LT5843B - Monoclonal antibodies against major capsid protein vp1 of merkel cell polyomavirus - Google Patents

Abstract

The present invention describes new monoclonal antibodies against major capsid protein VP1 of Merkel cell polyomavirus. Produced monoclonal antibodies can be used for diagnosis of infection of Merkel cell polyomavirus or Merkel cell carcinoma caused by said virus. The present invention describes new hybridomas 11A2 and 9G6 producing monoclonal antibodies against major capsid protein VP1 of Merkel cell polyomavirus. Antibodies produced by hybridoma 11A2 recognize Merkel cell polyomavirus in the infected cells as determined by immunohistochemical method. Antibodies produced by hybridoma 9G6 does not react with the virus-infected cells.@@@

Description

Field of the Invention

The present invention relates to the field of protein biotechnology. Its essence is the development of new monoclonal antibodies against the primary capsid protein VP1 of the Merkel polyoma virus. Said monoclonal antibodies may find applications in medicine and other applications.

BACKGROUND OF THE INVENTION

The Merkel Polyoma Virus, which belongs to the Polyomaviridae virus family, was identified in 2008. Integrated into the cell genome, this virus is thought to cause a rare form of skin cancer, Merkel cell carcinoma (MCC), which occurs in older age or in immunosuppression [Feng H., Shuda M., Chang Y. et al., Science, 208 , 319, 1096-1100]. MCC is an aggressive, rapidly progressive disease for which there is no effective treatment other than surgery. Detection of Merkel's polio virus by molecular diagnostics would be important for confirmation of Merkel's polio virus infection and for the diagnosis of MCC and could be of great prognostic significance.

It is not easy to diagnose MCC, as the disease differs clinically from other skin malignancies and non-malignancies. A quick and accurate diagnosis would allow timely surgical intervention and prevent metastases and disease progression.

Currently, there are no clinical diagnostic methods available to make an accurate diagnosis of MCC. In scientific laboratories, Merkel's polyoma virus can be detected by the polymerase chain reaction (PCR) method or other methods based on viral DNA detection [Zampetti A., Feliciani C., Massi G. et al., J. Cutan. Med. Surg., 14, 2010, p. 51-61]. These techniques are quite complex, require special equipment and expertise in molecular biotechnology, and are difficult to implement in clinical settings.

As a result, alternative, simpler MCG diagnostic methods and tools that are easy to use in clinical diagnostic laboratories are under search. Detection of influenza virus, such as direct immunofluorescence, in cytological specimens of the nasopharynx, is considered the "gold standard" for the diagnosis of this infection [Gharabaghi F., Tellier R., Cheung R. et al., J.Clin Virol., 2008, 42,190-193].

Immunochemical methods for the detection of viruses require antibodies that can be raised against the native virus or against individual components of the virus. Antibodies against other polyoma viruses have been described. For example, antibodies specific for SV40 T antigen have been used to detect monkey SV40 in lymphoma tissue by immunohistochemistry [Toracchio S., Kozinetz C.A., Killen D.E. et al., J.Clin.

Virol., 46, 154-160]. Often, recombinant antigens synthesized in bacterial, yeast, or mammalian cells are used to generate antibodies. Recombinant Merkel polyoma virus capsid proteins (VP1, VP2, VP3) have been described, but their use in antibody development has not been described [WO 2009/079481]. Monoclonal antibodies have been developed against the recombinant hamster polyoma virus capsid protein

VP1, which can be used to detect this virus in infected cells [Žvirbliene A., Samonskyte L., Gedvilaite A. et al., J. Immunol. Meth., 2006; 311, 57-70]. Using recombinant antigens synthesized in baculoviruses, monoclonal antibodies have been generated against the BKV and JCV human polyoma virus SV40 capsid proteins of the monkey polioma virus. Some of these antibodies have been found to recognize conformational epitopes, have cross-specificity, and sometimes have virus-neutralizing activity [Randhavva P, Viscidi R, Carter JJ et al., J Gen Virol. 2009 90,634-639],

Monoclonal antibodies to the Merkel polyoma virus capsid protein VP1 are not described.

The object of the present invention was to create monoclonal antibodies against the Merkel polyoma virus core capsid protein VP1, which are suitable for the accurate and simple detection of this virus in a tissue sample.

The essence of the invention

The main objects of the invention are hybridoma 11A2 (MPV001 or DSM ACC3097) and hybridoma 9G6 (MPV002), which produce monoclonal antibodies against the Merkel polyoma virus capsid protein VPis, as well as monoclonal antibodies recognizing the amino acid sequence (SEQ NO. 1):

MAPKRKASSTCKTPKRQCIPKPGCCPNVASVPKLLVKGGVEVLSVVTGEDSITQ

IELYLNPRMGVNSPDLPTTSNWYTYTYDLQPKGSSPDQPIKENLPAYSVARVSL

PMLNEDITCDTLQMWEAISVKTEVVGISSLINVHYWDMKRVHDYGAGIPVSGV

NYHMFAIGGEPLDLQGLVLDYQTQYPKTTNGGPITIETVLGRKMTPKNQGLDP

QAKAKLDKDGNYPIEVWCPDPSKNENSRYYGSIQTGSQTPTVLQFSNTLTTVLL DENGVGPLCKGDGLFISCADIVGFLFKTSGKMALHGLPRYFNVTLRKRWVKNP YPVVNLINSLFSNLMPKVSGQPMEGKDNQVEEVRIYEGSEQLPGDPDIVRFLDK FGQEKTVYPKPSVAPAAVTFQSNQQDKGKAPLKGPQKASQKESQTQEL, or similar sequence of identity is at least 95%.

The monoclonal antibodies of the invention can be used to detect the VP1 protein of Merkel's polyoma virus in a tissue sample, and the monoclonal antibodies produced by hybridoma 11A2 (MPV001 or DSM ACC3097) can also be used to detect a Merkel cell carcinoma (MCC) in a tissue sample by immunohistochemical, immuno .

Other objects of the invention are a diagnostic composition for detecting Merkel's polyoma virus in a tissue sample comprising an effective amount of the monoclonal antibody of the invention and a diagnostic composition for detecting Merkel cell carcinoma (MCC) in a tissue sample comprising an effective amount of monoclonal antibody produced by hybridoma 11A2 (MPV001 or DSM ACC3097).

The monoclonal antibodies provided in the present invention are novel and different from analogs from other sources.

Brief description of the drawings

Figure 1 reproduces the immunoblotting pattern obtained with recombinant Merkel polyoma virus capsid protein VP1, human polyoma virus JCV capsid protein VP1, and yeast S.cerevisiae lysate using monoclonal antibodies produced by hybridomas 11A2 and 9G6 (see Example 2).

Figure 2 illustrates the ability of monoclonal antibodies produced by hybridomas 11A2 and 9G6 to recognize the Merkel polio virus in cells infected with this virus (see Example 3).

Detailed Description of the Invention

The inventors have succeeded in creating 2 hybridoma lines, which produce monoclonal antibody specificity that is suitable for the identification of the Merkel polyoma virus VP1 protein by different immunochemical methods.

Studies have shown that the created monoclonal antibodies react with both recombinant purified Merkel polio virus capsid protein VP1 synthesized in yeast S.cerevisiae and native Merkel polio virus in infected tissue. Thus, these antibodies proved to be suitable for Merkel's polio virus infection or

For MCC diagnostics.

In order to generate new monoclonal antibodies against the Merkel polyoma virus capsid protein VP1, the recombinant yeast S.cerevisiae yeast recombinant Merkel polyoma virus capsid protein VP1 was used and the following procedures were performed, namely:

1) Immunization: Purified Merkel polyoma virus capsid protein VP1 (antigen) was injected subcutaneously in BALB / c line mice. Prior to injection, the antigen solution was mixed with Freund's complete adjuvant in equal volumes;

2) re-immunization - after a period of time, the mice were re-injected with the same dose of antigen subcutaneously after mixing the antigen solution with incomplete Freund's adjuvant;

3) a few weeks later, the same dose of antigen was administered without adjuvant.

Immunizations were repeatedly tested for the presence of antibodies against the antigen in the serum of immunized mice. A known method for immunoassay (IFA) was used for screening;

4) A few days after the third immunization, mouse spleen cells were hybridized by fusion with cancer cells. This and other procedures were performed according to the previously described methodology used to obtain hybridomas [Kohler, Milstein, Nature 1975; 256: 495-497],

5) After obtaining the hybrid cell clones (hybridomas), their specificity, i.e. it is determined whether they produce antibodies against the capsid protein of the Merkel polio virus

VP1. The IFA method was used for this;

6) The resulting hybridomas were characterized by various traits:

- determining the isotype of the monoclonal antibody they produce;

- testing for antibody specificity by IFA, immunoblotting and immunohistochemistry.

7) After identification of the hybridomas that produce monoclonal antibodies to the Merkel polyoma virus core capsid protein VP1, they have been cloned, propagated and frozen for further storage;

(8) The monoclonal antibodies produced by the hybridomas were characterized in known ways:

IFA and immunoblotting using Merkel's polyoma virus capsid protein VP1 as an antigen by immunohistochemistry using tissue samples from Merkel cell carcinoma.

Thus, immunization of mouse BALB / c with recombinant Merkel polio virus capsid protein VP1 and fusion of immunized mouse spleen cells with myeloma cells resulted in hybridomas producing monoclonal antibodies to Merkel polio virus capsid protein VP1. Examination of the properties of the resulting monoclonal antibodies revealed that they react with native Merkel's polyoma virus in a sample of Merkel cell carcinoma tissue and do not react with normal tissue in the absence of said virus.

Two new hybridomas were obtained and analyzed as described, but only one of them proved suitable for recognition of the Merkel polio virus in the MCC histological sample. The following hybridomas are deposited in the Department of Immunology and Cell Biology, Institute of Biotechnology, Vilnius University:

1) Hybridoma 11A2, accession number MPV001, which produces a IgG2a subclass (subtype) monoclonal antibody to the primary capsid protein VP1 of the Merkel polyoma virus;

2) Hybridoma 9G6, accession number MPV002, which produces a IgGl subclass (subtype) monoclonal antibody to the Merkel polyoma virus core capsid protein VP1.

Hybridoma 11A2 is also deposited in the German Cell Culture Collection (DSMZ). It has been given a deposit number: DSM ACC3097.

Hybridomas 11A2 and 9G6 are characterized by the following features.

Morphological features. Hybridomas are spherical in shape with smooth nuclei and have a large nucleus. Multiplies in suspension and exhibits weak adhesion to glass or plastic surfaces.

Origin of cell culture. The hybridoma was obtained by fusing the spleen cells of immunized BALB / c mice (Mus.musculus) with the mouse (Mus.musculus) myeloma cell line Sp2 / 0.

Signs of cell culture. Hybridoma culture conditions: Growth medium - Dulbecco's 5 modified Eagl's medium (DMEM) with 2mM L-glutamine, 200 pg / mL gentamycin and

10% fetal calf serum. Hybridomas are grown in glass or plastic containers for cell cultures. Cells are transplanted every 3-4 days at a dose of 50100,000 cells / ml. Hybridomas are grown in a 5% CO2 incubator at 37 ° C. For long-term storage, hybridomas are frozen in embryonic serum with 10% DMSO and stored in liquid nitrogen. Freezing conditions: 1x10 ⁶ - 2x10 ⁶ cells per ml, temperature regime - 1 ° C per minute to -70 ° C. After a day, the ampoules with cells are transferred to liquid nitrogen. Ampoules were thawed in a 37 ° C water bath for culture revitalization. Cell viability after thawing - 70-80%.

Contamination. Bacterial and fungal hybridoma cultures were not found and mycoplasma test was negative.

Productivity of hybridomas. Monoclonal antibody secretion level in growth medium 10-30 pg / ml. Monoclonal antibody production remains up to 15 passages (cell culture grafts).

Product Characteristics. Hybridomas 11A2 and 9G6 produce monoclonal antibodies specific for the Merkel polyoma virus core capsid protein VP1. Monoclonal antibodies produced by hybridoma 11A2 are of the IgG class, a subclass of IgG2a. They recognize recombinant Merkel polio virus capsid protein VP1 and Merkel polio virus in infected tissue. Monoclonal antibodies produced by hybridoma 9G6 are of the IgG class, a subclass of IgG1. They recognize recombinant Merkel polyoma virus capsid protein VP1.

The monoclonal antibodies produced by hybridomas 11A2 and 9G6 differ from the described analogs, the monoclonal antibody to other polyoma virus proteins, in that they are specific for the Merkel polyoma virus capsid protein VP1, which is functionally and structurally different from other polyoma virus proteins. The Merkel polyoma virus capsid protein VP1 differs from other polyoma virus proteins by the protein's primary structure (amino acid sequence).

Monoclonal antibodies produced by hybridomas 11A2 and 9G6 recognize the following amino acid sequence (SEQNO: l):

MAPKRKASSTCKTPKRQCIPKPGCCPNVASVPKLLVKGGVEVLSVVTGEDSITQ

IELYLNPRMGVNSPDLPTTSNWYTYTYDLQPKGSSPDQPIKENLPAYSVARVSL

PMLNEDITCDTLQMWEAISVKTEVVGISSLINVHYWDMKRVHDYGAGIPVSGV NYHMFAIGGEPLDLQGLVLDYQTQYPKTTNGGPITIETVLGRKMTPKNQGLDP QAKAKLDKDGNYPIEVWCPDPSKNENSRYYGSIQTGSQTPTVLQFSNTLTTVLL DENGVGPLCKGDGLFISCADIVGFLFKTSGKMALHGLPRYFNVTLRKRWVKNP YPVVNLINSLFSNLMPKVSGQPMEGKDNQVEEVRIYEGSEQLPGDPDIVRFLDK

FGQEKTVYPKPSVAPAAVTFQSNQQDKGKAPLKGPQKASQKESQTQEL

The analogs described above, the monoclonal antibody against hamster polio virus core capsid protein VP1, differ from the antibodies produced by hybridomas 11A2 and 9G6 in that they are specific for hamster polio virus core capsid protein VP1, which differs in amino acid sequence from the Merkel polio virus capsid protein [ A., Samonskyte L., Gedvilaite A. et al., J. Immunol. Meth., 2006; 311, 57-70],

The analogs described above, the monoclonal antibody to the human polyoma virus BKV and JCV and the monkey polyoma virus SV40 capsid protein, differ from the antibodies produced by hybridomas 11A2 and 9G6 in that they are specific for the other polyoma virus capsid proteins VP1, which differ in amino acid sequence from Merko capsid protein VP1 [Randhawa P, Viscidi R, Carter JJ et al., J Gen Virol. 2009 90, 634–639].

The method of obtaining the novel monoclonal antibodies, their properties are described in more detail in Examples 1-3 below and in Table 1, which illustrate but do not limit the scope of the invention.

example

Development of hybridomas producing monoclonal antibodies to the Merkel polyoma virus capsid protein VP1

BALB / c mice were immunized with purified recombinant Merkel polyoma virus capsid protein VP1 expressed in yeast S.cerevisiae. Mice were immunized with 50 µg of antigen with Freund's complete adjuvant subcutaneously. One week after the first immunization, mice were repeatedly immunized with 50 pg of antigen with incomplete Freund's adjuvant. After a further 4 weeks, the mice were immunized with 50 pg of antigen without adjuvant. During immunizations, the titre of the antigen-specific antibodies was tested by the enzyme-linked immunosorbent assay (IFA). 3 days after the last immunization, hybridization was performed.

Hybridization of the immunized mouse spleen cells and mouse myeloma Sp 2/0 cells lacking an enzyme hypoxanthine-phosphoribosyl-GUAN, proliferating in a selective hypoxanthine-aminopterin-thymidine (HAT) medium (IO ^ M hypoxanthine, 1.6 x 10 ^"5 M thymidine , 4 x 10 ' ⁷ M aminopterin) and do not produce immunoglobulins or their chains. Cells must be in the logarithmic growth phase before hybridization. Hybridization was performed by treating cells with 50% polyethylene glycol (PEG-4000, Sigma, USA) for 2 minutes in Dulbecco's modified Eagl's medium (DMEM) with 10% dimethylsulfoxide (DMSO). Myeloma to spleen cells ratio 1: 5 during hybridization. After hybridization, cells were washed with serum-free DMEM, suspended in selective HAT medium, and plated (5x10 ⁵ cells / well).

Hybrid clones appeared 5-10 days after hybridization. The medium was tested for indirect IFA by testing the clones for antibodies against the VP1 protein of the Merkel polyoma virus capsid. Half-cell growth medium was replaced every 4-5 days. For the first time, HAT medium was changed to hypoxanthine-thymidine (HT) medium containing 10 ⁴ M hypoxanthine and 1.6 χ 10 ⁴ M thymidine. Subsequently, the HT medium was gradually changed to normal growth medium. Hybrid clones producing antibodies to the Merkel polyoma virus capsid protein VP1 were cloned by the limiting dilution method. The clones appeared after 4-7 days. They were tested by the indirect IFA method. Selected clones were propagated and further grown in culture or frozen and stored in liquid nitrogen.

Several hybridoma clones have been obtained that produce monoclonal antibodies against

Merkel polyoma virus capsid protein VP1. Their specificity was investigated and antibody isotypes were determined.

Hybridomas that produce monoclonal antibodies against the Merkel polyoma virus capsid protein VP1 have been named 11A2 and 9G6. They were deposited, as above, in the Laboratory of Immunology and Cell Biology, Institute of Biotechnology, Vilnius University (11A2 - deposit number MPV001, 9G6 - deposit number MPV001) and in the German Cell Culture Collection (DSMZ) (11A2 - deposit number DSM ACC3097).

example.

Monoclonal antibody specificity assay and isotype determination.

The specificity of the antibodies produced by hybridomas 11A2 and 9G6 was determined by immunoenzyme assay (IFA) and immunoblotting. Immunoglobulin isotypes were determined by the IFA method using the isotyping kit [Sigma, USA].

By these methods, antibody 11A2 was found to be of the IgG isotype, IgG2a subtype, and antibody 9G6 was of the IgG1 subtype. Both antibodies react with recombinant Merkel polyoma virus capsid protein VP1 (Fig. 1B, C). Immunoblotting revealed that monoclonal antibodies 11A2 and 9G6 react with a 60 kDa protein in the yeast S.cerevisiae lysate, which corresponds to the Merkel polyoma virus capsid protein VP1 but does not react with the yeast S.cerevisiae lysate protein (Fig. 1, B, C, tracks 3 and

4). Antibodies 11A2 and 9G6 also react with the purified Merkel polyoma virus (MPV) capsid protein VP1, but do not react with the human polyoma virus JCV capsid protein VP1 (Figures 1, B, C, lanes 1 and 2). This indicates that the resulting monoclonal antibodies are specific for the Merkel polyoma virus capsid protein VP1.

Table 1 provides data on the specificity and subclasses of antibodies 11A2 and 9G6 produced by hybridomas.

table

Hybridoma clone Isotype Reaction with different antigens: MPV VP1 JCV VP1 S.cerevisiae lysate 11A2 IgG2a + - - 9G6 IgGl + - -

FIG. 1 replicates protein electrophoresis (A) and immunoblotting (B, C) obtained with recombinant Merkel polyoma virus capsid protein VP1, human polyoma virus JCV capsid protein VP1, and yeast S.cerevisiae lysate using monoclonal antibodies produced by hybridomas 11A2 and 9G6. Immunoblotting was obtained with different antibodies: B - 11A2, C - 9G6.

lane - purified recombinant Merkel polyoma virus capsid protein VP1;

lane - purified human polyoma virus JCV capsid protein VP1;

lane - yeast S.cerevisiae lysate with Merkel's polyoma virus capsid protein VP1;

lane 1 - yeast S.cerevisiae lysate without Merkel's polyoma virus capsid protein VP1.

M is the molecular weight standard for proteins.

and in lanes 3, the visible band after incubation with the antibody corresponds to a 60 kDa protein which is the VP1 protein of the Merkel polio virus capsid.

example.

Monoclonal antibody specificity assay by immunohistochemistry

Immunohistochemistry was used to investigate whether antibodies produced by hybridomas 11A2 and 9G6 recognize Merkel's polio virus. Skin samples taken from cancerous tissue - Merkel cell carcinoma - were used for the study. This tissue was analyzed by polymerase chain reaction (PCR) and found to contain cells infected with Merkel's polio virus. Samples from normal (non-cancerous) tissue (healthy skin) were used as a negative control. The PCR method confirmed the absence of cells infected with Merkel's polio virus in this tissue. Tissue samples were incubated with antibodies 11A2 and 9G6 and tested for specific recognition of Merkel's polio virus in tissue containing cells infected with this virus.

Immunohistochemical analysis revealed that antibody 11A2 reacted with cells present in Merkel cell carcinoma tissue but did not react with normal (healthy) tissue cells (Fig. (Fig.2). 2). This indicates that the 11A2 antibody recognizes the Merkel polio virus in infected cells. Thus, this antibody differs from the analogue described above which is specific for other polyoma viruses and is suitable for the detection of Merkel's polyoma virus in a skin sample by immunohistochemistry. The 9G6 antibody was found not to react with cells present in Merkel cell carcinoma tissue (Fig. (Fig.2). 2). Therefore, this antibody is not suitable for immunohistochemical detection of Merkel's polioma virus.

FIG. 2 replicates the immunohistochemical assay obtained with Merkel cell carcinoma tissue and normal (normal skin) tissue using antibodies produced by hybridomas 11A2 and 9G6. Immunohistochemical images correspond to the following cases:

2.1 B08-18233 MCC biopsy specimen incubated with 11A2;

2.2 B08-18233 MCC biopsy specimen incubated with 9G6;

2.3 Non-MCC tissue sample incubated with 11A2;

2.4 Non-MCC tissue sample was incubated with 9G6.

The data obtained indicate that the newly created monoclonal antibodies 11A2 and 9G6 are specific for the Merkel polio virus core capsid protein VP1. These antibodies differ from the analogs described above which are specific for other polyoma virus proteins. Antibodies 11A2 and 9G6 specifically react with recombinant Merkel polyoma virus capsid protein VP1 and do not react with human polyoma virus JCV capsid protein VP1. One of the newly created antibodies, clone 11A2, recognizes it

Merkel Polyoma Virus in Infected Cells by Immunohistochemistry.

The monoclonal antibodies generated could be used to detect Merkel's polio virus by immunochemical methods for the diagnosis of this virus infection or its Merkel cell carcinoma. For example, these antibodies could be used for immunohistochemical, immunoblotting or

IFA method. Detection of samples containing Merkel's poliomavirus capsid protein VP1 would confirm Merkel's polio virus infection or Merkel's cell carcinoma.

The diagnostic composition for detecting Merkel's polio virus in a tissue sample may contain other suitable adjuvants in addition to an effective amount of the monoclonal antibodies of the invention. In an optimal embodiment of the invention, the diagnostic composition also contained a solution of sodium phosphate (0.01 M), sodium chloride (0.15 M) and bovine serum albumin (1 mg / ml) at pH 7.2.

A diagnostic composition for detecting Merkel cell carcinoma (MCC) in a tissue sample may contain other suitable adjuvants in addition to an effective amount of the monoclonal antibody produced by hybridoma 11A2 (MPV001 or DSM ACC3097). In an optimal embodiment of the invention, the diagnostic composition for detection of Merkel cell carcinoma (MCC) contained 0.01 mg / ml of hybridoma 11A2 (MPV001 or

Monoclonal antibodies produced by DSM ACC3097), as well as sodium phosphate (0.01 M), sodium chloride (0.15 M) and bovine serum albumin (1 mg / ml).

Claims (9)

DEFINITION OF INVENTION: A hybridoma 11A2 (MPV001 or DSM ACC3097) producing monoclonal 5 antibodies to the Merkel polyoma virus capsid protein VP1. Hybridoma 9G6 (MPV002), which produces a monoclonal antibody to the Merkel polyoma virus capsid protein VP1. 3. Monoclonal Antibodies Recognizing the Amino Acids (SEQ NO: 1) MAPKRKASSTCKTPKRQCIPKPGCCPNVASVPKLLVKGGVEVLSVVTGEDSITQ 10 IELYLNPRMGVNSPDLPTTSNWYTYTYDLQPKGSSPDQPIKENLPAYSVARVSL PMLNEDITCDTLQMWEAISVKTEVVGISSLINVHYWDMKRVHDYGAGIPVSGV NYHMFAIGGEPLDLQGLVLDYQTQYPKTTNGGPITIETVLGRKMTPKNQGLDP QAKAKLDKDGNYPIEVWCPDPSKNENSRYYGSIQTGSQTPTVLQFSNTLTTVLL DENGVGPLCKGDGLFISCADIVGFLFKTSGKMALHGLPRYFNVTLRKRWVKNP 15 YPVVNLINSLFSNLMPKVSGQPMEGKDNQVEEVRIYEGSEQLPGDPDIVRFLDK FGQEKTVYPKPSVAPAAVTFQSNQQDKGKAPLKGPQKASQKESQTQEL or similar sequence with at least 95% identity. The monoclonal antibody of claim 3, produced by hybridoma 11A2 (MPV001 or DSM ACC 3097) of claim 1. 20th The monoclonal antibody of claim 3, produced by hybridoma 9G6 (MPV002) of claim 2. Monoclonal antibodies according to any one of claims 3 to 5 for use in the detection of Merkel's polio virus. The monoclonal antibody of claim 4 for use in Merkel cell carcinoma 25 (MCC) setting. Use of the monoclonal antibody of claim 6 or 7 for immunohistochemical, immunoblotting or IFA histological examination. A diagnostic composition for detecting Merkel's polio virus in a tissue sample comprising an effective amount of the monoclonal antibody of any one of claims 3-5. A diagnostic composition for detecting Merkel cell carcinoma (MCC) in a tissue sample comprising an effective amount of the monoclonal antibody of claim 4.