FIELD OF THE INVENTION

The present invention refers to the medical field. Particularly, a specific epitope has been identified in the present invention, which is herein used for detecting and/or quantifying autoantibodies against alpha-fetoprotein (AFP) in a biological sample, and consequently for the in vitro diagnosis of hepatocellular carcinoma (HCC) or liver cirrhosis.

STATE OF THE ART

The HCC is the second cause of mortality related to cancer, and the sixth most common cause of cancer worldwide. Most patients diagnosed of HCC have liver cirrhosis as a baseline disease, thus mandatory HCC screening is performed with ultrasound every six months in all these patients. However, most patients, even those included in a screening program are diagnosed at late stages, when survival prognosis is worst. Accordingly, the survival rate at 5 years in patients with HCC is less than 16%. In order to improve the survival of these patients, it is very important to develop new tools to better diagnose HCC at early stages, when curative treatments are possible. Discovery of new biomarkers or the improvement of those already described to detect HCC at early stages will be essential to develop better screening tests. The American Association for the Study of Liver Diseases and the European Association for the Study of the Liver call for the need to establish screening tests for at-risk patients, mainly those suffering from cirrhosis. The screening protocol approved by both associations consists of abdominal ultrasound every 6 months. Other associations like British and Asian add AFP determination to ultrasound. Although, a recent meta-analysis sets the ultrasound sensitivity to detect HCC in 84%, the sensitivity drops to 47% when used for early detection. This meta-analysis also studies the comparison of sensitivity between ultrasound with or without serum AFP detection as screening test. Results show that ultrasound without serum AFP detection has a sensitivity of 78% to detect HCC, while the sensitivity increases to 97% when both ultrasound and serum AFP detection are concurrently used. However, early stage HCC detection shows a sensitivity of 45% when using ultrasound without serum AFP detection, and a sensitivity of 63%; when adding serum AFP detection to the ultrasound. Also, serum AFP detection alone shows a sensitivity of 46-59% and a specificity of 87-93% to early detect HCC. Appropriate screening leads to early diagnosis, which leads to better management options, a higher proportion of treatable lesions, and better outcomes, including survival. Screening at-risk population to develop HCC also has been demonstrated to be cost-effective if the expected HCC risk exceeds 1.5% per year in patients with hepatitis C and 0.2% per year in patients with hepatitis B.

The immune system plays an important role controlling cancer. At early tumorigenesis stages tumor cells release proteins, peptides to the cell surface that produce a humoral response against them. These proteins and peptides are known as tumor-associated antigens (TAA). TAAs could be key proteins in transformation of cancer and transition to malignancy, thus becoming important as a therapeutic target molecule. Importantly, antibodies against TAAs could be detected few months or years before cancer diagnosis, helping diagnosis at early stages. 80-90% of HCC have liver cirrhosis, thus antibodies against TAAs could be detected in cirrhotic patients at high risk to develop HCC. Although, there are many TAAs associated to HCC, they are not used in clinical practice due to their low sensitivity, thus not suitable for screening HCC in at-risk population. In contrast, AFP could be a good TAA to detect serum autoantibodies due to; 1) its high specificity in the diagnosis of HCC, 2) its use in immunoassays, and 3) its similar sensitivity compared to other TAAs previously reported to detect serum autoantibodies. Consequently, the present invention aims to identify, and immunogenicity-optimize the AFP immunodominant epitope as a TAA to detect serum AFP autoantibodies in patients with HCC and liver cirrhosis.

DESCRIPTION OF THE INVENTION Brief description of the invention

As explained above, a specific epitope has been identified in the present invention, which is herein used for detecting and/or quantifying autoantibodies against alpha-fetoprotein (AFP) in a biological sample, and consequently for the in vitro diagnosis of hepatocellular carcinoma (HCC) or liver cirrhosis.

Such as it is shown in the Examples, after a screening process based on immunoassay techniques of different amino acid peptides included in protein AFP, immunogenicity- optimized epitopes have been identified in the present invention.

Of note, SEQ ID NO: 1 of peptide 9.7.1 is present in several amino acid sequences which show good results. For instance, the SEQ ID NO: 1 of peptide 9.7.1 is included in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 12 and SEQ ID NO: 26 to 38. So, this sequence is herein presented as a key sequence for conferring immunogenicity to the epitope. Moreover, a validation process was carried out as shown in the Examples and peptides 9.7.8 of SEQ ID NO: 2 and, particularly, peptide 9.7 of SEQ ID NO: 3, were confirmed as good epitopes for AFP determination and/or quantification.

Kindly note that SEQ ID NO: 1 is included in SEQ ID NO: 2 which is in turn included in SEQ ID NO: 3.

So, the first embodiment of the present invention refers to an epitope, or to an antigen comprising the epitope, wherein the epitope consisting of SEQ ID NO: 1 or a sequence having an identity of at least 95% with the SEQ ID NO: 1. In a preferred embodiment, the epitope consists of SEQ ID NO: 2 or a sequence having an identity of at least 95% with the SEQ ID NO: 2. In a still preferred embodiment, the epitope consists of SEQ ID NO: 3 or a sequence having an identity of at least 95% with the SEQ ID NO: 3.

The second embodiment of the present invention refers to an in vitro method for detecting and/or quantifying autoantibodies against alpha-fetoprotein (AFP) in a biological sample, wherein the autoantibodies bind to an epitope which comprises or consists of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 and it is immobilized on a solid support. In a preferred embodiment, the autoantibodies bind to an antigen comprising an epitope which in turn comprises or consists of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 and it is immobilized on a solid support. In a preferred embodiment, the detection and/or quantification of the autoantibodies is carried out by an immunochemical technique, preferably by ELISA, and most preferably by indirect ELISA, in which the antigen is bound by the primary antibody to be detected, that binds directly to the antigen, which then is detected by a labeled secondary antibody. So, the indirect ELISA is a two-step ELISA which involves two binding process of primary antibody and labeled secondary antibody. The primary antibody to be detected is incubated with the antigen followed by the incubation with the secondary antibody. Initially, micro-well plates are incubated with antigens characterized by comprising the epitope of the invention, washed up and blocked with BSA. After that, biological samples which may comprise the antibodies to be detected are added and washed. Moreover, enzyme linked secondary antibody are added and washed. A substrate is then added, and enzymes on the antibody elicit a chromogenic or fluorescent signal.

So, in a preferred embodiment, the present invention refers to an in vitro method which comprises: a) Immobilizing an epitope comprising or consisting of the SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, or an antigen comprising an epitope which in turn comprises or consists of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 on a solid support, b) Adding the biological sample to be analysed on the solid support, c) Adding an antibody, conjugated with a detectable labelling agent, which reacts against the autoantibody which may be present in the biological sample, d) Detecting and/or quantifying the complex obtained, for example with a composition containing a chromogenic, fluorogenic and/or chemiluminescent indicator substrate.

In a preferred embodiment the in vitro method described above further comprises treating the epitope or the antigen comprising the epitope, before it is immobilized on a solid support, by: a. Adding at least one charged molecule to a solution comprising the epitope consisting of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, or the antigen comprising the epitope consisting of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; and b. Adding at least one amino acid to the biomolecule solution.

In a preferred embodiment the charged molecule is SDS.

In a preferred embodiment the SDS is at a concentration of between 0.1% and 10% (w/v). In a preferred embodiment the amino acid is lysine, arginine, histidine or combinations thereof.

In a preferred embodiment the amino acid is added to obtain a concentration between 12.5% (w/v) to 20% (w/v).

In a preferred embodiment, the sample is obtained from a subject who may be suffering from hepatocellular carcinoma or liver cirrhosis.

In a preferred embodiment, the sample is blood, serum or plasma.

The third embodiment of the present invention refers to an in vitro method for the diagnosis of hepatocellular carcinoma or liver cirrhosis which comprises detecting and/or quantifying autoantibodies against AFP by following the above described method.

The fourth embodiment of the present invention refers to the in vitro use of an epitope comprising or consisting of the SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, or an antigen comprising an epitope which in turn comprises or consists of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 for detecting and/or quantifying autoantibodies AFP. In a preferred embodiment said epitope or antigen is used for the diagnosis of hepatocellular carcinoma or liver cirrhosis.

The fifth embodiment of the present invention refers to a kit for detecting and/or quantifying autoantibodies against AFP in a biological sample which comprises: a) An epitope comprising or consisting of the SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 or an antigen comprising an epitope which in turn comprises or consists of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. b) At least a secondary antibody which reacts against the autoantibody which may be present in the biological sample.

In a preferred embodiment this kit is intended for the diagnosis of hepatocellular carcinoma or liver cirrhosis.

The seventh embodiment of the present invention refers to a monoclonal antibody, or fragment thereof, specifically binding the epitope consisting of SEQ ID NO: 3 or an antigen comprising an epitope consisting of SEQ ID NO: 3.

In a preferred embodiment, the monoclonal antibody is characterized in that it comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein said VFl comprises HCDR1, HCDR2 and HCDR3 polypeptides and VL comprises LCDR1, LCDR2 and LCDR3 polypeptides and wherein HCDR1 consists of the sequence SEQ ID NO: 39, HCDR2 consists of the sequence SEQ ID NO: 40, HCDR3 consists of the sequence SPY, LCDR1 consists of the sequence SEQ ID NO: 41, LCDR2 consists of the sequence SEQ ID NO: 42 and LCDR3 consists of the sequence SEQ ID NO: 43.

In a preferred embodiment the sequence of the monoclonal antibody is as follows:

Heavy chain

EVQLQESGPGLVAPSQSLSITCTVSGFSLTSYAVSWVRQPPGKGLEWLGIIWGD GSTNYHSPLISRLSISKYNSKSQVFLKLNSLQTDDTATYYCASSPYWGQGTLVT VS A (SEQ ID NO: 44).

The CDR sequences are as follows:

• HCDR1 (SEQ ID NO: 39): SYAVS.

• HCDR2 (SEQ ID NO: 40): IIWGDGSTNYHSPLIS.

• HCDR3 : SPY.

Light chain Protein DVVMTQTPLSLPVSLGDQASISCRSSQRLVHSNGATYLHWYLQKPGQSPKLLIY KVSKRF SGVPDRF SGSGSGTDFTLKISRVEAEDLGVYFC SQ STHVPWTF GGGTK LEIKR (SEQ ID NO: 45)

The CDR sequences are as follows:

• LCDR1 (SEQ ID NO: 41): RSSQRLVHSNGATYLH.

• LCDR2 (SEQ ID NO: 42): KVSKRF S.

• LCDR3 (SEQ ID NO: 43): SQSTHVPWT.

In a preferred embodiment, the present invention refers to an in vitro method which comprises: a) Immobilizing an epitope comprising or consisting of the SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, or an antigen comprising an epitope which in turn comprises or consists of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 on a solid support, b) Adding the biological sample to be analyzed on the solid support, c) Assessing the presence in the biological sample of autoantibodies against the epitope or antigen described above, by determining whether antigen-antibody complexes have been formed, wherein, preferably, the antibody characterized by the sequences SEQ ID NO: 39, 40, SPY, 41, 42 and 43 is used as a calibrator or positive control.

In a preferred embodiment, the present invention is a computer-implemented invention, wherein a processing unit (hardware) and a software are configured to: a) Receive data/information about the signal emitted when a complex is formed between autoantibodies (which may be present in the biological sample) and the epitopes or antigens characterized by the SEQ ID NO: 1, 2 or 3, b) Process the data/information generated in step a) for finding substantial variations or deviations with respect to the signal emitted when a complex is formed between the antibody characterized by the sequences SEQ ID NO: 39, 40, SPY, 41, 42 and 43 (which is used as a calibrator or positive control) and the epitopes or antigens characterized by the SEQ ID NO: 1, 2 or 3, c) Provide an output through a terminal of any statistically significant variation or deviation identified according to step b) wherein, if the signal identified when the biological sample is added to the support is at least the same than the signal emitted by the complex formed when the antibody characterized by the sequences SEQ ID NO: 39, 40, SPY, 41, 42 and 43 is used, this is an indication that the sample comprises autoantibodies against the above cited epitopes or antigens.

The present invention also refers to the in vitro use of any of the antibodies described above for the diagnosis of hepatocellular carcinoma or liver cirrhosis.

The present invention also refers to any of the antibodies described above for use in a method for the diagnosis of hepatocellular carcinoma or liver cirrhosis.

For the purpose of the present invention the following terms are defined:

• The term "comprising" means including, but not limited to, whatever follows the word "comprising". Thus, use of the term "comprising" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present.

• By "consisting of’ is meant including, and limited to, whatever follows the phrase “consisting of’. Thus, the phrase "consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present.

Detailed description of the invention

The present invention is illustrated by the Examples set below which merely serve as a proof of concept without the intention of limiting its scope.

Example 1. Study subjects

We selected 100 consecutive patients from outpatient hepatology unit of Parc Tauli University Hospital (68 males, 32 females, within the ages of 43-93 years old and median age of 74 years old). 58 patients had confirmed HCC following the criteria of the American Association for the Study of Liver Diseases (AASLD), 42 patients had confirmed hepatic cirrhosis from any etiology according to the criteria of AASLD. 40 non-hepatic diseases serum donors from Banc de Sang i Teixits de Barcelona were selected as a control group. The HCC group of patients was followed-up during 36.2±29.8 months and cirrhosis group was followed-up during 32.5±33.3 months. The ethical committee’s approval was obtained, and all samples collected at our Hospital were accompanied by patient’s consent. All patients signed an informed consent.

Example 2. First phase of the epitope mapping

AFP is a 609 amino acid protein. It binds copper, nickel, and fatty acids as well as, and bilirubin less well than, serum albumin. Amino acids 1-18 correspond to the signal peptide and positions 19-609 correspond to the AFP chain.

To make the first epitope mapping, 48 amino acid biotinylated peptides were synthesized with overlapping of 8 amino acids.

The peptides that were synthesized are included in Table 1:

Table 1

To carry out epitope mapping, 9 serum samples from patients with HCC, 3 serum samples from patients with cirrhosis who developed HCC in its follow-up, and 4 serum samples from blood donors were used. The different peptides were sensitized in an ELISA plate to perform an immunoassay following the protocol below:

-Streptavidin-ELISA plates preparation:

• Nunc ELISA plates maxisorp H4BX (Nunc).

• Sensitized 100 pL of streptavidin (Merck) at 20 pg/mL in Carbonate-Bicarbonate 50 mM pH9.

• Incubate overnight at 4°C.

• Discard streptavidin.

• Block ELISA plates with BSA 5% (Merck) in PBS (Inova Diagnostics) 2 hours at room temperature.

-ELISA Plate sensitization with peptides:

• Prepare a dilution of biotinylated peptide at 10 pg/mL in Tris-HCl at 0.15M pH7.3.

• Add 100 pL of peptide per well.

• Incubate 1 hour at room temperature.

• Wash 3 times with Washing solution (Grifols).

-ELISA assay: • Serum samples were diluted 1/100 with PBS-Tween 20 0.05% (Diluent buffer, Grifols).

• 100 pL of diluted serum samples were added per well and incubated for 1 hour with shaking at room temperature.

• Perform 3 washes with PBS-Tween 20 0.05% (Washing solution, Grifols).

• Anti-human IgG antibody conjugated with HRP (Thermofisher Scientific) were diluted at 1/5000 in diluent buffer (Grifols).

• 100 pL of anti -human IgG were incubated for 1 hour at room temperature

• Perform 3 washes with PBS-Tween 200.05% (Washing solution, Grifols)

• 100 pL of substrate (Thermofisher) were incubated for 30 minutes at room temperature.

• Addition of 100 pL of stop solution (sulfuric acid 25% or Grifols) Spectrophotometer reading at 450 nm and reference filter at 620 nm.

Example 3. First assay results

The results are expressed in optic density obtained from the ELISA described above and are summarized in Table 2 below, wherein the 15 peptides disclosed above were analyzed. The mean of optical densities obtained by the different peptides in the group of patients was analyzed. This mean was higher for peptide 9 (0,320), which indicates that a greater number of antibodies bind to this sequence compared to the rest of the analyzed sequences. Therefore peptide 9 (SEQ ID NO: 12) showed the best results.

Table 2

Table 2 (cont.)

HCC (Hepatocellular carcinoma patients); Cirr (Cirrhotic patients who develop HCC); NHS (Normal human serum from blood donors)

Example 4. Second phase of epitope mapping

Once the peptide 9 of SEQ ID NO: 12 was selected as the most interesting starting point, the second phase of epitope mapping was performed by decomposing the peptide 9, which comprises 48 amino acids, into shorter sequences of 20 amino acids with 16 overlapping amino acids. According with this strategy, the peptides which were synthesized are included in Table 3. Table 3

Then, the same immunoassay was performed with the same samples as described above in Example 2.

Example 5. Second assay results

The optic density obtained in the ELISA are summarized in Table 4 below. The mean of optical densities obtained by the different peptides in the group of patients was analyzed. This mean was higher for peptide 9.7 (0,381), which indicates that a greater number of antibodies bind to this sequence compared to the rest of the analyzed sequences. Therefore, the peptide that shows the best results is peptide 9.7 of SEQ ID NO: 3.

Table 4

HCC (Hepatocellular carcinoma patients); Cirr (Cirrhotic patients who develop HCC); NHS (Normal human serum from blood donors)

Example 6. Third phase of epitope mapping

Since the linear epitopes recognized by B lymphocytes comprise 5 to 20 amino acids, 15 more peptides were synthesized according to the results obtained in the second phase of epitope mapping to identify the epitope with the lowest number of amino acids (see Table

5)· Table 5

Then, the same immunoassay was performed with the same samples as described above in Example 2. Example 7. Third assay results

The optic density obtained in the ELISA are summarized in Table 6 below. The mean of optical densities obtained by the different peptides in the group of patients was analyzed. This mean was higher for peptide 9.7.8 (0,217), which indicates that a greater number of antibodies bind to this sequence compared to the rest of the analyzed sequences. Therefore, peptide that shows the best results is peptide 9.7.8 (SEQ ID NO: 2).

Table 6 Table 6 (cont.)

HCC (Hepatocellular carcinoma patients); Cirr (Cirrhotic patients who develop HCC); NHS (Normal human serum from blood donors) With the present results, we can conclude that the immunodominant epitope recognized by the samples of patients with HCC corresponds to the sequence of the peptide 9.7 of SEQ ID NO: 3, which include the SEQ ID NO: 2 and in turn the SEQ ID NO: 1.

Example 8. Comparison between recombinant human AFP and AFP identified epitope

Subsequently, we performed the immunoassay with the 9.7 peptide with the methodology described above and a capture immunoassay with the recombinant human AFP in patients with HCC as a pathologic population and active chronic hepatitis without cirrhosis as a control population.

To perform the capture immunoassay for recombinant human AFP we followed the protocol below:

ELISA Plate sensitization with anti -AFP mouse monoclonal antibody:

• Non-covalent binding ELISA plate for hydrophilic amino acids Immulon 4 HBX (Thermofisher Scientific) for sandwich ELISA

• 1/100 dilution of anti-AFP mouse monoclonal antibodies (mAh) (Bio-Rad) to have a concentration of 20 pg/mL, in 50 mM carbonate-bicarbonate buffer pH 9 were prepared.

• Addition of 100 pL of anti-AFP mouse mAh dilution were incubated overnight at 4°C (3 pg/well)

• After that, discard mouse mAh dilution and blocked the ELISA plate with 5% of bovine serum albumin in PBS

• Perform 3 washes with PBS-Tween 20 0.05%

Antigen addition:

• 100 pL of rhAFP at 10 pg/mL in PBS were added per well (1 pg/well)

• Incubation of AFP for 1 hour at room temperature

• Perform 3 washes with PBS Perform ELISA assay:

• Patients and control sera were diluted 1/100 with PBS-Tween 20 0.05%

• 100 pL of diluted sera were added for rAFP treated and not treated per well and incubated for 1 hour at room temperature • Perform 3 washes with PBS-Tween 200.05%

• 100 pL of anti -human IgG for cardiolipins (In ova Diagnostics) were incubated for 1 hour at room temperature

• Perform 3 washes with PBS-Tween 200.05%

• 100 pL of substrate (Inova Diagnostics) were incubated for 30 minutes at room temperature

• Addition of 100 pL of ELISA STOP solution (Inova Diagnostics)

• Spectrophotometer reading at 450 nm and reference filter at 620 nm The optic density obtained in the ELISA are summarized in Table 7 below:

Table 7

HCC (Hepatocellular carcinoma patients); CH (Chronic hepatitis patients without cirrhosis) A ROC curve was performed with the optic density obtained from HCC patients and controls to establish the cut-off point to perform the analysis of the sensitivity, specificity and accuracy of both assays shown in Table 8:

Table 8

Example 9. Validation assay of the epitope of SEQ ID NO: 3 and antibody with CDRs consisting of SEQ ID NO: 39, SEQ ID NO: 40, SPY, SEQ ID NO: 41, SEQ ID NO: 42 and SEQ ID NO: 43

5 mice were immunized with SEQ ID NO: 3 to obtain monoclonal antibodies against said sequence from the hybridomas generated. To choose the hybridoma with the highest antibody response against SEQ ID NO: 3, an ELISA test was performed with a standard protocol and with a modified protocol.

ELISA standard protocol:

-Streptavidin-ELISA plates preparation:

• Nunc ELISA plates maxisorp H4BX (Nunc).

• Sensitized 100 pL of streptavidin (Merck) at 20 pg/mL in 50 mM Carbonate- Bicarbonate pH9.

• Incubate overnight at 4°C.

• Discard streptavidin.

• Block ELISA plates with BSA 5% (Merck) in PBS (Inova Diagnostics) 2 hours at room temperature.

-ELISA Plate sensitization with peptides:

• Prepare a dilution of biotinylated peptide at 10 pg/mL in Tris-HCl at 0.15M pH7.3. • Add 100 pL of peptide per well.

• Incubate 1 hour at room temperature.

• Wash 3 times with Washing solution (Grifols).

-ELISA assay:

• Supernatant of 10 negative hybridomas for SEQ ID NO: 3 were diluted 1/2 and 10 positive hybridomas for SEQ ID NO: 3 were diluted 1/100 with PBS-Tween 20 0.05% (Diluent buffer, Grifols).

• 100 pL of diluted supernatant hybridoma samples were added per well and incubated for 1 hour with shaking at room temperature.

• Perform 3 washes with PBS-Tween 20 0.05% (Washing solution, Grifols).

• Anti-human IgG antibody conjugated with HRP (Thermofisher Scientific) were diluted at 1/2000 in diluent buffer (Grifols).

• 100 pL of anti -human IgG were incubated for 1 hour at room temperature

• Perform 3 washes with PBS-Tween 20 0.05% (Washing solution, Grifols)

• 100 pL of substrate (Thermofisher) were incubated for 30 minutes at room temperature.

• Addition of 100 pL of stop solution (sulfuric acid 25% or Grifols)

ELISA modified protocol:

-Streptavidin-ELISA plates preparation:

• Nunc ELISA plates maxisorp H4BX (Nunc).

• Sensitized 100 pL of streptavidin (Merck) at 20 pg/mL in 50 mM Carbonate- Bicarbonate pH9.

• Incubate overnight at 4°C.

• Discard streptavidin.

• Block ELISA plates with BSA 5% (Merck) in PBS (Inova Diagnostics) 2 hours at room temperature.

-Peptide treatment:

• The amount of peptide of SEQ ID NO: 3 sufficient to perform the ELISA (final concentration of 10 pg/mL). A 1/2 dilution (v/v) was performed with 2% SDS- Tris-HCl 0.15M pH7.3 (Merck).

• The mix was incubated with shaking for 15 minutes at 4°C. • 3 volumes of 20% arginine solution in Tris-HCl at 0.15M pH7.3 (Merck) was added to the peptide-SDS mixture.

• The new mix was incubated with shaking for 15 minutes at 4°C.

-ELISA Plate sensitization with peptides:

• Add the necessary volume of 0.15M Tris-HCl pH7.3 to bring the biotinylated peptide-SDS-arginine mixture to 10 pg/mL.

• Add 100 pL of peptide per well.

• Incubate 1 hour at room temperature.

• Wash 3 times with Washing solution (Grifols).

-ELISA assay:

• Supernatant of 10 negative hybridomas for SEQ ID NO: 3 were diluted 1/2 and 10 positive hybridomas for SEQ ID NO: 3 were diluted 1/100 to 1/5000 with PBS- Tween 200.05% (Diluent buffer, Grifols).

• 100 pL of diluted supernatant hybridoma samples were added per well and incubated for 1 hour with shaking at room temperature.

• Perform 3 washes with PBS-Tween 200.05% (Washing solution, Grifols).

• Anti-human IgG antibody conjugated with HRP (Thermofisher Scientific) were diluted at 1/2000 in diluent buffer (Grifols).

• 100 pL of anti -human IgG were incubated for 1 hour at room temperature

• Perform 3 washes with PBS-Tween 20 0.05% (Washing solution, Grifols)

• 100 pL of substrate (Thermofisher) were incubated for 30 minutes at room temperature.

• Addition of 100 pL of stop solution (sulfuric acid 25% or Grifols)

Table 9 Table 10

Table 11

Table 12

NA: not applicable EXAMPLE 10. Diagnosis of liver cirrhosis

The study of the presence of autoantibodies against SEQ ID NO: 3 was carried out in 102 patients with liver cirrhosis and 48 patients with active chronic hepatitis. For this, an ELISA was performed with the protocol explained in Example 9. The results were the following:

Table 13

Table 14