MXPA03003794A - Detection of infectious agents using antigen mimics. - Google Patents

Abstract

A method for making a diagnosis of an antigen, comprising identifying the binding specificity of the anti-antigen antibody molecules in the serum by the Antibody Detection by Antigen Mimics (ADAM) methodology, comprising screening phage libraries using sera from antigen-infected patients and non-infected individuals, identifying peptides binding antibodies (ligands) specifically associated with said antigen. Improvements of the method are given by in vitro maturation strategies; linking the ligands to a common core, such as MAP. In particular the method applies to HCV.

Description

DETECTION OF INFECTIOUS AGENTS USING IMITATORY ANTIGENS DESCRIPTION OF THE INVENTION The present invention relates to a diagnostic assay for detecting infectious agents, in particular viral agents, more particularly the human hepatitis C virus (hereinafter abbreviated as HCV), for its acronyms in English), peptides that bind an antibody against infectious agent useful in the assay, as well as procedures for preparing such peptides and equipment to perform such an assay. The present invention relates in particular to HCV, peptides that bind the antibody against HCV useful for diagnostic assays, processes for the preparation thereof and equipment for performing such assays.

BACKGROUND OF THE INVENTION Hepatitis C virus (HCV) is the main etiologic agent of parenterally transmitted hepatitis of the non-A, non-B type. This virus very often causes persistent infection and often induces the development of chronic hepatitis and liver cirrhosis, which is a major worldwide cause of chronic liver disease (Boyer N, Marcellin P, REF: 146409 Pathogenesis, diagnosis and manangement of hepatitis C, J Hepatol 2000; 32: 98-112). Viral infection was diagnosed by detecting antibodies against HCV in the serum or by returning evident viral AR using nucleic acid amplification methods (Robbins DJ, Pasupuleti V. Cuan J, Chiang CS, Reverse transcriptase PCR quantitation of hepatitis C virus, Clin Lab Sci 2000 Winter; 13: 23-30). The latter methods are very sensitive but expensive and susceptible to technical or laboratory error. In addition, the reliability and specificity of the PCR technique have not been standardized (Gretch DR, Diagnostic test for hepatitis C, Hepatology 1997 SEP; 26 (3 Suppl 1): 43S-47S). The present invention relates to the diagnosis of infectious diseases, such as viral infections, in particular HCV infections, by detecting antibodies generated against the infectious agent, in particular antibodies against HCV. Very recently a system for the sensitive detection of HCV core protein has been reported (Komatsu F, Takasaki K, Liver 1999 Oct; 19 (5): 375-80). An impressive number of both patent and non-patent literature relates to diagnostic methods and equipment for the detection of HCV. The document of E.U.A. 5,985,542 assigned to Somitomo - - Chemical Company, provides equipment for detection of liver diseases, such as hepatitis C and alcoholic cirrhosis containing an antibody capable of recognizing cytochrome P450. The document of E.U.A. 5,972,347, assigned to Baxter Aktiengeselleschaf, provides a composition for the treatment of HCV infection comprising inactivated HCV bound to neutralizing human HCV antibodies, wherein the antibodies are generated against at least one protein that is selected from the group consisting of HCV core protein and the NS3 protein. The documents of E.U.A. 5,939,262, 5,919,625 and 5,677,124, assigned to Ambion and Cenetron, provide a very broad method for determining the presence of a proven nucleic acid sequence in a sample, where obtaining a nuclease resistant nucleic or ribonucleic acid segment is involved. ribonuclease. The document of E.U.A. 5,866,139, assigned to Institut Pasteur, provides equipment to determine the presence of specific antibodies E-1 of HCV. See also document E.U.A. 5,854.001 for Abbott. The document of E.U.A. 5,800,982, assigned to Tonen, describes antigenic peptides capable of reacting specifically with antibodies directed against group II of HCV. JP 10019897 for Tonen provides a kit containing a peptide having an amino acid sequence of at least five continuous amino acids that constitute a protein of the 4A region without structure (NS4A, for its acronym in English) of HCV and a peptide that has the amino acid sequence of at least five continuous amino acids that constitute the protein of a 4B region without structure (NS4B, for its acronym in English) of HCV. In order to acquire antecedents with respect to the state of the art see also the documents of E.U.A. 5,871,904, 5,869,253, 5,750,331, 5,747,241, 5,667,992, 5,645,983, 5,625,034, 5,610,009, WO 9707400, WO 9637783, EP 593291, EP 593290, EP 586065, JP 4349885. The detection of antibodies against HCV in serum is still by far the test most widely used to determine HCV infection. Since 1987, when the genome was cloned for HCV, several improved versions of diagnostic tests for HCV detection have been developed. Currently available diagnostic kits show serum antibodies against a mixture of four recombinant antigens that correspond to part of the HCV core, non-structural 3 regions, and non-structural 5 regions of the HCV polypeptide (Gretch DR, Diagnoatic testa for hepatitis C hepatology 1997 Sep; 26 (3 Suppl 1): 43S-47S). Samples that are classified as positive by screening by ELISA are thus confirmed by an immunoblot assay where the presence of antibodies for the same four antigens is detected separately. A diagnosis with a positive result requires the detection of antibodies against at least two recombinant antigens. For a relevant part of the population with antibodies against HCV, reactivity towards only one antigen occurs, which makes the conclusive diagnosis impossible to formulate. An additional problem is derived from the use of recombinant antigens which can be recognized by antibodies not related to HCV and generate a diagnosis with a false positive result. In this way, additional research and long-term surveillance is required to reach a conclusive diagnosis. The contact of a foreign antigen with an organism activates a specific immune response. The antigen image that is detected by the humoral response is defined by the epitopes recognized by the host antibodies. Assuming that the antibody binding site is the negative image of the epitope, a molecule that specifically binds the paratope must represent the positive image of the epitope. This line of reasoning suggests that, with respect to the humoral response, an antigen can be described accurately by specific ligands that bind to the antigen-specific antibodies. Identifying these ligands can provide a way to detect the specific humoral response against an antigen, regardless of whether or not the same antigen is known or available. These concepts are applied to develop a diagnostic assay for the detection of antibodies related to the infection, comprising HCV infection in humans. Screening of phage libraries with HCV-positive sera identifies peptide ligands that specifically bind antibodies against HCV. This is obtained by adopting selection strategies for that purpose, given that sera from infected patients that contain specific antibodies to the virus spread among a very large population other antibodies with different binding specificities. The screening of peptides with a large number of negative sera eliminates those ligands which detect antibodies in negative samples (false positive result), which leads to the selection of a set of highly specific peptides. The incidence of nonspecific reactivities is also reduced by the use of short peptide sequences and outside the context of the natural antigen. This technique is described in EP 0 698 091 Bl, published on 02.22.1996. see also J. Mol.

Biol. (1991), 222, 301-310; Gene, 128 (1993), 51-57; Gene, 148 (1994), 7-13; The EMBO Journal, vol. 13, no. 9, 2236-2243; 1994; Bacterial Protein Toxins, Zb. Bakt. Suppl 24, 415-425 (1994); The Journal of Immunology (1996) 4504-4513; Methods in Molecular Biology, vol. 87, Humana Press Inc. p. 195-208; Combinatorial Librarles (R. Cortese ed.) Alter de Gruyter 1996, chapter 8; Methods in Enzymology, (1996) vol. 267, 116-129; Biol. Chem. Vol. 378, 495-502, June 1997; The EMBO Journal Vol. 17, No. 13, 3521-3533, 1998; Nature Biotechnolohy Volume 16, November 1998, 1068-1073 for a review of the technology of the mimotopes and fagotopos described in EP 0 698 091 described in the foregoing and the methods for carrying out the same. These selection strategies identify those peptide structures which best bind antibodies against immunodominant HCV. In addition, the multivalent display of the ligand contributes a remarkable avidity effect for detection sensitivity. Despite these considerations, the ADA mixture test -HCV (antibody detection by antigen mimic, ADAM, for its acronym in English) in a group of sera that has not been used for screening, results at a sensitivity lower than 100%, although very high. The particular feature of the humoral response against HCV can explain this result, since it does not involve an immunodominant - principal epitope but rather it is directed towards several different viral determinants. The existence of different viral genotypes complicates the issue more. The currently available diagnostic kits show serum antibodies against four recombinant antigens corresponding to large regions of the HCV polypeptide (Gretch DR., Diagnostic test for hepatitis C, Hepatology 1997 Sep; 26 (3 Suppl 1); 43S-47S). An adequate diagnosis requires the detection of serum antibodies against at least two recombinant antigens: for this reason, it is impossible to formulate a conclusive diagnosis for an important part of the population with antibodies against HCV, since reactivity can be shown only against a antigen. The ADAM-HCV EIA (enzyme-linked immunosorbent assay) uses primers from several different immunodominant epitopes of different antigens and thus ensures greater resolution power in the analysis. As an immediate result, the frequency of indeterminate samples is significantly reduced. An important effort has been developed for the technical development of the ADAM-HCV assay (using a strategy described basically in the document mentioned before EP 0 698 091). Peptides selected from phage libraries are displayed as fusions in the N-terminal part of the major capsid protein pVIII. Using the phage as a diagnostic reagent has many advantages: it is an extremely flexible reagent, which in itself generates different types of immunoassay (Dente et al., 1994, F. Felici, G. Galfré, A. Luzzago, P. Monaci, A. Nicosia and R. Cortese "Phage-displayed peptides as tools for the characterization of human sera" Methods in Enzymology 267, 116-129 - 1996, Bartoli et al., Nature Biotechnology Volume 16, November 1998, 1068-1073 ), and production in small quantities is easy and cheap. Despite these advantages, the size of the phage particle limits the concentration of peptide molecules that can be obtained in the assay. The interference of serum antibodies against the phage capsid requires the addition of a carrier phage in the assay mixture to sequester antibodies against the phage. Finally, the large-scale production of this, as well as of any biological reagent, faces problems of microbiological contamination, reproduction capacity, quality control, purification and production costs. Simple linear synthetic peptides derived from peptide sequences shown in the phage in most cases do not retain their sensitivity or specificity to obtain an efficient antibody detection.

SUMMARY OF THE INVENTION It has now been found that an objective of the present invention is a method for making a diagnosis of infectious diseases, such as viral infections, in particular hepatitis C, which comprises identifying the binding specificity of the antibody molecules against antigen in the serum by the methodology of detection of antibody by antigen mimic (ADAM, for its acronym in English) comprising screening phage libraries using sera from patients infected with antigen and uninfected individuals, identify antibodies that bind peptides (ligands) related specifically with such an antigen. In a particularly preferred embodiment, the present invention relates to HCV infection. DETAILED DESCRIPTION OF THE INVENTION In a first preferred embodiment, in the method according to the present invention, such ligands are improved by in vitro maturation strategies. In a second preferred embodiment, in the method according to the present invention, such ligands are synthetic peptides. In a third preferred embodiment, such ligands are attached to a common core, in a particularly preferred embodiment, such ligands, together with the common core, is MAP, according to the definition given below. Another objective of the present invention is a collection of specific ligands for HCV that can be obtained by the process comprising: a) first panning (scanning or performing an examination and selection) of a phage library in n positive sera to generate a first series of n phage deposits; b) prepare n mixtures of deposits containing n-1 deposits; c) select by affinity each of the n mixtures against the serum that generated the phage-excluded deposit, to provide a second series of n phage deposits, and optionally d) additionally pack each of the second series of n phage deposits in a mixture consisting of all n original sera, except those used for the first panning; e) immunoregistering the second series resulting from n phage deposits using a mixture of all the n original sera to provide positive clones; f) testing the individual reactivity of all positive clones with a group of positive and negative sera by using an ordered array of clones as phage-secreting colonies; g) generate replicas of phage-secreting colonies; h) screen each replicate to determine its reactivity with positive and negative sera, reveal clones that react specifically with positive sera; i) using each of the phages that specifically react as ligated to affinity purify antibodies from a positive serum; j) test the antibodies to determine their reactivity with previously identified HCV peptides; k) particularize clones by detecting serum antibodies. The simple peptides that come from the above process are also an object of the present invention. In particular, in the preferred embodiment of the present invention applied to HCV, the following peptides are a further objective of the present invention: YSREQLNKLFGIDMT; YSREQLNKMFGIEIS; - YSREQLSKLFGIEPM; NSRWLSKAHGIEGM; YSREQLNKLFGIEVM; YSREQLSKLFGIDTQ; KSREQLSKLHGVDTS; - RSREQLSKLFGIDLT; M RTWLMKTHGIES; MLRTWLMKYQGIESW; YSRSWLMKAHGLELG; MMRSYLMKAHGIESL; MSRLWLMKAHGISSE; KHSEWLNKARGIESW; MSRTFLMKAHGIESW; MSRTWL KAHGIES; AEGEKKLRRST WGDPAK; AEGEFKTRRQTNYQDPAK; AEGEFKTLRNA RLDPAK; AEGEFKTLRMSMRLDPAK; AEGEFKKFPGSSTPKDPAKAAFDSL; AEGEFPQDARFPGGGDÁKAAFDSL; PQDARFPGGGDPAKAAFDSL; AEGEFKGAGGAQTVDWALLVDPAK; AEGEFMQKHFGGAQWINGDPAK; AEGEFLSKGSGGGQLRALVDPAK; AEGEFLSLKGSGGAQLRALVDPAK; AEGEFYLLKRSSPPDPAKAAFDSL; AEGEFPILVGPYLLPRRSREEAVDPAK; AEGEFPILVGPYLLPRRSREEAVDPAKGK AEGEFRLGVRAPRKALD A; AEGEFRLGVRALRKALDPAK; AEGEFRLGVRALRKAPDPAK; - - RLGVRALRKAPDPAK; AEGEFTQPRGHSYQDPAK; AEGEFLKERAEMSARKTLGADPAK; AEGEFFYQIPRRMETKYGDPAK; AEGEFSREQLNKLFGIEGDPA; AEGEFNSREWLSKAHGIEGMDPAK; AEGEFRSREQLSKLFGIDLTDPAK; AEGEFYSREQLN LFGIDMTDPAK; AEGEFYSREQLNKMFGIETSDPAK; AEGEFYSREQLKLFGIEVMDPAK; AEGEFKSREQLRKLHGFDTSDPAK; AEGEFKMRNYL KAFGIEMDPAK; AEGEFRSREQLSKLFGIELTDPAK; AEGEFSRREYSNKAFGIETQDPAK; AEGEFRRREYLNKAFGIEGGDPAK; AEGEFSRREWL KRFGIEYLDPAK; AEGEF SRTWL KAHGIESWDPAK; AEGEFYSPEWL ARGIDRSDPAK AEGEFKSREQLSKLHGVDTSDPAK; AEGEFYSREQ N MFGIElSDPAK; AEGEFYSREWLMKAHGLELDGPAK; AEGEFMMRSYLMKAHGIESKLDPAK; AEGEFMSRLWLMKAHGISSEDPAK; AEGEFPQPQEVHVYREQKLGLDPAKAAFDSL; AEGEFGEVLYRGFDEVGGDPAKAAFDSL; - - AGEPYVIERGMQDPAK; AEGEFTTASPRHFLVPLDPA AAFDSL; AEGEFTTASPAHFLVPLDPAKAAFDSL; AEGEFTTASPSHFLVPLDPAKAAFDSL; AEGEFATAPPRHYSWDPAK; AEGEFATAPPAHYSWDPAK; AEGEFATAPPSHYSDPAK; AEGEFRFW VPDYDPPAAGGDPAK; AEGEFTESSVSSTLADLASKTFGSADPAK; AEGEFTLADLATMTFGSTDPAK; AEGEFGLADLATLTFGSPDPAK; For the collection of the present invention, a preferred phage library of step a) is pVIII-12aa. For the collection of the present invention, the inserts of the preferred clones particularized from step k), have the sequences 1. SREQLNKLFGIEG; 2. RATLSNEHGITIG; 3. DQREN F YHGFG; 4. EWRRYMSDIHGYG; 5. DSLRYMYVMPGFG. In the collection of the present invention, a phage library is preferably generated in which the clones that react, most preferably the clones that react best, are partially mutagenized so that each amino acid of the clone sequence is independently replaced by another amino acid. In the collection of the present invention, preferably such clones have a preferred insert sequence: SREQLNKLFGIEG. In the collection of the present invention, in the phage library, the random sequence may be flanked by two cysteine residues. This aspect may be applied in the general use of the present invention and may not be limited to the case of HCV. An objective of the present invention is the use of the above collection for the preparation of a diagnostic assay for detecting infectious agents, such as viruses, in particular HCV in a subject suspected of being affected by infectious agents, such as viruses, in particular HCV. An objective of the present invention is a device for diagnostic purposes, comprising the above collection. One objective of the present invention is the available immunogenic peptides, either in the form of a collection or as a single peptide of the process described herein. Such peptides are useful as an immunogenic material, and are therefore useful for the preparation of vaccines, in particular against HCV. Conveniently, the peptides are in the form of the aforementioned equipment. Advantageously, in contrast to antigen-based systems, the diagnostic assay we tested has an implicit amelioration capacity: selections can be made for the purpose with those sera for which no response is detected. A similar methodology can be used to identify a group of peptides that differentiate between the different HCV serotypes, thus replacing costly and labor-intensive PCR methods with an EIA test that is cheaper and faster. The present invention will be described with additional details also by means of examples and figures, wherein in the latter: Figure 1 represents the characterization of clones derived from screening. The amino acid sequences of the selected clones are presented with the single-letter code. The upper and lower groups show sequences derived from the original or secondary library, respectively. The pVIII sequences flanking the foreign epitope are (H2) AEGEF [foreign epitope] DPAK. The gray rectangles indicate residues that occur more frequently at any given position in the clones of the original library. The residues that contribute to the consensus sequence of the peptides of the secondary library are indicated with bold. The data presented are average values of two independent tests and are related to the difference between the absorbance (A = A450 nm - A62o nm) measured in the indicated phage and measured in phage wt pC89 (Felici et al., 1991 ). the symbol * indicates that the serum was not tested. Figure 2 represents the identification of a phage that mimics the same antigenic determinant. Antibodies purified by affinity for positive C65 serum by PA1 clones, PA3, PA8, PA12 or P18 (which are included in the first column) are tested in ELISA to determine au reactivity against the same phage (and included in the first row). The data presented are average values of two independent tests and refer to the difference between the absorbance (A = A450 nm - A62o nm) measured in the indicated phage and the measurement in phage wt pC89 (Felici et al., 1991 ). Figure 3 represents the reactivity in ELISA of the deposit derived from the selection of the pVIIIA12 library. The phage deposit derived from the library is panning (scanning or examining and selecting) pVIIIA12 in positive sera C76 is tested in ELISA to determine its reactivity with positive sera C12, C13, C29, C40, C47, C65, C73, C74, C76, C83 and C85. The white, gray and black bars indicate the reactivity of the wt phage, the pVIIIA12 library and the p76? I deposit, respectively. The results of ELISA are expressed as A = A405 nm - A620 nm- The data presented are average values of two independent tests. Figure 4A represents. Reactivity of the ADAM-HCV mixture with sera. The discriminatory value) CO = 0.232 is calculated as CO = N + 5s where N and s are the average and the standard deviations, respectively, of the data obtained with negative sera. Figure 4B depicts EIA of ADAM-HCV in a group of sera obtained from the Italian red cross. The discriminatory value is calculated as CO = N + 5o where N and o are the average and the standard deviations, respectively, of the data obtained from five negative control sera. The ELISA test, as described in materials and methods, detects the binding of peptides to antibodies present in human sera. Average values are collected from two independent experiments. The results are expressed as the relationship between the measured signal and the discriminatory value (S / CO). The number of sera tested for each group of sera is indicated. Figure 5 EIA of ADA / HCV in a group of sera not yet determined. The left column indicates the name of the HCV peptides tested, grouped according to their binding specificity. The following four columns present the reactivities of the peptides that are included with positive (c25 and rl5) and negative (r6 and rl3) control sera. Each additional column presents the reactivities of the peptides in the list, with the sera not yet determined. The binding of antibodies to HCV peptides present in human sera is detected by ELISA as described in Materials and Methods. The average values of two independent experiments have been determined. For each peptide the discriminatory value (CO) is calculated as CO = N + 5 where N and are the average and the standard deviation, respectively, of the data obtained from 31 negative control sera. The results are expressed as the relationship between the measured signal and the discriminatory value (S / CO). Figure 6 is ADAM-HCV / SIA on positive, negative and undetermined sera. The following ADAM-HCV peptides are grouped according to their binding specificity and immobilized on a nylon membrane to obtain 10 bands ml909.2 and ml913.2 (A); ml901.31, m3322.3, m3362.3 (B); ml977.1 (C); m3551.3 (D); m3566.3 (E); m858, mF78 and H1 (F); mA12.1, mA12.2 and mA1 .12 (G); mB11.17 (H); mg21.2 (I); ml929A3.1, ml929C3.4 and ml929.21 (J). An additional line containing purified human IgG is included as a positive internal control. The binding of antibodies to HCV peptides present in human sera is detected as described in Materials and Methods. In the most preferred embodiment of the present invention, multiple antigen (MAP) peptides, in which the C-terminal part of eight identical peptide sequences join a common core (Tam, JP: Synthetic peptide vaccine design: aynthesis and properties of a high-density multiple antigenic peptide system, Proc.Nat.Acid Sci.85 (1988) 5409-5413), are synthesized, provide reagents that maintain a specificity capacity comparable to the peptides generated by phages. The separation of the phage scaffold allows a higher peptide concentration of the immobilized ligand, which results in greater sensitivity. Preferably, the group of HCV-specific ligands available according to the present invention includes peptides that mimic NS3 of HCV, an immunodominant antigen of HCV. In the case of the preferred embodiment, as far as we know, this is the first time that a short peptide mimicking an immunodominant determinant of NS3 has been described. Pepscan analysis of the NS3 protein reveals larger sequences which rarely react with positive sera (Khudyakov, Y., et al., 1995. Virology, 206: 666-672). In contrast, other solutions (Santini C. Brennan D, Mennuni C. Hoess RH, Nicosia A, Cortese R, Luzzago A, Efficient display of an HCV cDNA expression library as C-terminal fusion to the capsid protein D of bacteriophage lambda. Mol Biol 1998 SEP 11; 282 (1): 125-35; Pereboeva J.

- - Med. Virol 1998 Cot; 56 (2): 105-11) have isolated large protein domains which retain antigenic properties. The solution we have developed in this way contains the intrinsic properties and is not based on the use of information regarding HCV antigens, and can even be applied to systems where the antigen is unknown, thus generating a process that leads to the discovery of the antigen. Random peptide libraries of phages displayed (phage libraries) represent a useful tool for identifying ligands which are specifically recognized by serum antibodies against HCV. Phage-generated peptides have a broad imitation potential, since they are capable of mimicking linear, conformational and even non-proteinaceous epitopes (for a review see Felici F, Luzzago A, Monaci P, Nicosia A, Sollazzo M, Traboni C Peptide and protein display on the surface of filamentous bacteriophag Biotechnol Annu Rev. 1995; 1: 149-83; Z ick MB, Shen J, Scott JK.Phage-displayed peptide librarles.Curr Opin Biotechnol., 1998 Aug; 9 (4 ): 427-36). In the present invention, it is reported the identification of a broader collection of effective ligands specific for HCV and the development of a novel type of diagnostic equipment for the detection of antibodies against HCV in the serum.

- - The peptides provided in the present invention are useful as diagnostic materials or as immunogens or vaccines. The pharmaceutical compositions comprising immunogens and vaccines according to the present invention are conventionally prepared as commonly understood by those skilled in the art. For example, they can be prepared as described in EP 0 698 091. The following example further illustrates the invention. EXAMPLE Identification of HCV specific ligands, with novel binding specificities The pVIII-12aa phage library is panned (screened or examined and selected) on 8 positive sera (C13, C14, C27, C29, C40, C47, C62 and C65) to generate 8 phage deposits (indicated as P131, pl4 ?, P271,? 29 ?,? 401, P471, P621 and p65 ?, respectively). Eight mixtures are prepared, each with a different combination of seven of the eight phage deposits. Each mixture is selected by affinity using the serum that generates the phage-excluded deposit: for example, the p65 mixture mix consisting of the P131, pl4x, p27x, p29x,? 40 ?, p47J and p62x deposits, the C65 serum is panned. Each of the eight resulting phage deposits (indicated as P1311, P1411, etc.) are individually paneled against a substituted mixture of all the original sera except those used for the pre-selection. For example, reservoir 1311 is panned over the mixture of sera C14, C27, C29, C40, C47, C62 and C65. The immuno-labeling of the eight resulting phage deposits using a mixture of all eight of the original sera shows a large number of positive clones. We tested the individual reactivity of each of these clones with a group of positive and negative sera by generating an ordered distribution of the clones as phage secretory colonies. After growth, the entire clone group is tapped as an array ordered on a nitrocellulose filter using a multi-tip device. This procedure is repeated to generate many copies, which are then screened individually and simultaneously to determine their reactivity with positive and negative sera, showing many clones which react specifically with positive sera. Each of these phages is used as a binder to affinity purify antibodies from a positive serum. These antibodies are then tested in ELISA to determine their reactivity with any of the four groups of HCV peptides previously identified (ref: Prezzi, C, Nuzzo, M., Mela, A., Delmastro, AR, Galfre ', C, Cortese, R., Nicosia, A. and Monaci, P. 1996, 1. Immunology 156: 4504-4513.

- - Bartoli et al., Nature Biotechnology Volume 16, November 1998; 1068-1073). This analysis separates 12 clones that detect serum antibodies with novel binding specificity and in this way indicate that they are antigenic determinants that are different from those already identified. The sequence analysis shows 5 different sequences. The culture supernatants of each of these 5 clones (PAl, PA3, PA8, PA12 and PAl8) are prepared and their reactivity is tested by ELISA with 30 different positive and 24 negative sera (figure 1). Only clones PA8 and PA12 show statistically significant reactivity with positive sera (p <0.2). The PAI phage is then used to immunopurify antibodies from C65 serum. These purified antibodies react specifically with clones PA3, PA8, PA12, PA18 as well as with the PAI clone itself, indicating that all these phages can be grouped into a single class recognized by antibodies of the same specificity (Figure 2). No reactivity is detected with any of the above clones when the wild type is used as a ligand or when the antibodies are affinity purified from a negative serum. When a different clone of the same group is used to immunopurify serum antibodies or when a different positive serum is used, we obtain results - - consistent with the reactivities presented in figure 1. For example, when phage PA3 is used to immunopurify antibodies from the same serum, these antibodies react with clones PA8 and PA12, in addition to the same PA3. These results indicate that clones PA1, PA3, PA8, PA12 and PA18 detect antibodies that react with the same B lymphocyte epitope, as suggested by the weak similarity between the peptide sequences (figure 1). Affinity maturation of the HCV peptide A phage library is generated in which the sequence of clone PA12 is partially mutagenized. In this "secondary" library called pVlllA12, oligonucleotides are synthesized so that each amino acid of the sequence SREQLNKLFGIEG is independently substituted by another amino acid: in theory, a substitution at each position would occur at a frequency of 20 percent. In addition, a random residue is included at both sites of the foreign peptide sequence. The pVlllAl2 library is paired twice with 12 positive sera (C8, CIO, C12, C13, C22, C58, C60, C76, C83, C85, C141 and C177). When tested in ELISA, the p76IX, 14111 and pl77IT phage deposits (derived from the selection using sera C76, C141 and C177, respectively) show the highest and most extensive reactivity with a group of positive sera and were further analyzed. (figure 3). On the basis of this reactivity, p76n, P14111 and p777 phage deposits are immunocruted by the use of sera C40, C141 and C177 respectively. For each deposit, this analysis takes several clones that are then individually tested to determine their reactivity with many different sera, positive and negative, by means of a radiofrequency protocol, as described above. This final screening separates 51 clones that react specifically with positive sera. The sequence analysis shows 16 different sequences (8, 5 and 3 derived from the deposit P7611, pl41i: r and plll11, respectively). The culture supernatants of each of these clones are prepared and their reactivity is tested by ELISA with 33 different sera positive and 24 negative (figure 1) · Clones P40.17 and P40.7 react with most of the positive sera tested (42% each). Its combination with clones P141.7 and P177.22 qualify 70% of the group's positive serum. The alignment of the selected peptides defines a consensus sequence (M / Y) SRE (W / Q) L (M / N) K (A / L) (H / F) GIES (W / M).

Identification of additional ligands specific for HCV Carried out similar selection strategies with the objective of identifying ligands with novel binding specificities different from those previously identified. Phage libraries of various lengths are subjected to screening in which the random sequence is completely random or flanked by two cysteine residues that limit the conformation of the peptide shown. Various combinations of sera and phage deposits are used to select phage libraries. Phage deposits show interesting reactivity profiles with positive sera and are further analyzed. The determination of the reactivity of a large number of individual clones by means of copy screening separates phages showing a specific reactivity with positive sera. We focus our attention on clones with interesting reactivity patterns. Probing these clones with purified affinity antibodies of the HCV peptides removes these clones which mimic antigenic determinants of the previously identified peptides. The clones that survive these selection stages are tested in ELISA with a large number of positive and negative sera to statistically define their specificity for HCV. These selected clones are further enhanced by creating and screening secondary libraries in which the sequence of the original clone or a population of clones is subjected to partial mutagenicity and screened again to take variants with improved binding properties. This stage is carried out by adopting different strategies that have already been described (reference to the Urbanelli document, Zhu). This repetitive effort identifies 7 novel groups of ligands which specifically bind specific HCV-specific serum antibodies with different binding specificities. The screening of a repertoire exhibited in phage of variant HVR1 using peptides isolated from serum that react specifically with a large number of positive sera (Puntoriero et al.; Nicosia unpublished). A set of peptides generated by phage HVR1 derived from this screening is analyzed to determine its reactivity with our group of sera. Three peptides are identified with the highest and most specific reactivity frequency, with positive sera (mF78, mHl and m858). In summary, 12 groups of ligands are identified, which include the 4 groups previously identified (Bartoli et al., Nature Biotechnology Volume 16, November 1998, 1068- - - 1073, Prezzi, C, Nuzzo, M., Melala, A., Delmastro, AR, Galfre ', C, Cortese, R., Nicosia, A. and Monaci, P. 1996, 1. Immunology. 156: 4504-4513.); see FIG. 5, groups A to L. Of the peptide generated in phage to synthetic Twenty-two peptide sequences derived from the 12 groups of HCV ligands are synthesized as multiple antigen peptides with eight branches (DAM-HCV peptides). In this molecule, eight identical peptide sequences are linked through a lysine fork to a common core to form a multiple display similar to that of the peptides fused to pVIII in the phage capsid. The sequences of the ADAM-HCV peptides are presented below, m858 ETYTTGGAAARTTSGLTSLFSPGPSQN ml901.31 AEGEFKKFPGSSTP DPAKAAFDSL ml901.34 AEGEFPEDTFPGSKLILSGDPAKAAFDSL mi909.2 AEGEFKTRRNTNYQDPAK mi913.2 AEGEFKTLRNTNRLDPAK ml929.21 AEGEFATASPTHYTSELDPAK ml929A3. AEGEFTTASPTHFLVPKDPAK ml929C3.4 AEGEFATAPPSHY? WDPAK mi977.1 AEGEFPYLLPRRSREEAVDPAK m3322.3 AEGEFPQDARFPGGGDPAK m3362.3 AEGEFLSLKGSGGGQLRALVDPAK m3351.3 AEGEFRLGVRALRKAPDPAK m3566.3 AEGEFKTSVRSVPRARPPINGDPAK MA12 MA12 .2 .1 AEGEFNSREWLSKAHGIEG DPAK AEGEFRSREQLSKLFGIDLTDPAK MA12 .13 .17 AEGEFMSRTWLMKAHGIESWDPA MBLL AEGEFRELLYEAFDDMEGDPA mF78 QTHTTGGQAGHQAHSLTGLFSPGAKQN MG21 .2 AGEPYVIEQGM DPAK mHl QTHTTGGWGHATSGLTSLFSPGPSQN m 15. .3 AEGEFGLADLATLTFGSTDPAK mS48, .5 AEGEFRF KVPDYDPPAAGGDPAK the data regarding its performance summary in table 1, below.

TABLE 1 Table 1. Reactivity of the ADAM-HCV peptides N, number of tested sera; S, measured signal; CO, discriminatory value (CO = neg + 3, where neg and are the mean and the standard deviation of the negative serum data) Sera negative positive sera peptide group N N% S / CO ^ l mean S / C0 > 1 A ml913.2 33 21 100 45.0 ml909.2 33 21 100 50.3 B ml901.31 39 38 61 3.2 ml901.34 39 38 39 5.4 ?? 3322.3 30 18 78 10.1 m3362.3 31 37 84 4.4 C ml977.1 32 28 96 13.6 D ni3551.3 37 26 46 3.0 E m3566.3 14 12 50 2.1 F m858 40 41 73 8.9 mHl 60 60 68 9.8 mF78 64 51 80 5.8 G mA12.1 37 45 64 8.4 mA12.2 37 45 33 8.6 mA12.13 38 45 31 4.4 H mBll .17 35 24 46 2.7 I mG21.2 34 34 26 4.0 J ml929C3.4 35 33 27 7.1 ml929A3.1 43 25 40 3.9 ml929.21 13 18 72 9.7 K mS48.5 29 9 67 7.0 L m 15.3 36 21 10 15.2 In many cases, the pVIII sequences flanking the foreign epitope (NH2AEGEF and / or DPAK-COOH) are shown to be relevant to the binding specificity of the corresponding peptide. A mixture containing these 22 ADAM-HCV peptides (mixture of ADAM-HCV) is used to detect the presence of antibodies against HCV by EIA (EIA of ADAM-HCV). The ADAM-HCV mixture of peptides is immobilized by passive coating on the bottom of the multi-well ELISA plate and incubated with diluted serum samples 1:40 for 40 min. Human antibodies bound to the peptides are detected by incubating for 20 min with antibody against human conjugate and measured by the chromogenic enzymatic reaction. As reported in Figure 4A, the ADA-HCV EIA discriminates effectively between positive and negative sera. ADAM-HCV / EIA A group of HCV positive and HCV negative sera are tested for the presence of antibodies against HCV by ADAM-HCV-EIA (Figure 4B). The test identifies all positive sera included in the group and also shows 100% specificity when identifying all negative samples. Undetermined samples A collection of sera that are diagnosed as not determined is obtained from various sources, according to the commercially available HCV confirmatory assay. The 23 ADAM-HCV peptides are tested individually in ELISA to determine their reactivity with 31 samples (FIG. 5) . 6 samples do not react with any of the MAPs tested and therefore qualify as negative. 8 samples recognize only one antigen, and therefore confirm the indeterminate analysis. Finally, 17 samples present two or more reactions against different peptide groups, and are therefore identified as positive.

ADAM-HCV Strip Immunoblot Assay (ADAM-HCV / SIA) The ADAM-HCV peptides covalently immobilized on an activated nylon membrane to obtain a strip with ten bands. Each line includes different peptides with the same binding specificity, as detailed in the legend of Figure 6. Also included as an internal positive control is a control line containing purified human IgG. A selected number of samples from the group of indeterminate sera is tested to determine their reactivity with immobilized antigens by incubating serum samples with the strip. Antibodies against HCV retained by individual antigens visualized by incubating the strips with antibodies against human, conjugated with enzyme followed by a colorimetric enzymatic reaction. The reactivity of the samples with the peptide bands is determined by visually comparing the intensity of each band with that of an internal positive control. The ADAM-HCV / SIA test shows the reactivity of all of the 8 positive sera tested for the various different viral determinants imitated by the ADAM-HCV peptides. No reactivity is detected when 8 negative sera tested (figure 6). We also analyzed by ADAM-SIA a selected number of samples not determined. As shown in Figure 6, the analysis of 8 undetermined sera confirms the results obtained by ADAM-HCV / EIA using individual peptides, indicating a comparable sensitivity of the two assays.

Materials and Methods Phage Libraries Four libraries of random peptides displayed by phage used as a source of ligands: pVIII9aa, pVIII9aa_cys, pVIII12aa, pVIII15aa and pVIIIA12. pVIII9aa (Felici et al., 1991), pVIII12aa and pVIIUSaa three different libraries made up of groups with 9 units, 12 units and 15 random units, respectively, which shown in filamentous phages as fusion of the terminal H2 part of the protein of main coating pVIII. pVIII9aa_cys is a library in which the random nonapeptide is flanked by two cysteine residues (Luzzago et al., 1993). In this last library, the cysteines promote the formation of a disulfide bridge that limits to a certain extent the conformation of the peptide exhibited. The pVIIIA12 library is constructed by synthesizing an oligonucleotide that codes for the amino acid sequence SREQLNKLFGIEG. By adopting a split-resin synthesis methodology (Glaser et al., 1992), each - - amino acid position is replaced with an NNS triplet, with a frequency of 20 percent. In addition, a random residue is included at both sites of the foreign peptide sequence. All of the five libraries have been generated as described (Folgori et al., 1998). The complexity of the library, as derived from the number of individual clones obtained by bacterial transformation is approximately 1 x 108 for each of the five libraries.

Human serum The human sera used in this study randomly collected from rejected blood units from donors of transfusion centers and hospital departments and units of healthy volunteers. Specifically, many of the undetermined samples used in this study obtained from the Laboratory of Virology, Instituto Superiore di Sanita Rome (Italy) and the Sangue della Croce Rossa Italian Nafional Transfusion Center, Rome (Italy). The sera have been tested for the presence of antibodies to HCV by the second generation HCV ELISA test system (Ortho Diagnostic Systems, Bersee, Belgium) and confirmed with a test system for HCV RIBA dotted immunoassay. of the first generation (Chiron Co., Emeryville, CA). The sera also tested for the absence of antibodies to HBsAg and HIV-1 / HIV / 2 by the EIA test for AUSAB (Abbott Labs, Chicago, IL) and by the EIA test for HIV-1 / HIV / 2 of the third generation (Abbott Labs, South Pasadena, CA). Samples positive for the presence of antibodies against HCV, but negative for antibodies against HBsAg and against HIV, included in this study as positive sera for HCV. Negative sera for the presence of antibodies against all three antigens included in this study as negative sera for HCV.

Affinity and immuno-labeled selection HCV-positive sera are used to randomly select libraries of random peptides displayed on phage, as described (Folgori et al., 1998, Felici et al., 1996, Prezzi et al., 1996). Clones derived from affinity selection are subjected to immuno-labeling using sera as described (Prezzi et al., 1996, Minenkova et al).

ELISA that uses phage clones The ELISA test using phage supernatant and human sera is carried out as follows. The phage supernatants are prepared from DH5-F 'infected cells, as previously described (Felici et al., 1991). Multiple well plates (Immunoplate Maxisorp, Nunc, Roskilde, Denmark) are coated overnight at 4 ° C with 200 1 of monoclonal antibody against pIII, 57D1 (Dente et al., 1994) at a concentration of 1 g of antibody / ml in 50 mM NaHCO 3, pH 9.6. After discarding the coating solution, the plates are incubated at 37 ° C for 60 min with ELISA blocking buffer (0.1% casein, 1% Triton-X100 in PBS). The plates are washed several times with PBS / 0.05% Tween-20 (wash buffer). To each well is added a 1: 1 mixture of ELISA blocking buffer and purified phage supernatant, to allow attachment for 1 h at 37 ° C. Human serum diluted 1:40 is incubated for 30 min at room temperature with 5 x 1010 plaque forming units (pfu) / ml of phage fll.l (Dente et al., 1996), 25 1 / ml of an extract of DH5-F 'cell protein with phage fll.l (Dente et al.), and 25 1 / ml supernatant of unrelated rat hybridoma cells in ELISA blocker buffer. After discarding the mixture containing the phage supernatant, plates are washed with wash buffer and 200 1 of pre-incubated serum mixture are added to each well and incubated for 60 min at 37 ° C. The plates are then washed with a wash buffer and a 1: 20,000 dilution of goat antibody against each well is added to each well.

Human IgG conjugated to HRP (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) in secondary antibody blocking buffer (Triton 1%, horse serum 1%, fetal bovine serum 50% in PBS, Mab 5 1 supernatant / well) . After 30 min of incubation at 37 ° C, the plates are washed and the peroxidase activity is detected by incubation with 200 1 of a TMB liquid substrate system (SIGMA, St. Louis, O). After 15 minutes of development, the reaction is stopped by adding 25 1 of 2M H2SO4. The plates are read in an automated ELISA reader (Labsystems Multiskan Bichromatic, Helsinki, Finland) and the results are expressed as A = A450 nra - A62o nm- The ELISA data reported in the exhibition articles are the average values of two tests independent They are considered statistically significant if they are greater than the discriminatory value of 0.3 and differ by more than 3 (. {1/2 [p + 2w].} 1 2) of the background signal observed for phage wt. The p-value referred to clones PA8 and PA12 is the probability that the distributions of observed frequency reactivities of the positive and negative sera are statistically the same, according to the X2 test.

Affinity purification of serum phago- topo-specific antibodies A 60 mm diameter polystyrene petri dish (Becton Dickinson Labware, NJ) is coated overnight at 4 ° C with a solution of 1 x 1011 phage particle purified with CsCl / ml in 50 mM NaHCO 3, pH 9.6. After washing with ??? / Tween, the box is incubated for 60 min at 37 ° C with ELISA buffer buffer. A mixture of human serum (dilution 1/100 in ELISA blocking buffer), 1? 1012 fll.lpf / ml and 25 1 / ml protein extract of XLl-blue cells are incubated for 60 min at room temperature. After discarding the blocking solution, the pre-incubated mixture is added to the plate and incubated overnight at 4 ° C. The dilution of serum is discarded and the box is washed with washing buffer. The bound antibodies are eluted with glycine hydrochloride 0.1, pH 2.7 with 10 g / ml BSA, and neutralized.

Characterization of phage clones Affinity-purified antibodies are tested in the standard ELISA to determine their reactivity against phagocytes. Typically, multiple well plates are coated with 100 1 / well of a 1 x 1011 TU / ml solution of phage purified with CsCl in 50 mM NaHCO 3, pH 9.6 overnight, at 4 ° C. After washing with PBS / Tween, the plates are incubated for 60 min at 37 ° C with blocking buffer. Subsequently, 100 1 affinity purified antibodies are added to each well, and allowed to bind overnight at 4 ° C. The plates are then washed with cold PBS / Tween and 100 1 / well of goat antibody is added to human IgG (specific for Fe) conjugated with alkaline phosphatase (Sigma, St. Louis, MO) diluted 1/5000 in blocking buffer. After incubation at room temperature for 2 hours, the plates are washed and the alkaline phosphatase is revealed as described above.

Synthetic peptides We have used synthetic octa-branched multiple antigen (MAP) peptides: (Tam, 199X). The synthesis is carried out by the flow-polyamide method (Pessi et al., 1990). The peptides are dissolved in dimethyl sulfoxide.

ELISA that uses synthetic peptides Multiple well plates (Immuno piate Maxisorb, Nunc, Roskilde, Denmark) are coated overnight at 4 ° C with a MAP solution at a concentration of 10 g / ml in 50 mM NaHCO 3, pH 9.6. After discarding the coating solution, the plates are incubated at 37 ° C for 60 min with ELISA blocking buffer (0.1% casein, 1% Triton-X100). The plates are washed several times with PBS / 0.05% Tween-20 (wash buffer). Human diluted serum 1:40 is added to each well and incubated for 40 min at 37 ° C. The plates are then washed with wash buffer and a 1: 20,000 dilution of goat antibody against human IgG conjugated to HRP (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) in ELISA buffer is added to each well. After an incubation period of 20 min at 37 ° C, the plates are washed and the peroxidase activity is detected by incubation with a liquid substrate of TMB ((SIGMA, St. Louis, MO) After 15 minutes of development , the reaction is stopped by adding 2M H2SO4 The plates are read in an automated ELISA reader (Labsystems Mutiskan Bichromatic, Helsinki, Finland) and the results are expressed as A = A450nm - A62o ™ The ELISA data reported in articles of screen are average values of two independent tests.

References: Smith, C. P. and Petrenko, v.A. 1997. Phage display. Chem. Rev. 97: 391-410 Folgori, A., Tafi, R., Meola, A., Felici, F., Galfre, G., Cortese, R., Monaci, P. and Nicosia, A. 1994. A general strategy to identify mimotopes of pathological antigens uaing onlu random peptide libraries and human sera. EMBO 1. 13: 2236- "43. Prezzi, C, Nuzzo, M., Mela, A., Delmastro, AR, Galfre ', C, Cortese, R., Nicosia, A. and Monaci, P. 1996, 1 Immunology 156: 4504-4513, Alter, H. J. 1995. To C or not to C: these are the questiona., Blood 85: 1681. Felici, F., Castagnoli, L., Musacchio, A. , Jappelli, R. and Cesareni, C. 1991. Selection fo antibodies ligands from a large library of ilogopeptides expressed on a multivalent exposure vector, f.Mol. Biol. 222: 301-310 Luzzago, A., __Felici, F. , Tramontano, A., Pessi, A. and Cortese, R. 1993. Mimicking of discontinuous epitopes by phage displayed peptides, I. Epitope mapping of human H ferritin using a phage library of constrained peptides, Gene. 128: 51-57. Dente, L., Cesareni, G. Micheli, C, Felici, F., Folgori, A., Luzzago, A., Monaci, P., Nicosia, A. and Delmastro, P. 1994. Monoclonal antibodies that recognize filamentous phage Useful tools for phage display technology, Gene. 148: 7, Sambrook, J., Fritsch, T. and Maniatis, T. 198 9. Molecular Cloning: a laboratory manual (second edition) Takamizawa, A., Mori, C, Fuke, I., Manabe, S .; - - Murakami,?., Fujita, J., Onoshide, E., Andoh, T., Yoshida, I. and Okayama, H. 1991. Structure and organization of the Hepatitis C virus genome isolated from human carriers. I. Virol. 65: 1105-1113. Smith, D. B. 1993. Purification of glutathione S- Transferase fusion prteins. Methods in molecular and cellular biology. 4: 220-229. Frangioni, f.V. and Neel, B.J. 1993. Solubilization and purification of enzymatically active glutathione S-transferase (pGex) fusion proteins. Analyt. Biochem. 210: 179-187. Komatsu F, Takasaki F 1999 Determination of serum hepatitis C virus (HCV) core protein using a novel approach for quantitative evaluation of HCV viraemia in anti -HCV-positive patients. Liver 19: 375-80.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (38)

1. CLAIMS Having described the invention as above, the content of the following claims is claimed as property: 1. A method for making a diagnosis of the presence of an antigen, ccterized in that it comprises identifying the binding specificity of the antibody molecules against antigen in the serum by the methodology of detection of antibody by antigen mimic (ADAM, for its acronym in English) , ccterized in that it comprises screening phage libraries using sera from patients infected with antigen and uninfected individuals, identifying antibodies (ligands) that bind peptides specifically associated with such an antigen. 2. The method according to claim 1, ccterized in that the ligands are improved by in vitro maturation strategies. 3. The method of compliance with the claim 1 or 2, ccterized in that the ligands are synthetic peptides. 4. The method according to claim 1 or 2 or 3, ccterized in that the ligands are joined to a common core. 5. The method according to claim 4, ccterized in that the ligands together with the common core are MAP. 6. A collection of antigen-specific ligands, ccterized in that they are accessible by the process comprising: a) first panning (scanning or performing an examination and selection) of a phage library in n positive sera to generate a first series of n deposits of phages; b) prepare n mixtures of deposits containing n-1 deposits; c) select by affinity each of the n mixtures against the serum that generated the phage-excluded deposit, to provide a second series of n phage deposits, and optionally d) additionally pack each of the second series of n phage deposits in a mixture consisting of all n original sera, except those used for the first panning; e) immunoregistering the second series resulting from n phage deposits using a mixture of all the n original sera to provide positive clones; f) testing the individual reactivity of all positive clones with a group of positive and negative sera by using an ordered array of clones as phage-secreting colonies; g) generate replicas of the secretory colonies of f gos; h) screen each replicate to determine its reactivity with positive and negative sera, reveal clones that react specifically with positive sera; i) using each of the phages that specifically react as ligated to affinity purify antibodies from a positive serum; j) testing the antibodies to determine their reactivity with previously identified antigen-specific peptides; k) particularize clones by detecting serum antibodies. 7. A method for carrying out the diagnosis of hepatitis C, ccterized in that it comprises identifying the binding specificity of antibody molecules against HCV in the serum by the methodology of detection of antibody by antigen imitador (ADAM, for its acronym in English), which comprises screening phage libraries using sera from patients with HCV and uninfected individuals, identifying antibodies that bind peptides (ligands) specifically related to HCV infection 8. The method according to claim 7, ccterized in that the ligands are improved by in vitro maturation. 9. The method according to claim 7 or 8, ccterized in that the ligands are synthetic peptides. 10. The method according to claim 6, 7 or 8, ccterized in that the ligands are joined to a common core. 11. The method according to claim 8, ccterized in that the ligands together with the common core are an AP. 12. A collection of specific ligands for HCV accessible by the process ccterized in that it comprises: a) first panning (scanning or performing an examination and selection) of a phage library in n positive sera to generate a first series of n blood deposits;; b) prepare n mixtures of deposits containing n l l deposits; c) select by affinity each of the n mixtures against the serum that generated the phage-excluded deposit, to provide a second series of n phage deposits, and optionally d) additionally pack each of the second series of n phage deposits in a mixture consisting of all n original sera, except those used for the first panning, - e) immunoregistering the second series resulting from n phage deposits using a mixture of all the n original sera to provide positive clones; f) testing the individual reactivity of all positive clones with a panel of positive and negative sera by using an ordered array of clones as phage-secreting colonies; g) generate replicates of phage-secreting colonies; h) screen each replicate to determine its reactivity with positive and negative sera, reveal clones that react specifically with positive sera i) use each of the phages that react specifically as bound to affinity purification antibodies from a positive serum; j) test the antibodies to determine their reactivity with previously identified HCV peptides; k) particularize clones by detecting serum antibodies. 13. The collection according to claim 12, characterized in that the phage library of step a) is pVIII-12aa. 14. The collection according to claim 12 and 13, characterized in that the inserts of the particularized clones of step k) according to claim 12, have the following sequences: SREQLNKLFGIEG; RATLSNEHGITIG, DQRENWFKYHGFG, EWRRYMSDIHGYG, DSLRYMYVMPGFG. 15. The collection according to claim 12, characterized in that the phage library is generated in which the clones that react best are partially mutagenized so that each amino acid of the clone sequence is independently substituted by another amino acid. 16. The collection according to claim 12, characterized in that a phage library is generated in which the reactive clones are partially mutagenized so that each amino acid of the clone sequence is independently substituted by another amino acid. 17. The collection according to claim 15 or 16, characterized in that the clones have the following sequence of inserts: SREQLNKLFGIEG. 18. The collection according to any of claims 12 to 17, characterized in that in the phage library, the random sequence is flanked by two cysteine residues. 19. The collection according to any of claims 12 to 18, characterized in that the peptide sequence is joined in a common nucleus. 20. The collection according to claim 17, characterized in that the peptide together with the common nucleus is A.P. 21. The use of the collection, according to any of claims 12 to 20, for the preparation of a diagnostic assay for detecting HCV in a subject suspected of being affected by HCV. 22. A device for diagnostic purposes, characterized in that it comprises a collection according to claim 6. 23. A device for the diagnosis of HCV, characterized in that it comprises a collection according to any of claims 12 to 20. 24. The equipment according to claim 22 or 23, characterized in that it comprises strips where the collection is immobilized thereto. 25. The equipment according to claim 24, characterized in that the strip also contains an internal standard. 26. The peptide, characterized in that it is selected from the group consisting of: i) SREQLNKLFGIEG; ii) RATLSNEHGITIG; iii) DQRENWFKYHGFG; i) EWRRYMSDIHGYG; v) DSLRYMYVMPGFG; vi) YSREQLNKLFGIDMT; VÜ) YSREQLNKMFGIEIS; viii) YSREQLSKLFGIEPM; ix) NSRWLS AHGIEGM; x) YSREQLNKLFGIEVM; xi) YSREQLSKLFGIDTQ; xii) KSREQLSKLHGVDTS; xiii) RSREQLSKLFGIDL; xiv) MWRTWL KTHGIESW; xv) MLRTWLM YQGIESW; xvi) YSRS LMKAHGLELG; xvii) M RSYLMKAHGIESL; xviii) MSRLWLMKAHGISSE; xix) KHSEWLNKARGIESW; xx) MSRTFLMKAHGIESW; xxi) MSRT LMKAHGIES; xxíi) AEGEKKLRRSTNWGDPAK; xxiii) AEGEFKTRRQTNYQDPAK; xxiv) AEGEFKTLRNANRLDPAK; xxv) AEGEFKTLRNSNRLDPAK; xxvi) AEGEFKKFPGSSTPKDPAKAAFDSL; xxvii) AEGEFPQDARFPGGGDPÁKAAFDSL; xxviii) PQDARFPGGGDPA AAFDSL; xxix) AEGEFKGAGGAQTVDWALLVDPA; xxx) AEGEFMQKHFGGAQWIMGDPAK; xxxi) AEGEFLSKGSGGGQLRALVDPAK; xxxii) AEGEFLSLKGSGGAQLRALVDPAK; xxxiii) AEGEFYLLKRSSPPDPAKAAFDSL; xxxiv) AEGEFPILVGPYLLPRRSREEAVDPAK; XXX) AEGEFPILVGPYLLPRRSREEAVDPAKGK; xxxvi) AEGEFRLGVRAPRKALDPAK; xxxvii) AEGEFRLGVRALRKALDPAK; xxxviii) AEGEFRLGVRALRKAPDPAK; ????) RLGVRALRKAPDPAK; ??) AEGEFTQPRGHSYQDPAK; xli) AEGEFLKERAEMSARKTLGADPA; xlii) AEGEFFYQIPRRMETKYGDPAK; xliii) AEGEFSREQLNKLFGIEGDPAK; xliv) AEGEFNSREWLSKAHGIEGMDPAK; xlv) AEGEFRSREQLSKLFGIDLTDPAK; xlvi) AEGEFYSREQLNKLFGIDMTDPAK; xlvii) AEGEFYSREQLNKMFGIETSDPAK; xlviii) AEGEFYSREQLNKLFGIEVMDPAK; xlix) AEGEFKSREQLRKLHGFDTSDPAK; 1) AEGEFKMRNYLNKAFGIEGMDPAK; li) AEGEFRSREQLSKLFGIELTDPAK; lii) AEGEFSRREYSNKAFGIETQDPAK; liii) AEGEFRRREYLNKAPGIEGGDPAK; liv) AEGEFS REWLN RFGIEYLDPAK; lv) AEGEFMSRTWLMKAHGIESWDPAK; lvi) AEGEFYSPEWLNKARGIDRSDPAK; lvii) AEGEFKSREQLSKLHGVDTSDPAK; lviii) AEGEFYSREQLN FGIEISDPAK; lix) AEGEFYSRS L KAHGLELGDPAK; lx) AEGEFMMR? YLMKAHGIESLDPAK; lxi) AEGEF SRLWLMKAHGISSEDPAK; lxii) AEGEPPQPQEVHVYREQLGLDPAKAAFDSL; lxiii) AEGEFGEVLYRGFDEVGGDPAKAAFDSL; lxiv) AGEPYVIERGMQDPAK; lxv) AEGEFTTASPRHFLVPLDPAKAAFDSL; lxvi) AEGEPTTASPAHFLVPLDPAKAAFDSL; lxvii) AEGEFTTASPSHFLVPLDPAKAAFDSL; lxviii) AEGEFATAPPRHYS DPAK; lxix) AEGEFATAPPAHYSWDPAK; lxx) AEGEFATAPPSHYSDPAK; lxxi) AEGEFRFWKVPDYDPPAAGGDPAK; lxxii) AEGEFTESSVSSTLADLASKTFGSADPAK; lxxiii) AEGEFTLADLATMTFGSDPAK; lxxiv) AEGEFGLADLATLTFGSPDPAK; 27. The use of peptides, according to claim 26, in the method according to claims 7 to 11. 28. A collection, according to any of claims 12 to 20, characterized in that it comprises at least one of the peptides according to claim 26. 29. A device for detecting an infection, characterized in that it comprises the collection in accordance with the claim 6. The equipment, according to claim 29, characterized in that it comprises strips where the collection is immobilized therein. 31. The equipment, according to claim 30, characterized in that the strip also contains an internal standard. 32. A device for detecting an infection by HCV, characterized in that it comprises a collection according to any of claims 12 to 20. 33. A device for detecting an infection by HCV, characterized in that it comprises at least one peptide of the collection according to claim 25. 34. The equipment according to the claim 32 or 33, characterized in that it comprises strips where the collection is immobilized therein. 35. The equipment according to claim 34, characterized in that the strip also contains an internal standard. 36. The use of the collection according to claim 6, for the preparation of vaccines. 37. The use of the collection, according to any of claims 12 to 20, or of at least one peptide according to claim 26 for the preparation of vaccines against HCV. 38. A vaccine against HCV, characterized in that it comprises at least one collection according to any of claims 12 to 20, or at least one peptide according to claim 26.