JPH0411191B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Field of Industrial Application The present invention relates to apoprotein (hereinafter referred to as LS molecular weight of about 62,000 and about
34,000 to 37,000 apoproteins)
Produces IgG type mouse monoclonal antibodies,
This article relates to a mouse-mouse hybridoma and its production method. The purpose is to provide a hybridoma that produces a monoclonal antibody that recognizes LS apoprotein, which can be used as an indicator for the dynamics of lung surfactant and as a reagent for clinical and pathological diagnosis.

(b) Prior Art In the alveoli of animals, physiologically active substances mainly composed of phospholipids, called lung surfactants, exist. It covers the inner wall of the alveoli and has a protective effect on the alveolar epithelium, as well as an important physiological function in maintaining the respiratory function of animals. In other words, pulmonary surfactants have a unique surface activity that changes the surface tension on the inner surface of the alveoli during exhalation and inspiration, and contributes to the stability between alveoli, resulting in anti-inflammatory properties. It is said to have a pneumolung effect. When there is a lack of such pulmonary surfactants, the alveoli collapse, making it impossible to maintain stable ventilation capacity, and symptoms such as neonatal respiratory distress syndrome (IRDS) appear. Approximately 90% of pulmonary surfactant substances are lipids such as phospholipids and neutral lipids, but approximately 10% are proteins, and these exist as complexes of lipids and proteins, that is, lipoproteins.
When lipids are removed from lung surfactants, water-insoluble proteins are obtained, which are called apoproteins.
The main component is protein with a molecular weight of approximately 36,000 (36K). The structure, function, and substitution of apoprotein have been studied in detail, and immunological quantification using polyclonal antibodies and immunohistological studies have also been conducted. However, to date, LS apoprotein has not yet been widely used in clinical and pathological diagnosis as an indicator of the dynamics of pulmonary surfactants.

(c) Problems to be solved by the invention As markers for pulmonary surfactants, the L/S ratio (ratio of lecithin to sphingomyelin) in amniotic fluid;
The amount of dipalmitoylphosphatidylcholine in amniotic fluid and bronchopulmonary lavage fluid has been measured, but compared to these phospholipids, protein has superior specificity and can be detected with high sensitivity, so protein has been used as a marker for lung surfactant. is expected to be used.

For this purpose, it is necessary to obtain a monoclonal antibody against the LS apoprotein, but to date, neither such monoclonal antibody nor any hybridoma that produces it has been obtained.

(d) Means for Solving the Problems The present inventors have discovered, for example, pulmonary surfactants in pulmonary and/or bronchial lavage fluid in patients with alveolar proteinosis who accumulate large amounts of pulmonary surfactants. From bronchopulmonary lavage fluid, molecular weight approximately 62,000 and approximately 34,000 ~
By separating and purifying the 37,000 LS apoprotein, immunizing a mouse with it, and fusing the antibody-producing cells obtained from the mouse with mouse myeloma cells, we obtained a molecular weight of approximately 62,000 and approximately 34,000 to 34,000.
We were able to obtain a mouse-mouse hybridoma that produces a mouse monoclonal antibody that specifically recognizes 37,000 LS apoproteins.

When the thus obtained mouse-mouse hybrid and/or cell line derived therefrom is cultured by an appropriate method, an IgG type that specifically recognizes human LS apoprotein with a molecular weight of about 62,000 and about 34,000 to 37,000 is obtained from the culture. mouse monoclonal antibodies can be collected.

The pulmonary surfactant in the present invention is separated and collected from human lung and/or bronchial lavage fluid, preferably from bronchopulmonary lavage fluid from patients with alveolar proteinosis. Pulmonary surfactant is a complex (lipoprotein) of about 90% lipid and about 10% protein, and it can be obtained by known methods such as the method of Frosolonon (J.Lipid
Res. 11, 439-457 (1970)), and then lipids are removed to obtain the LS apoprotein of the present invention. The main components of LS apoprotein are proteins with a molecular weight of approximately 62,000 and approximately 36,000, but the protein with a molecular weight of approximately 36,000 is separated as a broad band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
It is thought that this includes proteins with molecular weights of approximately 37,000 and 34,000. Therefore, apoprotein in the present invention means these proteins or fragments thereof. In addition, the molecular weight of the protein was measured by SDS-PAGE.

A hybridoma that produces a monoclonal antibody that recognizes the LS apoprotein of the present invention can be produced by a cell fusion method, which is known per se. First, the molecular weight is about 62000 and about 34000 ~
Mice are immunized with 37,000 human LS apoproteins, antibody-producing cells (lymphocytes) are collected from the spleens and lymph nodes of these animals, and these cells are fused with mouse myeloma cells. Mouse myeloma cells include BALB/C mouse P3-X63-Ag8, P3-X63-Ag8-U1, P3-
NS1/1-Ag4-1, P3-X63-Ag8-6.5.3,
There are SP2/O-Ag14, FO, MPC11-45.6TG1.7, etc.

Conditions for cell fusion are, for example, as follows. Antibody-producing cells and myeloma cells at a ratio of 10:1 to 1:10,
Preferably, they are mixed at a ratio of 1:1 to 1:3, and a suitable cell fusion solution, such as about 35% polyethylene glycol (molecular weight about 1000 to 6000) and about 7.5%
Add RPMI1640 containing 10% dimethyl sulfoxide and stir at room temperature to 37°C for 1 to several minutes, then add 10% dimethyl sulfoxide.
After gradual dilution and washing with RPMI1640 with FCS
HAT (hypoxanthine-aminopterin-thymidine) selection culture medium with a cell concentration of 1 to 5 x 10 ⁵
Adjust so that the number of cells/ml is obtained. Add this 0.2ml each,
For example, it is dispensed into a 96-well plate and cultured for 2 to 3 weeks at 35 to 38°C in air containing 5% _CO2 . Only hybridomas exist in the HAT culture solution, and 8-azaguanine-resistant myeloma cells and myeloma-fused cells cannot survive (unfused antibody-producing cells die within a few days). Next, from this hybridoma colony, molecular weights of about 62,000 and about 34,000 ~
Only those that secrete monoclonal antibodies specific to 37,000 human LS apoproteins will be selected. This selection step (screening) can be carried out by examining whether the monoclonal antibodies produced by each hybridoma cause an antigen-antibody reaction with the target LS apoprotein using an enzyme antibody method. Hybridomas that secrete the monoclonal antibody of interest must then be cloned into cloned cells. Specifically, this step can be performed using the limiting dilution method. Approximately 2 to 3 weeks later, the colonies grown in the 96-well plate were picked, and the molecular weight was determined using the enzyme antibody method again.
62,000 and approximately 34,000 to 37,000 human LS apoproteins will be examined to select hybridomas. In the present invention, this process was repeated twice.

Selected by cloning, molecular weight approx.
Hybridomas producing antibodies that recognize 62,000 and about 34,000 to 37,000 human LS apoproteins can be frozen and stored, and can also be cultured in large quantities by appropriate methods. Such hybridoma cell lines or replicated cells are also within the scope of the present invention. In addition, as long as the cloned hybridoma produces antibodies against substantially the same LS apoprotein,
Mutant strains thereof are also included within the scope of the present invention.

(E) Effects of the Invention When the hybridoma of the present invention is cultured in large quantities using an appropriate method, monoclonal antibodies that recognize human LS apoprotein with a molecular weight of about 62,000 and about 34,000 to 37,000 can be obtained from the culture supernatant. Alternatively, this hybridoma can be transplanted into an animal to form a tumor, and the desired antibody can be obtained from the condensate or serum. Purification of such antibodies involves protein A
This is carried out by a method such as affinity chromatography using a method such as chromatography.

(F) Hereinafter, the present invention will be explained in detail with reference to Examples.

Example 1 (1) For therapeutic purposes, the lungs and bronchi of a patient with alveolar proteinosis were washed with 0.15M sodium chloride solution to obtain three volumes of bronchopulmonary lavage fluid (BALF). this
BALF was centrifuged at 300 xg for 10 minutes to remove cells and cell debris. Next, the obtained supernatant was centrifuged at 48,000×g for 20 minutes, and the precipitate fraction was collected.

This precipitate fraction was treated with 145mM sodium chloride,
The cells were suspended in 80 ml of 10 mM Tris buffer (pH 7.4) containing 1 mM disodium ethylenediaminetetraacetate, and centrifuged using a 0.25 M and 0.65 M discontinuous sucrose density gradient at 4000 x g for 60 minutes. . IB fractions of 0.25M and 0.65M sucrose solutions were collected and resuspended in 400 ml of the above buffer. and,
This suspension was centrifuged at 48,000×g for 30 minutes.
A precipitate (lung surfactant) was obtained.

To remove impurities such as albumin, the precipitate was treated with 1% Triton X-100 (polyoxyethylene alkyl phenyl ether) and 3mM.
Contains EDTA, 1mM phenylmethylsulfonyl fluoride, 0.5mM dithiothreitol
The mixture was gently dispersed in 27 ml of 5 mM Tris buffer (PH7.8, hereinafter referred to as Triton buffer). This dispersion was centrifuged at 150,000 xg for 60 minutes to obtain a precipitate (purified lung surfactant).

This precipitate was redispersed in 4 ml of the above Triton buffer, 4 ml of a butanol-ethanol mixture (volume ratio 6:1) was added to this redispersion, shaken vigorously, and left at 0°C for 10 minutes. After extracting and removing the lipids, the mixture was centrifuged at 2000 rpm for 15 minutes to obtain a precipitate (apoprotein). This operation was repeated three more times to obtain the final apoprotein precipitate.

This precipitate was suspended in 20 ml of Triton buffer,
Dialyzed against the same Triton buffer using a cellophane membrane (removal of butanol-ethanol),
Dialysis fluid was obtained. This dialysis fluid was heated to 150,000×g
The mixture was centrifuged for 60 minutes to obtain a supernatant. After solubilizing the precipitate by adding 20 ml of Triton buffer again,
The supernatant was obtained by centrifugation at 150,000×g for 60 minutes. This operation was repeated six more times and the supernatant was collected. The total amount of supernatant obtained was 150 ml.
Pour into a Blue Sepharose 4B column (diameter 1.8 cm x 3.5 cm) equilibrated with Triton buffer, elute with the same buffer, and collect the flow-through fraction to completely remove the albumin present. Removed.

This fraction was equilibrated with Triton buffer.
DEAE-Toyo Pearl Column (1.4cm diameter x
19 cm) and elute with 200 ml of the same buffer at a flow rate of 15 ml/hr. Next, the concentration of sodium chloride contained in the same buffer was adjusted from 0 to 0.5M.
A fraction with a sodium chloride concentration between 0.30M and 0.35M was obtained. The molecular weight of the protein contained in this fraction is approximately
62,000 and about 36,000.

(2) The above sodium chloride concentrations are 0.30M and 0.35M
The LS apoprotein contained in the fraction eluted between was used in the following experiment.

LS apoprotein was emulsified in Freund's complete adjuvant and administered intraperitoneally to BALB/C mice. The mice were then boosted 30 days later. Meanwhile, RPMI supplemented with 15% fetal bovine serum
1640 (manufactured by Gibco), myeloma cells P3−
X63-Ag8-U1 was maintained in culture. Three days after boosting, spleen cells obtained from mice and P3-
X63−Ag8−U1 was prepared using the method of Oi et al. (Selective
methods in cellular immunology 1980,
Cells were fused using polyethylene glycol 4000 (see p. 351-372) and plated in a 96-well microplate. After cell fusion, adjust the medium to 100 μM
Hypoxanthene, 0.4μM aminopterin,
It was replaced with RPMI medium (HAT medium) supplemented with 16 μM thymidine. Cultivating in HAT medium 2-3
Within weeks, only hybridomas of splenocytes and myeloma cells grew. Antibody activity in the hybridoma culture solution was examined using the ELISA method described below.

[Antibody screening] Attach LS apoprotein to an ELISA plate,
3% (w/
Blocking was carried out using the BSA-added solution of v). After blocking, hybridoma culture solution
50μ was added to the ELISA plate and incubated for 2 hours at room temperature or overnight at 4°C. Then biotinylated horse anti-mouse IgG immunoglobulin (2 μg/ml)
Add 50μ of secondary antibody and incubate at room temperature for 1 hour. To this, 50μ of horseradish peroxidase avidin D solution (1μg/ml) was added, and the antibody bound to LS apoprotein was added to 100μ of substrate solution (0.1μg/ml).
%O _- phenylenediamine, 0.015% _H2O2 , 0.1M
Detection was performed by adding citrate buffer, pH 4.6).

(3) Hybridomas producing antibodies against LS apoprotein were selected and further cloned by limiting dilution method, and finally two types of single clone hybridomas were obtained. This hybridoma was treated with pristane-administered BALB/C.
It was grown in the peritoneal cavity of mice to obtain ascitic fluid containing monoclonal antibodies. 50% saturated ammonium sulfate was added to the obtained ascites to precipitate the antibody, and this precipitate was diluted to 0.1M
It was dissolved in phosphate physiological saline (PH8.0). After dialysis, Protein A-Sepharose
Apply the antibody to a CL4B column (manufactured by Pharmacia) in a 0.2M glycine-hydrochloric acid buffer (PH3.0).
It was eluted and purified.

Monoclonal antibodies obtained from two types of hybridomas were named PC6 and PE10, respectively.

Claims (1)

[Scope of Claims] 1. A mouse that produces an IgG type mouse monoclonal antibody that recognizes human lung surfactant with a molecular weight of about 62,000 and apoprotein of about 34,000 to 37,000.
Mouse hybridomas and cell lines derived therefrom. 2. Immunize mice with human lung surfactant substance having a molecular weight of about 62,000 and apoprotein of about 34,000 to 37,000,
Production of an IgG type monoclonal antibody that recognizes human lung surfactant apoprotein with a molecular weight of approximately 62,000 and approximately 34,000 to 37,000, which is characterized by fusing antibody-producing cells obtained from the mouse with mouse myeloma cells. A method for producing hybridomas.