WO2008031190A1

WO2008031190A1 - Alpha-1-antitrypsin as a diagnostic/prognostic indicator for neurodegenerative diseases

Info

Publication number: WO2008031190A1
Application number: PCT/CA2007/001477
Authority: WO
Inventors: Hyman Schipper; Ajay Gupta
Original assignee: Osta Biotechnologies Inc.
Priority date: 2006-09-15
Filing date: 2007-08-27
Publication date: 2008-03-20

Abstract

This invention provides a method useful in aiding in the diagnosis/prognosis of a neurodegenerative disease, such as Alzheimer's Disease (AD), Mild Cognitive Impairment (MCI) and Parkinson's disease (PD) using alpha-1-antitrypsin (AAT) as a biological indicator. The method involves detecting the amount of AAT in the biological sample of a subject and correlating the amount detected to a disease state relative to a normal control. An amount of AAT that is elevated or reduced relative to a normal control represents a positive and more direct test for probable diagnosis in a subject suffering from a neurodegenerative disease. Diagnostic kits are also included in the invention.

Description

ALPHA-I-ANTITRYPSIN AS A DIAGNOSTIC/PROGNOSTIC INDICATOR FOR NEURODEGENERATIVE DISEASES

FIELD OF THE INVENTION

The present invention relates to a method for predicting, diagnosing and prognosticating a neurodegenerative disease, such as Alzheimer's disease (AD), Mild Cognitive Impairment (MCI) and Parkinson's disease (PD) using alpha-1-antitrypsin (AAT) as a diagnostic/prognostic indicator.

BACKGROUND OF THE INVENTION

Alzheimer Disease (AD) is a neurodegenerative disease that causes dementia. The terms "Alzheimer Disease" and "Alzheimer's Disease" are both utilized in the art, these terms being equivalent and are used interchangeably here and elsewhere. The period from first detection of AD to termination can range from a few years to 15 years, during which time the patient progressively suffers loss of both mental function and control of bodily functions. There is significant variability in the progress of the disease. While the majority of patients have a gradual, inexorable progression (losing on average 3 to 4 points on the 30 point Folstein mini-mental state score annually) , approximately 30% of AD cases have a prolonged stable initial plateau phase lasting several years (Haxby J. V. , et al . , Individual trajectories of cognitive decline in patients with dementia of the Alzheimer type, J. Clin. Exp. Neuropsychol 14:575-592, 1992.). A subgroup of patients has a fulminant, rapidly progressive downhill course over several years

(Mann, U., et al . , Heterogeneity in Alzheimer ' s disease: Progression rate segregated by distinct neuropsychological and cerebral metabolic profiles, J. Neurol. Neurosurg. Psychiatry 55:956-959, 1992). Other patients (about 10% of cohorts) remain slowly progressive, showing only gradual decline from year to year (Grossi, D., et al . , Senile dementias, II International Symposium (pp. 97-99), Paris: John Libbey Eurotext, 1988.). The pathological, chemical and molecular bases of this heterogeneity remain undetermined. Recognition of the variability of AD progression represents an important clinical insight, and may explain the diagnostic difficulties presented by "atypical" cases. While in certain cases, there is a familial manifestation of the AD disease, it appears that the majority of AD cases are non-familial, and until recently (see below) , no simple biological marker for the disease had been determined.

Current methods used to diagnose AD involve analysis of cerebrospinal fluid (CSF) or brain tissue obtained from postmortem patients. Thus, among the markers currently under consideration are those related to the proteins, which account for the features found in Alzheimer brains postmortem. The neurofibrillary tangle is composed primarily of a hyperphosphorylated tau protein, a cytoskeletal protein. The neuritic plaque contains a core of amyloid protein, much of which is a 42 -amino acid peptide (Aβ₄₂) derived from proteolytic cleavage of a larger precursor protein. Another form of this protein derived from the same precursor contains only 40 amino acids (Aβ₄₀) . Deposits of this protein are found in the brains of AD victims. However, alterations in tau and the beta amyloid peptides do not occur with sufficient frequency and magnitude so as to afford diagnostic value and therefore, blood tests based on these proteins do not seem to correlate well with AD. At present, there appears to be no satisfactory- diagnostic marker for existing AD, or for a subject, who although exhibiting normal cognitive responses, will inevitably, or most likely, develop AD.

Age-Associated Cognitive Decline (AACD) and Mild

Cognitive Impairment (MCI) are terms used to identify individuals who experience a cognitive decline that falls short of dementia. These terms are equivalent, MCI being a more recently adopted term, and are used interchangeably throughout this application. Satisfaction of criteria

(World Health Organization) for this diagnosis requires a report by the individual or family of a decline in cognitive function, which is gradual, and present at least 6 months. There may be difficulties across any cognitive domains (although memory is impaired in the vast majority of cases) , and these must be supported by abnormal performance on quantitative cognitive assessments for which age and education norms are available for relatively healthy individuals (i.e., the patient is compared to normal subjects his/her own age) . Performance must be at least

1 SD below the mean value for the appropriate population on such tests. Neither dementia, nor significant depression or drug effects may be present . No cerebral or systemic disease or condition known to cause cerebral cognitive dysfunction may be present. In Applicant's experience, all patients who were classified as CDR.5 ("questionable dementia") on the Clinical Dementia rating scale and who met these exclusions, also met the criteria for AACD/MCI . About 1/3 of Alzheimer's patients have had a clearly definable period of isolated memory deficit which preceded their more global cognitive decline. (Haxby J. V., et al . , Individual trajectories of cognitive decline in patients with dementia of the Alzheimer type, J. Clin. Exp. Neuropsychology 14:575-592, 1992.) Using AACD/MCI criteria, which look at other domains in addition to memory, the percentage with an identifiable prodrome is likely higher. Fortunately, not all AACD/MCI individuals seem to decline. It appears that a significant number of these subjects show a stable, non-progressive memory deficit on testing.

Idiopathic Parkinson disease (PD) is a common neurodegenerative disorder that affects more than 2% of the population over 65 years of age. PD is characterized pathologically by progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta, formation of intraneuronal fibrillar inclusions (Lewy bodies) in this cell population and variable depletion of noradrenergic neurons in the locus coeruleus and serotoninergic cells in the median raphe. Resting tremor, rigidity, hypokinesia and postural instability are the cardinal symptoms of this extrapyramidal disease. Many PD patients exhibit dementia in later stages of the illness, but the degree to which this represents spread of Lewy body pathology beyond the brain stem vs. intervening AD-type neuropatholological changes remains unclear. A growing body of evidence implicates oxidative stress and oxidatively-modified proteins (e.g. alpha-synuclein, DJ-I) in the pathogenesis of PD. Although pharmacotherapy, including the administration of L-DOPA, dopamine receptor agonists and anticholinergics, is useful in ameliorating symptoms of PD, there currently exists no treatment that unequivocally attenuates neuronal attrition and clinical decline in this condition. (Jenner, P., Oxidative stress in Parkinson' s disease. Ann Neurol, 2003. 53 Suppl 3: p. S26-36; discussion S36-8.)

The diagnosis of PD is rendered purely on clinical grounds. Resting tremor, rigidity, hypokinesia and postural instability are the cardinal symptoms of this extrapyramidal disease. Autonomic insufficiency, sleep disorders, and neuropsychological symptoms may complete the clinical pictures. There currently exist no known chemical or other biological markers that may assist in the diagnosis of this condition.

Attempts at predicting the onset of AD, MCI or PD, or monitoring their progression have met with limited success. It has been discovered by the inventors of this application that an amount of AAT in a biological sample obtained from a subject that deviates from a reference amount in a control person can be positively correlated to a neurological disease state. Thus, the correlation of the presence of AAT with the disease state represents a positive and more direct test for diagnosis in a patient suffering from one of the neurodegenerative diseases described above.

Accordingly, the invention provides an easily administered biological sample test for predicting, diagnosing, or prognosticating AD, MCI and PD using AAT as a diagnostic marker.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that an amount of alpha- 1 -antitrypsin (AAT) in a biological sample obtained from a subject suffering from AD or MCI is elevated compared to an amount of AAT in the biological sample obtained from a normal (i.e. healthy) control subject .

Alternatively, an amount of AAT in a biological sample obtained from a subject suffering from PD is reduced compared to an amount of AAT in the biological sample obtained from a normal control subject. The indication that the amount of AAT differs between these neurological diseases and normal controls, forms the basis for the development of a test for diagnosing AD, MCI or PD in a subject. As such, the methods for diagnosing AD, MCI or PD of the present invention by measuring the amount of AAT in patient sample will greatly improve current clinical diagnostic assessment for patients suffering from these neurodegenerative diseases.

Based on newly discovered differences in the amount of AAT present in a biological sample obtained from a patient compared to that of a normal control, a strong correlation of the amount of AAT can be made to a probable diagnosis of a neurodegenerative disease. A statistically significant elevation in the amount of AAT relative to control samples is reasonably predictive that the patient does have AD or MCI whereas a statistically significant reduction in the amount of AAT relative to control samples is reasonably predictive that the patient does have PD. A normal amount of AAT as determined by an amount of AAT characteristic of a control AAT sample isolated from a normal age-matched population indicates that the patient does not have a neurodegenerative diseases, such as AD, MCI or PD. A positive indication of a neurodegenerative disease based on an elevated or reduced amount of AAT in a biological sample relative to a normal control is generally considered together with other factors in making a definitive determination of a particular disease. Therefore, the elevated or reduced AAT levels of the subject being tested will usually be considered together with other accepted clinical symptoms of AD, MCI or PD-related conditions in making a determinative diagnosis of a neurodegenerative disease. Thus, according to a first aspect of the invention, there is provided a method for diagnosing probable Alzheimer's Disease (AD) or Mild Cognitive Impairment (MCI) in a subject, the method comprising:

(a) detecting the amount of alpha-1-antitrypsin

(AAT) , or its isoforms, in a biological sample obtained from said subject; and

(b) comparing the detected amount of AAT in the biological sample with an amount of AAT characteristic of a normal control;

whereby an elevated amount of AAT in said biological sample relative to the normal control is a positive indicator of AD or MCI .

According to another aspect of the invention, there is provided a method for diagnosing probable Parkinson's disease (PD) in a subject, the method comprising :

(a) detecting the amount of alpha-1-antitrypsin (AAT) , or its isoforms, in a biological sample obtained from said subject; and

whereby a reduced amount of AAT in said biological sample relative to the normal control is a positive indicator of PD.

According to a preferred embodiment of the invention, the biological sample is a fluid body sample such as serum, plasma, urine or cerebrospinal fluid. More preferably, the fluid body sample is plasma.

The amount of AAT detected or quantified in a biological sample from a subject can be accomplished by any means known in the art. Such means may include, but are not limited to, for example by immunoturbidimetric assay, immunofluorescence, immunodiffusion, enzyme-linked immunosorbent assay (ELISA) , radioimmunoassay (RIA) , Western Blot or protein activity assay. Also useful are high performance liquid chromatography (HPLC) , mass spectrometry (MS) and gas chromatography (GC) , as well as their various configurations, including gas chromatograph-mass spectrometry (GC-MS) , liquid chromatography-mass spectrometry (LC-MS) and liquid-chromatography-tandem mass spectrometry (LC-MS/MS) systems.

Preferably, the amount of AAT in the biological sample is detected using an antibody that binds to AAT in an immunoassay format. Thus, according to a preferred embodiment of the invention, there is provided a method of diagnosing a neurodegenerative disease in a subject, the method comprising:

(a) obtaining a biological sample from said subject ;

(b) contacting said biological sample with an antibody that binds to alpha-1-antitrypsin (AAT) , or its isoforms ;

(c) allowing the antibody and AAT to form an immune complex; and (d) detecting the amount of immune complex formed as an indication of the amount of AAT in said biological sample; and

(e) comparing the detected amount to a normal control;

whereby a detected amount that is elevated or reduced relative to the normal control is a positive indicator of a neurodegenerative disease.

According to yet a further aspect of the invention, there is provided a diagnostic kit for determining whether a subject is suffering from a neurodegenerative disease comprising an antibody that binds to AAT and an established standard of an amount of AAT characteristic of a normal control. Reagents and instructions for carrying out the assays may also be included.

DESCRIPTION OF THE EMBODIMENTS OF THE INVENTION

The present invention provides an efficient and rapid in vitro method for diagnosing a neurodegenerative disease by directly detecting an amount of AAT in a biological sample obtained from a subject and comparing the detected amount of AAT with an amount of AAT characteristic of a normal control . An elevated amount of AAT in the biological sample of the subject is a positive indication of AD or MCI whereas a reduced amount of AAT in the biological sample of the subject is a positive indication of PD. Thus, as described herein, it is demonstrated that AAT is consistently and significantly elevated in a biological sample of AD or MCI patients compared to normal controls, whereas AAT is consistently and significantly reduced in a biological sample of PD patients compared to normal controls. As such, the methods for diagnosing AD, MCI or PD of the present invention by detecting or quantifying the amount of AAT in a patient sample will greatly improve current clinical diagnostic assessment for patients suffering from these neurodegenerative diseases.

Accordingly, there is provided a method for assessing whether a subject may be suffering from AD, MCI or PD using AAT as a biological marker. AAT is a 51 kDa acute phase reactant with serine proteinase inhibitor (serpin) and anti-inflammatory activity (Churg A, Dai J, Zay K, Karsan A, Hendricks R, Yee C, Martin R, MacKenzie R, Xie C, Zhang L, Shapiro S, and Wright JL, Alpha-1 -antitrypsin and a broad spectrum metalloprotease inhibitor, RS113456, have similar acute anti -inflammatory effects, Lab Invest 81: 1119-1131, 2001) . Within the affected brain tissue, AAT predominantly localizes to astrocytes, senile plaques and neurofibrillary tangles (Gollin PA, Kalaria RN, Eikelenboom P, Rozemuller A, and Perry G., Alpha 1-antitrypsin and alpha 1-antichymotrypsin are in the lesions of Alzheimer ' s disease, Neuroreport 3: 201-203, 1992). It has been suggested that AAT and related serpins of systemic or local (glial) origin may impact the natural history of AD by suppressing β-amyloid fibrillogenesis (Bohrmann B, Tjernberg L, Kuner P, PoIi S, Levet-Trafit B, Naslund J, Richards G, Huber W, Dobeli H, and Nordstedt C, Endogenous proteins controlling amyloid beta-peptide polymerization . Possible implications for beta-amyloid formation in the central nervous system and in peripheral tissues, J Biol Chem 274 : 15990-15995, 1999), altering clearance of β-amyloid deposits within senile plaques (Abraham CR. , Reactive astrocytes and alphal-antichymotrypsin in Alzheimer ' s disease. Neurobiol Aging, 22: 931-936, 2001), and serving as broad-spectrum inhibitors of AD-associated neuroinflammation (Sun YX, Minthon L, Wallmark A, Warkentin S, Blennow K, and Janciauskiene S., Inflammatory markers in matched plasma and cerebrospinal fluid from patients with Alzheimer ' s disease, Dement Geriatr Cogn Disord 16: 136-144, 2003).

Isoforms of AAT which result from oxidation, peptide cleavage, polymerization, and complexation with proteases or other ligands have been observed (Hopkins, P. C, Stone, S. R., The contribution of the conserved hinge region residues of alpha-1 -antitrypsin to its reaction with elastase, Biochemistry, VoI 34, pp .15872-15879, 1995;

Vaughan L., Lorier M.A. , Carrell R. W. , Alpha-1 -antitrypsin microheterogeneity. Isolation and physiological significance of isoforms, Biochim Biophys Acta 701(3), pp.339-45, 1982). Multiple isoforms of a protein, such as AAT, can also be produced from a single gene by a variety of mechanisms, including alternative RNA splicing, post- translational proteolytic processing and cell type-specific glycosylation. Thus, the terms "alpha-1-antitrypsin" and "AAT" as used herein refer to AAT in its native form, as well as any of its isoforms.

Thus, according to a first aspect of the present invention, there is provided a method for diagnosing probable Alzheimer's Disease (AD) or Mild Cognitive Impairment (MCI) in a subject, the method comprising:

(a) detecting the amount of alpha-1-antitrypsin

(AAT), or its isoforms, in a biological sample obtained from said subject; and

(b) comparing the detected amount of AAT in the biological sample with an amount of AAT characteristic of a normal control; whereby an elevated amount of AAT in said biological sample relative to the normal control is a positive indicator of AD or MCI .

According to another aspect of the present invention, there is provided a method for diagnosing probable PD in a subject, the method comprising:

(a) detecting the amount of alpha- 1-antitrypsin (AAT), or its isoforms, in a biological sample obtained from said subject; and

The term "subject" refers to a mammal which is afflicted with, or suspected to be afflicted with a neurogenerative disease such as AD, MCI or PD. Preferably, "subject" refers to a human.

The term "biological sample" refers to any source of biological material, including, but are not limited to, peripheral blood, plasma, lymphocytes, cerebrospinal fluid, urine, saliva, epithelia, fibroblasts, or any other sample comprising AAT protein. In a preferred embodiment, the amount of AAT is detected in a body fluid sample obtained from a mammal, most preferably a human. The term "body fluid" refers to all fluids that are present in the human body including but not limited to blood, lymph, urine and cerebrospinal fluid (CSF) comprising AAT. The blood sample may include a plasma sample or a serum sample, or fractions derived from these samples. The sample can be treated prior to use, such as preparing plasma from blood, diluting viscous fluids, and the like. Preferably, the plasma sample is treated with an anti -coagulant , such as EDTA.

According to a preferred embodiment of the present invention, the amount of AAT is detected in a blood sample taken from the subject, more preferably a plasma sample. Thus, the present invention preferably relates to a method as described above, comprising the steps of: obtaining a plasma sample from said subject; detecting the amount of AAT in the plasma sample; comparing the detected amount of AAT in the plasma sample with the amount of AAT in a plasma sample from a normal control, whereby an elevated amount of AAT relative to the normal control is a positive indication of AD or MCI and a reduced amount of AAT relative to the normal control is a positive indication of PD.

Elevated amounts of AAT have been shown to correlate with and are useful in aiding the diagnosis of AD and MCI, whereas reduced amounts of AAT have been shown to correlate with and are useful in aiding the diagnosis of PD.

An "elevated amount" of AAT means that the amount of AAT detected in the subject is greater than the mean amount of AAT characteristic of a normal control beyond the range of experimental error, as known in the art. A "reduced amount" of AAT means that the amount of AAT detected in the subject is less than the mean amount of AAT characteristic of a normal control beyond the range of experimental error.

A "normal control" is a biological sample of the same type obtained from the subject, for example, that is obtained from at least one normal age-matched control person or from the patient at another time. In an embodiment, the normal control is from the patient at an earlier time. A normal control sample from a normal age-matched population should be isolated from an adequate population sample of healthy age matched controls with no history of AD, MCI or PD in their family. By way of example, a plasma AAT level higher than the control levels of AAT, as determined by an adequate control population sample size, is indicative of AD or MCI. In contrast, a plasma AAT level lower than the control levels of AAT is indicative of PD. One of skill in the art will appreciate that the sample from the subject to be diagnosed is assessed against a normal age-matched control and that a significant elevation or reduction in the amount of AAT in the subject's protein sample is determined based on comparison to the controls used in the given assay.

The amount of AAT detected or quantified in a subject's biological sample can be accomplished by any means known in the art. Such means may include, but are not limited to, for example by immunoturbidimetric assay, immunofluorescence, immunodiffusion, enzyme-linked immunosorbent assay (ELISA) , radioimmunoassay (RIA) , Western blot and protein activity assay. Also useful are high performance liquid chromatography (HPLC) , mass spectrometry (MS) and gas chromatography (GC) , as well as their various configurations, including gas chromatograph-mass spectrometry (GC-MS) , liquid chromatography-mass spectrometry (LC-MS) and liquid-chromatography-tandem mass spectrometry (LC-MS/MS) systems, to name a few.

While detection of AAT can be accomplished by methods known in the art for detecting peptides, the use of immmunological detection techniques using antibodies, antibody fragments, recombinant antibodies, and the like, is preferred. Therefore, such detection of AAT includes, but is not limited to, the use of antibodies, which specifically bind to AAT, and its isoforms, to form an immune complex, as well as reagents for detecting the formation of the immune complex. Particularly suitable detection techniques employing one or more antibodies include immunoturbidimetric assay, immunofluorescence, immunodiffusion, ELISA, RIA and the like.

Such antibodies may be polyclonal or monoclonal . Methods to produce polyclonal or monoclonal antibodies are well known in the art. For a review, see Harlow and Lane (Harlow, E. and Lane, D., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988) and Yelton et al . (Yelton D. E. and Scharff M. D. Monoclonal Antibodies: a powerful new tool in biology and medicine. Ann. Rev. Biochem. 50:657-680, 1981), both of which are herein incorporated by reference. For monoclonal antibodies, see Kohler and Milstein (Kohler G. and Milstein C, Continuous cultures of fused cells secreting antibody of predefined specificity, Nature 256:495-497, 1975), herein incorporated by reference. The antibodies of the invention are of any isotype, e.g., IgG or IgA, and polyclonal antibodies are of a single isotype or a mixture of isotypes.

According to a preferred embodiment of the invention, the anti-AAT antibody is a monoclonal antibody. Although anti-AAT antibodies are widely commercially available, antibodies for use in the various immunoassays described herein, can be produced according to standard methods (see, for example, U.S. Patent 5,114,863 to

McCombs et al . entitled "Immunosorbant assay for α-1- antitrypsin, kit employing said assay, monoclonal antibody to α-1-antitrypsin, and hybridoma for producing said monoclonal antibody"). Further, the monoclonal anti-AAT antibody is capable of recognizing AAT in its native form, as well as any of its isoforms. Thus, any monoclonal antibody that specifically recognizes AAT, including its isoforms, can be used in said method for the quantification of AAT. Also fragments derived from these monoclonal antibodies such as Fab, F(ab)_2/ ssFv (single chain variable fragment) and other antibody-like constructs that retain the variable region of the antibody, providing they have retained the original binding properties, can be used in a method of the present invention. Such fragments are commonly generated by, for instance, enzymatic digestion of the antibodies with papain, pepsin, or other proteases. It is well known to the person skilled in the art that monoclonal antibodies, or fragments thereof, can be modified for various uses. Thus, antibodies of the invention, may be recombinant, e.g., chimeric {e.g. , constituted by a variable region of murine origin associated with a human constant region) , humanized (a human immunoglobulin constant backbone together with hypervariable region of animal, e.g., murine, origin), and/or single chain.

An antibody specific for AAT, or its isoforms, used in a method of the present invention may be labelled by an appropriate label and identified in the biological sample based upon the presence of the label. The label allows for the detection of the antibody when it is bound to AAT. Examples of labels include, but are not limited to, the following: radioisotopes (e.g., ³ H, ¹⁴ C, ³⁵ S, ¹²⁵ I, ¹³¹I), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), luminescent labels, enzymatic labels (e.g., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescent , and biotinyl groups . Methods for conjugating or labelling the antibodies discussed above may be readily accomplished by one of ordinary skill in the art (see for example Inman, "Methods In Enzymology" , Vol. 34, Affinity Techniques, Enzyme Purification: Part B, Jakoby and Wichek (eds.), Academic Press, New York, p. 30, 1974; and Wilchek and Bayer, "The Avidin-Biotin Complex in Bioanalytical Applications," Anal. Biochem. 171:1-32, 1988).

For diagnostic applications, the anti-AAT antibody is either in a free state or immobilized on a solid support, such as a tube, a bead, or any other conventional support used in the field. Immobilization is achieved using direct or indirect means. "Direct means" include passive adsorption (non-covalent binding) or covalent binding between the support and the reagent. By "indirect means" is meant that an anti-reagent compound that interacts with a reagent is first attached to the solid support. Indirect means may also employ a ligand-receptor system, for example, where a molecule such as a vitamin is grafted onto the reagent and the corresponding receptor immobilized on the solid phase. This is illustrated by the biotin-streptavidin system.

Those skilled in the art will readily understand that an immune complex is formed between AAT in the biological sample and the antibody, and that any unbound material is removed prior to detecting the complex. It is understood that an antibody of the invention is used for quantifying an amount of AAT in the biological sample, such as, for example, blood, plasma, lymphocytes, cerebrospinal fluid, urine, saliva, epithelia and fibroblasts.

As is known in the art, the determination of such antibody binding can be performed using a great variety of immunoassay formats including, but not limited to immunoturbidimetric assay (agglutination) , enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA) (see, for example, "Principles and Practice of Immunoassay" (1991) Christopher P. Price and David J. Neoman (eds) , Stockton Press, New York, N. Y. and Ausubel et al . (eds) (1987) in "Current Protocols in Molecular Biology" John Wiley and Sons, New York, N. Y., both of which are incorporated herein by reference) . Detection may be by colormetic or radioactive methods or any other conventional methods known to one skill in the art. Other standard techniques known in the art are described in "Methods in Immunodiagnosis" , 2nd Edition, Rose and Bigazzi, eds., John Wiley and Sons, New York 1980 and Campbell et al . ; "Methods of Immunology", W. A. Benjamin, Inc., 1964; U.S. Patent Nos . 4,366,241; 4,376,110; 4,517,288; and 4,837,168, the disclosures of which are incorporated herein by reference. For a review of the general immunoassays, see also "Methods In Cell Biology", Vol. 37, Asai, ed. Academic Press, Inc. New York (1993) ; "Basic And Clinical Immunology" 7^th Edition, Stites & Terr, eds. (1991) .

Such assays for detecting AAT may be a direct, indirect, competitive, or noncompetitive immunoassay as described in the art (see, for example, "Principles and Practice of Immunoassay" (1991) Christopher P. Price and David J. Neoman (eds), Stockton Press, New York, N.Y.; Ausubel et al . (eds) (1987) in "Current Protocols in Molecular Biology" John Wiley and Sons, New York, N. Y.; and Oellirich, M. 1984. J. Clin. Chem. Clin. Biochem. 22: 895-904, incorporated herein by reference) .

Noncompetitive immunoassays are assays in which the amount of AAT is directly detected. In the "sandwich" assay, for example, the anti -AAT antibodies can be bound directly to a solid substrate where they are immobilized. These immobilized antibodies then capture the AAT present in the biological sample. The AAT thus immobilized is then bound by a labeling agent, such as a second human AAT antibody bearing a label.

In a competitive immunoassay, the amount of antigen present in the biological sample is determined indirectly following addition of a known amount of labeled antigen to the sample and detecting the amount of labeled antigen bound with antibodies. For example, a known amount of, in this case, labeled AAT is added to the biological sample and the sample is then contacted with anti-AAT antibodies. The amount of labeled AAT bound to the anti-AAT antibody is inversely proportional to the concentration of AAT in the biological sample. This is because the greater the amount of labeled AAT detected, the less the amount of AAT was available in the biological sample to compete with the labeled AAT.

Diagnostic kits for carrying out the assays for diagnosing AD, MCI or PD in a subject are also provided. Thus, the present invention can be practiced using a diagnostic kit that includes at least one antibody specific for AAT, and its isoforms, as described herein as well as any reagents necessary for the detection of antibody-AAT binding immune complexes. Generally, the kit may include a single antibody that specifically recognizes AAT, and its isoforms. On the other hand, the kit may include a primary antibody that specifically recognizes AAT, and its isoforms, as well as a secondary antibody that is conjugated with a signal -producing label and is capable of binding to the primary antibody, or at a site different from the site where the primary antibody binds. The signal -producing label linked to the secondary antibody may be, but is not limited to, an enzyme, such as horseradish peroxidase or alkaline phosphatase. The kits may further comprise other reagents for carrying out the assay such as buffers, a solid support, solutions and the like. The kit may also contain instructions for carrying out the method of the invention using one or more antibodies in diagnostic assays.

The present invention is further described by the following example. The example is provided solely to illustrate the invention by reference to specific embodiments. These examples, while illustrating certain specific aspects of the invention, do not portray the limitations or circumscribe the scope of the disclosed invention. Indeed, many modifications and variations of the invention will be apparent to those skilled in the art upon reading this specification and can be made without departing from its spirit and scope.

Example 1: Determination of the presence of AAT in plasma derived from NEC, MCI, AD and PD patients.

Materials and Methods

Subjects. This study was approved by the Research and

Ethics Committee of the Sir Mortimer B. Davis Jewish General Hospital (JGH) . Written informed consent was obtained from all patients or their primary caregivers. Patients meeting the National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer's Disease and Related

Disorders Association (NINCDS-ADRDA) criteria for probable sporadic AD and amnestic Mild Cognitive Impairment (MCI) were recruited from the JGH/McGill University Memory Clinic, a tertiary care facility for the evaluation of memory loss in Montreal. MCI individuals exhibited a score of 0.5 on the Clinical Dementia scale (Levy, R., Aging-associated cognitive decline, International Psychogeriatrics, 6:63-8, 1994; Hughes, CP, Berg, L, Danziger, WL, Coben, LA and Martin, RL, A new scale for the staging of dementia, British Journal of Psychiatry, 140:566-572, 1982). All AD and MCI patients underwent comprehensive neuropsychological testing by Memory Clinic neuropsychologists as previously described [Schipper HM, Chertkow H, Mehindate K, Frankel D, Melmed C, and Bergman H., Evaluation of heme oxygenase-1 as a systemic biological marker of sporadic AD, Neurology 54: 1297-1304, 2000] . Patients with various non-Alzheimer dementias (NAD) , idiopathic Parkinson disease (PD) and other non- neurodegenerative illnesses, were recruited from Neurology, Psychiatry and Internal Medicine Clinics at the JGH. Normal young controls less than 60 years of age (NYC) and normal elderly controls aged 60 years and over (NEC) were recruited from JGH Family Practice clinics. Normal subjects had no memory complaints and scored within one standard deviation of age- and education-standardised normal values on a series of memory and attention tests (CDR score 0.00) . Patients with PD were diagnosed by a general neurologist or Movement Disorders sub-specialist based on standard clinical criteria and staged using the modified Hoehn and Yahr rating scale [Hoehn HM, Yahr MD. Parkinsonism: onset, progression and mortality. Neurology 17:427-442, 1967].

Materials. All determinations were performed using standard kits for quantitative immunological determination of human AAT in serum and plasma (COBAS INTEGRA Tina-quant D αl-Antitrypsin ver.2 cassette for use on COBAS INTEGRA systems, Catl . # 03005771, System-ID 07 9008 7, Roche Diagnostics GmbH) . The AAT kit is developed using an anti- human AAT antibody (rabbit) for capturing and detecting AAT.

Plasma samples. Whole blood was collected by phlebotomy in EDTA tubes (BD Biosciences, San Jose, CA, USA) between 09:00-11 : 00h, layered over Ficoll Paque™(Amersham Biosciences, Baie d'Urfe, Canada) density gradient and centrifuged at 1800 rpm for 20 minutes. The top plasma layers were collected.

Statistical Methods. ATT levels were compared amongst various groups by Pearson Chi Square method with p<0.05 indicating significance. The closeness of the observed value to the expected value under the null hypothesis of no difference amongst various groups was evaluated using the Pearson chi square method.

Results. In a study involving a total of 81 subjects with 16 normal elderly controls (NEC), 26 MCI, 20 AD and 19 PD subjects, a majority of NEC, MCI and AD subjects were found to have plasma concentration of AAT higher than 1.23 mg/mL, whereas, majority of subjects with PD were found to have plasma concentration of AAT less than 1.23 mg/mL. Moreover, the plasma AAT concentrations of MCI subjects were found to be similar to those with AD. The mean plasma AAT concentration of PD subjects was found to be 1.18 + 0.19 mg/mL and was lower than the mean plasma AAT concentration of normal elderly controls who had a mean value of 1.33 +■ 0.12 mg/mL. The mean plasma AAT concentration of MCI subjects was found to be 1.44 +■ 0.30 mg/mL and that of the AD subjects was found to be 1.48 +_ 0.26 mg/mL which were both higher than those in PD and normal elderly control subjects. Table 1. Alpha-1-antitrypsin plasma (EDTA) levels in 81 subjects .

NEC = Normal Elderly Control MCI = Mild Cognitive Impairment

AD = Alzheimer's disease

PD = Parkinson's disease

Claims

CLAIMS :

1. A method for diagnosing probable Alzheimer's Disease (AD) or Mild Cognitive Impairment (MCI) in a subject, the method comprising:

(a) detecting the amount of alpha-1-antitrypsin

(AAT), or its isoforms, in a biological sample obtained from said subject; and

2. The method of claim 1, wherein said biological sample is serum, plasma, urine or cerebrospinal fluid.

3. The method of claim 2, wherein said biological sample is plasma.

4. The method of claim 1, wherein the amount of AAT is detected by immunoturbidimetric assay, immunofluorescence, immunodiffusion, enzyme-linked immunosorbent assay (ELISA) , radioimmunoassay (RIA) , Western blot, protein activity assay, high performance liquid chromatography (HPLC) , mass spectrometry (MS) , gas chromatography (GC), GC-MS, LC-MS, or LC-MS/MS.

5. The method of claim 4, wherein the amount of AAT is detected by immunoturbidimetric assay.

6. The method of claim 1, wherein the amount of AAT is detected using an antibody that specifically binds to AAT, or its isoforms.

7. A method for diagnosing probable Parkinson's disease (PD) in a subject, the method comprising:

(a) detecting the amount of alpha- 1 -antitrypsin (AAT) , or its isoforms, in a biological sample obtained from said subject; and

(b) comparing the detected amount of AAT in the biological sample with an amount of AAT characteristic of a normal control; '

whereby a reduced amount of AAT in said biological sample relative to the normal control is a positive indicator of

PD.

8. The method of claim 7, wherein said biological sample is serum, plasma, urine or cerebrospinal fluid.

9. The method of claim 7, wherein said biological sample is plasma.

10. The method of claim 7, wherein the amount of AAT is detected by immunoturbidimetric assay, immunofluorescence, immunodiffusion, enzyme-linked immunosorbent assay (ELISA) , radioimmunoassay (RIA) , Western blot, protein activity assay, high performance liquid chromatography (HPLC) , mass spectrometry (MS) , gas chromatography (GC), GC-MS, LC-MS, or LC-MS/MS.

11. The method of claim 10, wherein the amount of AAT is detected by immunoturbidimetric assay.

12. The method of claim 7, wherein the amount of AAT is detected using an antibody that specifically binds to AAT, or its isoforms.

13. A method of diagnosing a neurodegenerative disease in a subject, the method comprising: (a) obtaining a biological sample from said subject;

(b) contacting said biological sample with an antibody that binds to alpha- 1 -antitrypsin (AAT) , or its isoforms;

(c) allowing the antibody and AAT to form an immune complex;

(d) detecting the amount of immune complex formed as an indication of the amount of the AAT in said biological sample; and

(e) comparing the detected amount to a normal control ;

14. The method according to claim 13, wherein the detected amount that is elevated relative to the normal control is a positive indicator of AD.

15. The method according to claim 13, wherein the detected amount that is elevated relative to the normal control is a positive indicator of MCI.

16. The method according to claim 13, wherein the detected amount that is reduced relative to the normal control is a positive indicator of PD.

17. A kit for diagnosing a neurodegenerative disease comprising an antibody that binds to AAT and an established standard of an amount of AAT characteristic of a normal control .

18. The kit according to claim 17, wherein the neurodegenerative disease is AD.

19. The kit according to claim 17, wherein the neurodegenerative disease is MCI.

20. The kit according to claim 17, wherein the neurodegenerative disease is PD.

21. A method of using an antibody that binds to alpha- 1 -antitrypsin (AAT) , or its isoforms, as a biological marker for diagnosing probable Alzheimer's Disease (AD) in a subject, said method of using comprising contacting a biological sample obtained from said subject with said antibody to determine the amount of AAT in the sample.

22. A method of using an antibody that binds to alpha-1-antitrypsin (AAT), or its isoforms, as a biological marker for diagnosing probable Mild Cognitive Impairment (MCI) in a subject, said method of using comprising contacting a biological sample obtained from said subject with said antibody to determine the amount of AAT in the sample .

23. A method of using an antibody that binds to alpha-1-antitrypsin (AAT) , or its isoforms, as a biological marker for diagnosing probable Parkinson's disease (PD) in a subject, said method of using comprising contacting a biological sample obtained from said subject with said antibody to determine the amount of AAT in the sample.

24. A use of an antibody that binds to alpha-1- antitrypsin (AAT), or its isoforms, as a biological marker for diagnosing probable Alzheimer's Disease (AD) in a subject .

25. A use of an antibody that binds to alpha- 1- antitrypsin (AAT), or its isoforms, as a biological marker for diagnosing probable Mild Cognitive Impairment (MCI) in a subject .

26. A use of an antibody that binds to alpha-1- antitrypsin (AAT) , or its isoforms, as a biological marker for diagnosing probable Parkinson's disease (PD) in a subject .