JPS60188327A - Monoclonal antibody against swine insulin and human insulin and its preparation - Google Patents

Abstract

PURPOSE:Lymphocytes which produce antibody against swine or human insulin are fused with myeloma cells to form hybridoma, which is screened and cloned to give the titled antibody. CONSTITUTION:Lymphocytes producing antibody against swine or human insulin such as mouse lymphocytes are fused with myeloma cells (e.g., from mouse) to form a hybridoma. Then, the hybridoma which can secrete the antibody against the insulin is subjected to screening and cloning. Monoclonal antibody is collected from the resultant hybridoma. EFFECT:The objective antibody is infinitely produced with same levels of compatibility and potency, resulting in economical immunoassay of high reproducibility.

Description

DETAILED DESCRIPTION OF THE INVENTION (Technical Field) The present invention relates to a monoclonal antibody against porcine insulin or human insulin and a method for producing the same.

and somatic cell hybridomas that produce the antibodies.

(Prior art) Kohler and Milstein
reported in 1.975 that monoclonal antibodies against sheep erythrocytes were produced from fusion cells formed by fusing lung cells from immunized mice with mouse myeloma cells ( (Natur
e) Voβ, 256 pages 495-497 (
1975) ).

Since then, monoclonal antibodies produced by somatic cell association have been used as viral antigens (Japanese Patent Application Laid-Open No. 17185-1985).

Malignant tumors (Japanese Patent Application Laid-Open No. 54-1.43513), human T cells (Japanese Patent Application Publication No. 55-12'7321), drugs such as gentamicin (Japanese Patent Application Publication No. 57-53411), cyclic nucleotides (Japanese Patent Application Publication No. 57-1973) -146594) and other antigens. Regarding insulin as an antigen, a monoclonal antibody against bovine insulin was developed by J.
(Basic and
C11nical Aspects of Immun
ity to In5ulin, edited by
K.Keck.

P, Erb, 183-200. Walter de
Gruyter & Co.

Berlin-New York (1981))
.

By the way, measurement of the in vivo content of human insulin has been recognized to have important significance in the diagnosis of diabetes.

Radioimmunoassay (
redio immunoassay) method and enzyme immunoassay (enzyme immunoassay) method.
Immunoassay methods such as the ay) method are used. As the insulin antibody used in this immunoassay, an antiserum obtained by immunizing an animal such as a guinea pig with bovine insulin or porcine insulin is used. Such antiserum can only be obtained in small amounts, for example, 2 Qmn or less per guinea pig, and is quantitatively limited, and the affinity and titer for insulin vary among individual animals. Therefore.

For immunoassays, antisera are collected from a large number of animals, mixed together, and used to a certain quality. Even so, there are variations in affinity and potency between lots.

It is economically disadvantageous and has a quantitative limit.

There have been no reports of producing monoclonal antibodies reactive against human insulin using porcine insulin or human insulin as an immunogen.

Schroer's monoclonal antibody against bovine insulin has been used only for the purpose of determining the location of antigenic determinants on the maggot insulin molecule, and has not been used for the purpose of immunoassay of human insulin. Furthermore, in the amino acid sequence of seven molecules, bovine insulin has a
It differs from human insulin at the 8th and 10th positions from the end and at the C-terminus of the B chain. Therefore, when bovine insulin is used as an immunogen, monoclonal antibodies that are reactive with human insulin are more difficult to obtain than when pig insulin or human insulin is used as an immunogen.

OBJECTS OF THE INVENTION The objects of the invention have the same affinity and the same potency. The object of the present invention is to provide monoclonal antibodies against porcine insulin or human insulin. Another object of the present invention is to provide a method for stably producing the above antibodies of the same affinity and titer in large amounts. Yet another object of the present invention is to provide somatic cell hybridomas that produce the above-mentioned antibodies.

(Structure of the Invention) The method for producing a monoclonal antibody against porcine insulin or human insulin of the present invention comprises (1) forming a hybridoma by fusing lymphocytes that produce antibodies against porcine insulin or human insulin with myeloma cells; (2) screening for hybridomas that secrete antibodies against the insulin; (3) cloning the hybridomas obtained by screening; and (4) collecting monoclonal antibodies secreted from the cloned hybridomas. The above object is thereby achieved.

The method for producing the above antibody of the present invention also includes the steps of: (1) immunizing an animal with porcine insulin or human insulin;
(2) Generate antibodies against the insulin.

fusing lung cells from the immunized animal with myeloma cells to form a hybridoma; (3) screening for hybridomas that secrete antibodies against the insulin; and (4) cloning the hybridomas obtained by screening. and (5) collecting monoclonal antibodies secreted from the cloned hybridoma.

, thereby achieving the above objective.

Somatic cell hybridomas secreting the antibodies of the invention are formed by fusing mouse lung cells producing the antibodies with mouse myeloma cells.

The above objective is thereby achieved. The present invention will be explained below.

+a+ Preparation of antibody-producing cells Preparation of antibody-producing cells does not need to be special.

This can be carried out according to known methods. For example, any animal can be immunized with pig insulin or human insulin, and antibody-producing cells from that animal can be obtained.

Porcine insulin is different from bovine insulin.

Since the only difference is in the C-terminal amino acid of the B chain, a monoclonal antibody reactive against human insulin can be obtained using this as an immunogen.

Any porcine insulin that can be used as an antigen may be used as long as it contains porcine insulin.

For example, purified porcine neutral insulin injection (Novo Actrapid MC), crystalline porcine insulin (Sigma 1-3
505). In addition to human insulin itself, human insulin can be derived by chemical synthesis from pig insulin, or obtained from Escherichia coli or the like by genetic engineering techniques. Examples include Novo's human monocomponent insulin. Examples of animals to be immunized include mice, guinea pigs, and rabbits. However, it is preferable to use the same animal as the animal from which the myeloma cells used are derived, since the resulting hybridoma will be more stable. As an antibody producing cell.

Any cell containing lymphocytes of the immunized animal may be used; for example, lung cells, thymocytes, lymph node cells, and/or peripheral blood cells may be used. Lung cells are preferred because they contain large amounts of lymphocytes.

1 Preparation of fbl myeloma cells There are no particular restrictions on myeloma cells, and many mice.

Run 1～. Animal cell lines such as rabbit and human can be used. The cell line used may be either secretory or non-secretory. The secretory type is characterized by secreting immunoglobulins or fragments thereof, while the non-secreting cells are characterized by the fact that the resulting hybridoma secretes only the specific immunoglobulin produced by the parent lymphocytes. Therefore, it is more preferable that the cell line used is non-secreting. It is also preferably drug resistant, so that unfused myeloma cells cannot survive in the selective medium and only hybridomas can survive. The most commonly used are 8-azaguanine resistant cell lines. this is,
Due to the lack of hypoxanthine guanine phosphoribosyltransferase, hypoxanthine-aminopterin-thymidine (
HAT) has the property of not being able to grow in medium.

Two examples of myeloma cell lines effective in the present invention include:

P3-X63-8gB', P3-N5I from mouse
/12-Ag4-1. P3-X63-Ag8-TJI, 5p
210-8g14. X63-Ag3: 653; 210・RCY 3・Agl・2.3 derived from rat; and 81 (0-007, G former 50067G-
Examples include A12.

(C) Cell fusion Eagle minimal basic medium (MEM) or l? PM116
1-5 x 10' bone marrow 11ffl in medium such as 40
Cells and 1 to 7.5 x 10" antibody-producing cells are mixed at a mixing ratio of 1:4 to 1:15 to perform cell fusion. As a fusion promoter, polyethylene gel with an average molecular weight of 1000 to 6000 is used. Cole (PEG) is preferred. Its concentration is usually 30 to 50%. Other fusion promoters that can be used include Sendai virus and the like.

+d+ Screening of hybridomas using selective medium Cells that have completed cell fusion are placed in RPM containing 10% live fetal serum.
■Appropriately dilute with 1640 medium, etc., add IXLO5~106X105L1063 medium to a microculture plate, and then replace the selection medium appropriately. Cultivate. When cells lacking hypoxanthine guanine phosphoribosyltransferase are used as myeloma cells.

Unfused myeloma cells die in HAT medium within about 10 days. Unfused lymphocytes are normal cells and cannot grow for long periods of time. Therefore, cultures 10-14
Everything that is on the order of days is a hybridoma.

(e) Screening for insulin antibody-producing hybridomas Screening for insulin antibody-producing hybridomas is not particularly limited and may be performed by conventional methods. for example,
Collect a portion of the supernatant from the well where the hybridoma grew. Search for wells secreting the antibody of interest by reacting with labeled human insulin (or labeled porcine insulin may be used when the immunogen is porcine insulin) and examining the binding ability with labeled insulin. can do. Specifically, radioisotopes such as +251 and 3H, enzymes. After the reaction with insulin labeled with fluorescent substance 1 luminescent substance 4, etc., BF bone separation is performed on the 2 reaction solutions, and the presence and binding ability of insulin antibodies is assayed by measuring the amount of labeling in the antigen-antibody conjugate fraction. be done. Also.

An in-phase method using immobilized insulin and a labeled second antibody is also applicable.

(fl Cloning There is a possibility that two or more types of hybridomas are growing in each well. Therefore, cloning is performed by a limiting dilution method or the like to obtain a monoclonal antibody-producing hybridoma clone.

(Obtaining g+ antibodies The desired monoclonal antibodies can be harvested by two different methods. In one method.

Hybridoma clones are cultured in vitro in a suitable culture medium such as RP old 1640 medium containing 10% tallow serum,
The desired monoclonal antibody is obtained from the culture supernatant. Another method involves intraperitoneally administering a mineral oil such as Pliscone (2,6,10,14-tetramethylbentadecane) to an animal syngeneic to the animal from which the myeloma cells were derived, and then administering the hybridoma clone. , Vibe IJ in vivo
Proliferate P-ma in large quantities. In this case, the hybridoma forms an ascites tumor in about 10 to 20 days, and high concentrations of antibodies (approximately 1 to 20 mg/mρ) are produced in the serum and ascites. Remove this using an appropriate method.

(h) Purification of antibodies Antibodies from culture fluid or ascites fluid can be used as is, but if necessary, they may be subjected to dialysis, ion exchange chromatography, ammonium sulfate fractionation, affinity treatment such as insulin-conjugated Sepharose 4B, etc. It is used after being purified by an appropriate combination of chromatography and the like.

Example Next, two embodiments of the present invention will be described.

Fifi (j-111 Novoactrapid Mc injection solution (porcine insulin 40 units/m +2, sold by Kodama Co., Ltd.) was diluted with physiological saline, and this was mixed with complete Freund's adjuvant in a 1=1 ratio.
This was mixed with porcine insulin 6 and 100 μg was added to BALB/c 7 mice (male;
At the age of 8 weeks), two doses were administered intraperitoneally every 30 days. Finally, 100 μg of Novoact Rapid Mc injection, which was simply diluted with physiological saline without adding an adjuvant, was intraperitoneally administered as porcine insulin. Mouse lung cells were collected 4 days after the final immunization.

The cells were placed in a Petri dish (60 caliber) containing 5 mA of MEM.
11 and placed on a stainless steel sieve (sieve mesh 149 μm) in a depth 151), and the Lieba spheres were pushed out with a bottle set. This lymphocyte-containing fluid.

The cells were separated, washed three times with MEM, and then suspended in MEM.

On the other hand, mouse myeloma cells P3-X63-Ag8-
Ul (P 3 U 1 ) (Cold Spri
ng 1larbor Symp, Quant.

Biol, 41: 781-791 (1976)
, Current Topics in Micro
biology and immunologL 81
: 1-7 (1978)) are grown in RPM11640 medium containing 10% tallow serum, and the cells in the logarithmic phase are centrifuged. After washing twice with MEM, it was suspended in MEM.

7 108 lymphocytes and mouse myeloma cells P3U110
'7 were mixed and centrifuged, and the supernatant was aspirated and discarded. 5 of PEG #1,000 (Wako Pure Chemical Industries, Ltd. Product No. 165-09085) was added to this cell mixture.
Cell fusion was performed by adding 0.5 ml of 0% MBM solution and slowly rotating the centrifuge tube. More MEM 1 after 1 minute
mj! was added and the centrifuge tube was slowly rotated.

Further, add M E M l m R every 30 seconds while slowly rotating the centrifuge tube, and finally lQm j
! Closed. This was centrifuged again. P3U of the obtained pellet was added to RPM11640 medium containing 10% tallow serum.
105 pieces 10.2 mj as 1! It was suspended so that Add this to 96-well microplay 1~0.2m I
1 was dispensed into a total of 98 wells. CO2/
The cells were cultured for 1 day at 37°C in an atmosphere with an air ratio of 5/95.

After culturing, half of the medium in each well (0,tmp)
The medium was replaced with HAT medium, and the same operation was repeated every 3 days. Then, it was observed that hybridomas began to grow from around the 7th day, and growth of hybridomas was observed in all wells on the 12th day. Hybrid 8 0.1rr+7 of the supernatant of the well where dormers have grown!
Take this and add it to bovine serum γ-globulin corn fraction (Hani Chemical Co., Ltd. product No. 167-
22) 20mg/m (containing l 0,05
Diluted 10 times with molar Harbicool buffer (pl+ 8.5). 100 μp of the diluted solution and 12 μp in advance.
2.0 ng of 5I-labeled porcine insulin was added to raw serum albumin (Seikai 012-03 manufactured by Hanui Chemical Co., Ltd.) 5
Mix with 100 μl of a porcine insulin solution obtained by dissolving in 0.05 molar Harbicool buffer ? & (pH 8.5) containing 4 m
The reaction was allowed to proceed for 4 hours. Add 25% PEG #6,000 to this
(Wako Pure Chemical Industries, Ltd. Product No. 169-091
500 μp of the aqueous solution of 25) was added, centrifuged, and the radioactivity of the resulting precipitate was measured. Five wells were found to be positive for binding to porcine insulin.

The hybridomas in these positive wells were transferred to 11PM11 containing 10% tallow serum containing hypoxanthin and thymidine.
Cloning was performed in 640 medium (HT medium) using 96-well microplates containing 1×106 IIALB/c mouse thymocytes. Culture supernatants from wells in which cells had grown were checked for insulin antibody production using the previous i7R method, and a total of 10 clones were obtained. The cloned cells were successively scaled up and cultured from 96-well plates to 24-well plates and then to 25 cJ flasks using RPM11640 medium containing 10% tallow serum, and the two supernatants were collected. This-
The titer of insulin antibody obtained in the supernatant (dilution factor when 50% of 0.05 ng of +251-porcine insulin is bound) was 30-2.500.

Next, 2×10′ cells were treated with pristane (Al
drich Chemical Co, product) 0.5m
Ili was administered intracavitally to RALB/c mice that had been previously administered with ni to induce hydrolung tumors, and ascites was collected.

The insulin antibody titer at this time was 50,000 to 350,000.

One representative clone of this hybridoma is NS-2C.
It was named -11. 8 cc of ascites obtained by administering this intraperitoneally in the same manner was 0.0 cc of ascites. IMl-Lis-HCl buffer pl+7.
DEA dialyzed against 4 and equilibrated with the same buffer.
E0 Sephacel (Pharmacia) column (diameter 1.6 cm)
, height 16 cm), and the flow-through fractions 32c and 32c were collected.

This was then added to PBS saturated with ammonium sulfate (0
, 15 mol Na C12-0.01 mol phosphate buffer pl+7.4) was added and the resulting precipitate was heated to 1200 Or
Centrifuged at 4° C. for 15 minutes at pm. The obtained precipitate was dissolved in 32 cc of PBS, and an equal amount of saturated ammonium sulfate PBS was added again to perform salting out. The precipitate obtained by centrifugation in the same manner was dissolved in PBS 7.5 m It,
Extensive dialysis was performed against PBS. The total amount after dialysis is 8
．． It was 5mA.

-14, it was ]Omg/mβ. The titer of this was determined by the method described above to be 270,000.

Next, this clone was cryopreserved by a conventional method.

After 6 months, it was thawed and intraperitoneally administered to the mouse again.

The obtained ascites was purified in the same manner, and the TgG concentration was 10 mg.
/ml. Its titer was 290,000. Thawed clones were frozen again and thawed again after 6 months.

The mouse was administered again into the peritoneal cavity, and the obtained ascites was similarly purified to 1 and T g G ? ? The degree of s was set to 10 mg/rrw2. Its titer was 280,000. This fact indicates that monoclonal antibodies with the same affinity and titer can be produced indefinitely. In addition.

When we applied for deposit of these cells to the Institute of Microbial Technology, Agency of Industrial Science and Technology, we received a letter of refusal for deposit on the grounds that they were outside the scope of entrusted microorganisms. (Notification number: 58 Weiyobun No. 1224
issue, September 29, 1982).

The Novoactravit Mc injection used in Northern Comparison Example 1 and Complete Soloin adjuvant were mixed at a ratio of 1:1, and this was applied to 10 guinea pigs I- (300 g male).

A dose of 500 μg/mouse was administered subcutaneously to the back and legs of each mouse 4 times every 15 days. final immunity 7
Heart blood was collected after 1 day, and 6 c of serum was collected from 15 cc of blood per animal.
c/mouse were collected. This was purified using the purification method used in Example 1, and the IgC concentration was 10 mg/m7! I made it.

The titer was determined for serum from each guinea pig in the same manner as in Example 1. The results are shown in the table below.

2 As is clear from the table, a wide range of titers were observed in the 9-mormo throat samples despite having the same amount of IgG. This indicates that there are variations in the proportion of IgG as insulin antibodies per total IgC or differences in the affinity of antibodies for insulin. In any case, it is found that it is extremely difficult to obtain large quantities of antibodies having the same titer and the same affinity.

3 Actual drawing (cross-reactivity of porcine insulin monoclonal antibody and human insulin) fil Preparation of labeled porcine insulin: 2 ng of labeled porcine insulin was mixed with 0.5% raw serum and Halbicool buffer containing albumin (pH 8,5) 4 Labeled porcine insulin dissolved in m+2? Yong? Get the night.

(2) Preparation of porcine insulin monoclonal antibody: 10 mg/mj! purified from mouse ascites in which In5-2 C-11 was grown in Example 1! Insulin antibodies at a concentration of 2% live serum gamma globulin containing 0.05
Monoclonal antibodies are obtained by diluting 280,000 times with molparbital buffer.

(3) Preparation of standard insulin 20 Novoactrapid Mc injection solution (porcine insulin 40 units/mβ) was added to 0.
5.10.20.40.80.160 and 3201
Standard porcine insulin is obtained by dissolving the insulin in a 0.05 molar barbital buffer (pH 8.5) containing 0.5% bovine serum albumin so that the concentration is 0.5% bovine serum albumin. Or, ■ human monocomponent insulin (human insulin 264 units/mg, sold by Novo Research Institute Kodama Co., Ltd.) at 0. 5.10.20.40°80,
0 to be 1.60 and 320 μU/m Q
．． Standard human insulin is obtained in 0.05 molar halbital buffer (pl+ 8.5) containing 5% bovine serum albumin.

(4) Cross-reactivity between porcine insulin monoclonal antibody and human insulin: Labeled porcine insulin 2 prepared in (1) above
00 μl, 200 μl of the porcine insulin monoclonal antibody from (2) above, and 100 μe of the standard insulin prepared from (3) above were mixed, and after standing at 4°C for 24 hours, 25% PEG #6, 000 aqueous solution 1,0
00 μl was added and centrifuged. The radioactivity of the obtained precipitate was measured. Based on the measurements, the ratio of bound labeled insulin to total labeled insulin is plotted against the amount of standard insulin in the figure. From the figure, it can be seen that standard porcine insulin and standard human insulin are on almost the same curve, and they often intersect.

(Effects of the Invention) 5 According to the present invention, monoclonal antibodies reactive with human insulin and porcine insulin can be produced indefinitely while maintaining the same affinity and titer. Therefore, the problem of lot-to-lot variation during immunoassay using this is completely eliminated, and highly reproducible measurement values can be obtained.

It's also economical.

[Brief explanation of the drawing]

The figure is a graph showing the relationship between the ratio of bound labeled porcine insulin to labeled porcine insulin and the amount of standard porcine insulin and standard human insulin. Applicant: Sekisui Chemical Co., Ltd. 6 Obstruction per unit quantity (PVmQ) Procedural amendment (I'1 button June 3, 1985, Mr. Commissioner of the Japan Patent Office 1, Indication of case) Patent Application No. 44983 of 1982 2. Name of the invention Monoclonal antibody against porcine insulin or human insulin and its manufacturing method 3. Relationship with the amended person's case Patent applicant Postal code 530 Address Patent Department, 2-4-4 Nishitenma, Kita-ku, Osaka T.E.
I, (06) 365-2181 Patent Department Tokyo Station (
03) 434-95524, Subject of amendment (1) One or more drawings after amendment

Claims (1)

[Claims] 1. Monoclonal antibody against porcine insulin or human insulin. 211.1 The step of fusing lymphocytes that produce antibodies against porcine insulin or human insulin with myeloma cells to form hybridomas. (2) Screening for hybridomas that secrete antibodies against the insulin. (3) a step of cloning the hybridoma obtained by screening; and (4) a step of collecting a monoclonal antibody secreted from the cloned hybridoma. A method for producing a monoclonal antibody against porcine insulin or human insulin, including: 3. A hybridoma obtained by the fusion. 3. The method according to claim 2, further comprising the step of screening on a medium selective thereto. 4. The method according to claim 3, wherein the lymphocytes are lymphocytes obtained from a mouse, and the myeloma cells are mouse myeloma cells. 5. The mouse myeloma cells lack hypoxanthine guanine phosphoribosyltransferase, and the screening step in the selective medium contains hypoxanthine. 5. The method of claim 4, which is carried out on a medium containing aminopterin and thymidine, said medium being selective for hybridomas formed from said myeloma cells. 6. The method of claim 2, wherein said hybridoma is formed by fusion of said lymphocyte and said myeloma cell in the presence of polyethylene glycol. 7. The method according to claim 2, wherein the monoclonal antibody is collected by culturing the cloned hybridoma in a test tube and removing a culture solution containing the secreted monoclonal antibody. 8. 3. The method according to claim 2, wherein the monoclonal antibody is collected by introducing the cloned hybridoma into an animal and removing ascites fluid containing the secreted monoclonal antibody. 9, fl,) Immunizing the animal with porcine insulin or human insulin. (2) Generate antibodies against the insulin. The process of fusing lung cells from an immunized animal with myeloma cells to form hybridomas. (3) Screening for hybridomas that secrete antibodies against the insulin. (4) a step of cloning the hybridoma obtained by screening; and (5) a step of collecting a monoclonal antibody secreted from the cloned hybridoma. A method for producing a monoclonal antibody against porcine insulin or human insulin, including: 10. The method according to claim 9, further comprising the step of screening the hybridomas obtained by the fusion in a medium selective for the hybridomas. 11. the immunized animal is a mouse, and the bone It;
11. The method of claim 10, wherein the Itoma cells are mouse myeloma cells. ]2. the myeloma cells of said mouse are deficient in hyboxanthine guanine phosphoribosyltransferase, the screening step with said selective medium is carried out on a medium containing hyboxanthine, aminopterin and thymidine, and said medium is formed from said myeloma cells; 12. The method of claim 11, which is selective for hybridomas that have been identified. 13. When the hybridoma is in the presence of polyethylene glycol, the VIll! ! 10. The method of claim 9, formed by fusion of WA cells and said myeloma cells. 14. The method according to claim 9, wherein the monoclonal antibody is collected by culturing the cloned hybridoma in a test tube and removing the culture medium containing the secreted acid clonal antibody. 15. The method according to claim 9, wherein the monoclonal antibody is collected by introducing the cloned hybridoma into an animal and removing ascites fluid containing the secreted monoclonal antibody. 16. Somatic cell hybridoma secreting monoclonal antibodies against porcine insulin or human insulin. 17. The hybridoma of claim 16, which is formed by fusing mouse Ill visceral cells producing the antibody with mouse myeloma cells. 18. The mouse myeloma cells are P3-X63-Ag8
-Ul. The hybridoma according to claim 17.