JP2004513346A - Diagnosis method - Google Patents

Abstract

Screening of phage libraries using sera from antigen-infected and non-infected patients, including identifying the binding specificity of anti-antigen antibody molecules in serum by antibody detection by the antigen mimic (ADAM) method; A method for diagnosing an antigen, comprising identifying a peptide-bound antibody (ligand) specifically associated with the antigen. In vitro maturation strategy; method improvements are obtained by attaching ligands to a common core, eg, MAP. In particular, the method is applied to HCV.

Description

[0001]
(Technical field)
The present invention relates to diagnostic assays for detecting infectious agents, particularly viral agents, specifically human hepatitis C virus (abbreviated herein as HCV), anti-infectious agents useful in said assays. The present invention relates to an antibody-binding peptide, a method for preparing the peptide and a kit for performing the assay. The present invention relates to HCV anti-HCV antibody binding peptides, particularly useful in diagnostic assays, methods for preparing them and kits for performing the assays.
[0002]
Background of the invention
Hepatitis C virus (HCV) is a major etiological agent of non-A, non-B hepatitis that is transmitted parenterally. This virus often causes permanent infections, often leads to the development of chronic hepatitis and cirrhosis, and constitutes a major global cause of chronic liver disease (Boyer N, Marcellin P, Pathogenesis, diagnosis and management). of hepatitis C., J Hepatol 2000; 32: 98-112).
[0003]
Viral infection is diagnosed by detecting anti-HCV antibodies in serum or by elucidating viral RNA from nucleic acid amplification methods (Robbins DJ, Pasupuleti V, Cuan J, Chiang CS, Reverse transcriptase PCR quantification of infection). C virus, Clin Lab Sci 2000 Winter; 13: 23-30). The latter method is very sensitive, but expensive and prone to technical or experimental errors. In addition, the reliability and specificity of the PCR technique has not been standardized (Gretch DR, diagnostic tests for hepatitis C, Hepatology 1997 Sep; 26 (3 Suppl 1): 43S-47S).
[0004]
The present invention relates to the diagnosis of infectious diseases, such as viral infections, especially HCV infections, by a method for detecting antibodies raised against infectious agents, especially anti-HCV antibodies.
[0005]
Recently, a system for sensitive detection of HCV core protein has been reported (Komatsu F, Takasaki K, Liver 1999 Oct; 19 (5): 375-80).
[0006]
Much of the patent and non-patent literature is directed to diagnostic methods and kits for the detection of HCV.
[0007]
US 5,985,542 from Sumitomo Chemical Company provides a kit for liver disease, such as hepatitis C and alcoholic cirrhosis, which contains an antibody that can recognize cytochrome P450.
[0008]
Baxter Aktiengeselleschaft US 5,972,347 discloses an inactive binding neutralizing human HCV antibody, wherein the antibody is directed against at least one protein selected from the group consisting of HCV-core protein and NS3-protein. Provided are compositions for treating HCV infection, comprising HCV.
[0009]
Ambion and Cenetron US Pat. Nos. 5,939,262, 5,919,625 and 5,677,124 describe nucleic acid sequences tested in a sample, including (ribo) nuclease resistance, obtaining (ribo) nucleic acid segments. Provides a very broad way to determine the existence of
[0010]
US 5,866,139 of the Institut Pasteur provides a kit for determining the presence of HCV E-1 specific antibodies. See also Abbott's US 5,854,001.
[0011]
US Pat. No. 5,800,982 to Tonen discloses an antigenic peptide that can specifically react with an antibody directed against Group II of HCV. Tonen, JP 10019897, discloses a peptide having an amino acid sequence of at least 5 contiguous amino acids constituting the protein of non-structural region 4A (NS4A) of HCV and at least five peptides constituting the protein of non-structural region 4B (NS4B) of HCV. Provided is a kit containing a peptide having an amino acid sequence of consecutive amino acids.
[0012]
Regarding the state of the technical stage, US 5,871,904, US 5,869,253, US 5,750,331, US 5,747,241, US 5,667,992, US 5,645,983, US 5 , 625,034, US 5,610,009, WO 9707400, WO 9637783, EP 593291, EP 593290, EP 586065, JP 4349885.
[0013]
Detection of anti-HCV antibodies in serum is still used as the most widely used test to assess HCV infection. Since 1987, when the HCV genome was cloned, various improved versions of anti-HCV diagnostic assays have been developed. Currently available diagnostic kits present serum antibodies against a mixture of four recombinant antigens corresponding to the HCV core, non-structure 3, non-structure 4 and part of the non-structure 5 regions of the HCV polypeptide (Gretch). DR, Diagnostic tests for Hepatitis C, Hepatology 1997 Sep; 26 (3 Suppl 1): 43S-47S). Samples that were considered positive by this screening ELISA should be confirmed by an immunoblot assay in which the presence of antibodies to the same four antigens is separately detected. A positive diagnosis requires the detection of antibodies to at least two recombinant antigens.
[0014]
For relevant parts of the population with anti-HCV antibodies, reactivity to only one antigen is presented, making it impossible to formulate a final diagnosis. A further problem derives from the use of recombinant antigens that are recognized by HCV-unrelated antibodies and can give rise to false positive diagnoses. Therefore, supplemental surveillance and long-term monitoring of patients is needed to reach the final diagnosis.
[0015]
Contact between the foreign antigen and the organism activates a specific immune response. The antigen image detected by the humoral response is defined by the epitope recognized by the host antibody. Assuming that the antibody binding site is a negative image of the epitope, molecules that specifically bind the paratope should show a positive image of the epitope. According to this concept, as far as the humoral response is concerned, the antigen is faithfully described by a specific ligand which binds to the antigen-specific antibody. Identification of these ligands will provide a method of detecting a specific humoral response to the antigen, regardless of whether the same antigen is known and / or available.
[0016]
These ideas were used to develop a diagnostic assay to detect antibodies associated with the infection, including HCV infection in humans. Screening of phage libraries with HCV positive sera has identified peptide ligands that specifically bind anti-HCV antibodies. This was achieved by using a special selection strategy, as sera from infected patients contained virus-specific antibodies scattered among a very large population of different antibodies with varying binding specificities.
[0017]
Screening a large number of negative sera and peptides that excluded those ligands that detected antibodies in negative samples (false positives) led to the selection of a set of highly specific peptides. The range of non-specific activity was reduced by the use of short peptide sequences and those deviating from the native antigen. This technique is described in EP 0 698 091 B1, published on February 28, 1996. For a review of the mimotopes and phagotopes disclosed in EP 0 698 091 and methods of practicing them, see: 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. pp. 195-208; Combinatorial Libraries (R. Cortese ed.) Walter 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 Biotechnology Volume 16, November 1998, 1068-1073.
[0018]
These selection strategies have identified peptide structures that best bind immunodominant anti-HCV antibodies. In addition, multivalent display of ligands has contributed to strong affinities effective for detecting sensitivity. Despite these considerations, the ADAM-HCV (antibody detection by ADAM antigen mimic) mixture is very high but less than 100% sensitive when tested against a panel of sera not used for screening. Occurred. Certain features of the anti-HCV humoral response may explain this result, as it is not involved in the major immunodominant epitopes, but rather is directed against a number of different viral determinants. It is possible. The presence of various viral genotypes further complicates the problem.
[0019]
Currently available diagnostic kits have presented serum antibodies against four recombinant antigens corresponding to a wide region of the HCV polypeptide (Gretch DR, Diagnotic tests for hepatisis C, Hepatology 1997 Sep; 26 (3 Suppl 1): 43S. -47S). A positive diagnosis requires the detection of serum antibodies to at least two recombinant antigens: for this reason, the unique reactivity with only one antigen can be revealed, and the use of anti-HCV antibodies to It is not possible to formulate a final diagnosis for the relevant part. The ADAM / HCV Enzymatic Immuno Assay (EIA) uses mimics of several different immunodominant epitopes from a variety of antigens, thereby ensuring analytical capabilities during the analysis. As an immediate consequence, the frequency of uncertain samples decreases significantly.
[0020]
We have put a great deal of effort into developing the technology for the ADAM-HCV assay (basically using the strategy disclosed in EP 0 698 091 above). Peptides selected from the phage library are presented as N-terminal fusions to the major capsid protein pVIII. The use of phage as a diagnostic reagent has many advantages: it is a very flexible reagent and is useful for various types of immunoassays (Dente et al., 1994, F. Felici, G. Galfre, 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 small-scale production is easy and inexpensive. .
[0021]
Despite these advantages, the size of the phage particles limits the concentration of peptide molecule that can be achieved in the assay. Interference of serum antibodies against the phage capsid requires the addition of carrier phage in the assay mixture to sequester anti-phage antibodies. Ultimately, the mass production of this biological reagent in any case faces problems with microbial contamination, reproducibility, quality control and production costs. Simple linear synthetic peptides derived from peptide sequences displayed on phage did not retain, in most cases, either sensitivity or specificity to achieve effective antibody detection.
[0022]
SUMMARY OF THE INVENTION
It is an object of the present invention to identify the binding specificity of anti-antigen antibody molecules in serum by antibody detection by antigen mimic (ADAM) method, and to use phage using sera from antigen-infected patients and non-infected persons A method for diagnosing an infectious disease, for example, a viral infection, particularly hepatitis C, comprising screening a library and identifying a peptide-bound antibody (ligand) specifically associated with the antigen.
In a particularly preferred embodiment, the present invention relates to HCV infection.
[0023]
Detailed description of the invention
In a first preferred embodiment, in the method of the present invention, said ligand is modified by in vitro maturation strategies.
In a second preferred embodiment, in the method of the invention, said ligand is a synthetic peptide.
[0024]
In a third preferred embodiment, the ligand is attached to a common core. In a particularly preferred embodiment, the ligand with the common core is MAP. The MAP will be clarified later. Another object of the present invention is as follows:
a) first panning the phage library against n-positive sera to generate a first series of n-phage pools;
b) preparing an n-pool mixture containing the n-1 pool;
c) performing an affinity selection of each of the n mixtures on the sera that generated the excluded pool of phage to obtain a second series of n phage pools, and optionally,
d) further panning each of the second series of n phage pools against a mixture containing all n original sera except those used for the first panning;
e) using a mixture of all n original sera to immunoscreen a resulting second series of n phage pools to obtain positive clones;
f) testing the individual reactivity of all positive clones with a panel of positive and negative sera using an ordered array of said clones as phage secreting colonies;
g) generating a replica of the phage secreting colony;
h) screening each replica for reactivity with positive and negative sera to identify clones that specifically react with the positive sera;
i) using each of the phages that specifically react as ligands for affinity-purified antibodies from positive sera;
j) testing the antibody for reactivity with the previously identified HCV peptide;
k) selecting a clone that detects serum antibodies;
Is a collection of HCV-specific ligands obtained by a method comprising:
[0025]
A single peptide resulting from the above method is also an object of the present invention.
In particular, in a preferred embodiment of the invention subjected to HCV, the following peptides:
-YSREQLNKLFGIDMT;
-YSREQLNKMFGIEIS;
-YSREQLSKLFGIEPM;
-NSRWLSKAHGIEGM;
-YSREQLNKLFGIEVM;
-YSREQLSKLFGIDTQ;
-KSREQLSKLHGVDTS;
-RSREQLSKLFGIDLT;
-MWRTWLMKTHGIESW;
-MLRTWLMKYQGIESW;
-YSRSWLMKAHGLELG;
-MMRSYLMKAHGIESL;
-MSRLWLMKAHGISSE;
-KHSEWLNKARGIESW;
-MSRTFLMKAHGIESW;
-MSRTWLMKAHGIESW;
-AEGEKLRRSTNWGDPAK;
-AEGEFKTRRQTNYQDPAK;
-AEGEKTLRNANRLDPAK;
-AEGEKTLRNSNRLDPAK;
-AEGEKKKKPGSSTPKDPAKAAFDSL;
-AEGEFPQDARFPGGGDPAKAAFDSL;
-PQDARFPGGGDPAKAAFDSL;
-AEGEFKGAGGAQTVDWALLVDPAK;
-AEGEFMQKHFFGGAQWIMGDPAK;
-AEGEFLSLKGSGGGQLRALVDPAK;
-AEGEFLSLKGSGGAQLRALVDPAK;
-AEGEFYLLKRSSPPDPAKAAFDSL;
-AEGEFPILVGPYLLLPRRSREEAVDPAK;
-AEGEFPILVGPYLLLPRRSREEAVDPAKKGK;
-AEGEFRLGVRAPRKALDPAK;
-AEGEFRLGVRRALKALDPAK;
-AEGEFRLGVRRALKAPDPAK;
-RLGVRRALKAPDPAK;
-AEGEFTQPRGHSYQDPAK;
-AEGEFLKERAEMSARKTLGADPAK;
-AEGEFFYQIPRRMETKYGDPAK;
-AEGEFSREQLNKLFGIEGDPAK;
-AEGEFNSREWLSKAHGGIEGMDPAK;
-AEGFRSREQLSKLFGIDLTDPAK;
-AEGEFYSREQLNKLFFGIDMTDPAK;
-AEGEFYSREQLNKMFGIETSDPAK;
-AEGEFYSREQLNKLFGIEVMDPAK;
-AEGEFKSREQLRKLHGFDTSDPAK;
-AEGEFKMNRYLNKAFGIEGMDPAK;
-AEGEFRSREQLSKLFGGIELTDPAK;
-AEGEFSRREYSNKAFGIETQDPAK;
-AEGFRRREYLNKAFGIEGGDPAK;
-AEGEFSRREWLNKRFGIEYLDLPAK;
-AEGEFMSRTWLMKAHGIESWDPAK;
-AEGEFYSPEWLNKARGIDRSDPAK;
-AEGEFKSREQLSKLHGVDTSDPAK;
-AEGEFYSREQLNKMFGIIEISDPAK;
-AEGEFYSRSWLMKAHGLELGDPAK;
-AEGEFMMRSYLLMKAHGIESLDPAK;
-AEGEFMSRLWLMKAHGISSEDPAK;
-AEGEFPQPQEVHVYREQLGLDPAKAAFDSL;
-AEGEFGEVLYRGFDEVGGDPAKAAFDSL;
-AGEPYVIERGMQDPAK;
-AEGEFTTASPRHFLVPLDPAKAAFDSL;
-AEGEFTTASPAHFLVPLDPAKAAFDSL;
-AEGEFTTASPSHFLVPLDPAKAAFDSL;
-AEGFATAPRPHYSWDPAK;
-AEGEFATAPAHYSWDPAK;
-AEGFATAPPSHYSWDPAK;
-AEGEFRFWKVPDYDPPAAGGDPAK;
-AEGEFTESSVSSTLADLASKTFGSADPAK;
-AEGEFTLADATMFTGSTDPAK;
-AEGEFGLADLATTFGSPDPDAK;
Is a further object of the present invention.
[0026]
For the collection of the invention, the preferred phage library of step a) is pVIII-12aa.
[0027]
For the collection of the present invention, the insert of a preferred clone selected from step k) is the following sequence:
1. SREQLNKLFGIEG;
2. RATLSNEHGITIG;
3. DQRENWFKYHGFG;
4. EWRRYMSDIHGYG;
5. DSLRYMYVMPGFG.
[0028]
In the collections of the present invention, a phage library is preferably generated, where the reactive clones, preferably the most reactive, are cloned such that each amino acid in the clone sequence is independently replaced by any other amino acid. , Partially mutate.
[0029]
In the collection of the present invention, the preferred clone has a preferred insert sequence: SREQLNKLFGIEG.
[0030]
In the collection of the present invention, in a phage library, a random sequence may be flanked by two cysteine residues. This aspect is applicable to the broad development of the present invention and is not limited to HCV.
[0031]
It is an object of the present invention to use said collection in the preparation of a diagnostic assay for detecting an infectious agent, such as a virus, especially HCV, in a patient suspected of being affected by an infectious agent, such as a virus, especially HCV. .
[0032]
An object of the present invention is a kit for diagnostic purposes comprising said collection.
[0033]
It is an object of the present invention to obtain immunogenic peptides from the methods disclosed herein, either in collection form or in single peptide form. The peptides are effective as immunogenic and are useful for preparing vaccines, especially vaccines against HCV. Suitably, the peptide is in the form of a kit as described above.
[0034]
Advantageously, in contrast to antigen-based systems, the diagnostic assays we have tested have built-in upgradeability: specific selections can be made with serum in which no response is detected. Analogous methods can be used to identify a panel of peptides that discriminate between various HCV genotypes, thereby replacing expensive and labor-intensive PCR with inexpensive and rapid EIA.
[0035]
The invention is explained in more detail by means of examples and figures. In the latter:
FIG. 1 shows the characteristics of the clones derived from the screening. The amino acid sequence of the selected clone is reported in one letter code. The upper and lower panels present sequences derived from the original or secondary library, respectively. The pVIII sequence flanking the foreign epitope is (NH₂) AEGEF [foreign epitope] DPAK. Gray boxes indicate residues that occur frequently at any given position in clones from the original library. Residues that contribute to the consensus sequence of peptides from the secondary library are shown in black. Reported data is the average value from two independent assays and the absorbance measured for the indicated phage (A = A_450nm-A_{620 nm}) And the absorbance measured for wt phage pC89 (Felici et al., 1991). * Indicates untested serum.
[0036]
FIG. 2 shows the identification of phage mimicking the same antigenic determinant. The antibody affinity purified by positive serum C65 by clones PA1, PA3, PA8, PA12 or P18 (shown in column 1) was tested in ELISA for reactivity against the same phage (shown in column 1).
[0037]
Reported data is the average from two independent assays and the absorbance measured for the indicated phage (A = A_450nm-A_{620 nm}) And the absorbance measured for wt phage pC89 (Felici et al., 1991).
[0038]
FIG. 3 shows the ELISA reactivity of pools derived from selection of library pVIIIA12. A phage pool derived from the library panning library pVIIIA12 against positive serum C76 was tested for reactivity with positive sera C12, C13, C29, C40, C47, C65, C73, C74, C76, C83 and C85 by ELISA. Tested. White, gray and black bars indicate wt phage, pVIIIA12 library and pool p76.^IIShows the reactivity of each. The result of the ELISA is A = A_{405 nm}-A_{620 nm}Expressed as Reported data is the average from two independent assays.
[0039]
FIG. 4 shows A and B. A. Reactivity of ADAM-HCV mixture with serum. The cut-off value (CO = 0.232) was calculated as CO = N + 5σ. Where N and σ are the mean and standard deviation, respectively, of the data obtained with the negative control serum. B. ADAM / HCV EIA on a panel of sera from the Italian Red Cross. The cut-off value (CO = 0.252) was calculated as CO = N + 5σ. Where 5 N and σ are the mean and standard deviation, respectively, of the data obtained with the negative control serum. The binding between the antibody and the peptide present in human serum was detected by ELISA described in Materials and Methods. Mean values from two independent experiments were collected. The result is expressed as the ratio between the measured signal and the cut-off value (S / CO). The number of sera tested for each group of sera is indicated.
[0040]
FIG. 5 is an ADAM / HCV EIA for a panel of indeterminate sera. The left column shows the names of the HCV peptides grouped and tested according to binding specificity. The next four columns show the reactivity of the listed peptides with positive (c25 and r15) and negative (r6 and r13) control sera. Each additional column indicates the reactivity of the listed peptides with the indeterminate serum. Binding of the antibody to the HCV peptide present in human serum was detected by ELISA as described in Materials and Methods. The average from two independent experiments was determined. For each peptide, the cut-off value (CO) was calculated as CO = N + 5σ. Where N and σ are the mean and standard deviation, respectively, obtained from 31 negative control sera. The results are expressed as the ratio between the measured signal and the cut-off value (S / CO).
[0041]
FIG. 6 is ADAM-HCV / SIA for positive, negative and indeterminate sera. The following ADAM-HCV peptides were grouped according to binding specificity and immobilized on a nylon membrane to give 10 bands: m1909.2 and m1913.2 (A); m1901.31, m3322.3, m3362.3. (B); m1977.1 (C); m3551.3 (D); m3566.3 (E); m858, mF78 and mH1 (F); mA12.1, mA12.2 and mA12.12 (G); .17 (H); mG21.2 (I); m1929A3.1, m1929C3.4 and m1929.21 (J). An additional line containing purified human IgG is included as an internal positive control. Binding of the antibody to the HCV peptide present in human serum was detected as described in Materials and Methods.
[0042]
In a most preferred embodiment of the present invention, multiple antigenic peptides (MAPs) in which the C-termini of eight identical peptide sequences bind to a common core (Tam, JP: Synthetic peptide vaccine design: synthesis and properties of a high-tide). Density multiple antigenic peptide system. Proc. Natl. Acad. Sci. 85 (1988) 5409-5413) is synthesized, and a reagent is prepared that maintains the specificity at a level comparable to that of the phage-derived peptide. Removal of the phage-scaffold allows for high peptide concentrations of immobilized ligand, which results in high sensitivity.
[0043]
Preferably, the panel of HCV-specific ligands obtained according to the present invention comprises a peptide mimicking HCV NS3, an immunodominant HCV antigen. To our knowledge, in a preferred embodiment, this is the first time described for a short peptide that mimics the immunodominant NS3 determinant. Pepscan analysis of the NS3 protein revealed a relatively long sequence (Khudyakov, Y., et al. 1995. Virology, 206: 666-672) that rarely reacted with positive sera.
[0044]
In contrast, another approach (Santini C, Brennan D, Mennuni C, Hoess RH, Nicosia A, Cortese R, Luzzago A, Efficient display of a representation of the HCV cDNA expression scheme). Mol Biol 1998 Sep 11; 282 (1): 125-35; Pereboeva J. Med. Virol 1998 Oct; 56 (2): 105-11) isolated a large protein domain that retained antigenic properties. That is, this approach we have developed contains essential properties that do not depend on the use or information about the HCV antigen, is applicable to systems where the antigen is unknown, and leads to antigen discovery. Will create a process.
[0045]
Phage display random peptide libraries (phage libraries) are a powerful tool for identifying ligands that are specifically recognized by anti-HCV serum antibodies. Phage-derived peptides can mimic linear, conformational and non-proteinaceous epitopes and thus have a wide range of mimetic potentials (Felici F, Luzzago A, Monaci P, Nicosia A, Sollazo M). , Traboni C. peptide and protein display on the surface of filamentous bacteriophage, Biotechnol Annu Rev. 1995; 1:... 149-83; Zwick MB, Shen J, Scott JK phage-displayed peptide libraries Curr Opin Biotechnol 1998 Aug; 9 (4): 427-3 See).
[0046]
In this invention, we report the identification of an extensive collection of HCV-specific ligands, and the development of a new type of diagnostic kit for the detection of anti-HCV antibodies in serum.
[0047]
The peptides provided in the present invention are useful as diagnostic substances or immunogens or vaccines.
[0048]
Pharmaceutical compositions containing the immunogens and vaccines of the present invention are prepared in a conventional manner that can be understood by one skilled in the art. For example, they can be prepared as described in EP0698091.
[0049]
The following examples further illustrate the invention.
[0050]
Example
Identification of HCV-specific ligand with novel binding specificity
The phage library pVIII-12aa was panned against eight positive sera (C13, C14, C27, C29, C40, C47, C62 and C65) to generate eight phage pools (p13^I, P14^I, P27^I, P29^I, P40^I, P47^I, P62^IAnd p65^IRespectively). Eight mixtures were prepared, each containing seven different combinations of these eight phage pools. Each mixture was subjected to affinity selection using the serum that generated the excluded phage pool: eg, pool p13^I, P14^I, P27^I, P29^I, P40^I, P47^IAnd p62^I, Mixture Δp65 was panned against serum C65. Eight phage pools (p13^II, P14^II) Were again individually panned to a mixture consisting of all original sera except those used for the selection. For example, pool p13^IIWas panned with a mixture of sera C14, C27, C29, C40, C47, C62 and C65.
[0051]
Eight phage pools obtained using a mixture of all eight original sera were immunoscreened to reveal a number of positive clones. The individual reactivity of these clones with a panel of positive and negative sera was revealed by generating an ordered array of clones as phage secreting colonies. After their growth, a complete set of clones was spotted on nitrocellulose filters using a multipin device as an ordered array. The same procedure was repeated to generate a number of replicas, then screened individually and simultaneously for positive and negative sera and their reactivity, revealing a number of clones that reacted specifically with the positive sera. Each of these phages was used as a conjugate to affinity purified antibodies from positive sera. These antibodies were then tested by ELISA for reactivity with any of the four groups of HCV peptides previously identified (Prezzi, C., Nuzzo, M., Meola, A., Delmastro, ＾ R, Galfre '). , C., Cortese, R., Nicosia, A. and Monaci, P. 1996. 1. Immunology 156: 4504-4513., Bartoli et al. Nature Biotechnology, see Vol. ). By this analysis, 12 clones were detected that detected serum antibodies with novel binding specificities, ie, they displayed a different pseudoantigenic determinant than previously identified.
[0052]
Sequence analysis revealed five different sequences. Culture supernatants from each of these five clones (PA1, PA3, PA8, PA12 and PA18) were prepared and their ELISA reactivity was determined using 30 different positive and 24 negative sera (FIG. 1). Tested. Only clones PA8 and PA12 showed statistically significant reactivity with positive sera (p <0.2).
[0053]
The antibody from serum C65 was then immunopurified using phage PA1. These purified antibodies reacted specifically with clones PA3, PA8, PA12, PA18 and even clone PA1 itself, indicating that all these phages were in a unique class recognized by antibodies with the same specificity. It shows that grouping is possible (FIG. 2). No reactivity with any of the above clones was detected when using wild type as conjugate or when the antibody was affinity purified from negative serum.
[0054]
Using different clones from the same group and immunopurifying serum antibodies or using different positive sera, we obtained results consistent with the reactivity reported in FIG. For example, when phage PA3 was used for immunopurified antibodies from the same serum, these antibodies reacted with clones PA8 and PA12 in addition to the same PA3. These results indicate that clones PA1, PA3, PA8, PA12 and PA18 detect antibodies reactive with the same B-cell epitope, as suggested by weak similarities in the peptide sequences (FIG. 1).
[0055]
Affinity maturation of HCV peptides (Affinity (Maturation)
A phage library was generated in which the sequence of clone PA12 was partially mutated. In this "secondary" library, called pVIIIA12, the oligonucleotides were synthesized so that each amino acid of the SREQLNKLFGIEG sequence was independently replaced by another amino acid: In theory, the substitution at each site was 20% Happens at a frequency of In addition, random residues were included at both sites in the foreign peptide sequence. The pVIIIA12 library was panned twice with 12 positive sera (C8, C10, C12, C13, C22, C58, C60, C76, C83, C85, C141 and C177).
[0056]
When tested in ELISA, the phage pool p76^II, P141^IIAnd p177^II(Derived from selection with sera C76, C141 and C177, respectively) showed the highest and broadest reactivity with the panel of positive sera. This was further analyzed (FIG. 3). Based on this reactivity, the phage pool p76 was used with each of serum C40, C141 and C177.^II, P141^IIAnd p177^IIWas immunoscreened. For each pool, this analysis selected several clones independently tested for reactivity with various positive and negative sera by the filter replica method as indicated above. This final screen selected 51 clones that specifically reacted with positive sera.
[0057]
Sequence analysis revealed 16 different sequences (each of pool p76^II, P141^IIAnd p177^II8, 5 and 3). Culture supernatants from each of these clones were prepared and their ELISA reactivity was tested on 33 different positive and 24 negative sera (FIG. 1).
[0058]
Clones P40.17 and P40.7 reacted with higher positive sera tested (42% each). The combination with clones P141.7 and P177.22 scored 70% of the panel positive sera. Alignment of the selected peptides revealed the consensus sequence (M / Y) SRE (W / Q) L (M / N) K (A / L) (H / F) GIES (W / M).
[0059]
Identification of another HCV specific ligand
A similar selection strategy was undertaken to identify ligands with new binding specificities different from those previously identified. Phage libraries of various lengths were screened that were completely random or flanked by two cysteine residues whose random sequence restricted the conformation of the display peptide.
[0060]
Phage libraries were selected using various combinations of serum and phage pools. Phage pools showing a reactivity profile with the desired positive sera were further analyzed. The reactivity of a number of individual clones was evaluated by replica screening to select phage that displayed specific reactivity with positive sera.
[0061]
We focused on clones with the desired reactivity pattern.
To search for clones with antibody affinity purified from HCV peptides, clones mimicking the antigenic determinants of the previously identified peptides were excluded. Clones that survived these selection steps were tested in an ELISA with a number of positive and negative sera to statistically define their HCV specificity.
[0062]
These selected clones were further refined by creating and screening a secondary library. The sequence or clonal population of the original clone in the secondary library was partially mutated and rescreened to select for variants with improved binding specificity. This step was performed according to various suitable strategies already described (see the article by Urbanelli, Zhu). This extensive and repeated effort has identified seven new groups of ligands that specifically bind HCV-specific serum antibodies with different binding specificities.
[0063]
Serum was used to screen the phage display reporter of the HVR1 mutant, and peptides that specifically reacted with a number of positive sera were isolated (Puntoriero et al .; Nicosia unpublished). A set of HVR1 phage-derived peptides derived from this screen were analyzed for reactivity with our panel of sera. Three peptides (mF78, mH1 and m858) were identified by the highest and specific frequency of reactivity with positive sera.
[0064]
In summary, 12 ligands have been identified, encompassing the four groups previously identified (Bartoli et al. Nature Biotechnology Volume 16, November 1998, 1068-1073, Prezzi, C., Nuzzo, M., Meola, A .; , Delmastro, ＾ R, Galfre ', C., Cortese, R., Nicosia, A. and Monaci, P. 1996. 1. Immunology. 156: 4504-4513.); FIG. I want to.
[0065]
Phage − From origin to synthetic peptides
Twenty-two peptide sequences derived from twelve groups of HCV-ligands were synthesized as octa-branched multi-antigen peptides (ADAM-HCV peptides). In this molecule, eight identical peptide sequences were linked by a lysine fork to a common core forming a multiple display similar to that of the pVIII-fusion peptide of the phage capsid. The sequence of the ADAM-HCV peptide is reported below.
[0066]
m858 @ ETYTTGGAARTTSGLTSLFSPGPSQN
m1901.31 AEGEFKKFPGSSTPKDPAKAAFDSL
m1901.34 @AEGEFPEDTFPGSKLILSGDPAKAAFDSL
m1909.2 @ AEGEFKTRRNTNYQDPAK
m1913.2@AEGFKTLRNTNRLDPAK
m192.21@AEGFATSPTHYTSELDPAK
m1929A3.1@AEGEFTTASPTHFLVPLDPAK
m1929C3.4@AEGFATAPPSHYSWDPAK
m1977.1 @AEGEFPYLLLPRRSREEAVDPAK
m3322.3@AEGEFPQDARFPGGGDPAK
m3362.3@AEGEFLSLKGSGGGQLRALVDPAK
m3551.3@AEGEFRLGVRRALKAPDPAK
m3566.3 @ AEGEFKTSVRSVPRARPPINGDPAK
mA12.1@AEGEFNSREWLSKAHGIEGMDPAK
mA12.2@AEGFRSREQLSKLFGIDLTDPAK
mA12.13@AEGEFMSRTWLMKAHGIESWDPAK
mB11.17 AEGEFRELLYEAFDDMEGDPAK
mF78 @ QTHTTGGQAGHQAHSLTGLFSPGAKQN
mG21.2@AGEPYVIEQGMMDPAK
mH1 @ QTHTTGGVVGHATTSGLTSLFSPGPSQN
mN15.3 @ AEGEFGLADLATTFTGSTDPAK
mS48.5@AEGEFRFWKVPDYDPPAAGGDPAK
Meanwhile, the data for their analytical performance is summarized in Table 1 and attached herewith with the figures.
[0067]
In many cases, the pVIII sequences flanking the foreign epitope (NH2AEGEF and / or DPAK-COOH) have been shown to be involved in the binding specificity of the corresponding peptide.
[0068]
Using a mixture containing these 22 ADAM-HCV peptides (ADAM-HCV mixture), the presence of anti-HCV antibodies was detected by EIA (ADAM / HCV EIA). The ADAM-HCV mixture of the peptides was immobilized by a passive coating on the bottom of a multiwell ELISA plate, and a 1:40 dilution of the serum sample was incubated for 40 minutes. The human antibody bound to the peptide was detected by incubation with the anti-human conjugate for 20 minutes and measured by a colorimetric enzyme reaction.
[0069]
As reported in FIG. 4A, ADAM / HCV EIA efficiently distinguished between positive and negative sera.
[0070]
ADAM-HCV / EIA
A panel of HCV positive and HCV negative sera was tested for the presence of anti-HCV antibodies by ADAM / HCV-EIA (FIG. 4B). The test showed 100% specificity by identifying all positive sera contained in this panel and identifying all negative samples.
[0071]
Uncertain sample
According to a commercial HCV-confirmation assay, collections of sera diagnosed as indeterminate were obtained from various sources. Twenty-three ADAM-HCV peptides were individually tested for their reactivity using ELISA in 31 samples (FIG. 5). Six samples did not react with any of the MAPs tested. That is, it was evaluated as negative. Eight samples recognized only one antigen. That is, uncertain analysis was confirmed. Finally, 17 samples showed more than one response to the various groups of peptides, ie, were identified as positive.
[0072]
ADAM − HCV strip immunoblot assay ( ADAM − HCV / SIA )
The ADAM-HCV peptide was covalently immobilized on an activated nylon membrane, resulting in a strip with 10 bands. Each line included various peptides with the same binding specificity, as described in the legend of FIG. A control system containing purified human IgG is included as an internal positive control. A number of samples selected from a panel of indeterminate sera were tested for reactivity with immobilized antigen by incubating serum samples with strips. Anti-HCV antibodies supplemented by individual antigens were incubated with the anti-human enzyme conjugate on the strips and then imaged by a colorimetric enzyme reaction. The reactivity of the test strips with peptide bands was determined by visually comparing the intensity of each band with that of an internal positive control.
[0073]
ADAM-HCV / SIA revealed the reactivity of all eight positive sera tested against several different viral determinants mimicked by the ADAM-HCV peptide. No reactivity was detectable when eight negative sera were tested (FIG. 6).
[0074]
We analyzed a selected number of uncertain samples by ADAM-SIA. As shown in FIG. 6, analysis of eight indeterminate sera confirmed the results obtained by ADAM / HCV EIA with individual peptides. The results showed sensitivity comparable to the two assays.
[0075]
Materials and methods
Phage library
Four different phage display random peptide libraries: pVIII9aa, pVIII9aa_cys, pVIII-12aa, pVIII15aa and pVIIIA12 were used as ligand sources. pVIII9aa (Felici et al., 1991), pVIII12aa and pVIII15aa are the NH4s of the major coat protein pVIII.₂-Three different libraries composed of each of the random 9-, 12- and 15-mers displayed on filament-like phage as fused to the termini. pVIII9aa_cys is a random library in which a random nonapeptide is sandwiched between two cysteine residues (Luzago et al., 1993). In this latter library, cysteine promotes the formation of disulfide bridges that limit some of the conformation of the display peptide. The pVIIIA12 library was constructed by synthesizing an oligonucleotide encoding the amino acid sequence SREQLNKLFGIEG. By employing a resin-splitting synthesis method (Glaser et al., 1992), each amino acid position was replaced with an NNS triplet at a frequency of 20%. In addition, random residues encompass both sites of the foreign peptide sequence. All five libraries were generated as described (Folgori et al., 1998). The complexity of the library, as derived from the number of individual clones obtained in microbial transformation, was approximately 1 × 10 5 for each of the five libraries.⁸Met.
[0076]
Human serum
The human sera used in this study were randomly collected from rejection units of donor blood from transfusion centers and hospitals, and from units from healthy volunteers. That is, a number of uncertain samples used in this study were obtained from the Laboratory of Virology, Istituto Superiore di Sanita, Roma (Italy) and the Centro Nazionale Trassiona radia San Francisco, Italy. Serum was tested for the presence of antibodies to HCV by a second next generation HCV ELISA test system (Ortho Diagnostic Systems, Bersee, Belgium) and a first next generation dot blot immunoassay RIBA HCV test system (Chiron Co., Emeryville, CA, USA). ). Serum was also tested against HBsAg and HIV-1 / HIV-2 by the AUSAB EIA test (Abbott Labs, Chicago, IL) and the third generation HIV-1 / HIV-2 EIA test (Abbott Labs, South Pasadena, CA). Tested for absence of antibody. Samples that are positive for the presence of anti-HCV antibodies but negative for anti-HBsAg and anti-HIV antibodies are included in this experiment as HCV positive sera. Seronegative for the presence of antibodies to all three antigens is included in this study as HCV negative sera.
[0077]
Affinity selection and immunoscreening
A phage display random peptide library was used for affinity selection as described for HCV positive sera (Folgoli et al. 1998; Felici et al., 1996; Prezzi et al., 1996). Clones derived from the affinity selection were immunoscreened using the serum described (Prezzi et al. 1996, Minenkova et al).
[0078]
ELISA using phage clone
ELISA using phage supernatant and human serum was performed as follows. Phage supernatants were prepared from DH5α-F ′ infected cells as previously published (Felici et al., 1991). A multiwell plate (Immunoplate Maxisorp, Nunc, Roskilde, Denmark) was loaded with 200 μl of anti-pIII monoclonal antibody 57D1 (Dente et al., 1994) at 4 ° C. overnight at 50 mM NaHCO 3.₃ The antibody was coated at a concentration of 1 μg / ml in (pH 9.6). After removing the coating solution, the plates were incubated with ELISA blocking buffer (0.1% casein, 1% Triton-X100 PBS) at 37 ° C. for 60 minutes. Plates were washed several times with PBS / 0.05% Tween-20 (wash buffer). A 1: 1 mixture of ELISA blocking buffer and clear phage supernatant was added to each well and allowed to bind for 1 hour at 37 ° C. 1:40 diluted human serum at room temperature for 30 minutes at 5 × 10 5 per ml of phage f11.1¹⁰ Plaque forming unit (pfu) (Dente et al., 1996), 25 μl / ml of protein extracted from DH5α-F ′ cells infected with phage f11.1 (dente et al.) And rats not treated with ELISA blocking buffer Incubated with 25 μl / ml of supernatant from hybridoma cells.
[0079]
After removing the mixture containing the phage supernatant, the plates were washed with wash buffer, 200 μl of pre-incubated serum mixture was added to each well, incubated for 60 minutes at 37 ° C., and then washed with wash buffer. Goat anti-human IgG HRP-washed, diluted 1: 20.000 in secondary antibody blocking buffer (1% Triton, 1% horse serum, 50% fetal bovine serum, 5 μl / well Mab supernatant in PBS) Conjugates were added to each well (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). After incubation at 37 ° C. for 30 minutes, the plates were washed and peroxidase activity was detected by incubation with 200 μl of TMB liquid substrate system (SIGMA, St. Louis, MO). After 15 minutes of development, the reaction was diluted with 25 μl of 2M H₂SO₄Was stopped by adding. Plates were read on an automated ELISA reader (Labsystems Multiskan Bichromatic, Helsinki, Finland) and the results were expressed as A = A450 nm-A620 nm. The ELISA data listed in the columns are the average values from two independent assays. They are greater than the 0.3 cut-off value and are 3σ (σ = ｛1/2 [σ] from the background signal observed for wt phage.²p + σ²w]｝^1/2) If different, it is considered statistically significant.
The p values with reference to clones PA8 and PA12 indicate that the observed frequency distribution of the reactivity of positive and negative sera was²The probability that a test is statistically the same.
[0080]
Phagotope from serum − Affinity purification for specific antibodies
A 60 mm diameter Petri dish of polystyrene (Becton Dickinson Labware, NJ) was treated overnight at 40 ° C. with 50 mM NaHCO 3.₃1 × 10 in (pH 9.6)¹¹ Coated with CsCl-purified phage particles / ml solution. After washing with PBS / Tween, the dishes were incubated with ELISA blocking buffer at 37 ° C. for 60 minutes. Human serum (diluted 1/100 with ELISA blocking buffer), 1x10¹² A mixture of f11.1 pfu / ml and 25 μl / ml of XL1-blue cell protein extract was incubated for 60 minutes at room temperature. After removing the blocking solution, the pre-incubation mixture was added to the plate and incubated overnight at 4 ° C. Serum dilutions were removed and dishes were washed with wash buffer. Bound antibodies were eluted and neutralized with 0.1 M glycine-HCl (pH 2.7) supplemented with 10 μg / ml BSA.
[0081]
Phage clone characterization
Affinity purified antibodies were tested in a standard ELISA for their reactivity to phagotopes. Usually, a multiwell plate is prepared by adding 50 mM₃1x10 CsCl-purified phage in (pH 9.6)¹¹ Coated overnight at 4 ° C. with TU / ml solution, 100 μl / well. After washing with PBS / Tween, the plates were incubated for 60 minutes at 37 ° C. with blocking buffer. Thereafter, affinity purified antibody (100 μl) was added to each well and allowed to bind overnight at 4 ° C. The plates were then washed with cold PBS / Tween and 100 μl / well of goat anti-human IgG (FC specific) alkaline phosphatase conjugated antibody (Sigma, St. Louis, MO) diluted 1/5000 in blocking buffer was added. did. After incubation for 2 hours at room temperature, the plates were washed and exposed to alkaline phosphatase as described above.
[0082]
Synthetic peptide
Synthetic octa-branched multi-antigen peptides (MAPs) were used (Tam, 199X). The synthesis was performed by the flow polyamide method (Pessi et al., 1990). The peptide was dissolved in dimethyl sulfoxide.
[0083]
ELISA using synthetic peptides
A multiwell plate (Immuno plate Maxisorp, Nunc, Roskilde, Denmark) was loaded with 50 mM NaHCO₃Coated overnight at 4 ° C. with a 10 μg / ml MAP solution in (pH 9.6). After removing the coating solution, the plates were incubated with ELISA blocking buffer (0.1% casein, 1% Triton-X100 in PBS) at 37 ° C. for 60 minutes. Plates were washed several times with PBS / 0.05% Tween-20 (wash buffer). Human serum diluted 1:40 was added to each well and incubated for 40 minutes at 37 ° C. The plates were then washed with wash buffer and a 1: 20.000 dilution of goat anti-human IgG HRP-conjugate in ELISA blocking buffer was added to each well (Jackson ImmunoResearch Laboratories, Inc., West Grove, AZ). PA). After a 20 minute incubation at 37 ° C, the plates were washed and peroxidase activity was detected by incubation with a TMB liquid substrate system (SIGMA, St. Louis, MO). After 15 minutes of development, the reaction was diluted with 25 μl of 2M H₂SO₄Was stopped by adding. Plates were read on an automated ELISA reader (Labsystems Multiskan Bichromatic, Helsinki, Finland) and results were expressed as A = A450 nm-A620 nm. The ELISA data listed in the column are average values from two independent assays.
[0084]
Reference
Smith, C.E. P. and Petrenko, V .; A. 1997. Page display. Chem. Rev .. 97: 391-410
Folgori, A .; Tafi, R .; , Meola, A .; Felici, F .; Galfre, G .; Cortese, R .; , Monaci, P .; and Nicosia, A.J. 1994. A general strategy to identity mimotopes of pathological antigens using only random peptide libraries and human sera. EMBO 13: 2236- "43.
Prezzi, C.I. Nuzzo, M .; , Meola, A .; , Delmastro, 'R, Galfre', C.I. Cortese, R .; , Nicosia, A .; and Monaci, P.M. 1996. 1. Immunology. 156: 4504-4513.
Alter, H .; j. 1995. To Cor not to C: these are the questions'. Blood. 85: 1681.
Felici, F .; , Castagnoli, L .; , Musacchio, A .; Jappelli, R .; and Cesareni, C.I. 1991. Selection of antibodies ligands from a large library of oligopeptides expressed on a multiple exposure vector. f. Mol. Biol. 222: 301-310.
Luzzago, A. et al. , _Felici, F.R. , Tramontano, A .; , Pessi, A .; and Cortese, R.A. 1993. Micking of discontinuous epitopes by phase displayed peptides, I.M. Epitope mapping of human H ferritin using a phase library of constrained peptides. Cene. 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 phase. Useful tools for phage display technology. Gene. 148: 7
Sambrook, J .; Fritsch, T .; and Maniatis, T.W. 1989. Molecular Cloning: a laboratory manual (second edition)
Takamizawa, A .; , Mori, C .; Fuke, I .; Manabe, S .; , Murakami, S .; Fujita, J. et al. , Onoshi, 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.M. B. 1993. Purification of glutathione S-Transferase fusion proteins. Methods in molecular and cellular biology. 4: 220-229.
Frangioni, f. V. and Neel, B.C. J. 1993. Solubilization and purification of enzymatically active glutathione S-transferase (pGex) fusion proteins. Analyt. Biochem. 210: 179-187.
Komatsu F, Takasaki K 1999
Determination of serum hepatitis C virus (HCV) core protein using a novel application for quantitative evaluation of HCV viremiaia inti-vitiemia-antiviation-HCV. Liver 19: 375-80
[Brief description of the drawings]
FIG. 1 shows data on the analytical performance of ADAM-HCV peptides.
FIG. 1 shows the characteristics of clones derived from screening.
FIG. 2 shows the identification of phage mimicking the same antigenic determinant.
FIG. 3 shows the ELISA reactivity of pools derived from selection of library pVIIIA12.
FIG. 4 shows 4A. Reactivity of ADAM-HCV mixture with serum. 4B. Figure 9 shows ADAM / HCV EIA for a panel of sera obtained from the Italian Red Cross.
FIG. 5 is an ADAM / HCV EIA for a panel of indeterminate sera.
FIG. 6 is ADAM-HCV / SIA for positive, negative and indeterminate sera.

Claims (38)

Screening of phage libraries using sera from antigen-infected and non-infected patients, including identifying the binding specificity of anti-antigen antibody molecules in serum by antibody detection by the antigen mimic (ADAM) method; A method for diagnosing an antigen, comprising identifying a peptide-bound antibody (ligand) specifically associated with the antigen. 2. The method of claim 1, wherein the ligand is modified by an in vitro maturation strategy. 3. The method according to claim 1, wherein said ligand is a synthetic peptide. 4. The method of claim 1 or 2 or 3, wherein the ligands bind to a common core. 5. The method of claim 4, wherein said ligand with said common core is MAP. The following process:
a) first panning the phage library against n-positive sera to generate a first series of n-phage pools;
b) preparing an n-pool mixture containing the n-1 pool;
c) performing an affinity selection of each of the n mixtures on sera that generated an excluded pool of phage to obtain a second series of n phage pools, and optionally,
d) further panning each of the second series of n phage pools against a mixture containing all n original sera except those used for the first panning;
e) using a mixture of all n original sera to immunoscreen a resulting second series of n phage pools to obtain positive clones;
f) testing the individual reactivity of all positive clones with a panel of positive and negative sera using an array of said clones as phage secreting colonies;
g) generating a replica of the phage secreting colony;
h) screening each replica for reactivity with positive and negative sera to reveal clones that react specifically with positive sera;
i) using each of the phages that specifically react as ligands for affinity-purified antibodies from positive sera;
j) testing the antibody for reactivity with the previously identified antigen-specific peptide;
k) selecting a clone that detects serum antibodies;
A collection of antigen-specific ligands obtained by a method comprising: Screening phage libraries using sera from HCV patients and non-infected individuals, including identifying the binding specificity of anti-HCV antibody molecules in serum by antibody detection by the antigen mimic (ADAM) method, A method for diagnosing hepatitis C, comprising identifying a peptide-binding antibody (ligand) specifically associated with 8. The method of claim 7, wherein the ligand is modified by an in vitro maturation strategy. 9. The method according to claim 7, wherein said ligand is a synthetic peptide. 9. The method according to claim 6, wherein the ligand is attached to a common core. 9. The method of claim 8, wherein said ligand with said common core is MAP. The following process:
a) first panning the phage library against n-positive sera to generate a first series of n-phage pools;
b) preparing an n-pool mixture containing the n-1 pool;
c) performing an affinity selection of each of the n mixtures on sera that generated an excluded pool of phage to obtain a second series of n phage pools, and optionally,
d) further panning each of the second series of n phage pools against a mixture containing all n original sera except those used for the first panning;
e) using a mixture of all n original sera to immunoscreen a resulting second series of n phage pools to obtain positive clones;
f) testing the individual reactivity of all positive clones with a panel of positive and negative sera using an array of said clones as phage secreting colonies;
g) generating a replica of the phage secreting colony;
h) screening each replica for reactivity with positive and negative sera to reveal clones that react specifically with positive sera;
i) using each of the phages that specifically react as ligands for affinity-purified antibodies from positive sera;
j) testing the antibody for reactivity with the previously identified antigen-specific peptide;
k) selecting a clone that detects serum antibodies;
A collection of HCV specific ligands by a method comprising: 13. The collection of claim 12, wherein the phage library of step a) is pVIII-12aa. 14. The collection according to claims 12 and 13, wherein the insert of the clone selected from step k) in claim 12 has the following sequence: SREQLNKLFGIEG; 13. The collection of claim 12, wherein a phage library is generated and the most responsive clones are partially mutated such that each amino acid in the clone sequence is independently replaced by any other amino acid. 13. The collection of claim 12, wherein a phage library is generated and the clones that react there are partially mutated such that each amino acid in the clone sequence is independently replaced by another amino acid. 17. The collection of claim 15 or 16, wherein said clone has the following insert sequence SREQLNKLFGIEG. 18. The collection according to any of claims 12-17, wherein in the phage library the random sequence is flanked by two cysteine residues. 19. The collection according to any of claims 12-18, wherein said peptide sequence binds a common core. 18. The collection of claim 17, wherein said peptide having said common core is MAP. 21. Use of a collection according to any of claims 12-20 for preparing a diagnostic assay for detecting HCV in a subject suspected of being infected with HCV. A kit for diagnostic purposes, comprising the collection of claim 6. A kit for the purpose of diagnosing HCV, comprising a collection according to any one of claims 12-20. The kit according to claim 22 or 23, wherein the collection comprises an immobilized strip. 25. The kit of claim 24, wherein the strip further contains an internal standard. following:
i) SREQLNKLFGIEG;
ii) RATLSNEHGITIG;
iii) DQRENWFKYHGFG;
iv) EWRRYMSDIHGYG;
v) DSLRYMYVMPGFG;
vi) YSREQLNKLFFGIDMT;
vii) YSREQLNKMFGIEIS;
viii) YSREQLSKLFGIEPM;
ix) NSRWLSKAHGIEGM;
x) YSREQLNKLFGIEVM;
xi) YSREQLSKLFGIDTQ;
xii) KSREQLSKLHGVDTS;
xiii) RSREQLSKLFGIDLT;
xiv) MWRTWLMKTHGIESW;
xv) MLRTWLMKYQGIESW;
xvi) YSRSWLMKAHGLELG;
xvii) MMRSYLMKAHGIESL;
xviii) MSRLWLMKAHGISSE;
xix) KHSEWLNKARGIESW;
xx) MSRTFLMKAHGIESW;
xxi) MSRTWLMKAHGIESW;
xxii) AEGEKKLRSTNWGDPAK;
xxiii) AEGEFKTRRQTNYQDPAK;
xxiv) AEGEFKTLRNANRLDPAK;
xxv) AEGEFKTLRNSNRLDPAK;
xxvi) AEGEFKKFPGSSTPKDPAKAAFDSL;
xxvii) AEGEFPQDARFPGGGDPAKAAFDSL;
xxviii) PQDARFPGGGDPAKAAFDSL;
xxix) AEGEFKGAGGAQTVDWALLVDPAK;
xxx) AEGEFMQKHFFGGAQWIMGDPAK;
xxxi) AEGEFLSLKGSGGGGQLRALVDPAK;
xxxii) AEGEFLSLKGSGGAQLRALVDPAK;
xxxiii) AEGEFYLLKRSSPPDPAKAAFDSL;
xxxiv) AEGEFPILVGPYLLLPRRSREEAVDPAK;
xxxv) AEGEFPILVGGPYLLLPRRSREEAVDPAKKGK;
xxxvi) AEGEFRLGVRAPRKALDPAK;
xxxvii) AEGEFRLGVRRALKALDPAK;
xxxviii) AEGEFRLGVRALRKAPDPAK;
xxxix) RLGVRRALKAPDPAK;
xl) AEGEFTQPRGHSYQDPAK;
xli) AEGEFLKERAMSARKTLGADPAK;
xlii) AEGEFYQIPRRMETKYGDPAK;
xliii) AEGEFSREQLNKLFGIEGDPAK;
xliv) AEGEFNSREWLSKAHGGIEGMDPAK;
xlv) AEGFRSREQLSKLFGIDLTDPAK;
xlvi) AEGEFYSREQLNKLFFGIDMTDPAK;
xlvii) AEGEFYSREQLNKMFGIETSDPAK;
xlviii) AEGEFYSREQQLNKLGGIEVMDPAK;
xlix) AEGEFKSREQLRRKHLGFDTSDPAK;
l) AEGEFKMNRNYKAFGGIEGMDPAK;
li) AEGEFRSREQLSKLFGGIELTPDPAK;
lii) AEGEFSRREYSNKAFGIETQDPAK;
lii) AEGEFRRREYLNKAFGIEGGDPAK;
iv) AEGEFSRREWLNKRFGIEYLDLPAK;
lv) AEGEFMSRTWLMKAHGIESWDPAK;
lvi) AEGEFYSPEWLNKARGIDRSDPAK;
lvii) AEGEFKSREQLSKLHGVDTSDPAK;
lviii) AEGEFYSREQLNKMFGIIEISDPAK;
lix) AEGEFYSRSWLMKAHGLELGDPAK;
1x) AEGEFMMRSYLLMKAHGIESLDPAK;
lxi) AEGEFMSRLWLMKAHGISSEDPAK;
lxii) AEGEFPQPQEVHVYREQLGLDPAKAAFDSL;
lxiii) AEGEFGEVLYRGFDEVGGDPAKAAFDSL;
lxiv) AGEPYVIERGMQDPAK;
lxv) AEGEFTTASPRHFLVPLDPAKAAFDSL;
lxvi) AEGEFTTTASPAHFLVPLDPAKAAFDSL;
Ixvii) AEGEFTTASPSHFLVPLDPAKAAFDSL;
lxviii) AEGEFATAPRPHYSWDPAK;
Ixix) AEGEFATAPAHYSWDPAK;
lxx) AEGFATAPPSHYSWDPAK;
lxxi) AEGEFRFWKVPDYDPPAAGGDPAK;
lxxii) AEGEFTESSVSSTLADLASKTFGSADPAK;
lxxiii) AEGEFTLADLATTMTFGSTDPAK;
lxxiv) AEGEFGLADLATTFTSGPPDAK;
A peptide selected from the group consisting of: 27. Use of the peptide of claim 26 in the method of claims 7-11. A collection according to any of claims 12 to 20, containing at least one peptide according to claim 26. A kit for detecting an infection, comprising the collection of claim 6. 30. The kit of claim 29, wherein said collection contains immobilized strips. 31. The kit of claim 30, wherein the strip further contains an internal standard. A kit for detecting HCV infection, comprising a collection according to any one of claims 12-20. A kit for detecting HCV infection, comprising at least one peptide from the collection of claim 25. 34. The kit of claim 32 or claim 33, wherein the collection contains fixed strips. 35. The kit of claim 34, wherein the strip further contains an internal standard. Use of the collection of claim 6 for the preparation of a vaccine. Use of a collection according to any of claims 12 to 20 for the preparation of a vaccine against HCV. A vaccine against HCV, comprising at least one collection according to any of claims 12 to 20, or at least one peptide according to claim 26.

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