U.S. patent application number 10/877688 was filed with the patent office on 2004-12-16 for methods for aiding in the diagnosis of alzheimer's disease by measuring amyloid-beta peptide (x->41) and tau.
Invention is credited to Barbour, Robin, Schenk, Dale B., Seubert, Peter A., Vigo-Pelfrey, Carmen.
Application Number | 20040253643 10/877688 |
Document ID | / |
Family ID | 26991508 |
Filed Date | 2004-12-16 |
United States Patent
Application |
20040253643 |
Kind Code |
A1 |
Seubert, Peter A. ; et
al. |
December 16, 2004 |
Methods for aiding in the diagnosis of Alzheimer's disease by
measuring amyloid-beta peptide (x->41) and tau
Abstract
This invention provides methods useful in aiding in the
diagnosis of Alzheimer's disease. The methods involve measuring the
amount of amyloid-.beta. peptide (x-.gtoreq.41) in the
cerebrospinal fluid of a patient. High levels of the peptide
generally are inconsistent with a diagnosis of Alzheimer's. Low
levels of the peptide are consistent with the disease and, with
other tests, can provide a positive diagnosis. Other methods
involve measuring the amounts of both A.beta.(x-.gtoreq.41) and
tau. Low levels of A.beta.(x-.gtoreq.41) and high levels of tau are
a positive indicator of Alzheimer's disease, while high levels of
A.beta.(x-.gtoreq.41) and low levels of tau are a negative
indication of Alzheimer's disease.
Inventors: |
Seubert, Peter A.; (South
San Francisco, CA) ; Vigo-Pelfrey, Carmen; (Mountain
View, CA) ; Schenk, Dale B.; (Pacifica, CA) ;
Barbour, Robin; (Newark, CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Family ID: |
26991508 |
Appl. No.: |
10/877688 |
Filed: |
June 25, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10877688 |
Jun 25, 2004 |
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08466554 |
Jun 6, 1995 |
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08466554 |
Jun 6, 1995 |
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08419008 |
Apr 7, 1995 |
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08419008 |
Apr 7, 1995 |
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08339141 |
Nov 14, 1994 |
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6114133 |
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Current U.S.
Class: |
435/7.1 |
Current CPC
Class: |
C07K 16/18 20130101;
Y10S 436/811 20130101; G01N 2333/4709 20130101; C07K 14/4711
20130101; G01N 2800/2821 20130101; G01N 2500/00 20130101; G01N
33/6896 20130101 |
Class at
Publication: |
435/007.1 |
International
Class: |
G01N 033/53 |
Claims
1-41. (canceled).
42. A method for diagnosing probable Alzheimer's disease in a
patient, the method comprising: measuring the amount of one or more
soluble amyloid-.beta. peptide (x-.gtoreq.41)
("A.beta.(x-.gtoreq.41)") in a cerebrospinal fluid sample of the
patient; comparing the measured amount of the soluble
A.beta.(x-.gtoreq.41) with a predetermined indicator value of the
A.beta.(x-.gtoreq.41); measuring the amount of tau in a
cerebrospinal fluid sample of the patient; comparing the measured
amount of tau with a predetermined indicator value of tau; and
assessing patient status based on a difference between the measured
amounts and predetermined indicator values, wherein a measured
amount at or below the A.beta.(x-.gtoreq.41) indicator value and at
or above the tau indicator value provides a positive indication in
the diagnosis of probable Alzheimer's disease, and wherein a
measured amount above the A.beta.(x-.gtoreq.41) indicator value and
below the tau indicator value provides a negative indication in the
diagnosis of probable Alzheimer's disease.
43. The method of claim 42 wherein the amount of the soluble
A.beta.(x-.gtoreq.41) is measured by: capturing the soluble
A.beta.(x-.gtoreq.41) from the sample on a solid phase with a first
antibody or antibody fragment specific for an epitope within a
junction region of A.beta. disposed between amino acids 13-26; and
detecting capture of the soluble A.beta.(x-.gtoreq.41) using a
second antibody or antibody fragment specific for
A.beta.(x-.gtoreq.41).
44. The method of claim 42 wherein the amount of the soluble
A.beta.(x-.gtoreq.41) is measured by: capturing the soluble
A.beta.(x-.gtoreq.41) from the sample on a solid phase with a first
antibody or antibody fragment specific for A.beta.(x-.gtoreq.41);
and detecting capture of the soluble A.beta.(x-.gtoreq.41) using a
second antibody or antibody fragment that recognizes A.beta..
45. The method of claim 42 wherein the amount of the soluble
A.beta.(x-.gtoreq.41) is measured by: capturing the soluble
A.beta.(x-.gtoreq.41) from the sample on a solid phase with a first
antibody or antibody fragment specific for A.beta.; and detecting
capture of the soluble A.beta.(x-.gtoreq.41) using a second
antibody or antibody fragment specific for
A.beta.(x-.gtoreq.41).
46. The method of claim 42 wherein A.beta.(x-.gtoreq.41) is
A.beta.(x-42).
47. The method of claim 43 wherein the detecting step comprises
detecting binding between the second antibody or antibody fragment
and A.beta.(x-.gtoreq.41) using a third labeled antibody or
antibody fragment that recognizes the second antibody or antibody
fragment but not the first antibody or antibody fragment.
48. The method of claim 43 wherein the first antibody or antibody
fragment has the specificity of an antibody raised against
A.beta..sub.13-28.
49. The method of claim 43 wherein the second antibody or antibody
fragment has the specificity of an antibody raised against amino
acids 33-42 of A.beta..
50. The method of claim 44 wherein the detecting step comprises
detecting binding between the second antibody or antibody fragment
and A.beta.(x-.gtoreq.41) using a third labeled antibody or
antibody fragment that recognizes the second antibody or antibody
fragment but not the first antibody or antibody fragment.
51. The method of claim 44 wherein the first antibody or antibody
fragment has the specificity of an antibody raised against amino
acids 33-42 of A.beta..
52. The method of claim 44 wherein the second antibody or antibody
fragment is specific for an epitope within a junction region of
A.beta. disposed between amino acids 13-26.
53. The method of claim 45 wherein the detecting step comprises
detecting binding between the second antibody or antibody fragment
and A.beta.(x-.gtoreq.41) using a third labeled antibody or
antibody fragment that recognizes the second antibody or antibody
fragment but not the first antibody or antibody fragment.
54. The method of claim 45 wherein the second antibody or antibody
fragment has the specificity of an antibody raised against amino
acids 33-42 of A.beta..
55. The method of claim 47 wherein the label is an enzymatic
label.
56. The method of claim 49 wherein the first antibody or antibody
fragment has the specificity of an antibody raised against
A.beta..sub.13-28.
57. The method of claim 50 wherein the label is an enzymatic
label.
58. The method of claim 52 wherein the second antibody or antibody
fragment has the specificity of an antibody raised against
A.beta..sub.13-28.
59. The method of claim 53 wherein the label is an enzymatic
label.
60. A kit comprising: a first antibody or antibody fragment
specific for A.beta.(x-.gtoreq.41) that does not cross-react with
A.beta.(x-.ltoreq.40) and a second antibody or antibody fragment
specific for tau.
61. The kit of claim 60 wherein the first antibody or antibody
fragment has the specificity of an antibody raised against amino
acids 33-42 of A.beta..
62. The kit of claim 60 further comprising a third antibody or
antibody fragment specific for A.beta..
63. The kit of claim 60 wherein A.beta.(x-.gtoreq.41) is
A.beta.(x-42).
64. The kit of claim 60 wherein the first antibody and second
antibody are whole immunoglobulins.
65. The kit of claim 60 further comprising a third antibody or
antibody fragment that is labeled and that is specific for tau.
66. The kit of claim 62 wherein the third antibody or antibody
fragment is specific for an epitope within a junction region of
A.beta. disposed between amino acids 13-26.
67. The kit of claim 62 further comprising a fourth labeled
antibody or antibody fragment that recognizes the first antibody or
antibody fragment but not the third antibody or antibody
fragment.
68. The kit of claim 62 further comprising a fourth labeled
antibody or antibody fragment that recognizes the third antibody or
antibody fragment but not the first antibody or antibody
fragment.
69. The kit of claim 66 wherein the third antibody or antibody
fragment has the specificity of an antibody raised against
A.beta..sub.13-28.
70. The kit of claim 67 wherein the label is an enzymatic
label.
71. The kit of claim 68 wherein the label is an enzymatic
label.
72. The method of claim 51 wherein the second antibody or antibody
fragment recognizes an N-terminal region of A.beta..
73. The method of claim 54 wherein the first antibody or antibody
fragment recognizes an N-terminal region of A.beta..
74. The kit of claim 62 wherein the third antibody or antibody
fragment recognizes an N-terminal region of A.beta..
75. The method of claim 72 wherein the second antibody or antibody
fragment recognizes an epitope within amino acids 1-16 of
A.beta..
76. The method of claim 73 wherein the first antibody or antibody
fragment recognizes an epitope within amino acids 1-16 of
A.beta..
77. The kit of claim 74 wherein the third antibody or antibody
fragment recognizes an epitope within amino acids 1-16 of A.beta..
Description
[0001] This application is related to co-pending applications Ser.
No. 07/965,972, filed Oct. 26, 1992, Ser. No. 08/079,511, filed
Jun. 17, 1993, and Ser. No. 08/339,141, filed Nov. 14, 1994, all of
which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates generally to methods for
diagnosing or monitoring Alzheimer's disease. More particularly,
the present invention relates to measuring the amount of tau
protein and/or the amount of .beta. amyloid peptide (x-.gtoreq.41)
in patient fluid samples and using these amounts as a diagnostic
indicator.
[0004] Alzheimer's disease (AD) is a degenerative brain disorder
characterized clinically by progressive loss of memory, cognition,
reasoning, judgment and emotional stability that gradually leads to
profound mental deterioration and ultimately death. AD is a very
common cause of progressive mental failure (dementia) in aged
humans and is believed to represent the fourth most common medical
cause of death in the United States. AD has been observed in all
races and ethnic groups worldwide and presents a major present and
future public health problem. The disease is currently estimated to
affect about two to three million individuals in the United States
alone. AD is at present incurable. No treatment that effectively
prevents AD or reverses its symptoms or course is currently
known.
[0005] The brains of individuals with AD exhibit characteristic
lesions termed senile plaques, and neurofibrillary tangles. Large
numbers of these lesions are generally found in several areas of
the human brain important for memory and cognitive function in
patients with AD. Smaller numbers of these lesions in a more
restricted anatomical distribution are sometimes found in the
brains of aged humans who do not have clinical AD. Senile plaques
and amyloid angiopathy also characterize the brains of individuals
beyond a certain age with Trisomy 21 (Down's Syndrome) and
Hereditary Cerebral Hemorrhage with Amyloidosis of the Dutch-Type
(HCHWA-D). At present, a definitive diagnosis of AD usually
requires observing the aforementioned lesions in the brain tissue
of patients who have died with the disease or, rarely, in small
biopsied samples of brain tissue taken during an invasive
neurosurgical procedure. The principal chemical constituent of the
senile plaques and vascular amyloid deposits (amyloid angiopathy)
characteristic of AD and the other disorders mentioned above is an
approximately 4.2 kilodalton (kD) protein of about 39-43 amino
acids designated the amyloid-.beta. peptide (A.beta.) or sometimes
.beta.AP, A.beta.P or .beta./A4. A.beta. was first purified and a
partial amino acid sequence reported in Glenner and Wong (1984)
Biochem. Biophys. Res. Commun. 120:885-890. The isolation procedure
and the sequence data for the first 28 amino acids are described in
U.S. Pat. No. 4,666,829. Forms of A.beta. having amino acids beyond
number 40 were first reported by Kang et al. (1987) Nature
325:733-736.
[0006] Roher et al. (1993) Proc. Natl. Acad. Sci. USA 90:10836-840
showed that A.beta.(1-42) is the major constituent in neuritic
plaques (90%) with significant amounts of isomerized and racemized
aspartyl residues. The authors also showed that A.beta.(17-42) also
predominates in diffuse plaques (70%), while A.beta.(1-40) is the
major constituent in the meningovascular plaques, comprising 60% of
the total A.beta. and, in parenchymal vessel deposits A.beta.(1-42)
represents 75% of the total A.beta.. Iwatsubo et al. (1994) Neuron
13:45-53 showed that A.beta.42(43)-positive senile plaques are the
major species in sporadic AD brain.
[0007] Molecular biological and protein chemical analyses conducted
during the last several years have shown that A.beta. is a small
fragment of a much larger precursor protein, referred to as the
.beta.-amyloid precursor protein (APP), that is normally produced
by cells in many tissues of various animals, including humans.
Knowledge of the structure of the gene encoding APP has
demonstrated that A.beta. arises as a peptide fragment that is
cleaved from the carboxy-terminal end of APP by as-yet-unknown
enzymes (proteases). The precise biochemical mechanism by which the
A.beta. fragment is cleaved from APP and subsequently deposited as
amyloid plaques in the cerebral tissue and in the walls of cerebral
and meningeal blood vessels is currently unknown.
[0008] Several lines of evidence indicate that progressive cerebral
deposition of A.beta. plays a seminal role in the pathogenesis of
AD and can precede cognitive symptoms by years or decades (for
review, see Selkoe (1994) J. Neuropath. and Exp. Neurol. 53:438-447
and Selkoe (1991) Neuron 6:487). The single most important line of
evidence is the discovery in 1991 that missense DNA mutations at
amino acid 717 of the 770-amino acid isoform of APP can be found in
affected members but not unaffected members of several families
with a genetically determined (familial) form of AD (Goate et al.
(1991) Nature 349:704-706; Chartier Harlan et al. (1991) Nature
353:844-846; and Murrell et al. (1991) Science 254:97-99). Suzuki
et al. (1994) Science 264:1336-1340 showed that in persons with the
717 mutation, there is a higher percentage of A.beta.(1-42) than
A.beta.(1-40).
[0009] In addition, a double mutation changing
lysine.sup.595-methionine.s- up.596 to
asparagine.sup.595-leucine.sup.596 (with reference to the 695
isoform) found in a Swedish family was reported in 1992 (Mullan et
al. (1992) Nature Genet 1:345-347) and is referred to as the
Swedish variant. Genetic linkage analyses have demonstrated that
these mutations, as well as certain other mutations in the APP
gene, are the specific molecular cause of AD in the affected
members of such families. In addition, a mutation at amino acid 693
of the 770-amino acid isoform of APP has been identified as the
cause of the A.beta. deposition disease, HCHWA-D, and a change from
alanine to glycine at amino acid 692 appears to cause a phenotype
that resembles AD in some patients but HCHWA-D in others. The
discovery of these and other mutations in APP in genetically based
cases of AD argues that alteration of APP and subsequent deposition
of its A.beta. fragment can cause AD.
[0010] Neurofibrillary tangles are composed mainly of the
microtubule protein, tau. Z. S. Khachaturian (1985) Arch. Neurol.
42:1097-1105. Recent studies have shown that tau is elevated in the
CSF of Alzheimer's disease patients. M. Vandermeeren et al. (1993)
J. Neurochem. 61:1828-1834.
[0011] Despite the progress which has been made in understanding
the underlying mechanisms of AD, there remains a need to develop
methods for use in diagnosis of the disease. While the level of tau
is of some help in diagnosing Alzheimer's disease (M. Vandermeeren
et al., supra) more markers, and more specific markers would be
helpful. It would be further desirable to provide methods for use
in diagnosis of A.beta.-related conditions, where the diagnosis is
based at least in part on detection of A.beta. and related
fragments in patient fluid samples. Specific assays for A.beta.
detection should be capable of detecting A.beta. and related
fragments in fluid samples at very low concentrations as well as
distinguishing between A.beta. and other fragments of APP which may
be present in the sample.
[0012] 2. Description of the Background Art
[0013] Glenner and Wong (1984) Biochem. Biophys. Res. Commun.
120:885-890 and U.S. Pat. No. 4,666,829, are discussed above. The
'829 patent suggests the use of an antibody to the 28 amino acid
A.beta. fragment to detect "Alzheimer's Amyloid Polypeptide" in a
patient sample and diagnose AD. No data demonstrating detection or
diagnosis are presented.
[0014] Numerous biochemical electron microscopic and immunochemical
studies have reported that A.beta. is highly insoluble in
physiologic solutions at normal pH. See, for example, Glenner and
Wong (1984) Biochem. Biophys. Res. Commun. 122:1131-1135; Masters
et al. (1985) Proc. Natl. Acad. Sci. USA 82:4245-4249; Selkoe et
al. (1986) J. Neurochem. 46:1820-1834; Joachim et al. (1988) Brain
Research 474:100-111; Hilbich et al. (1991) J. Mol. Biol.
218:149-163; Barrow and Zagorski (1991) Science 253:179-182; and
Burdick et al. (1992) J. Biol. Chem. 267:546-554. Furthermore, this
insolubility was predicted by and is consistent with the amino acid
sequence of A.beta. which includes a stretch of hydrophobic amino
acids that constitutes part of the region that anchors the parent
protein (APP) in the lipid membranes of cells. Hydrophobic,
lipid-anchoring proteins such as A.beta. are predicted to remain
associated with cellular membranes or membrane fragments and thus
not be present in physiologic extracellular fluids. The
aforementioned studies and many others have reported the
insolubility in physiologic solution of native A.beta. purified
from AD brain amyloid deposits or of synthetic peptides containing
the A.beta. sequence. The extraction of A.beta. from cerebral
amyloid deposits and its subsequent solubilization has required the
use of strong, non-physiologic solvents and denaturants.
Physiologic, buffered salt solutions that mimic the extracellular
fluids of human tissues have uniformly failed to solubilize
A.beta..
[0015] Separate attempts to detect APP or fragments thereof in
plasma or CSF have also been undertaken. A large secreted fragment
of APP that does not contain the intact A.beta. region has been
found in human cerebrospinal fluid (Palmert et al. (1989) Proc.
Natl. Acad. Sci. USA 86:6338-6342; Weidemann et al. (1989) Cell
57:115-126; Henriksson et al. (1991) J. Neurochem. 56:1037-1042;
Palmert et al. (1990) Neurology 40:1028-1034; and Seubert et al.
(1993) Nature 361:260-263) and in plasma (Podlisny et al. (1990)
Biochem. Biophys. Res. Commun. 167:1094-1101). The detection of
fragments of the carboxy-terminal portion of APP in plasma has also
been reported (Rumble et al. (1989) N. Engl. J. Med.
320:1446-1452), as has the failure to detect such fragments
(Schlossmacher et al. (1992) Neurobiol. Aging 13:421-434).
[0016] Despite the apparent insolubility of native and synthetic
A.beta., it had been speculated that A.beta. might occur in body
fluids, such as cerebrospinal fluid (CSF) or plasma (Wong et al.
(1984) Proc. Natl. Acad. Sci. USA 92:8729-8732; Selkoe (1986)
Neurobiol. Aging 7:425-432; Pardridge et al. (1987) Biochem.
Biophys. Res. Commun. 145:241-248; Joachim et al. (1989) Nature
341:226-230; Selkoe et al. (1989) Neurobiol. Aging 10:387-395).
[0017] Several attempts to measure A.beta. in CSF and plasma have
been reported by both radioimmunoassay methods (WO90/12870
published Nov. 1, 1990) and sandwich ELISAs (Wisniewski in
Alzheimer's Disease, eds. Becker and Giacobini, Taylor and Francas,
N.Y. pg. 206, 1990; Kim and Wisniewski in Techniques in Diagnostic
Pathology, eds. Bullock et al., Academic Press, Boston pg. 106; and
WO90/12871 published Nov. 1, 1990). While these reports detected
very low levels of A.beta. immunoreactivity in bodily fluids,
attempts to directly purify and characterize this immunoreactivity
further and determine whether it represented A.beta. were not
pursued, and the efforts were abandoned. The possibility of A.beta.
production by cultured cells was neither considered nor
demonstrated.
[0018] Retrospectively, the inability to readily detect A.beta. in
bodily fluids was likely due to the presence of amyloid precursor
fragments with overlapping regions or fragments of A.beta. that
obscured measurements and to the lack of antibodies completely
specific for intact A.beta.. This is presumably because the
antibodies used by both groups would cross-react with other APP
fragments containing part of A.beta. known to be present in CSF
thereby interfering with the measurement, if any, of intact
A.beta.. These difficulties have been overcome with the use of
monoclonal antibodies specific to an epitope in the central
junction region of intact A.beta. (Seubert et al. (1992) Nature
359:325-327).
[0019] Seubert et al. (1992) Nature 359:325-327 and Shoji et al.
Science (1992) 258:126-129 provided the first biochemical evidence
for the presence of discrete A.beta. in bodily fluids. Vigo-Pelfrey
et al. (1993) J. Neurochem. 61:1965-1968 reported the
identification of many A.beta. species in cerebrospinal fluid.
SUMMARY OF THE INVENTION
[0020] The present invention provides methods useful for aiding in
the diagnosis and monitoring of A.beta.-related conditions in
patients, where the methods rely on the specific detection in
patient fluid samples of one or more soluble A.beta. or soluble
A.beta. fragments having amino acid residues beyond number 40 in
their carboxy-terminal end. These peptides are designated
"A.beta.(x-.gtoreq.41)" (A.beta. from amino acid number "x" to an
amino acid greater than or equal to amino acid number 41). In one
embodiment, the measured peptides belong to the class of
A.beta.(x-.gtoreq.41) that contain at least amino acids 13-41.
[0021] For the diagnosis and monitoring of A.beta.-related
conditions, the amount of the aforementioned peptides in a patient
fluid sample, especially cerebrospinal fluid (CSF), is measured and
compared with a predetermined value, such as an indicator value (in
the case of diagnosis) or a prior patient value (in the case of
monitoring). In the case of diagnosis, measured amounts of
A.beta.(x-.gtoreq.41) which are above the indicator value are
considered to be a strong indication that the patient is not
suffering from AD or other A.beta.-related condition. However, this
information may also be considered together with other factors in
making a determinative diagnosis. Measured amounts of
A.beta.(x-.gtoreq.41) which are at or below the indicator value are
considered to be a positive indication that the patient may be
suffering from AD or other A.beta.-related condition. The low
A.beta.(x-.gtoreq.41) status of the tested individual usually will
not by itself be considered a determinative diagnostic of an
A.beta.-related condition, but instead will be considered together
with other accepted clinical symptoms of A.beta.-related conditions
in making a diagnosis. In cerebrospinal fluid, an indicator value
of about 0.5 ng/ml is useful.
[0022] In a particular aspect, the present invention provides
specific binding assays which are useful for detecting soluble
A.beta.(x-.gtoreq.41) in fluid samples and which may be employed in
patient diagnostic and monitoring methods just described. Specific
binding assays according to the present invention employ two
binding substances specific for different epitopes or determinant
sites on the A.beta.(x-.gtoreq.41) molecule. One epitope or site is
generally not found on other fragments or degradation products of
the amyloid-.beta. precursor protein (APP), so as to avoid
cross-reaction with those fragments. Particularly useful are
antibodies which recognize a junction region within A.beta., where
the junction region is located about the site of normal proteolytic
cleavage of APP between residues Lys.sup.16 and Leu.sup.17 (Esch et
al. (1990) Science 248:492-495 and Anderson et al. (1991) Neuro.
Science Lett. 128:126-128), typically spanning amino acid residues
13 to 26. The other epitope or site contains at least one amino
acid beyond amino acid number 40 of A.beta. that is essential for
recognition, but does not cross-react with A.beta. or A.beta.
fragments whose carboxy-terminal amino acid is number 40 or less.
Exemplary specific binding assays include two-site (sandwich)
assays in which the capture antibody is specific for the junction
region of A.beta., as just described, and a second detectable
antibody is specific for an epitope or site containing at least one
A.beta. amino acid beyond number 40. In particular, the second
antibody can be produced by immunization with a hapten containing
A.beta. amino acids 33-42.
[0023] This invention also provides methods for aiding in the
diagnosis or monitoring of Alzheimer's disease in a patient
involving measurements of both A.beta.(x-.gtoreq.41) and the
microtubule protein tau. The methods involve measuring the amount
of one or more soluble A.beta.(x-.gtoreq.41) in a patient sample;
comparing the measured amount with a predetermined amount of
soluble A.beta.(x-.gtoreq.41); measuring the amount of tau in a
patient sample; comparing the measured amount with a predetermined
amount of said tau; and assessing patient status based on a
difference between the measured and predetermined amounts of
A.beta.(x-.gtoreq.41) and tau. Again, the predetermined amount can
be an indicator value or a prior patient value. A measured amount
at or below the A.beta.(x-.gtoreq.41) indicator value and at or
above the tau indicator value provides a positive indication in the
diagnosis of Alzheimer's disease, and wherein a measured amount
above the of the A.beta.(x-.gtoreq.41) indicator value and below
the tau indicator value provides a negative indication in the
diagnosis of Alzheimer's disease. Indicator values in the CSF of
about 0.5 ng/ml for A.beta.(x-.gtoreq.41) and about 0.3 ng/ml for
tau are useful.
[0024] This invention also provides kits for aiding in the
diagnosis of Alzheimer's disease. The kits include a binding
substance that binds A.beta.(x-.gtoreq.41) but that does not bind
to A.beta.(.ltoreq.40) and a binding substance that binds to tau.
In one embodiment, the kit contains four antibodies: a) an
un-labeled antibody that binds to the junction region of A.beta.;
b) a detectably labelled antibody that binds to an epitope
containing amino acids beyond number 40 in A.beta.; c) an
un-labelled antibody that binds to tau; and d) a detectably
labelled antibody that binds to tau.
[0025] In another aspect, the present invention provides a system
for detecting one or more soluble A.beta.(x-.gtoreq.41) in a fluid
sample. The system includes a first binding substance, typically an
antibody, specific for an epitope in a junction region of A.beta.,
as described above, and a second binding substance, typically an
antibody, specific for an epitope of A.beta. containing an amino
acid beyond amino acid number 40 of A.beta. at the carboxy-terminus
essential for recognition. The first binding substance is an
anti-A.beta. antibody bound to a solid phase, while the other is a
reporter antibody against the A.beta. carboxy-terminus. The
reporter antibody can, itself, be labeled, or can be detectable by
another antibody (e.g., a rabbit antibody recognizable by labeled
or enzyme-conjugated anti-rabbit antibodies.) The system can
further include substrate for an enzyme label. The system is useful
in performing enzyme-linked immunosorbent assays (ELISA) having
high specificity and sensitivity for the detection of
A.beta.(x-.gtoreq.41) in fluid samples.
[0026] In another aspect, this invention provides methods for
screening a compound to determine its ability to alter the amount
of A.beta.(x-.gtoreq.41) in the CSF. The methods involve measuring
a first amount of soluble A.beta.(x-.gtoreq.41) in the CSF of a
non-human animal used as a model of Alzheimer's disease;
administering the compound to the non-human animal; measuring a
second amount of soluble A.beta.(x-.gtoreq.41) in the CSF of the
non-human animal; and comparing the first amount with the second
amount. The difference indicates whether the compound increases
A.beta.(x-.gtoreq.41) in the CSF, in which case it might be useful
in the treatment of Alzheimer's; or decreases the amount, in which
case the compound might aggravate or hasten Alzheimer's. The
non-human animal preferably is a mammal, more preferably a rodent,
and most preferably a mouse.
[0027] In another aspect, this invention provides methods for
screening a compound to determine its ability to alter the amount
of both A.beta.(x-.gtoreq.41) and tau in the CSF involving
measuring a first amount of one or more soluble
A.beta.(x-.gtoreq.41) in the CSF of a non-human animal used as a
model of Alzheimer's disease; measuring a first amount of tau in
the CSF of the non-human animal; administering the compound to the
non-human animal; measuring a second amount of said one or more
soluble A.beta.(x-.gtoreq.41) in the CSF of the non-human animal;
measuring a second amount of tau in the CSF of the non-human
animal; and comparing the first amounts with the second amounts,
the difference indicating whether the compound increases,
decreases, or leaves unchanged the amount of soluble
A.beta.(x-.gtoreq.41) and increases, decreases, or leaves unchanged
the amount of tau in the CSF. The information is useful, as above,
to identify compounds that might be useful in treating Alzheimer's
or that might aggravate or hasten Alzheimer's. The non-human animal
preferably is a mammal, more preferably a rodent, and most
preferably a mouse.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] FIG. 1 shows the results of ELISA assays using antibody 266
(directed to the A.beta. junction region) and antibody 277/2
(directed to A.beta. amino acids 33-42) to detect A.beta.(42), but
not A.beta.(28), A.beta.(38), or A.beta.(40).
[0029] FIG. 2 shows the amounts of A.beta.(x-.gtoreq.41) in CSF of
control patients (C) and AD patients (AD) in Group A as detected by
ELISA.
[0030] FIG. 3 shows the amounts of A.beta.(x-.gtoreq.41) in CSF of
AD patients (AD), non-Alzheimer's neurological controls (NC) and
controls (C) in Group B as detected by ELISA.
[0031] FIG. 4 shows the amounts of A.beta.(x-.gtoreq.41) in CSF of
AD patients (AD), non-Alzheimer's neurological controls (ND) and
non-demented controls (NC) as detected by ELISA.
[0032] FIG. 5 shows the amounts of tau in CSF of Alzheimer's
disease patients (AD), non-Alzheimer's neurological controls (ND)
and non-demented control patients (NC).
[0033] FIG. 6 shows the amounts of A.beta.(x-.gtoreq.41) and tau in
CSF of Alzheimer's disease patients (AD), non-Alzheimer's
neurological controls (ND) and non-demented controls (NC). Data
from FIGS. 4 and 5 are combined to illustrate the effect of
simultaneous consideration of the two measures in discriminating
the AD group. Lines indicate optimized cut-offs. The high tau/low
A.beta.(x-.gtoreq.41) quadrant contains AD patients with only a
single exception (21/22 patients) whereas the low tau/high
A.beta.(x-.gtoreq.41) quadrant contains only control
individuals.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
[0034] The present invention results at least in part from the
discovery that the cerebrospinal fluid ("CSF") of individuals
suffering from Alzheimer's disease generally contains
A.beta.(x-.gtoreq.41) in amounts which are in the very low end of
the normal range present in the CSF of non-Alzheimer's individuals
and, in particular, below about 0.5 ng/ml. This discovery is
surprising because the bulk of A.beta. deposits in the brain tissue
of persons suffering from Alzheimer's disease is A.beta.(1-42), and
is significantly elevated compared to the amount of A.beta.(1-42)
in non-Alzheimer's individuals.
[0035] Based on this discovery the present invention provides
methods for diagnosing and monitoring Alzheimer's disease.
According to one method, a patient sample is first obtained. The
patient sample is usually a fluid sample and, preferably,
cerebrospinal fluid. Then the amount of soluble
A.beta.(x-.gtoreq.41) in the patient sample is measured. A
preferred method of measuring the amount is by using the sandwich
assay described herein. The measured amount is then compared with a
predetermined value, such as an indicator value in the case of
diagnosis, or a prior patient value in the case of monitoring. The
status of the patient is assessed based on the difference between
the two amounts.
[0036] As described in more detail below, the methods of the
present invention will be useful as both a positive and negative
indicator of AD and other A.beta.-related conditions in tested
individuals. The data in the Experimental section show that
individuals not suffering from Alzheimer's disease have CSF
concentrations of soluble A.beta.(x-.gtoreq.41) that range from
about 0.2 ng/ml to about 1.0 ng/ml. However, patients with
Alzheimer's disease have CSF concentrations of soluble
A.beta.(x-.gtoreq.41) generally below 0.5 ng/ml. Therefore, a
measured amount above the indicator value of about 0.5 ng/ml is a
very strong negative indication of Alzheimer's disease. That is,
individuals having such levels are considered to be less likely to
suffer from an A.beta.-related condition and, in particular,
Alzheimer's disease. An indicator value of 0.7 ng/ml will reduce
the number of false negatives detected and is also useful as a
predetermined amount. By contrast, a measured amount below the
indicator value of 0.5 ng/ml is a positive indicator of Alzheimer's
disease and individuals having these levels are considered to be
more likely to suffer from Alzheimer's disease. An indicator value
of 0.45 ng/ml reduces the number of false positives and is also
useful as a predetermined value. However, since values below 0.5
ng/ml and 0.45 ng/ml are at the low end of the normal range found
in non-Alzheimer individuals, a measured amount below the indicator
level does not, by itself, suffice to provide a diagnosis of
Alzheimer's disease. Therefore, the methods of the present
invention will be useful as part of a diagnosis procedure which
will also consider other known AD symptoms, such as those described
in the NINCDS-ADRDA criteria (e.g., clinical dementia and memory
impairment).
[0037] The invention also results in part from the discovery that a
finding of A.beta.(x-.gtoreq.41) in the low end of the normal range
together with a finding of higher than normal amounts of tau in the
CSF of an individual is a stronger positive indicator of
Alzheimer's disease than either finding alone, and that a finding
of high levels of A.beta.(x-.gtoreq.41) and low levels of tau in
the CSF of an individual is a very strong negative indicator of
Alzheimer's disease. Thus, the combined use of these two markers
appears to offer significant complementary diagnostic
information.
[0038] Data presented in FIG. 6 show that patients who exhibit high
tau (above about 0.3 ng/ml) and low A.beta.(x-.gtoreq.41) (below
about 0.5 ng/ml) had a 96% likelihood of having Alzheimer's disease
(22/23). Fifty-nine percent of the Alzheimer's disease patients in
this study (22/37) fall into this category. Conversely, patients
who exhibit low tau (below about 300 ng/ml) and elevated
A.beta.(x-.gtoreq.41) had a 100% likelihood of not having
Alzheimer's disease (28/28, FIG. 6). Slightly over half of the
non-Alzheimer's disease subjects (28/52, 54%) fall into this
category. Taken together, the combined analysis of CSF tau and
A.beta.(x-.gtoreq.41) was highly predictive of either the presence
or the absence of Alzheimer's disease in slightly over half of all
individuals enrolled in this study. The combined CSF tau and
A.beta.(x-.gtoreq.41) measurements were not informative in those
patients that fell into the low A.beta.(x-.gtoreq.41)/low tau
group. Nevertheless, the ability of any test to aid in the
inclusion or exclusion of Alzheimer's disease with high specificity
and even moderate sensitivity is greatly important.
[0039] According to a second method of this invention, the amount
of both soluble A.beta.(x-.gtoreq.41) and tau in a patient sample
is measured. One useful method of determining the amount of tau is
by ELISA as described in more detail below. The measured amounts of
A.beta.(x-.gtoreq.41) and tau are then compared with pre-determined
values for each. The status of the patient is assessed based on the
difference between the predetermined values and the measured
values.
[0040] As discussed below, indicator values can be calibrated based
on the particular binding substance used and the particular
A.beta.(x-.gtoreq.41) and tau protein to be detected. Calibration
involves testing the binding substance against standards from
individuals having an A.beta.-related disease and control standards
from those not having such a disease. Indicator values are selected
from these results based on the numbers of false positives or false
negatives the practitioner is willing to tolerate. It is expected
that indicator values using different binding substances and
directed against the targets described herein will be roughly the
same as the indicator values described herein. Indicator values
below 0.45 ng/ml for A.beta.(x-.gtoreq.41) and above 0.4 ng/ml for
tau decrease the number of false positive results; while indicator
values above 0.7 ng/ml for A.beta.(x-.gtoreq.41) and below 0.25
ng/ml for tau decreases the potential for a false indication of
freedom from Alzheimer's disease.
[0041] If the reason for reduced CSF A.beta.(x-.gtoreq.41) in AD is
indeed secondary to ongoing plaque deposition, it could explain why
a substantial number of neurological disease subjects and a few
control subjects presented with low A.beta.(x-.gtoreq.41) levels in
CSF. Plaque deposition has been hypothesized to precede cognitive
failure and a significant portion of these elderly non-AD subjects
would be expected to develop AD within the next several years (DMA
Mann et al. (1992) Neurodegeneration 1:201-215 and D L Price et al.
(1991) Neurobiol Aging 12:295-312). Longitudinal studies will
obviously be required to address the possibility that low
A.beta.(x-.gtoreq.41) levels are predictive of AD.
[0042] It was also found that levels of tau in AD CSF do not
correlate with age, MMSE, total A.beta., A.beta..sub.42, or ApoE
.epsilon.4. Although the precise reason for elevation of tau in AD
remains unclear, it is likely due to the increased tau levels in AD
brain tissue (S. Khatoon et. al. (1992) J Neurochem 59:750-753)
combined with the ongoing degeneration of neurons in the
disease.
[0043] The sandwich assay described in the Experimental section
used antibodies raised against the junction region of A.beta. and
against residues 33-42 of A.beta.. In-this assay, Alzheimer's
patients generally had levels of A.beta.(x-.gtoreq.41) below 0.5
ng/ml as detected by the antibodies. The indicator value of 0.5
ng/ml is, in part, a function of the particular peptides recognized
by the antibodies used as well as the peptide lot used in making
the calibration. Therefore, the practitioner may base the
predetermined amount on a re-calibration using reagents and
protocols to be used in measuring A.beta.(x-.gtoreq.41) in the
test.
[0044] In addition to initial diagnosis of the A.beta.-related
condition, the measured concentrations of A.beta. may be monitored
in order to follow the progress of the disease, and potentially
follow the effectiveness of treatment (when such treatments become
available). It would be expected that levels of
A.beta.(x-.gtoreq.41) would decrease as the disease progressed.
[0045] The term "amyloid-.beta. peptide," or "A.beta." as used
herein refers to an approximately 4.2 kD protein which, in the
brains of AD, Down's Syndrome, HCHWA-D and some normal aged
subjects, forms the subunit of the amyloid filaments comprising the
senile (amyloid) plaques and the amyloid deposits in small cerebral
and meningeal blood vessels (amyloid angiopathy). A.beta. can occur
in a filamentous polymeric form (in this form, it exhibits the
Congo-red and thioflavin-S dye-binding characteristics of amyloid
described in connection therewith). A.beta. can also occur in a
non-filamentous form ("preamyloid" or "amorphous" or "diffuse"
deposits) in tissue, in which form no detectable birefringent
staining by Congo red occurs. A portion of this protein in the
insoluble form obtained from meningeal blood vessels is described
in U.S. Pat. No. 4,666,829. A.beta. is an approximately 39-43 amino
acid fragment of a large membrane-spanning glycoprotein, referred
to as the .beta.-amyloid precursor protein (APP), encoded by a gene
on the long arm of human chromosome 21. Forms of A.beta. longer
than 43 amino acids are also contemplated herein. A.beta. is
further characterized by its relative mobility in
SDS-polyacrylamide gel electrophoresis or in high performance
liquid chromatography (HPLC). A sequence for a 43-amino
acid-version of A.beta. is:
1 1 Asp Ala Glu Phe Arg [SEQ ID NO: 1] His Asp Ser Gly Tyr 11 Glu
Val His His Gln Lys Leu Val Phe Phe 21 Ala Glu Asp Val Gly Ser Asn
Lys Gly Ala 31 Ile Ile Gly Leu Met Val Gly Gly Val Val 41 Ile Ala
Thr.
[0046] As used herein, A.beta. also refers to related polymorphic
forms of A.beta., including those that result from mutations in the
A.beta. region of the APP normal gene.
[0047] The term "A.beta. fragment" as used herein refers to
fragments and degradation products of A.beta. which are generated
at low concentrations by mammalian cells. Particular A.beta.
fragments have a molecular weight of approximately 3 kD and are
presently believed to include peptides with, for example, amino
acid residues 3-34, 6-27, 6-34, 6-35, 6-42, 11-34, 11-40, 17-40,
11-43 and 12-43 of A.beta..
[0048] As used herein, the term "A.beta.(x-.gtoreq.41)" refers to
A.beta. whose amino-terminus begins at amino acid number 1 of
A.beta. or which is truncated, and whose carboxy-terminus extends
beyond amino acid number 40. These peptides and fragments comprise
a heterogenous group. For example, A.beta.(6-42), A.beta.(11-43)
and A.beta.(12-43) all have been found in the CSF. However, this
list is not meant to be exclusive. Other peptides from among the
group are presumed to exist in the CSF and are detectable with the
methods described herein.
[0049] The particular peptides measured from among the group of all
A.beta.(x-.gtoreq.41) depends on the particular measuring method
used. In the case of using binding substances, such as antibodies,
the binding substance can be directed to one or more from among the
group of peptides. For example, an antibody raised against amino
acids 33-42 of A.beta. that does not cross react with A.beta.(1-40)
will bind to A.beta.(x-42). It also may bind to A.beta.(x-41) and
A.beta.(x-43). According to one embodiment of the invention, the
method involves determining the amount of A.beta.(x-.gtoreq.41)
having at least amino acids 13-41 of A.beta.. These species can be
measured using a sandwich assay employing antibodies that recognize
the junction region (amino acids 13-26) and antibodies produced by
immunization with a hapten having A.beta. amino acids 33-42, as
described in the Example.
[0050] The term "A.beta. junction region" as used herein refers to
a region of A.beta. which is centered at the site between amino
acid residues 16 and 17 (Lys.sup.16 and Leu.sup.17) which is a
target for proteolytic processing of APP. Such processing results
in a variety of APP fragments which may, for example, terminate at
amino acid 16 of A.beta. and which, therefore, are potentially
immunologically cross-reactive with antibodies to the intact
A.beta. molecule which are to be identified in the methods of the
present invention. Antibodies raised against a synthetic peptide
including amino acid residues 13-29 having been found to display
the requisite specificity.
[0051] The term "amyloid-.beta. precursor protein" (APP) as used
herein is defined as a polypeptide that is encoded by a gene of the
same name localized in humans on the long arm of chromosome 21 and
that includes A.beta. within its carboxyl third. APP is a
glycosylated, single-membrane-spanning protein expressed in a wide
variety of cells in many mammalian tissues. Examples of specific
isotypes of APP which are currently known to exist in humans are
the 695-amino acid polypeptide described by Kang et al. (1987)
Nature 325:733-736 which is designated as the "normal" APP; the
751-amino acid polypeptide described by Ponte et al. (1988) Nature
331:525-527 (1988) and Tanzi et al. (1988) Nature 331:528-530; and
the 770-amino acid polypeptide described by Kitaguchi et al. (1988)
Nature 331:530-532. Examples of specific variants of APP include
point mutations which can differ in both position and phenotype
(for review of known variant mutations see Hardy (1992) Nature
Genet. 1:233-234).
[0052] The term "A.beta.-related condition" as used herein is
defined as including Alzheimer's disease (which includes familial
Alzheimer's disease), Down's Syndrome, HCHWA-D, and advanced aging
of the brain.
[0053] As used herein, "tau" refers to the family of
microtubule-associated proteins. The paired helical filament of
neurofibrillary tangles in the brains of Alzheimer's disease
patients are composed of tau protein. (See, e.g., M. Goedert et al.
(1989) Neuron 3:519-526 and M. Goedert (1993) TINS 16:460-465,
incorporated herein by reference.) Goedert et al. (1989) also
presents a DNA and amino acid sequence for tau.
[0054] The term "body fluid" as used herein refers to those fluids
of a mammalian host which will be expected to contain measurable
amounts of A.beta., A.beta. fragments or tau protein, specifically
including cerebrospinal fluid (CSF), blood, urine, and peritoneal
fluid. The term "blood" refers to whole blood, as well as blood
plasma and serum.
[0055] The methods and systems of this invention involve the
ability to detect species of A.beta. extending beyond amino acid
number 40 at the carboxy-terminal end and, therefore, to
distinguish them from shorter species, such as A.beta.(40). While
detection of A.beta.(x-.gtoreq.41) can be accomplished by any
methods known in the art for detecting peptides, the use of
immunological detection techniques employing binding substances
such as antibodies, antibody fragments, recombinant antibodies, and
the like, is preferred. Particularly suitable detection techniques
include ELISA, Western blotting, radioimmunoassay, and the like.
Suitable immunological methods employing a single antibody are also
contemplated, for example, radioimmunoassay using an antibody
specific for .gtoreq.41 forms of A.beta., or single antibody ELISA
methods.
[0056] Thus, this invention also provides antibodies specific for
A.beta.(x-.gtoreq.41) that do not cross react with
A.beta.(.ltoreq.40). These antibodies can be made by immunizing
animals with synthetic peptides that include amino acids beyond
number 40 of A.beta.. For example, the synthetic peptide can
include amino acids 33-42. A specific example of the production of
such an antibody is provided in the Experimental section.
[0057] According to one embodiment of the invention, detection and
measurement of A.beta.(x-.gtoreq.41) peptides involves the use of
two antibodies, one specific for an epitope containing amino acids
beyond number 40 in A.beta., and another antibody capable of
distinguishing A.beta. and A.beta. fragments from other APP
fragments which might be found in the sample. In particular, it has
been found that antibodies which are monospecific for the junction
region of A.beta. are capable of distinguishing A.beta. from other
APP fragments. The junction region of A.beta. is centered at amino
acid residues 16 and 17, typically spanning amino acid residues
13-26, and such junction-specific antibodies may be prepared using
synthetic peptides having that sequence as an immunogen.
[0058] A preferred immunoassay technique is a two-site or
"sandwich"-assay employing a junction-specific antibody as the
capture antibody (bound to a solid phase) and a second antibody
which binds to an epitope containing amino acids beyond number 40
in A.beta.. Particular methods for preparing such antibodies and
utilizing such antibodies in an exemplary ELISA are set forth in
the Experimental section hereinafter and in related U.S. patent
application Ser. No. 07/965,972, supra.
[0059] Antibodies specific for A.beta. may be prepared against a
suitable antigen or hapten comprising the desired target epitope,
such as the junction region consisting of amino acid residues 13-29
and the carboxy terminus consisting of amino acid residues 33-42.
Conveniently, synthetic peptides may be prepared by conventional
solid phase techniques, coupled to a suitable immunogen, and used
to prepare antisera or monoclonal antibodies by conventional
techniques. Suitable peptide haptens will usually comprise at least
five contiguous residues within A.beta. and may include more than
six residues.
[0060] Synthetic polypeptide haptens may be produced by the
well-known Merrifield solid-phase synthesis technique in which
amino acids are sequentially added to a growing chain (Merrifield
(1963) J. Am. Chem. Soc. 85:-2149-2156). The amino acid sequences
may be based on the sequence of A.beta. set forth above.
[0061] Once a sufficient quantity of polypeptide hapten has been
obtained, it may be conjugated to a suitable immunogenic carrier,
such as serum albumin, keyhole limpet hemocyanin, or other suitable
protein carriers, as generally described in Hudson and Hay,
Practical Immunology, Blackwell Scientific Publications, Oxford,
Chapter 1.3, 1980, the disclosure of which is incorporated herein
by reference. An exemplary immunogenic carrier utilized in the
examples provided below is .alpha.-CD3.epsilon. antibody
(Boehringer-Mannheim, Clone No. 145-2C11).
[0062] Once a sufficient quantity of the immunogen has been
obtained, antibodies specific for the desired epitope may be
produced by in vitro or in vivo techniques. In vitro techniques
involve exposure of lymphocytes to the immunogens, while in vivo
techniques require the injection of the immunogens into a suitable
vertebrate host. Suitable vertebrate hosts are non-human, including
mice, rats, rabbits, sheep, goats, and the like. Immunogens are
injected into the animal according to a predetermined schedule, and
the animals are periodically bled, with successive bleeds having
improved titer and specificity. The injections may be made
intramuscularly, intraperitoneally, subcutaneously, or the like,
and an adjuvant, such as incomplete Freund's adjuvant, may be
employed.
[0063] If desired, monoclonal antibodies can be obtained by
preparing immortalized cell lines capable of producing antibodies
having desired specificity. Such immortalized cell lines may be
produced in a variety of ways. Conveniently, a small vertebrate,
such as a mouse, is hyperimmunized with the desired immunogen by
the method just described. The vertebrate is then killed, usually
several days after the final immunization, the spleen cells
removed, and the spleen cells immortalized. The manner of
immortalization is not critical. Presently, the most common
technique is fusion with a myeloma cell fusion partner, as first
described by Kohler and Milstein (1975) Nature 256:495-497. Other
techniques including EBV transformation, transformation with bare
DNA.beta. e.g., oncogenes, retroviruses, etc., or any other method
which provides for stable maintenance of the cell line and
production of monoclonal antibodies. Specific techniques for
preparing monoclonal antibodies are described in Antibodies: A
Laboratory Manual, Harlow and Lane, eds., Cold Spring Harbor
Laboratory, 1988, the full disclosure of which is incorporated
herein by reference.
[0064] In addition to monoclonal antibodies and polyclonal
antibodies (antisera), the detection techniques of the present
invention will also be able to use antibody fragments, such as
F(ab), Fv, V.sub.L, V.sub.H, and other fragments. In the use of
polyclonal antibodies, however, it may be necessary to adsorb the
anti-sera against the target epitopes in order to produce a
monospecific antibody population. It will also be possible to
employ recombinantly produced antibodies (immunoglobulins) and
variations thereof as now well described in the patent and
scientific literature. See, for example, EPO 8430268.0; EPO
85102665.8; EPO 85305604.2; PCT/GB 85/00392; EPO 85115311.4;
PCT/US86/002269; and Japanese application 85239543, the disclosures
of which are incorporated herein by reference. It would also be
possible to prepare other recombinant proteins which would mimic
the binding specificity of antibodies prepared as just
described.
[0065] Detection of tau also can be accomplished by any methods
known in the art for detecting peptides. However, the use of
immunological detection techniques employing binding substances is
preferred. Useful detection techniques include all those mentioned
above. ELISA assays involving a capture antibody and a labeled
detection antibody, both against tau, are particularly useful.
[0066] Antibodies against tau can be prepared by inoculating
animals with tau purified from AD brains or from recombinant
sources. Recombinant tau can be produced by expression in insect
cells from a baculovirus vector containing pVL941-tau-4 repeat
isoform as described by J. Knops et al. (1991) J Cell Biol
1991:114:725-733. Purified tau also is available from Immogenetics
(Zwijndrecht, Belgium). Antibodies against tau are available from
Sigma (St. Louis, Mo.). Additional sources can be identified in the
Lindscott directory.
[0067] Tau can be prepared from AD brain by the method of Mercken
et al. (1992) J Neurochem 58:548. Typically, 50 g of fresh brain is
cut into small pieces with scissors and homogenized 1:1 (wt/vol) in
buffer A (20 mM 2-[N-morpholino]ethanesulfonic acid, 80 mM NaCl, 2
mM EDTA, 0.1, mM EGTA, 1 mM MgCl.sub.2, and 1 mM
.beta.-mercaptoethanol, pH 6.75) with a Potter homogenizer equipped
with a Teflon plunger. The homogenate is centrifuged for 1 hour at
150,000 g at 4.degree. C., and the supernatant is heated for 5
minutes in boiling water and chilled again for 10 minutes on ice.
The slurry is centrifuged for 2 hours at 150,000 g at 4.degree. C.,
and the supernatant is collected thereafter. The heat-stable
cytosolic extract is made to 2.5% perchloric acid and centrifuged
for 1 hour at 150,000 g at 4.degree. C., after which the
supernatant is neutralized with 3 M Tris. The supernatant is then
dialyzed and concentrated in water in a Centiprep concentrator
(Amicon, Lausanne, Switzerland). The end product can be evaluated
in sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) (Laemmli method). This preparation is useful for
immunizing animals to produce anti-tau antibodies.
[0068] Tau also can be immunopurified from this preparation. Ten
milligrams of anti-tau monoclonal antibody is coupled to 1 g of
cyanogen bromide-activated Sepharose (Pharmacia) by the method
proposed by the manufacturer. Fifty milliliters of the heat stable
cytosolic extract described above is diluted 1:2 in 0.1 M phosphate
buffer (pH 8.5) and applied to the column. The column is washed
with 0.1 M phosphate, and tau is eluted with 0.1 M citric acid (pH
2.5) and neutralized immediately with 1 M NaOH. Fractions can be
evaluated by SDS-PAGE on 10% gels and in immunoblotting with
anti-tau antibodies.
[0069] This invention also provides kits for performing assays that
aid in the diagnosis of Alzheimer's disease. The kits include means
for detecting A.beta.(x-.gtoreq.41) and means for detecting tau.
The means can include any means known or described above, e.g.,
binding substances. Useful binding substances include molecules
containing the binding portion of an antibody, such as a full
antibody or an antibody fragment. The binding substances can be
monoclonal antibodies. In one embodiment the kit includes a binding
substance that binds A.beta.(x-.gtoreq.41) but that does not bind
to A.beta.(.ltoreq.40) and a binding substance that binds to
tau.
[0070] In one embodiment the kit includes antibodies or the like
for performing sandwich ELISAs to detect each compound, for
example, as described above. In one embodiment, the means to detect
A.beta.(x-.gtoreq.41) can include a binding substance that binds to
an epitope containing amino acids beyond number 40 in A.beta. and a
binding substance that binds A.beta. or a fragment of A.beta. but
that does not bind other fragments of APP. The means to detect tau
also can involve a sandwich ELISA. For example, the kit can include
a) an un-labeled binding substance that binds to the junction
region of A.beta.; b) a detectably labelled binding substance that
binds to an epitope containing amino acids beyond number 40 in
A.beta.; c) an un-labelled binding substance that binds to tau; and
d) a detectably labelled binding substance that binds to tau.
[0071] The detectable labels can be any known and used in the art
including, e.g., biotinylation, radioactive label, enzymes,
fluorescent labels and the like.
[0072] Animal models are currently being used to study Alzheimer's
disease. (See, e.g., International Patent Application WO 93/14200,
U.S. patent application Ser. No. 08/143,697, filed Oct. 27, 1993,
and U.S. Pat. No. 5,387,742 all of which are incorporated herein by
reference.) These models are useful for screening compounds for
their ability to effect the course of Alzheimer's disease, both to
ameliorate and aggravate the condition. Since AD is characterized
by a decrease in the amounts of A.beta.(x-.gtoreq.41) in the CSF,
it is expected that effective treatments for Alzheimer's disease
will result in an increase in amount of A.beta.(x-.gtoreq.41) in
the CSF, while agents that hasten progress of the disease will
result in a decrease in the amount of A.beta.(.gtoreq.41) in the
CSF.
[0073] Accordingly, this invention provides methods for screening
compounds that elevate or decrease the amount of
A.beta.(x-.gtoreq.41) in a fluid sample, in particular the CSF, and
that, therefore, are candidates for use in treating the disease, or
that hasten the disease and are to be avoided by humans. The
methods involve measuring a first amount of said one or more
soluble A.beta.(x-.gtoreq.41) in a sample of a non-human animal
used as a model of Alzheimer's disease; administering the compound
to the animal; measuring a second amount of one or more soluble
A.beta.(x-.gtoreq.41) in a sample of the animal; and comparing the
first amount with the second amount, the difference indicating
whether the compound increases, decreases, or leaves unchanged the
amount of soluble A.beta.(x-.gtoreq.41) in the sample. The dosage
level given to the animal and the amount of time that elapses
before measuring the second amount will, of course, depend on the
model system.
[0074] This invention also provides methods for screening compounds
that elevate the amount of A.beta.(x-.gtoreq.41) and decrease the
amount of tau in a fluid sample, particularly CSF, and that,
therefore, are candidates for use in treating the disease; or that
decrease the level of A.beta.(x-.gtoreq.41) and that increase the
level of tau and therefore, that hasten the disease and are to be
avoided by humans. The methods involve measuring a first amount of
said one or more soluble A.beta.(x-.gtoreq.41) and tau in a fluid
sample of a non-human animal used as a model of Alzheimer's
disease; administering the compound to the animal; measuring a
second amount of one or more soluble A.beta.(x-.gtoreq.41) and tau
in a fluid sample of the animal; and comparing the first amounts
with the second amounts, the difference indicating whether the
compound increases, decreases, or leaves unchanged the amount of
soluble A.beta.(x-.gtoreq.41) and tau in the fluid sample. The
dosage level given to the animal and the amount of time that
elapses before measuring the second amount will, of course, depend
on the model system.
[0075] One useful non-human animal model harbors a copy of an
expressible transgene sequence which encodes the Swedish mutation
of APP (asparagine.sup.595-leucine.sup.596) The sequence generally
is expressed in cells which normally express the
naturally-occurring endogenous APP gene (if present). Mammalian
models, more particularly, rodent models and in particular murine
and hamster models, are suitable for this use. Such transgenes
typically comprise a Swedish mutation APP expression cassette, in
which a linked promoter and, preferably, an enhancer drive
expression of structural sequences encoding a heterologous APP
polypeptide comprising the Swedish mutation.
[0076] The transgenic animals that harbor the transgene encoding a
Swedish mutation APP polypeptide are usually produced by
introducing the transgene or targeting construct into a fertilized
egg or embryonic stem (ES) cell, typically by microinjection,
electroporation, lipofection, or biolistics. The transgenic animals
express the Swedish mutation APP gene of the transgene (or
homologously recombined targeting construct), typically in brain
tissue. Preferably, one or both endogenous APP allele is
inactivated and incapable of expressing the wild-type APP.
[0077] The following examples are offered by way of illustration,
not by way of limitation.
EXPERIMENTAL
I. A.beta.(x-.gtoreq.41) is Decreased in Alzheimer's Patients
[0078] Materials and Methods
[0079] 1. Antibody Preparation.
[0080] a. Monoclonal Antibodies to the A.beta. Junction Region.
[0081] Monoclonal antibodies to the junction region of A.beta. were
prepared using a synthetic peptide spanning amino acid residues
13-31, except that AI, amino acids 30 and 31, were substituted with
GC. This peptide was called A.beta..sub.13-28. A.beta..sub.13-28
was conjugated to an immunogen (.alpha.-CD3.epsilon. antibody;
Clone No. 145-2C11, Boehringer-Mannheim) using
m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) according to
the manufacturer's (Pierce) instructions.
[0082] A/J mice were immunized initially intraperitoneally (IP)
with the A.beta. conjugate mixed with complete Freund's adjuvant.
Fourteen days later, the mice were boosted IP with the A.beta.
conjugate mixed with phosphate buffered saline (PBS) at 14 day
intervals. After six total boosts, the mice were finally boosted
intravenously with A.beta. conjugate mixed with incomplete Freund's
adjuvant and fused 3 days later. Fusion of spleen cells with P3.653
myeloma cells was performed according as described in Oi and
Herzenberg, Selective Methods in Cellular Immunology, Mishell and
Shigii, Eds., W.H. Freeman and Company, San Francisco, Chapter 17
(1980). Serum titers and initial screens were performed by the RIA
method described below. Several clones were expanded to a 24-well
plate and subjected to further analysis as described below. Clones
of interest were produced in mouse ascites.
[0083] The RIA method used to screen serum bleeds and fusion
hybridoma supernatants was based upon a method developed by Wang et
al. (1977) J. Immunol. Methods 18:157-164. Briefly, the supernatant
(or serum) was incubated overnight at room temperature on a rotator
with .sup.125I-labeled A.beta..sub.1-28 and Sepharose.RTM. 4B beads
to which sheep anti-mouse IgG had been coupled via cyanogen
bromide. The beads from each well were harvested onto glass fiber
filter discs with a cell harvester and washed several times with
PBS. The filter discs were then transferred to gamma tubes and the
bound radioactivity was counted in a gamma counter.
[0084] All hybridomas were tested for binding to A.beta..sub.1-28
using the method described above in the initial screen, and then
retested 3 days later. A.beta..sub.1-28 positive clones were
further characterized for reactivity to .sup.125I-labeled
A.beta..sub.1-16 using the RIA method described above. No clones
were found to bind A.beta..sub.1-16. In a peptide capture ELISA,
all clones were found to react with A.beta..sub.13-28 while no
clones reacted to A.beta..sub.17-28. Therefore, it was determined
that all clones had an epitope within the junction region spanning
amino acids 16 and 17.
[0085] Based on results of the above assays, several clones were
expanded into 24 well plates. These clones were further
characterized by saturation analysis. Supernatants at the 50% titer
point (as determined by the RIA method described above) were added
to wells containing Sepharose.RTM.-sheep anti-mouse IgG beads, a
constant amount of .sup.125I-labeled-A.beta..sub.1-28, and varying
amounts of unlabeled A.beta..sub.13-28 or A.beta..sub.17-28. The
concentration of cold peptide for 50% inhibition was determined for
each antibody. For the A.beta..sub.17-28, no inhibition was seen at
100 ng/well for any clones. The 50% inhibition point for
A.beta..sub.13-28 ranged from 10-80 ng/well. The clones were also
characterized based on reactivity in Western blots. Based on titer
point, sensitivity (as determined by the 50% inhibition point), and
reactivity on Western blot, several clones were produced in
ascites. Antibodies from hybridoma designated 266 was selected for
use as a capture antibody in the assays described below.
[0086] b. Polyclonal Antibodies to the C-terminal Epitope
Containing Amino Acids 33-42 of A.beta.
[0087] Polyclonal antibodies were generated against A.beta.(33-42)
as follows. Peptide 277-2 (C-aminoheptanoic-GLMVGGVVIA [SEQ ID
NO:2]) was conjugated to cationized BSA (Pierce activated
"Supercarrier") at a ratio of 5 mg of 277-2 peptide to 10 mg of
cationized BSA as follows. One vial of Pierce Supercarrier (10 mg)
was resuspended in 1 mL of deionized water. 5 mg of the 277-2
peptide was dissolved in 5 ml of 10 mM PO.sub.4 pH 8.0. The 277-2
peptide was added to the Supercarrier and incubated overnight at
room temperature. This was then concentrated and the EDTA
removed.
[0088] The immunogen (500 mg of peptide equivalent) was injected
subcutaneously in complete Freund's adjuvant. Rabbits received a
booster of 0.2-0.5 mg after three weeks and 0.2 to 0.5 mg at two to
four week intervals thereafter. Boosters were subcutaneously
administered in incomplete Freund's adjuvant. Twenty-five ml of
serum was collected one week after each boost. Bleeds were screened
as follows. Week 7 of the rabbit bleeds were titered by serial
dilution. ELISA plates were coated with A.beta. 1-42 overnight, and
then blocked with 3% gelatin. Serial dilutions of the rabbit bleeds
from {fraction (1/100)}-{fraction (1/200,000)} were incubated on
the plates for 2 hours at room temperature. The plates were then
washed and the anti rabbit HRP was added to each well. This
incubated for one hour. The plate was washed and TMB substrate was
used. ELISA titer of the rabbits was {fraction
(1/20,000)}-{fraction (1/200,000)}.
[0089] The ELISA positive rabbit bleeds were then titered in a
capture RIA to compare its ability to capture .sup.125I
A.beta.(1-42) versus .sup.125I A.beta.(1-40). Dilutions of rabbit
antiserum from {fraction (1/25)}-{fraction (1/675)} were incubated
with approximately the same number of cpm's of both tracers.
Protein A sepharose was used to precipitate the immune complexes
and they were then counted on a Microbeta scintillation counter.
277-2 rabbit D showed the highest titer to A.beta.(1-42) tracer and
no cross reaction with A.beta.(1-40) tracer. The highest titer
bleeds were then subjected to affinity purification of
antibodies.
[0090] To affinity purity anti-277-2 antibodies, a 277-2 affinity
matrix was prepared as follows: three ml of sulfo-link gel (Pierce)
was washed with six volumes of 50 mM Tris, 5 mM EDTA, pH 8.5. Three
mg of 277-2 peptide dissolved in 0.3 ml DMSO was brought to 3 ml
with 50 mM Tris, 5 mM EDTA pH 8.5. and added to the gel. After
gentle mixing for 15 minutes, the column resin was washed with six
volumes of 50 mM Tris, 5 mM EDTA, 0.5 M NaCl pH 8. The column resin
was then washed with 16 volumes of PBS/0.05% NaN.sub.3.
[0091] To affinity purify the antibodies, 20 ml of high titer serum
was diluted to 40 ml with PBS and an equal volume of saturated
(NH.sub.4).sub.2SO.sub.4 was slowly added while stirring at
4.degree.. The mixture was allowed to stir an additional 30 minutes
then spun for 15 minutes at 10,000 rpm in a Beckman JA17 rotor. The
pellets were resuspended in PBS, brought to a volume of 40 ml with
PBS and the (NH.sub.4).sub.2SO.sub.4 precipitation repeated as
above. The pellets were resuspended in a total of 20 ml of PBS and
dialyzed overnight against PBS at 4.degree..
[0092] The 277-2 column was washed with 10 ml of PBS. Then the
dialyzate was run over the column. The column was then washed with
50 ml of PBS. 0.1 M glycine, 0.5 M NaCl pH 2.5 was added 1 ml at a
time and fractions collected. The first four fractions containing
the majority of elated protein were pooled and neutralized with 0.4
ml of 1 M Tris pH 8.0. The pool was concentrated by membrane
filtration to slightly less than 2 ml. The initial column
flow-through was subjected to a second chromatographic step (after
first neutralizing the column and re-equilibrating it in PBS). The
second affinity-purified material was similarly neutralized and
concentrated, combined with the first material and then dialyzed
against PBS overnight, 4.degree.. The protein content was
determined (Pierce BCA method) and these antibodies were used in
ELISA experiments.
[0093] 2. ELISA Assay.
[0094] a. Binding of Capture Antibody to Microtiter Wells.
[0095] Monoclonal antibody 266 was diluted to a concentration of 10
.mu.g/ml in a buffer containing 0.23 g/L
NaH.sub.2PO.sub.4.H.sub.2O, 26.2 g/L Na.sub.2HPO.sub.4; 7H.sub.2O,
1 g/L NaN.sub.3, pH 8.5. One hundred .mu.l/well of this solution
was then dispensed in a 96 well white Dynatech Microlite 2, 96 well
flat-bottomed plate. The plates were sealed and incubated overnight
at room temperature. Following coating, the remaining solution was
aspirated and the non-specific binding sites were blocked with 200
.mu.L per well of (NaH.sub.2PO.sub.4H.sub.2O) 0.2 g/L,
Na.sub.2HPO.sub.4.7H.sub.2O 0.8 g/L, human serum albumin (HSA)
crystallized and lyophilized 2.5 g/L, pH 7.4. These plates were
blocked by incubating for 1 hour at room temperature in the
blocking solution.
[0096] b. Assay Protocol.
[0097] The calibrators were prepared from a stock solution of
A.beta..sub.1-42, 1 .mu.g/ml, in DMSO. In specimen diluent
((NaH.sub.2PO.sub.4.H.sub.2O) 0.2 g/L, Na.sub.2HPO.sub.4.7H.sub.2O
2.16 g/L, NaN.sub.3 0.5 g/L, bovine serum albumin (BSA) (globulin
free) 6 g/L, triton x-405 0.5 ml/L NaCl 8.5 g/L, pH 7.4.), the
highest calibrator, 1000 pg/ml (10 .mu.l A.beta..sub.1-42 stock (1
.mu.g/ml DMSO) in 10 ml casein specimen diluent) was prepared.
Sequential dilutions were made in specimen diluent to obtain 500,
250, 125, 62.5 and 31.25 pg/ml concentrations of A.beta.1-42.
[0098] CSF samples were prepared as follows. The CSF samples
(100-500 .mu.l) were boiled for 3 minutes. The boiled samples were
placed at 4.degree. C. for 10-14 hours before assaying. CSF samples
are assayed undiluted. Dilutions are only made if the initial
calculated value is above the highest calibrator (1000 pg/ml).
[0099] One hundred .mu.L per well calibrators or samples were
applied to the microtiter plates. The plates were sealed and
incubated for 1 hour at room temperature. The plates were then
washed three times with washing buffer (NaCl 80 g/L, KCl 3.85 g/L,
Tris-HCl 31.75 g/L, tween-20 0.5 ml/L, pH 7.5).
[0100] Anti-A.beta.(33-42) (antibody 277-2) was diluted in specimen
diluent to 1 .mu.g/ml and 100 .mu.l was added per well. The plate
was covered and incubated for 1 hour at room temperature. The plate
was washed three times with washing buffer. The alkaline
phosphatase affinity purified F(ab')2 fragment donkey anti-rabbit
IgG (H+L) (Jackson) was diluted 1:1000 in specimen diluent. One
hundred .mu.l/well was added. The plate was covered and incubated
for 1 hour at room temperature. The plate was washed three times
with washing buffer, then 100 .mu.l/well of chemiluminescent
substrate was added. The chemiluminescent substrate was prepared by
diluting the chemiluminescent reagent, AMPPD (Tropix), and an
enhancer, emerald green (Tropix), 1:1000 and 1:100 respectively in
1M diethanolemine buffer, pH 10, containing 1 mM MgCl.sub.2 and
0.2% NaN.sub.3. The plates were sealed and incubated for 10 to 15
minutes at room temperature. Solution was not aspirated. This time
may have to be optimized for different antibody lots.
[0101] Chemiluminescence was read and expressed as relative
chemiluminescence units (CLU) after 15 minutes using a Dynatech ML
1000.
[0102] Results
[0103] 1. A.beta.(x-<41) Assay Specificity
[0104] A.beta.(x-.gtoreq.41) ELISA does not cross-react with
A.beta.(1-28), (1-38), or (1-40) (FIG. 1).
[0105] 2. A.beta.(x-.gtoreq.41) Assay Sensitivity
[0106] The lower sensitivity limit for this assay is 31 pg/ml or
3.1 pg/well (0.7 fmol/well) (FIG. 1).
[0107] 3. A.beta.(x-.gtoreq.41) Levels in CSF
[0108] A.beta.(x-.gtoreq.41) has been verified in CSF using the
A.beta.(x-.gtoreq.41) ELISA. On occasion, two different groups of
CSF samples, designated Group A and Group B, were obtained from
various sources. Sometimes, two hundred .mu.L of the CSF samples
were boiled for 3 minutes prior to assay (boiling was found to
increase A.beta.(x-.gtoreq.41) immunoreactivity in some cases). The
results of this assay can be seen in FIG. 2 and FIG. 3.
2TABLE I AD DIAGNOSTICS A.beta.(x-.gtoreq.41) Data Groups A and B
CSF A.beta.1-42(pg/mL) SENSITIVITY SPECIFICITY GROUP CUTOFF FOR AD*
FOR AD Group A .ltoreq.362.7 50% 84% .ltoreq.588.0 93.8% 50.0%
Group B .ltoreq.367.4 50% 85% .ltoreq.504.4 97.4% 56.6% *Equal to
specificity for detecting that an individual does not have AD.
[0109] 4. A.beta.(x-.gtoreq.41) in CSF of Rodents and Dogs
[0110] A.beta.(x-.gtoreq.41) immunoreactivity was also detected in
CSF of guinea pigs and dogs (Table II).
3TABLE II A.beta. IMMUNOREACTIVITY IN THE CSF OF VARIOUS ANIMAL
SPECIES A.beta.(X-.gtoreq.41) SPECIES TOTAL A.beta. (ng/ml) (ng/ml)
% A.beta.(x-.gtoreq.41) Guinea Pig 4.5 0.242 5.4 Dog 4.4 0.59
13.4
[0111] This sandwich ELISA demonstrates the presence of
A.beta.(x-.gtoreq.41) in CSF. A.beta.(x-.gtoreq.41) is only a minor
component of the total A.beta. in CSF. The levels of
A.beta.(x-.gtoreq.41) in CSF are significantly lower in AD than
normal and neurological controls. Taking a 50% sensitivity limit,
the specificity is 93.8 for Group A and 97.4% for Group B. These
two independent groups show a remarkable similarity demonstrating
that measurements of A.beta.(x-.gtoreq.41) in CSF have diagnostic
utility.
II. Combined Measurements of A.beta.(x-.gtoreq.41) and Tau are
Highly Sensitive for Alzheimer's Disease
[0112] Materials and Methods
[0113] 1. Subjects
[0114] All subjects enrolled in this study underwent detailed
clinical and neurological evaluation at university medical centers
by neurologists expert in the diagnosis of dementia. Informed
consent was obtained from subjects, or their guardians, as
appropriate. The evaluation included medical history, physical and
neurological examinations, laboratory blood tests to exclude
metabolic causes of dementia, a neuroimaging study (head CT or MR
within the past 3 years for demented patients and neurological
controls), and detailed psychometric testing (this varied between
institutions). In addition, all subjects received the following
assessment instruments: the Mini-Mental State Examination (MMSE)
(American Psychiatric Association, Committee on Nomenclature and
Statistics: Diagnostic and Statistical Manual of Mental Disorders:
Revised Third Edition, Washington D.C. Am. Psych Associ. (1987)),
the Hamilton Depression. Inventory (V. C. Hachiniski et al. (1975)
Ann Neurol 32:632-637) and the Hachinski Ischemic Index (G. McKann
et al. (1984) Neurology 34:939-944). Patients with more than one
dementia diagnosis, recent stroke, head trauma, or significant
peripheral nervous system disorders were excluded. The following
diagnostic criteria were used:
[0115] i. AD (n=37): patients met NINCDS-ADRDA guidelines for
probable AD; those who met criteria for possible AD were excluded
(The Lund and Manchester Groups (1994) J Neurol Neurosurg Psychiatr
57:416-418). All patients were community dwelling and had mild to
moderate dementia.
[0116] ii. Neurological disease controls (ND; n=32): patients with
non-AD dementia or degenerative disorders affecting the central
nervous system. For neurological controls, a summary of clinical
records was also reviewed by a second neurologist (DG) to confirm
diagnoses and to ensure that co-existing AD was unlikely. Patients
with frontal lobe dementia were diagnosed according to the criteria
set forth by the Lund and Manchester groups (Kawasaki E. S., in:
PCR Protocols: A guide to methods and applications. Academic Press,
Inc., New York 1990 pp. 146-152).
[0117] iii. Non-demented controls (NC; n=20): Subjects were age 50
or older and lacked significant cognitive complaints, did not have
functional impairment, had normal findings on neurological
examination, and scored 28-30 on the MMSE. A subgroup of these
controls had symptoms of depression that did not result in
significant cognitive or functional impairment, and were judged not
to have AD or any organic neurological condition.
[0118] Lumbar punctures were performed in the mornings, after an
overnight fast. All CSF samples were collected into specimen tubes
provided to all sites. The first 2-3 ml of CSF was analyzed for
protein, glucose and cells at the local medical center laboratory,
and 4.5 mL were removed from original collection tubes and added to
8 mL Sarstedt tubes containing 500 .mu.L buffer (containing
additives such that the final CSF solution composition included: 20
mM sodium phosphate, 20 mM triethanolamine, 0.05% Triton X-100, 100
mM NaCl, 0.05% NaN.sub.3, 1 mM diethylene triamine penta acetic
acid, 1 mM EGTA, pH 7.4) and frozen at -20.degree. C. until
analysis. Assay operators were unaware of the subjects'
diagnoses.
[0119] 2. ApoE Genotyping
[0120] ApoE genotyping was performed on available blood samples,
which had been collected into EDTA vacutainer tubes. Samples were
prepared by the method of Kawasaki (Kawasaki E S, in: PCR
Protocols: A guide to methods and applications, Academic Press,
Inc., New York 1990 pp.146-152) and PCR analysis performed as
described by Wenham (P. R. Wenham et al. (1991) Lancet
337:1158-1159).
[0121] 3. Total A.beta. ELISA
[0122] Total A.beta. was measured in a sequential double monoclonal
antibody sandwich ELISA as described in Seubert et al. (1992)
Nature 359:325-327. Briefly, A.beta. in CSF was captured by
monoclonal antibody 266 (specific for A.beta. peptide residues
13-28) which had been pre-coated in microtiter plate wells.
Detection utilized a second A.beta. specific, biotinylated
monoclonal antibody 6C6 (recognizing A.beta. residues 1-16),
followed by reaction with an alkaline phosphatase-avidin conjugate.
After incubation with the fluorogenic substrate
4-methyl-umbellipheryl phosphate (MUP), the fluorescent product was
measured using a Millipore Cytofluor 2350 fluorometer.
[0123] 4. A.beta.(x-.gtoreq.41) ELISA
[0124] A.beta.(x-.gtoreq.41) was measured in a similarly formatted
assay using 266 as the capture antibody. The reporter polyclonal
antibody 277-2 was raised against a synthetic peptide which
included A.beta. residues 33-42 (GLMVGGVVIA) [SEQ ID NO:2], with
cysteine-aminoheptanoic-acid at its amino-terminus. It was
conjugated through the cysteine to cationized BSA (Pierce). The
antibody 277-2 was affinity purified using the synthetic peptide
conjugated to Sulfo-link resin (Pierce) and reacted strongly with
.sup.125I-A.beta..sub.1-42 as detected by precipitation of tracer.
It showed no detectable cross-reactivity with A.beta..sub.1-40 in
either immunoprecipitation or ELISA formats, indicating at least a
1,000-fold less sensitivity towards the A.beta..sub.1-40 peptide.
Synthetic A.beta..sub.1-42 was used as the standard. Detection of
the 277-2 reporter antibody was achieved using a donkey anti-rabbit
IgG-alkaline phosphatase conjugate and the AMPPD chemiluminescent
substrate with Emerald enhancer (Tropix) (C. Vigo-Pelfrey et al.
(1994) J Neurochem 61:1965-1968).
[0125] To eliminate inter-assay variability as a factor in the
A.beta.(x-.gtoreq.41) analysis, all samples were run in duplicate
on the same day with the same lot of standards. The intra-assay
variability was less than 10%. Prior to measure, aliquots of CSF
samples were heated to 100.degree. C. for three minutes and then
stored at 4.degree. overnight before assay. The heating step was
found to generally increase immunoreactivity in CSF samples,
independent of diagnosis, and was therefore included. It should be
noted that different lots of synthetic A.beta.(x-.gtoreq.41)
generate slightly different standard values, despite being
normalized by amino acid analysis. Values listed are based upon a
single standard used for the entire study. Studies involving
addition of synthetic A.beta.3(x-.gtoreq.41) to CSF demonstrated
that measured recovery was 80.+-.5%.
[0126] 5. Detection of Tau by ELISA
[0127] a. Purified Tau
[0128] Tau purified from human AD brain tissue and from recombinant
sources were used for characterization of the assay and antibodies.
Recombinant human tau was produced using the previously described
baculovirus vector containing the pVL941-tau-4-repeat isoform (J.
Knops et al. (1991) J Cell Biol 1991:114:725-733). High levels of
tau were expressed and purified from both SF9 and high five insect
cells. Maximally expressing cell cultures were harvested, washed
once in PBS, and chilled on ice. The cells were then sonically
disrupted in 0.1 M MES pH 6.5, 1 mM EGTA, 18 .mu.M EDTA, 0.5 mM
MgCl.sub.2, 5 .mu.g/ml leupeptin, 1 mM PMSF. Cell debris was
removed by low speed centrifugation and the supernatant adjusted to
0.75 M NaCl, 2% .beta.-mercaptoethanol. The samples were boiled 10
minutes in capped tubes, cooled in ice and clarified by
centrifugation at 100,000.times.g for 30 minutes. The supernatants
were then adjusted to 2.5% perchloric acid and spun for 15 minutes
at 13,000.times.g. The pellets were subjected to a second cycle of
boiling/acid precipitation and the pooled supernatants were
dialyzed against 100 mM KH.sub.2PO.sub.4 pH 6.9, 2 mM EDTA, 2 mM
EGTA, 2 mM .beta.-mercaptoethanol, 0.3 mM PMSF.
[0129] The recombinant tau was judged to be at least 85% pure by
SDS-PAGE stained with Coomassie blue and was used without further
purification. The concentrations of all tau standards were
estimated by amino acid analysis. To dephosphorylate tau, an
aliquot was dialyzed into 20 mM Tris-HCl pH 8.6, 2 mM MgCl.sub.2, 1
mM DTT, 10 .mu.M ZnCl.sub.2 buffer. To half of the sample, 0.1
units of alkaline phosphatase (Boehringer Mannheim) per .mu.g-tau
were added; the other half was similarly diluted with buffer alone
and the two samples were incubated from 5 hours at 37.degree..
[0130] b. Monoclonal Antibodies Against Tau
[0131] Monoclonal antibodies were prepared according to a
modification of the method of Kohler and Milstein (G. Kohler and C.
Milstein (1975) Nature 256:495-497). Tau used in all injections and
screening assays was purified from SF9 cells infected with the
tau-containing baculovirus construct. Six week old A/J mice were
injected with 100 .mu.g of purified tau at two week intervals. Tau
was emulsified in complete Freund's adjuvant for the first
immunization and in incomplete Freund's adjuvant for all subsequent
immunizations. Serum samples were taken three days after the third
injection to assess the titer of these animals. The highest titer
mouse was injected intravenously with 100 .mu.g of tau in 500 .mu.L
of PBS two weeks after receiving its third injection. The myeloma
fusion occurred three days later using SP2/0 as the fusion partner.
Antibodies 16G7 and 8C11 were obtained from this fusion while
antibodies 16B5 and 16C5 were isolated from a subsequent
fusion.
[0132] Supernatants from wells containing hybridoma cells were
screened for their ability to precipitate .sup.125I-labeled tau.
Tau was radio-iodinated using immobilized glucose oxidase and
lactoperoxidase according to the manufacturer's instructions
(Bio-Rad). Briefly, 10 .mu.g of purified recombinant tau was
radiolabeled with 1 mCi of Na.sup.125I to a specific activity of 20
.mu.Ci/.mu.g protein. 16G7, 8C11, 16B5 and 16C5 were identified as
the four highest affinity monoclonal antibodies specific to tau and
were cloned by limiting dilution. The isotypes on all four
monoclonal antibodies specific to tau were determined to be gamma 1
kappa.
[0133] c. Tau ELISA
[0134] The anti-tau monoclonal antibody 16G7 was suspended at 5
.mu.g/ml in TBS and 100 .mu.l/well coated into microtiter plates
(Dynatec Microlite 2). The coating was carried out overnight at
room temperature. The solution was then aspirated and the plates
blocked with 0.25% casein (w/v) in phosphate buffered saline (PBS).
The anti-tau antibody 16B5 was biotinylated with the
N-hydroxysuccinimide ester of biotin following the manufacturer's
instructions (Pierce). Samples of either 50 .mu.l CSF or
calibrators (50 .mu.l of 3-1000 .mu.g/ml human tau), were combined
with 50 .mu.l of the biotinylated anti-tau antibody (0.75 .mu.g/ml
in PBS-casein, 0.05% Tween 20) into the 16G7 coated wells and
incubated overnight at room temperature with constant shaking. The
solution was then aspirated and plates washed three times in TTBS.
Streptavidin alkaline phosphatase (Boehringer-Mannheim) was diluted
1:1000 in PBS-casein, 0.05% Tween 20 and 100 .mu.l added to each
well. After incubation for 1 hour at room temperature, the fluid
was aspirated and wells washed three times. The chemiluminescent
reagent, disodium 3-(4-methoxyspiro
(1,2-dioxetane-3,2.sup.1-tricyclo [3.3.1.1.sup.3,7] tdecan)-4-yL)
phenyl phosphate (AMPPD, Tropix) and an enhancer Emerald green
(Tropix) were diluted 1:1000 and 1:100 respectively in 1 M
diethanolamine buffer, containing 1 mM MgCl.sub.2, 0.02% NaN.sub.3,
pH 10. 100 .mu.l were added per well and the plates were read after
30 min. in a Dynatech ML 1000 chemiluminometer. The data reported
here used human tau isolated from brain as the calibrator.
[0135] 6. Statistical Analysis
[0136] Statistical analysis of data was performed by one way
analysis of variance (ANOVA) using InStat, Version 1.21.
[0137] Results
[0138] Comparison of the three patient groups (Table III) showed
that they were well matched for age and gender. The AD group had an
average MMSE of 17.5.+-.7.1 indicating mild to moderate cognitive
impairment. The neurological disease control group consisted of a
variety of disorders including vascular dementia (4), frontal lobe
dementia (7), depression (6), Parkinson's disease (3),
cortico-basal ganglionic degeneration (2), cerebellar ataxia (2),
progressive supranuclear palsy (1), normal pressure hydrocephalus
(1), grand mal seizure (1), Bell's palsy (1), age-associated memory
impairment (1), dementia with extrapyramidal signs (1), amnestic
syndrome (1), cerebellar degeneration (1). The control group
consisted of individuals who were free of neurological disease and
were cognitively normal (Table III).
4TABLE III SUMMARY OF PATIENT PROFILES AND MEASURED PARAMETERS
Alzheimer's Neurological Normal Disease Controls Controls (AD) (ND)
(NC) n 37 32 20 Age (mean .+-. SD) 70 .+-. 9.1 66 .+-. 9.1 70 .+-.
6.2 Sex (M %/F %) 48.6/51.4 59.4/40.6 50/50 MMSE (mean .+-. SD)
17.5 .+-. 7.1 23 .+-. 8.2 29.5 .+-. 0.6 CSF A.beta. (mean .+-. SD,
19.0 .+-. 6.9 17.9 .+-. 6.7 21.8 .+-. 6.9 ng/ml) APOE.epsilon.4
frequency.sup.1 0.58 0.26 0.21 A.beta..sub.42 (mean .+-. SD, 383
.+-. 76** 543 .+-. 177 632 .+-. 156 pg/ml) Tau (mean .+-. SD, 407
.+-. 241* 168 .+-. 63 212 .+-. 102 pg/ml) .sup.1ApoE genotypes were
determined on 30/37 AD, 19/32 neurological control and 17/20 normal
controls. **p < .0001 comparing AD group to either control
group. *p < .001 comparing AD group to either control group.
[0139] Analysis of total CSF A.beta. levels revealed no significant
differences among the different patient groups (Table III). The
mean values ranged from 19.0 ng/ml in the AD group to 17.9 ng/ml in
the NC group. There was significant overlap with no statistically
significant differences among the groups (p>0.05). Analysis of
the A.beta.(x-.gtoreq.41) form of the peptide, however,
demonstrated a reduction in the mean value in the AD group,
relative to both the ND and NC subjects (383 versus 543 and 632
pg/ml respectively) that was significant at the p<0.0001 level
(FIG. 4). The relatively small standard deviation (76 pg/ml) of the
AD group was particularly striking. Conversely, some of the ND
patients exhibited reduced A.beta.(x-.gtoreq.41) in their CSF. When
a cutoff was set at 505 pg/ml, 15 of 37 ND patients and only four
of 23 NC fell below this level. Alternatively, of the 35
individuals that have levels of A.beta.(x-.gtoreq.41) greater than
505 pg/ml, none was diagnosed with AD, suggesting the test is
highly specific for the absence of disease. AB(x-.gtoreq.41) was
measured as described in the text. All measures are the averages of
duplicate determinations, variation was .ltoreq.10%. Samples were
assigned randomly to plates and the operator was unaware of the
subject diagnoses. Reference standards, present on each microtiter
plate, were not significantly different between plates.
[0140] Tau levels in the same subjects' CSF samples were also
examined. Tau measurements were performed in duplicate. To ensure
consistency, several samples from previous assays were included on
subsequent plates and all samples were evaluated in at least
replicate measure. Replicate measures were within 15% of original
values. A significant difference exists between the AD group and
either control group (p<0.001). Human brain-derived tau was used
as the reference standard. AD patients had a mean value of 407
pg/ml versus 168 and 212 pg/ml in neurological and normal controls,
respectively (FIG. 5). This difference between the AD group and the
other groups is significant at p<0.001. Employing a cutoff of
312 pg/ml, individuals with values above this level had a very high
likelihood of Alzheimer's disease (22/24=92%). Only one NC and one
ND subject registered above this cutoff. Separate analysis of
average CSF A.beta., A.beta.(x-.gtoreq.41) or tau levels obtained
from each center did not reveal differences between centers that
were statistically significant for any of the disease categories as
revealed by one-way analysis of variance. Of particular interest
was the simultaneous analysis of A.beta.(x-.gtoreq.41) and tau
measurements in the same CSF samples (FIG. 6). FIG. 6 is divided
into four quadrants using the cutoffs for A.beta.(x-.gtoreq.41) and
tau previously described. The presence of both elevated tau and
reduced A.beta.(x-.gtoreq.41) (lower-right quadrant) was highly
predictive of AD (22/23=96%). Conversely, high
A.beta.(x-.gtoreq.41) and low tau (upper-left quadrant) was
represented entirely by control patients (FIG. 6). More than half
(58.7%) of all the individuals in this study fell into one of these
two quadrants. The remaining patients exhibited low
A.beta.(x-.gtoreq.41) and low tau levels (lower left quadrant).
[0141] Although the foregoing invention has been described in
detail for purposes of clarity of understanding, it will be obvious
that certain modifications may be practiced within the scope of the
appended claims.
[0142] 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.
Sequence CWU 1
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