JPH02177897A - Monoclonal antibody and immunoassay for biological substance using same - Google Patents

Abstract

NEW MATERIAL:A monoclonal antibody belonging to immunoglobulin G class 3, capable of specifically binding to at least sialyllacto N-fucopentaose II in the blood. USE:Reagents for use in immunoassay for sialyllacto N-fucopentaose II, pancreatic carcinoma diagnostics. PREPARATION:For example, Salmonella Minnesota bacteria incorporated with sialyllacto N-fucopentaose II is suspended into a phosphoric acid-buffered physiological saline solution, and the resulting suspension is injected into the abdomen of a BALB/C mouse to effect immunization. Three days after the final immunization, antibody-produced cells are collected from the spleen of the mouse, and fused with mouse myeloma cells followed by making a culture in a HAT medium into a hybridoma, which is then screened and put to cloning by e.g. critical dilution method followed by making a culture in the mouse abdomen and the resultant antibody is then separated from the ascites fluid, thus obtaining the objective monoclonal antibody.

Description

Detailed Description of the Invention (Industrial Field of Application) The present invention provides sialyl rough l-N-fucopentaose II.
The present invention relates to a monoclonal antibody that specifically binds to sialyl rough l-N-fucopentaose and an immunoassay method using the antibody.

(Background of the invention) Sialyl rough l-N-fucopentaose■ is Le
Rough l-N-fucopentaose (2), which is present in healthy individuals with type Ill, is sialylated by the action of activated sialyltransferase in cancerous cells. Therefore, serum or other Knowing the increase or decrease of sialyl rough h N-fucopentaose ■ in body fluids is useful for diagnosing various cancers, especially pancreatic cancer.

Conventionally, sialyl rough I-N-fucopentaose has been measured by enzyme immunoassay or radioimmunoassay, but the monoclonal antibody used in this case is immunoglobulin G (hereinafter abbreviated as IgG) It is an antibody belonging to class 1 (described in Table 1).

The present inventors have discovered that monochrome cells belonging to IgG subclass 1
The inventors of the present invention believed that even a monoclonal null antibody could be used for the measurement of sialyllacto N-fucopentaose, and as a result of intensive study, the present invention was completed. That is, the present invention firstly aims at reducing sialyl raft N- in blood at least.
specifically binds to fucopentaose II and binds to IgG
It is a monoclonal antibody that has the property of belonging to class 3, and secondly, it is a monoclonal antibody that has the property of belonging to class 3.
This is the standard method.

The present invention will be explained in detail below.

(Structure of the Invention) The monoclonal antibody of the present invention can be produced by a cell fusion method. More specifically, the monoclonal antibody of the present invention can be obtained through the following steps (1)'-(4), namely: (]) Preparation of antibody-producing cells] - step (2) Fusion, screening, cloning step (3)
Hybridoma culture step (4) Obtained by carrying out a purification step if necessary. Each step will be described in detail below, but the present invention is not limited to these examples.

(C-) Antibody-producing cell preparation process Antibody-producing cells can be collected from the lungs of mammals such as mice and rats that have been sufficiently immunized with the antigen sialyl rough l-N fucopentaose. Preferably, sialyl rough l-
Although it is better that the purity of N-fucopentaose ① is high, the purpose can be achieved even if the purity is not so high. for example,
The above-mentioned animals may be immunized using cells isolated and purified from human cancer cells or similar cells, or artificially synthesized. Immunization conditions, such as the amount of sialyllacto N-fucopentaose H used and the site of administration, can be the same as those used for obtaining conventional antiserum. Antibody producing cells can be harvested from the lungs of the immunized mammal 2-50 days after the final immunization.

(2) Fusion, screening, and cloning steps Fusion can be carried out by mixing the above antibody-producing cells and known myeloma cells (myeloma cells) in the presence of a conventional fusion promoter by a known method.

In general, myeloma cells, which may be derived from the immunized animal used in step (1) or from a different animal, are unable to grow in hybridoma selection medium and have antibodies against them. Preferably, those that do not produce Examples of such myeloma cells include commercially available mouse myeloma cells P3.X63.A.
Examples include g8/Ul or something equivalent to this.

The ratio of both cells is usually 1-20 antibody-producing cells to 1 myeloma cell. As the cell fusion promoter, for example, polyethylene glycol is used, and the molecular weight is 100.
0-7500 is preferred.

To culture fused cells, i.e., hybridomas, 200 µl of hybridoma i-00, which has been washed to remove the fusion promoter and suspended in hybridoma selection medium, is plated and incubated for about 30 minutes.
It is carried out in 5% carbon dioxide gas atmosphere at 7°C.

Screening for the target hybridoma is performed by measuring the antibody titer in the culture solution.

That is, the supernatant of a culture medium in which hybridomas have grown sufficiently is added to each well of an assay plate that has been bound with sialylrough l-N-fucopentaose and blocked with bovine serum albumin, and after sufficient incubation, an antigen-antibody reaction is carried out. Remove the supernatant and wash. Furthermore, this was treated with anti-mouse IgG3 antibody labeled with peroxidase, and after washing, AB containing hydrogen peroxide as a substrate was washed.
Add TS aqueous solution to develop color.

A desired hybridoma can be prepared by cloning the screened hybridoma using the limiting dilution method.

(3) The cloned hybridoma obtained in the pre-step of hybridoma cultivation step is in vitro
If cultured in vitro or in vivo, monoclonal antibodies with specificity of 1" can be produced.

This can be done by starting with culturing a few hybridomas in plates and gradually scaling up. In addition, in vivo culture can be carried out by inoculating the hybridoma into the peritoneal cavity of a mouse that has been treated with pristane to facilitate the proliferation of the fused cells.
-15 days later, ascites fluid containing monoclonal antibodies accumulates.

(4) Purification] - The purification step, which is carried out as necessary, is a purification step in which the monoclonal antibody obtained in the previous step is subjected to conventional physicochemical techniques such as salting out, centrifugation, dialysis, and ion exchange chromatography. This is done by combining means.

Next, regarding the second point of the present invention, is it done as described above? A standard method for sialyl raft l-N-fucopentaose Immunization 41 using a monoclonal antibody that specifically binds to sialyl raft N-fucopentaose II and has the property of belonging to IgG class 3 will be described.

Examples of all immunoassays include, but are not limited to, enzyme immunoassays and n-1-directed element immunoassays. Specifically, as described above? The method consists of using the monoclonal antibodies prepared in 11 as immobilized antibodies in immunoassays or as labeled antibodies.

For example, enzyme immunoassay (EIA) includes 1, a competitive method in which both an enzyme-labeled antigen and an unlabeled antigen are reacted competitively;
Another example is the Santowich assay method, in which the antibody is labeled with an enzyme and the antigen is sandwiched between the in-phase antibody and the enzyme-labeled antibody.

In the sandwich method, it may be difficult to sandwich one molecule of sialyllacto N-fucopentaose■ with two antibodies, or it may be that sialyllactone-N-fucopentaose■ normally exists in the blood in the form of multiple molecules or bound to carrier proteins. It is possible to detect the glycoprotein by sandwiching it.

When using the monoclonal antibody of the present invention as an immobilized antibody, the antibody may be bound to an insoluble carrier,
When used as a labeled antibody, a label such as an enzyme, a fluorescent substance, or a radioactive isotope may be bound to the antibody. The binding of the antibody to the carrier or the binding of the antibody to the label may be carried out according to known methods, and there are no particular limitations. Also,
For example, operations for determining 8+11 in competition law, sandwich law, etc. may be performed in accordance with known methods, and there are no particular restrictions.

(Effects of the Invention) According to the present invention, a monoclonal antibody is provided that specifically binds to sialyl rough I-N-fucopentaose II and has the property of belonging to IgG class 3. As described above, this antibody can be used for immunoassay of sialyllacto N-fucopentaose.

The monoclonal antibody of the present invention is different from conventionally used I
Specifically, it is a monoclonal antibody belonging to IgG class 3, which is different from a monoclonal antibody belonging to IgG class 1-. In today's field of immunology research, antibodies with higher antigen specificity than conventional antibodies, antibodies with different binding sites than conventional antibodies, or antibodies with some kind of different action than conventional antibodies are required. , the antibodies provided by the present invention belonging to a class different from conventional antibodies are of great significance.

[Example] Next, the present invention will be explained in further detail by giving examples.
The present invention is not limited to this.

Example 1 (Preparation of antibody-producing cells) 20 μg of sialyllacto N-fucopentaose was embedded in Salmonella minnesota cells, suspended in phosphate buffered saline, and injected intraperitoneally into BALB/c mice. It was tilted. Further, booster immunizations were given 4 days, 7 days, 12 days, 18 days, and 261 days after the initial immunization.

Three days after the final immunization, this mouse's IJII! Antibody-producing cells were collected from the viscera.

Example 2 (Fusion, Screening, Cloning) These liver cells (1,0,0XIC)8 cells) and mouse myeloma (2,0,X],07 cells) were fused by the method of Koehler and Milstein. After polymerization, the cells were suspended in DMEM (Dulbetzco Modified Eagles Medium) containing hypoxanthine, aminopterin, and thymidine to a density of 2.0×IO6 spleen cells/m1, and 2.0×105 cells/m were placed in a 96-well plate. The seeds were sown in wells. Once the hybridoma has grown,
Screening was conducted. For screening, a 96-well microtiter plate with 20 ng/well of sialyllacto N-fucopentaose was used, and an anti-sialyllactin-N-fucopentaose antibody was prepared using anti-mouse IgG3 as a secondary antibody. picked up. At this time, from five 96-well plates, anti-allyllac I-N-fucopentaose ■ antibody-positive hybrids were collected.
[Ma] stock was obtained. This hybridoma was cloned by the limiting dilution method three times to form a cell line.

Example 3 (Production of monoclonal antibody) Hybridoma 1 obtained in the previous section. 107 OX were injected intraperitoneally into BALB/c mice that had been previously treated with pristane. 1
Ascites containing monoclonal antibodies targeting 1 on day 05
m1/mouse was obtained.

Example 4 (Application to Enzyme Immunization 8+11 standard method (EIA)) The monoclonal antibody obtained as above was labeled with an enzyme. A labeling substance and horseradish peroxidase (manufactured by TOYOBO, hereinafter abbreviated as HRP) were used.

0.3M with HRP at a rate of 5 mg/m I
1% ethanolic solution of 1-fluoro-2,4-dinitrobenzene in sodium bicarbonate buffer (pH 8.1),
ml and reacted at room temperature for 1 hour, then 0.06M
1. Thorium periodate solution. ml was added and the reaction was continued for an additional 30 minutes. 0.16M ethylene glycol 1
ml to remove unreacted sodium periodate, add 0.01M sodium carbonate buffer (pH 9,5
)] 2 and dialyzed, followed by adding 5 mg of monoclonal antibody and reacting for 5 hours. Then, 5 mg of sodium borohydride was added, and the mixture was left at 4° C. overnight. To remove unreacted sodium borohydride, 10 mM sodium phosphate buffer (pH 7) containing 0.85% sodium chloride was added.
, 1) at 4°C overnight with stirring,
High performance liquid chromatography (manufactured by Tosoh Corporation, T
Purify the enzyme-labeled monoclonal antibody using SK Gel G-3000SW). 1 again.

Each well of an untreated 96-well microtiter plate (Nunc Immunoplate, Intermed) was injected with 5 μg/ml of unlabeled monochrome dissolved in O, 1 M sodium carbonate buffer (pH 9,6). 250 μl of null antibody was added and incubated at 37° C. to bind to the plate. After removing the reaction supernatant and washing, add phosphate buffered saline containing 1% bovine serum albumin and incubate at 4°C.
The mixture was allowed to react overnight and blocked. Sandwich assay was performed using the plates prepared as described above.

The culture supernatant of colon cancer cells was prepared in advance using commercially available sialyl rough l-N-
Fucopentaose ■Concentration with measurement kit if+11
The standard substance was diluted and 10 μl was added to each well. Next, 100 μl of a solution of 5 mg/ml of enzyme-labeled monoclonal antibody diluted 1000 times with phosphate buffered saline containing 1% bovine serum albumin was added to each well and incubated at 37°C for 2 hours. After washing, the solution was removed and the cells were washed three times with phosphate buffered saline containing 1% bovine serum albumin.

Furthermore, 100 μl each of a citrate buffer containing hydrogen peroxide and 0.6 mg/ml ABTs was added as a substrate and a coloring solution. After stopping the reaction, the wavelength was 41.5n for each well.
m, the absorption intensity at a reference wavelength of 600 nm was determined using a dynamic microtiter plate reader (MPR-A4; manufactured by Tosoh Corporation), and the concentration and absorption intensity of the standard substance were plotted to determine the antibody of the present invention. The sialyl rough I-N-fucopentaose used was subjected to immunoassay. As a result, it was confirmed that the signal (absorbance at 415 nm) correlated with the increase in the HHH degree of sialyl rough l-N-fucopentaose. The results are shown in Table].

Table 1 ] 5

Claims (2)

[Claims] (1) A monoclonal antibody having at least the following properties. (a) It specifically binds at least to sialyllacto N-fucopentaose II in the blood, and (b) It belongs to immunoglobulin G class 3. (2) Sialyllacto N-fucopentaose I in a sample using the monoclonal antibody according to claim (1)
I immunoassay.