JPH08231600A - Anti-tyrosinase monoclonal antibody f(ab')2 fragment - Google Patents

Abstract

PURPOSE: To obtain an F(ab')2 fragment of an anti-tyrosinase antibody recognizing at least a part of tyrosinase as an antigen and useful for the detection, structural analysis, etc., of tyrosinase effective for the diagnosis of grave dermatopathy such as pigmentation disorder. CONSTITUTION: This new anti-tyrosinase antibody F(ab')2 fragment recognizes a part of tyrosinase composed of at least a part of the amino acid sequence of the formula, etc., as an antigen and is useful for the detection and structural analysis, etc., of tyrosinase effective e.g. for the diagnosis of grave dermatopathy such as pigmentation disorder. The F(ab')2 fragment can be produced by administering a mammal with a compound obtained by bonding a carrier with a peptide containing an amino acid sequence of a region having low homology to the tyrosinase-relating protein among the amino acid sequences of tyrosinase, collecting the spleen cell from the mammal, fusing with a cultured cell to prepare a hybridoma, culturing the hybridoma, collecting an antibody, cutting the antibody into F(ab')2 and Fc by the partial digestion with a proteinase and separating the F(ab')2 fragment.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an anti-human tyrosinase antibody, particularly an F (ab ′) ₂ fragment of an anti-tyrosinase monoclonal antibody, which is useful for detecting human tyrosinase.
The present invention relates to a method for producing the same and a method for detecting tyrosinase using the method.

[0002]

BACKGROUND OF THE INVENTION Tyrosinase is the most important enzyme in biosynthesis of melanin pigment in vivo. It is produced by the ribosome of the rough endoplasmic reticulum, and then concentrated and activated by the endoplasmic reticulum associated with the Golgi apparatus. It is thought to be selectively transferred to the more produced premelanosomes.

It is known that inhibition of tyrosinase biosynthesis causes serious skin disorders. For example, dyschromatosis is caused by structural changes in tyrosinase itself or abnormalities in its biosynthesis process. It is said that there is. It is also possible that dyschromatosis is involved in abnormal selective transfer of tyrosinase to premelanosomes.

[0004] It is considered very useful to know the structure of tyrosinase and the behavior of tyrosinase in living organisms or cells in order to diagnose such diseases or to investigate their causes. Efforts have been made to know. In particular, the method using the antigen-antibody reaction is
Because of its high sensitivity, various studies have been made, and preparation of antibodies against tyrosinase has been made for a long time.

Based on such studies, the present inventors have prepared an anti-tyrosinase monoclonal antibody and found that it is useful for detecting tyrosinase. However, a typical antibody molecule, immunoglobulin G (IgG)
Vary depending on various animal species and are composed of proteins. Therefore, when a monoclonal antibody prepared using an animal such as a mouse is used for an animal of another species such as human, There is a high possibility that the antibody will be recognized as a foreign substance, cause allergies, or be neutralized and neutralized by the antibody against the monoclonal antibody, and there is room for improvement.

[0006]

SUMMARY OF THE INVENTION The present invention has been made in view of the above circumstances, and is a means for detecting tyrosinase that is difficult to recognize a foreign substance, and an anti-tyrosinase antibody, particularly anti-tyrosinase monoclonal antibody F necessary for that purpose.
It is an object to provide an (ab ') ₂ fragment and a method for producing the same.

[0007]

Means for Solving the Problems The present inventor has conducted extensive studies to solve the above problems, and in the process, a tyrosinase-related protein (TRP) which is a protein very similar to tyrosinase in the amino acid sequence of tyrosinase. The monoclonal antibody that specifically binds to tyrosinase was obtained by using a peptide having a sequence not found in (1) as an antigen. Here, IgG refers to two Fab fragments that recognize an antigen and bind to the antigen (the two Fab fragments bound to each other are referred to as F (ab ′) ₂ fragments),
It consists of two parts, an Fc fragment that serves as a marker and binds to immunocompetent cells on the living body side such as macrophages, but that the Fc fragment may not be necessary for the detection of tyrosinase, which is the object of the present invention. The strength of binding to the antigen is F (ab ') than that of the Fab fragment.
Considering that ₂ fragments would be stronger, F
It was confirmed that the (ab ′) ₂ fragment would be a powerful tool for detecting tyrosinase, and as a result of repeated studies, this was confirmed and the invention was completed.

That is, the present invention is an F (ab ′) ₂ fragment of an anti-tyrosinase antibody that recognizes at least a part of tyrosinase as an antigen. Examples of the recognition site for tyrosinase of the monoclonal antibody include at least a part of the amino sequence shown in SEQ ID NO: 1. Further, as a specific embodiment of the F (ab ′) ₂ fragment of the present invention, the above-mentioned F (ab ′) ₂ fragment in which the anti-tyrosinase antibody is an anti-tyrosinase monoclonal antibody, and the monoclonal antibody is IgG,
Above F (ab ') subclass is IgG _{1 2} fragments, a molecular weight of 90,000 to 120,000 above F (ab') ₂ fragment, the above F (ab ') ₂ fragment is a further tyrosinase human tyrosinase Can be mentioned.

The present invention also provides the above F including the following steps.
A method for producing an (ab ') ₂ fragment is provided. (A) After immunizing a mammal with a conjugate of a carrier and a peptide having at least a part of the amino acid sequence having a low homology with the amino acid sequence of a tyrosinase-related protein among the amino acid sequences of tyrosinase, the animal is immunized. Spleen cells are removed and fused with cultured cells to prepare a hybridoma, and (b) a hybridoma that produces a monoclonal antibody that binds to tyrosinase and does not bind to tyrosinase-related protein is selected, and (c) the hybridoma is appropriately selected. Antibody protein was collected from the culture supernatant obtained by culturing in a different medium, or ascites obtained by culturing the hybridoma in the abdominal cavity of a mouse, and (d) the antibody protein was partially digested with a proteolytic enzyme to produce F (ab '). Cleave into ₂ and Fc fragments to separate the F (ab ') ₂ fragment.

Further, the present invention uses the above-mentioned F (ab ') ₂ fragment or a labeled F (ab') ₂ fragment introduced with a label for the detection thereof, for immunological detection of tyrosinase. Provide the law.

The term "specifically binds to tyrosinase" as used herein means to specifically recognize tyrosinase as an antigen and bind to it. The term “specifically” as used herein has the meaning conventionally used in the technical field relating to antibodies, and particularly means that it binds to human tyrosinase but does not substantially bind to TRP.

The present invention will be described in detail below.

The cDNA sequence of tyrosinase represented by human tyrosinase is already known (Hiroaki Yamamo
to et. al., Jpn. J. Genet, 64, 121-135 (1989)), and the TRP cDNA sequence is already known (Sigeki).
Shibahara et. Al., TohokuJ. Exp. Med., 156, 403-4
14 (1988)). As a result of translating these cDNA sequences into amino acid sequences, the present inventors have found that the portion of the amino acid sequence of human tyrosinase having the amino acid sequence shown in SEQ ID NO: 1 has low homology with TRP. Furthermore, although this part does not completely match the same region of tyrosinase of other animals, for example, mouse, it has high homology.

Focusing on this point, the present inventors entrusted the peptide research institute to synthesize a peptide having the above sequence (hereinafter, also referred to as "tyrosinase-specific peptide"). After immunizing a mammal with the obtained peptide, spleen cells were taken out and fused with a cultured cell to prepare a hybridoma, and this hybridoma was successfully produced a monoclonal antibody specific for tyrosinase. Furthermore, by subjecting the antibody to enzyme treatment, an anti-tyrosinase antibody F (ab ′) ₂ fragment with high specificity and reactivity was obtained.

Below, the monoclonal antibody F (ab ')
An example of the method for producing ₂ fragments will be described along with the production procedure.

<1> Antigen As an antigen used for immunizing a mammal, a peptide having an amino acid sequence specific for human tyrosinase is preferable. As such an amino acid sequence, SEQ ID NO: 1
The sequences shown in are listed. The peptide having this amino acid sequence can be chemically synthesized by a conventional method. For example, using a commercially available peptide synthesizer,
By inputting the above sequence, the above peptide can be obtained. Alternatively, the peptide may be obtained by entrusting it to a company that provides a peptide synthesis service.

The peptide thus obtained (hereinafter, also referred to as "synthetic peptide") can be used as it is as an antigen, or may be used as a keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA). ), Ovalbumin (OVA), or the like. When such a carrier is used, the antigenicity can be enhanced even if the peptide alone has low or no antigenicity. In this case, antibodies against the carrier can be prepared, but they can be removed at the stage of selecting the hybridoma.

<2> Immunization of mammals The mammals to be immunized with the above-mentioned antigens are not particularly limited as long as they are mammals usually used in immunization experiments, and examples thereof include mice, rats and rabbits. In addition, the method of immunization is
It may be carried out according to a usual immunization method, and for example, a synthetic peptide or a synthetic peptide bound to a carrier is
mg / kg to 50 mg / kg with Freund's complete or incomplete adjuvant
Examples include a method in which administration is repeated several times every two weeks, and two or more weeks after the final administration, only the synthetic peptide is administered for final immunization.

The dose of the synthetic peptide or the synthetic peptide bound to the carrier at this time is 0.5 m per dose.
About g / kg to 50 mg / kg is suitable. If the dose is less than 0.5 mg / kg, the antibody may not be sufficiently produced. Further, even if it exceeds 50 mg / kg, no further immune effect can be expected, and further, immunosuppression occurs in vivo, and the desired antibody may not be obtained.

<3> Preparation of hybridoma The animal immunized as described above is sacrificed 3 to 4 days after the final immunization, the spleen is excised, and splenocytes are taken out. A splenocyte-cultured cell hybridoma can be obtained by fusing this cell with a cultured cell that is a fusion partner in the presence of a fusion promoter and selecting the resulting fused cell.

The cultured cells to be fused are not particularly limited as long as they can be fused with splenocytes, but myeloma cells (myeloma cells) are generally suitable. Further, in order to be able to distinguish unfused cells and fused cells after cell fusion, those having a specific drug marker for selection are preferable.

Examples include hypoxanthine / guanine / phosphoribosyltransferase-deficient ones. Such cells cannot grow in a medium (HAT medium) supplemented with hypoxanthine aminopterin and thymidine, but fused cells of these cells and normal cells are HAT.
It can grow in the medium and can be distinguished from unfused cells. Specifically, P3X63Ag8.653 strain, P3 / NSI / 1-AG4-1
Commercially available strains of myeloma cells such as strains, FO strains, SP2 / 0-Ag14 strains, etc. are mentioned, but not limited thereto.

Cell fusion is usually RPMI1640, MEM
The antibody-producing cells (spleen cells) and myeloma cells are mixed with a fusion promoter in a medium such as the above at a mixing ratio of 10: 1 to 2: 1. As a fusion accelerator,
There is no particular limitation as long as it is a fusion agent that is commonly used in cell fusion experiments, and specific examples thereof include Sendai virus and polyethylene glycol having an average molecular weight of 500 to 7,000. Alternatively, they may be fused by electric pulses.

The cells that have completed cell fusion are, for example, RPMI.
Cells that have been diluted with 1640 or MEM medium and washed by centrifugation are suspended in a selective medium such as HAT medium, dispensed on a multiplate or the like and cultured to grow only hybridomas.

<4> Selection of hybridoma The hybridoma obtained as described above is a mixture of strains of hybridomas that produce monoclonal antibodies against different antigenic determinant sites among peptides used as antigens or monoclonal antibodies against proteins used as carriers. Since it is the body, a hybridoma producing a monoclonal antibody that specifically binds to the synthetic peptide, that is, the peptide specific for tyrosinase is selected from these.

As a method for this selection, an enzyme immunoassay method (ELISA method) using a synthetic peptide used as an antigen
Is preferred. For example, the synthetic peptide is immobilized on a plastic plate or the like, and the hybridoma culture supernatant is further labeled with an enzyme, a fluorescent substance or a luminescent substance.
The amount of the antibody that binds to the synthetic peptide can be known from the amount of the label that is bound by adding the antibody. Alternatively, the antibody produced by the hybridoma may be solid-phased, and the synthetic peptide and the labeled second antibody may be sequentially added thereto and incubated.

One strain of the hybridoma obtained in this way is from the Institute of Biotechnology, Institute of Biotechnology, Institute of Industrial Science and Technology (Postal Code 305, 1-3-1 Higashi Tsukuba City, Ibaraki Prefecture, Japan) since May 27, 1994. No. FEPM P-14340, and on September 14, 1994, the original deposit was transferred to an international deposit under the Budapest Treaty, and the deposit number is FERM BP-4801.

<5> Preparation of anti-tyrosinase antibody F (ab ') ₂ fragment that specifically binds to tyrosinase and its utilization method A monoclonal antibody that specifically binds to tyrosinase is obtained from the hybridoma obtained as described above. For example, a monoclonal antibody specific to tyrosinase can be obtained by, for example, purifying an antibody-producing hybridoma by an ammonium sulfate fractionation, gel filtration, affinity chromatography and the like according to a conventional method. This is further digested with a proteolytic enzyme and the Fc fragment is removed on a protein A column to give an F (ab ') ₂ fragment. The proteolytic enzyme is not particularly limited as long as it can cleave F (ab ′) ₂ in a restricted manner by appropriately setting the environment such as pH, and examples thereof include pepsin and ficin. .

Further, an anti-tyrosinase polyclonal antibody can be obtained by immunizing a mammal with a synthetic peptide in the same manner as described above and preparing an immunoglobulin fraction from the blood in the same manner as in the purification of ordinary antibodies. The anti-tyrosinase polyclonal antibody thus obtained also specifically binds to tyrosinase, and the F (ab ′) ₂ fragment of the present invention can be obtained even by digesting with this proteolytic enzyme.

Since the anti-tyrosinase antibody F (ab ') ₂ fragment of the present invention which specifically binds to tyrosinase specifically reacts with tyrosinase, it is a chromosomal antibody for immunostaining of human-derived cultured cells, skin tissues, etc. Further, it can be used as an antibody for staining in Western blot or microautoradiography, and can be used as a detection reagent for qualitative or quantitative determination of human tyrosinase. At this time, as in the conventional immunological detection method for antigens using a monoclonal antibody, F
(Ab ') ₂ labeled introduced a label for the detection of fragments F (ab') can be used ₂ fragments.

Furthermore, it is also possible to use this F (ab ') ₂ fragment in combination with the Fc portion of human IgG as a chimeric monoclonal antibody by utilizing gene recombination technology.

[0032]

EXAMPLES The present invention will be described in more detail below with reference to examples, but it goes without saying that the present invention is not limited to these examples.

<1> Preparation of hybridoma The peptide synthesis and the binding to the carrier (KLH) were requested to Peptide Institute Inc.

100 μg of the above-mentioned carrier-bound synthetic peptide was dissolved in 100 μl of physiological saline, mixed with 100 μl of complete Freund's adjuvant (manufactured by SIGMA) and emulsified, and intraperitoneally administered to BALB / c mice (CLEA Japan, 8 weeks old). did. Then, 1 week and 2 weeks thereafter, 200 μl of the carrier-bonded synthetic peptide-incomplete Freund's adjuvant equivalent mixed emulsion was intraperitoneally administered. Two weeks after the third administration, 100 μl of physiological saline containing 50 μg of the synthetic peptide was intravenously injected.

After 3 days, the spleen was removed from the mouse, and the splenocytes were suspended in RPMI1640 medium and washed.
On the other hand, mouse myeloma cell line P3X63Ag8.653 (purchased from Dainippon Pharmaceutical Co., Ltd.) was cultured at the logarithmic growth phase in accordance with cell fusion and collected by centrifugation.

To 10 ⁸ splenocytes, 2 × 10 ⁷ myeloma were mixed in the above medium, and the cells were pelleted by centrifugation. After removing the supernatant, the temperature was kept at 37 ° C.
RPMI1 containing 0% PEG4000 (manufactured by SIGMA)
1 ml of 640 medium was gradually added dropwise over 1 minute and gently stirred for 1 minute. Furthermore, RPMI1640 medium 2 at 37 ° C
ml was added dropwise over 2 minutes and 8 ml over 3 minutes with stirring.

After completion of the dropping, the supernatant was removed by centrifugation and the cell pellet was added to 40 ml of GIT medium (manufactured by Wako Pure Chemical Industries).
100 μl per well was dispensed into four 96-well plates (Sumitomo Bakelite). The next day, 25 μl of HAT medium was added, and 4 days later, 2
5 μl HAT medium was added. After culturing for 1 week, half of the culture supernatant was removed, and 100 μl of GIT medium was added.

Approximately 2 weeks after cell fusion, only hybridomas in which myeloma and spleen cells were fused formed colonies, and the culture was continued until the diameter of the colonies became approximately 1 mm. At this point, the antibody secreted in the culture supernatant was
The above synthetic peptide and a commercially available secondary antibody (horseradish peroxidase (HRP) -labeled goat anti-mouse Ig's (γ
+ L) antibody: Sandwich E using TAGO)
It was assayed by the LISA method. Of these, the hybridoma in the well with the highest antibody titer was cloned by the limiting dilution method to obtain a monoclonal antibody-producing hybridoma strain. This strain has been deposited as BP-4801 at the Institute of Biotechnology, Industrial Technology Institute.

<2> Preparation of monoclonal antibody F (ab ') ₂ fragment that specifically binds to tyrosinase The hybridoma cell line obtained above was cultured and a monoclonal antibody that specifically binds to tyrosinase was collected. .

The above hybridoma was cultured in GIT medium, and when the cell concentration reached about 5 × 10 ⁶ cells / ml, the culture supernatant was recovered by centrifugation, filtered with a filter having a pore size of 0.22 μm, and filtered. The solution was purified with a protein A column kit (manufactured by Amersham Japan) to obtain an anti-tyrosinase monoclonal antibody.

When the class and subclass of the obtained monoclonal antibody were examined by an isotyping kit (manufactured by Amersham Japan), it was found that the class was IgG and the subclass was IgG ₁ .

The obtained antibody was partially digested with an IgG1F (ab ') ₂ preparation kit (Pierce), and the Fc fragment was removed from the digest by adsorption on a protein A column to give F (ab') _2. A fragment was obtained. The molecular weight of this product was 10500 when measured by electrophoresis.
It was 0. From this value, the obtained fragment is F
It was found to be an F (ab ') ₂ fragment, not the ab fragment.

<3> Evaluation of anti-tyrosinase monoclonal antibody F (ab ') ₂ fragment (1) Anti-tyrosinase monoclonal antibody F (ab') 2
Specificity of ₂ fragments for tyrosinase For the monoclonal antibody obtained above, MeWo
Using an extract of cells (human malignant melanoma-derived cultured cells) and HeLa cells (human cervical cancer-derived cultured cells),
The specificity was evaluated by Western blotting (enzyme immunoblotting).

MeWo and H cultured in culture flask
After removing the culture supernatant of eLa cells and washing several times with Dulbecco's PBS buffer (PBS), the cells were collected using a rubber policeman. Collected cells are again in PBS
The cells were washed several times with a cell lysis buffer (Triton X-1
00 was suspended in 0.1 M sodium phosphate buffer containing 0.5%, pH 6.9).

The cells were crushed by treating with an ultrasonic cell crusher (manufactured by Tosho Denki Co., Ltd.) for 30 seconds and crushed at 16,000 × G.
Centrifugation was performed for 0 minutes. The protein concentration contained in the obtained supernatant was measured and used as a sample protein. 5 to 20
% Density gradient polyacrylamide gel (PAJEL: AT
It was subjected to electrophoresis using TO). Apply amount is 1
The amount of protein per lane is 10 μg / lane.
The electrophoresis was performed according to a conventional method. After completion of the electrophoresis, the gel was taken out, one lane was cut out, and the activity staining of tyrosinase (DOPA staining) was performed to detect the position of tyrosinase on the gel.

On the other hand, the remaining lanes of the gel were transferred to transfer buffer (100 mM, Tris; 192 mM, g
After immersing in lycin), the electrophoretic material was transferred from the gel to a membrane (Immobilon; manufactured by Millipore) previously immersed in transfer buffer using a blotting device (semi-dry blotting device: manufactured by ATTO).

The membrane on which the electrophoretic material was transferred was immersed in PBS buffer for 15 minutes, and then the blocking buffer (5% skim milk or 1% BSA and 0.1% was added).
Soak in PBS buffer containing Tween 20 for 25
Shake gently for 1 hour at ℃.

Thereafter, the membrane was washed with 0.1% Tween.
It was washed with a PBS buffer containing 20 (TPBS buffer) (15 minutes once, 5 minutes twice), and the F (ab ′) ₂ fragment solution (5 μg / m) obtained in <2> above.
1 solution was immersed in about 2 ml) and gently shaken at 25 ° C. for about 1 hour. After the membrane was washed, it was further dipped in a commercially available secondary antibody solution (horseradish peroxidase (HRP) -labeled goat anti-mouse Ig's (γ + L) antibody: 10,000 times diluted with TAGO, about 2 ml), 25
It was shaken gently for about 1 hour at ° C.

After washing the membrane with TPBS buffer (15 minutes once, 5 minutes 4 times), a band of the electrophoresed product to which the anti-tyrosinase monoclonal antibody was bound using a commercially available HRP detection kit (Amersham ECL kit). Was detected. The band to which HRP is bound can also be detected by using 4-chloro-1-naphthol as a substrate.

As a result of the immunoblotting carried out as described above, a band was recognized at the same position as the DOPA-stained band considered to be tyrosinase from the result of the above-mentioned activity staining, and the monoclonal antibody F (ab ') obtained above was obtained.
It was found that the _two fragments bind to a protein band having human tyrosinase activity, that is, human tyrosinase. Furthermore, this monoclonal antibody is HeLa
It did not bind to cell extracts. As another animal species,
When the same treatment was performed on the mouse-derived Melanoma B-16 cells, it was bound to a protein band having a weak tyrosinase activity.

(2) Reactivity of anti-tyrosinase monoclonal antibody F (ab ') ₂ The monoclonal antibody F (ab') ₂ fragment obtained above was subjected to enzyme-labeled antibody assay (ELISA).
The non-specific staining property was compared with that of the enzyme-untreated IgG which was not treated with a protease.

The culture supernatant of MeWo cells cultured in a culture flask was removed, and Dulbecco's PBS buffer (PBS) was added.
After several washes with cells, cells were harvested using a rubber policeman. The collected cells are washed again with PBS several times,
Cell lysis buffer (Triton X-100 0.5%
0.1M sodium phosphate buffer containing, pH 6.9)
Suspended in water.

The cells were crushed by treating with an ultrasonic cell crusher (manufactured by Tosho Denki Co., Ltd.) for 30 seconds and crushed at 16,000 × G.
Centrifugation was performed for 0 minutes. The protein concentration contained in the obtained supernatant was measured and used as a cell extract. This was designated as an antigen protein.

Next, 50 μl of the antigenic protein (10 μg / ml) was placed in each well of an ELISA microplate (Sumilon typeH (Sumitomo Bakelite Co., Ltd.)) and allowed to stand at 37 ° C. for 1 hour. 100 μl of PBS containing serum-derived albumin was added and blocking was performed at 25 ° C. for 1 hour. It was washed twice with PBS to prepare an antigen solid phase well containing tyrosinase. The purified tyrosinase monoclonal antibody IgG (5
00 ng / ml) and F (ab ′) ₂ fragment (500 ng / ml) were added in an amount of 50 μl each, and incubated at 37 ° C. for 1 hour. After washing in the same manner as described above, horseradish peroxidase (HRP) -labeled goat anti-mouse Ig's (γ + L) antibody (manufactured by TAGO) as a secondary antibody
50 μl was added and incubated at 37 ° C. for 1 hour. After washing in the same manner, 75 μl of a coloring solution containing o-phenylenediamine hydrochloride was added and incubated at 37 ° C. for 30 minutes. Stop the reaction with 25 μl of 1M sulfuric acid and
The absorbance at 0 nm was measured.

As shown in Table 1, the reactivity to tyrosinase is higher in F (ab ') ₂ than in IgG, so that it can be used at a lower concentration in actual use and non-specific reaction can be prevented. You can Furthermore, this indicates that the Fc fragment is not required for tyrosinase recognition and binding. Therefore, the F (ab ′) 2 fragment of the present invention from which the Fc fragment has been removed can be expected to be less likely to be recognized as a foreign substance than the anti-tyrosinase monoclonal antibody itself, because the Fc portion is small.

[0056]

[Table 1]

[0057]

EFFECT OF THE INVENTION F (a of the monoclonal antibody of the present invention
The b ′) ₂ fragment is highly reactive with human tyrosinase and can prevent nonspecific binding. This monoclonal antibody is obtained by the method of the present invention.

[0058]

[Sequence Listing] SEQ ID NO: 1 Sequence length: 14 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Met Glu Lys Glu Asp Tyr His Ser Leu Tyr Gln Ser His Leu 1 5 10

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location G01N 33/563 G01N 33/577 B 33/573 A61K 39/395 P 33/577 9281-4B C12N 5 / 00 B // A61K 39/395 9162-4B 15/00 ZNAC (C12P 21/08 C12R 1:91) (72) Inventor Takashi Shibata 560 Kashio-cho, Totsuka-ku, Yokohama-shi, Kanagawa Pola Chemical Industry Co., Ltd. Totsuka Kenkyu Co., Ltd. (72) Inventor Tomomi Kato 560 Kashio-cho, Totsuka-ku, Yokohama-shi, Kanagawa Pola Chemical Industry Co., Ltd. Totsuka Laboratory

Claims (8)

[Claims] 1. An F (ab ′) ₂ fragment of an anti-tyrosinase antibody that recognizes at least part of tyrosinase as an antigen. 2. The F (ab ′) ₂ fragment according to claim 1, wherein the recognition site for tyrosinase of the antibody is at least a part of the amino sequence shown in SEQ ID NO: 1. 3. The F (ab ′) ₂ fragment according to claim 1 or 2, wherein the antibody is a monoclonal antibody. 4. The F (a) of claim 3, wherein the monoclonal antibody is IgG and the subclass is IgG _1.
b ') ₂ fragments. 5. The F (ab ′) ₂ fragment according to claim 3, wherein the monoclonal antibody is derived from mouse cells. 6. The F (ab ′) _{2 according} to any one of claims 1 to 5, wherein the tyrosinase is human tyrosinase.
Fragment. 7. The F (a) according to claim 3, including the following steps.
b ′) Method for producing ₂ fragment, (a) Using a conjugate of a peptide having at least a part of an amino acid sequence of a region having low homology with the amino acid sequence of a tyrosinase-related protein in the amino acid sequence of tyrosinase, and a carrier After immunizing a mammal, splenocytes of the animal are taken out, fused with cultured cells to prepare a hybridoma, and (b) a hybridoma producing a monoclonal antibody that binds to tyrosinase and does not bind to a tyrosinase-related protein is selected. And (c) an antibody protein is collected from a culture supernatant obtained by culturing the hybridoma in an appropriate medium, or ascites obtained by culturing the hybridoma in the abdominal cavity of a mouse, and (d) the antibody protein is treated with a protease. Partially digested and cleaved into F (ab ') ₂ and Fc fragments to obtain F (ab') ₂ fragment Separate the components. 8. The F according to any one of claims 1 to 7.
An immunological detection method for tyrosinase, which comprises using a (ab ′) ₂ fragment or a labeled F (ab ′) ₂ fragment into which a label for detecting the fragment has been introduced.