JPH07103977A - Immunological sandwich assay for slpi or slpi elastase complex - Google Patents

Abstract

PURPOSE:To accurately detect an SLPI and SLPI-elastase complecx by using antibodies for recognizing both [1-54] and [55-107] of the SLPI. CONSTITUTION:After a peptide having an amino acid sequence of C terminal [55-107] of human SLPI and an amino sequence of N terminal [1-54] thereof is synthesized, it is, for eaxmple, immunized on a mouse, and a lymphocyte of the mouse is fused with mouse myeloma cells to generate a hybridoma, then a mono-clonal antibody for recognizing the C and N terminals is obtained through a cloned hybridoma that the generated hybridoma is cloned. The antibody will be specifically united with the elastase joint part or non-joint part of the SLPI [1-54] and [55-107], so the kind of molecular such as an SLPI complete molecule or free SLPI not united with the can be selectively determined accurately. Therefore, a measuring system for detecting the condition of respiratory disease sensitively can be obtained.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an anti-human SLPI monoclonal antibody that specifically recognizes human SLPI, SLPI using the same, and a method for measuring a human SLPI-elastase complex.

[0002]

2. Description of the Related Art SLPI (Secretory Leukocyte Protea)
se Inhibitor) is known as a low molecular weight protease inhibitor present in exocrine fluid such as bronchial mucus, saliva, semen, cervical mucus, and nasal discharge. Recently, the structure of an inhibitor: SLPI purified from parotid fluid was clarified (RC Thompson et al., Proc. Natl. Acad. Sci.
USA, 83 , 6692-6696, 1986; Tokusho Sho-62-
501291), and an inhibitor purified from semen: HU
The structure of SI-I (Human Seminal Proteinase Inhibitor I) was also identified (U. Seemuller et al., FEBS Lett., 19
9, 43-48, 1986), all of which are the same substance, and are proteins consisting of 107 amino acid residues having a molecular weight of about 14,000. As a structural feature, SLPI is composed of two domains, the N-terminal domain (Ser ¹ -Pro ⁵⁴ ) has trypsin inhibitory activity, and the C-terminal domain (Asn ⁵⁵ -Ala ¹⁰⁷ ) has chymotrypsin and neutrophils. It was presumed to be responsible for the elastase and cathepsin G inhibitory activities.

Recently, by genetic engineering techniques, C
Terminal domain polypeptides are produced, and chymotrypsin inhibitory activity (see JP-A-62-259591) and neutrophil elastase inhibitory activity (WO89 / 06239) are produced.
No., Bio / Technology, 7 , 55-60, 198
9, Am. Rev. Respir. Dis., 139 , A201, 198.
(See 9) is becoming clear.

By the way, self-tissue damage due to neutrophil elastase released from neutrophils activated at the site of inflammation has been highlighted, but as mentioned above, SLPI is used.
Has a strong neutrophil elastase inhibitory activity, it is highly possible that SLPI functions as a physiological inhibitor at the site of inflammation.

Several groups are challenging the measurement of SLPI. K. Ohlsson et al. (Hopp
e-Seyler's Z. Physiol. Chem., 362 , 1273-
1277 (1981)) reported a radioimmunoassay by a competitive method using a polyclonal antibody against human SLPI, and that SLPI was increased in the serum of patients with pneumonia in the aggravating period compared with healthy persons using the assay system. (Eur. J. Respir. Dis., 65 , 20)
1 (1984)). In addition, JA Kramps et al. (Am. Rev. R
espir. Dis., 129 , 959-963 (1984))
Produced an enzyme-labeled antibody using a polyclonal antibody against human SLPI, reported a measurement system by the sandwich method, and measured the transition in sputum of patients with respiratory tract infection (Chest, 89, 731 ( 1986)).

In any of these methods, a measuring system is constructed using an antibody obtained by using a human SLPI whole molecule consisting of two domains as an antigen.

[0007]

However, the conventional measuring method has some problems. That is,
The point is that a polyclonal antibody against human SLPI consisting of two domains is used. In inflammatory diseases, self-tissue damage by neutrophil elastase, cathepsin G, which is a lysosomal enzyme released from activated neutrophils, is a problem. Therefore, when measuring inhibitors for these enzymes, it is necessary to accurately measure SLPI molecular species having an inhibitory active site. SLPI
It has been reported that molecular species that undergo intramolecular cleavage also exist physiologically (Pulmonary Emphysema and Pr.
oteolysis, p297 (1986) Academic Press In
c.) Since all SLPI molecules are measured by the conventional method, for example, a molecular species lacking the C-terminal domain or a part thereof having neutrophil elastase inhibitory activity will be detected. Therefore, it is presumed that it will be difficult to accurately detect the amount of the molecular species having the neutrophil elastase inhibitory activity to be measured.
The physiological significance of LPI in inflammatory diseases cannot be accurately reflected.

Therefore, the specific site of human SLPI is
Recognizing monoclonal antibody, region-specific SLPI assay system using the same, and elastase SLP
A measurement system for SLPI-elastase complex, which is a reaction intermediate captured by I, has been desired.

[0009]

Therefore, the present inventors have
In view of the problems of the prior art, as a result of earnest research to develop an assay system that recognizes only a specific molecular structure, as a result, human SLPI C-terminal domain [55-107] and N-terminal domain [1-54] By obtaining a monoclonal antibody that recognizes the C-terminal and the N-terminal by synthesizing a peptide having the following formula and constructing a region-specific SLPI assay system and SLPI-elastase complex assay system using these The present invention has been accomplished by making it possible and yet finding that this complex exists in human body fluids.

That is, the present invention relates to SLPI [1-5].
4] and an antibody recognizing [55-107] of SLPI are used, which is an immunological sandwich assay method for full-length SLPI.

The monoclonal antibody of the present invention can be obtained by immunizing with the following peptides, but the immunogen is not limited. [I] SLPI [1-54] ... A polypeptide having an amino acid sequence of 1 to 54 counting from the N-terminus of SLPI. [II] SLPI [1-107] ... A polypeptide having the entire amino acid sequence of SLPI.

The monoclonal antibody is a cell fusion method by G. Kohler and Milstein,
Nature (London), 256, 495-497 (197)
It is prepared by culturing and secreting the hybridoma prepared in 5)) and separating it from the culture solution.
That is, the human SL represented by the above formulas (I) and (II)
After immunizing a mouse with the N-terminal domain polypeptide of PI and the SLPI whole molecule, lymphocytes of this mouse are fused with mouse myeloma cells to prepare a hybridoma,
The obtained hybridoma is cloned, and the anti-human SLPI monoclonal antibody is separated from this cloned hybridoma.

The fragment equivalent to the anti-human SLPI antibody of the present invention means an anti-human SLPI antibody having the same level of activity as the antigen-binding activity of the anti-human SLPI antibody within a range that does not interfere with the immunoassay. And a fragment such as F (a
b ′) ₂ , Fab ′, Fab, Facb and the like can be mentioned. Such a fragment is, for example, F (a
In the case of b ′) ₂ or Fab ′, the polyclonal antibody or monoclonal antibody obtained as described above is separated by a known method, for example, the antibody is digested and cleaved with pepsin and separated to obtain an F (ab ′) ₂ fragment. Or F (a
b ′) ₂ Fragment is treated to reduce Fa
It is obtained by forming a b'fragment (A. Nisono
ff et al., Arch. Biochem. Biophys., 89 , 230
(1960): P. Parham., J. Immunol., 131 , 28.
95 (1983)).

By the way, it is known that, among the enzyme inhibitory activities, the enzyme inhibitory activity derived from a polypeptide is a requirement not only for the primary structure of the polypeptide but also for its higher-order structure to be active.

Therefore, the human SLPI N-terminal domain SLPI complete molecule represented by the above formulas (I) and (II) is also intramolecularly disulfide-bonded by a plurality of Cys present in the molecule, resulting in a high molecular weight. It is expected to form substructures.

Therefore, the anti-human SLPI antibody of the present invention or a fragment equivalent thereto is also included. Among them, as the anti-human SLPI antibody of the present invention or a fragment equivalent thereto, an antibody or the like obtained by using an antigen having a neutrophil elastase inhibitory activity, which forms a higher-order structure by an intramolecular disulfide bond, is an inflammatory disease or the like. Is preferable from the viewpoint of measurement significance.

The anti-human S of the present invention thus obtained
The LPI monoclonal antibody can be region specific for SLPI. For example, [1-54] area, [55-
The recognition site can be the elastase binding site or the elastase non-binding site in the [107] region. Therefore, an assay system that can be constructed using these monoclonal antibodies is, for example, an SLPI complete molecule assay system or a free SLPI assay system that does not bind to elastase or S.
It is considered to be excellent in that it is possible to construct a region-specific measurement system for SLPI from various existing forms in clinical specimens such as the LPI-elastase complex measurement system.

In the present invention, the aforementioned anti-human SLP is used.
Human SLP present in a sample using an I antibody or a fragment equivalent thereto (hereinafter abbreviated as “antibody etc.”)
Methods of immunologically measuring I or its elastase complex are provided. As the immunological measurement method, for example, a method known per se described in “Enzyme Immunoassay” (2nd edition, Eiji Ishikawa et al., Medical Shoin 1982) and the like,
A competitive method, a sandwich method or the like can be used.

Among these, the sandwich method is preferable as the immunological measurement method of the present invention.

In the case of the sandwich method, the antibody or the like (first antibody) of the present invention is immobilized on a suitable insoluble carrier (for example, a plastic container) (hereinafter referred to as "immobilized antibody"). Examples of the insoluble carrier include polymers such as polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluororesin, cross-linked dextran, and polysaccharides, as well as paper, glass, metal, agarose, and combinations thereof.

The shape of the insoluble carrier may be various shapes such as tray shape, spherical shape, fibrous shape, rod shape, plate shape, container shape, cell and test tube. 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 first antibody is immobilized is brought into contact with a sample for a certain period of time and temperature to react. During this period, the solid phase antibody (first antibody) is bound to the human / SLPI in the sample or the complex thereof.
Examples of specimen samples to be measured include human plasma, serum, sputum, saliva, nasal discharge, semen, uterine mucus, tears, urine, sweat, body fluids such as feces, tissue homogenates, and organ lavage fluids such as alveolar lavage fluid. Further, the above biological sample derived from a laboratory animal can also be used as a sample sample, in which case,
The immunological assay method of the present invention can also be used as a measurement system for preclinical evaluation in drug development.

Then, after washing with a suitable washing solution, the antibody of the present invention labeled with a suitable labeling substance (eg enzyme) (second
Antibody) (for example, an aqueous solution containing 1% BSA, 0.1% skim milk) is contacted with human SLPI bound to the solid phase antibody in the insoluble carrier or a complex thereof at a constant time and temperature for the second antibody. React with. This is washed with an appropriate washing solution, and then the amount of the labeling substance labeled with the second antibody which is bound to the solid phase antibody on the insoluble carrier through human SLPI and is present is measured.

Examples of the washing liquid include various surfactants, body fluids and protein fluids. Preferred examples include betaine-type, sulfobetaine-type, and phosphate-type amphoteric surfactants. Amphitol (registered trademark) is preferable, and it is preferable that the concentration of such a surfactant as a cleaning liquid is 1% (w / w) or less in the cleaning liquid, for example, 0.05% in the case of Amphitol (registered trademark).

Here, it is advantageous to use an enzyme, a fluorescent substance, a luminescent substance, a radioactive substance or the like as the labeling substance of the labeled antibody. Peroxidase, alkaline phosphatase, β-D-galactosidase as the enzyme, fluorescein isothiocyanate as the fluorescent substance,
As luminescent substances such as fucobiliprotein, isoluminol and lucigenin, and as radioactive substances
^{Although 125} I, ¹³¹ I, ¹⁴ C, ³ H and the like can be used, these are not limited to the exemplified ones, and other ones can be used as long as they can be used in the immunological assay method.

In particular, when the labeling substance is an enzyme, a substrate and, if necessary, a color former are used to measure the activity.

When peroxidase is used as the enzyme, H ₂ O ₂ is used as the substrate and _{2, 2} as the color former.
2'-azinodi- [3-ethylbenzthiazolinesulfonic acid] ammonium salt (ABTS), 5-aminosalicylic acid, O-phenylenediamine, 4-aminoantipyrine, 3,3 ', 5,5'-tetramethylbenzidine, etc.
When alkaline phosphatase is used as the enzyme, O-nitrophenyl phosphate or the like is used as the substrate and β- is used as the enzyme.
When D-galactosidase is used, fluorescein-di- (β-D-galactopyranoside), 4-
Methylumbelliferyl-β-D-galactopyranoside and the like can be used.

In the above reaction, a solid phase antibody, a labeled antibody and a sample specimen containing human / SLPI or a complex thereof may be mixed at the same time, and these three may be simultaneously contacted at a certain time and temperature for reaction. .

Thus, the value of human S in the sample is calculated from the value.
The amount of LPI or complex thereof can be calculated.

Although the sandwich method has been described above,
Also in the competitive method, the antibody of the present invention or a fragment equivalent thereto is similarly used, and human SLPI in a specimen sample is used.
Can be measured.

The measuring reagent for immunological measurement of human SLPI or its complex of the present invention comprises the above-mentioned solid phase antibody,
It consists of a labeled antibody.

Further, the kit for immunological measurement of human SLPI or its complex of the present invention comprises the above-mentioned measuring reagents and auxiliary agents for efficiently and conveniently using these measuring reagents, for example, A lysing agent for dissolving a solid reagent or a liquid sample, an antigen non-specifically bound to an insoluble carrier, a detergent used for washing an antibody,
When an antibody labeled with an enzyme is used, a substrate for measuring the enzyme activity and its reaction terminator, and other commonly used kits for immunological measurement can be mentioned.

The anti-human SLPI monoclonal antibody of the present invention can be used for immunohistostaining by directly labeling or using a labeled second antibody. As a labeling method, it is advantageous to use a fluorescent substance, an enzyme, a luminescent substance, a radioactive substance or the like.

[0034]

The monoclonal antibody of the present invention is SLP
A monoclonal antibody that specifically binds to the elastase binding site of [1-54] and [55-107] of I or the non-elastase binding site, and a fragment equivalent thereto. Therefore, by using this antibody or the like, it becomes possible to carry out a measurement which has been impossible in the past. For example, it is possible to perform selective quantification of molecular species such as SLPI complete molecule or free SLPI that is not bound with elastase. Therefore, it can be said that the present invention can provide a measurement system capable of sensitively reflecting the state of respiratory diseases.
Further, by using the monoclonal antibody of the present invention and combining it with an anti-elastase antibody, it is possible to detect the existing mode of the complex of human SLPI and elastase in a sample, which has been impossible in the past. Human S useful for the diagnosis and treatment of inflammatory diseases, especially lung diseases, by using these measurement systems
It becomes possible to provide an immunoassay method for LPI and its complex. In particular, it was found for the first time that the SLPI-elastase complex was present in sputum of patients with asthma, and it was revealed that its presence was useful for diagnosing asthma complications. Can be very usefully used for complications of respiratory diseases.

[0035]

The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. % In the examples means% by weight.

[0036]

Example 1 Acquisition of hybridoma (1) Preparation of antigen (a) Synthesis of SLPI · N-terminal [1-54] peptide (I) Amino acid sequence of SLPI · N-terminal [1-54] (Proc.
Natl. Acad. Sci. USA, 83, 6692-6696, 1
A DNA fragment encoding 986) was chemically synthesized. Leu-Val-Pro, which can be cleaved with thrombin, in the same manner as in the examples of WO89 / 06239.
-The human growth hormone gene was fused to the DNA encoding SLPI via the DNA sequence encoding Arg. The expression vector thus obtained (plasmid pGH-SLPI) was transformed into E. E. coli HB 101. Transformed cells (E. coli HB 101 strain (p
GH-SLPI)) was cultured to obtain an inclusion body, which was purified by thrombin treatment, SS rearrangement, and chromatography to obtain a target SLPI / N-terminal [1-54].

(A) Synthesis of SLPI [1-107] peptide (II) The amino acid sequence of SLPI ((Proc. Natl. Acad. Sci. U
SA, 83, 6692-6696, 1986) was chemically synthesized. WO89 / 06
In the same manner as in the example of US Pat. No. 239, SL was added to the human growth hormone gene via a DNA sequence encoding Leu-Val-Pro-Arg which can be cleaved by thrombin.
The DNA encoding PI was fused. The expression vector thus obtained (plasmid pGH-SLPI) was transformed into E. E. coli HB 101. Transformed cells (Escherichia coli HB 101 strain (pGH-SLPI))
C. to obtain a complex, which is purified by thrombin treatment, SS rearrangement and chromatography to obtain the desired S
Got the LPI. The inhibition constant (Ki) of the obtained SLPI for elastase was 2.5 × 10 ^-10 M, which was equivalent to the activity of SLPI isolated from nature (Table 62-5).
No. 01291).

(C) Preparation of Complex of Antigen ([I] and [II]) and Carrier Protein SLPI [1-54] or SLPI [1-107] 10 mg each is commercially available maleimidated KLH (Boehringer Mannheim) 10 mg And reacted in 2 ml of PBS for binding.

(2) Preparation of antigen-stimulated lymphocytes SLPI [1-5 was intraperitoneally injected into male Balb / c mice.
4] or a conjugate 9 of SLPI [1-107] and KLH
An emulsion of 0 μg and complete Freund's adjuvant was administered. SLPI 5 times at 3-4 week intervals thereafter
An emulsion of 40 to 50 μg of [1-54] or SLPI [1-107] / KLH conjugate and incomplete adjuvant was intraperitoneally administered. SLPI 14 days after the 5th administration
60 μg of [1-54] or SLPI [1-107] / KLH conjugate was dissolved in 1.0 ml of physiological saline and intravenously administered, and four days after that, the spleen was aseptically removed from the mouse,
L-glutamine 0.39 g / liter, kanamycin sulfate 0.
A splenocyte suspension suspended in RPMI-1640 (GIBCO) supplemented with 2 g / liter and NaHCO ₃ 2.0 g / liter was prepared. The floating cells were washed 3 times with the above medium, and then a splenocyte suspension was prepared.

(3) Cell fusion Mouse myeloma cells P3U1 contained 0.2 kanamycin sulfate.
The cells were cultured in a GIT synthetic medium (manufactured by Daigo Nutrition Chemical Co., Ltd.) supplemented with g / liter. Myeloma cells were in the exponential phase of cell division at the time of cell fusion. The spleen cells and the myeloma cells were suspended in serum-free RPMI-1640 medium at a cell number ratio of 3: 1 and centrifuged at 1300 rpm for 5 minutes.
After removing the medium, the sedimented cells had an average molecular weight of 1,50.
50% polyethylene glycol solution (pH 8.2)
A suspension was prepared by gently adding 1 ml. Then serum-free R
9 ml of PMI-1640 medium was added and the cells were shaken carefully. After that, centrifugation was performed at 1,000 rpm for 5 minutes to remove the supernatant medium, and then the cells were collected as a precipitate and GIT
Suspended in 40 ml of medium.

(4) Acquisition of cloned hybridoma The fused cell solution was distributed on 96 microplates (200 μl per well). This plate is 37 ℃, 5
Cultured in a% CO ₂ atmosphere. After 1 day and 2 days, half of the medium was replaced with fresh GIT / HAT medium and cultured at intervals of 2 days thereafter. After 11 days, the antibody in the culture supernatant of the hybridoma was screened by the enzyme immunoassay. The antigen used for the screening is S
The secondary antibody with LPI [1-54] and SLPI [1-107] is alkaline phosphatase-labeled goat anti-mouse I
It was a gG antibody.

Among the 192 wells into which the hybridomas were distributed, colonies of fused cells were found in 124 wells, of which 1 well was SLPI [1-54] antibody-positive and 2 wells were SLPI [1-107] antibody-positive. Met.

The hybridomas thus obtained in the antibody-positive wells were diluted to 0.9 cells per well of 96 microplates, and distributed to the plate containing Balb / c mouse thymocytes as feeder cells. , G supplemented with kanamycin sulfate 0.2 g / l
Cultured in IT medium. Observation under a microscope confirmed that it was definitely a single colony.

SLPI in culture supernatant of hybridoma
Antibodies against [1-54] and [1-107] were screened by enzyme immunoassay. Each well was antibody-positive and produced anti-human SLPI antibody.

Three cloned hybridomas were obtained as described above.

[0046]

Example 2 (1) Culture of hybridoma The cloned hybridoma obtained in Example 1 was used for 2
Balb / c mice intraperitoneally administered with 0.1 ml of pristane (Wako Pure Chemical Industries) a week before, the number of cells was 10 ⁶ to 10 ⁷ cells /
It was intraperitoneally administered to a rat. Ascitic fluid 2-3 on 7-10 days after that
ml / animal was collected.

(2) Purification of Monoclonal Antibody The collected ascites fluid was subjected to the method of Ey et al. (PL Ey et al., Immuno
chemistry, 15 , 429, 436 (1978)). That is, 0.1M phosphate buffer (pH
The ascites fluid 2 to 3 ml was applied to a Protein A-Sepharose column (gel volume 5 ml) equilibrated with 8.0), and then 0.1 M sodium citrate had a pH of 6.0 or 5.
A buffer solution of 0, 4.0 and 3.0 was sequentially flown to elute the monoclonal antibody from the column to obtain a purified monoclonal antibody.

[0048]

Example 3 Measurement of human SLPI by sandwich method EIA using monoclonal antibody (A) (1) Preparation of antibody-immobilized beads Obtained in Example 2 after thoroughly washing polystyrene beads (diameter 6 mm) Monoclonal antibody F10T1, E
PB with a concentration of 20 μg / ml of 9E5, F4E3
After leaving it in the S solution at a temperature of 4 ° C for 1 day and night, it was washed with PBS, and then left for 1 day in a PBS solution of 1% bovine serum albumin (BSA) at a temperature of 4 ° C for post-coating treatment. , F10T1, E9E5, and F4E3 antibody-immobilized beads were obtained, respectively.

(2) Preparation of HRP-labeled antibody C-terminal domain of human SLPI (Asn prepared in Japanese Patent Application No. 3-279862 filed by the present inventors)
⁵⁵ -2.0 of polyclonal antibody against Ala ¹⁰⁷ )
To 1 ml of a PBS solution of mg / ml, 100 μl of 1M acetate buffer (pH 4.2) and 2 μg of 40 μg pepsin were added.
It was dissolved in 0 μl of the same buffer and added, and the mixture was reacted at 37 ° C. for 4 hours.

After completion of the reaction, separation was carried out using a Sephadex G25 column (φ2 cm × 45 cm) equilibrated with PBS to collect F (ab ′) ₂ . F (ab ') ₂ of 1
mg / ml 0.01M phosphoric acid 0.15M NaCl
To 2 ml of the (pH 7.4) solution, 50 μl of a dimethylformamide solution having a concentration of 10 mg / ml MBS was added, and 2
The reaction was carried out at a temperature of 5 ° C for 30 minutes. Then, using a column packed with Ceradex G-25, gel filtration was performed with 0.1 M phosphate buffer (0.1 M PB) (pH 6.0) to separate the maleimidated antibody and unreacted MBS.

On the other hand, HRP of 10 mg / ml of 0.1M
To 2 ml of a PB (pH 6.5) solution, 120 μl of a 60 mg / ml dimethylformamide solution of S-acetylmercaptosuccinic anhydride was added, and the mixture was reacted at 25 ° C. for 2 hours.

Next, 0.1 M Tris-HCl buffer (pH
7.0) 800 μl, 0.1 M EDTA 160 μ
1, 1M hydroxylamine (1.6 ml) was added, and the mixture was reacted at 0 ° C. for 4 minutes. Then, the reaction solution was placed in a collodion bag, and 0.1 M PB (pH 6.0), 5 mM ED
The solution containing TA was dialyzed at 4 ° C. for 3 days to obtain thiolated HRP.

Next, 2 mg of the maleimidated antibody and 4 mg of thiolated HRP were mixed, concentrated using a collodion bag under ice cooling to a protein concentration of 4 to 10 mg / ml, and allowed to stand at 15 to 20 ° C. overnight. . The solution was subjected to gel filtration with a column packed with Ultrogel AcA44 (LKB) to obtain HRP-labeled anti-human SLPI [55-107] antibody.

(3) Sandwich EIA Assay System Each contains one F10T1, E9E5, F4E3 antibody-immobilized bead prepared in (1) and purified human SLPI (standard substance) in the range of 0 to 500 ng / ml. 1% BS
A containing 0.05 M TBS (pH 8.0) 200 μl and the HRP-labeled antibody prepared in (2) containing 1% BSA.
200 μl of 05M TBS (pH 8.0) solution was added to each test tube and incubated at a temperature of 25 ° C. for 2 hours. Next, after removing the solution in the test tube by suction, 0.0
After washing with 5M TBS (pH 8.0), 3,
3 ', 5,5'-tetramethylbenzidine hydrochloride 0.0
After adding 0.1M phosphoric acid / citrate buffer (pH 4.3) containing 2% and H ₂ O ₂ 2.5 mM to each test tube in an amount of 0.4 ml, the mixture was reacted at a temperature of 25 ° C. for 30 minutes. The enzyme reaction was stopped by adding 1 ml of 1N sulfuric acid aqueous solution as a reaction terminator. Then, the absorption intensity of this solution at a wavelength of 450 nm was measured using a spectrophotometer. A calibration curve is plotted in FIG. 1 corresponding to standard substance concentrations of 0 to 500 ng / ml. From this result, solid phase F
A good calibration curve is drawn only when 10T1 is used,
When F4E3 or F9E5, which is considered to have a close epitope, was used as a solid phase, the calibration curve was not good.

[0055]

[Example 4] Measurement of human / SLPI by sandwich method EIA using monoclonal antibody (B) (1) Preparation of antibody-immobilized beads E9E5 and F4E3 antibody-immobilized beads obtained in Example 3 (1) were used. .

(2) Preparation of HRP-labeled antibody Using the monoclonal antibody F10T1 obtained in Example 2, HRP-labeled F10T1 antibody was obtained according to Example 3 (2).

(3) Sandwich EIA assay system Immunoassay was performed according to Example 3 (3). The results are shown in Fig. 2. As shown in FIG. 2, both E9E5 and F4E3 antibody-immobilized beads gave good calibration curves. From this fact, it is possible to construct the measurement system for the complete molecule SLPI by this method.

[0058]

Example 5 Measurement of SLPI-elastase complex (1) Preparation of antibody-immobilized beads Complete molecule SLPI synthesized in (1) of Example 1 (1)
A rabbit was immunized with [1-107] to obtain a polyclonal antibody. The antiserum was Ig using a protein A column
Purified to G. This IgG is 20 μg / ml in PBS
It was diluted with and fixed to beads. In addition, 2 of Example 3 (1)
Seed monoclonal antibody-immobilized beads were also used in the next experiment.

(2) Sandwich EIA measurement system 0 to 100 ng of antibody-immobilized beads prepared in (1)
/ Ml range SLPI-elastase complex 200μ
1 was incubated for 2 hours at a temperature of 37 ° C. Next, each bead was washed, and 200 μl (5 μg / m) of HRP-labeled anti-human elastase antibody (Cappel, USA) was added.
1) added and incubated at 37 ° C. for 1 hour.

Next, after removing the solution in the test tube by suction,
After washing with 0.05M TBS (pH 8.0),
0.1 M containing 3,3 ', 5,5'-tetramethylbenzidine hydrochloride 0.02% and H ₂ O ₂ 2.5 mM
0.4 ml of phosphoric acid / citrate buffer solution (pH 4.3) was added to each test tube and reacted at a temperature of 25 ° C. for 30 minutes. Stopped. Then, the suction intensity of this solution was measured at a wavelength of 450 nm using a spectrophotometer. FIG. 3 shows a calibration curve obtained by plotting the values corresponding to standard substance concentrations of 0 to 100 ng / ml.

From these results, a good concentration-dependent curve was obtained using the polyclonal antibody against the complete molecule SLPI and the solid phase on which the monoclonal antibody F10T1 recognizing the SLPIN end [1-54] was immobilized. On the other hand, when using a solid phase on which a monoclonal antibody that recognizes the SLPIC terminus [55-107] was immobilized, a very good concentration-dependent curve was not obtained.
Among them, the F4E3 antibody-immobilized beads showed a slight concentration dependency, whereas the E9E5 antibody-immobilized beads did not show any concentration dependency. The above results are E9E5
Recognizes the elastase binding site of SLPI, while F
It was presumed that 4E3 recognizes the elastase non-binding part of SLPI.

Example 6 Measurement of SLPI-elastase complex in sputum of asthmatic patients SLPI in sputum of asthmatic patients using the method of Example 5
The elastase complex was measured. The results are shown in Fig. 4. From this result, it was found that the complex was detected in sputum of asthmatic patients with infection.

Thus, the following can be mentioned as embodiments of the present invention. [Item 1] A monoclonal antibody that recognizes the three-dimensional structure of human SLPI. [Item 2] A monoclonal antibody that recognizes [1-54] of SLPI. [Item 3] A monoclonal antibody that recognizes [55-107] of SLPI. [Item 4] The monoclonal antibody according to Item 3, which recognizes the elastase binding site of SLPI. [Item 5] The monoclonal antibody according to Item 3, which does not recognize the elastase binding site of SLPI. [Item 6] Antibodies and SLs that recognize [1-54] of SLPI
A method for immunologically measuring full-length SLPI using an antibody that recognizes [55-107] of PI. [Item 7] The method for measuring SLPI according to Item 6, which uses the monoclonal antibody according to Item 2 above. [Item 8] The method for measuring SLPI as described above in item 6, wherein the monoclonal antibody described in above item 3 is used. [Item 9] A measurement method for immunologically measuring full-length SLPI using a polyclonal antibody that recognizes full-length SLPI and a monoclonal antibody that recognizes SLPI. [Item 10] The method for measuring SLPI according to Item 9, which uses the monoclonal antibody according to Item 3 above. [Item 11] The method for measuring SLPI as described in [9] above, wherein the monoclonal antibody described in [3] above is used. [Item 12] A method for measuring a SLPI-elastase complex. [Item 13] The SL according to item 12, wherein an antibody that recognizes SLPI and an antibody that recognizes elastase are used.
Method for measuring PI-elastase complex. [Item 14] The method for measuring an SLPI-elastase complex according to item 13, wherein the monoclonal antibody according to item 2 is used. [Item 15] The method for measuring the SLPI-elastase complex according to item 13, which comprises using the monoclonal antibody according to item 5. [Item 16] The SLP according to item 13, wherein a polyclonal antibody that recognizes full-length SLPI is used.
Method for measuring I-elastase complex. [Item 17] A free SLPI that is not complexed with elastase, characterized by using the monoclonal antibody according to item 2 above and the monoclonal antibody according to item 4 above.
Immunologically measuring method.

[Brief description of drawings]

FIG. 1 shows a calibration curve for human SLPI measurement obtained when three types of monoclonal antibodies of the present invention were used as a solid phase and anti-SLPI [1-54] was used as an enzyme-labeled antibody.

FIG. 2 shows a calibration curve for human SLPI measurement obtained when two types of monoclonal antibodies of the present invention were used as a solid phase and another type was used as an enzyme-labeled antibody.

FIG. 3: Three monoclonal antibodies of the present invention and anti-SLP
Human SLPI obtained by using an anti-human elastase antibody as an enzyme-labeled antibody using a polyclonal antibody I
The calibration curve of an elastase complex is shown.

FIG. 4 shows the concentration of human SLPI-elastase complex in sputum of asthmatic patients.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Takashi Uemura 4-3, Asahigaoka, Hino-shi, Tokyo Inside Teijin Ltd. Tokyo Research Center (72) Takashi Kawamura 4-32, Asahigaoka, Hino-shi, Tokyo Teijin Limited Tokyo Research Center (72) Inventor Kenichi Masuda 4-3-2 Asahigaoka, Hino City, Tokyo Inside Teijin Limited Tokyo Research Center

Claims (5)

[Claims] 1. An immunological sandwich assay method for full-length SLPI, which comprises using an antibody that recognizes [1-54] of SLPI and an antibody that recognizes [55-107] of SLPI. 2. A SLPI-characterizing the use of an antibody that recognizes SLPI and an antibody that recognizes elastase.
Immunological sandwich assay method for elastase complex. 3. An antibody that recognizes the SLPI is SLPI.
3. The sandwich assay method according to claim 2, which is an antibody that recognizes [1-54]. 4. A method for immunological sandwich assay of free SLPI, which comprises using an antibody that recognizes [1-54] of SLPI and an antibody that recognizes the elastase binding site of SLPI. 5. The sandwich assay method according to claim 1, wherein the antibody is a monoclonal antibody.