JP2614822B2 - Method for producing antibody-producing hybridoma - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a monoclonal antibody,
In particular, the present invention relates to a method for producing a hybridoma that produces a monoclonal antibody that recognizes a sugar chain containing N-glycolylneuraminic acid.

[0002]

BACKGROUND OF THE INVENTION Glycolipids are components of the plasma membrane of cells,
Various molecular species exist depending on the type, number, and binding method of the constituent sugars, and show species-specific, organ-specific, and cell-specific distributions. Its function is to act as a receptor for bacterial toxins and hormones, as well as immunological determinants such as blood group substances, and also play an important role in the control of growth or differentiation and interaction between cells. It is becoming clear that they are performing. Furthermore, with the canceration of cells, qualitative and quantitative changes in composition occur, and it has been shown that some can be cancer antigens,
In addition, it has been suggested that the composition change may be directly involved in the canceration mechanism, such as an example in which a certain glycolipid acts as a regulator of a cell growth mechanism mediated by growth factors and protein kinases. On the other hand, Milstein et al. Reported a method for establishing a cell line producing a uniform antibody having specificity for one type of antigenic determinant [Nature]
256, 495-497 (1975)],
Qualitative and quantitative determination of trace substances has become possible. To search for cancer antigens, a number of monoclonal antibodies specific to cancer cells were produced using this technology.Some of these antibodies are those that recognize glycolipids or glycoprotein sugar chains. [Journal of National Cancer Institute (J. Natl.
Cancer Inst.) Vol. 71, pp. 231-251, (19
83)]. For example, GD ₂ ganglioside or GD _3, as a monoclonal antibody against melanoma human
Antibodies that react with glycolipids such as gangliosides have been obtained. In addition, monoclonal antibody N specific for pancreatic cancer
S19-9 reacts with a glycolipid having a sugar chain of sialosyl Lewis A type. These antibodies are useful for diagnosing cancer, observing the course of treatment, and are also being used for therapy. The qualitative and quantitative changes associated with glycolipid carcinogenesis are due to abnormal gene expression that alters the activities of various glycosyltransferases in the sugar chain biosynthesis mechanism, and as a result, are present in normal tissues. Such a sugar chain structure is created. The sugar chain structure can be used as a cancer marker. As described above, the importance and usefulness of glycolipids as clues for elucidating the mechanism of canceration and as cancer antigens and cancer markers are attracting attention, and their application to clinical fields such as diagnosis and treatment is expected.

[0003] Among glycolipids, those containing sialic acid, which is an acidic sugar, in their sugar chains are collectively referred to as gangliosides. The main types of sialic acids are N-acetylneuraminic acid and N-glycolylneuraminic acid.
Sialic acid is widely detected in various organs, cells and body fluids of animal species, but N-glycolylneuraminic acid has not been found so far in normal humans and chickens. Heterophilic antibodies that are detected in patients with serum sickness and agglutinate red blood cells from sheep, horses, pigs, rabbits, and guinea pigs are known as Hanganatziu-Deicher (H-analyte).
D). The antigen recognized by this antibody is called HD antigen. It has been reported that gangliosides containing N-glycolylneuraminic acid have HD antigen activity [Biochemical and Biophysical Research Communications (Biochem. B).
iophys. Res. Commun.) Vol. 79, pp. 388-395 (1977)], and the sugar chain structure of NeuGcα2-3Gal was identified as a main antigenic determinant. In recent years, HD
N-glycolyl GM ₃ ganglioside (II ³ NeuGc-LacCe) which is a ganglioside having antigen activity
immunizing chickens with r) to produce antibodies from serum that react with various gangliosides having HD antigen activity,
Using this antibody, it was reported that N-glycolylneuraminic acid was characteristically present in human cancer tissues [Viken
Journal (Biken J.), Vol. 25, pp. 47-50 (1982)]. In addition, several types of HD antigen-active N-glycolylneuraminic acid-containing gangliosides were detected in glycolipids extracted from human colon cancer tissues, and HD antigens were detected in teratoma tissues. Glycoproteins with active sugar chains were detected [Gann, Vol. 75,
1025 to 1029 (1984)]. Analysis of glycolipids having HD antigen activity detected from human colon cancer tissue using the antibody prepared in chicken showed that N-
Glycolyl GM ₂ Ganglioside, N-glycolyl GM
₃ Ganglioside, O-acyl-N-glycolyl GM ₃
Ganglioside, IV ³ NeuGc-nLcOse ₄ Ce
r, which were not detected in normal tissues [Cancer Res. 45, 379
6-3802 (1985)]. Gangliosides having HD antigen activity were also detected in a cell line of Marek's disease, a kind of chicken lymphoma, {Journal of Biochemistry (Tokyo), [J. Bioche
m. (Tokyo)] 95, 785-794 (198
4) ②.

[0004] N-glycolylneuraminic acid and N-glycolylneuraminic acid-containing sugar chains are considered to be cancer-related antigens, and it is extremely important to detect them with high sensitivity and accuracy in cancer diagnosis. In order to efficiently detect N-glycolylneuraminic acid or a sugar chain containing N-glycolylneuraminic acid, an immunoassay is considered to be excellent in terms of detection sensitivity and accuracy. Antibodies that react with N-glycolylneuraminic acid-containing sugar chains having HD antigen activity have conventionally been obtained by immunizing chickens with purified glycolipid antigens having HD antigen activity and separating them from serum. Was done.
However, this method has several disadvantages. That is, (1) a large amount of purified antigen is required each time to obtain an antiserum. (2) There are variations in affinity and titer mainly due to individual differences between immunized animals. (3) Since antibodies other than the target antibody are also present, complicated operations are required to purify the target antibody. (4) There is a drawback that the amount that can be manufactured at one time is limited. Therefore, in order to perform an immunological measurement accurately and with the maximum effect, it has been desired to be able to supply a large amount of a stable and uniform antibody of a quality free from contamination with other antibodies. The production of such antibodies has already been reported as a monoclonal antibody production technique.

[0005]

However, the HD
To date, there has been no report that a monoclonal antibody that reacts with an N-glycolylneuraminic acid-containing sugar chain having antigen activity and a hybridoma capable of producing the antibody have been successfully produced. An object of the present invention is to provide a method for producing a hybridoma that produces a monoclonal antibody that specifically recognizes an N-glycolylneuraminic acid-containing sugar chain having HD antigen activity.

[0006]

SUMMARY OF THE INVENTION The present invention is summarized as an invention relating to a method for producing a hybridoma, comprising a method for producing a monoclonal antibody-producing hybridoma that specifically recognizes an N-glycolylneuraminic acid-containing sugar chain. Wherein an autoimmune disease animal is used as the immunized animal.

[0007] In this specification, to specifically recognize an N-glycolylneuraminic acid-containing sugar chain means to recognize neither an N-acetylneuraminic acid-containing sugar chain nor a sialic acid-free sugar chain. Means

The present inventor has proposed an N-type having an HD antigen activity.
The present inventors have made intensive studies to produce a monoclonal antibody that specifically recognizes glycolylneuraminic acid-containing ganglioside and a hybridoma producing the monoclonal antibody. As a result, the present invention has been completed.

The structures of the glycolipids described in the present specification are shown in the following formulas (1) to (8).

[0010]

Embedded image

[0011]

Embedded image

[0012]

Embedded image

[0013]

Embedded image

[0014]

Embedded image

[0015]

Embedded image Asialo GM ₂ (GgOse ₃ Cer) GalNAcβ ₁ → 4Galβ ₁ → 4Glcβ ₁ → 1C
er

[0016]

Embedded image

[0017]

Embedded image CDH (LacCer) Galβ ₁ → 4Glcβ ₁ → 1Cer

Wherein Gal is galactose, Glc is glucose, GalNAc is N-acetylgalactosamine, GlcNAc is N-acetylglucosamine, Neu.
Gc is N-glycolylneuraminic acid, NeuAc is N-glycolylneuraminic acid.
Acetylneuraminic acid, Cer means ceramide]

The monoclonal antibody of the present invention has reactivity with one or several gangliosides having HD antigen activity, which is considered to be a human cancer-related antigen, and contains N-acetylneuraminic acid-containing ganglioside and sialic acid. Does not react with glycolipids containing no. The sugar chain having HD antigen activity recognized by the monoclonal antibody of the present invention exists not only in glycolipids but also in glycoproteins. Therefore, the monoclonal antibody of the present invention that recognizes these sugar chains considered to be human cancer-related antigens is extremely useful for cancer diagnosis.
The monoclonal antibody of the present invention is extremely useful for human cancer diagnosis by tissue, cell diagnosis, blood, urine diagnosis, image diagnosis, etc., and is also applied to missile therapy for binding a drug to the antibody or treatment using cytotoxicity. Is possible. Further, the monoclonal antibody of the present invention can be used for detecting an HD antibody. Further, the present invention can be applied to diagnosis and treatment of Marek's disease in chickens. The monoclonal antibody of the present invention can be used as a useful tool in basic research on the relationship between sugar chains and canceration mechanisms, the role of sugar chains in vivo, and the like.

The monoclonal antibody of the present invention can be obtained by the following method. First, a mammal is immunized with the immunogen.
At this time, the mammal to be immunized is preferably selected in consideration of compatibility with the myeloma cells used for cell fusion, and more preferably a mouse or rat is used. In the case of the N-glycolylneuraminic acid-containing glycolipid of interest in the present invention, it is more preferable to use an autoimmune disease animal, and it is particularly preferable to use an autoimmune mouse. Examples of usable autoimmune disease mice include NZB, NZW, B
/ WF ₁ , MRL / l, BXSB male, SL / Ni and the like.

In addition, normal mice such as Balb / c, whose autoantibody production ability has been enhanced by administering a polyclonal B cell activator (PBA) such as bacterial lipopolysaccharide (LPS) or dextran sulfate, have been autoimmunized. The diseased state may be used as an immunized animal.

N-glycolylneuraminic acid is present in many animals, including mice. It is known that N-glycolylneuraminic acid-containing glycolipids containing gangliosides having the HD antigen activity of interest in the present invention are widely present in mouse tissues. It is a self-antigen, and its immunogenicity is considered to be extremely weak. In a conventional method using a normal mouse such as a Balb / c mouse as an immunized animal, it is extremely difficult to obtain a monoclonal antibody-producing hybridoma against N-glycolylneuraminic acid-containing glycolipid. On the other hand, autoimmune disease mice are known to produce antibodies against self-antigens such as antinuclear antibodies and anti-red blood cell antibodies.

The present inventor has attempted to produce a hybridoma producing a monoclonal antibody against glycolipid having HD antigen activity, and found that the desired hybridoma can be produced very easily by immunizing an autoimmune disease mouse. The present invention has been completed.

According to the present invention, N-
A hybridoma producing a monoclonal antibody against a glycolyl neuraminic acid-containing sugar chain can be easily produced, and the monoclonal antibody can be obtained.

As the immunogen, any of cells having glycolipids containing N-glycolylneuraminic acid itself, cell membrane components separated from the cells, and glycolipids containing N-glycolylneuraminic acid separated from the cells are used. It is possible. Glycolipids can be used as liposomes together with phospholipids and cholesterol. Immunization is performed by a general method, and the above immunogen is treated with a phosphate buffer solution (hereinafter referred to as P
(Abbreviated as BS) or the like, and administered intraperitoneally or intravenously. At this time, the immunogen may be supported on a carrier such as bovine serum albumin (BSA) or cells, or an adjuvant such as Freund's adjuvant or cell adjuvant may be injected together.

The spleen cells collected from the immunized animal are fused with mouse myeloma cells. As myeloma cells, various cells already known, for example, NS-1, SP-2, X63.
3, P3-U1, etc. are used. The fusion method is performed according to a known method. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus (HV).
J) etc. are used. The usage ratio of spleen cells to myeloma cells is the same as in a general method, and is preferably 1: 1 to 10: 1. After completion of the fusion, hybridomas are selected by culturing in a normal selection medium. Since the above-mentioned myeloma cells cannot grow in a HAT medium (a medium containing hypoxanthine, aminopterin and thymidine), cells that grow in a HAT medium may be selected.

When the hybridoma colonies become sufficiently large, a strain producing and producing a target antibody is searched and cloned. The antibody producing strain is searched for by a method generally used for detecting an antibody, for example, an ELISA method [Method in Enzymol. (Meth. Enzymol.) 70, 419-439 (1980)], Method, RIA method, double immunodiffusion method and the like.

Specifically, after blocking the plate to which the purified glycolipid antigen has been attached with BSA, the plate is reacted with the culture supernatant of the test hybridoma, and further reacted with an antibody against an enzyme-labeled mouse antibody. The presence of the antibody bound to the antigen is confirmed by measuring the enzyme activity, and a desired antibody-producing strain is selected.

The cloning is performed by the limiting dilution method. That is, a single colony is grown on a 96-well microtiter plate so that the number of hybridomas is 1 or less per well. At this time, it is preferable to add mouse thymocytes as feeder cells. The above cloning is repeated to obtain a monoclonal hybridoma.

The hybridoma producing the monoclonal antibody of the present invention can be stored in liquid nitrogen for a long period of time, and is kept in a state that can be distributed.

To obtain the monoclonal antibody of the present invention,
There is a method in which the hybridoma is cultured in a medium and separated from the culture supernatant, or a method in which the hybridoma is administered intraperitoneally to a mouse and recovered from the ascites. Furthermore, it is also possible to purify using a general method, that is, ammonium sulfate precipitation, gel filtration, ion exchange column chromatography or the like.

[0032]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Example 1 1) Isolation and Purification of Antigen and Various Glycolipids Rabbit Thymus Cell Membrane Components Thymus tissues obtained from two rabbits 6 weeks old were homogenized in a phosphate buffer solution and centrifuged at 800 rpm for 5 minutes. separated. The supernatant was centrifuged at 10,000 g for 1 hour, and the obtained pellet was dispersed in 10 ml of PBS and used as an immunogen. Glycolipid Bovine kidney was homogenized in cold acetone, and a large amount of cold acetone was added. From the resulting precipitate, chloroform: methanol: water (10: 20: 1) (10: 1)
(0: 0) (20: 10: 1). The obtained crude glycolipid was subjected to DEAE-Sephadex A-25 column chromatography to separate it into an acidic fraction and a neutral fraction, and the acidic fraction was subjected to iatrobeads column chromatography to obtain N-glycolyl GM _2.
Ganglioside was obtained. Similarly, N-glycolyl GM ₃ ganglioside was obtained from horse erythrocytes, and IV ³ N was obtained from bovine erythrocytes.
euGc-nLcOse ₄ Cer and VI ³ NeuGc-
nLcOse ₆ Cer was purified from human brain with N-acetyl GM ₂
The gangliosides and N- acetyl GM ₃ ganglioside, to obtain a CDH from human erythrocytes. N-acetyl GM ₂ ganglioside was reacted in 1N formic acid at 100 ° C. for 1 hour, and subjected to DEAE-Sephadex A-25 column chromatography and then to Iatrobeads column chromatography to obtain asialo GM ₂ . An acidic glycolipid fraction was obtained from C3H / He mouse erythrocytes and used for enzyme immunostaining on TLC without purification. Glycolipid-containing liposomes having an immunizing effect include
mg, phosphatidylcholine 4mg, cholesterol 10mg
The solvent was completely removed under reduced pressure from a chloroform: methanol (1: 1) mixed solution in a flask in a volume ratio of PB
It was prepared by adding 5 ml of S and sonicating.

2) Immunization method and cell fusion First time The same amount of a PBS solution of rabbit thymocyte membrane component in Freund's complete adjuvant was emulsified and mixed, and 400 μl was injected intraperitoneally into NZB mice (female, 6 weeks old). Two weeks later, another injection was made using Freund's incomplete adjuvant. After 13 weeks, IV ³ NeuGc-nLcOse ₄ Ce
100 μl of the r-containing liposome PBS solution was injected intraperitoneally, and two weeks later, the liposome was similarly injected. Cell fusion Three days after the final immunization, the spleen was taken out of the mouse and loosened into single cells, and the spleen cells were washed with RPMI1640 medium. On the other hand, mouse myeloma cells X63.
6.5.3 was collected and washed with RPMI1640 medium.
Suspension of 7 × 10 ⁷ splenocytes and mouse myeloma 1.4
× 10 ⁷ suspensions were mixed and the medium was removed by centrifugation. Add 50 cells to the mixed cells in a water bath heated to 37 ° C.
% Polyethylene glycol-RPMI1640 medium 1 ml
Was gradually added over 1 minute, and the mixture was gently stirred for 1 minute to perform fusion. 2 ml of RPMI 1640 medium was added for 2 minutes, and 7 ml was further added for 2 minutes with gentle stirring. The medium was removed by centrifugation, and 20 ml of 10% fetal bovine serum-containing RPMI 1640 medium was added to the cells.
0.1 ml per well was distributed to two well plates. The next day, HAT medium (4 × 10 ⁻⁷ M aminopterin, 1.6
× 10 ⁻⁵ M thymidine, 1 × 10 ⁻⁴ M hypoxanthine, 1
RPMI 1640 medium containing 0% fetal calf serum) 0.1 ml
Was added to each well. The medium in each well was further replaced with a half amount of HAT medium every 3 or 4 days. Three weeks later, 80% of the wells showed hybridoma growth. Second time The PBS solution of the rabbit thymocyte membrane component and the same amount of Freund's complete adjuvant were emulsified and mixed, and 300 μl of the mixture was injected intraperitoneally into NZB mice (female, 6 weeks old). Thereafter, 20 μg of N-glycolyl GM ₃ ganglioside adsorbed on 80 μg of Salmonella minnesota bacteria treated with acid was added.
200 μl of the BS solution was injected intravenously five times every two weeks.
Three days after the final immunization, the spleen was removed from the mouse, and after disintegration into single cells, the spleen cells were washed with RPMI1640 medium. On the other hand, mouse myeloma cells X63.
6.5.3 was collected and washed with RPMI1640 medium.
A suspension of 4.7 × 10 ⁸ spleen cells and a suspension of 9.2 × 10 ⁷ mouse myeloma were mixed, and the medium was removed by centrifugation. In a water bath heated to 37 ° C., 2 ml of 50% polyethylene glycol-RPMI1640 medium was gradually added to the mixed cells over 1 minute, and the mixture was gently stirred for 1 minute to perform fusion. 4 ml of RPMI1640 medium was added for 2 minutes, and 14 ml was further added for 2 minutes with gentle stirring. The medium was removed by centrifugation, and 120 ml of 10% fetal bovine serum-containing RPMI1640 medium was added to the cells, which were distributed to 12 96-well plates at a rate of 0.1 ml per well. The following day, 0.1 ml of HAT medium (4 × 10 ⁻⁷ M aminopterin, 1.6 × 10 ⁻⁵ M thymidine, 1 × 10 ⁻⁴ M hypoxanthine, RPMI 1640 medium containing 10% fetal bovine serum) was added to each well. added. The medium in each well is
Further, every three or four days, half of the HAT medium was replaced. Three weeks later, 90% of the wells showed hybridoma growth. The third immunization was performed in the same manner as the second except that the intravenous injection was performed eight times. For cell fusion, 3.6 × 10 ⁸ spleen cells and 7.1 × 10 ⁷ mouse myeloma cells were used, and 100 ml of 10%
A bovine fetal serum-containing RPMI medium was added, and a 96-well plate 1
The same procedure was performed as the second time except that the sheets were distributed to 0 sheets. Three weeks later, 80% of the wells showed hybridoma growth.

3) Selection of hybridoma Antibodies in the hybridoma culture supernatant were searched for by ELISA. As an antigen, N- glycolyl GM ₂ ganglioside, N- glycolyl GM ₃ ganglioside and IV
³ NeuGc-nLcOse ₄ Cer was used. Antigen 5
00 ng was adsorbed to a microtiter plate for ELISA, blocked with a 1% BSA PBS solution, and the culture supernatant was reacted. Further, a target antibody was detected by reacting a peroxidase-labeled goat anti-mouse immunoglobulin antibody and measuring absorbance at 492 nm using orthophenylenediamine as a substrate. As a result, first immunization, in hybridomas prepared in cell fusion, N- glycolyl antibodies reacted strongly to the GM ₂ ganglioside was detected in one well, also the second immunization, in hybridomas produced by cell fusion , N-glycolyl GM ₂ ganglioside, N-glycolyl GM ₃
Ganglioside and IV ³ NeuGc-nLcOse ₄ C
An antibody that reacts strongly with er was detected in one well.
Further, the third immunization, in hybridomas prepared in cell fusion, N- into glycolyl GM ₂ ganglioside and strong antibody are two wells to react, N- glycolyl GM ₂ ganglioside, N- glycolyl GM ₃ ganglioside and IV ³ NeuGc antibodies that react strongly with -nLcOse ₄ Cer is detected in one well. These hybridomas were transferred to an HT medium from which aminopterin had been removed from the HAT medium, and then RPM containing 10% fetal calf serum (FCS).
The cells were transferred to I1640 medium and cultured. This hybridoma was cloned by the limiting dilution method. That is, 96
Dilute cells to a density of 0.8 cells per well in a well plate and culture with 4 × 10 ⁵ mouse thymocytes per well,
Two weeks later, antibody-producing cells were selected by ELISA.
Cloning is further repeated to obtain a stable hybridoma Py.
K-2, YHD-02, YHD-04, YHD-05 and YHD-07 were obtained. PyK-2 is the first YHD
-02 is the second YHD-04, YHD-05, YH
D-07 was obtained from the third immunization and cell fusion, respectively.
Monoclonal antibodies PyK-2, YHD-02, YHD
-04, YHD-05 and YHD-07 were tested by the double immunodiffusion method and the ELISA method, and the antibody class was Ig, respectively.
M, IgG _3, IgM, was determined to IgM and IgM.

Example 2 1) Measurement of antigen specificity of PyK-2 by ELISA method Using N-glycolyl GM ₂ ganglioside as an antigen,
The antigen in varying amounts was adsorbed onto the ELISA plate, and the 4-fold diluted hybridoma culture supernatant was reacted. After washing with PBS, a peroxidase-labeled goat anti-mouse immunoglobulin antibody was reacted, washed with PBS, and washed with orthophenylenediamine as a substrate.
The absorbance at 2 nm was measured to examine the reactivity of the antibody. The result is shown in FIG. In FIG. 1, the vertical axis represents the absorbance at 492 nm, and the horizontal axis represents the amount of antigen (ng). Using various glycolipids as antigens, 500 ng of ELISA
It was adsorbed on the A plate. The serially diluted hybridoma culture supernatant is reacted, and the antibody PyK of the present invention is similarly treated.
The reactivity with -2 was examined. The results are shown in FIG. In FIG. 2, the vertical axis represents absorbance, and the horizontal axis represents antibody dilution times (2
^-n ). Each symbol indicates the following. White circle: N-glycolyl GM ₂ ganglioside (II ³ Ne)
uGc-GgOse ₃ Cer) Black circle: N-glycolyl GM ₃ ganglioside (II ³ Ne)
UGC-LacCer) open triangle: IV ³ NeuGc-nLcOse ₄ Cer black triangle: VI ³ NeuGc-nLcOse ₆ Cer open squares: N- acetyl GM ₂ ganglioside (II ³ Ne
uAc-GgOse ₃ Cer) Asialo GM ₂ (GgOse ₃ Cer) As is apparent from FIG. 2, the antibody PyK-2 of the present invention is N-
Glycolyl GM ₂ ganglioside has a strong reactivity with N-glycolyl GM ₂ ganglioside from terminal N-
It was confirmed to react only weakly with glycolipids such as N- glycolyl GM ₃ ganglioside having acetylgalactosamine is removed structure. PyK-2 is N
- The acetyl GM ₂ ganglioside and asialo GM ₂ was confirmed that no reaction.

2) TLC (Thin Layer Chromatography)
Measurement of the reactivity of PyK-2 on the plate Various glycolipids were spotted with a width of 6 mm at a position 1 cm from the lower end of the TLC plate and developed with an appropriate solvent system. Of the two plates on which the same operation was performed, one plate was developed with an orcinol reagent. The other was subjected to enzyme immunostaining. That is, the antibody of the present invention was reacted, and further reacted with a peroxidase-labeled goat anti-mouse immunoglobulin antibody. Using 4-chloro-1-naphthol as a substrate, a blue-violet spot was detected. Figure 3 shows the results with N- glycolyl GM ₂ ganglioside and N- acetyl GM ₂ ganglioside as an antigen. Chloroform: methanol: 2.5N aqueous ammonia (55:45:10 volume ratio) as a developing solvent
Was used. A shows color development by orcinol reagent and B
Is a plate subjected to enzyme immunostaining. The 1 N- acetyl GM ₂ ganglioside, the 2 Expand N- glycolyl GM ₂ ganglioside. Obviously, the present invention antibody reacts with N- glycolyl GM ₂ ganglioside, it can be seen that does not react with N- acetyl GM ₂ ganglioside. . FIG. 4 shows the results obtained by using N-glycolyl GM ₂ ganglioside and a mouse erythrocyte crude acidic glycolipid fraction as antigens. As a developing solvent, chloroform: methanol: 0.2% calcium chloride (55:
45:10 volume ratio). A and B plates are shown in FIG.
Is the same as The 1 N-glycolyl GM ₂ ganglioside, the expansion of mouse erythrocyte crude acidic glycolipid fraction 2. C3H / the He Mice erythrocytes has been reported to contain many N- acetyl GM ₄ ganglioside and N- glycolyl GM ₂ ganglioside as the ganglioside {Journal of Biochemistry (Tokyo), [J. Biochem. (Tokyo)] Vol. 94, 327-
At page 330 (1983)｝, as a result of enzyme immunostaining, a colored spot was detected at ₂ in the same Rf value as N-glycolyl GM2. As a result of searching the reactivity of the antibody PyK-2 of the present invention using extracts from two different tissues, bovine kidney and mouse erythrocytes, N-glycolyl GM was determined.
₂ It was confirmed that it reacts with ganglioside itself.

3) YHD-02, Y by ELISA method
Measurement of antigen specificity of HD-04, YHD-05 and YHD-07 ELISA was performed using 0.2 nmol of various glycolipids as antigens. The antigen adsorbed on the plate was reacted with the hybridoma culture supernatant, and further reacted with a peroxidase-labeled goat anti-mouse immunoglobulin antibody. Using o-phenylenediamine as a substrate, the absorbance at 492 nm was measured to examine the reactivity of the monoclonal antibody to various antigens.
Table 1 shows the results.

[0039]

[Table 1]

[0040] YHD-04 and YHD-07 is the N- glycolyl GM ₂ ganglioside, also YHD-02 and YHD-05 is N- glycolyl GM ₂ ganglioside,
N- glycolyl GM ₃ ganglioside, IV ³ NeuGc
-NLcOse ₄ Cer and VI ³ NeuGc-nLcO
It was found to react with se ₆ Cer. Further, any of the monoclonal antibodies is N- acetylneuraminic acid-containing glycolipid N- acetyl GM ₃ ganglioside and N
- it does not react with acetyl GM ₂ ganglioside,
And do not contain sialic acid, was found not to react with asialo GM _2, and CDH.

4) YHD-02 on TLC plate,
Measurement of Reactivity of YHD-04, YHD-05 and YHD-07 Various glycolipids were spotted with a width of 6 mm at a position 1 cm from the lower end of the TLC plate, and developed with an appropriate solvent system. Of the two plates on which the same operation was performed, one plate was developed with an orcinol reagent. The other was subjected to enzyme immunostaining. That is, the antibody of the present invention was reacted, and further reacted with a peroxidase-labeled goat anti-mouse immunoglobulin antibody. Using 4-chloro-1-naphthol as a substrate, a blue-violet spot was detected. FIG. 5 shows the results using four gangliosides as antigens. Chloroform: methanol: 2.5N aqueous ammonia (55:45:10 volume ratio) as a developing solvent
Was used. A is the color developed by the orcinol reagent, B,
C, D and E are YHD-02, YHD-04 and YHD, respectively.
-05, a plate subjected to enzyme immunostaining using YHD-07. 1 has N-acetyl GM
₂ ganglioside, 2 has N-glycolyl GM ₂ ganglioside, 3 has N-acetyl GM ₃ ganglioside,
The 4 developed the N- glycolyl GM ₃ ganglioside. The monoclonal antibodies YHD-02 and YH of the present invention
D-05 is N-glycolyl GM ₃ ganglioside and N
- it is reacted with glycolyl GM ₂ ganglioside,
Further, YHD-04 and YHD-07 was that is Me probability of reacting with N- glycolyl GM ₂ ganglioside. Further, it was confirmed that none of the monoclonal antibodies of the present invention reacted with N-acetylneuraminic acid-containing glycolipid.

[0042]

As described above, a novel monoclonal antibody has been provided by the present invention. These antibodies are
It is very effective in elucidating, diagnosing and treating cancer generation mechanisms.

[Brief description of the drawings]

FIG. 1 shows the N-glycolyl GM ₂ of the antibody PyK-2 of the present invention.
It is the graph which showed the reactivity with respect to ganglioside by changing the amount of antigen in ELISA method.

FIG. 2 is a graph showing the reactivity of the antibody PyK-2 of the present invention with various glycolipids by changing the amount of the antibody in the ELISA method.

FIG. 3 is a spot diagram in which N-glycolyl GM ₂ ganglioside and N-acetyl GM ₂ ganglioside are used as antigens, developed on a plate, and the reactivity with the antibody PyK-2 of the present invention is detected by enzyme immunostaining. .

FIG. 4 is a spot diagram similar to FIG. 3 using N-glycolyl GM ₂ ganglioside and a crude glycolipid fraction of mouse erythrocyte as an antigen.

[5] N- glycolyl GM ₂ ganglioside, N- acetyl GM ₂ ganglioside, expand N- glycolyl GM ₃ ganglioside, the N- acetyl GM ₃ ganglioside on the plate, the present invention antibody YHD-02, YHD-
It is a spot figure which detected reactivity with 04, YHD-05, and YHD-07 by enzyme immunostaining.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification number Agency reference number FI Technical display location // (C12P 21/08 C12R 1:91)

Claims (6)

(57) [Claims] 1. A method for producing a monoclonal antibody-producing hybridoma that specifically recognizes an N-glycolylneuraminic acid-containing sugar chain, wherein an autoimmune disease animal is used as an immunized animal. . 2. The method for producing a hybridoma according to claim 1, wherein the sugar chain is a sugar chain having a Hangangachi-daiher antigen activity. 3. The method for producing a hybridoma according to claim 1, wherein the sugar chain is a component of a glycolipid. Wherein said antibody, according to claim 1 which is a monoclonal antibody recognizing the N- glycolyl GM ₂ ganglioside
4. The method for producing a hybridoma according to any one of items 3 to 3. 5. The method according to claim 5, wherein the antibody is N-glycolyl GM ₂ ganglioside, N-glycolyl GM ₃ ganglioside, IV ³
NeuGc-nLcOse ₄ Cer and VI ³ NeuGc
-NLcOse ₆ Cer method for manufacturing a hybridoma according to any one of recognizing claims 1-3 which is a monoclonal antibody. 6. The method for producing a hybridoma according to claim 1, wherein the immunized animal is an autoimmune disease mouse.