JPH08134098A - High-sensitivity measurement of human timp-1 - Google Patents

Abstract

PURPOSE: To enable high-sensitivity determination of human tissue inhibitor of metalloproteinase-1(TIMP-1) which is useful in diagnosis of pulmonary fibrosis or the like by using a labeled monoclonal antibody against the TIMP-1. CONSTITUTION: TIMP-1 in a specimen is determined in high sensitivity through a sandwich enzyme immunoassay technique by using a labeled anti-human TIMP-1 mouse clonal antibody which reacts with human TIMP-1, but not with human TIMP-2 and bovine TIMP-1 under a reduced and a non-reduced conditions in the immunoblotting after SDS electrophoresis of human TIMP-1. This antibody is an IgG which does not inhibit the collagenase activity of the TIMP-1, and, when combining the labeled antibody with another antibody immobilized to an immunoplate prepared by immobilizing an anti-human TIMP-1 mouse clonal antibody having its reactive site different from that of the above antibody, the human TIMP-1 in a specimen can determined in high sensitivity.

Description

Detailed Description of the Invention

[0001]

The present invention relates to TIMP-1 (tissue inhibitor of metalloproteinase-1, tissue inhibit), which has attracted attention in the fields of medicine and bioscience.
The present invention relates to a highly sensitive assay method for or of metalloproteinases-1), an anti-human TIMP-1 mouse monoclonal antibody used in the assay method, and a mouse hybridoma producing the mouse monoclonal antibody.

[0002]

The extracellular matrix (connective tissue between cells) has a complex structure mainly composed of collagen, proteoglycan, glycosaminoglycan, glycoproteins such as fibronectin and laminin, and elastin. There is. The main group of enzymes mainly involved in collagen degradation in the extracellular matrix metabolic process is collectively called matrix metalloproteinase (MMP).
These MMPs are secreted from cells as zymogens,
The peptide chain is cleaved and activated to exert a decomposition action. The enzymatic activity of these MMPs is controlled by various activators and inhibitors in the body (Matrisian (199
0) Trends Genet. 6 , 121-125; Murphy et al. (1991)
Br. J. Rheumatol. 30 ,, Suppl. 1, 25-31). Various M
A biological substance that comprehensively inhibits MP is known as Tissue Inhibitor of Metalloproteinase (TIMP), and at least TIMP-1 (Doch
erty et al. (1985) Nature 318, 66-69; Gasson et a
l. (1985) Nature 315, 768-771; Carmichael et al.
(1986) Proc. Natl. Acad. Sci. USA 83, 2407-241
1) and TIMP-2 (Boone et al. (1990) Proc. N
atl. Acad. Sci. USA 87 , 2800-2804; Stetler-Stev
enson et al. (1990) J. Biol. Chem. 265, 13933-1393
Two types of 8) are known.

TIMP-1 is a glycoprotein consisting of 184 amino acid residues and having a molecular weight of 28.5 kD, and has 6 -SS- bonds in the molecule. TIMP-1 inhibits these enzymatic activities by binding to active interstitial collagenase, active stromelysin, 92 kD type IV collagenase and the like at a molecular ratio of 1: 1 (Welgus et al. (198
3) J. Biol. Chem. 253, 12259-12264; Welgus et al.
(1985) Collagen Relat. Res. 5, 167-179; Wilhelm et
al. (1989) J. Biol. Chem. 264 , 17213-17221). Rheumatoid arthritis (Woolley et al., Arth. Rheum. 20: 1231-
1239, 1977), cancer cell invasion and metastasis (Mignatti
Et al., Cell 47: 487-498, 1986), pulmonary fibrosis (Montano et al.,
Chest 96: 1115-1119, 1989) is an active form of MM
It is said to occur when the balance between P and their inhibitor TIMP-1 is lost. Therefore, TI
Accurately quantifying the amount of MP-1 is extremely meaningful for elucidating the mechanism of these diseases or monitoring the therapeutic effect. As a method for quantifying TIMP-1, a method for measuring biological activity up to now (Terato et al. (1976)
Biochim. Biophys. Acta 445, 753), enzyme immunoassay (Clark et al. (1991) Matrix 11, 76-85; Kodama et a
l. (1990) J. Immunol. Meth. 127 , 103-108; Japanese Patent Application No. 62-42781). However, since these conventional methods have low sensitivity, it has been difficult to accurately measure low-level concentrations of TIMP-1 in urine. Therefore, it has been difficult to investigate the relationship between the TIMP-1 concentration in these low-concentration body fluids and the pathological condition.

[0004]

DISCLOSURE OF THE INVENTION The object of the present invention is to make it possible to measure the TIMP-1 concentration in a sample which cannot be measured because the sensitivity is low in the conventional measuring methods.
The purpose of the present invention is to provide a highly sensitive measurement method.

[0005]

Means for Solving the Problems As a result of intensive studies conducted by the present inventors in order to achieve the above object, the conventional method of bovine T
By using human TIMP-1 as an immunogen instead of IMP-1 (Japanese Patent Application No. 62-42781), human TIM
The present invention has been completed by producing mouse monoclonal antibodies that specifically react with P-1 and establishing a simple and highly sensitive assay method for human TIMP-1 using them. That is, the present invention has the following properties a to e: a. Does not inhibit the collagenase inhibitory activity of human TIMP-1; b. IgG; c. Human TIMP-under reducing and non-reducing conditions
Immunoblotting after SDS electrophoresis of 1
Reacts with human TIMP-1; d. Does not react with human TIMP-2; e. Anti-human T having no reaction with bovine TIMP-1
A highly sensitive method for measuring human TIMP-1 characterized by using an IMP-1 mouse monoclonal antibody as a labeled antibody, and the following properties a to f: a. Inhibit the collagenase inhibitory activity of human TIMP-1; b. IgG; c. Human TIMP-1 was subjected to SDS electrophoresis of human TIMP-1 under non-reducing conditions, followed by immunoblotting.
Reacts with 1; d. Human TIMP-1 was subjected to SDS electrophoresis of human TIMP-1 under reducing conditions, followed by immunoblotting.
Does not react with; e. Does not react with human TIMP-2; f. The present invention relates to a highly sensitive assay method for human TIMP-1, which comprises using an anti-human TIMP-1 mouse monoclonal antibody having no reaction with bovine TIMP-1 as an immobilized antibody.

In the present invention, the labeled antibody is TIM
-225 is preferable as the immobilized antibody, and TIM-293 is preferable because of their high reactivity. Furthermore, it is more preferable to use these two kinds of antibodies together as a labeled antibody and an immobilized antibody, respectively. TIM-225-, a mouse hybridoma that produces anti-human TIMP-1 mouse monoclonal antibodies TIM-225 and TIM-293
14 and TIM-293-21 were deposited at the Patent Microorganism Depositary Center, Institute of Biotechnology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry, on November 8, 1994, and the deposit number was FERM-.
P-14616 and FERM-P-14615. The mouse monoclonal antibody against human TIMP-1 can be prepared, for example, according to the method of Iwasaki et al. (Monoclonal antibody, Kodansha, 1983).
Further, the highly sensitive assay method of TIMP-1 is, for example, the method of Ishikawa (Ultrasensitive enzyme immunoassay method, Academic Publishing Center, 19
1993) can be applied.
Therefore, the present invention also relates to these mouse monoclonal antibodies and the mouse hybridomas that produce them. Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to these examples.

[0007]

【Example】

Example 1-Preparation of anti-human TIMP-1 monoclonal antibody- (a) of mouse splenocytes immunized with human TIMP-1
From the culture supernatant of HeLa cells, a prepared human cervical cancer cell line
Cawston et al. (Cawston et al. (1981) Biochem.
J., 195 , 159-165) and purified TIMP-1
The mixture was mixed with ibi adjuvant (Ribi Immunochem Research, Inc.), and 20 μg / mouse was injected into two intraperitoneal and subcutaneous sites of NZB mice (8 weeks old) for immunization. After that, booster immunization was performed twice every 3 weeks in the same manner as in the initial immunization, and 3 days after the final immunization, the spleen was extracted and the splenocytes were fused with myeloma cells as follows.

(B) Cell fusion 2 × 10 ⁷ mouse myeloma cell lines P3-X63-Ag8-U1 (P3U
1) and 1 × 10 ⁸ splenocytes were used to perform cell fusion in the presence of 50% polyethylene glycol 1500 (Boehringer Mannheim) according to the method of Iwasaki et al. (Monoclonal antibody, Kodansha, 1983). It was 15 cells after fusion
% ERD containing fetal bovine serum (MA Bioproducts)
The cells were suspended in F medium (Kyokuto Pharmaceutical Co., Ltd.) and cultured on a 96-well plate (Falcon). Next day, 100 μM
ERDF medium containing hypoxanthine, 0.4 μM aminopterin, 16 μM thymidine and 15% fetal bovine serum (hereinafter referred to as H
(AT medium) was added, and HAT medium was further added after 3 days. After 1 week, the medium was replaced with a medium obtained by removing aminopterin from the HAT medium (hereinafter referred to as HT medium), and then 3-4.
The HT medium was changed every other day. Approximately 2 weeks after the cell fusion, at the stage when the growth of the fused cells was visually confirmed, the anti-TIMP-1 antibody titer in the culture supernatant was measured by the enzyme immunoassay shown below to obtain the anti-TIMP-1 antibody. Screening for cells producing

(C) Anti-TIMP by enzyme immunoassay
Screening 1 antibody-producing strain In each well of the immunoplate (Maxisorp, Nunc),
50 μl of 1 μg / ml TIMP-1 solution was added and coated overnight at 4 ° C., phosphate buffered saline-0.05%
After washing once with Tween 20 (hereinafter referred to as PBS-Tween),
Blocking treatment was performed with PBS-Tween containing 1% bovine serum albumin. After the treatment, 50 μl of the culture supernatant of the hybridoma obtained in (b) above was added to the wells and reacted at 37 ° C. for 1 hour. After washing with PBS-Tween, peroxidase-labeled anti-mouse IgG goat antibody was added as a secondary antibody, and the mixture was reacted at 37 ° C for 1 hour. After washing, add 0.3 mg / ml 2,2'-azino substrate solution of peroxidase to the wells.
-Bis (3-ethylbenzothiazoline-6-sulfonic acid)
100 μl of diammonium salt-0.003% hydrogen peroxide-0.1 M phosphate citrate buffer (pH 4.0) was added and reacted for 30 minutes. After the reaction was stopped by adding 100 μl of a 1.5% oxalic acid solution, the absorbance at 415 nm was measured with a microplate absorptiometer (MPR-4, manufactured by Tosoh Corporation).

(D) High production of anti-TIMP-1 antibody
Cloning of Blidomas The cells in the wells in which the production of anti-TIMP-1 antibody was confirmed by the screening by the enzyme immunoassay method (c) above were then cloned by the limiting dilution method. That is, cells were seeded so that the number of cells contained in each well of a 96-well plate was 3, 1, and 0.3, and HT
Cultured in medium. After 10 to 14 days, when the growth of cells can be visually confirmed, the anti-TIM in the culture supernatant is
The P-1 antibody titer was measured by the enzyme immunoassay in the same manner as in (c) above. The cloning operation was repeated for the cells in the wells in which the production of anti-TIMP-1 antibody was observed, and the cloning was repeated until the production of anti-TIMP-1 antibody was finally confirmed in all the wells. In this way, finally 8 anti-TIMP-1 monoclonal antibody-producing hybridoma strains were obtained.

(E) Anti-TIMP-1 monoclonal antibody
Production of monoclonal antibodies by the hybridomas of the above 8 strains was carried out by adding E to each of the hybridoma strains to which fetal bovine serum was added.
It was carried out by culturing in an appropriate medium such as RDF medium or by culturing the hybridoma in the abdominal cavity of nude mice and then collecting ascites. When cultured in ERDF medium, the concentration of monoclonal antibody in the culture medium is 1 to 50 μg / ml.
Met. On the other hand, in culture in the peritoneal cavity of nude mice, pristane (2,6,10,14-tetramethylpentadecane) was intraperitoneally administered to each mouse in an amount of 0.5 ml, and after 1 to 3 weeks, 5 × 10 ⁶ hybridomas were intraperitoneally administered. If after administration of cells
The antibody concentration in the ascitic fluid on days 10 to 14 was 1 to 10 mg / ml.

(F) Purification of Monoclonal Antibody Monoclonal antibody contained in the culture supernatant or ascites fluid obtained in the above (e) is 0.
Protein A- equilibrated with 1 M phosphate buffer (pH 7.0)
Pour onto a Sepharose CL-4B column and wash with 0.1 M phosphate buffer (pH 7.0).
It was purified by eluting with 1 M glycine-hydrochloric acid buffer (pH 3.0).

Example 2- Identification of monoclonal antibody-The properties of the eight types of monoclonal antibodies thus obtained were examined by the methods described below. First, inhibition of TIMP-1 activity by each monoclonal antibody was analyzed by a collagenase activity measurement system using ¹⁴ C-collagen (K. Terato et al. (1976) Biochim. Biophys. Acta, 44
5 , 753-762). Next, human TIMP-1
Was subjected to SDS electrophoresis on a 12% polyacrylamide gel in the presence and absence of mercaptoethanol (UK Laemmli (1970) Nature, 227 , 680-685) and transferred to a nitrocellulose membrane, and each monoclonal antibody was used. Performed immunoblotting (edited by the Japanese Biochemical Society, sequel to Biochemistry Laboratory 2, Protein Chemistry (above) (198)
7) pp.41-57), and the reactivity of the antibodies was compared. Furthermore, the cross-reactivity of each monoclonal antibody with human TIMP-2 or bovine TIMP-1 was examined by the same enzyme immunoassay method as in (c) above. 40 per well
An immunoplate coated with ng of human TIMP-2 or bovine TIMP-1 was used, and the obtained monoclonal antibody was used as a primary antibody. As a substrate solution, 1 mg / ml o-phenylenediamine-0.012%
Hydrogen peroxide-50 mM phosphate citrate buffer (pH 5.
0), 1N sulfuric acid solution was used as a reaction stop solution. The measurement measured the light absorbency of 492 nm. From these results, 8
The respective monoclonal antibodies exhibited the characteristics shown in Table 1.

[0014]

[Table 1] Table 1 Properties of monoclonal antibody ──────────────────────────────────── Responsiveness Monochrome ───────────────────── TIMP-1 inhibitory activity Immunoblotting Enzyme immunoassay Null antibody ( _A492 ) Human bovine non-reduced reduced TIMP-2 TIMP-1 ──────────────────────────────────── ─ TIM-33 Yes Reaction reaction 0.00 0.34 TIM-211 No reaction No reaction 0.00 0.20 TIM-216 Yes Reaction No reaction 0.04 0.00 TIM-225 No reaction Reaction 0.00 0.00 TIM-280 No reaction No reaction 0.00 0.09 TIM-293 Yes No reaction 0.00 0.00 TIM-347 No reaction No reaction 0.00 0.00 TIM-625 No reaction 0.00 0.00 Clark monoclonal antibody RRU-T1 No reaction No reaction No reaction − RRU-T2 Yes Reaction reaction No reaction − ────────────────────────────────────

Example 3-Quantification of TIMP-1 (a) Combination of Monoclonal Antibodies For performing sandwich enzyme immunoassay of TIMP-1, a combination of the most suitable antibodies among the above eight types of monoclonal antibodies for measurement. I checked. Eight kinds of purified monoclonal antibodies (1 μ each) were added to the wells of the immunoplate.
After coating separately at a concentration of g / ml and blocking treatment, purified TIMP-1 was reacted. Next, each monoclonal antibody (labeled antibody or secondary antibody) biotinylated by the method of Kumagai and Okumura (Immunological Experiment Operation Method VIII, pp.2425-2430, 1979, edited by the Japanese Society of Immunology) was treated with peroxidase-labeled streptavidin ( (Manufactured by Vector Co., Ltd.) and reacted. A substrate solution was added to the washed well to carry out an enzymatic reaction, and the absorbance at 415 nm was measured. As a result, among 8 kinds of monoclonal antibodies, TIM-29
The monoclonal antibody of No. 3 was used as a immobilized antibody for coating, and biotinylated TIM-225 or TI was used.
The combination using the M-347 monoclonal antibody as the secondary antibody was the combination that enabled the most sensitive measurement.

(B) Enzyme labeling of monoclonal antibody The antibody of TIM-225 was prepared by the method of Imagawa et al. (Imagawa et al.
al. (1982) J. Appl. Biochem., 4 , 41).

(C) Sandwich enzyme immunoassay using monoclonal antibody TIM-293 was coated on the immunoplate at a concentration of 10 μg / ml, and the peroxidase-labeled TIM-225 of the above (b) was used as a secondary antibody and a substrate. Purified in the same manner as in Example 1 (c) using 1 mg / ml o-phenylenediamine-0.012% hydrogen peroxide-50 mM phosphate citrate buffer (pH 5.0) as a solution and 1N sulfuric acid solution as a reaction stop solution. The measurement of TIMP-1 was performed. The measurement is 49
Absorbance at 2 nm was measured. As a result, a method for measuring TIMP-1 that is 10 times more sensitive than the conventional method was established (FIG. 1). This measurement method uses 0.25 ng /
It is possible to accurately quantify TIMP-1 in an amount of ml or more. The TIMP-1 used by the present inventors for highly sensitive measurement
Since the mouse monoclonal antibodies TIM-225 and TIM-293 against TK differ from the mouse monoclonal antibody used by Clark et al. In their TIMP-1 inhibitory activity and reactivity in immunoblotting, they are considered to recognize different epitopes. It is considered that the difference produced 10 times higher sensitivity.

Example 4-Measurement of TIMP-1 in body fluids-Using the enzyme immunoassay method shown in Example 2,
When the amount of TIMP-1 in the serum of Nominari was measured, it was found to be 163
ng / ml and 221 ng / ml. Similarly, when the amount of TIMP-1 in urine of 5 healthy persons was measured, the results shown in Table 2 were obtained.

[0019]

[Table 2] Table 2 TIMP-1 concentration in urine of healthy subjects ──────────────────────── Test body TIMP-1 concentration ───── ─────────────────── A 1.8 ng / ml B 2.1 C 1.7 D 5.4 E 19.5 ────────── ───────────────

As described above, it is possible to accurately measure the concentration of TIMP-1 in body fluid, and because of its low concentration, it was impossible to measure TIMP-1 in urine by the conventional method.
It was revealed that the MP-1 concentration can be accurately measured by the measuring method according to the present invention.

[0021]

Industrial Applicability According to the present invention, it becomes possible to measure the concentration of human TIMP-1 contained in a large number of specimens easily, rapidly and specifically with high sensitivity in a minute range of 0.25 ng / ml or more. .

[Brief description of drawings]

FIG. 1 is a graph showing the results of the measurement method for TIMP-1 described in Example 3.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI Technical display location G01N 33/53 V 33/577 B // C12N 15/02 (C12P 21/08 C12R 1:91)

Claims (10)

[Claims] 1. The following properties a to e: a. Does not inhibit the collagenase inhibitory activity of human TIMP-1; b. IgG; c. Human TIMP-under reducing and non-reducing conditions
Immunoblotting after SDS electrophoresis of 1
Reacts with human TIMP-1; d. Does not react with human TIMP-2; e. A highly sensitive assay method for human TIMP-1, which comprises using an anti-human TIMP-1 mouse monoclonal antibody having no reaction with bovine TIMP-1 as a labeled antibody. 2. The human T according to claim 1, wherein the anti-human TIMP-1 mouse monoclonal antibody is TIM-225.
A highly sensitive method for measuring IMP-1. 3. The following properties af: a. Inhibit the collagenase inhibitory activity of human TIMP-1; b. IgG; c. Human TIMP-1 was subjected to SDS electrophoresis of human TIMP-1 under non-reducing conditions, followed by immunoblotting.
Reacts with 1; d. Human TIMP-1 was subjected to SDS electrophoresis of human TIMP-1 under reducing conditions, followed by immunoblotting.
Does not react with; e. Does not react with human TIMP-2; f. A high-sensitivity assay method for human TIMP-1, which comprises using an anti-human TIMP-1 mouse monoclonal antibody having no reaction with bovine TIMP-1 as an immobilized antibody. 4. The human T according to claim 3, wherein the anti-human TIMP-1 mouse monoclonal antibody is TIM-293.
A highly sensitive method for measuring IMP-1. 5. A highly sensitive assay method for human TIMP-1, which comprises using the antibody according to claim 1 as a labeled antibody and the antibody according to claim 3 as an immobilized antibody. 6. TIM-225 and TIM-293 are
The method for highly sensitive measurement of human TIMP-1 according to claim 5, which is used as a labeled antibody and an immobilized antibody, respectively. 7. An anti-human TIMP-1 mouse monoclonal antibody TIM-225. 8. An anti-human TIMP-1 mouse monoclonal antibody TIM-293. 9. A mouse hybridoma T producing an anti-human TIMP-1 mouse monoclonal antibody TIM-225.
IM-225-14. 10. A mouse hybridoma T producing anti-human TIMP-1 mouse monoclonal antibody TIM-293.
IM-293-21.