An assembly for the diagnosis of alzheimer's disease from non-cellular organic material from the skin surface

Abstract

The object of the invention is an assembly for the diagnosis of Alzheimer's disease from non-cellular organic material from the skin surface comprising a means (1) for collection of the organic material with an adhesive surface (2) for contacting the skin, a kit for the immunoassay detection of at least one Alzheimer's disease biomarker from the collected organic material, and an optical apparatus for capturing the result of the immunoassay reaction.

Description

[0001] An assembly for the diagnosis of Alzheimer's disease from non -cellular organic material from the skin surface

[0002] Technical Field

[0003] The invention relates to the field of clinical biochemistry, specifically the collection of organic material from the skin surface for the detection of biomarkers signaling neurodegenerative disease.

[0004] Background of the Invention

[0005] The development and research in the field of medical sciences is constantly progressing, wherein one of the priorities is to facilitate, simplify, and reduce patient discomfort during examination as much as possible.

[0006] Many diseases, especially dermatological ones, can be diagnosed from skin biopsies. In the prior art, mainly biopsy methods were used, i.e. , the removal of a small piece of tissue, which is, however, always an invasive, painful procedure for the patient.

[0007] Assuming that the evidence for determining the diagnosis can be found in detached skin cells, this determination can be performed instead of biopsy, after overcoming the problem of how to remove enough cells from the patient for laboratory determination, without any disruption to the integrity of the patient's skin tissue.

[0008] Based on current research, it can be expected that during skin regeneration, not only skin cells are detached, but organic material from the intercellular space is also brought to the skin surface. Biomarkers of neurodegenerative diseases, which accumulate not only in areas of the central nervous system but also elsewhere in the body, can be found not only in detached skin cells but also in the intercellular space itself. For example, some biomarkers are known to circulate in blood plasma and can be found in skin biopsies. These are mainly proteins typically found in the blood plasma of Alzheimer's patients, such as tau protein or amyloid beta and variants thereof. Blood plasma is a material that is relatively quick to clean and the biomarker content can depend on a number of circumstances and can fluctuate throughout the day. Blood plasma collection is invasive, stressful, and requires trained personnel. In contrast, accumulated biomarkers (e.g. stable proteins) in the skin may be easily accessible and their concentrations stable. Collecting them directly from the skin surface can be completely non-invasive and very easy and also very inexpensive.

[0009] It would be useful to present a solution that can be used to detect the presence of biomarkers of Alzheimer's disease, which will meet the requirements suitable for the deployment of large-scale screening of this disease - therefore, it should be inexpensive, non-invasive, comfortable for both the patient and medical staff, and at the same time not demanding in terms of equipment and storage conditions.

[0010] Summary of the Invention

[0011] Said object is achieved by an assembly for diagnosis of Alzheimer's disease from non-cellular organic material from skin surface comprising a means for collection of organic material with an adhesive surface for contacting the skin, a kit for immunoassay detection of at least one Alzheimer's disease biomarker from the collected organic material, and an optical apparatus for capturing the result of the immunoassay reaction.

[0012] Currently, detection of Alzheimer's disease is performed by detecting biomarkers in cerebrospinal fluid, clinical symptoms of dementia, and by PET detection of presence of amyloid or tau deposits in the brain. The idea of detecting biomarkers of Alzheimer's disease from detached organic materials from the intercellular space is based on studies trying to prove that, for example, tau proteins and their variants are to a certain extent detectable in blood plasma and also in skin tissue. There are biomarkers, proteins, circulating in blood plasma, but they are eliminated relatively quickly and are also present in very low concentrations. However, some protein biomarkers can deposit and accumulate in the skin and can be detected there well before the first symptoms of the disease appear. They can be determined from blood or skin biopsy, however, these methods are not very suitable for mass screening, either in terms of handling or storage of the biological material.

[0013] However, during the skin renewal process, the biomarkers are released onto the skin surface and can be collected - this requires the use of the means for collection of organic material from the skin surface which is applied to the cleaned skin surface of the patient and left on the skin for several hours, for example overnight. During this time, the skin naturally regenerates, skin cells peel off and material is released from the intercellular space. After the means is removed from the patient's skin, the detached skin components along with proteins from the intercellular space remain trapped on the means.

[0014] The clinical material is trapped either by adherence to the adhesive surface of the patch or by moistening the skin in the presence of absorbent patch pad material into which the dissolved / dispersed solid parts of the clinical material are absorbed.

[0015] The method of determining the biomarker from the non-cellular organic material from the intercellular space from the skin surface comprises the steps of applying the means for collection of the organic material to the cleaned skin, leaving it on the skin for at least 1 hour, preferably at least 4 hours, even more preferably at least 8 hours, removing the means for collection of cells from the skin, washing with biomarker buffer, processing the solution using the kit for the immunoassay detection of the biomarker, and detection by means of the optical apparatus for capturing the result of the immunoassay reaction.

[0016] An advantage of such skin cell acquisition is the increase in the volume of the obtained clinical sample at the time of the patch application, thus the possibility to use detection methods that otherwise lack sufficient sensitivity. With a standard, very small volume of skin biopsy, sufficiently sensitive determination by commonly used methods could not be possible. In addition, biomarkers are thought to be more stable in the environment outside the body as they are not degraded by enzymes in the body.

[0017] The method of determining Alzheimer's disease from the non-cellular organic material from the skin surface comprises the steps of applying the means for collection of the organic material to the cleaned skin, leaving the means for collection of the organic material on the skin for at least 1 hour, preferably at least 4 hours, more preferably at least 8 hours, removing the means for collection of the organic material from the skin, processing the means for collection of the organic material using the kit for performing the immunoassay method, and detection using the optical apparatus for capturing the result of the immunoassay reaction.

[0018] Thus, by using the presented means for collection of the skin cells, the patient can be spared not only a biopsy in case of skin diseases but also a lumbar puncture or at least repeated collection of blood in case of the Alzheimer's disease diagnosis.

[0019] The means for collection of the skin cells works based on adhesion to the skin. It can either be adhesive over the entire surface thereof facing the patient's skin surface, or it can be adhesive only around the edges and the central surface will be non-adhesive, less adhesive, or as adhesive as the edges of the means. To increase the yield, it is possible to use a means with a larger surface area or a larger number of these means.

[0020] One variant of the means for collection of the skin cells is a means with an adhesive surface over its entire surface, which is applied to the patient’s skin.

[0021] Another variant is a means in which the adhesive surface thereof comprises a moist surface and an absorbent surface. The moist surface releases a fluid into the space between the means and the skin that helps to dissolve and disperse the skin cells, which are then absorbed by the absorbent surface material.

[0022] Another variant is a means in which the adhesive surface comprises a surface with a lower adhesion, which allows the skin cells and organic material from the intercellular space to stick but also facilitates the removal of the means from the skin after the collection of the material is completed.

[0023] Another variant is a means comprising a solid substrate with immobilized antibodies to which the antigens of interest are bound. The solid substrate may be a grid of fibers or nanofibers and may be part of a moistened pad.

[0024] The basic requirement for the material of the surface intended for the skin cells collection is compatibility with the detection method used. The material of the surface intended for the collection of cells must not comprise substances inhibiting the detection method. An advantage of this means is its potential for widespread use and subsequent inclusion in screening methods, given that the use of this means is inexpensive and uncomplicated for the patient, easily performed at home or in doctors' offices. This advantage is very valuable, especially given the importance of early diagnosis, as Alzheimer's disease is still incurable, but if detected early, the progress thereof can be significantly slowed or even stopped by medication. Even in the context of the prevalence of Alzheimer's disease in the aging population, it seems very important to introduce a new technique for early diagnosis.

[0025] Additional advantage of said product is the additional variants of use for various molecular analyses of the skin microbiome as well as keratinocytes, T-cells, dendritic cells, melanocytes, and other skin cells involved in the pathology of various skin conditions andconditions where the skin can serve as a surrogate target organ, i.e. , for example, the already mentioned Alzheimer's disease and other neurodegenerative disorders.

[0026] For biomarker detection, the organic material is then analyzed using standard ELISA-type tests. The kit for performing such immunoassay method then comprises a wash solution for the given biomarker for washing the organic material from the means for collection of the organic material, a microtiter vessel with immobilized biomarkerspecific antibodies, a buffer for reaction with the secondary labelled antibody, a reagent with the secondary labelled antibody for the given biomarker, and a buffer for the final detection of the secondary antibody binding.

[0027] This method results in a visual change in the sample, such as a change in color or a change in fluorescence. This change is captured by the optical apparatus, the type of which is chosen based on the expected change in the sample. It can be a spectrometer, a fluorimeter, or even the camera unit of a camera, or other mobile device, whose control unit processes the image with the appropriate algorithm. Description of Drawings

[0028] A summary of the invention is further clarified using exemplary embodiments thereof, which are described with reference to the accompanying drawings, in which: fig. 1 shows the means for collection of the skin cells according to this invention in an embodiment with an adhesive layer over the entire surface of the means, fig. 2 shows the means for collection of the skin cells in an embodiment comprising a moistened and an absorbent part, fig. 3 shows the means for collection of the skin cells with a solid substrate.

[0029] Exemplary Embodiments of the Invention

[0030] Example 1

[0031] The means 1. for collection of the clinical material is a patch of an elastic material with an adhesive surface 2. The means 1. is applied to the skin surface, which is cleaned, rinsed with ethanol solution, and dried before application. The means 1_ is applied in the usual manner, i.e. , by applying it to the skin and leaving it on the skin for at least 10 hours, ideally overnight. The means 1. is then carefully removed and washed with a buffer suitable for the biomarker of interest. The buffer is applied to the means 1_, and after the time required for the release of the given biomarkers, it is aspirated and transferred to a tube. In this way, a sample of the buffer solution with the organic material that potentially contains the biomarker of interest is obtained.

[0032] The sample is transferred to a microtube or a well in a microtiter plate where antibodies specific for the biomarker or variant of the biomarker of interest are immobilized and the biomarker of interest is bound to these immobilized antibodies. The microtube or well is washed with wash solutions to remove unbound organic material. Subsequently, a buffer and a second reagent containing a secondary antibody specific for the biomarker of interest and labelled with the bound ligand are added. The sample is incubated, wherein the secondary antibodies are bound to the biomarkers of interest. Further wash solutions remove the unbound secondary antibody. The buffer solution for the final detection of the secondary antibody binding is added. A solution activating the color or fluorescence reaction of the ligand is added to the sample. An optical apparatus, for example an ELISA reader or similar fluorescence-detecting apparatus, is used to record this reaction, for detection of this color change or fluorescence.

[0033] The assembly of reagents and the design of the method are variable and allow the detection of binding of the antibody to the biomarker of interest to be performed by semiautomatic methods (manual pipetting steps, incubation apparatus and reader) or by a fully automatic apparatus from various manufacturers.

[0034] Example 2

[0035] The means 1. for collection of the skin cells is a patch with an adhesive surface 2 over the entire surface (Fig. 1 ) with which the means 1_ is applied to the patient's skin. The patch is applied to the patient's skin and left in place for 12 hours. After the removal of the patch, the biological material with the skin cells is rinsed from the adhesive surface 2 of the patch with purified water into a beaker and the solution is analyzed using ELISA technique to detect tau proteins and amyloid beta, or variants thereof, as biomarkers of Alzheimer's disease.

[0036] In the event that detection of other markers, proteins, or cells is required, another suitable detection method known in the state of the art is selected.

[0037] Example 3

[0038] The means 1. for collection of the skin cells is a patch that comprises a moistened surface 3 and an absorbent surface 4 on the adhesive surface 2, as can be seen in Fig. 2. In this arrangement, the three absorbent surfaces 4 are surrounded by the moistened surface 3. The purpose of the presence of the moistened surface 3 is to dissolve, disperse the solid parts of the biological material of the detached skin, which is transferred into the fluid originating from the moistened part, wherein this fluid is, together with the dispersed biological material, absorbed into the material of the absorbent surface 4. The patch is applied to the patient's skin and left in place for 12 hours. After removing the patch, the sample is rinsed from the absorbent material of the absorbent surface 4 with purified water into a beaker and the solution is analyzed by ELISA technique or other suitable detection method.

[0039] Example 4

[0040] The means 1_ for collection of the skin cells comprises a pad with a solution suitable for the interaction of antibodies and antigens and further comprises a grid 5 of a nanofibrous or other fibrous structure as shown in Fig. 3 on which antibodies against the antigen of interest are immobilized. The grid 5 is part of, or adjacent to, the pad with the fluid facilitating access of the skin cells to the grid 5. After application of the means 1. and after gradual release from the skin surface, the antigens enter the moist environment between the means 1. and the skin surface. In this environment, the antigen binds to the antibody immobilized on the grid 5. The means 1. is then removed from the patient's skin and the entire means 1_ or only the grid 5 are further analyzed either in a laboratory after transporting the grid 5 as a clinical sample or directly at the site of collection by an immunological detection method. This detection can be performed directly on the means 1. by adding a detection reagent, as in conventional immunological methods, for example using a fluorescently labelled antibody that binds to the antigen trapped on the grid, whether by the immobilized antibody or otherwise. The signal is detected by a film or a camera, or a mobile phone camera with an appropriate application to evaluate the captured data.

[0041] Example 5

[0042] The means 1. comprises a solid substrate in the form of a grid 5 of nanofibrous structure as in Example 4, but to which all particles from the biological material of the detached skin are non-specifically bound, wherein only specific antigens are detected, as in Example 3.

[0043] Example 6

[0044] The adhesive surface 2 of the means 1. for collection of the cells comprises a pad with a lower adhesion than the rest of the adhesive surface. The pad is of gel-like nature and its adhesion is sufficient for the skin cells to stick, yet it facilitates removal of the means from the patient's skin. It also facilitates washing of the stuck cells for further processing using the selected detection method. The sample is further processed in the same manner as in the previous cases. List of Reference Signs

[0045] 1 - Means

[0046] 2 - Adhesive surface

[0047] 3 - Moistened surface 4 - Absorbent surface

[0048] 5 - Grid

Claims

CLAIMS1. An assembly for the diagnosis of Alzheimer's disease from non-cellular organic material from the skin surface, characterized in that it comprises a means (1 ) for collection of the organic material with an adhesive surface (2) for contacting the skin, a kit for the immunoassay detection of at least one Alzheimer's disease biomarker from the collected organic material, and an optical apparatus for capturing the result of the immunoassay reaction.

2. The assembly for the diagnosis of Alzheimer's disease from non-cellular organic material from the skin surface according to claim 1 , characterized in that the adhesive surface (2) of the means for collection of the organic material comprises a moistened surface (3) and an absorbent surface (4).

3. The assembly for the diagnosis of Alzheimer's disease from non-cellular organic material from the skin surface according to claims 1 to 2, characterized in that the adhesive surface (2) of the means for collection of the organic material comprises a solid substrate with immobilized antibodies for the detected antigens.

4. The assembly for the diagnosis of Alzheimer's disease from non-cellular organic material from the skin surface according to claim 3, characterized in that the solid substrate is a fibrous material.

5. The assembly for the diagnosis of Alzheimer's disease from non-cellular organic material from the skin surface according to claim 3, characterized in that the solid substrate is a nanofibrous material.

6. The assembly for the diagnosis of Alzheimer's disease from non-cellular organic material from the skin surface according to claims 1 to 5, characterized in that the kit for the immunoassay detection comprises a wash solution for the biomarker for washing the organic material from the means (1 ) for collection of the organic material, a microtiter vessel with immobilized biomarker-specific antibodies, a buffer for reaction with the secondary labelled antibody, a reagent with the secondary labelled antibody for the given biomarker, a buffer for the final detection of the secondary antibody binding.

7. The assembly for the diagnosis of Alzheimer's disease from non-cellular organic material from the skin surface of claims 1 to 6, characterized in that the optical apparatus is a spectrometer or a fluorimeter.

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