JPS63258493A - Anti-ganglioside gm, monoclonal antibody, cell producing said antibody and reagent composed thereof - Google Patents

Abstract

NEW MATERIAL:An anti-ganglioside GM1 monoclonal antibody specifically recognizing only ganglioside GM1. USE:An assay reagent for ganglioside GM1 and a diagnosticum for cancer, systemic lupus erythematosus (SLE), etc. PREPARATION:For example, ganglioside GM1 separated from a bovine brain and purified is injected into a mouse with an adjuvant to immunize, and after the final immunization the spleen is taken out and antibody-producing cells are collected. Then these cells are fused to mouse myeloma cells, and cultivated in an HAT medium to obtain hybridomas. Further, a clone producing an antibody which specifically reacts with ganglioside GM1 is selected and cloned through a limiting dilution method, etc., to monoclonize, and cultured and afford the monoclonal antibody. By assaying GM1 in blood using this antibody, cancer, SLE, etc., can be diagnosed.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a monoclonal antibody against ganglioside GM, a cell that produces it, and a reagent comprising the same for detecting or quantifying ganglioside GM.

[Prior Art and Its Disadvantages] In this specification, the names of carbohydrates and lipids, and the indications of bonding modes, etc. are in accordance with the common or common names in research in this field.

Glycolipids are biological substances that have attracted particular attention in research on cell development, differentiation, and canceration. Among glycolipids, glycosphingolipids containing sialic acid are collectively called gangliosides, and ganglioside GM+ (hereinafter simply GM
) is one of them.

GM is a substance that is present in large amounts in humans and various animals, especially in nervous system tissue. Cancer patients or autoimmune disease patients
Species of systemic lupus erythematosus (hereinafter referred to as SLE)
It has been reported that the composition of gangliosides in the blood of patients is significantly different from that of healthy individuals (Journal o
f Biochemistry Volume 98, Page 843, 198
5 years). In other words, the gangliosides that exist in the form of immune complexes in the blood are mainly 0M3 in healthy people, but 0M3 is rarely seen in patients with cancer or SLE, and instead GMI and GDll. , is the main ganglioside. Therefore, by accurately quantifying GM in the blood, it is possible to predict whether a person is suffering from cancer or SLE.

In addition to conventional chemical and biochemical methods, immunological methods using specific antibodies against GM 1 have come to be used as a means for quantifying, identifying and purifying GM. Conventionally, antibodies against GM have been used that are derived from antisera obtained by immunizing rabbits with GM together with appropriate carriers and immune adjuvants. It is a so-called polyclonal antibody, and its specificity may not necessarily be constant depending on the lot of the antibody produced or the individual used for immunization, and it is desired to obtain a monoclonal antibody without this drawback.

[Problems to be solved by the invention] As a result of intensive research, the inventors of the present application succeeded in producing an anti-GM monoclonal antibody that is extremely specific to GM and has a high antibody titer. This invention was completed.

That is, the present invention provides an anti-GM monoclonal antibody that specifically recognizes only GM.

Furthermore, the present invention provides a hyfritoma, which is a fusion cell of an antibody-producing cell derived from an animal immunized with GMI and a myeloma cell, and which produces the above-mentioned anti-G M 1 monoclonal antibody.

The present invention also provides a cell line that is derived from lymph beads infected with EB virus and produces the above-mentioned anti-GM monoclonal antibody.

Furthermore, the present invention provides anti-GM, ganglioside GM consisting of a monoclonal antibody, and a reagent for detection or quantification.

[Effects of the Invention] The present invention provides an anti-GM monoclonal antibody that is highly specific and has a high antibody titer against GM.

As will become clear in the Examples below, this monoclonal antibody does not react with other gangliosides having sugar chains similar to GM 1, but reacts extremely specifically with GM. Therefore, it is possible to quantify GM with extremely high sensitivity using the anti-GM monoclonal antibody of the present invention. Therefore, GM in blood can be quantified with high sensitivity using the monoclonal antibody of this invention, and therefore, with the reagent of this invention containing the monoclonal antibody of this invention,
The probability that a subject has cancer or SLH can be known with high sensitivity.

Furthermore, the present invention provides novel cells that produce the above-mentioned monoclonal antibodies.

[Detailed Description of the Invention] The monoclonal antibody of the present invention specifically binds only to GM, and its specificity is extremely high. That is, as will become clear in the later examples, the monoclonal antibody of the present invention has an extremely high antibody titer of 216 or more against GM, while it has a sugar chain similar to that of GM□ or another having it. glycolipids, namely GalCer, LacC:er,
Gb3. Gb, , GA2, G A + , ganglioside 0M3. Ganglioside GM 2, Ganglioside GD, a, Ganglioside GD1a, Ganglioside GD1b, Ganglioside GTIb, Ganglioside G Q s b, Ganglioside Fuc-GM
, , nLc, and sialosyl nLc4 (Gal is galactose, Cer is ceramide, Lac is lactose, Fuc is fucose, and n1.c4 is paracrobocyto). , its antibody titer is 22 or less.

The monoclonal anti-GM and antibody of this invention can be produced by a method of producing bilitomas by fusion of antibody-producing cells of a mammal immunized with GM1 with myeloma cells (hereinafter referred to as the hybridoma method), or by a method of creating bilitomas by fusion of antibody-producing cells of a mammal immunized with GM1 (hereinafter referred to as the hybridoma method), or by a method of producing EB to human B lymphocytes. virus (hereinafter referred to as
A method of infecting EBV and causing transformation (
(hereinafter referred to as the EBV transformation method). That is, after selecting only anti-GM and antibody-producing clones from the antibody-producing cells given proliferative properties by the above method and making them monoclones, 1. From this, monoclonal antibodies having the above characteristics can be produced by isolating them.

The Hyfritoma method will now be described in detail. In this invention, the hyperfritomas are produced by a known method, for example, the method described in Nature, Vol. 256, p. 495, 1975, or a modified method thereof (Journal of Exp.
Erin+Ental Medicine, Vol. 150, p. 1008, 1979).

The animal species and organs from which GM is prepared for use as an immunogen are not specified, or generally GM is prepared from the brains of animals that contain it in large amounts, such as cows. Unless otherwise specified, GM used in Examples and Test Examples described later in this specification was also isolated and purified from bovine brain by known methods. In addition, as an immunogen,
In addition to purified GM1, various cells having it on the surface,
Furthermore, any substance or cell containing a sugar chain moiety that is an antigenic determinant of GM may be used.

The type of mammal to be immunized with GM is not particularly limited, or it is preferable to select it in consideration of compatibility with the myeloma cells used for cell fusion, and generally humans, mice, rats, or in some cases rabbits are selected. used.

In vivo and in vitro immunization of GM
Any of these methods can be used. For in vivo immunization, GM, physiological saline, phosphate buffer (hereinafter referred to as PB)
After diluting the drug to an appropriate concentration (referred to as S), it may be administered to animals by intravenous, subcutaneous, or intraperitoneal injection. To be more specific, for example, when using purified GM, it is diluted with PBS etc. to an appropriate concentration, and together with commonly used carriers such as Salmonella Minnesota strain and bovine serum alulamine (hereinafter referred to as BSA), This is administered to the animals several to several times every 4 to 14 days, and the total dose per animal per solid
It is preferable to adjust the amount to about 300 gg. In addition, when membrane components or cells themselves are used for in vivo immunization, the procedure is similar; for example, when membrane components are used, the total dose is 1 to 100 mg/solid, and when cells themselves are used, the total dose is 1 to 100 mg/solid. It is preferable to set the number to 10 6 to 10 9 pieces/solid. In this case, an appropriate immune adjuvant, such as Freund's complete adjuvant, may be used if necessary.

In the case of this in vivo immunization, the antibody-producing cells may be any of tile cells, lymph node cells, intraperitoneal lymphocytes, or peripheral blood lymphocytes, but it is most preferable to use tile cells 4 days after the final immunization.

In addition, in the case of immunization in an in vitro system,
So-called in vitro sensitization methods can be used. This is done by culturing appropriately selected lymphocytes from tile cells, lymph node cells, intraperitoneal lymphocytes, and peripheral blood lymphocytes with the immunogen GM□ for about a week in vitro, thereby culturing antibodies against GM. It is intended to cause production cells to appear. In this case, G.M.
If the product is a purified product, it is dissolved in a cell culture medium or adsorbed onto a suitable carrier such as sheep red blood cells, liposomes, or Salmonella minnesota strain, and cultured with lymphocytes.

In addition, when the cell itself containing G M 1 or its membrane component is used as an immunogen, it is dissolved or suspended in a medium and cultured together with lymphocytes. In addition, when using the cells themselves,
It is desirable to culture the cells with lymphocytes after treatment such as mitomycin treatment or radiation irradiation. The culture medium for lymphocytes is PRM11640, Talbetzkow's ME.
You can use any of those commonly used for lymphocyte culture, such as D-MEM (hereinafter referred to as D-MEM), or add 5 to 20% fetal calf serum (hereinafter referred to as FC3) when using them. It is desirable to add it to a concentration of . In addition, 2-mercaptoethanol (
2-ME) and pork lyde mitogen (hereinafter referred to as PWM) to a final concentration of 5 x 10 M and 5 to 30 g/ml, respectively, resulted in efficient in vitro sensitization of GM. It is possible to make it come true.

The cell concentration during lymphocyte culture may vary depending on the equipment used for culture, but is generally preferably 106 to 107/ml.

Immunogens such as purified GM, GM, containing cells, and their membrane components were each used at a concentration of 1 to 20 gg/+n1. 1-100
It is preferred to use a final concentration of 0.1 to 10 n+g/ml.

Subsequently, in vivo or in vitro as described above
r.

Antibody-producing cells obtained by immunization using this system are fused with myeloma cells.

As myeloma cells, various known cells such as N5-1, P3, P3-old, X45, and X6 in mice can be used.
3.6.5.3. , SF3, 711. in rats. Y3
, Ag1.2.3, etc. can be used.

Cell fusion can be carried out according to known methods, for example, by incubation in a fusion promoter-containing medium.

As a fusion promoter, for example, polyethylene glycol (
(hereinafter referred to as PEG), Sendai virus, etc. are used.
Furthermore, an adjuvant such as dimethyl sulfoxide may be added to increase the fusion efficiency.

The ratio of lymphocytes and myeloma cells to be used is the same as in general methods; for example, the ratio of lymphocytes to myeloma cells may be about 1 to 10 times.

As the medium for the above-mentioned fusion, any of the usual various media used for cell culture can be used, and it is usually preferable to exclude serum such as Fe2.

For fusion, predetermined amounts of the immune cells and myeloma cells are thoroughly mixed in the medium, centrifuged, the supernatant is removed, and a PEG solution (PEG has an average molecular weight of 10
00 to 6000) is usually added to the culture medium at a final concentration of about 30 to 6000 Sow/v% and mixed. Thereafter, hybridomas are formed by repeating the steps of sequentially adding an appropriate medium, centrifuging, and removing the supernatant.

Isolation of the desired hybridoma is carried out by culturing the above-described cell fusion cells in a conventional hybridoma selection medium. The myeloma cell lines described above contain hypoxanthine guanine phosphoribosyltransferase (HGPR).
T) is a deficient strain and therefore HAT medium (hyboxanthin, aminobutyric).

It does not grow in medium containing chlorine and thymidine). Therefore, cells that grow in HAT medium may be selected. The cells may be cultured in the HAT medium for a sufficient period of time, usually from several days to several weeks, to kill cells other than the target hybridoma.

The hybridomas obtained in this way are prepared using conventional cloning methods such as limiting dilution method or soft agar method.
A search for the desired antibody-producing strain and monocloning are performed.

The search for the antibody-producing strain can be performed, for example, by the ELISA method (Japanese).
nese Journal of Experiments
tal Medicine51@, 309 pages, 1981
) and plaque method, agglutination reaction method, octacony method,
This is carried out by various methods generally used for detecting antibodies, such as the RIA method.

In this way, a monoclonal anti-GM, antibody-producing cell line can be obtained by the Hyturidoma method.

Next, monoclonal anti-GM1 by EBV transformation method
The production of antibodies will be described. The final method is E to human B lymphocytes.
It is based on the principle that when infected with BV, each B lymphocyte not only proliferates but also produces antibodies. In that case, EBV does not act only on a specific B lymphocyte clone to bring about the above effects, but basically acts on all clones, causing them to proliferate and produce antibodies. Therefore, after infecting human B lymphocytes with EBV, only clones producing the desired antibody can be selected and used to obtain the desired monoclonal antibody.

Transformation of human lymphocytes by this method can be carried out according to known methods, such as the method described in Nature, Vol. 267, p. 52, 1979.

The organ from which B lymphocytes are obtained is not particularly limited as long as it is a lymphoid organ, or, considering the special nature of human-derived materials, peripheral blood lymphocytes from healthy individuals are generally used.

As a virus source, EBV persistent producing cells, such as EBV
B-95-8 cells, an infected marmoset leukocyte cell line (Proceedings of the Nation
nalAcadeIly of 5science of
USA Vol. 70, p. 190, 1973).

In this method, B lymphocytes are first infected with EBV, but in this case, it is not necessary to specifically separate only B lymphocytes from the lymphocyte fraction, and it is not necessary to specifically separate only B lymphocytes from the lymphocyte fraction; instead, it is necessary to infect lymphocytes obtained using a conventional method with EBV. Bye.

First, add the virus solution to the lymphocyte pellet and add 1 lymphocyte.
Add about 10ml to 10' pieces and mix, 37
Incubate for 1 hour at 15% CO2 at °C to infect lymphocytes with EBV.

The infected lymphocytes thus obtained are dispersed in a cell culture medium and cultured for 2 to 5 weeks at 37° C. and 5% CO□.

The culture medium is not particularly limited, and RPMI 1640, D
- Any conventional lymphocyte culture medium such as MEM may be used, but it is preferable to add Fe2 to a concentration of 10 to 20% at the time of use. Also,
Cells can be grown more efficiently by adding glutamine to the medium at a final concentration of 0.5 to IB/ml.

The thus obtained transformed B lymphocytes are searched for the desired antibody-producing strain and monocloned using the same method as in the Hyturidoma method.

Search for the antibody-producing strain can be performed by various methods generally used for detecting antibodies, such as ELISA and RIA, as in the case of the Hyfritomas method.

In this way, monoclonal anti-GM and antibody-producing cell lines can also be obtained by EBV transformation.

The monoclonal anti-GM antibody-producing cell line of this invention described in Section 2 can be obtained by the hyperfritomas method or the EBV method.
Regardless of the transformation method, subculture is possible in a normal medium, and long-term storage is easily possible in liquid nitrogen.

The inventor of the present application selected clone MX-1 obtained in Example 1 described below as a representative of the monoclonal anti-GM, antibody-producing strain.
Fermentation Research Institute (2-17 Jusaki-cho, Yodoyo-ku, Osaka City)
-85) (accession number IFO50115)
.

In order to obtain the monoclonal antibodies of the present invention from the specific cell lines obtained as described above, those cell lines can be cultured in large quantities according to conventional methods and separated from the culture supernatant, or A method can be employed in which the strain is injected into compatible mammals, allowed to grow, and isolated from the serum or ascitic fluid.

The monoclonal anti-GM antibody of the present invention specifically recognizes only GM (the sugar chain moiety that is its antigenic determinant) with extremely high sensitivity, and therefore can be used as a highly sensitive detection or quantitative reagent for GM. has.

As mentioned above, it has been reported that the ganglioside composition in the blood of cancer patients or SLE patients is significantly different from that of healthy individuals (Journal of Bioch.
emistry, Vol. 98, p. 843, 1985). Therefore, the monoclonal anti-GM antibodies of this invention also have use as cancer and SLE diagnostic reagents.

Immunological identification and quantification of GM□ using the quantitative reagent of the present invention comprising the above-mentioned monoclonal anti-GM and antibody of the present invention is carried out in the same manner as in general immunological quantification of in vivo substances. be able to. The method is not particularly limited; for example, radioimmunoassay (RIA), El
, ISA, thin layer chromatography (hereinafter referred to as TLC)-immunostaining, or immunohistochemistry can be used.

Furthermore, in RIA and ELISA, the so-called competition method or sandwich method can be used, but the method is not limited thereto.

Furthermore, the immunological identification and quantification of GM using the reagent of the present invention is not only applicable to GM in the blood of cancer patients and SLE patients, but also broadly applicable to GM in the blood of humans and various experimental materials. Needless to say, it is also applicable to GM in body fluids, organs, or excreta.

Furthermore, as shown in Test Example 3 below, the monoclonal anti-GM antibody of the present invention, like monoclonal antibodies against other glycolipids, recognizes the epitope of the carbohydrate chain of GM. It is a structure. Therefore, it goes without saying that the reagent of the present invention is useful not only for GM, but also for the detection and quantification of molecules having a GM sugar chain structure.

As will become clear in the Examples below, cancer patients and SLE
The aM1 concentration in the patient's body fluid increases, or the change is at most several times to several times lO compared to the normal state. Changes of this magnitude cannot be detected with high sensitivity using conventional polyclonal antibodies. Furthermore, since conventional chemical analysis requires large amounts of gangliosides, for example,
In 1985, patient blood was passed through an immobilized protein A column using an extracorporeal circuit, and immunochlorin G was adsorbed onto the column in the form of an immune complex with ganglioside. It is collected and gangliosides are obtained. However, it is practically impossible to form an extracorporeal circuit and circulate blood through it frequently for people with reduced physical strength, such as cancer patients.

The reagent of the present invention has extremely high sensitivity and allows GM□ to be quantified by collecting a small amount of blood from a patient. Therefore, this invention has made practical diagnosis of cancer and SLH possible for the first time.

[Examples of the invention] Example 1 Purified GM, 100 p, g and formalin-treated Salmonella enterica strain Minnesota (ATCC9700) 400 gg
was added to 4 ml of physiological saline kept at 40°C and stirred well to form a uniform suspension. The resulting suspension was intravenously administered to mice and rats every 4 days, each dose containing 10 gg of GM, for a total of 4 to 15 times. Four days after the final administration, the spleen was removed, and 3 x 10 spleen cells and 3 x 10 N5-1 mouse myeloma cells (ATCCTl818) were collected.
Cell fusion was performed in the presence of 50% polyethylene glycol. This hybridoma was grown in a 96-well flat-bottom plastic culture plate (Falcon (registered trademark)).
) and add 10% FC9 containing HAT medium D-
The cells were cultured in MEM medium under TE370C15% CO2 conditions. The presence or absence of anti-GM1 antibody in the culture supernatant was searched for wells in which growth of Hyturidoma was observed using the ELISA method described below. The ELISA method uses a 96-well plastic plate (Falcon®), and first injects 10 pg/g in ethyl alcohol.
Dispense 0.05 ml of GM dissolved to a concentration of 1 ml into each well, allow the solvent to evaporate naturally, and remove the GM with a brake.
- was adsorbed to. Each well is reacted with the test culture supernatant as a primary antibody, and after thorough washing, a peroxidase-labeled anti-mouse immunoglotulin antibody (if a mouse is used as the immunized animal) as a secondary antibody, or Anti-rat immunoglobulin antibody (when rat is used as the immunized animal)
reacted.

Subsequently, the diammonium salt of 2,2'-azinobis(3-ethylbenzthiasolinsul)onic acid (hereinafter referred to as B
Perform a peroxidase reaction using TS) as a substrate,
Adjust the color intensity of each well to your own limit or use 96-well ELI
It was read using an automatic photometer for SA (wavelength 414nIl).

Hyturidoma in wells in which anti-GM antibody titers were observed in the culture supernatant were further cloned by limiting dilution method to obtain single clones.

The monoclonal hybridoma thus obtained was cultured in large quantities in plastic culture flasks to obtain the desired monoclonal anti-GM and antibody-producing cell line. From the culture supernatant of these cell lines, or after transplanting these cell lines into nude mice pretreated with the immunosuppressant pristane (2,6,1,0°14-tetramethylpentadecane, manufactured by Aldrich). From ascites, monoclonal anti-GM
, antibodies were obtained. In addition, if necessary, the antibodies were purified by the 5 oz saturated ammonium sulfate method.

In this way, monoclonal anti-GM antibodies have been obtained. In the test examples described below, mouse-derived monoclonal anti-GM, antibodies MX-1, MX-2, and MX-3 were used. In addition, such monoclonal anti-G M + antibody M
X-], MX-2, and MX-3 were named hybrid-vMX-1, MX-2, and MX-3.

Example 2 2 mg of purified GM and 11 mg of BSA in physiological saline
A water-in-oil emulsion was prepared using an equal volume of Freund's complete adjuvant, and 0.21 drops (total of 0.8n +
l) Injected. Four weeks later, an emulsion containing GM was prepared in the same manner as in the first immunization, and a total of 1 ml of the emulsion was injected subcutaneously into the Hyturidoma for booster immunization. Four days later, the spleen was removed, and N5-1 myeloma cell fusion and anti-GM and antibody-producing bilitoma cloning were performed in the same manner as described in Example 1 to obtain a monoclonal antibody-producing strain l clone. Ta. After cell fusion, a 24-well plastic plate (Falcon® Catalog No. 38) was used for the growth of Hyturidoma in HAT medium.
47) was used. In addition, a peroxidase-labeled anti-heron immunoglotulin antibody was used as the secondary antibody used in the ELISA method.

Example 3 Lymphocytes (mononuclear cells) were prepared from human peripheral blood using Ficoll Pack (registered trademark of Pharmacia) in a conventional manner, and then suspended in a medium at a concentration of l x In'/1.

The medium used is RPMI 1640 medium supplemented with 10 pieces of FC3, and when used, 2-ME and PWM were added 5x each.
It was used at a final concentration of 10-5M and:I07pg/111L. Next, a lymphocyte suspension with a concentration of 1 x 10'/ml was poured into the inner cylinder of a Marfurtsk type culture bottle (Lancet vol. 2, p. 1279, 1967).
1. 10 ml of PWM-free medium was added to each outer cylinder, and a dialysis tune was placed at the boundary between the inner cylinder liquid and the outer cylinder liquid.

GM, the method of Uchimaru et al. (Journal of Bioc
The mixture was incorporated into a liposome composed of egg yolk lecithin and cholesterol prepared according to Hemistry 87@, p. 1829, 1000), and added to the inner cylinder of a culture bottle at a final concentration of 5 gg#nl of GM.

Thus, the lymphocytes were incubated with GM at 37°C, 5% CO2.
The cells were cultured for 6 days under these conditions. Subsequently, cell fusion with N5-1 and anti-GM
The antibody-producing hybridoma was cloned, and one monoclonal antibody-producing strain clone was obtained.

In the case of this example, a bar oxidase-labeled anti-human immunoglobulin antibody was used as the secondary antibody in the ELISA method.

Example 4 First, B-95-8 that continuously produces and releases EBV
Cells (ATCC CRL1612) were treated with 0.0% curcumin.
Contains a final concentration of 86 mg/ml, plus 20% F
The cells were suspended at a concentration of 3 x 105/ml in RPMT 1640 medium (hereinafter referred to as complete medium) supplemented with e2 and cultured at 37°C and 15% CO2. The culture supernatant obtained after 7 days was used as the virus solution used below.

Subsequently, based on the conventional method, Ficallback (registered trademark)
Add the virus solution to the pellet of lymphocytes obtained using
The mixture was added at a ratio of 10 ml to 1 x 10' lymphocytes, mixed, and incubated at 37°C and 15% CO2 for 1 hour. 2 x 105 to 6 EBV-infected lymphocytes
Suspend in complete medium at a concentration of x 105/ml, dispense it into each well of a flat-bottomed 96-well culture plastic plate (Falcon (registered trademark)), and store at 37°C and 15% CO2. After 4 days, 0.1 ml of fresh complete medium was added to each well.After that, half of the culture L supernatant was replaced with fresh complete medium every 3 to 4 days, and culture was started. After 2 to 4 weeks, anti-GM was detected in the culture supernatant of wells where cell proliferation was observed.

Antibody titers were measured using the ELISA method described in Example 1 above. In this case, a peroxidase-labeled anti-human immunoglobulin antibody was used as the secondary antibody.

Anti-GM in the supernatant, cells in wells in which antibody titer was observed were transferred to a 24-well culture plate, 6-well culture plate, 6c.
Anti-GM in culture of I11 plastic dates.
, those with antibody titers were cloned using the soft agar method according to conventional methods, and monoclonal anti-GM,
A production cell line clone was obtained, which was named cell line 11X-1.

From the culture supernatant obtained by mass culturing this cell line, 50
% saturated monoclonal anti-G by ammonium sulfate precipitation method.
M, antibody HX-1 was obtained.

Test Example 1 The monoclonal anti-GM antibody (MX-
1, MX-2 and MX-3) The class of immunoglobulin antibodies was determined by ELISA method. That is, the monoclonal antibody was reacted with a peroxidase-labeled antibody against each class of mouse immunochlorin, and then a peroxidase reaction was performed using ABTS as a substrate.

Sono result, MX-1 and MX-2 are both IgM,
MX-3 was found to belong to the IgG class.

[Test Example 2] The reactivity of the monoclonal anti-GM and antibodies obtained in Examples 1 and 4 to various glycolipids was examined using ELISA. The glycolipids used were GM, Ga1C:er (derived from bovine brain), and LacCer, which have similar structures.
(derived from bovine brain), atz (derived from human red blood cells), a
b<(derived from human red blood cells), GA2 (derived from bovine brain), GA
I (derived from bovine brain), GM.

(derived from bovine brain), 0M2 (derived from bovine brain), GD, a(
(derived from bovine brain), GD+b (derived from bovine brain), GT+b (derived from bovine brain), GQIb (derived from bovine brain), Fuc-GM
, (derived from rat red blood cells), nLc4 (derived from human red blood cells), and sialosyl nLc4 (derived from bovine red blood cells) (structures are shown in Table 3 at the end). The antibody titer for each glycolipid was expressed as the maximum dilution factor (hereinafter expressed in the form of a power of 2) at which color development was observed with the naked eye in ELISA.

The results are shown in Table 1 below, MX-1.

MX-2, MX-3 and HX-1 Noi Gap or GM,
showed high antibody titer against. However, none of the antibodies reacted with other glycolipids (all antibody titers were 22 or less).

Table 1 Test Example 3 Monoclonal anti-GM of each clone of MX-1, MX-2, MX-3 and HX-1 obtained in the above example, glycolipid specificity of antibody was determined by TLC-immunostaining method We further investigated using The final method involves fractionating glycolipids using TLC on silica gel, and then performing immunostaining on a thin layer plate using the same principle as the ELISA method.

In this method, the sensitivity of glycolipid detection is very high, and
It has the advantage of denying reactions to substances that are mixed in trace amounts with each glycolipid. Therefore, it is currently widely used in research in this field as one of the most excellent methods for examining the specificity of antibodies against glycolipids.

This test example uses the method reported by Jour et al.
nal of Biochemistry Volume 95,
TLC-enzyme immunostaining was performed according to (1984, p. 1517).

First, each glycolipid including GM (the origin is described in the section of Test Example 2) was placed on a thin layer plate of silica gel (Pine spp.
It was spotted on Bolitarum Sil G) manufactured by Nagel.

Development was carried out for about 25 minutes using a mixture of chloroform-methyl alcohol-0.25% potassium chloride (50:40:10.v/v) as a solvent. The position of each glycolipid after development was determined using orcinol.

In parallel with the orcinol reaction, development for immunostaining was performed at the same time, and staining was performed using the following method. First, MX- produced in Example 1 was used as a primary antibody on a thin layer plate after development.
L MX-2, MX-3, llX- produced in Example 4
1 or a polyclonal anti-G Mr rabbit antibody. For the polyclonal anti-GM rabbit antibody, an antiserum obtained by immunizing a rabbit with GM using the method described in Example 2 and collecting blood two weeks after the booster immunization was used.

Subsequently, an antibody against mouse, human or rabbit immunoglobulin (all labeled with peroxidase) was appropriately selected and reacted with the plate as a secondary antibody.

Furthermore, color development was performed by peroxidase reaction using 4-chloro-1-naphthol as a substrate.

As shown in Table 2, all of the anti-G M 1 monoclonal antibodies of the present invention reacted only with the G M 1 subant. Polyclonal anti-GM used as a control, the antibody was G
It reacted not only with Ms, but also with GA and GD, albeit weakly.

Table 2 1) - indicates no color development, + indicates weak color development, and +10 indicates strong color development.

From the above results, MX-], MX- obtained in Example 1
2, and MX-3, and HX-1 obtained in Example 4
Both monoclonal anti-GM antibodies were shown to be specific for GM only. Furthermore, in this respect, these monoclonal anti-GM antibodies show high significance over polyclonal anti-GM rabbit antibodies that have been produced and used by conventional methods. .

Furthermore, from the results of this example, the monoclonal anti-GM antibody of the present invention is Galβ]-+GalNAcβ1-+
4[5A(X 2-+ 3]Galβ1-+4GIc
It is presumed that it recognizes the sugar chain structure of

Test Example 4 A polystyrene ball immobilized with protein A was placed in a glass test tube, and serum 11 of a cancer or SLE patient was placed in the tube and left overnight at 4°C. After washing with a polyethylene ball, add 0.3M glycine-hydrochloric acid buffer (pH 2,
8), the immune complex is dissociated from protein A, and the immune complex itself is also dissociated into GM and immunoglobulin G. Remove the polystyrene ball from the test tube and add a mixture of chloroform and methyl alcohol (2:1.v) to the test tube.
/v) was added and shaken well. After centrifugation, both portions of the lower chloroform layer were separated and evaporated to dryness. This was dissolved in an appropriate amount of PBS containing 1% BSA, and the following GM
, which was used as a specimen for measurement.

GM was measured using the ELISA method described below. First, sample solution 100.1 was placed in each well of a 96-well microtitration plate (hereinafter referred to as plate), and then monoclonal anti-GM and antibody MX of the present invention were added.
-1 bound polystyrene balls were added and the plate was incubated at 37°C for 3 hours. After washing the plate, biotinylated anti-G M r antibody (biotinylation was performed in a conventional manner) dissolved in PBS containing 1% BSA and 0.05% Tween 20 (polyoxyethylene sorbitan monolaurate). 100.1 was added and reacted at room temperature for 1 hour. After further washing, add 1% BSA and 0.05
Bar oxidase-labeled avidin dissolved in PBS containing % Tween 20 was added and allowed to react at room temperature for 1 hour. After washing,
A peroxidase reaction was carried out using ABTS as a substrate, and the degree of color development of each well was measured using a 96-well ELISA automatic photometer (wavelength: 414 nm). Said Example 3
GM was encapsulated in liposomes using the method described in , and the concentration of GM was determined based on a standard curve using the same.

As shown in the figure, the results showed that GM was higher in cancer patients and SLE patients than in healthy individuals.

The value was shown. Furthermore, in this case, high levels of GM were observed regardless of the type of cancer. Note that in the figure, GM and density are expressed after being corrected to an arbitrary density unit (U/[01).

Thus, the monoclonal anti-GM, antibody, and blood GM measurement system using the same of the present invention are extremely useful in the diagnosis of cancer and SLE.

Furthermore, in Test Example 4, the monoclonal antibody of the present invention was used as the "insolubilized" antibody bound to the polystyrene ball and the biotin-labeled antibody.
In this case, extremely low G of 1 to 10 ng/ml is used.
A linear standard curve was obtained over the concentration range.

On the other hand, when the insolubilized antibody and labeled antibody used were polyclonal anti-GM and rabbit antibodies, both of which have been used conventionally, a linear standard curve was obtained in the concentration range of 10 to 1100 n/ml.

Moreover, as an insolubilized antibody, the monoclonal antibody M of this invention
When X-1, polyclonal anti-GM, and rabbit antibodies were used as labeled antibodies, a linear standard curve was obtained in the concentration range of 5 to 50 ng/ml.

As described above, in the measurement of GM by the so-called Santowich method used in Test Example 4, the measurement sensitivity was increased by using a monoclonal antibody.

Also, in the quantification of GM under the so-called competition law,
Polyclonal anti-GM, 10-]00 when using antibodies
A linear standard curve was obtained for the monoclonal anti-GM of this invention.

A linear standard curve was obtained using antibody MX-1 in the 3-30 ng/ml range.

As described above, when the monoclonal antibody of the present invention was used to measure GM, a measurement system with higher measurement sensitivity was obtained compared to the case of using a polyclonal antibody. As a result, it has become possible to detect changes in GM concentration in body fluids in disease states, which was previously impossible to measure with measurement systems using polyclonal antibodies. This is also an advantage of the monoclonal anti-GM antibody of the present invention over the conventional polyclonal anti-GM□ antibody.

[Brief explanation of drawings]

The figure shows various cancer patients and SLE determined by the ELISA method using the monoclonal anti-GM and antibody MX-1 of this invention.
It shows individual values of GM and concentration in patient's blood.

Claims (20)

[Claims] (1) Anti-ganglioside GM_1 monoclonal antibody that specifically recognizes only ganglioside GM_1. (2) The monoclonal antibody is ganglioside GM_1
Has an antibody titer of 2^1^6 or more against GalCer,
LacCer, Gb_3, Gb_4, GA_2, GA_
1, Ganglioside GM_3, Ganglioside GM_2
, ganglioside GD_1_a, ganglioside GD_
1_b, ganglioside GT_1_b, ganglioside GQ_1_b, ganglioside Fuc-GM_1, nL
c_4, and sialosyl nLc4 (Gal is galactose, Cer is ceramide, Lac is lactose,
2. The monoclonal antibody according to claim 1, which does not substantially react with Fuc (Fuc represents fucose and nLc_4 represents paragloboside). (3) The monoclonal antibody according to claim 2, which is MX-1. (4) The monoclonal antibody according to claim 2, which is MX-2. (5) The monoclonal antibody according to claim 2, which is MX-3. (6) The monoclonal antibody according to claim 2, which is HX-1. (7) A hybridoma that is a fusion cell of an antibody-producing cell derived from an animal immunized with ganglioside GM_1 and myeloma cells, and produces an anti-ganglioside GM_1 monoclonal antibody that specifically recognizes only ganglioside GM_1. (8) The hybridoma according to claim 8, which is hybridoma MX-1. (9) The hybridoma according to claim 7, which is hybridoma MX-2. (10) The hybridoma according to claim 7, which is hybridoma MX-3. (11) A cell line that is derived from lymphocytes infected with EB virus and produces an anti-ganglioside GM_1 monoclonal antibody that specifically recognizes only ganglioside GM_1. (12) The cell line according to claim 11, which is cell line HX-1. (13) A reagent for detecting or quantifying ganglioside GM_1, comprising an anti-ganglioside GM_1 monoclonal antibody that specifically recognizes only ganglioside GM_1. (14) The monoclonal antibody is ganglioside GM
Has an antibody titer of 2^1^6 or higher against _1 and GalCe
r, LacCer, Gb_3, Gb_4, GA_2, G
A_1, ganglioside GM_3, ganglioside GM
_2, ganglioside GD_1_a, ganglioside G
D_1_b, ganglioside GT_1_b, ganglioside GQ_1_b, ganglioside Fuc-GM_1,
nLc_4, and sialosyl nLc4 (however, Gal
The reagent according to claim 13, which does not substantially react with galactose, Cer is ceramide, Lac is lactose, Fuc is fucose, and nLc_4 is paragloboside. (15) The reagent according to claim 14, wherein the monoclonal antibody is MX-1. (16) The reagent according to claim 14, wherein the monoclonal antibody is MX-2. (17) The reagent according to claim 14, wherein the monoclonal antibody is MX-3. (18) The reagent according to claim 14, wherein the monoclonal antibody is HX-1. (19) The reagent according to any one of claims 13 to 18, which is a cancer and systemic lupus erythematosus diagnostic reagent. (20) A method for detecting and quantifying GM_1 using an anti-ganglioside GM_1 monoclonal antibody that specifically recognizes only ganglioside GM_1.