JPH0787798B2 - Method for producing monoclonal antibody - Google Patents

Description

Detailed Description of the Invention

[Field of Industrial Application] The present invention relates to a method for producing and producing a monoclonal antibody that reacts with at least N-glycolyl GM ₂ ganglioside by a hybridoma. [Prior Art] Glycolipids are constituent components of cell membranes.
Various molecular species exist depending on the binding method, and show species-specific, organ-specific, and cell-specific distribution. As its function, it plays an important role as a receptor for bacterial toxins, hormones, etc., and as an immunological determinant for blood group substances, as well as in controlling proliferation or differentiation and in cell-cell interactions. It is becoming clear that Furthermore, it has been shown that qualitative and quantitative compositional changes occur as cells become cancerous, and that some of them can become cancer antigens. In addition, certain glycolipids regulate the cell growth mechanism mediated by growth factors and protein kinases. It is also suggested that the compositional change, such as the case of working as a person, may be directly involved in the canceration mechanism. On the other hand, a method for establishing a cell line that produces a uniform antibody having specificity for one type of antigenic determinant was reported by Milstein et al. (Nature; Nature, 256 495-497, 1975),
It became possible to qualitatively and quantitatively analyze trace substances. This technique was used to search for cancer antigens and differentiation antigens, and many monoclonal antibodies specific for cancer cells and fetal cells undergoing differentiation were produced. Some of them were glycolipids or glycoproteins. It was revealed to be an antibody that recognizes the sugar chain of J. Natl. Cancer Inst. 71 231-251,198.
3). For example, as a monoclonal antibody against human melanoma, an antibody that reacts with a glycolipid such as GD ₂ ganglioside or GD ₃ ganglioside has been obtained. The pancreatic cancer-specific monoclonal antibody NS19-9 reacts with glycolipids and glycoproteins having a sialosyl Lewis A type sugar chain. These antibodies are useful for diagnosing cancer and observing the course of treatment,
Further, its use for treatment has also been attempted. The qualitative and quantitative changes associated with canceration of glycolipids are due to changes in the activity of various glycosyltransferases in the sugar chain biosynthesis mechanism due to abnormal gene expression, and as a result, they are present in normal tissues. A sugar chain structure that does not occur is created. The sugar chain structure can be used as a cancer marker. As described above, the importance and usefulness of glycolipids as cancer antigens and cancer markers and as cancer markers are attracting attention, and they are expected to be applied to clinical fields such as diagnosis and treatment. Among glycolipids, those containing sialic acid, which is an acidic sugar, in their sugar chains are collectively called gangliosides. The main types of sialic acid 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. In the following description, when it is necessary to distinguish between these types of sialic acid, N-glycolyl or N-acetyl will be added before the name of glycolipid to distinguish them. Detected in patients with serum sickness, sheep, horses, pigs, rabbits,
The heterophilic antibody that aggregates guinea pig erythrocytes is called Hanganatziu-Deicher (hereinafter abbreviated as HD) antibody. The antigen identified by this antibody is called the HD antigen. It has been reported that gangliosides containing N-glycolylneuraminic acid have HD antigenic activity [Biochemical and Biophysical Research Communications (Biochem.Biophys.Res.Co.
mmun.) 79, 388-395 (1977)], NeuGcα2-3
The sugar chain structure of Gal was identified as the major antigenic determinant. In recent years, N- which is a ganglioside having HD antigenic activity
Immunize chickens with glycolyl GM ₃ ganglioside (II ³ NeuGc-LacCer) to prepare antibodies that react with various gangliosides having HD antigen activity from serum. Using this antibody, N-glycolylneuraminic acid It was reported to be characteristically present in human cancer tissues [Biken J., Vol. 25, pp. 47-50 (1982)]. In addition, some of the glycolipids extracted from human colon cancer tissue
HD antigen-active N-glycolylneuraminic acid-containing ganglioside was detected, and HD antigen-active glycoprotein-containing glycoprotein was detected in the teratoma tissue [Gan (Gan
n), Vol. 75, pp. 1025-1029 (1984)]. When a glycolipid having HD antigen activity detected from human colon cancer tissue was analyzed using an antibody prepared in chicken, N-
Glycolyl GM ₂ ganglioside, N-glycolyl GM ₃ ganglioside, o-acyl-N-glycolyl GM ₃ ganglioside, IV ³ NeuGc-nLcOse ₄ Cer, which were not detected in normal tissues [Cancer Research (Canc.
er Res.), Vol. 45, pp. 3996-3802 (1985)]. Gangliosides with HD antigenic activity were also detected in a Marek's disease cell line, one of chicken lymphomas [Journal of Biochemistry (Tokyo); J. Biochem. (Tokyo)], No. 95, 785-794 (1984)]. Including N- glycolylneuraminic acid and N- glycolyl GM ₂ ganglioside sugar chain, N- glycolyl neuraminic acid containing sugar chain is considered to cancer-associated antigens, cancer can be detected accurately these high sensitivity It is extremely important for diagnosis. Including N- glycolylneuraminic acid or N- glycolyl GM ₂ ganglioside sugar chain to efficiently detect the N- glycolyl-neuraminic acid containing sugar chain, the detection sensitivity, in terms of accuracy, immunoassay The law is considered excellent. An antibody that reacts with an N-glycolylneuraminic acid-containing sugar chain having HD antigen activity has been conventionally obtained by immunizing a chicken with a purified glycolipid antigen having HD antigen activity and separating it from its serum [Molecular • Immunology (Molec.Jmmunol.) Vol. 19, pp. 87-94 (1982)]. N- glycolyl GM higher polyclonal antibody specificity to _ganglioside can be obtained by immunizing chicken to N- glycolyl GM ₂ ganglioside [Biochemical and Biophysical Research Communications (Biochem.) , First
29, 334-341 (1985)], N-glycolyl GM ₂
Polyclonal antibodies reactive against gangliosides and N- glycolyl GM ₃ ganglioside can be obtained by immunizing an N- glycolyl GM ₃ Gagurioshido chicken [Cancer Research (Cancer Res.)
Volume 45, pages 3996-3802 (1985)]. However, these methods have some drawbacks. That is, (1) A large amount of purified antigen is required each time to obtain antiserum. (2) There are variations in affinity and titer mainly due to individual differences in immunized animals. (3) Since antibodies other than the target antibody are mixed,
A complicated operation is required to purify the desired antibody. (4) There are drawbacks such as a limited amount that can be manufactured at one time. Therefore, in order to perform an immunological assay accurately and with the maximum effect, it has been desired to supply a large amount of a uniform antibody having a stable quality and free from the contamination of other antibodies. The production of such an antibody has already been reported as a technology for producing monoclonal antibodies. On the other hand, it has been reported that a human monoclonal antibody that specifically recognizes melanoma obtained by transforming phosphoplasm of melanoma patient with EB virus reacts with GM ₂ ganglioside (Proceedings Off
The National Akademyiofu Science's off-the-Yuesuei; Proc.Natl.Acad.Sci.USA, 80, 53
92-5396,1985). Also, even though GM ₂ ganglioside hardly present in normal erythrocytes, the human erythroleukemia cell, GM
Accumulation of ₂ gangliosides has been reported (Blood; Blood, 62 , 1230 to 1241, 1983). In addition, N obtained by immunizing mouse myeloma
- acetyl GM ₂ ganglioside and N- glycolyl GM ₂ ganglioside and with monoclonal antibodies recognizing GM
It has been shown that ₂ gangliosides are mainly expressed in cancer cells of neuroectodermal origin (Cancer Research; Cancer
Res., 46 , 4116-4120, 1986). Thus, N-acetyl GM ₂ ganglioside and N-acetyl
Glycolyl GM ₂ ganglioside is considered to be a cancer-associated antigen, and it is important to detect these with high sensitivity and accuracy for the diagnosis of cancer. As described above, N-glycolyl GM ₂ ganglioside and its related glycolipids and their sugar chain structures are extremely important as cancer-related antigens, and thus monoclonal antibodies having reactivity with N-glycolyl GM ₂ ganglioside,
For example, a monoclonal antibody having specific reactivity with N-glycolyl GM ₂ ganglioside and N-acetyl GM ₂ ganglioside, and specificity with a glycolipid containing N-glycolyl GM ₂ ganglioside and other N-glycolyl neuraminic acid Monoclonal antibodies having specific reactivity are useful for diagnosing various diseases, particularly tissue, cytodiagnosis or blood,
It is extremely useful for diagnosing human cancer by urine diagnosis or image diagnosis. Further, it can be applied to a missile therapy in which a drug is bound to an antibody, a therapy utilizing cytotoxicity, and a diagnosis by detecting an antibody against a sugar chain. Also, N
- Various monoclonal antibodies reactive with glycolyl GM ₂ ganglioside related and the sugar chain and carcinogenesis mechanisms,
It can be used as a useful tool in basic research on the structures of sugar chains and glycolipids, their role in vivo, and the like. Thus, it is considered extremely important to produce a monoclonal antibody that recognizes at least N-glycolyl GM ₂ ganglioside. However, N-glycolylneuraminic acid exists in many animal bodies including mice except humans and chickens, and the N-glycolylneuraminic acid-containing sugar containing N-glycolyl GM ₂ ganglioside, which is the object of the present invention, is included. It is known that lipids are widely present in mouse tissues,
For mice, these glycolipids are self-antigens,
Immunogenicity is considered to be extremely weak. Therefore, in the conventional method using normal mice such as Balb / c mice as immunized animals, monoclonal antibodies against N-glycolylneuraminic acid-containing glycolipids including N-glycolyl GM ₂ ganglioside are used. Obtaining a producing hybridoma was extremely difficult. On the other hand, animals with autoimmune diseases are known to produce antibodies against autoantigens such as antinuclear antibodies and anti-erythrocyte antibodies [Immunological Review (Immunol. Rev.), Vol. 55, Vol.
121-154 (1981)]. The present inventor can enhance the immunoreactivity to an N-glycolylneuraminic acid-containing glycolipid, which is considered to be a self-antigen, by immunizing an autoimmune disease animal, particularly a mouse having an autoimmune disease, and thus producing a hybridoma. I thought it would be possible, tried this, and achieved its purpose. In this process, in particular, it was found that a hybridoma producing a monoclonal antibody that recognizes N-glycolyl GM ₂ ganglioside can be easily prepared, and the present invention has been completed. [Object of the Invention] An object of the present invention is to provide a method for producing a monoclonal antibody that specifically recognizes at least N-glycolyl GM ₂ ganglioside. [Structure of the Invention] (Means for Solving Problems) The present invention relates to a method for producing a monoclonal antibody that recognizes at least N-glycolyl GM ₂ ganglioside, and fuses myeloma cells with plasma cells of an autoimmune animal. It is characterized in that it is produced by the hybridoma. The structures of glycolipids described below are shown below. N-glycolyl GM ₂ ganglioside (II ³ NeuGc-GgOse ₃ Ce
r) N-glycolyl GM ₃ ganglioside (II ³ NeuGc-Lac Ce
r) N-acetyl GM ₂ ganglioside (II ³ NeuAc-GgOse ₃ Ce
r) N-Acetyl GM ₃ Ganglioside (II ³ NeuAc-LacCer) Asialo GM ₂ (GgOse ₃ Cer) GalNAcβ1 → 4Galβ1 → 4Glcβ1 → 1Cer CDH (Lac Cer) Galβ1 → 4Glcβ1 → 1Cer In the formula, Gal is galactose, Glc is glucose, GalNAc is N-acetylgalactosamine, and GlcNAc is N-acetylglucosamine. NeuGc is N-glycolylneuraminic acid, Neu
Ac means N-acetylneuraminic acid, and Cer means ceramide. A method for producing a hybridoma that produces the monoclonal antibody will be described below. An autoimmune disease animal is used as a mammal for obtaining plasma cells, and it is preferable to select it in consideration of compatibility with myeloma cells used for cell fusion, and mice and rats are preferable. Available autoimmune disease mice are NZB, NZW, B / WF ₁ ,
MRL / 1, BXSB male, SL / N1 mouse of each strain, etc.,
Examples of autoimmune disease rats include spontaneously hypertensive rats. In addition, normal mice such as Balb / c whose autoantibody-producing ability was enhanced by administering a polyclonal B cell activator (PBA) such as gram-negative bacterial lipid polysaccharide (LPS) and dextran sulfate were treated for autoimmune disease. May be used. The immunogen can be used any of the cells themselves, N- glycolyl GM ₂ ganglioside isolated from cell membrane components and the cells isolated from the cells with N- glycolyl GM ² ganglioside of interest. It is also possible to use glycolipids as liposomes together with phospholipids and cholesterol. Immunization is performed by a general method, and the above immunogen may be diluted with a phosphate buffer solution (hereinafter referred to as PBS) or the like and administered intraperitoneally or intravenously.
At that time, the immunogen may be carried on a carrier such as bovine serum albumin (BSA) or bacterial cells, or may be injected together with an adjuvant such as Freund's adjuvant or bacterial cell adjuvant. Further, in the method of the present invention using an autoimmune disease mouse, it is not always necessary to use an immunogen. The presence of N-glycolyl GM ₂ ganglioside in mouse erythrocytes [J. Biochem. (Tokyo)], Vol. 91, 1039-1
046 (1982)], and since autoimmune disease mice produce autoantibodies including anti-erythrocyte antibodies, autoimmune disease mice are treated with anti-erythrocyte autoantibodies or other autoantibodies against N-glycolyl GM ₂ ganglioside. Is considered to be produced or is likely to be produced. Therefore, when using an autoimmune disease mouse that has never been immunized with N-glycolyl GM ₂ ganglioside, its sugar chain, or a substance containing them, N-glycolyl GM ₂ ganglioside is used without using an immunogen. It is possible to produce a reactive monoclonal antibody-producing hybridoma. When no immunogen is used, no injection is required for autoimmune disease mice, but various adjuvants such as Freund's complete adjuvant, Freund's incomplete adjuvant, pertussis killed cells, and Salmonella killed cells are used. It is preferable to inject it to enhance the immune response capacity of autoimmune disease mice. Plasma cells collected from the animal are fused with myeloma cells.
Plasma cells are generally obtained as splenocytes. As the myeloma cells, mouse myeloma cells are preferable, and various known cells such as NS-1, SP-2, X63.6.5.3, P3-U1 and the like can be used. The fusion method is performed according to a known method. Examples of fusion promoters include polyethylene glycol (PE
G), Sendai virus (HVJ), etc. are used. The ratio of splenocytes to myeloma cells used was the same as in the general method.
1 to 10 to 1 is preferable. After completion of the fusion, hybridomas are selected by culturing in a usual selection medium. The myeloma cells described above are HAT
Since it cannot grow in a medium (medium containing hypoxanthine, aminopterin and thymidine), cells that grow in a HAT medium may be selected. When the colony of the hybridoma becomes sufficiently large, the production strain of the desired antibody is searched and cloned. The antibody-producing strain is searched by a method generally used for antibody detection, for example, an ELISA method (method in enzymology; Meth. Enzymol., 70 , 419 to 439, 1980), an agglutination reaction method, a RIA method, a double method. It is performed by an immunodiffusion method or the like. Specifically, after blocking the plate on 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 enzyme-labeled mouse antibody antibody to the antigen. The presence of the bound antibody is confirmed by measuring the enzyme activity, and the desired antibody-producing strain is selected. Moreover, 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 monocloned hybridoma. To obtain the monoclonal antibody produced by the hybridoma, there is a method of culturing the hybridoma in a medium and separating it from the culture supernatant, or a method of administering the hybridoma into the abdominal cavity of a mouse and collecting it from the ascites. Further, it can be purified by a general method such as ammonium sulfate precipitation, gel filtration, ion exchange column chromatography and the like. [Examples] Hereinafter, the present invention will be described in more detail with reference to Examples, but these Examples do not limit the scope of the present invention. Example 1 1) Isolation and Purification of Antigen and Various Glycolipids Rabbit Thymus Cell Membrane Components Thymus tissues obtained from two 6-week-old rabbits were homogenized in PBS and centrifuged at 800 rpm for 5 minutes. 1 supernatant
Centrifuge at 0,000g for 1 hour and use the resulting pellet in PBS.
It was dispersed in 10 ml and used as an immunogen. Glycolipid Homogenize bovine kidney in cold acetone and add a large amount of cold acetone. From the resulting precipitate, volume ratio chloroform: methanol: water (10: 20: 1) (10: 10: 0) (20 :Ten:
Extraction operation was sequentially performed with each mixed solution of 1). The crude glycolipid thus obtained was subjected to DEAE-Sephadex A-25 column chromatography to divide 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 from horse red blood cells, IV ³ NeuGc-nLcOse ₄ Cer from bovine red blood cells, and N-acetyl from human brain.
CDH was obtained from human erythrocytes for GM ₂ ganglioside and N-acetyl GM ₃ ganglioside. N-acetyl GM ₂ ganglioside was reacted in 1N formic acid at 100 ° C for 1 hour to give DEAE-
Sephadex A-25 column chromatography followed by iatro beads column chromatography
Got GM ₂ . The glycolipid-containing liposome for immunity has 1 mg of glycolipid, 4 mg of phosphatidylcholine and 10 mg of cholesterol.
Volumetric ratio chloroform: methanol (1: 1) mixed solution in a flask under reduced pressure to completely remove the solvent, then PBS5
It was prepared by adding ml and applying ultrasonic waves. 2) Immunization method and cell fusion First time, PBS solution of eel thymus cell membrane component and the same amount of Freund's complete adjuvant were emulsified and mixed, and 400 μl was intraperitoneally injected into NZB mouse (female, 6 weeks old). Two weeks later, the same injection was performed using Freund's incomplete adjuvant.
13 weeks later, IV ³ NeuGc-nLcOse ₄ Cer-containing liposome PBS solution
100 μl was intraperitoneally injected, and two weeks later, liposomes were similarly injected. Cell fusion Three days after the final immunization, the spleen was taken out from the mouse, dissociated into single cells, and the splenocytes were washed with RPMI 1640 medium.
On the other hand, mouse myeloma cells X63.6.5.3 in the logarithmic growth phase were collected and washed with RPMI1640 medium. A suspension of 7 × 10 ⁷ splenocytes and a suspension of 1.4 × 10 ⁷ mouse myeloma were mixed and centrifuged to remove the medium. 50% polyethylene glycol-RPMI was added to the mixed cells in a water bath heated at 37 ℃.
1 ml of 1640 medium was gradually added over 1 minute to gently stir for 1 minute to perform fusion. 2 ml of RPMI1640 medium
Over a period of 2 minutes, another 7 ml was added over a period of 2 minutes with gentle stirring. Remove the medium by centrifugation and add 10% to the cells.
20 ml of RPMI1640 medium containing fetal bovine serum was added, and 0.1 ml per well was distributed to two 96-well plates. Next day, HAT medium (4
× 10 ^-7 M aminopuritene, 1.6 × 10 ^-5 M thymidine, 1 × 10
^-4 M hypoxanthine, RPMI1640 medium containing 10% fetal bovine serum) 0.1 ml was added to each well. Half of the medium in each well was replaced with HAT medium every 3 or 4 days. Three
After a week, growth of hybridomas was observed in 80% of the wells. Second time Rabbit thoracic cell membrane component PBS solution and the same amount of Freund's complete adjuvant were emulsified and mixed, and 300 μl was intraperitoneally injected into NZB mice (female, 6 weeks old). Then, N- adsorbed on 80 μg of Salmonella minetabacteria treated with acid
Glycolyl GM ₃ Ganglioside 20μg in PBS solution 200μl
Was intravenously injected 5 times every 2 weeks. Three days after the final immunization, the spleen was taken out from the mouse, dissociated into single cells, and the splenocytes were washed with RPMI1640 medium. On the other hand, mouse myeloma cells X63.6.5.3 in the logarithmic growth phase were collected and washed with RPMI1640 medium. A suspension of 4.7 × 10 ⁸ splenocytes and a suspension of 9.2 × 10 ⁷ mouse myeloma were mixed and centrifuged to remove the medium. Mix in cells in a water bath heated to 37 ℃ to add 50% polyethylene glycol-RPMI to the cells.
2 ml of 1640 medium was gradually added over 1 minute, and gently stirred for 1 minute to perform fusion. 4 ml of RPMI1640 medium was added for 2 minutes, and 14 ml was added for 2 minutes with gentle stirring. The medium was removed by centrifugation, 120 ml of RPMI1640 medium containing 10% fetal bovine serum was added to the cells, and 96-well plate 1
0.1 ml was distributed to each of the two holes. Next day, HAT medium (4
× 10 ^-7 M aminopuritene, 1.6 × 10 ^-5 M thymidine, 1 × 10
^-4 M hypoxanthine, RPMI1640 medium containing 10% fetal calf serum) 0.1 ml was added to each well. Half of the medium in each well was replaced with HAT medium every 3 or 4 days.
After 3 weeks, 90% of well hybridoma growth was observed. Third immunization method was performed in the same manner as the second immunization except that the intravenous injection was performed eight times. Cell fusion is 3.6 × 10 ⁸ splenocytes, mouse myeloma 7.1
× 10 ⁷ cells was used, plus 10% fetal bovine serum-containing RPMI medium 100 ml, except that distributed in 96-well plates 10 sheets was carried out in the same manner as the second. After 3 weeks, growth of hybridomas was observed in 80% of the wells. 3) Selection of hybridoma The antibody in the hybridoma culture supernatant was searched by ELISA. As an antigen, N-glycolyl GM ₂ ganglioside, N-
Glycolyl GM ₃ Ganglioside and IV ³ NeuGc-nLcOse ₄ Cer
Was used. 500 ng of each antigen was adsorbed on an ELISA microtiter plate, blocked with a 1% BSA / PBS solution, and then the culture supernatant was reacted. Furthermore, a target antibody was detected by reacting a peroxidase-labeled goat anti-mouse immunoglobulin antibody and using orthophenylenediamine as a substrate and measuring the absorbance at 492 nm. As a result, activity was detected in one well in the hybridoma prepared by the first immunization and cell fusion, and activity was detected in two wells in the hybridoma prepared by the second immunization and cell fusion. . In addition, the activity was detected in four wells of the hybridoma prepared by the third immunization and cell fusion. These hybridomas were transferred from HAT medium to HT medium lacking aminopuritene, and further transferred to RPMI1640 medium containing 10% fetal calf serum (FCS) and cultured. Example 2 Measurement of Antigen Specificity of Each Monoclonal Antibody by ELISA Method: ELISA method was carried out using 0.2 nmol of various glycolipids as an antigen. The hybridoma culture supernatant was reacted with the antigen adsorbed on the plate, and further with peroxidase-labeled goat anti-mouse immunoglobulin antibody. Using ortho-phenylenediamine as a substrate, the absorbance at 492 nm was measured to examine the reactivity of the monoclonal antibody with various antigens. The results are shown in the table below. Among the obtained monoclonal antibodies, PyK-2, YHD-04 and YHD-07 show strong reactivity with N-glycolyl GM ₂ ganglioside, and YHD-02, YHD-03 and YHD-05 have N-glycolyl GM. _In addition to ₂ gangliosides, reactivity with N-glycolyl GM ₃ ganglioside and IV ³ NenGL-nLcOse ₄ Cer was also shown. In addition, YHD-06 showed a reaction with N-glycolyl GM ₂ ganglioside as well as N-acetyl GM ₂ ganglioside. That is, PyK-2, YHD-04, YHD-06 and YHD-07 do not show reactivity with IV ³ NenGc-nLcOse ₄ Cer or N-glycolyl GM ₃ ganglioside used as an immunogen, and N-
Glycolyl GM ₂ showed strong reactivity with ganglioside. Example 3 Examination of glycolipids in rabbit pleural tissue on thin layer chromatography (hereinafter referred to as “TCL”) plates: N-glycolyl GM was contained in the rabbit pleural tissue used as an immunogen in Example 1. ₃ Ganglioside or IV ³ NenGc-nLcOse ₄ C
It has been reported that many glycolipids containing N-glycolylneuraminic acid, such as er, are present [Biochim, Biophys.Acta, No. 66].
5: 205-213 (1981)] is unknown about the existence of N-glycolyl GM ₂ ganglioside. The confirmation was tried using the monoclonal antibody. Rabbit thoracic tissue was sequentially extracted with mixed solutions of chloroform: methanol: water (10: 20: 1), (10: 10: 0), (20: 10: 1) in volume ratio, and obtained. The crude glycolipid
DEAE-Sephadex A-25 column chromatography was performed to obtain an acidic fraction containing ganglioside. The obtained acidic fraction was subjected to enzyme immunostaining on TLC using the monoclonal antibody PyK-2. Spot the sample with a width of 6 mm 1 cm from the bottom edge of the TLC plate and use the solvent system chloroform: methanol:
It was developed with 2.5N ammonia water (55:45:10 volume ratio). Of the two plates subjected to the same operation, one plate was colored with the orcinol reagent. The other plate was reacted with PyK-2, and further with a peroxidase-labeled goat anti-mouse immunoglobulin antibody. Using 4-chloro-1-naphthol as a substrate, a blue-violet colored spot was detected. The results are shown in the figure. A is a plate developed with an orcinol reagent, and B is a plate subjected to enzyme immunostaining. N-glycolyl for 1 and 3
₂ nmol and 30 pmol of GM ₂ ganglioside were spotted, respectively, and 2 and 4 were developed by spotting a chloroform / methanol-extracted acidic fraction of rabbit thymus tissue having a sialic acid content of 2 nmol. Rabbits thymus tissue used in Example 1 of the present invention as an immunogen is that no N- glycolyl GM ₂ ganglioside is confirmed by this example, N- glycolyl GM ₂ ganglioside created by Example 1 It was confirmed that a hybridoma producing an antibody reactive with was produced without injection of N-glycolyl GM ₂ ganglioside into NZB mice.

【The invention's effect】

According to the present invention, various monoclonal antibodies having reactivity with N-glycolyl GM ₂ ganglioside can be provided. The antibody is very effective for elucidating the mechanism of cancer development, diagnosis and treatment, and is also useful for basic research on glycolipids and glycoproteins.

[Brief description of drawings]

The figure is a diagram showing the reactivity of the monoclonal antibody RyK with N-glycolyl GM ₂ ganglioside, and an acidic fraction of rabbit thoracic tissue tissue chloroform-methanol extraction.

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Claims (4)

[Claims] 1. A monoclonal antibody which reacts with at least N-glycolyl GM ₂ ganglioside is produced by a hybridoma prepared by fusing myeloma cells and plasma cells of an autoimmune disease animal. Production method. 2. The method for producing a monoclonal antibody according to claim 1, wherein the myeloma cell is a mouse myeloma cell. 3. The method for producing a monoclonal antibody according to claim 1 or 2, wherein the animal having an autoimmune disease is a mouse having an autoimmune disease. 4. A mouse having autoimmune disease is N-glycolyl GM _2.
The method for producing a monoclonal antibody according to claim 3, wherein the mouse is an autoimmune disease mouse that has never been immunized with ganglioside, its sugar chain, or a substance containing them.