WO2018047069A1

WO2018047069A1 - Peptide for the differential diagnosis of crohn's disease

Info

Publication number: WO2018047069A1
Application number: PCT/IB2017/055357
Authority: WO
Inventors: Margherita Morpurgo; Giacomo Carlo STURNIOLO; Sonia FACCHIN; Liboria DIGIGLIO; Renata D'INCA'; Andrea Buda
Original assignee: Universita degli Studi di Padova
Current assignee: Universita degli Studi di Padova
Priority date: 2016-09-06
Filing date: 2017-09-06
Publication date: 2018-03-15
Anticipated expiration: 2019-03-06
Also published as: IT201600090052A1

Abstract

The present invention relates to a peptide that is able to selectively bind to biopsies of inflamed intestinal tissue taken from patients affected by Crohn's disease, for the differential diagnosis of this pathology, thus allowing to distinguish it from ulcerative colitis. A method for diagnosing Crohn's disease, and a diagnostic kit are also described.

Description

Peptide for the differential diagnosis of Crohn's disease

DESCRIPTION

FIELD OF THE INVENTION

STATE OF THE ART

Inflammatory bowel disease ("IBD") comprises Crohn's disease and ulcerative colitis (UC). These are pathologies of particular clinical relevance, both for the severity of the symptoms, and the degree of diffusion in the population. It is estimated that about 200,000 people are today affected by these pathologies in Italy. Over the past 10 years, the diagnosis of new cases and the number of patients have increased by about 20 times. IBD affects both sexes with equal frequency, with a clinical outbreak typically ranging from 15 to 45 years.

Crohn's disease (CD), or Crohn disease, can occur in any part of the alimentary canal, from the mouth to the anus, although in most cases it affects the final part of the small intestine or variable tracts of the colon, or both zones (ileo-colitis). The affected intestinal segments are thickened, inflamed, edema-like, and ulcerated throughout the wall thickness. The establishment of these phenomena is slow, but steady and progressive.

Ulcerative colitis (UC) essentially affects the rectum, sometimes it can extend, partially or totally, to the colon, while it never affects other parts of the intestine. The mucosa of the area(s) affected by UC is inflamed and ulcerated, and this results in the onset of all the characteristic symptoms.

IBD is an idiopathic disease and, as such, its origin is not uniquely determined. Causes are sought in the living environment, genetic make-up, intestinal microbiota composition, and organism immune response.

The differential diagnosis of UC and CD is based on clinical laboratory, radiological, endoscopic, and histological evidence. However, due to histologic overlapping features in 10-15% of patients (especially pediatric patients (4)) and a similar localization of the inflammation, the differentiation between these two pathologies is not always certain. In addition, currently available genetic or serological markers do not provide conclusive information: at a genetic level, 163 genetic loci associated with IBD have been identified, of which only 23 are specific for UC, and 30 for CD (1 ), and it was observed that different genetic factors predispose to IBD through different inflammatory mechanisms (2, 3).

Serological testing to search for ASCA (5) and pANCA (6) antibodies, although considered useful for the differential diagnosis, are also characterized by low specificity and sensitivity (7). Similarly, other recently identified serologic markers (e.g. anti-pancreatic antibodies (8, 9) anti-erythrocyte antibodies (10), anti- endothelial cell antibodies (1 1 )) are characterized by insufficient specificity.

The low specificity of the currently available serological markers means that, to date, there is no universally recognized diagnostic test for the differential diagnosis of either CD or UC.

Therefore, the aim of the present invention is to provide a method suitable for rapid and early differential diagnosis of either CD or UC, useful to avoid the risk of prescribing inappropriate treatments, thus causing health issues to the patient, and increasing the costs for the treatment of the disease.

SUMMARY OF THE INVENTION

The invention therefore concerns the use of a peptide having the sequence of SEQ ID NO.1 as a biological marker. The inventors have surprisingly found that such a protein enables the diagnosis of patients affected by Crohn's disease distinguishing these patients from those affected by ulcerative colitis or various types of intestinal inflammation.

The peptide according to the present invention is able to selectively bind to biopsies of inflamed intestinal tissue taken from patients affected by CD, but not from UC, or non-IBD inflammation, supplementing the currently available diagnostic tools for IBD pathologies, especially when the current diagnostic approaches are not able to distinguish between the two pathologies.

The present invention also relates to a method for the diagnosis of Crohn's disease, comprising the steps of:

a. providing a biological sample; b. providing the peptide having the sequence of SEQ ID NO.1 immobilized on a solid support to obtain the substrate;

c. contacting the sample of step a. with the substrate of step b.; and d. detecting the reaction signal, wherein when a signal is detected, Crohn's disease is identified.

Under another aspect, the invention relates to a diagnostic kit comprising the peptide having the sequence of SEQ ID NO.1 , acting as an antigen, and a reagent for separate, simultaneous, and subsequent use for the diagnosis of Crohn's disease, and instructions for use by the method comprising the steps of:

a. providing a biological sample;

b. providing the peptide having the sequence of SEQ ID NO.1 immobilized on a solid support to obtain the substrate;

In the present invention, when using the definition:

- "ulcerative colitis" (UC), or colitis ulcerosa, or ulcerative rectocolitis are meant to include the chronic inflammatory disease that primarily affects the rectum, and may involve part or all of the colon. The causes of this inflammation are still unknown. The main clinical symptoms are diarrhea, often with blood and mucus, and abdominal pain;

- "Crohn's disease" (CD), or Crohn disease, means a chronic inflammatory disease that can affect the entire gastrointestinal tract. Causes are still unknown. It is characterized by intestinal ulcers, often alternating with healthy intestinal tracts, and, if not properly treated, can lead to complications, such as stenoses or fistulas, that may require surgery. Symptoms may vary from abdominal pain, to chronic diarrhea, weight loss, or low-grade fever. It may also affect the anal region with fistulas or abscesses. In most cases, immunosuppressive therapy and regular monitoring enable to control the disease and its progression.

Further possible embodiments of the present invention are specified in the dependent claims.

DESCRIPTION OF THE FIGURES The invention will now be described in detail, and with reference to the attached Figures in which:

Figure 1 shows the graph of the differential affinity results of the phage carrying the peptide of SEQ ID NO: 1 (TVRTSAD peptide): CD-ph, in mucosa samples of patients affected by various inflammatory pathologies of the colon.

The number of colony forming units (CFUs) in CD-ph (TVRTSAD peptide) from different types of mucosa biopsies is reported. The experiments were performed on patients biopsies: Crohn's disease in remission Sigma (CDSR), Crohn's disease Inflammed Sigma (CDSI), Crohn's disease in remission Ileum (CDIr), Crohn's disease inflamed Ileum (CDII), ulcerative colitis in remission (UCR), inflamed ulcerative colitis (UCI); lleo and Sigma in patients without IBD. The statistical analysis was performed using the t-test (P-value <0.05).

Figure 2A: Scrambled and Synthetic CD Peptide (SEQ ID NO 1 : TVRTSAD), Figure 2B: Multimerization process using colloidal poly-avidin nanoparticles (Ananas nanotech),

Figure 2C: Diagram of the procedure followed for the bioaffinity experiment on a biopsy from a patient affected by CD, followed by ELISA quantification.

Figure 3 shows the graph of the effective S/P values obtained by performing the ELISA test with a derivative of the CD-pep peptide (SEQ ID NO: 1 : TVRTSAD) multimerized on ANANAS nanoparticles, and performing the test on mucosa of patients (total 67), affected by UC in an inflamed state (21 ) or in remission (15), CD in an inflamed state (12) or in remission (15), and non-IBD intestinal inflammatory disease (4).

Figure 4: Confocal fluorescence microscopy (40X) of biopsies of CD inflamed mucosa (4A, 4B, and 4C) treated with a derivative of CD-pep peptide (SEQ ID NO

1 : TVRTSAD), multimerized on ANANAS nanoparticles.

The box 4D represents a magnification (64X) of the area in the box of panel 4C.

Figure 5: Results showed that only patients affected by CD generated a positive response, according to the following formula:

Effective Abs = Abs on CD well - Abs on Scr well

DETAILED DESCRIPTION OF THE INVENTION

Given the current limits in the diagnostic tests for the differential diagnosis of CD and UC based on serological markers, we focused our attention on searching for potential mucosal markers.

For this purpose, we used a screening method based on the Peptide Phage Display technique. Starting from a random library of peptides expressed on bacteriophages surface (M13 phage library commercially available from New England BioLabs) (Morpurgo M. et al J. Biochem. Biophys. Methods 2002) and, performing subsequent serial biopanning experiments on biopsies taken from patients with confirmed ulcerative colitis diagnosis, we have identified a phage clone (CD-ph) having selective affinity/adhesiveness for the inflamed mucosa under investigation, and obtained the sequence of the peptide (CD-pep: SEQ ID NO: 1 ) expressed on its surface, and responsible for this affinity.

The peptide of SEQ ID NO:1 is present on the phage, and in the diagnostic test, in a cyclic form, given the presence of two cysteines at the head and tail of the sequence. The recognition specificity for the inflamed mucosa of patients affected by CD was then validated by using the isolated phage UC-ph, and counting the number of phages that bind to the mucosal biopsies of patients affected by different types of intestinal inflammation.

The invention therefore concerns a peptide having the sequence of SEQ ID NO.1 , or a fluorescent derivative thereof, for use as a biological marker. The inventors have surprisingly found that such a protein enables the diagnoses of patients affected by Crohn's disease, distinguishing such patients form those affected by ulcerative colitis, or different types of intestinal inflammation.

The use of a fluorescent derivative of the peptide of SEQ ID NO:1 or NP fluorescence for tissue analysis by fluorescence microscopy has surprisingly been found useful.

In the field of Inflammatory Bowel Diseases (IBD) there is currently no diagnostic marker of disease, that could be used in diagnostic, able to distinguish with reasonable confidence the presence of ulcerative colitis (UC), Crohn's disease (CD), or other types of inflammation. When the inflammation is localized at colic level, it is therefore difficult to diagnose the presence of one or the other pathology, thus resulting in the risk of prescribing inappropriate treatments.

This invention relates to the identification of a peptide sequence being for the first time able to selectively bind to biopsies of inflamed intestinal tissue taken from patients affected by CD (SEQ ID NO:1 : TVRTSAD), but not by UC or non-IBD inflammation, and the use of this peptide or derivatives thereof in diagnostic testing for the the rapid and selective diagnosis of this pathology.

Potentially, this method could supplement currently available diagnostic tools for IBD pathologies, especially when the current diagnostic approaches are not able to distinguish between the two pathologies.

The peptide according to the present invention is therefore a biological marker to distinguish between patients affected by Crohn's diseases and patients affected by ulcerative colitis.

a. providing a biological sample;

Advantageously, the method according to the invention enables to distinguish patients affected by Crohn's disease from those affected by ulcerative colitis, or different types of intestinal inflammation.

In one embodiment, in the method for the diagnosis of Crohn's disease according to the present invention, said peptide having the sequence of SEQ ID NO.1 is immobilized on the surface of the solid support of step b.

The peptide according to the present invention is able to selectively bind to biopsies of inflamed intestinal tissue taken from patients affected by CD, but not by UC or non-IBD inflammation, supplementing the currently available diagnostic tools for IBD pathologies, especially when the current diagnostic approaches are not able to distinguish between the two pathologies.

Such a solid support allows the multimerization of the peptide, and it is selected from the group consisting of nanoparticles, polyfunctionalizable polymers, or by adsorption on plastic surfaces (e.g. polystyrene). The nanoparticles described in WO2009003951 may advantageously be used. In a further embodiment, in the method according to the present invention, the detection step d. is carried out by a technique selected from the group consisting of ELISA assay, radioimmunoassays (RIA), immunoassays, preferably Western blot and LINE blot, assays with protein or DNA microarrays, and surface plasmon resonance detection (SPR).

Surprisingly, then, using a commonly used laboratory technique such as the, ELISA assay, it is possible to distinguish between the different pathologies, and proceed with correct and timely treatment of the patient.

To date, there are in fact no diagnostic tests for IBD applicable to human biopsies, other than histological research. The latter, in addition to require a long time, is able to highlight the specific cytological features of the mucosa examined, but not the presence of any specific markers. If the intestinal inflammation is restricted to the colic compartment, the histology is not always able to distinguish with sufficient certainty between these two pathologies.

In a further embodiment, in the method according to the present invention, when the reaction signal of said step d. is detected, Crohn's disease is diagnosed.

In a further embodiment, in the method according to the present invention, said biological sample of step a. is a biopsy, preferably an intestinal tissue biopsy, more preferably a biopsy of inflamed intestinal tissue, or patient's serum.

The biopsy may, for example, derive from the colon mucosa, or the pouch region of operated patients.

a. providing a biological sample;

b. providing the peptide having the sequence of SEQ ID NO.1 immobilized on a solid support, to obtain the substrate;

c. contacting the sample of step a. with the substrate of step b.; and d. detecting the reaction signal, wherein when a signal is detected, Crohn's disease is identified. Advantageously, in the kit according to the present invention, said peptide having the sequence of SEQ ID NO:1 is immobilized on a solid support, preferably on a nanoparticle, or a polyfuctionalized colloidal support.

The following are Examples of embodiments of the present invention provided for illustrative purposes.

EXAMPLES

Example 1

Validation of the sequences inserted on the surface of a M13 phage (fused to the N-terminus of the coat III protein) for the diagnosis of either UC or CD.

Mucosal biopsies were taken from the colon or ileum of patients affected by intestinal inflammatory pathologies of different types, after informed consent. The patients were subjected to colonoscopy evaluation or, when scheduled, they underwent surgery.

After collection, the biopsies were stored in DMEM at 4°C, and used within 2 hours.

For the evaluation, the biopsies were incubated with 4x10¹⁰ CFU of CD-ph suspended in 1 imL of PBS buffer, under constant mild stirring. After 30 minutes, the biopsies were subjected to subsequent washing cycles (1 x PBST + 4x PBS) and dried by aspiration. 100 μΙ_ of 0,2M glycine-HCI buffer, pH 2.2, were then added to the biopsies, and after 5 minutes the solution was aspirated and immediately neutralized with 15 μΙ_ of 1 M TRIS buffer, pH 9.1 .

The bacteriophage content in the neutralized solution was measured by inoculating different dilutions of E.co//^'-agar suspension in a Petri dish, and counting the number of lysis plaques formed after 16h of incubation at 37°C. Figure 1 shows the average of the values obtained carrying out the test on a cohort of different patients.

It is observed that the number of CD-ph CFU expressing on their surface the sequence TVRTSAD (SEQ ID NO:1 ) systematically obtained from biopsies of patients affected by CD is an order of magnitude higher compared to when the same phage is incubated with biopsies of non-CD patients, including UC patients and non-IBD inflamed patients.

Example 2 Use of sequences obtained synthetically for the differential diagnosis of either UC or CD through an ELISA procedure.

Mucosal biopsies taken from the colon or ileum of patients with confirmed diagnosis of intestinal inflammatory pathologies of different types, were incubated with a derivative of CD-pep, or a peptide having the same amino acid composition but amino acids arranged in a different order (scrambled peptide -SCR-pep) immobilized on the surface of colloidal poly-avidin nanoparticles (ANANAS nanotech, PD, Italy) through a biotin linker (30% of biotin binding sites saturated, 100 μg/mL in avidin) suspended in 1 imL of PBS buffer, under constant mild stirring. After 30', the biopsies were subjected to subsequent washing cycles (1 x PBST + 4x PBS), and then dried by aspiration. PBS percentage and ANANAS concentration may be varied, without compromising the assay functioning. The nanoparticles affinity-bound to the biopsies were removed by acidic treatment with 100 μΙ_ of 2M glycine buffer, pH 2.0, and after 5 minutes the acidic solution was transferred into a second test tube, and immediately neutralized with 15 μΙ_ of 1 M TRIS buffer, pH 9.1 . The nanoparticles content in the various washing solutions, and in the detachment solution, was obtained through an ELISA test. For the testing, the solutions were incubated in microwells of an ELISA plate (50 μίΛ/νβΙΙ), pre-conditioned so as to expose immobilized biotin on the surface. After 3 hours incubation at RT, the microwells were washed extensively, and added with 2 μg/mL (50 μΙ_/ννβΙΙ) of biotin-HRP (ANANAS nanotech, PD, Italy). After 30 minutes, the biotin-HRP solution was removed, and the plate developed with a solution of tetramethylbenzidine (TMB), blocking the enzymatic reaction after 20 minutes by addition of 0.5M H2SO4. The absorbance intensity generated in each well was measured spectrophotometrically at 450 nm, and compared to that generated in the well of the respective positive control, in which the corresponding nano- assembled ANANAS-peptide was incubated at a known concentration. For the analysis and the calculation of the test specificity and sensitivity, the absorbance data were processed according to the following formula:

Eff S/P =[S/P(CD-pep S/P(Scrambled)], wherein:

- S/P CD-pep indicates the ratio between the signal intensity generated by the neutralized elution solutions (S) and the corresponding positive control

(P);

- S/P Scrambled corresponds to the same value generated by performing the test on a second biopsy with a peptide having the same amino acid composition, but whose amino acids have been aligned in inverted order.

Figure 2 shows the sequences of the biotinylated peptides used in the test, the multimerization procedure using ANANAS particles, and the test operation scheme based on the use of multimerized synthetic peptides combined with the ELISA method.

Figure 3 summarizes the effective S/P values obtained by performing the test with CD-pep e UC-pep peptides on the mucosa of patients (67 in total) affected by UC in inflamed state (21 ) or in remission (15), CD in inflamed state ( 2) or in remission (15), and non-IBD inflammatory intestinal diseases(4).

The results of the statistical analysis of the same data by ROC (receiver operating characteristic) curves is summarized in the table below.

Table 1 :

Summary ROC UC and CD pep / ANANAS-ELISA

Peptide Associated Disease used as Sensitivity Specificity

AUC

Pathology Control (%) (%)

CD-pep CD inflamed All colon+ileum

100 66.70 0.89 inflammations except UC

Example 3

Use of sequences obtained synthetically for the differential diagnosis of UC through a fluorescence microscopy procedure.

Confocal fluorescence microscopy images were obtained from a series of UC samples, and from biopsies of normal mucosa, treated with the multimerized peptide in the same way as in the ELISA assays, and fixed before the acidic elution phase.

All synthetic peptides also had the fluorescein tag for fluorescence-based analysis.

The results shown in Figure 4 confirm the high specificity and selectivity of the general approach. While no fluorescence signal was observed in healthy samples, the specific fluorescence signal is evident in UC biopsies when the peptide of SEQ ID NO: 1 UC1 -PEP-ANANAS is used. More specifically, in the UC mucosa (panels A, B), there are areas that show inflammatory alterations (crypt, transmural inflammation with lymphoid follicles).

An important information obtained from fluorescence is the position of interactions at a tissue level. In particular, fluorescence is localized at the epithelial surface level, while no signal is observed at the lumen cells level, clearly indicating that targets are on the surface.

Example 4

Differential analysis of sera of CD vs UC patients using the multimerized CDpep peptide on a polystyrene surface

Microwells of a polystyrene ELISA plate (Nunc Maxissorp) were conditioned with streptavidin and, subsequently, after blocking with BSA, half of them with a biotinylate derivative of CD peptide, and the other half with scrambled peptide. The sera of 2 patients affected by ulcerative colitis (UC), 2 patients affected by Crohn's disease (CD), and one patient affected by non-IDB colic inflammation were diluted 1 to 500 with 0.1 % PBS+BSA, and incubated both in the microwells pre-conditioned with scrambled peptide and in those conditioned with CD peptide. After 1 h, the solutions were removed, the wells were washed with PBST, and added with a solution of anti-human IgG, HRP conjugate, antibody. After 1 h incubation, the wells washed with PBST were developed with a solution of tetramethylbenzidine/H2O2 (50 μΙ_Λ/νβΙΙ). After 20 minutes, the enzymatic reaction was blocked with 50 μΙ_ of 1 M H2SO4, and the absorbance at 450 nm was detected by a microplate reader.

From the detailed description and the Examples above, the advantages of the peptide of the present invention are apparent. In particular, this peptide has been shown, surprisingly and advantageously, to be suitable for the diagnosis of ulcerative colitis. At the same time, this method, being fast and extremely easy to perform, can be conveniently carried out in any type of laboratory. BIBLIOGRAPHIC REFERENCES

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3. Leong RW, Armuzzi A, Ahmad T, Wong ML, Tse P, Jewell DP, et al. NOD2/CARD15 gene polymorphisms and Crohn's disease in the Chinese population. Alimentary pharmacology & therapeutics. 2003;17(12):1465-70. Epub 2003/06/26.

4. Tremaine WJ. Is indeterminate colitis determinable? Current gastroenterology reports. 2012;14(2):162-5. Epub 2012/02/09.

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6. Eggena M, Targan SR, Iwanczyk L, Vidrich A, Gordon LK, Braun J. Phage display cloning and characterization of an immunogenetic marker (perinuclear anti-neutrophil cytoplasmic antibody) in ulcerative colitis. Journal of immunology. 1996;156(10):4005-1 1 . Epub 1996/05/15.

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9. Seibold F, Weber P, Jenss H, Wiedmann KH. Antibodies to a trypsin sensitive pancreatic antigen in chronic inflammatory bowel disease: specific markers for a subgroup of patients with Crohn's disease. Gut. 1991 ;32(10):1 192-7. Epub 1991 /10/01 . 10. Berberian LS, Valles-Ayoub Y, Gordon LK, Targan SR, Braun J. Expression of a novel autoantibody defined by the VH3-15 gene in inflammatory bowel disease and Campylobacter jejuni enterocolitis. Journal of immunology. 1994;153(8):3756- 63. Epub 1994/10/15.

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Claims

1 . Use of a peptide having the sequence of SEQ ID NO.1 or a fluorescent derivative thereof, as a biological marker for the diagnosis of Crohn's disease.

2. The use according to claim 1 , wherein said peptide is a biological marker for distinguishing patients having Crohn's disease from patients with ulcerative colitis.

3. Method for the diagnosis of Crohn's disease, comprising the steps of:

a. providing a biological sample;

4. The method according to claim 3, wherein said peptide having the sequence of SEQ ID NO.1 is immobilized on the surface of the solid support of step b., and is selected from the group consisting of nanoparticles, polyfunctionalizable polymers, or by adsorption on plastic surfaces (e.g. polystyrene).

5. The method according to claim 3, wherein the detection step d. is carried out by a method selected from the group consisting of ELISA assay, radioimmunoassays, immunoassays, preferably Western blot and LINE blot, assays with protein or DNA microarrays, and surface plasmon resonance detection.

6. The method according to claim 3, wherein when the reaction signal of said step d. is detected, Crohn's disease is diagnosed.

7. The method according to claim 3, wherein said biological sample of step a. is a biopsy, preferably an intestinal tissue biopsy, more preferably a biopsy of inflamed intestinal tissue, or patient's serum.

8. The method according to claim 3, wherein said biopsy is a biopsy of the colon mucosa, or of the pouch region of operated patients.

9. Diagnostic kit comprising the peptide having the sequence of SEQ ID NO.1 , acting as a marker, and a reagent for separate, simultaneous, and subsequent use, for the diagnosis of Crohn's disease, and instructions for use by the method comprising the steps of: a. providing a biological sample;

10. The kit according to claim 9, wherein said peptide having the sequence of SEQ ID NO.1 is immobilized on a solid support, preferably on a nanoparticle, or a polyfunctionalizable colloidal support.