JPH0715469B2 - Method for measuring human pulmonary surfactant - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION TECHNICAL FIELD The present invention relates to a method for measuring human pulmonary surfactant using an antibody against the apoprotein of human pulmonary surfactant.

BACKGROUND ART: The alveoli of animals contain a physiologically active substance called pulmonary surfactant, which is primarily composed of phospholipids. This substance coats the inner walls of the alveoli, protecting the alveolar epithelium and also plays an important physiological role in maintaining the animal's respiratory function. Specifically, pulmonary surfactant possesses a unique surface activity that alters the surface tension of the alveolar lining during breathing and expiration, contributing to the stability of the alveoli and exhibiting an anti-atelectatic effect. A lack of pulmonary surfactant leads to alveolar collapse, making it impossible to maintain stable ventilation, resulting in symptoms such as neonatal respiratory distress syndrome (IRDS).

To prevent the birth of newborns with such diseases,
The amount of pulmonary surfactant in the amniotic fluid, which correlates with the maturity of the fetal lungs, can be measured, and if the fetus is about to be born with immature lungs, it is possible to administer intrauterine treatment, such as steroid administration to promote the secretion of pulmonary surfactant.

Several methods have been proposed to measure or estimate the amount of pulmonary surfactant in amniotic fluid. For example, the L/S ratio (ratio of lecithin to sphingomyelin) in amniotic fluid and the amount of dipalmitoylphosphatidylcholine (DPPC) in amniotic fluid have been measured as markers of pulmonary surfactant. However, the former method does not measure phospholipids, which are the main components of pulmonary surfactant, and has the disadvantage of low correlation with IRDS, while the latter method has the disadvantage of low sensitivity.

Furthermore, a deficiency of pulmonary surfactant in adults can lead to symptoms such as adult respiratory distress syndrome (ARDS). To diagnose these lung diseases, the amount of pulmonary surfactant in lung lavage fluid, such as DPPC and phosphatidylglycerol (PG) as surfactant markers, is measured, but this method lacks sufficient sensitivity.

On the other hand, measurements using physicochemical methods of lung lavage fluid include:
Surface tension and other parameters are also measured, and sputum excreted by patients is also used for diagnostic purposes. Specifically,
Measurements of sputum viscosity, color, etc., and fucose, which reflects the amount of airway mucus in the sputum, are performed. Compared to lung lavage fluid collection, sputum is easier to collect, which gives it an advantage as a liquid to be measured.

Approximately 90% of pulmonary surfactant is lipid, such as phospholipids and neutral lipids, while approximately 10% is protein, which exists as a lipid-protein complex, i.e., lipoprotein.
When lipids are removed from pulmonary surfactant, a water-insoluble protein is obtained, called apoprotein (LSP), whose main component is a protein with a molecular weight of approximately 36,000 (36K). Compared to phospholipids, proteins have superior specificity and can be detected with high sensitivity, so the use of proteins as pulmonary surfactant markers has been considered, and immunological quantification using so-called polyclonal antibodies has also been carried out. However, conventional methods have had problems such as the long measurement time required and insufficient sensitivity.

Therefore, the present inventors have already prepared a monoclonal antibody against the apoprotein constituting the pulmonary surfactant and proposed a method for measuring the activity using this antibody (Japanese Patent Laid-Open Publication No. 62-64956).

However, when performing immunoassays as described above, differences in reactivity were observed between the standard apoprotein and pulmonary surfactant in samples such as amniotic fluid and sputum. This is because the pulmonary surfactant in a sample is composed of apoprotein and a 10-fold amount of phospholipids (in the case of sputum, it also contains airway viscosity). Therefore, when measuring apoprotein using antibodies, the immune response is inhibited by phospholipids and airway mucus. To avoid this, the immune response must be carried out for a very long time, which is inconvenient for diagnosing emergency conditions such as IRDS and ARDS.

Disclosure of the Invention The present inventors have investigated various conditions for immunoassay using two types of monoclonal or polyclonal antibodies specific to apoproteins, as described in Japanese Patent Application Laid-Open No. 62-64956. As a result, they have found that, depending on the type of surfactant added to the immunoreaction buffer, in some cases apoproteins and lipoproteins in the pulmonary surfactant of a sample can behave equivalently in immunoassay in a short period of time, i.e., apoproteins in lipoproteins can be correctly detected, leading to the present invention.

That is, the present invention provides a method for measuring human pulmonary surfactant in a sample by an immunological method comprising an antigen-antibody reaction, characterized in that the immune reaction is carried out in the presence of both an anionic surfactant and a nonionic surfactant in a buffer solution used for the immune reaction.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the measured values of pulmonary surfactant (lipoprotein) in amniotic fluid samples at different SDS concentrations in the buffer solution (2% Triton) used for diluting amniotic fluid samples (immunoreaction at 37°C,
1hr).

FIG. 2 shows the measured values of pulmonary surfactant (lipoprotein) in amniotic fluid samples at different Triton concentrations in the buffer solution (% SDS) for diluting amniotic fluid samples (immunoreaction 37°C, 1
hr).

FIG. 3 shows the measured values of pulmonary surfactant (lipoprotein) in amniotic fluid samples at different SDS concentrations in the buffer solution (2% Triton) for diluting amniotic fluid samples (immunoreaction at 45°C, 3
0 minutes).

FIG. 4 shows the measured values of pulmonary surfactant (lipoprotein) in amniotic fluid according to the time course of immune reaction.

FIG. 5 shows the results of measuring pulmonary surfactant in sputum samples at varying SDS and Triton concentrations.

FIG. 6 shows the amount of pulmonary surfactant (LSP) and the LSP/fucose ratio in patients with chronic airway disease and bronchoalveolar carcinoma.

BEST MODE FOR CARRYING OUT THE INVENTION The method of the present invention makes it possible to carry out a short-term solid-phase immune reaction, for the following reasons: When a nonionic surfactant is used alone, it has a degrading effect on the immune reaction but has an insufficient surfactant effect. On the other hand, when an anionic surfactant is used alone, it has a strong surfactant effect but causes protein denaturation of the antibody, thereby inhibiting the antigen-antibody reaction. However, by using a nonionic surfactant together with an anionic surfactant, the advantages of each surfactant are utilized, maintaining the strong surfactant effect of the anionic surfactant while providing the protein protective effect of the nonionic surfactant, which is thought to have made a short-term immune reaction possible.

In the present invention, the specimen means human amniotic fluid, sputum, lung lavage fluid, and the like.

The antibody used in the immunological method of the present invention, i.e., in the antigen-antibody reaction, is not particularly limited, and polyclonal or monoclonal antibodies against the apoprotein of pulmonary surfactant can be used. These antibodies can be prepared by known methods.

The surfactants used in the present invention include, as nonionic surfactants, polyoxyethylene alkyl ethers, polyoxyethylene alkylphenol ethers, polyoxyethylene fatty acid esters (mono) and polyoxyethylene alkyl thioethers, and as anionic surfactants, higher alcohol sulfates, alkyl sulfonates, alkyl benzene sulfonates, dinaphthylmethane disulfonates,
Examples include alkyl sulfosuccinates, polyoxyethylene alkyl ether sulfates, and polyoxyethylene alkylphenol sulfates.

Examples of nonionic surfactants include Triton (Ro
Preferred are polyoxyethylene alkylphenol ethers such as those available from H&H Co. (trade name), and preferred anionic surfactants are alkylsulfonates such as sodium dodecyl sulfate (SDS).
The concentration of the former is 0.25 to 4% by weight, and the latter is 0.2 to 3% by weight.
It is preferable to use about 1.0% by weight of Triton/SDS, and the concentration ratio (weight ratio) of the former to the latter is particularly preferably 1.0 to 4.0. When the Triton/SDS concentration ratio is 1.0 or less, SDS, which has a strong surfactant effect, is used.
DS becomes dominant, causing protein denaturation of antibodies and tending to inhibit antigen-antibody reactions.

On the other hand, when the Triton/SDS concentration ratio is 4.0 or higher, the protein-protecting effect of the nonionic surfactant becomes apparent, but the effect of the anionic surfactant, which disperses the phospholipids and airway mucus surrounding the apoproteins, is masked.

In the present invention, it is preferable to immobilize the primary antibody on a carrier. A known method can be used for immobilization.
As the carrier, a solid phase such as balls, beads, gears or microplates made of polystyrene, polyethylene, polyacrylate, Teflon, polyacetal or the like is preferably used.

The labeling method and the detection method and the detection method are not limited in any way, and the measurement can be carried out by a known method or means, for example, a secondary reaction with an anti-immunoglobulin antibody or Staphylococcus aureus protein A labeled with a radioactive substance, an enzyme, or a fluorescent substance. As a labeling agent, enzymes such as horseradish peroxidase, β-D-galactosidase, alkaline phosphatase, etc. are used in the enzyme immunoassay (EIA), and enzymes such as β-D-galactosidase, alkaline phosphatase, etc. are used in the radioactive substance immunoassay (RIA).
In the method using fluorescent substances (FIA), ²⁵ I, ³ H, etc. are usually used, and in the method using fluorescent substances (FIA), fluorescein isothiocyanate, etc. are usually used, but other labeling agents may also be used as long as the activity of the labeling agent can be measured.

When the labeling agent is an enzyme, a substrate is used to measure its activity. For example, the substrate for horseradish peroxidase is 2,2'-azinodi[3-ethylbenzthiazoline sulfonic acid]ammonium acid ( _ABTS ) _-H2O2,5 -aminosalicylic acid _{-H2O2 ,} O
-Phenylenediamine _{- H2O2} , 4-aminoantipyrine-
H ₂ O ₂ and the like are used as substrates for β-D-galactosidase.
Examples include fluorescein-di-(β-D-galactopyranoside), O-nitrophenol-β-D-galactopyranoside, etc. In addition to these reagents, known reagents such as dissolving agents, detergents, and reaction stoppers are also used for the measurement.

The method of the present invention allows for the rapid and simple measurement of apoproteins in pulmonary surfactant in a sample, and is useful for the treatment of IRDS and ARDS.
It is expected that this will provide a useful measurement method for urgent diseases such as those listed above.

The present invention will be described in detail below with reference to examples.

In the examples, "%" means "% by weight."

Example 1 (1) Monoclonal antibody-insolubilized beads were thoroughly washed and then left overnight in a PBS (pH 7.4) solution containing 20 μg/ml of monoclonal antibody PC6 at 4° C. As a result, the beads were washed with PBS solution and then left overnight in a 0.5%
Bovine serum albumin (BSA) was dissolved in water at 4°C for 1
The mixture was left overnight for post-coating treatment to obtain monoclonal antibody-insolubilized beads.

(2) Preparation of horseradish peroxidase-labeled monoclonal antibody: A 1.0 mg/ml PBS solution of monoclonal antibody PE10 was added to N-
(m-Maleimidobenzoic acid)-N-succinimide ester (MBS) in dimethylformamide at a concentration of 10 mg/ml
50 ml of the solution was added and reacted at 25°C for 30 minutes. Then, a column packed with Sephadex G-25 was used to separate the solution.
Gel filtration was carried out using 1 M phosphate buffer (pH 6.0) to separate the maleimide-modified monoclonal antibody from unreacted MBS.

On the other hand, horseradish peroxidase (HRP)
In a 1.0 mg/ml PBS solution, N-succinimidyl-3-
10 mg/m of (2-pyridylthio)propionate (SPOP)
A 1 ml ethanol solution was added and the mixture was allowed to react at 25°C for 30 minutes. The mixture was then purified by gel filtration using a Sephadex G-25 column with 0.01 M acetate buffer (pH 4.5). The fraction containing pyridyl disulfide-conjugated HRP was collected and concentrated approximately 10-fold in a collodion bag under constant cooling. Next, 1 ml of 0.1 M acetate buffer (pH 4.5) containing 0.85% NaCl and 0.1 M dithiothreitol was added, and the mixture was stirred at 25°C for 30 minutes to reduce the pyridyl disulfide group introduced into the HRP molecule. The mixture was then subjected to gel filtration on a Sephadex G-25 column to obtain the thiolated HRP.
A fraction containing

The maleimide-modified monoclonal antibody obtained as described above was mixed with thiolated HRP, concentrated to a protein concentration of 4 mg using a collodion bag under constant cooling, left to stand at 4°C overnight, and then gel-filtered using a column packed with Ultrogel AcA44 to obtain an HRP-labeled monoclonal antibody.

(3) Measurement of pulmonary surfactant in amniotic fluid samples by varying SDS concentration (immunoreaction temperature: 37°C). A mixture containing 2.0% Triton X-100 and a predetermined concentration of SDS (0, 0.25, 0.5, 0.75, 1.0, 2.0, 3.0, 4.0%) was used as the amniotic fluid sample dilution buffer. This mixture containing the amniotic fluid sample and a diluted solution of HRP-labeled monoclonal antibody PE10 were each added in 200 μm portions to a monoclonal antibody PC6-immobilized ball in a test tube, and the immunoreaction was carried out (37°C, 1 hour). Next, the solution in the test tube was aspirated, washed with saline, and then left for 3,
A 3/7 (V/V) mixture of 1% 3',5,5'-tetramethylbenzidine in methanol _and 0.015% _H2O2 in 1M phosphate-citrate buffer (pH 4.5) was added to each test tube in an amount of 0.4 ml each, and the tubes were incubated at room temperature for 15 minutes.
The enzyme reaction was stopped by adding 2 ml of 1.5N H ₂ SO ₄ aqueous solution as a reaction stopper to each well, and the absorption intensity of this solution at a wavelength of 450 nm was measured using a spectrophotometer.

On the other hand, apoprotein was used as a standard substance, and the same mixture as above was used as a dilution buffer, and a calibration curve corresponding to each concentration of SDS was prepared in the same manner as above.
Using the calibration curve of SDS concentration, the 4
The concentration of apoprotein (LSP) was determined from the absorption intensity at 50 nm. The LPS concentration obtained corresponding to each SDS concentration is shown in Figure 1.

From FIG. 1, it can be seen that in the presence of 2% Triton, the antigen (LSP) in the amniotic fluid sample was recognized relatively well by the antibody in the range of SDS from 0.25% to 3.0%.

Example 2 Measurement of lung surface substances in amniotic fluid samples by changing Triton concentration (immunoreaction temperature: 37°C) Using amniotic fluid samples, 1% SDS and various concentrations of Triton X-1 were used.
A similar experiment to that described in Example 1(3) was conducted using a mixture containing 0.00 (0, 0.25, 0.5, 1.0, 2.0, 3.0, 4.0, and 5.0%) as a diluent. The relationship between Triton concentration and LPS is shown in Figure 2. As is clear from Figure 2, antigens in amniotic fluid samples could be successfully measured in the presence of 1% SDS at Triton concentrations ranging from 0.25% to 4.0%.

Example 3 Measurement of pulmonary surfactant in amniotic fluid samples by changing SDS concentration (immunoreaction temperature: 45°C) Using amniotic fluid samples, 2% Triton X-100 and various concentrations of SDS were used.
The immunoreaction was carried out in the same manner as in Example 1 (3) using a mixture containing SDS (0, 0.2, 0.4, 0.6, 0.8, 1.0%) as a diluent at 45°C for 30 minutes. The relationship between SDS concentration and LPS is shown in Figure 3. As is clear from Figure 3, the immunoreaction temperature of 45°C, 2
In the presence of % Triton, SDS showed good results up to 0.2% to 1.0%, with 0.6% SDS producing the highest immune response.

Example 4 Time course of pulmonary surfactant measurement values in amniotic fluid samples (immunoreaction temperature: 45°C) As dilutions of the standard substance (apoprotein) and amniotic fluid samples,
Immunoreactions were carried out at 45°C in the same manner as in Example 1(3), using (a) 2% Triton X-100 and 0.6% SDS buffer, (b) 2% Triton X-100 only, and (c) 0.6% SDS only buffer, for 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, and 5 hours, respectively. Using each calibration curve, the concentration of pulmonary surfactant in the amniotic fluid was read as a function of the immunoreaction time and plotted in Figure 4.

As shown in Figure 4, the immune reaction did not progress at all with only 0.6% SDS (c), and progressed slowly with 2% Triton (b). In contrast, in the Triton/SDS mixed solvent system (a), the measured values reached an equilibrium point in 30 minutes, and the immune reaction progressed slowly with 2% Triton (b).
The effect of using a mixed solvent system of PEG/SDS is clear.

Example 5 Using the monoclonal antibody PC6 obtained in Example 1 and the labeled monoclonal antibody PE10, pulmonary surfactant in sputum samples was measured at varying SDS and Triton concentrations.

The sputum was doubled in volume with saline and homogenized.
SDS concentrations of 0, 1.5, 1, 1.5, and 2% were used. Triton 0, 1, 2,
Solutions of 3% and 4% concentrations were prepared (25 types in total), and the saline diluted sputum solution was diluted 10 times with these 25 diluted solutions.

The mixture containing the specimen and the diluted HRP-labeled monoclonal antibody PE10 were each added in 200 μm portions to the monoclonal antibody PC6-immobilized ball in the test tube, and an immune reaction was carried out (37°C, 1 hour).
After washing with saline, the tissue was immersed in 3/7 (V/V) of a 1 M phosphate-citrate buffer solution (pH 4.5) containing 1% 3,3',5,5'-tetramethylbenzidine in methanol and 0.015% _{H2O2 .}
V) Add 0.4 ml of each mixed solution to each test tube and let stand at room temperature.
After 15 minutes of incubation, the reaction was stopped with 1.5NH ₂
The enzyme reaction was stopped by adding 2 ml of an aqueous solution of _SO4 to each well. The absorbance of this solution was measured at a wavelength of 450 nm using a spectrophotometer. The absorbance values obtained for each Triton/SDS concentration ratio (by weight) are shown in Figure 5.

From FIG. 5, it can be seen that the antigen (LSP) in sputum is well recognized by the antibody when the Triton/SDS concentration ratio is in the range of 1.0 to 4.0 (W/W).

Example 6 Measurement of pulmonary surfactant in sputum samples using latex agglutination method with varying SDS and Triton concentrations. 25 types of Triton/SDS mixed solutions were mixed with 50 ml of sputum in equal amounts.
The final concentrations were adjusted to 0, 0.5, 1, 1.5, and 2% SDS and 0, 1.2, 3, and 4% Triton, respectively. 100 μl of a latex suspension mixed with PC6 and PE10, which recognize the apoprotein of pulmonary surfactant, was added to the sputum.
The mixture was mixed with 100 μl of diluted SDS solution, gently stirred for 15 minutes, and the degree of agglutination was recorded as -, ±, or +. The results are shown in Table 1.

Example 7 Measurement of pulmonary surfactant in sputum The concentration ratio of LSP to fucose was determined for each sample from patients with membranous airway diseases (n=17) and bronchoalveolar carcinoma (n=3) under the conditions determined in Example 5 (Triton 3.0%, SDS 1%), and the values were divided according to each patient group. The results are shown in Figure 6.

As can be seen from Figure 6, the LSP/fucose ratio of sputum from patients with bronchoalveolar carcinoma is higher than that of viscous and purulent sputum from patients with chronic respiratory tract diseases, making this method useful for differentiating between these two conditions.

MS refers to viscous sputum from patients with chronic airway diseases, PS refers to purulent sputum from patients with chronic airway diseases, and BAC refers to sputum from patients with bronchoalveolar carcinoma.

──────────────────────────────────────────────────── Continued from the front page (56) References: Japanese Patent Application Publication No. 61-275654 (JP, A) Japanese Patent Application Publication No. 62-64956 (JP, A) Japanese Patent Application Publication No. 61-277699 (JP, A) Japanese Patent Application Publication No. 61-277700 (JP, A) Japanese Patent Application Publication No. 58-187862 (JP, A) Japanese Patent Application Publication No. 59-97057 (JP, A)

Claims (6)

[Claims] [Claim 1] A method for measuring human pulmonary surfactant in a specimen by an immunological method comprising an antigen-antibody reaction, comprising the steps of: adding an anionic surfactant and a nonionic surfactant in a concentration ratio (weight ratio) of 1:1 to 1:4 to a buffer solution used in the immunological reaction;
The immune reaction is caused by coexistence of
Methods for measuring pulmonary surfactant in humans. [Claim 2] A method for measuring human pulmonary surfactant according to claim 1, characterized in that the apoprotein is measured using a first monoclonal antibody that recognizes the apoprotein of human pulmonary surfactant and a labeled second monoclonal antibody that recognizes the apoprotein but binds to an antigenic site different from that of the first monoclonal antibody. 3. A method for measuring human pulmonary surfactant according to claim 1, characterized in that the apoprotein of human pulmonary surfactant is measured using latex particles bound to an antibody that recognizes the apoprotein as an immune reaction. 4. The method for measuring human pulmonary surfactant according to claim 1, wherein the anionic surfactant is an alkylsulfonate. 5. The method according to claim 1, wherein the nonionic surfactant is a polyoxyethylene alkylphenol ether.
3. A method for measuring human pulmonary surfactant according to claim 2. 6. The composition according to claim 1, wherein the anionic surfactant is an alkyl sulfonate salt and the nonionic surfactant is a polyoxyethylene alkylphenol ether.
3. A method for measuring human pulmonary surfactant according to claim 2.