JP2848705B2 - Monoclonal antibody against human lung surfactant apoprotein D and its use - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a monoclonal antibody that specifically binds to human lung surfactant apoprotein D and a use thereof.

BACKGROUND ART The lungs are luminal organs, and the surface of the alveoli that is in direct contact with the outside air is covered with an outer membrane called an alveolar coating layer. The main component of the alveolar covering layer is a pulmonary surfactant (pulmonary surfactant) rich in phospholipids.

Pulmonary surfactant is a lipoprotein consisting of phospholipids (mainly dipalmitoyl phosphatidylcholine: DPPC and phosphatidylglycerol: PG) and proteins (containing about 10%), and the surface of the air-liquid interface of the alveoli. Phospholipids are arranged and act to reduce the surface tension of the alveoli. For this reason, for example, the amount of lung surfactant in amniotic fluid is considered to reflect the maturity of the fetal lung.

The protein portion of the lung surfactant is called lung surfactant apoprotein (to date, four types of lung surfactant apoproteins A, B, C and D are known), and the expression and metabolism of lung surfactant functions It is being revealed that it plays an important role in regulation and biological defense mechanisms.

Neonatal respiratory distress syndrome (IRDS) and adult respiratory distress syndrome (ARDS) have been reported as diseases involving pulmonary surfactant. Newborns with IRDS cannot maintain stable respiratory function because the alveoli collapse due to reduced pulmonary surfactant content in the alveoli.

Therefore, the lung surfactant content in amniotic fluid is measured before parturition, and it is determined whether or not the fetus to be born has IRDS.If the fetus has IRDS, a surfactant liposome formulation or the like is administered immediately after birth. It is possible.

Conventionally, methods for measuring the amount of pulmonary surfactant in amniotic fluid include the ratio of lecithin to sphingomyelin (L / S
These methods focus on phospholipids, such as the measurement of ratio) and the amount of DPPC. However, these methods have low correlation with disease, lack quantitativeness, and are difficult to operate. It had problems and was not a satisfactory method.

Attempts to detect or measure pulmonary surfactant using antibodies against this protein, focusing on the protein part of the pulmonary surfactant, namely pulmonary surfactant apoprotein, to solve the problems of the method focusing on phospholipids have also been reported. (For example, see JP-A-62-64956)
, JP-A-4-9665, WO89 / 02075, WO89 / 01624, etc.).

As described above, four types of human lung surfactant apoproteins A to D are known. Pulmonary surfactant apoprotein A is a hydrophilic protein having a molecular weight of 28 to 38 kDa (reducing conditions) and is mainly involved in the regulation of pulmonary surfactant metabolism. Pulmonary surfactant apoproteins B and C have a molecular weight of 8 kDa (reducing conditions) and It is a hydrophobic protein of 3 to 4 kDa (reducing conditions) and is mainly involved in the expression of functions of lung surfactant itself.

Pulmonary surfactant apoprotein D is a hydrophilic protein with a molecular weight of 43 kDa (reducing conditions). Although its function has not been fully elucidated, it has been reported that pulmonary surfactant apoprotein D may have an action different from that of pulmonary surfactant apoproteins AC. I have. In addition, the time-dependent change in lung surfactant apoprotein D in amniotic fluid is also different from that of lung surfactant apoprotein A,
Interest in detecting pulmonary surfactant apoprotein D in lung tissue and quantifying pulmonary surfactant apoprotein D in blood, bronchoalveolar lavage, and amniotic fluid and accurately measuring changes over time in relation to its function Is leaning.

Therefore, an object of the present invention is to provide a monoclonal antibody capable of specifically detecting or measuring human lung surfactant apoprotein D. Another object of the present invention is to provide a human lung surfactant apoprotein using the obtained monoclonal antibody. An object of the present invention is to provide a method and a kit for specifically detecting or measuring protein D.

DISCLOSURE OF THE INVENTION As a result of repeated studies to achieve the above object, the present inventors succeeded in efficiently obtaining a monoclonal antibody meeting the above object, and by using this monoclonal antibody, The inventors have found that protein D can be specifically detected or measured, and completed the present invention.

Accordingly, the present invention relates to a monoclonal antibody that specifically binds to human lung surfactant apoprotein D.

The present invention also relates to a method and a kit for measuring human lung surfactant apoprotein D, wherein the above monoclonal antibody is used as a measuring reagent.

Further, the present invention relates to a method and a kit for detecting the presence of human lung surfactant apoprotein D in human lung tissue, wherein the monoclonal antibody is used as an antibody reagent.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the specificity of a monoclonal antibody by immunoblotting.

FIG. 2 shows the reactivity of the monoclonal antibody (6B2).

FIG. 3 shows the reactivity of the monoclonal antibody (7C6).

FIG. 4 shows the cross-reactivity of the monoclonal antibodies (6B2 and 7C6) in the immunoblotting method.

FIG. 5 shows the cross-reactivity of the monoclonal antibodies (6B2 and 7C6) in a sandwich ELISA.

FIG. 6 shows a calibration curve in the sandwich ELISA.

FIG. 7 shows the results of a dilution test in a sandwich ELISA.

Fig. 8 shows lung surfactant apoprotein D in amniotic fluid of pregnant women
(SP-D) shows the results of measuring the concentration by sandwich ELISA.

FIG. 9 shows the results of measuring the concentration of SP-D in bronchoalveolar lavage fluid of patients with alveolar proteinosis and healthy subjects by sandwich ELISA.

Figure 10 shows various respiratory diseases (lung squamous cell carcinoma, lung adenocarcinoma, small cell lung cancer, sarcoidosis, tuberculosis, emphysema, pneumonia)
Of serum SP-D concentration in patients and healthy subjects with the above, which was measured by sandwich ELISA.

Fig. 11 shows patients with various respiratory diseases (idiopathic interstitial pneumonia, interstitial pneumonia with collagen disease, alveolar proteinosis, bronchial asthma, bronchiectasis, panbronchiolitis, Hashimoto's disease, Basedow's disease) And SP-D concentration in healthy human serum were measured by sandwich ELIS
It shows the result measured in A.

FIG. 12 shows the results of immunostaining of adenocarcinoma tissue using a monoclonal antibody (6B2).

FIG. 13 shows the results of immunostaining of lung squamous cell carcinoma tissue using the monoclonal antibody (6B2).

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.

1. Monoclonal Antibody (1) Characteristics The monoclonal antibody of the present invention is a monoclonal antibody that specifically binds to human lung surfactant apoprotein D, and other characteristics are not limited at all. Has the following properties: By using such a monoclonal antibody, it has become possible for the first time to specifically detect or measure human lung surfactant apoprotein D in a sample.

Specificity The specificity of the antibody was determined by the immunoblotting method described in the Examples below.
Specifically reacts with

Reactivity When the reactivity of the antibody is examined by the ELISA method described in the Examples described later, the antibody reacts with human lung surfactant apoprotein D depending on the concentration of the antibody.

Cross-reactivity The reactivity of the antibody was examined by immunoblotting and sandwich ELISA as described in the Examples below, and it was determined that the lung surfactant apoproteins A, B and C originated from humans.
Does not substantially react, or even if it does, has no effect on the measurement of human lung surfactant apoprotein D.

Species specificity The reactivity of the antibody was examined by the immunoblotting method and the sandwich ELISA method described in the Examples below, and the lung surfactant apoprotein D derived from other animals (rats) was determined.
Does not substantially react, or even if it does, has no effect on the measurement of human lung surfactant apoprotein D.

(2) Production Method The above-mentioned monoclonal antibody of the present invention can be produced by appropriately applying a known method.

The immunogen used is human lung surfactant apoprotein D
This is, for example, from human bronchoalveolar lavage fluid, preferably from bronchoalveolar lavage fluid of patients with alveolar proteinosis.
It can be prepared by the method of Persson et al. (J. Biol. Chem. 265, 5755, 1990). The degree of purification of the immunogen is not particularly limited.

Alternatively, a peptide corresponding to a part of the amino acid sequence of human lung surfactant apoprotein D may be prepared chemically by a known method and used as an immunogen. When the synthesized peptide has poor antigenicity, it is preferable to use, as the immunogen, a complex of a hapten antigen such as bovine serum albumin and keyhole limpet hemocyanin with a polymer carrier commonly used for producing antibodies. Furthermore, a recombinant human lung surfactant apoprotein D prepared using a known DNA recombination technique may be used as an immunogen.

Animals to which the immunogen is administered include cows, horses, sheep, goats, rats, mice, guinea pigs, dogs, pigs,
Any of rabbits, monkeys, pigeons, chickens and the like may be used, and mice, rats, guinea pigs, rabbits, goats and the like are particularly convenient for use.

Administration of the immunogen to such an animal may be performed according to a conventional method, for example, complete Freund's adjuvant, incomplete Freund's adjuvant, alum adjuvant,
An emulsion of various adjuvants such as aluminum hydroxide adjuvant and B. pertussis adjuvant and the above-mentioned immunogen may be prepared and administered to the animal intravenously, intraperitoneally, subcutaneously or intradermally.

The dose is 0.01 to 10 mg / animal as a protein amount when using rabbits and guinea pigs as animals, or mice,
When a rat is used, about 0.001 to 1 mg / animal is suitable.

After the initial administration, an additional 1 to 5 booster immunizations as described above are performed every 1 to 4 weeks to induce antibody production against human lung surfactant apoprotein D in the animal body.

Next, antibody-producing cells, such as spleen cells, lymph node cells, and peripheral blood lymphocytes, are obtained from animals in which antibody production has been induced by a conventional method.

Myeloma cells fused with antibody-producing cells include:
Cell lines derived from various animals such as mice, rats, and humans and generally available to those skilled in the art are used. The cell line to be used is preferably a cell line which has drug resistance, cannot survive in a selection medium in an unfused state, and can survive only in a state fused to an antibody-producing cell. Usually 8-
An azaguanine-resistant strain was used, and this cell line was hypoxanthine guanine phosphoribosyl transferase (hypoxanthine guanine phosphoribosyl transferas).
e) cannot be grown on hypoxanthine / aminopterin / thymidine (HAT) medium. In addition, a so-called non-secretory cell line that does not secrete immunoglobulin as a cell property is preferable.

Specific examples of myeloma cell lines include P3x63Ag8 (ATCC
TIB-9) (Nature, 256 , 495-497 (1975)), P3x63Ag
8U.1 (P3U1) (ATCC CRL-1597) (Current Topics in
Microbiology and Immunology. 81 , 1-7 (1978)), P3
x63Ag8.653 (ATCC CRL-1580) (J. Immunology, 123 , 154)
8-1550 (1979)), P2 / NSI / 1-Ag4-1 (ATCC TIB-1
8) (Europian J. Immunology, 6, 511-519 (1976)), S
p2 / 0-Ag14 (ATCC CRL-1581) (Nature, 276 , 269-270
(1978)) and other mouse myeloma cell lines, 210.RCY.Ag
l.2.3 (Y3-Ag1.2.3) (ATCC CRL-1631) (Nature, 27
7 , 131-133 (1979)) and the like.
U-266-AR1 (Proc. Natl. Acad. Sci. USA, 77 , 5429 (1
980)), GM1500 (Nature, 288 , 488 (1980)), KR-4
(Proc. Natl. Acad. Sci. USA, 79 , 6651 (1982)).

For cell fusion, a myeloma cell suitable for the antibody-producing cell is selected.

Cell fusion, Eagle minimum essential medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), RPMI-1640 medium in an animal cell culture medium such as 10 ⁶ to 10 ⁸ cells / ml of myeloma cells and antibody-producing cells At a mixing ratio of 1: 4 to 10 at 37 ° C.
The cells can be efficiently fused by contacting the cells with each other for 1 to 10 minutes. To promote cell fusion,
Polyethylene glycol with an average molecular weight of 1,000 to 6,000 (PE
G), polyvinyl alcohol, a fusion promoter such as Sendai virus can be used. Alternatively, the antibody-producing cells and myeloma cells can be fused using a commercially available cell fusion device utilizing electric pulses.

As a means for selecting a target hybridoma from cells after the cell fusion treatment, a method utilizing selective growth of cells in a selective medium can be used. For example, a cell suspension was prepared using RPMI-1 containing 15% fetal calf serum (FCS).
After appropriately diluting with 640 medium or the like, 10 ³
10 ⁶ cells / well of about Maki, selective medium to each well (e.g., HAT medium etc.) was added, the culture is performed by appropriately replacing the selective medium. When an 8-azaguanine-resistant strain is used as a myeloma cell and a HAT medium is used as a selection medium, unfused myeloma cells are killed by about 10 days of culture, and antibody-producing cells, which are normal cells, are in vitro ( in vitro).
In vitro ), cells cannot grow for a long period of time, and cells growing from day 10 to 14 of culture can be obtained as hybridomas.

Hybridomas producing monoclonal antibodies recognizing human lung surfactant apoprotein D were searched for by enzyme immunoassay (EIA, ELISA) and radioimmunoassay (RI
A) and so on. For example, 96-well EL1S adsorbing human lung surfactant apoprotein D
The culture supernatant containing the monoclonal antibody is added to the microplate for A and reacted with human lung surfactant apoprotein D, and then the bound specific antibody is reacted with an enzyme-labeled anti-immunoglobulin antibody, or a biotin-labeled anti-immunoglobulin antibody After the reaction, the avidin D-enzyme labeled product is reacted, and in each case, an enzyme substrate is added to each well to develop color. By selecting a culture supernatant that develops color only in the wells on which human lung surfactant apoprotein D is immobilized, a hybridoma that produces an antibody that specifically reacts with human lung surfactant apoprotein D can be searched.

The human lung surfactant apoprotein D used in the above screening is preferably highly purified. For example, by using a human lung surfactant apoprotein D having a purity of 90% or more, the monoclonal antibody of the present invention can be efficiently screened. Can be.

Hybridoma cloning can be performed by a limiting dilution method, a soft agar method, a fibrin gel method, a fluorescence excitation cell sorter method, or the like.

As a method for producing a monoclonal antibody from the thus obtained hybridoma, a normal cell culture method, an ascites formation method, or the like can be employed.

In cell culture, hybridomas are
An antibody can be obtained from the culture supernatant by culturing in an animal cell culture medium such as S-containing RPM1-1640 medium or serum-free medium by an ordinary method.

The method of recovery from ascites involves the use of pristane (2,6,10,14-
After intraperitoneal administration of a mineral oil such as tetramethylpentadecane), for example, in the case of a mouse, a hybridoma is intraperitoneally administered at about 10 ⁶ cells / animal. Hybridoma 10-1
It forms ascites tumor in about 8 days and produces high concentration of antibody in serum and ascites.

If antibody purification is required, ammonium sulfate precipitation, DE
It can be purified by properly selecting and combining known methods such as ion exchange chromatography using an anion exchanger such as AE cellulose, affinity chromatography using protein A-sepharose, etc., and molecular sieve chromatography. .

2. Measuring Method and Kit The measuring method of the present invention is characterized by using the above-described monoclonal antibody of the present invention as a reagent, and is not limited by the measurement principle, conditions, and the like.

As a classification according to the reaction mode, a competitive reaction method and a non-competitive reaction method (immunometric assay) are known, and any method can be employed in the present invention.

As a classification by the detection method, a non-labeling method (nephelometry and the like) for directly detecting the result of the antigen-antibody reaction and a labeling method for detection using some marker are known. In the present invention, either method is used. Is also good.

Heterogeneous methods that require BF separation and homogeneous methods that do not require BF separation are known, and any of these methods may be applied to the present invention.

As a classification according to the reaction phase, a liquid phase method in which the whole reaction is performed in a liquid phase and a solid phase method in which the partner of the immune reaction is immobilized to perform the reaction are known, and any method can be employed in the present invention. .

A method suitable for the purpose of the measurement method of the present invention may be appropriately selected from these known general methods.

The details of the general method are described in detail in the following documents, for example.

Edited by Hiroshi Irie, "Radio Immunoassay" (Kodansha Co., Ltd., issued on May 1, 1979) Eiji Ishikawa et al., "Enzyme Immunoassay" (Second Edition) (Co., Ltd.)
Medical Shoin, published December 15, 1982) Clinical Pathology Special Issue No. 53 “Immunoassay for Clinical Testing-Technology and Application” (Clinical Pathology Publishing Association, 1983)
Published in 2010) "Encyclopedia of Biotechnology" (CMC, Inc.
October 9, 1986) "Methods in ENZYMOLOGY Vol.70" (Immunochemical techniques (Part A)) "Methods in ENZYMOLOGY Vol.73" (Immunochemical techniques (Part B)) "Methods in ENZYMOLOGY Vol.74" (Immunochemical) techniques (Part C)) "Methods in ENZYMOLOGY Vol.84" (Immunochemical techniques (Part D: Selected Immun
oassay)) "Methods in ENZYMOLOGY Vol.92" (Immunochemical techniques (Part E: Monoclonal Ant)
ibodies and General Immunoassay Methods)) Moreover, the mode of use of the monoclonal antibody of the present invention used in the measurement method of the present invention may be appropriately derived according to the measurement method employed. Specific examples include a labeled antibody and an immobilized antibody.

The antibody used may be the antibody itself, but it is preferable to use an active fragment of the antibody from the viewpoint of preventing nonspecific adsorption.

The active fragment of the antibody may be any as long as it retains the characteristics of the antibody (for example, various fragments such as F (ab ') ₂ , Fab', and Fab). The preparation of these active fragments involves purifying the purified antibody with papain,
A known method such as a method of limited degradation using a protease such as pepsin or trypsin can be applied (for example, “Research Methods for Immunobiochemistry (Seismic Chemistry Experimental Course 5)”, edited by The Biochemical Society of Japan, p. 89). (1986)).

Labeling agents to be bound to the antibody include radioisotopes (eg, ³² P, ³ H, ¹⁴ C, ¹²⁵ I, etc.), enzymes (eg, β-
Galactosidase, peroxidase, alkaline phosphatase, glucose-6-phosphate dehydrogenase,
Catalase, glucose oxidase, lactate oxidase, alcohol oxidase, monoamine oxidase, etc.), coenzyme / prosthetic groups (eg, FAD, FMN, AT
P, biotin, heme, etc.), fluorescein derivatives (eg, fluorescein isothiocyanate, fluorescein thiol bamil, etc.), rhodamine derivatives (eg, tetramethylrhodamine B isothiocyanate, etc.), umbelliferone and 1-anilino-8-naphthalene sulfone Fluorescent dyes such as acids, luminol derivatives (for example, luminol, isoluminol, N- (6-
Aminohexyl) -N-ethylisoluminol) and the like. The binding between the antibody or the active fragment thereof and the labeling agent is described in a textbook [for example, “Seizoku Chemistry Experimental Course 5 Immunobiochemical Research Method”, Tokyo Chemical Industry Co., Ltd.
(Published in 1986), pp. 102-112] may be appropriately selected from known methods.

As the material of the carrier substance for immobilizing the antibody, for example, polyvinyl chloride, polystyrene, styrene-divinylbenzene copolymer, styrene-maleic anhydride copolymer, nylon, polyvinyl alcohol, polyacrylamide, polyacrylonitrile, polypropylene, Polysaccharides such as synthetic organic polymer compounds such as polymethylene methacrylate, dextran derivatives (such as Sephadex), agarose gel (such as Sepharose and biogel), and cellulose (such as paper disc and filter paper); and inorganic inorganic compounds such as glass, silica gel, and silicone. There may be mentioned molecular compounds, which may have a functional group such as an amino group, an aminoalkyl group, a carboxyl group, an acyl group or a hydroxyl group introduced therein. The material of the carrier substance is preferably one having a low protein binding ability, and examples of such a material include untreated polystyrene and polyvinyl chloride.

The carrier material can be in the form of a flat plate (microtiter plate, disk, etc.), particulate (beads, etc.), tubular (test tube, etc.), fibrous, membrane, fine particles (latex particles, etc.), capsule, vesicle The shape of the carrier is exemplified, and a carrier having an appropriate shape according to the measurement method can be selected. In addition, liposomes (monolayer or multilayer lipid membrane) and the like can also be used as a carrier substance for immobilizing antibodies.

The binding between the monoclonal antibody and the carrier substance can be performed by a known method such as physical adsorption, ionic bonding, covalent bonding, and inclusive method (for example, "immobilized enzyme" (edited by Chiba Ichiro, March 20, 1975, (See Kodansha Co., Ltd.)). In particular, the physical adsorption method is preferred because it is simple. In addition, the above-described binding may be performed directly, or may be performed via another substance between the two substances.

The sample to be measured is not particularly limited as long as it contains human lung surfactant apoprotein D. For example, amniotic fluid, alveolar lavage fluid, blood, serum, plasma and the like can be exemplified.

The kit used in the above-mentioned assay is characterized by containing the monoclonal antibody of the present invention as one of the constituent reagents of the kit, and other reagent configurations of the kit may differ depending on the assay employed. Good.

In the case of a kit based on the competitive reaction method, for example, the following reagent configuration can be adopted.

Immobilized antigen (antibody) Labeled antibody (antigen) solution Antigen solution of known concentration In the case of a kit based on the sandwich method, for example, the following reagent configuration can be adopted.

Immobilized first antibody Second antibody solution Labeled anti-immunoglobulin antibody solution Antigen solution of known concentration In the above kit, "antibody" is the monoclonal antibody of the present invention, and "antigen" is human lung surfactant apoprotein. Needless to say, it is D. Further, human lung surfactant apoprotein D is a polyvalent antigen, and in the case of a kit based on the sandwich method, the "first antibody" and "second antibody" use the same antigenic determinant on human lung surfactant apoprotein D. It may be one that recognizes, or one that recognizes a different antigenic determinant.

Further, as a modification of the kit based on the sandwich method, for example, the following reagent configuration can be adopted.

Immobilized first antibody Labeled second antibody solution Antigen solution of known concentration 3. Detection method and kit The detection method of the present invention is a method for detecting lung surfactant apoprotein D in lung tissue. It is characterized in that an antibody is used as an antibody reagent. Therefore, the labeling method, the labeling method of the antibody, and the detection method using the labeled antibody can be directly applied to the method commonly used in immunohistological diagnosis.

That is, as the labeling agent, the above-mentioned radioisotopes, enzymes, coenzyme / prosthetic groups, fluorescent dyes, luminol derivatives and the like can be used. The antibody to which such a labeling agent is bound may be the antibody itself or a fragment thereof. The binding between the antibody or the fragment thereof and the labeling agent can be carried out by a conventional method. The labeling of the antibody may be performed indirectly using a labeled anti-immunoglobulin antibody or the like.

The labeled antibody thus prepared is allowed to act on a lung tissue specimen according to a conventional method, and by visualizing the labeling agent bound to the antibody, lung surfactant apoprotein D in the lung tissue can be detected.

When an enzyme is used as a labeling agent in the above detection method,
The kit may include the following constituent reagents.

Enzyme-labeled antibody substrate solution If the biotin-avidin method is adopted as a modification of the above kit, the kit is composed of the following reagents.

Biotinylated antibody Avidinating enzyme substrate solution In the above-mentioned kit, it goes without saying that the “antibody” is the monoclonal antibody of the present invention.

 Hereinafter, the present invention will be described specifically with reference to Examples.

Example 1 Human Lung Surfactant Apoprotein D (SP-
D) Preparation of Mouse Monoclonal Antibody Against D) Preparation of Monoclonal Antibody-Producing Hybridoma Known Methods (Persson. A. atal, J. Biol. Chem. 265, 575)
5, (1990)) was dissolved in physiological saline (0.4 mg / ml), and complete Freund's adjuvant was added.
BALB / c was prepared by mixing at a 1: 1 ratio to form an emulsion.
Mice (female, 6 weeks old) were intraperitoneally administered (ip) at 20 μg / 100 μl for the first immunization. After the initial immunization, immunization (ip) is performed several times every two weeks by the same method, and then a physiological saline solution of human SP-D is injected into the tail vein of the mouse as a final immunization.
μg / 200 μl was administered (iv).

Three days after the final immunization, the spleen of the mouse was removed, and RPMI-
The suspension was washed with 1640 medium to prepare a spleen cell suspension. At the same time, mouse myeloma P3x63Ag8U1 (P3U1) (ATCC CRL-1597)
Was washed with RPMI-1640 medium, spleen cells and P3U1 were mixed at 10: 1, and 1 ml of RPMI-1640 medium solution containing 50% polyethylene glycol (PEG) 1000 was gradually added to the pellet obtained by centrifugation. Cell fusion was performed. Further, RPMI-1640 medium solution was added to make 10 ml, and the pellet obtained by centrifugation was
The P3U1 was suspended at 3 × 10 ⁴ cells / 0.1 ml in RPMI-1640 medium containing% fetal calf serum (FCS), and 0.1 ml of each well was dispensed into a 96-well microtiter plate. On the next day, 0.1 ml of RPMI-1640 medium containing hypoxanthine-thymidine-aminopterin (HAT medium) was added, and thereafter, every 3 to 4 days, half of the medium was replaced with a new HAT medium.

On day 14 after the fusion, hybridomas were screened. That is, human SP-D (10 μg / ml) is coated in advance and contains 25% Block Ace (Dainippon Pharmaceutical Co., Ltd.)
50 μl of the culture supernatant was added to a 96-well microtiter plate blocked with PBS, and reacted at room temperature for 1 hour. After washing three times with 200 μl of PBS, biotinylated anti-mouse
50 μl of an IgG (manufactured by Vector) solution was added, and the mixture was further reacted at room temperature for 1 hour. After the reaction, the plate was washed three times with PBS, 50 μl of avidin D-peroxidase (manufactured by Vector) solution was added, and the mixture was reacted at room temperature for 30 minutes. Further, the plate was washed similarly with PBS, and the substrate solution (4-aminoantipyrine (0.25 mg / m
l), phenol (0.25 mg / ml), 200 μl of 0.425 M hydrogen peroxide) was added and reacted at room temperature, and the absorbance at 550 nm was measured to detect an antibody specifically reacting with human SP-D.
Hybridomas producing specific antibodies were selected (Table 1).

Each of the hybridomas thus selected is further cloned using the limiting dilution method, and 9 hybridomas (1G11, 3E4, 3H4, 5A4, 6B2, 7A10, 7C6, 9E1, 10H1
1) established.

Preparation and Purification of Monoclonal Antibody
3 × 10 ⁶ mice were intraperitoneally administered to the mice treated with 5 ml,
About 2 weeks later, ascites was collected. Next, Ig from the above ascites
To purify G, affinity chromatography using a protein A-Sepharose column was performed. First, protein A-Sepharose (Pharmacia)
20 ml was packed in a 1.5 × 20 cm glass column, and equilibrated with 1.5 M glycine buffer (pH 8.9) containing 3 M sodium chloride. Next, an equal volume of the above ascites was added to the same glycine buffer,
After diluting twice and adding to the column, washing off the non-adsorbed protein with a sufficient amount of the same glycine buffer, the adsorbed IgG was eluted with 0.1 M citrate buffer (pH 3.0). IgG obtained in this way
Fractions were immediately dialyzed overnight against PBS to avoid denaturation.

Example 2 Analysis of Properties of Monoclonal Antibody (Search for Class and Type) Human SP-D (10 μg / ml) was coated and subjected to blocking treatment with PBS containing 25% Block Ace (Dainippon Pharmaceutical) 96 The culture supernatant of the hybridoma or the purified monoclonal antibody solution was added to the well microtiter plate, and the class and type of the antibody were searched using a MonoAb-ID EIA kit (Zymed). The results are as shown in Table 2.

Example 3 Analysis of Properties of Monoclonal Antibody (Antigen Specificity of Monoclonal Anti-Human SP-D Antibody) Hybridomas (IG11, 3E4, 3H4, 5A4, 6B2, 7A10,
7C6, 9E1, 10H11) produced monoclonal antibody (the name of the antibody is the same as the name of the hybridoma producing it)
The antigen specificity was confirmed by an immunoblotting method using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and an enzyme immunoassay (ELISA).

(A) Specificity analysis by immunoblotting method SDS-PAGE was performed according to the method of Lammli (Nature, 227 : 680,1972). The basic operation of the immunoblotting method is as follows. That is, the separation gel obtained by SDS-PAGE was placed on a nitrocellulose membrane, and electricity was transferred to the nitrocellulose membrane at 60 V for 12 hours. The nitrocellulose membrane thus obtained was cut into strips along the sample migration line, and a part thereof was stained with amide black for protein. The other membrane was subjected to a blocking treatment by immersing it in PBS containing 0.5% Triton X-100 and 2% skim milk at 37 ° C. for 1 hour, and reacted with a monoclonal antibody solution appropriately diluted with PBS at room temperature for 1 hour. After washing thoroughly with PBS,
A peroxidase-labeled anti-mouse IgG antibody was reacted at room temperature for 1 hour. Further, the nitrocellulose membrane is washed similarly and reacted with a substrate solution (containing 30 mg of color developer (manufactured by Bio-Rad), 10 ml of methanol, 50 ml of PBS, and 30 μl of 30% hydrogen peroxide solution). To stop the reaction.

Human SP-D shows a molecular weight of 43 kDa under reducing conditions. The human SP-D fraction was subjected to electrophoresis under reducing conditions, and the reaction specificity of the monoclonal antibody was examined by immunoblotting. As a result, as shown in FIG. 1, nine types of monoclonal antibodies (lane 1: 1G11, lane 2: 3E4, lane 3: 3H4,
Lane 4: 5A4, Lane 5: 6B2, Lane 6: 7A10, Lane 7: 7C
6, lane 8: 9E1, lane 9: 10H11) are all 3kD molecular weight
a) showed very strong reactivity with human SP-D. In addition, the monoclonal antibody (7C6) also showed extremely strong reactivity against a degradation product of human SP-D having a molecular weight of about 38 kDa, which was considered to have been derived at the time of preparation.

(B) Specificity analysis by ELISA 5 mM Tris solution of purified human SP-D (1.0 μg / ml, pH7.
4) was added to each well of a 96-well microtiter plate in an amount of 50 μl, left at 4 ° C. overnight, and then washed three times with PBS. 200 μl of PBS containing 0.5% Triton 100-X and 2% skim milk was added to each well, and left at room temperature for 1 hour to perform blocking. After washing three times with PBS, 50 μl of the monoclonal antibody solution was added and reacted at room temperature for 1 hour. After washing three times with 200 μl of PBS, 50 μl of a biotinylated anti-mouse IgG (manufactured by Vector) solution was added, and the mixture was further reacted at room temperature for 1 hour. After the reaction, the plate was washed three times with PBS, and 50 μl of avidin D-peroxidase (Vector) solution
Was added and reacted at room temperature for 30 minutes. Further, the plate was washed similarly with PBS, and the substrate solution (O-phenylenediamine (0.2 mg / m
l) and 0.1 M citrate buffer containing 0.425 M hydrogen peroxide, pH 5.
0) After adding 100 μl and reacting at room temperature, 2N sulfuric acid 100 μl
1 was added to stop the reaction, and the absorbance at 492 nm was measured. As shown in FIGS. 2 and 3, the reactivity of the monoclonal antibodies (6B2 and 7C6) with human SP-D (1.0 μg / ml) was concentration-dependent. The results are not shown,
Other monoclonal antibodies (1G11, 3E4, 3H4, 5A4, 7A1
0, 9E1, 10H11) have confirmed the same phenomenon.

Example 4 Analysis of Properties of Monoclonal Antibody (Cross-reactivity of Monoclonal Anti-human SP-D Antibody) Monoclonal antibody (6B2
And 7C6) were checked for cross-reactivity with human SP-A and rat SP-D by immunoblotting using SDS-PAGE and sandwich ELISA.

For human and rat SP-D, the method of Persson et al. Described above, and for human SP-A, the method of Kuroki et al. (Proc. Natl.
Acad. Sci. USA 85, 5566, 1988).

(A) Analysis of cross-reactivity by immunoblotting method Human SP-D fraction, human SP-A fraction, rat SP-D fraction and human amniotic fluid (38 weeks of gestation) were electrophoresed under reducing conditions, The cross-reactivity of the monoclonal antibody was examined by blotting.

From the results of blotting (lane 1: human SP-D, lane 2: human SP-A, lane 3: rat SP-D, lane 4: human amniotic fluid), the respective monoclonal antibodies (6B2 and 7C)
6) showed extremely strong reactivity with human SP-D having a molecular weight of 43 kDa, a protein of about 90 kDa which seems to be a dimer of human SP-D, and SP-D in human amniotic fluid. In addition, the monoclonal antibody (7C6) also showed extremely strong reactivity with a human SP-D degradation product having a molecular weight of about 38 kDa, which is considered to have been derived during the preparation of human SP-D. In contrast, both monoclonal antibodies show a human SP-A showing a molecular weight of about 26-38 kDa under reducing conditions.
Showed no reactivity at all, and only slightly reacted with human SP-D having a molecular weight of 43 kDa which seems to be present in a trace amount in the human SP-A fraction. On the other hand, both monoclonal antibodies showed little reactivity with rat SP-D (reducing, showing a molecular weight of about 43 kDa) (FIG. 4).

(B) Analysis of cross-reactivity by sandwich ELISA Human SP of monoclonal antibodies (6B2 and 7C6) of the present invention
Cross-reactivity with -A and rat SP-D was examined by sandwich ELISA using both monoclonal antibodies.
The basic operation of the sandwich ELISA is as follows.

Monoclonal antibody (6B2) PBS solution 10μg / ml 50μ
Each l was added to each well of a 96-well microtiter plate, left at 4 ° C. overnight, and washed three times with PBS.
PBS 200μ containing 0.5% Triton X-100 and 2% skim milk
1 was added to each well and left at room temperature for 1 hour to perform blocking. After washing three times with PBS, human SP-
50 μl of a PBS solution of D was added and left at 4 ° C. overnight. P
After washing three times with 200 μl of BS, a biotinylated monoclonal antibody (7C6) in PBS solution (0.5% Triton X-100 and 0.1%) was added.
% Skim milk) and add 50 μl of 10 μg / ml and add
Allowed to react for hours. After the reaction, the plate was washed three times with PBS, 50 μl of avidin D-peroxidase (manufactured by Vector) solution was added, and the mixture was reacted at room temperature for 30 minutes. Further, the plate was washed with PBS in the same manner, and a substrate solution (0.2 M citrate buffer containing 3 mM 3,3 ', 5,5'-tetramethylbenzidine and 0.005% hydrogen peroxide, pH
3.8) Add 100 μl and react at room temperature.
After stopping the reaction by adding μl, the absorbance at 450 nm was measured.

 The cross reactivity (%) was calculated by the following equation.

As a result, the cross-reactivity with human SP-A was 0.2% or less, and the cross-reactivity with rat SP-D was 0.25.
% Or less (FIG. 5, Table 3).

In the analysis of cross-reactivity by immunoblotting using the same sample, both antibodies (6B2 and 7C6)
It shows no reactivity against 26-38 kDa SP-A,
The actual cross-reactivity with human SP-A is considered to be even lower because it reacts with SP-D which seems to be present in a trace amount in the -A fraction.

(C) The monoclonal antibody of the present invention comprises human SP as described above.
-D-specifically, among which immobilized monoclonal antibody (7C6) and the method of Nakane et al. (I
mmunoassays in the clinical laboratory.Alan R. Liss
Inc., New York, 81, 1979), a specific and sensitive sandwich ELISA against human SP-D was established by using a combination of horseradish peroxidase-labeled monoclonal antibody (6B2). The basic operation method of the sandwich ELISA is as follows.

100 μg / ml of PBS solution of monoclonal antibody (7C6)
Each μl was added to each well of a 96-well microtiter plate, left at 4 ° C. overnight, and washed three times with PBS. 200 μl of PBS containing 1% bovine serum albumin (BSA) was added to each well, and left at room temperature for 1 hour to perform blocking. After washing three times with PBS, human SP-D
100 μl of a PBS solution containing 0.5% Triton X-100 was added, and the mixture was allowed to stand at room temperature overnight. After washing three times with 200 μl of PBS, a horseradish peroxidase-labeled monoclonal antibody (6B
2) 2μ of PBS solution containing 0.5% Triton X-100 and 1% BSA
100 μl of g / ml was added and reacted at room temperature for 2 hours. After the reaction, the plate is washed three times with PBS, and the substrate solution (0.3 mM 3,3 ', 5,5'
-Contains tetramethylbenzidine and 0.005% hydrogen peroxide
After adding 100 μl of 0.2M citrate buffer (pH 3.8) and reacting at room temperature for 20 minutes, the reaction was stopped by adding 100 μl of 2N sulfuric acid, and the absorbance at 450 nm was measured.

Calibration curve 0.5 of purified human SP-D prepared by the method of Persson et al.
Sandwich ELISA was performed using a PBS solution containing% Triton X-100 as a standard substance. As a result, the concentration is 3.13ng / ml ~ 200ng /
In the ml range, a good calibration curve dependent on human SP-D concentration was obtained (FIG. 6).

Addition / recovery test and dilution test In order to evaluate the basic performance of the sandwich ELISA of the present invention, an addition / recovery test of purified human SP-D with respect to a human amniotic fluid sample and a dilution test of a human amniotic fluid sample were performed. In the spike recovery test, 0.5% of purified human SP-D was added to the human amniotic fluid sample.
A PBS solution containing Triton X-100 (0, 12.5, 25, 50 ng / ml) was added, and the measurement was performed by sandwich ELISA. As a result, good recovery (94.4 to 111.2%) of purified human SP-D was shown (Table 4). On the other hand, a dilution test using a human amniotic fluid sample showed good linearity between the sample dilution ratio and the SP-D measurement value (FIG. 7).

Cross-reactivity To examine the cross-reactivity of the sandwich ELISA for the human SP-D analog, human SP-A and rat SP-D,
Further, a serially diluted solution of human SP-D was prepared as a control,
The reactivity in the sandwich ELISA was examined, and each cross reactivity was calculated by the following equation.

As a result, the cross-reactivity to human SP-A was 0.3% or less, and the cross-reactivity to rat SP-D was 0.6%.
(Table 5).

In the analysis of cross-reactivity by immunoblotting using the same sample, both antibodies (6B2 and 7C6)
Human SP which does not show any reactivity with human SP-A of 26-38 kDa and seems to be present in a trace amount in the SP-A fraction
Since it reacts with -D, the actual cross-reactivity to human SP-A is considered to be even lower.

Example 5 Measurement of SP-D concentration in biological sample According to the sandwich ELISA of Example 4 (c), pregnant women's amniotic fluid, bronchoalveolar lavage fluid of patients with alveolar proteinosis, interstitial pneumonia / alveolar proteinosis and others The SP-D concentration in the serum of a patient with respiratory disease was measured.

(A) SP-D concentration in pregnancy amniotic fluid As a result of measuring SP-D concentration in 21 specimens of pregnancy amniotic fluid from 26 weeks to 40 weeks of pregnancy, in the amniotic fluid of more than 30 weeks of pregnancy (13 cases),
The SP-D concentration clearly showed a higher value than amniotic fluid (8 cases) less than a week (FIG. 8). This indicates that the SP-D concentration in amniotic fluid reflects the maturation of the fetal lung, and that the measurement of the SP-D concentration in amniotic fluid may be useful for diagnosis of a fetus with IRDS.

(B) SP-D concentration in bronchoalveolar lavage fluid Alveolar proteinosis patients (13 cases), idiopathic pulmonary fibrosis (IPF),
As a result of measuring SP-D concentration in bronchoalveolar lavage fluid of sarcoidosis (Sar) and healthy subjects (13 cases), SP-D concentration is clearly higher in patients with alveolar proteinosis than in healthy subjects. As shown (FIG. 9). This indicated that the measurement of SP-D concentration in bronchoalveolar lavage fluid may be useful for diagnosis of patients with alveolar proteinosis. The values of IPF and Sar were almost the same as those of healthy subjects.

(C) SP-D concentration in serum of patients with various respiratory diseases Idiopathic interstitial pneumonia, interstitial pneumonia with collagen disease, alveolar proteinosis, lung squamous cell carcinoma, lung adenocarcinoma, small cell lung cancer Serum SP-D levels were measured in patients with various respiratory diseases such as sarcoidosis, pulmonary tuberculosis, emphysema, pneumonia, bronchial asthma, bronchiectasis, panbronchiolitis, and healthy subjects as controls. As a result, patients with interstitial pneumonia such as idiopathic interstitial pneumonia and interstitial pneumonia with collagen disease and alveolar proteinosis showed clearly higher values than those in healthy subjects (FIGS. 10 and 11). This indicates that measurement of serum SP-D concentration is useful for differential diagnosis of the above various diseases.

Example 6 Immunohistological Staining Lung cancer tissues (adenocarcinoma, squamous cell carcinoma, large cell carcinoma, small cell carcinoma) and other organ carcinoma tissues obtained by surgical excision and necropsy were fixed in formalin, embedded in paraffin, and monoclonal antibodies were used. The immunohistological examination by the ABC method used was performed.

That is, the tissue section was sufficiently deparaffinized using xylene, hydrated by gradually changing the ethanol concentration, and washed with water. Next, endogenous peroxidase activity was removed by soaking in methanol containing 0.3% hydrogen peroxide for 30 minutes at room temperature, followed by washing by soaking in PBS for 5 minutes, and this washing was repeated three times. Next contains 10% horse serum
After blocking by immersing in PBS for 30 minutes at room temperature, monoclonal antibody human SP-D appropriately diluted with PBS
Drop the antibody or monoclonal anti-human SP-A antibody,
After reacting at room temperature for 30 minutes, it was washed similarly with PBS. Next, a biotinylated anti-mouse IgG antibody (manufactured by Vector) was added dropwise, reacted at room temperature for 30 minutes, washed in the same manner with PBS, and then ABC reagent (manufactured by Vector) was added dropwise and reacted at room temperature for 30 minutes. I let it. After washing three times with PBS, a peroxidase substrate solution (manufactured by Vector) is added dropwise and reacted at room temperature.
When the color develops appropriately, wash with water to stop the color reaction,
Sections were mounted.

As a result, SP-D was positive in 25 out of 36 cases of lung adenocarcinoma and 4 out of 5 cases of lung squamous cell carcinoma, respectively, and was negative in all other types of lung cancer and other organ cancers. . Regarding SP-A, which was performed as a reference, 18 of 36 cases of lung adenocarcinoma were positive, and 31 of 36 cases where at least one of SP-D and SP-A was positive. From this, SP-D
The use of a combination of antibodies against SP-A and SP-A improved the positive rate, confirming that it is more useful in diagnosing lung adenocarcinoma. In addition, monoclonal anti-human SP-
Typical examples of immunohistochemical staining of lung tissue derived from lung adenocarcinoma and lung squamous cell carcinoma using the D antibody (6B2) are shown in FIGS. 12 and 13.

INDUSTRIAL APPLICABILITY The monoclonal antibody of the present invention specifically binds to human lung surfactant apoprotein D. By using such a monoclonal antibody as an antibody reagent, human monoclonal surfactant apoprotein D can be specifically identified. For the first time, it has become possible to detect or measure it.
For this reason, the monoclonal antibody of the present invention is useful as one tool for elucidating the function of human lung surfactant apoprotein D, and the detection method or measurement method of the present invention and kits used therefor are IRDS, ARDS, pulmonary Vesicle proteinosis,
It seems to be useful for the diagnosis of respiratory diseases such as interstitial pneumonia, lung adenocarcinoma, and lung squamous cell carcinoma.

──────────────────────────────────────────────────続 き Continuation of the front page (72) Shosaku Abe, Inventor 5-4-1, Shinkawa Sanjo, Kita-ku, Sapporo, Hokkaido (56) References JP-A-61-277699 (JP, A) JP-A-62-64956 (JP) , A) JP-A-4-9665 (JP, A) Arch. Biophys 290 [1] (1991) p. 116-126 (58) Field surveyed (Int. Cl. ⁶ , DB name) WPI (DIALOG) BIOSIS (DIALOG)

Claims (17)

(57) [Claims] 1. A monoclonal antibody that specifically binds to human lung surfactant apoprotein D, having the following characteristics: Specificity: Human lung surfactant is substantially non-reactive with other human-derived antigenic substances. Reacts only with apoprotein D; Reactive Reacts with human lung surfactant apoprotein D depending on the concentration of antibody; Alternating human lung surfactant apoproteins A, B and C of human origin
Does not substantially react with pulmonary surfactant apoprotein D from rat. 2. A monoclonal antibody according to claim 1 and an antigen-antibody reaction of human lung surfactant apoprotein D in a biological sample, and human lung surfactant apoprotein D is measured based on the obtained reaction product. A method for measuring human lung surfactant apoprotein D. 3. The method according to claim 2, wherein the biological sample is amniotic fluid, bronchoalveolar lavage fluid, blood, serum, or plasma. 4. The method according to claim 2, for determining a respiratory disease. 5. The method according to claim 4, wherein the respiratory disease is IRDS, ARDS, alveolar proteinosis, or interstitial pneumonia. 6. The method according to claim 2, wherein the biological sample is serum and the determination is for interstitial pneumonia. 7. The method according to claim 2, wherein the biological sample is serum, and the method is used for judging idiopathic interstitial pneumonia or interstitial pneumonia with collagen disease. 8. Reacting the monoclonal antibody according to claim 1 with a biological sample derived from human lung tissue, and detecting the presence of human lung surfactant apoprotein D in human lung tissue based on the obtained reaction product. A method for detecting human lung surfactant apoprotein D, comprising: 9. The detection method according to claim 8, wherein the biological sample is lung tissue. 10. The detection method according to claim 8, which is for judging a respiratory disease. 11. The detection method according to claim 10, wherein the respiratory disease is lung adenocarcinoma or lung squamous cell carcinoma. 12. A kit for measuring human lung surfactant apoprotein D, comprising the monoclonal antibody according to claim 1 as one constituent reagent. 13. The kit according to claim 12, for determining a respiratory disease. (14) The respiratory disease is IRDS, ARDS, alveolar proteinosis,
14. The kit according to claim 13, wherein the kit is interstitial pneumonia. 15. A kit for detecting the presence of human lung surfactant apoprotein D in human lung tissue, comprising the monoclonal antibody according to claim 1 as one constituent reagent. 16. The kit according to claim 15, for determining a respiratory disease. 17. The kit according to claim 16, wherein the respiratory disease is lung adenocarcinoma or lung squamous cell carcinoma.