JPH0672894B2 - Method for measuring human lung surface-active substance and reagent kit used therefor - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Field of Industrial Application The present invention uses a monoclonal antibody against human lung surfactant apoprotein, and a method for measuring human lung surfactant. The present invention relates to a reagent kit.

(B) Conventional Technology In the alveoli of animals, there is a physiologically active substance containing phospholipids as a main component, which is called a lung surface active substance. It covers the inner wall of the alveoli, has a protective effect on the alveolar epithelium, and has important physiological potential for maintaining the respiratory function of animals. In other words, lung surface-active substances have a unique surface activity such as changing the surface tension of the inner surface of the alveoli during exhalation and inspiration, contributing to the stability of the alveoli and contributing to anti-atelectasis. It is said to exhibit action. If the lung surfactant is deficient, the alveoli collapse and the stable ventilation capacity cannot be maintained. For example, symptoms such as neonatal respiratory distress syndrome (IRDS) appear.

To prevent the birth of a newborn baby with such a disease,
When the amount of lung surfactant in amniotic fluid, which is associated with the maturity of fetal lung, is measured, and the fetus is trying to be born in an immature lung, for example, steroid administration promotes secretion of lung surfactant, Intrauterine treatment is possible.

Then, conventionally, several methods have been proposed as methods for measuring or estimating the amount of lung surface-active substances in amniotic fluid. For example, the L / S ratio in amniotic fluid (the ratio of lecithin to sphingomyelin) and the amount of dipalmitoylphosphatidylcholine (DPPC) in amniotic fluid have been measured as markers for lung surface-active substances. It does not measure phospholipids, which are the main component of the active substance, and has the drawback of low correlation with IRDS, and the latter method has the problem of poor sensitivity. By the way, about 90% of lung surface-active substances are lipids such as phospholipids and neutral lipids, but about 10%
Are proteins, and these exist as a complex of lipid and protein, that is, lipoprotein. When lipids are removed from lung surface-active substances, a water-insoluble protein is obtained, which is called apoprotein, which is mainly composed of a protein having a molecular weight of about 36,000 (36K). Since proteins have superior specificity and can be detected with high sensitivity as compared with phospholipids, the use of proteins as markers for lung surface-active substances has also been investigated, and immunological quantification using so-called polyclonal antibodies has been performed. However, the method using a polyclonal antibody has problems that it takes a long time for measurement and the sensitivity is not sufficient.

(C) Problems to be Solved by the Invention In order to solve the above problems and to measure a minute amount of a pulmonary surface-active substance present in a sample such as amniotic fluid with high sensitivity in a short time, a pulmonary surface-active substance is required. Although it is necessary to obtain a monoclonal antibody against the apoprotein that constitutes E. coli, no such monoclonal antibody has been obtained to date.

(D) Means for Solving the Problems The present inventors have found that the molecular weight of lung surfactant of about 62,000 and / or about 34,000 is obtained from bronchoalveolar lavage fluid of patients with alveolar proteinosis in which a large amount of lung surfactant is accumulated. ~ 37,000 apoproteins were separated and purified, two types of monoclonal antibodies specific to the apoproteins were prepared, and immunological assay methods were examined using these. As a result, the inventors have found that the two monoclonal antibodies according to the present invention are extremely useful as reagents for measuring lung surface-active substances, and arrived at the present invention.

That is, the present invention provides a method for measuring human lung surfactant in a sample by an immunological method, wherein the molecular weight of the human lung surfactant is about 62,000 and / or about 34,000-3.
In the specimen, a first monoclonal antibody that recognizes 7,000 apoproteins and a second monoclonal antibody that recognizes the apoproteins but binds to an antigenic site different from the first monoclonal antibody are used. Is a method for measuring human lung surfactant. Among the monoclonal antibodies in the present invention, particularly preferable in terms of reactivity and stability,
An antibody that recognizes an apoprotein having a molecular weight of about 62,000 and / or about 34,000 to 37,000 is used. As the monoclonal antibody in the present invention, IgG type mouse antibody is preferable.

The monoclonal antibody of the present invention is preferably prepared by fusing myeloma cells with antibody-producing cells of an animal immunized with a human lung surfactant apoprotein having a molecular weight of about 62,000 and / or about 34,000 to 37,000. , A hybridoma producing a monoclonal antibody that recognizes the apoprotein is obtained, and then the hybridoma and / or a cell line derived from the hybridoma is cultured, and a monoclonal antibody that recognizes the human lung surface-active substance apoprotein is obtained from the culture. Obtained by collecting.

The lung surface-active substance in the present invention is separated and collected from human lung and / or bronchial lavage fluid, preferably bronchopulmonary lavage fluid of patients with alveolar proteinosis. Lung surfactant is
It is a complex of about 90% lipid and about 10% protein (lipoprotein), and the lung surface activity is determined by these known methods, for example, Frosolono's method (see J. Lipid Res. 11, 439-457 (1970)). Obtaining the substance and then removing the lipids gives the apoprotein according to the invention. Apoprotein has a molecular weight of about 62,000
, And a protein with a molecular weight of about 36,000 is the main component, but a protein with a molecular weight of about 36,000 is separated as a broad band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). It is thought to contain 34,000 proteins. Therefore, the apoprotein in the present invention means these proteins or their fragments. The molecular weight of the protein is SDS-PAG.
Measured by E.

A hybridoma producing a monoclonal antibody that recognizes an apoprotein can be produced by a cell fusion method known per se. First, monkeys with apoprotein,
Animals such as horses, cows, goats, sheep, rabbits, rats, and mice are immunized, and then antibody-producing cells (lymphocytes) are collected from the spleen, lymph nodes, etc. of these animals, and these cells are human or animal. Fused with myeloma cells.
It is convenient to use mouse myeloma cells as myeloma cells, examples of which include BALB / C mouse P3-X63-Ag8, P3-X63-Ag8-U1, P3-NS1 / 1-Ag4-1. , P3-
X63-Ag8 6.5.3, SP2 / 0-Ag14, FO, MPC 11-45.6TG 1.7 etc.

The conditions for cell fusion are as follows, for example. Antibody-producing cells and myeloma cells are 10: 1 to 1-10, preferably 1: 1 to 1:
Mix in a ratio of 3 and mix with a suitable cell fusion solution, e.g.
% Polyethylene glycol (Molecular weight: 1,000 to 6,000)
And RPMI 1640 containing about 7.5% dimethylsulfoxide and stirred at room temperature to 37 ° C for 1 to several minutes, then 10% FC.
The cells are gradually diluted with S-supplemented RPMI1640, washed, and then adjusted with a HAT (hypoxanthine-aminopterin-thymidine) selective culture medium to a cell concentration of 1 to 5 × 10 ⁵ cells / ml. this
0.2 ml each is dispensed into, for example, a 96-well plate and cultured in air containing 5% CO ₂ at 35 to 38 ° C. for 2 to 3 weeks. Only hybridomas exist in the HAT culture medium, and 8-azaguanine-resistant myeloma cells and fused cells of myeloma cells cannot exist (unfused antibody-producing cells die in a few days). Next, from this hybridoma colony, only those secreting a monoclonal antibody specific to the apoprotein are selected. This selection step (screening) can be carried out by investigating whether or not the monoclonal antibody produced by each hybridoma reacts with the target apoprotein by an antigen-antibody method by an enzyme-antibody method. The hybridoma secreting the desired monoclonal antibody must then be cloned into cloned cells. This step can be carried out specifically by using the limiting dilution method. After about 2 to 3 weeks, colonies grown in a 96-well plate were picked up, and the hybridomas selected by investigating the antibody activity against apoprotein by the enzyme antibody method were cultured again to obtain a monoclonal antibody specific to the desired apoprotein. To generate.

Another method for obtaining monoclonal antibodies is to use Epstein-Barr virus (Epstein-Barr virus) in antibody-producing cells.
-Barr Virus, hereinafter abbreviated as EB virus) to produce a transformed cell, culturing the transformed cell and / or a cell line derived from the transformed cell, and determining the property of binding to apoprotein from the culture. This is a method of collecting the monoclonal antibody possessed.

E-B virus is a virus belonging to the herpesvirus group, which is said to be a causative virus for Burkitt lymphoma and nasopharyngeal cancer. The antibody-producing cells are infected with E-B virus and cultured in a 5% CO ₂ incubator for about 2 to 3 weeks to form transformed cells composed of many heterogeneous colonies. Next, from the transformed cells, only those secreting a monoclonal antibody specific to the apoprotein are selected by the same method as described above. Then, in the same manner as described above, a cloned transformed cell can be obtained.

Next, in the present invention, the selected hybridoma or transformed cell is cultured to produce a desired specific monoclonal antibody. Selected by cloning,
The hybridoma or transformant producing an antibody that recognizes the lung surfactant apoprotein can be frozen and stored, or it can be cultured in a large amount by an appropriate method. Then, a monoclonal antibody that specifically binds to apoprotein can be obtained from the culture supernatant. It is also possible to transplant such cells into an animal to form a tumor and obtain the desired antibody from the ascites or serum. Purification of the monoclonal antibody in the present invention is carried out by a method such as affinity chromatography using protein A or the like.

In the present invention, two types of monoclonal antibodies PC6 and PE10 that recognize the human lung surface-active substance apoprotein were obtained by the method described above. These were monoclonal antibodies that recognize different antigenic sites of apoprotein.

Therefore, a solid-phase enzyme-linked immunosorbent assay (ELISA) using a sandwich of both antibodies became possible.

In the examination of the ELISA method, the combination of solid phase CP6 and biotinylated PE10 was able to measure apoprotein most sensitively. Lung apoprotein of each animal species was measured using this sandwich ELISA method. As a result, only human lung and human amniotic fluid were measurable, and none was measured in other animal species lung and human serum. That is, it was revealed that both antibodies are specific to human lung and amniotic fluid, and that the apoprotein of the present invention is not derived from serum.

In addition, a two-site simulta neous i
muanoassay), that is, by allowing the first monoclonal antibody (primary antibody) immobilized on the microplate, the antigen (apoprotein), and the biotinylated second monoclonal antibody (secondary antibody) to react simultaneously, It could be shortened. Moreover, the background could be markedly reduced by using skim milk (skimmed milk) as the blocking agent.

In the present invention, it is preferable to immobilize the primary antibody on a carrier, but a known method can be used for immobilization.
As the carrier, solid phase, for example, balls, beads, gears and microplates using polystyrene, polyethylene, polyacrylate, Teflon, polyacetal and the like are preferably used.

Further, the labeling method and means, its detection method and means are not limited at all, known methods and means, for example, anti-immunoglobulin antibodies or grapes labeled with radioactive substances or enzymes or fluorescent substances. It can be measured by a secondary reaction with coccus protein A. As a labeling agent, in a method using an enzyme (EIA), an enzyme such as horseradish peroxidase, β-D-galactosidase, alkaline phosphatase, etc. is used in a method using a radioactive substance (RI.
In A), ¹²⁵ I, ³ H, etc. are usually used, and in the method using a fluorescent substance (FIA), fluorescein isothiocyanate, etc. are usually used, but if the activity of the labeling agent can be measured, other It may be one.

When the labeling agent is an enzyme, the substrate is used to measure its activity. Substrates for example, as a substrate for horseradish peroxidase, 2,2 'Ajinoji [3 ethylbenzthiazoline sulfonic acid] ammonium acid _{(ABTS) -H 2 O 2,} 5- aminosalicylic acid -H ₂ O _2, O
- phenylenediamine -H ₂ O _2, 4- aminoantipyrine -
H ₂ O _{2 and the} like, as a substrate for β-D-galactosidase,
Examples thereof include fluorescein-di- (β-D-galactopyranoside) and O-nitrophenol-β-D-galactopyranoside. In addition to these reagents, known reagents such as a lysing agent, a cleaning agent, and a reaction terminator are used for the measurement.

In the present invention, a combination of a biotinylated antibody and an enzyme-labeled avidin is preferably used. Avidin is a basic glycoprotein having a molecular weight of about 68,000 existing in egg white, and is known to have an extremely high affinity (affinity constant 10 ¹⁵ M ⁻¹ ) with biotin known as vitamin H. Although avidin is known to be composed of four subunits, the avidin of the present invention also includes these subunits.

The present invention also includes within its scope the kits of the above-described antibodies and reagents. This preferred example includes (1) a first monoclonal antibody (1) immobilized on a carrier, which recognizes human lung surfactant apoprotein.
Secondary antibody), (2) a labeled second monoclonal antibody (secondary antibody) that recognizes the apoprotein but binds to an antigenic site different from the primary antibody, and (3) if desired, A reagent kit for measuring a lung surface-active substance present in human amniotic fluid, human lung, or bronchial lavage fluid by an immunological method, which comprises a labeled antibody detection reagent as a main component.

(E) Effect of the Invention According to this method, the amount of apoprotein in lung surfactant is 10-640 ng /
It can be measured in the range of ml, and the coefficient of variation (Variation coeffici
ent) was 6% or less. Human amniotic fluid is 0.2 by this method.
It can be measured in ml. According to this method, as shown in Table 1 below, the apoprotein concentration before 30 weeks gestation was very low, but it was 6.5 times this value between 34 and 36 weeks, and after 37 weeks. It increased by 15.5 times. That is, the apoprotein concentration of lung surfactant in amniotic fluid increases dramatically before delivery. And the pattern of amniotic fluid apoprotein in this pregnancy is
It was in good agreement with the pattern of amniotic fluid phospholipid (L / S ratio, DPPC) increase in embryonic period.

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

Example 1 (1) The lungs and bronchi of a patient with alveolar proteinosis were washed with 0.15 M sodium chloride solution for the purpose of treatment to obtain 3 bronchopulmonary lavage fluids (BALF). The BALF was centrifuged at 300 × g for 10 minutes to remove cells and cell debris. Then, the obtained supernatant was centrifuged at 48,000 × g for 20 minutes to collect a sediment fraction.

This sediment fraction was suspended in 80 ml of 10 mM Tris buffer (ph7.4) containing 145 mM sodium chloride and 1 mM ethylenediaminetetraacetate and centrifuged by a discontinuous sucrose density gradient of 0.25M and 0.65M. , 40,000 × g for 60 minutes.

IB fractions of 0.25 M and 0.65 M sucrose solutions were collected and resuspended in 400 ml of the above buffer. And this suspension, 48000 ×
After centrifugation at g for 30 minutes, a precipitate (lung surface-active substance) was obtained.

1% of this precipitate to remove impurities such as albumin
Triton X-100 (polyoxyethylene alkyl phenyl ether), 3 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol in 5 mM Tris buffer (pH 7.8, hereinafter referred to as Triton buffer) 27 ml
Gently dispersed in. This dispersion was centrifuged at 150,000 × g for 60 minutes to obtain a precipitate (purified lung surface-active substance).

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. Then, the lipid was extracted and removed, and then centrifuged at 2000 rpm for 15 minutes to obtain a precipitate (apoprotein). Repeat this operation 3
Repeated times to obtain the final precipitate of apoprotein.

This precipitate was suspended in 20 ml of Triton buffer and dialyzed against the same Triton buffer using a cellophane membrane (removal of butanol ethanol) to obtain a dialysate. The dialyzed solution was centrifuged at 150,000 × g for 60 minutes to obtain a supernatant. After adding 20 ml of Triton buffer to the precipitate to solubilize it,
Centrifugation at 0000 xg for 60 minutes gave a supernatant. This operation was repeated 6 times and the supernatant was collected. The total of the obtained supernatants is 15
It was 0 ml. This supernatant was added to a Blue Sepharose 4B column (diameter 1.8 cm x 3.5 cm) equilibrated with Triton buffer, eluted with the same buffer, and the mixed fraction was collected by collecting the flow-through fraction. Completely removed.

This fraction was added to a DEAE-Toyopearl column (diameter 1.4 cm × 19 cm) equilibrated with Triton buffer, and first,
Elute with 200 ml of the same buffer at a flow rate of 15 ml / hr.
The concentration of sodium chloride contained in the same buffer should be 0 to 0.5M.
Elution was carried out while increasing linearly and continuously until a fraction with a sodium chloride concentration of 0.30 M and 0.35 M was obtained. The molecular weights of the proteins contained in this fraction were about 62000 and about 36000.

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

Emulsify LS apoprotein in Freund's complete adjuvant
It was intraperitoneally administered to LB / C mice. Then, 30 days later, the mice were boosted. On the other hand, with RPMI 1640 (manufactured by Gibco) supplemented with 15% fetal bovine serum, myeloma cells P3-X6
3-Ag8-U1 was maintained and cultured. Three days after the booster immunization, spleen cells obtained from the mouse and P3-X63-Ag8-U1 were treated with the method of Oi et al. (Selective methods in cellular immunology 1980,
Cell fusion was performed using polyethylene glycol 4000 according to p351-372, and seeded in a 96-well microplate. After cell fusion, the medium was replaced with RPMI medium (HAT medium) supplemented with 100 µM hypoxanthene, 0.4 µM aminopterin, and 16 µM thymidine. Only a hybridoma of spleen cells and myeloma cells grew within 2-3 weeks in the HAT medium. The antibody activity in the culture medium of the hybridoma was examined by the ELISA method described below.

[Screening of Antibody] LS apoprotein was attached to a plate for ELISA, and blocking was performed with a solution containing 3% (w / v) BSA in 10 mM phosphate saline (pH 7.4). After blocking, 50 μ of the hybridoma culture solution was added to the ELISA plate and cultured at room temperature for 2 hours or at 4 ° C. overnight. Then, biotinylated horse anti-mouse IgG immunoglobulin (2μg / ml) 50μ
Secondary antibody was added and reacted at room temperature for 1 hour.
μ horseradish peroxidase avidin D solution (1
μg / ml) and added the antibody bound to LS apoprotein to 100μ
Substrate solution (0.1% O-phenylenediamine, 0.015% H
_It was detected by adding ₂ O ₂ , 0.1 M citrate buffer, pH 4.6).

(3) Hybridomas producing an antibody against LS apoprotein were selected and further cloned by the limiting dilution method to finally obtain two kinds of single clone hybridomas. Each of the hybridomas was proliferated in the abdominal cavity of pristane-administered BALB / C mice to obtain ascites containing the monoclonal antibody. 50% saturated ammonium sulfate was added to the obtained ascites to precipitate the antibody, and the precipitate was dissolved in 0.1M phosphate saline (pH 8.0). And after dialysis, protein A
-Applied to a Sepharose CL4B column (Pharmacia), and the antibody was purified by elution with 0.2 M glycine-hydrochloric acid buffer (pH 3.0).

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

(4) Properties of Monoclonal Antibodies By Western blotting, PC6 and PE10 were added to the bronchoalveolar lavage fluid of patients with alveolar proteinosis.
It recognized both 36K and 62K apoproteins obtained from the B fraction.
Both antibodies recognized 37K, 34K and 62K apoproteins in human amniotic fluid and normal human bronchopulmonary lavage fluid. 36K protein is SD
It was separated by S-PAGE as a wide band, and 37K and 34
Since it contains K protein, 36K protein and 37K, 34K protein are the same protein. PC6 and PE10 are linked by dot-immuno binding.
The results of the cross-reaction by the binding method showed that they recognize different epitopes (antigen sites) that are close to each other. These antibodies are specific to the human lung surface active substance apoprotein and do not react with the rat lung surface active substance apoprotein of rats, pigs, rabbits, etc., and also with human serum proteins. There wasn't.

[Western blotting method] The antigen specific to the monoclonal antibody was identified by the Western blotting method according to the method of Towbin et al. (Pro. NAS 76 4350-4354). First, the antigen having the lung surfactant apoprotein was subjected to SDS-PAGE. SDS-PA
After GE Electrolyte buffer is 25 mM Tris-HCl, pH 8.3,192 mM
Glycine, 20% (v / v) methanol, with a voltage ramp of 7V
The protein was transferred from the slab gel to the nitrocellulose sheet under the condition of / cm for 2 hours. Each lane of the nitrocellulose sheet was cut off. 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) BSA / PBS, a monoclonal antibody (PC6 or PE10) was added as a primary antibody. Biotinylated horse anti-mouse IgG immunoglobulin was added as a secondary antibody. Each sheet is 0.05% (v / v) Tween (Twee
n) Washed with PBS containing 20. After incubation with horseradish peroxidase avidin D, detection was performed by addition of a substrate solution (0.05% diaminobenzidine, 0.03% H ₂ O ₂ , 0.01MPBS) for identification.

Example 2 (1) Preparation of Insolubilized Monoclonal Antibody (Immobilization of Monoclonal Antibody on Carrier) Untreated Microtiter Plate (Dynatech Laborator)
ies INC), 10 wells of monoclonal antibody PC6 in each well
200 ml of 0.1 M sodium hydrogen carbonate aqueous solution of μg / ml was added, and the mixture was incubated at 20 ° C. overnight. Then, after removing the solution from each well by suction, 2% skim milk and 1%
PBS containing Triton X-100 (0.01% containing 0.85% NaCl)
M phosphate buffer, pH7.4) solution (hereinafter referred to as TX / SM / PBS solution) is added, and post-coating treatment is performed at room temperature for 30 minutes. After the treatment is completed, the microtiter plate is washed with the above solution, Stored at -20 ° C until use.

(2) Preparation of biotin-labeled monoclonal antibody Monoclonal antibody PE10 in 0.1 mg / ml in PBS was added to 0.1M.
After dialysis with an aqueous solution of sodium hydrogen carbonate, 1 volume of a 1.0 mg / ml dimethyl sulfoxide solution of N-hydroxysuccinimide biotin (manufactured by Pierce Chemicals) was added to 10 volumes of the monoclonal antibody, and the mixture was incubated at room temperature for 4 hours. Then, it was dialyzed against a 50 mM PBS solution to obtain a biotin-labeled monoclonal antibody.

(3) Quantification of lung surfactant apoprotein by two-point simultaneous immunoassay The microtiter plate on which the monoclonal antibody PC6 was immobilized was returned to room temperature and washed with TX / SM / PBS solution, and then purified lung surfactant 100 μl of a TX / SM / PBS solution containing 10 ng / ml to 640 ng / ml of apoprotein of was added to each well as a standard substance solution. Separately, a 2-256-fold diluted TX / SM / PBS solution of human amniotic fluid was used as a measurement sample.
0 μ was added to each well of another plate. Then, the biotin-labeled monoclonal antibody PE10 was added at 2 μg / ml.
TX / SM / PBS solution containing at a concentration of 100 μm was added to each well filled with the standard substance solution or the measurement sample solution, incubated at 37 ° C. for 90 minutes, and then the solution was removed by suction and then TX / Washed with SM / PBS solution.

Then, add avidin D (Vector Laboratories, IN) labeled with horseradish peroxidase (HRP) to each well.
C.) was diluted 1200 times with TX / SM / PBS solution to give a solution of 200μ
Each was added and incubated at room temperature for 20 minutes, the solution was suctioned off, and then washed with a PBS solution containing 1% Triton X-100.

Further, a substrate solution consisting of 0.1M citrate buffer (pH 4.6) containing 0.1% O-phenylenediamine and 0.015% H ₂ O ₂ was added to each well, and the mixture was reacted at room temperature for 30 minutes, and then 2M. The reaction was stopped by adding 100 μl of sulfuric acid. After stopping the reaction, the absorption intensity at a wavelength of 500 nm to 610 nm was measured for each well with an automatic microplate reader, and the concentration and absorption intensity of the standard substance were plotted to prepare a calibration curve as shown in FIG. Then, the concentration of each measurement sample was determined using this calibration curve. Using such a method, the apoprotein concentration in 59 amniotic fluid samples from the 23rd to 41th week of pregnancy was measured.
As shown in the table, the results showed that the apoprotein concentration increased as the number of gestational weeks increased.

Example 3 (1) Preparation of Monoclonal Antibody-Insolubilized Beads After polystyrene beads were thoroughly washed, they were placed in a PBS (pH7.4) solution containing 20 μg / ml of monoclonal antibody PC6 at a temperature of 4 ° C. for one day. I left it. After that, the beads are washed with PBS solution, and then 0.5% bovine serum albumin (BS
A) Post-coating treatment was carried out by leaving it in the aqueous solution at a temperature of 4 ° C. for one day and night to obtain monoclonal antibody-insoluble beads.

(2) Preparation of Horseradish Peroxidase-Labeled Monoclonal Antibody Monoclonal antibody PE10 was added to a 1.0 mg / ml PBS solution to give N-
A solution of (m-maleimidobenzoic acid) -N-succinimide ester (MBS) in dimethylformamide at a concentration of 10 mg / ml.
50 ml was added and reacted at a temperature of 25 ° C. for 30 minutes. Then, using a column packed with Sephadex G-25,
Gel filtration was performed with 1 M phosphate buffer (pH 6.0) to separate maleimidated monoclonal antibody and unreacted MBS.

Meanwhile, horseradish peroxidase (HRP)
To a 1.0 mg / ml PBS solution, N-succinimidyl-3-
10 mg / m of (2-pyridylthio) propionate (SPDP)
An ethanol solution having a concentration of 1 was added and reacted at 25 ° C. for 30 minutes. Then, using a column packed with Sephadex G-25, the product was purified by gel filtration with 0.01 M acetate buffer (pH 4.5), and the fraction containing pyridyl disulfide HRP was collected and collected in a collodion bag. It was concentrated about 10 times under ice cooling. Next, 1 ml of 0.1 M acetate buffer (pH 4.5) containing 0.85% NaCl and 0.1 M dithiothreitol was added to this, and the mixture was stirred at 25 ° C for 30 minutes, and pyridyl disulfide introduced into the HRP molecule was added. The group was reduced. Then, it was subjected to gel filtration through a Sephadex G-25 column to obtain a fraction containing thiolated HRP.

The maleimidated monoclonal antibody obtained as described above and thiolated HRP were mixed, concentrated to a protein concentration of 4 mg under ice cooling using a collodion bag, and allowed to stand at 4 ° C for 1 day, then Ultrogel AcA44 was added. Gel filtration was performed using the packed column to obtain an HRP-labeled monoclonal antibody.

(3) Quantification of apoprotein by two-point simultaneous immunoassay (simultaneous sandwich method) One bead each insolubilized with PC6 monoclonal resistance,
Purified lung surfactant apoprotein 10 ng / ml-640 ng / ml
(Standard substance) containing 0.5% BSA and 1% Triton X-
200μ of PBS solution containing 100 (pH7.4), 0.5% BSA and 1% Triton X containing HRP-labeled monoclonal antibody PE10
200 μl of -100-containing PBS solution (pH 7.4) was added to each test tube and incubated at a temperature of 37 ° C. for 1 hour. next,
After removing the solution in the test tube by suction, 1% Triton X-10
After washing with 0-containing PBS, 2,2'-azinodi-3-ethylbenzthiazoline-6-sulfonic acid (ABTS) 0.05%, H
0.1M phosphate-citrate buffer containing 0.003% ₂ O ₂ (pH
4.5) was added to each test tube in an amount of 0.5 ml and incubated at a temperature of 37 ° C. for 30 minutes, and then an enzymatic reaction was stopped by adding 1 ml of 0.2 M oxalic acid aqueous solution as a reaction terminator.

Next, the absorption intensity of this solution at a wavelength of 420 nm was measured using a spectrophotometer, and this was plotted against the concentration of the standard substance, whereby a calibration curve with good concentration dependence was obtained.
The results are shown in Fig. 2.

[Brief description of drawings]

1 and 2 show the calibration curve of the two-point simultaneous immunoassay using the monoclonal antibody of the present invention.

Claims (8)

[Claims] 1. A method for measuring human lung surfactant in a sample by an immunological method, wherein the molecular weight of the human lung surfactant is about 62,000 and / or about 34,000 to 37,000.
In a sample, characterized by using a first monoclonal antibody that recognizes the apoprotein of and a second monoclonal antibody that recognizes the apoprotein but binds to an antigenic site different from the first monoclonal antibody. Method for measuring human lung surfactant. 2. The method for measuring a human lung surface-active substance according to claim 1, wherein the first monoclonal antibody is immobilized on a carrier and the second monoclonal antibody is labeled. 3. The method for measuring a human lung surface-active substance according to claim 1 or 2, wherein the second monoclonal antibody is a biotinylated antibody. 4. The method for measuring a human lung surface-active substance according to any one of claims 1 to 3, wherein skim milk is added as a blocking agent to the measurement system. 5. A reagent kit for measuring a human lung surfactant in a sample by an immunological method, comprising: (1) a human lung surfactant having a molecular weight of about 62,000 and / or about 3.
A first monoclonal antibody immobilized on a carrier that recognizes 4,000 to 37,000 apoproteins, and (2) a labeled antibody that recognizes the apoproteins but binds to an antigenic site different from the first monoclonal carrier A reagent kit comprising a second monoclonal antibody and (3) if necessary, a detection reagent for the labeled antibody as a main component. 6. The reagent kit according to claim 5, wherein the monoclonal antibody is an IgG type mouse antibody. 7. The method according to claim 5 or 6, wherein the labeled second monoclonal antibody is a biotinylated antibody.
Reagent kit according to the item. 8. The reagent kit according to claim 7, wherein the detection reagent for labeled antibody is enzyme-labeled avidin.