EP2097755A1 - In vitro method for the diagnosis and early diagnosis of neurodegenerative disorders - Google Patents
In vitro method for the diagnosis and early diagnosis of neurodegenerative disordersInfo
- Publication number
- EP2097755A1 EP2097755A1 EP07856296A EP07856296A EP2097755A1 EP 2097755 A1 EP2097755 A1 EP 2097755A1 EP 07856296 A EP07856296 A EP 07856296A EP 07856296 A EP07856296 A EP 07856296A EP 2097755 A1 EP2097755 A1 EP 2097755A1
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- European Patent Office
- Prior art keywords
- disease
- determination
- dementia
- diagnosis
- alzheimer
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- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
- G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
-
- 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 present invention relates to a novel in vitro method for the diagnosis and, in particular, early diagnosis of neurodegenerative diseases, in particular dementias such as Alzheimer's disease and its precursors.
- diagnosis is used as a generic term for medical determinations which, depending on the clinical condition of the patient in which the determination is carried out, can be based on different questions and those of recognition and, in the present case, especially serve the early detection, the determination of severity and the course assessment, including the therapy-accompanying course assessment, and the prognosis of the future course of a disease.
- a diagnosis can also be a negative diagnosis in which, for example, the presence of a particular disease is rendered unlikely on account of the fact that a certain disease-typical concentration of biomarker in a blood sample of a patient can not be detected.
- the diseases to be diagnosed in the present invention are rather slow-developing, chronic neurodegenerative diseases of noninfectious etiology, especially dementia disorders.
- Dementia diseases are generally referred to diseases for which a common feature is that acquired intellectual ability, v. A. the memory, and the normal personality level are impaired as a result of brain damage. Dementias are usually relatively slow-developing diseases of chronic nature. If dementia occurs before old age in middle age, it is referred to as pre-senile dementia. In the case of dementia patients, a distinction is made between the following diseases or groups of diseases on the basis of the symptoms typical for them and changes in the brain pathology:
- Alzheimer's disease (Alzheimer's disease) is the most common neurodegenerative dementia disorder, accounts for 2/3 of all cases of dementia and is also the most important therapeutic area for the present invention
- Post mortem pathological features that can be ascertained with certainty: the formation of amyloid plaques and neurofibrillary tangles and the loss of nerve cells (for review see (1)), references in the description in the form of numbers refer to the description following bibliography).
- Amyloid plaques consist of extraneuronal aggregates of the amyloid ⁇ -protein, while the neurofibrillary tangles mainly contain tau protein and neurofilaments. It is believed that plaque and neurofibrillary formation is the cause of nerve cell death.
- AD Alzheimer's disease
- Dementia with Lewy bodies is the second leading cause of dementia after Alzheimer's disease.
- DLB is characterized by the appearance of so-called Lewy bodies in the brainstem and in the cortex. These Lewy bodies consist predominantly of aggregates of the presynaptic protein ⁇ -synuclein and ubiquitin. Lewy body pathology may be associated to varying degrees with Alzheimer's and Parkinson's-type neuropathological changes.
- DLB also causes the formation of beta-amyloid and senile plaques, but not neurofibrillary tangles (for review see (6)).
- Lewy bodies are also present in the brain of patients with Parkinson's disease, albeit in a different distribution.
- DLB Frontotemporal dementia
- tauopathies which are characterized by an over- or underexpression of a subtype of subtypes or by the expression of a mutated tau protein.
- vascular dementia refers to diseases in which dementia is triggered by circulatory disorders in the brain.
- VAD multi-infarct dementia
- subcortical VAD also referred to as Binswanger's disease
- Binswanger's disease is a slowly progressive dementia that is pathologically characterized by cerebrovascular lesions in the cerebral white matter. Clinically, this results in behavioral problems such as agitation, irritability, depression and euphoria as well as a mild memory disorder.
- the MuIti infarction dementia gradually develops as a result of several small strokes, also known as transient ischemic attacks (TIA), which led to the destruction of brain tissue in the cortex and / or subcortical areas.
- TIA transient ischemic attacks
- the strokes may have gone completely unnoticed, the dementia is the first noticeable in this case Episode.
- MID transient ischemic attacks
- cognitive abilities associated with severe depression, mood swings, and epilepsy.
- a diagnosis of dementia diseases today is predominantly based on neuropsychological examinations and the observation of disease development and its course, using exclusion criteria for certain forms of dementia. These studies provide very ambiguous results in many cases, which the o.g. Explain numbers for the underdiagnosed dementia forms or incorrectly diagnosed cases.
- the disease-typical brain changes naturally can not be detected directly in living patients, and apparatus-medical examinations of brain functions by means of e.g. Computer or magnetic resonance tomography are complex and expensive.
- the diagnostic sensitivity and the specificity of at least 80% should be given.
- the disease-specific change of the biomarker should be manifested at the earliest possible stage of the disease in order to begin appropriate therapies (8).
- biomarker in the circulation (i.e., in the blood or in blood preparations such as serum or plasma) of a patient that could be used in clinical practice to improve the early and differential diagnosis of AD and all the above. Criteria met.
- inflammatory markers such as IL-6 and TNF ⁇
- markers of oxidative stress such as 3-nitrotyrosine
- markers associated with characteristic pathological changes in AD such as amyloid ⁇ , which is a major component of amyloid plaques
- tau protein which is an essential component of the neurofibrillary tangles (see the overview in (7); (9)).
- AD Alzheimer's disease
- suitable substances in blood or plasma samples and in patients who are suspected of Existence of dementia, in particular of AD is supportive of an early positive diagnosis and / or suitable for a negative exclusion diagnosis.
- the present invention provides such an assay method in the form of an in vitro method for detection and early detection, for determining the severity and for evaluating the course of disease and prognosis of neurodegenerative diseases, which comprises administering to a patient in a serum or plasma sample suffers from objectively detectable cognitive disorders, determines the Apo C I immunoreactivity using an immunodiagnostic assay, and based on the concentration for the occurrence of, or a typical early form of, neurodegenerative disease or the course of the disease and / or the success of efforts to alleviate or prevent it.
- the present invention is based on the experimental finding that in the blood (serum, plasma) of patients with cognitive disorders up to the diagnosis "possibly Alzheimer's” or "probably Alzheimer's” in a characteristic manner reduced concentrations of the immunoreactive Apo CI are measured in comparison with control persons ,
- analyte is defined as it using an immunoassay, in particular an immunoassay, as described in the experimental part of the present application or in German patent application DE 103 43 815 Al the applicant is described in more detail, can be determined.
- hydrophobic molecular structures eg the octyl residues of a hydrophobic octyl sepharose chromatography material, from which it is under typical conditions for the elution of proteins , in particular with dilute acetic acid, can be eluted.
- free apolipoprotein CI does not necessarily mean that the material which can be separated by hydrophobic interaction chromatography must be completely free in the original samples.
- said immunoassay in the blood of cancer patients also recognizes an apo C-I fraction which does not bind to octylsepharose and whose occurrence appears to be characteristic of tumor diseases. Possibly. Therefore, in the context of the overall diagnosis for dementia disease or its precursors using the measured values obtained in the determination of the immunoreactive Apo C-I, it would be ruled out that the measured values in the respective patient would be falsified by a cancer. This can be done, for example, in the manner described in DE 103 43 815 A1 by checking whether the measured concentration values are obtained by contact of the sample with e.g. Octyl Sepharose change.
- the immunoreactive Apo CI to be determined according to the present invention comprises, in addition to analytes which represent or contain the complete peptide Apo CI, also "Apo CI derivatives". These include in particular immunoreactive apo CI fragments and aggregates, in particular those which behave in the said sandwich immunoassay as the free protein apolipoprotein CI.
- the "derivatives" may be, for example, apolipoprotein C-1 molecules shortened by individual amino acids or amino acid sequences, or also, for example, by aggregation, sterically or conformally altered complete apolipoprotein C-I molecules.
- Apo C-1 derivatives should also be encompassed by the invention takes into account the known facts that immunoassays normally can not differentiate between different analytes if the differences are outside the molecular segments used for antibody binding, i. in the case of a sandwich immunoassay in end regions of the detected molecule outside the amino acid sequence delimited by the two antibody binding sites. Furthermore, the further known fact is to be taken into account that when peptidic analytes occur in the circulation, the appearance of their degradation products in the form of peptide fragments must always be expected.
- concentrations of such fragments are usually approximately proportional to the concentration of the starting peptide, the differences in concentration differences in stability or different rates of "clearing" of the peptide or fragment by further proteolytic degradation or by discharge from the circulation, e.g. reflect on the kidneys.
- the determination according to the invention of immunoreactive Apo C-I in the context of the diagnosis of neurodegenerative diseases is proposed in particular as a measurement which supplements the parallel determination of physiological or clinical and neuropsychological parameters and other biomarkers and refines them for differential diagnostic purposes.
- the method is therefore preferably carried out within the scope of a multiparameter determination, in which at least one additional one for the respective clinical picture is simultaneously detected.
- meaningful biochemical or physiological parameter is determined and in which a measurement result is obtained in the form of a set of at least two measured variables, which is evaluated for the fine diagnosis of the neurodegenerative disease.
- the immunoreactive Apo C-I e.g. at least one other biochemical parameter selected from the groups of natriuretic peptides, inflammatory mediators, complement components, cytokines, chemokines, blood coagulants and fibrinolytic factors, acute phase proteins and free radical compounds.
- the co-parameters to be mentioned in particular include those parameters which are described in the earlier German patent applications of the applicant with the file references 10 2005 034 174.8 (midregionales Proadrenomedullin), 10 2005 036 094.7 (Liquorodiagnostic determination of procalcitoline), 10 2006 027 818.6 and 10 2006 023 175.9 and the parameter CPS-I (carbamoyl phosphate synthetase 1), to which relevant discussions with regard to neurodegenerative diseases can be found in the applications EP 05023420.2 and DE 10 2006 021 406.4.
- the method according to the invention can also be carried out as a simultaneous determination by means of a chip technology measuring device or an immunochromatographic measuring device and the evaluation of the complex measurement results possibly obtained can take place with the aid of a suitable computer program.
- FIG. 1 shows the results of the measurement of the concentrations of the immunoreactive Apo CI determinable with the sandwich immunoassay described in the experimental section, namely in EDTA plasmas of 60 healthy control persons, and of 196 patients with cognitive disorders of various severity corresponding to the above-mentioned groups (b), (c) and (d), ie the groups 11 SKS “(50 patients),” LKS mgl AD “(46 patients) and” ws AD "(100 patients).
- a sandwich-type immunoassay was constructed from the components described below:
- a) Coated Tubes Polystyrene tubes (Greiner) were coated with a commercially available polyclonal affinity-purified antibody against apolipoprotein C-I (source: Acris Antibody, Bad Neuheim, Germany). The antibody was obtained according to the manufacturer by immunization of rabbits with human Apo C-I and purified on a Sepharose affinity column with human apolipoprotein C-I. For coating, 0.2 ⁇ g of antibody in 300 ⁇ l of PBS was bound to polystyrene tubes (Greiner, Germany) coated with sheep anti-rabbit IgG antibody (Sigma). The binding was stopped after 18 hours at room temperature.
- the tubes were washed twice with 3 ml each of 0.5% bovine serum albumin (BSA) in PBS. After drying in vacuo, the tubes were used as the solid phase for the apolipoprotein C-I immunoassay.
- BSA bovine serum albumin
- Acridinium ester-labeled antibody 100 ⁇ g of another antibody to human apolipoprotein CI (from Rabbit, reference: Academy Bio-Medical Company, Texas, USA) in 100 ⁇ l of PBS was incubated with 10 ⁇ g of acridinium-NHS ester (in 10 ⁇ l of acetonitrile ) implemented. After a 10 minute incubation at room temperature, the purification was carried out of the labeled antibody with removal of unreacted constituents of the reaction mixture by HPLC on SW 300 (Waters).
- Serum or plasma samples were diluted 1:10 000 with PBS, 0.5% BSA. Of these, 300 ⁇ l each were added to the o.g. coated with the immobilized antibody. Pipette and then incubated for 4 hours at room temperature with shaking (300 U / min on a Heidolph orbital shaker). The tube contents were then washed out with PBS (4 times filling and decanting with 1 ml PBS each), and apolipoprotein CI bound to the tube wall was incubated within 20 hours at room temperature and 300 rpm with 300 ⁇ l per tube of acridinium ester labeled anti -Apolipoprotein C-I antibody reacted. Thereafter, unbound labeled antibody was removed by washing 5 times with 1 ml PBS each time, and the remaining chemiluminescence was measured in a known manner in a Berthold Luminometer 952T.
- concentrations of apo C-I which can be determined immunologically in plasmas, which can be identified by the values for the medians of the various patient groups, decrease with the severity of the symptoms in the direction:
- apo CI is not a brain-specific parameter
- the measurement of apo C-I immunoreactivity in the circulation is very well suited for the purpose of assisting AD early diagnosis, given the above-mentioned correlations.
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Abstract
In vitro method for the detection and early detection of, for the determination of the degree of severity of, and for the assessment of the course of and prognosis of neurodegenerative disorders, in which an immunodiagnostic determination method is used to determine the apolipoprotein C-I (Apo C-I) immunoreactivity in a serum or plasma sample from a patient suffering from subjective or objectively verifiable cognitive disorders.
Description
In vitro Verfahren zur Diagnose und Frühdiagnose von neuro- degenerativen Erkrankungen In vitro diagnostic and early diagnosis of neurodegenerative diseases
Die vorliegende Erfindung betrifft ein neues in vitro Verfahren für die Diagnose und insbesondere Frühdiagnose von neurodegenerativen Erkrankungen, insbesondere von Demenz- erkrankungen wie der Alzheimer Krankheit und deren Vorstufen.The present invention relates to a novel in vitro method for the diagnosis and, in particular, early diagnosis of neurodegenerative diseases, in particular dementias such as Alzheimer's disease and its precursors.
Im Rahmen der vorliegenden Erfindung wird dabei der Begriff "Diagnose" als Oberbegriff für medizinische Bestimmungen gebraucht, denen je nach dem klinischen Zustand des Patienten, bei dem die Bestimmung durchgeführt wird, unterschiedliche Fragestellungen zugrunde liegen können und die der Erkennung und im vorliegenden Falle insbesondere auch der Früherkennung, der Bestimmung des Schweregrads und der Verlaufsbeurteilung, auch der therapiebegleitenden Verlaufsbeurteilung, und der Prognose des zukünftigen Verlaufs einer Erkrankung dienen. Dabei ist im vorliegenden Zusammenhang von besonderer Bedeutung, dass eine Diagnose auch eine Negativdiagnose sein kann, bei der z.B. aufgrund der Nicht - feststellbarkeit einer bestimmten krankheitstypischen Bio- markerkonzentration in einer Blutprobe eines Patienten das Vorliegen einer bestimmten Erkrankung unwahrscheinlich gemacht wird.
Für die Negativdiagnose sind auch Biomarker von großem Wert, für die bei mehreren verschiedenen Krankheiten pathologisch veränderte Konzentrationen festgestellt werden können und die daher allein, für sich genommen, noch keine positive Diagnose einer spezielle Krankheit ermöglichen - obwohl sie in der Regel bei Hinzuziehung weiterer klinischer oder biochemischer Parameter auch für die Positivdiagnose entscheidend sein können.In the context of the present invention, the term "diagnosis" is used as a generic term for medical determinations which, depending on the clinical condition of the patient in which the determination is carried out, can be based on different questions and those of recognition and, in the present case, especially serve the early detection, the determination of severity and the course assessment, including the therapy-accompanying course assessment, and the prognosis of the future course of a disease. In the present context, it is of particular importance that a diagnosis can also be a negative diagnosis in which, for example, the presence of a particular disease is rendered unlikely on account of the fact that a certain disease-typical concentration of biomarker in a blood sample of a patient can not be detected. For the negative diagnosis are also biomarkers of great value, for which in several different diseases pathologically altered concentrations can be detected and therefore alone, taken alone, do not allow a positive diagnosis of a specific disease - although they usually in consultation with other clinical or biochemical parameters may also be decisive for the positive diagnosis.
Die Erkrankungen, um deren Diagnose es bei der vorliegenden Erfindung geht, sind sich eher langsam entwickelnde, chronische neurodegenerative Erkrankungen von nichtinfektiöser Ätiologie, insbesondere Demenz-Erkrankungen.The diseases to be diagnosed in the present invention are rather slow-developing, chronic neurodegenerative diseases of noninfectious etiology, especially dementia disorders.
Als Demenz-Erkrankungen (Dementia) werden generell Krankheiten bezeichnet, für die ein gemeinsames Merkmal ist, dass erworbene intellektuelle Fähigkeiten, v.a. das Gedächtnis, und das normale Persönlichkeitsniveau als Folge von Hirnschädigungen beeinträchtigt sind. Demenzerkrankungen sind in der Regel sich relativ langsam entwickelnde Krankheiten von chronischem Charakter. Treten Demenzerscheinungen vor dem hohen Alter im mittleren Lebensalter auf, werden sie als präsenile Demenz-Erkrankungen bezeichnet. Bei Demenz-Erkran- kugnen unterscheidet man auf der Basis der für sie typischen Symptome und hirnpathologischen Veränderungen insbesondere die folgenden Erkrankungen bzw. Gruppen von Erkrankungen:Dementia diseases are generally referred to diseases for which a common feature is that acquired intellectual ability, v. A. the memory, and the normal personality level are impaired as a result of brain damage. Dementias are usually relatively slow-developing diseases of chronic nature. If dementia occurs before old age in middle age, it is referred to as pre-senile dementia. In the case of dementia patients, a distinction is made between the following diseases or groups of diseases on the basis of the symptoms typical for them and changes in the brain pathology:
Die Alzheimer Demenz (AD) (Alzheimer Krankheit; Morbus Alzheimer) ist die häufigste neurodegenerative Demenz-Erkrankung, macht 2/3 aller Demenzfälle aus und stellt auch das praktisch wichtigste Anwendungsgebiet für die vorliegende Erfindung dar. AD zeichnet sich durch drei wichtige, allerdings erst post mortem mit Sicherheit feststellbare pathologische Merkmale aus: die Bildung von Amyloid-Plaques und neurofibrillären Bündeln sowie den Verlust an Nervenzellen (Übersicht s. (1); Literaturangaben in der Beschreibung in Form von Zahlen beziehen sich auf die der Beschreibung
folgende Literaturliste) . Amyloid-Plaques bestehen aus extraneuronalen Aggregaten des Amyloid-ß-Proteins, während die Neurofibrillenbündel hauptsächlich Tau-Protein und Neurofilamente enthalten. Es wird vermutet, dass die Plaque- und Neurofibrillenbildung die Ursache für das Absterben von Nervenzellen ist.Alzheimer's disease (AD) (Alzheimer's disease) is the most common neurodegenerative dementia disorder, accounts for 2/3 of all cases of dementia and is also the most important therapeutic area for the present invention Post mortem pathological features that can be ascertained with certainty: the formation of amyloid plaques and neurofibrillary tangles and the loss of nerve cells (for review see (1)), references in the description in the form of numbers refer to the description following bibliography). Amyloid plaques consist of extraneuronal aggregates of the amyloid β-protein, while the neurofibrillary tangles mainly contain tau protein and neurofilaments. It is believed that plaque and neurofibrillary formation is the cause of nerve cell death.
Die wichtigsten Symptome von AD sind zunehmende Merkfähig- keits- und Denkstörungen bei relativ lang anhaltender Ge- mütsansprechbarkeit , wobei diese Symptome von weiteren weniger spezifischen Störungen begleitet werden, die die Abgrenzung der AD von anderen Demenzformen erschweren.The most important symptoms of AD are increasing memory and thinking disorders with relatively long-lasting gait responsiveness, accompanied by other less specific disorders that make it difficult to differentiate AD from other types of dementia.
Beobachtungen an AD-Patienten und Patienten, die im Laufe ihrer langjährigen klinischen Beobachtung AD entwickelten, führten zur Formulierung von Kriterien für gegeneinander abgrenzbare Patientengruppen, die die ganze Breite vonObservations in AD patients and patients who developed AD in the course of their long-term clinical observation led to the formulation of criteria for distinct groups of patients covering the full breadth of
(a) Personen ohne subjektive und objektive kognitive Störungen (die im Rahmen der vorliegenden Anmeldung die Kontrollgruppe repräsentieren) , über(a) Persons without subjective and objective cognitive disorders (who represent the control group in the context of the present application)
(b) Patienten, die über subjektiv empfundene kognitive Leistungseinbußen klagen, bei denen jedoch keine kognitiven Defizite festgestellt werden können (im Rahmen der vorliegenden Anmeldung ist das die Gruppe der "SKS" -Patienten, wobei "SKS" für "subjektive kognitive Störungen" steht) , weiter über(b) Patients who complain of perceived cognitive impairment but who can not identify cognitive deficits (in this application, this is the group of "SKS" patients, where "SKS" stands for "subjective cognitive disorders" ), continue over
(c) Patienten, bei denen leichte kognitive Störungen feststellbar sind und bei denen dann, wenn keine anderen demenzverursachenden Erkrankungen vorliegen, die Diagnose "möglicherweise AD" ("mgl AD") gestellt wird (im Rahmen der vorliegenden Anmeldung ist das die Gruppe "LKS mgl AD", wobei "LKS" für "leichte kognitive Störungen" steht) bis hin
- A -(c) Patients diagnosed with mild cognitive impairment who are diagnosed with "possibly AD"("mglAD") if there are no other dementia-causing diseases (in the present application this is the group "LCS mgl AD ", where" LKS "stands for" mild cognitive disorders ") - A -
(d) zur Gruppe der Patienten mit dem typischen Bild erheblicher kognitiver Störungen, die schleichend begonnen haben und langsam progredieren, für die dann, wenn andere Demenz-Ursachen ausgeschlossen werden können, die Diagnose "wahrscheinlich AD" gestellt wird (im Rahmen der vorliegenden Anmeldung ist das die Gruppe "ws AD", wobei die Abkürzung für "wahrscheinlich Alzheimer" steht) , abdecken.(d) to the group of patients with the typical picture of significant cognitive disorders that have begun to creep slowly and slowly, for which, when other dementia causes can be excluded, the diagnosis "probably AD" is made (in the context of the present application this is the group "ws AD", where the abbreviation stands for "probably Alzheimer's").
Bezüglich der Zuordnung von Patienten mit subjektiven und/- oder objektiven kognitiven Störungen zu verschiedenen Gruppen wird ergänzend verwiesen auf (2) , (3) , (4) und (5) .With regard to the assignment of patients with subjective and / or objective cognitive disorders to different groups, reference is additionally made to (2), (3), (4) and (5).
Die Demenz mit Lewy-Körperchen (dementia with lewy-bodies: DLB) ist nach der Alzheimer Demenz die zweithäufigste Ursache für eine demenzielle Erkrankung. Neuropathologisch ist die DLB durch das Auftreten von sogenannten Lewy-Körperchen im Hirnstamm und im Cortex charakterisiert. Diese Lewy-- Körperchen bestehen überwiegend aus Aggregaten des präsynaptischen Proteins α-Synuclein und Ubiquitin. Die Lewy-- Body-Pathologie kann in unterschiedlichem Ausmaß mit Alzheimer und Parkinson-typischen neuropathologischen Veränderungen assoziiert sein. So kommt es auch bei der DLB zur Bildung von beta-Amyloid und senilen Plaques, jedoch nicht zu Neurofibrillenbündeln (Übersicht s. (6)). Lewy-Körperchen sind auch im Gehirn von Patienten mit Morbus Parkinson vorhanden, wenn auch in einer unterschiedlichen Verteilung.Dementia with Lewy bodies (dementia with lewy-bodies: DLB) is the second leading cause of dementia after Alzheimer's disease. Neuropathologically, DLB is characterized by the appearance of so-called Lewy bodies in the brainstem and in the cortex. These Lewy bodies consist predominantly of aggregates of the presynaptic protein α-synuclein and ubiquitin. Lewy body pathology may be associated to varying degrees with Alzheimer's and Parkinson's-type neuropathological changes. For example, DLB also causes the formation of beta-amyloid and senile plaques, but not neurofibrillary tangles (for review see (6)). Lewy bodies are also present in the brain of patients with Parkinson's disease, albeit in a different distribution.
Kernsymptome von DLB sind eine progrediente kognitive Störung, Verwirrtheitsepisoden mit fluktuierender Aufmerksam- keits- und Bewusstseinslage, Parkinsonismus, häufige Stürze und Synkopen (anfallsartige, kurz dauernde Bewusstlosig- keit) . Die Sensitivität und Spezifität der diagnostischen Kriterien zeigen durchgehend eine hohe Spezifität, aber zum Teil eine sehr niedrige Sensitivität. Das bedeutet, dass die DLB im klinischen Alltag häufig nicht diagnostiziert wird.
Die Frontotemporale Demenz (FTD) wird auch als Pick' sehe Krankheit bezeichnet und macht ca. 20% der präsenilen Demenzerkrankungen aus. FTD ist teilweise genetisch bedingt und zählt zu den sogenannten Tauopathien, die sich durch eine Über- oder Unterexpression eines Tauprotein-Subtyps bzw. durch die Expression eines mutierten Tauproteins auszeichnen. Neuropathologisch kommt es zu einer lokalen Atrophie des frontalen und/oder temporalen Cortex sowie der Substantia Nigra und der Basalganglien . Dies hat unterschiedlich ausgeprägte Sprachstörungen, eine Wesensänderung sowie Verhaltensauffälligkeiten zur Folge. Insgesamt ist die FTD mit einer Sensitivität von 93% bei einer Spezifität von nur 23% unterdiagnostiziert, wobei die AD die häufigste Fehldiagnose darstellt.The core symptoms of DLB are progressive cognitive impairment, confusion episodes with fluctuating attention and consciousness, parkinsonism, frequent falls and syncope (seizure-type, short-lasting unconsciousness). The sensitivity and specificity of the diagnostic criteria show consistently high specificity, but in part a very low sensitivity. This means that DLB is often not diagnosed in clinical practice. Frontotemporal dementia (FTD) is also referred to as Pick's disease and accounts for approximately 20% of pre-senile dementias. FTD is partly genetic and is one of the so-called tauopathies, which are characterized by an over- or underexpression of a subtype of subtypes or by the expression of a mutated tau protein. Neuropathologically there is a local atrophy of the frontal and / or temporal cortex as well as the substantia nigra and the basal ganglia. This results in different pronounced speech disorders, a change in nature and behavioral problems. Overall, FTD is under-diagnosed with a sensitivity of 93% with a specificity of only 23%, with AD being the most common misdiagnosis.
Unter dem Begriff vaskuläre Demenz (vascular dementia; VAD) werden Erkrankungen zusammengefasst , bei denen eine Demenz durch Durchblutungsstörungen im Gehirn ausgelöst wird. Es gibt unterschiedliche Typen der VAD, von denen die Multi-- Infarkt -Demenz (MID) und die subcorticale VAD (auch als Binswanger' sehe Erkrankung bezeichnet) die häufigsten Formen darstellen.The term vascular dementia (vascular dementia) refers to diseases in which dementia is triggered by circulatory disorders in the brain. There are several types of VAD, of which multi-infarct dementia (MID) and subcortical VAD (also referred to as Binswanger's disease) are the most common forms.
Bei der Binswanger' sehen Erkrankung handelt es sich um eine langsam progrediente demenzielle Entwicklung, die pathologisch durch cerebrovasculäre Läsionen in der weißen Hirnsubstanz charakterisiert ist. Klinisch resultiert dies in Verhaltensauffälligkeiten wie Agitation, Reizbarkeit, Depression und Euphorie sowie einer leichten Gedächtnisstörung.Binswanger's disease is a slowly progressive dementia that is pathologically characterized by cerebrovascular lesions in the cerebral white matter. Clinically, this results in behavioral problems such as agitation, irritability, depression and euphoria as well as a mild memory disorder.
Die MuIti-Infarkt -Demenz entsteht allmählich als Folge von mehreren kleinen Schlaganfällen, auch als transiente ischämische Attacken (TIA) bezeichnet, die zum Untergang von Hirngewebe im Cortex und/oder subcortikalen Arealen führten. Die Schlaganfälle können auch gänzlich unbemerkt geblieben sein, die Demenz ist in diesem Falle die erste spürbare
Folge. Bei Vorliegen einer MID kommt es zur stufenweisen Abnahme kognitiver Fähigkeiten, verbunden mit schweren Depressionen, StimmungsSchwankungen und Epilepsie.The MuIti infarction dementia gradually develops as a result of several small strokes, also known as transient ischemic attacks (TIA), which led to the destruction of brain tissue in the cortex and / or subcortical areas. The strokes may have gone completely unnoticed, the dementia is the first noticeable in this case Episode. In the presence of MID, there is a gradual decrease in cognitive abilities associated with severe depression, mood swings, and epilepsy.
Eine Diagnose von Demenz-Erkrankungen erfolgt heutzutage überwiegend auf der Basis neuropsychologischer Untersuchungen und der Beobachtung der Krankheitsentwicklung und ihres Verlaufs, unter Heranziehung von Ausschlusskriterien für bestimmte Demenzformen. Diese Untersuchungen liefern in sehr vielen Fällen mehrdeutige Ergebnisse, die die o.g. Zahlen für die unterdiagnostizierten Demenzformen oder unrichtig diagnostizierten Fälle erklären. Die krankheitstypischen Gehirnveränderungen können an lebenden Patienten naturgemäß nicht direkt festgestellt werden, und apparatemedizinische Untersuchungen der Gehirnfunktionen mittels z.B. Computeroder Magnetresonanz-Tomographie sind aufwändig und teuer.A diagnosis of dementia diseases today is predominantly based on neuropsychological examinations and the observation of disease development and its course, using exclusion criteria for certain forms of dementia. These studies provide very ambiguous results in many cases, which the o.g. Explain numbers for the underdiagnosed dementia forms or incorrectly diagnosed cases. The disease-typical brain changes naturally can not be detected directly in living patients, and apparatus-medical examinations of brain functions by means of e.g. Computer or magnetic resonance tomography are complex and expensive.
Es wäre daher wünschenswert, die Erkennung und insbesondere Früherkennung von Demenzerkrankungen durch die Messung von aussagekräftigen Biomarkern, die mit Hilfe eines relativ einfachen Testverfahrens z.B. in einer Blutprobe (Serumprobe, Plasmaprobe) eines Patienten bestimmt werden können, ergänzen und dadurch erheblich verbessern zu können.It would therefore be desirable to provide for the detection and, in particular, early detection of dementia diseases by the measurement of meaningful biomarkers determined by a relatively simple assay, e.g. in a blood sample (serum sample, plasma sample) of a patient can be determined complement and thereby significantly improve.
Für die Alzheimer-Diagnostik veröffentlichten das Ronald und Nancy Reagan Institut der Alzheimer Association und die NIA-Working Group Richtlinien für die Kriterien, die an einen idealen Biomarker zur Detektion der AD gestellt werden (7) . Folgende Kriterien sollen im Idealfall durch den Biomarker erfüllt werden:For Alzheimer's diagnostics, the Ronald and Nancy Reagan Institute of the Alzheimer's Association and the NIA Working Group published guidelines for the criteria for an ideal biomarker for the detection of AD (7). The following criteria should ideally be met by the biomarker:
1. Er sollte hirnspezifisch sein und ein fundamentales Merkmal der Neuropathologie dieser Erkrankungen detek- tieren.1. It should be brain specific and detect a fundamental feature of the neuropathology of these disorders.
2. Die diagnostische Sensitivität und die Spezifität von mindestens 80% sollte gegeben sein.2. The diagnostic sensitivity and the specificity of at least 80% should be given.
3. Die krankheitsspezifische Veränderung des Biomarkers
sollte sich in einem möglichst frühen Stadium der Erkrankung manifestieren, um mit geeigneten Therapiemaßnahmen beginnen zu können (8) .3. The disease-specific change of the biomarker should be manifested at the earliest possible stage of the disease in order to begin appropriate therapies (8).
Bis jetzt gibt es jedoch keinen in der Zirkulation (d.h. im Blut bzw. in Blutpräparaten wie Serum oder Plasma) eines Patienten nachweisbaren Biomarker, der im klinischen Alltag zur Verbesserung der Früh- und Differentialdiagnose von AD herangezogen werden könnte und alle o.g. Kriterien erfüllt. Aktuell werden verschiedene potentielle Markerkandidaten untersucht, darunter Inflammationsmarker wie IL-6 und TNFα, Marker für oxidativen Stress wie 3 -Nitrotyrosin, sowie Marker, die mit charakteristischen pathologischen Veränderungen der AD assoziiert sind, wie Amyloid ß, das einen Hauptbestandteil der Amyloid-Plaques darstellt, und das Tau-Protein, das einen wesentlichen Bestandteil der Neurofi- brillenbündel darstellt (vgl. die Übersicht in (7); (9)) .However, to date, there is no detectable biomarker in the circulation (i.e., in the blood or in blood preparations such as serum or plasma) of a patient that could be used in clinical practice to improve the early and differential diagnosis of AD and all the above. Criteria met. Currently, several potential candidate markers are being investigated, including inflammatory markers such as IL-6 and TNFα, markers of oxidative stress such as 3-nitrotyrosine, and markers associated with characteristic pathological changes in AD, such as amyloid β, which is a major component of amyloid plaques , and the tau protein, which is an essential component of the neurofibrillary tangles (see the overview in (7); (9)).
Es besteht ein aktueller Bedarf nach ergänzenden, valide Laborbefunde liefernden Untersuchungsmethoden, die auf einer Bestimmung von als Biomarker für Demenzerkrankungen, insbesondere für die Alzheimer Demenz (AD) , geeigneten Substanzen in Blut- bzw. Plasmaproben beruhen und bei Patienten, bei denen der Verdacht auf Vorliegen einer Demenzerkrankung, insbesondere von AD, besteht, unterstützend für eine frühe Positivdiagnose und/oder für eine negative Ausschlussdiagnose geeignet sind.There is a current need for complementary, valid laboratory test-providing research methods that are based on a determination of biomarkers for dementia, in particular for Alzheimer's disease (AD), suitable substances in blood or plasma samples and in patients who are suspected of Existence of dementia, in particular of AD, is supportive of an early positive diagnosis and / or suitable for a negative exclusion diagnosis.
Die vorliegende Erfindung stellt eine solche Untersuchungs- methode in Form eines in vitro Verfahrens zur Erkennung und Früherkennung, zur Bestimmung des Schweregrads und zur Verlaufsbeurteilung und Prognose von neurodegenerativen Erkrankungen bereit, bei dem man in einer Serum- oder Plasmaprobe eines Patienten, der an subjektiven oder objektiv nachweisbaren kognitiven Störungen leidet, mit Hilfe eines immundiagnostischen Bestimmungsverfahrens die Apo C- I -Immunreaktivität bestimmt, und bei dem man auf der Basis der
dafür gemessenen Konzentration Schlüsse hinsichtlich des Vorliegens einer neurodegenerativen Erkrankung oder einer für diese typischen Frühform davon oder hinsichtlich des Verlaufs der Erkrankung und/oder des Erfolgs der Bemühungen zu ihrer Abmilderung oder Verhinderung zieht.The present invention provides such an assay method in the form of an in vitro method for detection and early detection, for determining the severity and for evaluating the course of disease and prognosis of neurodegenerative diseases, which comprises administering to a patient in a serum or plasma sample suffers from objectively detectable cognitive disorders, determines the Apo C I immunoreactivity using an immunodiagnostic assay, and based on the concentration for the occurrence of, or a typical early form of, neurodegenerative disease or the course of the disease and / or the success of efforts to alleviate or prevent it.
Vorteilhafte bzw. bevorzugte Ausgestaltungen eines Verfahrens gemäß Anspruch 1 sind in den Unteransprüchen 2 bis 8 wiedergegeben .Advantageous or preferred embodiments of a method according to claim 1 are given in the dependent claims 2 to 8.
Grundlage der vorliegenden Erfindung ist der experimentelle Befund, dass im Blut (Serum, Plasma) von Patienten mit kognitiven Störungen bis hin zur Diagnose "möglicherweise Alzheimer" oder "wahrscheinlich Alzheimer" im Vergleich mit Kontrollpersonen auf charakteristische Weise verminderte Konzentrationen des immunreaktiven Apo C-I gemessen werden.The present invention is based on the experimental finding that in the blood (serum, plasma) of patients with cognitive disorders up to the diagnosis "possibly Alzheimer's" or "probably Alzheimer's" in a characteristic manner reduced concentrations of the immunoreactive Apo CI are measured in comparison with control persons ,
Wenn im Rahmen der vorliegenden Anmeldung von "immunreaktivem Apo C-I" oder "immundiagnostisch bestimmbarem Apo C-I" gesprochen wird, wird damit ein Analyt definiert, wie er mit Hilfe eines Immunoassays, insbesondere eine Immunoassays, wie er im experimentellen Teil der vorliegenden Anmeldung bzw. in der deutschen Patentanmeldung DE 103 43 815 Al der Anmelderin näher beschrieben wird, bestimmbar ist.If in the context of the present application of "immunoreactive Apo CI" or "immunodiagnostic determinable Apo CI" is spoken, so that an analyte is defined as it using an immunoassay, in particular an immunoassay, as described in the experimental part of the present application or in German patent application DE 103 43 815 Al the applicant is described in more detail, can be determined.
Wie in der genannten DE 103 43 815 Al - auf deren gesamten Inhalt, soweit dieser die Assaydurchführung und die Diskussion zu den damit bestimmbaren Apo C-I Molekülen bzw. Apo C- I -Abkömmlingen betrifft, zur Ergänzung der Offenbarung der vorliegenden Anmeldung ausdrücklich Bezug genommen wird - erläutert wird, werden mit einem Immunoassay der genannten Art in Blut von gesunden Kontrollpersonen solche Apo C-I- Anteile bestimmt, die eine Bindefähigkeit gegenüber hydrophoben Molekülstrukturen bzw. hydrophoben Oberflächen aufweisen und die durch Umsetzung mit einem geeigneten Material für die hydrophobe Ausschlusschromatographie (z.B. Octylse- pharose) aus der Probe entfernbar sind.
Das auf die beschriebene Weise bestimmbare Apo C-I bzw. der entsprechende Apo C- I -Abkömmling wird dabei auch als "freies Apolipoprotein C-I" bezeichnet. Es weist die Eigenschaften auf, aus einer Serum- oder Plasma-Probe (in PBS Verdünnung) heraus an hydrophobe Molekülstrukturen, z.B. die Octylreste eines hydrophoben Octylsepharose-Chromatographie-Materials, gebunden zu werden, von dem es unter typischen Bedingungen für die Elution von Proteinen, insbesondere mit verdünnter Essigsäure, eluiert werden kann. Die Verwendung des Begriffs "freies Apolipoprotein C-I" bedeutet jedoch nicht notwendigerweise, daß das durch hydrophobe Interaktionschromatographie abtrennbare Material in den Ursprungsproben völlig frei vorliegen muss. Assoziate bzw. Aggregate, auch mit Lipiden, die unter den Versuchsbedingungen beim Kontakt mit Octylse- pharose unter Bindung des Apolipoproteins C-I an das Chroma- tographiermaterial aufgebrochen werden, sind ebenfalls als "freies Apolipoprotein C-I" im Sinne der Verwendung dieses Begriffs anzusehen.As in the aforementioned DE 103 43 815 Al - to the entire contents thereof, insofar as this relates to the assay procedure and the discussion of the thus determinable Apo CI molecules or Apo C- I-derivatives, to supplement the disclosure of the present application expressly incorporated by reference is explained with an immunoassay of the type mentioned in blood of healthy controls such Apo CI shares determined which have a binding capacity to hydrophobic molecular structures or hydrophobic surfaces and by reaction with a suitable material for hydrophobic exclusion chromatography (eg Octylse- pharose) are removable from the sample. The Apo CI or the corresponding Apo C-1 derivative which can be determined in the manner described is also referred to as "free apolipoprotein CI". It has the properties of being bound from a serum or plasma sample (in PBS dilution) to hydrophobic molecular structures, eg the octyl residues of a hydrophobic octyl sepharose chromatography material, from which it is under typical conditions for the elution of proteins , in particular with dilute acetic acid, can be eluted. However, the use of the term "free apolipoprotein CI" does not necessarily mean that the material which can be separated by hydrophobic interaction chromatography must be completely free in the original samples. Associates or aggregates, also with lipids, which are broken up under the experimental conditions on contact with octyl-pharose with binding of the apolipoprotein CI to the chromatographic material, are likewise to be regarded as "free apolipoprotein CI" within the meaning of the use of this term.
Wie der genannten DE 103 43 815 Al zu entnehmen ist, erkennt der genannte Immunoassay im Blut von Krebspatienten auch eine Apo C- I -Fraktion, die nicht an Octylsepharose bindet und deren Auftreten für Tumorerkrankungen charakteristisch zu sein scheint. Ggf. wäre daher im Rahmen der Gesamtdiagnose auf eine Demenzerkrankung bzw. auf deren Vorstufen unter Verwendung der bei der Bestimmung des immunreaktiven Apo C-I gewonnenen Messwerte auszuschließen, dass die Messwerte bei dem jeweiligen Patienten durch eine Krebserkrankung verfälscht werden. Das kann beispielsweise auf die in der DE 103 43 815 Al beschriebene Weise erfolgen, indem man prüft, ob sich die gemessenen Konzentrationswerte durch einen Kontakt der Probe mit z.B. Octylsepharose verändern.As can be deduced from the aforementioned DE 103 43 815 A1, said immunoassay in the blood of cancer patients also recognizes an apo C-I fraction which does not bind to octylsepharose and whose occurrence appears to be characteristic of tumor diseases. Possibly. Therefore, in the context of the overall diagnosis for dementia disease or its precursors using the measured values obtained in the determination of the immunoreactive Apo C-I, it would be ruled out that the measured values in the respective patient would be falsified by a cancer. This can be done, for example, in the manner described in DE 103 43 815 A1 by checking whether the measured concentration values are obtained by contact of the sample with e.g. Octyl Sepharose change.
Das gemäß der vorliegenden Erfindung zu bestimmende immunreaktive Apo C-I umfasst neben Analyten, die das vollständige Peptid Apo C-I darstellen oder enthalten, auch "Apo C-I Abkömmlinge" . Darunter sind insbesondere immunreaktive Apo
C-I -Fragmente und Aggregate zu verstehen, insbesondere solche, die sich in dem genannten Sandwich- Immunoassay wie das freie Protein Apolipoprotein C-I verhalten. Die "Abkömmlinge" können z.B. um einzelne Aminosäuren oder Aminosäuresequenzen verkürzte Apolipoprotein C- I -Moleküle sein, oder auch - z.B. durch Aggregation - sterisch oder konformatio- nell veränderte vollständige Apolipoprotein C- I -Moleküle .The immunoreactive Apo CI to be determined according to the present invention comprises, in addition to analytes which represent or contain the complete peptide Apo CI, also "Apo CI derivatives". These include in particular immunoreactive apo CI fragments and aggregates, in particular those which behave in the said sandwich immunoassay as the free protein apolipoprotein CI. The "derivatives" may be, for example, apolipoprotein C-1 molecules shortened by individual amino acids or amino acid sequences, or also, for example, by aggregation, sterically or conformally altered complete apolipoprotein C-I molecules.
Dass auch die Bestimmung solcher Apo C- I -Abkömmlinge von der Erfindung umfasst sein soll, trägt den bekannten Tatsachen Rechnung, dass Immunoassays normalerweise nicht zwischen unterschiedlichen Analyten unterscheiden können, wenn die Unterschiede außerhalb der für die Antikörperbindung genutzten Molekülabschnitte liegen, d.h. im Falle eines Sandwich- Immunoassays in Endbereichen des erfassten Moleküls außerhalb der von den beiden Bindungsstellen für die Antikörper begrenzten Aminosäuresequenz. Ferner soll der weiteren bekannten Tatsache Rechnung getragen werden, dass dann, wenn peptidische Analyten in der Zirkulation auftreten, stets auch mit dem Auftreten ihrer Abbauprodukte in Form von Peptidfragmenten zu rechnen ist. Die Konzentrationen derartiger Fragmente sind in der Regel in etwa proportional zu der Konzentration des Ausgangspeptids, wobei die Konzentrationsunterschiede Stabilitätsunterschiede bzw. unterschiedliche Geschwindigkeiten des "Clearings" der Peptide bzw. Fragment durch weiteren proteolytischen Abbau oder durch Ausschleusung aus dem Kreislauf z.B. über die Nieren widerspiegeln.That the determination of such Apo C-1 derivatives should also be encompassed by the invention takes into account the known facts that immunoassays normally can not differentiate between different analytes if the differences are outside the molecular segments used for antibody binding, i. in the case of a sandwich immunoassay in end regions of the detected molecule outside the amino acid sequence delimited by the two antibody binding sites. Furthermore, the further known fact is to be taken into account that when peptidic analytes occur in the circulation, the appearance of their degradation products in the form of peptide fragments must always be expected. The concentrations of such fragments are usually approximately proportional to the concentration of the starting peptide, the differences in concentration differences in stability or different rates of "clearing" of the peptide or fragment by further proteolytic degradation or by discharge from the circulation, e.g. reflect on the kidneys.
Soweit in der Literatur eine Diskussion von Apo C-I im Zusammenhang mit der Alzheimer Krankheit gefunden werden kann, handelt es sich ausschließlich um Untersuchungen zur Apolipoprotein C-I Expression in Extrakten von Gehirngewebe Verstorbener, jedoch nicht in der Zirkulation lebender Patienten (vgl. 10). Angesichts der Existenz der Blut-Hirn- Schranke (blood-brain barrier, BBB) , die einen Schrankeneffekt für den Übergang von z.B. proteinischen Substanzen
zwischen dem Gehirn und der Zirkulation ausübt, können aus Untersuchungen mit Gehirngewebeextrakten keine Schlüsse bezüglich des Auftretens bzw. der Konzentration von proteinischen Substanzen in der Zirkulation gezogen werden.Insofar as a discussion of Apo CI in the context of Alzheimer's disease can be found in the literature, it exclusively deals with studies of apolipoprotein CI expression in extracts of brain tissue of deceased, but not in the circulation of living patients (see Figure 10). Given the existence of the blood-brain barrier (BBB), which is a barrier effect for the passage of eg proteinaceous substances between the brain and the circulation, conclusions about the appearance or concentration of proteinaceous substances in the circulation can not be drawn from studies with brain tissue extracts.
Soweit im Rahmen der in der DE 103 43 815 Al beschriebenen chromatographischen Untersuchungen auch Fraktionen aus Seren von zwei Alzheimer-Patienten vermessen wurden, wurden keine statistisch signifikanten Ergebisse erhalten, die einen Schluss bezüglich eines Zusammenhangs zwischen der Konzentration des immunreaktiven Apo C-I und verschiedenen Stufen von Demenzerkrankungen bzw. der Alzheimer-Krankheit erlauben würden .Insofar as fractions from sera from two Alzheimer's patients were measured within the scope of the chromatographic examinations described in DE 103 43 815 A1, no statistically significant results were obtained concluding a relationship between the concentration of the immunoreactive Apo CI and various stages of Dementia or Alzheimer's disease would allow.
Die nachfolgend im experimentellen Teil beschriebenen Messergebnisse in EDTA-Plasmaproben von 60 augenscheinlich gesunden Normalpersonen (symptomfreien Kontrollen) und 196 Patienten mit leichten bis schweren kognitiven Störungen gemäß den eingangs beschrieben Gruppen (b) bis (d) ergab erstmals eine klare, diagnostisch signifikante Korrelation zwischen den für immunreaktives Apo C-I gefundenen Konzentrationen und der Schwere der Demenzsymptome in Form kognitiver Störungen, wobei die gemessenen Konzentrationen in signifikanter Weise mit der Schwere von AD-Vorstufen korrelierten und damit die klinische Unterscheidung der verschiedenen Patientengruppen widerspiegelten.The measurement results in EDTA plasma samples from 60 apparently healthy normal persons (symptom-free controls) and 196 patients with mild to severe cognitive disorders according to the groups (b) to (d) described in the experimental part resulted for the first time in a clear, diagnostically significant correlation between the levels found for immunoreactive Apo CI and the severity of the dementia symptoms in the form of cognitive disorders, the measured concentrations significantly correlating with the severity of AD precursors and thus reflecting the clinical distinction of the different patient groups.
Die erfindungsgemäße Bestimmung von immunreaktivem Apo C-I im Rahmen der Diagnostik von neurodegenerativen Erkrankungen wird insbesondere als Messung vorgeschlagen, die die parallele Bestimmung physiologischer bzw. klinischer und neuro- psychologischer Parameter und anderer Biomarker ergänzt und für differentialdiagnostische Zwecke verfeinert.The determination according to the invention of immunoreactive Apo C-I in the context of the diagnosis of neurodegenerative diseases is proposed in particular as a measurement which supplements the parallel determination of physiological or clinical and neuropsychological parameters and other biomarkers and refines them for differential diagnostic purposes.
Das Verfahren wird daher bevorzugt im Rahmen einer Multipa- rameter-Bestimmung durchgeführt, bei der gleichzeitig mindestens ein weiterer für das jeweilige Krankheitsbild aus-
sagekräftiger biochemischer oder physiologischer Parameter bestimmt wird und bei der ein Messergebnis in Form eines Satzes von mindestens zwei Messgrößen gewonnen wird, der zur Feindiagnostik der neurodegenerativen Erkrankung ausgewertet wird.The method is therefore preferably carried out within the scope of a multiparameter determination, in which at least one additional one for the respective clinical picture is simultaneously detected. meaningful biochemical or physiological parameter is determined and in which a measurement result is obtained in the form of a set of at least two measured variables, which is evaluated for the fine diagnosis of the neurodegenerative disease.
Neben der Bestimmung des immunreaktiven Apo C-I kann z.B. wenigstens ein weiterer biochemischer Parameter bestimmt werden, der aus den Gruppen der natriuretischen Peptide, Entzündungsmediatoren, Komplementkomponenten, Cytokine, Chemokine, der Blutkoagulanzien und fibrinolytischen Faktoren, Akutphasenproteine und radikalischen Verbindungen ausgewählt ist. Als Co-Parameter sind insbesondere auch zu nennen diejenigen Parameter, die in den älteren deutschen Patentanmeldungen der Anmelderin mit den Aktenzeichen 10 2005 034 174.8 (midregionales Proadrenomedullin) , 10 2005 036 094.7 (liquordiagnostische Bestimmung von Procalcito- nin) , 10 2006 027 818.6 und 10 2006 023 175.9 diskutiert werden sowie der Parameter CPS-I (Carbamoylphosphat Syn- thetase 1) , zu dem sich relevante Diskussionen im Hinblick auf neurodegenerative Erkrankungen in den Anmeldungen EP 05023420.2 und DE 10 2006 021 406.4 finden.Besides the determination of the immunoreactive Apo C-I, e.g. at least one other biochemical parameter selected from the groups of natriuretic peptides, inflammatory mediators, complement components, cytokines, chemokines, blood coagulants and fibrinolytic factors, acute phase proteins and free radical compounds. The co-parameters to be mentioned in particular include those parameters which are described in the earlier German patent applications of the applicant with the file references 10 2005 034 174.8 (midregionales Proadrenomedullin), 10 2005 036 094.7 (Liquorodiagnostic determination of procalcitoline), 10 2006 027 818.6 and 10 2006 023 175.9 and the parameter CPS-I (carbamoyl phosphate synthetase 1), to which relevant discussions with regard to neurodegenerative diseases can be found in the applications EP 05023420.2 and DE 10 2006 021 406.4.
Es ist dabei vorgesehen, dass das erfindungsgemäße Verfahren auch als Simultanbestimmung mittels einer Chiptechnologie- Messvorrichtung oder einer immunchromatographisehen Messvorrichtung erfolgen kann und die Auswertung der dabei ggf. erhaltenen komplexen Messergebnisse mit Hilfe eines geeigneten Computerprogramms erfolgen kann.It is thereby provided that the method according to the invention can also be carried out as a simultaneous determination by means of a chip technology measuring device or an immunochromatographic measuring device and the evaluation of the complex measurement results possibly obtained can take place with the aid of a suitable computer program.
Obwohl die Untersuchungen bisher auf Plasmaproben von Patienten beschränkt waren, die Anzeichen von Vorstufen von AD zeigten bzw. für die die Diagnose "wahrscheinlich Alzheimer" gestellt worden war, gehen die Erfinder davon aus, dass - möglicherweise mit unterschiedlichen typischen Konzentrationsbereichen - auch bei anderen neurodegenerativen Erkrankungen, insbesondere bei vaskulärer Demenz (VAD) und
Demenz mit Lewy-Körperchen (DLB) , charakteristische Veränderungen der Konzentrationen des immunreaktiven Apo C-I in Patientenplasmen feststellbar sein könnten.Although investigations have been limited to plasma specimens from patients who have had evidence of AD precursors or who have been diagnosed as "likely to have Alzheimer's disease," the inventors believe that - possibly with different typical ranges of concentration - in other neurodegenerative disorders as well Diseases, especially in vascular dementia (VAD) and Dementia with Lewy bodies (DLB), characteristic changes in the levels of immunoreactive Apo CI in patient plasmas could be detectable.
Nachfolgend wird die Erfindung anhand von Messergebnissen und einer Figur noch näher erläutert .The invention will be explained in more detail with reference to measurement results and a figure.
Figur 1 zeigt die Ergebnisse der Messung der Konzentrationen des immunreaktiven Apo C-I, wie sie mit dem im experimentellen Teil beschriebenen Sandwich- Immu- noassay bestimmbar sind, und zwar in EDTA-Plasmen von 60 gesunden Kontrollpersonen, und von 196 Patienten mit kognitiven Störungen verschiedener Schwere, die den o.g. Gruppen (b) , (c) und (d) , d.h. den Gruppen 11SKS" (50 Patienten), "LKS mgl AD" (46 Patienten) und "ws AD" (100 Patienten) entsprachen.
FIG. 1 shows the results of the measurement of the concentrations of the immunoreactive Apo CI determinable with the sandwich immunoassay described in the experimental section, namely in EDTA plasmas of 60 healthy control persons, and of 196 patients with cognitive disorders of various severity corresponding to the above-mentioned groups (b), (c) and (d), ie the groups 11 SKS "(50 patients)," LKS mgl AD "(46 patients) and" ws AD "(100 patients).
Experimenteller TeilExperimental part
1. Apolipoprotein C-I -Immunoassay1. Apolipoprotein C-I immunoassay
Die direkte Messung von immundiagnostisch bestimmbarem Apo C-I in Plasma erfolgte mit dem gleichen Sandwich- Immunoassay, wie er bereits in der deutschen Patentanmeldung DE 103 43 815 Al der Anmelderin beschrieben wird. Im einzelnen ist dieser Assay wie folgt aufgebaut:The direct measurement of immunodiagnostic determinable Apo C-I in plasma was carried out with the same sandwich immunoassay as already described in the German patent application DE 103 43 815 A1 of the applicant. Specifically, this assay is constructed as follows:
Für die direkte immundiagnostische Bestimmung von Apolipoprotein C-I im Serum wurde aus den nachfolgend beschriebenen Bestandteilen ein Immunoassay vom Sandwich-Typ aufgebaut :For the direct immunodiagnostic determination of apolipoprotein C-1 in serum, a sandwich-type immunoassay was constructed from the components described below:
a) Coated Tubes : Polystyrolröhrchen (Greiner) wurden mit einem kommerziell erhältlichen polyklonalen affinitätsgerei- nigten Antikörper gegen Apolipoprotein C-I (Bezugsquelle: Acris Antibody, Bad Neuheim, Deutschland) beschichtet. Der Antikörper war nach Herstellerangaben durch Immunisierung von Kaninchen mit humanen Apo C-I erhalten und über eine Sepharose-Affinitätssäule mit humanem Apolipoprotein C-I aufgereinigt worden. Zur Beschichtung wurden 0,2 μg Antikörper in 300 μl PBS an Polystyrolröhrchen (Greiner, Deutschland) gebunden, die mit Schaf-Anti -Kaninchen IgG-Antikörper (Sigma) beschichtet waren. Die Bindung wurde nach 18 Stunden bei Raumtemperatur beendet. Anschließend wurden die Röhrchen 2 mal mit je 3 ml 0,5% Rinderserumalbumin (BSA) in PBS gewaschen. Nach ihrer Trocknung in Vakuum wurden die Röhrchen als Festphase für den Apolipoprotein C- I -Immunoassay verwendet .a) Coated Tubes: Polystyrene tubes (Greiner) were coated with a commercially available polyclonal affinity-purified antibody against apolipoprotein C-I (source: Acris Antibody, Bad Neuheim, Germany). The antibody was obtained according to the manufacturer by immunization of rabbits with human Apo C-I and purified on a Sepharose affinity column with human apolipoprotein C-I. For coating, 0.2 μg of antibody in 300 μl of PBS was bound to polystyrene tubes (Greiner, Germany) coated with sheep anti-rabbit IgG antibody (Sigma). The binding was stopped after 18 hours at room temperature. Subsequently, the tubes were washed twice with 3 ml each of 0.5% bovine serum albumin (BSA) in PBS. After drying in vacuo, the tubes were used as the solid phase for the apolipoprotein C-I immunoassay.
b) Akridiniumester-markierter Antikörper: 100 μg eines anderen Antikörpers gegen humanes Apolipoprotein C-I (aus Kaninchen; Bezugsquelle: Academie Bio-Medical Company, Texas, USA) in 100 μl PBS wurden mit 10 μg Akridinium-NHS- Ester (in 10 μl Acetonitril) umgesetzt. Nach einer 10-minütigen Inkubation bei Raumtemperatur erfolgte die Reinigung
des markierten Antikörpers unter Abtrennung unumgesetzter Bestandteile der Reaktionsmischung durch HPLC an SW 300 (Waters) . Für seine Verwendung im Immunoassey wurde der markierte Antikörper in PBS mit 0,5% BSA und 1 mg/ml Kaninchen IgG zur Absättigung der Röhrchenwände auf ca. 1 Mio. RLU/300 μl (RLU = relative Lichteinheiten) eingestellt.b) Acridinium ester-labeled antibody: 100 μg of another antibody to human apolipoprotein CI (from Rabbit, reference: Academie Bio-Medical Company, Texas, USA) in 100 μl of PBS was incubated with 10 μg of acridinium-NHS ester (in 10 μl of acetonitrile ) implemented. After a 10 minute incubation at room temperature, the purification was carried out of the labeled antibody with removal of unreacted constituents of the reaction mixture by HPLC on SW 300 (Waters). For its use in Immunoassey, the labeled antibody in PBS with 0.5% BSA and 1 mg / ml rabbit IgG was set to approximately 1 million RLU / 300 μl (RLU = relative light units) to saturate the tube walls.
2. Durchführung der immundiagnostischen Bestimmung von Apolipoprotein C-I in Serum- bzw. Plasma-Proben2. Carrying out the immunodiagnostic determination of apolipoprotein C-I in serum or plasma samples
Serum- bzw. Plasmaproben wurden 1:10 000 mit PBS, 0,5% BSA verdünnt. Davon wurden jeweils 300 μl in die mit dem immobilisierten Antikörper beschichteten o.g. Röhrchen pipettiert und anschließend für 4 Stunden bei Raumtemperatur unter Schütteln (300 U/min auf einem Heidolph-Kreisschüttler) inkubiert. Der Röhrcheninhalt wurde anschließend mit PBS ausgewaschen (4 -maliges Befüllen und Dekantieren mit jeweils 1 ml PBS) , und an die Röhrchenwand gebundenes Apolipoprotein C-I wurde innerhalb von 20 Stunden bei Raumtemperatur und 300 U/min mit 300 μl je Röhrchen des akridiniumestermarkier- ten Anti-Apolipoprotein C- I -Antikörpers umgesetzt. Danach wurde ungebundener markierter Antikörper durch 5 -maliges Waschen mit je 1 ml PBS entfernt, und die verbleibende Chemilumineszenz wurde auf bekannte Weise in einem Berthold Luminometer 952 T gemessen.Serum or plasma samples were diluted 1:10 000 with PBS, 0.5% BSA. Of these, 300 μl each were added to the o.g. coated with the immobilized antibody. Pipette and then incubated for 4 hours at room temperature with shaking (300 U / min on a Heidolph orbital shaker). The tube contents were then washed out with PBS (4 times filling and decanting with 1 ml PBS each), and apolipoprotein CI bound to the tube wall was incubated within 20 hours at room temperature and 300 rpm with 300 μl per tube of acridinium ester labeled anti -Apolipoprotein C-I antibody reacted. Thereafter, unbound labeled antibody was removed by washing 5 times with 1 ml PBS each time, and the remaining chemiluminescence was measured in a known manner in a Berthold Luminometer 952T.
Zur Kalibrierung des Assays wurde ein synthetisches Apolipoprotein C-I verwendet. Eine typische Standardkurve, wie sie für den obigen Assay erhalten wird, ist in Fig. 3 der bereits genannten DE 103 43 815 Al gezeigt.To calibrate the assay, a synthetic apolipoprotein C-I was used. A typical standard curve, as obtained for the above assay, is shown in Fig. 3 of the already mentioned DE 103 43 815 A1.
3. Messung der Apo C- I -Immunreaktivität im Plasma von gesunden Kontrollen und Patienten mit kognitiven Störungen verschiedenen Schweregrads3. Measurement of apo C-I immunoreactivity in plasma of healthy controls and patients with cognitive disorders of varying severity
Zur Ermittlung eines Bezugswerts für die Konzentration des immundiagnostisch bestimmbaren "freien" Apo C-I wurde eine
Messung in EDTA- Plasmen von 60 symptomfreien Kontrollpersonen durchgeführt, die weder Symptome kognitiver Störungen zeigten noch an irgendeiner anderen erkennbaren Erkrankung (Tumorerkrankungen; schwere Infektion oder Entzündung) litten, für die bekannt ist, dass sie die Apo C-I-Spiegel in der Zirkulation beeinflussen. Für die Kontrollgruppe wurde ein Mittelwert (Median) für die gemessene Apo C-I-Konzentra- tion von 332 μg/ml ermittelt.To determine a reference value for the concentration of the immunodiagnostic determinable "free" Apo CI was a Measurement was performed in EDTA plasmas of 60 symptom-free controls who neither showed symptoms of cognitive impairment nor suffered from any other identifiable disease (tumors, severe infection, or inflammation) known to affect circulating Apo CI levels. For the control group, a mean (median) for the measured Apo CI concentration of 332 μg / ml was determined.
Als Patientenkollektiv dienten Patienten mit Demenzerscheinungen in Form kognitiver Störungen verschiedener Schwere, aufgrund derer eine Zuordnung der einzelnen Patienten zu einer der o.g. Gruppen (b) , (c) bzw (d) erfolgt war.Patients with symptoms of dementia in the form of cognitive disorders of various severity, due to which an assignment of the individual patients to one of the above-mentioned patients, served as a patient collective. Groups (b), (c) and (d) were done.
Die gemessenen Konzentrationen an immunreaktivem Apo C-I im Plasma von gesunden Kontrollen und Patienten mit kognitiven Störungen sind in Figur 1 gezeigt .The measured levels of immunoreactive plasma Apo C-I of healthy controls and patients with cognitive disorders are shown in FIG.
Die ermittelten Zahlenwerte in Form der sog. Mediane für die verschiedenen Patientengruppen waren wie folgt :The calculated numerical values in the form of the so-called medians for the different patient groups were as follows:
Die Konzentrationen des in Plasmen immundiagnostisch bestimmbaren Apo C-I, erkennbar an den Werten für die Mediane der verschiedenen Patientengruppen, sinken mit der Schwere der Symptome in der Richtung:The concentrations of apo C-I, which can be determined immunologically in plasmas, which can be identified by the values for the medians of the various patient groups, decrease with the severity of the symptoms in the direction:
Kontrollen > SKS > LKS mgl AD = ws AD klar ab.Checks> SKS> LKS mgl AD = ws AD clear off.
Aus der bereits erwähnten älteren DE 103 43 815 Al ist es bekannt, dass die messbaren Konzentrationen von Apo C-I auch bei anderen Krankheiten oder physiologischen Zuständen
Abweichungen von den bei Kontrollpersonen messbaren Werten zeigen; die meisten einschlägigen Erkrankungen wie z.B. Sepsis oder Tumoren sind jedoch klinisch in der Regel einfach diagnostisch von Demenzerkrankungen abgrenzbar und ihren evtl. Auswirkungen können daher bei der Stellung einer Diagnose angemessen berücksichtigt werden.It is known from the earlier mentioned DE 103 43 815 A1 that the measurable concentrations of Apo CI are also found in other diseases or physiological conditions Show deviations from the values measurable by control persons; However, most relevant diseases, such as sepsis or tumors, are generally clinically easily delineated diagnostically from dementia, and their potential effects may therefore be adequately considered when making a diagnosis.
Obwohl Apo C-I somit daher kein gehirnspezifischer Parameter ist, eignet sich die Messung der Apo C- I -Immunreaktivität in der Zirkulation (Plasma, Serum) angesichts der o.g. Korrelationen sehr gut für Zwecke der unterstützenden AD-Frühdia- gnostik.
Therefore, although apo CI is not a brain-specific parameter, the measurement of apo C-I immunoreactivity in the circulation (plasma, serum) is very well suited for the purpose of assisting AD early diagnosis, given the above-mentioned correlations.
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10. C. PETIT-TURCOTTE, S. M. STOHL, U. BEFFERT, J. S. COHN, N. AUMONT, M. TREMBLAY, D. DEA, LIN YANG, J. POIRIER, and N. S. SHACHTER (2001). Apolipoprotein C-I Expression in the Brain in Alzheimer's Disease, Neurobiology of Disease 8, 953-963.
Claims
1. In vitro Verfahren zur Erkennung und Früherkennung, zur Bestimmung des Schweregrads und zur Verlaufsbeurteilung und Prognose von neurodegenerativen Erkrankungen, bei dem man in einer Serum- oder Plasmaprobe eines Patienten, der an subjektiven oder objektiv nachweisbaren kognitiven Störungen leidet, mit Hilfe eines immundiagnostischen Bestimmungsverfahrens die Konzentration des immunreaktiven Apolipoproteins Apo C-I bestimmt, und bei dem man auf der Basis der gemessenen Konzentration Schlüsse hinsichtlich des Vorliegens einer neurodegenerativen Erkrankung oder einer für diese typischen Frühform davon oder hinsichtlich des Verlaufs der Erkrankung und/oder des Erfolgs der Bemühungen zu ihrer Abmilderung oder Verhinderung zieht.1. In vitro methods for detection and early detection, for determining the severity and for the assessment and prognosis of neurodegenerative diseases, in which one in a serum or plasma sample of a patient suffering from subjective or objectively detectable cognitive disorders, using an immunodiagnostic determination method determining the concentration of the immunoreactive apolipoprotein Apo CI and concluding, on the basis of the measured concentration, conclusions as to the presence or typical early form of a neurodegenerative disease or the course of the disease and / or the success of efforts to alleviate it Prevention pulls.
2. Verfahren nach Anspruch 1, dadurch gekennzeichnet, dass das immundiagnostische Bestimmungsverfahren ein Immunoassay vom Sandwichtyp ist .2. The method according to claim 1, characterized in that the immunodiagnostic determination method is a sandwich-type immunoassay.
3. Verfahren nach einem der Ansprüche 1 oder 2, dadurch gekennzeichnet, die neurodegenerative Erkrankung eine Demenzerkrankung ist, die ausgewählt ist aus einer Gruppe, die die Alzheimer Demenz (AD) , Demenz mit Lewy-Körperchen (DLB) , frontotemporale Demenz (FTD) und verschiedene Formen der vaskulären Demenz (VD) umfaßt.3. The method according to any one of claims 1 or 2, characterized in that the neurodegenerative disease is a dementia disease selected from a group comprising Alzheimer's disease (AD), dementia with Lewy bodies (DLB), frontotemporal dementia (FTD) and various forms of vascular dementia (VD).
4. Verfahren nach Anspruch 4, dadurch gekennzeichnet, dass es im Rahmen der Alzheimer-Diagnostik zur Erkennung von Frühformen der Alzheimer Demenz (AD) durchgeführt wird.4. The method according to claim 4, characterized in that it is performed in the context of Alzheimer's diagnosis for the detection of early forms of Alzheimer's disease (AD).
5. Verfahren nach einem der Ansprüche 1 bis 4, dadurch gekennzeichnet, dass es im Rahmen einer Multiparameter-Bestim- mung durchgeführt wird, bei der gleichzeitig mindestens ein weiterer für das jeweilige Krankheitsbild aussagekräftiger biochemischer oder physiologischer Parameter bestimmt wird und bei der ein Messergebnis in Form eines Satzes von mindestens zwei Messgrößen gewonnen wird, der zur Feindiagnostik der neurodegenerativen Erkrankung ausgewertet wird.5. The method according to any one of claims 1 to 4, characterized in that it is carried out in the context of a multi-parameter determination, in which at least one other simultaneously more meaningful for the respective clinical picture biochemical or physiological parameter is determined and in which a measurement result is obtained in the form of a set of at least two measured variables, which is evaluated for the fine diagnosis of neurodegenerative disease.
6. Verfahren nach Anspruch 5, dadurch gekennzeichnet, dass im Rahmen der Multiparameter-Bestimmung neben der Bestimmung des immunreaktiven Apo C-I wenigstens ein weiterer biochemischer Parameter bestimmt wird, der aus den Gruppen der natriuretischen Peptide, Entzündungsmediatoren, Komplement - komponenten, Cytokine, Chemokine, der Blutkoagulanzien und fibrinolytischen Faktoren, Akutphasenproteine und radikalischen Verbindungen ausgewählt ist.6. The method according to claim 5, characterized in that in the context of multiparameter determination in addition to the determination of the immunoreactive Apo CI at least one further biochemical parameter is determined, consisting of the groups of natriuretic peptides, inflammatory mediators, complement - components, cytokines, chemokines, of blood coagulants and fibrinolytic factors, acute phase proteins and free radical compounds.
7. Verfahren nach Anspruch 5 oder 6, dadurch gekennzeichnet, dass die Multiparameter-Bestimmung als Simultanbestimmung mittels einer Chiptechnologie-Messvorrichtung oder mittels einer immunchromatographisehen Messvorrichtung erfolgt .7. The method according to claim 5 or 6, characterized in that the multiparameter determination is carried out as a simultaneous determination by means of a chip technology measuring device or by means of an immunochromatographisehen measuring device.
8. Verfahren nach Anspruch einem der Ansprüche 5 bis 7, dadurch gekennzeichnet, dass die Auswertung des komplexen Messergebnisses der Multiparameter-Bestimmung mit Hilfe eines Computerprogramms erfolgt . 8. The method according to claim one of claims 5 to 7, characterized in that the evaluation of the complex measurement result of the multiparameter determination by means of a computer program.
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| DE102006056337A DE102006056337A1 (en) | 2006-11-29 | 2006-11-29 | In vitro diagnostic and early diagnosis of neurodegenerative diseases |
| PCT/EP2007/010331 WO2008064883A1 (en) | 2006-11-29 | 2007-11-28 | In vitro method for the diagnosis and early diagnosis of neurodegenerative disorders |
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| EP (1) | EP2097755A1 (en) |
| JP (1) | JP2010511159A (en) |
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| GB201616691D0 (en) * | 2016-09-30 | 2016-11-16 | King S College London | Assay |
| CN111650372A (en) * | 2019-03-04 | 2020-09-11 | 中国医学科学院药物研究所 | Application of apolipoprotein C1 as a biomarker for the diagnosis and prognostic evaluation of gastric cancer |
| CN118475840A (en) * | 2022-06-28 | 2024-08-09 | 株式会社Mcbi | Determination support information generation method, determination support information generation system, and information processing apparatus |
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| DE69333461T2 (en) * | 1992-10-13 | 2005-03-24 | Duke University | METHOD FOR DETECTING Alzheimer's disease |
| US20050266501A1 (en) * | 2001-04-10 | 2005-12-01 | Shigeo Ota | Reagent for detecting risk factor of alzheimer's disease, detection kit therefor and method of detecting risk factor of alzheimer's disease using the same |
| DE10343815A1 (en) | 2003-09-22 | 2005-04-14 | B.R.A.H.M.S Ag | Diagnosis, particularly of cancer and sepsis, by determining abnormal contents of apolipoprotein C-I in serum or plasma, also therapeutic use of apolipoprotein C-I |
| EP2369347A1 (en) * | 2003-11-07 | 2011-09-28 | Ciphergen Biosystems, Inc. | Biomarkers for Alzheimer's disease |
| EP1780287A1 (en) * | 2005-10-26 | 2007-05-02 | B.R.A.H.M.S. Aktiengesellschaft | In vitro method for diagnosing neurodegenerative diseases |
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| JP2010511159A (en) | 2010-04-08 |
| US20100035286A1 (en) | 2010-02-11 |
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