MXPA06004451A - Quick test for the diagnosis of alzheimer'disease - Google Patents

Abstract

The invention relates to method for the diagnosis of Alzheimer's disease or the early stages thereof or a predisposition to said disease. Said method is based on quantitative determination of a mitogenically expressible surface marker, in particular CD69, and peripherally accessible cells, e.g. skin cells or lymphocytes, (a) prior to and (b) after mitogenic stimulation. A specific stimulation index a:b is an indication of Alzheimer's disease or early stages thereof or of a predisposition to said disease. The invention also relates to kits which are suitable for carrying out the inventive method of diagnosis.

Description

RAPID TEST FOR THE DIAGNOSIS OF ALZHEIMER'S DISEASE X • > ! • BACKGROUND The present invention relates to a method of diagnosing Alzheimer's disease, the presence of this at an early stage or a predisposition. The method is based on the quantification of mitogenically expressed surface markers, preferably CD69 of peripherally accessible cells, such as skin cells or lymphocytes (a) before and (b) after undergoing mitogenic stimulation, a special rate of stimulation a: b is shown as a sign of Alzheimer's disease, early stage of the disease or predisposition.

The present invention also relates to portable equipment for carrying out the diagnostic method according to the invention.

Alzheimer's disease can not be diagnosed with absolute certainty by clinical means or by available para-clinical means or device-based methods or technology of this type. Verification by autopsy is always required. Diagnostic differentiation in relation to other causes of dementia is often complicated, especially in the early stages of the disease. In these early stages, however, efficient diagnosis is important for the following reasons: first, it allows for the diagnostic differentiation of potentially treatable forms of dementia and thus can be subjected to effective treatment and, on the other hand, On the other hand, it is a precondition for any form of therapeutic intervention in the neurodegenerative process of Alzheimer's disease, which can be successful only in these early stages. This type of certainty in diagnosis can only be guaranteed by biomarkers of Alzheimer's disease, such as changes easily determined biologically with sensitivity and specificity appropriate for this disease.

The biomarkers of Alzheimer's disease have diagnostic value and can therefore help to safely identify risk groups and patients in preclinical stages or in early clinical stages. The Biomarkers also contribute to the subsequent check-up and therefore help with the prognosis and with the patient's response to therapeutic interventions.

The model biomarkers must comply with certain theoretical and practical requirements. These include high sensitivity and specificity, the ability to identify preclinical stages, and a high positive and negative predictive value. Biomarkers should be determined, if possible, in a non-invasive manner and should not bother or scare the patient. The analyzes must be economic and adapted to be carried out easily and, if possible, in the practice of a family doctor. Unfortunately none of the biomarkers of known Alzheimer's disease meet the above requirements. Considering the lower sensitivity and specificity of the If there are known biomarkers, these "are not suitable as diagnostic means.

Other diagnostic examinations with more sensitivity and specificity present complicated technical preconditions and therefore are not suitable for local use with a larger group of patients.

Thus, the invention is fundamentally based on the technical problem of providing a simple method for the diagnosis of Alzheimer's disease, the detection of preclinical stages of the disease and the diagnostic differentiation of Alzheimer's disease from other dementias with adequate sensitivity and specificity.

This technical problem was solved by providing the presentations included in the claims.

•] DETAILED DESCRIPTION OF THE INVENTION A diagnostic method based on the determination of the mitogenic index (activation index) could be developed using patient cells that are peripherally accessible, such as skin cells or blood lymphocytes, either with or without mitogenic stimulation, as it may be after immunomagnetic cell separation. The activation of these cells is accompanied by the presentation of activation surface markers that can be quantitatively detected, preferably by means of antigen-antibody interactions, magnetic particles preferably covered with the used antibodies, which allows cellular and magnetic separation. Subsequently, the quantification of the number of cells bearing the surface marker before and after the mitogenic stimulation.

This trait shows typical deviations of the disease in relation to the normal conclusions. Thus, the method of the invention allows the diagnosis of Alzheimer's disease, the detection of preclinical stages of the disease and the diagnostic differentiation of Alzheimer's disease from other dementias.

Thus, the present invention relates to a method for diagnosing Alzheimer's disease, an early stage or predisposition to the disease by means of a sample of the patient. The method consists of the following steps: a) Mitogenic stimulation of peripherally accessible cells in the sample; b) Quantification of mitogenically stimulated cells within the cell population before and after step (a), through one or more surface markers expressed after mitogenic stimulation, cells carrying surface markers separated from the cells that they do not carry them using antibodies directed against surface markers; and c) Determination of the stimulation index as a ratio between the number of cells carrying the surface marker (s) before and after step (a), A stimulation index that reaches at least 10 times, a maximum of 100, the unstimulated control sample as a sign of a predisposition, early stage or presence of Alzheimer's disease.

A skilled person knows suitable measures to obtain samples of patients that are functional to the method of the invention, those that contain sufficient mitogenic stimulable cells. For example, suitable samples are skin tissue, blood samples - preferably intravenously -, urine cells, and brain spinal fluid cells.

In the preferred embodiment of the diagnostic method employed in the invention, when a blood sample is used, an anticoagulant compound (sodium citrate or heparin) is added for stabilization before the other steps of the method.

The term "diagnosis of Alzheimer's disease", as used herein, also includes monitoring and prognosis, monitoring the efficiency of therapeutic interventions and the diagnostic differentiation of the disease from other dementias.

The term "peripherally accessible cells" as used herein refers to cells that can be removed without surgical intervention or in a minimally invasive form of the human organism containing skin cells and blood lymphocytes, which are preferred for the method of the invention.

Mitogenic stimulation to obtain the expression of surface markers can be done by known stimulators, such as phytohemagglutinin (PHA), protein A, PWM or other compounds with mitogenic effects. The stimulation can be carried out by adding the individual compounds or by a compound sum.

The skilled person knows suitable experimental conditions for this type of stimulation, as is the case of the concentration of mitogens used, the duration of the stimulation and other incubation conditions. The stimulation must be carried out in suitable vessels that allow the exchange of appropriate gas. The concentrations of the respective stimulation agents should be within the physiological range of 1 ug / ml at 20 ug / ml per PHA, 1 ug / ml at 50 ug / ml per PVM and 10 ug / ml at 200 ug / ml per protein A. The period of stimulation depends on the range of expression of the molecule examined. However, stimulation periods of 2 to 24 hours may be necessary for some examinations. In the case of CD69, a stimulation period of 4 hours is optimal. The stimulation must be carried out under physiological conditions and can be conducted in a gas incubator at 37 C and with 5% CO2, for example.

The specialist also knows suitable surface markers by which a mitogenic stimulation can be manifested, such as CD69, CD25, CD45RO, CD63 and HLA-Dr, with the surface marker CD69 as preferred. For the purposes of the invention, it is also possible to carry out a determination of a combination of surface markers or the subsequent specification of the separated cells through a certain CD69 surface marker, as future subpopulations, by means of (CD4 +) and / or CD8 + and / or CD19 + and / or C056 +) subpopulations.

The stimulation index (activation index) comes from the ratio of the number of cells carried by the marker or surface markers before and after the stimulation. A stimulation index that reaches at least 10 times, at most 100 times, the unstimulated control sample is a sign of predisposition, early stages or Alzheimer's disease. Cells that carry surface markers can be determined according to conventional methods, such as Western blot, ELISA, RIA, FACS, LSC, etc.

To determine the cells carried by the surface markers, they are preferably separated from cells that do not carry a surface marker or other surface markers through other characteristic cellular features.

In the diagnostic method of the present invention, the cells that carry the surface markers are separated from the cells that do not carry surface markers by antibodies directed against the desired surface markers. Antibodies suitable for this purpose may be monoclonal, polyclonal or synthetic antibodies or fragments derived therefrom. In this connection, the term fragment means all parts of the monoclonal antibody (Fab, Fv or single chain of Fv fragments) having the same epitope-specific as the whole antibody. The production of said fragments is known to specialists, many antibodies directed against surface markers that are also commercially available.

In the preferred embodiment of the diagnostic method according to the invention, the antibody or antibodies specific to the surface markers are tied to magnetic particles, such as paramagnetic beads (available from DYNAL AS, PO Box 158 Skoyen, N-0212 Oslo, Norway) , which allows the separation of cells with the corresponding surface markers through the immunomagnetic separation corresponding to the current methods.

The stimulation index can be specified by determining the number of cells separated by means of the desired surface marker based on their nucleic acid and / or protein content using current methods, such as after lysis of the cells by spectrophotometric determination of acid. nucleic acid or protein content or after staining the nucleic acid using specific dyes such as bromidium etidium, propidium iodide, orange acridine, DAPI, etc., through photometric quantification. The number of cells can be calculated by the protein and / or the nucleic acid content of the sample by means of the calibration curves. X. ' The present invention also relates to a package that carries out the diagnostic method of 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 equipment of the invention also preferably contains a) at least one reaction vessel b) an anticoagulant compound and / or a buffer for cell lysis; c) a buffer to repair the cells; d) substances required for DNA quantification and / or protein concentration and solutions ready for the calibration curve; e) a magnet for separating the cells attached to the magnetic particles (contained if an antibody attached to a magnetic particle is used); and f) a reagent for removing bound magnetic particles (content if an antibody attached to a magnetic particle is used). • In a preferred embodiment of the package corresponding to the invention, the antibody is an anti-CD69 antibody. In addition, the package may contain, in place of the anti-CD69 antibody, an anti-CD4 and / or an anti-CD8 antibody.

Finally, the equipment corresponding to the invention can present, in appropriate cases, in combination with one or more suitable detection agents, such as primary double fluorescence antibodies, secondary antibodies, detection agents for proteins and / or nucleic acids, such as a dye. interleaver, etc.

Example Determination of the mitogenic stimulation index by CD69 in patients with Alzheimer's disease.

The determination of the features known to date of Alzheimer's disease, which can be carried out in living patients (biomarkers), shows only sensitivity and insufficient specificity or is not appropriate for examinations with a large number of cases due to costs or the compeljidad of the arrangements for the tests. By clinical means, the certainty of the diagnosis is only 80% to 90% and is particularly complicated in the early stages of the disease as indicated by the diagnostic differentiation. The detection of preclinical stages of the disease is currently not possible due to the lack of an adequate biomarker.

The neurodegenerative changes are based on altered processes of intracellular mediation of tropic and mitogenic signals in the case of Alzheimer's disease. These dysfunctions of the intracellular translocation signal are not limited to the nervous system.

They can also be found in skin cells and lymphocytes in the bloodstream of these patients. Considering the specificity of the disease, this alternation has diagnostic value and is appropriate as a biomarker.

In the example below, the question of whether there is a typical dysfunction of Alzheimer's disease of the intracellular mediation of tropic and mitogenic signals was determined by the immunomagnetic cell separation of CD69 presenting lymphocytes before and after mitogenic stimulation.

Blood is collected intravenously using your blood collection system from SARSTEDT. There the blood is stabilized during the withdrawal by means of anicoagulants integrated into 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 reaction vessels that can be aerated, as in the case of a suspension plate 24 of the company Greiner bio-one. For this, the mitogens phytohemagglutinin (PHA), protein A and pokeweed mitogen (PWM) are used separately or in different combinations for 400 ul of blood each. The final concentrations of the respective mitogens were found within the physiological range and were 12 ug / ml for PHA, 50 ug / ml for protein A and 4 ug / ml for PWM in this example. The stimulation was carried out under physiological conditions at 37 degrees centigrade and with a CO2 concentration of 5% in a gas incubator for 4 hours. 100 ul of the stimulated blood was incubated with magnetic particles of different antibodies. In this example, anti-CD4 and anti-CD8 varnished magnetic particles from the company DYNAL were used. The corresponding magnetic particles were added to the particular sample in excess (magnetic particle in suspension) to ensure complete isolation of the corresponding subpopulation of lymphocytes. After an incubation period of 30 minutes at 4 degrees Celsius, the corresponding subpopulation of lymphocytes was separated magnetically and after subsequent steps of washing converted into 100 ul defined medium, in this example RPM1640, mixed with 1% fetal calf serum ( SBF).

The magnetic particles attached were removed in this example using ul DETACHaBEAD of the company DYNAL. After an incubation period of 45 minutes at room temperature, the magnetic particles removed were separated and the cell suspension was taken in a defined medium, in the example RPMl 1640, after several washing steps. By adding a specific lyisis buffer the cells were broken up, the DNA was labeled with specific dyes such as ethidium bromide, iodine propidium, orange acridine or DAPI and subsequently photometrically quantified. The content Protein samples were compared by the Bradford protein determination method. The number of cells was calculated from the DNA and / or protein content of the sample through the calibration curves. This procedure allowed a direct conlusion on the number of cells. The calculation of the quotient derived from the number of CD69 cells presented before and after the mitogenic stimulation (stimulation index) enriched the information on alterations of the mitogenic stimulation of these cells.

A stimulation index that reaches at least 10 times and a maximum of 100 times, the control sample without being stimulated is a sign of Alzheimer's disease, early stage or predisposition. A stimulation index less than 10 times of the unstimulated control sample is not a sign of predisposition, early stage or Alzheimer's disease.

In another experiment, the protein content of the sample was determined and the DNA content was determined without the addition of substances that stain the DNA for the quantification of CD69 cells. In this case, the absorption of light with a certain wavelength (260 nm or 280 nm) by DNA or protein was measured.

Claims (13)

1. A method to diagnose Alzheimer's disease, predisposition or early stages of this through a sample of the patient, characterized because it consists of the following steps: a) mitogenic stimulation of peripherally accessible cells in the sample; b) quantification of mitogenically stimulated cells within the cell population before and after passage (á) through one or more of the expressed surface markers after mitogenic stimulation, the cells carrying the surface markers are separated from cells that do not carry them through antibodies directed against surface markers; c) determining the stimulation index as a ratio of the number of cells bearing the marker or surface markers before and after step (a), a stimulation index that reaches at least 10 times, maximum 100 times, the sample of unstimulated control as a sign of Alzheimer's disease, early stages or predisposition to it.

2. The method according to claim 1, further characterized in that the sample is a blood sample and the cells are lymphocytes.

3. The method according to any of claims 1 or 2 further characterized in that the surface marker is CD69.

4. The method according to claim 3, further characterized in that the CD69 cells are specified below with respect to the CD4 + and / or CD8 + subpopulations.

5. The method corresponding to any of claims 1 to 4, further characterized in that the blood is stabilized by one or more anticoagulant compounds before step (a).

6. The method corresponding to any of claims 1 to 5, further characterized in that the cells are stimulated by PHA, protein A or PWM.

7. The method according to claim 1, further characterized in that the antibodies in step (b) are tied to magnetic particles and the separation is carried out by immunomagnetic separation.

8. The method corresponding to any of claims 1 to 7, further characterized in that the stimulation index is determined by specifying the protein content and / or nucleic acid content of the cells bearing surface markers before and after step (a) .

9. A team for the diagnosis of Alzheimer's disease, early stage or predisposition to it, characterized because it contains the following elements: a) a compound for mitogenic stimulation; and b) at least one antibody directed against a surface marker expressed after mitogenic stimulation.

10. The equipment according to claim 9, characterized in that it contains: c) an anticoagulant compound; and / or d) a buffer for cell lysis

11. The equipment corresponding to any of claims 9 or 10, further characterized in that e! The antibody is attached to a magnetic particle.

12. The equipment corresponding to any of claims 9 to 11, further characterized in that the antibody is a CD69 antibody.

13. The kit according to any of claims 9 to 12, further characterized in that it also contains an anti-CD4 and / or an anti-CD8 antibody.