WO1992022571A1

WO1992022571A1 - Immunoassay for non-a non-b hepatitis

Info

Publication number: WO1992022571A1
Application number: PCT/US1992/003635
Authority: WO
Inventors: David C. Leahy; John A. Todd; Michael E. Jolley
Original assignee: Baxter Diagnostics Inc.
Priority date: 1991-06-13
Filing date: 1992-04-29
Publication date: 1992-12-23
Also published as: JPH06500796A; EP0544861A4; CA2087974A1; JP3350729B2; EP0544861A1; AU648912B2; AU1972092A

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.

Description

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 5¹ 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 (non- structural) 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. , 60:

167 (1990) have recently described the 5¹ 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 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 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 5¹ 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 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. 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 amino acid homology between the Arima and the 5¹ 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 IA 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 IA, No. 9, is the Arima sequence 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 IB 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 20. The amino acids depicted in Figure 1C are in registration with the sequence of Figure IB. A glycine was added to the 5' end of the common sequence to prevent cyclization of the glutamine residue at amino acid position 1. Figure ID is a non-structural HCV sequence disclosed in EP 0 318 216 and is located within the C-100 protein. Figure IE 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 IF 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 probably because some spacing is necessary to prevent steric interference with the correct conformational presentation of the epitope. The results also indicate that the HCV 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 identify 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 (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, ribosome- binding, 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. In an alternative embodiment, the peptides of the present invention may be coated onto microtiter plate wells in the classical enzyme linked im unosorbent assay (ELISA) , incubated with sample, aspirated, and an enzyme- conjugated 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. 81:1684 (1984); PCFIA, Jolley, et al., J. Immunol. Meth. 67:21 (1984). It should be emphasized that minor changes in sequence, e.g., 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 having strict homology to the sequence of the original polypeptide.

Brief Description of Drawings

Figure 1:

A. The amino acid sequence of full length Arima clone 14 peptide (#9) and fragments thereof are described. In addition, the sequence of clone 14 peptide modified at amino acid position 20 (peptide #8) and fragments thereof are described. The dashed line represents the actual peptide synthesized.

B. The amino acid sequence 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 no. 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 non- structural 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 acids 109-133 and peptide no. 22 covers amino acids 133-169. The amino acid sequence of a peptide representing HCV capsid amino acids 1-38 is presented as peptide no. 25. Two other peptides representing amino acids 29-38 (peptide no. 23) and amino acids 19-38 (peptide no. 24) are described as well. Dashed line represents the peptide synthesized.

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 Inc.) according to the following procedure: 250 ul of 5% weight/volume paramagnetic particles were pelleted in a microfuge at 5000 rpm for 5 minutes. The supernatant was removed and the particle pellet was resuspended with 500 ul of 70% 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)-1- propane 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 urn) water, resulting in a peptide concentration of 10 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 rpm for 3.5 minutes, the supernatant was removed and particles were 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 follows: 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 15% 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°C-42°C for 30 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, 0.5 ml Tween-20, 8.76 g sodium chloride, and 1.0 g sodium azide per liter; pH 7.4). 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. Particles in each well were resuspended in 30 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 7.4). 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 7.5, 0.5M sodium chloride, 5% glycerol, 2.3 mM magnesium chloride, 0.1% sodium azide and 20% 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°C-42°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-methyl- umbelliferyl-B-D-galactopyranoside, 3.58 g tricine, 5.1 ml dimethyl sulfoxide, 30 ml methyl alcohol, 0.20 g sodium aide, 0.5 ml

Tween-20, per liter, pH 8.5). The presence of B-galactosidase (i.e., conjugate) in the wells triggered the cleavage of MUG to generate a fluorescent coumarin product. Fluorescence (excitation wavelength 365 nm/emission wavelength 450 nm) was measured at two timed intervals (i.e., 2 and 14 minutes) post MUG addition. The plate was incubated at 37°C-42°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 cou arin 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 IA 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 non- reactive sera. As the peptide length was increased stepwise from 10 to 19 amino acids in length (i.e., 1 thru 4), positive signal was observed (Table 1) . 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, 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.

Xable 1

_Reactivity of peptide fragments 1-6 with HCV positive and negative sera. e Pe tide Number

Avg. Negative-^

11 12 13 12 11 11 HCV Positive/HCV Negative Avg.⁴

KC3 0.9 0.9 0.9 19 35 31

5397 0.6 0.6 0.6 41 47 31

1. Results are represented as nM coumarin. 2. Samples positive or non-reactive to HCV 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.

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 20 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.

Peptide #18 comprised the common amino acid sequence shared by clone 14 and the HCV capsid sequence first described by Okamoto. Peptides #16 and #17 are identical to #18 except that the serine of #18 at position 20 was substituted with a glutamine or asparagine for peptides #16 and #17, respectively (Figure IB). 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 (i.e., #6 and #10). 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. Table 2

Reactivity of peptide fragments #6, 10, 16-18 with HCV positive and negative sera.¹

EXAMPLE 5 Three fragments (#13-15) corresponding to the first 28 amino acids of HCV capsid as described by Okamoto and depicted in Figure IB 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 3) . However, as the peptide length was increased to include the clone 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.

Table 3

Reactivity of peptide fragments 13-15 with HCV positive and negative sera.

1. Results are presented as nM coumarin obtained with peptides 13, 14 and 15. 2. Signal/Noise = Results represent nM coumarin HCV reactive sample/nM coumarin Avg. of 10 HCV negative samples for peptides 13, 14 and 15. 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 IB) 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. 50 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 to asparagine substitution. Therefore, some samples display immunoreactivity to peptide 15 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 15. 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 15.

To further identify the nature of the 10 amino acid fragment (peptide fragment 13) inhibition, eighteen HCV capsid reactive samples were tested for inhibition of binding to peptide 15 using fragment 13. The results presented in Table 8 show that fragment 13 completely inhibited all samples binding to peptide 15. This finding was unexpected, since some to these samples have been shown to have immunoreactivity 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.

Table 4

Use of peptide fragments to inhibit HCV positive sample binding to peptide #15.^!

15

1. Results are presented as % of control (competing no peptide addition) sample reactivity remaining after preadsorbtion with indicated peptide. For the control (None) column the nM coumarin values are indicated in parenthesis.

Table 5

Use of peptide fragments to inhibit HCV positive sample binding to peptide #15.¹

Sample Peptide

None 2. 10 £ 11 15.

AR54 ( 880)100% 85% 78% 90% 75% 3%

AR191 (5000)100% 100% 100% 100% 100% 4%

AR222 ( 830)100% 100% 63% 102% 111% 6% AR225 ( 492)100% 108% 57% 61% 58% 6%

1. Results are presented as % of control (none) sample reactivity remaining after preadsorbtion with indicated peptide fragment. For the control column (none) the nM coumarin values are indicated in parenthesis.

Table 6

Sample Peptide

None 16 11 18 12 15.

AR89 (5000) 100% 100% 100% 100% 100% 1%

AR44 (5000) 100% 100% 100% 100% 100% 1%

Table 7

30

Table 8

Use of peptide fragment #13 to inhibit HCV positive sample binding to peptide #15.

nM Coumarin¹ Percent

1. Results are presented as nM coumarin generated in the absence and presence of fragment #13.

2. % inhibition:

1 - nM coumarin f+ fragment)

X 100 nM coumarin {- fragment} 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 IA, peptide 9) , clone 14 fragments (peptides 6 and 8) , HCV capsid 1-28 (peptide 15) , or HCV nonstructural protein C- 100 (peptide 19) , Figures IA, IB 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 IA, 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, 10 and 15) . Even the 1-28 amino acid peptide corresponding to HCV capsid (peptide 15) 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 IA (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 solid phase target in inhibition experiments. As shown in Table 10, peptides corresponding to the common sequence (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 15 (i.e., 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 20) 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 (i.e., #19, of HCV nonstructural protein C-100, Figure ID) 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. 33

Table 9

Use of peptide fragments to inhibit HCV sample binding to peptide #9.¹

Table 10

Use of peptide fragments to inhibit HCV positive sample binding to peptide #6.^!

1. Results are presented as % of control (no peptide added) sample reactivity remaining after preadsorbtion with indicated peptide fragment. For the control column (none) the nM coumarin values are indicated in parenthesis.

Table 11

Use of peptide fragment #13 to inhibit HCV positive sample binding to peptides 9, 8, 15, 19.

Table 11 (cont ' d)

nM Percent

Coumarin¹ Inhibition^

-#13 +#13

1372 1542 0%

252 253 0%

5000 5000 0%

5000 5000 0%

1. Results are presented as nM coumarin generated in the absence (-) and presence (+) of fragment #13.

2. % inhibition:

1 - nM coumarin f+ fragmentt

X 100 nM coumarin {- fragment}

Example 8

Four peptides (#15, 20, 21 and 22) corresponding to sequences of the HCV capsid and depicted in Figures IB and IE were tested for immunoreactivity as according to Example 2. As shown in Table 12, immunoreactivity with HCV positive sera was strongest for peptide #15 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 15 provides the largest value for every sample. Thus, the greatest assay utility in 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. Table 12

Reactivity of peptides 15, 20-22, with HCV positive and negative sera.

Peptide 21 21

ND-

49 91 Table 12 (con't)

1. Results are represented as nM coumarin.

2. Samples reactive or non-reactive to HCV capsid antigen.

3. ND = Sample not tested.

4. Positive/Negative = HCV Positive/HCV Negative Mean from 5 negative samples. Example 9 To further define the immunoreactivity of HCV capsid peptides, peptides spanning amino acid positions 1-28 (number 15, Figure IB) , 29- 38(number 23, Figure IF), 19-38 (number 24, Figure IF) , 1-38 (number 25, Figure IF) , and 39-74 (number 20, Figure IE) were evaluated in the paramagnetic microparticle assay described in Example 2. The peptide particle combinations were tested with three different NA BH 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 and 25 were reactive with all specimens indicated by Boston Biomedica to be c22 capsid reactive and were non-reactive with the remaining specimens. The reactivity found with peptide number 25 covering amino acids 1- 38 was generally stronger than that found with peptide 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 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 #15 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 # , peptide 15 gave signal approximately 2 times the cutoff value of 200 nM coumarin, while peptide 25 gave signal greater than 25 times the cutoff. Even though this sample was from a blood donor implicated in the transmission of NANBH, it was non-reactive with peptides 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 25 results in a significant increse in signal distance between positive and negative specimen signal. Table 13

Reactivity of peptides 15 , 20 , 23 , 24 and 25 with Boston Biomedica low titre HCV panel.¹

1. Results are represented as nM coumarin. Values greater than 200 are reactive.

2. HCV capsid reactivity reported by Boston Biomedica, Inc. : R= reactive; N = non-reactive; I = Indeterminate. Table 14

Reactivity of peptides 15,20,24,25,and 30 with Boston Biomedica Inc, Mixed Titre HCV panel.¹

l - O -

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

1. Results presented as nM Coumarin. Values greater than 200 are reactive. Table 14 (con't )

2. Interpretation: R = reactive; N = non-reactive; I = indeterminate with recombinant c22capsid as reported by Boston Biomedica, Inc.

Table 15

Reactivity of peptides 15 , 20 , 23 , 24 and 25 with the Harvey Alter NANBH performance panel . ¹

ID Peptide Diagnosis

15 20 22 24 5

A 5000 5000 13 37 5000 NANBH-chronic B 28 22 7 6 27 Normal control

C 5000 5000 70 281 5000 Implicated Donor D 39 26 8 10 24 Control E 5000 188 49 44 5000 NANBH-Chronic F 65 80 30 30 71 NANBH-Acute G 31 151 24 27 36 Control H 46 30 26 24 27 Control I 5000 5000 37 1843 5000 Implicated Donor J 418 92 49 134 5000 Implicated Donor K 67 86 23 26 48 Normal Control L 48 44 40 46 51 Alcoholic M 155 72 126 115 171 Normal Control N 5000 5000 13 45 5000 NANBH-Chronic 0 22 28 15 14 38 Normal Control P 487 5000 Implicated Donor Q 13 26 Control R 52 5000 NANBH-Chronic S 27 80 NANBH-Acute T 15 47 Control U 24 26 Control V 2046 5000 Implicated Donor W 147 5000 Implicated Donor X 27 45 Normal Control Y 27 24 Alcoholic z

82 71 Normal Control

1. Results presented as nM coumarin. Values greater than 200 are reactive

2. Diagnosis provided by Harvey Alter. SEQUENCE LISTING (1) GENERAL INFORMATION:

(i) 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: 25 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Baxter Diagnostics Inc.

(B) STREET: One Baxter Parkway, DF2-2E

(C) CITY: Deerfield

(D) STATE: Illinois

(E) COUNTRY: USA (F) ZIP: 60015

(V) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentin Release #1.0,

Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US 7/718,052

(B) FILING DATE: 20-JUN-1991 (C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Barta, Kent

(B) REGISTRATION NUMBER: 29,042

(C) REFERENCE/DOCKET NUMBER: PA-4148 CIP (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 708/948-3308

(B) TELEFAX: 708/948-2642 (2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 34 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: Arg Glu Gin Asp Gin lie Lys Thr Lys Asp Arg Thr 1 5 10

Gin Gin Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg

15 20

Arg Ser Lys Asn Glu Lys Lys Lys Lys Lys 25 30 (2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 34 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Arg Glu Gin Asp Gin lie Lys Thr Lys Asp Arg Thr 1 5 10 Gin Gin Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg 15 20

Arg Ser Lys Asn Glu Lys Lys Lys Lys Lys 25 30

(2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 34 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Arg Glu Gin Asp Gin lie Lys Thr Lys Asp Arg Thr 1 5 10

Gin Gin Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg

15 20

Arg Ser Lys Asn Glu Lys Lys Lys Lys Lys 25 30

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 34 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide ( i) SEQUENCE DESCRIPTION: SEQ ID NO:4: Arg Glu Gin Asp Gin lie Lys Thr Lys Asp Arg Thr 1 5 10

Gin Gin Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg

15 20

Arg Ser Lys Asn Glu Lys Lys Lys Lys Lys 25 30 (2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 34 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: Arg Glu Gin Asp Gin lie Lys Thr Lys Asp Arg Thr 1 5 10

Gin Gin Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg 15 20

Arg Ser Lys Asn Glu Lys Lys Lys Lys Lys

25 30

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

Arg Glu Gin Asp Gin lie Lys Thr Lys Asp Arg Thr 1 5 10

Gin Gin Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg 15 20 Arg Ser Lys Asn Glu Lys Lys Lys Lys Lys 25 30

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 34 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Arg Glu Gin Asp Gin lie Lys Thr Lys Asp Arg Thr 1 5 10

Gin Gin Arg Lys Thr Lys Arg Asn Thr Asn Arg Arg 15 20

Arg Ser Lys Asn Glu Lys Lys Lys Lys Lys

25 30

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

Arg Glu Gin Asp Gin lie Lys Thr Lys Asp Arg Thr 1 5 10

(2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 34 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: Arg Glu Gin Asp Gin lie Lys Thr Lys Asp Arg Thr 1 5 10

Gin Gin Arg Lys Thr Lys Arg Ser Thr Asn Arg Arg 15 20

Arg Ser Lys Asn Glu Lys Lys Lys Lys Lys 25 30

(2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

Arg Glu Gin Asp Gin lie Lys Thr Lys Asp Arg Thr 1 5 10

Gin Gin Arg Lys Thr Lys Arg Ser Thr Asn Arg Arg 15 20 Arg Ser Lys Asn Glu Lys Lys Lys Lys Lys 25 30

(2) INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 34 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: Arg Glu Gin Asp Gin lie Lys Thr Lys Asp Arg Thr 1 5 10

Gin Gin Arg Lys Thr Lys Arg Ser Thr Asn Arg Arg 15 20

Arg Ser Lys Asn Glu Lys Lys Lys Lys Lys 25 30

(2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide ( i) SEQUENCE DESCRIPTION: SEQ ID NO:12:

Arg Glu Gin Asp Gin lie Lys Thr Lys Asp Arg Thr 1 5 10

(2) INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: Met Ser Thr lie Pro Lys Pro Gin Arg Lys Thr Lys 1 5 10

Arg Asn Thr Asn Arg Arg Pro Gin Asp Val Lys Phe 15 20

Pro Gly Gly Gly 25

(2) INFORMATION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

Met Ser Thr lie Pro Lys Pro Gin Arg Lys Thr Lys 1 5 10

Arg Asn Thr Asn Arg Arg Pro Gin Asp Val Lys Phe 15 20 Pro Gly Gly Gly 25

(2) INFORMATION FOR SEQ ID NO:15: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: Met Ser Thr lie Pro Lys Pro Gin Arg Lys Thr Lys 1 5 10

Arg Asn Thr Asn Arg Arg Pro Gin Asp Val Lys Phe 15 20

Pro Gly Gly Gly 25

(2) INFORMATION FOR SEQ ID NO:16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: unknown

(D) 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 10

(2) INFORMATION FOR SEQ ID NO:17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: unknown

(D) 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 10

(2) INFORMATION FOR SEQ ID NO:18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: Gly Gin Arg Lys Thr Lys Arg Ser Thr Asn Arg Arg 1 5 10

(2) INFORMATION FOR SEQ ID NO:19: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 42 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: lie lie Pro Asp Arg Glu Val Leu Tyr Arg Glu Phe 1 5 10

Asp Glu Met Glu Glu Cys Ser Gin His Leu Pro Tyr

15 20 lie Glu Gin Gly Met Met Leu Ala Glu Gin Phe Lys 25 30 35 Gin Lys Ala Leu Gly Leu

40 (2) INFORMATION FOR SEQ ID NO:20: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 36 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: Arg Arg Gly Pro Arg Leu Gly Val Arg Ala Thr Arg 1 5 10

Lys Thr Ser Glu Arg Ser Gin Pro Arg Gly Arg Arg 15 20

Gin Pro lie Pro Lys Ala Arg Arg Pro Glu Gly Arg 25 30 35

(2) INFORMATION FOR SEQ ID NO:21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: unknown

(D) 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 10

Lys Val lie Asp Thr Leu Thr Cys Gly Phe Ala Asp 15 20 Leu 25

(2) INFORMATION FOR SEQ ID NO:22: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 37 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: Leu Met Gly Tyr lie Pro Leu Val Gly Ala Pro Leu 1 5 10

Gly Gly Ala Ala Arg Ala Leu Ala His Gly Val Arg 15 20

Val Leu Glu Asp Gly Val Asn Tyr Ala Thr Gly Asn 25 30 35

Leu

(2) INFORMATION FOR SEQ ID NO:23: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 38 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: Met Ser Thr lie Pro Lys Pro Gin Arg Lys Thr Lys 1 5 10

Arg Asn Thr Asn Arg Arg Pro Gin Asp Val Lys Phe 15 20

Pro Gly Gly Gly Gin He Val Gly Gly Val Tyr Leu 25 30 35

Leu Pro (2) INFORMATION FOR SEQ ID NO:24: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 38 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide ( i) SEQUENCE DESCRIPTION: SEQ ID NO:24: Met Ser Thr He Pro Lys Pro Gin Arg Lys Thr Lys 1 5 10

Arg Asn Thr Asn Arg Arg Pro Gin Asp Val Lys Phe 15 20

Pro Gly Gly Gly Gin He Val Gly Gly Val Tyr Leu 25 30 35

Leu Pro

(2) INFORMATION FOR SEQ ID NO:25: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 38 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: Met Ser Thr He Pro Lys Pro Gin Arg Lys Thr Lys 1 5 10

Arg Asn Thr Asn Arg Arg Pro Gin Asp Val Lys Phe 15 20

Pro Gly Gly Gly Gin He Val Gly Gly Val Tyr Leu 25 30 35

Leu Pro

Claims

CLAIMS WHAT IS CLAIMED IS:

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 5¹ 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 5Open 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 consisting of QRKTKRNTNRR 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.

5. The assay of claims 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 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.