JPH01279900A - Anti-human lipoprotein lipase monoclonal antibody - Google Patents

Abstract

NEW MATERIAL:An anti-human lipoprotein lipase monoclonal antibody having the following properties. (A) Recognizes the human lipoprotein lipase, (B) substantially unreactive with a human liver triglyceride lipase, human pancreatic lipase and human pseudocholine esterase. USE:Useful in the field of a clinical diagnosticum and biochemical reagent. PREPARATION:An antibody-producing cell is collected from pancreas of an animal immunized with a human lipoprotein lipase as an antigen. Then the antibody-producing cell and a myeloma cell are dispersed in a serum-free medi um for cell-fusion in the presence of a fusion-promoting agent, mixed, fused and then subjected to screening and cloning. Thus obtained cloned hybridoma is cultured in vitro or in vivo to afford the aimed monoclonal antibody.

Description

[Detailed Description of the Invention] Industrial Application Field The present invention relates to human lipoprotein lipase (hereinafter referred to as LPL).
The present invention relates to a monoclonal antibody against and a method for producing the same, and is useful in the fields of clinical diagnostic agents and biochemical tests.

Conventional technology and problems to be solved
It is an enzyme that hydrolyzes human lipoproteins such as L). Blood In
Knowing the increase and decrease of LPL in human body fluids is viewed as an indicator for elucidating various diseases, especially hyperriboproteinemia, supernatant fluids, and shoulder problems such as arteriosclerosis.

Anti-LPL polyclonal antibodies can be obtained by parenterally administering (immunizing) LPL to mice or rabbits, storing the antiserum, and checking the antibody titer. In this case, antibodies with different antibody titers and properties can be obtained depending on the manufacturing loft. We obtained the minimum amount of antiserum required from immunized rabbits (
By keeping rabbits alive as long as possible while collecting polyclonal antibodies as needed, polyclonal antibodies with more or less consistent properties have been obtained on a somewhat continuous basis. However, with this method, the death of the rabbit results in the loss of a source of antibody, so it is not an advantageous method. In addition, the antigen LP
L is rarely found in the plasma of healthy individuals and is only present at low i5[f in the plasma of humans injected intravenously with heparin solutions. In addition, LPL does not gradually lose its enzymatic activity when left at -20°C or freeze-dried, and is easily stored in glass containers in the absence of other proteins. As described above, it is extremely difficult to secure LPL as an antigen. Therefore, it is impractical to prepare polyclonal antibodies each time they are used.

Because of this, anti-LPL monoclonal antibodies! ! I
There is an urgent need to build one. However, the inventors
1. An attempt was made to create anti-LPL monoclonal antibody-producing cells using ILT's cell fusion technique, but the desired product was not obtained.

Therefore, the present inventors investigated in detail all the steps of the cell fusion technique, and by intentionally not adding feeder cells, which is normally done when culturing fused cells, the monoclonal antibody-producing fused cells (hyperdomas) of the present invention were obtained. The present invention was completed based on the knowledge that it could be created.

Means for Solving the Problems The present invention can be used as an LPL quantitative reagent or a biochemical reagent.
An anti-LPL monoclonal antibody having the following properties for use in: ■ Recognizing human lipoprotein lipase; ■ Substantially not reacting with the following: human hepatic triglyceride lipase, human pancreatic lipase, human pseudocholinesterase.

The monoclonal antibody of the present invention is produced through the following steps: (1) antibody-producing cell preparation step; ■ fusion/screening/cloning step; (3)
It is obtained by carrying out a monoclonal antibody production process and (4) a monoclonal antibody purification process. Each step will be explained below.

(1) Antibody-producing cell preparation process Antibody-producing cells are obtained from animals sufficiently immunized with the antigen LPL.
It can be collected from f[.

It is most preferable to use a large amount of highly purified LPL as an antigen.For LPL, human plasma (heparin-perfused plasma) in which a heparin solution has been administered into the D layer is treated with heparin-Sepha0-se, for example. Gel II with hydroxyapatite treatment, Sephadex G-150! It is purified by a rational combination of treatments such as filtration, and 5DS
- An LPL with a single band in polyacrylamide electrophoresis is obtained.

Immunization can be performed by administering LPL as an antigen to mammals such as mice and rats. The immunization conditions, such as the amount of LPL used as an antigen, the site of administration, and the amount of [7] used in adjuvant 7, are the same as those used for obtaining conventional anti-blood 1n. Immunization is usually carried out by parenterally administering an emulsion containing Freund's complete adjuvant 7 and LPL to a mammal such as a mouse, followed by boosting several times at 3-week intervals. Immunization is preferably performed following purification of LPL, taking into consideration the inactivation of LPL as an antigen. Antibody-producing cells can be collected from the lungs of sufficiently immunized mammals 2 to 5 days after the final immunization.

■ Fusion/screening/cloning process fusion is
In the presence of a fusion promoter, the above antibody-producing cells and known self-proliferating bone NH cells (hereinafter referred to as myeloma cells) are placed in a known bloodless 7n medium for fusion! ! This can be done by clouding and mixing.

Generally, myeloma cells are preferably derived from the same species as the immunized animal used in step (1), cannot grow in a hybridoma selection medium, and do not themselves secrete antibodies. As such myeloma cells,
For example, commercially available mouse myeloma cells P3-X6
Examples include 3-^[8-Ul or its equivalent.

The mixing ratio of both cells is usually 1 to 20 antibody-producing cells to 1 myeloma cell. As the cell fusion promoter, for example, polyethylene glycol is used, and the molecules ff11,
000 to 7,500 is preferred.

Culturing of the fused cells, ie, hybridomas, is carried out at least twice. The first culture is performed in a 96-well culture dish. In the first culture, 96 hybridomas were washed to remove the fusion promoter and suspended in myeloma cell medium.
This can be done by seeding the culture in a well-cultured dish and placing it in an atmosphere of 5% carbon dioxide gas and air at about 37°C. During culture, HAT
A known hybridoma selection medium such as culture medium is added and the proportions are gradually increased. Such medium exchange kills cells other than hybridomas.

Screening for the target hybridoma (primary screening) can be performed by examining the antibody titer in the culture solution. In the primary screening, a 96-well culture plate with a nitrocellulose membrane was prepared with immobilized LPL as an antigen, culture supernatant was added to this to perform an antigen-antibody reaction, and anti-1gG-biotin complex was added to this, and then an anti-1gG-biotin complex was added. This can be carried out by sequentially reacting an avidin-biotin-peroxidase complex and measuring the peroxidase activity by a conventional method. If peroxidase activity is observed, it is determined that the antibody titer is positive.

The second fusion cell culture is performed in a 24-well culture dish. In other words, for the second culture, the culture that was determined to be positive in the first culture was sown in a 24-well culture dish.
This can be carried out by culturing in the same manner as the first time.

Supporting cells (such as mouse lung cells and thymocytes) that maintain a constant cell density and promote proliferation of the fused cells interfere with the assay of antibody titers in the culture;
It is not added throughout the second culture. Secondary and tertiary screening is performed on the second culture medium tn that has grown under such controlled conditions.

The secondary screening is the same as the primary screening.
Prepare a hole with a PL-fixed nitrocellulose membrane and a hole without it, add the second culture supernatant to these holes, and perform the same operation as the first screening.
Select cultures with positive holes in the LPL-fixed membrane and negative holes in other holes.

The primary and secondary screening is a method using an LPL-immobilized nitrocellulose membrane, and l'J that immobilizes LPL does not lose the antigenicity of LPL, but its lipase activity is lost. Therefore, in the tertiary screening, we perform antibody titer assay using lipase activity as an indicator.
That is, in the tertiary screening, the second culture supernatant is reacted with LPL to form an antigen-antibody complex, this complex is captured as a precipitate using a second antibody, and the lipase of LPL in the supernatant is This can be carried out by measuring activity, and it is determined that the lower the lipase activity, the higher the antibody titer. The lipase activity of LPL can be measured using a substrate labeled with a radioactive element, such as "C triolein."Measurement of lipase activity using a synthetic group [nALB] disclosed in Japanese Patent Publication No. 57-21998 method cannot be applied because the lipase activity in the supernatant is weak.

By applying the limiting dilution method to the culture screened in this way, a cloned hybridoma that produces the desired anti-LPL monoclonal antibody can be created. This hybridoma can be preserved semi-permanently by subculturing or freezing.

(3) The cloned hybridoma obtained in the pre-process of the monoclonal antibody manufacturing process is in vitro or i
The monoclonal antibody of the present invention can be produced by culturing in nViVO.

In vNro culture can be performed by starting from culturing a few hybridomas in a 96-well culture dish and gradually scaling up. In addition, in ViVO culture is performed using blistane (p) to facilitate the proliferation of fused cells.
This can be carried out by intraperitoneally inoculating hybridomas into mice treated with (Ristane), and ascites containing monoclonal antibodies accumulates 10 to 20 days later.

In general, in vHro culture is performed when it is desired to obtain monoclonal antibodies of high purity, and in ViVO culture is performed when it is desired to obtain large amounts of monoclonal antibodies. Ascites fluid accumulated through in ViVO culture can be used as it is for quantifying LPL, but it may be further purified if necessary.

(4) Monoclonal antibody purification step: in VitrO culture or in ViV as necessary
Preparative purification of monoclonal antibodies from concentrated ascites in culture of O. This can be done by rationally combining means such as column chromatography.

The monoclonal antibody of the present invention thus obtained is particularly
It is useful as a reagent for quantifying PL. Quantification of LPL using the monoclonal antibody of the present invention can be carried out by the Santoifuchi EIA method using an enzyme-labeled anti-LPL monoclonal antibody and an insolubilized washed LPL monoclonal or polyclonal antibody.

Concrete example The present invention will be explained in more detail by giving Examples.

In addition, the following culture medium was used below.

■ Serum-free medium for cell fusion (Flow Inc.) RPIJI
A medium prepared by adding the following to -1840 medium.

50U/ml Beninri 7G 50Mg7@1 streptomycin ■ +1 A T medium RP) A medium prepared by adding the following to Jl-1040 medium.

15% 5% run blood ll'? (Contains non-essential amino acids) 2m
M Glutamine 1 mM Sodium Pyruvate 500/en! Penicillin G 50Mg/ml Streptomycin 13.131-gel Hyboxanthin 0.18 m
g/l Aminopterin 3.881g 1l Thymidine Example 1 Monoclonal antibody production LPL, the antigen produced by hybridoma 'A, was perfused with heparin 1 mf according to a conventional method.
fl 2+ 1 was treated with two heparin-bound Sepharose columns, hydroxyapatite column treatment and Sef 1.
The antigen purified by DEFUCS G-150 column treatment was immediately used as an immunizing antigen. In addition, purified LPL is 5DS
- shows a single band in polyacrylamide electrophoresis;
Its molecular weight was estimated to be 61 kilodaltons.

Preparation of Antibody-Producing Cells Add an equal volume of Freund's complete adjuvant (Difco) and 2 drops of mannide monooleate solution to O, Oml of 0.15M NaC+ 0.01M phosphate buffer (pl+ 7.2) containing LPL. Emulsified by adding 0.
5 l (contains 80 Mg of LPL as protein)
LII/c mice (Shizuoka Experimental Animal Cooperative) are administered intra- and subcutaneously, and immunized in the same manner three times at three-week intervals.

Four days after the final immunization, the lungs are removed and antibody-producing cells are collected.

Mouse myeloma cells r'3-XG3 in logarithmic growth phase after fusion and first culture
-Ag8-Ul (ATCCh 9 Log number CRLI5
97) No5 x 10' and lXl0 of antibody producing cells
Mix 0.9% sodium chloride 10.1 17
Centrifuge (400 x g, 1
0 minutes) After washing, 0.5μl of polyethylene glycol 1500 (Wako Pure Chemical Industries) kept at 37°C -RPM+-
Gradually add 1640 medium (1:1) and stir slowly. After 90 seconds, 10 ml of medium kept at 37°C is added in the same manner. After 10 minutes, add 10 mj) (D) medium, centrifuge (400Xg, 10 minutes), and
Remove 8. 200■ for the beret you get! ■ Gradually add the medium. This 0.1 liter is sown in a 96-well culture dish and cultured at 37°C in 5% carbon dioxide gas/air. 5.10.After 15 days, 0.05 ml of new medium
Add each to the hole. Hybridoma growth started 7 days after the start of culture, and growth was observed in 80% of the holes on day 15.

Assay of antibody titer (primary screening) After 15 days of culture, the antibody titer of the culture fl solution was examined as follows.

96-well culture dish with nitrocellulose membrane (milliliter 1
1A: Nippon Millipore Co., Ltd.) is suction-washed with Tris buffer (20 mM) Lis, 50 (1+1 JNaCl, pH 7,5), and the membrane is equilibrated with the same buffer. Add to this 30ng of LP
Add 2μβ of 0.1% nSA solution containing L, leave it for 1 hour at room temperature, and add 400μβ of the same Tris G Uj solution containing Ly-720 (0.05%). Wash with suction twice. 1 for this
After adding 250 μl of the same Tris buffer containing % BSA to the wells,
Aspirate and add 400μ of the same Tris rA buffer containing Tween 20!
Wash with suction twice. Add 100 μl of the culture supernatant to the well, leave it overnight at room temperature, and wash with suction with 400 μl of the same Tris buffer containing Soy-720. Add 45μ of anti-mouse IgG antibody-biotin complex solution (Vector) to the hole! Tween content Tris buffer I low! 100% solution diluted with
μ! After suction, leave at room temperature. After 1 hour, add 400μ of the same Tris warm solution containing Tween 20! Wash with suction three times, and fill the hole with 90μ of Abin-biotin-peroxidase complex solution (A B C Reagent Ni-Vector).
! Add 100 μl of a solution diluted with Tween-containing Tris buffer Ionl and leave at room temperature for 1 hour. After suction, peroxidase activity is measured according to the procedure using a reagent for measuring peroxidase activity manufactured by Bio-Rad.

Cultures with a positive antibody titer in the second culture primary screening are
The cells are cultured in a 24-well culture dish in the same manner as in the case of incubation. Hybridoma growth started on the 3rd day after the start of culture and 7 days later, the growth of the hybridoma reached 9.
Growth is observed in 0% or more of the holes.

Secondary Screening 2 [ij1] A secondary screening was performed on the first culture supernatant by the method described above, and culture 120 was positive in the LPL-immobilized nitrocellulose membrane and negative in the LPL-non-immobilized membrane. Select seeds.

Tertiary Screening The following tertiary screening will be performed on the 120 types of cultures selected in the secondary screening.

Second culture supernatant 100μl and 20μl! of LPL solution (5 ng of LPL dissolved in 0.1% BSA aqueous solution) and left on ice for 2 hours, then 20μ! prepared according to the instructions. A second antibody (anti-mouse IgG rabbit antibody) solution (Miles) was added and left on ice. 2 hours later,
Centrifuge (7500×g, 10 minutes) to obtain supernatant. Supernatant 110μ! C-) lioolein solution containing 40 μl of apoprotein C-■ prepared according to the instructions) and 150 μβ of IO% B S
Mix A-0.4M) Liss buffer (pl+8.2) and incubate at 37°C for 20 minutes.

Heptane-chloroform-methanol (6:
7.5: 8.4) Mixed liquid 3■! Add 0.5 ml of 0.5N aqueous sodium hydroxide solution and shake well. Centrifuge the mixed solution (7500X z, 10 minutes) to remove the aqueous layer (
The supernatant) is separated and the free IC fatty acids are measured using a liquid scintillator method.

The 15 types of cultures that were positive in Cloni/G's 1st to 2nd screening and determined to have high antibody titers in the 3rd screening were collected from 96
Dilute with ① medium so that 1 hybridoma/well is placed in a well culture dish and culture. Cloning is performed by repeating such limiting dilution culture to obtain a cloned hybridoma.

■ Preparation of monoclonal antibodies Add 0.5 ml of Prista/(Altrifti) to lXl0' of the hybridomas obtained in the previous section! 11 Inoculated intraperitoneally into BAL mouth/C mice. 1
Day 55: 8 m of ascites containing the desired monoclonal antibody
Obtain l/mouse.

(3) Properties of monoclonal antibody The properties of the monoclonal antibody thus obtained are as follows.

■ Recognize LPL.

■ Does not substantially react with the following lipases.

Human hepatic triglyceride lipase, human pancreatic liver enzyme, human pseudocholinesterase. The same operation was performed except for the addition, and the lipase activity of the resulting supernatant was measured in the same manner, and the recovery rate was confirmed to be approximately 100%.

Patent applicant: Dainippon Pharmaceutical Co., Ltd.

Claims (2)

[Claims] (1) An anti-human lipoprotein lipase monoclonal antibody having the following properties: [a] recognizes human lipoprotein lipase; [b] does not substantially react with the following: human hepatic triglyceride lipase, human pancreatic lipase, human pseudocholinesterase. (2) Fuse antibody-producing cells and self-proliferating myeloma cells, culture them, select the monoclonal antibody-producing fused cells according to claim 1, and apply the monoclonal antibody according to claim 1 to the selected fused cells. 2. The method for producing a monoclonal antibody according to claim 1, wherein the fused cells are cultured substantially in the absence of supporting cells.