JPH04221398A - Antibody to non-a, non-b hepatitis virus and immunochemically reactive peptide - Google Patents

Abstract

PURPOSE: To provide a peptide immunochemically reactive with an antibody to non A, non B type hepatitis virus (NANBH virus), a detection method of NANBH or anti-NANBH in a testing liquid, and an immunochemical reagent and a test kit to be used in the detection.

CONSTITUTION: The polypeptide includes 15 amino acid sequence, Arg-Thr-Gln- Gln-Arg-Lys-Thr-Lys-Arg-Ser-Thr-Asn-Arg-Arg-Arg as a main component, which is immunochemically reactive without antibody to NANBH.

COPYRIGHT: (C)1992,JPO

Description

[Detailed description of the invention]

0001

FIELD OF THE INVENTION The present invention relates to a peptide that immunochemically reacts with antibodies against non-A, non-B hepatitis virus (NANBH virus).

The present invention further relates to a method for detecting NANBH or anti-NANBH in a test solution, and immunochemical reagents and test kits to be used when applying this detection method.

0003

BACKGROUND OF THE INVENTION Non-A, non-B hepatitis, which may or may not be caused by the hepatitis C virus (HCV), is a group of transmissible or virus-induced diseases. This non-A, non-B hepatitis is a disease caused by known hepatitis viruses, namely hepatitis A virus (HAV), hepatitis B virus (HBV) and hepatitis delta virus (HDV), as well as cytomegalovirus (CMV) or Epstein-mediated hepatitis. It may be different from other forms of viral liver disease, including hepatitis induced by Barr virus (EBV). N
ANBH was first identified in individuals who received blood transfusions. When chimpanzees were inoculated from humans and serially passaged in chimpanzees, NANBH was found to be caused by one or more transmissible infectious agents.

Epidemiological evidence is suggestive that NANBH may be of three types: waterborne epidemic, blood- or needle-borne, and sporadic (community acquired). . However, many of the factors that can cause NANBH are unknown.

[0005]

[Problems to be Solved by the Invention] Clinical diagnosis and identification of NANBH was first performed by excluding other viral markers. Methods used to detect putative NANBH antigens and antibodies include agar gel diffusion, counterimmunoelectrophoresis, immunofluorescence microscopy, immunoelectron microscopy,
There are radioimmunoassays and enzyme-linked immunosorbent assays. However, none of these tests
It was found that the sensitivity, specificity and reproducibility were insufficient for use as a diagnostic test for NBH.

However, for the development of specific sensitive methods that would allow reliable diagnosis to be carried out at various stages of NANBH infection, it is very important to identify immunodominant viral epitopes of this type. is important.

[0007]

[Means for Solving the Problems] We have discovered a peptide consisting of 15 amino acids that is particularly immunochemically reactive with NANBH antibodies. The amino acid sequence is shown in the sequence listing.

The present invention further relates to a fragment of the pentadecapeptide that immunochemically reacts with a NANBH antibody, and a polypeptide containing the pentadecapeptide as a main component or a fragment thereof that immunochemically reacts with a NANBH antibody.

[0009] The amino acid sequence is Arg-Lys-Thr-
The nonapeptide of Lys-Arg-Ser-Thr-Asn-Arg is preferred as the pentadecapeptide fragment of the invention.

The invention further relates to immunochemical reagents containing at least one of the peptides of the invention.

The present invention further relates to a method for detecting antibodies against NANBH in a test solution using at least one of the peptides of the present invention.

The present invention further relates to a method for detecting NANBH in a test solution using at least one of the peptides of the present invention.

Finally, the invention relates to a test kit to be used for carrying out immunoassays. This test kit contains at least one immunochemical reagent of the invention.

[0014] The above-mentioned peptide is the NANB in the test solution.
It is particularly suitable for use in diagnostic methods for determining the presence of H or NANBH antibodies.

In contrast to natural NANBH, the peptides of the invention have the great advantage of being a safe, non-infectious source.

The peptides of the invention are produced by one of the known organic chemical methods for peptide synthesis or by using recombinant DNA technology. This recombinant DNA technique consists of producing the desired peptide by expressing a recombinant polynucleotide with a polynucleotide sequence encoding one or more peptides in question in a suitable microorganism as host. .

[0017] Organic chemical methods for peptide synthesis are believed to involve the attachment of the necessary amino acids via condensation reactions in a homogeneous phase or in the so-called solid phase.

The condensation reaction can be carried out as follows.

a) In the presence of a condensing agent, a compound (amino acid, peptide) containing a free carboxyl group and a protected other reactive group is converted into a compound (amino acid, peptide) containing a free amino group and a protected other reactive group ( Condenses with amino acids, peptides).

b) Compounds containing an activated carboxyl group and other free or protected reactive groups (amino acids, peptides) are converted into compounds containing free amino groups and other free or protected reactive groups (amino acids, peptides). peptide).

Among others, the carboxyl group can be modified by converting the carboxyl group into an acid halide, an acid azide, an acid anhydride, an acid imidazolide or an activated ester (for example, N-hydroxysuccinimide, N-hydroxybenzotriazole or p-nitrophenyl ester). groups can be activated.

The most common methods of the condensation reaction are the carbodiimide method, the azide method, the mixed anhydride method and the P
eptides, Analysis, Synthe
sis, Biology, Vol. 1-3 (Ed.
Gross, E. and Meienhofer,
J. ) 1979, 1980, 1981 (Acad
emic Press, Inc. This method uses an activated ester as described in ).

The production of the above-described peptide of the present invention using a "solid phase" is described, for example, in J. Amer. Chem.
Soc. 85, 2149 (1963) and In
t. J. Peptide Protein Res
．． 35, 161-214 (1990). The attachment of amino acids of the peptide to be produced usually starts from the carboxyl terminal side. This method requires a solid phase on which there are reactive groups or into which such groups can be introduced. This can be, for example, a copolymer of benzene and divinylbenzene containing reactive chloromethyl groups, or a polymeric solid phase which is reacted with hydroxymethyl or amine functions.

Particularly suitable solid phases are described, for example, by Wang, J.; Am. Chem. Soc. 95,1328
(1974) is a p-alkoxybenzyl alcohol resin (4-hydroxymethylphenoxymethylcopolystyrene-1% divinylbenzene resin). After synthesis, the peptide can be separated from this solid phase under mild conditions.

After synthesis of the desired amino acid sequence, the peptide is separated from the resin, for example with trifluoromethanesulfonic acid or with methanesulfonic acid dissolved in trifluoroacetic acid. The peptide can also be removed from the carrier by transesterification with lower alcohols, preferably methanol or ethanol. In this case, the lower alkyl ester of the peptide is produced directly. Similarly, separation using ammonia produces amides of the peptides of the invention.

Reactive groups which cannot take part in the condensation reaction are, as mentioned above, substantially protected by groups which can be very easily removed again by acid, base or reductive hydrolysis. Thus, the carboxyl group may be substantially protected by esterification with, for example, methanol, ethanol, tert-butanol, benzyl alcohol or p-nitrobenzyl alcohol and an amine attached to a solid support.

Groups capable of substantially protecting an amino group include an ethoxycarbonyl group, a benzyloxycarbonyl group, a t-butoxycarbonyl group, a p-methoxybenzyloxycarbonyl group, or an acid group obtained from a sulfonic acid (for example, a benzenesulfonyl group or a p-methoxycarbonyl group). -toluenesulfonyl) but also other groups, such as substituted or unsubstituted aryl or aralkyl groups (e.g. benzyl and triphenylmethyl), or orthonitrophenylsulfenyl and 2-benzoyl-1-methylvinyl. You can also use Particularly suitable α-amino protecting groups are, for example, base-sensitive 9-fluorenylmethoxycarbonyl (F
moc) group [Carpino & Han (1970
)J. Amer. Chem. Soc. 92, 57
48].

[0028] The Peptides, Analysis
sis, Synthesis, Biology,
Vol. 1-9 (Eds.
1979-
1987 (Academic Press, Inc.
) provides a more extensive explanation of possible protecting groups.

It is also necessary to protect the ε-amino group of lysine, which is appropriate for the guanidyl group of arginine. In this case, the customary protecting groups are the Boc group for lysine, the Pmc group, Pms group, Mbs group for arginine.
group or Mtr group.

Depending on the nature of the particular group, the protecting group is removed by various conventional methods, for example using trifluoroacetic acid, or by mild reduction, for example with hydrogen and a catalyst (eg palladium) or with HBr in glacial acetic acid. can be separated.

As already mentioned above, the peptides of the invention can likewise be produced using recombinant DNA technology. This possibility is particularly important when incorporating peptides into repeating sequences ("tandems") or when peptides can be produced as components of (much larger) proteins or polypeptides. Therefore, this type of peptide production is also within the scope of the invention. For this purpose, a polynucleotide encoding a peptide of the invention is used as a component of a recombinant DNA, which is furthermore substantially free of polynucleotide segments flanking the aforementioned polynucleotide sequences in the natural NANBH genome.

Polynucleotides of this type encoding the peptides of the invention and recombinant DNA into which this polynucleotide has been incorporated are likewise within the scope of the invention.

Although this is not specifically stated in the claims, it is obvious that one or more amino acids in the peptides of the present invention can be substituted with other amino acids.

In general, (a) an acid addition salt of a peptide; (b) an acid addition salt of a peptide;
) amides, especially C-terminal amides, (c) esters, especially C-terminal esters, and (d) N-acyl derivatives of peptides,
Functional derivatives of these peptides, meaning in particular N-terminal acyl derivatives, especially N-acetyl derivatives, are also considered as peptides according to the invention.

The "immunochemical reagent" of the present invention usually consists of one or more peptides of the present invention and a suitable support or labeling substance.

Supports that can be used are, for example, the inner walls of microtest wells or cuvettes, tubes or capillaries,
Membranes, filters, test strips, or particle surfaces such as latex particles, red blood cells, dye sols, metal compounds as metal sols or sol particles, carrier proteins such as BSA or KLH.

Labeling substances that can be used are in particular radioisotopes, fluorescent compounds, enzymes, metal compounds which are dye sols, metal sols or sol particles.

[0038] In the method for detecting antibodies against NANBH in a test solution, the immunochemical reagent of the present invention is brought into contact with the test solution. Thereafter, the presence of an immune complex formed between the peptide and the antibody in the test solution is detected, and by this detection, the presence of NANBH antibodies in the test solution can be confirmed and quantified.

The immunochemical reaction that occurs is a so-called sandwich type reaction, agglutination reaction, competitive reaction or inhibition reaction, depending on the type and further characteristics of the immunochemical reagent.

For the detection of NANBH in the test solution, the immunochemical reagent of the present invention is contacted with the test solution and anti-NANBH, and the presence of the formed immune complex is then detected, thereby detecting the presence of NANBH in the test solution. can be determined.

A particularly suitable method for the detection of NANBH in a test solution is based on a competitive reaction between the peptide of the invention provided with a labeling substance and the NANBH antigen (present in the test solution), whereby the peptide and antigen are separated. and compete with an antibody against NANBH bound to a solid support.

The test kit of the present invention contains the above-mentioned immunochemical reagents as essential components. NANB
When performing a sandwich reaction for detection of H antibody, the test kit includes, for example, the peptide of the present invention coated on a solid support (for example, the inner wall of a micro test well), and the labeled peptide or labeled antibody of the present invention. may include.

When carrying out a competitive reaction, the test kit contains the peptide of the invention coated on a solid support and NANB.
The peptide may contain a labeled antibody against H.H., preferably a monoclonal antibody against said peptide.

For agglutination reactions, the test kit contains an immunochemical reagent consisting of a peptide of the invention coated onto particles or sols.

A test kit for the detection of NANBH antigen contains, for example, a labeled peptide of the invention and an antibody against NANBH coated on a solid support.

[0046]

EXAMPLES Example 1 A pentadecapeptide having the sequence shown in the sequence listing was produced by stepwise solid phase peptide synthesis. Using a VEGA Coupler 250 C automatic peptide synthesizer or a Labortec SP640 semi-automatic peptide synthesizer, p-benzyloxybenzyl alcohol resin (Wang resin; 0.6-0.7 mm
les/g, Bachem AG, Swit
The synthesis was carried out using an amino acid protected with N (α-position)-Fmoc (Fmoc refers to 9-fluorenylmethyloxycarbonyl).

DDC (dicyclohexylcarbodiimide, 1 eq.), HOBt (1-hydroxybenzotriazole, 2 eq.) and DMAP (N,N-dimethylaminopyridine, 1 eq.) were dissolved in DMF-dichloromethane (1:1).
, vol/vol) for 18 h at 4°C.
The synthesis was initiated by coupling -Arg(Pmc)-OH to the resin. Unreacted alcohol functionality on the resin was then blocked by benzoylation with benzoyl chloride-pyridine for 2 hours.

The resulting Fmoc-Arg(Pmc)-resin (0.38 mmol/g) was treated with 20% piperidine in DMF three times for 6 minutes successively to remove the Fmoc group. The protected pentadecapeptide was then prepared by successive coupling steps of Fmoc-amino acids on H-Arg(Pmc)-resin, as indicated by the amino acid sequence. The following side chain protecting groups were used. That is, in the case of Arg, -Pmc (2,2,5,7,8-pentamethylchroman-6-sulfonyl-), in the case of Thr and Ser, -tBu (t-butyl-), and in the case of Lys, Bo
c (t-butyloxycarbonyl), Asn and Gl
In the case of n, Trt (trityl) was used.

3 equivalents of each Fmoc-amino acid,
BOP (=benzotriazolyloxy-tris(dimethylamino)-phosphonium hexafluorophosphate) and HOBt and 4.5 equivalents of DIPEA (=N,N
-diisopropylethylamine) for 15 minutes using 12-15 milliliters of DMF per gram of resin, followed by three cycles of washing with DMF and ethanol (each cycle is 1 minute). was carried out. The completeness of the binding reaction was monitored by Kaiser's ninhydrin test (Anal. Biochem.
34, 595-598, 1970). A positive ninhydrin reaction was observed after binding of Ser10 and Arg5. In each case, one equivalent of the corresponding Fmoc-
The coupling reaction using amino acids, BOP, HOBt and DIPEA was repeated again for 30 minutes. It was then acetylated with acetic anhydride-DMF (5:59; vol/vol) for 10 min, followed by washing with DMF and ethanol (1 min each) to block any remaining free amino groups.

After each synthesis cycle, DM
N(α-position)-Fmoc by treatment with 25% piperidine in F
Protecting groups were removed.

After completion of the synthesis, the obtained fully protected pentadecapeptide resin was mixed with trifluoroacetic acid/water/phenol/thioanisole/ethanedithiol (82:5
:5:5:2.5vol/vol) mixture at room temperature for 18 hours to separate the peptide from the resin and simultaneously remove all protecting groups. The crude peptide was isolated after precipitation of the reaction mixture with diethyl ether. HPL of C18 silica using an acetonitrile gradient in 0.1 M phosphate buffer at pH 2.1
The pentadecapeptide was purified by C.

Example 2 A pentadecapeptide consisting of an amino acid sequence as shown in the sequence listing was added to a 100mM phosphate buffer solution with a pH of 8.0 (7.5%).
Dissolved at μg/ml. Microtiter plates were pretreated with 135 μl/well of 0.2% glutaraldehyde in pH 5.0 phosphate buffer for 4 hours at room temperature with continuous shaking. The plate was then emptied and 135 μl of the above peptide solution was applied to each well. The peptide was allowed to bind to the microtiter plate by standing at 37°C for 3 hours. Freeze the plate at -20°C
It was stored overnight.

The plate was then thawed and emptied, and the remaining binding sites were removed with 0.2M Tris pH 7.4/0.2M Na
5 in 0.05% Tween20® solution in Cl.
Blocked for minutes at room temperature. Then, 0.2M Tris (
pH 7.4)/washed once with 0.2M NaCl, 0.
The plates were washed twice with 0.04M Tris (pH 7.4) (250 μl per well). For identification of antibodies specific for non-A, non-B hepatitis, serum samples were pipetted into wells (100 μl per container) using a sample diluent (phosphate-buffered saline (PBS)/20% normal goat serum). /1%
Triton X100) and incubated at 37°C for 1 hour. Fill wells with PBS/0.05%T
After washing with ween20®, peroxidase (100μ per well) diluted in sample diluent was added.
Bound human antibodies were detected by goat anti-human immunoglobulin labeled with 1 hour at 37°C. Plates were washed four times with PBS/0.05% Tween20®. TM as a substrate for peroxidase enzymes
When adding B (100 μl per well), 30 μl at room temperature
The reaction was allowed to proceed for minutes. 100μl 2M H2SO4
was added to each well to stop the reaction. Organon
The yellow color was read at 450 nm on a Teknika microelisa reader.

[0054] Using serum from patients with non-A, non-B hepatitis,
Extinction coefficients ranging from 1.8 to 3.0 were measured. The average extinction coefficient of 20 normal human sera was 0.301 (standard deviation 0.07). As a control, the procedure was repeated with two unrelated pentadecapeptides. In all cases, no significant difference was observed in the extinction coefficients obtained from normal human serum and NANBH patient serum samples.

Example 3 The pentadecapeptide fragment shown in the sequence listing was
Reactivity with serum from patients with NBH was also tested again.

[0056] The following nonapeptides 2 to 8 were synthesized,
It was attached to a solid support essentially as described in Examples 1 and 2. Immunoreactivity with serum samples obtained from patients with NANBH was investigated as described in Example 2. Nonapeptides 5, 6, and 7 showed specific reactions with antibodies in the serum of NANBH patients. These results are shown in Table 1.

[0057]

[Table 1] Peptide 2: Amino acid sequence of SEQ ID NO: 2.

Peptide 3: Amino acid sequence of SEQ ID NO:3.

Peptide 4: Amino acid sequence of SEQ ID NO: 4.

Peptide 5: Amino acid sequence of SEQ ID NO: 5.

Peptide 6: Amino acid sequence of SEQ ID NO: 6.

Peptide 7: Amino acid sequence of SEQ ID NO: 7.

Peptide 8: Amino acid sequence of SEQ ID NO: 8.

[0064]

[Sequence list] Sequence number: 1 Sequence length: 15 Sequence type: Amino acid sequence type: Peptide sequence SEQ ID NO: 2 Sequence length: 9 Sequence type: Amino acid sequence type: Peptide sequence SEQ ID NO: 3 Sequence length: 9 Sequence type: Amino acid sequence type: Peptide sequence SEQ ID NO: 4 Sequence length: 9 Sequence type: Amino acid sequence type: Peptide sequence SEQ ID NO: 5 Sequence length: 9 Type: Amino acid sequence type: Peptide sequence SEQ ID NO: 6 Sequence length: 9 Sequence type: Amino acid sequence type: Peptide sequence Arg-Lys-Thr-Lys-Arg-Ser
-Thr-Asn-Arg Sequence number: 7 Sequence length: 9 Sequence type: Amino acid sequence type: Peptide sequence Sequence number: 8 Sequence length: 9 Sequence type: Amino acid sequence type: Peptide sequence

Claims (5)

[Claims] Claim 1: A pentadecapeptide consisting of an amino acid sequence as shown in SEQ ID NO: 1, a fragment thereof that immunochemically reacts with a NANBH antibody, and a polypeptide containing the pentadecapeptide as a main component or a fragment thereof that immunochemically reacts with a NANBH antibody. fragment. 2. An immunochemical reagent comprising the peptide according to claim 1. 3. A method for detecting antibodies against NANBH in a test solution, comprising: contacting the immunochemical reagent according to claim 2 with the test solution, and detecting the immunity generated between the peptide and the antibody in the test solution; The presence of the complex is detected, which indicates that N in the test solution
A method characterized in that it is a method for measuring the presence of ANBH antibodies. 4. A method for detecting NANBH in a test solution, comprising adding the immunochemical reagent according to claim 2 to the test solution and anti-N
A method characterized in that the presence of NANBH in the test liquid is determined by contacting with ANBH and subsequently detecting the presence of the generated immune complex. 5. A test kit to be used in the immunoassay according to claim 3 or 4.