JP3350729B2 - Immunoassay for non-A non-B hepatitis - Google Patents

Description

This application is a continuation-in-part of our Patent Application No. PA-4148, filed June 13, 1991.

BACKGROUND OF THE INVENTION Most evidence for the nature of the causative agent of non-A non-B hepatitis (NANBH) comes from the conclusion that it is a Flaviviridae or Flaviviridai virus with a genome containing a single open reading frame. Matches. This virus is a hepatitis C
Virus (HCV). The proposed structural gene encoding the capsid, matrix and envelope proteins of this virus is organized at the 5 'end of the genome, and the proposed regulatory non-structural gene is located at the 3' end.

EP0318216 (Houghton) is based on Bradley et al., Semina
r in Liver Disiese, 6:56 (1986)
Disclosed is the cloning of a portion of the HCV viral genome from an enriched plasma source donated by D. Bradley. All of the sequences disclosed in EP0318216 appear to belong to the regulatory (non-structural) part of the genome. EP 0388232 supplemented the '216 application by disclosing additional nucleotide and polypeptide sequences that appeared to represent structural genes at the 5' end of the genome.

Okamoto et al., Japan. J. Exp. Med., 60: 167 (1990) describe the 5 'of the HCV virus genome corresponding to a structural protein containing amino acids 110 to 190, which is recently considered a capsid protein. The amino acid sequence is described. EP0388232
A description of the core (known as a capsid) and the envelope region corresponding to that described in (Houghton) is provided by Kataheuchi et al., Nuc. Acids Res., 18: 4626 (199).
0). None of the aforementioned patent applications indicate which particular part of the HCV structural sequence forms an antigenic determinant. Okamoto et al., Japan.J.Exp.Med., 60: 167 (1990)
Describes at least three areas of local hydrophilicity that span amino acid positions 1-120. In one of these areas, Okamoto et al., Japan. J. Exp. Med., 60: 223 (1990) identified an amino acid sequence (aa # 39-74) that had utility in immunoassays when synthesized as a synthetic peptide. did.

Additional sequences for non-A non-B hepatitis are described in EP0363025 (Arima). Computer analysis of Arima nucleotides indicates that they are not homologous to other sequences previously described in patent applications or the literature. However, a comparison of the predicted amino acid sequences shows that a short sequence for the capsid region where 10 out of 11 amino acids in the Arima sequence are identical and homologous to the corresponding sequence in the European '232 application and the article cited above. Reveal the map. Applicants have since referred to this homologous region as
Call.

SUMMARY OF THE INVENTION Applicants have identified amino acid 3 from the beginning of the HCV open reading frame.
Synthetic peptides have been created that surround up to eight amino acid sequences and surround a "consensus sequence." Peptides of varying lengths formed within this region are characterized by Arima, which contains a consensus sequence.
The 34 amino acid polypeptide was compared in an immunoassay.

According to the present invention, an epitope group having the predicted peptide sequence encoded by the first 114 contiguous open reading frame 5 'nucleotides of the previously published HCV genomic sequence was identified. These epitopes contained in the capsid protein of this virus are the first having the amino acid sequence QRKTKRNTNRR or QRTKTKRSTNRR.
And the amino acid sequence PQDVKF, which is adjacent to the first epitope at the C-terminus of the first epitope
It contains a second epitope with PGGG or PQDVKFPGGGQIVGGVYLLP.

The above defined peptide comprising the epitope group, or a substantially equivalent thereof, derived from the predicted amino acid sequence encoded by the first 114 contiguous open reading frame 5 'nucleotides of the virus, is an antiserum of the patient's serum. HC
It can be conveniently incorporated into an immunoassay for the determination of the presence of the V antibody. Such assays involve contacting a sample containing such antibodies with a solid substrate on which the peptide is immobilized, separating unbound antibodies from antibodies bound to the solid substrate, and binding on the solid substrate. Detecting the presence of the antibody.

In another aspect of the invention, a competitive inhibition assay for detecting an HCV-specific antibody comprises a sample comprising an antibody having specificity for an epitope group comprising the first 28 capsid amino acids; A solid phase substrate on which an epitope group-containing peptide is immobilized, and a substantially amino acid sequence
Contacting in the presence of a competing amount of the peptide having PQDVKFPGGG, separating the antibody bound to the peptide immobilized on the solid substrate from the antibody that did not so bind, and determining the amount of bound antibody Including.

In another embodiment, the peptide can be labeled with a fluorophore such as fluorescein. The labeled fluorophore is incubated with the sample to form an antibody-peptide complex and the increased fluorescence polarization is measured. Thus, a homogeneous assay is provided that avoids the separation step.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Peptides containing protein epitopes, preferably produced by synthetic chemistry techniques, may be more desirable in immunoassays than the whole protein itself. In assays utilizing a solid substrate on which the target antigen is adsorbed, the adsorbed surface area is the limiting parameter of the assay. For this reason, it is more efficient to adsorb more peptide molecules containing a critical epitope per unit surface area than the unreacted portion of the large protein occupying the adsorption site.

In the present invention, the selection of the amino terminus of the HCV capsid protein was performed on the following basis. Arima array and Ho
The complete lack of nucleic acid homology between the sequences disclosed in the ughton application suggested that 12 completely different viruses could be involved in NANBH. However, there is a high degree of reactivity against antigens expressed from both source clones, which cannot be explained but have the same serum panel.

An 11 amino acid segment containing residues 8 to 18 (HCV, Ho
ughton) and Arima at the polypeptide level
The finding that there is a 10 amino acid homology between the 5'Houghton sequences suggested some correlation of the virus. The findings also suggested that the consensus sequences form a common epitope. Analysis of the immunoreactivity of a panel of HCV positive sera against the peptide encoding the consensus sequence and peptides containing flanking sequences of Arima and Houghton of various lengths confirmed the presence of an epitope within the consensus sequence.

FIG. 1A shows the sequence of a number of peptides obtained from the Arima clone 14 sequence. In this series Nos. 1-8, serine was replaced with asparagine so that the consensus sequence precisely corresponds to Houghton. In this figure, the Arima amino acid sequence is arranged horizontally in a number of vertical rows. Dotted lines between them indicate the corresponding ranges of the individual synthetic peptides. Thus, for example, in row No. 1, the peptide is intended to extend from amino acid residues 25 to 34. Fig.1A, N
o.9 is an Arima sequence with a serine at position 20 as described by Arima. Peptides No. 11 and 12 were No.
Serine at position 20 in 11 and position 20 in No. 12
Is a consensus sequence having asparagine at the end.

FIG. 1B shows a synthetic peptide containing Houghton (and Okamoto) sequences of various lengths. FIG. 1C shows the consensus sequence with various amino acid substitutions at position 20. The amino acids shown in FIG. 1C correspond to the sequence in FIG. 1B. Glycine was added to the N-terminus of the consensus sequence to prevent cyclization of the glutamine residue at amino acid position 1. FIG. 1D is the non-structural HCV sequence disclosed in EP0318216 and is located within the C-100 protein. FIG. 1E provides the sequence for the capsid fragment selected by Okamoto et al., Extending from amino acid residue number 39 to all 74. In addition, sequences corresponding to the two structural peptides (aa109-133 and 133-169) have been submitted. FIG. 1F shows HCV covering amino acids 1-38.
Figure 2 shows the peptide sequence and its two additional carboxy terminal peptide fragments.

The results of experiments in which these various peptides were immobilized on an immobilized substrate and tested in an immunoassay on a panel of sera containing antibodies to HCV are detailed in the examples. Generally, amino acids 1-38
The complete peptide of the Okamoto sequence covering the best gave the best signal to background ratio. The consensus sequence exerted some immunoreactivity, however, if the carboxy terminal sequence was present, the immunoreactivity was significantly improved. This is probably due to the need for some spacing to prevent steric hindrance to providing the correct conformation of the epitope.

The results also indicate that the HCV capsid peptide extending from residues 1 to 8 and from residues 28 to 38 correctly identifies a greater number of HCV-reactive sera than the Mare clone 14 peptide. In contrast, Arima peptide does not identify additional positive sera that were not identified by the indicated HCV capsids 1-28 or 1-38. This means
A carboxy-flanking sequence comprising PQDVKFPGGG is meant to form a second epitope following the C-terminus of the first epitope contained in the consensus sequence. All peptide fragments were Millgen, using a fluorenylmethoxycarbonyl (FMOC) amino protection strategy and 1,3-diisopropylcarbodiimide coupling chemistry.
-Synthesized in amide form on a Biosearch 9600 model peptide synthesizer. The amide form of the sequence was adopted because it could be expected to more closely mimic the biologically active analog than the free acid form. The activated amino acids were linked to 2,4-dimethoxybenzhydrylamine resin. Peptide synthesis was monitored by ninhydrin analysis for all amino acids except proline where the isatin test was performed. The synthesized peptide contains a reagent R containing trifluoroacetic acid, thioanisole, ethanedithiol and anisole in a volume ratio of 90: 5: 3: 2.
Cleaved from the resin.

Peptides cleaved from the resin were purified by high performance liquid chromatography (HPLC) and characterized by the Porton PI 2090E integrated microsequencing system to confirm correct sequence. Purity was checked by HPLC on a reversed phase column for 35 minutes using a linear gradient from 5% to 40% acetonitrile in trifluoroacetic acid. Absorbance was tracked at 230 nm.

Alternatively, recombinant peptides can be produced biologically using cloning techniques, promoter manipulation, ribosome binding, and translation, which are routinely available to those of skill in the art.

The peptides of the present invention can be advantageously used in any assay system that uses a protein target.
In a preferred embodiment, the target peptide fragment is coated onto the paramagnetic microparticle in a passive or covalent coating method. After the incubation step in the presence of the anti-HCV antibody, the bound antibody-peptide complex is separated from the unbound antibody by magnetic separation, and the amount of antibody in the antibody-peptide complex is determined.

Conveniently, detection of the conjugated anti-HCV antibody can be performed by further reacting the conjugate with an enzyme-linked anti-human antiserum. Upon separation of the labeled complex on the paramagnetic microparticles by magnetic separation and washing, a fluorogenic substrate is added. The amount of fluorescence measured is therefore directly proportional to the amount of anti-NANBH antibody present in the sample.

In an alternative embodiment, the peptides of the invention are coated onto a classical enzyme-linked immunosorbent assay (ELISA) microtiter plate, incubated with the sample, aspirated, and the conjugated anti-human antiserum is added. Can be. Glass fiber filters may be used as solid substrates in the round partition chromatography scheme.
Detection is conventionally performed by adding the appropriate substrate and / or chromogen, and measuring the product formed. For a general discussion of ELISA, see Langone et al.,
Immuno Techniques, Part D Immunoassay.Methods
See In Enzymology, p. 84 (1982).

Other assay schemes that can be applied to the peptides of the invention include:
Western blot, Towbin et al., Proc. Nat.
ci., 76: 4350 (1979); Radioimmunoassay (RIA), W
alsh et al., J. Infect. Dis., 21: 550 (1970); Competition assay, Diamandis, Clin., Biochem., 21: 139 (1988); Non-competition assay, Crook et al., J. Gen. Virol., 46:29 (19
80); Immunoprecipitation, Tojo et al., Clin.
Chem. 34: 2423 (1988) and dot blots, Jahn et.
al., Proc. Nat'1. Acad. Sci. 81: 1684 (1984); PCFIA, Jo
lley et al., J. Immunol. Meth. 67:21 (1984).

It should be emphasized that minor changes in the sequence, such as amino acid substitutions, additions or deletions, do not appreciably affect the assay performance as the epitopes do not change significantly. Thus, such insignificant changes in structure are considered to be peptide equivalents with strict homology to the sequence of the original polypeptide.

BRIEF DESCRIPTION OF THE FIGURES FIG. 1: The full length amino acid sequence of A. Arima clone 14 peptide (No. 9) and fragments thereof are described. In addition, the sequence of the clone 14 peptide modified at amino acid position 20 (peptide No. 8) and fragments thereof are described. The dotted line represents the peptide actually synthesized.

B. Amino acid sequence of the HCV capsid covering the first 28 amino acids is described. The dotted line represents the peptide actually synthesized.

C. Amino acids 8-18 of HCV capsid (or clone 1
The amino acid sequences of three peptides representing four amino acids 14-24) are presented. Glycine was added to the N-terminus for all peptides. Peptides No. 16 and No. 18
It contained an amino acid substitution at HCV capsid amino acid position 14 (or amino acid position 7 in FIG. 1C). The dotted line represents the synthesized peptide.

D. Amino acid sequence of a peptide corresponding to a certain region in HCV nonstructural region 4 (C-100 protein) is presented. This peptide is the HCV amino acid 1 described by Houghton.
693-1735.

E. Amino acid sequences of three different HCV capsid peptides are presented. Peptide No. 20 is amino acids 39-74
, Peptide No. 21 covers amino acids 109-133, and peptide No. 22 covers amino acids 133-169.

F. The amino acid sequence of the peptide representing HCV capsid amino acids 1-38 is provided as Peptide No. 25. Amino acids 29-38 (peptide No. 23) and amino acids 19-38
Two other peptides representing (Peptide No. 24) have also been described. The dotted line represents the synthesized peptide.

Example 1 Peptides corresponding to the sequences shown in FIGS. 1A to 1E were made as indicated above. Peptides were passively coated on paramagnetic polystyrene microparticles (4 micron diameter, Baxter, Diagnostics, Inc., Pandex Division) according to the following procedure. 250 μm of 5% w / v paramagnetic particles were pelleted for 5 minutes at 5000 rpm in a microcentrifuge. The supernatant was removed and the particle pellet was resuspended in 500 μl of 70% ethanol for 15 minutes. The particles were then pelleted as above and the supernatant was removed. The particles were treated with a 0.1 M CAPS buffer [(3-cyclohexylamino) -1-propanesulfonic acid],
Resuspended in. The particles were pelleted as before and the supernatant was removed.

The lyophilized peptide is weighed and sterile filtered (0.22μ
m) Resuspended in water at a peptide concentration of 10 mg / mL and allowed to dissolve in solution for 30 minutes at room temperature. The dissolved peptide was allowed to stabilize for 20 minutes at room temperature. 250 μ of this peptide solution was transferred to the washed particles. The particles were resuspended and shaken at room temperature for 12-16 hours.

The passively adsorbed peptide particles are then 3.5 rpm at 5000 rpm.
Pellet for minutes, remove supernatant, and remove particles to 0.05%
Resuspended in isotonic buffered saline with Tween20. The particles were then washed once with isotonic buffered saline with 0.05% Tween 20 and three times with isotonic buffer using centrifugation at 5000 rpm (3.5 minutes). The coated particles were then resuspended in isotonic buffered saline at a final particle concentration of 0.025% w / v.

Example 2 Paramagnetic particle assays were performed using the peptide fragment coated particles described in FIG. 1 as follows.

Human serum or plasma was added to a 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)
1: 100 dilution. 50 μl of the diluted sample was added to each well of a black plastic microtiter plate.
Samples were tested in at least two replicates. Paramagnetic particles coated with the peptide described in Example 1 were added to each well (20μ). Plate then at 37 ° C to 42 ° C
For 30 minutes.

At the end of the incubation, remove 100 particles from the wells.
μPBS and Tween 20 (2.06 g of dibasic sodium phosphate, 0.318 g of monobasic sodium phosphate per liter, Tween 20
0.5m, sodium chloride 8.76g, sodium azide 1.0g, p
H7.4). During the washing step, the paramagnetic particles were retained in the microtiter plate well by applying a magnetic field to the bottom of the plate. The particles were washed five times in this manner.

Reconstitute the particles in each well with 30μ of particle resuspension buffer (4.346 g sodium phosphate dibasic, 0.524 g sodium phosphate monobasic, 8.76 g sodium chloride, 1 g sodium azide, pH 7.4 per liter). Suspended. Next, a goat anti-human IgG (H + L) conjugated with β-galactosidase was added to a conjugate dilution buffer (0.1 M Tris-HCl pH 7.5, 0.5 M sodium chloride, 0.1 M
20μ diluted 1: 1,000 in 20% neonatal calf blood (% sodium azide) was then added to the wells.

Any human IgG or IgM bound to the particles was recognized by the conjugate and related. The conjugate solution was designed to provide maximum liquid stability and reactivity. In particular, newborn calf serum is preferred over calf serum. After incubation with the conjugate at 37 ° -42 ° C. for 15 minutes, the particles in the wells were washed five times with PBS and Tween 20 as described above to remove substantially all unbound conjugate. Tween 20 in the wash accelerated the washing process and removed non-specifically bound conjugates.

Finally, 50 μl of a substrate solution of 4-methylumbelliferin-β-D-galactoside (MUG) was added to each well (0.178 g of 4-methylumbelliferyl-β-D-galactoside and 3.58 g of tricine per liter). , Dimethyl sulfoxide 5.1m, methyl alcohol 30m, sodium azide
0.20 g, Tween 20 0.5 m, pH 8.5). The presence of β-galactosidase (ie, conjugate) in the well triggered the cleavage of MUG, generating a fluorescent coumarin product. Fluorescence (excitation wavelength 365 nm / emission wavelength 450 nm) was measured 2 hours after MUG addition (ie 2 minutes and 14 minutes). The plate was incubated at 37 ° C to 42 ° C during the fluorescence measurement. The difference between the two values was a kinetic measure of fluorescent product generation and a direct measurement of conjugate and human IgG / IgM bound to particles. Dynamic fluorescence values were converted to nM coumarin values using various concentrations of coumarin itself and its resulting fluorescence to establish a standard curve. The results were calculated as nM coumarin generated over 12 minutes. Dynamic values greater than 5000 are referred to as 5000 in subsequent examples. The cut-off for the response was determined to be 200 nM coumarin by testing 300 random donor EDTA plasma samples and HCV capsid seroconverted samples.

Example 3 The immunoreactivity of the clone 14 peptide fragment was determined as follows. The peptide fragments 1 to 6 shown in FIG. 1A were coated on paramagnetic particles according to Example 1.
The resulting peptides were evaluated in the assay described in Example 2 using three HCV capsid reactive sera and three non-HCV reactive sera. A positive signal was observed when the peptide length was increased stepwise from 10 to 19 amino acids in length (ie 1 to 4) (Table 1). Fragments shorter than 19 amino acids were not reactive. Increasing the peptide length by adding 23 amino acids did not increase the signal any further. Thus, in this assay design, the immunoreactivity of the peptide is approximately at the amino acid position
Exists between 12 and 19. Negative samples remained non-reactive with increasing peptide length, and therefore the optimal assay performance measured with the positive / negative sample signal was at amino acid position 10-
34 were observed in fragment Nos. 4-6.

Example 4 The immunoreactivity of the clone 14 peptide fragment (FIG. 1) was further evaluated in four HCV capsids and two HCV negative sera using the paramagnetic microparticle assay described in Example 2. As seen in Table 2, peptide N
o.6 shows stronger reactivity than peptide No.10. Both peptides contain clone 14 amino acids 10-34 and peptide No. 6
Are identical except that amino acid position 20 was changed from serine to asparagine. This single amino acid substitution increased the positive signal reactivity without increasing the negative signal.

Peptide No. 18 contained a consensus amino acid sequence occupied by clone 14 and the HCV capsid sequence first described by Okamoto. Peptides No. 16 and No. 17
Indicates that No. 18 serine at position 7 is No. 16 and No. 17
Is identical to No. 18 except that in Example 1 was replaced with glutamine and asparagine, respectively (FIG. 1C). Table 2
Although these consensus sequences alone show some reactivity, they are not as immunoreactive as the longer peptides containing the consensus sequence (ie, Nos. 6 and 10). In addition, modification of position 20 with glutamine significantly reduces immunoreactivity compared to the unmodified clone 14 fragment. Three common peptides (No. 16-1
8) was synthesized with glycine at the N-terminus to prevent glutamic acid cyclization. Thus, the low observed immunoreactivity was not due to glutamate cyclization.

Example 5 Three fragments (Nos. 13-15) corresponding to the first 28 amino acids of the HCV capsid described by Okamoto and illustrated in FIG. 1B were tested for HCV capsid reactivity according to Example 2. The immunoreactivity was found to correspond to amino acid positions 19-28 and to be present in fragment 13 outside the co-sequence of clone 14 (Table 3). However, when the peptide chain length was increased to include the clone 14 consensus sequence, immunoreactivity was greatly increased. Further increases in peptide chain length to the aal position (peptide No. 15) gave maximal reactivity and optimal assay performance. Although immunoreactivity was demonstrated by any of these three peptides, the best performance measured by positive / negative signals was that of peptide N covering amino acid positions 1-28.
o.15.

Example 6 To further identify the immunoreactive region of the first 28 amino acids of the HCV capsid, a series of inhibition experiments was performed. Peptide No. 15 corresponding to HCV capsid aa1-28
(FIG. 1B) was coated on paramagnetic particles as described in Example 1 and then tested in a modified immunoassay as described below using HCV capsid reactive serum. The immunoassay of Example 2 is based on the first diluted sample (1:
(100 dilution) Potentially competing peptide (100 µg / m)
And modified by incubation for 30 minutes. Diluted samples containing potentially competing peptides
Add 50μ to the plastic plate, then peptide N
o.15 coated particles were added. Except for this modification, the assay was performed exactly as described in Example 2. Samples were tested with or without peptide addition, and the results were calculated to determine the% activity remaining in the presence of potentially competing peptides. Thus, 100% means that no competition occurred and that the tested peptides do not have the same immunoreactivity as peptide 15, while 6% means strong competition and exceptionally high common immunity Indicate the level.

As presented in Table 4, binding of some samples to peptide 15 can be strongly or completely inhibited by the clone 14 fragment and the common clone 14 / HCV capsid fragment. This inhibition occurs even when amino acid position 20 of clone 14 has been modified by a serine to asparagine substitution. Therefore, some samples show immunoreactivity to peptide 15 localized only to the consensus sequence. On the other hand, as shown in Tables 4, 5 and 6, some samples were completely or only partially inhibited by the peptide containing the consensus sequence fragment, and the immunoreactivity in peptide 15 also occurred outside the consensus sequence Indicates that It should be noted that the addition of glycine to the N-terminus of the consensus sequence did not enhance peptide inhibition (peptides Nos. 16-18). Thus, the lack of inhibition for peptide 12 shown in Table 6 was not due to cyclization of the N-terminal glutamic acid.

Inhibition of peptide 15 outside the clone 14 / capsid consensus sequence was further investigated using fragments of peptide 15. As shown in Table 7, capsid fragment No. 13 (aa-19-28) completely blocked binding of peptide 4 to the four capsid-reactive samples. Extension of the length of this fragment over positions 7-28 gave the same observation. Thus, some samples are only immunoreactive with the 10 amino acid fragment of peptide 15.

To further identify the identity of this 10 amino acid fragment (peptide fragment 13) inhibition, 18 HCV capsid reactive samples were tested with fragment 13 for inhibition of binding to peptide 15. The results presented in Table 8 indicate that fragment 13 completely inhibited binding of all samples to peptide 15. This finding was unexpected, as it showed that some of these samples were immunoreactive with regions of peptide 15 other than fragment 13. For example, sample A83 was shown to be completely inhibited by the consensus sequence in Table 4, but in this experiment it was completely inhibited by a 10aa sequence distinct from the consensus sequence.

Example 7 To further define the immunoreactive region of clone 14, a series of competition experiments was performed. These experiments were performed on clone 14 (Fig. 1A, peptide 9), clone 14 fragment (peptides 6 and 8), HCV capsid 1-28.
(Peptide 15) or HCV protein C-100 (peptide
19), performed as described in Example 6, except that FIGS. 1A, 1B and 1D were bound to paramagnetic particles and used as targets for inhibition.

In the first series of experiments, peptide 9 corresponding to the full length of clone 14 was used as target. As shown in Table 9, the 11 amino acid clone 14 / capsid consensus sequence (Figure 1A, peptide 11) was able to potently inhibit the immunoreactivity of eight HCV capsid-reactive samples.
The same finding was observed with longer peptides containing this consensus sequence (peptides 9, 10 and 15). Even the 1-28 amino acid peptide corresponding to the HCV capsid (peptide 15) gave the same effect. However, Peptide 2 consisting of the C-terminal 13 amino acids of clone 14 showed little reactivity. Peptide 2 contains the C-terminal 3 amino acids of the clone 14 / capsid consensus sequence in its sequence, and these 8
The reactivity of the sample was approximately aa14 of the consensus sequence shown in FIG.
It appears to be against -21 (peptide 11).

To further support the conclusion that the majority of reactivity to clone 14 was due to this consensus sequence, peptide 6 was used as a solid phase target in inhibition experiments. Table 10
As shown in the figure, peptides corresponding to the consensus sequence (12,16,17
And 18) completely inhibited HCV sample binding to peptide 6. This inhibition was complete even if amino acid 20 was serine, asparagine or glutamine.

The peptide fragment corresponding to the C-terminal 10 amino acids of peptide 15 (ie, peptide 13, FIG. 1B) was unexpectedly reactive in Example 6 and was therefore evaluated for possible inhibitory reactivity to clone 14. . Table 11 shows that the peptides were No. 9 (clone 14), No. 8 (clone 14 at position 20 with serine replaced by asparagine) and No. 15 of 9 different HCV samples.
(HCV capsid amino acids 1-28) was completely inhibited. This inhibition is located outside the capsid region
HCV peptide (ie, HCV non-structural protein C-100 No.
19, FIG. 1D) was not non-specific because it was not inhibited. These results, combined with the results of Example 6, indicate that peptide 13 can potently inhibit sample reactivity binding to the clone 14 / HCV capsid consensus sequence and can be used in a competitive HCV antibody immunoassay.

Example 8 Corresponds to the sequence of the HCV capsid and in FIGS. 1B and 1
The four peptides illustrated in E (Nos. 15, 20, 21 and 22) were tested for immunoreactivity according to Example 2. Table 12
As shown in the figure, peptide No. 15 corresponding to the first 28 amino acids of HCV capsid had the strongest immunoreactivity with HCV positive serum. Peptide No. 20 (amino acids 39-74) showed some reactivity with these samples, while Peptide No. 1
It was smaller than 5. Peptides No. 21 and 22 are the same HCV
It showed little immunoreactivity with positive samples. Assay performance is measured as a positive signal divided by a negative signal. As seen in Table 12, peptide 15 provides the maximum value for all samples. HCV positive and HCV
Peptide 15 is the most useful assay for discriminating negatives
Was obtained. Similar experiments using peptides 15 and 20 were performed using publicly obtained HCV reactive sera.
Tables 13 and 14 show mixed HCV and Bston Biomedica, In
These results are shown using low titer panels obtained from c (Boston, Mass.). The results obtained for these panels are described in detail in Example 9.

Example 9 HCV Capsid Peptide, Amino Acid Positions 1-28 (No.
15 Fig. 1B), 29-38 (No. 23, Fig. 1F), 19-38 (No. 24, Fig. 1
F), peptides ranging from 1-38 (No. 25, FIG. 1F) and 39-74 (No. 20, FIG. 4) were evaluated in the paramagnetic microparticle assay described in Example 2. Peptide particle conjugates are publicly available and well characterized three different NANBH
And tested on HCV panels. The first two panels were obtained from Boston Biomedica Inc. and were low mixing titer HCV panels. Along with each panel, the manufacturer provided the reactivity of each sample to recombinant capsid c22. This reaction is a RI sold by Ortho Diagnostics (Raritan, NJ)
It is obtained using the BA2 HCV supplemental test system and is a measure of sample reactivity to full length capsid protein. As submitted in Tables 13 and 14, peptides 15 and 25 were reactive with all specimens indicated by Boston Biomedica to be c22 capsid reactive and non-reactive with the remaining specimens. Was. The reactivity seen with peptide No. 25 covering amino acids 1-38 was generally stronger than the reactivity seen with peptide No. 15 consisting of amino acids 1-28. In fact, some specimens that were mildly reactive with peptide 15 (eg, low titer panel specimen 12 and mixed titer panels 22 and 18) were strongly reactive with peptide 25. It is important to note that although their amino acid sequences are present in peptide No. 25, little reactivity was obtained with peptides 23 and 24. In addition, in the Boston Biomedica panel, peptides No. 15 and 25 significantly outperformed peptide No. 20 in immunoreactive signal strength. In addition, ten recombinant HCV capsid reactive samples from two panels were non-reactive with peptide No. 20, but reactive with peptides No. 15 and 25. None of the samples were reactive with peptide No. 20 and non-reactive with peptide No. 15 or 25. These four peptides are Harvey
Tested using the Alter's NANBH performance panel (NIH, Bethesda, MD) and yielded findings similar to those described above for the Boston Biomedica panel.
In particular, peptide 25 gave the best assay performance (as measured by positive signal intensity), and peptide 15 was second best. For example, for specimens J and W, peptide 15 is 20
Peptide 25 was 25 times greater than the cut-off value, although it gave a signal approximately twice the cut-off value of 0 nM coumarin.
This sample was from a blood donor suspected of transmitting NANBH, but was non-reactive with peptides 20, 23 and 24. Note that the values for the negative control samples are similar to those for both samples, and therefore the increased signal intensity observed with peptide 25 results in a significant increase in the signal distance between the positive and negative sample signals This is very important.

Sequence Listing (1) General Information (i) Applicant: Leahy, David C Todd, John A Jolley, Michael E (ii) Title of the Invention: IMMUNOASSAY FOR NON-A NON-B HEPATITIS (iii) Number of Sequences: 25 ( iv) Contact address: (A) Address: Baxter Diagnostics Inc. (B) Street: One Baxter Parkway, DF2-2E (C) City: Deerfield (D) State: Illinois (E) Country: USA (F) ZIP code : 60015 (v) Computer readable format: (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: June 20, 1991 (C) Classification: (viii) Agent information: (A) Name: Barta , Kent (B) Registration number: 29,042 (C) See / Docket number: PA-4148 CIP (ix) Telecommunication information (A) Telephone: 708 / 948-3308 (B) Telefax: 708 / 948-2462 (2) Information about SEQ ID NO: 1 (i) Sequence characteristics: (A) Length: 34 amino acids (B ) Type: amino acid (C) number of chains: unknown (D) topology: unknown (ii) type of molecule: peptide (ix) description of sequence: SEQ ID NO: 1 (2) Information about SEQ ID NO: 2 (i) Sequence characteristics: (A) Length: 34 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule Type: Peptide (ix) Sequence description: SEQ ID NO: 2 (2) Information on SEQ ID NO: 3 (i) Sequence characteristics: (A) Length: 34 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule Type: peptide (ix) Sequence description: SEQ ID NO: 3 (2) Information on SEQ ID NO: 4: (i) Sequence characteristics: (A) Length: 34 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule Type: peptide (ix) Sequence description: SEQ ID NO: 4 (2) Information on SEQ ID NO: 5 (i) Sequence characteristics: (A) Length: 34 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule Type: peptide (ix) Sequence description: SEQ ID NO: 5 (2) Information on SEQ ID NO: 6: (i) Sequence characteristics: (A) Length: 34 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule Type: peptide (ix) Sequence description: SEQ ID NO: 6 (2) Information about SEQ ID NO: 7: (i) Sequence characteristics: (A) Length: 34 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule Type: peptide (ix) Sequence description: SEQ ID NO: 7 (2) Information on SEQ ID NO: 8: (i) Sequence characteristics: (A) Length: 34 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule Type: peptide (ix) Sequence description: SEQ ID NO: 8 (2) Information on SEQ ID NO: 9: (i) Sequence characteristics: (A) Length: 34 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule Type: peptide (ix) Sequence description: SEQ ID NO: 9 (2) Information about SEQ ID NO: 10: (i) Sequence characteristics: (A) Length: 34 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule Type: peptide (ix) Sequence description: SEQ ID NO: 10 (2) Information on SEQ ID NO: 11: (i) Sequence characteristics: (A) Length: 34 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule Type: peptide (ix) Sequence description: SEQ ID NO: 11 (2) Information about SEQ ID NO: 12: (i) Sequence characteristics: (A) Length: 34 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) molecule Type: peptide (ix) Sequence description: SEQ ID NO: 12 (2) Information about SEQ ID NO: 13: (i) Sequence characteristics: (A) Length: 28 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule Type: peptide (ix) Sequence description: SEQ ID NO: 13 (2) Information on SEQ ID NO: 14: (i) Sequence characteristics: (A) Length: 28 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule Type: peptide (ix) Sequence description: SEQ ID NO: 14 (2) Information about SEQ ID NO: 15: (i) Sequence characteristics: (A) Length: 28 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule Type: peptide (ix) Sequence description: SEQ ID NO: 15 (2) Information on SEQ ID NO: 16: (i) Sequence characteristics: (A) Length: 12 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule Type: peptide (ix) Sequence description: SEQ ID NO: 16 (2) Information on SEQ ID NO: 17: (i) Sequence characteristics: (A) Length: 12 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule Type: peptide (ix) Sequence description: SEQ ID NO: 17 (2) Information about SEQ ID NO: 18: (i) Sequence characteristics: (A) Length: 12 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule Type: peptide (ix) Sequence description: SEQ ID NO: 18 (2) Information on SEQ ID NO: 19: (i) Sequence characteristics: (A) Length: 42 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule Type: Peptide (ix) Sequence description: SEQ ID NO: 19 (2) Information on SEQ ID NO: 20: (i) Sequence characteristics: (A) Length: 36 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule Type: peptide (ix) Sequence description: SEQ ID NO: 20 (2) Information on SEQ ID NO: 21: (i) Sequence characteristics: (A) Length: 25 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule Type: peptide (ix) Sequence description: SEQ ID NO: 21 (2) Information about SEQ ID NO: 22: (i) Sequence characteristics: (A) Length: 37 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule Type: Peptide (ix) Sequence description: SEQ ID NO: 22 (2) Information about SEQ ID NO: 23: (i) Sequence characteristics: (A) Length: 38 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule Type: peptide (ix) Sequence description: SEQ ID NO: 23 (2) Information about SEQ ID NO: 24: (i) Sequence characteristics: (A) Length: 38 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule Type: peptide (ix) Sequence description: SEQ ID NO: 24 (2) Information about SEQ ID NO: 25: (i) Sequence characteristics: (A) Length: 38 amino acids (B) Type: amino acids (C) Number of chains: unknown (D) Topology: unknown (ii) Molecule Type: Peptide (ix) Sequence description: SEQ ID NO: 25

Continuing the front page (72) Inventor Todd, John, A United States 94596 California, Walnut Creek, Santos Lane 2955, number 307 (72) Inventor Jolly, Michael, E. United States 60073 Illinois, Round Drake, North Circle Drive 3449 (56 JP-A-4-159298 (JP, A) JP-A-3-284696 (JP, A) JP-A-4-330098 (JP, A) JP-A-5-262792 (JP, A) JP-A-4-99799 (JP, A) JP-A-4-221398 (JP, A) JP-A-4-36185 (JP, A) JP-A-6-506669 (JP, A) JP-A-5-503722 (JP, A) A) JP-T6-500925 (JP, A) JP-T5-506230 (JP, A) WO 92/9634 (WO, A1) Proc. Natl. Acad. Sc i. U. S. A. 88 (9), 3647-3651 (1991)

Claims (6)

(57) [Claims] 1. A peptide having an epitope contained in an HCV capsid protein, wherein the peptide is a first epitope consisting of an amino acid sequence selected from the group consisting of QRKTKRNTNRR and QRKTKRSTNRR, and a peptide consisting of PQDVKFPGGG and PQDVKFPGGGQIVGGVYLLP. A second amino acid sequence which is continuous with the first epitope at the C-terminus of the first epitope.
A peptide comprising an epitope of 2. A first epitope comprising an amino acid sequence selected from the group consisting of QRKTKRNTNRR and QRKTKRSTNRR,
And a second amino acid sequence consisting of an amino acid sequence selected from the group consisting of PQDVKFPGGG and PQDVKFPGGGQIVGGVYLLP, which is contiguous to the first epitope at the C-terminus of the first epitope
Contacting a sample containing an antibody specific for a peptide consisting of an epitope with the peptide immobilized on a solid substrate; separating unbound antibodies from antibodies bound to the solid substrate; Detecting the presence of the bound antibody above. An assay method for detecting antibodies specific for HCV antigens. 3. The assay method according to claim 2, wherein said solid substrate is selected from the group consisting of a surface of a microtiter plate, a glass fiber filter, paramagnetic particles, and latex particles. 4. The assay method according to claim 2, wherein said bound antibody is detected by an anti-human specific antibody conjugated with an enzyme. 5. The amino acid sequence QRKTKRNTNRR or QRKTKRSTN.
Contacting a sample containing an antibody against a peptide consisting of RR with the peptide immobilized on a solid substrate in the presence of a competitive amount of a peptide consisting of the amino acid sequence PQDVKFPGGG, to the peptide immobilized on the solid substrate. Separating the bound antibodies from the antibodies that did not so bind; detecting the presence of the bound antibodies on the solid substrate; detecting an antibody specific for the HCV antigen. Competitive Inhibition Assay Method 6. An assay method for detecting an antibody specific to HCV antigen, comprising incubating a sample with the peptide of claim 1 labeled with a fluorophore and measuring an increase in fluorescence polarization.