JPH07113000A - Monoclonal antibody against anti-mink-growth hormone and hybridoma - Google Patents

Abstract

PURPOSE:To obtain a monoclonal antibody which can safely determine mink growth hormone in a shortened time. CONSTITUTION:An animal is immunized with mink growth hormone and the antibody-producing cell from the animal is fused with a cell of a high cell proliferation line to give a hybridoma. The monoclonal antibody of IgM type against the growth hormone produced from the hybridoma and the hybridoma which can produce the IgM type anti-mink growth hormone monoclonal antibody.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an anti-mink growth hormone monoclonal antibody and a hybridoma producing the same.

[0002]

2. Description of the Related Art Conventionally, as a method for quantifying mink growth hormone, for example, a method for quantifying growth hormone of other animals, that is, a method using ratGH [ ¹²⁵ I] assay system (manufactured by Amersham) and the like can be mentioned. To be

However, according to the above method, a special facility is required to use the radioisotope,
In addition, there are some drawbacks such as the danger and the need for a long time of about 1.5 days. Therefore, it has been desired to develop a monoclonal antibody that does not have such drawbacks.

[0004]

Therefore, the present inventors
As a result of various examinations in view of the above circumstances, from a fused cell of antibody-producing cells and highly proliferative cells obtained from an animal immunized with recombinant mink growth hormone, a target antigen, that is, to mink growth hormone It is possible to produce a highly specific antibody by selecting a clone that produces an antibody that specifically reacts with it and monocloning it, and it is safe to quantify mink growth hormone using this antibody. Moreover, they have found that mink growth hormone can be measured within a few hours, and completed the present invention.

That is, the present invention is a monoclonal antibody reactive with mink growth hormone. Furthermore, the present invention relates to an IgM type anti-mink growth hormone monoclonal antibody produced from a hybridoma formed by immunizing an animal with mink growth hormone and fusing antibody-producing cells from the animal with highly proliferative cells. Is. Furthermore, the present invention provides a hybridoma having the ability to produce an IgM type anti-mink growth hormone monoclonal antibody, which is formed by immunizing an animal with mink growth hormone and cell fusion of antibody-producing cells from the animal with highly proliferative cells. Is.

The present invention will be described in detail below. In order to achieve the object of the present invention, a novel monoclonal clone hybridoma producing an antibody is established. The method for establishing this hybridoma is 1. Preparation of antigen, 2. Immunity, 3. Cell fusion, and 4. It consists of four steps: selection of hybridoma and monocloning. Next, each of these steps will be described. 1. The antigen mink growth hormone may be derived from any of natural mink growth hormone and recombinant mink growth hormone. 2. The animal to be immunized is determined by the tumor cell line used for cell fusion, and for example, mice and rats are used, and among the types of mice, the BALB / c strain in which a tumor cell line that does not produce immunoglobulin is established is preferable. The above-mentioned antigen is dissolved in an isotonic buffer solution or a physiological saline solution, and in the case of a mouse, 10 to 300 μg is administered once per mouse. Usually, immunization is divided into several times, and the first immunization is administered together with an adjuvant. As adjuvants, alum, killed tuberculosis bacteria, nucleic acids and the like are commonly used. Immunization is performed every 2 to 4 weeks, and final immunization is performed without using adjuvant. In the case of mice, the administration method is generally intraperitoneal or subcutaneous, and intravenous administration or the like can also be performed. Final immunity 2
~ 4 days later, the lymph node or spleen is removed, and the obtained lymphocytes are subjected to cell fusion with highly proliferative cells. As highly proliferative cells, for example, P3X63Ag8U.1 (U1), P3X63Ag
8.653 (653), P3 / NS1 / 1-Ag4-1 (NS-1), SP2 / 0-Ag14 (SP2)
Etc.

3. Cell fusion is described, for example, in [G. Galfre, Natu
re, 266, 550 (1977)] or a method analogous thereto. At this time, use 30-50% polyethylene glycol (average molecular weight 1,000-4,000)
It is suitable to react at a temperature of 30 to 40 ° C. for about 1 to 3 minutes. As the feeder cells, thymocytes of BALB / c mice, cell growth promoting components such as human interleukin VI and the like can be used. 4. The fused cells obtained by cell fusion are subjected to screening of clones producing the desired monoclonal antibody according to a conventional method. That is, the cells are cultured in, for example, a microplate (96-well culture plate, etc.), and the antibody titer in the culture supernatant of the hole in which proliferation is observed is measured by, for example, the enzyme-antibody method (ELISA), Obtain holes producing the appropriate antibody. From such holes, for example, FACS (Fluoresent Activated Cell Sorter), Sof
A clone is obtained by cloning by a method of picking up a colony using t Agar or a commonly used limiting dilution method. This clone is, for example, transplanted into the abdominal cavity of BALB / c mouse or nude mouse to which pristane was previously administered, and after 7 to 14 days, ascites containing a high concentration of monoclonal antibody is collected and assayed. Alternatively, it can be grown in a serum-free medium containing bovine serum albumin (0.1 to 1%). That is, it is grown in a serum-free medium containing 0.5% bovine serum albumin (for example, RITC55-9 medium),
Collect the culture supernatant. Monoclonal antibodies produced by the selected clones are recovered by conventional ammonium sulfate fractionation methods, polyethylene glycol fractionation methods, ethanol fractionation methods, anion exchangers, immunoglobulin affinity columns, etc. It can be easily done by applying. Thus, the present invention provides an antibody obtained by intraperitoneal administration to mice using mink growth hormone as an antigen. That is, an anti-mink growth of IgM type that recognizes an antigen specific to mink growth hormone, its binding to mink growth hormone is inhibited by mink growth hormone, and does not react with a control (ELISA plate blocked only with milk protein). Provided are 3 hybridoma clones producing hormone monoclonal antibodies, MGH-1 (11E), MGH-3 (14B6) and MGH-3 (15B10).

[0007]

INDUSTRIAL APPLICABILITY The present invention provides a novel monoclonal antibody reactive with mink growth hormone and a hybridoma producing the same. And this monoclonal antibody is not only for mink growth hormone but also for pigs,
It is also reactive with bovine and human growth hormone.
If mink growth hormone is quantified using this monoclonal antibody, mink growth hormone can be quantified safely and in a short period of time without using a special facility that has been used to use radioactive isotopes. . In addition, porcine, bovine and human growth hormones other than mink can be quantified. Therefore, the present invention is extremely useful industrially.

[0008]

EXAMPLES The present invention will be specifically described below with reference to examples. However, the technical scope of the present invention is not limited to these examples. Example Preparation of hybridoma and antibody producing anti-mink growth hormone monoclonal antibody: Preparation of Recombinant Mink Growth Hormone Recombinant mink growth hormone was obtained by the method described in the example of JP-A-5-30974. 2. Immunorecombinant mink growth hormone 500 in borate buffer
Dilute to μg / ml, mix 1 ml of diluted solution and 1 ml of Freund's complete adjuvant to make an emulsion,
200,100 μl thereof was intraperitoneally administered to BALB / c mice (female, 8 weeks old). After 14 days, the same amount of antigen was intraperitoneally administered together with Freund's incomplete adjuvant to perform booster immunization. On the 28th day, 3 days before the fusion, the same amount of the antigen was intraperitoneally administered without an adjuvant for the final immunization. 3. Preparation of thymocyte suspension A thymocyte suspension used as feeder cells for increasing the rate of hybridoma formation was prepared. ddY mouse, 4-6
At the age of a week, females were killed by cervical dislocation and the thymus was removed,
Thymocytes were carefully loosened in a plastic petri dish containing 10 ml of a live cell washing solution (MEM medium) (for 3 to 4 mice). The loosened thymocytes were transferred to a centrifuge tube after mesh filtration, centrifuged at 2,000 rpm for 5 minutes, and the supernatant was aspirated, followed by washing with 30 ml of a live cell washing solution, centrifugation, and HAT medium (hypoxanthine 13.6 mg / l, Aminopterin 0.176 mg /
l, hybridoma growth medium containing thymidine 3.8 mg / l [HAT solution]), and a suspension 100 m containing 5 × 10 8 thymocytes
got l. The cell suspension was transferred to a 200 cm2 flask and cultured in a carbon dioxide gas culture device for 1 day. The hybridoma growth medium was prepared by adding 20% FCS-IMDM medium containing antibiotics to L-glutamic acid, 2-mercapto ethanol,
This is a medium supplemented with transferrin and insulin.

4. Cell fusion 4 days after the final immunization, the spleen of the mouse was excised, and spleen lymphocytes were carefully extruded in a plastic petri dish containing 10 ml of a living cell washing solution (MEM medium) (mouse 3
~ 4). The loosened spleen lymphocytes are transferred to a centrifuge tube after mesh filtration, and a live cell washing solution is added to bring the total volume to 50 ml.
And washed twice by repeating centrifugation at 1,600 rpm for 5 minutes to obtain 50 ml of a suspension containing 1-2 × 10 ⁸ spleen lymphocytes. A suspension of 6-thioguanine-resistant myeloma Sp-2 / O-Ag14 cells (containing 2 × 10 ⁷ cells) and a suspension containing 1 × 10 ⁸ spleen lymphocytes were mixed and spun at 1,600 rpm for 5 minutes. I made it into a pellet. The supernatant medium was removed by suction and the pellet was carefully loosened. PEG solution (PEG # 4,000 + DMSO, etc.) 1 ml
Was added slowly over 1 minute and allowed to fuse for 1 minute at 37 ° C. with stirring with the pipette used. Continue to basic medium (IMD
1 ml of M medium) was added at 37 ° C. for 1 minute. After repeating the same operation once more, 8 ml of the basic medium was added at 37 ° C. for 3 minutes. The resulting fused cell suspension was added to the thymocyte (feeder cell) HAT medium suspension prepared in item 3 and well suspended, and this cell suspension was added to 10 to 14 96-well culture plates at 0.1 per well. The solution was dispensed in ml and cultured in a carbon dioxide incubator. 5. HAT selection On the 1st day after culturing, 0.15 ml of HAT medium was added per well, and half of the medium was removed by suction on the 3rd, 6th, and 10th days, and 0.1 to 0.15 ml of HAT medium was added per well. added. When the growth of the hybridoma was confirmed on the 10th day, the growth in almost all the holes was observed.

6. Selection of hybridoma 12 to 13 days after the fusion, the culture supernatant was collected and the antibody positive hole was selected by the ELISA method. 100 μl of recombinant mink growth hormone was dispensed into a 96-well plate for ELISA and left at 4 ° C. overnight or at room temperature for 1 hour to immobilize the antigen on the plate. To remove the liquid well and avoid non-specific adsorption of protein in the culture supernatant, 100 μl of 2% skim milk blocking liquid was dispensed into each well and left at room temperature for 5 minutes. After removing the liquid well, add the above cell culture supernatants
100 μl was dispensed and left at room temperature for 1 hour. As a negative control, 100 μl of 20% FCS-IMDM medium was dispensed. Then 0.0
Wash 3-5 times with buffer containing 2% Tween 20 (wash),
100 μl of mouse immunoglobulin antibody-horseradish peroxidase complex solution was dispensed to the plate, left at room temperature for 1 hour, and washed 3-5 times with the same washing solution, followed by ABTS (2,2'-Azino-
bis (3-etylbenzthiazoline) -6-sulfonic acid) solution (0.6
(100 mg / ml), and after reacting for 1 hour at room temperature, 100 μl of enzyme reaction stop solution (Sodium dodecyl aulfate 5% solution)
The addition reaction was stopped and OD405 nm was measured to quantify the peroxidase activity. 10 cells in 672 holes
Mink growth hormone antibody production was observed in the hole. 7. Scale expansion of culture Once it was determined which hole was producing mink growth hormone antibody, HT medium (hybridoma growth medium containing hypoxanthine 13.6 mg / l and thymidine 3.8 mg / l [HT solution]) was dispensed 24 The replanting scale was expanded to a well culture plate. That is, 500 μl of HT medium was preliminarily dispensed in a 24-well culture plate, and the cell suspension in the antibody-producing hole in the 96-well culture plate was transplanted into each well, and the cells were cultured in a carbon dioxide culture device. It was Culture 2
After 3 days, 1 ml of HT medium was further added to each well, and the supernatant of each well was retested for antibody activity by ELISA.
The cell holes for cloning were reselected.

8. Monocloning As a cloning medium, a hybridoma growth medium containing human IL-6 (1 U / ml) was used. Anti-mink growth hormone antibody-producing hybridomas were counted and added to 1 ml of cloning medium.
Diluted to include 5 cells. 200 this suspension
Each 96 μl culture plate was seeded in an amount of μl and cultured in a carbon dioxide culture device. On the 10th day, while checking the growth, the antibody activity of the supernatant was measured by ELISA, and 1-2 cell wells with large colonies and high antibody activity were selected.
After culturing and recloning in the same manner, 3 clones of anti-mink growth hormone antibody-producing hybridoma,
MGH-1 (11E), MGH-3 (14B6) and MGH-3 (15B10) were obtained. The hybridoma MGH-3 (15B10) has been deposited as FERM P-13892 at the Institute of Biotechnology, Institute of Biotechnology, AIST. 9. Production of Monoclonal Antibody Each of the obtained antibody-producing hybridomas was sequentially expanded and cultured in a growth medium to obtain a large amount of culture supernatant. An example will be described for hybridoma MGH-3 (15B10). Hybridoma MGH-3 (15B10) was added to 1,300 ml of ASF104 medium and incubated at 37 ° C for 240
The culture is obtained by culturing for a period of time. 1,000 rpm for this culture
After centrifuging for 5 minutes, the solution was subjected to salt folding treatment with a saturated ammonium sulfate solution to obtain about 10 mg of the monoclonal antibody. 10. Determination of Class of Monoclonal Antibody The class of immunoglobulin produced by each hybridoma clone is determined by isotype determination kit of mouse monoclonal antibody (RPN29 manufactured by Amersham Japan).
/ Western blotting). As a result, all three strains have immunoglobulin type IgM and L
The chain was kappa. 11. Purification of Monoclonal Antibody The culture supernatant obtained from the hybridoma was purified by salting out with ammonium sulfate.

12. Monoclonal antibody cross-reactivity To investigate the cross-reactivity of anti-mink growth hormone monoclonal antibody, cross-reactivity with several animal growth hormones, which are similar compounds of recombinant mink growth hormone,
It was examined by ELISA. The measurement method was performed according to the hybridoma selection method described in the above item 6. Specifically, each concentration of recombinant mink growth hormone, mink growth hormone (natural type), pig growth hormone (natural type), bovine growth hormone (natural type) and human growth hormone (natural type) 5 μg / ml each After 100 μl of each was adsorbed to the wells of a 96-well microplate, the reactivity with each antibody was developed by a conventional ELISA method and measured.
Then, the reactivity (reactivity) with the recombinant mink growth hormone, which is the antigen of each antibody, was defined as 100%, and the reactivity with other growth hormones was calculated. As a result, as is clear from Fig. 1, some antibodies showed a wide range of cross-reactivity with growth hormones of other animals [MGH-3 (15B10) strain], and other strains were mink (natural type), pig, bovine. And the cross-reactivity was reduced in the order of human growth hormone [MGH-1 (11E) strain]. Furthermore, a strain that does not cross-react with mink (natural type) and human growth hormone and does not cross-react with bovine growth hormone was also obtained [MGH-3 (1
4B6) Stock].

[0013]

[Brief description of drawings]

FIG. 1 is a monoclonal antibody of the present invention, MGH-1 (11E),
MGH-3 (14B6) and MGH-3 (15B10) are recombinant mink growth hormone analogs, mink growth hormone (natural type), porcine growth hormone (natural type), bovine growth hormone (natural type) and human growth It is a figure which shows the crossability with respect to a hormone (natural type).

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location C12P 21/08 9161-4B G01N 33/53 B 33/577 B (72) Inventor Mamoru Kikuchi Chiba Prefecture 339 Noda, Noda City Kikkoman Corporation

Claims (3)

[Claims] 1. A monoclonal antibody having reactivity with mink growth hormone. 2. Immunizing an animal with mink growth hormone,
An IgM type anti-mink growth hormone monoclonal antibody produced from a hybridoma formed by cell fusion of antibody-producing cells from the animal with highly proliferative cells. 3. Immunizing an animal with mink growth hormone,
A hybridoma capable of producing an IgM type anti-mink growth hormone monoclonal antibody, which is formed by cell fusion of an antibody-producing cell from the animal with a highly proliferative cell.