JP3017591B2 - Production method of anti-human TIMP-2 monoclonal antibody and use thereof - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a human tissue inhibitor of metalloproteinase-2 (TIMP-2).
The present invention relates to the production of the monoclonal antibody, the monoclonal antibody obtained thereby, and TIMP-2 present in human blood, body fluids, tissues, and human-derived tissue culture fluids using the monoclonal antibody. The present invention relates to a method of analyzing and measuring. More specifically, the present invention relates to an anti-human TIMP-2 monoclonal antibody, a method for producing the same, and a method for sensitively quantifying human TIMP-2 by an enzyme immunoassay based on a sandwich assay using the monoclonal antibody. The present invention relates to a method for measuring human TIMP-2, which comprises using two kinds of anti-human TIMP-2 monoclonal antibodies, an antibody to be bound to a solid support and an antibody to give an enzyme label.

[0002]

BACKGROUND ART Tissue inhibitor of metalloproteinase (TIMP-1) is used in cultured cells of humans and other animals (fibroblasts, tumor cells, chondrocytes, smooth muscle cells, endothelial cells, etc.), platelets, monocytes, It is a glycoprotein with a molecular weight of about 30 kDa produced by macrophages, dental pulp and the like. Conventionally, TIMP-1 has been called a collagenase inhibitor because it inhibits stromal collagenase activity. However, it is apparent that TIMP-1 inhibits various metalloproteinases (72 kDa gelatinase, 92 kDa gelatinase, and stromelysin) in addition to stromal collagenase. And TIMP-1 (Tissue Inhibitor of
Metalloproteinase). (Docher
ty, A. J. P. Et al., Ann. Rheum. Dis.
49.469-479, (1990); E. FIG. Caw
stone: in “Proteinase Inhib
itors ", ed. by AJ Barrett
Et al., Elsevier, Amsterdam, (198
6) p589. reference).

Recently, a metalloproteinase inhibitor called Tissue Inhibitor of Metalloproteinase-2 (TIMP-2) having a different molecular weight from TIMP-1 has been discovered (Steller-
Stevenson, W.C. G. FIG. J. et al. Biol. Ch
em. 246, 17374-17378, 1989; G
Oldberg, G .; I. Proc. Natl. A
cad. Sci. U. S. A. 86, 8207-821
1, 1989; Williamson, R .; A. Et al., B
iochem. J. 268, 267-274, 1990
reference).

[0004] The difference between TIMP-2 and conventional TIMP-1 is distinguished by the fact that it does not bind to the Con-A-Sepharose column in addition to the molecular weight. Human TIM
The primary structure of P-2 has been revealed by cDNA cloning (Boone, TC et al., Pro.
c. Natl. Acad. Sci. U. S. A. 87,
2800-2804, 1990). according to it,
Human TIMP-2 consists of 194 amino acid residues and has 38% homology with human TIMP-1.

[0005] Human TIMP-2 is a human TIMP.
It has the same 12 cysteine residues as -1 and its position is well conserved.

On the other hand, it has been recognized that TIMP-1 forms a complex with pro-92 kDa gelatinase and 92-kDa gelatinase, and that TIMP-2 forms a complex with pro-72 kDa gelatinase. No further details are disclosed (Wilhelm, SM et al., J. Biol. Ch.
em. 264, 17213-17221, 1989; W
ard, R .; V. Et al., Biochem. J. 278, 1
79-187, 1991).

[0007]

DISCLOSURE OF THE INVENTION The present inventors have succeeded in producing an anti-human TIMP-2 monoclonal antibody. When this anti-human TIMP-2 monoclonal antibody is used, human TIMP-2 present in a biological sample can be assayed by enzyme immunoassay by a sandwich method. Accordingly, the present invention relates to a monoclonal antibody having specificity for human TIMP-2, comprising DSGNDIYGNPIKR
IQ, DTLSTTQKKSLNHRYQ or YRG
A monoclonal antibody having reactivity with a polypeptide having the sequence of APPKQEFLDIED, and having no cross-reactivity with human TIMP-1, and a method for producing the monoclonal antibody. Using a combination of monoclonal antibodies, using a small amount of biological samples of human origin, with high accuracy,
An object of the present invention is to provide a simple and rapid method for specifically quantifying TIMP-2 present in a sample. This method comprises quantifying human TIMP-2 by an enzyme immunoassay using an anti-human TIMP-2 monoclonal antibody as an antibody to be bound to a solid phase carrier and an antibody to give an enzyme label, respectively. As the enzyme immunoassay according to the present invention, one exemplary method is shown as an example described later. For example, as a solid support, polystyrene beads, polycarbonate microplates, polypropylene microplates are used. Any suitably selected material such as a plate, a stick, and a test tube can be used.

On the other hand, anti-human TIMP-
2 As a monoclonal antibody, the antibody-containing substance was fractionated by adding ammonium sulfate, and then fractionated on an anion exchange gel such as DEAE-Sephacel or Pro.
IgG fraction purified by tein A column chromatography and F (a) obtained after pepsin digestion
b ') ₂ and the specific binding moiety Fab' obtained by reducing it can also be used.

Examples of labels that can be further used include enzymes (such as peroxidase, alkaline phosphatase, glucose oxidase and β- _D -galactosidase), chemical substances, fluorescent substances and radioisotopes.

As described above, human TIMP-2 is analyzed by a solid phase enzyme immunoassay using a combination of two types of monoclonal antibodies as an antibody to be bound to a solid support and an antibody to give an enzyme label. Can be measured. Hereinafter, the present invention will be described specifically with reference to examples. However, the present invention is not limited by these examples.

Example 1 Preparation of Anti-Human TIMP-2 Monoclonal Antibody (a) Preparation of Human TIMP-2 Polypeptide Boone, T .; C. Predict the sequence of the human TIMP-2 polypeptide from the sequence of the cDNA (Pr
oc. Natl. Acad. Sci. U. S. A. ,
87; 2800-2804, 1990). From the predicted amino acid sequence structure, the three polypeptides (P-1, P-2, and P-3) shown in Table 1 were each synthesized with a peptide synthesizer 9600 (Milligen / Biosearch).
, Followed by the C of each of their polypeptides.
A cysteine residue was introduced at the end. The obtained three kinds of crude polypeptides were each used in μBondasphere.
(Waters, 5μ, C18-100Å, 3.9 × 1
Purification was performed by high performance liquid chromatography using a 50 mm) column.

(B) Preparation of human TIMP-2 polypeptide and keyhole limpet hemocyanin complex A complex was prepared for each of the three polypeptides obtained in (a) as follows. 2mg keyhole limpet hemocyanin (KLH, Calbioc)
hem. ) With 1 ml of 0.1 M phosphate buffer (pH 7.
5) and 1.85 mg N- (ε-male)
imidocaproyloxy) succinimi
de was dissolved in 200 μl of dimethylformamide, and the mixture was incubated at 30 ° C. for 30 minutes. Next, the above mixture was added to a 0.1 M phosphate buffer (pH
Gel filtration was performed on PD-10 (Pharmacia) equilibrated in (7.0). Fractionating KLH to which maleimide is bound,
It was concentrated to 1.5 ml or less. Maleimide-bonded K
A 50-fold molar amount of each of the polypeptides synthesized in the above (a) with respect to LH was added to 1 ml of a 0.1 M phosphate buffer (pH 7.0).
0) was mixed with that dissolved. After incubation at 4 ° C for 20 hours, three kinds of polypeptide-KLH complexes were prepared.

[0013]

[Table 1]

(C) Preparation of antibody-producing cells 250 μg of each complex prepared by the method of the above (b) together with complete Freund's adjuvant was used for 8 weeks old Balb / c.
Each female mouse was intraperitoneally administered and immunized for the first time. Fifteen
On the day after, 200 μg of each complex dissolved in 0.1 M phosphate buffer (pH 6.0) was intraperitoneally administered to each mouse that had been initially immunized, and boosted. Further, after 38 days, 70 μg of each complex was intravenously and 130 μg as in the case of the booster.
g was intraperitoneally administered for final immunization. Three days later, the spleen was removed to prepare a spleen cell suspension.

(D) Cell fusion (1) The following materials and methods were used.

RPMI 1640 medium: RPMI 16
40 (Flow Lab.) With sodium bicarbonate (2
4 mM), sodium pyruvate (1 mM), potassium penicillin G (50 U / ml), streptomycin sulfate (50 μg / ml) and amikacin sulfate (100 μm).
g / ml), adjust the pH to 7.2 with dry ice,
The cells were sterilized and filtered through a 0.2 μm Toyo membrane filter.

NS-1 medium: fetal calf serum (MA Biopro) filtered and sterilized from the above RPMI 1640 medium
ducts) was added to a concentration of 15% (v / v).

PEG 4,000 solution: RPMI 16
Polyethylene glycol 4,000 (PE
G 4,000, Merck & Co. ) 50%
(W / w) to prepare a serum-free solution.

8-azaguanine-resistant myeloma cell SP
2 (SP2 / O-Ag 14) was
ted Method in Cellular Im
Munology (eds. Mishell, BB).
and Shihigi, S .; M. ), W.C. H. Free
man and Company 351-372, 1
980, with some modifications.

(2) Each nucleated spleen cell (viable cell rate: 100%) prepared in (c) was fused with myeloma cells (viable cell rate: 100%) at a ratio of 5: 1. In this case, the spleen cells and the myeloma cells were separately washed with the above-mentioned RPMI 1640 medium, and then both were suspended in the same RPMI 1640 medium and mixed at the above ratio. For each mixed medium, add a new RPM
I 1640 medium was prepared using the peptides P-1, P-2, P
-3, 25.8 ml for each complex, 28.
5 ml and 34.5 ml were added, and 10 × at 400 × g using a 50 ml polypropylene centrifuge tube (Iwaki Glass).
After centrifugation for a minute, the supernatant was completely removed by suction. A PEG 4000 solution heated at 37 ° C. was added to each of the obtained precipitated cells, and peptide P was added.
In the case of each complex of -1, P-2, and P-3, 2.3 ml, 4.8 ml, and 5.0 ml were added dropwise in 1 minute while stirring gently, and then 1 ml.
After stirring for minutes, each cell was resuspended and dispersed. To each of the obtained suspensions, the RPMI 1640 medium heated at 37 ° C. was added to 4.6 ml, 9.6 ml, and 10 ml of the respective complexes of the peptides P-1, P-2, and P-3. 0.0ml
After adding dropwise for 2 minutes using the amount of, the same fresh medium was further added to each of the complexes of the peptides P-1, P-2, and P-3 at 16.0 ml and 33.6 ml, respectively.
Using 35.0 ml, the cells were added dropwise for 2 to 3 minutes with constant stirring to disperse the cells. Each of the obtained suspensions was centrifuged at 400 × g for 7 minutes, and the supernatant was completely removed by suction. Next, the NS-1 medium heated at 37 ° C. was added to each of the obtained precipitated cells at 37 ° C. for 22.7 ml for each case of each complex of peptides P-1, P-2, and P-3.
0 ml, 40.0 ml were added immediately and the large clumps of cells were carefully dispersed by pipetting using a 10 ml pipette. For each of the obtained dispersions,
NS-1 medium heated at 37 ° C. was added to peptides P-1, P-2 and P-2.
-45.3 m for each case of each complex
1, 104 ml and 110 ml were added and diluted, and 6.0 × 10 ⁵ cells / 0.1 ml of cells were added per well to a polystyrene 96-well microwell (Iwaki Glass). Then, this was heated at 37 ° C. in 7% carbon dioxide / 93% air.
The culture was performed under a humidity of 100%.

(E) Selective growth of hybridoma on selective medium (1) The medium to be used is as follows. HAT medium: In addition to the NS-1 medium described in (d) above, hypoxanthine (100 μM) aminopterin (0.
4 μM) and thymidine (16 μM) were added. HT medium: HAT as described above except that aminopterin was removed.
It has the same composition as the medium.

(2) The next day (1) after the start of the culture of (d)
Day), two drops (about 0.1 ml) of HAT medium were added to the cells with a 10 ml pipette. On days 2, 3, 5, 8, and 11, half (0.1 ml) of the medium was replaced with fresh HAT medium, and on day 11, half of the medium was replaced with fresh HT medium. Usually, sufficient hybridoma growth is observed in about 2 weeks. Positive wells of all the hybridoma-grown wells were checked by the solid phase-antibody binding test (ELISA) described in the following section (f). Then the HT medium 1ml containing 10 ⁷ mouse thymocytes as feeder used after added to a polystyrene 24-well cell well (Iwaki Glass), it was transferred the entire contents of each positive hybridoma has been detected above. This is 7% as in the above (d).
The cells were cultured at 37 ° C. for about 2 to 3 days in the presence of carbon dioxide. When the hybridomas had grown sufficiently, the positivity was confirmed again by ELISA, and each was cloned by the limiting dilution method described in the following section (g). The residual solution used for cloning was transferred to a polystyrene 25 cm ² tissue culture flask (Iwaki Glass) to prepare a cryopreservation sample.

(F) Anti-human TIM by ELISA
Search for hybridoma producing P-2 antibody Anal. Biochem. 104, 205-214,
A slightly modified version of the method of Rennard et al. This method is suitable for detecting a hybridoma antibody. A 96-well microtitration plate (FlowLab.) Was coated with 50 ng of each polypeptide obtained in Example 1 (a). (E)
A part of the supernatant of the hybridoma growing well obtained in the above was added and incubated at room temperature for about 1 hour. Horseradish peroxidase-labeled goat anti-mouse immunoglobulin (Cappel Lab.) Was added as a secondary antibody, and the mixture was further incubated at room temperature for about 1 hour. Next, the degree of brown color generated by adding hydrogen peroxide and o-phenylenediamine as substrates was determined by measuring absorbance at 492 nm using a microplate reader (MPR-A4, Toyo Soda).

(G) Cloning After the above operation (e), since there is a possibility that two or more types of hybridomas may be growing in each well, cloning is performed by the limiting dilution method to obtain monoclonal antibody-producing hybridomas. get. NS-1 medium 1ml per feeder cloning medium containing 10 ⁷ mouse thymocytes were prepared as 36 wells of a 96-well micro-well, five wells per 36 well and 24-well, 1
And 0.5 hybridomas were added. On day 5, about 0.1 ml of NS-1 medium was added to all wells.
Sufficient hybridoma growth was observed 11 to 14 days after the start of cloning, and 12 wells forming colonies were selected and subjected to ELISA. If all the tested wells are not positive, the number of colonies in the antibody-positive well is confirmed, and two wells in which one colony is confirmed are selected from the wells and one of them is recloned. Finally, as shown in Table 2, P1-peptide, P2-peptide, P
A total of 51 clones producing monoclonal antibodies for each of the 3-peptides were obtained.

(H) In Vitro Propagation and In Vivo Propagation of Monoclonal Antibody Proliferation of the monoclonal antibody is carried out by a conventional method. That is, each of the obtained hybridomas is cultured in an appropriate culture medium such as NS-1 medium (in vitro propagation), and the culture supernatant is used for 10 to 10 minutes.
A monoclonal antibody having a concentration of 100 μg / ml was obtained. On the other hand, in order to obtain a large amount of antibodies, animals of the same strain as those derived from spleen cells and myeloma cells (Balb / c
Mice) were intraperitoneally administered with 0.5 ml of a tumor formation promoter pristane (2,6,10,14-tetramethylpentadecane, Aldrich Chem. Co.) per mouse. After 1 to 3 weeks, each hybridoma 1 × 10
^Seven were similarly intraperitoneally administered, and one to two weeks later, ascites containing 4 to 7 mg / ml of the monoclonal antibody produced in vivo could be obtained.

(I) Heavy chain and light chain of monoclonal antibody According to the above-mentioned ELISA method, the microtitration plate coated with each of the above-mentioned P1-peptide, P2-peptide and P3-peptide is obtained in (g) above. The culture supernatant of each monoclone was added. Next, PB
After washing with S, isotype-specific rabbit anti-mouse Ig antibody (Zymed Lab.) Was added. PBS
After washing with horseradish peroxidase, goat anti-rabbit IgG (H + L) antibody was added, and the heavy chain and light chain, respectively, using hydrogen peroxide and 2,2'-azino-di (3-ethylbenzothiazoline sulfate) as substrates. The chain was determined. The results are summarized in Table 2 below.

(J) Purification of Monoclonal Antibody Each ascites fluid obtained in the above (h) was fractionated with 40% saturated ammonium sulfate, and then 0.5% for IgG class antibody.
Adsorbed on a Protein A Affigel (Bio-Rad Lab.) Column equilibrated with a 1.5 M glycine-NaOH buffer (pH 8.9) containing M sodium chloride, washed with the above washing solution, and then washed with a 0.1 M citrate buffer ( pH5.
Purified by eluting with 0).

[0028]

[Table 2]

Example 2 Immunoblotting (a) Preparation of Material A human dermal fibroblast culture solution producing TIMP and TIMP-2 was used as a material. For culturing cells, MEME powder medium (Minimum Essential Media) is used.
m Eagle, Flow lab. ) Was dissolved in distilled water, 26.2 mM sodium bicarbonate was added, the pH was adjusted to 7.2 with dry ice, and the mixture was sterilized and filtered through a 0.2 μm Toyo membrane filter. America
n Type Culture Collection
Human skin fibroblasts CCD-41Sk purchased from
5% fetal calf serum and NEAA medium (NonE
sensual Amino Acids, Flow
lab. 5% CO 2/95 in MEME medium containing
The cells were cultured for 5 to 8 days at 37 ° C. in 100% humidity in 100% humidity. The cells collected by centrifugation at 800 rpm for 5 minutes were combined with 0.2% lactalbumin hydrolyzate, NEAA medium, 1.7 mM sodium bicarbonate and 10 U / m
ME containing recombinant human interleukin 1-α
The cells were similarly cultured in ME medium for 5 to 7 days. 800
After centrifugation at 5 rpm for 5 min, the supernatant was
The sample was concentrated to 0 times to obtain a sample for immunoblotting.

(B) Immunostaining The sample prepared in Example 2- (a) was subjected to polyacrylamide electrophoresis containing sodium dodecyl sulfate, and then subjected to the method of Tabe described in Cell Engineering 1 & 2, 1061-1068, 1983. Perform Western blotting,
After reacting with each monoclonal culture supernatant, peroxidase-labeled goat anti-mouse immunoglobulin (Cappel La
b. ) Was used to perform immunostaining by an indirect method.

Among the monoclonal antibodies obtained in Example 1 (j), CCD-
When a sample prepared from a 41Sk cell culture was used, clone numbers 68-6H4, 69-8F1, 69-9E were used.
6, 69-10B11, 69-24H6 and 67-4
Positive bands were confirmed in the antibodies of the six clones of H11.
Note that the CCD-41Sk cell culture solution contained in
Anti-human T invented by Hayakawa et al.
IMP monoclonal antibody (clone number 7-6C1)
There is TIMP-1 having a molecular weight of about 30 kDa that reacts with TIMP-1. However, among the monoclonal antibodies obtained in Example 1 (j) above, clone numbers 68-6H4, 69-8F
1, 69-9E6, 69-10B11, 69-24H6
And those obtained from 67-4H11 have a molecular weight of about 24.
specifically reacts with kDa TIMP-2 and has a molecular weight of about 30
It did not react with kDa TIMP-1. The results of the immunoblotting are shown in FIG.

Example 3 Sandwich Enzyme Immunoassay (a) Method for Placing Monoclonal Antibody on Solid Phase Example 2 of the IgG fraction obtained in Example 1- (j)
Clone numbers 67-4H11, 68-6H4 and 69-24 from the six fractions that were recognized as positive in (b)
H6 were selected and dissolved in a 0.1 M phosphate buffer (pH 7.5), respectively, and then dissolved in 100 μg / ml (A
₂₈₀ = 0.150), and then put on a microplate (module plate F-8H, Nunc).
The mixture was dispensed in μl portions and allowed to stand at 4 ° C. for 24 hours. The monoclonal antibody was then aspirated off and 0.1% bovine serum albumin (BSA), 0.1 M sodium chloride and 0.00
200 mM 10 mM phosphate buffer containing 5% thimerosal
1 and stored at 4 ° C.

(B) Enzyme-labeled monoclonal antibody (I
(1) Introduction of SH Group in IgG Fraction The above three IgG fractions were mixed with a 0.1 M phosphate buffer (pH
6.5), and S-acetylmercaptosuccinic anhydride was added at 100 times the molar amount of each IgG fraction.
Allowed to react for minutes. Next, 0.1 M Tris-HCl buffer (pH
7.0), 0.1 M ethylenediaminetetraacetic acid (EDT
A) (pH 6.0) and 1M hydroxylamine (p
H7.0) was 1/5, 1/10, 1
The reaction was stopped by adding a / 5-fold volume, and the SH group-introduced IgG fraction was separated by gel filtration using a Sephadex G50 column (Pharmacia) equilibrated with 0.1 M phosphate buffer (pH 6.0) containing 5 mM EDTA. I took it.

(2) Introduction of Maleimide Group of Peroxidase Apart from the above operation (1), a maleimide group was introduced into horseradish peroxidase (POD, Boehringer Mannheim) as described below. That is, POD
At a concentration of 10 mg / ml in a 0.1 M phosphate buffer (pH
7.0), and 25-fold molar amount of N- (ε-maleimidocaproyloxy) succinimide (EMCS, Dojin Kagaku) is added as a dimethylformamide solution to the POD, and the mixture is added at 30 ° C. for 30 minutes. Reacted. This was subjected to gel filtration using a Sephadex G-50 column (Pharmacia) equilibrated with a 0.1 M phosphate buffer (pH 6.0) to collect a maleimide group-introduced POD fraction.

(3) Preparation of IgG-POD complex The SH-group-introduced IgG fraction prepared as in (1) above was combined with the maleimide group-introduced POD fraction prepared as in (2) above. 1: added in a molar ratio of POD 3;
After the mixture was reacted at 4 ° C. for 20 hours, N-ethylmaleimide in an amount 10 times the molar amount of IgG was added to block unreacted SH groups. This was added to a 0.1 M phosphate buffer (pH 6.
After fractionating the IgG-POD complex fraction by gel filtration using an Ultrogel AcA34 column (Pharmacia) equilibrated in 5), 0.1% BSA and 0.005% thimerosal were added and stored at 4 ° C.

(C) Two-step sandwich assay A TIMP-2-containing human skin fibroblast (CCD-41Sk cell) culture solution prepared in Example 2- (a) was concentrated about 100-fold by ultrafiltration. After that, the mixture was diluted with 10 mM phosphate buffer (pH 7.5) containing 1% BSA and 0.1 M sodium chloride, and 100 μl of the mixture was added to the monoclonal antibody-binding microplate prepared in the above (a).
Was added. After reacting for 1 hour at room temperature, the reaction solution was removed,
10 mM phosphate buffer containing 0.1 M sodium chloride (p
H7.0) After washing three times with 300 μl, the above (b) (3)
The IgG-POD complex prepared in the section was diluted with the above-mentioned buffer solution containing BSA to 1 μg / ml, and 100 μl each was dispensed to the microplate where the reaction was completed. 1 at room temperature
After standing for a period of time, the reaction solution was removed, and 300 μl of 10 mM phosphate buffer (pH 7.0) containing 0.1 M sodium chloride was added.
Washed twice. Further, o-phenylenediamine (Sigma Chem. Co.), which is a POD substrate, dissolved in a citric acid-phosphate buffer (pH 4.6) containing 0.02% hydrogen peroxide at a concentration of 2 mg / ml was microplated. Was dispensed at 100 μl.

After standing at room temperature for 15 minutes, 2N sulfuric acid 10
The reaction was stopped by dispensing 0 μl each. The _A492 value of the reaction stop solution was measured using a microplate reader (MPR-A4, Toyo Soda). Antibodies from clone no 67-4H11 as shown in Table 3 as IgG-POD conjugate, when using antibodies from clone no 68-6H4,69-9E6 and 69-10B11 as a solid _{phase, A 492} value It was confirmed to show a high value of 1.0 or more.

[0038]

[Table 3]

Example 4 Quantification of Human TIMP-2 (a) Selection of Antibody As described in Table 3, as a combination of an antibody for solid phase plate and an antibody for enzyme labeling, clone No. 68-6H4:
67-4H11, 69-9E6: 67-4H11, 69
-10B11: 67-4H11, 68-6H4: 69-
24B6 were selected as candidates. In order to determine which of the four types of combinations is suitable for the one-step sandwich measurement method, see Example 2 above.
Using a human fibroblast culture supernatant containing TIMP-2 of item (a) as a sample, one-step sandwich measurement was performed by the method shown in Table 4. As a result, clone number 68-6H4
Clone No. 67-4H using the antibody from
The _A492 value was highest when the antibody from 11 was used as an IgG-POD conjugate.

The above-mentioned hybridoma of clone number 67-4H11 was obtained from NIKKEI No. 12690 (FERM).
P-12690) and the clone number 68-
The 6H4 hybridoma has been deposited with the institute under the accession number of NIKKEI No. 12691 (FERMP-12691).

[0041]

[Table 4]

1) 1 μg / ml IgG-POD complex solution (lysis buffer: 30 mM phosphate buffer (pH 7.0) containing 1% BSA, 0.1 M sodium chloride and 10 mM EDTA) 2) physiological saline 3) 2 mg / Ml o-phenylenediamine solution (dissolution buffer: citric acid-phosphate buffer containing 0.02% hydrogen peroxide (pH 4.6)) 4) 2N sulfuric acid

(B) Separation and purification of human TIMP-2 Human placenta was used as a material. Five placentas were minced, 1 mM calcium chloride, 0.1 M sodium chloride and 0.005
% Brij35 in 20 mM Tris-HCl buffer (p
H7.5), thoroughly mixed with a spatula, and centrifuged (10,000 rpm, 40 minutes, 4 ° C.). The supernatant obtained is 80% saturated ammonium sulfate salting-out, Ultrogel AcA54
Cam (Pharmacia), Red Agarose Column (S
igmaChem. Co. ), Cu ²⁺ saturated chelating Sepharose column (Pharmacia), anti-human TI
MP-1 monoclonal antibody-conjugated Sepharose 4B column (using clone number 7-21B12;
19392) and high performance liquid chromatography (reverse phase column, S
yncropak RP-4 (Synchrom)) to obtain human TIMP-2. At the time of each separation and purification step, the aforementioned clone No. 68-6 was added to the solid phase plate.
Using antibodies from H4, using antibodies from IgG-POD conjugate the clone number 67-4H11 to measure the ₄₉₂ value _A by 1 step sandwich assay was it as an index.

The TIMP-2 obtained by the purification is described in J. Am.
Mol. Biol. 80, 579 to 599; a single band with a molecular weight of about 24 kDa was shown by sodium dodecyl sulfate-polyacrylamide electrophoresis according to the method of Laemmli et al.

(C) Preparation of standard curve A standard curve was prepared using the human TIMP-2 obtained in the above (b). As shown in FIG. 2, 6.3-50 ng /
Linearity was observed in the range of ml (63-500 pg / well). The quantitative sensitivity was about 16 pg.

(D) TIMP- in serum and synovial fluid
Quantification of TIM in serum of 2 samples from healthy volunteers, 1 sample of serum from patients with liver disease, 1 sample of synovial fluid from patients with meniscal injury, 1 sample from synovial fluid from patients with rheumatoid arthritis, and 1 sample from synovial fluid from osteoarthritis
P-2 was quantified. Table 5 shows the results.
The synovial fluid was measurable above the quantitative sensitivity. However, the specimen is not limited to these.

[0047]

[Table 5]

[Brief description of the drawings]

FIG. 1 is a western blot diagram of a culture medium of human skin fibroblast CCD-41Sk, which shows an anti-human TIMP-1 monoclonal antibody (clone number 7-6C1, lane 1) and an anti-human TIMP-2 monoclonal antibody (clone number 68). -6H4, lane 2), anti-human TIMP-
2 monoclonal antibody (clone number 69-8F1, lane 3), anti-human TIMP-2 monoclonal antibody (clone number 69-9E6, lane 4), anti-human TIMP
-2 monoclonal antibody (clone number 69-10B1
1, lane 5), anti-human TIMP-2 monoclonal antibody (clone no. 69-24H6, lane 6), and anti-human TIMP-2 monoclonal antibody (clone no. 6)
7-4 is a photograph instead of a drawing, showing each lane stained using 7-4H11, lane 7).

FIG. 2 shows that an antibody from clone No. 68-6H4 was used as a solid phase, and an antibody from clone No. 67-4H11 was used as an IgG.
FIG. 4 shows a standard curve of human TIMP-2 subjected to a one-step sandwich assay using the POD complex.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FIC12R 1:91) (72) Inventor Noboru Fujimoto 4-18 Higashiyukicho, Tonami-shi, Toyama Prefecture (72) Inventor Zhang Jian Takaoka, Toyama Prefecture 555-4, Ichinamioka (72) Kayoko Yonezawa, Inventor Kayoko Yonezawa 405 steps, one step, Fukuoka-cho, Nishitonami-gun, Toyama Prefecture No. 335-1, Nomura, Takaoka-shi, Toyama (72) Inventor Shinichi Yoshida 4-13-16 Nakajima, Toyama-shi, Toyama (72) Inventor Choichiro Shimazaki 23-14, Southern No. 1, Omachi 1, Toyama-shi, Toyama (56) Reference Document WO 90/14363 (WO, A1) WO 90/11287 (WO, A1) (58 ) investigated the field (Int.Cl. ^7, DB name) BIOSIS (DIALOG) CA (STN ) REGISTRY (S N) WPI (DIALOG)

Claims (2)

(57) [Claims] 1. Specificity for human TIMP-2 obtained by culturing a hybridoma obtained by fusing mouse antibody-producing cells with mouse myeloma cells and purifying a monoclonal antibody from a culture solution or mouse ascites A monoclonal antibody having DSGNDIYGNP
A monoclonal antibody reactive with a polypeptide having the sequence of IKRIQ, DTLSTTQKKSLNHRYQ or YRGAAPPKQEFLDIED, and having no cross-reactivity with human TIMP-1. 2. A method for quantifying human TIMP-2 by an enzyme immunological method by a sandwich method, wherein a combination of any two monoclonal antibodies according to claim 1 is used. Law.