JP2000060551A - Anti-prion antibody - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an antibody that has specific and stable reactivity with human prion and is useful as a reagent for studying the diseases relating to prion and as a diagnostic agent therefor by specifically linking to a peptide having a specific amino acid sequence. SOLUTION: The objective antibody specifically bonds to the peptide having the amino acid sequence given in the formula. The antibody originates from mouse and is a monoclonal antibody and belongs to the IgG1 in the immunoglobulin subclass. The antibody is collected from the body fluid of the animals immunized with the peptide as an antigen and/or from the cells producing the antibody. In addition, it is preferred that the antibody is combined with an anti-IgG antibody labeled with a labeling substance for example, biotin, fluorescein isothiocyanate or the like to prepare a kit.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an antibody which specifically binds to a peptide consisting of a specific amino acid sequence present in a prion.

The present invention is also characterized in that it is collected from a body fluid and / or antibody-producing cells of an animal immunized with a peptide consisting of a specific amino acid sequence present in prion as an antigen and specifically binds to prion. The present invention relates to antibodies.

[0003]

2. Description of the Related Art A technique closest to the present invention will be described below.

[0007] WO97 / 10505 discloses the entire amino acid sequence of human prion (SEQ ID NO: 2). In addition, the monoclonal antibody "3F against prion
There is a description suggesting that "4" binds to a partial amino acid sequence of human prion (amino acid numbers 109 to 112 in SEQ ID NO: 2).

Japanese Patent Publication No. 7-501798 discloses an antibody which specifically binds to a partial amino acid sequence of human prion (amino acid numbers 86 to 114 in SEQ ID NO: 2).

However, there is no description or suggestion of an antibody that specifically binds to the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1. Further, it is also suggested that the antibody is characterized by being collected from a body fluid and / or antibody-producing cells of an animal immunized with the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 as an antigen and specifically binding to prion. Nor.

[0007]

[Problems to be Solved by the Invention] If an antibody that specifically binds to a peptide consisting of an amino acid sequence that is characteristic of prion and has high antibody production efficiency can be obtained, reagents and diagnostics for research of diseases associated with prion can be obtained. It can be used as a drug and the production cost of the antibody can be significantly reduced.

[0008]

Means for Solving the Problems The present inventor has conducted extensive studies in order to solve the above problems. First, a known human prion sequence was diligently analyzed to identify a site at which antibody production could be induced most efficiently, and an antibody was prepared using this site as an antigen. The inventors have found that the antibody specifically binds to the specific site and further specifically binds to human prion, and completed the present invention.

That is, the present invention provides an antibody which specifically binds to a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 (hereinafter, also referred to as antibody 1 of the present invention).

Further, the present invention is characterized in that it is collected from a body fluid and / or antibody-producing cells of an animal immunized with a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 as an antigen, and specifically binds to prion. Antibody (hereinafter,
The present invention antibody 2) is also provided. (Hereinafter, the antibodies of the present invention 1 and 2 are also referred to simply as the antibodies of the present invention)

The present invention also provides the antibody of the present invention labeled with a labeling substance (hereinafter also referred to as the labeled antibody of the present invention).

The present invention further comprises the antibody 1 or 2 of the present invention,
Provided is a kit containing an anti-IgG antibody labeled with a labeling substance (hereinafter, also referred to as the kit of the present invention).

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described below.

<1> Antibody of the Present Invention Antibody 1 of the present invention is an antibody that specifically binds to a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1.

The antibody 2 of the present invention is collected from the body fluid and / or antibody-producing cells of an animal immunized with the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 as an antigen, and specifically binds to prion. It is a characteristic antibody.

The "body fluid" in the antibody 2 of the present invention is
It is not particularly limited as long as it is a body fluid in which an antibody is present, but blood, serum, plasma, milk, etc. are exemplified, and among these, serum is preferable.

The "antibody producing cell" in the antibody 2 of the present invention is not particularly limited as long as it is an antibody producing cell. In the present specification, "antibody-producing cell"
The term includes not only antibody-producing cells directly obtained from the immunized animal but also cells obtained by modifying the antibody-producing cells directly obtained from the immunized animal into a proliferative state. Examples of cells obtained by modifying antibody-producing cells directly obtained from an immunized animal into a proliferative state include, for example, cells obtained by fusing antibody-producing cells directly obtained from an immunized animal with a myeloma cell that is a tumor cell line. Examples are (hybridomas) and cells obtained by transforming antibody-producing cells directly obtained from an immunized animal with cancer virus or the like.

Among these, hybridomas of antibody-producing cells directly obtained from the immunized animal and myeloma cells are preferable. Among them, hybridomas of antibody-producing cells directly obtained from an immunized animal and myeloma cells derived from an animal of the same species as the animal are more preferable.

The antibody-producing cells directly obtained from the immunized animal include spleen cells and lymphocytes. Further, examples of hybridomas of antibody-producing cells and myeloma cells directly obtained from the immunized animal include hybridomas of spleen cells and myeloma cells.

Of these, hybridomas of spleen cells and myeloma cells are preferable.

The amino acid sequence represented by SEQ ID NO: 1 corresponds to the amino acid sequence represented by amino acid numbers 96 to 114 in SEQ ID NO: 2 (human prion amino acid sequence), and the amino acid sequences 96 to 114 in SEQ ID NO: 2.
The amino acid sequence shown by is found as a result of keen analysis of the amino acid sequence of a known human prion by the present inventor, is an extremely preferable amino acid sequence as an antigen when producing a prion antibody, and has all of the following features. There is. (1) It exists in both normal and abnormal prions. (2) The antigenicity is hardly changed by the abnormalization of prions. (3) A sequence characteristic of humans. (4) This is a highly hydrophilic region.

This amino acid sequence is an amino acid sequence that is considered to be most efficient as an antigen for producing an antibody that specifically binds to human prion, and a peptide having such an amino acid sequence should be used as the antigen. This makes it possible to obtain an antibody which has specific and stable reactivity with human prions and is extremely easy to produce.

The method for producing the antibody of the present invention will be described below.

The antibody of the present invention can be obtained by a conventional antibody production method using a peptide comprising the amino acid sequence represented by SEQ ID NO: 1 (hereinafter also referred to as an antigen peptide) as an antigen.

Since the amino acid sequence of this antigen peptide has been disclosed by the present invention, a known chemical synthesis method of the peptide based on the sequence (eg, liquid phase synthesis method, solid phase synthesis method; Nobuo Izumiya, Tetsuo Kato) , Toshihiko Aoyagi, Michinori Waki, “Basics and Experiments of Peptide Synthesis” 1985, Maruzen Co., Ltd.).

It is also possible to produce a polynucleotide (DNA or RNA) corresponding to the amino acid sequence of the antigen peptide and produce it by a genetic engineering technique using the polynucleotide.

The produced antigenic peptides are isolated from proteins generally known in the field of protein chemistry,
It can be purified by a purification method. In particular,
For example, extraction, recrystallization, salting out with ammonium sulfate (ammonium sulfate) or sodium sulfate, centrifugation, dialysis, ultrafiltration, adsorption chromatography, ion exchange chromatography, hydrophobic chromatography, normal phase chromatography, reverse phase chromatography. Examples include processing operations such as chromatography, gel filtration, gel permeation chromatography, affinity chromatography, electrophoresis, countercurrent distribution, and combinations thereof.

The amino acid sequence of the produced antigen peptide can be determined by a known amino acid sequence determination method (for example, Edman degradation method) to confirm whether or not the antigen peptide was produced correctly.

The antibody of the present invention may be either polyclonal or monoclonal, but is preferably a monoclonal antibody.

The method for producing the polyclonal antibody of the present invention and the monoclonal antibody of the present invention will be described in detail below.

Method for Producing Polyclonal Antibody of the Present Invention: Antigen peptide is subcutaneously, intraperitoneally, or footpad of an immunized animal such as mouse, rat, guinea pig, rabbit, goat, sheep, horse, pig, dog, cat, chicken. (footpad) etc. Of these immunized animals, it is preferable to use mice. Among the mice, Balb / c mouse is preferably used.

When immunizing an animal to be immunized, it is desirable to use an auxiliary agent in combination because it activates antibody-producing cells. In addition, antiserum with high titer can be obtained by performing booster immunization by a conventional method 2-3 weeks after the first immunization. About one week after the final immunization, blood is collected and serum is separated. The serum may be heat-treated to inactivate complement, and then the immunoglobulin fraction may be purified by a usual antibody purification method.

Method for Producing Monoclonal Antibody of the Present Invention: The monoclonal antibody of the present invention is produced by Kohler and Milstein.
Method (Nature 256, 495-497 (1975)).

For example, the antigenic peptide may be mouse, rat, guinea pig, rabbit, goat, sheep, horse, pig, dog,
Intraperitoneal, subcutaneous, or footpad (f) of immunized animals such as cats and chickens
Ootpad) etc. Of these immunized animals, it is preferable to use mice. Also in the mouse
It is preferable to use Balb / c mice.

Spleen cells, lymphocytes, peripheral blood, etc. are collected from the immunized animal, and these are fused with myeloma cells, which are tumor cell lines, to prepare hybridomas.
As the myeloma cells used for cell fusion, cell lines of various mammals can be used, but it is preferable to use cell lines of animals of the same species as the immunized animal. In addition, myeloma cells can be distinguished from unfused cells and fused cells after cell fusion, so that unfused myeloma cells cannot survive and only hybridomas can grow.
It is preferable to use one having a marker. For myeloma cells, it is preferable to use a strain that does not secrete a proper immunoglobulin, since it becomes easy to obtain the desired antibody from the culture supernatant of the hybridoma.

The obtained hybridomas are continuously grown,
A hybridoma strain that continuously produces an antibody that specifically binds to the antigenic peptide is selected.

By culturing the hybridoma strain thus selected in a suitable medium, the monoclonal antibody of the present invention can be obtained in the medium. In addition, a large amount of the monoclonal antibody of the present invention can be produced by culturing the hybridoma strain in a living body such as the abdominal cavity of a mouse and isolating it from ascites. The thus obtained monoclonal antibody of the present invention may be purified by a conventional antibody purification method.

As a method for purifying the antibody, sodium sulfate,
Ion exchangers such as salting out with ammonium sulfate, low temperature alcohol precipitation and selective precipitation fractionation with polyethylene glycol or isoelectric point, electrophoresis, DEAE (diethylaminoethyl) -derivative, CM (carboxymethyl) -derivative were used. Examples include ion exchange chromatography, affinity chromatography using protein A or protein G, hydroxyapatite chromatography, immunoadsorption chromatography with immobilized antigen, gel filtration method and ultracentrifugation method.

The antibody of the present invention was used as an antigen binding site (Fa
b) may be treated with a protease that does not decompose (for example, plasmin, pepsin, papain, etc.) to give a fragment containing Fab.

If the nucleotide sequence of the gene encoding the antibody of the present invention or the amino acid sequence of the antibody of the present invention is determined, a fragment containing a Fab of the antibody of the present invention or a chimeric antibody (for example, the Fab portion of the antibody of the present invention) is genetically engineered. A chimeric antibody or the like) can also be prepared. Such Fab-containing fragments of the antibody of the present invention and chimeric antibodies are also included in the antibody of the present invention as long as they specifically bind to the antigenic peptide or prion.

Examples of Fab-containing fragments of the antibody include Fabc, (Fab ′) _{2 and the} like, in addition to Fab.

The antibody of the present invention thus obtained preferably has an immunoglobulin class of IgG1.
Antibodies whose immunoglobulin class is IgG1 are anti-Ig
It can be obtained by screening using the G1 antibody.

The antibody of the present invention may contain other components as long as the action of the antibody is not substantially impaired. For example, an excipient, a buffering agent, a stabilizer, a preservative and the like used in the preparation of ordinary reagents can be included. Specifically, phosphate buffered saline (PBS), sodium azide (Na
N ₃ ), bovine serum albumin and the like are exemplified. Formulation examples of these components will be described in Examples below.

<2> Labeled Antibody of the Present Invention The labeled antibody of the present invention is a labeled antibody of the present invention. The labeling substance that can be used for labeling is not particularly limited as long as it can be used for labeling ordinary proteins, and examples thereof include enzymes (peroxidase, alkaline phosphatase, β-galactosidase, luciferase, acetylcholinesterase, etc.), radioactive isotopes. ( ¹²⁵ I,
¹³¹ I, ³ H, etc., fluorescent dyes (fluorescein isothiocyanate (FITC), 7-amino-4-methylcoumarin-3-acetic acid
(AMCA), dichlorotriazinylaminofluorescein (D
TAF), tetramethylrhodamine isothiocyanate (TRI
TC), Lissamine Rhodamine
B), Texas Red, Phycoerythrin (Ph
ycoerythrin (PE), umbelliferone, etc.), chemiluminescent substances (luminol, etc.), haptens, biotin, avidin (eg, streptavidin, etc.) and the like.

Among these labeling substances, biotin and fluorescein isothiocyanate (hereinafter referred to as FIT
C)) or peroxidase is preferable, and biotin or FITC is more preferable.

The method of labeling the antibody of the present invention with a labeling substance is as follows:
Known methods suitable for labeling substances, for example, glutaraldehyde method when labeling with enzyme, periodate crosslinking method, maleimide crosslinking method, carbodiimide method, activated ester method, etc., and chloramine T method when labeling with radioactive isotope ,
Lactoperoxidase method, etc.
"Protein Chemistry (2)", Tokyo Kagaku Dojin (1987)
Year))).

Such a labeled antibody of the present invention can be used in an immunoassay for detecting or measuring human prions. More specifically, examples of the immunoassay include enzyme immunoassay, radioimmunoassay (radioimmunoassay), fluorescent immunoassay, luminescent immunoassay, and the like. Among these immunoassays, the labeled antibody of the present invention is used. It can be appropriately selected depending on the labeling substance bound to.

Further, the labeling substance in the labeled antibody of the present invention can be appropriately selected according to known antigen detection means (for example, Western blotting, dot blotting, immunohistological staining, flow cytometry, etc.).

As a method for detecting the labeled antibody of the present invention in such an immunoassay or an antigen detection means, a known detection means can be appropriately selected according to the labeling substance used for labeling the labeled antibody of the present invention. For example, when biotin is used as the labeling substance, an enzyme to which streptavidin is bound (such as peroxidase) is added to bind the streptavidin moiety to biotin, and hydrogen peroxide (enzyme, for example) is used as a substrate for the enzyme. Is a peroxidase) and a color-forming substance such as tetramethylbenzidine, diaminobenzidine, etc., and the degree of color development of the product of the enzymatic reaction is measured by absorbance. Further, for example, when a fluorescent substance or a chemiluminescent substance is used as the labeling substance, a method of measuring fluorescence or luminescence of the solution after the reaction is exemplified.

The labeled antibody of the present invention may contain other components as long as the action of the antibody is not substantially impaired. For example, an excipient, a buffering agent, a stabilizer, a preservative and the like used in the preparation of ordinary reagents can be included. Specifically, phosphate buffered saline (PBS), sodium azide (Na
N ₃ ), bovine serum albumin and the like are exemplified. Formulation examples of these components will be described in Examples below.

<3> Kit of the Present Invention The kit of the present invention is a kit containing the antibody of the present invention and an anti-IgG antibody labeled with a labeling substance. Specifically, the above-mentioned antibody 1 or 2 of the present invention and anti-Ig labeled with a labeling substance
An example is a form in which the G antibody is filled in each independent container, and these are provided as a set.

The material and form of the container filled with the antibody are not particularly limited and can be appropriately selected by those skilled in the art.

Further, the antibody of the present invention or the anti-IgG antibody labeled with a labeling substance may contain other components as long as the action of the antibody is not substantially impaired. For example, an excipient, a buffering agent, a stabilizer, a preservative and the like used in the preparation of ordinary reagents can be included. Specific examples include phosphate buffered saline (PBS), sodium azide (NaN ₃ ), bovine serum albumin and the like.

The labeling substance used for labeling the anti-IgG antibody, the method of labeling the anti-IgG antibody with the labeling substance, and the like are the same as in the above-mentioned "<2> Labeled antibody of the present invention". The anti-IgG antibody labeled with a labeling substance is also not particularly limited, but an anti-mouse IgG antibody is preferable.

A commercially available anti-IgG antibody labeled with a labeling substance may be used.

The kit of the present invention can be used for detecting prions, for example. Specifically, (1) by contacting the "antibody of the present invention" in the kit of the present invention with a sample, it is allowed to bind to prion in the sample, (2) then this "anti-IgG antibody labeled with a labeling substance By contacting the antibody of the present invention bound to prion, and (3) then "anti-IgG antibody labeled with a labeling substance".
The prion can be detected by detecting the labeling substance. The kit of the present invention can be used, for example, in immunohistological staining.

If necessary, the kit of the present invention may further include an enzyme substrate, a coloring substance and the like.

Specific examples of the constitution of the kit of the present invention will be described in the examples below.

[0059]

The present invention will be described in more detail with reference to the following examples, but the examples are merely examples of the present invention, and the present invention is not limited thereto.

<1> Preparation of Antigen Peptide Outsourced to Iwaki Glass Co., Ltd., the following antigen peptide was produced by solid phase synthesis. His Ser Gln Trp Asn Lys Pro Ser Lys Pro Lys Thr As
n Met Lys His Met AlaGly (SEQ ID NO: 1)

<2> Production of Antibody of the Present Invention The antigen peptide produced in the above <1>
It was bound to a carrier protein by the method (hereinafter, referred to as an immunizing peptide). This was mixed with Freund's complete adjuvant and administered to Balb / c mice for priming immunization. Four weeks after the priming immunization, booster immunization was performed with an immunizing peptide mixed with an incomplete adjuvant, and one week after the booster immunization, blood was collected and the increase in antibody titer was confirmed by an ELISA method using prion as an antigen.

Immunizing peptides were intravenously injected into mice in which an increase in antibody titer was observed, and 3 days after the intravenous injection, spleen cells were collected and fused with PAI mouse myeloma cells by the polyethylene glycol method to obtain HAT (hypoxanthine). -Fused cells were selected using (aminopterin-thymidine) medium.

The supernatant of the hybridoma was searched by an ELISA method using the antigen peptide as an antigen to obtain a hybridoma clone producing an antibody that specifically binds to the antigen peptide. After subcloning this clone by the limiting dilution method, it was transferred into the abdominal cavity of Balb / c mice to collect ascites, and the monoclonal antibody was purified by the ammonium sulfate precipitation method and protein A affinity column.

As a result of determining the immunoglobulin subclass of this monoclonal antibody using a mouse monoclonal antibody isotyping kit (Immunotype (trade name); manufactured by Sigma), it was IgG1.

<3> Production of labeled antibody of the present invention <3> -1 Production of antibody of the present invention labeled with biotin The antibody of the present invention produced in <2> above is mixed with 0.1M carbonate buffer (pH).
It was dialyzed against 8.5) to prepare an antibody solution. In addition, 0.1M carbonate buffer (pH 8.5) or dimethyl sulfoxide (DMSO)
To prepare a 1 mg / ml biotin (PIERCE) solution.

While the antibody solution was being stirred, the biotin solution containing biotin which was one-tenth the weight of the antibody was added, and the mixture was reacted at room temperature for 4 hours with stirring. After this reaction, the solution is 0.01M without calcium and magnesium.
The antibody of the present invention labeled with biotin was produced by dialysis against phosphate buffered saline (PBS (-)).

<3> -2 Preparation of FITC-labeled antibody of the present invention The antibody of the present invention prepared in <2> above was mixed with 0.1M carbonate buffer (pH).
It was dialyzed against 9.0) to prepare an antibody solution. Also in DMSO
A 10 mg / ml FITC (SIGMA) solution was prepared.

To the above antibody solution, the above FITC solution containing 1/2 weight of the FITC was slowly added, and the mixture was allowed to stand at room temperature for 4 hours for reaction. The solution after this reaction was applied to a PD-10 column and unreacted FITC and FITC were used.
It was separated into C-labeled antibody. The FITC-labeled antibody was dialyzed against PBS (-) to produce the FITC-labeled antibody of the present invention.

<4> Use of the antibody of the present invention and the labeled antibody of the present invention <4> -1 Immunohistological staining using the antibody of the present invention Formalin-fixed paraffin-embedded from brain tissue of a patient with Creutzfeldt-Jakob disease (CJD). A section was prepared. After deparaffinization, endogenous peroxidase was blocked by incubation in methanol containing 0.3% hydrogen peroxide for 30 minutes. The tissue section was immersed in hydrochloric acid, autoclaved at 121 ° C. for 10 minutes, and then washed with water and 0.05 M Tris-HCl (pH 7.6).
The antibody of the present invention (1 μg / ml) produced in <2> above was added dropwise to this, and the mixture was incubated at 37 ° C. for 30 minutes.

After incubation, the plate was washed with phosphate buffered saline (PBS), a secondary antibody (biotin-labeled anti-mouse IgG antibody) was added dropwise, and the mixture was incubated at 37 ° C. for 30 minutes. After washing with PBS, peroxidase-labeled streptavidin was added dropwise and incubated at 37 ° C for 30 minutes.

After the incubation, the plate was washed with PBS and then DAB (3,3'-diaminobenzidine) was developed.
DAB color development was performed by the following method. 40 ml 0.1 M Tris (p
H7.6) and 40 μl of 30% H ₂ O ₂ were mixed and sufficiently stirred, 40 ml of 1 mg / ml DAB was added thereto, and the mixture was well shielded from light with an aluminum foil and well stirred.

The solution was left in contact with the sections until they were stained. When the staining progressed moderately, the section was washed with cold pure water, 70% ethanol (3 minutes x 1 time), 80% ethanol (3 minutes x 1 time), 90% ethanol (3 minutes x 1 time),
100% ethanol (3 minutes x 1 time), xylene (3 minutes x 2)
And then 15 minutes x 1 time) and then sealed.

The results of observation with an optical microscope are shown in FIG.
As a result, it was shown that the antibody of the present invention stains "Kouru plaque" characteristic of CJD patients, suggesting that the antibody of the present invention recognizes prions.

<4> -2 Western blotting using the antibody of the present invention The method of Laemmli (Laemmli, UK (1970) Nature, 227, 68) using recombinant prion protein (100 ng / lane) as a sample.
0-685), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed.

After SDS-PAGE, the gel was transferred to a transfer buffer (25 mM Tris containing 20% methanol, 192 mM glycine) at 12 ° C.
Medium, 100 V for 3 hours, polyvinylidene difluoride film (PV
DF film). This membrane is 5% bovine serum albumin
3 in PBS containing (BSA) and 0.1% Tween 20 (trade name)
Blocking was achieved by incubating for 0 minutes. The antibody of the present invention (1 μg /
ml) were contacted and incubated at room temperature for 2 hours.

After the incubation, the membrane was washed with 0.1% Tween2.
After washing with PBS containing 0 (trade name), secondary antibody (1000
It was contacted with a double-diluted peroxidase-labeled anti-mouse IgG antibody) and incubated at room temperature for 2 hours. After the incubation, the membrane was washed with PBS containing 0.1% Tween 20 (trade name), and then ECL Western blotting detection reagents
Was used for color development.

As a result, one band appeared in the recombinant prion protein lane. From this result, it was shown that the antibody of the present invention binds to the prion protein, and it is suggested that it binds to the following amino acid sequence portion in the prion protein. His Ser Gln Trp Asn Ly
s Pro Ser Lys Pro Lys Thr Asn Met Lys His Met AlaG
ly (SEQ ID NO: 1)

<4> -3 Flow cytometry using the labeled antibody of the present invention The cell surface or the inside of HL-60 cells (a kind of promyelocytic cell line) produced by the above <3> -2. Flow cytometry was performed after staining with the invention labeled antibody (FITC labeled).

The results of staining the cell surface are shown in FIG. 2A and the results of staining the inside of the cell are shown in FIG. 2B. In FIG. 2, the horizontal axis represents the fluorescence intensity of FITC and the vertical axis represents the number of cells. In addition, the points indicated by "b" in FIGS. 2A and 2B show the maximum fluorescence intensity of the control (when the labeled antibody of the present invention is not used), and the fluorescence intensity higher than that (ie, on the horizontal axis extending from "b"). Cells showing fluorescence intensity in the range indicated by parallel straight lines were designated as "positive".

As a result, when the cell surface of HL-60 cells was stained, about 43% of the cells became positive. Also HL
When the inside of -60 cells was stained, about 89% of the cells became positive.

From this result, the antigen (the antigen containing the amino acid sequence represented by SEQ ID NO: 1) that binds to the labeled antibody of the present invention (and the antibody of the present invention) is expressed on the cell surface and inside the cells of HL-60 cells. It was shown that this antigen was highly expressed inside the cells.

<5> Production Example of Composition Including Antibody of the Present Invention or Labeled Antibody of the Present Invention <5> -1 Production Example of Composition Including Antibody of the Present Invention A solution composition containing the antibody of the present invention was produced as follows. .
A solution composition containing the antibody of the present invention (final concentration 1.0 mg / mL) was prepared by dissolving the antibody of the present invention (produced in <2> above) in PBS (-) containing 0.02% NaN _3. , 0.2 mL each was dispensed into vials and sealed.

<5> -2 Production Example of Composition Including Labeled Antibody of the Present Invention A solution composition containing a labeled antibody of the present invention labeled with biotin was produced as follows. The labeled antibody of the present invention (produced in <3> -1 above) was dissolved in PBS (-) containing 0.05% NaN ₃ and 1% BSA to give the labeled antibody of the present invention (final concentration).
A solution composition containing 0.5 mg / mL) was prepared, and 0.2 mL each was dispensed into vials and sealed.

<5> -3 Production Example of Composition Containing Labeled Antibody of the Present Invention A solution composition containing the labeled antibody of the present invention labeled with FITC was produced as follows. The labeled antibody of the present invention (produced in <3> -2 above) was dissolved in PBS (-) containing 0.05% NaN ₃ and 1% BSA to give the labeled antibody of the present invention (final concentration).
A solution composition containing 0.25 mg / mL) was prepared, and 0.4 mL was dispensed into each vial and sealed.

<6> Production Example of Kit of the Present Invention Vial in which a solution composition containing the antibody of the present invention is enclosed
The kit of the present invention is produced by combining (the one produced in the above <5> -1) and the “vial enclosing a solution composition containing an anti-IgG antibody labeled with a labeling substance” produced by the following: did. Prepare a solution composition containing the biotin-labeled anti-mouse IgG antibody (final concentration 1.0 mg / mL) by dissolving the biotin-labeled anti-mouse IgG antibody with PBS (-) containing 0.02% NaN _3, and add 0.2 mL per vial. Aliquot
Enclosed.

[0086]

INDUSTRIAL APPLICABILITY The antibody of the present invention and the labeled antibody of the present invention are antibodies having specific and stable reactivity with human prions, and can be used as reagents for studying diseases related to prions and diagnostic agents. Can be expected. Further, since the antibody of the present invention and the labeled antibody of the present invention can be produced very efficiently, the production cost of the antibody can be significantly reduced, and a large amount of the antibody can be provided at extremely low cost.

[0087]

[Sequence list] <110> Seikagaku Corporation <120> Anti-prion antibody <130> 10-12 <160> 2 <210> 1 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide having an amino acid sequence represented by amino acid numbers 96 to 114 in SEQ ID NO: 2. This peptide was used as an antigen. <400> 1 His Ser Gln Trp Asn Lys Pro Ser Lys Pro Lys Thr Asn Met Lys His   1 5 10 15 Met Ala Gly

[0088] <210> 2 <211> 253 <212> PRT <213> Homo sapiens <400> 2 Met Ala Asn Leu Gly Cys Trp Met Leu Val Leu Phe Val Ala Thr Trp   1 5 10 15 Ser Asp Leu Gly Leu Cys Lys Lys Arg Pro Lys Pro Gly Gly Trp Asn              20 25 30 Thr Gly Gly Ser Arg Tyr Pro Gly Gln Gly Ser Pro Gly Gly Asn Arg           35 40 45 Tyr Pro Pro Gln Gly Gly Gly Gly Trp Gly Gln Pro His Gly Gly Gly      50 55 60 Trp Gly Gln Pro His Gly Gly Gly Trp Gly Gln Pro His Gly Gly Gly  65 70 75 80 Trp Gly Gln Pro His Gly Gly Gly Trp Gly Gln Gly Gly Gly Thr His                  85 90 95 Ser Gln Trp Asn Lys Pro Ser Lys Pro Lys Thr Asn Met Lys His Met             100 105 110 Ala Gly Ala Ala Ala Ala Gly Ala Val Val Gly Gly Leu Gly Gly Tyr         115 120 125 Met Leu Gly Ser Ala Met Ser Arg Pro Ile Ile His Phe Gly Ser Asp     130 135 140 Tyr Glu Asp Arg Tyr Tyr Arg Glu Asn Met His Arg Tyr Pro Asn Gln 145 150 155 160 Val Tyr Tyr Arg Pro Met Asp Glu Tyr Ser Asn Gln Asn Asn Phe Val                 165 170 175 His Asp Cys Val Asn Ile Thr Ile Lys Gln His Thr Val Thr Thr Thr             180 185 190 Thr Lys Gly Glu Asn Phe Thr Glu Thr Asp Val Lys Met Met Glu Arg         195 200 205 Val Val Glu Gln Met Cys Ile Thr Gln Tyr Glu Arg Glu Ser Gln Ala     210 215 220 Tyr Tyr Gln Arg Gly Ser Ser Met Val Leu Phe Ser Ser Pro Pro Val 225 230 235 240 Ile Leu Leu Ile Ser Phe Leu Ile Phe Leu Ile Val Gly                 245 250

[Brief description of drawings]

FIG. 1 is a view showing a result of immunohistochemical staining of CJD patient's brain with the antibody of the present invention (micrograph).

FIG. 2 shows the results of flow cytometry when the surface of HL-60 cells was labeled with the labeled antibody of the present invention (A) and when the inside of HL-60 cells was labeled with the labeled antibody of the present invention (B). It is a figure.

Claims (8)

[Claims] 1. An antibody which specifically binds to a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1. 2. The antibody according to claim 1, which is a monoclonal antibody. 3. The antibody according to claim 1, which is a mouse-derived antibody.
The described antibody. 4. The antibody according to claim 1, wherein the immunoglobulin subclass is IgG1. 5. A body fluid of an animal immunized with a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 as an antigen, and / or
Alternatively, an antibody that is collected from antibody-producing cells and specifically binds to prion. 6. The antibody according to any one of claims 1 to 5, which is labeled with a labeling substance. 7. The antibody according to claim 6, wherein the labeling substance is biotin or fluorescein isothiocyanate. 8. A kit containing the antibody according to claim 1 and an anti-IgG antibody labeled with a labeling substance.