JP2003104999A - Method for measuring herpes simplex virus antibody - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a peptide useful for diagnosing the presence of human herpes simplex infection, and especially a peptide useful in a method with which the type 1 herpes simplex antibody and the type 2 herpes simplex antibody are distinguished and diagnosed, and to provide a method for such diagnosis. SOLUTION: A peptide having a specific amino acid sequence, and another peptide having a specific amino acid sequence. Further, a method for detecting the existence of the type 1 herpes simplex virus antibody and the type 2 herpes simplex virus antibody in a specimen by bringing the peptides into contact with the specimen.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for measuring HSV antibody for diagnosing herpes simplex virus (hereinafter referred to as “HSV”) infection. More particularly, the present invention relates to herpes simplex virus type 1 (HSV type 1,
(HSV-1)) and herpes simplex virus type 2 (HSV type 2, hereinafter "HSV-2") infections that can be distinguished and diagnosed, and peptides and reagent compositions used in this method Regarding

[0002]

2. Description of the Related Art HSV includes type 1 (HSV-1) and type 2 (HSV-2). Among them, HSV-2 is known as the causative virus of genital herpes. In recent years, an increase in sexually transmitted diseases (STD) caused by HSV-1 has also been reported, and it has been desired to develop a method for determining the type of virus infection of both.

HSV infection can be diagnosed by detecting an anti-HSV antibody possessed by a subject. However, HSV-1
And each constituent protein of HSV-2 has high homology in their amino acid sequences, and since about 80% or more is retained, even if any virus-extracted protein is used as an antigen,
Anti-HSV-1 specific antibody and anti-HSV-2 specific antibody cannot be detected separately.

Recently, a reagent for distinguishing and detecting anti-HSV-1 antibody and anti-HSV-2 antibody has been developed by using glycoprotein G (gpG), which is one of the above-mentioned constituent proteins, as an antigen. However, this gpG also has a common amino acid sequence, and therefore the reagent is still insufficient in terms of its specificity and sensitivity.

[0005] In addition, several HSV-2 specific monoclonal antibodies were used to clone the peptide reactive with the antibody by biopanning method using a random peptide display library to obtain an epitope (antigenic determinant or antigenic determinant). Site), and this epitope is HSV
-2 gpG (gpG2) has been reported to be present in the 525-587th amino acid sequence (A. Grabowska,
et al., Jaurnal of General Virology, 80 , pp.1789-1
798 (1999)). In the report, an ELISA system was constructed using a synthetic peptide corresponding to the 551-570th amino acid sequence of gpG2, and anti-HSV-2 antibody positive sera and negative sera confirmed by Western blot were used in this ELISA system. It is described that the sensitivity was 77% and the specificity was 98% as a result of the measurement.

[0006]

The object of the present invention is to provide anti-HS.
V-specific antibody-binding peptides, especially anti-HSV-1 that specifically reacts with HSV-1 patient serum and HSV-2 patient serum respectively
To provide a specific antibody-binding peptide and an anti-HSV-2 specific antibody-binding peptide.

Another object of the present invention is a method for detecting HSV-1 specific antibody and HSV-2 specific antibody with high sensitivity and high specificity (detecting HSV-1 infection and HSV-2 infection). Method to distinguish and diagnose).

[0008]

The above object can be achieved by the present invention having the following gist.

That is, the present invention is a peptide having the amino acid sequence shown in SEQ ID NO: 1 or a peptide having an amino acid sequence in which one or more amino acids in the amino acid sequence are modified by substitution, deletion or addition. A peptide having specific reactivity with the anti-HSV-1 antibody is provided.

The present invention is also a peptide having the amino acid sequence shown in SEQ ID NO: 2 or a peptide having an amino acid sequence in which one or more amino acids in the amino acid sequence are modified by substitution, deletion or addition. A peptide having specific reactivity with anti-HSV-2 antibody is provided.

Further, the present invention provides a reagent composition for diagnosing HSV infection, which comprises each of the above peptides as an active ingredient, particularly,
Reagent composition for diagnosing HSV infection in which the peptide is a multi-antigenic peptide form, and HS containing both diagnostic reagent compositions
Provided is a reagent composition for distinguishing and diagnosing V-1 infection and HSV-2 infection.

[0012] In addition, the present invention is a method for diagnosing HSV-1 infection and HSV-2 infection in a sample by distinguishing them from each other, wherein both peptides (peptides having specific reactivity with anti-HSV-1 antibody) And a peptide having a specific reactivity with the anti-HSV-2 antibody) are brought into contact with the sample, and the anti-HSV-1 antibody and anti-HS in the sample
Provided is a method including the step of detecting the presence or absence of a V-2 antibody.

The abbreviations of amino acids, peptides, base sequences, nucleic acids and the like in this specification refer to IUPAC, IUB, "Guidelines for preparing specifications including base sequences or amino acid sequences" (edited by the Japan Patent Office). ) And conventional symbols in the field.

The DNA and amino acid sequences of gpG of HSV-1 and HSV-2 are known, and the amino acid sequence numbers used in this specification are according to this known sequence (McGeoch, et al.
al., J. Mol. Biol., (1985), 181: 1-13; McGeoch, e.
t al., J. Gen. Virol., (1987), 68: 19-38).

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION The peptide of the present invention is characterized in that it reacts only with either an anti-HSV-1 antibody of an HSV-1 infected patient or an anti-HSV-2 antibody of an HSV-2 patient, and does not react with the other. To do. Therefore, by using these peptides, it is possible to specifically detect the anti-HSV-1 antibody and the anti-HSV-2 antibody in the sample, and thus whether the sample is infected with HSV-1 and HSV-1. -It can be diagnosed by distinguishing whether or not it is infected with -2. The present invention provides a method of diagnosing such HSV-1 and / or HSV-2 infections, particularly a simple,
An extremely accurate and efficient diagnostic method is provided.

(1) Peptide of the Present Invention Hereinafter, the peptide of the present invention having specific reactivity with an anti-HSV-1 antibody (hereinafter, also referred to as “HSV-1 peptide”) and a book having specific reactivity with an anti-HSV-2 antibody Invention peptide (hereinafter referred to as "HSV-
2 ").

The HSV-1 peptide of the present invention is a peptide having the amino acid sequence represented by SEQ ID NO: 1 or a peptide having an amino acid sequence in which one or more amino acids in the amino acid sequence are modified by substitution, deletion or addition. And is characterized by having specific binding properties to anti-HSV-1 antibody.

Further, the HSV-2 peptide of the present invention has SEQ ID NO: 2
A peptide having the amino acid sequence shown in, or a peptide having an amino acid sequence modified by substitution, deletion or addition of one or more amino acids in the amino acid sequence, and having specific binding properties to anti-HSV-1 antibody Is characterized by having

The term antibody is used here as a common term in the art. Therefore, an anti-HSV-1 antibody and an anti-HSV-2 antibody are an antibody that recognizes HSV-1 (or an antibody that binds to HSV-1) and an antibody that recognizes HSV-2 (or an antibody that binds to HSV-2, respectively). ) Is defined. These antibodies are preferably antibodies elicited in HSV-1 infected patients (anti-H
SV-1 antibody) and antibodies induced by HSV-2 infected patients (anti-HS
V-2 antibody).

The amino acid sequence shown in SEQ ID NO: 1 is HSV
-1 is the 69-83 amino acid sequence of gpG (gpG1), the peptide consisting of this amino acid sequence has specific binding to anti-HSV-1 antibody, This is a preferred specific example.

The amino acid sequence shown in SEQ ID NO: 2 is the 558-566th amino acid sequence of gpG (gpG2) of HSV-2, and the peptide consisting of this amino acid sequence is anti-HSV-2.
It has a specific binding property to an antibody and is a preferred specific example of the HSV-2 peptide of the present invention.

The peptides of the present invention include, in addition to the peptides having the above-mentioned respective amino acid sequences, those having a partial amino acid in these respective amino acid sequences or an amino acid sequence in which the amino acid sequence is modified, that is, in each amino acid sequence. Those having an amino acid sequence in which one or more amino acids are substituted, deleted or added are included.

Here, the degree and position of modification of the amino acid sequence, that is, the degree of amino acid substitution, deletion or addition and their position are determined by the peptide (or protein) of the modified amino acid sequence before modification. There is no particular limitation as long as it has the same effect as the peptide having the same property, that is, as long as it retains the specific binding property to the anti-HSV-1 antibody or the anti-HSV-2 antibody.

Confirmation of the same effect as above, that is, the one having the specific binding property to the anti-HSV-1 antibody or the anti-HSV-2 antibody is confirmed by the peptide of the present invention and the anti-HSV-1 antibody or It can be carried out by testing the reactivity or cross-reactivity with the anti-HSV-2 antibody according to a conventional method. One specific example of this test will be described in detail in Examples below.

When modified by the addition of amino acids, the HSV-1 peptide of the present invention retains its specific binding property to the anti-HSV-1 antibody, and undesired cross-reactivity with the anti-HSV-2 antibody. In order to avoid sex, addition of the amino acid sequence or amino acid sequence of gpG of HSV-1 following 68-85th amino acid sequence of gpG, particularly preferably 69-83th amino acid sequence thereof, may be excluded. is important.

In the case of the HSV-2 peptide of the present invention, the HSV-peptide retains its specific binding property to the anti-HSV-2 antibody and avoids undesired cross-reactivity with the anti-HSV-1 antibody. 2 g
The gpG in question following the 558th to 566th amino acid sequence of pG
It is important to exclude the addition of amino acids or amino acid sequences of

The peptide of the present invention preferably has a length of at least 5 amino acid sequences in consideration of its antigenicity and the like.

The peptide of the present invention may, if desired, also have a form in which its antigenicity is enhanced, such as a multi-antigenic peptide (MAP:
multiple antigen peptide) form.

(2) Production of Peptide of the Present Invention The HSV-binding peptide of the present invention can be produced according to its amino acid sequence by a general chemical synthesis method.
The method includes a conventional liquid phase method and a solid phase method for peptide synthesis.

More specifically, this peptide synthesis method is based on the amino acid sequence information provided by the present invention. A stepwise erosion method in which each amino acid is sequentially bonded one by one to extend the chain, and several amino acids are used. Fragment synthesis method in which each fragment is pre-synthesized, and then each fragment is subjected to a coupling reaction. The peptide of the present invention can be synthesized by any of them.

The condensation method used for peptide synthesis can also follow various known methods. As a concrete example,
For example, azide method, mixed acid anhydride method, DCC method, active ester method, redox method, DPPA (diphenylphosphoryl azide)
Method, DCC + Additives (Additives: 1-hydroxybenzotriazole, N-hydroxysuccinamide, N-hydroxy-5-
Norbornene-2,3-carboximide, etc.), Woodward method and the like. The solvent that can be used in each of these methods can also be appropriately selected from general solvents that are well known to be used in this type of peptide condensation reaction.
Examples thereof include, for example, dimethylformamide (DMF),
Dimethyl sulfoxide (DMSO), hexaphosphoroamide,
Examples thereof include dioxane, tetrahydrofuran (THF), ethyl acetate and the like, a mixed solvent thereof and the like.

In the above peptide synthesis reaction, the amino acid not involved in the reaction and the carboxyl group in the peptide are generally esterified to form a lower alkyl ester such as methyl ester, ethyl ester and tertiary butyl ester, such as benzyl ester, p -It can be protected as an aralkyl ester such as methoxybenzyl ester and p-nitrobenzyl ester.
Further, an amino acid having a functional group in its side chain, for example, a hydroxyl group of Tyr may be protected with an acetyl group, a benzyl group, a benzyloxycarbonyl group, a tertiary butyl group, or the like. It is not necessary to provide such protection. Furthermore, for example Arg
The guanidino group of is protected by a suitable protecting group such as nitro group, tosyl group, 2-methoxybenzenesulfonyl group, methylene-2-sulfonyl group, benzyloxycarbonyl group, isobornyloxycarbonyl group, adamantyloxycarbonyl group, etc. be able to.

The deprotection reaction of these protecting groups in the amino acids having the above protecting groups, peptides and finally the peptide of the present invention can also be carried out by a conventional method such as catalytic reduction method, liquid ammonia / sodium, hydrogen fluoride, It can be carried out according to a method using hydrogen bromide, hydrogen chloride, trifluoroacetic acid, acetic acid, formic acid, methanesulfonic acid or the like.

The peptide of the present invention obtained by the above-mentioned method is a peptide obtained by a conventional method such as ion exchange resin, partition chromatography, gel chromatography, affinity chromatography, high performance liquid chromatography (HPLC), and countercurrent partition method. The purification can be appropriately performed according to a method generally used in the field of chemistry.

The peptide of the present invention can also be produced according to a genetic engineering technique using a DNA sequence encoding the peptide.

This genetic engineering method can follow a conventional method. For example, DNA synthesis, production of an expression vector of the DNA, expression method of the vector in a host cell, etc.
Both can be performed according to general genetic engineering methods or in accordance with these (Molecular Cloning 2
nd. Ed., Cold Spring Harbor Lab. Press (1989); Continuation Biochemistry Laboratory "Gene Research Methods I, II, III", edited by The Biochemical Society of Japan (1986), etc.).

For example, DNA encoding the peptide of the present invention
Can be prepared according to a conventional method based on the amino acid sequence information of the peptide of the present invention (for example, Science, 224,
1431 (1984); Biochem. Biophys. Res. Comm., 130, 6
92 (1985); Proc. Natl. Acad. Sci., USA., 80, 5990
(1983)).

More specifically, DNA can be synthesized by a chemical synthesis method such as phosphoramidite method and triester method. Alternatively, it can be carried out using, for example, a commercially available automatic oligonucleotide synthesizer. Double-stranded fragments can be obtained by annealing a chemically synthesized single-stranded product with a chemically synthesized complementary strand under appropriate conditions, or by adding a complementary strand using a DNA polymerase with an appropriate primer sequence. You can

If desired, the DNA synthesized above can be modified in its encoded amino acid sequence.
Modification of the DNA for this modified amino acid sequence can be performed by, for example, site-directed mutagenesis using an oligonucleotide (Zolle
r, M., et al., Nucl. AcidsRes., 10, 6487-6500 (198
2)), cassette mutagenesis (Well, J., et al., Gene, 34,
315-323 (1985)) and the like.

Production and expression of a desired peptide using the above DNA can be carried out according to a technique well known in the art (for example, Science, 224, 1431 (1984); Biochem. B).
iophys. Res. Comm., 130, 692 (1985); Proc. Natl. A
cad. Sci. USA., 80, 5990 (1983), etc.).

Further, when the peptide of the present invention is produced and expressed as a fusion peptide or a fusion protein, for example, the method of Ohno et al., "Protein Experiment Protocol 1 Functional Analysis, Cell Engineering Separate Volume, Experimental Protocol Series, 1997, Shujunsha Co., Ltd. , Etc. can be used as a reference.

The desired peptide is subjected to various separation operations utilizing its physical and chemical properties (eg, “Biochemical Data Book II”, pages 1175-1259, 1st edition, 1st edition, 19).
June 23, 80 Tokyo Kagaku Doujinshi; Biochemistry,
25 (25), 8274-8277 (1986); Eur. J. Biochem., 163,
313-321 (1987), etc.) for separation and purification. Specific examples of the separation and purification operations include ordinary reconstitution treatment, treatment with a protein precipitating agent (salting out method), centrifugation, osmotic shock method, ultrasonic disruption, ultrafiltration, molecular sieve chromatography. Examples thereof include various liquid chromatographys such as chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, high performance liquid chromatography (HPLC), dialysis method, and combinations thereof.

If desired, the peptide of the present invention can be in a form with enhanced immunogenicity, for example, a MAP form. This MAP form is characterized as a form in which a plurality of peptides of the present invention are presented on the basic molecule.

The peptide of the present invention in the MAP form can be easily produced, for example, by using a dendrimer as a basic molecule or a skeleton.

Dendrimers are generally known as molecules having a spherical or other structure having a dendritic to star-shaped configuration. The molecule is also characterized by a branch (repeating unit) having a plurality of functional groups (see, for example, Table 6
0-500295; JP-A-63-99233; JP-A-3-2634
31; U.S. Pat. No. 4,507,466; U.S. Pat. No. 4,568,737; Polymer Journal, 17, p. 117 (1985); Angewan.
dte Chem. Int. Engl., 29, 138-175 (1990); Macromol
ecures, 25, p. 3247 (1992), etc.).

As one specific example of the peptide of the present invention in the form of MAP, for example, a nitrogen atom is used as a starting nucleus,
Examples of the dendrimer having a repeating unit (branch) composed of a —CH ₂ CH ₂ CONHCH ₂ CH ₂ — structure bonded to the core include a plurality of peptides of the present invention bound to the outermost ends of each branch. it can. Other specific examples include, for example, Lys, Arg,
Use any of the amino acids such as Glu and Asp as the starting nucleus.
An example is one in which the same amino acid is used as a repeating unit that is directly bonded to the core portion, and a plurality of peptides of the present invention are similarly bonded to each branch end.

The above-mentioned dendrimer having a nitrogen atom as a starting nucleus can be produced by a conventional method. In addition, the structure (a dendrimer raw material to be bound with the peptide of the present invention before fusion or ligation) is also available as a commercial product (Polyscience
s, Inc., 400 Vally Road, Warrington, PA, 18976 U.
SA). A dendrimer having the other amino acid as a starting nucleus portion can be produced, for example, according to the above-described chemical synthesis method of peptide. Also, for example, Fmoc ₈ -Lys ₄ -Lys ₂ -Lys-
It can also be produced by using a commercially available dendrimer raw material such as βAla-Alko resin (manufactured by Watanabe Chemical Industry Co., Ltd.).

More specifically, the dendrimer raw material can be produced as follows. That is, a resin for solid-phase peptide synthesis is subjected to a condensation reaction of α, ω-diamino acid in which two amino groups are protected with the same or different protecting groups, with or without a spacer, and then protected. The group is removed, and the same condensation reaction and deprotection reaction of protected α, ω-diamino acid are repeated.

As the resin for solid phase peptide synthesis, any resin commonly used in ordinary peptide synthesis can be used. Examples thereof include chloromethyl group, 4- (hydroxymethyl) phenoxy group, 4-((α-2 ′, 4 ′) at the end of polystyrene resin, polyacrylamide resin, polystyrene polyethylene glycol resin, etc.
Examples thereof include those having a -dimethoxyphenyl) -9-fluorenylmethoxycarbonylaminomethyl) phenoxy group and the like. Spacers can include one or more amino acids. Examples of α, ω-diamino acids include lysine, ornithine, 1,4-diaminobutyric acid, and 1,3-diaminopropionic acid.

Examples of the protecting group are as described in the section of the production of the peptide of the present invention. The reaction for removing the protecting group can also follow the method described in the section of the production of the peptide of the present invention. The number of branches is n for condensation of repeating units and removal of protecting groups.
By repeating this, 2n is obtained. The number of branches is preferably in the range of 2 to 16.

By binding the peptide of the present invention to the functional group (amino group, carboxyl group or hydroxyl group) at each branch end of the resulting dendrimer raw material, a desired MAP can be obtained. This binding reaction can follow the peptide synthesis method described above.

The MAP form of the peptide of the present invention can be prepared according to a conventional method by using a suitable matrix such as Sefacryl S-300.
(Pharmacia) and the like can be purified by a chromatography operation using a resin.

In the above MAP, H attached to each branch end
The SV-1 peptides or HSV-2 peptides do not have to be the same and can be any combination belonging to the HSV-1 peptide of the invention or any combination belonging to the HSV-2 peptide of the invention.

(3) Diagnosis of HSV infection The HSV-1 peptide of the present invention functions as an antigen that specifically binds to the anti-HSV-1 antibody, and the HSV-2 peptide of the present invention is
It functions as an antigen that specifically binds to anti-HSV-2 antibody.
Therefore, by using the peptide of the present invention, anti-HSV-1 antibody and anti-HSV-2 antibody produced by HSV infection can be selectively selected.
It can be detected (distinguishingly).

The present invention provides a method for diagnosing HSV infection, which comprises such an HSV antibody detection step.

The diagnostic method of the present invention uses blood (serum, plasma), urine, sweat, saliva, semen, from a subject suspected of being infected with HSV.
Various body fluids such as cerebrospinal fluid, preferably serum, is collected, and the peptide of the present invention is used as a sample for contact with the sample to detect the presence or absence of anti-HSV-1 antibody and / or anti-HSV-2 antibody in the sample. It is essential to include the step of

The method for detecting an antibody is not particularly limited as long as the peptide of the present invention is used as an antigen in a measuring system. Various conventional methods can be widely adopted as the antibody detection method.

Specifically, standard electrophoretic techniques and immunodiagnostic techniques including immunoassay techniques such as a competitive method, a direct reaction method, a solid-phase sandwich assay method and the like can be employed. Such immunoassay techniques include:
For example, Western blot, agglutination test, enzyme labeling such as ELISA and enzyme-mediated immunoassay, biotin / avidin type assay, radioimmunoassay, immunoelectrophoresis, immunoprecipitation, etc. are included.

For example, when the solid phase sandwich method using human serum as a test sample is taken as an example, the target antibody can be measured by the following method. That is, first, the peptide of the present invention, which is the antigen to be measured, is immobilized on a solid phase, and a serum sample as a test sample is added thereto. As a result, an antigen-antibody reaction occurs between the immobilized peptide and the antibody in the sample, and the target antibody present in the sample binds to the immobilized peptide. Next, the bound target antibody is detected using a normal human antibody detection reagent such as an anti-human IgG antibody, whereby the target antibody present in the test sample can be detected and measured.

The person skilled in the art is well aware of the selection of various means in these measuring methods and their modification, and any of these methods can be adopted in the present invention ["Clinical Test Method Recommendation"]. , Kinbara Publishing, 19
See 1995 etc.].

When the solid phase method is adopted in the above-mentioned antibody detection method, the antigen of the measurement system, the human antibody detection reagent and the like can be immobilized on the solid phase and used according to a conventional method. The solid phase is not particularly limited as long as it is an insoluble and inert carrier, and commonly used ones are widely used. For example, glass, cellulose powder, sephadex, sepharose, polystyrene, filter paper, carboxymethyl cellulose, ion exchange resin, dextran, plastic film, plastic tube, nylon, glass beads, silk, polyamine-methyl vinyl ether-maleic acid copolymer, amino acid Widely used are sticks, beads, microplates, test tubes and the like made of various materials such as copolymers and ethylene-maleic acid copolymers.

The method of immobilizing the antigen and the like is not particularly limited, and methods using both physical and chemical bonds can be appropriately adopted. For example, as a covalent bond method, diazo method, peptide method (acid amide derivative method, carboxychloride resin method, carbodiimide resin method, maleic anhydride derivative method, isocyanate derivative method, cyanogen bromide activated polysaccharide method, cellulose carbonate derivative method , A method using a condensation reagent), an alkylation method, a carrier binding method using a crosslinking reagent (glutaraldehyde as a crosslinking reagent,
Hexamethylene isocyanate, etc.), chemical reaction such as carrier binding method by Ugi reaction, or ion binding method using carrier such as ion exchange resin: physical adsorption method using porous glass such as glass beads as carrier And the like can be exemplified as preferable ones.

In immobilizing the antigen on the carrier, the antigen can be first made into a form having better binding properties according to a conventional method. As the antigen in the form,
For example, the peptide of the present invention in the MAP form, the peptide of the present invention in the carrier-bound form, and the like can be exemplified as preferable ones.

Suitable carriers used in the production of the above carrier-bound peptides include various proteins such as serum albumin, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin and the like. Other carriers include polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids,
Amino acid copolymers and the like are included. Such carriers and methods for binding them to antigens are also well known to those skilled in the art.

Especially as a labeling agent in each measuring system,
However, the present invention is not limited to these, and those known in the art or those that may be used in the future
Any of can be used. Specifically, for example
³H, ¹⁴Radioisotopes such as C; Alkaline phosphaters
Enzymes such as ze, peroxidase (POX); fluorescein
Isothiocyanate (FITC), tetramethylrhodamine
Fluorescent substances such as isothiocyanate (RITC); and 1N-
(2,2,6,6-tetramethyl-1-oxyl-4-piperidyl) -5N-
(Aspartate) -2,4-dinitrobenzene (TOPA); Dye
Examples include sol; metal sol; latex particles and the like. This
Immunoassays that use these as labeling agents are
Geoimmunoassay, Enzyme Immunoassay, Full
Oleumnoassay, Spin Immunoassay, Float
Rou assay and immunochromatographic assay
It In the present invention, the viewpoints of simplicity, safety, sensitivity, etc.
From this point of view, it is preferable to use an enzyme as a labeling agent.
The Muimmunoassay is adopted.

As the enzyme labeling substance for enzyme labeling,
For example, in addition to the above, examples include microperoxidase, chymotrypsinogen, procarboxypeptidase, glyceroaldehyde-3-phosphate dehydratase, amylase, phosphorylase, D-nase, P-nase and the like. The labeling method using these labeling substances is
It can be carried out according to a method known per se (“Monoclonal antibody” Tatsuo Iwasaki et al., Kodansha Scientific,
1984; "Enzyme-linked immunosorbent assay" 2nd edition, Eiji Ishikawa et al., Medical Institute, 1982, etc.).

The enzyme activity can be measured by a known method depending on the type of enzyme used. For example, when peroxidase is used as the labeling enzyme,
ABTSJ [2,2'-azinobi (3'-ethylbenzthiazoline sulfonic acid)] was used, and when alkaline phosphatase was used, p-nitrophenyl phosphate was used as a substrate, and the enzyme was incubated in these substrates. Examples include a method of measuring the decomposition of the substrate using a spectrophotometer (see "Enzyme-linked immunosorbent assay" 2nd edition, Eiji Ishikawa et al., Medical Institute, 1982).

When a label such as a radioisotope or a fluorescent substance is used instead of the enzyme label, the labeled substance can be measured according to a known method.

As the solvent used in each of the above measurement systems,
Any substance that does not adversely affect the reaction may be used. Specific examples of commonly used solvents include citrate buffer,
PH of phosphate buffer, Tris-HCl buffer, acetate buffer, etc.
An example of the buffer solution is about 5-9.

The immune reaction (binding) conditions are not particularly limited, either.
Generally, the usual conditions used in this type of measurement method are adopted. Generally, the reaction can be carried out under a temperature condition of about 45 ° C. or lower, preferably about 4-40 ° C. for several tens of minutes to several hours.

Thus, the anti-HSV-1 antibody and anti-HSV-2 antibody in the subject sample can be detected. Detection (presence) of anti-HSV-1 antibody indicates that the subject is infected with HSV-1, and detection (presence) of anti-HSV-2 antibody indicates that the subject is infected with HSV-2. The detection (presence) of both the anti-HSV-1 antibody and the anti-HSV-2 antibody indicates that the subject is infected with both HSV-1 and HSV-2 (superinfection).

In carrying out the diagnosis of HSV infection of the present invention,
It is convenient to use the reagent composition (or reagent kit) for antibody detection or diagnosis described above, and the present invention also provides a reagent composition that can be used for such HSV diagnosis.

The reagent composition of the present invention comprises, as an active ingredient, at least one HSV-1 peptide of the present invention which is an antigenic reagent specific to an anti-HSV-1 antibody, and is a reagent for diagnosing HSV-1 infection. It is a composition.

Further, the reagent composition of the present invention comprises, as an active ingredient, at least one kind of the HSV-2 peptide of the present invention which is an antigen reagent specific to anti-HSV-2 antibody, and is diagnostic for HSV-2 infection. It is a reagent composition for use.

Furthermore, the reagent composition of the present invention contains the above HSV-
1 A diagnostic reagent composition for HSV infection, which comprises both a diagnostic reagent composition for infection and a diagnostic reagent composition for HSV-2 infection.

These reagent compositions of the present invention are a combination of at least one selected from a solid phase, a labeling agent, and a substrate (detection reagent) corresponding to the labeling agent, in addition to the active ingredient. It may be a set.

When the set includes a solid phase, an arbitrary component depending on the measurement system may be immobilized in advance on the solid phase. Also, when a labeling agent is included in the set, similarly, an arbitrary component may be previously conjugated with the labeling agent. Examples of the labeling agent include various compounds such as the above-described radioisotopes, enzymes and fluorescent substances, preferably enzymes.

Further, the reagent composition of the present invention may contain various diluents, standard antibodies, buffer solutions, washing solutions, substrate solutions, reaction stopping solutions, etc., which are suitable for the convenience of performing the measurement. .

[0079]

EXAMPLES In order to explain the present invention in more detail below, first, a conventional method for detecting an antibody using HSV culture antigen, HSV-1 (gpG1) recombinant protein antigen and HSV-2 (gpG2) recombinant protein antigen is described. An example of the peptides of the present invention and an antibody detection method using them will be given below.

Specimens Serum samples (Jikei University School of Medicine Dermatology Department) were 29 sera of herpes patients diagnosed with HSV-1 infection by clinical diagnosis, HSV
-2 sera of 42 herpes patients diagnosed as infected, HSV-1 and HSV
-3 sera of herpes patients and 96 sera of healthy subjects diagnosed with -2 superinfection.

Detection of Antibodies Anti-HSV antibodies (IgG) in these serum samples were detected using a herpes IgG-EIA kit (Denka Seiken) according to a conventional method using HSV culture antigens.

For all cases of HSV patient specimens and healthy specimen specimens in which serum anti-HSV antibody (IgG) was confirmed to be positive, type 1 specific using HSV-1 glycoprotein G (gpG1) recombinant protein antigen Detection of antibody (IgG) and detection of type 2 specific antibody (IgG) using HSV-2 glycoprotein G (gpG2) recombinant protein antigen were performed using commercial kits (Pri
mier Type specific HSV-1 or HSV-2 IgG ELISA Test;
Meridian Diagnostics, Inc.).

The obtained results are shown in FIGS.

FIG. 1 is a graph showing the detection results of anti-HSV antibody according to the conventional method using HSV cultured antigen, and FIG. 2 is gpG1.
FIG. 4 is a graph showing the detection results of the antibody when using the recombinant protein antigen, and FIG. 3 is a graph showing the detection results of the antibody when using the gpG2 recombinant protein antigen.

In each figure, the horizontal axis represents the HSV-infected patient or healthy person, and the vertical axis in FIG. 1 represents the absorbance (OD450-650 nm).
And the vertical axis in FIG. 3 is the relative ELISA value (Rerative EL
ISA Volue: shows the value obtained by dividing the OD value of the sample by the OD value of the control serum.

From the results shown in these figures, serum
The HSV antibody (IgG) positive rate was 86% (25%
/ 29) and 90% (38/42) in the serum of HSV-2 infected patients,
The sera of both co-infected patients were 100% (3/3) and 57% (55/96) in healthy controls.

The type 1 specific antibody (IgG) was highly reactive with sera of HSV-1 infected patients, but was confirmed to be reactive with several sera of anti-HSV antibody negative healthy individuals ( (See Figure 2). In addition, the type 2 specific antibody (IgG) was highly reactive with sera of HSV-2 infected patients, but was confirmed to be reactive with sera of two anti-HSV antibody-positive healthy individuals (Fig. See 3).

[0088]

Example 1 (1) HSV-2 Peptide A library of random peptides (consisting of 9 amino acids) was prepared based on the phage vector pTV119N (Takara Shuzo) by the p8 protein display method of M13 phage. The obtained library was transformed into Escherichia coli JM109 strain by the electroporation method to prepare a random peptide display phage library.

Serum biopanning using the above-mentioned samples (serum of healthy subjects, serum of HSV-1 infected patients and serum of HSV-2 infected patients) showed reactivity with a plurality of HSV-2 infected patient sera and showed HSV-1 infection. "HSV-2c5" and "HV-2c5" as phage clones showing no reactivity to patient serum and healthy person serum
Two clones named "SV-2g60.3" were selected.

(2) Phage ELISA Anti-M13 phage p8 protein antibody (Pharmacia) 1 μg / ml
A 96-well microtiter plate in which was immobilized was used. Add 90 μl of phage ELISA buffer (0.5% B
SA, phosphate buffer containing 10% normal goat serum and 0.05% Tween20) and 10 μl peptide display phage culture medium
(Prepared as described below) was added, and the mixture was reacted at 37 ° C. for 1 hour (primary reaction), and then unreacted phages were washed 4 times and removed. <Preparation of Peptide Display Phage Culture Solution> A single colony was cultured in 50 μl of Luria-Bertani medium containing ampicillin at 37 ° C. for 8 hours, and then the helper phage M13K0 was added thereto.
Infection was carried out by adding 50 μl of 7 liquid (10 ¹² cfu), and then liquid medium (Luria containing ampicillin, kanamycin and IPTG.
-Bertani medium) (400 μl) and the mixture was cultured at 37 ° C. overnight to prepare a peptide display phage culture solution.

Then, each well of the plate after the completion of the primary reaction was diluted with the phage ELISA buffer solution 101-fold and serum sample 10
0 μl was added and reacted at 37 ° C for 1 hour (secondary reaction)
After that, the unreacted antibody was removed by washing 4 times.

Further, 100 μl of enzyme-labeled anti-human IgG (Fc region-specific) antibody diluted 40,000 times with phage ELISA buffer was added to each well and reacted at 37 ° C. for 1 hour (tertiary reaction).
After that, the unreacted antibody was removed by washing 4 times.

Finally, 100 μl of TMB solution was added to each well and allowed to stand at room temperature for 10 minutes to cause the color reaction, and then 100 μl of 1N sulfuric acid solution was added to stop the color reaction. The absorbance (OD450-650nm) of each well was measured.

The results of the above ELISA reactivity (phage ELISA) obtained using both clones selected in (1) above are shown in FIG.

In FIG. 4, the upper row shows the result using the HSV-2c5 clone, and the lower row shows the result using the HSV-2g60.3 clone. In each figure, the horizontal axis represents each patient or healthy individual, and the vertical axis represents the OD value (NET OD).

From the results shown in FIG. 4, it is clear that both phage clones show similar reactivity to the serum of each patient and the serum of healthy subjects.

Hereinafter, the reaction inhibition test using the cultured antigens of HSV-1 and HSV-2 conducted to confirm that these two clones react with the antibody specific to the HSV-2 infected patient is described in detail. To do.

(3) Reaction Inhibition Test HSV-1 was added to the secondary reaction solution of the phage ELISA shown in (2) above.
Alternatively, HSV-2 culture antigen was added and the reactivity between each clone and the antibody was measured in the same manner.

The results are shown in FIG.

FIG. 5-A shows the result when HSV-2c5 was used, and FIG. 5-B shows the result when HSV-2g60.3 was used.
In each figure, the horizontal axis represents the reaction-inhibiting antigen concentration (ng / mL), and the vertical axis represents B / B0 (%). Further, in each figure, (1) is the result when E. coli culture antigen was added, (2) is the result when HSV-1 culture antigen was added, and (3) is the HSV-2 culture antigen. It is the result when is added.

From the results shown in FIG. 5, both clones have HSV-2.
It is clear that the reactivity is inhibited only when the culture antigen of 1 is added.

The results show that HSV-2c5 and HSV-2g60.3
Is a peptide that mimics the HSV-2 specific antigen.

(4) Determination of peptide sequence In order to analyze the peptides displayed by the phage clones HSV-2c5 and HSV-2g60.3, both clones were analyzed.
The DNA sequence was determined and the peptide sequence was determined.

As a result, the sequence of the peptide displayed by HSV-2c5 is shown in SEQ ID NO: 3,
It was confirmed that the sequence of the peptide displayed by -2g60.3 is shown in SEQ ID NO: 4. Comparing these amino acid sequences, 7 of 9 amino acids were in agreement. Since the reactivity of both clones was the same as described above, it was considered that each of these clones mimics the same region of HSV-2.

For the purpose of determining the mimicking region of both clones,
Homology analysis was performed centering on the sequence retained between the clones.

As a result, homology was confirmed in the 6 amino acid sequence of 558-563 of gpG2 (SEQ ID NO: 5). C of both clones
The terminal 3 amino acids showed no homology downstream of the above 6 amino acid sequence of gpG2, but this 9 amino acid sequence (5 of gpG2
The 58-566th position (sequence shown in SEQ ID NO: 2) is a sequence retained between both clones and is a sequence important for binding to a patient antibody, and it was presumed to be a central epitope.

(5) Production of Peptide of the Present Invention in MAP Form The peptide of the present invention having the amino acid sequence shown in SEQ ID NO: 2 (hereinafter referred to as “2gpG58-66”) and the peptide described in the prior art for comparison (SEQ ID NO: 6) The amino acid sequence shown in the following, and this peptide is hereinafter referred to as "2gpG51-70") was synthesized as follows.

That is, using a fully automatic peptide synthesizer (ACT357, manufactured by Advanced Chemtech), according to the program of the company, Fmoc / NMP, HOBt method [Fmoc: 9-fluorenylmethoxycarbonyl group, NMP: N- Methylpyrrolidone, HOBt:
Solid phase synthesis of peptides with 1-hydroxybenzotriazole] was performed.

According to the amino acid sequence information, 0.25 mmol of Fmoc-amino acid-Alko resin corresponding to the C-terminal amino acid was sequentially subjected to condensation reaction with Fmoc-amino acids corresponding to the second and subsequent amino acids from the C-terminal according to a synthetic program to extend the chain. I went.

After each reaction, according to the program, the N-terminal F
The deprotection reaction of the moc group was performed.

Each of the obtained peptide-resins was collected on a polypropylene mini-column (manufactured by Assist), washed with methanol and dried in vacuum. The peptide was cleaved from the resin by the following procedure to remove the side chain. A protective reaction was performed.

That is, Reagent K: 82.5% TF was added to each resin.
A, 5% phenol, 5% H ₂ O, 5% thioanisole
le) and 2.5% ethane di-thiol) 2m
l was added and reacted in the mini column for 60 minutes.

Then, the reaction solution was cooled with 8 ml of diethyl ether.
The reaction was stopped by dropwise addition to precipitate the peptide.
Wash the mini column with 2 ml of TFA and cool 5m of diethyl ether.
l was added, the mixture was centrifuged, and the precipitate was washed 4 times with 10 ml of diethyl ether, then the peptide was solubilized with about 5 ml of 50% acetonitrile, and freeze-dried. Further, solubilization and freeze-drying operations were repeated twice to obtain a desired crude freeze-dried product.

This crude dried product was put into an octadecyl column (diameter 2
The desired peptide was isolated by fractionation by reverse phase high performance liquid chromatography (HPLC) using 0 × 250 mm, YMC).

The resins and amino acid derivatives used in the above were manufactured by Watanabe Chemical Industry Co., Ltd.

Each peptide thus isolated was identified by amino acid sequence analysis and molecular weight measurement by mass spectrometry.

By using the peptide of the present invention and the comparative peptide obtained above, the multi-antigenic peptide (MAP) was labeled with Fmoc-
It was synthesized using MAP-Alko resin (Watanabe Chemical Industry Co., Ltd.).

Fmoc-MAP-Alko resin (Fmoc ₈ -Lys ₄ -Lys ₂ -Lys-
The reaction between the βAla-Alko resin) and the starting peptide was carried out in the same manner as in the above solid phase synthesis method.

The structure of the obtained MAP is as follows.

(2gpG58-66) ₈ -Lys ₄ -Lys ₂ -Lys-βAla (2gpG51-70) ₈ -Lys ₄ -Lys ₂ -Lys-βAla (6) MAP antigen ELISA In each well of a 96-well microtiter plate Dispense 100 μl of MAP solution adjusted to 1 μg / ml with phosphate buffer, and
The reaction was carried out overnight at 0 ° C to solidify.

The obtained solid phased plate was blocked in 300 μl of phosphate buffer containing 1% BSA after removing the MAP solution from each well.

100 μl of a serum sample diluted 101-fold with the primary reaction buffer was added to each well, reacted at 37 ° C. for 1 hour (primary reaction), and then unreacted antibody was washed four times to be removed.

Then, add 50% of each well with the secondary reaction buffer.
Enzyme-labeled anti-human IgG (Fc region-specific) antibody 100 diluted 100 times
After adding μl and reacting at 37 ° C. for 1 hour (secondary reaction),
Unreacted antibody was removed by washing 4 times.

Finally, 100 μl of TMB solution was added to each well and allowed to stand at room temperature for 10 minutes to cause the color reaction, and then 100 μl of 1N sulfuric acid solution was added to stop the color reaction. The absorbance (OD450-650nm) of each well was measured.

[0125] For 41 specimens which were determined to be anti-HSV antibody negative by the test using the herpes IgG-EIA kit, the M
AP antigen ELISA was performed.

The obtained results are shown in FIG. 6 as in FIG. In addition, cut-off (Cut-of) of the average OD value of measurement results + 0.4
f) The value.

From the results shown in FIG. 6, 2gpG58-66 (FIG. 6-
It was found that both MAP antigens utilizing A) and 2gpG51-70 (Fig. 6-B) show high reactivity with the serum of HSV-2 infected patients.

However, as shown in FIG. 6-B, 2
MAP antigen of gpG51-70 was used in serum samples from patients other than HSV-2 infected patients.
The example also showed reactivity. The 6 cases showing this reactivity were HSV
-1 in 2 sera of infected patients (shown as # 1 and # 2 in Figure 6-B) and 3 in healthy subjects with positive HSV antibody (# 3, # 4, # 5 in Figure 6-B)
1) and a HSV antibody-negative healthy subject (Fig. 6-
B is shown as # 6).

The HSV-1 infected patient sera designated # 1 in FIG. 6-B was
It also showed reactivity with the MAP antigen of 2gpG58-66 (Fig. 6-A).
(See # 1). In addition, the HSV antibody-positive healthy subject serum shown as # 5 in Fig. 6-B also showed reactivity with the MAP antigen of 2gpG58-66 (see # 5 in Fig. 6-A) and the gpG2 recombinant protein antigen described above. .

(7) Reaction Inhibition Test The reactivity of the above 6 cases was confirmed by the following reaction inhibition test using HSV-2 cultured antigen.

That is, after a mixed solution of 25 μg of HSV-2 cultured antigen added to each diluted serum sample was reacted at 37 ° C. for 1 hour, the reaction solution was mixed with a MAP antigen plate of 2gpG51-70 or MAP of 2gpG58-66.
It was added to the antigen plate and measured according to the MAP antigen ELISA method of (6) above.

The results are shown in Table 1 below.

[0133]

【table 1】

As shown in Table 1, 2gp G51-70 MAP antigen and 2gp
Serum of HSV-1 infected patient who showed reactivity with MAP antigen of G58-66 #
1 and 2 gpG51-70 MAP antigen and 2gpG58-66 MAP antigen and g
In HSV antigen-positive healthy sera # 5 that showed reactivity with the pG2 recombinant protein antigen, inhibition of reactivity was confirmed in both antigen plates. Therefore, these serum samples # 1 and # 5 are considered to have anti-HSV-2 antibody.

However, no inhibition of reactivity was observed in the other 4 samples. Therefore, 2gpG51-7
It was confirmed that the reactivity of these four specimens that were positive with the 0 MAP antigen was not a reaction with the anti-HSV-2 antibody but a non-specific reaction.

Table 2 below shows the results of the above-mentioned ELISA in each sample in which serum anti-HSV antibody was positive.

[0137]

[Table 2]

[0138] In Table 2, "recombinant" is the measurement result by a commercial kit using the gpG2 recombinant protein antigen.

In addition, anti-HSV was confirmed by the reaction inhibition test in (7) above.
The two cases (# 1 and # 5) that proved the presence of -2 antibody were excluded from the analysis in Table 2.

The results shown in Table 2 show the following.

That is, the positive rate of MAP antigen of 2gpG51-70 for comparison was 82% (31/38) in the serum of HSV-2 infected patients, 67% (2/3) in the serum of superinfected patients, HSV-1. 4% (2/5
5), 4% (2/54) in anti-HSV antibody-positive healthy sera, anti-HSV antibody
It is 2% (1/41) in the serum of antibody-negative healthy subjects.

On the other hand, the positive rate of 2gpG58-66 according to the present invention in the MAP antigen was 74% (28/38) in the serum of HSV-2 infected patients,
It was 33% (1/3) in the serum of superinfected patients. Furthermore, the present invention MA
In the MAP antigen ELISA using P antigen, no positive case was detected in the sera of HSV-1 infected patients, the sera of anti-HSV antibody-positive healthy subjects and the sera of anti-HSV antibody-negative healthy subjects.

From the above results, the MAP antigen of 2gpG51-70 was
Although the positive rate is high but contains a few% of non-specific reactivity, M of 2gpG58-66 of the present invention showing no false positive reaction at all.
The excellent usefulness of the AP antigen was confirmed.

[0144]

Example 2 (1) HSV-1 Peptide Based on the information of the amino acid sequence (SEQ ID NO: 5) of 558-563 of gpG2 found in Example 1, the corresponding HSV-1 glycoprotein G ( The sequence of gpG1) and its neighboring sequences were selected, and their availability as HSV-1 peptides was analyzed.

The analyzed region of gpG1 is the 68th-85th region (the amino acid sequence of which is shown in SEQ ID NO: 7;
5)), and the 92-108th region, 113-132th region (hereinafter referred to as “1gpG113-132”) located downstream thereof,
There are 5 regions, 126-145th region and 146-165th region.

Peptides corresponding to each of the above regions were used in the examples.
Synthesis as MAP according to 1, and MAP in the same manner as in Example 1- (6)
Reactivity to those specimens was tested by antigen ELISA.

As a result of the above-mentioned measurement by ELISA, 1gpG68-85
The MAP antigen and the MAP antigen of 1gpG113-132 showed high reactivity with sera of HSV-1 infected patients and sera of normal anti-HSV antibody-positive individuals. No specific reactivity was confirmed with the other three MAP antigens.

1gpG68-8 which showed the most excellent reactivity above
For 25 kinds of peptides in which the sequence of the 5 region is shortened by 1 amino acid from the N-terminal side and the C-terminal side, MAP is similarly synthesized, and each of these MAPs is used as an inhibitor, Example 1- (7) The reaction inhibition test was carried out in accordance with the above, and its central epitope was determined. The reaction inhibition test consisted of a serum sample diluted 101-fold with primary reaction buffer and 10 μg (10 μl) of each MAP.
After reacting at 37 ° C. for 1 hour, 1 gpG68-85 was added to a MAP antigen-immobilized plate and reacted at 37 ° C. for 1 hour.

Detection of the antigen-antibody complex was performed in the same manner as in Example 1- (7).

As a serum sample, 32 healthy sera showing reactivity with the 1gpG68-85 MAP antigen were used.

The results obtained are shown in Table 3 below. In Table 3, the upper column shows the amino acid sequence of the MAP antigen used as a reaction inhibitor (indicated by one letter), and the lower column shows the reactivity (B / B0%) with the serum sample according to the following criteria. . B / B0 ≦ 30% +++ 30% <++ ≧ 50% 50% <+ ≧ 70% 70% <± ≧ 80% 80% <−

[0152]

[Table 3]

From the results shown in Table 3 above, the following is clear.

That is, when MAP of the N-terminal deleted peptide was used as a reaction-inhibiting antigen, the MAP antigen of 1gpG68-85 (in Table 3,
The reactivity of the serum sample to the inhibitor No. 0) is 3 out of 32 cases by deleting the second histidine (H),
By deleting the third threonine (T), 19 out of 32 cases showed recovery of 70% (B / B0) or more, respectively. Therefore, the N-terminal side was judged to require up to the second H.

On the other hand, when MAP, a peptide deleted at the C-terminal side, was used as a reaction-inhibiting antigen, the reactivity of the serum sample to the MAP antigen of 1gpG68-85 was determined by deleting the fourth glutamic acid (E) in 32 cases. In one case, recovery was 50% (B / B0) or more. Also, by removing the 5th E, out of 32 cases
Reactivity was recovered by 50% (B / B0) or more in 8 cases and in 32 cases
In 7 cases, the reactivity recovered more than 70% (B / B0). Therefore,
The C-terminal side decided that the 4th E was necessary.

From the above, the shortest epitope of the 1gpG68-85 region is the 15th amino acid at the 69-83th position (hereinafter referred to as “1gpG69-83”).
It was decided).

To confirm the reactivity of 1gpG69-83, 1gpG6
9-83 MAP was synthesized in the same manner as described above, and this MAP antigen was used for measurement by MAP antigen ELISA in the same manner as in Example 1- (6).

The obtained results are shown in Table 4 below in accordance with Table 2.

[0159]

[Table 4]

From the results shown in Table 4, MAP of 1gpG69-83
The antigen was as reactive as the MAP antigen of 1gpG68-85.
The positive rate was 92% (23/25) in HSV-1 infected patient sera, 100% (3/3) in superinfected patient sera, and anti-HSV antibody-positive healthy sera.
89% (49/55) and 0% in anti-HSV antibody-negative healthy sera. This positive rate was superior to that using the gpG1 recombinant protein antigen.

From the above, by using the HSV-1 peptide and HSV-2 peptide of the present invention, a specific detection system for anti-HSV-1 antibody and anti-HSV-2 antibody has been established, and these are clinically used. Considered useful.

[0162]

INDUSTRIAL APPLICABILITY According to the present invention, HSV-1 infected patients and H
Peptides that specifically recognize antibodies specific to SV-2 infected patients are provided. These peptides are useful as a diagnostic reagent for HSV infection, particularly as a reagent capable of distinguishing and diagnosing HSV-1 infection and HSV-2 infection. Using these peptides and diagnostic reagents, HSV-1 infection and HSV-2
The presence or absence of infection can be distinguished and accurately diagnosed.

[0163]

[Sequence list]                           SEQUENCE LISTING <110> Otsuka Pharmaceutical Co. Ltd., <120> A method for mesuring anti-herpes simplex virus antibodies <130> 2EF01JP <160> 7 <170> PatentIn Ver. 2.1 <210> 1 <211> 15 <212> PRT <213> phage library <400> 1 His Thr Pro Pro Met Pro Ser Ile Gly Leu Glu Glu Glu Glu Glu   1 5 10 15 <210> 2 <211> 9 <212> PRT <213> phage library <400> 2 Arg Gly Gly Pro Glu Glu Phe Glu Gly   1 5 <210> 3 <211> 9 <212> PRT <213> phage library <400> 3 Arg Gly Gly Pro Ser Glu Tyr His Val   1 5 <210> 4 <211> 9 <212> PRT <213> phage library <400> 4 Arg Gly Gly Pro Gly Glu Tyr Asp Val  1 5 <210> 5 <211> 6 <212> PRT <213> phage library <400> 5 Arg Gly Gly Pro Glu Glu   1 5 <210> 6 <211> 20 <212> PRT <213> phage library <400> 6 Ala Pro Pro Pro Pro Glu His Arg Gly Gly Pro Glu Glu Phe Glu Gly   1 5 10 15 Ala Gly Asp Gly              20 <210> 7 <211> 18 <212> PRT <213> phage library <400> 7 Asp His Thr Pro Pro Met Pro Ser Ile Gly Leu Glu Glu Glu Glu Glu   1 5 10 15 Glu Glu

[Brief description of drawings]

FIG. 1 is a diagram showing the results of measuring an anti-HSV antibody (IgG) in a sample with a herpes IgG-EIA kit using an HSV culture antigen.

FIG. 2 is a diagram showing the results of measuring the anti-HSV-1 antibody (IgG) in a sample by an ELISA method using a gpG1 recombinant protein antigen.

FIG. 3 is a diagram showing the results of measuring the anti-HSV-2 antibody (IgG) in a sample by an ELISA method using a gpG2 recombinant protein antigen.

FIG. 4 Phage clone HSV- selected in (1) of Example 1.
FIG. 3 is a diagram showing the results of measuring the reactivity of each of 2c5 and HSV-2g60.3 with an anti-HSV antibody in a sample by an ELISA method.

FIG. 5: Cultured antigen of HSV-1 or H according to (3) of Example 1
It is a figure which shows the result of the reaction inhibition test using the culture antigen of SV-2.

FIG. 6 is a view showing a measurement result by MAP antigen ELISA according to (6) of Example 1.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Toshikazu Saito             463-30 Kagasuno, Kawauchi Town, Tokushima City, Tokushima Prefecture             Tsuka Pharmaceutical Imakiri Dormitory E-117 (72) Inventor Tetsuya Tachikawa             13-6 Higashi-no-Kamimoto, Takafusa, Kitajima-cho, Itano-gun, Tokushima Prefecture (72) Inventor Koichi Ogino             197-3 Higashihama, Minamihama, Naraya-cho, Naruto City, Tokushima Prefecture (72) Inventor Riki Tanaka             42-13 Sumiyoshi Chidorigahama, Aizumi-cho, Itano-gun, Tokushima Prefecture (72) Inventor Takao Taki             8-4 Sannomiya, Ejiri, Kitajima-cho, Itano-gun, Tokushima Prefecture F-term (reference) 4H045 AA10 AA30 BA15 BA17 DA86                       EA53 FA33

Claims (7)

[Claims] 1. A peptide having the amino acid sequence shown in SEQ ID NO: 1, or a peptide having an amino acid sequence in which one or more amino acids in the amino acid sequence have been modified by substitution, deletion or addition, which is of simple type 1. A peptide having specific reactivity with a herpesvirus antibody. 2. A peptide having the amino acid sequence shown in SEQ ID NO: 2, or a peptide having an amino acid sequence in which one or more amino acids are modified by substitution, deletion or addition in the amino acid sequence, which is simple type 2. A peptide having specific reactivity with a herpesvirus antibody. 3. A reagent composition for diagnosing herpes simplex virus infection, which comprises the peptide according to claim 1 as an active ingredient. 4. A reagent composition for diagnosing herpes simplex virus infection, which comprises the peptide according to claim 2 as an active ingredient. 5. The diagnostic reagent composition according to claim 3 or 4, wherein the peptide is in the form of a multi-antigenic peptide. 6. A diagnostic for distinguishing between type 1 herpes simplex virus infection and type 2 herpes simplex virus infection, which comprises the reagent composition according to claim 3 and the reagent composition according to claim 4. Reagent composition. 7. A method for distinguishing between a herpes simplex virus type 1 infection and a herpes simplex virus type 2 infection in a sample, which comprises the peptide according to claim 1 and the peptide according to claim 2. A method comprising the step of contacting a sample and detecting the presence or absence of the type 1 herpes simplex virus antibody and the type 2 herpes simplex virus antibody in the sample.