JP3532945B2 - Anti-human tyrosinase monoclonal antibody - Google Patents

Description

Description: BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an anti-human tyrosinase monoclonal antibody useful for detecting human tyrosinase and a method for producing the same. [0002] Tyrosinase is the most important enzyme for biosynthesizing melanin pigment in vivo, and it is known that inhibition of tyrosinase biosynthesis causes serious skin damage. . For example, tyrosinase is thought to be produced in the ribosomes of the rough endoplasmic reticulum, then enriched and activated in the Golgi organ-associated endoplasmic reticulum, and selectively transferred to premelanosomes produced by the Golgi organ. [0003] It is known that a serious skin disorder occurs when tyrosinase biosynthesis is inhibited. For example, dyschromia is known to be caused by mutations in the structure of tyrosinase itself or abnormalities in its biosynthesis process, and may involve abnormalities in the selective transfer of tyrosinase to premelanosomes. . In diagnosing such diseases or investigating the causes, it is considered to be very useful to know the behavior of tyrosinase in living organisms or cells. Efforts have been made. In particular, methods using an antigen-antibody reaction have been studied variously because of their high sensitivity, and polyclonal antibodies against tyrosinase have been produced for a long time. [0005] However, polyclonal antibodies are produced each time by immunizing an animal and extracting it from the blood.
There is a major drawback in that the antibody titer varies greatly from lot to lot and the reproducibility is poor, so that it is not practical and has not been commercialized. For this reason, attempts have been made to immunize an animal using tyrosinase as an antigen, and then fuse the lymphocytes of this animal with cultured cells such as myeloma to produce a hybridoma and use this 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
5)) However, it was later revealed that the anti-tyrosinase monoclonal antibody described 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 tyrosinase-related protein (hereinafter, referred to as "TRP") having a very similar amino acid sequence and molecular weight to tyrosinase exists in a living body, and therefore tyrosinase is completely separated from TRP. This is extremely difficult, and in the conventional technique of immunizing an animal with tyrosinase purified from cells or tissues as an antigen, the contaminating TRP becomes an antigen and an anti-tyrosinase-related protein antibody is generated. [0007] Therefore, at present, 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 it is an object of the present invention to provide a monoclonal antibody that does not recognize TRP as an antigen and specifically recognizes human tyrosinase as an antigen. Means for Solving the Problems The inventors of the present invention have conducted intensive studies in order to solve the above-mentioned problems, and as a result, a peptide having a sequence having low homology with the sequence of TRP in the amino acid sequence of human tyrosinase Using TRP as an antigen, TRP
The present inventors have found that a monoclonal antibody that specifically binds to human tyrosinase without binding to tyrosinase can be obtained, thereby completing the present invention. That is, the present invention provides an anti-human tyrosinase monoclonal antibody which recognizes, as an antigen, a region having low homology to the amino acid sequence of a tyrosinase-related protein in the amino acid sequence of human tyrosinase, and a tyrosinase in the amino acid sequence of human tyrosinase. After immunizing a mammal with a conjugate of a carrier and a peptide having at least a part of the amino acid sequence of a region having low homology to the amino acid sequence of the related protein, spleen cells of the animal are removed and fused with cultured cells. A method for producing a monoclonal antibody specific to human tyrosinase, which comprises preparing a hybridoma, and culturing the hybridoma and collecting an antibody protein from the culture. [0010] In the present specification, "specifically binds to human tyrosinase" refers to specifically recognizing human tyrosinase as an antigen, and particularly to not binding to TRP. Hereinafter, the present invention will be described in detail. The cDNA sequence of human tyrosinase is already known (Hiroaki
Yamamoto et.al., Jpn.J. Genet, 64, 121-135 (198
9)), and the cDNA sequence of human TRP is already known (Sigeki Shibahara et. Al., Tohoku J. exp. Med., 1).
56, 403-414 (1988)). We have determined that these cDNAs
The sequences were translated into amino acid sequences and compared (FIG. 1). FIG.
, The upper lane shows the amino acid sequence of human tyrosinase, and the lower lane shows the amino acid sequence of TRP with one-letter code. The (:) shown between the lanes indicates that the amino acids are completely identical,
(.) Indicates the same kind of amino acids (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 in FIG. 1)
It was found to be one of the parts having low homology with RP. The present inventors have paid attention to this point, and have synthesized a synthetic peptide having the above sequence (hereinafter also referred to as “peptide specific to human tyrosinase”) (Peptide Research Institute, Inc.) ). After immunizing a mammal with the obtained peptide, spleen cells were taken out and fused with cultured cells to prepare a hybridoma, and the hybridoma succeeded in producing a tyrosinase-specific monoclonal antibody. Hereinafter, a monoclonal antibody specific to human tyrosinase and a method for producing the same will be described along the production procedure. <1> Antigen The peptide specific to human tyrosinase used for immunizing a mammal includes the amino acid sequence shown in SEQ ID NO: 1. Among the amino acid sequences of human tyrosinase, tyrosinase-related proteins are included. As long as it has at least a part of the amino acid sequence of a region having low homology to the amino acid sequence of the present invention, 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 is preferably chemically synthesized by a conventional method. For example, by inputting the above sequence using a commercially available peptide synthesizer, the above peptide can be obtained. Alternatively, it may be obtained by entrusting 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, or can be used as a keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA). ), Ovalbumin (OVA) and the like. When such a carrier is used, the antigenicity can be increased even when the peptide alone has low antigenicity or no antigenicity. In this case, antibodies to the carrier can be used, but they can be removed at the stage of selecting the hybridoma. <2> Immunization of mammal The mammal to be immunized with the above-mentioned antigen is not particularly limited as long as it is a mammal usually used for immunization experiments, and examples thereof include mice, rats, rabbits and the like. Also, how to immunize
The immunization may be performed in accordance with a general immunization technique. For example, a synthetic peptide or a synthetic peptide bound to a carrier may be used in an amount of 0.5
mg / kg to 50 mg / kg with Freund's complete or incomplete adjuvant
There is a method in which administration is repeated several times every two weeks, and at least two weeks after the final administration, only the synthetic peptide is administered to perform final immunization. At this time, the dose of the synthetic peptide or the synthetic peptide bound to the carrier is 0.5 m / time.
The appropriate amount is about g / kg to 50 mg / kg. If the dose is less than 0.5 mg / kg, the antibody may not be produced sufficiently. Further, even if the amount exceeds 50 mg / kg, no further immunological effect can be expected, and furthermore, immunosuppression may occur 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 removed, and the spleen cells are removed. The spleen cell-cultured cell hybridoma can be obtained by fusing the cell and a cultured cell as a fusion partner in the presence of a fusion promoter and selecting the obtained fused cell. The culture cell of the fusion partner is not particularly limited as long as it fuses with spleen cells, but myeloma cells (myeloma cells) are generally suitable. In addition, 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. For example, those lacking hypoxanthine / guanine / phosphoribosyltransferase can be mentioned. Such cells cannot grow in a medium supplemented with hypoxanthine / aminopterin and thymidine (HAT medium).
The cells can grow in the medium, and can be distinguished from unfused cells. Specifically, P3X63Ag8.653 strain, P3 / NSI / 1-AG4-1
Strain, FO strain, SP2 / 0-Ag14 strain, and other commercially available strains of myeloma cells, but are not limited thereto. Cell fusion is usually performed using RPMI 1640, MEM
In a medium such as the above, antibody-producing cells (splenocytes) and myeloma cells are mixed at a mixing ratio of 10: 1 to 2: 1 with a fusion promoter. As a fusion promoter,
There is no particular limitation as long as it is a fusion agent commonly used in cell fusion experiments, and specific examples include Sendai virus and polyethylene glycol having an average molecular weight of 500 to 7000. The fusion may be performed by an electric pulse. The cells after 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, dispensed into a multiplate, etc., and cultured, and only the hybridoma is cultured. <4> Selection of hybridoma The hybridoma obtained as described above is a hybridoma that produces a monoclonal antibody against a different antigenic determinant site among peptides used as an antigen or a monoclonal antibody against a protein used as a carrier. , 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 of this selection, an enzyme-linked immunosorbent assay (ELISA) 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 amount of the antibody that binds to the synthetic peptide can be known from the amount of the hybridoma culture supernatant, enzyme, fluorescent substance, or luminescent substance bound thereto. Alternatively, the antibody produced by the hybridoma may be immobilized and then sequentially incubated with a labeled second antibody using a synthetic peptide, an enzyme, or the like. One strain of the hybridoma obtained in this manner was sent to the Institute of Biotechnology and Industrial Technology,
Deposited as MP-13937. <5> Preparation of Monoclonal Antibody That Binds Specifically to Human Tyrosinase and Use Thereof To obtain a monoclonal antibody that specifically binds to human tyrosinase from the hybridoma obtained as described above, for example, Purification of the produced hybridoma by ammonium sulfate fractionation, gel filtration, affinity chromatography or the like according to a conventional method yields a monoclonal antibody specific to human tyrosinase. Since the monoclonal antibody of the present invention which specifically binds to human tyrosinase reacts specifically with human tyrosinase, it can be used as a chromosome antibody for immunostaining of human-derived cultured cells, skin tissues, etc. It can be used as a staining antibody in microautoradiography and can be used as a detection reagent for qualitative or quantitative determination of human tyrosinase. Embodiments of the present invention will be described below. <1> Preparation of Hybridoma Synthesis of peptide and binding to carrier (KLH) were requested to Peptide Research Institute, Ltd. The above carrier-bound synthetic peptide (100 μg) was dissolved in physiological saline (100 μl), mixed and emulsified with complete Freund's adjuvant (SIGMA) (100 μl), and administered to the abdominal cavity of an 8-week-old BALB / c mouse (CLEA Japan). did. After 1 week and 2 weeks, 200 μl of the above-mentioned carrier-bound synthetic peptide-incomplete Freund's adjuvant mixed emulsion was intraperitoneally administered. Further, two weeks after the third administration, 100 μl of physiological saline in which 50 μg of the synthetic peptide was dissolved was intravenously injected. Three days later, the spleen was removed from the mouse, and the spleen cells were suspended in RPMI1640 medium and washed.
On the other hand, the mouse myeloma cell line P3X63Ag8.653 (purchased from Dainippon Pharmaceutical Co., Ltd.) was cultured in the logarithmic growth phase in accordance with the cell fusion, and collected by centrifugation. For 10 ⁸ spleen cells, 2 × 10 ⁷ myeloma were mixed in the above medium, and the cells were pelleted by centrifugation. After removing the supernatant, the mixture was kept at 37 ° C. 5
RPMI1 containing 0% PEG4000 (manufactured by SIGMA)
1 ml of 640 medium was gradually added dropwise over 1 minute, followed by gentle stirring for 1 minute. Furthermore, RPMI 1640 medium 2 at 37 ° C.
ml are added dropwise over 2 minutes and a further 8 ml over 3 minutes with stirring. After 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).
And dispensed 100 μl per well into four 96-well plates (Sumitomo Bakelite). The next day, 25 μl of HAT medium was added, and 4 days later, 2
5 μl of HAT medium was added. After culturing for one week, half of the culture supernatant was removed, and 100 μl of GIT medium was added. About two weeks after the cell fusion, only the hybridoma in which the myeloma and spleen cells were fused formed a colony, and the culture was continued until the colony became about 1 mm in diameter. At this point, the antibody secreted into the culture supernatant is
The above synthetic peptide and a commercially available secondary antibody (horseradish peroxidase (HRP) -labeled goat anti-mouse Ig's (γ
+ L) Antibody: Tag E)
The test was performed by the LISA method. Among them, the hybridomas in the wells having a high antibody titer were cloned by the limiting dilution method to obtain a monoclonal antibody-producing hybridoma strain. This strain has been deposited with the National Institute of Advanced Industrial Science and Technology as a FERM P-13937. <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 hybridoma was cultured in a GIT medium, and when the cell concentration reached about 5 × 10 ⁶ cells / ml, the culture supernatant was collected by centrifugation, and this was filtered through a filter having a pore size of 0.22 μm. The solution was purified using a protein A column kit (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 extracts of cells (cultured cells derived from human malignant melanoma) and HeLa cells (cultured cells derived from human cervical cancer),
The specificity was evaluated by enzyme immunoblot. MeWo and H cultured in a culture flask
After removing the culture supernatant of eLa cells and washing with PBS several times, the cells were collected using a rubber policeman. The collected cells were washed several times with PBS again, and then washed with a cell lysis buffer (0.1 M containing 0.5% Triton X-100).
The suspension was suspended in a sodium phosphate buffer (pH 6.9). The cells were crushed by treating them with an ultrasonic cell crusher (manufactured by Tosho Denki) for 30 seconds, and the cells were crushed at 16,000 × G for 1 hour.
Centrifugation was performed for 0 minutes. The protein concentration contained in the obtained supernatant was measured to obtain a cell extract. This is 5-2
0% density gradient polyacrylamide gel (Pagel: A
(Manufactured by TTO). The amount of applied
The amount of protein per lane is 10 μg / lane. The electrophoresis was performed according to a conventional method. After the electrophoresis, the gel was taken out, one lane was cut out, and tyrosinase activity staining (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 a transfer buffer (100 mM, Tris; 192 mM, g).
After immersion in lycin), the electrophoresis material was transferred from the gel to a membrane (Immobilon; manufactured by Millipore) previously immersed in a transfer buffer using a blotting device (semi-dried blotting device: manufactured by ATTO). The membrane onto which the electrophoresis material was transferred was placed in a TBS buffer (20 mM, Tris; 500 mM, NaCl).
(pH 7.5) for 15 minutes, then immersed in a blocking buffer (5% skim milk or TBS buffer containing 1% BSA and 0.1% Tween 20) and gently shaken at 25 ° C. for 1 hour. Thereafter, the membrane was washed with 0.1% Tween.
After washing with a TBS buffer containing 20 (TTBS buffer) (once for 15 minutes and twice for 5 minutes), the monoclonal antibody solution obtained in the above <2> (10-fold dilution station is about 2
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: 10,0, manufactured by TAGO)
The resultant was immersed in about 2 ml of a 00-fold diluted solution and gently shaken at 25 ° C. for about 1 hour. After washing the membrane with TTBS buffer (once for 15 minutes, 4 times for 5 minutes), using a commercially available HRP detection kit (ECL kit manufactured by Amersham), the electrophoreses bound to the anti-human tyrosinase monoclonal antibody were used. Band was detected. The band to which HRP was bound was
It can be detected even when 4-chloro-1-naphthol is used as a substrate. FIG. 2 shows the results of the activity staining and the results of the enzyme immunoblot method. As is clear from these results, 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 The monoclonal antibody obtained above was analyzed using MeWo.
The specificity was evaluated by immunoprecipitation using the extract. After removing the culture supernatant of the MeWo cells cultured in the culture flask and washing with PBS several times, the cells were collected using a rubber policeman. The collected cells were again washed several times with PBS and suspended in a cell lysis buffer. The cells were crushed by treatment with an ultrasonic cell crusher (manufactured by Tosho Denki) for 30 seconds, and the cells were crushed at 16,000 × G for 1 second.
Centrifugation was performed for 0 minutes. The protein concentration contained in the obtained supernatant was measured to obtain a cell extract. [0045] Protein A-Sepharose
Sepharose (manufactured by Sigma) (20 μl) was placed in a microtube, and washed using 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 the binding buffer was added, and the mixture was gently inverted at 4 ° C. for 1 hour to mix the monoclonal antibody with the protein. A-Sepharose was bound. This was washed three times with a cell lysis buffer, and after adding the above cell extract (about 25 μg protein), the mixture was gently mixed at 4 ° C. for 2 hours or more. After completion of the mixing, the mixture was centrifuged at 10,000 × g for 1 minute, and the tyrosinase activity of the supernatant (the resulting supernatant was hereinafter referred to as an antibody-treated MeWo extract) was measured. Note that the tyrosinase activity was measured using MBTH (3
-Methyl-2-benzothiazolinone hydrazone) method. The method is described below. Above-treated antibody MeWo
The extract and the MeWo extract (30 μl) were heated at 37 ° C. for 10 minutes, and then DO preheated to 37 ° C.
PA solution (12 mM, MBTH; 4%, dimethyl
(formamide; 2 mM, L-DOPA) 30 μl
Was added to each to initiate the reaction. Reaction 3
The reaction was performed at 7 ° C., and after 30 minutes, the reaction was stopped by transferring onto ice. As the reaction proceeds, the absorbance of the reaction solution at 505 nm increases, and the tyrosinase activity was examined by measuring this value. Table 1 shows the results. [Table 1] As can be seen from the table, the monoclonal antibody obtained above reduced 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 between the monoclonal antibody obtained above and TRP, the antibody-treated M
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>. The results are shown in Table 2. [Table 2] As can be seen from the table, there is no difference in the ELISA values obtained using the antibody-treated MeWo extract and the untreated MeWo extract as antigens. It was found that it did not bind to the monoclonal antibody (anti-TRP monoclonal antibody). That is, it was revealed that the monoclonal antibody obtained above was completely different from the conventional anti-tyrosinase monoclonal antibody and did not bind to TRP. The present monoclonal antibody specifically binds to human tyrosinase. This monoclonal antibody
Obtained by the method of the present invention. [Sequence List] SEQ ID NO: 1 Sequence length: 14 Sequence type: Amino acid topology: Type of linear sequence: Peptide sequence Met Glu Lys Glu Asp Tyr His Ser Leu Tyr Gln Ser His Leu 1510 [0056]

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the homology of amino acid sequences of human tyrosinase and TRP. FIG. 2 is a photograph showing the results of electrophoresis by tyrosinase activity staining and enzyme immunoblot.

Continuation of front page (56) References Jpn. J. Genet. Vol. 64 (1989) p. 121-135 Tohoku J .; Exp. Med. , Vol. 156 (1988) p. 403-414 J.C. Biol. Chem. 264 [6] (1989) p. 3397-3403 Biol. Chem. , Vol. 266 [2] (1991) p. 1147-1156 (58) Field surveyed (Int. Cl. ⁷ , DB name) C07K 16/40 C12P 21/08 JICST file (JOIS) BIOSIS / WPI (DIALOG) PubMed

Claims (1)

(57) [Claim 1] Consists of the amino acid sequence shown in SEQ ID NO: 1.
Anti-human tyrosinase mono recognizing peptide as antigen
Clonal antibodies.