AU2003221653A1 - Haptoglobin fragments diagnostic of alzheimers disease - Google Patents

Description

WO 03/087835 PCT/CAO3/00540 1 HAPTOGLOBIN FRAGMENTS DIAGNOSTIC OF ALZHEIMERS DISEASE FIELD OF THE INVENTION This invention relates broadly to the field of characterizing the existence of a disease state; more particularly to the utilization of mass spectrometry to elucidate particular biopolymer markers which are diagnostic, e.g. indicative or predictive of a 5 particular disease state, particularly to specific biopolymer markers whose up regulation, down-regulation, or relative presence in disease vs. normal states has been determined to be useful in disease state assessment and therapeutic target recognition, development and validation; and most particularly to the characterization of a particular group of haptoglobin fragments which have been evidenced in the bodily 10 fluid of patients suffering from Alzheimers disease and have been deemed to be of diagnostic significance. BACKGROUND OF THE INVENTION Alzheimer's disease, also referred to as Alzheimer's dementia or AD is a progressive neurodegenerative disorder that causes memory loss and serious mental 15 deterioration. Diagnosticians have long sought a means to definitively identify AD during the lifetime of demented patients, as opposed to histopathological examination of brain tissue, which is the only present means available for rendering an ultimate diagnosis of AD. AD is the most common form of dementia, accounting for more than half of all dementias and affecting as many as 4 million Americans and nearly 15 20 million people worldwide. Dementia may start with slight memory loss and confusion, but advances with time reaching severe impairment of intellectual and social abilities. At age 65, the community prevalence of AD is between 1B2 %. By age 75, the figure rises to 7 %, and by age 85 it is 18 %. The prevalence of dementia in all individuals over age 65 is 8 %. Of those residing in institutions, the prevalence is about 50 %, at 25 any age. The social impact of this disease is enormous, caused by the burden placed on caregivers, particularly in the latter stages of the disease. The substantial economic costs are largely related to supportive care and institutional admission. The rapidly increasing proportion of elderly people in society means that the number of individuals WO 03/087835 PCT/CAO3/00540 2 affected with AD will grow dramatically, therefore finding an early accurate diagnosis and a cure for AD is becoming an issue of major importance world wide. When an individual is suspected of AD, several recommended tests are performed: (1) Mini Mental State Examination (MMSE) B an office-based 5 psychometric test in the form of a Functional Assessment Questionnaire (FAQ) to examine the scale for functional autonomy, (2) Laboratory tests B complete blood count, measurement of thyroid stimulating hormone, serum electrolytes, serum calcium and serum glucose levels, (3) Neuroimaging B most commonly used is computed tomography (CT) which has a role in detecting certain causes of dementia such as 10 vascular dementia (VaD), tumor, normal pressure hydrocephalus or subdural hematoma. However, neuroimaging is less effective in distinguishing AD or other cortical dementias from normal aging. In primary care settings, some suggest that CT could be limited to atypical cases, but others recommend routine scanning. Magnetic resonance imaging (MRI) currently offers no advantage over CT in most cases of 15 dementia. In comparison to other disease areas, the field of dementia raises questions concerning the value of diagnosis, since there is currently no cure or effective therapy available. In dementia, as in all other branches of medicine, the certainty of a diagnosis has an important impact on the management of the patient. While AD cannot be cured 20 at present time, there is symptomatic treatment available and the first drugs (acetylcholinesterase inhibitors) for the temporary improvement of cognition and behavior are now licensed by the U.S. Food and Drug Administration. Other drugs are at different stages of clinical trials: (1) Drugs to prevent decline in AD B DESFERRIOXAMINE, ALCAR, anti-inflammatory drugs, antioxidants, estrogen, (2) 25 Neurotrophic Factors : NGF, (3) Vaccine : the recent most exciting report by Schenk et al. (Nature 1999;400:173-7) raises the hope of a vaccine for AD. The specificity of the various therapies thus require sophisticated diagnostic methodologies, having a high degree of sensitivity for AD, in order to insure their success. 30 Currently there are a multitude of tests available which aid in the diagnosis of AD. However, the only true existing diagnosis is made by pathologic examination of postmortem brain tissue in conjunction with a clinical history of dementia. This WO 03/087835 PCT/CA03/00540 3 diagnosis is based on the presence in brain tissue ofneurofibrillary tangles and of neuritic (senile) plaques, which have been correlated with clinical dementia. Neuritic plaques are made up of a normally harmless protein called amyloid-beta. Before neurons begin to die and symptoms develop, plaque deposits form between neurons 5 early on in the disease process. The neurofibrillary tangles are interneuronal aggregates composed of normal and paired helical filaments and presumably consist of several different proteins. The internal support structure for brain neurons depends on the normal functioning of a protein called tau. In Alzheimer's disease, threads of tau protein undergo alterations that cause them to become twisted. The 10 neurohistopathologic identification and counting of neuritic plaques and neurofibrillary tangles requires staining and microscopic examination of several brain sections. However, the results of this methodology can widely vary and is time-consuming and labor-intensive. Given the ability of both current and prospective pharmacological therapies to 15 forestall and/or reverse the onset and/or progress of Alzheimer's dementia, an early diagnosis of AD will assist to better manage the care of patients. There are many cases where non-AD dementia could be confused with AD dementia. Such examples include small, undetected strokes which temporarily interrupt blood flow to the brain. Clinically depressed patients or those with Parkinson's disease can also experience 20 lapses in memory. Many older people are on a variety of medications which as a side effect may, alone or in conjunction, impair their ability to perform cognitive tasks. Thus, if diagnostic techniques for the early differentiation of AD could be provided, physician's would achieve an enhanced ability to prescribe appropriate therapeutic intervention at an early stage in the pathogenesis of this disease. 25 Various biochemical markers for AD are known and analytical techniques for the determination of such markers have been described in the art. As used herein the term "marker" "biochemical marker" or "marker protein" refers to any enzyme, protein, polypeptide, peptide, isomeric form thereof, immunologically detectable fragments thereof, or other molecule that is released into the circulation or is present therein, e.g. 30 derived from the brain, during the course of AD pathogenesis. Such markers include, but are not limited to, any unique proteins or isoforms thereof that are particularly associated with the brain.

WO 03/087835 PCT/CA03/00540 4 There are a number of different potential uses for biomarkers in AD evaluation, and each use could involve a different marker or set of markers. Such uses may include, but are not limited to, the use of a marker to distinguish AD from other causes of dementia; distinguishing dementia from the non-pathological effects of aging; 5 monitoring the progress of the disease after clinical symptoms become apparent; utilization of a surrogate to monitor the efficacy of the forthcoming therapies for AD; and isolating markers which have utility as risk assessment factors for AD; and identifying both the earliest biological changes occurring in the brain and other changes that occur as the disease progresses. Ideally, it would be preferable to isolate a single 10 marker to fulfill all requirements with a high degree of sensitivity and specificity, however this may be an unreasonable goal. Any individual marker needs to be assessed by sensitivity, specificity, reliability and validity for the type of clinical situation to which it is meant to apply. A marker which is poor at distinguishing AD from other causes of dementia, could nevertheless be an excellent marker for 15 monitoring the progression of the disease process or the response to therapy. With regard to diagnostic devices, the clinical evaluation and use of point-of care tests utilizing biological markers are valuable tools for evaluating risk, monitoring disease progression and guiding therapeutic interventions. The advantages which flow from the use of biological markers as diagnostic tools include strengthening the 20 certainty of the clinical diagnosis, distinguishing AD from other causes of dementia, and quantifying the severity of the disease and rate of progression. In addition, tests using biological markers should be rapid, non-invasive, simple to perform and inexpensive. What is lacking in the art is a relatively non-invasive method and device 25 therefore effective for definitively diagnosing Alzheimer's dementia in living patients. Additionally, a definitive method of assessing the risk of developing AD is greatly needed. Generally, most scientific papers tend to focus on the peptide, 3-amyloid, since it is postulated to be a major determinant of AD. This is supported by the observation 30 that certain forms of familial AD mutations result in the over production of BBamyloid, particularly the longer form (1-42) which aggregates more readily than the shorter form. Hensley et al. (Proc. Natl. Acad. Sci., (1994), 91, pp3270- 3 2 74 ) examine the WO 03/087835 PCT/CA03/00540 5 neurotoxicity based on free radical generation by the peptide B-amyloid in its aggregation state. Several synthetic fragments of the peptide are tested for resulting neurotoxicity. Based on the fact that oxygen seems to be a requirement for radical generation and glutamate synthetase and creatine kinase enzymes are oxidation 5 sensitive biomarkers, the inactivation of these enzymes are utilized as indicators of active attack on biological molecules by these fragmented B-amyloid aggregates. In U.S. Pat. No. 5,508,167, Roses et al. describe methods for diagnosing AD involving the detection of an apolipoprotein E type 4 (ApoE4) isoform or DNA encoding ApoE4. The methods can use blood samples and are analyzed by an 10 immunochemical assay. The blood sample is optionally combined with a reducing agent to reduce the disulfide bond in cysteine residues to the corresponding reactive sulfhydryl groups. Roses et al. further describes a kit for detection of the ApoE4 isoform. The test is based on the differences in the amino acid sequences of the three major ApoE isoforms. The test is not specific for haptogloin, nor does it have 15 sensitivity in differentially diagnosing AD versus non-AD dementia. In U.S. Patent 5,429,947, a method is taught for detecting elevated levels of the middle isoform of a-2 haptoglobin, a-2FS haptoglobin in bodily fluids. The patent discloses that elevated levels of specific haptoglobin proteins serve as a diagnostic marker of Alzheimer's disease. 20 U.S. Patent 6,287,793 teaches a diagnostic method for Alzheimer's disease utilizing an antibody binding assay which recognizes a-chain haptogloin in the biological fluid of a patient suffering from Alzheimer's disease. JP2190767A2, entitled "Method for Determining Globin", is directed toward measuring the concentration of nonhem-GL conjugated with haptoglobin. 25 Electrophoresis, immunoassay and HPLC are disclosed methods of measurement; usefulness of the method in diagnosing dementia is also disclosed. Contrary to the instant invention, the prior art references fail to contemplate (or demonstrate an ability for) isolation of distinct fragments of the particular biopolymer marker of interest, in this case haptoglobin, so as to elucidate the presence, absence, or 30 change in relative concentrations, of fragments of the diagnostic marker. This is important, in that various disease processes will cause fragmentation of the marker in a predictable fashion, and armed with this knowledge, diagnostic methods which are WO 03/087835 PCT/CA03/00540 6 determinative of these isolated marker fragments take on added significance. Thus the instant invention is directed toward the identification of isolated biopolymer fragments which provide an ability to diagnose, monitor the progression, and validate therapeutic avenues for Alzheimer's disease. 5 Methods utilizing mass spectrometry for the analysis of a target polypeptide have been taught wherein the polypeptide is first solubilized in an appropriate solution or reagent system. The type of solution or reagent system, e.g., comprising an organic or inorganic solvent, will depend on the properties of the polypeptide and the type of mass spectrometry performed and are well-known in the art (see, e.g., Vorm et al. 10 (1994) Anal. Chem. 66:3281 (for MALDI) and Valaskovie et al. (1995) Anal. Chem. 67:3802 (for ESI). Mass spectrometry of peptides is further disclosed, e.g., in WO 93/24834 by Chait et al. In one prior art embodiment, the solvent is chosen so that the risk that the molecules may be decomposed by the energy introduced for the vaporization process is considerably reduced, or even fully excluded. This can be 15 achieved by embedding the sample in a matrix, which can be an organic compound, e.g., sugar, in particular pentose or hexose, but also polysaccharides such as cellulose. These compounds are decomposed thermolytically into CO 2 and H 2 0 so that no residues are formed which might lead to chemical reactions. The matrix can also be an inorganic compound, e.g., nitrate of ammonium which is decomposed practically 20 without leaving any residues. Use of these and other solvents are further disclosed in U.S. Pat. No. 5,062,935 by Schlag et al. Prior art mass spectrometer formats for use in analyzing the translation products include ionization (I) teelmiques, including but not limited to matrix assisted laser desorption (MALDI), continuous or pulsed electrospray (ESI) and related methods (e.g., IONSPRAY or THERMOSPRAY), or massive cluster 25 impact (MCI); these ion sources can be matched with detection formats including linear or non-linear reflection time-of-flight (TOF), single or multiple quadropole, single or multiple magnetic sector, Fourier Transform ion cyclotron resonance (FTICR), ion trap, and combinations thereof (e.g., ion-trap/time-of-flight). For ionization, numerous matrix/wavelength combinations (MALDI) or solvent 30 combinations (ESI) can be employed. Subattomole levels of protein have been detected, for example, using ESI (Valaskovic, G. A. et al., (1996) Science 273:1199 1202) or MALDI (Li, L. et al., (1996) J. Am. Chem. Soc. 118:1662-1663) mass WO 03/087835 PCT/CA03/00540 7 spectrometry. ES mass spectrometry has been introduced by Fenn et al. (J. Phys. Chem. 88, 4451-59 (1984); PCT Application No. WO 90/14148) and current applications are summarized in recent review articles (R. D. Smith et al., Anal. Chem. 62, 882-89 (1990) and B. Ardrey, Electrospray Mass Spectrometry, Spectroscopy 5 Europe, 4, 10-18 (1992)). MALDI-TOF mass spectrometry has been introduced by Hillenkamp et al. ("Matrix Assisted UV-Laser Desorption/Ionization: A New Approach to Mass Spectrometry of Large Biomolecules," Biological Mass Spectrometry (Burlingame and MeCloskey, editors), Elsevier Science Publishers, Amsterdam, pp. 49-60, 1990). With ESI, the determination of molecular weights in femtomole amounts 10 of sample is very accurate due to the presence of multiple ion peaks which all could be used for the mass calculation. The mass of the target polypeptide determined by mass spectrometry is then compared to the mass of a reference polypeptide of known identity. In one embodiment, the target polypeptide is a polypeptide containing a number of repeated amino acids directly correlated to the number of trinucleotide 15 repeats transcribed/translated from DNA; from its mass alone the number of repeated trinucleotide repeats in the original DNA which coded it, may be deduced. U.S. Patent No. 6,020,208 utilizes a general category of probe elements (i.e., sample presenting means) with Surfaces Enhanced for Laser Desorption/Ionization (SELDI), within which there are three (3) separate subcategories. The SELDI process 20 is directed toward a sample presenting means (i.e., probe element surface) with surface associated (or surface-bound) molecules to promote the attachment (tethering or anchoring) and subsequent detachment of tethered analyte molecules in a light dependent manner, wherein the said surface molecule(s) are selected from the group consisting of photoactive (photolabile) molecules that participate in the binding 25 (docking, tethering, or crosslinking) of the analyte molecules to the sample presenting means (by covalent attachment mechanisms or otherwise). PCT/EP/04396 teaches a process for determining the status of an organism by peptide measurement. The reference teaches the measurement of peptides in a sample of the organism which contains both high and low molecular weight peptides and acts 30 as an indicator of the organism's status. The reference concentrates on the measurement of low molecular weight peptides, i.e. below 30,000 Daltons, whose distribution serves as a representative cross-section of defined controls. Contrary to the WO 03/087835 PCT/CA03/00540 8 methodology of the instant invention, the '396 patent strives to determine the status of a healthy organism, i.e. a "normal" and then use this as a reference to differentiate disease states. The present inventors do not attempt to develop a reference "normal", but rather strive to specify particular markers whose presence, absence or relative 5 strength/concentration in disease vs. normal is diagnostic of at least one specific disease state or whose up-regulation or down-regulation is predictive of at least one specific disease state, whereby the presence of said marker serves as a positive indicator useful in distinguishing disease state. This leads to a simple method of analysis which can easily be performed by an untrained individual, since there is a 10 positive correlation of data. On the contrary, the '396 patent requires a complicated analysis by a highly trained individual to determine disease state versus the perception of non-disease or normal physiology. Richter et al, Journal of Chromatography B, 726(1999) 25-35, refer to a database established from human hemofiltrate comprised of a mass database and a sequence database. The goal of Richter et al was to analyze the 15 composition of the peptide fraction in human blood. Using MALDI-TOF, over 20,000 molecular masses were detected representing an estimated 5,000 different peptides. The conclusion of the study was that the hemofiltrate (HF) represented the peptide composition of plasma. No correlation of peptides with relation to normal and/or disease states is made. 20 Definitions: As used herein, "analyte" refers to any atom and/or molecule; including their complexes and fragment ions. The term may refer to a single component or a set of components. In the case of biological molecules/macromolecules or "biopolymers", such analytes include but are not limited to: polypeptides, polynucleotides, proteins, 25 peptides, antibodies, DNA, RNA, carbohydrates, steroids, and lipids, and any detectable moiety thereof, e.g. immunologically detectable fragments. Note that most important biomolecules under investigation for their involvement in the structure or regulation of life processes are quite large (typically several thousand times larger than

H

2 0). As used herein, the term "molecular ions" refers to molecules in the charged or 30 ionized state, typically by the addition or loss of one or more protons (HI). As used herein, the term "molecular fragmentation" or "fragment ions" refers to breakdown products of analyte molecules caused, for example, during laser-induced desorption WO 03/087835 PCT/CA03/00540 9 (especially in the absence of added matrix). As used herein, the term "solid phase" refers to the condition of being in the solid state, for example, on the probe element surface. As used herein, "gas" or "vapor phase" refers to molecules in the gaseous state (i.e., in vacuo for mass spectrometry). As used herein, the term "analyte 5 desorption/ionization" refers to the transition of analytes from the solid phase to the gas phase as ions. Note that the successful desorption/ionization of large, intact molecular ions by laser desorption is relatively recent (circa 1988)--the big breakthrough was the chance discovery of an appropriate matrix (nicotinic acid). As used herein, the term "gas phase molecular ions" refers to those ions that enter into the gas phase. Note that 10 large molecular mass ions such as proteins (typical mass=60,000 to 70,000 times the mass of a single proton) are typically not volatile (i.e., they do not normally enter into the gas or vapor phase). However, in the procedure of the present invention, large molecular mass ions such as proteins do enter the gas or vapor phase. As used herein in the case of MALDI, the term "matrix" refers to any one of several small, acidic, light 15 absorbing chemicals (e.g., CHCA (alpha-cyano-4-hydroxy-cinnamic acid), nicotinic or sinapinic acid) that is mixed in solution with the analyte in such a manner so that, upon drying on the probe element, the crystalline matrix-embedded analyte molecules are successfully desorbed (by laser irradiation) and ionized from the solid phase (crystals) into the gaseous or vapor phase and accelerated as intact molecular ions. For the 20 MALDI process to be successful, analyte is mixed with a freshly prepared solution of the chemical matrix (e.g., 10,000:1 matrix:analyte) and placed on the inert probe element surface to air dry just before the mass spectrometric analysis. The large fold molar excess of matrix, present at concentrations near saturation, facilitates crystal formation and entrapment of analyte. 25 As used herein, "energy absorbing molecules (EAM)" refers to any one of several small, light absorbing chemicals that, when presented on the surface of a probe, facilitate the neat desorption of molecules from the solid phase (i.e., surface) into the gaseous or vapor phase for subsequent acceleration as intact molecular ions. The term EAM is preferred, especially in reference to SELDI. Note that analyte desorption by 30 the SELDI process is defined as a surface-dependent process (i.e., neat analyte may be placed on a surface composed of bound EAM or EAM and analyte may be mixed prior to placement on a surface). In contrast, MALDI is presently thought to facilitate analyte WO 03/087835 PCT/CA03/00540 10 desorption by a volcanic eruption-type process that "throws" the entire surface into the gas phase. Furthermore, note that some EAM when used as free chemicals to embed analyte molecules as described for the MALDI process will not work (i.e., they do not promote molecular desorption, thus they are not suitable matrix molecules). 5 As used herein, "probe element" or "sample presenting device" refers to an element having the following properties: it is inert (for example, typically stainless steel) and active (probe elements with surfaces enhanced to contain EAM and/or molecular capture devices). As used herein, "MALDI" refers to Matrix-Assisted Laser 10 Desorption/Ionization. As used herein, "TOF" stands for Time-of-Flight. As used herein, "MS" refers to Mass Spectrometry. As used herein, "MS/MS" refers to multiple sequential mass spectrometry. As used herein "MALDI-TOF MS" refers to Matrix-assisted laser desorption/ionization 15 time-of-flight mass spectrometry. As used herein, "ESI" is an abbreviation for electrospray ionization. As used herein, "chemical bonds" is used simply as an attempt to distinguish a rational, deliberate, and knowledgeable manipulation of known classes of chemical interactions from the poorly defined kind of general adherence observed when one chemical substance (e.g., matrix) is placed on another substance (e.g., an 20 inert probe element surface). Types of defined chemical bonds include electrostatic or ionic (+/-) bonds (e.g., between a positively and negatively charged groups on a protein surface), covalent bonds (very strong or "permanent" bonds resulting from true electron sharing), coordinate covalent bonds (e.g., between electron donor groups in proteins and transition metal ions such as copper or iron), and hydrophobic interactions (such as 25 between two noncharged groups), weak dipole and London force or induced dipole interactions. As used herein, "electron donor groups" refers to the case of biochemistry, where atoms in biomolecules (e.g, N, S, O) "donate" or share electrons with electron poor groups (e.g., Cu ions and other transition metal ions). As used herein, the term "biopolymer markers indicative or predictive of a 30 disease state" is refers to biopolymer markers which have diagnostic value, and is interpreted to mean that a biopolymer marker which is strongly present in a normal individual, but is down-regulated in disease is predictive of said disease; while WO 03/087835 PCT/CA03/00540 11 alternatively, a biopolymer marker which is strongly present in a disease state, but is down-regulated in normal individuals, is indicative of said disease state. Biopolymer markers which are present in both disease and normal states are indicative/predictive based upon their relative strengths in disease vs. normal, along with the observation 5 regarding when their signal strengthens/weakens relative to disease manifestation or progression, thereby embodying a valuable diagnostic tool. As used herein, the term "disease state assessment" is interpreted to mean quantitative or qualitative determination of the presence/absence of the disease, with or without an ability to determine severity, rapidity of onset, or resolution of the disease 10 state, e.g. a return to a normal physiological state. As used herein, the term "therapeutic target recognition, development, and validation" refers to any concept or method which enables an artisan to recognize, develop, or validate the efficacy of a therapeutic moiety which is effected in conjunction with a chemical or physical interaction with one or more of the biopolymer 15 markers of the instant invention. As used herein, the term "polypeptide" is interpreted to mean a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof linked via peptide bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs 20 thereof. Synthetic polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. The term "protein" typically refers to large polypeptides. The term "peptide" typically refers to short polypeptides. "Polypeptide(s)" refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds. "Polypeptide(s)" refers to both short chains, 25 commonly referred to as peptides, oligopeptides and oligomers and to longer chains generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene encoded amino acids. "Polypeptide(s)" include those modified either by natural processes, such as processing and other post-translational modifications, but also by chemical modification techniques. Such modifications are well described in 30 basic texts and in more detailed monographs, as well as in a voluminous research literature, and they are well-known to those of skill in the art. It will be appreciated that the same type of modification may be present in the same or varying degree at several WO 03/087835 PCT/CA03/00540 12 sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains, and the amino or carboxyl termini. Modifications include, for example, acetylation, acylation, ADP-ribosylation, 5 amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, 10 glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins, such as arginylation, and 15 ubiquitination. See, for instance, PROTEINS--STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993) and Wold, F., Posttranslational Protein Modifications: Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York (1983); Seifter et al., 20 Meth. Enzymol. 182:626-646 (1990) and Rattan et al., Protein Synthesis: Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663: 48-62 (1992). Polypeptides may be branched or cyclic, with or without branching. Cyclic, branched and branched circular polypeptides may result from post-translational natural processes and may be made by entirely synthetic methods, as well. 25 As used herein, the term "polynucleotide" is interpreted to mean a polymer composed of nucleotide units. Polynucleotides include naturally occurring nucleic acids, such as deoxyribonucleic acid ("DNA") and ribonucleic acid ("RNA") as well as nucleic acid analogs. Nucleic acid analogs include those which include non-naturally occurring bases, nucleotides that engage in linkages with other nucleotides other than 30 the naturally occurring phosphodiester bond or which include bases attached through linkages other than phosphodiester bonds. Thus, nucleotide analogs include, for example and without limitation, phosphorothioates, phosphorodithioates, WO 03/087835 PCT/CA03/00540 13 phosphorotriesters, phosphoramidates, boranophosphates, methylphosphonates, chiral methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs), and the like. Such polynucleotides can be synthesized, for example, using an automated DNA synthesizer. The term "nucleic acid" typically refers to large polynucleotides. The 5 term "oligonucleotide" typically refers to short polynucleotides, generally no greater than about 50 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which "U" replaces T. As used herein, the term "detectable moiety" or a "label" refers to a 10 composition detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include 32 p, 3"S, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin-streptavadin, dioxigenin, haptens and proteins for which antisera or monoclonal antibodies are available, or nucleic acid molecules with a sequence 15 complementary to a target. The detectable moiety often generates a measurable signal, such as a radioactive, chromogenic, or fluorescent signal, that can be used to quantitate the amount of bound detectable moiety in a sample. The detectable moiety can be incorporated in or attached to a primer or probe either covalently, or through ionic, van der Waals or hydrogen bonds, e.g., incorporation of radioactive nucleotides, or 20 biotinylated nucleotides that are recognized by streptavadin. The detectable moiety may be directly or indirectly detectable. Indirect detection can involve the binding of a second directly or indirectly detectable moiety to the detectable moiety. For example, the detectable moiety can be the ligand of a binding partner, such as biotin, which is a binding partner for streptavadin, or a nucleotide sequence, which is the binding partner 25 for a complementary sequence, to which it can specifically hybridize. The binding partner may itself be directly detectable, for example, an antibody may be itself labeled with a fluorescent molecule. The binding partner also may be indirectly detectable, for example, a nucleic acid having a complementary nucleotide sequence can be a part of a branched DNA molecule that is in turn detectable through hybridization with other 30 labeled nucleic acid molecules. (See, e.g., P. D. Fahrlander and A. Klausner, Bio/Technology (1988) 6:1165.) Quantitation of the signal is achieved by, e.g., scintillation counting, densitometry, or flow cytometry.

WO 03/087835 PCT/CAO3/00540 14 As used herein, the term "antibody or antibodies" includes polyclonal and monoclonal antibodies of any isotype (IgA, IgG, IgE, IgD, IgM), or an antigen-binding portion thereof, including but not limited to F(ab) and Fv fragments, single chain antibodies, chimeric antibodies, humanized antibodies, and a Fab expression library. 5 "Antibody" refers to a polypeptide ligand substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, which specifically binds and recognizes an epitope (e.g., an antigen). The recognized immunoglobulin --genes include the kappa and lambda light chain constant region genes, the alpha, gamma, delta, epsilon and mu heavy chain constant region genes, and the myriad 10 immunoglobulin variable region genes. Antibodies exist, e.g., as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases. This includes, e.g., Fab' and F(ab)' 2 fragments. The term "antibody," as used herein, also includes antibody fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant 15 DNA methodologies. It also includes polyclonal antibodies, monoclonal antibodies, chimeric antibodies and humanized antibodies. "Fc" portion of an antibody refers to that portion of an immunoglobulin heavy chain that comprises one or more heavy chain constant region domains, CH, CH 2 and CH3, but does not include the heavy chain variable region. 20 As used herein, the term "moieties" refers to an indefinite portion of a sample. A "ligand" is a compound that specifically binds to a target molecule. A "receptor" is a compound or portion of a structure that specifically binds to a ligand. A ligand or a receptor (e.g., an antibody) "specifically binds to" or "is 25 specifically immunoreactive with" a compound analyte when the ligand or receptor functions in a binding reaction which is determinative of the presence of the analyte in a sample of heterogeneous compounds. Thus, under designated assay (e.g., immunoassay) conditions, the ligand or receptor binds preferentially to a particular analyte and does not bind in a significant amount to other compounds present in the 30 sample. For example, a polynucleotide specifically binds under hybridization conditions to an analyte polynucleotide comprising a complementary sequence; an antibody specifically binds under immunoassay conditions to an antigen analyte WO 03/087835 PCT/CA03/00540 15 bearing an epitope against which the antibody was raised; and an adsorbent specifically binds to an analyte under proper elution conditions. As used herein, the term "pharmaceutically effective carrier" refers to any solid or liquid material which may be used in creating formulations that further include 5 active ingredients of the instant invention, e.g. biopolymer markers or therapeutics, for administration to a patient. As used herein, the term "agent" is interpreted to mean a chemical compound, a mixture of chemical compounds, a sample of undetermined composition, a combinatorial small molecule array, a biological macromolecule, a bacteriophage 10 peptide display library, a bacteriophage antibody (e.g., scFv) display library, a polysome peptide display library, or an extract made from biological materials such as bacteria, plants, fungi, or animal cells or tissues. Suitable techniques involve selection of libraries of recombinant antibodies in phage or similar vectors. See, Huse et al. (1989) Science 246: 1275-1281; and Ward et al. (1989) Nature 341: 544-546. The 15 protocol described by Huse is rendered more efficient in combination with phage display technology. See, e.g., Dower et al., WO 91/17271 and McCafferty et al., WO 92/01047. As used herein, the term "isolated" is interpreted to mean altered "by the hand of man" from its natural state, i.e., if it occurs in nature, it has been changed or 20 removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living organism is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herein. As used herein, the term "variant" is interpreted to mean a polynucleotide or 25 polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties. A typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid 30 substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, WO 03/087835 PCT/CA03/00540 16 differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A substituted or inserted amino acid residue 5 may or may not be one encoded by the genetic code. A variant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to skilled artisans. 10 As used herein, the term "biopolymer marker" refers to a polymer of biological origin, e.g. polypeptides, polynucleotides, polysaccharides or polyglycerides (e.g., di-or tri-glycerides), and may include any fragment, e.g. immunologically reactive fragments, variants or moieties thereof. As used herein, the term "fragment" refers to the products of the chemical, 15 enzymatic, or physical breakdown of an analyte. Fragments may be in a neutral or ionic state. As used herein, the term "therapeutic avenues" is interpreted to mean any agents, modalities, synthesized compounds, etc., which interact with a biopolymer marker in any manner that facilitates a therapeutic benefit, including 20 immunotherapeutic intervention, e.g. modalities such as administration of an immunologically reactive moiety capable of altering the course, progression and/or manifestation of the disease, as a result of interfering with the disease manifestation process, for example, at the early stages focused upon by the identification of the disease, such as by supplying a moiety capable of modifying the pathogenicity of 25 lymphocytes specific for the biopolymer marker or related components. As used herein, the term "interacting with a biopolymer marker" includes any process by which a biopolymer marker may physically or chemically relate with an organism, particularly when this interaction results in the development of therapeutic avenues or in modulation of the disease state. 30 As used herein, the term "therapeutic targets" may thus be defined as those analytes which are capable of exerting a modulating force, wherein "modulation" is defined as an alteration in function inclusive of activity, synthesis, production, and WO 03/087835 PCT/CA03/00540 17 circulating levels. Thus, modulation effects the level or physiological activity of at least one particular disease related biopolymer marker or any compound or biomolecule whose presence, level or activity is linked either directly or indirectly, to an alteration of the presence, level, activity or generic function of the biopolymer 5 marker, and may include pharmaceutical agents, biomolecules that bind to the biopolymer markers, or biomolecules or complexes to which the biopolymer markers bind. The binding of the biopolymer markers and the therapeutic moiety may result in activation (agonist), inhibition (antagonist), or an increase or decrease in activity or production (modulator) of the biopolymer markers or the bound moiety. Examples of 10 such therapeutic moieties include, but are not limited to, antibodies, oligonucleotides, proteins (e.g., receptors), RNA, DNA, enzymes, peptides or small molecules. With regard to immunotherapeutic moieties, such a moiety may be defined as an effective analog for a major epitope peptide which has the ability to reduce the pathogenicity of key lymphocytes which are specific for the native epitope. An analog is defined as 15 having structural similarity but not identity in peptide sequencing able to be recognized by T-cells spontaneously arising and targeting the endogeneous self epitope. A critical function of this analog is an altered T-cell activation which leads to T-cell anergy or death. With the advent of mass spectrometric methods such as MALDI and SELDI 20 and ESI, researchers have begun to utilize a tool that holds the promise of uncovering countless biopolymers which result from translation, transcription and post translational transcription of proteins from the entire genome. Operating upon the principles of retentate chromatography, SELDI MS involves the adsorption of proteins, based upon their physico-chemical properties at a 25 given pH and salt concentration, followed by selectively desorbing proteins from the surface by varying pH, salt, or organic solvent concentration. After selective desorption, the proteins retained on the SELDI surface, the "chip", can be analyzed using the CIPHERGEN protein detection system, or an equivalent thereof Retentate chromatography is limited, however, by the fact that if unfractionated body fluids, e.g. 30 blood, blood products, urine, saliva, cerebrospinal fluid, luymph and the like, along with tissue samples, are applied to the adsorbent surfaces, the biopolymers present in the greatest abundance will compete for all the available binding sites and thereby WO 03/087835 PCT/CA03/00540 18 prevent or preclude less abundant biopolymers from interacting with them, thereby reducing or eliminating the diversity of biopolymers which are readily ascertainable. Thus, if it were possible to particularly identify and characterize discrete fragments which can be confirmed as originating from a biopolymer marker known to 5 be indicative of a particular disease state; then a more thorough understanding of the pre- and post-disease processes can be realized, leading to more definitive tests, e.g. prognostic, risk-assessment and the like; and additionally more definitive validation of therapeutic moieties may be carried out, as well as assessment of disease state and/or progressio/regression. 10 SUMMARY OF THE INVENTION The present invention is directed toward determination of particular fragments of haptoglobin which are found in the circulation of patients suffering from Alzheimer's dementia. Via utilization of a combination of preparatory steps, e.g. chromatography and 15 1 -D tricine polyacrylamide gel electrophoresis, a variety of fragments of differing molecular weights may be elucidated from bodily fluids, e.g. serum. Subsequently, additional investigative techniques are used, e.g. a gel is stained, e.g. with Coomasie blue, silver or rubidium and bands are selected from the gels for further study. Next, tryptic digestion may be carried out on each of the bands, concluding with the 20 extraction of tryptic peptides from the digest. An illustrative, albeit non-limiting examples of such an extraction may be accomplished utilizing C18 ZIPTIPs, or organic extract and dry technique followed by MALDI Qq TOF (Maldi Quadrupole Quadrupole Time of Flight) processing. Additional methodologies may include SELDI MS, 2-D gel technology, 25 MALDI MS/MS and time-of-flight detection procedures to maximize the diversity of biopolymers which are verifiable within a particular sample. The cohort of biopolymers verified within a sample is then compared to develop data indicating their presence, absence or relative strength/concentration in disease vs normal controls, and further studied to determine whether the up-regulation or down-regulation of a single 30 biopolymer, group of biopolymers, or discrete fragments thereof, are indicative of a disease state or predictive of the development of said disease state.

WO 03/087835 PCT/CA03/00540 19 Additionally, biopolymers recognized as being indicative or predictive of a disease state in accordance with the instant invention are useful in therapeutic intervention, e.g. as therapeutic modalities in their own right, in the course of therapeutic target recognition, in the development and validation of efficacious 5 therapeutic modalities, e.g when interrogating or developing phage display libraries, and as ligands or receptors for use in conjunction with therapeutic intervention. A diagnosis of Alzheimer's disease is more readily arrived at in the later stages of the disease, but pharmaceutical intervention aimed at symptomatic control at a late stage is extremely difficult, and often problematic. Thus, an ability to diagnose the 10 onset of the disease process so as to maximize the benefit of any therapeutic product is much more effective at an earlier stage. In a further contemplated embodiment of the invention, samples may be taken from a patient at one point in time, as a single sample or as multiple samples, or at different points in time such that analysis is carried out on multiple samples for 15 ongoing analysis. Typically, a first sample is taken from a patient upon presentation with possible symptoms of a disease and analyzed according to the invention. Subsequently, some period of time after presentation, for example, about 3 - 6 months after the first presentation, a second sample is taken and analyzed according to the invention. The data can be used, by way of example, to diagnose or monitor a disease 20 state, determine risk assessment, identify therapeutic avenues, or determine/validate the therapeutic efficacy of an agent, such as a pharmaceutical. Subsequent to the isolation of particular disease state marker sequences as taught by the instant invention, the promulgation of various forms of risk assessment tests are contemplated which will allow physicians to identify asymptomatic patients 25 before they suffer irreversible damage. More particularly, biopolymer markers elucidated via methodologies of the instant invention find utility related to broad areas of disease therapeutics. Such therapeutic avenues include, but are not limited to: 1) utilization and recognition of said biopolymer markers, variants or moieties thereof as direct therapeutic modalities, either alone or in conjunction with an 30 effective amount of a pharmaceutically effective carrier; 2) validation of therapeutic modalities or disease preventative agents as a function of biopolymer marker presence or concentration; WO 03/087835 PCT/CA03/00540 20 3) treatment or prevention of a disease state by formation of disease intervention modalities; e.g. formation of biopolymer/ligand conjugates which intervene at receptor sites to prevent, delay or reverse a disease process; 4) use of biopolymer markers or moieties thereof as a means of elucidating 5 therapeutically viable agents, e.g. from a bacteriophage peptide display library, a bacteriophage antibody library or the like; 5) instigation of a therapeutic immunological response; and 6) synthesis of molecular structures related to said biopolymer markers, moieties or variants thereof which are constructed and arranged to therapeutically 10 intervene in the disease process. A process for identifying or developing therapeutic avenues related to a disease state utilizing any of the above examples may follow results obtained from conducting an analysis inclusive of interacting with a biopolymer including the sequence of the particular disease specific marker or at least one analyte thereof of the present 15 invention. Such treatment or prevention of a disease state by formation of disease intervention modalities may be by the formation of biopolymer/ligand conjugates which intervene at receptor sites to prevent, delay, or reverse a disease process. In addition, a means of elucidating therapeutically viable agents may include the use of a bacteriophage peptide display library or a bacteriophage antibody library. The 20 therapeutic avenues may regulate the presence or absence of the biopolymer including the sequence of the particular disease specific marker or at least one analyte thereof in the present invention. Accordingly, it is an objective of the instant invention to characterize fragments of haptoglobin which are useful in evidencing and categorizing Alzheimer's disease in 25 a patient. It is an additional objective of the instant invention to develop methods and means of disease therapy, including but not limited to: 1) utilization and recognition of said haptoglobin fragments, variants or moieties thereof as direct therapeutic modalities, either alone or in conjunction with an 30 effective amount of a pharmaceutically effective carrier; 2) validation of therapeutic modalities or disease preventative agents as a function presence or concentration of said haptoglobin fragments; WO 03/087835 PCT/CA03/00540 21 3) treatment or prevention of a disease state by formation of disease intervention modalities; e.g. formation of haptoglobin fragment/ligand conjugates which intervene at receptor sites to prevent, delay or reverse a disease process; 4) use of haptoglobin fragments or moieties thereof as a means of 5 elucidating therapeutically viable agents, e.g. from a bacteriophage peptide display library, a bacteriophage antibody library or the like; 5) instigation of a therapeutic immunological response; and 6) synthesis of molecular structures related to said haptoglobin fragments or moieties or variants thereof which are constructed and arranged to therapeutically 10 intervene in the disease process, e.g. by directly determining the three-dimensional structure of said fragments directly from an amino acid sequence thereof. It is another objective of the instant invention to evaluate samples containing a plurality of biopolymers for the presence of Alzheimer's related haptoglobin fragments (disease specific markers) which evidence a link to said disease state. 15 It is a further objective of the instant invention provide at least one purified antibody which is specific to at least one of said isolated haptoglobin fragments or immunologically reactive moieties thereof. It is yet another objective of the instant invention to teach a monoclonal antibody which is specific to at least one of said isolated haptoglobin fragments or 20 immunologically reactive moieties thereof. It is a still further objective of the invention to teach polyclonal antibodies raised against at least one of said isolated haptoglobin fragments or immunologically reactive moieties thereof. It is yet an additional objective of the instant invention to teach a diagnostic kit 25 for determining the presence, concentration, or relative strength/concentration of at least one of said isolated haptoglobin fragments or immunologically reactive moieties thereof It is a still further objective of the instant invention to teach methods for characterizing disease state based upon the identification of at least one of said isolated 30 haptoglobin fragments or immunologically reactive moieties thereof. Other objects and advantages of this invention will become apparent from the following description taken in conjunction with the accompanying drawings wherein WO 03/087835 PCT/CA03/00540 22 are set forth, by way of illustration and example, certain embodiments of this invention. The drawings constitute a part of this specification and include exemplary embodiments of the present invention and illustrate various objects and features thereof 5 BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a partial 2D map of DEAE fractionated sera proteins from Alzheimer's patients; Figure 2 is a partial 2D map of DEAE fractionated sera proteins from age matched controls; 10 Figure 3 is an MS spectrum of the tryptic peptides recovered from spot A of Fig. 1, having molecular weights of 1439, 1580, 1708 and 1815 daltons. DETAILED DESCRIPTION OF THE INVENTION 25[iL sera samples from AD patients and age-matched controls were diluted 20 15 fold with 20mM Tricine buffer, pH 8.5, containing 100 mM NaC1. The diluted samples were loaded on mini DEAE columns equilibrated with sample buffer. After washing, the bound proteins were eluted with the tricine buffer containing 500 mM NaCl and separated by 2D gel electrophoresis. Only the spots that were significanatly different between AD and controls were cut, in-gel tryptic digested, and sequenced 20 using QSTAR PULSARi. The MSMS spectra were analyzed with Mascot software. Two spots of about 20 kDa, labeled A and B in Figure 1, were observed in all AD samples, but were not visible in all age-matched controls, as seen in Figure 2. In accordance with Figure 3, MS spectrum of tryptic peptides of spot A are shown. Four peptides with molecular weights of 1439 (Sequence ID 1), 1708 (Sequence ID 2), 1815 25 (Sequence ID 3) and 1580 (Sequence ID 4) were sequenced and all matched to haptoglobin. Spot B of figure 1 was found to have the same MS soectra as spot A, which indicates that A and B are the same protein. Both the sequence data and molecular eight of the two spots, about 20 kDa, indicate that the two spots are fragments of haptoglobin, derived from the N-terminal, as observed from comparing 30 the full sequence for haptoglobin. As a result of these procedures, the disease specific haptoglobin fragments as WO 03/087835 PCT/CA03/00540 23 identfied were found to be A method for evidencing and categorizing Alzheimer's disease is therefore disclosed. The steps taken include obtaining a sample from a patient, preferably human, and conducting MS analysis on the sample. As a result, at least one 5 haptoglobin fragment sequence is isolated from the sample which undergoes evidencing and categorizing and is compared to the biopolymer marker sequence as disclosed in the present invention. The step of evidencing and categorizing is particularly directed to biopolymer markers or analytes thereof linked to at least one risk of disease development of the 10 patient or related to the existence of a particular disease state. In addition, various kits are contemplated for use by the present invention. One such kit provides for determining the presence of the disease specific biopolymer marker, e.g. fragments of haptoglobin as identified by Sequence ID 1-4. At least one biochemical material is incorporated which is capable of specifically binding with a 15 biomolecule which includes at least the disease specific biopolymier marker or analyte thereof, and a means for determining binding between the biochemical material and the biomolecule. The biochemical material for any of the contemplated kits, by way of example an antibody or at least one monoclonal antibody specific therefore, or biomolecule may be immobilized on a solid support and include at least one labeled 20 biochemical material which is preferably an antibody. The sample utilized for any of the kits may be a fractionated or unfractionated body fluid or a tissue sample. Non limiting examples of such fluids are blood, blood products, urine, saliva, cerebrospinal fluid, and lymph. Further contemplated is a kit for diagnosing, determining risk-assessment, and 25 identifying therapeutic avenues related to a disease state. This kit includes at least one biochemical material which is capable of specifically binding with a biomolecule which includes at least one biopolymer marker including at least one fragment sequence selected from the particularly isolated haptoglobins as set forth herein, or an analyte thereof related to the disease state. Also included is a means for determining 30 binding between the biochemical material and the biomolecule, whereby at least one analysis to determine a presence of a marker, analyte thereof, or a biochemical material specific thereto, is carried out on a sample. As previously described, analysis may be WO 03/087835 PCT/CA03/00540 24 carried out on a single sample or multiple samples. In accordance with various stated objectives of the invention, the skilled artisan, in possession of the specific disease specific marker as instantly disclosed, would readily carry out known techniques in order to raise purified biochemical materials, e.g. 5 monoclonal and/or polyclonal antibodies, which are useful in the production of methods and devices useful as point-of-care rapid assay diagnostic or risk assessment devices as are known in the art. The specific disease markers which are analyzed according to the method of the invention are released into the circulation and may be present in the blood or in any 10 blood product, for example plasma, serum, cytolyzed blood, e.g. by treatment with hypotonic buffer or detergents and dilutions and preparations thereof, and other body fluids, e.g. CSF, saliva, urine, lymph, and the like. The presence of each marker is determined using antibodies specific for each of the markers and detecting specific binding of each antibody to its respective marker. Any suitable direct or indirect assay 15 method may be used to determine the level of each of the specific markers measured according to the invention. The assays may be competitive assays, sandwich assays, and the label may be selected from the group of well-known labels such as radioimmunoassay, fluorescent or chemiluminescence immunoassay, or immunoPCR technology. Extensive discussion of the known immunoassay techniques is not required 20 here since these are known to those of skilled in the art. See Takahashi et al. (Clin Chem 1999;45(8):1307) for a detailed example of an assay. A monoclonal antibody specific against at least one of the disease marker sequences isolated by the present invention may be produced, for example, by the polyethylene glycol (PEG) mediated cell fusion method, in a manner well-known in the 25 art. Traditionally, monoclonal antibodies have been made according to fundamental principles laid down by Kohler and Milstein. Mice are immunized with antigens, with or without, adjuvants. The splenocytes are harvested from the spleen for fusion with immortalized hybridoma partners. These are seeded into microtiter plates where they 30 can secrete antibodies into the supernatant that is used for cell culture. To select from the hybridomas that have been plated for the ones that produce antibodies of interest, the hybridoma supernatant are usually tested for antibody binding to antigens in an WO 03/087835 PCT/CA03/00540 25 ELISA (enzyme linked immunosorbent assay) assay. The idea is that the wells that contain the hybridoma of interest will contain antibodies that will bind most avidly to the test antigen, usually the immunizing antigen. These wells are then subcloned in limiting dilution fashion to produce monoclonal hybridomas. The selection for the 5 clones of interest is repeated using an ELISA assay to test for antibody binding. Therefore, the principle that has been propagated is that in the production of monoclonal antibodies the hybridomas that produce the most avidly binding antibodies are the ones that are selected from among all the hybridomas that were initially produced. That is to say, the preferred antibody is the one with highest affinity for the 10 antigen of interest. There have been many modifications of this procedure such as using whole cells for immunization. In this method, instead of using purified antigens, entire cells are used for immunization. Another modification is the use of cellular ELISA for screening. In this method instead of using purified antigens as the target in the ELISA, 15 fixed cells are used. In addition to ELISA tests, complement mediated cytotoxicity assays have also been used in the screening process. However, antibody-binding assays were used in conjunction with cytotoxicity tests. Thus, despite many modifications, the process of producing monoclonal antibodies relies on antibody binding to the test antigen as an endpoint. 20 The purified monoclonal antibody is utilized for immunochemical studies. Polyclonal antibody production and purification utilizing one or more animal hosts in a manner well-known in the art can be performed by a skilled artisan. Another objective of the present invention is to provide reagents for use in diagnostic assays for the detection of the particularly isolated disease specific marker 25 sequences of the present invention. In one mode of this embodiment, the marker sequences of the present invention may be used as antigens in immunoassays for the detection of those individuals suffering from the disease known to be evidenced by said marker sequence. Such assays may include but are not limited to: radioimmunoassay, enzyme-linked 30 immunosorbent assay (ELISA), "sandwich" assays, precipitin reactions, gel diffusion immunodiffusion assay, agglutination assay, fluorescent immunoassays, protein A or G immunoassays and immunoelectrophoresis assays.

WO 03/087835 PCT/CA03/00540 26 According to the present invention, monoclonal or polyclonal antibodies produced against the disease specific marker sequence of the instant invention are useful in an immunoassay on samples of blood or blood products such as serum, plasma or the like, cerebrospinal fluid or other body fluid, e.g. saliva, urine, lymph, and 5 the like, to diagnose patients with the characteristic disease state linked to said marker sequence. The antibodies can be used in any type of immunoassay. This includes both the two-site sandwich assay and the single site immunoassay of the non-competitive type, as well as in traditional competitive binding assays. Particularly preferred, for ease and simplicity of detection, and its quantitative 10 nature, is the sandwich or double antibody assay of which a number of variations exist, all of which are contemplated by the present invention. For example, in a typical sandwich assay, unlabeled antibody is inmmnobilized on a solid phase, e.g. microtiter plate, and the sample to be tested is added. After a certain period of incubation to allow formation of an antibody-antigen complex, a second antibody, labeled with a 15 reporter molecule capable of inducing a detectable signal, is added and incubation is continued to allow sufficient time for binding with the antigen at a different site, resulting with a formation of a complex of antibody-antigen-labeled antibody. The presence of the antigen is determined by observation of a signal which may be quantitated by comparison with control samples containing known amounts of antigen. 20 Antibodies may also be utilized against the disease specific markers, as haptens, to create an antibody response against the protein to which it binds, thereby identifying targets for treatment of the disease or a sub-class thereof. Lastly, the markers and associated antibodies provide a tool for monitoring the progress of a patient during a therapeutic treatment, so as to determine the usefulness 25 of a novel therapeutic agent. All patents and publications mentioned in this specification are indicative of the levels of those skilled in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by 30 reference. It is to be understood that while a certain form of the invention is illustrated, it is not to be limited to the specific form or arrangement herein described and shown. It WO 03/087835 PCT/CAO3/00540 27 will be apparent to those skilled in the art that various changes may be made without departing from the scope of the invention and the invention is not to be considered limited to what is shown and described in the specification and drawings/figures. One skilled in the art will readily appreciate that the present invention is well 5 adapted to carry out the objectives and obtain the ends and advantages mentioned, as well as those inherent therein. The oligonucleotides, peptides, polypeptides, biologically related compounds, methods, procedures and techniques described herein are presently representative of the preferred embodiments, are intended to be exemplary and are not intended as limitations on the scope. Changes therein and other 10 uses will occur to those skilled in the art which are encompassed within the spirit of the invention and are defined by the scope of the appended claims. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out 15 the invention which are obvious to those skilled in the art are intended to be within the scope of the following claims.

Claims (38)

CLAIMS What is claimed is:
1. Claim 1. A biopolymer marker which is an isolated haptoglobin fragment selected from the group consisting of Sequence ID 1 , Sequence ID 2, Sequence ID 3, Sequence ID 4, or at least one analyte thereof useful in diagnosing at least one particular disease state.
2. Claim 2. The biopolymer marker of claim 1 wherein said disease state is Alzheimers disease.
3. Claim 3. A method for evidencing and categorizing Alzheimer's disease comprising: obtaining a sample from a patient; conducting mass spectrometric analysis on said sample; evidencing and categorizing at least one biopolymer marker sequence or analyte thereof isolated from said sample; and, comparing said at least one isolated biopolymer marker sequence or analyte thereof to the biopolymer marker sequence as set forth in claim 1 ; wherein correlation of said isolated biopolymer marker and said biopolymer marker sequence as set forth in claim 1 evidences and categorizes Alzheimer's disease.
4. Claim 4. The method of claim 3, wherein said step of evidencing and categorizing is particularly directed to biopolymer markers or analytes thereof linked to at least one risk of disease development of said patient.
5. Claim 5. The method of claim 3 , wherein said step of evidencing and categorizing is particularly directed to biopolymer markers or analytes thereof related to the existence of a particular disease state.
6. Claim 6. The method of claim 3, wherein the sample is an unfractionated body fluid or a tissue sample.
7. Claim 7. The method of claim 3, wherein said sample is at least one of the group consisting of blood, blood products, urine, saliva, cerebrospinal fluid, and lymph.
8. Claim 8. The method of claim 3, wherein said mass spectrometric analysis is selected from the group consisting of Surface Enhanced Laser Desorption Ionization (SELDI) mass spectrometry (MS), Maldi Qq TOF, MS/MS, TOF-TOF, and ESI-Q-TOF or an ION-TRAP.
9. Claim 9. The method of claim 3, wherein said patient is a human.
10. Claim 10. A diagnostic assay kit for determining the presence of the biopolymer marker or analyte thereof of claim 1 comprising: at least one biochemical material which is capable of specifically binding with a biomolecule which includes at least said biopolymer marker or analyte thereof, and means for determining binding between said biochemical material and said biomolecule; whereby at least one analysis to determine a presence of a marker, analyte thereof, or a biochemical material specific thereto, is carried out on a sample.
11. Claim 11. The diagnostic assay kit of claim 10, wherein said biochemical material or biomolecule is immobilized on a solid support.
12. Claim 12. The diagnostic assay kit of claim 10 including: at least one labeled biochemical material.
13. Claim 13. The diagnostic assay kit of claim 10, wherein said biochemical material is an antibody.
14. Claim 14. The diagnostic assay kit of claim 12, wherein said labeled biochemical material is an antibody.
15. Claim 15. The diagnostic assay kit of claim 10, wherein the sample is an unfractionated body fluid or a tissue sample.
16. Claim 16. The diagnostic assay kit of claim 10, wherein said sample is at least one of the group consisting of blood, blood products, urine, saliva, cerebrospinal fluid, and lymph.
17. Claim 17. The diagnostic assay kit of claim 10, wherein said biochemical material is at least one monoclonal antibody specific therefore.
18. Claim 18. A kit for diagnosing, determining risk-assessment, and identifying therapeutic avenues related to a disease state comprising: at least one biochemical material which is capable of specifically binding with a biomolecule which is an isolated haptoglobin fragment selected from the group consisting of Sequence ID 1, Sequence ID 2, Sequence ID 3, Sequence ID 4, or at least one analyte thereof related to said disease state; and means for determining binding between said biochemical material and said biomolecule; whereby at least one analysis to determine a presence of a marker, analyte thereof, or a biochemical material specific thereto, is carried out on a sample.
19. Claim 19. The kit of claim 18, wherein said biochemical material or biomolecule is immobilized on a solid support.
20. Claim 20. The kit of claim 18 including: at least one labeled biochemical material.
21. Claim 21. The kit of claim 18, wherein said biochemical material is an antibody.
22. Claim 22. The kit of claim 20, wherein said labeled biochemical material is an antibody.
23. Claim 23. The kit of claim 18, wherein the sample is an unfractionated body fluid or a tissue sample.
24. Claim 24. The kit of claim 18, wherein said sample is at least one of the group consisting of blood, blood products, urine, saliva, cerebrospinal fluid, and lymph.
25. Claim 25. The kit of claim 18, wherein said biochemical material is at least one monoclonal antibody specific therefore.
26. Claim 26. The kit of claim 18, wherein said diagnosing, determining risk assessment, and identifying therapeutic avenues is carried out on a single sample.
27. Claim 27. The kit of claim 18, wherein said diagnosing, determining risk assessment, and identifying therapeutic avenues is carried out on multiple samples such that at least one analysis is carried out on a first sample and at least another analysis is carried out on a second sample.
28. Claim 28. The kit of claim 27, wherein said first and second samples are obtained at different time periods.
29. Claim 29. Polyclonal antibodies produced against a marker which is an isolated haptoglobin fragment selected from the group consisting of Sequence ID 1, Sequence ID 2, Sequence ID 3, Sequence ID 4, or at least one analyte thereof in at least one animal host.
30. Claim 30. An antibody that specifically binds a biopolymer including a marker which is an isolated haptoglobin fragment selected from the group consisting of Sequence ID 1, Sequence ID 2, Sequence ID 3, Sequence ID 4, or at least one analyte thereof.
31. Claim 31. The antibody of claim 30 that is a monoclonal antibody.
32. Claim 32. The antibody of claim 30 that is a polyclonal antibody.
33. Claim 33. A process for identifying therapeutic avenues related to a disease state comprising: conducting an analysis as provided by the kit of claim 18; and interacting with a biopolymer which is an isolated haptoglobin fragment selected from the group consisting of Sequence ID 1, Sequence ID 2, Sequence ID 3, Sequence ID 4, or at least one analyte thereof; whereby therapeutic avenues are developed.
34. Claim 34. The process for identifying therapeutic avenues related to a disease state in accordance with claim 33, wherein said therapeutic avenues regulate the presence or absence of the biopolymer which is an isolated haptoglobin fragment selected from the group consisting of Sequence ID 1, Sequence ID 2, Sequence ID 3, Sequence ID 4, or at least one analyte thereof.
35. Claim 35. The process for identifying therapeutic avenues related to a disease state in accordance with claim 33, wherein said therapeutic avenues developed include at least one avenue selected from a group consisting of 1 utilization and recognition of said biopolymer markers, variants or moieties thereof as direct therapeutic modalities, either alone or in conjunction with an effective amount of a pharmaceutically effective carrier; 2)validation of therapeutic modalities or disease preventative agents as a function of biopolymer marker presence or concentration; 3)treatment or prevention of a disease state by formation of disease intervention modalities; 4)use of biopolymer markers or moieties thereof as a means of elucidating therapeutically viable agents, 5)instigation of a therapeutic immunological response; and 6) synthesis of molecular structures related to said biopolymer markers, moieties or variants thereof which are constructed and arranged to therapeutically intervene in said disease state.
36. Claim 36. The process for identifying therapeutic avenues related to a disease state in accordance with claim 35, wherein said treatment or prevention of a disease state by formation of disease intervention modalities is the formation of biopolymer/ligand conjugates which intervene at receptor sites to prevent, delay or reverse a disease process.
37. Claim 37. The process for identifying therapeutic avenues related to a disease state in accordance with claim 35, wherein said means of elucidating therapeutically viable agents includes use of a bacteriophage peptide display library or a bacteriophage antibody library.
38. Claim 38. A process for regulating Alzheimer's disease by controlling the presence or absence of a biopolymer which is an isolated haptoglobin fragment selected from the group consisting of Sequence ID 1 , Sequence ID 2, Sequence J-D 3, Sequence ID 4, or at least one analyte thereof.