JPH0380097A - Monoclonal antibody capable of recognizing myristoylglycylpeptide - Google Patents

Abstract

NEW MATERIAL:A monoclonal antibody capable of specifically recognizing myristoylglycyloligopeptide. USE:Diagnostics for examining the cause of canceration for carcinomatous cells. PREPARATION:For example, myristoylglycyloligopeptide of the formula (Myr is myristoyl group), an N-terminated amino acid sequence as a manifestation product of carcinogenic gene P60SRC is reacted with hemocyanin in the presence of a water-soluble carbodiimide to prepare an antigen for immunization. This antigen is administered into the abdominal cavity of a Balb/c mouse to effect immunization, and after the final immunization, spleen cells are collected and fused with mouse myeloma cells using polyethylene glycol followed by making a culture in a HAT medium to obtain hybridoma. Thence, the hybridoma is screened and cloned by e.g. the limiting dilution analysis into single cell clone, which is then put to culture, thus obtaining the objective monoclonal antibody.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a monoclonal antibody that specifically reacts with myristoylglycyl oligopeptide, a method for producing the same, and a method for using the antibody.

In recent years, a certain type of protein phosphate enzyme (Adenos 1n
e3'5'-phosphate dependent
t protein kinase type+1)
It was discovered that the N-terminal glycine of [Proc, Natl, Acad,
Sci.

USA Vol, 79', p6128 (1982)
, Biochemi'5try Vol.

22 p3702 (1983)], and many studies on various myristoylated proteins have been conducted since this work. In particular, a report on the biological significance of myristoylation states that myristoylation of N-terminal glycine has important implications for the functional expression of certain proteins involved in cell transformation and growth regulation [5ciencc
Vol, 227. p427 (1985)], and has also recently been linked to various diseases involving retroviruses, such as acquired immunodeficiency syndrome (AIDS), adult T-cell leukemia (ATL), and myristoylation of causative viral proteins. It is attracting a lot of attention.

Although the general role of protein myristoylation has not yet been elucidated in detail, as mentioned above, there is no doubt that it plays an important role in biological systems, and it is therefore important to track this reaction. It is believed that if a method to do this can be established, it will be possible to further clarify the biological significance of myristoylation, and in particular to elucidate its relationship with pathological conditions, which in turn will pave the way for the diagnosis, prevention, and treatment of diseases. . However, at present, a method for easily detecting myristoylated proteins has not yet been established.

The present inventors+3 related to each of the above reports, and in the course of their own research, they immunized mice with myristoylglycyl oligopeptide as an immunization antigen, and the spleen cells and spleen cells obtained from these mice. , prepared a hybridoma obtained by fusion with mouse myeloma (myeloma) cells, succeeded in obtaining a monoclonal antibody that specifically recognizes myristoylglycyl oligopeptide from this hybridoma, and completed the present invention. . Furthermore, it was confirmed that such a monoclonal antibody of the present invention specifically reacts only with cells in which the SRC gene is strongly involved in cell canceration.

The anti-myristoylglycyl peptide monoclonal antibody of the present invention is an extremely highly specific monoclonal antibody that can easily detect and identify a specific myristoylglycyl protein.

The monoclonal antibody that identifies anti-myristoyl glycyl oligopeptide obtained by the present invention (hereinafter referred to as the present monoclonal antibody) is useful for analyzing the properties of myristoyl glycylated proteins produced in biological systems. can serve as a means for isolating myristoylglycyl peptides with For example, by using affinity chromatography using the present monoclonal antibody as a ligand, it is possible to selectively separate the target myristoylglycyl peptide.

Furthermore, it is also possible to detect and measure trace amounts of myristoylglycyl peptide through antigen-antibody reactions using this monoclonal antibody. That is, by subjecting samples extracted from body fluids and tissues to immunodouble diffusion, immunoelectrophoresis, radioimmunoassay, and enzyme immunoassay using this monoclonal antibody, myristoylglycyl in the sample can be detected. Peptides can be detected and quantified. Also,
Immunohistochemical means using this monoclonal antibody (
For example, by using the enzyme-labeled monoclonal antibody of the present invention, it is also possible to directly detect myristoylglysylated proteins in living tissues.

A representative example of the myristoylglycyl oligopeptide referred to in the present invention is N-Myr-Gl, which is the N-terminal amino acid sequence of the expression product of the oncogene p6QsRc gene.
y-Ser-Ser-Lys (N- indicates the N-terminus, Myr- indicates a myristoyl group), and the antibody that recognizes the above-mentioned myristoylglycyl oligopeptide in the present invention is useful for diagnosis to investigate the cause of cancerous cells. It is extremely effective. In other words, if an immune reaction is performed on the target cancerous cells using this monoclonal antibody, and a substance that reacts with this monoclonal antibody is confirmed in the cancerous cells, the cause of the canceration of the cells can be determined. p6QSRC
Indicates that it is a gene. Furthermore, in the future, if this monoclonal antibody can be introduced into such cancerous cells, it can be expected to be used as an antibody that can prevent canceration of cells.

Hereinafter, a method for preparing an antigen for immunization according to the present invention, a method for preparing a hybridoma producing a monoclonal antibody according to the present invention,
and the method for producing monoclonal antibodies.

1. Preparation of antigen for immunization Commercially available myristoylglycyl oligopeptides used in the present invention can be used depending on the purpose.

An immunizing antigen is prepared by binding such myristoylglycyl oligopeptide to a suitable carrier protein, such as BSA (bovine serum albumin), KT, H (-ff fellin hemocyanin), etc. In this case, by using hemocyanin as a carrier protein, it becomes possible to immunize mice to efficiently induce the desired antibodies.

2 Preparation of hybridoma: Mice were immunized with the myristoylglycyl peptide antigen prepared above according to a conventional immunization method, and after the final immunization, II'! 'Get visceral cells. These spleen cells and mouse myeloma cells, typically P3U cells, are used for cell fusion according to a conventional method.

From the fused cells (hybridoma) thus obtained, the desired hybridoma can be selected using a selective medium such as HAT medium.

3. Screening of hybridomas: Among the hybridomas thus obtained, hybridomas producing antibodies that recognize the myristoyl oligopeptide of interest are further screened. The selection method is to use a microplate coated with the same antigen as the myristoylglycyl oligopeptide used for immunization, and measure the culture supernatant of each hybridoma by ELISA to find a hybridoma that produces the desired monoclonal antibody. can be screened.

By further inoculating such a hybridoma into the peritoneal cavity of a mouse and culturing it in vivo, a single hybridoma and the monoclonal antibody produced by it can be quantitatively amplified, and the mouse is kept alive for 1 to 2 weeks. Hybridomas and monoclonal antibodies can be obtained by culturing them in the body and collecting them as ascites from mice.

Purified monoclonal antibodies can be obtained from this ascites according to conventional antibody purification methods.

The monoclonal antibody of the present invention thus obtained can be used for various purposes as already mentioned above, and is particularly useful in clinical diagnosis of cancerous cells.

Hereinafter, the present invention will be explained in more detail according to Examples.
The present invention is not limited to the following examples in any way.

Water-soluble carbodiimide (30 μmol) was added to commercially available NM.
yr-Gly-Ser-Ser-Lys (20p,
mol ) of 5 (H dimethyl sulfoxide solution 10 m)
was added to the solution, and allowed to react at room temperature for 5 minutes. Next, this activated N-Myr-Gly-Ser-Ser9-Lys was dissolved in 30 mg of hemocyanin in a mixed solvent Me.
The mixture was added to 25 ml of 2SOC1(sC)I-H2O, stirred, and reacted overnight at room temperature. Centrifuge the insoluble substances (18,000
x g for 15 min at 4°C), and the supernatant was dialyzed against deionized water for 3 days and then lyophilized. Thus, myristoyl oligopeptide antigen (1l-
14yr-Gly'5er-Ser-Lys-KLH)
I got it.

2 N-Myr-Gly-Ser prepared by the above (1) of Hub 1'-Ma
-Ser-LysHLll solution was added to pure mouse Ba
7 to give 200 μ9/mouse for l b/c mice.
It was administered intraperitoneally four times at intervals. Thereafter, the final immunization was carried out at intervals of 30 days after the completion of the basic immunization, and the antigen amount was 40μ.
It was administered through the tail vein at a rate of 9/mouse. Three days later, the mouse gave me III! ! Visceral cells were obtained and fused with mouse myeloma cells P3U1 using polyethylene glycol in a conventional manner. Hybridomas thus obtained were selected using HAT medium.

3. Myristoyl oligopeptide (N-Myr-Gly-
(SerScr-Lys) and (N-Myr-GI
y-Ser-Ser-LysBSA) and Tris-1 (C1 buffer (50mM Tris-1 (C1pH8,0,10+n
M MgCh, 0. IXTween80),
Add this to a 96-well microplate at 50β/well at 37°C.
, and the antigens were allowed to be in phase for 2 hours. After washing the plate with the above buffer and water, add 1x ovalbumin solution at 10%
0〆/hole was added and masking was performed overnight at 4°C. This was washed and dried to form an antigen plate.

Next, the culture supernatant of the hybridoma obtained above was added to this antigen plate at 50 wells/well and allowed to react at 37°C for 1.5 hours. After washing, peroxidase-labeled anti-mouse IgG was added to the antigen plate.
Add rabbit serum solution (1:5000) 50 times/hole, 3
The reaction was carried out at 7°C for 30 minutes. TMBZ after cleaning (3'3
“5'5'-Tetramethylber+zid
Ine) was used as a substrate to develop color and absorbance at λ450/λ630 was measured.

Hybridomas that were positive for antibody production were cloned by the limit dilution method.

For primary cloning, 3 hybridomas/well 11 were cultured in a culture plate, and only those forming one colony per well were examined for production of specific antibodies by ELISA. Here, the hybridomas that were positive for antibody production were seeded on a culture plate at one cell/well, and recloning was performed. After screening,
Anti-N-Myr-Gly-
The cells were established as Ser-Ser-Lys monoclonal antibody producing cells.

Anti-N-Myr-Gly-Ser thus obtained
-SerLys monoclonal antibody-producing hybridomas were treated with Blistane by intraperitoneal administration one week beforehand.
Hybridomas were grown by intraperitoneally injecting 1×10 7 cells/mouse into a1b/c mice. Here, we were able to obtain a large amount of ascites by pre-administering Bristan.

After 7 to 10 days, the mouse's abdomen was opened, ascites fluid together with the hybridoma was collected, and the cells were removed by centrifugation to obtain ascites fluid.

The ascites obtained above was mixed with an antibody purification gel, Affi-Gel Protein A (2-conjugated with Protein A).
(Commercially available product) was bonded to Kara I\ under the following conditions.

All operations were performed at 4°C.

Column Afgel protein A (2, Ox 12
cm) Mffl liquid A・], M(NH+>2sOApl
(9,0 buffer B: 0.3M citrate buffer pH 3,
0 Elution method: M* Wash with solution A and then elute with buffer B Detection method ・Measure the absorbance at U, V, 280 nm M'ffr
After salting out both fractions eluted with solution B with ammonium sulfate, 0.1M Tris-
1 (CI pH 8.0 solution) overnight to obtain a purified monoclonal antibody.

To identify subclasses of monoclonal purified monoclonal antibodies, anti-γ1, anti-γ2a, anti-γ2b, anti-γ3, anti-α, and anti-]1 antibodies were used to identify subclasses of the heavy chain; The subclass of the L chain was determined by the Ophthalony method. As a result of the precipitation immunoreaction, the subclass of the purified monoclonal antibody of the present invention was determined to be γ1 for the 4 chain, and subclass for the L chain.

3. Various cancer cells were reacted with the monoclonal antibody of the present invention as described below, and the results were observed.

Human colon cancer cells DLD-1, W i Dr, CO
I, 0320D11, C0LO201 and LoVo(J
oseph B, Tlolen ej al, Pr
ocN, A, S, USA Vol, 84 p2251
-2255. (1987) and hepatoma cells in 1% Triton, 1x deoxycoforic acid, 1 mM, respectively.
50mM Tris-1 (CI Buf) containing PMSF
fer (pH 7°4), remove the precipitate by centrifugation, and collect the supernatant to prepare lysade.
- subjected to polyacrylamide gel electrophoresis. After electrophoresis,
Transplot was performed from this gel onto a nitrocellulose membrane. To this, N-Myr-Gly-Ser- of the present invention
A monoclonal antibody that recognizes Ser-Lys was reacted.

Next, peroxidase-labeled anti-mouse IgG rabbit serum was reacted, and a diaminobenzidine solution was further reacted as a substrate to stain the gel. All of the above operations were performed according to the usual Western blot method. In addition, a similar nitrocellulose membrane was prepared using anti-SRC protein rabbit serum (TBS: commercially available product) instead of the 4-monoclonal antibody described above, and anti-heron IgG goat serum instead of the anti-mouse IgG rabbit serum. A Western plot was made.

As a result, Western blot using the monoclonal antibody of the present invention revealed that only in lysates 1 to 5 of the above five types of human colon cancer cells, in which the p6QsRc gene is known to have a causal relationship to canceration, a molecular weight of 60 was detected. A band was detected at the position. On the other hand, no such band was detected in lysates of hepatoma cells in which the RAS gene is thought to have a causal relationship with canceration (see Figure 1). Further, by Western blotting using an anti-SRC antibody, it was confirmed that the band detected above was p6QSRC protein.

These results confirmed that the monoclonal antibody of the present invention can specifically detect only cancer cells caused by the p6QsRc gene in various cancerous cells.

[Brief explanation of drawings]

5- FIG. 1 shows a schematic diagram of the results of Western blot using the monoclonal antibody of the present invention performed in Example (6). Lane 1 is DID-1, lane 2 is WiDr,
Lane 3 is C0LO320DM, Lane 4 is C0LO2
01, lane 5 is the electrophoresed lysate of LoVo, and lane 6 is the electrophoresed lysate of hepatoma cells. FIG. 2 shows a schematic diagram of the results of Western Blot 1 using anti-SRC protein rabbit serum performed in Example (6). Each lane represents the same sample electrophoresed as the corresponding lane in FIG. ]6-Figure 2

Claims (1)

[Scope of Claims] (1) A monoclonal antibody characterized by specifically recognizing myristoylglycyl oligopeptide. (2) Myristoylglycyl oligopeptide is N-M
The above ( yr-Gly-Ser-Ser-Lys)
Monoclonal antibody described in section 1). (3) A monoclonal antibody characterized by culturing a hybridoma obtained by fusing mouse myeloma cells with spleen cells derived from a mouse immunized with a protein in which myristoylglycyl oligopeptide is bound to a carrier as an immunizing antigen. Production method. (4) Myristoylglycyl oligopeptide is N-M
The above ( yr-Gly-Ser-Ser-Lys)
3) The method for producing a monoclonal antibody described in section 3). (5) Myristoylglycyl oligopeptide binding protein is N-Myr-Gly-Ser-Ser-Lys-K
The method for producing the monoclonal antibody according to item (3) above, which is LH. (6) A method for identifying cancer cells, which comprises performing an antigen-antibody reaction on an extract of cancer cells to be tested using a monoclonal antibody that specifically recognizes the following myristoylglycyl oligopeptide. N-Myr-Gly-Ser-Ser-Lys (7) Item (6) above, wherein the antigen-antibody reaction comprises reacting a cell extract with the monoclonal antibody and then reacting with an anti-mouse IgG enzyme-labeled antibody. Identification method described.