Link:  Pharm/Biotech Resources

Title:  Purified antigen for Alzheimer's disease and methods of obtaining and using same

United States Patent:  6,864,062

Issued:  March 8, 2005

Inventors:  Zinkowski; Raymond P. (Northbrook, IL); Kerkman; Daniel J. (Lake Villa, IL); Kohnken; Russell E. (Skokie, IL); DeBernardis; John F. (Lindenhurst, IL); Davies; Peter (Rye, NY)

Assignee:  Molecular Geriatrics Corporation (Vernon Hills, IL)

Appl. No.:  017822

Filed:  December 12, 2001

Abstract

The invention relates, among other things, a preparation comprising Alzheimer's disease antigen (A68), as well as methods of obtaining this purified antigen, and methods of using this purified antigen, for instance, for diagnosing Alzheimer's disease and for detecting human autoantibodies to the Alzheimer disease antigen. The antigen preparation according to the invention is purified in that it is substantially free of immunoglobulin G. The invention further relates to methods of making Alzheimer disease antigens that can be used instead of or along with the A68 antigen preparation (e.g., for diagnosing AD), such as recombinant human tau, tau isolated from various species including human, and phosphorylated recombinant human tau or isolated tau, as well as A68 anti-idiotypic antibodies.

Description of the Invention

TECHNICAL FIELD OF THE INVENTION

The invention relates to a preparation comprising Alzheimer's disease antigen (A68), to methods of obtaining this purified antigen, and to methods of using this purified antigen preparation, for instance, in diagnosing Alzheimer's disease. The antigen preparation according to the invention is purified in that it is substantially free of immunoglobulin G. The invention further relates to methods of making Alzheimer disease antigens that can be used instead of or along with the A68 antigen preparation (e.g., for diagnosing AD), such as recombinant human tau, tau isolated from various species including human, and phosphorylated recombinant human tau or isolated tau, as well as A68 anti-idiotypic antibodies.

BACKGROUND OF THE INVENTION

Alzheimer's disease (AD) is a progressive neurodegenerative disorder affecting 7% of the population over 65 years of age and characterized clinically by progressive loss of intellectual function and pathologically by a continuing loss of neurons from the cerebral cortex. This pathological impairment usually is correlated with increased numbers of neuritic plaques in the neocortex and with the loss of presynaptic markers of cholinergic neurons. Neuritic plaques are composed of degenerating axons and nerve terminals, as well as possible astrocytic elements, and these plaques often exhibit a central amyloid core.

Another characteristic pathological feature of Alzheimer's disease is development of neurofibrillary tangles. A neurofibrillary tangle is an intraneuronal mass composed of normal intermediate filaments and paired helical filaments having unusual properties, which twist and form tangles. Neurofibrillary tangles are comprised of several different proteins.

Neurochemical studies confirm that neurotransmitter systems are deleteriously affected by Alzheimer's disease. The most consistently and severely affected system is that of the cholinergic neurons located in the Nucleus Basalis of Meynert. In addition, a reduction in somatostatin, substance P, and corticotropin releasing factor are observed.

None of the above-mentioned pathologic states such as neurochemical alterations, neuritic plaques or neurofibrillary tangles are unique to Alzheimer's disease. These impairments also occur in the brains of normal aged individuals and are associated with other diseases such as Guam Parkinson's Disease, Dementia Pugilistica and Progressive Supra-nuclear Palsy. For example, paired helical filaments, the twisted filaments that form the tangles and fill the neurites of plaques, also occur in certain other diseases. In fact, immunologic studies have shown that AD epitopes of paired helical filaments exist in Pick bodies, the spherical structures found in affected neurons in the temporal cortex of brains affected by Pick's Disease. In addition, the densities of neurofibrillary tangles and neuritic plaques within the cerebral cortex of an Alzheimer's disease patient correlates only weakly with the stages of the illness.

Accordingly, the diagnosis of Alzheimer's disease has been extremely difficult. Ante-mortem diagnosis of the disease is performed primarily by exclusion of other diseases. An article entitled, "The Neurochemistry of Alzheimer's Disease and Senile Dementia", by Peter Davies in Medicinal Research Reviews, Vol. 3, No. 3, pp. 221-236 (1983), discusses Alzheimer's disease and at page 223 states:

The problem in the diagnosis of Alzheimer's disease is that there is no positive test: the clinician has to rule out other causes of dementia such as strokes, microvascular disease, brain tumors, thyroid dysfunction, drug reactions, severe depression and a host of other conditions that can cause intellectual deficits in elderly people. Only when all of these problems have been eliminated as a cause of the symptoms should a diagnosis of Alzheimer's disease be accepted.

Post-mortem diagnosis of Alzheimer's disease has been based on determination of the number of neuritic plaques and tangles in brain tissue using specialized staining techniques. However, such diagnostic methods, based on neurohistopathological studies, require extensive staining and microscopic examination of several brain sections. Moreover, the plaques and tangles are not confined to individuals having Alzheimer's disease, but also may occur in the brains of normal, elderly individuals or individuals with other diseases. Thus, a more definitive and reliable method for making the diagnosis is needed.

U.S. Pat. No. 4,666,829 issued to Glenner et al. discloses attempts to identify an antigen specific for Alzheimer's disease. However, the antigen described by Glenner et al. also is present in adults of advanced age who do not have Alzheimer's disease (see Ghanbari et al., Journal of the American Medical Association, 263, pp. 2907-2910 (1990)). Therefore, a need still exists for a method of diagnosing Alzheimer's disease as distinct from other diseases or age-related indicia.

Similarly, U.S. Pat. No. 5,492,812 issued to Voorheis et al. describes a diagnostic method for Alzheimer's disease that is carried out by screening for tau peptides in the blood of a patient. This method calls for the use of an antibody or Fab fragment that specifically binds tau peptide derived from either the amino terminal 200 amino acids or carboxy terminal 50 amino acids of a tau protein. This method requires that "the whole of the 200 amino acid N-terminal residues of the various tau proteins as well as some portion of their 50 amino acid most C-terminal residues will be released when cleaved from the filaments by ubiquitin-recognizing proteases or other proteases during degeneration and rupture of the affected neurons" (col. 5, lines 12-19). The method further requires that the cleaved segments find their way into body fluids outside the brain (col. 5, lines 19-22). Accordingly, the method is dependent upon, and is ineffective in the absence of, proteolytic fragmentation of the tau complex of proteins. The method further is dependent upon, and is ineffective in the absence of, the subsequent release of the proteolytic fragments into body fluids. It thus is desirable that a direct means of assay for Alzheimer disease-associated antigens be identified, particularly a means that does not require proteolytic fragmentation and subsequent release into the bloodstream of fragments.

Along these lines, PCT International Application WO 96/20218 of Ghanbari et al. describes the isolation of an antigen associated with Alzheimer's disease and a monoclonal antibody directed against this antigen. This antigen is specific for Alzheimer's disease, being present in high quantities in Alzheimer's Disease patients, and being nearly non-detectable in non-Alzheimer's Disease patients. However, the antigen is described as being only "partially purified" in the preparation of Ghanbari et al., consisting of an aggregate of proteins, with the predominant protein having a molecular weight of about 68,000 daltons, and including tau and hyperphosphorylated tau. Accordingly, for some applications, a more purified preparation of this Alzheimer's Disease antigen may be desirable and/or required.

It therefore is an object of this invention to provide, among other things, a purified preparation of an Alzheimer's disease antigen. It is another object of this invention to provide, among other things, a method of diagnosing Alzheimer's disease using the purified preparation. It is a further object of this invention to provide, methods of obtaining the purified preparation of the Alzheimer's disease antigen. These and other objects and advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.

BRIEF SUMMARY OF THE INVENTION

The invention provides, among other things, a preparation comprising Alzheimer's disease antigen (A68), as well as methods of obtaining this purified antigen (Ag), and methods using the purified Ag, for instance, for diagnosing Alzheimer's Disease (AD). This Ag is purified in that it is substantially free of immunoglobulin G (IgG). The invention additionally provides methods of making AD Ags that can be used instead of or along with A68 (e.g., for binding AD autoantibodies), such as recombinant human tau, tau isolated from various species including human, and phosphorylated recombinant human tau or isolated tau, as well as A68 anti-idiotype antibodies (Abs). The invention further describes treatments of these Ags that enhance their reactivity with autoantibodies directed against A68. These treatments include treatment with hypcricin, free fatty acids, and/or hydroxynonenal or other advanced glycation end products.

The invention also describes methods using a bovine microtubule-associated protein preparation (MAPf) for diagnosing AD. The invention describes analysis of autoantibody reactivity with both A68 and MAPf such that either a quantitative or a qualitative analysis of these reactivities provides a diagnosis for AD.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

By "individuals with Alzheimer's disease" is meant individuals or patients suffering from, affected by, or manifesting the clinical symptoms of the disease. According to the invention, a diagnosis of "Alzheimer's disease" is based on accepted standards of clinical diagnosis. Preferably, an individual according to the invention diagnosed with Alzheimer's disease is one that has met most (i.e., a majority) of the generally accepted criteria for diagnosis of Alzheimer's disease. Desirably, because of certain atypical features, such an individual may be described by a physician as `possible Alzheimer's disease`, and at least 75% to 85% of these individuals would be found at autopsy to have suffered from Alzheimer's disease. Even more preferably, an individual according to the invention diagnosed with Alzheimer's disease is one that has met the best available clinical criteria: for the diagnosis of Alzheimer's disease, and may be described by a physician as "probable Alzheimer's disease`. When such a diagnosis is made by one skilled in the art, optimally about 90% of these patients would be found on autopsy to have Alzheimer's disease.

Alzheimer's Disease Antigen Preparation

The present invention pertains, inter alia, to the Alzheimer's disease antigen designated A68. The antigen has been described as obtained from the brains and cerebrospinal fluid (CSF) of Alzheimer patients, but is referred to herein as the "Alzheimer antigen" regardless of where it is found in a person if the antigen has the properties set forth herein. It has been discovered that one such Alzheimer antigen is a protein, so that the Alzheimer antigen is also referred to herein as an "Alzheimer protein" when the protein property is a prominent factor in the discussion. The autoantibodies which are immunologically reactive with the Alzheimer antigen are referred to herein as "Alzheimer antibodies." The Alzheimer's disease antigen also refers to components of a preparation containing this antigen that have been modified to increase their reactivity with antibodies directed against the antigen, as further described below.

The identification, isolation, and characterization of the Alzheimer's disease antigen as a "partially purified preparation of A68 antigen" has been described in PCT International Application WO 96/20218. In particular, PCT International Application WO 96/20218 describes the confinement of this antigen to Alzheimer's disease patients as compared to normal individuals, and individuals having other impairments. Generally, the Alzheimer antigen is found in Alzheimer patients while being present in much reduced (or non-measurable) quantities in non-Alzheimer patients, including patients suffering from other neurologic diseases.

The antigen of the invention which is associated with Alzheimer's disease (i.e., the Alzheimer's antigen) is an aggregate of several proteins, and the major protein species have an apparent molecular weight of about 68,000 daltons on a reducing SDS gel. The aggregate migrates electrophoretically as a band or bands on sodium dodecyl sulfate polyacrylamide gel with an apparent M_r of from about 60 to about 70 kDa. A68 desirably is prepared from human brain (typically frozen), most often from cerebral cortex from an Alzheimer's disease patient. Other properties of the A68 antigen are set forth in PCT International application WO 96/20218, and include, but are not limited to, the following characteristics: immunologically reactive with a monoclonal antibody produced by the hybridoma cell line identified as ATCC No. HB9205 (i.e., ALZ-50, further described below); has an isoelectric point of about 6 in reduced or non-reduced form; binds to an affi-Blue column; is at least 50% soluble in a solution of 0.01 M sodium phosphate, 0.14 M sodium chloride and 1 mM phenyl methyl sulfonyl flouride at pH 6.8, and precipitates in 50% saturated ammonium sulfare at 4^oC.

Since its first description, the Alzheimer's antigen has been additionally referred to as A68, tau, hyperphosphorylated tau (Lee et al., Science 251: 675-678, 1991), abnormally phosphorylated tau (Grundke-Iqbal et al., Proc. Natl. Acad. Sci. 83: 4913-4917, 1986), soluble PHF (Greenberg and Davies, Proc Natl. Acad. Sci 87: 5827-5831, 1990), PHF tau (Greenberg et al., J. Biol. Chem. 267: 564-569, 1992), and Alzheimer's Disease Associated Protein (ADAP) (Ghanbari et al., JAMA 263: 2907-2910, 1990) All terms are deemed to be equivalent when referring to the Alzheimer's antigen herein. It contains tau and phosphorylated tau. Thus, according to the invention, A68 refers to a form of the microtubule-associated protein tau which, in Alzheimer's disease, is the primary protein constituent of paired helical filaments Relative to normal tau (also a microtubule protein), it is hyperphosphorylated and exhibits an altered conformation.

A68 is obtained in only a "partially purified" form as described in Example 1 of PCT International Application WO 96/20218. By comparison, as described herein, in the process of obtaining a purified preparation of A68 antigen, it was discovered that, not only did the preparation comprise expected elements that needed to be removed (e.g., proteins, etc.), but also, that the preparation surprisingly contained immunoglobulin G (IgG). Removal of such IgG substantially and surprisingly increases the effectiveness of the antigen preparation, and its effectiveness for use in an assay.

Accordingly, the present invention provides a protein preparation consisting essentially of an antigen that is immunologically reactive with a monoclonal antibody produced by the hybridoma cell line identified as ATCC No. HB9205 (i.e., Al2-50), wherein the preparation is substantially free of immunoglobulin G. As used herein, "substantially free of immunoglobulin G" means a preparation having an amount of immunoglobulin G that preferably is equal to or less than about 0.05% of the total protein present in the preparation, and even more preferably is less than or equal to about 0.0015% of the total protein present in the preparation, and/or a total immunoglobulin amount that desirably is less than about 500 pg of immunoglobulin G per .mu.g of A68 (i.e., when A68 amount is assessed by Western analysis with use of the Alz-50 antibody, or by other appropriate means), and optimally is less than about 15 pg of immunoglobulin G per .mu.g of A68. In particular, by substantially free of immunoglobulin G desirably is meant a level of immunoglobulin that does not interfere with the assays of the invention.

Further, the present invention desirably provides a purified preparation of antigen, which preparation is a diagnostic marker of Alzheimer's disease, wherein the antigen preferably comprises a major polypeptide species that:

(a) has an isoelectric point of about 6 in reduced or non-reduced form;

(b) binds to an affi-Blue column;

(c) is at least 50% soluble in a solution of 0.01 M sodium phosphate, 0.14 M sodium chloride and 1 mM phenyl methyl sulfonyl fluoride (PMSF) at pH 6.8, and precipitates in 50% saturated ammonium sulfate at 4^oC.;

(d) is immunologically reactive with a monoclonal antibody produced by the hybridoma cell line identified as ATCC No. HB9205 (i.e., Alz-50); and

(e) is substantially free of immunoglobulin G (i.e., as described above).

Process for Obtaining an Alzheimer Antigen Protein Preparation

The present invention thus provides a process for obtaining an Alzheimer antigen protein preparation. As a first step, desirably a partially purified preparation is obtained. A partially purified A68 preparation desirably is obtained by a method that comprises:

(a) obtaining a sample of cortical brain tissue containing the antigen;

(b) homogenizing the sample in buffer to obtain a homogenate;

(c) removing particulate matter from the homogenate;

(d) removing the Ag from the homogenate by contacting the homogenate with an antibody (i.e., immobilized AD antibody, as in affinity purification) under conditions wherein the antigen and antibody bind to form an antigen-antibody complex; and

(e) eluting the antigen from the antigen-antibody complex to obtain a partially purified protein preparation.

More specifically, this method of preparation desirably is carried out by homogenization of tissue in about 5 volumes of an aqueous buffer such as Tris buffered saline (TBS), preferably where the buffer further contains protease and phosphatase inhibitors. The homogenate optimally is fractionated, e.g., by centrifugation at about 27,000xg for about 60 minutes at about 4^oC. Desirably, the supernatant is collected, and passed over an affinity column, preferably in an iterative fashion for about 16 hours at about 4^oC. Optimally the affinity column is a MC1 column, desirably which is prepared by using a purified mouse monoclonal antibody (e.g., MC1 antibody) which reacts specifically with A68, and coupling the antibody to Affigel-10, according to manufacturer's instructions. Preferably, A68 is specifically removed from the supernatant by attaching to the MC1 column matrix. Following extensive washing with TBS, optimally A68 is eluted from the MC1 column using, for instance, 3 M KSCN. The A68 preparation preferably is subsequently dialyzed against buffer (e.g., TBS) and stored at -80^oC. Alternative means of obtaining partially purified A68 preparations are set out, for example, in PCT International Application WO 96/20218.

The preparation obtained by this process is highly enriched in A68, but contains amounts of other proteins. In particular, this preparation contains endogenous human immunoglobulins that comprise approximately from about 1 to about 5% of the total protein. These immunoglobulins interfere with the ability to detect serum autoantibodies to A68 by either Western analysis or ELISA. Such an A68 protein preparation is not substantially pure according to the invention, but is only partially purified.

Thus, it is necessary according to the invention to remove the contaminating immunoglobulins prior to analysis of serum autoantibodies, or prior to use of the A68 protein preparation. The invention accordingly provides an additional step (f) in the process described above which step comprises removing immunoglobulin G from the eluent to obtain the antigen preparation that is substantially free of immunoglobulin G. This is accomplished, for instance, by incubation of the A68 preparation, desirably with either Protein A or Protein G, and preferably with both Protein A and Protein G, optimally which have first been immobilized on agarose beads. Desirably, about 1 ml of a partially pure A68 preparation is added to packed Protein A beads (generally about 75 .mu.l) and packed Protein G beads (generally about 75 .mu.l). The sample preferably is placed on a rotator, optimally for about 8 hours at 4^oC. After incubation, desirably the beads are spun out of solution, e.g., using a microcentrifuge at about 14,000xg for about 3 minutes. The A68 supernatant preferably then is transferred to a new tube containing packed Protein A and Protein G beads (generally about 75 .mu.l of each) and allowed to incubate, optimally for up to 16 hours on a rotator at 4^oC. Subsequently, the Protein A and G beads are pelleted, optionally using a microcentrifuge at about 14,000xg for about 3 minutes. The A68 supernatant is then stored (e.g., in 250 .mu.l aliquots at -80^oC.).

Alternately, immunoglobulin G desirably is removed by incubation of the protein preparation with an immunoglobulin G removal method that is substantially equivalent to use of both Protein A and Protein G, such as, for instance, use of fixed bacteria or Pansorbin.

Total protein concentration of the purified A68 antigen preparation desirably is determined by a Coomassie Blue protein assay, for instance, using Coomassie Plus Protein Assay Reagent (Pierce catalog #23236) and the microassay as described by the manufacturer with known concentrations of BSA as a standard. Subsequently, reactivity of the antigen preparation with the A68 specific monoclonal antibody MC15 desirably is determined by chemiluminescent indirect ELISA, and the activity expressed as relative light units (rlu)/ng protein. Protein concentrations of subsequent A68 antigen preparations desirably can be estimated by comparing MC15 reactivity of the subsequent antigen preparations against MC15 reactivity of the initial lot. This method circumvents problems of variability typically encountered when A68 protein concentration is measured using a Coomassie Blue protein assay.

Determination of the immunoglobulin G content of the Protein A/G treated A68 (or the partially purified A68 preparation) desirably can be done by chemiluminescent indirect ELISA using purified human IgG (Sigma, St. Louis, Mo.) as a standard, or by other appropriate means.

Using these methods of the invention, an A68 antigen preparation can be obtained that is substantially free of immunoglobulin G--i.e., which has an amount of immunoglobulin G that preferably is equal to or less than about 0.05% (and even more preferably is equal to or less than about 0.0015%) of the total protein of the preparation, and/or desirably which has less than about 500 pg of immunoglobulin G (and even more preferably has less than about 15 pg of immunoglobulin G) per .mu.g of A68. When the antigen is used for Western blot analysis, the purified A68 preparation preferably contains less than about 500 pg of IgG, and more desirably contain less than about 15 pg IgG per amount of antigen loaded per gel lane. It further is envisioned that substantially immunoglobulin G-free antigen preparations also can be achieved using methods for "blocking" or "typing-up" any IgG present, or other appropriate means of removal of the IgG. Such methods include, but are not limited to: caprylic acid precipitation of A68; adsorption using an anti-human IgG resin; and use of Pansorbin.

Assays Making Use of the Alzheimer's Antigen Protein Preparation

This invention is directed inter alia to the detection of antibodies (autoantibodies) specific for Alzheimer's disease-associated antigen, which antigen is present in individuals with Alzheimer's disease and substantially absent from individuals who do not have Alzheimer's disease. The present invention provides specific and sensitive assays for diagnosis of Alzheimer's disease (i.e., for detecting the presence of autoantibodies to the AD antigen). The methods of the invention overcome the drawbacks of the prior art which require a diagnosis based on a process of elimination of other disorders, and thus provide clarity to an assessment of treatment options.

Thus, this invention desirably is directed to detection of antibodies (i.e., autoantibodies) towards an Alzheimer's disease-associated antigen present in individuals with Alzheimer's disease and substantially absent from individuals who do not have Alzheimer's disease. This invention is also directed to the detection of autoantibodies specific for tau proteins from human, as well as, other species (i.e., desirably a mammalian species), such as bovine tau. Such tau protein antibodies are present in individuals who do not have AD, and are substantially absent from individuals with AD. The present invention provides a specific and sensitive assay for diagnosis of AD. Diagnosis is made based on the relative levels of Alzheimer's antibodies and MAPf autoantibodies present in body fluids, such as serum, plasma, and cerebrospinal fluid, such that individuals with substantial levels of Alzheimer's antibodies, and without substantial levels of autoantibodies to MAPf, are diagnosed as having AD.

This method accordingly provides for the use of Protein A/G treated A68 (i.e., an A68 preparation that is substantially free of immunoglobulin G) as an antigen for detecting autoantibodies which are diagnostic for Alzheimer's disease. It is a novel and unexpected finding of the invention that A68 purified according to the invention to be substantially free of IgG, but not partially purified A68 preparations, can be employed in various methods (e.g., Western blot analysis, chemiluminescent sandwich ELISA assay, chemiluminescent indirect ELISA assay, direct ELISA assay, immunoprecipitation assays, and others) to detect autoantibodies specific for Alzheimer's disease. It will be apparent to one skilled in the art that these assays may be conducted in many ways including direct and/or indirect ELISA, sandwich ELISA, Western blot analysis, etc. Furthermore, it will be apparent that competition assays with any of the various Alzheimer's disease antigens and/or their precursors or related proteins, either alone or in combination, can aid in the detection of autoantibodies diagnostic for Alzheimer's disease.

In these assays according to the invention, desirably, antibodies directed against the Alzheimer disease antigen can be employed. These antibodies include monoclonal antibodies (e.g., as described in PCT International Application WO 96/20218) as well as serum autoantibodies. However, one particularly preferred antibody is ALZ-50 secreted by hybridoma No. HB9205, which was deposited under the Budapest Treaty on Sep. 17, 1986 with the American Type Culture Collection, 10801 University Blvd., Manassas, Va. 20110-2209. ALZ-50 has become the standard reagent for detecting the presence of Alzheimer's disease in this field. (See, for example, Wood et al., Histochemical Journal, 21, No. 11, pp. 659-662 (1989); Itagaki et al., Annals of Neurology, 26, No. 5, pp.685-689 (1989); Beach et al., Brain Research, 501, No. 1, pp. 171-175 (1989); Love et al., Journal of Neuropathology and Experimental Neurology, 47, No. 4, pp. 393-405 (1988); Nukina et al., Neuroscience Letters, 87, No. 3, pp. 240-246 (1988); and Hyman et al., Brain Research, 450, pp. 392-397 (1988).)

In the assay methods described herein, the sample used in the assay of the invention is preferably selected from the group consisting of brain tissue, pre or post-mortem, cerebrospinal fluid, urine and blood. In a preferred embodiment, the sample comprises serum. The methods described herein for use with serum are applicable to CSF and urine. The following is another test procedure is believed to be suitable for detecting the presence of autoantibodies to Alzheimer antigen in the blood or other body fluids of a person having Alzheimer's disease. The procedure is similar to the procedure used in the detection of HTLV-III as disclosed in "Immunoassay for the Detection and Quantitation of Infectious Human Retrovirus, Lymphadenopathy-Associated Virus (LAV)", by J. S. McDougal et al., Journal of Immunological Methods, 76, pp. 171-183 (1985). Preferably from about 0.1 .mu.l to about 100 .mu.l of serum is utilized, more preferably 0.25 .mu.l to 10 .mu.l of serum is utilized.

Thus, in terms of a Western analysis for detection of autoantibodies that are present in Alzheimer's disease, the present invention provides such a method that optimally comprises:

(a) obtaining an A68 purified protein preparation according to the invention, and a sample being tested for the presence of the autoantibodies;

(b) electrophoresing the protein preparation on a gel;

(c) transferring the electrophoresed protein preparation to a membrane (e.g., nitrocellulose);

(d) contacting the membrane with a sample being tested for the presence of the autoantibodies such that an antigen-autoantibody complex can form; and

(e) detecting the autoantibodies by the formation of the complex.

Similarly, in the instant invention, a method is provided for determining the presence of autoantibodies specific to Alzheimer's disease in a sample, thereby diagnosing Alzheimer's disease. The method optionally comprises contacting a sample from an individual suspected of having Alzheimer's disease with a purified A68 antigen preparation according to the invention. In terms of a sandwich ELISA assay, this contacting optimally is done after the sample has been allowed to bind to Protein A/G (preferably which has been immobilized on beads, plates, nitrocellulose, fixed bacteria, Pansorbin, and the like). This contacting optionally is done such that the autoantibody is free in solution, and immobilized subsequent to contacting the Ag. Subsequently (e.g., following washing), the mixture desirably is contacted with an antibody specific for an antigenic determinant on the Alzheimer's antigen (e.g., ALZ-50 or monoclonal TG5 specific for A68) and capable of binding so as to produce a complex. According to the invention, the "antibody" can be a portion of an antibody (e.g., a Fab fragment, etc.). The resulting complex then optionally can be separated and recovered from the sample, but preferably, is detected by an appropriate means, e.g. chemiluminescent means, etc.

Optionally, A68 is labeled, as with biotin or radioactive markers by standard protocols, and the complex desirably is measured by detection of that label. This is accomplished, for instance, with reagents such as streptavidin conjugated to horseradish peroxidase in the case of biotin-labeled A68, or through capture of the complex and detection of radioactivity in that complex. Labeling means and means of detecting labels are well known to those skilled in the art.

Accordingly, the invention further provides a method for detecting autoantibodies that are present in Alzheimer's disease comprising:

(a) obtaining a purified A68 protein preparation according to the invention;

(b) contacting the protein preparation with a sample being tested for the presence of the autoantibodies such that an antigen-autoantibody complex can form; and

(c) detecting the autoantibodies by the formation of the complex.

As previously described, the method desirably can be carried out where the presence of the autoantibodies is determined by the presence of the complex (i.e., a qualitative test). Optionally, the method can be carried out where the amount of the complex is measured, and the amount of the autoantibodies is determined by the amount of the complex (i.e., a quantitative test).

Additionally, the method optionally can comprise the further step of contacting the complex with an antibody that is immunologically reactive with an antigenic determinant found on either the autoantibody or the protein preparation such that an antigen-antibody or antibody-autoantibody complex is formed.

The antibodies employed in the methods of the present invention optimally can be made detectable by attaching an identifiable label thereto. The antibody preferably is made detectable by attaching to it an enzyme conjugated to an appropriate substrate which, in turn, catalyzes a detectable reaction. The enzyme may be horseradish peroxidase, beta-galactosidase or alkaline phosphatase. Other means of detection of the antibody include attaching a fluorescent, chemiluminescent, or radiolabel thereto. Alternatively, the antibody may be detected by use of another antibody directed to it, the other antibody being labeled or having an enzyme substrate bound to it. The presence of the detectable antibody (e.g., as an indicator of the complex) may be readily detected using well-known techniques. Thus, if the detectable antibody is linked to an enzyme and introduced to an appropriate substrate, the optical density of the detectable bound antibody is determined using a quantum spectrophotometer. If the detectable antibody is fluorescently labeled, the fluorescent emission may be measured or detected using a fluorometer technique. In a similar manner, if the detectable antibody is radioactively labeled, the bound antibody may be detected using radioactivity detection techniques. By comparing the results obtained using the above-described methods on the test sample with those obtained using the methods on a control sample, the presence of the purified A68 protein preparation/autoantibody complex specific to Alzheimer's disease may be determined. The elevated amount of purified A68 protein preparation/autoantibody specific to Alzheimer's disease is thereby detected and may optionally be quantitated.

The methods for qualitatively or quantitatively determining the Alzheimer's disease antigen/autoantibody complex may be used in the diagnosis of Alzheimer's disease. Utilization of the methods of the present invention is advantageous over prior art methods because the present invention provides simple, sensitive, very specific methods for detecting Alzheimers antigen/autoantibody complex. The Alzheimer's antigen is well-suited for sandwich immunoassay complex formation since it is present in aggregate form and, hence, is multiepitopic. This is in contrast to cross-reactive proteins, which are soluble and usually contain one epitope per protein.

Variations of these and other standard methods for detection of autoantibodies and autoantigens would be evident to one skilled in the art, and are contemplated by the invention.

Method of Making Alzheimer Disease Antigens for Detecting Autoantibodies

The invention also desirably provides a method of increasing the ability of an Alzheimer's disease antigen to detect autoantibodies that are present in Alzheimer's disease, wherein the antigen is tau isolated from various species including human, or is recombinant human tau. The resulting antigen preparation optionally can be employed instead of (or in addition to) the substantially pure A68 preparation of the invention.

One such method comprises phosphorylating the antigen. Preferably the phosphorylation is done using a cell extract prepared from a central nervous system (CNS) cell line, e.g. neuroblastoma cells (especially MSN neuroblastoma cells), optionally which has been treated with a phosphatase inhibitor, such as okadaic acid. Also, desirably the phosphorylation is done using a purified or partially purified kinase which has been associated in the literature with tau phosphorylation. Such kinases which can be used in the context of the invention include, but are not limited to, PKA, GSK, cdc2, cdc25, casein kinase I and II, MAP kinase, and PHF kinase.

The invention further provides a method of increasing the ability of an Alzheimer's disease antigen to detect autoantibodies that are present in Alzheimer's disease, preferably wherein the antigen is tau isolated from various species including human, or is recombinant human tau, or phosphorylated recombinant human tau (Ptau) or phosphorylated isolated tau, and the method comprises optionally treating the antigen with hypericin.

Similarly, the invention desirably provides a method of increasing the ability of an Alzheimer's disease antigen to detect autoantibodies that are present in Alzheimer's disease, wherein the antigen is tau isolated from various species including human, or is recombinant human tau (rht), or is phosphorylated recombinant human tau (phospho-rht) or phosphorylated isolated tau, and the method comprises treating the antigen with free fatty acids. Preferably the fatty acids are unsaturated fatty acids, particularly oleic or linoleic acids, and most preferably arachidonic acid.

Moreover, the invention also provides a method of increasing the ability of an Alzheimer's disease antigen to detect autoantibodies that are present in Alzheimer's disease, wherein the antigen is tau isolated from various species including human, or is recombinant human tau, or phosphorylated is recombinant tau (phospho-rht) or phosphorylated isolated tau, and the method optionally comprises treating the antigen with advanced glycation endproducts, especially where the advanced glycation endproduct is the lipid peroxidation product 4-hydroxy-2-nonenal (HNE).

Anti-Idiotypic Antibodies

The invention further provides a means of obtaining so-called "anti-idiotypic antibodies", which are antibodies that recognize amino acid differences in, and hence are specifically directed to, particular immunoglobulins. In particular, the invention preferably provides means of identifying anti-idiotypic antibodies to A68 Alzheimer's disease antigen-reactive immunoglobulins, starting from either monoclonal antibodies to A68 or human serum autoantibodies to A68. These anti-idiotypic antibodies desirably are employed in the methods of the invention for assaying for Alzheimer's disease.

Accordingly, the invention desirably provides an antibody (e.g., especially a monoclonal antibody) that is immunologically reactive with an antibody (e.g., especially a monoclonal antibody, or human serum autoantibody) directed against A68 antigen. Such an anti-idiotypic antibody, especially an antibody that is immunologically reactive with a monoclonal antibody or human serum autoantibody directed against A68 antigen, desirably is obtained by:

(a) obtaining sera from individuals having high titers of anti-A68 autoantibodies, combining to create a pool, and isolating antibodies from said pool, or, obtaining isolated monoclonal antibodies to A68 antigen;

(b) immunizing mice with the isolated antibodies;

(c) obtaining serum from the mice (i.e., after sufficient time and under sufficient conditions for antibodies to be produced); and

(d) testing the serum to identify mice having high levels of antibodies that are immunologically reactive with an antibody (e.g., a monoclonal antibody or human serum autoantibody) directed against A68 antigen.

The method optionally can be carried out comprising the further steps:

(a) obtaining the spleens of the mice having high levels of antibodies that are immunologically reactive with a monoclonal antibody or human serum autoantibody directed against A68 antigen;

(b) fusing the spleens with myeloma cells and plating onto tissue culture plates;

(c) selecting for fused cells by HAT resistance; and

(d) testing said fused cells for production of antibodies that are immunologically reactive with an antibody (e.g., a monoclonal antibody or human serum autoantibody) directed against A68 antigen. Optimally the method further comprises testing the fused cells for production of antibodies that are not immunologically reactive with antibodies not directed against A68 antigen. Suitable variations of these methods will be apparent to those skilled in the art.

Bovine Tau (MAPf) Preparation and Assays

This invention further desirably provides for the use of a bovine microtubule-associated protein preparation (i.e., MAPf) in conjunction with A68, for instance, in Western blot analysis of sera. Bovine MAPf contains, among other things, 70% MAPs 1 & 2, 20% other MAPs, and 10% Tau MAP isoforms. The current invention, as described herein, makes use of the six Tau MAP isoforms that migrate in the 40-65 kD range on a 10% SDS polyacrylamide gel. The methods for electrophoresis, Western transfer, sera incubation, and detection of bound autoantibody are well known to those skilled in the art.

Thus, desirably according to the invention, an individual can employ for electrophoresis alternating lanes of A68 and MAPf. MAPf preferably is used at the concentration of 1.5 ug total protein/lane. Following electrophoresis, the protein is transferred to an appropriate support, e.g., nitrocellulose, or other membrane. Desirably, patient sera is incubated with the strips of nitrocellulose containing purified A68 protein preparation and strips of nitrocellulose containing MAPf; and bound autoantibodies are then visualized as previously described. Under these conditions, bound autoantibodies to purified A68 protein preparation and the tau isoforms in MAPf desirably are obtained.

Accordingly, the present invention also desirably provides a method for detecting autoantibodies that are present in Alzheimer's disease comprising the steps of:

(a) obtaining a purified A68 protein preparation as previously described, a bovine microtubule associated protein preparation, and a sample being tested for the presence of autoantibodies;

(b) electrophoresing the A68 protein preparation and the bovine microtubule associated protein preparation on separate lanes on a gel;

(c) transferring the electrophoresed A68 protein preparation and the bovine microtubule associated protein preparation to a membrane;

(d) contacting the membrane with a sample being tested for the presence of said autoantibodies such that an autoantibody complex can form with antigen present in the A68 protein preparation and/or with antigen present in the bovine microtubule associated protein preparation; and

(e) detecting the autoantibodies by the formation of the complex(es).

Two methods of analysis preferably are used to assign a diagnosis to each serum tested. In the first method, the total optical density (OD)xmm signal from purified A68 protein preparation is divided by the total ODxmm signal from the tau isoforms. In general, the sample is assigned the diagnosis of AD, or non-AD on the basis of this ratio.

The second method optimally takes into account not only optical density measurements, but also the number of MAPf tau isoforms identified by a given serum. In this method of analysis, if a bovine MAPf tau signal is present in conjunction with purified A68 protein preparation, desirably the sample is assigned a diagnosis of AD if the tau signal contains less than three isoforms, and a diagnosis of non-AD if the sample identifies 3 or more isoforms of tau. In addition, preferably the sample is classified as non-AD if it lacks purified A68 protein preparation signal, regardless of the number of tau bands. Quantification of the MAPf tau signal in this instance takes on the formula: (Sum ODx(n-2)). For this formula, "Sum OD" is the sum of ODxmm measurements of all tau isoforms and "n" is the total number of bands present of the six tau MAPf isoforms. Thus, samples that give a purified A68 protein preparation signal and lack a substantial tau signal by this method are termed AD, and all other combinations (A68 signal+tau signal, tau signal alone, or absence of both signals) are diagnosed as non-AD.

Preferably the use of tau isoforms is not limited to use of bovine tau isoforms found in MAPf. Other forms of tau protein desirably are used, including but not limited to, tau purified from brain, and recombinant tau, either as a single molecule or as a mixture of tau isoforms. Additionally, the invention is not limited to tau from a bovine species. Purified tau or MAP from brains or cultured cells of other species may be used such as human, rodent, or other mammalian sources, as well as preparations from avian and reptiles.

Similarly, autoantibodies reactive with purified A68 protein preparation and bovine tau may also be detected in an indirect ELISA assay wherein the antigen is immobilized in a microtiter plate in which the bottom of each well is nitrocellulose. This support allows the antigen to be displayed in a manner which more closely resembles the Western blot than does a polystyrene support. Millipore MHAB plates are prewet with BBS for 1 minute, then the buffer is drawn through the filter under vacuum. Antigen is applied to the wells in BBS at 0.01 to 10 .mu.l per well (0.3 to 300 ng), and allowed to bind for 3 hours at 24^oC. In these experiments, the antigen may be purified A68 protein preparation, bovine tau (MAPf), or purified A68 protein preparation analogues such as phosphorylated rht. The antigen solution is drawn through the filter under vacuum, and the filters are blocked with 5% non-fat dry milk in BBS for 1.5 hr at 24^oC. Subsequently, all incubation solutions are removed by plate washer (Nunc) and washed with 0.1% tween 20 in tbs (defined earlier) rather than by drawing through the membrane under vacuum. 1% serum is added to wells in 100 .mu.l of 1% non-fat dry milk, 5% normal goat serum, BBS, and incubated for 16 hr at 4^oC. Bound human Ig is detected by addition of HRP-conjugated goat anti-human Ig in 1% casein/tbs for 2 hr at 24^oC. followed by addition of 90 .mu.l LumiGlo (Kirkegaard and Perry) chemiluminescent substrate.

Claim 1 of 5 Claims

What is claimed is:

1. A method for detecting autoantibodies that are present in Alzheimer's disease comprising:

(a) obtaining a protein preparation consisting essentially of an antigen that is immunologically reactive with a monoclonal antibody produced by the hybridoma cell line identified as ATCC No. HB9205, said preparation being substantially free of immunoglobulin G, a bovine microtubule associated protein preparation, and a sample being tested for the presence of said autoantibodies;

(b) electrophoresing said protein preparation and said bovine microtubule associated protein preparation on separate lanes on a gel;

(c) transferring said electrophoresed protein preparation and said bovine microtubule associated protein preparation to a membrane;

(d) contacting said membrane with a sample being tested for the presence of said autoantibodies such that an autoantibody complex can form with antigen present in said protein preparation and/or with antigen present in said bovine microtubule associated protein preparation; and

(e) detecting said autoantibodies by the formation of said complex(es).

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