JPS6264956A - Method for detecting lung surface active substance and reagent kit therefor - Google Patents

Abstract

PURPOSE:To detect the presence of the lung surface active substance in human lung tissue, by acting monocronal primary antibody on lung tissue and treating the same with a biotinized secondary antibody before acting enzyme labelled avidine thereon. CONSTITUTION:About 90% of a lung surface active substance is lipid such as phospholipid or neutral lipid and about 10% thereof is protein. when lipid is removed from the lung surface active substance, water-insoluble protein called as apoprotein is obtained. Because this protein is excellent in specifity as compared with phospholipid and can be detected with high sensitivity, it is suitable as the marker of the lung surface active substance. A monocronal antibody is one specifically recognizing said apoprotein. Hereupon, a monocronal primary antibody recognizing the apoprotein of the lung surface active substance is acted on lung tissue at first and, subsequently, this lung tissue is treated with the biotinized secondary antibody recognizing the primary antibody and, thereafter, enzyme labelled avidine is acted thereon. By this method, the presence of the lung surface active substance in human lung tissue can be detected by an enzyme-antibody method.

Description

Detailed Description of the Invention (a) Industrial Application Field The present invention relates to a method for detecting the presence of pulmonary surfactant in human lung tissue by an enzyme antibody method using a monoclonal antibody, and a reagent used for the method. It's about the kit.

(0) Conventional techniques and problems Diagnostic methods using monoclonal antibodies in the clinical field include:
In addition to serological diagnosis and image diagnosis using isotope-labeled antibodies, there is immunohistological diagnosis.

The use of tissue-specific markers in immunohistodiagnosis can be used, for example, in cases where tumor metastasis is discovered but its primary source is unknown, or when the origin of an ift tumor is unknown. It may be valid. It may also be effective in diagnosing the cause of stillborn babies (for example, suffocation or IRDS) and the forensic cause of death (for example, heart disease or lung disease).

Conventionally, polyclonal antibodies commonly used for immunohistochemistry include antibodies against various hormones, immunoglobulin, prostate-specific acid phosphatase, myoglobin, and the like. However, many of these tissue-specific antigens are tissue and cell differentiation antigens, so l! Because dedifferentiation occurs as the tumor becomes malignant, tumors that are histopathologically hypoplastic and difficult to differentially diagnose tend to exhibit less antigenicity. Therefore, it has been desired to produce monoclonal antibodies against organ- or tissue-specific antigens that can remain even after dedifferentiation and reduced differentiation antigenicity.

(c) Means for solving the problem The present inventors have discovered that pulmonary surfactants accumulate significantly differently in the lungs.
We isolated and purified apoprotein, a pulmonary surfactant, from the bronchial lavage fluid of patients with proteinosis, produced two types of monoclonal antibodies specific to the apoprotein, and investigated immunoassay methods using these. . As a result, it was found that the two heptagonal 9-nal antibodies according to the present invention are specific to apoprotein, a human lung surfactant, and are extremely useful as reagents for immunohistodiagnosis. Reached.

That is, the present invention provides a method for detecting the presence of lung surfactant in human lung tissue using an enzyme antibody method, in which: (1) a monoclonal primary antibody that recognizes the apoprotein of lung surfactant is applied to the lung tissue; and then ('2J
A method characterized in that the lung tissue is treated with a biotinylated secondary antibody that recognizes the primary antibody, and then +3) wI-labeled avidin is applied, or in the same method, (1) the lung surface is This method is characterized by reacting with a monoclonal biotinylated primary antibody that recognizes the active substance apoprotein, and then (2) reacting with an enzyme [1 avidin].

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, riboproteins. When lipids are removed from lung surfactant, a water-insoluble protein is obtained, which is called apoprotein, and has a molecular weight of approximately 36,000.
The main component is 0 (36K) glycoprotein. Compared to phospholipids, proteins have superior specificity and can be detected with high sensitivity, making them suitable as markers for lung surfactant substances. The monoclonal antibody used in the present invention is an antibody that specifically recognizes this apoprotein.

Among the monoclonal antibodies included in the present invention, particularly preferred in terms of reactivity and safety is Molecular Injury Ladle 62,000.
and/or an antibody that recognizes apoprotein of about 34°OOO to 37,000, and a mouse antibody of the 10G2b type is preferred.

The monoclonal antibody of the present invention is preferably produced by fusing myeloma cells with antibody-producing cells of an animal immunized with the apoprotein of human lung surfactant, thereby producing a monoclonal antibody that recognizes the apoprotein. It is obtained by obtaining a hybridoma, then culturing the hybridoma and/or a cell line derived therefrom, and collecting a monoclonal antibody that recognizes the apoprotein of human lung surfactant from the culture.

The pulmonary surfactant in the present invention is separated and collected from human lung and/or bronchial lavage fluid, preferably from bronchial lavage fluid from patients with alveolar proteinosis.

Pulmonary surfactant is a complex of about 90% lipid and about 10% protein (lipoprotein), and it can be obtained by known methods such as the method of Frosolono (J, LipidR (2003)).
! S, 11. 439-457 (1970)) to remove pulmonary surfactant by 1!7 and then remove lipids, the apoprotein in the present invention is reduced to 19. The main components of apoprotein are proteins with molecular ω of approximately 62,000 and approximately 36,000, but one protein with a molecular weight of approximately 36,000 is widely recognized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Separate as a band,
It is thought that this contains approximately 37,000 molecules and approximately 34,000 proteins. Therefore, apoprotein in the present invention refers to these glycoproteins and all other apoproteins or fragments thereof. In addition, the molecular size of the protein was measured by 5DS-PAGE.

Hybridomas that produce monoclonal antibodies that recognize apoproteins are created using a well-known cell fusion method! It is possible to build J. First, apoproteins are found in monkeys, horses, cows, goats, and sheep.

We immunized animals such as Tsubogi, rats, and mice, and then produced 111ff of antibodies from the spleens and lymph nodes of these animals.
1 (lymphocytes) are collected and the cells are fused with human or animal myeloma cells.゛Myeloma fJ
As Il)d, it is convenient to use mouse miniroma 1ll1 cells; examples of these include BAL B
/C mouse P3-X63-Ag8. P3-X63-
Ag8-Ul, P3-NS1/1-Ag4-1.
P3-X63-△! 118-6.5.3.5P210
-i14, FO, MPC11-45,6TG 1.
7 etc.

Conditions for cell fusion are, for example, as follows. Antibody-producing cells and mini 0-ma cells are mixed at a ratio of 10:1 to 1 to 10°, preferably 1:1 to 1:3, and mixed with a suitable cell fusion solution, such as about 35% polyethylene glycol (molecule B i
, ooo ~ 6,000) and RPM I 16/10 containing about 1.5% dimethyl sulfoxide;
Stir for 1 to several minutes at room temperature to 37°C, then add 10% FC8
After gradual dilution with RPM I 1640 and washing, HA
Adjust the cell concentration to 1 to 5 x 105 cells/lll1 using T (hyboxanthin-aminobuterin-dimidine) selective culture medium. Add this in 0.2ae increments, for example 9
Dispense into 6-well plates and incubate for 35 minutes in air containing 5% CO2.
Culture in the dark for 2-3 weeks at ~38°C.

In the 1-IAT culture solution, only hybridomas exist, and 8-azaguanine-resistant myeloma am and myeloma-fused cells cannot survive (unfused antibody-producing cells die within a few days). Next, from this hybridoma population, only those that secrete monoclonal antibodies specific to apoprotein are selected.

This selection step (screening) can be carried out by examining whether the monoclonal antibodies produced by each hybridoma undergo an antigen-antibody reaction with the target 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 limit dilution.

Approximately 2 to 3 weeks later, the colonies grown in the 96-well plate 1 were picked up, and the antibody activity against the apoprotein was again examined using the enzyme antibody method. Generate antibodies.

Another method for obtaining monoclonal antibodies is to infect antibody-producing cells with Epstein-Barr virus (Eps).
tein-B arr V 1rus (hereinafter abbreviated as E-8 virus) to create transformed g+ cells, the transformed 411111 and/or cell lines derived therefrom were cultured, and the apoprotein was extracted from the culture. This is a method for collecting monoclonal antibodies that have binding properties.

The E-B virus is a member of the herpes virus group, which is thought to cause Burkitt's lymphoma and pharyngeal cancer.
It is a virus that does Antibody-producing cells are infected with E-8 virus and cultured for about 2 to 3 weeks in a 5% CO2 incubator to form transformed cells (l-transformed cells) consisting of many different N* cells. Next, this transformation! ! From the 2 cells, only those that secrete monoclonal antibodies specific to apoprotein are selected in the same manner as described above.

Then, cloned transformed cells can be obtained in the same manner as described above.

Next, in the present invention, the selected hybridomas or transformed cells are cultured to produce the desired specific L-plople antibody. selected by cloning,
Hybridomas or transformed hybridomas that produce antibodies that recognize the apoprotein of lung surfactant can be frozen and preserved, and can also be cultured into large seedlings by an appropriate method.

A monoclonal antibody that specifically binds to apoprotein can then be obtained from the culture supernatant. Alternatively, such cells can be transplanted into a primary tumor to form a tumor, and the desired antibody can be obtained from the ascites or serum.

Purification of the monoclonal antibody of the present invention is carried out by methods such as affinity chromatography using protein A and the like.

In the present invention, two types of monoclonal antibodies PC6 and PE10, which recognize the apoprotein of the actual pulmonary surfactant, were obtained by the method described above, and were monoclonal antibodies that recognized the antigenic site.

In the present invention, such monoclonal antibodies are used as primary antibodies. There are no restrictions on the type of secondary antibody that recognizes the primary antibody and the method for producing it. As the biotinylated secondary antibody, various commercially available products can be used.

In the present invention, lung tissue is treated with a primary antibody, then treated with a biotinylated secondary antibody, and then treated with a known enzyme-labeled avidin.

Avidin is a basic protein with about 68,000 molecules present in egg white, and is known to have extremely high affinity (affinity constant 10 M-1) with biotin, also known as vitamin H. Avidin is known to be composed of four subunits, and the avidin of the present invention also includes these subunits.

Examples of enzyme labeling agents include horseradish bar oxidase, β-D-galactosidase, and alkaline phosphatase. The enzyme-labeled avidin in the present invention includes:
It also includes the so-called ABC complex (avidin with a tetravalent reactive group and HR biotinylated at multiple sites).
A lattice-shaped ABC complex that is obtained by mixing P in an appropriate ratio in advance, contains a large number of HRP, and leaves some unreacted avidin groups. Addition of a substrate is necessary to measure the activity of the enzyme labeling agent. As a substrate, for example, horseradish peroxidase (HRP)
2,2' Azimedi-13-ethylbenzthiazoline sulfonosalicyl M-H202,0-phenylenediamine-1'202,4-aminoantipyrine-8202 and the like. Examples of substrates for β-D-galactosidase include fluorescein-d (β-D-galactopyranoside) and O-nitrophenol-β-D-galactopyranoside.

In addition to these reagents, a lysis agent is also required for measurement.

Known reagents such as detergent 1 reaction terminator are used.

The scope of the present invention also includes kits containing the antibodies and reagents described above. A preferred example of this is a reagent kit for detecting the presence of pulmonary surfactant in human lung tissue by enzyme-linked immunosorbent assay, comprising (1)
) a monoclonal primary antibody that recognizes the apoprotein of human lung surfactant, (2) a biotinylated secondary antibody that recognizes the primary antibody, (3) enzyme-labeled avidin, and (4) a substrate for the enzyme. A reagent kit comprising the main components, or (1) recognizing apoprotein of human lung surfactant;
This is a reagent kit whose main components are a monoclonal exposed primary antibody, (2) enzyme-labeled avidin, and (3) a substrate for the enzyme.

(2) Reference example (1) For the purpose of treatment, the lungs and bronchi of a patient with alveolar proteinosis were washed with a 0.15 M sodium chloride solution, and bronchopulmonary lavage fluid (BALF) was washed with 3J21? Ta. This BALF
were centrifuged at 300 x g for 10 minutes to remove cells and cell debris. The resulting supernatant was then heated at 48,000x
The mixture was centrifuged at 100 g for 20 minutes, and the submerged fraction was collected.

This precipitate fraction was treated with 145111M sodium chloride, 1
10 containing mM disodium ethylenediaminetetraacetate.
The suspension was suspended in 111 M Tris buffer (ph 7.4) for 80 d, and centrifugation was performed using a discontinuous sucrose density gradient of 0.25 M and 0.65 M at 40,000× for 60 minutes.

18 fractions of 0.25 M and 0.65 M sucrose solutions were collected and added to the above buffer 400 #li! resuspended in. This suspension was then centrifuged at 48,000 x g for 30 minutes to obtain a precipitate (lung surfactant).

The precipitate was dissolved in 5 mM Tris buffer (pH 7) containing 1% Triton ,8,
(hereinafter referred to as 1 to lydone buffer solution) was gently dispersed in 27 m. This dispersion was centrifuged at 150,000 x g for 60 minutes to obtain a precipitate.

This precipitate was redispersed in the above Triton buffer solution 4-, and 4 sl. Butanol-ethanol mixture (volume ratio 6:1) was added, shaken vigorously, and left at 0°C for 10 minutes to extract and remove lipids.
om, for 15 minutes to obtain a precipitate (apoprotein). This operation was repeated three more times to obtain the final apoprotein precipitate.

Triton this sediment! ! ! The suspension was suspended in 20% of aqueous solution and dialyzed against the same Tri-N buffer using a cellophane membrane (to remove butanol-ethanol), to obtain a dialysis solution. This dialyzed fluid was centrifuged at 150,000×g for 60 minutes to obtain a supernatant. Add 20ml of Triton buffer to the precipitate again.
After solubilization, centrifugation was performed at 150,000 x g for 60 minutes to obtain a supernatant. This operation was repeated six more times and the supernatant was collected. The obtained supernatant was ringed and the volume was 150ml! Met. This supernatant was applied to a blue Sepharose column (diameter 1.8α x 3.5c11) equilibrated with Triton 1lili solution.
) and the same! 1W77J solution was used for elution, and the flow-through fraction was collected to remove contaminating albumin.

These two fractions were combined into a DEAE-Toyovar column (diameter 1.4cJRx 19cm) equilibrated with Triton buffer.
), first, the same! A flow rate of 15ad using 1 buffer solution of 200d! /hr, and then elute while linearly and continuously increasing the concentration of sodium chloride contained in the Ii buffer from 0 to 0.5M until the sodium chloride concentration was 0.30M and 0.35M. I got both parts between M. The molecular weights of the proteins contained in these two portions are approximately 62,000 and approximately 360.
00 and had a sugar chain.

(2 The above sodium chloride concentrations are 0.30 M and 0.3
The LS apoprotein contained in both fractions eluted between 5 M was used in the following experiment.

Emulsify LS apoprotein in Freund's complete adjuvant,
It was administered intraperitoneally to BALB/C mice.

The mice were then boosted 30 days later. on the other hand,
RPMI 1640 supplemented with 15% fetal bovine serum (
Gibco), myeloma cells P3-X63-A (+
8-LJI was maintained in culture. Three days after boosting, spleen cells obtained from mice and P3-X63-Ag8-
tJl by the method of Qi et al.
1ethOd3in cellular iIllm
unology 1980. Cells were fused using polyethylene glycol 4000 (see p. 351-372) and seeded in a 96-well microplate.

After cell fusion, the medium was supplemented with 100 μM hyboxanthin.

The medium was replaced with RPMI medium (HAT medium) supplemented with 0.4 μM aminopterin and 16 μM thymidine.

During 2 to 3 weeks of culture in HAT medium, only hybridomas of splenic cells and myeloma cells grew.

Antibody activity in the hybridoma culture solution was examined using the ELISA method described below. .

[Antibody screening] LS apoprotein was attached to an ELISA plate, and 1
3% (W) in 0 n+M phosphate saline (pH 7,4)
Blocking was performed with a solution to which BS8 of /V) was added. After blocking, add 50μ of hybridoma culture to the ELISA plate and incubate for 2 hours at room temperature or at 4°C.
Cultured overnight. Then, biotinylated Tuma anti-mouse Ig
G-Immunog Purine (2 μg/d) and 50 μl of secondary antibody were added and allowed to react at room temperature for 1 hour. To this, 50μ of horseradish peroxidase avidin D solution (1μ9/d
) and the antibody bound to LS apoprotein was added to 100 μl of substrate solution (0.1% 0-phenylenediamine).

0.015% H202, 0.1M citrate buffer,
Detection was performed by adding 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.These hybridomas were each administered pristane. BALB/C71 mice were grown in the peritoneal cavity to obtain ascites containing monoclonal antibodies. 50% saturated ammonium sulfate was added to the obtained ascites to precipitate the antibodies, and the precipitate was mixed with 0.1M phosphate saline (1). H8,0). After dialysis, Protein A-Sepharose CL4B column (
(manufactured by Pharmacia), and the antibody was diluted with 0.2M glycine.
'FR was prepared by elution with hydrochloric acid buffer (1) 83.0).

Monoclonal antibodies obtained from two types of hybridomas were named PO2 and PE10, respectively.

(4) Properties of monoclonal antibodies Western blotting
tting) method, PO2 and pE10 were determined using 3G
Both apoproteins at K and 62 were expressed. Additionally, both antibodies were present in human amniotic fluid and normal human bronchopulmonary lavage fluid at 37 and 34.
Apoprotein was recognized at K and 62. The protein in 36 is 5DS
- Separated as a broad band by PAGE, in which 37
Ni and 34 contain protein, so 36 contains protein and 37K.

The protein in 34 is the same protein. PO2 and P E 10
is the dot-immune binding (dat-iIl+a+uno
The results of cross-reaction using the binding method showed that different epitopes (antigenic sites) that are close to each other are recognized. These antibodies are specific to the apoprotein of human lung surfactant, and do not react with the apoprotein of lung surfactant of animals such as rats, pigs, and rabbits, nor do they react with human serum proteins. There wasn't.

Furthermore, the subclass of 1gG was 10G2b for both PO2 and PE10.

[Western blotting method] The antigen specific to the monoclonal antibody is
Methods such as n (Pro, N, A, S, 76 43
50-4354) using the Western blotting method. First, the antigen bearing lung surfactant apoprotein was subjected to 5DS-PAGE. 5O8-PA
Post-GE electrolyte buffer was 25 sM Tris-WR.

pt-18,3,192mM glycine, 20% (V
/V) methanol, and the protein was transferred from the slab (5lab) gel to the nitrocellulose sheet under conditions of a voltage gradient of 7V/α for 2 hours. Nitro separated each lane of the Lurose sheet. One of the sheets was subjected to protein staining with amido black, and the other was subjected to the following enzyme immunoassay.

After blocking with 3% (w/v) BSΔ/PBS, monoclonal antibody (PO2 or P E 1
0) was added. Biotinylated anti-mouse IOG immunoglobulin was added as a secondary antibody.

Each sheet contains 0.05% (v/v) Tween (1w6e
n) Washed with PBS containing 20. After incubation with horseradish peroxidase Agogin D, the substrate solution (
0.05% diaminobenzidine.

0.03% fi202, 0.01 MPBS)
It was detected and identified by Wokaelco.

(5) Preparation of biotinylated monoclonal antibody Monoclonal antibody P E 10 of 1.0 Rg/II dl P
After the BS solution was dialyzed with a 0.1 M aqueous solution of liptrium carbonate, N-hydroxysuccinimide biotin (P 1 crce C
1. Oay/m1! A biotinylated monoclonal antibody was obtained by adding and mixing 1 volume of dimethyl sulfoxide solution, incubating at room temperature for 4 hours, and then dialyzing against 50 mM PBS solution.

Note that commercially available products can be used for biotinylated secondary antibodies such as biotinylated sheep anti-heron IgG antibody and enzyme-labeled avidin such as HRP-labeled avidin and ABC complex.

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

(Wolf) Example 1 Immunohistological study of the involvement of alveolar ■-type cells (pulmonary surfactant-producing cells) in lung adenocarcinoma.

Fifteen cases of lung adenocarcinoma were selected from autopsy cases of lung cancer, and these tissues were treated with the monoclonal antibody PE described in (3) of the reference example.
10, and was stained by the ABC method. That is, approximately 400 mm of formalin-fixed paraffin section tissue was
0 saline solution 0. IIIl (concentration 2.4μg/
d) was added, and then, when the antibody was recognized, it was treated with a Δtin secondary compound (commercially available kit), and then ABC complex (commercially available kit) was allowed to act for 30 minutes at room temperature.

As a control, lung squamous cell carcinoma and metastatic adenocarcinoma were stained at the same time.

As a result, 15 cases of lung adenocarcinoma (4 cases of tubular type, 11 cases of papillary type,
Among the bronchioloalveolar type (3 cases), most of the m tumor cells were positive (++): 3 cases, and some tumor cells were positive (+): 4 cases. Two of the three (++) cases were classified as bronchioloalveolar type, and one case was classified as classifier ductal type, but the area that did not show positive was papillary proliferation.

These three cases (→-+) involve alveolar ■-type cells and are judged to be adenocarcinomas showing differentiation into ■-type alveolar epithelium. The control group was negative, indicating that the method of the present invention is useful for elucidating the histological development of lung adenocarcinoma.

In the four (+) cases, it is possible that normal type ■ alveolar epithelium remained within the tumor, and its developmental origin could not be determined solely by the method of the present invention.

Example 2 Immunohistological examination of alveoli Immunohistological examination of various alveoli by the enzyme pile method was carried out in the same manner as in Example 1 using monoclonal antibodies PC6 or PE10.

The results showed clear positivity on the alveolar surface and within the alveolar ■-type cells of normal human adult lungs. However, neither antibody showed any reaction in the lungs of a 13-week-old fetus. Furthermore, no reaction with both antibodies was observed in the lungs of RDS deaths. From the above results, PO2 and P E +.

It is clear that it can be used as a marker for searching for vJfA, a pulmonary surfactant, in histopathology, and therefore for diagnosing the cause of death.

Claims (1)

[Scope of Claims] 1. A method for detecting the presence of lung surfactant in human lung tissue using an enzyme antibody method, which includes: A method characterized by treating the lung tissue with an antibody, then (2) treating the lung tissue with a biotinylated secondary antibody that recognizes the primary antibody, and then (3) treating the lung tissue with enzyme-labeled avidin. 2. In a method for detecting the presence of lung surfactant in human lung tissue using an enzyme antibody method, (1) a monoclonal biotinylated primary antibody that recognizes the apoprotein of lung surfactant is applied to the lung tissue; A method characterized by: (2) allowing enzyme-labeled avidin to act. 3. A reagent kit for detecting the presence of lung surfactant in human lung tissue using an enzyme-linked immunosorbent method, comprising: (1) a monoclonal primary antibody that recognizes the apoprotein of human lung surfactant; A reagent kit whose main components are 2) a biotinylated secondary antibody that recognizes the primary antibody, (3) enzyme-labeled avidin, and (4) a substrate for the enzyme. 4. The reagent kit according to claim 3, wherein the monoclonal antibody recognizes apoprotein having a molecular weight of about 62,000 and/or about 34,000 to 37,000. 5. The reagent kit according to claim 3 or 4, wherein the monoclonal antibody is a 1gG2b type mouse antibody. 6. A reagent kit for detecting the presence of lung surfactant in human lung tissue using an enzyme-linked antibody method, comprising: (1) a monoclonal biotinylated primary antibody that recognizes the apoprotein of human lung surfactant; A reagent kit whose main components are (2) enzyme-labeled avidin and (3) a substrate for the enzyme.