JP2003337134A - Nonspecific reaction inhibition peptide, nonspecific reaction inhibition method using the same and antibody measurement method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a means for preventing diagnosis errors by inhibiting a nonspecific reaction and accurately measuring in a syphilis diagnosis in an immunological measurement method for an antitreponemal pallidum antidody using a treponemal pallidum antigen. <P>SOLUTION: A peptide comprises all or a part of amino-acid sequences described in a sequence number 1 and includes at least five continuous amino- acid sequences in the sequence number 1. Or a peptide includes the peptide and has activity for inhibiting the nonspecific reaction in the immunological measurement method for the antitreponemal pallidum antibody using the antigen including a treponemal pallidum 47K dalton antigen protein. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a substance which suppresses non-specific reaction in the measurement of anti-TP antibody using Treponema Pallidum (hereinafter sometimes abbreviated as TP) antigen, and a non-specific substance using the substance. The present invention relates to a reaction suppressing method, an anti-TP antibody measuring reagent containing the substance, and an anti-TP antibody measuring method using these. The present invention is applied to life science fields such as clinical examination, immunology and medicine,
It is useful in the fields of chemistry such as analytical chemistry, food hygiene, and environmental hygiene.

[0002]

2. Description of the Related Art Syphilis is an infectious disease caused by Treponema pallidum. Whether syphilis is infected or not is determined mainly by detecting syphilis antibody by syphilis serum reaction. For this syphilis serum reaction, an anti-lipid antibody measuring method using a phospholipid such as cardiolipin or lecithin as an antigen (serologic test for)
syphilis (hereinafter sometimes abbreviated as STS method) and a method for measuring anti-TP antibody using TP cells or cell components as an antigen.

The STS method includes a glass plate method, an RPR method and the like, and the anti-TP antibody measuring method includes a fluorescent antibody method (fl).
uorecent responder antibo
dyabsorption test; FTA-ABS
Method), passive hemagglutination reaction (TP hemagglut
Ination test; TPHA method, particle agglomeration method (Treponema Pallidum Particulate)
le Agglutination; TPPA method), enzyme immunoassay method (enzyme-linkedimmu)
nosorbent assay; ELISA method), latex agglutination method (TP latex agglutina)
(TPLA method) and immunochromatography method are used for daily inspection. In particular, the anti-TP antibody measuring method using the TP antigen has high specificity for syphilis and is widely used for daily examination.

TP used in this anti-TP antibody measuring method
As the antigen, those purified from TP cells have been used. For example, the surface antigen protein of TP antigen can be used as an antigen. Such surface antigen proteins include 15K dalton antigen protein, 17K
The existence of K Dalton antigen protein, 47K Dalton antigen protein and the like are known, and these can be used.

In recent years, TP antigen has been produced in host cells such as Escherichia coli by using gene recombination technology. Since the amino acid sequences of these surface antigen proteins and the nucleotide sequences of their genes have already been reported (15K dalton antigen protein: Molecular
Microbiology Volume 4, 1371-1379
Page, 1990; 17K Dalton Antigen Protein: In
section and Immunity Volume 61, 12
02-1210, 1993; 47K Dalton Antigen Protein: Infection and Immunity.
y 60, pp. 1568-1576, 1992), it has been possible to produce these surface antigen proteins using genetic recombination techniques.

[0006] However, in the anti-TP antibody measuring method using these TP antigens, depending on the sample to be measured, a false positive may be shown due to a non-specific reaction even though the anti-TP antibody is not present in the sample. There was a risk of misdiagnosing the disease of patients. This phenomenon is considered to be caused by the reaction of components other than the antibody contained in the sample with the antigen and the like.

Various methods have been provided as methods for suppressing this non-specific reaction. For example, JP-A-58-14
Japanese Patent No. 4748 discloses a technique of adding bovine serum albumin, horse serum albumin or the like to a reagent in order to suppress a non-specific reaction, and Japanese Patent Application Laid-Open No. 8-105897 discloses a method for suppressing a non-specific reaction. Techniques for adding sodium chloride to do so have been shown. JP-A-8-176195
The publication discloses a method of using a chemically modified reduced albumin as a blocking agent to suppress a non-specific reaction. Further, Japanese Patent Publication No. 3-59382 discloses a technique of using a host bacterial component which is of the same species as that used for gene recombination in order to suppress a non-specific reaction but has not been genetically modified. . Furthermore,
Japanese Patent No. 3206311 discloses a technique of using a cultured cell component containing a vector of the same species as that used for antigen production, which does not contain a gene encoding the antigen. However, these techniques have not always been sufficient to suppress nonspecific reactions.

[0008]

As mentioned above, the TP
In the measurement of anti-TP antibody using an antigen, there is a problem that a false positive may be shown due to a non-specific reaction depending on the sample to be measured, even though the anti-TP antibody is not present in the sample. Therefore, an object of the present invention is to prevent an erroneous diagnosis by suppressing non-specific reaction in an immunoassay method for an anti-TP antibody using a TP antigen and performing an accurate measurement at the place of diagnosis of syphilis. Is to provide.

[0009]

Means for Solving the Problems As a result of intensive studies to solve the above-mentioned problems, the present inventors have shown that the TP antigen protein does not show the antigenicity but has a nonspecific reaction suppressing effect. The inventors have found that the above problems can be solved by using a fragment of an antigen protein as a non-specific reaction inhibitor, and have completed the present invention.

That is, the present invention includes the following inventions. (1) A peptide comprising the amino acid sequence of any of SEQ ID NOs: 4 to 36, which is Treponema pallidum 4
A peptide having an activity of suppressing non-specific reaction in an immunoassay method for an anti-Treponemal paridum antibody using an antigen including a 7K dalton antigen protein.

(2) Consists of all or part of the amino acid sequence of SEQ ID NO: 1 and at least 5 in SEQ ID NO: 1
A peptide containing one continuous amino acid sequence, or a peptide containing the peptide and having an activity of suppressing non-specific reaction in an immunoassay method for an anti-Treponemal paridum antibody using an antigen containing the Treponema pallidum 47K Dalton antigen protein.

(3) At least 5 of SEQ ID NO: 2, which consists of all or part of the amino acid sequence of SEQ ID NO: 2
A peptide containing one continuous amino acid sequence, or a peptide containing the peptide and having an activity of suppressing non-specific reaction in an immunoassay method for an anti-Treponemal paridum antibody using an antigen containing the Treponema pallidum 47K Dalton antigen protein.

(4) A peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence consisting of a part thereof, which has a deletion, addition, substitution or modification of one or several amino acid residues. Treponema
A peptide having an activity of suppressing a non-specific reaction in an immunoassay method for an anti-Treponemal pallidum antibody using an antigen including a Palidam 47K Dalton antigen protein.

(5) A peptide consisting of the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence consisting of a part thereof, wherein the amino acid sequence has one or several amino acid residues deleted, added, substituted or modified. Treponema
A peptide having an activity of suppressing a non-specific reaction in an immunoassay method for an anti-Treponemal pallidum antibody using an antigen including a Palidam 47K Dalton antigen protein.

(6) Treponema pallidum 47K Dalton antigenic protein which does not show antigenicity, (1) to
The peptide according to any one of (5). (7) A non-specific reaction inhibitor containing the peptide according to any one of (1) to (6), for use in an immunoassay method for an anti-Treponemal paris dame antibody that uses the Treponema pallidum 47K Dalton antigen protein.

(8) For use in an immunoassay method for an anti-Treponemal pallidum antibody using Treponema pallidum 47K Dalton antigen protein, (1) to
An anti-Treponemal paridum antibody measuring reagent containing the peptide according to any one of (6). (9) The reagent according to (8), which further contains Treponema pallidum 47K Dalton antigen protein.

(10) The reagent according to (9), wherein the Treponema pallidum 47K Dalton antigen protein is produced by gene recombination technology. (11) In an immunoassay method for an anti-Treponemal parisum antibody using Treponema pallidum 47K Dalton antigen protein, a method of contacting the sample according to any one of (1) to (6) with a sample to suppress a nonspecific reaction. .

(12) A method for immunotreating an anti-Treponemal paris dame antibody using a 47 K Dalton antigen protein of Treponema pallidum, which comprises contacting the peptide according to any one of (1) to (6) with a sample. Immunoassay method. (13) Treponema pallidum 47K Dalton antigen protein is produced by gene recombination technology,
The immunoassay method according to (12). (14) An immunoassay kit for measuring an anti-Treponemal paridum antibody, which comprises the peptide according to any one of (1) to (6) and a carrier on which the Treponema pallidum 47K Dalton antigen protein is immobilized.

[0019]

BEST MODE FOR CARRYING OUT THE INVENTION The peptide of the present invention has an activity of suppressing non-specific reaction in an immunoassay method for measuring anti-TP antibody in a sample using Treponema pallidum antigen protein. The immunoassay method that can use the peptide of the present invention is not particularly limited as long as it is a method including the use of Treponema pallidum 47K Dalton antigen protein. In the present invention, the immunoassay method has an ordinary meaning in the art, and means an immunoassay method for detecting an anti-TP antibody in a sample by utilizing an antigen-antibody reaction between an antigen and an antibody.

Examples of such immunoassays include latex nephelometry; indirect agglutination assay such as latex agglutination assay; enzyme immunoassay, fluorescent immunoassay, radioimmunoassay or luminescent immunoassay. Immunoassay using a labeling substance such as a method; Western blotting;
ELSA method (enzyme-linked ligan)
dsorbent assay) [Dahlbeack
Et al., Thromb. Haemost. , Volume 79, 767
~ 772, 1998; WO98 / 23963]; immunochromatography method; or JP-A-9-2
No. 29936 and Japanese Patent Application Laid-Open No. 10-132819, a carrier having a surface coated with a specific binding substance for a test substance and a specific binding substance for the test substance are immobilized. Examples include a method of using particles and measuring depending on whether or not the particles are collected on the surface of the carrier. When the labeled immunoassay method is used, the present invention can be applied to any method such as a sandwich method, a competitive method or a homogeneous method (homogeneous method). In the present invention, the latex nephelometry, or JP-A-9-229936 and JP-A-10-13.
No. 2819, etc., a carrier having a surface on which a specific binding substance for a test substance is immobilized and a particle on which a specific binding substance for the test substance is immobilized are used. It is preferable to use a method in which the measurement is performed depending on whether or not they are collected on the above surface.

The measurement may be performed manually or
You may perform using apparatus, such as an analyzer. The measurement may be performed by a one-step method (one-reagent method), or 2
It may be carried out by a method including a plurality of operation steps such as a step method (two-reagent method).

In the present invention, the TP antigen used in the above-described immunoassay method for anti-TP antibody is not particularly limited as long as it contains at least TP47K Dalton antigen protein. In addition to the TP47K Dalton antigen protein, it may contain other antigen proteins. It is preferable to include a surface antigen protein of Treponema pallidum, for example, a TP15K dalton antigen protein or a TP17K dalton antigen protein. The antigen protein used as an antigen may be one TP47K Dalton antigen protein or a mixture of several kinds of antigen proteins, but it is preferable to use a mixture of several kinds of antigen proteins. You may use those in the state in which a plurality of these antigen proteins were bound. These antigenic proteins may be purified from Treponema pallidum cells or may be obtained using gene recombination technology. As the TP47K Dalton antigen protein, a homogeneous one can be continuously and inexpensively prepared, and therefore it is preferable to use the one obtained by gene recombination technology.

When the antigenic protein is produced by the gene recombination technique, a method usually used in the art can be used. For example, by cloning a gene encoding an antigen protein, incorporating the obtained gene into an expression vector such as a plasmid, introducing this expression vector into a host cell such as Escherichia coli, and culturing the obtained transformant. Can be expressed. Examples of the method for cloning a gene include a PCR method, a recombinant PCR method, a ligation method, a linker ligation method, and the like.

The TP47K dalton antigen protein artificially prepared by this gene recombination technique is
It may be fused with another protein. Examples of other proteins to be fused include glutathione-S-transferase (GST), maltose binding protein (MBP), thioredoxin,
Examples include β-galactosidase, lactase, biotinylated protein, protein A, gene 10 and the like.

When the antigen protein is purified from Treponema pallidum cells, it can be obtained by extracting from the cells with a surfactant or ultrasonic disruption. Further, the antigen protein extracted from the bacterial cells can be obtained by gel filtration, ion exchange, or purification with hydroxyapatite or the like.

The present invention is a peptide having 3 or more amino acids, preferably 3 to 200, more preferably 50 to 200, which contains all or part of the amino acid sequence shown in SEQ ID NO: 1, and which is a TP47K dalton antigen protein. The present invention relates to a peptide having an activity of suppressing a non-specific reaction in an immunoassay method for an anti-Treponemal paridum antibody used and having no antigenicity of a TP antigen protein. Sequence number 1 here
The amino acid sequence described in is 4 of Treponema pallidum.
1 in the amino acid sequence of the 7K dalton antigen protein
It is the amino acid sequence corresponding to the 26th to 295th positions. Examples of such peptides include peptides containing the amino acid sequences set forth in SEQ ID NOS: 4 to 36 below. (1) EGAVALADRA (SEQ ID NO: 4) (2) LADRASSSFMV (SEQ ID NO: 5) (3) SSFMVDSEEY (SEQ ID NO: 6) (4) DSEEYKITNV (SEQ ID NO: 7) (5) KITNVKVHGM (SEQ ID NO: 8) (6) KVHGMKFVPV ( SEQ ID NO: 9 (7) KFVPVAVPHE (SEQ ID NO: 10) (8) AVPHELKGIA (SEQ ID NO: 11) (9) LKGIAKEKFH (SEQ ID NO: 12) (10) KEKFHFVEDS (SEQ ID NO: 13) (11) FVEDSRVTEN (SEQ ID NO: 14) 12) RVTENTNGLK (SEQ ID NO: 15) (13) TNGLKTMLTE (SEQ ID NO: 16) (14) TMLTEDSFSA (SEQ ID NO: 17) (15) DSFSARKVSS (SEQ ID NO: 18) (16) RKVSSMESP (SEQ ID NO: 19) (17) MESPHDLVVD (SEQ ID NO: 20) (18) DLVVDTVGTG (SEQ ID NO: 21) (19) TVGTGYHSRF (SEQ ID NO: 22) (20) YHSRFGSDAE (SEQ ID NO: 23) (21) GSDAEASVML (SEQ ID NO: 24) (22) ASVMMLKRADG (SEQ ID NO: 25) (23) KRADGSELSH (SEQ ID NO: 26) (24) SELSHREFID (SEQ ID NO: 27) (25) REFIDYVMNF (SEQ ID NO: 28) (26) YVMNFNTVRY (SEQ ID NO: 29) (27) NTVRYDYYGD (SEQ ID NO: 25) SEQ ID NO: 30 (28) DYYGDDASYT (SEQ ID NO: 31) (29) DASYTNLMAS (SEQ ID NO: 32) (30) NLMASYGTKH (SEQ ID NO: 33) (31) YGTKHSA SW (SEQ ID NO: 34) (32) SADSWWKTGR (SEQ ID NO: 35) (33) WKTGRVPRIS (SEQ ID NO: 36)

Of the above peptides, a peptide containing the amino acid sequence of SEQ ID NO: 2 corresponding to the 165th to 244th positions in the amino acid sequence of the 47K Dalton antigen protein of Treponema pallidum is preferred. A peptide containing the amino acid sequence of SEQ ID NO: 1 is more preferable. The peptide of the present invention comprises an amino acid sequence having the deletion, addition, substitution or modification of one or several amino acid residues in the amino acid sequence shown in SEQ ID NO: 1 or 2 or an amino acid sequence consisting of a part thereof. A peptide,
Treponemal paris dame has an activity of suppressing non-specific reaction in an immunoassay method for an anti-treponemal paris dame antibody using an antigen containing a 47K dalton antigen protein,
Also included are peptides that do not have the antigenicity of the TP47K Dalton antigen protein. Here, several means 1 to 10, preferably 1 to 5. The length of these peptides is not particularly limited, but is 3 or more amino acids, preferably 3 to 200 amino acids, and more preferably 5 amino acids.
It is 0 to 200. In the present invention, the peptide means a peptide in which two or more amino acids are linked by a peptide bond, and includes oligopeptides, polypeptides and proteins.

Furthermore, the peptide used in the present invention may be any other protein, peptide, carbohydrate or lipid residue as long as the effect of suppressing the non-specific reaction is maintained and the fusion itself does not cause the non-specific reaction. Alternatively, it may be one to which an amino acid or the like is bound. Here, other proteins include proteins for efficiently expressing or purifying the peptide of the present invention, for example, glutathione-S-transferase (GST), maltose binding protein (MBP), thioredoxin, β-galactosidase, lactase. , Biotinylated protein, protein A, gene 10 and the like.

The peptide of the present invention as described above can be prepared by a method commonly used in the art, and such a method is not particularly limited, and examples thereof include chemical synthesis method and gene recombination technology. Any method can be used. When using a gene recombination technique, for example, a gene encoding a target peptide is first cloned, and the obtained gene is incorporated into an expression vector such as a plasmid. The desired peptide can be expressed by introducing this expression vector into a host cell such as Escherichia coli and culturing the obtained transformant.

As a method for cloning a gene,
For example, a PCR method, a recombinant PCR method, a ligation method, a linker ligation method, etc. can be mentioned. For example, when the PCR method is used, the target nucleotide sequence can be amplified using a primer.

For example, in order to express a peptide consisting of the entire amino acid sequence of SEQ ID NO: 1, first,
It is obtained by amplifying the base sequence of the gene encoding the amino acid sequence of SEQ ID NO: 1 in the amino acid sequence of TP47K Dalton antigen protein using the PCR method using a primer. The nucleotide sequence of the gene thus obtained is incorporated into an expression vector by a ligation method or the like, the expression vector is introduced into Escherichia coli, and the obtained transformant is cultured to give the amino acid sequence of SEQ ID NO: 1. It is possible to express a peptide consisting of an amino acid sequence corresponding to the entire amino acid sequence.

When the peptide of the present invention is used to suppress the non-specific reaction in antibody measurement, the method of using the peptide is particularly so long as the sample to be measured can be brought into contact with the non-specific reaction-suppressing peptide of the present invention. Not limited.
For example, a method may be mentioned in which the peptide of the present invention contained in a buffer or the like is brought into contact with a sample before the antibody measurement reaction to suppress non-specific reaction in advance. Alternatively, when the peptide of the present invention is contained in a reagent in which an antigen containing a TP47K dalton antigen protein is present, the reagent is brought into contact with the sample to perform an antibody measurement reaction, and the reaction between the anti-TP antibody in the sample and the antigen is performed. Alternatively, the peptide of the present invention may be allowed to coexist. Alternatively, a method combining these two methods may be used.

In the present invention, the sample means an object in which anti-TP antibody may be present, and the presence or absence or content (concentration) of the anti-TP antibody is to be measured. Body fluids such as blood, serum, plasma, urine, saliva and the like can be mentioned. The amount of peptide used is anti-TP
The concentration can be changed depending on the strength of the non-specific reaction that occurs during antibody measurement, and a concentration sufficient to absorb the non-specific reaction may be appropriately set. For example, the concentration of the peptide of the present invention present at the time of antibody measurement is preferably 0.0001% (w /
v) or more, more preferably 0.0001 to 1% (w /
v), more preferably 0.01 to 1% (w / v).

The above-mentioned buffer containing the peptide of the present invention is not particularly limited. For example, various buffers such as tris (hydroxymethyl) aminomethane buffer, phosphate buffer or phosphate buffered saline can be used. Can be mentioned. The buffer solution may further contain additives such as a reaction accelerator and a stabilizer. It can also be used in combination with other non-specific reaction inhibitors, etc. as used in the prior art.

For example, when the latex turbidimetric method is used as the anti-TP antibody measuring method and the measurement is carried out by the two-step method, the peptide of the present invention is contained in the first reagent consisting of a buffer solution, and this is used as a sample in advance. By bringing them into contact, the non-specific reaction can be suppressed before the measurement of the anti-TP antibody, and then this can be brought into contact with the second reagent in which the latex particles having the TP antigen immobilized thereon are present. Further, the peptide of the present invention is contained in the second reagent containing the latex particles having the TP antigen immobilized thereon, and the second reagent is brought into contact with the mixture of the sample and the first reagent to carry out the antibody measurement reaction. Alternatively, the peptide of the present invention may coexist during the measurement reaction of the anti-TP antibody. Alternatively, these two methods may be used in combination. When a measuring method using a carrier is used as the antibody measuring method, the type and shape of the carrier used are not particularly limited, and those usually used in the art can be used.

The carrier that can be used in the latex nephelometry is not particularly limited, but examples thereof include polystyrene / latex particles, styrene / styrene sulfonate copolymer / latex particles, and acrylonitrile / butadiene / styrene copolymer / latex. Particles, vinyl chloride-acrylic acid ester copolymer / latex particles, vinyl acetate-
Acrylic acid copolymer / latex particles, polyacrolein / latex particles, styrene-methacrylic acid copolymer / latex particles, styrene-glycidyl (meth) acrylic acid copolymer / latex particles, methacrylic acid polymer / latex particles, or Examples thereof include latex particles in which synthetic polymer particles such as acrylic acid polymer / latex particles are uniformly suspended.

The carrier usable in the indirect agglutination measurement method such as the particle agglutination reaction measurement method or the latex agglutination reaction measurement method is not particularly limited, and examples thereof include polystyrene, liposome, latex, gelatin, polyacrylamide, microcapsule or Examples thereof include particles made of an organic polymer such as emulsion, particles made of an inorganic polymer such as glass, silica gel, carbon or bentonite, and other artificial carriers.

The above-mentioned carrier particles may be colored by coating a dye or dispersing or encapsulating the dye in the particles. The particle size of the carrier particles is not particularly limited, but the average particle size is 0.01 to 100 μm.
Is preferably within the range, and more preferably within the range from 0.1 to 10 μm. The specific gravity of these particles is preferably in the range of 1 to 10,
More preferably, it is within the range of 2.

Further, in the indirect agglutination reaction measuring method, for example, a test tube made of glass, polystyrene, polyvinyl chloride or polymethacrylate, a container such as a microplate (microtiter plate) or a tray may be used as a carrier. it can. The shape of the solution containing portion of such a container, that is, the shape of the bottom surface of the well of the microplate is not particularly limited, but is U-shaped, V-shaped or UV-shaped.
It is preferable to have a shape having a slope from the center of the bottom surface to the periphery like a mold.

Further, enzyme immunoassay, fluorescent immunoassay,
The carrier that can be used in a labeled immunoassay using a labeling substance such as a radioimmunoassay or a luminescence immunoassay is not particularly limited, but includes, for example, polystyrene, polycarbonate, polyvinyltoluene, polypropylene,
Particles made of materials such as polyethylene, polyvinyl chloride, nylon, polymethacrylate, polyacrylamide, latex, liposome, gelatin, agarose, cellulose, sepharose or glass, microcapsules, beads, microplates (microtiter plates), test tubes, Examples thereof include sticks and test pieces.

Further, a carrier having a surface coated with a substance specifically binding to a test substance (test substance) described in JP-A-9-229936 and JP-A-10-132819. Also, the carrier that can be used in the measurement method using particles having a specific binding substance immobilized to a test substance (test substance) is not particularly limited, and examples thereof include polystyrene, glass, polyvinyl chloride, polyacrylate, and polypropylene. ,Nylon,
Examples of the carrier include non-water-absorbing materials such as polyethylene, polycarbonate and polymethacrylate. In the present invention, a magnetic carrier or the like prepared by coating the above carrier with a ferromagnetic material or incorporating a ferromagnetic material in molding the carrier can also be used.

The label used in the label immunoassay is not particularly limited as long as it is one commonly used in the art, but it is preferable to use an enzyme label. As the enzyme label, horseradish peroxidase, alkaline phosphatase, glucose oxidase, β-galactosidase, luciferase, urease, lysozyme,
Glucose-6-phosphate dehydrogenase, ribonuclease and the like can be mentioned.

In the present invention, the anti-TP antibody measuring reagent is
It means a reagent for use in the above-mentioned immunoassay method for anti-TP antibody. Examples of the anti-TP antibody measurement reagent include a reagent containing the peptide of the present invention and a reagent containing TP47K Dalton antigen protein. The antigen protein may be immobilized on a carrier. The anti-TP antibody measurement reagent may contain additional components such as other antigenic proteins. The amount of the peptide of the present invention contained in the anti-TP antibody measurement reagent is not particularly limited as long as it does not inhibit the reaction in the measurement of anti-TP antibody and has a nonspecific reaction suppressing effect, but preferably 0.0001 to 1 % (W /
v), more preferably 0.01 to 1% (w / v).

The anti-TP antibody measuring reagent of the present invention is not particularly limited to these, but for example, proteins such as bovine serum albumin (BSA), human serum albumin (HSA), casein or salts thereof; various salts; Various sugars; skim milk powder; various animal sera such as normal rabbit serum;
Various preservatives such as sodium azide or antibiotics;
One or more of an activator; a reaction accelerating substance; a sensitivity-increasing substance such as polyethylene glycol; and a non-specific reaction-suppressing substance may be appropriately contained. The concentrations of these components in the anti-TP antibody measuring reagent are not particularly limited, but are preferably 0.001 to 10% (w / v),
Particularly, 0.01 to 5% (w / v) is preferable.

In the production of the anti-TP antibody measuring reagent of the present invention, various aqueous solvents can be used as the solvent. The aqueous solvent is not particularly limited, and examples thereof include purified water, physiological saline, or various buffers such as tris (hydroxymethyl) aminomethane buffer, phosphate buffer or phosphate buffered saline. You can

The pH of the anti-TP antibody measuring reagent may be appropriately selected and used, and there is no particular limitation, but a pH in the range of pH 3 to 12 is usually selected. In the present invention, it is preferable to use a pH in the alkaline range. pH is pH 7.3 or higher, for example, pH
It is more preferably 8 to 10.

The anti-TP antibody measuring reagent of the present invention may be used alone or in combination with other reagents. Other reagents include, for example, buffer, sample diluent,
Examples thereof include a reagent diluent, a reagent containing a labeling substance, a reagent containing a substance that produces a signal such as color development, or a reagent containing a substance for performing calibration. Using the anti-TP antibody measuring reagent in the present invention,
In order to measure the anti-TP antibody in the sample, the operation may be performed according to the operation method based on the measurement principle of the immunoassay method used.

The present invention also includes a kit for immunoassay in which an anti-TP antibody measuring reagent, the above-mentioned other reagents, a carrier and the like are appropriately combined based on the principle of the measuring method used.
As such a kit, for example, when used in a labeled immunoassay, a kit containing an anti-TP antibody measuring reagent, a labeled antigen, an antibody diluent, etc. is preferable, and when used in an indirect agglutination assay, Those containing a TP antibody measuring reagent, a particle dispersion liquid and the like are preferable. Hereinafter, the present invention will be further described with reference to examples, but the present invention is not limited thereto.

[0049]

EXAMPLES Example 1 Preparation of Peptide Having Activity to Suppress Nonspecific Reaction Treponema pallidum cells were extracted from the crushed rabbit testicles obtained by subculturing Treponema pallidum to extract genes. The nucleotide sequence of the TP47K dalton antigen protein is already known as described above, and the entire amino acid sequence is as shown in SEQ ID NO: 3. This amino acid sequence is divided into the five regions shown below, and each TP47
The K Dalton antigen protein fragment was prepared by the following method. Example 1: Peptide consisting of the 126th to 295th sequence of SEQ ID NO: 3 Comparative example 1: Peptide consisting of the 1st to 164th sequence of SEQ ID NO: 3 Comparative example 2: consisting of the 244th to 415th sequence of SEQ ID NO: 3 Peptide Comparative Example 3: Peptide consisting of the 1st to 295th sequence of SEQ ID NO: 3 Comparative Example 4: Peptide consisting of the 126th to 415th sequence of SEQ ID NO: 3

First, about 20 bases of oligonucleotides at both ends of the base sequence encoding the TP47K Dalton antigen protein fragment were synthesized as primers. Here, using the above-mentioned nucleotide sequence divided into 5 regions as a template and the above-mentioned primers, 5 kinds of TP4 were prepared by PCR.
A nucleotide sequence encoding a 7K Dalton antigen protein fragment was amplified. Next, 5 was obtained in a vector containing a nucleotide sequence encoding glutathione-S-transferase (GST) (Pharmacia; pGEX4T3).
Each type of base sequence was inserted. This vector was introduced into Escherichia coli and the cells were cultured. Then, Escherichia coli in which a fragment of the TP47K dalton antigen protein was sufficiently expressed was disrupted by ultrasonication, centrifuged to solubilize the precipitate with 8M urea, and then dialyzed to remove urea.
This was further centrifuged and the supernatant was collected to obtain 5 kinds of TP47K Dalton antigen protein fragments having a purity of 50% or more.

[Example 2] The effect of suppressing the non-specific reaction of the above peptide fragment in the measurement of an antibody (anti-TP antibody) against Treponema pallidum was confirmed using the ELISA method. (1) Preparation of TP47K Dalton Antigen Protein Immobilized Plate Preparation of Wash Solution Phosphate buffered saline containing 0.02% (w / v) Tween 20 was prepared and used as a wash solution. Preparation of blocking solution Phosphate buffered saline containing 0.5% (w / v) sodium caseinate was prepared as a blocking solution. Preparation of TP47K Dalton Antigen Protein Immobilized Plate

The 47K Dalton antigenic protein of Treponema pallidum was transformed into L. Weigel, M .; Norga
rd et al. (Infection and Immun
(Vol. 60, 1568-1576, 1992)
According to the method described above, it was prepared by a gene recombination technique. This TP
The 47K Dalton antigen protein was added to phosphate buffered saline at 2 μg / mL. 50 μL of this was dispensed into each well of a 96-well microtiter plate (Nunc), and then left overnight at 4 ° C. to prepare TP.
The 47K Dalton antigen protein was immobilized on the inner wall of each well.

Next, the liquid in each well of this 96-well plate was removed, and the plate was washed twice with the above-mentioned washing liquid. Then, 200 μL of the blocking solution was dispensed into each well to block each well. Then, the liquid in each well of this plate was removed, and the plate was washed twice with the washing liquid. TP4 prepared by the above operation
The plate was used as a 7K Dalton antigen protein-immobilized plate.

(2) Preparation of peroxidase (POD) -labeled antibody POD-labeled rabbit anti-human IgG antibody solution (manufactured by Dako; P
0406) was diluted 1000 times with phosphate buffered saline containing 0.5% (w / v) sodium caseinate.

(3) Preparation of sample diluent A phosphate buffered saline containing 0.5% (w / v) sodium caseinate was prepared, and Example 1 and Comparative Examples 1 to 1 prepared in Example 1 were prepared. 4 peptide fragments at a final concentration of 0.0
It was added to be 2% (w / v). A control example was one in which neither peptide was added.

(4) Effect of suppressing non-specific reaction in anti-TP antibody measurement False-positive serum (1 sample) or anti-TP antibody-positive serum (14 samples) diluted 200 times with the sample diluent prepared in (3) 5
0 μL was added to each well of the TP47K Dalton antigen protein-immobilized plate prepared in (1) above,
The reaction was carried out at 37 ° C for 4 hours. After that, the serum solution in the well of this plate was removed, and the plate was washed with the above washing solution. Furthermore,
50 μL of the POD-labeled rabbit anti-human IgG antibody solution prepared in (2) above was added to each well and reacted at room temperature for 4 hours. Then, each well was washed with the washing solution.

After this, a coloring solution (4 mM o-phenylenediamine, 103 mM disodium hydrogen phosphate, 49 m
33 μL of 0. 3 for 1 mL of buffer containing M citric acid.
3% hydrogen peroxide added immediately before use) 100 μL
Was added to each well and reacted at room temperature for 20 minutes, then 4
50 μL of N-sulfuric acid aqueous solution was added to each well to stop the reaction. Then, using a microplate reader (BioRad; model 3550), 490 nm of each well
The absorbance of was measured. The obtained absorbance is shown in Table 1.

[0058]

[Table 1]

(5) Discussion From Table 1, when the peptides of Comparative Example 1 and Comparative Example 2 were used, the reactivity with the sample in the case of measuring the false positive sample was 100% in the control example. In Comparative Example 1, 8
6.4%, and Comparative Example 2 was 85.9%, which shows that the non-specific reaction could not be suppressed. Furthermore, when a positive sample is measured, some samples have absorbed the anti-TP antibody contained in the positive sample, so that the absorbance is reduced as compared with the control example.
That is, the peptides of Comparative Example 1 and Comparative Example 2 have no activity of suppressing the non-specific reaction, and moreover, the antigenicity as the TP47K Dalton antigen protein remains, which causes a decrease in sensitivity and allows accurate measurement. I know there isn't.

When the peptides of Comparative Example 3 and Comparative Example 4 were used, the reactivity with the sample in the case of measuring the false positive sample was that of Comparative Example 3 when the control example was 100%.
35.6% in Comparative Example 4 and 14.5% in Comparative Example 4, and non-specific reactions can be suppressed, but when a positive sample is measured, it is contained in the positive sample depending on the sample. Since the anti-TP antibody has been absorbed, the absorbance is reduced as compared with the control example. That is, it can be seen that the peptides of Comparative Example 3 and Comparative Example 4 still retain the antigenicity as the TP47K Dalton antigenic protein, which causes a decrease in sensitivity and an inaccurate measurement cannot be performed.

On the other hand, when the peptide of Example 1 was used, the reactivity with the sample when the false positive sample was measured was 20.4% when the control example was 100%. It can be seen that the non-specific reaction can be suppressed. Furthermore, even when a positive sample is measured, the reactivity with the positive sample is maintained, although there are variations depending on the sample, and the peptide of the present invention has almost no antigenicity as a TP47K Dalton antigen protein. I understand.

Therefore, by using the peptide of the present invention, the non-specific reaction can be suppressed and the sensitivity can be kept high without absorbing the anti-TP antibody contained in the sample. Therefore, it was confirmed that the anti-TP antibody in the sample can be accurately measured.

From the results of the present invention, Comparative Example 1 and Comparative Example 2, the amino acid sequences of the peptides of the present invention were not included in the amino acid sequences of the TP47K Dalton antigen protein fragments of Comparative Example 1 and Comparative Example 165. It is considered that the sequence corresponding to the ~ 244th position is involved in suppression of non-specific reaction.

[Example 3] Using the measuring method described in JP-A-9-229936 and JP-A-10-132819, the peptide of the present invention in the measurement of an antibody (anti-TP antibody) against Treponema pallidum was used. The effect of suppressing non-specific reaction was confirmed.

(1) Preparation of peptide In the same manner as in Example 1, the peptide of the present invention (Example 1)
Was prepared. (2) Preparation of anti-TP antibody measuring instrument Acrylic resin extruded plate 1 (width 10 mm, depth 38 mm,
1mm from the front edge of [Acry Sunday]
2 μg / mL of TP47K Dalton antigenic protein prepared by gene recombination in the same manner as in (1) of Example 2 in the portion 5 mm to 18 mm (position 3 in FIG. 1).
Was loaded with a pipette and allowed to stand at 37 ° C. for 3 hours to immobilize the antigen.

Then, after the solution is removed by suction with a pipette,
Phosphate buffer containing 0.5% (w / v) casein (pH
7.5, 50 mM) (Hereinafter, this is referred to as diluent A)
Add 0.3 mL to this, leave it at 4 ° C overnight, and mask by pipetting it off.
The determination unit 3 is used.

Next, as shown in FIG. 1A, a cover 2 (width 12 m) made of a transparent plate having two frames 4 (depth 16 mm, height 0.5 mm, interval between frames 5 mm) formed in the center.
m, depth 40 mm, thickness 2.5 mm) and the sample absorption part 8 (Berclin E2, manufactured by Kanebo Co., Ltd., having a thickness of 2 mm, cut into a width of 4 mm and a depth of 5 mm) of the acrylic resin extruded plate 1 described above. By combining the above as shown in FIG. 2A, the height is 0.5 mm and the depth is 16 m surrounded by the acrylic resin extruded plate 1 and the cover 2 shown in FIG. 2A.
A passage 6 having a space of m and a width of 5 mm was provided. And
The opening 5 provided in the cover 2 (width 5 mm, depth 2 m
m, thickness 2.5 mm) becomes the sample supply unit 5. 1B is a cross-sectional view of the cover 2, and FIG. 1C is a view of the cover 2 viewed from the back side.

Further, as shown in FIG. 1A, a transparent obstacle having a hole of the same size as that of the sample supply section at the center and being formed so that one side surface of the hole spreads upwards. 7 (width 7 mm, depth 8 mm, height 1 mm) was stuck on the sample supply part 5 with an acrylic adhesive. This was used as an anti-TP antibody measuring instrument. 2B is a vertical sectional view of the anti-TP antibody measuring instrument.

(3) Preparation of TP47K Dalton Antigen Protein Immobilized Particles (Magnetic Particles) [Dynabeads M-450 u
ncoated, manufactured by Dynal Co., particle size: 4.5 μm, particle size C.I. V. 5% or less, specific gravity 1.5, concentration 6% (w /
v)] and T prepared in the same manner as in Example 1 (1)
P47K Dalton Antigen protein (pH 7.0, 10 m
1 mL dissolved in M phosphate buffer at a concentration of 100 μg / mL) was mixed and reacted at 37 ° C. for 1 hour. Phosphate buffer containing 0.5% (w / v) casein (p
(H7.5, 50 mM) was added in an amount of about 20 times to perform masking, and the obtained particles were washed with the diluent A. The particles were redispersed in glycine buffer (pH 9.5, 50 mM) so that the concentration became about 0.2% (w / v). In this way, a TP47K Dalton antigen protein-immobilized particle dispersion liquid was prepared.

(4) Nonspecific Reaction Suppressing Effect of Peptide in Anti-TP Antibody Measurement The TP47K Dalton antigen protein-immobilized particle dispersion prepared in (3) above was prepared with the peptide prepared in (1) above at a final concentration of 0.2. % (W / v) was added. Moreover, the thing which does not add a peptide was made into the control example. False positive serum (3 samples) or anti-TP antibody positive serum (1 sample) 5
0 μL each was placed on each sample supply unit 5 of the anti-TP antibody measuring instrument prepared in (2) above with a pipette to bring it into contact with the determination unit 3.

As a result, the sample is placed in the passage 6 of the measuring instrument.
After moving inside and coming into contact with the determination unit 3, the sample is absorbed by the sample absorption unit 8. After the false positive sample or the positive sample brought into contact with the determination unit 3 is absorbed and removed by the sample absorption unit 8, the TP47K Dalton antigen protein-immobilized particle dispersion liquid prepared in (3) above is used as the anti-TP antibody measuring instrument. The sample supplying section 5 was placed on each of the sample supplying sections 5 using a pipette so as to be brought into contact with the judging section 3.

After that, by arranging a magnet in the side surface direction in which the determination portion 3 of this anti-TP antibody measuring instrument is formed,
A magnetic field having a magnetic flux density of 40 to 60 gauss was generated in the direction from the sample supply unit 5 to the sample absorption unit 8 so that the TP47K Dalton antigen protein-immobilized particles moved.

Table 2 shows the result of determination 1 minute after the magnetic field was generated. In addition, + indicates a case where an image of particles is observed in the judging section 3, + W indicates a case where a weak image of particles is recognized in the judging section 3, and − indicates a case where an image of particles is not detected in the judging section 3. ing.

[0074]

[Table 2]

(5) Consideration As shown in Table 2, in the control example, the determination result of the false positive sample 1 is + (a particle image was observed in the determination portion 3),
The determination results of the false positive samples 2 and 3 showed + W (a weak image of particles was observed in the determination part 3), and it was confirmed that the nonspecific reaction could not be suppressed.

On the other hand, when the peptide of the present invention was used, all the determination results of the false positive samples 1 to 3 showed- (no image of particles was observed in the determination part 3), It was confirmed that the non-specific reaction could be suppressed. Furthermore,
The judgment result when measuring a positive sample also shows +,
It can be seen that the non-specific reaction suppressing peptide of the present invention does not retain the antigenicity as the TP47K Dalton antigen protein.

Therefore, by using the peptide of the present invention, the non-specific reaction can be suppressed, and the sensitivity can be kept high without absorbing the anti-TP antibody contained in the sample. Therefore, it was confirmed that the anti-TP antibody in the sample can be accurately measured.

Example 4 The inhibitory effect of the peptide of the present invention on the non-specific reaction in the measurement of an antibody (anti-TP antibody) against Treponema pallidum was confirmed using the latex agglutination method. (1) Preparation of first reagent The following first reagent was prepared as a diluent for the sample. 4%
50 containing (w / v) BSA, 0.2% (w / v) polyethylene glycol and 15 mM sodium azide
The peptide of the present invention prepared in Example 1 was added to mM tris (hydroxymethyl) aminomethane-hydrochloric acid buffer (pH 8.5) at a final concentration of 0.008% (w / v) or 0.0, respectively.
It was added so as to be 8% (w / v). Moreover, the thing which does not add a peptide was made into the control example.

(2) Preparation of the second reagent The 47K Dalton antigen protein of Treponema pallidum was prepared by Weigel, M .; The method of Nogard et al. (Infection and Immunity
60, 1568-1576, 1992),
It was prepared by gene recombination technology. 47 of Treponema pallidum prepared above
The final concentration of K Dalton antigen protein is 50 μg / m
50 mM glycine buffer (pH 9.
0) and dissolved.

Latex particles [Sekisui Chemical Co., Ltd., average particle size 0.4 μm, polystyrene] were added to 0.5% (w /
50 mM glycine buffer (pH 9.
0) and suspended. 0.2 mL of the above-mentioned solution of Treponema pallidum 47K Dalton antigen protein was mixed with 0.2 mL of the above-mentioned suspension of latex particles. Then 3
The mixture was allowed to stand at 7 ° C. for 3 hours to immobilize the 47 K dalton antigen protein of Treponema pallidum to the latex particles.

4 of Treponema Paris Dam
50m containing 1% (w / v) BSA in a suspension of latex particles immobilized with 7K Dalton antigen protein
1 mL of M tris (hydroxymethyl) aminomethane-hydrochloric acid buffer solution (pH 7.2) was added, and the mixture was allowed to stand at 37 ° C. for 2 hours to perform blocking treatment (masking treatment) on the surface of the latex particles as a carrier. .

After the blocking treatment (masking treatment), centrifugation was performed at a rotation speed of 14,000 rpm at 10 ° C. for 20 minutes. After centrifugation, the supernatant was removed by decantation to obtain precipitate latex particles (latex particles having 47K Dalton antigen protein of Treponema pallidum immobilized). 300 mM sodium chloride containing 0.25% (w / v) latex particles prepared above, 1% (w / v)
v) 5 containing BSA and 15 mM sodium azide
A 0 mM tris (hydroxymethyl) aminomethane-hydrochloric acid buffer solution (pH 7.2) was prepared and used as a second reagent.

(3) Suppressive Effect of Peptide on Non-Specific Reaction in Anti-TP Antibody Measurement 87.5 μL of the above-mentioned first reagent was added to the well of a 96-well microtiter plate (Nunc), and then false positive 10 μL each of serum (2 samples) or anti-TP antibody positive serum (1 sample) was added and mixed. Further to this well, 12.5 μL of the second reagent described above
Immediately after adding and mixing, the absorbance of the mixed solution in this well (main wavelength: 490 nm, sub wavelength: 655 nm)
Was measured with a microplate reader (3350 type, manufactured by Bio-Rad).

Immediately after the measurement of the absorbance, the microtiter plate was allowed to stand at 37 ° C. Ten minutes after the measurement of the absorbance in the above, the absorbance of the reaction mixture in the well was measured in the same manner as in the above case. The absorbance measured above was subtracted from the absorbance measured above to obtain the difference in absorbance. This difference in absorbance is proportional to the amount (concentration) of the anti-TP antibody contained in the sample.

(4) Measurement results Table 3 shows the absorbance differences obtained by measuring the anti-TP antibody in the sample in (3) above.

[0086]

[Table 3]

(5) Discussion As shown in Table 3, in the control example, the absorbance difference was 0.0021 in the false positive sample 1 and 0.0069 in the false positive sample 2.
It was confirmed that the non-specific reaction could not be suppressed.

On the other hand, in the case of using the peptide of the present invention, in the false positive sample 1, when the concentration of the peptide of the present invention was 0.008%, the absorbance difference was 0.0001 and the concentration was 0.1. In the case of 08%, the difference in absorbance was −0.0001, which confirmed that the nonspecific reaction could be suppressed. Further, in the false-positive sample 2, the absorbance difference was 0.0044 when the concentration of the peptide of the present invention was 0.008%, but the absorbance difference was 0.004 when the concentration was increased to 0.08%. 0006 is shown, and it is confirmed that the non-specific reaction can be suppressed. Furthermore, when a positive sample was measured, the absorbance difference was 0.0032 when the concentration of the peptide of the present invention was 0.008%, and 0.0034 when the concentration was 0.08%. . This is the difference in absorbance (0.003) in the control example which did not contain the peptide of the present invention.
It is almost the same value as 0), and it can be seen that the nonspecific reaction inhibitor of the present invention has no antigenicity as a TP47K Dalton antigen protein.

Therefore, by using the non-specific reaction inhibitor of the present invention, the non-specific reaction can be suppressed and the sensitivity is high without absorbing the anti-TP antibody contained in the sample. Anti-T in the sample because it can be kept
It was confirmed that the P antibody can be accurately measured.

[0090]

INDUSTRIAL APPLICABILITY According to the present invention, a non-specific reaction can be suppressed in the measurement of anti-TP antibody, and further, the anti-TP antibody is not absorbed in the measurement to inhibit the immune reaction. Accurate measurement can be performed in the field.

[0091]

[Sequence list]                                SEQUENCE LISTING <110> Shino-Test Corporation <120> A peptide inhibiting nonspecific reaction, a method for inhibiting  non specific reaction and an assay for measuring antibodies using the p eptide <130> P02-04-181 <160> 36 <170> PatentIn Ver. 2.1 <210> 1 <211> 170 <212> PRT <213> Treponema pallidum <400> 1 Glu Gly Ala Val Ala Leu Ala Asp Arg Ala Ser Ser Phe Met Val Asp   1 5 10 15 Ser Glu Glu Tyr Lys Ile Thr Asn Val Lys Val His Gly Met Lys Phe              20 25 30 Val Pro Val Ala Val Pro His Glu Leu Lys Gly Ile Ala Lys Glu Lys          35 40 45 Phe His Phe Val Glu Asp Ser Arg Val Thr Glu Asn Thr Asn Gly Leu      50 55 60 Lys Thr Met Leu Thr Glu Asp Ser Phe Ser Ala Arg Lys Val Ser Ser  65 70 75 80 Met Glu Ser Pro His Asp Leu Val Val Asp Thr Val Gly Thr Gly Tyr                  85 90 95 His Ser Arg Phe Gly Ser Asp Ala Glu Ala Ser Val Met Leu Lys Arg             100 105 110 Ala Asp Gly Ser Glu Leu Ser His Arg Glu Phe Ile Asp Tyr Val Met         115 120 125 Asn Phe Asn Thr Val Arg Tyr Asp Tyr Tyr Gly Asp Asp Ala Ser Tyr     130 135 140 Thr Asn Leu Met Ala Ser Tyr Gly Thr Lys His Ser Ala Asp Ser Trp 145 150 155 160 Trp Lys Thr Gly Arg Val Pro Arg Ile Ser                 165 170 <210> 2 <211> 80 <212> PRT <213> Treponema pallidum <400> 2 Glu Leu Lys Gly Ile Ala Lys Glu Lys Phe His Phe Val Glu Asp Ser   1 5 10 15 Arg Val Thr Glu Asn Thr Asn Gly Leu Lys Thr Met Leu Thr Glu Asp              20 25 30 Ser Phe Ser Ala Arg Lys Val Ser Ser Met Glu Ser Pro His Asp Leu          35 40 45 Val Val Asp Thr Val Gly Thr Gly Tyr His Ser Arg Phe Gly Ser Asp      50 55 60 Ala Glu Ala Ser Val Met Leu Lys Arg Ala Asp Gly Ser Glu Leu Ser  65 70 75 80 <210> 3 <211> 415 <212> PRT <213> Treponema pallidum <400> 3 Cys Gly Ser Ser His His Glu Thr His Tyr Gly Tyr Ala Thr Leu Ser   1 5 10 15 Tyr Ala Asp Tyr Trp Ala Gly Glu Leu Gly Gln Ser Arg Asp Val Leu              20 25 30 Leu Ala Gly Asn Ala Glu Ala Asp Arg Ala Gly Asp Leu Asp Ala Gly          35 40 45 Met Phe Asp Ala Val Ser Arg Ala Thr His Gly His Gly Ala Phe Arg      50 55 60 Gln Gln Phe Gln Tyr Ala Val Glu Val Leu Gly Glu Lys Val Leu Ser  65 70 75 80 Lys Gln Glu Thr Glu Asp Ser Arg Gly Arg Lys Lys Trp Glu Tyr Glu                  85 90 95 Thr Asp Pro Ser Val Thr Lys Met Val Arg Ala Ser Ala Ser Phe Gln             100 105 110 Asp Leu Gly Glu Asp Gly Glu Ile Lys Phe Glu Ala Val Glu Gly Ala         115 120 125 Val Ala Leu Ala Asp Arg Ala Ser Ser Phe Met Val Asp Ser Glu Glu     130 135 140 Tyr Lys Ile Thr Asn Val Lys Val His Gly Met Lys Phe Val Pro Val 145 150 155 160 Ala Val Pro His Glu Leu Lys Gly Ile Ala Lys Glu Lys Phe His Phe                 165 170 175 Val Glu Asp Ser Arg Val Thr Glu Asn Thr Asn Gly Leu Lys Thr Met             180 185 190 Leu Thr Glu Asp Ser Phe Ser Ala Arg Lys Val Ser Ser Met Glu Ser         195 200 205 Pro His Asp Leu Val Val Asp Thr Val Gly Thr Gly Tyr His Ser Arg     210 215 220 Phe Gly Ser Asp Ala Glu Ala Ser Val Met Leu Lys Arg Ala Asp Gly 225 230 235 240 Ser Glu Leu Ser His Arg Glu Phe Ile Asp Tyr Val Met Asn Phe Asn                 245 250 255 Thr Val Arg Tyr Asp Tyr Tyr Gly Asp Asp Ala Ser Tyr Thr Asn Leu             260 265 270 Met Ala Ser Tyr Gly Thr Lys His Ser Ala Asp Ser Trp Trp Lys Thr         275 280 285 Gly Arg Val Pro Arg Ile Ser Cys Gly Ile Asn Tyr Gly Phe Asp Arg     290 295 300 Phe Lys Gly Ser Gly Pro Gly Tyr Tyr Arg Leu Thr Leu Ile Ala Asn 305 310 315 320 Gly Tyr Arg Asp Val Val Ala Asp Val Arg Phe Leu Pro Lys Tyr Glu                 325 330 335 Gly Asn Ile Asp Ile Gly Leu Lys Gly Lys Val Leu Thr Ile Gly Gly             340 345 350 Ala Asp Ala Glu Thr Leu Met Asp Ala Ala Val Asp Val Phe Ala Asp         355 360 365 Gly Gln Pro Lys Leu Val Ser Asp Gln Ala Val Ser Leu Gly Gln Asn     370 375 380 Val Leu Ser Ala Asp Phe Thr Pro Gly Thr Glu Tyr Thr Val Glu Val 385 390 395 400 Arg Phe Lys Glu Phe Gly Ser Val Arg Ala Lys Val Val Ala Gln                 405 410 415 <210> 4 <211> 10 <212> PRT <213> Treponema pallidum <400> 4 Glu Gly Ala Val Ala Leu Ala Asp Arg Ala   1 5 10 <210> 5 <211> 10 <212> PRT <213> Treponema pallidum <400> 5 Leu Ala Asp Arg Ala Ser Ser Phe Met Val   1 5 10 <210> 6 <211> 10 <212> PRT <213> Treponema pallidum <400> 6 Ser Ser Phe Met Val Asp Ser Glu Glu Tyr   1 5 10 <210> 7 <211> 10 <212> PRT <213> Treponema pallidum <400> 7 Asp Ser Glu Glu Tyr Lys Ile Thr Asn Val   1 5 10 <210> 8 <211> 10 <212> PRT <213> Treponema pallidum <400> 8 Lys Ile Thr Asn Val Lys Val His Gly Met   1 5 10 <210> 9 <211> 10 <212> PRT <213> Treponema pallidum <400> 9 Lys Val His Gly Met Lys Phe Val Pro Val   1 5 10 <210> 10 <211> 10 <212> PRT <213> Treponema pallidum <400> 10 Lys Phe Val Pro Val Ala Val Pro His Glu   1 5 10 <210> 11 <211> 10 <212> PRT <213> Treponema pallidum <400> 11 Ala Val Pro His Glu Leu Lys Gly Ile Ala   1 5 10 <210> 12 <211> 10 <212> PRT <213> Treponema pallidum <400> 12 Leu Lys Gly Ile Ala Lys Glu Lys Phe His   1 5 10 <210> 13 <211> 10 <212> PRT <213> Treponema pallidum <400> 13 Lys Glu Lys Phe His Phe Val Glu Asp Ser   1 5 10 <210> 14 <211> 10 <212> PRT <213> Treponema pallidum <400> 14 Phe Val Glu Asp Ser Arg Val Thr Glu Asn   1 5 10 <210> 15 <211> 10 <212> PRT <213> Treponema pallidum <400> 15 Arg Val Thr Glu Asn Thr Asn Gly Leu Lys   1 5 10 <210> 16 <211> 10 <212> PRT <213> Treponema pallidum <400> 16 Thr Asn Gly Leu Lys Thr Met Leu Thr Glu   1 5 10 <210> 17 <211> 10 <212> PRT <213> Treponema pallidum <400> 17 Thr Met Leu Thr Glu Asp Ser Phe Ser Ala   1 5 10 <210> 18 <211> 10 <212> PRT <213> Treponema pallidum <400> 18 Asp Ser Phe Ser Ala Arg Lys Val Ser Ser   1 5 10 <210> 19 <211> 10 <212> PRT <213> Treponema pallidum <400> 19 Arg Lys Val Ser Ser Met Glu Ser Pro His   1 5 10 <210> 20 <211> 10 <212> PRT <213> Treponema pallidum <400> 20 Met Glu Ser Pro His Asp Leu Val Val Asp   1 5 10 <210> 21 <211> 10 <212> PRT <213> Treponema pallidum <400> 21 Asp Leu Val Val Asp Thr Val Gly Thr Gly   1 5 10 <210> 22 <211> 10 <212> PRT <213> Treponema pallidum <400> 22 Thr Val Gly Thr Gly Tyr His Ser Arg Phe   1 5 10 <210> 23 <211> 10 <212> PRT <213> Treponema pallidum <400> 23 Tyr His Ser Arg Phe Gly Ser Asp Ala Glu   1 5 10 <210> 24 <211> 10 <212> PRT <213> Treponema pallidum <400> 24 Gly Ser Asp Ala Glu Ala Ser Val Met Leu   1 5 10 <210> 25 <211> 10 <212> PRT <213> Treponema pallidum <400> 25 Ala Ser Val Met Leu Lys Arg Ala Asp Gly   1 5 10 <210> 26 <211> 10 <212> PRT <213> Treponema pallidum <400> 26 Lys Arg Ala Asp Gly Ser Glu Leu Ser His   1 5 10 <210> 27 <211> 10 <212> PRT <213> Treponema pallidum <400> 27 Ser Glu Leu Ser His Arg Glu Phe Ile Asp   1 5 10 <210> 28 <211> 10 <212> PRT <213> Treponema pallidum <400> 28 Arg Glu Phe Ile Asp Tyr Val Met Asn Phe   1 5 10 <210> 29 <211> 10 <212> PRT <213> Treponema pallidum <400> 29 Tyr Val Met Asn Phe Asn Thr Val Arg Tyr   1 5 10 <210> 30 <211> 10 <212> PRT <213> Treponema pallidum <400> 30 Asn Thr Val Arg Tyr Asp Tyr Tyr Gly Asp   1 5 10 <210> 31 <211> 10 <212> PRT <213> Treponema pallidum <400> 31 Asp Tyr Tyr Gly Asp Asp Ala Ser Tyr Thr   1 5 10 <210> 32 <211> 10 <212> PRT <213> Treponema pallidum <400> 32 Asp Ala Ser Tyr Thr Asn Leu Met Ala Ser   1 5 10 <210> 33 <211> 10 <212> PRT <213> Treponema pallidum <400> 33 Asn Leu Met Ala Ser Tyr Gly Thr Lys His   1 5 10 <210> 34 <211> 10 <212> PRT <213> Treponema pallidum <400> 34 Tyr Gly Thr Lys His Ser Ala Asp Ser Trp   1 5 10 <210> 35 <211> 10 <212> PRT <213> Treponema pallidum <400> 35 Ser Ala Asp Ser Trp Trp Lys Thr Gly Arg   1 5 10 <210> 36 <211> 10 <212> PRT <213> Treponema pallidum <400> 36 Trp Lys Thr Gly Arg Val Pro Arg Ile Ser   1 5 10

[Brief description of drawings]

FIG. 1A is an exploded perspective view of an anti-TP antibody measuring instrument of the present invention. B is a cross-sectional view of the cover 2, and C is a view of the cover 2 viewed from the back side.

FIG. 2 shows a perspective view and a longitudinal sectional view of an anti-TP antibody measuring instrument of the present invention.

FIG. 3 is a graph showing the results of Table 1 as% reactivity with respect to the reactivity of a control example.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Yuki Nakamura             2-29-14 Onodai, Sagamihara City, Kanagawa Prefecture             Shinotest Co., Ltd. Sagamihara Office F-term (reference) 4H045 AA10 BA10 BA15 CA11 EA52                       FA74

Claims (14)

[Claims] 1. A non-specific reaction in an immunoassay method for an anti-Treponemal paridum antibody, which comprises a peptide containing the amino acid sequence of any one of SEQ ID NOs: 4 to 36, wherein the antigen comprises Treponema paridum 47K Dalton antigen protein. A peptide having the activity of suppressing 2. A peptide comprising all or a part of the amino acid sequence set forth in SEQ ID NO: 1 and containing at least 5 consecutive amino acid sequences in SEQ ID NO: 1, or a peptide containing the peptide, and a Treponema pallidum 47K Dalton antigen protein. A peptide having an activity of suppressing non-specific reaction in an immunoassay method for an anti-Treponemal paridum antibody using an antigen containing the peptide. 3. A peptide comprising all or part of the amino acid sequence set forth in SEQ ID NO: 2 and containing at least 5 consecutive amino acid sequences in SEQ ID NO: 2, or a peptide containing the peptide, which comprises the Treponema pallidum 47K Dalton antigen protein. A peptide having an activity of suppressing non-specific reaction in an immunoassay method for an anti-Treponemal paridum antibody using an antigen containing the peptide. 4. A peptide comprising an amino acid sequence having the deletion, addition, substitution or modification of one or several amino acid residues in the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence consisting of a part thereof. , A peptide having an activity of suppressing non-specific reaction in an immunoassay method for an anti-Treponemal paridum antibody using an antigen containing Treponemal paridum 47K Dalton antigen protein. 5. A peptide comprising an amino acid sequence of SEQ ID NO: 2 or an amino acid sequence consisting of a part thereof, the amino acid sequence having one or several amino acid residues deleted, added, substituted or modified. , A peptide having an activity of suppressing non-specific reaction in an immunoassay method for an anti-Treponemal paridum antibody using an antigen containing Treponemal paridum 47K Dalton antigen protein. 6. The peptide according to any one of claims 1 to 5, which does not exhibit the antigenicity of Treponema pallidum 47K Dalton antigen protein. 7. The method according to claims 1 to 6 for use in an immunoassay method for an anti-Treponemal pallidum antibody using the Treponema pallidum 47K Dalton antigen protein.
A non-specific reaction inhibitor comprising the peptide according to any one of 1. 8. A method according to claims 1 to 6 for use in an immunoassay method for anti-Treponemal paris dame antibodies using the Treponema pallidum 47K Dalton antigen protein.
An anti-Treponemal paridum antibody measuring reagent containing the peptide according to any one of 1. 9. The reagent according to claim 8, further comprising Treponema pallidum 47K Dalton antigen protein. 10. The reagent according to claim 9, wherein the Treponema pallidum 47K Dalton antigen protein is produced by using a gene recombination technique. 11. An immunoassay method for an anti-Treponemal paris dam antibody, which uses the Treponema paris dam 47K Dalton antigen protein, according to any one of claims 1 to 6.
A method for suppressing the nonspecific reaction by contacting the peptide according to the item with a sample. 12. An immunoassay method for anti-Treponemal paris dame antibody using Treponema paris dame 47K Dalton antigen protein, which comprises any one of claims 1 to 6.
An immunoassay method comprising contacting the peptide according to the item with a sample. 13. The immunoassay method according to claim 12, wherein the Treponema pallidum 47K Dalton antigen protein is produced by gene recombination technology. 14. An anti-treponemal which comprises the carrier according to any one of claims 1 to 6 and a carrier on which the Treponema pallidum 47K Dalton antigen protein is immobilized.
Immunoassay kit for Palidam antibody measurement.