AU648912B2 - Immunoassay for non-A non-B hepatitis - Google Patents

Description

OPI DATE 12/01/93 APPLN. ID 19720/92 ACJP DATE 11/03/93 PCT NUMBER PCT/US92/03635 llllllllllllllllll llllllllllllll AU9219720

,PCT)

(51) International Patent Classification 5 (11) International Publication Number: WO 92/22571 C07K 7/00, C12Q 1/70 Al (43) International Publication Date: 23 December 1992 (23.12.92) (21) International Application Number: (22) International Filing Date: Priority data: 714,471 13 June 1 718,052 20 June 1 PCT/US92/03635 29 April 1992 (29.04.92) 991 (13.06.91) 991 (20.06.91) (71)Applicant: BAXTER DIAGNOSTICS INC. [US/US]; One Baxter Parkway, Deerfield, IL 60015 (US).

(72) Inventors: LEAHY, David, C. 22 Georgetown, Cary, IL 60013 TODD, Johr, A. 2955 Santos Lane, #307, Walnut Creek, CA 94596 JOLLEY, Michael, E.

3449 N. Circle Drive, Round Lake, IL 60073 (US).

(74) Agents: BARTA, Kent, S. et al.; One Baxter Parkway, Deerfield, IL 60015 (US).

(81) Designated States: AT (European patent), AU, BE (European patent), CA, CH (European patent), DE (European patent), DK (European patent), ES (European patent), FR (European patent), GB (European patent), GR (European patent), IT (European patent), JP, LU (European patent), MC (European patent), NL (European patent), SE (European patent).

Published With international search report.

648912 (54)Title: IMMUNOASSAY FOR NON-A NON-B HEPATITIS (57) Abstract An assay for antigens of hepatitis C virus utilizes a synthetic peptide comprising the first 38 amino acids of the capsid region containing at least two immunodominant epitopes. The assay detects antibodies in the sera of patients infected with the Non-A Non-B hepatitis virus. Of particular efficacy is a competitive inhibition assay, which incorporates in the liquid phase an inhibitor consisting of a peptide containing only one of the immunodominant capsid epitopes, which is capable of inhibiting binding of antibodies to all target epitopes present on the solid substrate.

WO 92/22571 PCT/US92/03635 IMMUNOASSAY FOR NON-A NON-B HEPATITIS CROSS-REFERENCE TO RELATED APPLICATION This application is a continuation-in-part of our prior copending application filed June 13, 1991, docket number PA-4148.

BACKGROUND OF THE INVENTION Most evidence concerning the identity of the causative agent of Non-A Non-B hepatitis (NANBH) is consistent with the conclusion that it is a flavivirus or flavi-like virus having a genome comprising a single open reading frame.

This virus has been tentatively named hepatitis C virus (HCV). The proposed structural genes encoding the capsid, matrix, and envelope proteins of the virus are organized at the end of the genome, and the proposed regulatory, nonstructural genes are located at the 3' end.

EP 0 318 216 (Houghton) discloses the cloning of portions of the HCV viral genome from an enriched plasma source contributed by D. Bradley as described in Bradley et al., Seminars in Liver Disease, 6: 56 (1986). All of the sequences disclosed in EP 0 318 216 appear to belong to the regulatory (nonstructural) portion of the genome. EP 0 388 232 supplements the '216 application in disclosing additional nucleotide and polypeptide sequences which appear to represent structural genes of the 5' end of the genome.

Okamoto, et al., Japan. J. Exp. Med., 167 (1990) have recently described the 5' amino acid sequence of HCV viral genome corresponding to the structural proteins including the 110 to 190 amino acid residues thought to be the capsid protein. A description of the core (also WO 92/22571 PCT/US92/03635 2 known as capsid) and envelope regions corresponding to those disclosed in EP 0 388 232 (Houghton) is given in Kataheuchi, et al., Nuc. Acids Res., 18: 4626 (1990). None of the foregoing patent applications point out which specific portions of the HCV structural sequences define antigenic determinants.

Okamoto, et al., Japan J. Exp. Med., 60:167 (1990) describe at least 3 areas of local hydrophilicity spanning amino acid positions 1- 120. In one of these areas Okamoto, et al., Japan J. Exp. Med., 60,223 (1990) further identified an amino acid sequence (aa #39-74) that when synthesizd as a synthetic peptides has utilitity in an immunoassay.

Additional sequences for Non-A Non-B hepatitis are set forth in EP 0 363 025 (Arima). Computer analysis of the nucleotide sequences of Arima indicates they are not homologous to other sequences heretofore described in the patent applications or literature. However, comparison of the predicted amino acid sequences reveals a short sequence mapping to the capsid region in which 10 of 11 amino acids in the Arima sequence are identical and homologous to the corresponding sequence in the European '232 application and the articles cited hereinabove. Applicants refer hereinafter to this region of homology as the "common sequence".

SUMMARY OF THE INVENTION Applicants prepared a synthetic peptide encompassing the amino acid sequence from the beginning of the HCV open reading frame to amino acid 38, and encompassing the "common WO 92/22571 PCT/US92/03635 3 sequence". Peptides of varying length defined within this region were compared in immunoassay to the 34 amino acid polypeptide of Arima containing the common sequence.

In accordance with the present invention, an epitope group was identified having the predicted peptide sequence encoded by the first 114 consecutive open reading frame nucleotides of the heretofore published sequence of the HCV genome. These epitopes, contained in the capsid protein of the virus, comprise a first epitope having an amino acid sequence QRKTKRNTNRR or QRKTKRSTNRR, and a second epitope contiguous to the first at the 3' end of the first epitope having the amino acid sequence PQDVKFPGGG or

PQDVKFPGGGQIVGGVYLLP.

The peptide defined above containing the epitope group, derived from the predicted amino acid sequence encoded by the first 114 consecutive open reading frame 5' nucleotides of the virus, or its substantial equivalents, may conveniently be formatted into an immunoassay for the determination of the presence of anti-HCV antibodies in patient sera. Such an assay comprises contacting a sample containing such antibodies with a solid substrate to which the peptide is immobilized, separating unbound antibodies from those bound to the solid substrate, and detecting the presence of bound antibodies on the solid substrate.

In another aspect of the present invention, a competitive inhibition assay for detecting HCV specific antibodies comprises WO 92/22571 PT/US92/03635 4 contacting a sample containing such antibodies with a specificity to the epitope group containing in the first 28 capsid amino acid residues, to a solid substrate to which the epitope group-containing peptide is immobilized, with such contacting being carried out in the presence of competing amounts of a peptide having substantially the amino acid sequence: PQDVKFPGGG, separating the antibodies binding to said peptide immobilized on the solid substrate from those antibodies not so bound, and determining the amount of bound antibodies.

In a further embodiment, the peptide can be tagged with a fluorophor such as fluorescein. The tagged peptide is incubated with the sample to form an antibody peptide complex, followed by measurement of the increased fluorescence polarization. Thus, a homogeneous assay is provided which avoids a separation step.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT A peptide, preferably produced by synthetic chemical techniques, which contains the epitopes of a protein, may be more desirable in an immunoassay than the whole protein itself. In assays utilizing a solid substrate upon which the target antigen is adsorbed, the surface area for adsorption is a limiting parameter of the assay.

Thus it is more efficient to adsorb a greater number of the peptide molecules containing the critical epitopes per unit surface area than for unreactive portions of the larger protein to occupy adsorption sites.

WO 92/22571 P@r/US92/03635 In the present invention, the selection of the amino-terminus of the HCV capsid protein was made on the following basis. The complete lack of nucleic acid homology between the Arima sequences and those disclosed in the Houghton applications suggested that two completely different viruses may be involved in NANBH. However, inexplicably there is a high degree of reactivity to antigens expressed from the clones of both sources with the same serum panels.

The discovery that there is 10 amir j acid homology between the Arima and the 5' Houghton sequence at the polypeptide level, over an eleven amino acid segment from residue 8 to 18 inclusive (HCV, Houghton) suggested some interrelatedness of the viruses. It also suggested the possibility that the common sequence defines a common epitope.

Analysis of the immunoreactivity of HCV positive serum panels to peptides encoding the common sequence and peptides containing various lengths of the flanking sequences of Arima and Houghton confirmed the existance of an epitope in the common sequence.

Figure 1A shows the sequence of a number of peptides derived from the Arima clone 14 sequence.

In this series, nos. 1-8, an asparagine is substituted for the serine so that the common sequence is precisely homologous to Houghton. In this figure, the amino acid sequence of Arima is set out horizontally in a number of vertical rows with dashed lines therebetween indicating the corresponding extent of the individual synthetic peptide. Thus, for example, in row no. 1 the peptide intended extends from amino acid residue 25 to 34. Figure 1A, No. 9, is the Arima sequence WO 92/22571 PCr/US92/03635 6 with the serine at position 20 as described by Arima. Peptide numbers 11 and 12 are the common sequence only with serine at postion 20 in no. 11 and asparagine at position 20 in no. 12.

Figure 1B shows synthetic peptides containing various lengths of the Houghton (and Okamoto) sequence. Figure 1C is the common sequence with various amino acid substitutions at position The amino acids depicted in Figure 1C are in rp, "tration with the sequence of Figure IB. A 6, .ine was added to the 5' end of the common sequence to prevent cyclization of the glutamine residue at amino acid position 1. Figure 1D is a non-structural HCV sequence disclosed in EP 0 318 216 and is located within the C-100 protein.

Figure 1E gives the sequence for the capsid fragment selected by Okamoto, et al. extending from amino acid residue number 39 to 74 inclusive.

In addition, sequences corresponding to 2 structural peptides (aa 109-133 and 133-169) are presented. Figure 1F shows the HCV capsid peptide sequence covering amino acids 1-38 and two additional carboxy terminal peptide fragments thereof.

The results of experiments in which these various peptides were immobilized onto a solid substrate and tested in an immunoassay against a panel of sera containing antibodies to HCV are set forth in detail in the Examples. In general the complete peptide of the Okamoto sequence covering amino acids 1 to 38 gave the best signal to background ratio. The common sequence exhibited some immunoreactivity, however, the immunoreactivity was signifiantly improved if a carboxy-terminal sequence was present. This was WO 92/22571 PCT/US92/03635 7 probably because some spacing is necessary to prevent steric interference with the correct conformational presentation of the epitope.

The results also indicate that the HC\ capsid peptide from residues 1 to 8 and extending to residue 28 to 38 will correctly identify a larger number of HCV reactive sera than the Arima clone 14 peptide. Conversely, the Arima peptide fails to -dentify any additional positive sera not identified by the HCV capsid 1-28 or 1-38 peptides indicated.

This means that the carboxy-flanking sequence comprising PQDVKFPGGG defines a second epitope contiguous on the 3' end of the first epitope contained in the common sequence. All peptide fragments were synthesized in the amide form on a Milligen-Biosearch 9600 model peptide synthesizer using fluorenylmethoxy carbonyl (FMOC) amino protection scheme and 1-3 diisopropylcarbodiimide coupling chemistry.

The amide form of the sequence was adopted because it could be expected to more closely mimic the biologically active analogue than the free acid form. Activated amino acids were coupled to a 2, 4-dimethoxy benzhydrylamine resin. Peptide synthesis was monitored by ninhydrin analysis for all amino acids except proline for which an Isatin test was performed.

The synthesized peptide was cleaved from the resin by Reagent R, which comprises trifluoroacetic acid, thioanisole, ethanedithiol and anisol in a volumetric ratio of 90:5:3:2.

Peptides cleaved from resins were purified by high performance liquid chromatography WO 92/22571 PCT/US92/03635 8 (HPLC), and characterized by Porton PI 20 90 E Integrated Micro-Sequencing System to confirm the correct sequence. Purity was ascertained by HPLC on a reverse phase column using a linear gradient in 0.1% trifluoroacetic acid from 5 to 40% acetonitrile over 35 minutes.

Absorbance was followed at 230nm.

Alternatively, recombinant peptides can be produced biologically by using cloning techniques, manipulation of promoter, ribosomebinding, translation terminator sites, expression systems and purification methods commonly available to those skilled in the art.

The peptides of the present invention may be conveniently used in any assay system utilizing a protein target. In the preferred embodiment, the target peptide fragment is coated onto a solid matrix, such as paramagnetic microparticles, by passive or covalent coating methods. Following an incubation step in the presence of anti-HCV antibodies, the bound antibody peptide complex is separated from any unreacted antibodies by magnetic separation, and the amount of antibody in the antibody peptide complex is determined.

Conveniently, detection of complexed anti- HCV antibody can be carried out by further reacting the complex with anti-human antisera to which an enzyme is attached. Upon separation of the tagged complex on paramagnetic particles, by magnetic separation and washing, a fluorescence-producing substrate is added. The amount of fluorescence measured is thus directly proportional to the amount of anti-NANBH antibody present in the sample.

WO 92/22571 PCT/US92/03635 9 In an alternative embodiment, the peptides of the present invention may be coated onto microtiter plate wells in the classical enzyme linked immunosorbent assay (ELISA), incubated with sample, aspirated, and an enzymeconjugated anti-human antisera added. Glass fiber filters may also be utilized as the solid substrate in a radial partition chromatography format. Detection is conventionally carried out by adding the appropriate substrate/chromogen and measuring the resultant product. For a general discussion of ELISA see Langone, et al., Immunological Techniques, Part D Immunoassays. Methods in Enzymology, p. 84 (1982).

Further alternative assay formats which are applicable to the present peptides include Western Blot, Towbin, et al., Proc. Nat. Acad.

Sci., 76:4350 (1979); Radioimmune Assay (RIA), Walsh, et al., J. Infect. Dis., 21:550 (1970); Competitive Assays, Diamandis, Clin., Biochem., 21:139 (1988); Noncompetitive Assays, Crook, et al., J. Gen. Virol., 46:29 (1980); Immunoprecipitation, Tojo, et al., Clin. Chem 34:2423 (1988) and Dot Blots, Jahn, et al., Proc. Nat'l. Acad. Sci. 8311684 (1984); PCFIA, Jolley, et al., J. Immunol. Meth. 67:21 (1984).

It should be emphasized that minor changes in sequence, amino acids substitutions, additions or deletions may not appreciably affect assay performance because the epitope is not significantly altered. Thus, peptides having such minor changes in structure are considered to be the equivalents of peptides WO 92/22571 PCr/US92/03635 having strict homology to the sequence of the original polypeptide.

WO 92/22571 PC/U92/03635 11 Brief Description of Drawings Fiqurp1.: A. The amino acid sequence of full length Arima clone 14 peptide and fragments thereof are described. Ii addition, the sequence of clone 14 peptide modified at amino acid position 20 (peptide and fragments thereof are described. The dashed line represents the actual peptide synthesized.

B. The amino acid secaence of HCV capsid covering the first 28 amino acids is described. The dashed line represents the actual peptide synthesized.

C. The amino acid sequence of 3 peptides representing amino acids 8-18 of HCV capsid (or clone 14 amino acids 14-24) are presented. To all peptides, glycine was added to the N terminus. Peptides ;io. 16 and no. 18 contained amino acid substitutions at HCV capsid amino acid position 14 (or amino acid position 7 within Figure 1C). Dashed line represents the peptide synthesized.

D. The amino acid sequence of a peptide corresponding to a region within HCV nonstructural region 4 (C-100 protein) is presented. This peptide corresponds to HCV amino acids 1693-1735 as described by Houghton.

E, The amino acid sequences of three different HCV capsid peptides is presented. Peptide no. 20 covers amino acids 39-74, peptide no. 21 covers amino WO 92/22571 PC/US92/036355 12 acids 109-133 and peptide no. 22 covers amino acids 133-169.

F. The amino acid sequence of a peptide representing HCV capsid amino acids 1-38 is presented as peptide no. 25. Two other peptides representing amnino acids 29-38 (peptide no. 23) and amino acids 19-38 (peptide no. 24) are described as well.

Dashed line represents the peptide iu synthesized.

WO 92/22571 PC/ US92/03635 13 EXAMPLE 1 Peptides were prepared as indicated above corresponding to the sequences depicted in Figures 1A-1E. Peptides were then passively coated onto paramagnetic polystyrene microparticles (4 micrometers in size, Pandex Division, Baxter Diagnostics IMic.) according to the following procedure: 250 ul of weight/volume paramagnetic particles were pelleted in a microfuge at 5000 rpm for minutes. The supernatant was removed and the particle pellet was resuspended with 500 ul of ethanol for 15 minutes. The particles were then pelleted as before and the supernatant was removed. The particles were resuspended in 500 ul of 0.1 M CAPS buffer [(3-Cyclohexyamino)-1propane sulfonic acid] at pH 11.0. The particles were pelleted as before and supernatant removed.

Lyophilized peptide was weighed out and resuspended in sterile filtered (0.22 um) water, resulting in a peptide concentration of mg/mL and allowed to dissolve into solution for 30 minutes at room temperature. The dissolved peptide was further diluted to 500 ug/mL in 0.1 M CAPS buffer at pH 11.0 and allowed to stabilize for 20 minutes at room temperature. 250 ul of this peptide solution was then transferred to the washed particle pellet. The particles were resuspended and then tumbled for 12 to 16 hours at room temperature.

The passively adsorbed peptide particles were then pelleted at 5000 rnm for 3.5 minutes, the supernatant was removed and particles were WO 92/22571 PCT/US92/03635 14 resuspended in isotonic buffered saline with 0.05% Tween 20 detergent. The particles were then washed once with isotonic buffered saline with 0.05% Tween-20 and then 3 times with isotonic buffered saline using centrifugation at 5000 rpm (3.5 minutes). The coated particles were then resuspended in isotonic buffered saline at final particle concentration of 0.025% weight to volume.

EXAMPLE 2 A paramagnetic particle assay using particles coated with peptide fragments described in Figure 1 was performed as foi. c-s* Human serum or plasma was diluted 1:100 in well buffer (0.103 M Tris-HCl, pH 7.4, 1.05 M sodium chloride, 0.33% NP-40, 0.09% sodium azide, and newborn calf serum). 50 ul of the diluted sample was added to each well of a black plastic microtiter plate. Samples were tested in replicates of at least 2. Paramagnetic particles, coated with peptides as described in Example 1, were added to each well (20 ul).

The plate was then placed at 37 0 C-42 0 C for minutes.

Upon completion of the incubation, the particles in the wells were washed with 100 ul PBS and Tween-20 (2.06 g sodium phosphate dibasic, 0.318 g sodium phosphate monobasic, ml Tween-20, 8.76 g sodium chloride, and 1.0 g sodium azide per liter; pH During the wash steps, the paramagnetic particles were held in the microtiter plate well via a magnetic field applied to the bottom of the plate. Particles were washed in this manner five times.

WO 92/22571 PC/US92/03635 Particles in each well were resuspended in ul of Particle Resuspension Buffer (4.346 g sodium phosphate dibasic, 0.524 g sodium phosphate monobasic, 8.76 g sodium chloride, and 1 g sodium azide per liter; pH 20 ul of goat anti-human IgG (H L) conjugated with B-galactosidase (conjugate) and diluted 1:1,000 in conjugate dilution buffer (0.1 M Tris-HCl pH 0.5M sodium chloride, 5% glycerol, 2.3 mM magnesium chloride, 0.1% sodium azide and newborn calf sera) was then added to the wells.

Any human IgG or IgM that was bound to the particles was recognized by and associated with conjugate. The conjugate solution was designed to give maximum liquid stability and reactivity. In particular, newborn calf serum is preferred over calf serum. After incubation with conjugate for 15 minutes at 37 0 C-42 0 C, the particles in the wells were washed five times with 100 ul of PBS and Tween-20 as described above to remove essentially all of the unbound conjugate. The Tween-20 in the wash solution enhanced the washing process and removed nonspecifically bound conjugate.

Finally, 50 ul of a substrate solution of 4-methyl-umbelliferyl-B-D-galactoside (MUG) was added to each well (0.178 g 4-methylumbelliferyl-B-D-galactopyranoside, 3.58 g tricine, 5.1 ml dimethyl sulfoxide, 30 ml methyl alcohol, 0.20 g sodium aide, 0.5 ml per liter, pH The presence of B-galactosidase conjugate) in the wells triggered the cleavage of MUG to generate a fluorescent coumarin product. Fluorescence (excitation wavelength 365 nm/emission WO 92/22571 PCT/JS92/03635 16 wavelength 450 nm) was measured at two timed intervals 2 and 14 minutes) post MUG addition. The plate was incubated at 37 0 C-42 0

C

between fluorescence determinations. The difference between the two values was a kinetic measurement of fluorescent product generation and was a direct measurement of conjugate and human IgG/IgM bound to the particles. Kinetic fluorescent values were converted to nM coumarin values using various concentrations of coumarin itself and its resultant fluorescence to establish a standard curve. Results were calculated as nM coumarin produced over a 12 minute timed interval. Kinetic values greater than or equal to 5000 are presented as 5000 in the following examples,. The cutoff for reactivity was determined to be 200 nM coumarin by testing over 300 random donor EDTA plasma specimens and HCV capsid seroconversion specimens.

EXAMPLE 3 The immunoreactivity of clone 14 peptide fragments was determined as follows. Peptide fragments 1-6 as depicted in Figure 1A were coated onto paramagnetic particles according to Example 1. The resultant peptides were evaluated in the assay described in Example 2 using 3 HCV capsid reactive sera and 3 HCV nonreactive sera. As the peptide length was increased stepwise from 10 to 19 amino acids in length 1 thru positive signal was observed (Table Fragments shorter than 19 amino acids in length displayed no reactivity.

Increasing the peptide length past 23 amino acids did not increase signal further. Thus, WO 92/22571 PCT/US92/03635 17 in this assay format, immunoreactivity of the peptide resides between approximately amino acid positions 12 and 19. The negative samples remained non-reactive as peptide length increased, therefore, optimal assay performance as measured in positive/negative sample signal was observed with fragments #4-6 which span amino acid positions 10-34.

PCr/US92/03635 WO 92/22571 PTU9/33 18 Table 1 Reactivity of peptide fragments 1-6 with HCV positive and negative sera.

1 Saml e HCV Positive 2 KC3 539*7 HCV Negative 2 LS2 LS 6 LS8 Avg. Negative 3 Pent ide Number 1 3 4 6 233 389 344 491 513 441 11 12 13 12 11 11 HCV PosiLtive/HCV KC3 5397 Negative Avg.

4 0.9 0.9 0.9 19 0.6 0.6 0.6 41 I Results are represented as nM coumarin.

2. samples positive or non-reactive to IiCV capsid antigen.

3. Average value in nM coumarin for the 3 HCV negative samples.

4. Values represent nM coumarin HCV positive sample/nM coumarin HCV negative average.

WO 92/22571 PC/US92/03635 19 EXAMPLE 4 The immunoreactivity of clone 14 peptide fragments (Figure 1) was further evaluated using the paramagnetic microparticle assay described in Example 2 with 5 HCV capsid reactive and 2 HCV negative sera. As seen in Table 2, peptide #6 displays stronger reactivity than peptide #10. Both peptides span clone 14 amino acid positions 10-34 and are identical except that amino acid position was altered in peptide #6 wherein an asparagine was substituted for a serine. This single amino acid substitution increased positive signal reactivity without an increase in negative signal.

Poptide #18 comprised the common amino ac:id sequence shared by clone 14 and the HCV capsid sequence first described by Okamoto.

Peptides #16 and #17 are identical to #18 excerpt that the serine of #18 at position was substituted with a glutamine or asparagine for peptides #16 and #17, respectively (Figure 1B). As presented in Table 2, the common sequences do demonstrate some reactivity alone, however, they are not as immunoreactive as the longer length peptides that contain the common sequence #6 and In addition, modification of position 20 with a glutamine significantly reduces immunoreactivity compared to the unmodified clone 14 fragments. The 3 common peptides (#16-18) were synthesized with a glycine residue at the N terminus to prevent glutamic acid cyclization. Thus, the lower observed immunoreactivity was not due to glutamic acid cyclization.

PCr/US92/03635 WO 92/22571 PrU9/33 Table 2 Reactivity of peptide fragments 10, with HCV positive and negative sera.

1 16-18 am HCV Positive 5397 5000 5402(12) 2363(12) HCV Nagative

LSS

Pentide 6 is 1-7 18 751 5000 5000 5000 454 3510 5000 3044 34 164 5000 171 21 90 5000 75 31 42 1773 37 1. Results are represented as nM coumarin.

WO 92/22571 PC~T/US92/03635 21 EXAMPLE Three fragments (#13-15) corresponding to the first 28 amino acids of HCV capsid as described by Okamoto and depicted in Figure 1B were tested for HCV capsid immunoreactivity as according to Example 2. Immunoreactivity was found to reside in fragment 13 which corresponds to amino acid position 19-28 and is outside the common sequence shared with clone 14 (Table However, as the peptide length was increased to include the clon 14 common sequence, immunoreactivity greatly increased. Increasing peptide length even further to aa position 1 (peptide 15) yielded maximum reactivity and optimal assay performance. Thus, even though immunoreactivity was demonstrated for each of the three peptides, best performance as measured by positive/negative signal was obtained with peptide 15 which covers amino acid positions 1-28.

WO 92/22571 PC/US92/03635 22 Table 3 Reactivity of peptide fragments 13-15 with HCV positive and negative sera.

Peptide 13 14 15 13 14 Sample nM Coumarin 1 Sianal/Noise 2 HCV Reactive 5397(1:16) 67 1272 3927 2 53 164 2563 1855 1856 5000 77 77 208 2450(1:4) 39 698 2420 2 29 101 2453(1:4) 25 119 362 1 5 5402(1:8) 1084 1687 5000 45 70 208 5380(1:16) 10 117 155 0 5 6 2363(1:16) 67 66 270 3 3 11 2368 2147 1434 3078 89 59 123 5370 53 88 570 2 4 5402(1:32) 84 166 1171 4 7 51 AR83 41 477 2885 2 20 125 Avg. Negative 24 24 23 1. Results are presented as nM coumarin obtained with peptides 13, 14 and 2. Signal/Noise Results represent nM coumarin HCV reactive sample/nM coumarin Avg. of HCV negative samples for peptides 13, 14 and WO 923,22571 PCT/US92/03635 23 EXAMPLE 6 To further identify the immunoreactive regions of the first 28 amino acids of HCV capsid, a series of inhibition experiments were performed. Peptide #15 corresponding to HCV capsid aal-28 (Figure lB) was coated onto paramagnetic microparticles as described in Example 1 and then tested in a modified immunoassay described below using HCV capsid reactive sera. The immunoassay of Example 2 was modified by first incubating the diluted sample (1:100 dilution) with a potentially competing peptide (100 ug/ml) for 30 minutes.

ul of the diluted sample containing potentially competing peptide was added to the plastic plate and then peptide #15 coated particles were added. Except for this modification, the assay was performed exactly as described in Example 2. Samples were tested with and without the competing peptide and the results were calculated to determine the percent activity remaining in the presence of the potentially competing peptide. Thus, 100% means no competition occurred and that the peptide tested has no immunoreactivity in common with peptide 15 whereas values of 6 percent would indicate strong competition and an exceptionally high level of common immunoreactivity.

As presented in Table 4, the binding of some samples to peptide 15 can be strongly or completely inhibited by clone 14 fragments and the common clone 14/HCV capsid fragment. This inhibition takes place even when amino acid position 20 of clone 14 is modified by a serine WO 92/22571 PCr/I1JS92/03635 24 to asparagine substitution. Therefore, some samples display immunoreactivity to peptide that is localized solely to the common sequence. On the other hand as shown in Tables 4, 5, and 6, some samples were not inhibited at all or were only partially inhibited by peptides containing the common sequence fragments demonstrating that immunoreactivity in peptide 15 does take place outside of the common sequence as well. It is important to note that addition of a glycine to the N terminus of the common sequence does not enhance peptide inhibition (peptides #16-18).

Thus the lack of inhibition for peptide 12 as shown in Table 6 was not due to the N terminal glutamic acid being cyclized.

Immunoreactivity to peptide 15 outside the clone 14/capsid common sequence was further investigated using fragments of peptide 15. As presented in Table 7, fragment #13 (aal9-28) of capsid) completely inhibited the binding of four capsid reactive samples to peptide Increasing the length of this fragment, spanning positions 7-28 gave the same finding.

Thus, some samples possess immunoreactivity only to this 10 amino acid fragment of peptide To further identify the nature of the amino acid fragment (peptide fragment 13) inhibition, eighteen HCV capsid reactive samples were tested for inhibition of binding to peptide 15 using fragment 13. Th results presented in Table 8 show that fragment 13 completely inhibited all samples binding to peptide 15. This finding was unexpected, since WO 92/22571 PCr/US92/03635 some to these samples have been shown to have iimunoreactivity to peptide 15 regions other than fragment 13. For example, sample A83 was shown in Table 4 to be completely inhibited by the common sequence, yet in this experiment it was completely inhibited by the 10 aa sequence distinct from the common sequence.

WO 92/22571PC, S9/65 PCf/US92/03635 26 Table 4 Use of peptide fragments to inhibit HCV positive sample binding to peptide #15.1 are AR83 AR2 25 L9 BB17 AR2 22 (1874)100% 6% 493)100% 63% 800)100% 22% (318S)100% 63% (826)100% 86% £Penide 11 2 6% 1~ 65% 7% 25% 6% 66% 2% 9 5%1 6 1. Results are presented as peptide addition) sam~ple of control (competing no reactivity remaining after preadsorbtion with indicated peptide. For the control (NoL. column the rnM coumarin valu~es are indicated ir, parwithes is.

WO 92/22571 PfU9/33 PCr/US92/03635 27 Table Use of peptide fragments to inhibit HCV positive sample binding to peptide #15.1 _apl Nolne ARS4 (880)100% AR191 (5000)100% AR222 830)100% AR225 492)100% 85% 100% 100% 108% 2.

78% 100% 63% 57% 90% 100% 102% 61% a. 11 75% 3% 100% 4% 111% 58% 1. Results are presented as of control (none) samiple reactivity remaining after preadaorbtion with indicated peptide fragm~ent. For the control colqmn (none) the nM cournarin values are indicated in parenthesis.

PCT/US92/03635 WO 92/22571 28 Table 6 Use of peptide fragments to inhibit HCV positive sample binding to peptide #15.1 Samplte AR8 9 AR4 4 None 16 17 18 12 (5000) 100% 100% 100% 100% 100% 1% (5000) 100% 100% 100% 100% 100% 1% 1. Results are presented as of control (none) sample reactivity remaining after preadsorbtion with indicated peptide fragment. For the control column (none) the nM coutnarin values are indicated in parenthes is.

WO 92/22571 PIU9133 PCT/US92/03635 29 Table 7 Use of peptide fragments to inh-ibit HCV positive sample binding to peptide #15.1 Sample AR8 9 AR4 4 AR191 ar14-I No~ne (1769) 100% (3582) 100% (3329) 100% (3106) 100% 3% 1% 3% 6% Pep-tide 2% 2% 1% 1% 2% 3% 6% 6% 76% 110% 103% 74% 1. Results are presented as of control (none) sample reactivity remaining after preadsorbtion with indicated peptide fragment. For the control column (none) tha nM coumarin values are indicated in parenthesis.

WO 92/22571 Table 8 Use of peptide fragment #13 to inhibit HCV positive sample binding to peptide pC/US92/03635 Samplie 1 2 3 4 6 8 11 12 13 14 16 18 L9 A240 145: A83 nM Coumarinl f raga 13 frag. 13 5000 73 5000 35 722 13 5000 174 292 18 3376 33 5000 70 5000 75 685 15 154 23 5000 267 5000 19 2344 21 5000 23 5000 44 836 68 J. 91 Percent Inbhitioqr2 99% 99% 98% 911% 94% 99% 99% 98% 98% 99% 99% 99% 99% 92% 94% 98% 1824 1. Results are presented as nM coumarin generated in the absence and presence of fragment #13.

2. inhibition: 1 n14 coumarin (+fragmnent) n.M coumarin {-fragment) WO 92/22571 PC/US92/03635 31 EXAMPLE 7 To further identify the immunoreactive regions of clone 14, a series of competition experiments were performed. These experiments were executed as described in Example 6, except that clone 14 (Figure 1A, peptide clone 14 fragments (peptides 6 and HCV capsid 1-28 (peptide 15), or HCV nonstructural protein C- 100 (peptide 19), Figures 1A, 1B and ID were coupled to the paramagnetic particles and used as the target for inhibition.

In the first series of experiments, peptide #9 corresponding to full length clone 14 was used as the target. As presented in Table 9, the 11 amino acid clone 14/capsid common sequence (Figure 1A, peptide 11) was able to strongly inhibit immunoreactivity of 8 HCV capsid reactive samples. The same finding was observed with larger peptides that contained the common sequence (peptides 9, and 15). Even the 1-28 amino acid peptide corresponding to HCV capsid (peptide gave the same effect. However, peptide 2 which comprises the c terminal 13 amino acids of clone 14 displayed little reactivity. Since peptide 2 contains in its sequence the 3 c terminal amino acids of the clone 14/capsid common sequence and since it displays only limited inhibition, it appears that the reactivity of these 8 samples is to approximately aa 14-21 of the common sequence shown in Figure 1A (peptide 11).

To further support the conclusion that the majority of reactivity to clone 14 is due to the common sequence, peptide 6 was used as the PC/US92,*03635 WO 92/22571 32 solid phase target in inhibition experiments.

As shown in Table 10, peptide corresponding to the common sequenr (12, 16, 17 and 18) completely inhibited HCV sample binding to peptide 6. This inhibition was complete whether amino acid 20 was a serine, asparagine of glutamine.

Since the peptide fragment corresponding to the c terminal 10 amino acids of peptide peptide 13, Figure IB) was shown to react unexpectedly in Example 6, experiments were performed to evaluate its potential for inhibiting reactivity to clone 14. Table 11 shows that this peptide completely inhibited the binding of 9 different HCV samples to peptides #9 (clone 14), #8 (clone 14 with serine to asparagine substitution at position and #15 (HCV capsid amino acid 1-28). This inhibition was not non-specific because specimen reactivity to an HCV peptide located outside of the capsid region #19, of HCV nonstructural protein C-100, Figure lD) was not inhibited. These results taken together with those of Example 6 indicate that peptide 13 can strongly inhibit sample reactivity (binding to) the clone 14/HCV capsid common sequence and will lend utility to competitive HCV antibody immunoassays.

WO 92/22571 PTU9/33 PCT/US92/03635 33 Table 9 Use of peptide fragments binding to peptide #9.1 _anl to inhibit HC"T sample AR148 BB17 L9 ARS 3 AR1 41 AR191 ALR225 No~ne (1408) 100% (3350) 100% 404)100% (2406) 100% (5000) 100% (5000) 100% (451)100% (459)100% z.

78% 108% 83% 74% 100% 100% 87 90% 6% 3% 17% 4% .eptid 2% 3% 2% 2% 10% 12% 4% 4% 23% 28% 4% 7% 3% 2% 26% 4% 24% 7% 1. Results are presented as reactivity remaining after of control (none) sample preadsorbtion with indicated peptide fragment. For the control column (none) the nM coumarin values are indicated in parenthesis. WO 92/22571 PTU9/33 PCr/US92/03635 34 Table Use of peptide fragments to inhibit HCV positive sample binding to peptide #6.1 J ePent ide Non 16 17 iZ 1 6 AR89 (5000)100% 1% 1% 1% 1% 1% AR44 (5000)100% 1% 1% 1% 1% 1% 1. Results are presented as of control (no peptide added) sample reactivity remaining after preadsorbtion with indicated peptide fragment. For the controli column (none) the nM coumarin values are indicated in parenthesis.

WO 92/22571PCPS9/35 PCT/"S92/03635 Table 1.1 Use of peptide fragment #13 to inhibit HCV positive sample binding to peptides 9, 8, 19.

Peptide Sampole on particles n14 Percent ,Coumarinl Inhibition 2 #13 +#113 5000 40 99% 5000 29 99% 520 17 97% 3826 24 99% 265 26 5000 29 99% 5000 42 99% 5000 48 99% 5000 39 99% 5000 38 99% 729 17 98% 5000 27 99% 591. 26 96% 5000 35 99% 5000 45 99% 5000 43 99% 5000 914 82% 5000 32 99% 795 29 96% 5000 165 97% 317 25 92 5000 68 99% 5000 67 99% 5000 5000 0% 5000 5000 0% 5000 5000 0% 5000 5000 0% WO 92/22571 prU9/33 PCr/US92/03635 Table 11 (cont'd) Peptide SamTple on particles rim Coumarinl -#13 +#L3.

1372 1542 252 253 5000 5000 5000 5000 Percent Inhibftion 2 0% 0 IS 0.% 0% 1. Results are presented as nM absence and presence(+ 2. %inhibition: 1- no, coumarin fracment) coumarin generated in the of fragment f13.

X 100 nM cournarin J- fragment) WO 92/22571 PCr/US92/03635 37 Example 8 Four peptides 20, 21 and 22) corresponding to sequences of the HCV capsid and depicted in Figures 1B and 1E were tested for immunoreactivity as according to Example 2.

As shown in Table 12, immunoreactivity with HCV positive sera was strongest for peptide which corresponds to the first 28 amino acids of the HCV capsid. Peptide #20 (amino acids 39-74) displayed some reactivity with these specimens, however, the reactivity was less than for peptide #15. Peptides #21 and #22 displayed little immunoreactivity with the same HCV positive samples. Assay performance is measured as positive signal divided by negative signal. As seen in Table 12, peptide provides the largest value for every sample.

Thus, the greatest assay utility ir differentiating HCV positive and negative sera was obtained with peptide #15. Similar experiments using peptides #15 and #20 were performed using HCV reactive sera panels that are publicly available. Tables 13 and 14 display these results using the HCV mixed and low titre panels available from Boston Biomedica,Inc (Boston, MA). The results obtained with these panels are explained in more detail in Example 9.

WO 92/22571 PCr/US92/03635 38 Table 12 Reactivity of y,eptides 15, 20-22, With ,lCV positive ay negative sera.

1 Peyptide HcV oji2 a 1 2 2363 2580 3825 18 i 2450 5000 5000 35 17 5378 5000 1283 109 911A 259 107 90 17 9 1.1 3165 211 44 18 2563 5000 5000 121 23 2367 soonl 5000 68 24 2368 5000 5000 70 21.

5370 561 364 74 19 2374 242 30 47 5402 5000 5000 76 21 2453 5000 5000 57 21 14391-44 2172 1447 70 23 FP19558 5000 134 54 31 14947-44 5000 5000 106 41 10519-046 5000 5000 156 31 5397 910 22 93 126 2190-92888 5000 77 ND 3 322 208030-9888 515 41 ND 3 122 H-CV Negative2 LS1 16 24 15 68 LS2 55 95 38 153 12 20 io 181 LS11 11 20 87 26 LS12 10 24 68 27 HCV Nectat ive M; 21 37 49 91 WO 92/22571 PTU9/33 PCT/US92/03635 Table 12 (can't) Pptv,/Neqat ive 4 2363 2450 5378 911 lA 91 1H- 2563 2367 2368 5370 2374 5402 24527 14391-44 FP19558 1.4947-44 1053.9-046 5397 2190-9188 20GO30-9888 Reptide 124 241 241 12 152 241 241 241 27 2 241 105 241 241 241 44 105 137 3 6 137 137 137 13 13 7 403 4 1.37 37 1 1.- 2.

3.

4.

Resuldts are relpreanted as nil ccnmrari,%.

Samples reactive or non-reactive to Hj(V capsid antigen.

ND =Sampl~e not tested.

Positive/Negative HCV Positive/HCV Negative Mean froft 5 negative samples, WO 92/22571 PCT/US92/03635 Example 9 To further define the immunoreactivity of HCV capsid peptides, peptides spanning amino acid positions 1-28 (number 15, Figure 1B), 29- 38(number 23, Figure IF), 19-38 (number 24, Figure IF), 1-38 (number 25, Figure IF), and 39-74 (number 20, Figure 1E) were evaluated in the paramagnetic microparticle assay described in Example 2. The peptide particle combinations were tested with three different NANBH and HCV panels that are publically available and are well characterized. The first 2 panels were obtained from Boston Biomedica, Inc. and were the low and mixed titre HCV panel. Along with each panel, the manufacturer supplied the reactivity of each sample to recombinant capsid c22. This reactivity was obtained using the RIBA2 HCV supplemental test system marketed by Ortho Diagnostics (Raritan, NJ) and is a measure of specimen reactivity to the whole length capsid protein. As presented in Tables 13 and 14, peptides 15 a d 25 were reactive with all specimens iricated by Boston Biomedica to be c22 capsid reactive and -are non-readtive with the remaining specimens. The reactivity found with peptide number 25 covering amino acids 1- 38 was genera.ly stronger than that found with paotide 15 which is comprised of amino acids 1- 28. In fact some specimens that were only moderately reactive with peptide 15 (eg: specimens 12, low titer panel and 22 and 18, mixed titer panel) were strongly reactive with peptide 25. It is important to note that little reactivity was obtained with peptides 23 WO 92/22571 PCT~US92/03635 41 and 24, even though their amino acid sequences are present in the longer peptide number 25. In addition, with both Boston Biomedica panels, peptides #15 and #25 significantly outperformed peptide #20 in immunoreactive signal strength.

Furthermore, 10 recombinant HCV capsid reactive samples, from the two panels, were non-reactive with peptide #20 yet reactive with peptides and 25. No samples were peptide #20 reactive and peptide #15 or 25 non-reactive. These four peptides were tested using Harvey Alter's NANBH performance panel(National Institutes of Health, Bethesda, MD) with similar findings to those described above with the Boston Biomedica panels. In particular, peptide 25 gave the best assay performance (as measured by positive signal intensity) and peptide 15 was second best. For example, with specimens #J and #W, peptide 15 gave signal approximately 2 times the cutoff value of 200 nM coumar.n, while peptide 25 gave signal greater th 25 times the cutoff. Even though this saml. Aras from a blood donor implicated in the trar ion of NANBH, it was non-reactive with p. des 20, 23 and 24. It is important to note that the values for negative control specimens were similar for both specimens, therefore, the increased signal strength observed with peptide results in a significant increse in signal distance between positive and negative specimen signal.

WO 92/22571 WO 9222571PCY/1JS92/03635 42 Table 13 Reactivity of peptides 15, 20, 23, 24 and with Boston Biomedica low titre HCV panel.

1 1 2 3 4 6 7 8 9 11 12 13 14 729 19 32 5000 109 3134 3260 3857 3160 5000 1280 755 1516 1409 5000 Pent ide 21 2A 20 16 15 77 22 23 53 27 21 2764 46 52 51 72 41 146 31 32 56 30 100 141 27 27 823 43 28 5000 21 31 250 41 38 44 22 87 62 29 40 442 25 23 56 70 75 215- 642 37 62 5000 146 5000 5000 5000 3258 5000 73 5000 1320 3414 5000 1. Results are represented as riM coumarin. Values greater than 200 are reactive.

2. HCV capsid reactivity reported by Boston Biomedica, Inc.: R= reactive; N =non-reactive; I Indeterminate.

WO 92/22571 PCT/US92/03635 43 Table 14 Reactivity of peptides 15,20,24,25,and 30 with Boston Biomedica Inc, Mixed Titre HCV panel.

1

IQ

1 2 3 4 6 7 8 9 11 12 13 14 16 17 18 19 21 22 23 24 Peptide 20 5000 5000 5000 5000 2068 2595 11 20 1629 27 1746 601 40 74 45 32 5000 5000 31 134 5000 1151 933 88 3453 1601 5000 579 5000 1972 5000 5000 3732 5000 521 45 21 28 40 62 5000 3426 683 104 5000 445 5000 5000 5000 5000 23 17 50 37 22 18 23 18 30 27 37 24 22 82 29 38 232 35 37 24 42 32 78 67 30 43 HCV Capsid 2 24 27 5000 242 5000 35 5000 19 32 48 5000 28 5000 24 28 28 96 51 5000 30 179 25 5000 20 1081 84 5000 19 5000 55 5000 329 5000 39 5000 43 3429 24 38 91 88 5000 85 5000 80 5000 47 5000 49 5000 1. Results presented as nM Coumarin. Values greater than 200 are reactive.

WO 92/22571 PCT/US92/03635 Table 14 (eon't) 2. Interpretation: R reactive; N =non-reactive; I indeterminate with recombinant c22capsid as reported by Bcuston Biomedica, Inc.

WO 92/22571 PCT/US92/03635 Table Reactivity of peptides 15, 20, 23, 24 and with the Harvey Alter NANBH performance panel.

1

ID

Peptide Diaanosis 200 A 5000 5000 23 13 7 22 B 28 C 5000 D 39 E 5000 F 65 G 31 H 46 I 5000 J 418 K 67 L 48 M 155 N 5000 O 22 P 5000 Q 41 R 5000 S 47 T 22 U 35 V 5000 W 442 X 54 Y 47 Z 103 1. Results greater 5000 26 188 80 151 30 5000 92 86 44 72 5000 28 5000 44 194 4 35 32 5000 141 71 36 109 70 8 49 30 24 26 37 49 23 40 126 13 15 146 14 64 37 25 26 67 74 35 97 173 24 37 6 281 10 44 30 27 24 1843 134 26 46 115 45 14 487 13 52 27 15 24 2046 147 27 27 82 5000 27 5000 24 5000 71 36 27 5000 5000 48 51 171 5000 38 5000 26 5000 80 47 26 5000 5000 45 24 71 NANBH-chronic Normal control Implicated Donor Control NANBH-Chronic NANBH-Acute Control Control Implicated Donor Implicated Donor Normal Control Alcoholic Normal Control NANBH-Chronic Normal Control Implicated Donor Control NANBH-Chronic NANBH-Acute Control Control Implicated Donor Implicated Donor Normal Control Alcoholic Normal Control presented as nM coumarin. Values than 200 are reactive 2. Diagnosis provided by Harvey Alter.

WO 92/22571 PCT/US92/03635 46 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Leahy, David C Todd, John A Jolley, Michael E (ii) TITLE OF INVENTION: Immunoassay For Non-A Non-B Hepatitis (iii) NUMBER OF SEQUENCES: (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Baxter Diagnostics Inc.

STREET: One Baxter Parkway, DF2-2E CITY: Deerfield STATE: Illinois COUNTRY: USA ZIP: 60015 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: US 7/718,052 FILING DATE: 20-JUN-1991

CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION: NAME: Barta, Kent REGISTRATION NUMBER: 29,042 REFERENCE/DOCKET NUMBER: PA-4148 CIP (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: 708/94j-3308 TELEFAX: 708/948-2642 WO 92/22571 PC/US92/03635 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 34 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: Arg Glu Gin Asp Gin Ile Lys Thr Lys Asp Arg Thr 1 Gin Arg Gin Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg Ser Lys Asn Glu Lys Lys Lys Lys Lys INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 34 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Arg Glu Gin Asp Gin Ile Lys Thr Lys Asp Arg Thr 1 5 Gin Gin Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg Arg Ser Lys Asn Glu Lys Lys Lys Lys Lys INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 34 amino acids TYPE: amino acid WO 92/22571 PCr/US92/03635 STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Glu Gin Asp Gin Ile Lys Thr Lys Asp Arg Thr Arg 1 Gin Arg 25 (2) Arg 1 Gin Arg (2)

I

Gin Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg Ser Lys Asn Glu Lys Lys Lys Lys Lys INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 34 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Glu Gin Asp Gin Ile Lys Thr Lys Asp Arg Thr Gin Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg Ser Lys Asn Glu Lys Lys Lys Lys Lys INFORMATION FOR SEQ ID SEQUENCE CHARPCTERISTICS: LENGTH: 34 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide WO 92/22571 PCT/ US92/03635 49 (xi) SI UENCE DESCRIPTION: SEQ ID Arg Glu G-n Asp Gin Ile Lys Thr Lys Asp Arg Thr 1 5 Gin Gln Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg 15 Arg Ser Lys Asn Glu Lys Lys Lys Lys Lys INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 34 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Arg Glu Gin Asp Gin Ile Lys Thr Lys Asp Arg Thr 1 5 Gin Gin Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg Arg Ser Lys Asn Glu Lys Lys Lys Lys Lys INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 34 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide WO 92/22571 PCr/US92/03635 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Arg Glu Gin Asp Gin Ile Lys Thr Lys Asp Arg Thr 1 5 Gin Gin Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg 15 Arg Ser Lys Asn Glu Lys Lys Lys Lys Lys INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 34 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Arg Glu Gin Asp Gin Ile Lys Thr Lys Asp Arg Thr 1 5 Gin Gin Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg Arg Ser Lys Asn Glu Lys Lys Lys Lys Lys INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 34 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide WO 92/22571 PC/U92/03635 51 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: Arg Glu Gin Asp Gin Ile Lys Thr Lys Asp Arg Thr 1 5 Gin Gin Arg Lys Thr Lys Arg Ser Thr Asn Arg Arg 15 Arg Ser Lys Asn Glu Lys L Ly Lys Lys Lys INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 34 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID Arg Glu Gin Asp Gin Ile Lys Thr Lys Asp Arg Thr 1 5 Gin l3n Arg Lys Thr Lys Arg Ser Thr Asn Arg Arg Arg Ser Lys Asn Glu Lys Lys Lys Lys Lys INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 34 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide WO 92/22571 C/U9/35 PCIMS92/03635 (xi) SEQUENCE DESCRIPTION: SEQ ID, NO:ii: Arg Giu Gin Asp Gin Ile Lys Thr Lys Asp Arg Thr 1 5 Gin Gin Arg Lys Thr Lys Arg Ser Thr Asn Arg Arg 15 Arg Ser Lys Asn Gi' .P ys Lys Lys Lys Lys INFORM4ATION FOR SFQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 34 amino acids TYPE: amiino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TrYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: Arg Giu Gin Asp Gin Ile Lys Thr Lys A Arg Thr 1 5 1 Gin Gin Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg Arg Ser Lys Asn Giu Lys Lys Lys Lys Lys INFORMATION FOR SEQ ID NO:i3: ()SEQUENCE CHARVACTERISTICS: LENGTH: 28 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (iMOLECULE TYPE: peptide WO 92/22571 C/S2(35 PC*r/US92/03635 Met 1 Arg Pro (2) Met 1 Arg Pro (2) (xi) P' IENCE DESCRIPTION: SEQ ID NO:13: Ser Thr Ile Pro Lys Pro Gin Arg Lys Thr Lys Asn Thr Asn Arg Arg Pro Gin Asp Val. Lys Phe 15 Gly Gly Giy INFORMATION FOR SE1"Q ID NO:14: SEQUENCE CHAACTERISTICS: LENGTH: 28 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUIENCE DESCRIPTION: SEQ ID NO:14: Ser Thr Ile Pro Pro Gin Arg Lys Thr Lys Asn Tbr Asn Arg Arg Pro Gin Asp Val. Lys Phe 113 Gly Gly Gly INFOFYd'ATION FOR SEQ ID SEQUENCE CHTER_1T'_Q.,S: LENGTH: 28 amino acids (rp) TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide WO 92/22571 PC/US92/03635 54 (xi) SEQUENCE DESCRIPTION: SEQ ID Met Ser Thr Ile Pro Lys Pro Gin Arg Lys Thr Lys 1 5 Arg Asn Thr Asn Arg Arg Pro Gin Asp Val Lys Phe 15 Pro Gly Gly Gly INFORMATION FOR SEQ 1. SEQUENCE CHARACTEK 3: LENGTH: 12 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: Gly Gin Arg Lys Thr Lys Arg Gin Thr Asn Arg Arg 1 5 INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 12 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: Gly Gin Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg 1 5 INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 12 amino acids TYPE: amino acid STRANDEDNESS: unknown WO 92/22571 PCT/US92/03635 TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: Gly Gin Arg Lrs Thr Lys Arg Ser Thr Asn Arg Arg 1 5 INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 42 amino acids (B TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: Ile Ile Pro Asp Arg Glu Val Leu Tyr Arg Glu Phe 1 5 Asp Glu Met Glu Glu Cys Ser Gin His Leu Pro Tyr Ile Glu Gin Gly Met Met Leu Ala Glu Gin Phe lys 30 Gin Lys Ala Leu Gly Leu INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 36 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide WO 92/22571 PCTr/US92/03635 56 (xi) SEQUENCE DESCRIPTION: SEQ ID Arg Arg Gly Pro Arg Leu Gly Val Arg Ala Thr Arg 1 5 Lys Thr Ser Glu Arg Ser Gin Pro Arg Gly Arg Arg 15 Gin Pro Ile Pro Lys Ala Arg Arg Pro Glu Gly Arg 30 INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 25 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: Pro Thr Asp Pro Arg Arg Arg Ser Arg Asn Leu Gly 1 5 Lys Val Ile Asp Thr Leu Thr Cys Gly Phe Ala Asp Leu INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 37 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide WOv 92/22571 PCT/US92/03635 57 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: Leu Met Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu 1 5 Gly Gly Ala Ala Arg Ala Leu Ala His Gly Val Arg 15 Val Leu Glu Asp Gly Val Asn Tyr Ala Thr Gly Asn 30 Leu INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 38 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: Met Ser Thr Ile Pro Lys Pro Gin Arg Lys Thr Lys 1 5 Arg Asn Thr Asn Arg Arg Pro Gin Asp Val Lys Phe 15 Pro Gly Gly Gly Gin Ile Val Gly Gly Val Tyr Leu 30 Leu Pro INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 38 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide WO 92/22571 WO 9222571PCT/US92/03635 58 (xi) SEQUENCE DESCRIPTION: 8LI ID NO:24: Met Ser Thr Ile Pro Lys Pro Gin Arg Lys Thr Lys 1. 5 Arg Asn Thr Asn Arg Arg Pro Gin Asp Val Lys Phe 15 Pro Gly Giy Gly Gin Ile Val Giy Gly Val. Tyr Leu 30 Leu Pro INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 38 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID Met Ser Thr Ile Pro Lys Pro Gin Arg Lys Thr Lys 1 5 Arg Asn Thr Asn Arg Arg Pro Gin Asp Val. Lys Phe 15 Pro Gly Giy Gly Gin Ile Val. Giy Giy Val. Tyr Leu 30 Leu Pro

Claims (9)

1. An epitope group useful in the diagnosis of Non-A Non-B hepatitis comprising a synthetic peptide having substantially the amino acid sequence encoded by the first 84 consecutive open reading frame 5' nucleotides of the hepatitis C virus genome.

2. Epitopes contained in the capsid protein of HCV comprising a first epitope having an amino acid sequence selected from the group consisting of QRKTKRNTNRR and QRKTKRSTNRR; and a second epitope contiguous thereto at the 3' end of the first said epitope having the amino acid sequence selected from the group consisting of PQDVKFPGGG and PQDVKFPGGGIVGGVYLLP.

3. An assay for detection of antibodies specific for HCV antigens comprising contacting a sample containing antibodies to a synthetic peptide having substantially the amino acid sequence encoded by the first 114 consecutive 5'open reading frame nucleotides of the HCV viral genome, with said peptide immobilized upon a solid substrate, separating unbound antibodies from those bound to the said solid substrate, and detecting the presence of bound antibodies on the solid substrate.

4. An assay for detection of antibodies specific for HCV antigens comprising contacting a sample containing antibodies specific for a peptide having an epitope having an amino acid sequence selected from the group WO 92/22571 PCT/US921%3635 consisting of QRITKRNTNRR and QRKTKRSTNRR and a second epitope having the amino acid sequence selected from the group consisting of PQDVKFPGGG and PQDVKFPGGGIVGGVYLLP separating unbound antibodies from those bound to the said solid substrate, and detecting the prescence of bound antibodies on the solid substrate.

The assay of ciaims 3 or 4 wherein the said solid substrate is selected from the group consisting of the surfaces of a microtiter plate, a glass fiber filter, paramagnetic microparticles, and latex particles.

6. The assay of claims 3 or 4 wherein the said bound antibodies are detected by antispecies specific enzyme-conjugated antibodies.

7. A competitive inhibition assay for detecting antibodies specific for HCV antigens comprising contacting a sample containing antibodies to a synthetic peptide having substantially the amino acid sequence encoded by the first 84 consecutive 5'open reading frame nucleotides of hepatitis C virus, with said peptide immobilized upon a solid substrate, said contacting being carried out in the presence of competing amounts of a peptide having substantially the amino acid sequence: PQDVKFPGGG, separating the antibodies binding to said peptide immobilized on said solid substrate from those antibodies not so bound, and detecting the presence of bound antibodies on said solid substrate.

8. A competitive inhibition assay for detecting antibodies specific for HCV antigens comprising WO 92/22571 PCr/US92/03635 61 contacting a sample containing antibodies to a peptide comprising amino acids 1-28 inclusion of the amino terminus of the hepatitis C capsid protein, or portions thereof selected from the group consisting of QRKTKRNTNRR, QRKTKRSTNRR, and PQDVKFPGGG, with said peptide immobilized upon a solid substrate, said contacting being carried out in the presence of competing amounts of a peptide having substantially the amino acid sequence: PQDVKFPGGG, separating the antibodies binding to said peptide immobilized on said solid substrate from those antibodies not so bound, and detecting the presence of bound antibodies on said solid substrate.

9. An assay for detection of antibodies specific for HCV antigens comprising incubating a sample with the peptides of claim 1 or 4 tagged with a fluorophor, and measuring an increase in fluorescence polarization.