JPH08231599A - Anti-tyrosinase monoclonal antibody fab fragment - Google Patents

Abstract

PURPOSE: To obtain an Fab 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 Fab fragment recognizes at least a part of tyrosinase composed of e.g. 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 Fab fragment can be produced by administering a mammal with a compound obtained by bonding a carrier with a peptide containing an amino acid sequence having low homology to the amino acid sequence of a 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 Fab and Fc by the partial digestion with a proteinase and separating the fragments.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an anti-tyrosinase antibody, particularly a Fab fragment of an anti-tyrosinase monoclonal antibody, which is useful for detecting tyrosinase and structural analysis, and a method for producing the same.

[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.

[0003]

It is known that inhibition of tyrosinase biosynthesis in vivo causes serious skin disorders. For example, dyschromatosis is a structural mutation of tyrosinase itself or its production. It is said that the cause is an abnormality in the synthesis process. 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. However, tyrosinase is difficult to be recognized by an antibody as an antigen, and because there is a tyrosinase-related protein that is extremely close to tyrosinase in terms of physical properties, anti-tyrosinase monoclonal antibodies were produced by overcoming numerous failures. It's just recently. Therefore, detailed studies have not been conducted on the antigenic site recognized by the anti-tyrosinase monoclonal antibody, and structural analysis of the recognition site on the antibody side has been desired to clarify them.

Further, immunoglobulin γ (IgG), which is a typical antibody molecule, is Fab or F that recognizes an antigen.
It is known that it is composed of the (ab ′) ₂ portion and the Fc portion that binds to macrophages and the like. However, depending on the structure of the Fab portion, a non-specific reaction may be induced, and The Fab fragment was also required to be analyzed in order to prove the specificity of the tyrosinase antibody.

Further, since the Fc portion binds to macrophages, it is unreasonable to use the anti-tyrosinase antibody itself in an environment where many macrophages exist.
A portion that does not include the c portion, that is, Fab or F (ab ') ₂
Was required.

On the other hand, the Fab fragment of the anti-tyrosinase antibody which specifically recognizes tyrosinase has not been obtained yet.

[0008]

DISCLOSURE OF THE INVENTION The present invention has been made from the above viewpoints, and in order to identify the characteristics of anti-tyrosinase antibody, a Fab fragment of the anti-tyrosinase antibody that recognizes at least a part of tyrosinase as an antigen, That is, it is an object to provide a fragment obtained by removing the Fc portion that binds to macrophages and the like from an anti-human tyrosinase antibody and to provide a method for producing the same.

[0009]

Means for Solving the Problems As a result of extensive studies to solve the above problems, the present inventors have found that tyrosinase-related protein (TRP), which is a protein very similar to tyrosinase in the amino acid sequence of tyrosinase, is selected. It was found that a monoclonal antibody that recognizes tyrosinase can be obtained by using a peptide having a sequence not recognized as an antigen, and succeeded in obtaining this Fab fragment by partially digesting this with a protease.
The present invention has been completed.

That is, the present invention is a Fab 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 antibody include at least a part of the amino sequence shown in SEQ ID NO: 1. Further, the Fab of the present invention
In a specific embodiment of the fragment, the above Fa wherein the anti-tyrosinase antibody is an anti-tyrosinase monoclonal antibody
b fragment, the above Fab fragment in which the monoclonal antibody is IgG, and the subclass is IgG ₁ , and the Fab in which the molecular weight is 40,000 to 60,000
Fragments, and also the above Fab fragments in which the tyrosinase is human tyrosinase.

The present invention also includes the above-mentioned F including the following steps.
A method for making an ab 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 protease to produce a Fab fragment and an Fc fragment. Cleave and cut the Fab fragment.

The present invention will be described in detail below. Among the tyrosinase, the cDNA sequence of human tyrosinase is already known (Hiroaki Yamamoto et. Al., Jpn. J. Gene.
t, 64, 121-135 (1989)), and the cDNA sequence of TRP is already known (Sigeki Shibahara et. al., Toho
ku J.exp. Med., 156, 403-414 (1988)). The present inventors have translated these cDNA sequences into amino acid sequences,
It was found that the portion of the amino acid sequence of tyrosinase having the amino acid sequence shown in SEQ ID NO: 1 has low homology with TRP. This sequence is peculiar to human tyrosinase, but it is also TR with tyrosinase from experimental animals such as mice.
It has much higher homology than P.

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. This antibody strongly recognized human tyrosinase, and also weakly recognized mouse tyrosinase. Furthermore, by subjecting the antibody to an enzyme treatment, an anti-tyrosinase antibody Fa having high specificity and reactivity is obtained.
The b fragment was obtained. Below, the monoclonal antibody F
An example of a method for producing an ab fragment will be described according to 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 an antigen as it is, and keyhole limpet hemocyanin (KLH: Keyhole Limpet Hemocyanin), bovine serum albumin (BSA: Bovine SerumAlbumin) can be used. ), 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 for 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 / phosphoribosyl transferase deficient. 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.
The cells, which are diluted with 1640 or MEM medium and washed by centrifugation, are suspended in a selective medium such as HAT medium and dispensed to a multiplate or the like for culturing to grow only hybridoma.

<4> Selection of hybridomas The hybridomas obtained as described above are hybridomas that produce 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, a peptide specific to human 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 used.
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 the Institute of Biotechnology, Institute of Biotechnology, Institute of Industrial Science (Postal Code 305, 1-3-1 Higashi, Tsukuba, Ibaraki, 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 Fab fragment that specifically binds to tyrosinase and its utilization method To obtain a monoclonal antibody that specifically binds to tyrosinase from the hybridoma obtained as described above, for example, The antibody-producing hybridoma may be purified by ammonium sulfate fractionation, gel filtration, affinity chromatography and the like according to a conventional method. The monoclonal antibody obtained in the Examples described later specifically and strongly binds to human tyrosinase and weakly and specifically to mouse tyrosinase. This is because in the amino acid sequence of tyrosinase, the sequence of SEQ ID NO: 1 is specific to human, and in mouse tyrosinase, the corresponding portion differs by one amino acid. From these facts, this region of tyrosinase is highly homologous although it varies slightly depending on the animal species, and it can be expected that the above-mentioned monoclonal antibody recognizes tyrosinase to some extent in any animal species. Furthermore, a slight change in this sequence allows each animal to recognize a unique tyrosinase. The Fab fragment of the present invention includes Fab fragments of these anti-tyrosinase monoclonal antibodies.

Thus, an anti-tyrosinase monoclonal antibody is obtained, which is further partially digested with a proteolytic enzyme and then Fc using a protein A column or the like.
Fab fragments are obtained by removing the fragments.
The enzyme is not particularly limited as long as it can cleave the Fab fragment by appropriately setting conditions such as pH, and examples thereof include insolubilized papain 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 thus-obtained anti-tyrosinase polyclonal antibody also specifically binds to tyrosinase, and the Fab fragment of the present invention can be obtained by digesting the anti-tyrosinase polyclonal antibody with the above-mentioned protease.

Since the anti-tyrosinase antibody Fab fragment which specifically binds to tyrosinase of the present invention specifically reacts with human tyrosinase, human-derived cultured cells,
It can be used as a chromosomal antibody in immunostaining of skin tissue or the like, and as a staining antibody in western blot or microautoradiography, and can be used as a detection reagent for qualitative or quantitative determination of human tyrosinase. Furthermore, since it does not contain the Fc portion, it is less susceptible to macrophages. Further, since it also binds to animal tyrosinase much more specifically than TRP, the same experiment as above can be performed using animals.

[0030]

EXAMPLES The present invention will be described in detail below with reference to examples of the present invention. However, 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 Co., Ltd.

100 μg of the above-mentioned carrier-bound synthetic peptide was dissolved in 100 μl of physiological saline and mixed and emulsified with 100 μl of complete Freund's adjuvant (manufactured by SIGMA) and administered intraperitoneally to BALB / c mice (CLEA Japan, Inc.) of 8 weeks of age. 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 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 splenocytes 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. Furthermore, from hybridomas producing a monoclonal antibody that binds to a synthetic peptide, a hybridoma producing a monoclonal antibody that binds to human tyrosinase and mouse tyrosinase but does not substantially bind to a tyrosinase-related protein was selected. This strain was submitted to the Institute of Biotechnology, Institute of Industrial Science, under the contract number FERMBP-4.
Deposited as 801.

<2> Preparation of monoclonal antibody Fab 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 collected 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-human tyrosinase monoclonal antibody.

When the class and subclass of the obtained monoclonal antibody were examined with 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 IgG ₁ Fab preparation kit (manufactured by Pierce), and the Fc fragment was removed from the digest by adsorption on a protein A column.
The Fab fragment was obtained. The molecular weight of this product was about 50,000 as measured by electrophoresis. From this value, it was found that the obtained fragment was a Fab fragment, not an F (ab ′) ₂ fragment.

<3> Evaluation of Anti-Tyrosinase Monoclonal Antibody Fab Fragment (1) Specificity of Anti-Tyrosinase Monoclonal Antibody Fab Fragment to Human Tyrosinase Regarding the monoclonal antibody Fab fragment obtained above, MeWo cells (cultured cells derived from human malignant melanoma) ) And HeLa cells (cultured cells derived from human cervical cancer) were used to evaluate the specificity 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 (hereinafter simply referred to as “PBS”), the cells were collected using a rubber policeman. The collected cells were washed again with PBS several times to obtain a cell lysis buffer (0.1 M containing Triton X-100 at 0.5%).
Suspended in sodium phosphate buffer, 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 lane of the gel was transferred to a 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 ℃.

Then, the membrane was washed with 0.1% Tween.
Washing with PBS buffer containing 20 (TPBS buffer) (15 minutes once, 5 minutes twice), immersing in the Fab fragment solution (5 μg / ml solution of about 2 ml) obtained in <2> above, It was gently shaken at 25 ° C. for about 1 hour. After washing the membrane, a commercially available secondary antibody solution (horseradish peroxidase (HRP) -labeled goat anti-mouse Ig's (γ + L) antibody: manufactured by TAGO, 10,
The 000-fold diluted solution was immersed in about 2 ml) and gently shaken at 25 ° C. for about 1 hour.

After washing the membrane with TPBS buffer (15 minutes once, 5 minutes 4 times), the anti-human tyrosinase monoclonal antibody Fab fragment was bound using a commercially available HRP detection kit (Amersham ECL kit). A band of the electrophoretic material 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 observed 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 Fab fragment obtained above was human. It was found to bind to a protein band having tyrosinase activity, that is, human tyrosinase. Furthermore, this Fab fragment did not bind to extracts of HeLa cells.

(2) Reactivity of anti-tyrosinase monoclonal antibody Fab fragment The monoclonal antibody Fab fragment obtained above was analyzed by enzyme-labeled antibody assay (ELISA).
Non-specific staining was compared with IgG that was not treated with proteolytic enzyme.

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 μ of the antigen protein (10 μg / ml) was added to each well of an ELISA microplate (Sumilon typeH (Sumitomo Bakelite)).
After adding 1 liter each and standing at 37 ° C for 1 hour, 100 μl of PBS containing 1% bovine serum-derived albumin was added, and the mixture was allowed to stand at 25 ° C for 1 hour.
Time blocking was performed. It was washed twice with PBS to prepare an antigen solid phase well containing tyrosinase. The purified tyrosinase monoclonal antibody IgG (50
0 ng / ml) and Fab fragment (500 ng)
/ Ml) was added to each and the mixture was incubated at 37 ° C for 1 hour. After washing as described above, horseradish peroxidase (HRP) -labeled goat anti-mouse I was used as a secondary antibody.
50 μl of g's (γ + L) antibody (manufactured by TAGO) was added and incubated at 37 ° C. for 1 hour. Similarly after washing,
-75 μl of a coloring solution containing phenylenediamine hydrochloride was added and incubated at 37 ° C. for 30 minutes. Two
The reaction was stopped with 5 μl of 1M sulfuric acid, and the absorbance at 420 nm was measured.

As shown in Table 1, Fab has a higher reactivity to tyrosinase than IgG, and it can be used at a lower concentration in actual use, and a non-specific reaction can be prevented.

[0054]

[Table 1]

Although the Fab fragment of the present invention was weak, it was found that the Fab fragment of the present invention specifically reacts with melanoma B-16, which is a mouse melanoma cell, by the same procedure.

[0056]

The Fab fragment of the monoclonal antibody of the present invention has high reactivity to tyrosinase and can prevent non-specific binding. Further, since it does not contain the Fc portion, it is less likely to be affected by macrophages. This monoclonal antibody can be obtained by partial digestion of the anti-tyrosinase monoclonal antibody of the present invention with a proteolytic enzyme.

[0057]

[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 number Office reference number FI technical display location // G01N 33/573 9281-4B C12N 5/00 B 33/577 9162-4B 15/00 ZNAC ( (72) Inventor Takashi Shibata 560 Kashio-cho, Totsuka-ku, Yokohama-shi, Kanagawa Pola Chemical Industry Co., Ltd. Totsuka Laboratory (72) Inventor Tomomi Kato 560, Kashio-cho, Totsuka-ku, Yokohama-shi, Kanagawa Toka Laboratory, Pola Chemical Industry Co., Ltd.

Claims (8)

[Claims] 1. A Fab fragment of an anti-tyrosylase antibody which recognizes at least a part of tyrosinase as an antigen. 2. The Fab 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 Fab fragment according to claim 1 or 2, wherein the antibody is a monoclonal antibody. 4. The Fab according to claim 3, wherein the monoclonal antibody is IgG and the subclass is IgG _1.
Fragment. 5. The Fab fragment according to claim 3 or 4, wherein the monoclonal antibody is derived from mouse cells. 6. The Fab fragment according to claim 1, wherein the tyrosinase is human tyrosinase. 7. The method for producing a Fab fragment according to claim 3, which comprises the steps of: (a) at least a part of the amino acid sequence of a region having low homology with the amino acid sequence of the tyrosinase-related protein in the amino acid sequence of tyrosinase; After immunizing a mammal with a conjugate of the peptide and a carrier, the splenocytes of the animal are taken out and fused with a cultured cell to prepare a hybridoma. (B) Binding to tyrosinase and tyrosinase-related protein Selecting a hybridoma producing a monoclonal antibody that does not bind to (c) a culture supernatant obtained by culturing the hybridoma in an appropriate medium, or collecting antibody protein from ascites obtained by culturing the hybridoma in the abdominal cavity of a mouse, (D) The antibody protein is partially digested with a protease and the Fab fragment and Fc fragment are digested. And cut into the door, F
Isolate the ab fragment. 8. The method according to claim 7, wherein the tyrosinase is human tyrosinase.