Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/28—Neurological disorders
  • G01N2800/2814—Dementia; Cognitive disorders
  • G01N2800/2821—Alzheimer

Definitions

• the invention relates to an antigen associated with Alzheimer's disease, to antibodies specific for said antigen and to methods for diagnosing Alzheimer's disease utilizing said antigen and said antibodies.
• Alzheimer's disease 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. More specifically, this pathological impairment usually is correlated with numbers of amyloid containing 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 core. Another characteristic pathological feature of Alzheimer's disease is development of neurofibrillary tangles. A neurofibrillary tangle is an intraneuronal mass composed of paired helical filaments having unusual properties, which twist and form tangles. Neurofibrillary tangles are comprised of several different proteins.
• Alzheimer's disease causes a reduction in somatostatin, substance P and corticotropin releasing factor.
• Alzheimer's disease 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 which has been stained to visualize these plaques and tangles.
• diagnostic methods based on neurohistopathological studies, require extensive staining and microscopic examination of several brain sections.
• the plaques and tangles also may occur in the brains of normal, elderly individuals or individuals with other diseases, a more definitive and reliable method for making the diagnosis on brain tissue is desirable.
• It is another object of ti is invention to provide antibodies which can be used in the diagnosis of Alzheimer's disease.
• This invention is directed to antibodies 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 a specific, sensitive and simple sandwich immunoassay for diagnosis of Alzheimer's disease.
• 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.
• this invention is directed to an Alzheimer's disease antigen present in individuals with Alzheimer's disease and substantially absent from individuals who do not have Alzheimer's disease.
• the antigen of the invention is comprised of several proteins and some of which have a molecular weight of about 68,000 daltons.
• this invention is directed to a partially purified preparation of antigen which is a diagnostic marker for Alzheimer's disease.
• the antigen of the invention has an isoelectric point of about 6 in reduced or non-reduced form, binds to an affi-blue column, is at least fifty percent soluble in a solution of 0.01 M sodium phosphate, 0.14 M sodium chloride and ImM phenyl methyl sulfonyl flouride at pH 6.8, and precipitates in fifty percent saturated ammonium sulfate at 4 C.
• Alzheimer antigen is detectable in cerebrospinal fluid from Alzheimer patients by western blot analysis but absent from cerebrospinal fluid from nondemented patients. In the methods described herein, 3.5 ml of cerebrospinal fluid is utilized; however, this volume could be readily reduced. Fluid volumes of from about 0.1 to 10 ml of fluid can be employed in the metiiods of the invention.
• This invention is further directed to a method for determining the presence of antigen specific to Alzheimer's disease in a sample comprising the steps of contacting a sample with a first antibody specific for a first anugenic determinant being a complex of proteins containg a 68,000 dalton antigen and related proteins found in individuals having Alzheimer's disease such that a first antibody-antigen complex is formed; separating the first complex from the sample; contacting the first complex with at least one second antibody specific for a second antigenic determinant on said Alzheimer's antigen, wherein said second antigenic determinant may be the same as said first antigenic determinant, such that said second antibody binds to the first complex to produce a second complex; and detecting the presence of the second complex, the presence of said second complex indicating the presence of antigen specific to Alzheimer's disease in the sample.
• This invention is additionally directed to use of the above-described method for diagnosing Alzheimer's disease using pre-mortem or post-mortem brain tissue or cerebrospinal fluid from an individual as a sample, and utilizing the Alzheimer's disease antigen and antibodies of the invention.
• the samples from the patient include, in addition to brain tissue or cerebrospinal fluid, other tissues, blood, urine, and serum.
• the quantitative data presented also shows that detection of Alzheimer antigen in cerebrospinal fluid can distinguish between Alzheimer's disease and other dementing illnesses such as Pick's Disease, multi-infarct dementia or Huntington's Disease because of the selective and quantitative accumulation of Alzheimer antigen in Alzheimer's disease.
• Figure 1 represents a two dimensional gel of die Alzheimer's antigen of the invention
• Figure 2 represents a sandwich immunoassay configuration of the present invention for the diagnosing Alzheimer's disease
• Figure 3 represents the relationship between absorbance and micrograms IgG/ bead
• Figure 4 represents titration curves for Alzheimer's disease brain homogenates and normal brain homogenates
• Figure 5B represents a y-axis 10 fold expansion of Figure 5 A
• Figure 6 represents the immunoreactivity of various monoclonal antibodies of the invention to the Alzheimer's antigen
• Figures 7A-7M represent brain assays for the Alzheimer's antigen utilizing various monoclonal antibodies, wherein one monoclonal antibody is used to capture the antigen and the second monoclonal antibody is used for detection purposes;
• Figure 8A represents a cerebrospinal fluid assay for determining the presence of
• Figure 8B represents a cerebrospinal fluid assay for determining the presence of Alzheimer's antigen in both Alzheimer's disease (AD) individuals and normal (N) individuals wherein the capture monoclonal antibody is either TG5 or MCI and the detection antibody is TG4 or MG15.
• Figure 9 represents an assay of the invention for detecting Alzheimer's antigen wherein the capture antibody is on a solid phase;
• Figure 10 represents a cerebrospinal fluid assay for the detection of Alzheimer's antigen wherein the capture antibody is PHF-1 and the detection antibody is TG3; and
• Figure 11 represents levels of Alzheimer's antigen in various individuals utilizing a cerebrospinal fluid assay and both monoclonal and polyclonal antibodies.
• DETAILED DESCRIPTION OF THE INVENTION By “individuals with Alzheimer's disease” is meant individuals or patients suffering from, affected by, or manifesting the clinical symptoms of the disease.
• NFTs Neurofibrillary Tangles
• PHFs tightly packed paired helical filaments
• This NFT form of PHF is highly insoluble in solvent conditions used to isolate the soluble Alzheimer's antigen of the invention.
• Soluble Alzheimer's antigen derives largely from abnormal neurites (sometimes referred to as "neuropil threads"). These abnormal neurites comprise the predominent pathology in Alzheimer's Disease.
• Alzheimer antigen in the cortex of the average Alzheimer case is present in neuronal processes (abnormal neurites) rather than in the plaque or tangle (Wolozin, Ann. Neurol. 22: 521-526, 1987).
• sensile plaques are actually two distinct types of structures, the classical plaques described by Alzheimer that is composed of degenerating neuronal elements (dystrophic neurites) surrounding a central core of amyloid, and die "diffuse” or ''primitive” plaques consisting of deposits of amyloid without the halo of degenerating neurites (Tagliavini et al., Neurosci. Lett. 22: 191-196, 1988; Dickson et al., Am. J. Path. 122: 86-101, 1988). Plaques containing degenerating neurites are now referred to as "neuritic plaques".
• the degenerating neurites in tiiese structures contain Alzheimer antigen and stain with the antibodies of the invention.
• Primitive or diffuse plaques do not contain Alzheimer antigen nor do they immunostain with the antibodies of the invention.
• Diffuse plaques can be found in large numbers in die brains of die majority of the very elderly (over 80 years), and are only rarely associated with dementia when present without evidence of degenerating neurites (Davies et al., Neurology 28: 1688-1693, 1988; Delkaere et al., Neurosci. Lett. _ l_ : 87-93, 1990; Dickson et al., Neurobiol. Aging 1 : 179-189, 1991).
• Alzheimer antigen 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.
• Alzheimer antibodies The antibodies which are immunologically reactive with the Alzheimer antigen are referred to herein as “Alzheimer antibodies.”
• the invention provides antibodies capable of substantial immunoreactivity with an antigen present in elevated amounts specific to Alzheimer patients, i.e., Alzheimer antibody.
• the invention is also d e use of Alzheimer antibody to diagnose Alzheimer's disease.
• the Alzheimer antigen is a protein complex with a major species having an apparent molecular weight of about 68,000 daltons and is found in Alzheimer patients while being present in much reduced quantities in non-Alzheimer patients, including patients suffering from other neurologic diseases.
• a method is provided for determining the presence of elevated levels of an antigen specific to Alzheimer's disease in a sample, thereby diagnosing Alzheimer's disease.
• the method comprises contacting a sample from an individual suspected of having Alzheimer's disease with a first antibody specific for an antigenic determinant on the Alzheimer's antigen and capable of binding to the antigen so as to produce a first complex.
• the resulting first complex then is separated and recovered from die sample and contacted with at least one second antibody specific for a second antigenic determinant on the antigen.
• the second antigenic determinant may be the same as the first antigenic determinant due to die multiepitopic nature of the antigenic entity.
• the second antibody is capable of binding to die antigen present in die first complex such that when the second antibody binds the antigen, a second complex is formed, which consists of antigen bound to the first and second antibodies.
• the presence of the second complex is detected and, thereby, the presence and/or level of die antigen specific to Alzheimer's disease in the sample is determined and, optionally, quantitated.
• 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.
• the sample comprises cerebrospinal fluid.
• both the first and second antibodies are monoclonal antibodies.
• a preferred monoclonal antibody of the invention is ALZ-50 secreted by hybridoma No. HB9205, which was deposited under the Budapest Treaty on September 17, 1986 with die American Type Culture Collection, Rockville, Maryland. Viability was confirmed on September 24, 1986.
• ALZ-50 has become the standard reagent for detecting die presence of Alzheimer's disease in this field.
• Wood et al. Histochemical Journal. Vol. 21, No. 11, pp. 659-662 (1989); Itagaki et al., Annals of Neurology. Vol. 26, No. 5, pp.685-689 (1989); Beach et al., Brain Research. Vol. 501, No. 1, pp. 171-175 (1989); Love et al., Journal of Neuropathology and Experimental Neurology. Vol. 47, No. 4, pp. 393-405 (1988); Nukina et al., Neuroscience Letters. Vol. 87, No. 3, pp. 240-246 (1988); and Hyman et al., Brain Research. Vol. 450, pp. 392-397 (1988).
• the antigen of the invention which is associated witii Alzheimer's disease is an aggregate of several proteins (see Figure 1) and die major protein species have an apparent molecular weight of about 68,000 daltons on a reducing SDS gel. 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. £2: 4913-4917, 1986), soluble PHF (Greenberg and Davies, Proc. Natl. Acad. Sci.
• Alzheimer's Disease Associated Protein ADAP
• All terms are deemed to be equivalent when referring to die Alzheimer's antigen herein. It contains tau and phosphorylated tau. This antigen can be used to raise monoclonal antibodies which can be used in a diagnostic assay for Alzheimer's disease.
• the first antibody can be attached to a suitable solid matrix, such as, for example polystyrene beads.
• a sample is obtained and contacted with a suitable amount of first antibody to produce a first complex.
• the contact preferably involves adding the sample to a column of polystyrene beads coated with the first antibody.
• the complex which results from contacting the sample witii the first antibody is separated from the sample by elution methods known to tiiose skilled in d e art.
• the separated first complex is contacted witii at least one second antibody specific for an antigenic determinant on the antigen and capable of binding to the antigen in the complex.
• the antigenic determinant to which die second antibody is directed may be die same one as tiiat to which the first antibody is directed due to d e multiepitopic nature (i.e. repeating epitopes) of the antigenic entity. The conditions for effecting such contact are described herein and known to those skilled in the art.
• the first or second antibody of the methods of the present invention may be made detectable by attaching an identifiable label thereto.
• die second antibody is made detectable.
• 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 or radiolabel thereto.
• die antibody may be detected by use of another antibody directed to it, d e other antibody being labeled or having an enzyme substrate bound to it.
• the presence of the detectable antibody bound to die antigen of the complex consisting of antigen bound to the first and second antibody may be readily detected using well-known techniques.
• the detectable antibody is linked to an enzyme conjugated to an appropriate substrate, die optical density of the detectable bound antibody is determined using a quantum spectrophotometer.
• die optical density of the detectable bound antibody is determined using a quantum spectrophotometer.
• the detectable antibody is fluorescently labeled, die fluorescent emission may be measured or detected using a fluorometer technique.
• the detectable antibody is radioactively labeled, the bound antibody may be detected using a radioactivity detection techniques. Chemileuminescent techniques may also be employed.
• first and second antibody may be monoclonal antibodies.
• the first or the second detectable antibody can be a polyclonal antibody.
• the methods for qualitatively or quantitatively determining die Alzheimer's disease antigen may be used in the diagnosis of Alzheimer's disease. Utilization of die methods of the present invention is advantageous over prior art mediods because die present invention provides simple, sensitive, very specific methods for detecting Alzheimer's antigen.
• the Alzheimer's antigen is well-suited for sandwich immunoassay 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 antibody.
• the methods described herein for use with cerebrospinal fluid are applicable to blood and urine.
• the following is anodier test procedure believed to be suitable for detecting the presence of Alzheimer antigen in die blood or otiier body fluids of a person having Alzheimer's disease.
• the procedure is similar to the procedure used in die detection of HTLV-i ⁇ 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. Vol. 76, pp. 171-183 (1985).
• die Alzheimer antibody is of die IgM class, however, Alzheimer antibodies of die IgG class have been created and are specifically included in die term Alzheimer antibody.
• Alzheimer antibodies of the IgM class were used to generate Alzheimer antibodies of die IgGi class.
• Alzheimer antibodies of the IgGj class showed substantial reductions in non-specific binding as compared to Alzheimer antibodies of the IgM class.
• IgM antibodies display more non-specific binding than do IgG antibodies.
• Alzheimer antibodies of the IgGj class from two hybridomas were compared to IgM Alzheimer antibody using homogenates of tiiree Alzheimer brains and tiiree normal brains.
• Alzheimer antibodies showed much less binding to normal brain tissue than the IgM Alzheimer antibody.
• the other IgG ⁇ Alzheimer antibody showed no significant reactivity with die normal tissue.
• Another advantage of die invention is die use of the Alzheimer antibody to carry a label, such as a radioactive label or the like to portions of the brain which are stained by die Alzheimer antibody. Thereafter a "map" of die brain can be made from the label using known methods in order to provide additional information on the brain.
• a conventional method is used so tiiat die Alzheimer antibody can be combined widi a suitable label so tiiat the Alzheimer antibody acts as a carrier.
• the combined Alzheimer antibody and label are introduced into the brain using conventional techniques.
• the Alzheimer antibody attaches the label to sites of Alzheimer antigen.
• the procedure used included the production of monoclonal antibodies to homogenates of ventral forebrain tissue taken during autopsies of four patients witii Alzheimer's disease.
• the resulting antibodies were screened on die basis of tiieir ability to differentiate brain tissue from Alzheimer patients from tissue from normal patients in both immunochemical and immunocytochemical prodecures.
• the resulting monoclonal antibodies are initially assayed according to their ability to bind to Alzheimer brain homogenate using a known technique referred to in die prior art as enzyme linked immunosorbent assay (ELISA).
• ELISA enzyme linked immunosorbent assay
• Antibodies showing greater than a fifty percent increase or decrease in binding to homogenates of Alzheimer's brain relative to normal tissue are studied further.
• One particular antibody was found to be highly selective for brain tissue from Alzheimer patients.
• ALZ-50 antibody ALZ-50
• ALZ-50 is deposited with d e American Type Culture Collection, Rockville, Maryland as ATCC No. under deposit No. HB9205.
• This monoclonal antibody binds to die region on Tau shown in Table A below.
• ALZ-50 does not cross-react witii proteins found in normal tissue under die specific assay conditions described herein. 2.
• the ALZ-50 monoclonal antibody described above in part 1 of this example was used to produce otiier antibodies specific for the Alzheimer's antigen of the invention. This was done by immunoaffinity chromatography. Large quantities of antigen were prepared from brain tissues from cases of Alzheimer's disease using ALZ-50 antibody. This material was used to immunize mice, and the spleens from these mice were used to produce new cell lines producing monoclonal antibodies reactive with the Alzheimer's antigen.
• the supernatant fraction was loaded onto the P42 column, and die column was washed after loading. Bound antigens were eluted with a solution of 3M potassium diiocyanate, and die eluate was dialysed against Tris-buffered saline.
• mice were then immunized witii antigen purified on die P42 column by intraperitoneal injection of 10 to 20 micrograms of protein per mouse per injection. Mice were immunized 4 or 5 times before removal of spleens for the production of hybridoma cells by standard protocols.
• Hybridomas were tested for the production of specific antibodies by ELISA and immunocytochemistry.
• Hybridomas secreting monoclonal antibodies having die basic property of selective reactivity with brain tissue from cases of Alzheimer's disease by ELISA, Western blotting and immunocytochemistry were obtained.
• the monoclonal antibodies obtained are: TG3, TG4, TG5, MCI, MC2, MC3, MC5,
• FIG. 6 shows reactivity of some of tiiese monoclonal antibodies with 6 different purified Alzheimer's antigen preparations. Some of these antibodies (e.g., antibody MCI) are very similar in specificity to ALZ-50. Other antibodies react with higher affinity and detect fragments of die Alzheimer's antigen (e.g., antibodies MC6, MCI 5, TG4 and TG5).
• the specificity of the antibodies of die invention for the Alzheimer's antigen is as follows. Under certain conditions ALZ-50 reacts with normal and recombinant tau; however, in the same assay configuration under similar conditions, its reactivity against die Alzheimer antigen is much greater. In fact under other modified conditions, such as the ELISA configurations described in this invention, ALZ-50 only reacts with the Alzheimer antigen. Similarly, the TG-3 epitope can be generated on recombinant tau by appropriate phosphorylation; its reactivity against the Alzheimer antigen is also vastly greater than with phospho recombinant tau. Indeed, the reactivity of many of the antibodies of die invention have epitopes on die tau molecule that are conformationally derived.
• Alzheimer antigen epitope mapping Since tau is known to be a major component of the Alzheimer antigen, many of die Alzheimer antibodies would bind to eitiier recombinant tau or phosphorecombinant tau. Although the affinities for die recombinant protein would undoubtedly be less tiian the antibody affinity for die Alzheimer antigen, useful information could still be obtained as to die sites of interaction of the antibodies.
• Alzheimer antigen epitopes were identified dirough deletion analysis of htau40, the full- length, 441-residue form of human central nervous system tau. First, a library of 42 internal deletions spanning the entire tau molecule was created through restriction and religation of htau40 cDNA.
• the mutants were placed into a bacterial expression vector and transformed into E. coli.
• the resultant library of tau deletions was processed like otiier bacterial expression libraries (Helfman et al., Focus 6_: 1-05, 1984), but was "probed” witii die monoclonal antibodies described herein.
• membrane-bound lysates of bacterial colonies containing individual clones of the expression library were incubated witii a crude mixture of protein kinases in a solid-phase phosphorylation assay (Carmel and Kuret, Anal. Biochem. 2Q2: 274-280, 1992).
• regions of the tau molecule essential or contributory to antibody binding were identified.
• a second series of deletions of tau sequences also show complete loss of reactivity of tau with ALZ-50 and MCI. Deletions which result in die loss of sequences from amino acids 312- 342 completely abolish reactivity of tau with both antibodies.
• binding of ALZ-50 and MCI to tau in PHF requires that tau be folded to bring together d e first 18 amino acids and the sequence from amino acids 312-342. Since these sequences are not adjacent to each otiier in tau prepared from normal brain, there is no reactivity of these antibodies with ti is protein.
• a suggested alignment of the sequences is shown below:
• the hybridoma secreting monoclonal antibody PHF-1 was deposited under die Budapest Treaty with the American Type Culture Collection, Rockville, Maryland on October 26, 1994, catalogued as ATCC No. HB 11743 and found viable on November 4, 1994. This monoclonal antibody binds to die region on tau shown in Table A.
• the hybridoma secreting monoclonal antibody MCI 6 was deposited under die Budapest Treaty with die American Type Culture Collection, Rockville, Maryland on October 26, 1994, catalogued as ATCC No. HB 11742 and found viable on November 4, 1994.
• the hybridoma secreting monoclonal antibody MC6 was deposited under the Budapest Treaty with die American Type Culture Collection, Rockville, Maryland on October 26, 1994, catalogued as ATCC No. HB 11740 and found viable on November 4, 1994.
• the hybridoma secreting monoclonal antibody MCI was deposited under the Budapest Treaty with the American Type Culture Collection, Rockville, Maryland on October 26, 1994, catalogued as ATCC No. 11736 and found viable on November 4, 1994. This monoclonal antibody binds to die region on tau shown in Table A.
• the hybridoma secreting monoclonal antibody TG5 was deposited under the Budapest Treaty with the American Type Culture Collection, Rockville, Maryland on October 26, 1994, catalogued as ATCC No. HB 11746 and found viable on November 4, 1994.
• the hybridoma secreting monoclonal antibody MC2 was deposited under the Budapest Treaty with the American Type Culture Collection, Rockville, Maryland on October 26, 1994, catalogued as ATCC No. HB 11737 and found viable on November 4, 1994. This monoclonal antibody binds to the region on tau shown in Table A.
• the hybridoma secreting monoclonal antibody MC3 was deposited under die Budapest
• the hybridoma secreting monoclonal antibody MC5 was deposited under die Budapest Treaty with the American Type Culture Collection, Rockville, Maryland on October 26, 1994, catalogued as ATCC No. HB 11739 and found viable on November 4, 1994.
• the hybridoma secreting monoclonal antibody MCI 5 was deposited under the Budapest Treaty with the American Type Culture Collection, Rockville, Maryland on October 26, 1994, catalogued as ATCC No. HB 11741 and found viable on November 4, 1994. This monoclonal antibody binds to the region on tau shown in Table A. 3.
• Rabbit anti-ALZ IgG Rabbit anti-ALZ IgG
• Alzheimer's disease brain homogenates were enriched for Alzheimer's antigen by multiple differential centrifugation steps and detergent extractions. Rabbits were immunized with highly enriched Alzheimer's antigen fraction and boosted according to a monthly schedule. Bleeds were screened by using ELISA in which die immunogen preparation was dried in die wells by forced air at 37 * C. After desired titers were achieved, rabbit sera * were purified by protein-A affinity chromatography.
• the Protein A purified IgG from the serum bleeds of these rabbits was used to coat beads used in the assays described below.
• Example 2 Isolation and Characterization of Alzheimer's Disease Antigen Monoclonal antibodies specific for the Alzheimer's disease antigen were assayed so as to determine tiieir ability to bind to Alzheimer brain homogenate using a known technique referred to in the prior art as enzyme linked immunosorbent assay (ELISA).
• the brain homogenate was prepared using 2.5g of Alzheimer cortex homogenized into 10ml of phosphate buffered saline (PBS) containing lmM phenyl methyl sulfonyl fluoride by passes of a glass pesde in a Dounce homogenizer.
• PBS phosphate buffered saline
• the brain homogenate was centrifuged in a BECKMAN JA-21 CENTRIFUGE (Beckman) for twenty minutes at a temperature of about 4 * C at a rate of about 15,000 rpm to produce about 27,200g.
• the supernatant of the brain homogenate was removed and loaded onto a chromatographic column containing 350 ml of the molecular sieve SEPH AROSE 6B (Pharmacia).
• the carrier used for die chromatographic procedure was PBS and was run through the chromatographic column at the rate of 6ml per hour. Fractions of 3.1 ml were collected.
• the Alzheimer's disease antigen largely elutes from the chromatographic column in the void volume.
• the presence of die antigen in the void volume was identified by use of die enzyme linked immunosorbent assay (ELISA). Fifty microliters of each void volume fraction were tested. Each fifty microliters was diluted widi water in a ratio of 1:100 and fifty microliters of this diluted solution was dried onto 96-well polyvinyl microtiter plates (NUNC) for the ELISA. Each dried sample of the antigen was combined widi 200 microliters per well of a blocking solution of PBS with 5 percent (v/w) powdered non-fat dry milk, and was incubated in the wells for the ELISA for one hour at room temperature.
• NUNC polyvinyl microtiter plates
• the antibody to be tested for the antigen was diluted to a ratio of 1:5 with the blocking solution. Fifty microliters of the diluted antibody solution were added to each well and allowed to incubate for one hour at room temperature. The diluted antibody solution was removed and die polyvinyl microtiter plates were washed five times with an aqueous mixture of 0.01 M TRIS (Sigma) and 0.1 percent TWEEN-20 (National Diagnostic) at a pH of 7.4. The dried sample was then incubated for one hour at room temperature with a secondary antibody comprising peroxidase-conjugated goat antibody to mouse immunoglobulins (Kirkegaard & Perry).
• Table 1 shows the results of antigen measurements from a pool of temporal cortices of both patients who died of Alzheimer's disease and nc>rrnal individuals. Ten Alzheimer cases and six normal cases were used for die analysis. The Alzheimer's disease cases were typical in both clinical and neuropathological features. Brain homogenates were prepared from normal individuals dying in hospitals from lung or heart diseases. These patients were not demented prior to deatii and had no history of neurologic or psychiatric disease. Neuropad ologic studies failed to reveal Alzheimer's disease pathology in the normal individuals.
• Alzheimer's disease patient had an optical density of 75, whereas the temporal cortex homogenate from a normal patient had an optical density of only 10. Hence, die Alzheimer's disease antigen is
• Immunochemical reagents used included peroxidase-coupled goat antimouse antibody (Biorad Labs.), 125 Iodine-coupled goat antimouse antibody (Amersham Corp.) and die peroxidase substrate 2,2'-Azino-di(3-ed ⁇ ylbenzd ⁇ iazoline sulfonate (ABTS, Biorad Labs.).
• ELISA enzyme linked immunosorbent assay
• Immunocytochemistry was performed using vibratome sections (40 microns thick) cut from tissue fixed in formalin. The sections were washed twice in phosphate-buffered saline (PBS), and incubated for 30 minutes in 0.25% Triton X-100-3% hydrogen peroxide. After a PBS wash, die sections were treated as described above for the ELISA assay. The peroxidase was visualized by reaction with 0.45mg/ml diaminobenzidine and 0.44mM hydrogen peroxide in 0.1M Tris, pH 7.4.
• PBS phosphate-buffered saline
• PBS-2mM EDTA insoluble material was dissolved in 5% SDS-5% beta-mercaptoedianol and fractionated on 10% SDS-polyacrylamide gels. Proteins were transferred to nitrocellulose, and the Alzheimer antibody staining was performed as described above, except that 125 ⁇ _ ⁇ a beled goat anti-mouse immunoglobulin was used as die secondary antibody, and the pattern of reactivity was visualized by autoradiography.
• the amount of immunoreactivity present in Alzheimer's tissue was far greater than tiiat found in tissue affected by Guam Parkinson Disease (GPD), Pick's Disease, Parkinson's Disease with cortical plaque padiology (PD) or normal tissue.
• GPD Guam Parkinson Disease
• PD Parkinson's Disease with cortical plaque padiology
• the amount of immunoreactivity in each disease is, respectively, 100, 3.0, 3.1, 2.4 and 1.8.
• the immunocytochemistry of the Alzheimer antibody was examined in cases representing the diseases described in Table 2, sections A & B.
• One of die dramatic characteristics of Alzheimer antibody staining is the extensive staining of abnormal neurites. When observed grossly, the neuritic staining accounts for most of die visible staining. Staining of neurons was found in GPD and Hallervorden-Spatz Disease (HSD) and staining of Pick bodies was found in Pick's disease. In contrast to d e extensive neuritic staining present in tissue from cases affected by Alzheimer's disease, tissues affected by GPD, HSD or Pick's Disease exhibit minimal neuritic staining.
• PD cortical tissue though pathologically similar to Alzheimer's tissue, exhibited only a small amount of staining of plaques in one case and no staining in die odier two cases.
• hippocampal tissue exhibited pyramidal layer neuronal staining, and thioflavin S stains revealed die presence of both neuritic plaques and neurofibrillary tangles.
• the number of tangles seen in Alzheimer's disease tissue was less than die number of Pick bodies present in the inferior temporal cortex of die cases of Pick's Disease, and more dian triple tiiat present in GPD or HSD tissue.
• the amount of immunoreactivity present did not differ in affected neurons of different diseases or in Pick bodies. Hence, the difference in levels of immunoreactivity is due to abundant staining of abnormnal neurites present in Alzheimer tissue.
• the total amount of immunocytochemical staining correlated with the total amount of immunoreactivity seen by ELISA.
• the small amount of immunoreactivity in other disease tissues corresponds to the low level presence of die Alzheimer's disease antigen. This was determined using Western blot analysis with the Alzheimer antibody.
• die samples were prepared in the presence of 2 mM EDTA, which decreases the solubility of the Alzheimer protein up to 90%. This decreased solubility was discovered when an attempt was made at stabilizing Alzheimer antigen by the addition of a cocktail of protease inhibitors, including EDTA.
• Alzheimer antibody reactivity was virtually absent from supernatant fractions prepared in die presence of EDTA, but was quantitatively recovered in die pellets.
• the addition of 2mM EDTA to homogenates thus provides a convenient enrichment of antigen.
• the PBS-2 mM EDTA insoluble fractions were examined by Western blot analysis widi Alzheimer antibody.
• large amounts of protein were loaded on the gel, and incubated overnight widi Alzheimer antibody, and 1-125 labeled secondary antibody.
• the immunoreactivity in Alzheimer's disease tissue corresponded to a 68,000-62,000 dalton protein doublet.
• faint higher molecular weight bands were also seen above die doublet.
• Normal tissue contained a great deal of lower molecular weight immunoreactivity. This results from d e use of large amounts of crude material. These bands did not appear when partially purified samples are examined and no Alzheimer's disease antigen was detected in purified fractions from normal or otiier neurologic diseases.
• the lower molecular weight cross-reactive proteins account for the normal brain tissue immunoreactivity measured by ELISA with overnight incubations. There was no reactivity in the 68,000 dalton region of d e immunoblot in preparations from normal tissues. Tissue from Pick's Disease cases contained mosdy 62,000 dalton protein and lower molecular weight material. A faint band in the 68,000 dalton region was also seen. GPD tissue gave a sharp immunoreactive band at 62,000 daltons and diffuse reactivity in die 68,000 dalton region.
• the selective elevation of die abundance of die Alzheimer antigen in Alzheimer ussue can be exploited in diagnostic testing for Alzheimer's disease.
• the usefulness of this antigen is shown below in the following examples.
• the Alzheimer's disease antigen of the invention is recognized by Alzheimer's disease-specific antibodies, and is present in high quantities in Alzheimer's disease patients and present in vanishingly small amounts in non-Alzheimer's disease patients, including patients suffering from other neurologic diseases.
• the Alzheimer's disease antigen is at least about 50 percent soluble in PBS and largely elutes from a molecular sieve column such as SEPHACRYL
• Alzheimer's disease antigen is an aggregate of several proteins.
• the inventors have discovered tiiat some of the proteins that make up the Alzheimer's disease antigen have molecular weights of about 53,000, 62,000,
• Alzheimer's disease antigen contains tau and phosphorylated tau, binds tightly to Affi-blue columns and does not elute off with aqueous 0.5M sodium chloride but can be removed widi aqueous 2M sodium chloride.
• the molecular weight of the major protein comprising the Alzheimer's disease antigen is about 68,000 daltons after denaturation with sodium dodecyl sulfate (SDS) and reduction with mercaptoethanol.
• SDS sodium dodecyl sulfate
• the apparent molecular weight of the Alzheimer's antigen in n ⁇ nreduced form after denaturation with SDS is much greater than 68,000 daltons, being greater than about 300,000 daltons. In nonreduced form, the Alzheimer's disease antigen will not enter a 10 percent acrylamide gel.
• the pi of the Alzheimer's disease antigen in reduced or nonreduced form is about 6 as determined by isoelectric focusing.
• the antigen precipitates in 50 percent saturated aqueous ammonium sulfate at 4 * C, and co-purifies widi and is thought to be a kinase which will phosphorylate molecules in the presence of magnesium (Mg " * " " 1 ”) and adenosine triphosphate (ATP).
• Mg " * " " 1 ” magnesium
• ATP adenosine triphosphate
• the Alzheimer's disease antigen does not co-purify with neurofilaments.
• An Alzheimer antigen is recognized by Alzheimer antibody. 2.
• An Alzheimer antigen is present in Alzheimer patients and substantially absent from non- Alzheimer patients, including patients suffering from other neurologic diseases.
• An Alzheimer antigen is at least about 50 percent soluble in PBS.
• An Alzheimer antigen largely elutes from a molecular sieve column such as SEPHACRYL S-400 or SEPHADEX G-25 or SEPHAROSE 6B at the void volume which corresponds to a molecular weight of at least 2 x 10 ⁇ daltons. This suggests that the size of the Alzheimer antigen may represent an aggregated form of the native protein.
• Alzheimer antigen binds tightly to Affi-blue columns and does not elute off with aqueous 0.5M sodium chloride but can be removed with aqueous 2M sodium chloride. 6. The apparent molecular weight of one Alzheimer antigen subunit recognized by the
• Alzheimer antibody is about 68,000 daltons after denaturation with sodium dodecyl sulfate (SDS) and reduction with mercaptoethanol.
• SDS sodium dodecyl sulfate
• the apparent molecular weight of the Alzheimer antigen recognized by die Alzheimer antibody in nonreduced form after denaturation with SDS is much greater than 68,000 daltons, being greater than about 300,000 daltons. In nonreduced form, the Alzheimer antigen subunit will not enter a 10 percent acrylamide gel.
• An Alzheimer antigen precipitates in 50 percent saturated aqueous ammonium sulfate at4°C.
• the Alzheimer antigen co-purifies with a protein kinase which will phosphorylate molecules in the presence of magnesium (Mg ++ ) and adenosine triphosphate (ATP).
• Mg ++ magnesium
• ATP adenosine triphosphate
• An Alzheimer antigen does not copurify with neurofilaments.
• An Alzheimer antibody reacts with antigen present in the cerebrospinal fluid of Alzheimer patients and absent from the cerebrospinal fluid of normal patients.
• Alzheimer antibody reacts with antigen present in Alzheimer's patients and largely absent from patients with Huntington's Chorea, Diffuse Lewy Body Disease, Creutzfeldt Jakob
• aqueous PBS and 2mM ethylene diamine tetraacetic acid solution has a solubility of about 10% for Alzheimer antigen.
• an aqueous 2mM ethylene diamine tetra acetic acid solution prevents Alzheimer antigen from being extracted from brain tissue.
• Alzheimer antigen was similarly elevated in the nucleus basalis and hippocampus of Alzheimer patients.
• the cortex, nucleus basalis and hippocampus are known to contain neuritic plaques, abnormal neurites, and neurofibrillary tangles in brains of patients with Alzheimer's disease.
• Tests made on brain areas known to be less affected by the disease, such as caudate, thalamus, and cerebellum demonstrated little or no reactivity.
• the procedure described herein enables the identification of an antigen (Alzheimer antigen) closely associated with Alzheimer's disease and antibodies (Alzheimer antibodies) which identifies the antigen.
• the concentration of Alzheimer antigen in a sample from an Alzheimer patient can be increased by at least K)3 times and preferably at least i times to simplify the study and detection of die Alzheimer antigen.
• a concentrate of the Alzheimer protein is obtained using the following procedure: A portion of an Alzheimer brain is homogenized at 4 * C for 30 seconds in 4 volumes of a buffer comprising PBS and ImM phenyl methyl sulfonyl fluoride, having a pH of 6.8. This amounts to a ratio of 1 gram of brain per 4ml.
• the homogenization is carried out in a teflon-glass homogenizer with a minimum of three strokes of a piston turning at 2,000 rpm.
• the brain homogenate is centrifuged for 23 minutes at 4 * C at 27,200g. After the centrifuging, die supernatant is loaded into a chromatographic column containing SEPHACRYL-S-400 (Pharmacia) and eluted widi the PBS.
• the Alzheimer antigen appears in the void volume from the chromotagraphic column and is monitored using the ELISA. Subsequently, the void volume is run over an Affi-blue column (Bio-Rad). The Affi-blue column is washed with 20 column volumes of the PBS.
• the Alzheimer protein remains bound to the Affi-blue column.
• the Alzheimer protein is e ⁇ uted off of die Affi-blue column with 2M aqueous sodium chloride. That is, the concentration of sodium chloride in the buffer increases during die elution from 0.5M to 2M.
• the presence of die Alzheimer antigen can be monitored using die ELISA.
• the sodium chloride is removed from die Alzheimer antigen by using a chromatographic column comprising SEPHADEX G-25 (Pharmacia) and eluting with buffer PBS.
• the Alzheimer protein elutes largely in the void volume.
• the void volume containing die Alzheimer protein is loaded onto an Alzheimer antibody immunoaffinity column and the column is washed with at least 20 column volumes of the PBS.
• the Alzheimer antigen is eluted with aqueous 3M KSCN.
• the elution is monitored for die presence of die Alzheimer protein.
• the eluted solution widi the Alzheimer protein is desalted using the SEPHADEX G-25 column with PBS.
• a buffer solution containing 0.001M sodium phosphate and 0.014M sodium chloride at pH 6.8 can be used to provide a more concentrated elution of Alzheimer antigen. Further concentration of die Alzheimer antigen, as much as 10 fold, can be accomplished by removal of water from the eluted sample by heating under vacuum.1 e resulting concentration is about 1 percent by weight.
• the major band for die normal brain void volume had a molecular weight of 59,000 daltons and a doublet at 245,000 daltons.
• die Alzheimer antigen has a molecular weight of about 62,000 to about 68,000 daltons, and is distinctively different from the antigen having a molecular weight of 59,000 daltons from brain showing no signs of Alzheimer's disease.
• TBS tris-buffered saline
• Alzheimer antigen proteins having subunits with molecular weights of about 68,000 daltons are distinguishable from the Alzheimer antigen. These proteins are in two families: in the cytoskeletal family there are neurofilaments and normal tau proteins, and in the cholinergic family there is choline acetyltransferase. None of these proteins exhibit an elevated concentration in Alzheimer brains as compared to normal brains. If the Alzheimer protein is related to die protein having a molecular weight of about 59,000 daltons and found in normal brains, then the Alzheimer antigen is unlikely to be neurofilament, normal tau protein, or choline acetyltransferase.
• brain tissue samples were fixed in fomialin until the tests were carried out.
• the immunocytochemistry study was performed using die Alzheimer antibody on brain tissue from Guam Parkinson's disease, Picks disease, Alzheimer's disease, Alzheimer's/Parkinson's disease and normal subjects.
• the Alzheimer antibody stained neurons, neuritic plaques and abnormal neurites in the Alzheimer brain sample.
• One of the characteristics of the Alzheimer antibody is that the staining is dramatic and extensive for the abnormal neurites. When observed closely, the neuritic staining accounts for most of the visible staining. Staining of some neurons is found in Guam Parkinson's brain tissue and staining of d e Pick bodies is found in Pick's brain tissue.
• the Guam Parkinson's brain tissue exhibits no neuritic staining and Pick's brain tissue exhibits little neuritic staining.
• the Alzheimer's-Parkinson's cortical tissue is pathologically similar to Alzheimer's brain tissue but exhibited only a small amount of stained neuritic plaques in one case and no staining in two other cases.
• Hippocampal tissue from one case exhibited pyramidal layer neuronal staining but there was little neuritic staining. No staining is observed for normal brain tissue even when a small number of neuritic plaques are present.
• the total amount of i munocytochemical staining correlates well with the total amount of immunoreactivity observed by die ELISA. The primary difference in die levels of immunoreactivity was due to die neuritic (abnormal neurite) staining for Alzheimer's brain tissue.
• Alzheimer's disease-specific antibodies of die invention react with Alzheimer's antigen which is present in the cerebrospinal fluid of Alzheimer's disease patients and absent from the cerebrospinal fluid of normal patients and patients widi Huntington's Chorea, Diffuse Lewy Body Disease, Cruetzfeldt Jakob Disease, Progressive Supra-nuclear Palsy and Multi-Infarct Disease.
• Immunocytochemistry of the Alzheimer ALZ-50 antibody on formalin-fixed brain tissue showed that the antibody was highly selective for abnormal neuritic components in the Alzheimer brain. Many neurons in the pyramidal layer of die hippocampus were stained. The Alzheimer antigen was found to be present in cell bodies and in abnormal neurites.
• neuritic plaques were strongly stained by die Alzheimer antibody. The staining was confined to the neuritic meshwork present in plaques. Strongly stained neurons and neuritic plaques were found throughout Alzheimer hippocampus and cortex. In contrast, there was virtually no staining of normal brain. This pattern of specificity was observed in 40 brains from Alzheimer patients and 16 brains from normal subjects.
• the relationship between the antibody staining of neurons and the presence of neurofibrillary tangles was determined using a double staining technique. Vibratome sections of formalin-fixed Alzheimer tissue react with die Alzheimer antibody and the immunoreactivity was visualized widi the use of the peroxidase conjugated goat antibody to mouse immunoglobulins. The peroxidase reaction was made visible by die use of 4-chloronaphthol. 4-Chloronaphthol is a peroxidase substrate and reacts to produce a product that precipitates in an aqueous solution but is soluble in organic solvents. The tissue section was photographed and die 4-chloronaphd ⁇ ol was removed by dehydration and xylene treatment.
• plaques and tangles were stained widi thioflavin S, a known sensitive histologic reagent for the demonstration of these lesions, and he section was photographed again.
• Study of die photographs and the staining patterns indicated that many neurons are stained botii by die Alzheimer antibody and by tiiioflavin S. Some neurons are darkly stained by the Alzheimer antibody and do not appear to contain neurofibrillary tangles. A small fraction of neurons contain tangles and are thioflavin-positive but are not positive for the Alzheimer's disease antigen.
• the staining of plaques by Alzheimer antibody was also studied by this method. The Alzheimer antibody revealed more plaques dian did die thioflavin.
• the plaques positive for Alzheimer antibody contained dystrophic neurites and hence were neuritic plaques.
• TBS tris-buffered saline
• TBS-SDS sodium dodecyl sulfate
• Solubility was tested by vortexing Alzheimer cortex homogenate for two minutes in TBS or TBS-SDS. The brain homogenate was then centrifuged for 10 minutes at 10,000g.
• the supernatant and pellet from the centrifugation were separated and d e pellet was washed twice by homogenizing and centrifuging as in the first instance.
• the supernatant and pellet were homogenized in water and various amounts of each sample were dried onto die polyvinyl plates for the ELISA. The amounts were 10, 3 and 1 microgram per 50 microliters.
• the presence of die Alzheimer antigen was determined using the ELISA.
• the reactivity of the insoluble paired helical filaments was monitored using a known antibody to paired helical filaments.
• the Alzheimer antibody showed reactivity in die TBS supernatant and die reactivity was quantitatively recovered in die TBS-SDS supernatant whereas die paired helical filaments reactivity remained in die pellet after the TBS-SDS extraction. Therefore, it can be concluded tiiat die Alzheimer antibody immunoreactivity is soluble and segregates away from insoluble paired helical filament immunoreactivity.
• Alzheimer antigen when viewed in d e electron microscope, consists of soluble paired helical filaments (Vincent and Davies, Proc. Natl. Acad. Sci. £ : 2878-2882, 1992). Although also consisting, at least in part, of Alzheimer antigen, the insoluble PHF will not readily dissolve under the solution conditions described above.
• Alzheimer antigen will dissolve under these solvent conditions.
• Alzheimer antigen epitope is not phosphatase sensitive, this does not establish that die Alzheimer antigen is not phosphorylated. It is known that many antibodies to neurofilament epitopes that identify neurofibrillary tangles fail to react after phosphatase treatment. In addition, this suggests that phosphorylation of the Alzheimer antigen epitope does not account for the ability of the Alzheimer antibody to distinguish between the Alzheimer antigen and normal brain antigens.
• Frozen brain specimens were thawed at room temperature until they had a firm consistency to prevent splintering during cutting. About 50 to 150 mg of cortical tissue was placed into a microfuge tube. Frontal and temporal brain cortical tissue consistently has higher Alzheimer's antigen levels.
• Cold homogenization buffer O.lmM Tris, 150 mM NaCl containing EGTA Gentamicin sulfate and Nipasept as preservatives
• the tissue was homogenized in the microfuge tube for about one minute using the matching pestie driven by hand or mechanical motor.
• the paniculate matter was removed from die homogenate by centrifuging it for one minute in a TDx centrifuge or the equivalent. The tissue was processed at 4 * C throughout the preparation. (-70'C is recommended for longer storage of die homogenate).
• the brain regions to be processed were identified and isolated.
• the tissue was stored at -80°C and placed at -20 * C overnight. When ready, the tissue was slightly thawed at room temperature in a biohazard hood to aid in dissection. However, the tissue was kept on ice at all times.
• the dissected tissue sample tiien was accurately weighed and placed in a polyethylene container surrounded by ice.
• Four (4) volumes (ml g) of cold homogenate buffer [lx] were added to tissue sample.
• PMSF was added to a total volume at a 1:100 dilution, ImM final concentration.
• die Polytron homogenizer was cleansed with several alternating washes of distilled water, followed by ethanol and a final water rinse.
• the tissue and buffer were homogenized using a Polytron with 15-20 second blasts with equal intervals, at a setting of 5-6 on the instrument dial. (Adequate homogenization of larger samples may require longer blasts or a higher dial setting.)
• the sample vessel was surrounded by ice and kept as cool as possible during the homogenization process.
• Protein- A purified IgG from the serum bleeds of die rabbits was used to coat beads.
• Tris Buffered Saline pH 8.0
• BSA phosphate buffered saline
• Beads tiien were washed, overcoated witii gelatin/sucrose, dried, and stored at room temperature.
• the assay of die present invention detects and measures Alzheimer's antigen.
• the Alzheimer's antigen effectively is captured by the polyclonal IgG coated on the beads.
• ALZ-50 used as the detection antibody, is allowed to specifically bind to die immobilized antigen and the bound ALZ-50 is subsequently quantified by using an enzyme conjugated anti-mouse IgM (e.g., a horseradish peroxidase linked goat anti IgM and an appropriate substrate).
• an enzyme conjugated anti-mouse IgM e.g., a horseradish peroxidase linked goat anti IgM and an appropriate substrate.
• Figure 2 depicts the configuration of the assay.
• Post-mortem samples tissue brain homogenates free of paniculate matters
• sample buffer 1% BSA in PBS, pH 7.5, containing Gentamicin and Nipasept
• 50 mL of the sample homogenate 50 mL of the sample homogenate and 150 mL of the sample buffer.
• sample buffer 1% BSA in PBS, pH 7.5, containing Gentamicin and Nipasept
• sample buffer 1% BSA in PBS, pH 7.5, containing Gentamicin and Nipasept
• the beads were washed with distilled water twice using the QwikWash System, and then incubated with 200 mL of an ALZ-50 solution containing about 0.35 mg/ml of IgM (in PBS pH 7.5 containing 0.1% casein, 0.5% Tween 20, Gentamicin and Nipasept) for 30 minutes at 37 * C.
• the beads were washed again (twice), incubated with 200 mL of the HRPO conjugated goat anti mouse IgM diluted in the ALZ-50 diluent buffer containing 1% goat serum and incubated again for 30 minutes at 37 * C.
• the beads were washed twice and transferred to die reaction test tubes according to instructions on the packaging.
• the samples producing a background corrected absorbance of 0.5 or higher may be diluted serially and die dilution providing the highest absorbance under 0.5 may be used, making sure that die appropriate corrections are made for die dilutions.
• tiiis assay normalizes the absorbance values to wet tissue weight, the data per unit protein can be expressed for Alzheimer antigen measurement.
• Figure 4 shows tiiat tiiere is no significant reactivity in die normal brain homogenate whereas ALZ-EIA activity in Alzheimer's disease dementia ("AD") brain homogenate titers down.
• the linear range of the curve is from 0.05 to 0.50.
• AD 1 is a pool of AD brain homogenates.
• AD 2 is a homogenate from one AD brain specimen.
• Normal 1 is a pool of nbn-AD brain homogenates and Normal 2 is a homogenate from one non-AD brain.
• Background represents assay runs in which homogenization buffer was used instead of brain homogenate. The mean is expressed as absorbance units. The data was accumulated over a period of 2 days. A total of 5 replicates were run per plate and mean of within plate CV's are reported.
• the concentration of an Alzheimer Antigen was measured in post-mortem brain tissue samples of temporal or frontal cortex from 111 human brains. Each of the patients from which the tissue was taken was evaluated clinically (e.g. diagnosis, age, gender and post mortem delay) and all die specimens used were evaluated pathologically prior to die biochemical analyses. There were 27 normal controls (NC), 28 neurological disease controls (NDC), and 53 Alzheimer's disease (AD) of which seven were designated as Senile Dementia of Alzheimer's Type (SDAT), and tiiree older Down's Syndrome with Alzheimer's neuropathology D/AD.
• NC normal controls
• NDC neurological disease controls
• AD Alzheimer's disease
• SDAT Senile Dementia of Alzheimer's Type
• D/AD tiiree older Down's Syndrome with Alzheimer's neuropathology
• the AD group included die following categories: AD, SDAT and D/AD (Down's with AD neuropathology) and the non-AD group included die following categories: NC (normal control) and NDC.
• AD cases were older tiian NC cases (75.8 vs 60.7 years), but NDC cases were comparable to AD group (69.8 vs. 75.8). Considering only cases that were 65 or older, die average ages becomes comparable, 74.4,, 76.7, and 78.8 years for NC, NDC, and AD categories respectively. D/AD cases were all older than 50 (average age, 53). Post mortem time varied from as short as 1 hour to as long as 96; some cases did not have this information available.
• NC Normal Control
• NDC Neurological Disease Control
• AD Alzheimer's disease
• SDAT Senile Dementia of Alzheimer's Type
• Alzheimer Antigen 1.39(0.03-2.0) 0.01(0.0-0.64) 2.0(0.70-2.0)
• the Alzheimer's disease group had significant Alzheimer antigen concentrations (expressed as per protein or per 10 mg of tissue weight), which is in contrast to both the normal and otiier neurological disease groups.
• the normal brain group and die group of odier neurological diseases essentially had no detectable Alzheimer antigens.
• the Alzheimer antigen concentrations in AD category had a median of 1.39 and ranged from 0.03 to 2.0 absorbance/10 mg tissue.
• the D/AD category mimicked AD, but the SDAT category demonstrated a wide range of the Alzheimer antigen concentrations (from 0 to 0.64).
• the Alzheimer antigen levels in NC and NDC categories were not significantly different (Student-t test, p >0.797).
• AD group had an overall median of 0.77 and ranged from 0.0 to 2.0 absorbance units per 10 mg. tissue.
• Figure 5 represents a scattergram plot of die Alzheimer antigen concentration for die AD/SDAT versus the combined normal and other neurological group at two different scales.
• the group of normal brains and the group of other neurological diseases are presented togedier in Figure 5 due to the lack of difference in the Alzheier antigen concentrations (Dunnet's Test, p 0.05).
• An absorbance value of 0.1 was used as an empirically derived cut off between Normal range and AD range.
• Alzheimer antigen concentrations for all non-AD cases fell under tiiis line whereas Alzheimer antigen concentrations for 48 out of 56 cases in AD group fell above this line. Only three cases in AD category overlapped widi NC and NDC cases.
• the data also was analyzed by a stepwise multiple regression analysis widi forward selection technique (using dummy variables for diagnosis, gender, and patiiological findings) and by Dunnet's Test when comparing die AD/SDAT and the other neurological diseases groups' Alzheimer antigen levels to those of the normal group.
• the regression analysis considered the variables for diagnosis, age, sex, post-mortem delay and pathological report and explained the variance of the Alzheimer antigen concentration (separate statistical analysis for each Alzheimer antigen concentration unit category).
• the variation was further explained by considering the neurofibrillary tangles and plaque counts to the extent of 7 and 5% (p 0.01), respectively. Finally, age was found to contribute 4 and 3%, respectively, to each variation but was inversely correlated (p 0.05). Thus, the total correlation coefficient (R2) was found to be 26 and 46%, respectively. While Alzheimer's disease is associated with dementia, neuritic plaques and aging, dementia alone was not associated with an increase in Alzheimer antigen concentration, as there were 24 demented cases (16 NDC's, 3 AD's, 5 SDAT's) in the normal range for Alzheimer antigen (Table 4 and Figures 5A and 5B).
• plaques were not always associated widi die presence of Alzheimer antigen since some of them were probably diffuse plaques lacking the dystrophic neurites containing Alzheimer antigen. There were 29 cases (5NC's, l ⁇ NDC's, 3AD's, 5 SDAT's) with plaques which had practically no detectable Alzheimer antigen levels (see Table 4, and Figures 5 A and 5B).
• the Spearman Correlation Test showed a 15% (not significant) probability of Alzheimer antigen concentration being negatively correlated widi age.
• the oldest NDC case was a 90 year old female with multi-infarct dementia showing moderate plaques, and yet widi Alzheimer antigen concentration of 0.01 absorbance unit/10 mg tissue, while the youngest AD case was 46 years old and had an Alzheimer antigen concentration of more than 2.0.
• die claimed immunoassay distinguishes AD/SDAT from both normal and odier neurological diseases. Furthermore, based on tiiis data, clinical dementia, plaques, and old age per se do not appear to be associated with the increased Alzheimer antigen levels in the AD group. Thus, using this assay, regional variations can be examined, as well as the relationship of the Alzheimer antigen concentration to duration of die disease.
• the following diagnostic assay employs monoclonal antibodies as both die first and second antibodies.
• the first antibody is referred to as the capture antibody
• die second antibody is referred to as the detection antibody.
• Various combinations of capture antibodies and detection antibodies were utilized.
• Figures 7A-7M botii Alzheimer's disease brain extract and normal brain extract were utilized with the various antibody combinations. The brain extracts were obtained as described herein.
• the Y axis represents optical density, and shows die level of Alzheimer's disease antigen in the brain tissue extracts.
• the X axis indicates die amount of brain protein used in die assay in nanograms. In all of the assays performed, no significant level of Alzheimer's antigen was detected in normal brain. However, in every assay performed on Alzheimer's disease brain, Alzheimer's antigen was detected, Hence, die monoclonal antibodies of die invention can be used in various combinations to produce sensitive and specific assays which detect the presence of Alzheimer's antigen, and thereby diagnose Alzheimer's disease.
• CSF cerebrospinal fluid
• SAS saturated ammonium sulfate
• the solution was then centrifuged at 5000 g and die pellet was dissolved in 40 ml of 0.01 M sodium phosphate and dialyzed into PBS pH 7.4 overnight.
• the sample was dissolved in 5% SDS-5% beta-mercaptoethanol, fractionated on a 10% SDS-polyacrylamide gel, and transferred to nitrocellulose.
• the nitrocellulose was incubated for 1 hr in PBS pH 7.4 plus 5% nonfat dry milk and 1% human serum. Overnight incubations with Alzheimer antibody followed by a 3 hour incubation with peroxidase coupled goat antimouse antibody both diluted in blocking solution containing 1% human serum was performed. Presence of peroxidase was visualized by reacting with 4-chloro-l-naphd ⁇ ol (0.2 mg/ml) plus 0.44 mM hydrogen peroxide in 0.1 M Tris pH 4.5.
• Alzheimer antigen was readily detected in CSF.
• the nine CSF samples examined were obtained by lumbar puncture from presumptive Alzheimer patients and one CSF sample obtained by cisternal magna puncture obtained from a deceased patient whose diagnosis of Alzheimer's disease was later confirmed by neuropadiologic analysis.
• Eight of die nine samples from the living patients and one sample from die deceased patient yielded a Western blot pattern showing the characteristic 68,000 dalton doublet.
• CSF was also obtained by lumbar puncture from 6 nondemented patients. These samples did not show d e 68,000 dalton doublet.
• a CSF sample was examined from one patient who died with an initial clinical diagnosis of Alzheimer's disease. Western blot analysis of die CSF did not reveal a 68,000 dalton doublet. Subsequent analysis by neuropatiiology showed no neuritic plaque or neurofibrillary tangle pathology indicating that die patient did not die from Alzheimer's disease.
• This example shows that the high degree of accumulation of Alzheimer antigen and die high degree of Alzheimer antibody immunoreactivity is unique to Alzheimer's disease.
• Quantitation by ELISA shows that under conditions favoring high affinity antibody binding, tiiere was 56 times more Alzheimer antibody immunoreactivity present in brains of Alzheimer's disease patients than in brains of normal patients, and that tiiere was 33 times more Alzheimer antibody immunoreactivity in brains from Alzheimer cases than in brains from cases with Pick's Disease or GPD.
• Immunocytochemistry suggested tiiat this increased reactivity is largely the result of the abundant reactivity of abnormal neurites present in brains affected by Alzheimer's disease. Other diseases possess very little abnormal neuritic reactivity.
• the selective increase in neuritic reactivity in Alzheimer's disease explains die specificity of plaque staining by Alzheimer antibody. Only in Alzheimer's disease does Alzheimer antibody immunoreactivity extend from the affected cell body into the neurites and into the neuritic plaques.
• Alzheimer antigen is precipitated from 3.5 ml of cerebrospinal fluid by incubation with an equal volume of saturated aqueous ammonium sulfate at 4 * C.
• the samples are spun in a centrifuge at about 20,000g for about 20 minutes at 4 * C.
• the supernatant is removed and die pellet is broken up in 50 microliters of aqueous 0.01 M sodium phosphate at pH 6.8.
• Western blot analysis is used to detect the Alzheimer antigen.
• the samples are run on a 10 percent SDS-PAGE gel.
• the Alzheimer antigen is transferred to nitrocellulose for three hours at 125 mA and the buffer contains 19.2mM glycine, 2.5mM trizma base and 20 percent medianol at pH 8.3.
• Successive treatments are carried out with a blocking solution of 0.01M TBS with 5% v/w dried milk at pH 7.4 for one hour and then Alzheimer antibodies and the blocking solution for one hour.
• Cerebrospinal Fluid Assays Utilizing Monoclonal Antibodies Alzheimer's disease antigen was concentrated from cerebrospinal fluid by immunoaffinity chromatography. Cultures of the ALZ-50 cell line were screened to isolate an IgGi secreting variant. Class-switch variants arose spontaneously in cultures of IgM secreting cells. The variant was designated P42, and was shown to retain die ALZ-50 binding properties using
• the P42 IgGi was purified by chromatography on protein A columns, and attached to Affi-gel 10 (Biorad Laboratories) following the manufacturers protocol. Columns were prepared on P42 Affi-gel 10. Cerebrospinal fluid from cases of Alzheimer's disease and from non-demented individuals was run dirough the column, and the column was then washed widi Tris-buffered saline. Bound antigens were eluted with a solution of 3M potassium diiocynate. When cerebrospinal fluid from cases of Alzheimer's disease was used, small quantities of protein (less than 1 microgram per ml of fluid used) was detected in the eluate. No detectable protein was eluted from columns used with cerebrospinal fluid from non-demented individuals.
• cerebrospinal fluid can be concentrated prior to being utilized in die assay of the invention. However, it is not necessary to concentrate cerebrospinal fluid prior to utilizing it in d e assay of the invention.
• Cerebrospinal fluid from both normal and Alzheimer's disease individuals was assayed for the presence of Alzheimer's antigen on a solid support, as described herein, in order to diagnose Alzheimer's disease.
• Monoclonal antibody TG5 was used as die first antibody to capture the Alzheimer's antigen in cerebrospinal fluid.
• Monoclonal antibody MCI 5 was used to detect die captured antigens (Figure 8A). Cerebrospinal fluid was not concentrated in this assay.
• Figure 8B illustrates additional data utilizing cerebrospinal fluid from botii normal (N) and Alzheimer's disease (AD) individuals.
• the first antibody i.e., die antibody used to capture die Alzheimer's antigen found in cerebrospinal fluid
• the second antibody i.e., die antibody used to detect the captured antigens, was either monoclonal antibody TG4 or MC15.
• die assay can be built out from several solid phases (i.e., nitrocellulose or PVDF paper).
• the first antibody is an anti-Fc IgG specific antibody which is non-covalendy bound to the paper.
• die uncovered sites on the paper are blocked widi a variety of blocking agents; e.g., BSA, casein, gelatin.
• the capture antibody is then added, and die sample is allowed to bind to die paper.
• die detection antibody is added.
• An anti- ⁇ chain specific biotinylated antibody is added, and an avidin HRP conjugate is added.
• the substrate used can be any of several, including ECL, ABTS and TMB.
• the presence of Alzheimer's antigen is then determined, the presence of said antigen correlating with a positive diagnosis of Alzheimer's disease.
• die capture monoclonal antibody is of the IgG isotype, and is selected from the group consisting of PHF-1, TG5 and MCI.
• the detection monoclonal antibody is of die IgM isotype, and is selected from die group consisting of TG3, TG4 and MCI 5.
• Figure 10 shows a CSF assay which indicates tiiat Alzheimer's disease antigen is present, wherein the capture antibody used in the assay is PHF-1, and the detection antibody used in the assay is TG3.
• Alzheimer's antigens can be detected in cerebrospinal fluid utilizing both polyclonal and monoclonal antibodies in die assay of d e invention.
• Polyclonal antibody was used to capture the
• Alzheimer antigens and monoclonal antibody PHF-1 was used to detect the bound antibodies.
• Cerebrospinal fluid samples were obtained from individuals visiting clinics specializing in the diagnosis and treatment of memory disorders. Approximately 140 samples were tested, and individuals were grouped according to accepted standards of clinical diagnosis. The individuals were grouped as follows:
• AD-1 These individuals met the best available clinical criteria for the diagnosis of
• Alzheimer's disease and are generally described as probably AD.
• AD Alzheimer's disease
• AD-2 These individuals met most, but not all, of the generally accepted criteria for diagnosis of Alzheimer's disease. Because of certain atypical features, they are described as possible AD, and 75% to 85% of tiiese individuals would be found at autopsy to have suffered from Alzheimer's disease.
• PC These individuals were diagnosed as suffering from one or more psychiatric disorders, such as depression, manic-depressive illness or schizophrenia.
• NC These individuals were diagnosed as being free from dementia at die time of examination, and are considered to be normal controls. Two individuals in this group had somewhat increased levels of immunoreactivity. Both patients were elderly women widi some impairment of cognitive function; howeyer, this impairment was insufficient to warrant a diagnosis of dementia.
• NDC This group of individuals was diagnosed as having a neurologic disorder other than Alzheimer's disease. Included in this group are individuals with Parkinson's disease, Huntington's Chorea or multi-infarct dementia.
• MUX The clinical diagnosis in this group of individuals suggested d e presence of more than one neurologic condition, die majority of the individuals having a combination of Alzheimer's disease, multi-infarct dementia and/or Parkinson's disease.

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