AU643743B2 - Method for diagnosing neurodegenerative diseases - Google Patents

Description

METHOD OF DIAGNOSING NEURODEGENERATIVE DISEASES

TECHNICAL FIELD

The present invention relates to diagnosis of certain diseases. More particularly, the invention relates to novel methods of diagnosing neurodegenerative diseases in living persons.

BACKGROUND' OF THE INVENTION There are many diseases that involve malfunctoning of cells in the brain and that are manifested by abnormalities in behavior or mental activities, such as slurring of speech, loss of control of bodily functions, difficulties in ambulation, difficulty in learning or memory loss, but are not diagnosable in living persons by any physical or chemical abnormality that is readily detectable, except, in some cases, by analysis of brain tissue obtained by extremely risky and costly brain biopsy. Among such diseases are neurodegenerative diseases, which are diseases that involve degeneration and, ultimately • death, of neurons in the brain. Neurodegenerative diseases include Alzheimer's Disease (AD) (which, for purposes of the present application, includes senile dementia of the Alzheimer's Type) , Pick's Disease, progressive supranuclear palsy (PSP) , Parkinson's Disease, diffuse Lewy body diseases, and multiple-infarct dementia (MID) .

Diagnosis of neurodegenerative diseases has been based primarily, and, in some cases, solely on evaluation of behavior and mental activities. The diagnoses are subject to a high rate of error, because there is a significant subjective component in such evaluations; significant overlap of behavioral characteristics and mental capabilities among persons afflicted with such diseases, especially early in the development thereof, and those not so afflicted; and significant overlap of the behavioral characteristics and mental capabilities of persons suffering from different ones of such diseases.

Misdiagnosis of such diseases, either in incorrectly identifying a disease, from which a person is suffering, as a neurodegenerative disease, when it is not, or in incorrectly identifying the neurodegenerative disease, from which a person is suffering, is a serious problem. Misdiagnosis often results in inappropriate treatment, which has serious adverse consequences for affected individuals and their loved ones and for society as a whole. Inappropriate treatment, due to misdiagnosis, can entail unwarranted confinement of patients, at very high cost, in financial and psychological terms, to the patients and their loved ones, and very high economic cost to - society. Inappropriate treatment, arising from mis¬ diagnosis, can also involve withholding of physical, psychological or drug therapy that could be effective in slowing the development, or controlling the symptoms, of a disease if it were correctly diagnosed. Inappropriate withholding of therapy is also sometimes accompanied by misprescription of therapy, which, while having no beneficial effect related to the disease which the patient actually has, can adversely affect the health of a patient. A major problem, to which the present invention is especially directed, is the misdiagnosis as having AD of persons who are actually suffering from treatable and, in some cases, correctable conditions such as various metabolic diseases (e.g., vitamin B12 deficiency, malnutrition) or depression, which are not neurodegenerative at all, or MID.

Thus, there is a need for methods to improve the accuracy of diagnoses of neurodegenerative diseases. Such improvements should entail measuring characteristics, such as physical, physiological or biochemical characteristics, of a person that can be measured objectively, without great risk to life or health and without great cost, and that can more definitively differentiate the presence of such a disease from the absence, thereof than evaluation of behavior or mental capabilities can. Satisfying this need requires the discovery of such objectively determinable charcteristics that correlate well with the presence or absence of a particular disease or type of disease. The present invention is directed to meeting this need with respect to diagnosing whether a person is suffering from a neurodegenerative disease of a particular type or not. In recent years, it has been found, in studies of brain tissue taken postmortem, that certain neurode¬ generative diseases are characterized, at the cellular level, by the presence in brain neurons of abnormal, fila¬ mentous material that includes ubiquinated components (i.e., components that are coupled to molecules of the protein ubiquitin) . Such filamentous material includes the paired helical filaments of the neurofibrillary tangle characteristic of AD; the straight filaments of the Lewy body characteristic of Parkinson's Disease; the straight filaments of the Pick body characteristic of Pick's

Disease; and the straight filaments of the neurofibrillary tangle characteristic of PSP. Mori et al., Science 235, 1641 (1987); Perry et al., Proc. Natl. Acad. Sci. (USA) 84, 3033 (1987); Manetto et al., Proc. Natl. Acad. Sci. (USA) 85, 4501 (1988) ; Shaw and Chau, Proc. Natl. Acad. Sci. (USA) 85, 2854 (1988); Lowe et al., J. Pathol. 155, 9 (1988); Kuzuhara et al., Acta Neuropathol. (Berlin) 75, 345 (1988); Nature 337, 687 (1989). Diseases such as these, characterized by abnormal neuronal filaments with ubiquinated components, are thought to involve some defect in production or processing of ubiquitin, at least in the affected neurons. Such diseases are referred to herein as "ubiquitin-associated neurodegenerative diseases" or "UAND's". On the other hand, there are neurodegenerative diseases, such as MID, which do not involve any defect in production or processing of ubiquitin and are, therefore, not UAND's.

A monoclonal antibody, that recognizes paired helical filaments ("PHF's") isolated from, as well as in, neurofibrillary tangles in brain tissue obtained postmortem from persons with Alzheimer's Disease, has been reported to recognize an antigen that had an average concentration, in cerebral spinal fluids ("CSF's") of a group of persons diagnosed, on the basis of behavior and mental activities, as having Alzheimer's Disease, that was significantly greater than the average concentration in CSF's of another group of persons, of approximately the same age on the average as the group diagnosed as having Alzheimer's Disease but said to be suffering from stroke, seizures, multiple sclerosis, and other neorological conditions but apparently not Alzheimer's Disease. Mehta et al., The Lancet, July 6, 1985, p. 35. There was a very large overlap in the data reported by Mehta et al. , supra, for the amounts of PHF-immunoreactive material found in the CSF's of persons thought to be suffering from AD and the amounts of such material found in the CSF's of such persons thought to not be suffering from that disease.^"

A number of monoclonal antibodies that recognize paired helical filaments, isolated from neurofibrillary tangles of brains obtained postmortem from persons afflicted with Alzheimer's Disease, have been reported.

See, e.g., Wang et al., Acta Neuropathol. (Berlin) 62, 268 (1984). Among these is one designated "5-25", a murine IgG antibody. Wang et al., supra. Monoclonal antibody 5-25 is also known to recognize mammalian ubiquitin. Perry et al._Λ "Ubiquitin in Alzheimer Paired Helical Filaments: a Study with Monoclonal Antibodies," Abstract, November, 1987 Meeting of the Society for Neuroscience.

Ubiquitin is a 76-amino acid protein found in all eukaryotes. The sequences of the protein are very highly conserved among all of the many species, from yeast to

H. sapiens, for which the sequences have been determined. Ubiquitin is thought to have an important role in regula¬ tion of protein turnover but apparently has other functions as well. Ubiquitin's role, or the role of its production or processing, in UAND's remains obscure.

Ubiquitin of many species, including many mammalian species, is readily available. A common source is bovine erythrocytes. Bovine and human ubiquitin are identical in sequence. Given the ready availability of ubiquitins, anti-ubiquitin antisera, which comprise polyclonal anti- ubiquitin antibody, can be readily obtained from any mammalian species, including mice, rats, rabbits, goats and sheep, using standard immunological methods. Polyclonal antibody (e.g., as part of an antiserum) prepared with ubiquitin from one mammalian species will recognize ubiquitin from all other mammlian species. Similarly, employing standard techniques, hybridomas (e.g., rat or murine hybridomas) that produce monoclonal, anti-ubiquitin antibodies are readily prepared and cultured to produce the antibodies. With rare exceptions, given the highly conserved sequences of ubiquitins among mammalian species, a monoclonal antibody prepared against the ubiquitin from one mammalian species will recognize, with identical or very closely similar avidity, the ubiquitin of any other mammalian species. The exceptions would occur when the epitope to which the monoclonal antibody binds, on the ubiquitin that was used to prepare the hybridoma that produces the antibody, is one that, because of a rare difference in amino acid sequence, differs in sequence or three-dimensional conformation in the ubiquitin from another species; in such a situation, the monoclonal antibody might bind with very much lower avidity to the ubiquitin not used in preparation of the hybridoma. Any anti-ubiquitin monoclonal antibody from an hybridoma prepared using bovine ubiquitin would recognize human ubiquitin, because the sequences of the two ubiquitins are identical.

Since before the priority date of this application, the aforementioned monoclonal antibody 5-25 has been available from the Institute for Basic Research of the New York State Research Foundation for Mental Hygiene (Staten Island, New York, USA) and Senetek PLC (Aarhus, Denmark).

The antibody remains, and will remain, available from these sources. Viable cultures of an hybridoma which produces antibody 5-25 were deposited, prior to the priority date of this application, at the American Type Culture Collection, Rockville, Maryland, USA under the terms of the Budapest Treaty on the Deposit of Microorganisms for Purposes of Patent Procedure and the Regulations promulgated thereunder; and samples of these deposited cultures will be available in accordance with the Treaty and Regulations once a now pending United States patent application or a now pending Canadian patent application, which refers to said deposit, is granted.

SUMMARY OF THE INVENTION

It has now been realized that soluble substances with ubiquitin epitopes (i.e., soluble substances recognizable by anti-ubiquitin antibodies) are present in the CSF's of living persons suffering from UAND's at significantly higher concentrations than in the cerebral spinal fluids of living persons not suffering from such diseases. Thus, provided hereby is a method of diagnosing whether a living person has a UAND, which method comprises assaying ari aliquot of the CSF from the person for the presence of a soluble substance recognizable by an anti- ubiquitin antibody. The presence of a significant concentration of such substance(s) , which could be free - ubiquitin or some other soluble antigen covalently bound by ubiquitin, in the CSF is consistent with the person's being afflicted with an UAND, provides support for an indication of such a disease provided by other criteria, heretofore usually involving the highly error-prone evaluation of behavior and mental activities, and is inconsistent with a conclusion, based on such other criteria, that such a disease is not present. The absence of a significant concentration of such ubiquitin-epitope-bearing substances in the CSF is inconsistent with the person's being afflicted with an UAND, provides support for an indication that such a disease is not present, based on other criteria, and is inconsistent with a conclusion, based on other criteria, that such a disease is present.

Thus, by providing an objectively and relatively simply measureable criterion for whether a living person is suffering from an UAND, the present invention provides significantly improved methods for diagnosing such diseases in living persons.

Among other advantages of the improved methods of the present invention, the methods can be carried out more simply and inexpensively and with more uniform reliability because a mammalian ubiquitin, readily available in consistently high quality and readily handled without dimunition in quality, is employed as the standard in the methods. By the use of a mammalian ubiquitin as "the standard" in the methods is meant that such an ubquitin is used as (1) the competing substance in such methods that entail competitive assays, (2) the control substance, and (3) the substance employed to standardize the measurement of concentration of the ubiquitin-epitope-bearing substances assayed for in CSF samples.

Among the other benefits provided by the invention, the methods of the invention provide an advantageously facile and much needed means to more reliably distinguish, than has been possible heretofore, AD from other diseases, such as MID, depression and metabolic diseases, which have similar clinical manifestations in behavior and mental activities.

DETAILED DESCRIPTION OF THE INVENTION In one of its aspects, the present invention is a method of diagnosing an UAND in a living person which comprises assaying an aliquot of CSF from the person for soluble, ubiquitin-immunoreactive substances in an assay wherein a mammlian CSF is employed as the standard. In another of its aspects, the present invention is an improvement, in a process of diagnosing whether a living person is suffering from an UAND, which improvement comprises assaying an aliquot of CSF from the person for soluble, ubiquitin-immunoreactive substances in an assay wherein a mammlian CSF is employed as the standard. The present invention relates to ubiquitin- associated, neurodegenerative diseases ("UAND's") generally. Thus, the presence of a significant concentration of soluble, ubiquitin-immunoreactive substances (i.e., 'substances which have epitopes recognized by anti-ubiquitin antibodies) in the cerebral spinal fluid ("CSF") indicates that the person, from whom the fluid- as taken, is suffering from an UAND but, by itself, does not indicate which one, of such diseases, the person is suffering from. Similarly, the absence of a significant amount of soluble, ubiquitin-immunoreactive substances in the CSF of a person indicates that the person is either not afflicted with any UAND or not afflicted with any such disease that has progressed to the stage where significant numbers of neurons have degenerated. In cases where the presence of significant amounts of soluble, ubiquitin- immunoreactive substances are found in the CSF of a person, indicating that the person is afflicted with some UAND, additional diagnostic steps, known to the skilled in evaluating such diseases, can be employed to define with greater specificity the UAND afflicting the person, or, if the disease is one of a group for which specific diagnosis is not possible, the group of UAND's which includes the one afflicting he person.

In any case, even if an UAND, from which a person may be suffering, cannot be identified specifically, it is very important to know, with a much higher degree of certainty than provided by methods available in the art heretofore, whether or not a person, and particularly a person suspected of suffering from some UAND, is suffering from any such disease. Thus, the method and improvement of the present invention are a significant advance in the art by virtue of providing an objective, biochemically based, simple-to-measure, and accurately measureable criterion that is indicative with greater certainty than prior art methods of whether or not a person is suffering from an UAND and that provides an important check on the accuracy of diagnoses, based on prior art methods, that a person is or is not suffering from such a disease.

By an ubiquitin-associated neurodegenerative disease in the present specification is meant a disease that entails degeneration of neurons in the brain and that is characterized by the presence, in affected neurons, of abnormal filaments that comprise ubiquinated components. As the skilled understand, the different ones of such diseases entail degeneration of neurons in different areas of the brain and, consequently, have different manifes¬ tations in abnormalities of behavior and mental activities. Among the UAND's are Alzheimer's Disease (including senile dementia of the Alzheimer's type), Parkinson's Disease, Pick's Disease, PSP, and diffuse Lewy body diseases. For these diseases, it has not heretofore been recognized that the degeneration of neurons that underlies neurodegenera- tive diseases is accompanied by release of ubiquitin- immunoreactive substance(s) into the cerebral spinal fluid and that such ubiquitin-immunoreactive substance(s) (including possibly free ubiquitin) remain(s)- sufficiently stable in the cerebral spinal fluid to be detectable therein through the ubiquitin epitopes thereon or in assays wherein an mammalian ubiquitin is the standard.

The aliquot of cerebral spinal fluid ("CSF") taken from a living person for assay in accordance with the present invention is obtained by, or under the supervision of, a physician by any standard procedure, preferably lumbar puncture. Preferably, a portion of the fluid after the 4th mL taken from the person is employed in an assay in accordance with the invention. This portion of the fluid is, preferably immediately after being taken from the person, centrifuged for about 5 minutes to about 20 minutes (peferably about 10 minutes) at low speed (about 500 x g to about 1000 x g, preferably about 700 x g) , in, e.g., any standard, bench-top centrifuge, to remove cells and particulate matter, especially blood cells.

It is important that the aliquot of CSF on which the assay in accordance with the invention is carried out be "blood-cell-free," i.e., have blood cells removed as by the centrifugation procedure just described and be "hemoglobin-free," , i.e., have less hemoglobin than is required to make the aliquot blood-colored to the naked eye. The blood-cell-free supernatant from the centrifugation is then, preferably immediately after the centrifugation, frozen at below - 20 °C until just before use. A blood-cell-free CSF sample should be used in an assay according to the invention preferably after, at most, one such freezing with subsequent thawing; it has been found that repeated freezing and thawing may reduce the • concentration of soluble ubiquitin-immunoreactive substances that can be detected in a sample of CSF.

The assay for ubiquitin-immunoreactive substances in a sample of CSF is by any immunoassay method in the art for detecting human ubiquitin-epitopes specifically. Preferred are immunoassay methods employing monoclonal antibodies specific for human ubiqutin (or any ubiquitin with the same amino acid sequence as that of humnan ubiquitin) , polyclonal anti-ubiquitin antisera or any antibody (monoclonal or polyclonal) , whether prepared using ubiquitin as the antigen or not, which recognizes human ubiquitin with high avidity.

Anti-ubiquitin monoclonal antibodies can be provided by hybridomas of any species, such as rats or mice, cultures of which^' have been prepared by any standard technique with a ubiquitin (preferably one with the same amino acid sequence as human ubiquitin) as antigen. Any of the various anti-ubiquitin monoclonal antibodies available in the art or prepared with antigens other than pure ubiquitin can also be employed. One such antibody is that designated "5-25" (also sometimes referred to as "525") , which is described hereinabove and made by an hybridoma that was prepared using as antigen paired helical filaments isolated from neurofibrillary tangles obtained postmortem from brains of persons with Alzheimer's Disease.

The immunoassay methods that can be employed include homogeneous and heterogeneous methods as well as competitive and non-competitive methods, as well known in the immunoassay art. Heterogeneous immunosorbent and sandwich assay methods ^"can be employed. In such an immunosorbent assay, particularly the most preferred immunoassay method in accordance with the invention, an enzyme-linked immunosorbent assay (ELISA) , ubiquitin is bound to a solid support, such as latex beads or the inside wall of a plastic tube or of a well of a standard millititer or microtiter plate, and competes with soluble ubiquitin-immunoreactive substances, that may be present in an aliquot of a sample of CSF being analyzed, for binding by anti-ubiquitin antibody. In the immunosorbent assay, the anti-ubiquitin antibody is present at a concentration determined to ensure maximum sensitivity; this concentration is readily determined by the skilled immunologist for any source of antibody (e.g., murine ascites, hybridoma culture supernatant) given an estimate of the maximum concentration of epitope for the antibody in samples to be assayed (in the assay method described in the Example, the assumption that 200 ng/ml ubiquitin presents the maximum amount of ubiquitin-immunoreactive epitope that will occur in an aliquot of CSF assayed has been found to be acceptable) . In the immunosorbent assay, antibody is first incubated with an aliquot of sample, whereupon some antibody becomes complexed with ubiquitin-immunoreactive substances that might be present, then the solution (with antibody and sample) is brought into contact with the solid-support, to which ubiquitin is bound (and is present in great excess relative to antbody concentration) , and antibody which did not bind to ubiquitin-immunoreactive substances in the sample binds to the ubiquitin on the solid support. Then, after washing to remove antibody not bound to solid-support-bound ubiquitin, the antibody so bound is detected using a second antibody, that recognizes the anti-ubiquitin antibody and that is labeled for detection, such as (in an ELISA) with an enzyme, that can be detected, for example (and preferably) through catalysis of a reaction that produces a spectrophotomertrically detectable substance, or in some other of numerous ways known to the art for detecting antibody (e.g., with a radioactive isotope, using an avidin-biotin detection method, using colloidal gold, using agglutination methods, etc.). Alternatively, a sandwich method can be employed wherein anti-ubiquitin antibody is bound to the solid support and wherein ubiquitin from a sample binds to this antibody and is then detected by binding of a second, anti- ubiquitn antibody that is labeled for detection. Radio- immunoassays, both heterogeneous and homogeneous, employing radiolabelled ubiquitin to compete for binding by an anti- ubiquitin antibody with ubiquitin that may be present in an aliquot of a sample of CSF being assayed, can also be employed, including immuneprecipitation assays, wherein ubiquitin from a sample and radiolabelled ubiquitin compete in a solution for binding to an anti-ubiquitin antibody and then the antibody (including that complexed with labeled and unlabeled ubiquitin) is separated from the solution by precipitation with an antibody against the anti-ubiquitin antibody and the amount of radioactivity (due to radio¬ labeled ubiquitin complexed by the anti-ubiquitm antibody) from the precipitate is measured.

As the skilled understand, in whatever immunoassay method is employed, appropriate controls and standards, using solutions of known concentrations of a mammalian ubiquitin, preferably one with the same amino acid sequence as that of human ubiquitin, must be used to obtain a meaningful result from an immunoassay. Second antibodies, that recognize the anti-ubiquitin antibodies and are used in various immunoassay methods, both polyclonal (which are adequate and preferred) and monoclonal, are readily and widely available to the skilled in the immunoassay art from, for example, numerous commercial sources. The second antibody will generally be of a species different from that of the first antibody and will recognize antibody of the same isotype as that of the first antibody. Proteins, other than antibodies, that bind specifically to antibodies, such as S. aureus protein A, can be employed in place of a second antibody and are also readily available in the art. Further, how to label such second antibodies (or other proteins) for detection and use them in the various methods are well known to the skilled ^'in the immunoassay art.

Practice of the invention is further taught in the following example.

EXAMPLE This example describes a method for assaying- in accordance with the invention an aliquot of CSF taken from a person for the presence of ubiquitin-immunoreactive substances. The method is a competitive ELISA. Generally, in a pre-incubation step, primary antibody (i.e., anti- ubiquitin antibody) and an aliquot of CSF are combined in a solution, which is then incubated to allow ubiquitin- immunreactive substances that might be present in the aliquot of CSF to bind to the antibody. Then the solution from the pre-incubation step is placed in a microtiter plate well that has been pre-coated with ubiquitin and a further incubation is carried out, whereupon a portion, depending on the concentration of ubiquitin-immunoreactive substance(s) in the CSF, of the primary antibody will bind to ubiquitin on the wall of microtiter plate. After this second incubation, and washing steps to remove from the microtiter plate well any primary antibody that is not complexed with ubiquitin bound to the wall of the well, a solution of an enzyme-labeled (e.g., alkaline-phosphatase- labeled) polyclonal antibody (in the form of antiseru ) against the primary antibody is added to the microtiter plate well and incubation carried out again to allow binding of labeled antibody to antibody complexed with ubiquitin bound to the wall of the microtiter plate well. After this incubation, the well is washed to remove unbound, labeled secondary antibody (i.e., anti-primary antibody antibody) and a chromogen solution is added and incubated in the well for a specified period of time at a specified temperature, after which the optical density of the solution in the well is measured at a suitable wavelength as a measure of the concentration of a product formed in a reaction catalyzed by the enzyme labelling the secondary antibody.

The chromogen solution is a solution of a substrate for a reaction, catalyzed by the enzyme label on the secondary antibody, that yields a colored product.- The concentration of colored product (and, consequently, the optical density of the chromogen solution at the appropri¬ ate wavelength after the incubation with enzyme-labelled, secondary antibody) is related to the quantity of secondary antibody present in the well, which in turn is related to the quantity of primary antibody, which in turn is inversely related to the concentration of ubiquitin- immunoreactive substance(s) in the aliquot of CSF that was assayed. The higher the concentration of ubiquitin- immunoreactive substance(s) that are recognized by the primary antibody in an aliquot of CSF, the more the optical density, at an appropriate wavelength for detecting the product of the reaction catalyzed by the enzyme label on the secondary antibody, will be reduced (i.e., color development "inhibited") in the chromogen solution after incubation in the microtiter plate well relative to the optical density obtained with a solution known to be free of ubiquitin in place of the aliquot of CSF.

100 x [1 - (optical density with aliquot of CSF/optical density with aliquot of ubiquitn-free solution) ] , where the optical densities are corrected for background, is called the "percent inhibition" or "% I" of the aliquot of CSF. The higher the %I for an aliquot of CSF, the higher the concentration of ubiquitin-immuno¬ reactive substance(s) recognized by the primary antibody in the CSF being assayed. By running assays of aliquots of CSF's in parallel with assays of solutions of known concentrations of ubiquitin, a standard curve can be obtained that can be used to estimate the concentration of ubiquitin epitopes recognized by the primary antibody in the CSF's being assayed. Microtiter well plates are prepared as follows: 200 xL of Ub stock solution (made by dissolving 10 mg of bovine erythrocyte ubiquitin in 1 mL of 1 % sodium dodecyl sulfate (SDS) and placing in boiling water for 2 minutes, followed by adding water to make a total of 10 mL) is mixed with 9.8 mL of coating buffer (50 mM sodium carbonate, pH 9.8) and 100 μ,L of the resulting solution is added to each well of the 96-well plate and the plate is incubated overnight at 4 °C. After the incubation, the plate is washed 3 times, each with GUB ("general use buffer") (10 mM Tris, 0.05 % Tween- 20^®, 0.85 % NaCl, pH 7.4). After the wash with GUB, the wells are blocked with 200 - 300 μL of 1 % bovine serum albumin (BSA) in GUB for 3 hours at 37 °C. (Alternatively, the BSA can be dissolved in phosphate-buffered saline (PBS) in place of GUB for the blocking step.) Then the wells are again washed 3 times with GUB as above. Finally, the excess of GUB is shaken from the wells and they are allowed to dry at 37 °C for 30 minutes.

Experiments were carried out using, as primary antibody, murine monoclonal antibody 5-25 obtained in murine ascites fluid after culturing an hybridoma, that makes the antibody, in vivo in mice after interperitoneal injection of hybridoma cells, in accordance with procedures standard in the immunological arts. As the skilled will understand, the concentration of antibody will vary somewhat from ascites preparation to ascites preparation, and the concentration of the antibody (which is critical in the assay) , at least in the solution combined with sample for the incubation during which the antibody binds with ubiquitin-immunoreactive substances that might be present in the sample, must be determined to provide maximum sensitivity and range, as described above and well known to the skilled. Stock ascites, stored until use at -70 °C, was raw ascites taken from a mouse diluted 1:800 with GUB. Primary antibody solution employed in the assay was then prepared from stock ascites by diluting stock ascites with PBS-BSA (PBS with 0.1 % BSA and 0.05 % sodium azide) to an extent to provide maximum sensitivity and range, assuming that 200 ng/ml of ubiquitin represents the maximum concentration of epitope reactive with 5-25 in a CSF sample.

Each aliquot of CSF to be assayed was diluted 1:1 with GUB (100 μL CSF (taken from after the 4th L of CSF taken from the patient by lumbar puncture and after centri¬ fugation as described above and thawing as described above) , mixed with 100 μL GUB) in a polypropylene tube, vortexed briefly and set aside on ice. 200 μL of primary antibody solution was added to each CSF-containing tube, the tube was vortexed briefly and then placed on ice.

At least four standards wee employed: 0 standard, which was GUB with 0.05 % sodium azide; low standard, which was 10 ng/ml in GUB supplemented with 0.05 % sodium azide of bovine erythrocyte ubiquitin; medium standard, which was 75 ng/ml in GUB supplemented with 0.05 % sodium azide of bovine erythrocyte ubiquitin; and high standard, which was 400 ng/ml in GUB supplemented with 0.05 % sodium azide of bovine erythrocyte ubiquitin. Other standards, with, e.g., 240 ng/ml, 120 ng/ml and 60 ng/ml of the ubiquitin in GUB supplemented with 0.05 % sodium azide can also be employed. When used as described further below, the standards have typically been found to provide approximately the following % inhibitions: Standard Approximate % I

(Ub cone, in ng/ml)

0 0 10 10

75 50

400 92

240 85

120 69 60 45

For each of the standards, 200 μL was placed, like the CSF samples, in a polypropylene tube, 200 μL of primary antibody solution was added, the tube was vortexed briefly and then placed on ice. For measurement of background, 400 μL of GUB supplemented with 0.05 % sodium azide was placed in a propylene tube on ice. (No antibody) .

All tubes were allowed to stand on ice for at least 5 minutes to insure temperatuure equilibration. Then all the tubes were removed from the ice and allowed to incubate for 90 minutes at 20 - 23 °C (preferably at constant 20 °C in a closed incubator) . The time and temperature of incubation in this step have been found to be important, thus both need to be controlled carefully. Just prior to use, a microtiter well plate, prepared as described above, was washed three times, with 1 minute incubation each time, with wash buffer and then "slap- dried." The "slap-drying" procedure was employed after all of the subsequent washings and was carried out by slapping the plate firmly face-down on to lint-free paper towels, holding the plate frame tightly during the process.

After the incubation of antibody with samples and standards, as described above, 100 μL of each incubating mixture (including that for "background") was added to each of three wells of the microtiter well plate (i.e., each was run in triplicate) . The plate was then allowed to incubate in a moistened incubation chamber for 90 mins. at room temperature (20 - 23 °C) . After the 90 min. incubation, the plate was washed three times in wash buffer (GUB with 0.5 M NaCl) , slap drying the plate after each wash.

After the wash, 100 μL of alkaline-phophatase conjugate was added to each well and the plate was incubated in the moistened incubation chamber either for 3 hours at room temperature or overnight at 4°C. Alkaline phosphatase conjugate was made by diluting 1:1000 with GUB supplemented with 0.05 % sodium azide a solution, purchased commercially, of polyclonal goat anti-mouse IgG (TAGO,

Inc., Catalog No. 6550) coupled to alkaline phosphatase. (Polyclonal rabbit anti-mouse IgG coupled to alkaline phosphatase (DAKO, Inc.,- Catalof No. D314) was also sometimes used, also diluted 1:1000 with GUB and 0.05 % sodium azide. *

Chromogen solution was prepared by dissolving para- nitrophenyl phosphate (PNPP) , obtained in tablet form from Sigma Chemical Co., St. Louis, Missouri, USA, to 2 mg/ml in 1 M aqueous diethylamine. (1 standard PNPP tablet as purchased dissolved in 1 ml of solution made by diluting 1:5 with water a 5 M, pH 9.5, aqeuous diethylamine solution.)

After incubation of the plate with alkaline- phosphatase conjugate, the plate was washed three times with wash buffer, with slap drying after each wash.

After this washing, 100 μL of chromogen solution was added to each we_l of the plate. The plate was incubated in the moistened incubation chamber at room temperature. The absorbance of 0 control wells was monitored periodically at _,405 nm (400 - 410 nm) using a standard microtiter plate reader until the absorbance reached 1.0. This usually occurred in 45 - 60 minutes. Then the absorbances of all of the wells were read.

The %I was then determined for each of the wells. The %I for a CSF sample or standard is the mean of the %I's for the three triplicates thereof. For persons with no known neurodegenerative disease (including persons for whom this status was confirmed by examination of brain tissue postmortem), %I's below the low standard, i.e., below 5 - 10 % and not significantly different from 0, have been found. It has been discovered in connection with the present invention that CSF's taken from such persons more than 2 - 3 hours portmortem do show significant amounts of ubiquitin-immunoreactive material, presumably arising from breakdown of cells in the brain. Because such persons have no observable, ubiquinated filaments characteristic of neurodegenerative disease in their brains, the ubiquitin in their CSF's postmortem is apparently free. Thus, the discovery suggests that much of the ubiquitin-immunoreactive material in the CSF's of persons suffering from UAND's is also free ubiquitin. The CSF's of persons suffering from Alzheimer's Disease have been found by the method described in -this Example to have %I's of more than 30 % and usually in the range of 50 -70 %. The CSF's of persons suffering from Parkinson's- Disease have been found by the method of this Example to have % I's in the 30 - 60 % range.

The CSF's of persons diagnosed as having amyelotrophic lateral sclerosis, which is not a neurodegenerative disease, have been found by the method of this Example to have %I's of 6 - 26 %.

Thus, a %I of greater than about 30 % indicates a significant concentration of ubiquitin-immunoreactive material in the CSF, indicating an UAND. While the present invention has been described herein with some specificity, those of skill will recognize many variations and modifications of what has been described that are still within the spirit of the invention. It is intended that such variations and modifications are also encompassed by the invention as described and claimed herein.

Claims (8)

1. A method of diagnosing an UAND in a living person which comprises assaying an aliquot of cerebral spinal fluid from the person for ubiquitin-immunoreactive substances.

2. A method according to Claim 1, wherein said disease is selected from the group consisting of Alzheimer's Disease, Pick's Disease, Parkinson's Disease, and progressive supranuclear palsy.

3. A method .according to Claim 1 wherein the ^'assay of the cerebral spinal fluid is by a competitive ELISA employing a monoclonal anti-mammalian ubiquitin antibody.

4. A method according to Claim 2 wherein the assay of the cerebral spinal fluid is by a competitive ELISA employing a monoclonal anti-mammalian ubiquitin^' antibody.

5. In a process of diagnosing whether^'a living person is suffering from an UAND, the improvement which comprises assaying an aliquot of cerebral spinal fluid from the person for ubiquitin-immunoreactive substances.

6. The improvement according to Claim 5, wherein the UAND, for which the person is being diagnosed, is selected from the group consisting of Alzheimer's Disease, Pick's Disease, Parkinson's Disease, and progressive supranuclear palsy.

7. The improvement according to Claim 5, wherein the assay of the cerebral spinal fluid is by a competitive ELISA employing a monoclonal anti-mammalian ubiquitin antibody.

8. The improvement according to Claim 6, wherein the assay of the cerebral spinal fluid is by a competitive ELISA employing a monoclonal anti-mammalian ubiquitin antibody.