JPH07138300A - Anti-human tyrosinase monoclocan antibody - Google Patents

Abstract

PURPOSE:To obtain a monoclonal antibody capable of specifically recognizing a human tyrosinase as an antigen. CONSTITUTION:This monoclonal antibody is obtained by immunizing a mammal with a peptide having an amino acid sequence in a region having low homology with the amino acid sequence of a tyrosinase-related protein in the amino acid sequence of a human tyrosinase as an antigen, fusing a cell of the spleen of the mammal to a cultured cell, preparing a hybridoma, culturing the prepared hybridoma and collecting the antibody from the resultant culture solution.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an anti-human tyrosinase monoclonal antibody useful for detecting human tyrosinase and a method for producing the same.

[0002]

2. Description of the Related Art Tyrosinase is the most important enzyme for biosynthesizing melanin pigment in vivo, and it is known that inhibition of tyrosinase biosynthesis causes serious skin disorders. For example, tyrosinase is considered to be produced in the ribosome of the rough endoplasmic reticulum, then concentrated and activated in the endoplasmic reticulum associated with the Golgi apparatus, and selectively transferred to premelanosomes produced by the Golgi apparatus.

Inhibition of tyrosinase biosynthesis is known to cause serious skin damage. For example, dyschromatosis is known to occur due to structural changes in tyrosinase itself and abnormalities in its biosynthesis process, and it is possible that abnormalities in selective transfer of tyrosinase to premelanosomes are involved. .

[0004] It is considered very useful to know the behavior of tyrosinase in a living body or a cell for the diagnosis of such a disease or the investigation of the cause. Efforts have been made. In particular, the method using an antigen-antibody reaction has been variously studied because of its high sensitivity, and polyclonal antibodies against tyrosinase have been produced for a long time.

However, a polyclonal antibody is produced by immunizing an animal each time and taking out from the blood,
Since the antibody titer varies greatly from lot to lot and the reproducibility is poor, it is not practical and has not been commercialized. Therefore, it has been attempted to immunize an animal with tyrosinase as an antigen, and then fuse the lymphocytes of this animal with cultured cells such as myeloma to prepare a hybridoma, and then to produce an anti-tyrosinase monoclonal antibody. (Margherita Cuo-mo et.al., J. Inve
st. Dermato., 96 (4), 446-451 (1991); Yasushi Tomita
et. al., J. Invest. Dermato., 85 (5), 426-430 (198
Five))

[0006]

However, it was subsequently clarified that the anti-tyrosinase monoclonal antibody in the above report was a monoclonal antibody against a tyrosinase-related protein (Yasushi Tomita et.
al., J. Invest. Dermato., 96 (4), 500-504, 199
1). This is because there is a protein called a tyrosinase-related protein (hereinafter referred to as "TRP") that has a very similar amino acid sequence and molecular weight to tyrosinase in the living body, and therefore tyrosinase is completely separated from TRP. This is because, in the conventional technique of immunizing an animal with tyrosinase purified from cells or tissues as an antigen, the contaminated TRP serves as an antigen to produce an anti-tyrosinase-related protein antibody.

Therefore, the present situation is that a monoclonal antibody that specifically recognizes tyrosinase as an antigen has not yet been obtained. The present invention has been made from such a viewpoint, and an object thereof is to provide a monoclonal antibody that does not recognize TRP as an antigen but specifically recognizes human tyrosinase as an antigen.

[0008]

Means for Solving the Problems As a result of intensive studies for solving the above problems, the present inventor has identified a peptide having a sequence having a low homology with the TRP sequence in the amino acid sequence of human tyrosinase as an antigen. By using TRP
It was found that a monoclonal antibody that specifically binds to human tyrosinase without being bound to Escherichia coli was obtained, and the present invention was completed.

That is, the present invention provides an anti-human tyrosinase monoclonal antibody that recognizes, as an antigen, a region of human tyrosinase amino acid sequence having low homology with the amino acid sequence of tyrosinase-related protein, and tyrosinase amino acid sequence of human tyrosinase. After immunizing a mammal with a conjugate of a peptide having at least a part of the amino acid sequence having a low homology with the amino acid sequence of the related protein and a carrier, the splenocytes of the animal are taken out and fused with the cultured cells. A method for producing a monoclonal antibody specific for human tyrosinase, which comprises producing a hybridoma by culturing the hybridoma and culturing the hybridoma and collecting an antibody protein from the culture.

The term "specifically binds to human tyrosinase" as used herein means to specifically recognize human tyrosinase as an antigen, and specifically not to bind to TRP.

The present invention will be described in detail below. The cDNA sequence of human tyrosinase is already known (Hiroaki
Yamamoto et.al., Jpn. J. Genet, 64, 121-135 (198
9)), and the human TRP cDNA sequence is already known (Sigeki Shibahara et. Al., Tohoku J. exp. Med., 1
56, 403-414 (1988)). We have these cDNAs
The sequences were translated into amino acid sequences and compared (Figure 1). Figure 1
In the above, in the upper lane, the amino acid sequence of human tyrosinase is shown, and in the lower lane, the amino acid sequence of TRP is shown by a one-letter code. The (:) shown between the lanes is the portion where the amino acids are completely the same,
(.) Is the same type of amino acid (hydrophobic, hydrophilic, basic, etc.)
Is the part that matches. As a result, in the amino acid sequence of human tyrosinase, the portion having the amino acid sequence shown in SEQ ID NO: 1 (underline portion in FIG. 1) was T
It was found to be one of the parts having low homology with RP.

Focusing on this point, the present inventors synthesized a synthetic peptide having the above sequence (hereinafter, also referred to as “human tyrosinase-specific peptide”) (requested synthesis to Peptide Institute Inc.). ). 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. Hereinafter, a human tyrosinase-specific monoclonal antibody and a method for producing the same will be described along with the production procedure.

<1> Antigen The peptide specific to human tyrosinase used for immunizing mammals includes the sequence shown in SEQ ID NO: 1 as its amino acid sequence. Among the amino acid sequences of human tyrosinase, tyrosinase-related protein As long as it has at least a part of the amino acid sequence of a region having low homology with the amino acid sequence of, it can be used in the present invention. Such a peptide can be partially obtained, for example, by isolating it from an enzymatic degradation product of tyrosinase, but it is preferably chemically synthesized by a conventional method. For example, the above peptide can be obtained by inputting the above sequence using a commercially available peptide synthesizer. 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 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 diluted with, for example, RPMI1640 or MEM medium, and the cells washed by centrifugation are suspended in a selective medium such as HAT medium and dispensed on a multiplate or the like to culture, and only hybridomas are cultured. Grow

<4> Selection of hybridoma The hybridoma obtained as described above is a mixture of strains of hybridomas that produce monoclonal antibodies against different antigen-determining sites of the peptides used as antigens or monoclonal antibodies against the 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.

The selection method is an enzyme-linked immunosorbent assay (ELISA) using a synthetic peptide used as an antigen.
Is preferred. For example, the amount of the antibody bound to the synthetic peptide can be known from the amount of the hybridoma culture supernatant and the amount of the enzyme, fluorescent substance or luminescent substance bound to the synthetic peptide, which has been immobilized on a plastic plate or the like. Alternatively, the antibody produced by the hybridoma may be solid-phased and then sequentially incubated with a labeled secondary antibody using a synthetic peptide, an enzyme or the like.

One strain of the hybridoma obtained in this way was sent to the Institute of Biotechnology, Institute of Industrial Science and Technology, FER
Deposited as MP-13937.

<5> Preparation of monoclonal antibody that specifically binds to human tyrosinase and method of using the same To obtain a monoclonal antibody that specifically binds to human tyrosinase from the hybridoma obtained as described above, for example, antibody A monoclonal antibody specific to human tyrosinase can be obtained by purifying the produced hybridoma by ammonium sulfate fractionation, gel filtration, affinity chromatography and the like according to a conventional method.

Since the monoclonal antibody of the present invention which specifically binds to human tyrosinase specifically reacts with human tyrosinase, it is a chromosomal antibody in immunostaining of human-derived cultured cells, skin tissue, etc. It can be used as an antibody for staining in microautoradiography, and can be used as a detection reagent for qualitative or quantitative determination of human tyrosinase.

[0026]

EXAMPLES Examples of the present invention will be described below. <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 carrier-bonded 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, a mouse myeloma cell line P3X63Ag8.653 (purchased from Dainippon Pharmaceutical Co., Ltd.) was cultured at a 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 in 2 minutes and 8 ml in 3 minutes with stirring.

After completion of 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. This strain has been deposited as FERM P-13937 at the Institute of Biotechnology, Industrial Technology Institute.

<2> Preparation of monoclonal antibody that specifically binds to human tyrosinase The hybridoma cell line obtained above was cultured and a monoclonal antibody that specifically binds to human 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 through 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.

<3> Evaluation of anti-human tyrosinase monoclonal antibody (1) Reactivity of anti-human tyrosinase monoclonal antibody with human 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 enzyme immunoblotting.

MeWo and H cultured in culture flask
After removing the culture supernatant of eLa cells and washing several times with 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 cell extract. 5-2
0% density gradient polyacrylamide gel (Pagel: A
It was subjected to electrophoresis using TTO). The applied amount is
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 transferred to a TBS buffer (20 mM, Tris; 500 mM, NaCl).
After immersing in pH 7.5) for 15 minutes, it was immersed in a blocking buffer (TBS buffer containing 5% skim milk or 1% BSA and 0.1% Tween 20) and gently shaken at 25 ° C. for 1 hour.

Then, the membrane was washed with 0.1% Tween.
After washing with TBS buffer containing 20 (TTBS buffer) (15 minutes once, 5 minutes twice), the monoclonal antibody solution obtained in the above <2> (10 times dilution station approximately 2 times).
ml) and 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, 10,0) was used.
The 00-fold diluted solution was immersed in about 2 ml) and gently shaken at 25 ° C. for about 1 hour.

After washing the membrane with TTBS buffer (15 minutes once, 5 minutes 4 times), a commercially available HRP detection kit (Amersham ECL kit) was used to bind an anti-human tyrosinase monoclonal antibody Band was detected. The band to which HRP is bound is
It can also be detected by using 4-chloro-1-naphthol as a substrate.

The results of the activity staining and the results of enzyme immunoblotting are shown in FIG. As is clear from this result, the monoclonal antibody obtained above binds to a protein band having human tyrosinase activity, that is, human tyrosinase. Furthermore, this monoclonal antibody does not bind to extracts of HeLa cells.

(2) Analysis of Antigen Binding to Anti-Human Tyrosinase Monoclonal Antibody Regarding the monoclonal antibody obtained above, MeWo
The extract was used to assess specificity by immunoprecipitation.

After removing the culture supernatant of MeWo cells cultured in a culture flask and washing several times with PBS, the cells were recovered using a rubber policeman. The collected cells were washed again with PBS several times and suspended in the cell lysis buffer.

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.

Protein A-Sepharose
20 μl of sepharose (manufactured by Sigma) was placed in a microtube and washed with a binding buffer (binding buffer: 1.5 M glycine, 3 M NaCl, pH 8.9).

To the washed protein A-Sepharose, an equal volume mixture of the above-obtained monoclonal antibody solution and binding buffer was added, and gently mixed by inversion at 4 ° C. for 1 hour to allow the monoclonal antibody to bind to the protein. It was bound to A-Sepharose. This was washed 3 times with a cell lysis buffer, the above cell extract (about 25 μg protein) was added, and then gently mixed for 2 hours or more at 4 ° C. After the mixing was completed, the mixture was centrifuged at 10,000 × g for 1 minute, and the tyrosinase activity of the supernatant (the supernatant thus obtained is hereinafter referred to as antibody-treated MeWo extract) was measured.

The tyrosinase activity is MBTH (3
-Methyl-2-benzothiazolinone hydrazone) method. The method is shown below. Antibody-treated MeWo as described above
30 μl of the extract and MeWo extract were heated at 37 ° C. for 10 minutes, and then DO was previously heated to 37 ° C.
PA solution (12 mM, MBTH; 4%, dimethyl)
formamide; 2 mM, L-DOPA) 30 μl
Was added to each to start the reaction. Reaction is 3
The reaction was stopped at 70C for 30 minutes and then transferred to ice. Since the absorbance of the reaction solution at 505 nm increases as the reaction proceeds, the tyrosinase activity was examined by measuring this value. The results are shown in Table 1.

[0048]

[Table 1]

As can be seen from this table, the monoclonal antibody obtained above reduces the tyrosinase activity in the cell extract by about half. That is, it is clear that the monoclonal antibody obtained above binds to human tyrosinase.

On the other hand, in order to examine the reactivity of the monoclonal antibody obtained above with TRP, the above antibody treatment M
Using eWo extract and untreated MeWo extract as antigens,
Prior art anti-tyrosinase monoclonal antibodies (anti-TR
P monoclonal antibody) and a commercially available secondary antibody described in Example <1> were used for sandwich ELISA. The results are shown in Table 2.

[0051]

[Table 2]

From the table, there is no difference in the values in the ELISA using the antibody-treated MeWo extract solution and the untreated MeWo extract solution as antigens. Therefore, the antigen binding to the monoclonal antibody obtained above was the anti-tyrosinase of the prior art. It was found that it did not bind to the monoclonal antibody (anti-TRP monoclonal antibody).

That is, it was revealed that the above-obtained monoclonal antibody is a completely different antibody from the conventional anti-tyrosinase monoclonal antibodies and does not bind to TRP.

[0054]

INDUSTRIAL APPLICABILITY The present monoclonal antibody specifically binds to human tyrosinase. This monoclonal antibody
Obtained by the method of the present invention.

[0055]

[Sequence list]

 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

[0056]

[Brief description of drawings]

[0057]

FIG. 1 is a diagram showing the homology of amino acid sequences of human tyrosinase and TRP.

[0058]

FIG. 2 is a photograph showing the results of electrophoresis by tyrosinase activity staining and enzyme immunoblotting.

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Claims (3)

[Claims] 1. An anti-human tyrosinase monoclonal antibody that recognizes, as an antigen, a region having a low homology with the amino acid sequence of a tyrosinase-related protein in the amino acid sequence of human tyrosinase. 2. The anti-human tyrosinase monoclonal antibody according to claim 1, wherein the region is at least a part of the amino acid sequence shown in SEQ ID NO: 1 in Sequence Listing. 3. Immunizing a mammal with a conjugate of a peptide having at least a part of the amino acid sequence of a region having low homology with the amino acid sequence of tyrosinase-related protein among the amino acid sequences of human tyrosinase after immunizing a mammal The production of a monoclonal antibody specific for human tyrosinase, characterized in that splenocytes of the animal are taken out, fused with cultured cells to prepare hybridomas, and the hybridomas are cultured and antibody proteins are collected from the cultures. Law.