. 83(1): p. 245-51;
Jazayeri et al., Am. J. Obstet. Gynecol., 2000. 183(1): p. 188-90;
Jazayeri et al., J. Perinatol., 1999. 19(4): p. 255-9) and its
specific degradation in acidic conditions (protonation of the
N-terminal amino acid (Goolcharran and Borchardt, J. Pharm. Sci.,
1998. 87(3): p. 283-8) and the relative importance for proline in
position 2 of the primary amino acid sequence) to yield AP-DKP.
[0074] Subjects for the study were selected from patients referred
to a Maternal-Fetal Medicine (MFM) practice with complicated
pregnancies. Inclusion criteria for the study were:
[0075] Estimated fetal weight <10.sup.th percentile for
gestational age by ultrasound in addition to: [0076] an amniotic
fluid index (AFI)<8 or, [0077] a ratio of blood flow velocity
during systole to diastole (S/D) in the umbilical artery as
measured with pulse-wave Doppler >3 or, [0078] preeclampsia, as
defined by standard clinical criteria.
[0079] There were 12 patients in the study group, including 11
singletons and one twin gestation. There were 5 patients in the
control group including 1 twin gestation. Gestational ages in the
study group at time of delivery were between 26.3-38 weeks with an
average gestational age of 30.2 weeks versus 38 weeks in the
control group. Average birth weights were 1016 grams in the study
groups versus 3114 grams in the control group. Birth weight
percentages for the study group averaged <10% versus 43% in the
control group. Umbilical artery Doppler flow studies were obtained
in 10 of the 12 study patients; of these, all were abnormal, with 2
patients having reversed end-diastolic flow, 6 having absent
end-diastolic flow, and 2 having an S/D ratio>3.0. Nine of the
12 study patients had preeclampsia. Two of the 12 study patients
had HELLP syndrome.
[0080] Recombinant .beta.HCG (Sigma) was incubated in phosphate
buffer 0.1M, pH 7.4 at 60.degree. C. for 12 hours and analyzed for
the presence of GL-DKP (MW 170.21) by liquid chromatography (LC)
followed by ESI+ mass spectrometry (ESI+/MS). The results are
presented in FIG. 1.
[0081] Similarly, recombinant erythropoietin (Amgen) was incubated
in phosphate buffer 0.1M, pH 7.4 at 60.degree. C. for 12 hours and
analyzed for the presence of AP-DKP (MW 168.18) by LC and ESI-/MS.
The results are presented in FIG. 3.
[0082] Plasma samples were taken from study group patients and the
control group patients and processed by LC followed by ESI/MS. The
results for one study group patient (patient 4) are presented in
FIGS. 2 and 4. As can be seen, GL-DKP (derived from (3HCG) and
AP-DKP (derived from fetal erythropoietin) were detected.
Example 2
Classification of MS Patients Using Liquid Chromatography-Mass
Spectrometry and Clustering
[0083] A novel method of determining the Multiple Sclerosis (MS)
status of patients is presented here along with some results of a
small test set of both MS and normal patients. In the present
method, blood samples were collected from both normal and MS
patients and analyzed by a liquid chromatography-mass spectrometry
(LC-MS) method to determine the concentration of several putative
MS markers. The resulting data were analyzed by a mathematical
clustering technique that finds natural groupings within the data
to see whether there were any simple relationships between the
levels of these putative markers and the presence, absence or
status of MS.
Patients
[0084] Patients with MS were diagnosed by accepted clinical and
laboratory standards. Neurological signs and symptoms, magnetic
resonance imaging evidence of demyelination, presence of
oligoclonal bands in cerebrospinal fluid, white cell enumeration
and IgG synthesis rates were used to make the diagnosis. Active
disease was defined based on the above in the presence of acute or
progressive neurological manifestations.
Sample Preparation
[0085] Blood samples were collected in heparinized tubes. The blood
samples were separated via centrifugation into plasma and red
cells. The red cells were discarded, and the plasma was further
refined by passing it through a size exclusion filter (Centricon 3)
to remove all components that were greater than 3,000 daltons. The
resulting filtrates were analyzed immediately or frozen for later
analysis.
LC-MS Method
[0086] The samples were run on an HPLC (a Waters 2975 system) to
separate the various components. The column used was an Amersham
mono-Q anion exchange. The mobile phase was a 50 mM solution of
ammonium acetate, pH 6.7, run at 1 ml/min. The flow was split 4:1
post column leaving a 250 ul/min stream which was routed to a
Micromass LCT mass spectrometer operated in negative electrospray
ionization (ESI-) mode using a cone voltage of 20v. Because of the
high flow rate and high aqueous content of the solvent, the
desolvation temperature was set to 400.degree. C. Standards of
DA-DKP and EA-DKP were run with each set of data to calibrate for
transient differences in instrument sensitivity. The standard
concentrations used are 500, 100, 20, and 4 ng/ml of each DKP.
Detection of the DKPs by the mass spectrometer was found to be
linear in this range of concentrations (r2>0.998).
Data Preparation
[0087] While there are no calibrants for some of the putative
markers described below, it was assumed that the sensitivity of the
instrument was linear across the spectrum and, thus, all masses for
which there was no calibrant were normalized to the level of 500
ng/ml DA-DKP.
Mathematical Analysis
[0088] Clustering is a classification technique that identifies
groups of similar objects where similarity is derived solely on the
basis of the variables that describe the data. Ideally, the groups
are formed in such a way that objects within a group are similar to
each other, while objects in different groups as are as dissimilar
as possible.
[0089] When one tries to cluster raw data from experimental data
whose variables are poorly scaled, components with large magnitude
will dominate any distance metric, resulting in a disproportionate
weighting of those variables. Since one has no a priori knowledge
of each variable's importance, one scales to give equal weight to
each of the variables. Dimensional scaling is employed so each
variable is shifted and scaled. As a result, the means are zero and
the variances are equal.
[0090] Often, experiments generate high-dimensional data sets which
may have strong dependencies. In order to maximize the likelihood
of a nontrivial classification, one wishes to minimize the number
of dimensions by extracting the most relevant information from the
data while minimizing noise. Methods of feature extraction include
wavelet decomposition, Fourier transformation, factor analysis and
independent component analysis.
[0091] In this work, feature extraction was performed using a
variant of factor analysis called principal component analysis
(PCA). In PCA, the data are represented as coefficients of the
eigenvectors of the covariance matrix that describes the data.
Moreover, the relative strengths of each of the eigenvectors (also
known as principal components) are given by the eigenvalues.
Eigenvectors with corresponding eigenvalues that are below some
threshold can often be omitted as noise.
[0092] After choosing a set of 10 putative masses for analysis, the
data were analyzed with a clustering toolset in Matlab written by
Raphael Bar-Or, DMI BioSciences, Inc., Englewood, Colo. Other
suitable clustering software is available commercially. Trial and
error analysis revealed that 2 masses of 185 and 199 appearing
early in the runs had some power to separate the data into 2
groups, one of which is active MS and the other non-active MS and
normals. In a subset of MS patients and normal patients, the
settings of the clustering toolkit were optimized to achieve good
separation between active MS and all other diagnoses. The settings
for this analysis are given in Table 4.
[0093] Samples from a total of 37 patients were run. Of these, 24
were in cluster #1 (8 normals and 16 non-active MS), and 13 were in
cluster #2 (active MS). There appear to be few, if any,
misclassifications, and an inspection of the clustering dendrogram
(FIG. 5) reveals that the space is quite separable, meaning that
there is sufficient space between the clusters so that this
separation is not likely to be the product of chance alone. A small
bootstrap (leave-one-out analysis) confirmed that the separation is
indeed stable (95% by Rand's Statistic).
[0094] The groups found by the clustering method were used as a
classifier. In this small data set, a sensitivity of 100% and a
specificity of 84.6% for active MS were found.
[0095] The two masses of 185 and 199 were identified to be Asp-Ala
DKP (DA-DKP) and N-acetyl-Ala-Ser DKP (NAS-DKP), respectively.
These two DKPs are the degradation products of two important
central nervous system proteins, namely beta amyloid and myelin
basic protein (see Table 1).
[0096] It was noted that a marker labeled "175 @ 8.5 mins" appears
to be deficient in all of the suspected MS patients and unusually
high in Alzheimer's patients. A similar pattern was observed in
another mass labeled "145 @ 12.7 mins". Addition of this variable
to the clustering analysis would surely improve the separation, but
data beyond 6 minutes is available for only a small subset of
patients (only 14 samples were run for longer than 6 minutes, and
only 10 of these had also been run for the shorter time). An
analysis of this smaller group revealed that a simple threshold on
the level of "175 @ 8.5 mins" was sufficient to quite accurately
separate MS from normals and non-MS patients without distinguishing
between the active and non-active forms. While there is
insufficient data to conclude that this 175 marker is definitive,
the evidence suggests that, together with the two markers (185 and
199) used in the above clustering analysis, there is a strong
likelihood for an algorithm that can accurately separate MS
patients from normals and non-MS patients and that the MS patients
can be further categorized into active and non-active MS.
TABLE-US-00004 TABLE 4
%*********************************************************** %****
EDIT THE VALUES IN THIS BLOCK *********************
%***********************************************************
standout=[ ]; % elements to color differently so that they stand
out logdata=1; % convert to log data scaling=0; % 1 = dimensional
scaling % 0 = no dimensional scaling numberOfClusters=2; % the
number of desired clusters (should be =>2) convertPca=1; % 1 =
convert to pca space % 0 = no conversion (original space)
keepVariation=0.95; % the amount of variability to keep in the pca
conversion clusterAlgorithm=`hierarchical`; % type of clustering
desired options are: % `kplane` % `kmeans` % `kmedians` %
`fuzzy_cmeans` % `hierarchical` % `gravity` gravorder=15; % only
applicable if cluster type is gravity gravtol=2e-3; % only
applicable if cluster type is gravity addmasses=1; % only
applicable if cluster type is gravity heirarchicalMetric=`Euclid`;
% n/a if clusterAlgorithm is not `hierarchical` or `permutation` %
options are: % `Euclid`=Euclidean distance (default) %
`SEuclid`=Standardized Euclidean distance % `Mahal`=Mahalanobis
distance % `CityBlock`=City Block metric % `Minkowski`=Minkowski
metric % `Correlation`=1-Correlation Coefficients
heirearchicalLinkage=`ward`; %`single`=Shortest distance
%`complete`=Largest distance %`average`=Average distance
%`centroid`=Centroid distance (approximate, computed using a
formula that is exact if Y contains Euclidean distances)
%`ward`=Incremental sum of squares displayClusterInfo=1; % 1 =
display cluster info % 0 = no cluster info display
displayScatterplot=1; % 1 = display scatterplot of first three
components % 0 = no scatterplot displayClusterGeneResponse=1; % 1 =
display gene response representation for each cluster % 0 = no
display typeResponseRep=`line`; % n/a if displayClusterGeneResponse
is disabled % options are: % `box` % `line` % `bar` % `area`
crossValidation=0; % leave one out cross-validation (can take a
VERY long time) % 1 = do cross validation % 0 = no cross
validation
Example 3
Analysis Of An MS Patient Using Liquid Chromatography-Mass
Spectrometry
[0097] A blood sample was collected from an MS patient with active
MS and processed and analyzed by LC-MS as described in Example 2.
The following DKP' s were found: DA-DKP (from N-terminus of
beta-amyloid), NAS-DKP (from N-terminus of myelin basic protein),
N-acetyl-Ala Phospho-Ser DKP (from N-terminus of myelin basic
protein), Gln-Asn DKP (from C-terminus of beta-amyloid) and Arg-Arg
DKP (from C-terminus of myelin basic protein).
Example 4
Diagnosis of Alzheimer's Disease
[0098] As noted in Example 2, a marker labeled "175 @ 8.5 mins" was
found to be present in unusually high amounts in the plasma of
Alzheimer's patients. It is expected that this marker will be
useful in the diagnosis of Alzheimer's disease.
[0099] In addition, a marker at mass 186.15, which DA-DKP, has been
found elevated in the plasma of Alzheimer's patients. It appears to
be diagnostic of the disease.
[0100] Finally, another possible marker of mass 200 (actual mass
201) has been found. It has not yet been identified, but a likely
candidate is NAS-DKP.
[0101] The above description of the invention, including the
Examples, is intended to be merely illustrative of the invention
and is not intended to limit the invention. Various changes and
modifications can be made by those skilled in the art without
departing from the spirit and scope of the invention.
* * * * *