JPH05503153A - How to diagnose neurodegenerative diseases - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Regarding malfunction of cells in the brain, e.g., slurred speech, impaired control of bodily functions , due to abnormalities in behavior or mental activity, such as difficulty walking, learning difficulties, or memory loss. is expressed, but in some cases by very dangerous and expensive brain biochemistry. Physical or chemical abnormalities that are readily detectable except by analysis of brain tissue obtained There are many diseases that are undiagnosable in living humans. Such a disease Neurodegenerative diseases, which are diseases that include degeneration and eventual death of neurons in the brain exists. Neurodegenerative diseases include Alzheimer's disease (AD) (for the purposes of this application) , including senile dementia of the Alzheimer type), Pick's disease ease), progressive supranuclear palsy (PSP), Parkinson's disease, diffuse Lewy bodies disease, and multiple-infarct dementia ia) (MID).

Diagnosis of neurodegenerative diseases is primarily, and sometimes solely, based on the assessment of behavior and neural activity. It was based on value. Significant subjective factors in such evaluations: In the early stages of a disease, there are differences between people who are affected by the disease and those who are not. Significant overlap in behavioral characteristics and mental abilities; as well as differences in the There is an overlap in the behavioral characteristics and mental abilities of humans affected by these diseases. , this diagnosis is prone to high misdiagnosis rates.

Mislabeling a human disease as a neurodegenerative disease when it is not a neurodegenerative disease. This is either an incorrect diagnosis or an incorrect diagnosis of a neurodegenerative disease affecting humans. Misdiagnosis of cancerous diseases is an important problem. Misdiagnosis often results in inappropriate treatment and have significant adverse consequences for affected individuals, their loved ones and society as a whole. vinegar. Inappropriate treatment due to misdiagnosis can have financial and emotional consequences for patients and their loved ones. of patients who have a very high cost in taste and a very high economic cost to society. entails unproven confinement. Inappropriate treatment due to misdiagnosis also May be effective in delaying disease onset or suppressing symptoms if diagnosed This may result in the patient being asked to refrain from physical therapy, psychotherapy, or drug therapy. inappropriate suppression of treatment; Sometimes, the system has no effective effect on the disease the patient actually has, and the patient's health may be compromised. with inappropriate prescribing of therapy, which may adversely affect the patient.

The main problem with which this invention is particularly concerned is in fact treatable and in some cases curable. For example, various metabolic diseases9! (e.g. vitamin B12 deficiency, malnutrition) A human suffering from depression or MID-like symptoms that is not neurodegenerative at all. It is a misdiagnosis that it is D.

Therefore, methods are needed to improve the accuracy of diagnosing neurodegenerative diseases. like this Improvements can be made without significant risk to life or health and without significant cost. This disease can be assessed objectively and more clearly than assessments of behavior or mental capacity. characteristics, such as physical, physiological or biochemical characteristics, that can determine the presence or absence of entails measurement. To meet this need, specific diseases or types of diseases must be met. It requires the discovery of objectively measurable characteristics that correlate well with the presence or absence of disease.

The present invention relates to the diagnosis of whether a human is suffering from a particular type of neurodegenerative disease. The purpose is to meet the need for leverage.

In recent years, studies of postmortem brain tissue have shown that certain neurodegenerative diseases is a ubiquinated component (i.e., a protein) at the cellular level. Presence of abnormal fibrous material in brain neurons, including components that bind to ubiquitin molecules It has been found that it is characterized by These fibrous substances contain Paired hemical filament of neurofibrillary tangle straight fibrils of Lewy bodies characteristic of Parkinson's disease. filaments, linear filaments of Pick bodies characteristic of Pick's disease, and characteristic of PsP. There are linear filaments of neurofibrillary tangles. Science such as sled (Mori) Science 235.1641 (1987); Perry et al. Proc, Natl, Acad, Sci, (Proc, Natl, Acad, Sci, ) (USA) 84.3033 (1987):? Magnetto Blogs such as Nattle, Akado, 4t, (USA) 85.4501 (1988 ); Shaw and Chau's block Natl Akado, Sai, (USA) 85.2854 (1988): Lowe et al. I, Pathol (J, Pathol,) 155.9 (1988); Kazuhara (Ka Acta Neuropathol (zuhara) etc. ) (Berlin) 75.345 (1988); Fuichi +- (Nature) 3 37.687 (1989). Abnormal neuronal filaments containing ubiquinated components Diseases such as these, characterized by This is thought to be related to some defect in the production or course of ubiquitin in the brain. this Such diseases are referred to here as “ubiquitin-related neurodegenerative diseases” or rUANDJ. I'll decide.

On the other hand, it does not involve any defects in the production or course of ubiquitin, i.e. There are neurodegenerative diseases like MID that are not AND.

Neurofibrillary tangles in postmortem brain tissue from humans with Alzheimer's disease Paired helical filaments in as well as neurofibrillary tangles isolated from Monoclonal antibody that recognizes (rPHPJ) has Alzheimer's disease This group was about the same age as the group diagnosed with Alzheimer's disease, but Humans who are said to be suffering from stroke, seizures, multiple sclerosis, and other neurological conditions. The brains of the group of A group of humans diagnosed on the basis of behavior and mental activity as having Zheimer's disease have been reported to recognize antigens with an average concentration in C3F of . Meter ( Mehta et al., The Lancet, July 6, 198 5.35 pages. I believe that I am suffering from AD as reported by Meter et al. in the above literature. The amount of PHF immunoreactive substances detected in C8F of humans affected by this disease The amount of such substances detected in human C3F, which is thought to be There is a huge overlap in the data.

Tangled nerve fibers from the brain removed postmortem from a human with Alzheimer's disease A monoclonal anti- body has been reported. For example, actors such as Wang, Neuropattle ( Berlin) 62.268 (1984). These include r5-25J, juniper There is something called murine IgG antibody. See the above literature by Wamp et al. .

Monoclonal antibody 5-25 is also known to recognize mammalian ubiquitin. There is. "Alzheimer's Paired Helical Phila" by Perry et al. Ubiquitin in the human body: Studies using monoclonal antibodies n　Alzheimer　Pa1red　Herical　Filaments ea 5tudy with Monoclonal Antibody) J, November 1987, Neuroscience Society Conference, Abstracts.

Ubiquitin is a heptad amino acid protein found in all eukaryotic cells. this The base sequences of proteins have been determined, from yeast to modern humans. It is highly conserved in all species. Ubiquitin is a protein turnover It is thought that it plays an important role in the regulation of turnover, but other mechanisms It also clearly has the ability. Role of ubiquitin or its production in UAND The role of time course is still unclear. In many species, including many mammalian species, ubiquitin is readily available. A common source is bovine red blood cells.

Bovine and human ubiquitin have the same base sequence. Easy availability of ubiquitin When considering sex, anti-ubiquitin antiserum containing polyclonal anti-ubiquitin antibodies standard immunizations from mammalian species including mice, rats, rabbits, goats and sheep. It can be easily obtained using scientific methods. Ubiquitin from one mammalian species Polyclonal antibodies (e.g., as part of an antiserum) produced using recognizes ubiquitin from all mammalian species. Similarly, monoclonal Hybridomas that produce biquitin antibodies (e.g., rat or murine hybrids) (doma) are easily produced and cultured to produce antibodies. highly in mammalian species Considering the ubiquitin base sequences possessed, the ubiquitin base sequence from one mammalian species is Monoclonal antibodies produced against Chin almost universally have the same affinity (a ubiquity in other mammalian species with very closely similar affinities. recognize the On ubiquitin used to produce hybridomas that produce antibodies The epitopes that monoclonal antibodies bind to have almost no differences in their amino acid sequences. Therefore, the base sequence or three-dimensional structure of ubiquitin differs from other species. In situations such as this, monoclonal antibodies are It is thought to bind to chitin with very low affinity. Using Uno's ubiquitin Anti-ubiquitin monoclonal antibodies produced from hybridomas are human. It recognizes ubiquitin because both ubiquitins have the same base sequence.

Prior to the priority date of this application, the monoclonal antibody 5-25 was Institute for Ba5icRe search of the New York 5tate Re5earc h Foundation for Mental Hygiene) (USA, Station Island, New York) and Senetech B.E. It was available from Enetek PLC (Arms, Denmark).

This antibody is and will continue to be available from these sources.

Viable cultures of hybridomas producing antibody 5-25 are a priority of this application. Prior to the date of entitlement, the provisions of the Butapest Treaty on the Deposit of Microorganisms for the Purposes of Patent Procedures and Samples of these deposited cultures deposited under the provisions promulgated under this Treaty. The pull may be based on currently co-pending U.S. patent applications or currently co-pending U.S. patent applications relating to said deposit. Once a U.S. patent application is granted, it is available pursuant to this Treaty and its provisions. Soluble substances containing ubiquitin epitopes (i.e. by anti-ubiquitin antibodies) (recognizable soluble substances) in the CSF of living humans with UAND. present in significantly higher concentrations than in the cerebrospinal fluid of living human brains that do not suffer from the disease. It has now become clear that this is the case.

Therefore, here we provide a method for diagnosing whether a living human has UAND. This method allows an aliquot of C3F from humans to be recognized by anti-ubiquitin antibodies. including analyzing for the presence of soluble substances. Free ubiquitin in C5F may be other soluble antigens to which ubiquitin is covalently attached; The presence of such concentrations is consistent with humans suffering from UAND, and until now the normal behavior and other criteria, including highly fallible assessments of mental activity. This argument supports the labeling of such diseases and that such diseases do not exist. This contradicts conclusions based on other criteria such as Such ubiquity in C3F The absence of significant concentrations of epitope-containing substances indicates that humans are susceptible to UAND. The absence of such a disease, which is inconsistent with having the disease and based on other criteria support for the indication that such a disease exists, based on other criteria. This contradicts the conclusions drawn.

Therefore, it is possible to objectively and relatively easily measure whether a living person is suffering from UAND. By providing criteria that can be used to determine such The present invention provides a significantly improved method for determining various diseases.

Another advantage of the improved methods of the present invention is that they are easy and inexpensive to implement. and as a standard for the method - consistently high quality, readily available and of high quality. Uniform reliability as ubiquitin is used which is easily handled without degradation It can be implemented with Mammalian species as standard J of the method The use of biquitin makes it difficult to determine whether such ubiquitin (1) (2) as a control substance, and (3) in C5F samples. used to standardize the concentration measurement of ubiquitin-epitope-containing substances to be analyzed. It means to use it as a substance for personal use.

In another advantage provided by the present invention, the method of the present invention improves behavior and mental activity. and other diseases with similar clinical manifestations, such as MID, depression and metabolic diseases. An advantageously easier method for distinguishing AD from AD more reliably than was previously possible. , provides a much-needed tool.

Detailed description of the invention In one of its embodiments, the present invention provides a method for diagnosing UAND in living humans. soluble shrimp chitin in an assay using mammalian C3F as a standard. in a method comprising evaluating a C3F aliquot from a human for epidemic-reactive substances. be.

In another of its embodiments, the invention provides a method for determining whether a living human is suffering from UAND. Improvements in this diagnostic method have been made in analyzes using mammalian C5F as a standard. Assessing C5F aliquots from humans for soluble ubiquitin immunoreactive substances This is an improvement that includes

The present invention generally relates to ubiquitin-related neurodegenerative diseases (UAND). Therefore, the brain soluble ubiquitin-immunoreactive substances (i.e., anti-ubiquitin) in the cerebrospinal fluid (C5F). The presence of significant concentrations of substances with epitopes recognized by human antibodies in the brain indicates that the person whose cerebrospinal fluid was collected is suffering from UAND, but the does not indicate which of these diseases a person is suffering from. Similarly, The absence of significant amounts of soluble shrimp chitin immunoreactive material in human C8F The person does not have UAND or a significant number of neurons degenerate. This indicates that the patient is not suffering from such a disease that has progressed to a serious stage. Dissolution The presence of significant amounts of sexually transmitted shrimp chitin immunoreactive material has been detected in human C3F; If it appears that a number of people are suffering from some kind of UAND, then this in humans using additional diagnostic steps known to those skilled in the evaluation of diseases such as The prevalence of UAND can be confirmed with great specificity, or if the disease has a specific diagnosis. If it is one of the groups in which it is impossible to You can check the UNAD group including D.

In any case, in cases where UAND affecting humans cannot be specifically identified, In many cases, humans can , especially in humans suspected of suffering from some form of UAND. It is very important to know whether you are doing so or not. Therefore, the method and improvements of the present invention Determines whether a person has UAND with greater accuracy than prior art methods The prior art shows that humans are suffering from or not suffering from such diseases. Easy to measure and accurate measurement that is an important check for the accuracy of diagnosis based on the surgical method Significant advances in technology have been made to provide possible objective, biochemically based criteria. It's Ayumu.

As used herein, ubiquitin-related neurodegenerative diseases refer to degeneration of neurons in the brain. As a result, affected nuclei of abnormal filaments containing ubiquinated components - refers to a disease characterized by the presence of As a skilled person would understand, this Various diseases, such as As a result, they exhibit different manifestations of abnormalities in behavior and mental activity. to UAND Alzheimer's disease (including Alzheimer's type senile dementia), Parkinson's disease disease, Bick's disease, PSP, and diffuse Lewy body disease. for these diseases Therefore, degeneration of neurons, which is the basis of neurodegenerative diseases, is caused by the release of shrimp chitin immunoreactive substances. brain with release into the cerebrospinal fluid, and such shrimp chitin immunoreactive substances (fearfully (containing free ubiquitin) remains sufficiently stable in the cerebrospinal fluid of the brain, and By ubiquitin-epitope or by mammalian ubiquitin-based analysis. It has never been shown that the brain can be detected in cerebrospinal fluid.

Brains collected from living humans for analysis according to the present invention are collected from cerebrospinal fluid (CSF). The coat is removed by standard methods, preferably by lumbar puncture, under the direction of a physician or under supervision. That's what you get. Preferably, 7 parts of the fluid after the 4th mL taken from a human is treated with the present invention. used for analysis. This portion of the fluid is preferably collected immediately after being collected from the human. For example, in a standard bench-top ill heart separator, about 5 minutes to about 20 minutes (preferably about 10 minutes) at low speed (about 500 x g to about 10 Centrifuge at 1000 x preferably about 700 x g) to remove cells and particulate matter, especially blood. Remove the sphere.

The C8F aliquot on which the analysis according to the invention is carried out is "free of blood cells", i.e. Blood cells have been removed by the above centrifugation, and "hemoglobin-free", i.e. i.e. less than is required to stain the aliquot blood-colored to the naked eye. It is important to have hemoglobin. Blood cell-free supernatant from centrifugation The solution is then frozen, preferably immediately after centrifugation, at below -20°C until ready for use. do. Analyzes according to the invention preferably involve such freezing, followed by thawing. In most cases, a C3F sample that does not contain blood cells after one treatment should be used. freezing and thawing Repeats of soluble shrimp chitin immunoreactivity that can be detected in C8F samples It has been found to reduce the concentration of sexual substances.

Analysis of soluble eviquitin immunoreactive material in C5F samples - By immunoassay methods in the art that specifically detect epitopes. human Ubiquitin (or ubiquitin with the same amino acid sequence as human ubiquitin) Monoclonal antibodies specific for Polyclonal anti-ubiquitin that recognizes human ubiquitin with high affinity Immunoassay using antiserum or antibodies (monoclonal or polyclonal) The Sei method is preferred.

Anti-ubiquitin monoclonal antibodies can be used in cultures where ubiquitin (preferably ubiquitin, which has the same amino acid sequence as human ubiquitin). hybrids of species, such as rats or mice, produced by can be formed by the user. Technically available or pure ubiquitous Any of the various anti-ubiquitin monoclonal antibodies produced with antigens other than ubiquitin Both can be used. One such antibody has the name r5-25J mentioned above ( (sometimes called r525J), which is associated with Alzheimer's disease. Paired helices isolated from neurofibrillary tangles postmortem removed from human brain Produced by hybridomas produced using filaments as antigens .

Available immunoassay methods include homogeneous and heterogeneous methods, as well as immunoassay methods. There are competitive and non-competitive methods that are well known in the field of research. Immunosolvents such as (immunosorbent) assays, particularly the most preferred immunosorbent assays according to the invention. In the enzyme-linked immunosorbent assay (ELISA), which is an assay method, , ubiquitin is present on latex beads or on the inner wall of plastic tubes or on standard media. Retiter (millititer) or microtitre (microtiter) iter) adheres to a solid support, such as the inner wall of the pores of a plate, and the anti-ubiquitin The solubility present in an aliquot of the analyzed C5F sample with respect to binding by the body. Competes with shrimp chitin immunoreactive substances. In immunosorbent assays, anti-ubiquitous Chitin antibodies have been tested at concentrations that have been found to guarantee maximum sensitivity. this concentration is the maximum concentration of the epitope for the antibody in the sample being analyzed. Antibodies from any source (e.g., murine ascites, Brydoma supernatant) is also easily determined by an experienced immunologist ( In the analysis method described in Examples, ubiquitin 200 ng/m1 is analyzed as C8 Gives the maximum amount of shrimp chitin-immunoreactive epitopes occurring in the F aliquot It turns out that this assumption is acceptable). In immunosorbent assays, anti- The body is first incubated with an aliquot of the sample, in which case some anti- The body then receives this solution, which is complexed by the shrimp chitin-immunoreactive substance present. When brought into contact with a solid support, ubiquitin is attached to it (relative to the antibody concentration). shrimp chitin in the sample (present in great excess), which does not bind to immunoreactive substances. The antibody binds to ubiquitin on the solid support. Next, wash and solid After removing antibodies that did not bind to port-bound ubiquitin, In this case, the detected antibody is detected using a second antibody that recognizes the anti-ubiquitin antibody. The second antibody produces a substance detectable by e.g. (preferably) spectrophotometry. can be detected by the catalysis of a reaction (in EIjSA) by an enzyme or by an anti- Other methods among the myriad methods known in the art for body detection (e.g. radioactive isotope using colloidal gold using an aviin-biotin detection method. (e.g., using a collection method). Or a sandwich In this method, anti-ubiquitin antibodies are bound to a solid support. Ubiquitin from the sample then binds to this antibody, which is labeled for detection. Detected by a second antibody. Present in an aliquot of the analyzed C3F sample ubiquitin and competing radiation that competes for binding by anti-ubiquitin antibodies. Both heterogeneous and homogeneous ubiquitin assays using functionally labeled ubiquitin Samples can also be used in immunoprecipitation assays, including immunoprecipitation assays. Ubiquitin and radiolabeled ubiquitin are combined with anti-ubiquitin antibodies in solution. Competing for binding, antibodies (complexed by labeled and unlabeled ubiquitin) ) are separated from the solution by precipitation with antibodies against anti-ubiquitin antibodies, The amount of radioactivity from the precipitate (radiolabeled ubiquitin complexed by anti-ubiquitin antibody) ) is measured.

As the skilled person will understand, no matter which immunoassay method is used, To obtain meaningful results from immunoassays, known concentrations of mammalian ubiquitous ubiquitin, preferably a mammalian ubiquitin having the same amino acid sequence as human ubiquitin. appropriate understanding and standards should be used.

A second antibody that recognizes anti-ubiquitin antibodies used in various immunoassay methods , both polyclonal (suitable and preferred) and monoclonal, e.g. Easily and widely available to those skilled in the immunoassay field from a myriad of commercial sources available. The second antibody is generally from a different species than the first antibody and is Recognizes antibodies with the same isotope as the antibody. For example, Staphylococcus aureus (S, aureus ) A second antibody is a protein other than the antibody that specifically binds to the antibody, such as protein A. It can also be used in place of , which is also readily available in the art.

Furthermore, a labeling method for detecting such a second antibody (or other protein 1it) and Their use in various methods is well known to those skilled in the immunoassay art.

The following examples further illustrate the practice of the invention.

Example This example demonstrates how C3F aliquots taken from humans were treated with ubiquitin-immunoreactive compounds. Explain how to analyze the existence of quality. This method is competitive ELI SA .

Generally, at the pre-incubation stage The first antibody (i.e., antibody ubiquitin antibody) and an aliquot of C8F are added to a solution. The solution is then incubated to remove the units present in the C3F aliquot. Biquitin - binds the immunoreactive substance to the antibody. Next, play incubator The solution from the ubiquitin step was transferred to a microtiter plate pre-coated with ubiquitin. the first antibody and then incubate it. Some microtiters depend on the concentration of ubiquitin-immunoreactive substances in C5F. Binds to ubiquitin in the wall soil of the tarplate. This Tsuta 2 incubation and The first antibody, which is not complexed by ubiquitin bound to the pore wall, is added to the microtiter. After washing to remove it from the plate holes, an enzyme label for the first antibody (e.g. Microtiter the polyclonal antibody (e.g. alkaline phosphatase labeled). Add the plate well, repeat the incubation, and transfer to the microtiter plate. Labeled antibody is attached to the antibody complexed by ubiquitin bound to the pore wall. . After this incubation period, the pores are washed and the unbound labeled second antibody is removed. That is, the anti-first antibody) is removed, a chromogen solution is added, and the chromogen solution is incubated in the hole for a predetermined period of time. Incubate at a predetermined temperature and then touch with an enzyme that labels the second antibody. Optical density in the pores as a measure of the concentration of products formed in a mediated reaction is measured at an appropriate wavelength.

The chromogen solution is catalyzed by the enzyme label on the second antibody to produce a colored A solution of a substrate for a reaction that produces a product. colored products concentration (therefore, at the appropriate wavelength after incubation with the enzyme-labeled second antibody) The optical density of the chromogen solution (optical density of the chromogen solution) is proportional to the amount of the second antibody present in the pores, and the amount of the second antibody is inversely proportional to the degree of ubiquitin-immunoreactive material in the analyzed CSF aliquot. do. Ubiquitin-immunoreaction recognized by first antibody in CFS aliquot The higher the concentration of the sex substance, the more ubiquitin can be substituted for the C8F aliquot. compared to the optical density obtained by solutions known to be In the chromogen solution after incubation in the plate wells, the suitable for detecting the products of reactions catalyzed by enzyme labels. The optical density at wavelengths is increasingly reduced (i.e., color development is suppressed). ).

100X [1-(Optical density by C3F aliquot/Ubiquitin-free solution) optical density by liquid aliquot) (in this case the optical density is relative to the bank ground) (corrected) is referred to as the "% inhibition" or "%I" of the C3F aliquot. C8F a The higher the %I of the recoat, the more likely it is to be recognized by the first antibody in the C5F being analyzed. The concentration of ubigitin, an immunoreactive substance, increases. Known concentrations of ubiquitin Running analysis of the C3F aliquot in parallel with the analysis of the solution showed that the analyte C8 Used to calculate the concentration of ubiquitin epitope recognized by the first antibody in F. A possible standard curve can be obtained.

A microtiter one-well plate is prepared as follows: Ub stock solution (1 10 mg bovine red blood cell ubiquitin in 1 ml sodium dodecyl sulfate (SDS) Prepared by dissolving >200 μl and adding water until the total volume is 10 ml. Coating buffer (50mM sodium carbonate) Thorium, pH 9.8) and 10 μl of the resulting solution were added to a 96-well plate. The plate was partially incubated at 4°C. incu After hydration, each plate was washed with GUB [general purpose buffer (gener buffer)]. aI use buffer) (10mM Tris, 0.05% Tween 20. Wash 3 times with 0.85% NaCL pH 7,4). By G.U.H. After washing, fill the holes with 200-300 μl of 1% bovine serum albumin (BSA) in GUB. Block for 3 hours at 37°C. [Alternatively, block BSA GUH can be dissolved in phosphate buffered saline instead (BSA )] The holes were then washed again with GUB three times as above. Finally, excessive The GUB is shaken out of the hole and the hole is dried for 30 minutes at 37°C.

The experiment was carried out using ibridoma as the first antibody according to standard methods in the field of immunology. Antibodies are produced in mice in vivo after intraperitoneal injection of cells/1 Murine monoclonal antibody 5- obtained in mouse ascites fluid after breeding bullioma culture It was carried out using 25. As the skilled artisan will appreciate, antibody concentrations are determined from ascitic fluid samples (p depending on the sample size of the incubation The concentration of antibody in the solution combined with the pull (which is critical in the analysis) is The antibody binds to ubiquitin-immunoreactive substances present in the sample. However, as mentioned above and as is well known to those skilled in the art, it provides maximum sensitivity and range. It must be determined that Stored at -70°C until use Raw ascitic fluid was collected from mice diluted 1:800 with GUB. It was ascites. Next, the first antibody solution used for analysis was added from the stock ascites to the stock abdominal fluid. Water P B S B SA xu, ryu, Ju+hwu, u uzu / /11. +/　l　　　/　/　+w ccxy t　wS) 200 n g/ml of ubiquitin was present in the C3F sample to the extent that it gave a range of Dilute, estimating to give the maximum concentration of epitope reactivity with 5-25 of Manufactured by.

Each aliquot of C3F to be analyzed was diluted 1=1 with GUB in a polypropylene tube. [C3F (taken after the 4th mL of C8F taken from the patient by lumbar puncture) GUB Mix 100 μl], vortex briefly and leave on ice.

Add 200 μl of the first antibody solution to each C3F-containing tube, vortex the tube briefly, and ice placed on top.

4 criteria were used: GUB containing 0.05% sodium azide Standard: Bovine red blood cell ubiquitin in GUB supplemented with 5% sodium azide Low standard of 10 ng/ml; in GUB supplemented with 0.05% sodium azide Bovine red blood cell ubiquitin 75 n g/m 1 medium standard: 005% sodium azide Bovine erythrocyte ubiquitin in GUB supplemented with thorium at 400 ng/ml Certain high standards. Other standards, e.g. GUB supplemented with 0.05% sodium azide Ubiquitin inside is 240ng/m! , 120 ng/mI and 60 ng/m] is also available. When used as further described below, the criterion is It is typically found to give approximately the following % inhibition: Standard: Approximate %r (Ub concentration, ng/ml) 200 μl of each standard was placed in a polypropylene tube, similar to the C3F sample. 1 antibody solution was added, the tube was vortexed briefly, strained, and placed on ice.

For background measurements, G 400 μl of UB was placed in a propylene tube (no antibody) on ice.

All tubes were left on ice for at least 5 minutes to ensure temperature equilibration. Next, all Remove all tubes from ice and incubate at 20-23°C (preferably in closed incubation). The cells were allowed to incubate for 90 minutes (at a constant temperature of 20° C. in a refrigerator). At this stage Incubation time and temperature are known to be important and therefore both needs to be carefully controlled.

Immediately before use, place the microtiter one-well plate prepared as above in a washing buffer. Wash in a vacuum cleaner three times for 1 minute each time, then wash the slap - dried. Use a “slap-dry” process after all consecutive washes. Make sure to slide the plate face down onto a lint-free paper towel. During this process, hold the plate frame firmly. It was carried out.

As described above, after incubating the antibodies with the sample and standards, each Incubate mixture (including “bank ground” mixture) 1001’ I want to add it to each of the three holes of the microtiter hole plate. ). The plates were then placed in a high humidity incubation chamber for 90 minutes at room temperature. (20-23°C).

After incubating for 90 minutes, wash the plate with washing buffer (0.5M Na The plates were slumped dry after each wash.

After washing, add 100 μl of alkaline-phosphatase conjugate to the hole and remove the plate. in a humid incubation chamber at room temperature for 3 hours or in portions at 4°C. Incubated. The alkaline-phosphatase conjugate is an alkaline-phosphatase conjugate. Polyclonal goat anti-mouse IgG conjugated with Tase [TAGO, I nC3), Catalog No. 6550] with 0.05% azyl Prepared by diluting 1:1000 with GUB supplemented with sodium chloride. did.

Polyclonal rabbit anti-mouse IgG that binds alkaline phosphatase DAK○, Inc., Catalog No. 3141 sometimes has a territory of 0.5%. 1 with Ct, +2 supplemented with adi f-hysodium. Dilute to vnn○ and use. ·Ta.

The chromogen solution was manufactured by Sigma Chemical Co. Para-nitrophenyl phosphene obtained in tablet form from St. Louis, Missouri, USA. (PNPP) by dissolving it in 1M diethylamine to 2mg/m+. It was manufactured. (Dilute 5M pH 9,5 noethylamine aqueous solution to 15 with water. Dissolve one standard PNPP tablet as purchased in 1 ml of the solution prepared by I understood).

After incubating the plate with alkaline-phosphatase conjugate , plates were washed three times with wash buffer and slap dried after each wash.

After this washing, 100 μl of chromogen solution was added to the holes of the plate. plate high Incubated at room temperature in a humidity incubation chamber. 0 control hole Adjust the luminous intensity to 405 nm (400-410 nm) using a standard microtiter plate. Using a photo reader, the absorbance is 1. Monitored until it reached 0. This is usually 4 This occurred between 5 and 60 minutes. The absorbance of all pores was then read.

Next, the %I of the holes was measured. %I of C8F sample or standard is measured in triplicate. is the average value of a given %I.

Humans without known neurodegenerative disease (postmortem examination of brain tissue confirms this condition) (including humans), below the low standard, i.e. less than 5-10%, with O! different from %■ was detected. In connection with the present invention, from such patients 2-3 hours after death. C3F collected over a period of time showed significant amounts of ubiquitin-immunoreactive material. However, this is thought to be due to the destruction of brain cells. These humans have This may be due to the lack of observable ubiquinated filaments characteristic of neurodegenerative diseases. Ubiquitin in their postmortem C5F is clearly liberated. Therefore, this release This suggests that it is a release of ubiquitin.

C8F from humans suffering from Altnosimer disease was obtained by the method described in this example. , is found to have a %1 greater than 30%, usually in the range of 50-70%. Ta.

C5F from humans suffering from Parkinson's disease was obtained by the method described in this example. It was found to have a %■ in the range of 30-60%.

C3F in humans diagnosed with amyotrophic lateral sclerosis, which is not a neurodegenerative disease, , can have a %I in the range of 6-26% by the method described in this example. found.

Therefore, a %I greater than about 30% is a significant concentration in C8F that is indicative of UAND. ubiquitin--an immunoreactive substance.

Although the invention has been described here in some specificity, the skilled person will understand that what has been described herein is , it is believed that the reader will appreciate the many modifications and variations that still fall within the scope of the invention. All such modifications and variations are intended to be encompassed by the invention herein described and claimed. .

Engraving (no changes to the content) abstract Ubiquitin-related neurodegenerative diseases in living humans, e.g. A method for diagnosing Parkinson's disease, Bick's disease, Parkinson's disease, and progressive supranuclear palsy is provided. child The method uses aliquots of brain cerebrospinal fluid from humans to detect ubiquitin-immunoreactive substances. and analysis. There are no living humans who do not have this disease. In living humans with diseases such as Biquitin - translation of odor correction book based on the surprising discovery of the existence of an immunoreactive substance statement submission form (Article 184-8 of the Patent Act) Delivery on June 8, 1992

Claims (8)

[Claims] 1. A method for diagnosing UAND in living humans, the method comprising: A method comprising analyzing the coat for ubiquitin-immunoreactive substances. 2. The disease is Alzheimer's disease, Pick's disease, Parkinson's disease and progressive supranuclear paralysis. The method of claim 1, wherein the method is selected from the group consisting of paralysis. 3. Analysis of cerebrospinal fluid was performed using competitive E using monoclonal anti-mammalian ubiquitin antibodies. The method according to claim 1, wherein the analysis is by LISA. 4. Analysis of cerebrospinal fluid was performed using competitive E using monoclonal anti-mammalian ubiquitin antibodies. 3. The method according to claim 2, wherein the analysis is by LISA. 5. In a method for diagnosing whether a living human is suffering from UAND, Analyzing an aliquot of cerebrospinal fluid for ubiquitin-immunoreactive substances Improvement method. 6. UAND, which is a diagnostic target for humans, is used to diagnose Alzheimer's disease, Pick's disease, and Park's disease. The improved method according to claim 5, wherein the method is selected from the group consisting of Nson's disease and progressive supranuclear palsy. . 7. Analysis of cerebrospinal fluid was performed using competitive E using monoclonal anti-mammalian ubiquitin antibodies. The improved method according to claim 5, wherein the analysis is performed by LISA. 8. Analysis of cerebrospinal fluid was performed using competitive E using monoclonal anti-mammalian ubiquitin antibodies. The improved method according to claim 6, wherein the analysis is performed by LISA.