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
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
• • 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
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
  • G01N33/5047—Cells of the immune system
  • G01N33/505—Cells of the immune system involving T-cells
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
  • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
  • G01N2333/70514—CD4
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
  • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
  • G01N2333/70517—CD8
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
  • G01N2333/7056—Selectin superfamily, e.g. LAM-1, GlyCAM, ELAM-1, PADGEM
• • 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 method for diagnosing Alzheimer's disease or an early stage or a predisposition for this disease, which is based on the quantitative determination of mitogenically expressible surfaces of ark, preferably CD69, peripherally accessible cells, e.g. Skin cells or lymphocytes, (a) before and (b) after mitogenic stimulation, a specific stimulation index a: b being a sign of Alzheimer's disease or an early stage or a predisposition to this disease.
• the present invention also relates to kits which are suitable for carrying out the diagnostic method according to the invention.
• Alzheimer's disease cannot be made with absolute certainty using clinical means and the available paraclinic and technical equipment alone and therefore always requires autoptical verification.
• it is often difficult to differentiate between other causes of dementia.
• a reliable diagnosis is important for two reasons.
• it allows the demarcation of potentially treatable forms of dementia and can thus lead to effective therapy
• it is a prerequisite for any form of therapeutic intervention in the process of neurodegeneration of Alzheimer's disease, which can only be successful in these early stages.
• Such diagnostic certainty can only be achieved through biomarkers of Alzheimer's disease, ie through easy to determine biological changes with a sensitivity and specificity sufficient for the disease.
• biomarkers of Alzheimer's disease have a diagnostic value and are intended to help in particular to reliably identify risk groups and patients in preclinical and early clinical stages.
• biomarkers serve to monitor the progress and thus the prognosis and the control of the responsiveness to therapeutic interventions.
• Ideal biomarkers should meet certain theoretical and practical requirements. In particular, this includes a high degree of specificity and sensitivity, the ability to identify preclinical stages, as well as a high positive and negative predictive value.
• the determination of the biomarkers should be as non-invasive as possible and should not burden or frighten the patient.
• the analyzes should be inexpensive and simple, if possible under the conditions of the family doctor's practice.
• none of the currently known biomarkers of Alzheimer's disease meet the above requirements. In particular due to the low sensitivity and specificity of the known biomarkers, they are unsuitable as diagnostic aids. Other diagnostic tests with higher sensitivity and specificity require complex technical requirements and are therefore not suitable for decentralized use in a larger group of patients.
• the invention is essentially based on the technical problem of providing a simple method for diagnosing Alzheimer's disease, which allows the diagnosis of Alzheimer's disease, the detection of preclinical disease phases and the differential diagnosis of Alzheimer's disease against other dementias with sufficient sensitivity and specificity.
• a diagnostic procedure could be developed which is based on the determination of the mitogenic index (activation index) on peripherally accessible cells, such as skin cells or blood lymphocytes, of the patient with and without mitogenic stimulation, for example after immunomagnetic cell separation.
• the activation of these cells is accompanied by the surface presentation of activation markers which can be detected quantitatively, preferably on the basis of antigen-antibody interactions, preferably using magnetic particles coated with antibodies, which allows the magnetic cell separation and subsequent quantification of the number of cells, that carry this surface marker before and after mitogenic stimulation.
• This characteristic shows disease-specific deviations from normal findings.
• the method according to the invention thus allows the diagnosis of Alzheimer's disease, the detection of preclinical phases of the disease and the differential diagnosis of Alzheimer's disease against other dementias.
• the present invention thus relates to a method for diagnosing Alzheimer's disease or an early stage or a predisposition for this disease on the basis of a sample from a patient, said method comprising the following steps: (a) mitogenic stimulation of the peripherally accessible cells in the sample; (b) quantitative determination of the mitogenically stimulated cells within the cell population before and after step (a) on the basis of one or more surface markers expressed after mitogenic stimulation, the cells bearing the surface markers from the cells bearing no surface markers using cells which are directed against the surface markers Antibodies are separated; and (c) Determination of the stimulation index as a ratio of the number of cells carrying the surface area marker (s) before and after step (a), wherein a stimulation index that reaches at least 10 times and at most 100 times the unstimulated control sample is an indication of a Alzheimer's disease or an early stage or predisposition to this disease.
• suitable samples are skin tissue samples, blood samples, preferably of venous blood, cells from the cerebrospinal fluid, and cells from urine.
• a coagulation-inhibiting compound e.g. sodium citrate or heparin, is added for stabilization before the further method steps.
• diagnosis of Alzheimer's disease also includes the follow-up and thus prognostics, the control of the efficiency of therapeutic measures and the differential diagnosis of the disease from other dementias.
• peripheral blood refers to cells that can be removed from the human organism without surgical intervention or (minimally) invasively and include, for example, skin cells and lymphocytes of the peripheral blood, the latter for the method according to the invention are preferred.
• Mitogenic stimulation to achieve the expression of the expression of surface markers can be carried out by known stimulators, such as phytohemagglutinin (PHA), protein A, PWM or other trophic or mitogenic compounds. The stimulation can take place by adding the individual compounds or by combined addition.
• PHA phytohemagglutinin
• PWM phytohemagglutinin
• mitogenic compounds can take place by adding the individual compounds or by combined addition.
• suitable experimental conditions for such stimulation e.g. with regard to the concentration of the mitogens used, duration of the stimulation and other incubation conditions.
• the stimulation should take place in suitable vessels that allow sufficient gas exchange.
• concentrations of the respective stimulation agents should be in the physiological range, e.g. for PHA l ⁇ g / ml to 20 ⁇ g / ml, for PWM l ⁇ g / ml to 50 ⁇ g / ml and for protein A lO ⁇ g / ml to 200 ⁇ g / ml.
• the duration of the stimulation depends on the speed of expression of the molecule to be examined.
• stimulation times of 2 to 24 hours may also be required; in the case of CD69, a stimulation period of 4 hours is optimal.
• the stimulation should take place under physiological conditions and can e.g. be carried out in a fumigation incubator at 37 ° C and 5% C02.
• suitable surface markers on the basis of which a mitogenic stimulation is manifested for example CD69, CD25, CD45RO, CD63 or HLA-DR, the surface marker CD69 being preferred.
• the stimulation index results from the ratio of the number of cells carrying the surface marker or markers before and after stimulation.
• a stimulation index that is at least 10 times and at most 100 times the unstimulated control sample is a sign of Alzheimer's disease or an early stage or a predisposition to this disease.
• a stimulation index that is less than 10 times The unstimulated control sample does not indicate any signs of Alzheimer's disease or an early stage or a predisposition to this disease.
• the cells carrying surface markers can be determined by conventional methods, for example Western blot, ELISA, RIA, FACS, LSC etc.
• the cell carrying surface markers For the determination of the cell carrying surface markers, it is preferable to separate them from the cells bearing no surface markers or other surface markers on the basis of characteristic cell features.
• the separation of the cells bearing surface markers from the cells not bearing surface markers is carried out by antibodies directed against the desired surface marker.
• the suitable antibodies can be monoclonal, polyclonal or synthetic antibodies or fragments thereof.
• fragment means all parts of the monoclonal antibody (e.g. Fab, Fv or “single chain Fv” fragments) which have the same epitope specificity as the complete antibody. The preparation of such fragments is known to the person skilled in the art, and many antibodies directed against surface markers are also commercially available.
• the surface margin (s) is Ker-specific antibodies are bound to magnetic particles, for example paramagnetic beads (for example available from DYNAL AS, POBox 158 Sk0yen, N-0212 Oslo, Norway), which makes the separation of the cells with the corresponding surface markers via immuno-magnetic separation according to the usual method Allow procedure.
• paramagnetic beads for example available from DYNAL AS, POBox 158 Sk0yen, N-0212 Oslo, Norway
• the stimulation index can then be determined by determining the amount of cells separated by means of the desired surface marker on the basis of their nucleic acid content and / or protein content by means of common methods, e.g. after lysis of the cells by spectrophotometric determination of the nucleic acid or protein content or after staining of the nucleic acid using specific dyes, e.g. Ethidium bromide, propidium odid, acridine orange, DAPI etc. via photometric quantification. Using calibration curves, the cell number can be calculated from the protein and / or nucleic acid content of the sample.
• the present invention also relates to a kit which is suitable for carrying out the diagnostic method according to the invention and contains at least the following components: (a) a compound for mitogenic stimulation; (b) at least one antibody directed against a surface marker expressed after mitogenic stimulation, preferably an antibody bound to a magnetic particle.
• the kit according to the invention preferably also contains (a) at least one reaction vessel; (b) an anticoagulant compound and / or a buffer for cell lysis; (c) a buffer to fix the cells; (d) substances which are required for the quantitative determination of the DNA or protein concentration, as well as ready-made solutions for producing a calibration curve; (e) a magnet for separating the cells bound to the magnetic particles (included if an antibody bound to a magnetic particle is used); and (f) a reagent for detaching bound magnetic particles (included if an antibody bound to a magnetic particle is used)
• the antibody is an anti-CD69 antibody.
• the kit may also contain an anti-CD4 and / or anti-CD8 antibody in addition to or instead of the anti-CD-69 antibody.
• kit according to the invention can optionally be combined with one or more suitable further detection means, e.g. fluorescence-coupled primary antibodies, secondary antibodies, detection means for proteins and / or nucleic acids, e.g. an intercalating dye, etc.
• suitable further detection means e.g. fluorescence-coupled primary antibodies, secondary antibodies, detection means for proteins and / or nucleic acids, e.g. an intercalating dye, etc.
• biomarkers Determinations of previously known features of Alzheimer's disease that can be carried out on living patients (biomarkers) show only insufficient sensitivity and specificity or are not suitable for investigations with high numbers of cases for reasons of cost or the high complexity of the test arrangement. With clinical means, the dia- Gnostic certainty only 80% to 90% and causes differential diagnostic difficulties, especially in the early stages of the disease. The detection of preclinical disease phases is currently not possible due to the lack of a suitable biomarker.
• Alzheimer's disease the neurodegenerative changes are based on impaired processes of intracellular mediation of trophic and mitogenic signals. These disorders of intracellular signal transduction are not limited to the nervous system. They can also be found in a similar manner on skin cells and on lymphocytes in the peripheral blood of these patients. Because of its disease specificity, this change has diagnostic value and is suitable as a biomarker.
• lymphocytes presenting CD69 presenting immuno-magnetic cell separation before and after mitogenic stimulation it was determined whether the disturbance in intracellular mediation of trophic and mitogenic signals typical of Alzheimer's disease is present by lymphocytes presenting CD69 presenting immuno-magnetic cell separation before and after mitogenic stimulation.
• the blood was obtained by venipuncture using a SARSTEDT blood collection system.
• the blood is stabilized during the withdrawal by anticoagulants integrated in the blood collection system, such as sodium citrate or sodium heparin. In this form it can be stored at room temperature for 24 to 48 hours.
• anticoagulants integrated in the blood collection system such as sodium citrate or sodium heparin. In this form it can be stored at room temperature for 24 to 48 hours.
• the stimulation experiments were carried out in well-ventilated reaction vessels, such as a 24 well suspension culture plate from Greiner bio-one.
• the mitogens phytohaemagglutinin (PHA), protein A and Pokeweed mitogen (PWM) were used individually or in different combinations for 400 ⁇ l of stabilized whole blood.
• the final concentrations of the respective mitogens were in the physiological range and in this example was 12 ⁇ g / ml for PHA, 50 ⁇ g / ml for Protein A and 4 ⁇ g / ml for PWM.
• the stimulation was carried out under physiological conditions at 37 ° C. and a CO 2 concentration of 5% in a fumigation incubator for a period of 4 hours.
• 100 ⁇ l of the stimulated whole blood were incubated with various antibody-coated magnetic particles.
• anti-CD4 and anti-CD8 coated magnetic particles from DYNAL were used.
• the corresponding magnetic particles were added to the respective sample in excess (10 ⁇ l magnetic particle suspension) in order to ensure complete isolation of the corresponding lymphocyte subpopulation.
• the corresponding lymphocyte subpopulation was magnetically separated and, after subsequent washing steps, transferred to 100 ⁇ l of defined medium, in this example RPMI1640, mixed with 1% fetal calf serum (FCS).
• FCS fetal calf serum
• the bound magnetic particles were detached using 10 ⁇ l DETACHaBEAD from DYNAL. After an incubation time of 45 minutes at room temperature, the detached magnetic particles were separated and the cell suspension was taken up in a defined medium, in this example RPMI1640, after washing several times.
• the cells were disrupted by adding a specific lysis buffer, the DNA was labeled with specific DNA dyes, such as, for example, ethidium bromide, propidium iodide, acridine orange or DAPI, and these were then quantified photometrically.
• DNA dyes such as, for example, ethidium bromide, propidium iodide, acridine orange or DAPI.
• the protein content of the samples was compared using the Bradford protein determination method. Using calibration curves, the cell number was calculated from the DNA and / or protein content of the sample. This procedure allowed a direct conclusion to be drawn about the cell number.
• the calculation of the quotient from the number of cells presenting CD69 before and after mitogenic stimulation provided information about changes in mitogenic stimulability of these cells.
• a stimulation index that is at least 10 times and at most 100 times the unstimulated control sample is a sign of Alzheimer's disease or an early stage or a predisposition to this disease. • A stimulation index less than 10 times that of the unstimulated control sample does not indicate any signs of Alzheimer's disease or early stage or predisposition to this disease.
• the protein content of the sample was determined, and the DNA content was determined without the addition of DNA-staining substances for the quantitative determination of the CD69-presenting cells.
• the absorption of DNA or protein by light of a certain wavelength e.g. 260 nm or 280 nm was measured.

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