JPH05312807A - Lung tissue lesion marker in blood - Google Patents

Abstract

PURPOSE:To diagnose lung tissue lesion by quantifying lectin constituting a complex of the lectin and immunoglobulin existing in Homo sapiens blood. CONSTITUTION:A monoclonal antibody being a primary antibody against lung surfactant apoprotein A is immobilized to an soluble carrier, such as a plastic vessel, and the surface of the insoluble carrier is covered by, for instance, ox serum albumin. The insoluble carrier is made to coexist simultaneously with a specimen, anti-lung surfactant apoprotein, an antibody marked by an enzyme being a secondary antibody, a nonionic anionic surface active agent, and, for instance, protein of 16-50 thousands molecular weight and of 1.0-5.0 isoelectric points in immune reaction liquid so as to react catalyticaly for a fixed time and at a fixed temperature. After washing this, the amount of marked substance marked to the insoluble carrier is determined. The amount of the lectin constituting a lectin-immunoglobulin complex in Homo sapiens blood is computed from the value thereof.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a lectin-immunoglobulin complex present in human blood, a complex thereof, and a method for measuring lectin present in the complex.
More specifically, a pulmonary tissue damage marker in blood and an antibody of immunoglobulin or an antibody of lectin are used, and these are combined to form an immunological measurement system, and preferably two types of surfactants coexist in the reaction system. A method for measuring a marker for lung tissue damage in human blood, which comprises:

[0002]

2. Description of the Related Art Pulmonary diseases such as interstitial pneumonia (IPF) often cause death. However, there are many inadequate diagnostic methods at present. For example, 67 Ga scintigram may reflect the active phase of TPF, but it is not always lung disease specific, and because it uses radioactive substances, it harms the living body. The blood concentration measurement of erythrocyte sedimentation, circulating immune complex, type 3 procollagen peptide and the like reflects inflammation but is not always lung-specific.

[0003] Other lung diseases such as sarcoidosis, pneumonia, pulmonary tuberculosis, lung adenocarcinoma, and squamous cell carcinoma of the lung are known, and lung disease markers that can be used for diagnosis and treatment are known. Its existence in blood is unknown.

[0004]

Means for Solving the Problems As a result of intensive studies by the present inventors, Bio-Ge was found in the serum of patients with lung diseases such as IPF.
In gel filtration using an A15m column (Bioland KK), it was found that a substance having a molecular weight of 60 to 2,000,000 daltons was present, and the present invention was reached.

That is, the present invention diagnoses lung tissue damage by quantifying the complex of lectin and immunoglobulin present in human blood, and the lectin constituting the complex, or by quantifying the self-confidence of the complex. It is an immunological assay method for performing the same or a measurement kit therefor.

First, the present invention is a complex of lectins and immunoglobulins present in human blood, that is, a marker for lung tissue damage, which is a composite fire resistance of lectins and immunoglobulins.

In the present invention, as a lectin-immunoglobulin complex which can be a lung disease marker present in human blood, lung surfactant apoprotein A as a lectin, and IgG and IgM as an immunoglobulin were found in serum of IPF patients. It was Then the complex
It was found by the inventor that it is also present in the serum of patients with lung adenocarcinoma. It is not clear yet why this complex is elevated in serum of patients with interstitial pneumonia or lung adenocarcinoma, for example. It was revealed that SP-A has a C-type lectin-like structure. The C-type lectin is a lectin having a collagen-like structure on the N-terminal side and a global head structure on the C-terminal side and having the ability to bind a sugar chain at this head.

The collagen-like structure on the N-terminal side of the above structure is
It has the ability to bind to FC receptors. Therefore, lectins are expected to play an effective role in eliminating foreign foreign substances having sugar chains from the living body. SP-
A seems to play an irreplaceable role in eliminating foreign bodies in the local lung. There is no clear answer as to why the SP-A / immunoglobulin complex appears in the blood of patients with these interstitial pneumonia and lung adenocarcinoma, but S
PA is released into the blood due to damage of the alveoli and binds to immunoglobulin in the blood due to damage of the alveoli in the lung inflammation, etc., and as a result, forms a complex, or binds to immunoglobulin in the alveoli to form a complex. Either it will form the body and the complex will appear in the blood. Free SP-A does not exist in blood.

According to the present invention, as a result of affinity purification of this complex using mannose Sephare, 2-
SDS-PAG in the presence of mercaptoethanol was used
In Western Blotting, the same band as SP-A was recognized at molecular weights of 36,000 and 62,000. On the other hand, antiIg
G (Heavy chain speif) and antiIgM (μ chain
Western blotting using a spedific)
It was confirmed that n and γ chains existed in this complex.

In the present invention, in particular, a nonionic and anionic surfactant and a protein having a molecular weight of 16 to 50000 and an isoelectric point of 1.0 to 5.0 are simultaneously present in the reaction system. Preferably.

The presence of such a surfactant and protein has made it possible to measure the lung disease marker protein in blood with particularly high sensitivity, for the following reasons. The marker protein is considered to be a lipoprotein that forms a complex with phospholipids like SP-A existing in amniotic fluid and sputum. In the case of coexistence with a nonionic / anionic surfactant, lipoprotein in blood is sufficiently solubilized, and the marker protein is bound by an antibody involved in an immune reaction. However, since there is no protein component that can suppress non-specific adsorption,
A decrease in sensitivity occurs, which makes it impossible to correctly measure the marker protein having a low abundance in blood.

On the other hand, in the presence of only such a protein capable of suppressing nonspecific adsorption and a nonionic surfactant, the lipoprotein in blood is insufficiently solubilized, and the marker protein is an antibody involved in an immune reaction. Therefore, the marker protein in blood cannot be measured. However, due to the coexistence of nonionic / anionic surfactants and specific proteins,
The advantages of each are utilized, that is, the reduction of non-specific adsorption by proteins and the solubilization of lipoproteins allow the apoproteins to be efficiently recognized by antibodies, and the marker protein present at a very low concentration in blood can be measured. It will be.

The two antibodies that recognize different epitopes for the pulmonary surfactant apoprotein used in the present invention include the monoclonal antibodies PC-6, PE-10, etc. already disclosed in JP-A-61-277699. .. The measuring method is described in JP-A-61-275.
As described in No. 654, the antibodies that recognize different epitopes may be polyclonal antibodies.

In the present invention, the proteins that can be simultaneously present in the reaction system are those having a molecular weight of 1.6 to 50000 and an isoelectric point of 1.0 to 5.0.

Examples of such proteins in the present invention include pepsin, ovoglycoprotein, orosomucoid, casein, and skim milk which is a mixture of casein and an inorganic substance. When a protein with a molecular weight of 16,000 or less is used, non-specific adsorption is increased, and with a molecular weight of 50,000 or more, the reduction of immunospecific reaction is insufficient and specific. As a result of a decrease in the immune response,
The molecular weight used in the present invention was determined to be 16-50,000.

Regarding the isoelectric point, when a protein having an isoelectric point of 5.0 or more is added, nonspecific adsorption increases, and below the isoelectric point of 1.0, a specific reaction is suppressed. The range of the isoelectric point of the protein used for was determined to be 1.0 to 5.0.

Among the above proteins, skim milk is particularly preferable.

In the present invention, such a protein preferably has a final concentration in the immune reaction solution of 0.01 to 0.9% by weight. For example, adjusting the concentration of skim milk in the buffer to such a range must satisfy two essential conditions for high sensitivity of the immunoassay (that is, reduce nonspecific reaction while maintaining specific reaction). Will be easier. Skim milk is insoluble in water at a concentration higher than 0.9%, and because its suspension is used for blocking, a large insoluble matter of skim milk covers the antigen from a microscopic point of view, so that the antibody cannot come close to the result. As a result, the antigen-antibody reaction is likely to be largely inhibited. If it is 0.01% or less, it is difficult to obtain a sufficient effect of reducing nonspecific adsorption.

As the surfactant used in the present invention for coexisting with the above protein in the immune reaction solution, nonionic surfactants include polyoxyethylene alkyl ethers, polyoxyethylene alkylphenol ethers, and polyoxyethylene alkylphenol ethers. Oxyethylene fatty acid ester (mono)
, Polyoxyethylene alkyl thioethers, and as anionic surfactants, higher alcohol sulfates, alkyl sulfonates, alkylbenzene sulfonates, dinaphthylmethane disulfonates, alkyl sulfosuccinates, poly Examples thereof include oxyethylene alkyl ether sulfuric acid esters and polyoxyethylene alkylphenol sulfuric acid ester salts.

The nonionic surfactant is preferably a polyoxyethylene alkylphenol ether such as Triton (trade name of Rohm & Haas Co.), and the anionic surfactant is sodium dodecyl sulfate (SDS). Alkyl sulfonates such as) are preferred. Regarding the concentration, it is particularly preferable to use about 0.25 to 4% by weight for the former and about 0.2 to 3% by weight for the latter.

When Triton is 0.25% by weight or more,
The protective effect of the immune reaction appears, and if it exceeds 4% by weight,
It tends to destabilize the immune response. Also, S
When DS is 0.2% by weight or more, the surface active effect appears.
When SDS exceeds 3% by weight, its surface-active effect is too strong, and even if the concentration of Triton is increased, its immune-reaction protective effect is difficult to be exerted.

In the present invention, it is preferable to use a nonionic and anionic surfactant in combination, but the weight ratio in the case of the combined use is anionic surfactant / nonionic surfactant =
0.25 to 1.5 is preferable.

In the present invention, an antibody against human lung surfactant apoprotein or an antibody against immunoglobulin is used as a primary antibody and a secondary antibody, respectively. It is preferable to immobilize such a primary antibody on a carrier, but a known method can be adopted as the immobilizing method, and a solid phase carrier such as polystyrene, polyethylene,
Fluorine resin such as polypropylene, polyester, polyacrylonitrile, polyacrylate and Teflon, polymer such as polyacetal, cross-linked dextran and polysaccharide, other paper, glass, metal, agarose and combinations thereof, tray-like, spherical Various shapes such as fibrous, rod-shaped, disk-shaped, container-shaped, cells, and test tubes are mentioned, and balls, beads, gears, microplates and the like are preferably used.

The secondary antibody is preferably labeled, but the method and means for labeling the secondary antibody and the method and means for detecting the secondary antibody are not limited at all, and known methods and It can also be measured by means such as a secondary reaction with an anti-immunoglobulin antibody labeled with a radioactive substance or an enzyme or a fluorescent substance, or Staphylococcal protein A. Examples of the labeling agent include enzymes, fluorescent substances, luminescent substances and radioactive substances. Method using enzyme (EIA)
Enzyme such as horseradish peroxidase, β-D-galactosidase, alkaline phosphatase is ¹²⁵ I in the method using radioactive material (RIA),
¹³¹ I, ¹⁴ C, ³ H and the like are methods using a fluorescent substance (FI
In A), fluorescein isothiocyanate, phycobiliprotein, etc., and in the method using a luminescent substance, isorucinol, lucigenin, etc. are usually used, but if the activity of the labeling agent can be measured, other It may be.

Of the above methods, it is desirable to use a secondary antibody directly labeled with an enzyme. This is because the indirect method increases the immune reaction by one step and is not suitable for practical use.

When the labeling agent is an enzyme, a substrate and, if necessary, a coloring agent are used to measure its activity.
Examples of the substrate include 2,2'-azinodi [3-ethylbenzthiazolinesulfonic acid] ammonic acid (ABT) as a substrate for horseradish peroxidase.
S) -H ₂ O ₂ , 5-aminosalicylic acid-H ₂ O ₂ , O
- phenylenediamine -H ₂ O _2, 4-aminoantipyrine -H ₂ O _2, also benzidine (3,3 ', 5,
5'-tetramethylbenzidine etc.)-H ₂ O ₂ etc.
As a substrate for β-D-galactosidase, fluorescein-di- (β-D-galactopyranoside), O-nitrophenol-β-D-galactopyranoside, 4-methylumbelliferyl-β-D-galacto Pyranoside, etc.
As a substrate for alkaline phosphatase, O-nitrophenyl phosphate and the like can be mentioned. In addition to these reagents, known reagents such as a lysing agent, a cleaning agent, and a reaction terminator are used for the measurement.

Immunological Assay for Lung Tissue Damage Marker Protein Present in Human Blood According to the Present Invention (1) Lectin in complex of SP-A which is lectin present in human blood and immunoglobulin SP-A
Immunological assay method for lung monoclonal antibody (primary antibody; PC-6) against pulmonary surfactant apoprotein A (SP-A) is immobilized on an appropriate insoluble carrier (for example, plastic container) (hereinafter referred to as "solid phase antibody"). “”). The surface of the insoluble carrier is then coated with a suitable substance (eg bovine serum albumin) to avoid non-specific binding between the insoluble carrier and the reagent or analyte sample to be measured.

The thus obtained insoluble carrier on which the primary antibody is immobilized is used as a sample and an enzyme-labeled anti-pulmonary surfactant apoprotein antibody (secondary antibody, PE-10).
Non-ionic / anionic surfactant (labeled antibody) and a protein having a molecular weight of 1.6 to 5.0 and an isoelectric point of 1.0 to 5.0 are simultaneously added to an immune reaction solution, for example, for an immunoreaction. Coexist in a buffer solution, and contact them for a certain time and at a certain temperature to react. The immune reaction temperature may be 0 to 50 ° C, but it is used at a relatively high temperature. However, in the low temperature immune reaction,
The reaction time is long, and the immunoreaction time is short in the immune reaction at a relatively high temperature. From a practical point of view, the temperature is from room temperature (20 ℃) to 45
° C is preferred. This is washed with an appropriate washing solution, and then the amount of the labeling substance labeled on the secondary antibody present on the insoluble carrier is measured.

From the value thus obtained, the lectin (S-A) -ignoglobulin complex lectin (S in human blood)
The amount of PA) can be calculated.

(2) Lectin present in human blood (SP
-A) -Immunoglobulin Complex Immunoassay Method A monoclonal antibody against pulmonary surfactant apoprotein (primary antibody; PC-6) is immobilized on an appropriate insoluble carrier (for example, a plastic container). "Phase antibody"). The surface of the insoluble carrier is then coated with a suitable substance (eg bovine serum albumin) to avoid non-specific binding between the insoluble carrier and the reagent or analyte sample to be measured.

The insoluble carrier on which the primary antibody is thus obtained is allowed to react with the sample for a certain period of time to allow the complex to bind to the solid phase. Then, after washing, the enzyme-labeled anti-immunoglobulin was simultaneously immunized with a nonionic / anionic surfactant and a protein having a molecular weight of 1.6 to 5.0 and an isoelectric point of 1.0 to 5.0. The reaction solution, for example, a buffer solution for immunoreaction, is allowed to coexist and is allowed to react for a certain period of time and at a certain temperature for reaction. The immune reaction temperature may be 0 to 50 ° C, but it is used at a relatively high temperature. However, an immune reaction at a low temperature has a long reaction time, and an immune reaction at a relatively high temperature has a short reaction time. From a practical point of view, normal temperature (20 ° C) to 45 ° C is preferably used. This is washed with a suitable washing solution, and then the amount of the labeling substance labeled on the secondary antibody present on the insoluble carrier is measured.

Thus, from the value, the lectin in human blood
The amount of immunoglobulin complex can be calculated.

(3) Composition of Assay Reagent and Kit The assay reagent used in the method for assaying a lung disease marker protein in human blood of the present invention comprises two antibody reagents, immunoglobulins, which recognize different epitopes for human lung surfactant apoprotein. It consists of an antibody reagent against (human IgG, human IgM), a nonionic and anionic surfactant, and a protein having a molecular weight of 16 to 50000 and an isoelectric point of 1.0 to 5.0.

Of these, a measurement reagent comprising a solid-phase antibody reagent in which two antibodies are bound to the insoluble carrier described above, a labeled antibody reagent, and an immunoreaction buffer containing the surfactant and protein described above is preferable. ..

Further, the kit for measuring a lung tissue disorder marker protein in human blood of the present invention comprises the above-mentioned measurement reagents and, for example, solid reagents as auxiliary agents for efficiently and conveniently using these measurement reagents. Alternatively, a lysing agent for dissolving a liquid sample, an antigen nonspecifically bound to an insoluble carrier,
When using a detergent used to wash the antibody and an antibody labeled with an enzyme, it is usually used as a substrate for measuring enzyme activity and its reaction terminator, and as a kit for other immunological measurements. The one used is mentioned.

In such a kit, the nonionic and anionic surfactants according to the invention and a molecular weight of 1.6 to 5.0 are used.
It is desirable to add a protein having an isoelectric point of 1.0 to 5.0 to a solubilizer for dissolving a sample.

[0037]

INDUSTRIAL APPLICABILITY According to the present invention, a lung tissue disorder marker protein present in human blood or the like can be measured with extremely high sensitivity, and therefore can be easily measured with a small amount of sample in a short time.

As described above, lung cancer and other lung diseases can be diagnosed by measuring the lung disease marker protein in blood.

[0039]

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

[0040]

[Reference example]

The structure of pulmonary surfactant apoprotein A (SP-A) and the structure of sugar-binding SP-A are shown in FIG. become a trimer and one su
Form a bunit structure (Fig. 2). This becomes a hexamer,
A C-type lectin structure is shown (FIG. 3).

[0041]

Example 1 SP-A in Serum / SP in Immunoglobulin Complex-
A Measurement method (1) After thoroughly washing the monoclonal antibody insolubilized beads, the monoclonal antibody PC-6 was left in a PBS (pH 7.4) solution having a concentration of 20 μg / ml at a temperature of 4 ° C. for one day. Then, the beads were washed with a PBS solution, and then left in a 0.5% bovine serum albumin (BSA) aqueous solution at a temperature of 4 ° C. for one day to perform a post-coating treatment to obtain monoclonal antibody-insoluble beads. It was

(2) Preparation of Horseradish Peroxidase-Labeled Monoclonal Antibody 1.0-10 mg / ml of monoclonal antibody PE-10 in PBS
To the solution was added 50 ml of a solution of N- (m-maleimidobenzoic acid) -N-succinimide ester (MBS) in dimethylformamide having a concentration of 10 mg / ml, and the mixture was reacted at a temperature of 25 ° C for 30 minutes. Next, Sephadex G-25
Using a column packed with 0.1M phosphate buffer (pH
Gel filtration was performed at 6.0) to separate the maleimidated monoclonal antibody and unreacted MBS.

On the other hand, to a 1.0 mg / ml PBS solution of horseradish peroxidase (HRP) was added an ethanol solution of N-succinimidyl-3- (2-pyridylthio) propionate (SPOP) at a concentration of 10 mg / ml. The mixture was reacted at 25 ° C for 30 minutes. Then, using a column packed with Sephadex G-25, 0.01
The product was purified by gel filtration with M acetate buffer (pH 4.5), and the fraction containing pyridyl disulfide HRP was collected and concentrated about 10 times under ice cooling in a collodion bag. Then, a 0.1 M acetate buffer solution (p containing 0.85% NaCl and 0.1 M dithiothreitol) was added thereto.
H4.5) 1 ml and stirred at 25 ° C. for 30 minutes,
The pyridyl disulfide group introduced into the HRP molecule was reduced. Then, gel filtration was performed on 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 one day and night. Ultrogel AcA44
Gel filtration was performed using a column filled with to obtain an HRP-labeled monoclonal antibody.

(3) The marker protein in serum (pulmonary disease
Measurement of serum protein) As a serum sample dilution buffer, 0.6% SDS, 2.0%
Liton X-100, mixed solution containing 0.1% skim milk
Using this mixture and HRP containing a dilution series of serum sample
Labeled monoclonal antibody PE-10 diluted solution, 2 for each
Monoclonal antibody PC-6 solid in a test tube of 00 μm each
Immune reaction was carried out by adding to a fixed bowl (37 ° C, 2:00
while). Next, after removing the solution in the test tube by suction, use saline solution.
After washing, 3,3 ', 5,5'-tetramethylben
Methanol solution containing 1% dizine / H ₂O₂0.015%
1M phosphate-citrate buffer (pH 4.5) containing
0.4% of each 3/7 (V / V) mixed solution of each test
Add it to the tube and incubate at room temperature for 15 minutes.
1.5NH as a stop agent₂SO_FourAdd 2 ml of aqueous solution
Therefore, the enzymatic reaction was stopped. And using a spectrophotometer
The absorption intensity at a wavelength of 450 nm of the leverage was measured.

On the other hand, an apoprotein purified from alveolar proteinosis was used as a standard substance, the same mixed solution as the above was used as a dilution buffer solution, and a calibration curve corresponding to each concentration of SDS was prepared in the same manner as above. I was there. Then, using the calibration curve of the corresponding SDS concentration, from the absorption intensity at 450 nm obtained as described above, the marker protein (pulmonary disease marker protein)
Was determined.

The amount of the complex in blood and the amount of apoprotein purified from the alveolar lavage fluid for alveolar proteinosis are different substances, and are therefore expressed as a unit.

(4) Examination of ratio of anionic / nonionic surfactant in serum sample dilution buffer in immunoreaction As a serum sample dilution buffer, Triton X-100, 1,
SDS of 1.75, 1.5,
Using a mixed solution containing 1.25, 1.0, and 0.75%, each of this mixed solution containing a dilution series of a serum sample and HRP-labeled monoclonal antibody PE-10 diluted to 200
Monoclonal antibody PC-6 in microliters in test tubes
Immune reaction was performed by adding to fixed balls (37 ° C, 90
Minutes). Hereinafter, according to Example 1 (3), the absorbance was measured after completion of the immune reaction and plotted against the anionic / nonionic ratio of the surfactant. The result is shown in FIG.

In the figure, ○-○ Triton X-100, 1%, ●-
● Indicates the measured value when using the same 2%, △-△ same 3%, and ▲-▲ same 4%.

From FIG. 4, it can be seen that the optimum weight ratio of SDS / Triton X-100 is 0.25 to 1.5.

[0051]

Example 2 Characterization of SP-A Complex in Human Serum (1) Identification of Molecular Weight of SP-A Complex in Serum Using Bio-Gel A15m Column Alveolar proteinosis serum was treated with Bio-Gel A15m. It was applied to a column (16 x 80 cm) and 1 cc of each was fractionated.
Each fraction was applied to the measurement system constructed in Example 1 (FIG. 5 (a)). On the other hand, the purified human SP-A was applied to a Bio-Gel A15m column in the same manner and subjected to Example 1 and the measurement system (FIG. 5 (b)). Molecular weight markers were applied as well. The molecular weight markers are as follows.

Blue dextran (arrow a, 2 million daltons) thyroglobulin (arrow b, 670,000 daltons) ferritin (arrow c, 440,000 daltons) From FIG. 5 (a), the molecular weight of SP-A complex in blood is 60.
Compared to ~ 2 million daltons, purified SP-A
It was distributed between 440,000 and 670,000 daltons, and it was shown that their molecular weights were clearly different.

(2) Western blotting of SP-A complex using PE-10 affinity column The serum of a patient with alveolar proteinosis was PE-10 identified Sepharos.
Apply to e4B column, elute the bound substance, and perform SDS-PAGE and Western Blotting under reducing conditions.
I went. The results are shown in FIGS. 6A and 6B ((A) 10% gel, (B) 15% gel).

From FIG. 6A, P1 has a molecular weight of 80 kD.
a, P2 has a molecular weight of 50 kDa, P3 has a molecular weight of 28 kDa
Met. From Fig. 6 (B), P1 is IgMμchain (la
It was found that ne4) and P2 are IgGγ chains (lane 3). In addition, antiSP-A PE-10 and antiS
Western Blotting with lane IgG (lane 6, lane
From 8), it was confirmed that this complex contained SP-A, IgG, and IgM.

(3) Examination of human IgG binding property of purified SP-A Human IgG was used as a solid phase of a plastic microplate to examine IgG reactivity of purified SP-A. The result is shown in FIG. 7.

As shown in FIG. 7, S to solid phase human IgG was
The high binding property of PA was observed. In addition, tick antigen,
Anti-mite antigen is a control experiment.

[0057]

Example 3 Measurement of Sample Using the measurement system established in Example 1, alveolar proteinosis (PAP), interstitial pneumonia (IPF), sarcoidosis (SA), pneumonia (Pn), pulmonary tuberculosis (Tbc). The pulmonary tissue damage marker value in the serum was measured for these patients. As a control, it was also measured in the serum of a healthy person.

The results are shown in FIG. Based on these results, the serum levels of patients with interstitial pneumonia and alveolar proteinosis were clearly high.

On the other hand, sarcoidosis, pulmonary tuberculosis (Tb
No positive result was shown in c), indicating that the method for measuring a lung disease marker protein in human blood of the present invention may be useful for the differential diagnosis of the above various diseases and the monitoring of those diseases.

[Brief description of drawings]

FIG. 1 is a structure of SP-A.

FIG. 2 is a trimer subunit structure of SP-A.

FIG. 3 is a C-type lectin structure of SP-A.

FIG. 4 shows measured values when an immunoreaction was carried out by changing the weight ratio of the surfactant in (4) of Example 1.

FIG. 5 shows serum SP-A complex (A) and purified S.
The gel filtration pattern of PA (B) is shown ((1) of Example 2).

FIG. 6 shows the results of immunoblot analysis of the serum SP-A complex after affinity purification in (2) of Example 2.

FIG. 7 shows the results of examining the binding property of SP-A to human IgG in (2) of Example 2.

FIG. 8 shows a patient sample (alveolar proteinosis (PA
P), interstitial pneumonia (IPF), sarcoidosis (S
A), pneumonia (Pn), tuberculosis (Tbc)) are shown by the results of the immunoassay of the present invention.

Claims (15)

[Claims] 1. A complex of a lectin and an immunoglobulin present in human blood. 2. The complex according to claim 1, wherein the lectin is a C-type lectin. 3. The lectin is lung surfactant apoprotein A (SP-A).
The described complex. 4. The complex according to claim 1, wherein the immunoglobulin is IgG. 5. The complex according to claim 1, wherein the immunoglobulin is IgM. 6. An immunological measurement method for specifically measuring a lectin in a complex of a lectin and immunoglobulin present in human blood. 7. The measuring method according to claim 6, wherein the lectin is a C-type lectin. 8. The lectin is lung surfactant apoprotein A (SP-A).
The measurement method described. 9. The measuring method according to claim 6, wherein the immunoglobulin is IgG. 10. The measuring method according to claim 6, wherein the immunoglobulin is IgM. 11. A method for immunologically measuring a lectin-immunoglobulin complex present in human blood. 12. The measuring method according to claim 6 or 11, wherein nonionic and anionic surfactants are simultaneously present in the immune reaction system. 13. The measuring method according to claim 12, wherein the anionic surfactant is an alkyl sulfonate. 14. The nonionic surfactant is a polyoxyethylene alkylphenol ether.
The measurement method described. 15. The measuring method according to claim 12, wherein the anionic surfactant is an alkyl sulfonate, and the nonionic surfactant is a polyoxyethylene alkylphenol ether.