JPH0673471B2 - Monoclonal antibody - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a monoclonal antibody, particularly a monoclonal antibody that specifically recognizes an N-glycolylneuraminic acid-containing sugar chain.

[Conventional technology]

Glycolipids are constituents of the plasma membrane of cells.
A variety of molecular species exist depending on the number and the binding method, and show species-specific, organ-specific, and cell-specific distribution. As its function, in addition to acting as a receptor for bacterial toxins, hormones, etc., and as an immunologically determinative amount for blood group substances,
It is becoming clear that it plays an important role in the control of proliferation or differentiation and the interaction between cells. Furthermore, it has been shown that qualitative and quantitative compositional changes occur as cells become cancerous, and some of them can become cancer antigens. In addition, certain glycolipids act as growth factors and protein kinases in the mechanism of cell growth. It has also been suggested that the compositional changes, such as the case of acting as a regulator of, may be directly involved in the carcinogenesis mechanism.

Meanwhile, Milstein et al. Reported a method for establishing a cell line that produces a uniform antibody having specificity for one type of antigenic determinant [Nature, Vol. 256, 495].
Pp. 497 (1975)], qualitative and quantitative determination of trace substances became possible. Use this technology to search for cancer antigens,
Many monoclonal antibodies specific to cancer cells have been produced, but it was revealed that some of them were antibodies that recognize the sugar chains of glycolipids or glycoproteins [Journal of National Cancer Institute]. (J.Natl.Cancer Inst.) Volume 71, Volume 231
~ 251 (1983)].

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. Further, the pancreatic cancer-specific monoclonal antibody NS19-9 reacts with a glycolipid 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. A sugar chain structure that does not exist is created. The sugar chain structure can be used as a cancer marker.

Thus, the importance and usefulness of glycolipids as cancer antigens and cancer markers and as cancer markers and cancer markers are drawing attention, and their application in clinical fields such as diagnosis and treatment is expected.

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.

Detected in patients with serum sickness, sheep, horses, pigs, rabbits,
A heterophilic antibody that aggregates guinea pig erythrocytes is called a Hanganatziu-Deicher (hereinafter abbreviated as HD) antibody. The antigen recognized 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 Comunications (Biochem.
hys.Res.Commun.) Vol. 79, pp. 388-395 (1977)], N
The sugar chain structure of euGc α2-3 Gal was identified as the major antigenic determinant.

In recent years, N, which is a ganglioside having HD antigenic activity
-Glycolyl GM ₃ ganglioside (II ³ NeuGc-LacCer)
Are immunized into chickens to produce antibodies that react with various gangliosides having HD antigenic activity from serum, and N-glycolylneuraminic acid is characteristically present in human cancer tissues using this antibody. [Biken J., Vol. 25, pp. 47-50 (1982).
Year)〕. In addition, several types of HD antigen-active N-glycolylneuraminic acid-containing gangliosides were detected in glycolipids extracted from human colon cancer tissue, and HD antigen was detected in the tissue of teratoma. A glycoprotein having an active sugar chain was detected [Gann, Vol. 75, 1025-10].
29 (1984)]. Glycolipids having HD antigenic activity detected from human colon cancer tissues were 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-n
LcOse ₄ Cer, which was not detected in normal tissues [Cancer Res. Vol. 45,
Pages 3996-3802 (1985)].

In addition, a ganglioside having HD antigenic activity was also detected in a cell line of Marek's disease, which is one of chicken lymphomas [J. Biochem. (Tokyo)]. Volume 95, Volumes 785-79
Page 4 (1984)}.

N-glycolylneuraminic acid and N-glycolylneuraminic acid-containing sugar chains are considered to be cancer-related antigens, and it is extremely important to detect these with high sensitivity and accuracy in cancer diagnosis.

In order to efficiently detect N-glycolylneuraminic acid or N-glycolylneuraminic acid-containing sugar chains, immunoassays are considered to be excellent in terms of detection sensitivity and accuracy.

An antibody that reacts with an N-glycolylneuraminic acid-containing sugar chain having HD antigenic activity is conventionally obtained by immunizing a chicken with a purified glycolipid antigen having HD antigenic activity and separating it from the serum. Was given. However, this method has several drawbacks. That is, (1) A large amount of purified antigen is required each time to obtain the antiserum. (2) Mainly due to individual differences in immunized animals,
There are variations in affinity and potency. (3) Since antibodies other than the target antibody are also mixed, a complicated operation is required to purify the target antibody. (4) There is a drawback that the amount of material that can be manufactured at one time is limited. 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 is
It has already been reported as a technique for producing a monoclonal antibody.

[Problems to be solved by the invention]

However, it has not been reported until now that a monoclonal antibody specifically reacting with an N-glycolylneuraminic acid-containing sugar chain having HD antigenic activity and a hybridoma having the antibody-producing ability have been successfully produced.

A first object of the present invention is to provide a monoclonal antibody that specifically recognizes an N-glycolylneuraminic acid-containing sugar chain having HD antigenic activity, and a second object thereof is HD
It is intended to provide a method for producing a hybridoma that produces a monoclonal antibody that specifically recognizes an N-glycolylneuraminic acid-containing sugar chain having antigenic activity.

[Means for Solving Problems] The present invention will be summarized. The first invention of the present invention relates to a monoclonal antibody, which recognizes an N-glycolylneuraminic acid-containing sugar chain and It is characterized in that it does not recognize a neuraminic acid-containing sugar chain or a sialic acid-free sugar chain.

Hereinafter, in the present specification, specifically recognizing an N-glycolylneuraminic acid-containing sugar chain means recognizing neither an N-acetylneuraminic acid-containing sugar chain nor a sialic acid-free sugar chain. means.

The present inventors have made extensive studies as a result of attempting to produce a monoclonal antibody that specifically recognizes an N-glycolylneuraminic acid-containing ganglioside having HD antigenic activity, and a hybridoma producing the monoclonal antibody. completed.

The structures of the glycolipids described herein are shown below.

[In the formula, Cal is galactose, Glc is glucose, CalNAc
Is N-acetylgalactosamine, GlcNAc is N-acetylglucosamine, Neu Gc is N-glycolyl neuraminic acid, Neu Ac is N-acetyl neuraminic acid, and Cer is ceramide. It has reactivity with one or several kinds of gangliosides having HD antigenic activity, which are considered to be antigens, and does not react with N-acetylneuraminic acid-containing gangliosides and glycolipids containing no sialic acid.

The sugar chain having HD antigenic activity recognized by the monoclonal antibody of the present invention exists not only in glycolipid but also in glycoprotein. Therefore, the monoclonal antibody of the present invention that recognizes these sugar chains that are considered to be human cancer-related antigens,
It is extremely useful for cancer diagnosis.

INDUSTRIAL APPLICABILITY The monoclonal antibody of the present invention is extremely useful for human cancer diagnosis by tissue, cell diagnosis or blood, urine diagnosis, image diagnosis and the like, and is also applied to a missile therapy for binding a drug to an antibody or a therapy utilizing cytotoxicity. Is possible. Furthermore, the monoclonal antibody of the present invention can also be used for detecting HD antibodies. It can also be applied to the diagnosis and treatment of Marek's disease of chickens.

INDUSTRIAL APPLICABILITY 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 mechanism, 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 an immunogen. At this time, the mammal to be immunized is preferably selected in consideration of compatibility with myeloma cells used for cell fusion, and it is more preferable to use mouse or rat. In the case of the N-glycolylneuraminic acid-containing glycolipid targeted by the present invention, it is more preferable to use it for an autoimmune disease animal, and it is particularly preferable to use an autoimmune mouse. Available mice for autoimmune diseases are NZB, NZW, B / WF ₁ , MRL / 1, BXSB male, SL / N
i etc. are mentioned.

Also, bacterial lipopolysaccharide (LPS),
Normal mice, such as Balb / c, whose autoantibody-producing ability has been enhanced by administering a polyclonal B cell activator (PBA) such as dextran sulphate may be brought into an autoimmune disease state and used as an immunized animal.

N-glycolylneuraminic acid is present in many animal bodies, including mice.

It is known that the N-glycolylneuraminic acid-containing glycolipid containing a ganglioside having an HD antigenic activity, which is a target of the present invention, is widely present in mouse tissues,
In mice, these glycolipids are self-antigens,
Immunogenicity is considered to be extremely weak. It is extremely difficult to obtain a monoclonal antibody-producing hybridoma against an N-glycolylneuraminic acid-containing glycolipid by the conventional method using a normal mouse such as Balb / c mouse as an immunized animal. On the other hand, mice with autoimmune diseases are known to produce antibodies against self-antigens such as antinuclear antibodies and anti-erythrocyte antibodies.

The present inventor has tried to produce a monoclonal antibody-producing hybridoma against a glycolipid having HD antigenic activity, found that the desired hybridoma can be produced extremely easily by immunizing a mouse with an autoimmune disease, and the present invention completed.

According to the present invention, a hybridoma producing a monoclonal antibody against an N-glycolylneuraminic acid-containing sugar chain which is considered to be an autoantigen can be easily produced, and the monoclonal antibody can be obtained.

As the immunogen, it is possible to use the cell itself having the N-glycolylneuraminic acid-containing glycolipid, the cell membrane component separated from the cell and the N-glycolylneuraminic acid-containing glycolipid separated from the cell. Is. 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 abbreviated as PBS) or the like and administered intraperitoneally or intravenously. At that time, the immunogen is bovine serum albumin (BSA),
It may be supported on a carrier such as bacterial cells, or Freund's adjuvant or adjuvant such as bacterial adjuvant may be injected together.

Splenocytes collected from immunized animals are fused with mouse myeloma cells. As myeloma cells, various already known cells,
For example, NS-1, SP-2, X63.6.5.3, P3-U1 and the like are 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 hybridoma colony becomes sufficiently large, the target antibody-producing strain is searched and cloned.

The antibody-producing strain is searched by a method generally used for detecting an antibody, for example, an ELISA method [Method in Enzymology (Meth. Enzymol.) 70, 419-439 (198).
0 years)], agglutination reaction method, RIA method, double immunodiffusion method, etc.

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 to react with 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.

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

In order to obtain the monoclonal antibody of the present invention, 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 intraperitoneally to a mouse and collecting it from the ascites. Furthermore, it is also possible to perform purification using a general method, that is, ammonium sulfate precipitation, gel filtration, ion exchange column chromatography and the like.

〔Example〕

Hereinafter, the present invention will be described more specifically with reference to 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 6-week-old rabbits were homogenized in a phosphate buffer solution and centrifuged at 800 rpm for 5 minutes. 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 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 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. Obtained. Similarly, N-glycolyl GM ₃ ganglioside was obtained from horse red blood cells and IV ³ NeuGc-nLcOse ₄ Cer and IV ³ NeuGc- from bovine red blood cells.
nLcOse ₆ Cer, N-acetyl GM ₂ ganglioside and N-acetyl GM ₃ ganglioside were obtained from human brain, and CDH was obtained from human erythrocyte. N-acetyl GM ₂ ganglioside was reacted in 1N formic acid at 100 ° C. for 1 hour, DEAE-Sephadex A-25 column chromatography, and then iatrobeads column chromatography were performed to obtain asialo GM ₂ . An acidic glycolipid fraction was obtained from C3H / He mouse erythrocytes and directly used for enzyme immunostaining on TLC without purification.

Glycolipid-containing liposomes for immunity include a glycolipid 1 mg, phosphatidylcholine 4 mg, and cholesterol 10 mg volume ratio chloroform: methanol (1: 1) mixed solution in a flask under reduced pressure, after completely removing the solvent, Add 5 ml of PBS,
It was prepared by applying ultrasonic waves.

2) Immunization method and cell fusion The first time, a rabbit thymus cell membrane component PBS solution and the same amount of Freund's complete adjuvant were emulsified and mixed, and 400 μ of 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 100μ
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 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. The suspension containing 7 × 10 ⁷ splenocytes and the suspension containing 1.4 × 10 ⁷ mouse myeloma were mixed and centrifuged to remove the medium. In a water bath heated to 37 ° C,
50% polyethylene glycol-RPMI1640 in mixed cells
1 ml of the medium was gradually added over 1 minute to gently stir for 1 minute to perform fusion. 2 ml of RPMI1640 medium was added for 2 minutes, and further 7 ml was added for 2 minutes with gentle stirring. The medium was removed by centrifugation, 20 ml of RPMI1640 medium containing 10% fetal bovine serum was added to the cells, and 0.1 ml per well was distributed to two 96-well plates. The next day, HAT medium (4 x 10
RPMI1640 medium containing ^-7 M aminopuritene, 1.6 x 10 ^-5 M thymidine, 1 x 10 ^-4 M hypoxanthine and 10% fetal bovine serum).
1 ml was added to the wells. Half of the medium in each well was replaced with HAT medium every 3 or 4 days. 3 weeks later, 8
Hybridoma growth was observed in 0% of the wells.

Second time, PBS solution of rabbit thymocyte membrane component and the same amount of Freund's complete adjuvant were emulsified and mixed, and 300 μm was intraperitoneally injected into NZB mouse (female, 6 weeks old). Thereafter, N adsorbed on 80 μg of Salmonella minnesota bacteria treated with acid
-Glycolyl GM ₃ Ganglioside 20μg in PBS solution 200μ
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. Add 50% polyethylene glycol-RPMI to the mixed cells in a water bath heated to 37 ℃.
2 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 14 minutes, another 14 ml was added over 2 minutes with gentle stirring. Remove the medium by centrifugation and add 10% to the cells.
120 ml of RPMI1640 medium containing fetal bovine serum was added, and 0.1 ml per well was distributed to 12 96-well plates. The next day, HAT medium (4 x 10 ^-7 M aminopterin, 1.6 x 10 ^-5 M thymidine, 1
RPMI164 containing × 10 ^-4 M hypoxanthine and 10% fetal bovine serum
0 medium) 0.1 ml was added to each well. The medium of each well is
Further, the HAT medium was replaced by half every 3 or 4 days. After 3 weeks, growth of hybridomas was observed in 90% of the wells.

Third immunization method was performed in the same manner as the second immunization except that eight intravenous injections were performed.

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, similar to the second except that distributed in 96-well plates ten KoTsuta.

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 the ELISA method.

As an antigen, N-glycolyl GM ₂ ganglioside, N-
Glycolyl GM ₃ ganglioside and IV ³ NeuGc-nLcOse ₄ Ce
r was used. 500 ng of the antigen was adsorbed on a microtiter plate for ELISA, and the mixture was 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, in the hybridoma prepared by the first immunization and cell fusion, an antibody that strongly reacts with N-glycolyl GM ₂ ganglioside was detected in one well, and in the hybridoma prepared by the second immunization and cell fusion. , antibodies that react strongly with N- glycolyl GM ₂ ganglioside, N- glycolyl GM ₃ ganglioside and IV ³ NeuGc-nLcOse ₄ Cer is detected in one well.

Further, the third immunization, in hybridomas prepared in cell fusion, N- 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 Ne
An antibody that strongly reacts with uGc-nLcOse ₄ Cer was detected in one well.

These hybridomas were transferred from HAT medium to HT medium without aminopterin, and further transferred to RPMI1640 medium containing 10% fetal calf serum (FCS) and cultured.

This hybridoma was cloned by the limiting dilution method. That is, cells were diluted in a 96-well plate at a density of 0.8 cells per well, cultured with 4 × 10 ⁵ mouse thymocytes per well, and 2 weeks later, antibody-producing cells were selected by ELISA. Stable hybridomas PyK-2, YHD-02, YHD-04, YHD-05 and YHD-0 were further cloned.
Got 7.

PyK-2 for the first time, YHD-02 for the second time, YHD-04, YHD
-05 and YHD-07 were obtained from the third immunization and cell fusion, respectively.

Monoclonal antibody PyK-2, YHD-02, YHD-04, YHD-
The antibody classes of 05 and YHD-07 were determined to be IgM, IgM ₃ , IgM, IgM and IgM by double immunodiffusion method and ELISA method, respectively.

Example 2 1) Measurement of antigen specificity of PyK-2 by ELISA ELISA using N-glycolyl GM ₂ ganglioside as an antigen
The antigen, the amount of which was changed stepwise, was adsorbed onto the plate for culture and the 4-fold diluted hybridoma culture supernatant was reacted. After washing with PBS, a peroxidase-labeled goat anti-mouse immunoglobulin antibody was reacted and washed with PBS, and then the absorbance at 492 nm was measured using orthophenylenediamine as a substrate to examine the reactivity of the antibody. The results are shown in FIG. In the figure, 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 thereof was adsorbed on the plate for ELISA. The serially diluted hybridoma culture supernatant was reacted, and the reactivity with the antibody of the present invention PyK-2 was examined in the same manner as. Results are shown in FIG. In Fig. 2, the vertical axis represents the absorbance and the horizontal axis represents the antibody dilution (2 ^- n). Moreover, each code | symbol shows the following.

White circle: N-glycolyl GM ₂ ganglioside (II ³ NeuGc-GgO
se ₃ Cer) Black circle: N-glycolyl GM ₃ ganglioside (II ³ NeuGc-Lac
Cer) White triangle: IV ³ NeuGc−nLcOse ₄ Cer Black triangle: VI ³ NeuGc−nLcOse ₆ Cer White square: As is clear from FIG. 2, the antibody PyK-2 of the present invention is N-
Has a strong reactivity to glycolyl GM ₂ ganglioside, confirmed that the reaction only weakly with glycolipids such as N- glycolyl GM ₃ ganglioside having N- glycolyl GM ₂ ganglioside from terminal N- acetylgalactosamine is removed structure Was given. It was also confirmed that PyK-2 does not react with N-acetyl GM ₂ ganglioside and asialo GM ₂ .

2) Measurement of PyK-2 Reactivity on TLC (Thin Layer Chromatography) 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 that had been subjected to the same operation, one plate was colored with the orcinol reagent. Enzyme immunostaining was performed on the other sheet. That is, the antibody of the present invention was reacted, and further a peroxidase-labeled goat anti-mouse immunoglobulin antibody was reacted. 4-chloro- as a substrate
Using 1-naphthol, blue-violet colored spots were detected.

FIG. 3 shows the results using N-glycolyl GM ₂ ganglioside and N-acetyl GM ₂ ganglioside as antigens. Chloroform: methanol: 2.5N aqueous ammonia (55:45:10 volume ratio) was used as a developing solvent. A is a plate developed with an orcinol reagent, and B is a plate subjected to enzyme immunostaining. N-acetyl for 1
GM ₂ ganglioside 2 was developed with N-glycolyl GM ₂ ganglioside. Apparently, the antibody of the present invention reacts with N-glycolyl GM ₂ ganglioside, but N-acetyl GM ₂
It can be seen that it does not react with ganglioside.

FIG. 4 shows the results using N-glycolyl GM ₂ ganglioside as an antigen and a mouse red blood cell crude acidic glycolipid fraction.

Chloroform: methanol: 0.2% calcium chloride (55:45:10 volume ratio) was used as a developing catalyst. The plates A and B are the same as those in FIG. N-glycolyl GM ₂ ganglioside was developed in 1 and mouse erythrocyte crude acidic glycolipid fraction was developed in 2. N-acetyl GM ₄ ganglioside and N-glycolyl G as gangliosides in C3H / He mouse erythrocytes
It has been reported that M ₂ gangliosides are contained in large amounts [Journal of Biochemistry (Tokyo), [J. Biochem. (Tokyo)] Vol. 94, 327-330 (1983)}, Enzyme Immuno. The result of the staining,
In 2, the color spot was detected at the same Rf value as N-glycolyl GM ₂ .

As a result of searching the reactivity of the antibody PyK-2 of the present invention with extracts from two different tissues of bovine kidney and mouse erythrocytes, it was confirmed that it reacts with N-glycolyl GM ₂ ganglioside itself.

3) YHD-02, YHD-04, YHD-05 and YHD by ELISA method
Measurement of antigen specificity of -07 ELISA method was performed using 0.2 nmol of various glycolipids as an antigen. 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 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 Table 1.

YHD-04 and YHD-07 are N-glycolyl GM ₂ gangliosides, and YHD-02 and YHD-05 are N-glycolyl GM ₂ gangliosides, N-glycolyl GM ₃ gangliosides, IV ³
It was found to react with NeuGc-nLcOse ₄ Cer and VI ³ NeuGc-nLcOse ₆ Cer.

Further, none of the monoclonal antibodies react with N-acetylneuraminic acid-containing glycolipids N-acetyl GM ₃ ganglioside and N-acetyl GM ₂ ganglioside, and sialic acid-free asialo GM ₂ and CDH
I knew it wouldn't react with.

4) Measurement of the reactivity of YHD-02, YHD-04, YHD-05 and YHD-07 on TLC (Thin Layer Chromatography) plates. Various glycolipids with a width of 6 mm were placed at a position 1 cm from the lower end of the TLC plate. Spotted and developed in an appropriate solvent system.
Of the two plates that had been subjected to the same operation, one plate was colored with the orcinol reagent. Enzyme immunostaining was performed on the other sheet. That is, the antibody of the present invention was reacted, and further a peroxidase-labeled goat anti-mouse immunoglobulin antibody was reacted. 4-chloro- as a substrate
Using 1-naphthol, blue-violet colored spots were detected.

FIG. 5 shows the results using four types of gangliosides as antigens. Chloroform: methanol: 2.5 as developing solvent
N-ammonia water (55:45:10 volume ratio) was used. A is the color developed by the orcinol reagent, and B, C, D and E are YHD, respectively.
This is a plate subjected to enzyme immunostaining using -02, YHD-04, YHD-05, and YHD-07. 1 for N-
Acetyl GM ₂ Ganglioside, 2 is N-glycolyl GM ₂
Ganglioside 3 was developed with N-acetyl GM ₃ ganglioside, and 4 was developed with N-glycolyl GM ₃ ganglioside.

The monoclonal antibodies YHD-02 and YHD-05 of the present invention are N-
Reacting with glycolyl GM ₃ ganglioside and N-glycolyl GM ₂ ganglioside also yields YHD-04 and YHD
-07 It has been Me probability of reacting with N- glycolyl GM ₂ ganglioside. Further, it was confirmed that none of the monoclonal antibodies of the present invention react with the N-acetylneuraminic acid-containing glycolipid.

〔The invention's effect〕

As described above, the present invention provides a novel monoclonal antibody. These antibodies are very effective in elucidating the mechanism of cancer development, diagnosis and treatment.

[Brief description of drawings]

FIG. 1 is a graph showing the reactivity of the antibody PyK-2 of the present invention with respect to N-glycolyl GM ₂ ganglioside by changing the amount of antigen in the ELISA method, and FIG. 2 is various glycolipids of the antibody PyK-2 of the present invention. The graphs showing the reactivity of glycerin against N-glycolyl GM ₂ ganglioside, N-
Acetyl GM ₂ ganglioside and mouse erythrocyte crude glycolipid fraction were developed on a plate and the reactivity with the antibody PyK-2 of the present invention was detected by enzyme immunostaining. A spot diagram and FIG. 5 show N-glycolyl GM ₂ ganglioside. , N-acetyl GM ₂ ganglioside, N-glycolyl G
M ₃ ganglioside and N-acetyl GM ₃ ganglioside were developed on a plate, and the antibodies of the present invention YHD-02, YHD-04, YH.
It is a spot diagram which detected the reactivity with D-05 and YHD-07 by enzyme immunostaining.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display location G01N 33/574 B 9015-2J 33/577 B 9015-2J (C12P 21/08 C12R 1:91) (56) References Nature, 276, 269-270 (1978) Mol. Immunol. , 19 (1), 87-94 (1982) J. Am. Biochem. (Tokyo), 95 (3), 785-794 (1984) Gann, 75 (11), 1025-1029 (1984) Biochem. Biophys. Re s. Commun. , 129 (2), 334-341 (1985) Cancer Res. , 45 (8), 3796-3802 (1985)

Claims (5)

[Claims] 1. A monoclonal antibody which recognizes an N-glycolylneuraminic acid-containing sugar chain and does not recognize an N-acetylneuraminic acid-containing sugar chain and a sialic acid-free sugar chain. 2. The monoclonal antibody according to claim 1, wherein the N-glycolylneuraminic acid-containing sugar chain is a sugar chain having Hanganachiu-Daiher antigenic activity. 3. The monoclonal antibody according to claim 1 or 2, wherein the sugar chain containing N-glycolylneuraminic acid is a constituent of glycolipid. 4. The antibody according to claim 1, which recognizes N-glycolyl GM ₂ ganglioside.
The monoclonal antibody according to any one of paragraphs. 5. The antibody comprises N-glycolyl GM ₂ ganglioside, N-glycolyl GM ₃ ganglioside, IV ³ Neu Gc-
The monoclonal antibody according to any one of claims 1 to 3, which recognizes nLcOse ₄ Cer and VI ³ Neu Gc-nLcOse ₆ Cer.