PATHOLOGY

FIELD OF THE INVENTION

The present invention is in the field of the rapid diagnosis of a pathology, e.g. of a cancer pathology such as melanoma using a kit, stable at room temperature. In particular, the invention refers to a method that can also be performed in environments outside the laboratory, "patient side" with immediate response of a chemocolorimetric, bioluminescent or digital reader for the diagnosis and/or prediction of the risk of developing melanoma comprising the detection in cell extracts of certain cancer markers using immunoassays, such as ELISA (enzyme linked immunosorbent assay).

PRIOR ART

Melanoma is a cancer that affects the skin, among all types of skin cancers, it is the least common but also the most dangerous because it can grow quickly and also invade the surrounding tissues. It is a naked eye visible cancer and it originates from a pre-existing naevus that changes shape or colour or from the appearance of a new naevus on intact skin. It is always a malignant neoplasm, in fact it is never possible to define a benign melanoma, at most we can speak of a benign naevus that does not have the characteristics of a melanoma. Cutaneous melanomas originate both on intact skin and from pre-existing naevi, which are present from birth or early childhood (congenital) or appear during the course of life (acquired). Cutaneous melanoma accounts for 9% of juvenile cancers in men and 7% in women; it is quite rare in children and mainly affects around 45-50 years of age, although the average age at diagnosis has dropped in recent decades. In Italy there are an estimated 7,300 new cases each year among men and 6,700 among women, the incidence is constantly growing and has even doubled in the last 10 years.

The precancerous formations of melanoma do not cause symptoms but can be identified with careful monitoring of skin naevi. A useful and easy to remember method for recognizing a suspicious naevus is the abbreviation ABCD which lists its characteristics: A as an asymmetry of the shape, B as irregular edges, C as a variable colour, D as an increasing size both in width and in thickness. In general, congenital naevi are rounded, have a uniform colour and do not undergo changes over time.

Primary melanoma refers to the first appearance of melanoma and can usually be seen on the skin surface. Melanoma can quickly spread to any part of the body, so it's important to spot and treat it in its early stage. When melanoma is identified in its early stages, there is a very good chance that all of the cancer can be surgically removed before it spreads to other parts of the body and in that case the chances of long-term survival are excellent. Rapid screening therefore becomes of primary importance to direct the doctor towards the treatment plan. There are currently no scientifically reliable, non-invasive screening methods for the prevention of melanoma.

Currently, the clear diagnosis of cutaneous melanoma requires a biopsy, wherein a tissue sample is taken and then analysed under a microscope. Therefore, there is a need to provide a method for an early diagnosis of melanoma that allows to identify subjects at risk of developing the pathology and to subject them to a diagnosis of certainty, based on histological sampling and anatomopathological confirmation.

In general, the preceding paragraph relating to diagnosis applies to numerous other cancer and non-cancer pathologies.

DESCRIPTION OF THE INVENTION

The present invention refers to an innovative kit with ELISA technique and chemocolorimetric, bioluminescent or digital result for the early diagnosis of a pathology, preferably of a cancer pathology such as a melanoma. The kit uses reagents that are stable at room temperature, is rapid, sensitive, specific, transportable, cheap and non-invasive. In particular, the kit of the invention allows the determination of a specific antigen associated with a pathology of interest, in the case of cancer pathologies such as melanoma, PRAME (PReferentially expressed Antigen Melanoma) by means of a double use of primary antibodies to ensure unequivocal the specificity and sensitivity of the signal. In particular, a first polyclonal primary antibody e.g. rabbit anti-PRAME will be adhered to a PVDF membrane, a second primary monoclonal antibody e.g. of mouse anti- PRAME will be used in test tube. Subsequently, secondary anti-mouse antibodies conjugated with an enzymatic signal amplification system, in particular alkaline phosphatase or peroxidase, will be used. In order to react with such enzyme systems, the kit comprises at least one substrate such as ABTS (2,2'-Azinobis [3-ethylbenzothiazoline- 6-sulfonic acid] -diammonium salt), OPD (o-phenylenediamine dihydrochloride), TMB (3 , 3 ', 5,5'-tetramethylbenzidine), p-Nitrophenyl Phosphate (PNPP).

The kit uses dried or lyophilized antibodies, stable at room temperature, which at the time of use are solubilized in a buffer solution present in the kit.

The present invention is configured as an effective diagnostic aid for a pathology e.g. a cancer pathology such as melanoma, which does not require laboratory aids and provides immediate answers.

The invention also refers to the creation of an innovative kit built by assembling commercially available and custom made semi-finished products. The kit comprises a hard bristle cytobrush useful for taking the biological sample e.g. at the level of the epidermis and the necessary to allow the professional (Doctor or person entitled to use by the regulations in force in the area of use) to analyse it and verify the presence of the biomarker e.g. of the cancer biomarker or to process it through a specific automatic development device. The product has an extremely simple method of use.

The present invention is described below in non-limiting examples:

Components: kit equipped with a cytobrush or other known device eg. stick intended for the collection of biological material and, strip or strips of PVDF (pol vinylidene fluoride) suitably mounted on a rigid support and preloaded test tubes of powders of protease inhibitors in the dried state, lyophilized and/or dried antibodies, tubes preloaded with liquids, liquids for washing, developing and detecting the enzymatic signal and devices for the solubilization of preloaded reagents, in particular two-phase break-out capsules. As an alternative to the cytobrush, a sterile medical patch can be used, e.g. comprising an adhesive support in non-woven fabric. Optionally there is also a gauze, preferably also of non- woven fabric, combined with a substance with antibacterial activity, e.g. 0.5% chlorhexidine digluconate.

Figure 1. Kit operation diagram: dissolve the reagents present in the wells/station P in the dried and or lyophilized state upon opening the kit by perforating the overlying re ervoirs S having function as break-out cap (figure above). Collection of potentially malignant cells from the dermis with the specific tools to carry out the collection; immersion of the same in the well/station zero, containing a protease inhibitor and a buffer lysing the cells collected by means of the collection device.

Transfer of the lysate from the well/station zero to the well/station one (generic embodiment example in the figure below) preferably empty when the user opens the kit; immersion of the PVDF strip in the well/station one for the recognition of the cancer antigen by the specific primary antibodies immobilized on the PVDF strip. In well/station one the formation of immunecomplexes takes place on the strip if there is the presence of the cancer protein in the sample collected.

Immersion of the PVDF strip in the well/station number two. The PVDF strip with the immobilized immunecomplexes is immersed into well/station number two containing a primary target solution equally specific for the antigen e.g. cancer, but of a different species from those adsorbed on the membrane. The use of the second on the series of primary antibodies has the purpose of allowing a specific recognition by the secondary antibody used in the next phase, preventing the latter from binding to all the useful primary adsorbed on the PVDF strip. Washings with T-PBS will take place in wells/stations number three and four.

Immersion in the well/station number five of the PVDF strip on which the immunecomplexes antigen/primary antibody are present to allow the interaction with the secondary antibodies conjugated to an enzymatic detection system; washings with T-PBS will take place in wells/stations number six and seven;

Bioluminescent colorimetric expression using the substrate stable at room temperature in well/station number eight.

Reading and interpretation of the result.

According to an embodiment (Figures 2 and 3), the wells and/or reservoirs are preferably produced based on aromatic hydrocarbons so as to be hydrophobic and allow a total convergence of the quantities of fluid at the bottom of the recesses and, even more preferably, produced as a monolayer for example of polystyrene. Furthermore, each well or recess, again in order to favour the convergence towards the bottom of the liquid substances in use, has convex walls observing the inside of the recesses. For example, the support 10 of figure 2 defines recesses 11 for liquid substances and recesses 12 for powdered or dried substances e.g. protease inhibitor/dried antibodies. Furthermore, the kit comprises a support 20 superimposed on the support 10 and having corresponding recesses 21 containing pre-defined quantities i.e. dosed liquids e.g. a lysis buffer and a buffer liquid, to define a two-phase container in use. In this regard, recesses 12 house recesses 21 when support 20 rests on support 10. Support 20 is preferably produced by thermoforming like support 10 and, even more preferably, support 20 is of the same polymeric material as support 10.

Furthermore, support 10 is protected by a peelable covering layer 13 to hermetically close recesses 11. Preferably, peelable layer 13 comprises a gripping appendage 14 e.g. having a smaller transverse dimension but, in any case, such as to close a recess 11.

Peelable layer 13 has one or more openings A to surround, when it adheres to support 10, recesses 12 and thus allow recesses 21 to be housed in the latter when support 20 rests on support 10.

Support 20 is also closed by means of a corresponding peelable closing layer 22 bearing symbols SI, S2, S3 to identify, each one, a preferred point for breaking layer 22 and underlying recess 21 in order to perform the mixing of the substances. In particular, recesses 21 contain a pre-defined quantity of liquid and this helps the complete gravity fall into recess 12 of the liquid: this is particularly important when, as in this case, the metered quantities are of a few micrograms and/or as in this case of 280 picograms. Furthermore, as a whole, symbols SI, S2, S3 provide a perforation sequence, which is preferably performed by means of a suitable tool 30 (Figure 3). Preferably, the tool has a shaped grip, for example triangular, having a dimension such as to contact during the perforation an edge of recesses 21, also having a calibrated geometry and dimension. In such position, the tool is in the position of maximum advancement towards the bottom of corresponding recess 12 and also a height between a perforation head of the tool and the abutment position between the shaped grip and the edge of recess 21 is pre-defined in so as to perforate recess 21 in a pre-defined manner and depth independent of the user that performs the operation. Figure 3 further illustrates a stick 40 on which the primary antibodies, e.g. through a membrane facing a window 41, are applied. Through the window 41, the antibodies are exposed to the substances contained in recesses 11, 12 to define any antigens, e.g. cancer antigens.

Figure 3 further illustrates an exploded view of the main components of the kit, i.e. supports 10 and 20 and a base 50 preferably with a double basin. Supports 10 and 20 are preferably arranged in pocket 51 so that the recesses 11, 12 are suspended when support 10 abuts against an edge of the pocket. Stick 40, perforation tool 30, the collection tool and a Pasteur pipette are preferably arranged in pocket 52.

Base 50 is preferably closed by means of a preferably square-shaped band casing which slides laterally with reference to the point of view of Figure 3.

With reference to what is illustrated in Figures 2 and 3, the kit can be adapted e.g. by number of biphasic containers and/or recesses, dehydrated or dried substances and liquids, to test methods made for other diseases.

Again with reference to Figure 3, preferably, support 20 or in an embodiment not shown support 10, comprises one or more spacer projections D having a calibrated vertical dimension and such as to define the adapted position wherein recesses 21 are housed in the corresponding recesses 12. In this way, it is easily identified e.g. by abutting the relative vertical position to perform the perforation by means of tool 30. Furthermore, as again visible in Figure 3, recesses 21 have a vertical dimension greater than that of the spacer projections D so that an end portion T of recesses 21 is within recesses 12. According to a preferred embodiment, pocket 52 houses at least one pipette, preferably a Pasteur pipette, in order to transfer the liquid from one recess 11, 12 to the other. Preferably, pocket 51 has a stepped profile and, correspondingly, support 10 has a lower width LI so as to rest a first step G1 of a width L2 of support 20. The latter abuts on a step G2 arranged above the step G1 when the kit is placed on a table.

EXAMPLES

EXAMPLE 1 - The cancer marker PRAME

The method for the early diagnosis and monitoring of melanoma of the present patent provides for the detection of the antigen expressed mainly in human melanomas (PRAME) in samples collected from suspected naevi of the subject under examination. The PRAME marker in the context of the present invention also includes variants, isoforms etc. Said marker is characterized by an NCBI Access number and the antibodies used for the preparation of the device preferably refer to the following collected purely by way of example: PRAME Monoclonal Antibody (CL5148) (Product # MA5-31409); PRAME Polyclonal antibody, N-term Cat No. GTX45142; Mouse IgG (Fc fragment) antibody, F (ab') 2 fragment, pre-adsorbed (AP) Cat No. GTX25880 (Goat, Polyclonal) or Goat Anti-Mouse IgG antibody (HRP) Cat No. GTX 213211-01.

EXAMPLE 2 - PVDF strip preparation protocol

The steps for preparing the PVDF strip armed with the primary antibodies of interest in sequence are as follows: hydration of the PVDF strip (ThermoFisher Scientific Catalog Number LC2002) with methanol for 5 min; washing with water for 5 min; two washes with PBS for 5 min; incubation with protein A from Staphylococcus aureus (Sigma- Aldrich, 10 pg / mL) for 1 h in PBS; two washes with PBS; blocking with 3% BSA solution in PBS for 1 h; three washes with PBS for 5 min; assembly of the PVDF; incubation with rabbit antibody solution (PRAME Rabbit Polyclonal antibody, N-term Cat No. GTX45142), stirred O.N .; three washes with PBS for 5 min; two washes of the filter with PBS TEA 0.2M; incubation with 25mM DMP in 0.2M TEA HC1 pH 8.2; incubation with 0.2 M TEA + 20 mM ethanolamine; two washes with PBS for 5 min; storage in 0.02 NaN3 in PBS; after appropriate cutting of the filter, the individual strips, with dimensions of approximately 0.4 x 0.3 cm, will be mounted on the device support. Then it will proceed with the ELISA method in order to highlight the specific antigen present in the cytological sample tested. The Positive Control (CTR +) is an anti-actin antibody (Sigma - Aldrich, A2066).

EXAMPLE 3 - Device description

The various components of the kit equipped with cytobrush for the collection of biological material and support for the PVDF (Immobilon) strip armed with the primary antibodies of interest are described below.

The kit box is organized in rows of nine wells/stations containing buffers, lysing systems, detection systems stable at room temperature and dried/lyophilized antibodies stable at room temperature (Fig. 1).

EXAMPLE 4 - How to use the kit The following are the operational steps which in sequence allow the diagnosis of melanoma or another pathology: collecting the sample from the suspect naevus, using the collection tools within the kit; immersing the biological sample for 15 min in the well/station number zero containing the lysis buffer; extracting the tool and transfering the lysate to the well/station number one; opening the package of the support to which the PVDF membrane is adhered; immersing the support with the PVDF strip for 5 min in the first well/station number one to allow the interaction of the proteins (antigens) with the primary antibodies adhered to the PVDF strip, in order to form the immunocomplex (purely by way of example: PRAME Polyclonal antibody, N-term Cat No. GTX45142; subsequently, the PVDF membrane is i mersed in well/station number two containing in solution the second primary mouse antibody (purely by way of example: PRAME Monoclonal Antibody (CL5148) (Product # MA5-31409) for the reaction with the immobilized immunocomplex; proceed with 2 washes of the support with the PVDF strip in wells containing T-PBS-buffer to eliminate the non- specifically attached proteins to the immunocomplex; immersion of the support with the PVDF strip for 10 min in well/station number five containing the mouse secondary antibody (purely by way of example: Mouse IgG (Fc fragment) antibody, F (ab') 2 fragment, pre-adsorb and (AP) Cat No. GTX25880 (Goat, Polyclonal) equipped with a detector enzymatic system (alkaline phosphatase or peroxidase); 2 washes of the support with the PVDF strip in wells/stations number six and seven containing T-PBS-buffer to eliminate excess secondary antibody; immersion of the support with the PVDF strip in the well/station number eight containing the substrate (BCIP / NBT, stable at room temperature or Luminol ECL substrate) necessary for the colorimetric reaction; interpretation of the result.

EXAMPLE 5 - Signal amplification as a function of temperature.

The colorimetric test will be carried out at room temperature, corresponding to a temperature range between 20° and 30° C. The use, in the kit, of a secondary antibody having a detector enzymatic system (alkaline phosphatase or peroxidase) has in this temperature range the maximum enzymatic activity, thus allowing an optimal signal amplification. EXAMPLE 6 - Use of the kit of the invention for the identification of an individual with melanoma.

In the I.L.S. mode the Operator, after having highlighted the suspected lesion by means of an epilluminescence examination, places the cytobrush on it and performs the sampling maneuver. The maneuver consists in rotating the cytobrush on itself 4-5 times, exerting constant pressure. This method of cell collection is totally safe and free from any iatrogenic type problem. This technique can also be used to perform mass screening on populations, given that such neoplasm ranks fourth by incidence in the world. Thanks to this protocol it is possible to implement the correct principles for primary prevention.

The exam can be performed by qualified personnel after having performed a learning trial on the collection method.