JPH06300757A - Immunoassay for complex of human interstitial collagenase and inhibitor and application to clinical diagnosis - Google Patents

Abstract

PURPOSE:To determine a human active MMP-1-TIMP-1 complex in a sample material by using a monoclonal antibody which connects specifically with each of human MMP-1 and TIMP-1. CONSTITUTION:A monoclonal antibody specifically bonding with human MMP-1 is prepared by using purified pro-MMP-1 as an immune source which is obtained by purifying human pro MMP-1 from a supernatant fluid of normal dermoblasts. The monoclonal antibody reacts specifically to the pro-MMP-1 and active MMP-1. For a solid carrier at the determination according to this immunoassay, a ball of polystyrene or the like well adsorbing protein such as the antibody, a test tube, etc., are optionally selected and used. Meanwhile, an antibody for labelling is an IgG fraction which is obtained by fractioning a substance including the antibody by ammonium sulfate and purifying with an anion exchanger, for instance, an enzyme, a chemical substance or the like. A human active MMP-1-TIMP-1 complex in the blood serum of a patient like a cancer patient who shows sthenia in the acitivity of collagenase is thus determined and the method is applied to diagnose a group of diseases.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to medical and physiological fields, and human interstitial collagenase [enzyme number: EC 3.
4.24.7, human matrix metalloproteinase-1, human MMP-1 (Matrix, 1 (Supp
l), 421-424, 1992)] and the immunological quantification method of the complex with the inhibitor.

More specifically, the present invention relates to human MM
By using a monoclonal antibody that specifically binds to P-1 and a monoclonal antibody that specifically binds to inhibitor-1 or -2 (TIMP-1 or TIMP-2) of matrix metalloproteinases (MMPs), Human active MMP-1-TIMP-1
Recombinant or human active MMP-1-TIMP-2
It relates to a method for immunologically quantifying a complex.

[0003]

BACKGROUND ART The extracellular matrix is composed of adhesive glycoproteins such as collagen, proteoglycan, elastin, fibronectin and laminin (Shimooka et al., 34, 1719-1724). Matrix metalloproteinases (MMPs) are involved in the degradation of these matrix components, and one of them, MMP-1, is released extracellularly as a latent form.
The latent MMP-1 itself does not participate in the decomposition of matrix components, and plasmin and stromlysin-in the body
1 (MMP-3) activated by limited decomposition, or activated in the laboratory by a thiol group-reactive organomercury compound to become active MMP-1.
It is known to have degrading activity for type II, type II and type III collagens, and active type MMP-1
Is an active MMP-1-TIMP-1 complex or an active MMP-1-TIMP-2 by the binding of TIMP-1 or the binding of TIMP-2 that specifically inhibits α ₂ -macroglobulin and MMPs activity. It is known that a complex is formed and its MMP-1 activity is inhibited (Clark et al., Matrix, 12, 108-11).
5, 1992).

Measurement of MMP-1-TIMPs complex in human body fluid was carried out by active MM which was involved in decomposition of matrix components.
It can be a means to know the P-1 amount. Therefore MM
By quantifying the P-1-TIMPs complex, MM
Diseases in which P-1 activity is enhanced, such as arthrosis, cancer invasion,
It becomes possible to diagnose or monitor pathological conditions such as metastasis and pulmonary fibrosis.

Conventionally, as a method for measuring MMP-1, it has been known that measurement is indirectly performed by measuring collagenolytic activity. However, in tissue or body fluid,
Since other MMPs and TIMPs coexist, and they also have a collagen degradation activity or a degradation inhibitory activity thereof, in order to measure the amount of MMP-1 with good accuracy, M
It is necessary to separate MP-1 with high purity, but the separation requires complicated operations, and it is difficult to obtain a highly pure product even by the complicated operations.

Recently, Omoto et al. Have carried out quantification of proMMP-1 using an antibody against proMMP-1 having a molecular weight of 52 kilodaltons (kDa) (quantitative sensitivity: about 3.1 ng).
/ Ml, measurement time 3.5 hours). (International publication number WO 91/12333, 1991). In that report it is not mentioned whether the antibody used recognizes MMP-1 in complex with TIMPs. In addition, Cooksley et al. Used a monoclonal antibody against MMP-1 and a monoclonal antibody against TIMP-1 by the streptavidin peroxidase-biotin complex method (SABC) method, and MM.
The P-1-TIMP-1 complex was measured, and it was reported that the measurement range was 4.5 to 72.5 ng / ml (Matrix, 10, 285-291, 1990).
In this report, activated MMP-1 and T were used as standard substances.
Quantitation is carried out using TIMP-1 as a standard using a solution in which IMP-1 is simply mixed at a ratio of 1: 1, but this method is clearly incomplete as a quantification method. Clark et al. Used a polyclonal antibody against porcine MMP-1, a monoclonal antibody against human TIMP-1, and a polyclonal antibody against immunoglobulin of rabbit to react with MM for about 6 hours,
The P-1-TIMP-1 complex was measured, and it was reported that the measurement range was 5 to 50 ng / ml (Ma.
trix, 12, 108-115, 1992). However, the method in this report uses a solution in which activated porcine MMP-1 and TIMP- are simply mixed as a standard substance, and is based on TIMP-1 and porcine MMP-1. To MMP in human blood
This is clearly incomplete as a method for quantifying the -1-TIMP-1 complex. Further, since all of these methods are SABC methods or methods using a rabbit immunoglobulin antibody for the purpose of increasing the sensitivity, the operation is complicated and requires a long time.

On the other hand, active MMP-1 involved in the decomposition of matrix components also forms a complex with TIMP-2,
The ratio of the combined amount of TIMP-1 and TIMP-2 is still unknown. In addition, to date, active MMP-1-
There is no report on a method for detecting the TIMP-2 complex or a quantification method.

The present inventors previously obtained a monoclonal antibody against a polypeptide of human MMP-1 (JP-A-4-183397), and further purified proMMP-1 purified from human dermal fibroblast culture supernatant. The monoclonal antibody obtained as an immunogen was successfully obtained.

The object of the present invention is (1) a monoclonal antibody which specifically binds to human MMP-1 and TIMP-
A human active MMP-1-TIMP-1 in a test sample using a monoclonal antibody that specifically binds to 1
To provide an excellent method of quantifying the complex,
(2) Using a monoclonal antibody that specifically binds to human MMP-1 and a monoclonal antibody that specifically binds to TIMP-2, human active M in a test sample
An object is to provide an excellent method for quantifying MP-1-TIMP-2 complex. Further, it is an object of the present invention to provide a diagnostic method and a diagnostic agent capable of diagnosing diseases such as arthrosis, various cancers and metastatic cancers using the above-mentioned quantitative method.

[0010]

DISCLOSURE OF THE INVENTION The present invention provides the following quantitative methods 1, 2.

1. Prepared from human active collagenase and TIMP-1 using a monoclonal antibody that specifically binds to human active collagenase and a monoclonal antibody that specifically binds to TIMP-1 (inhibitor of metalloproteinase-1). Human active collagenase-TIMP-1 complex is characterized by immunologically measuring the human active collagenase-TIMP-1 complex in human body fluid using the human active collagenase-TIMP-1 complex as a standard substance. Complex quantification method.

2. Prepared from human active collagenase and TIMP-2 using a monoclonal antibody that specifically binds to human active collagenase and a monoclonal antibody that specifically binds to TIMP-2 (inhibitor of metalloproteinase-2). Human active collagenase-TIMP-2 complex is characterized by immunologically measuring the human active collagenase-TIMP-2 complex in human body fluid using the human active collagenase-TIMP-2 complex as a standard substance. How to quantify the body.

Human M used in the quantification method of the present invention
A monoclonal antibody that specifically binds to MP-1 is a monoclonal antibody prepared by purifying human pro MMP-1 from a normal skin fibroblast culture supernatant and using this purified pro MMP-1 as an immunogen. -1 and activated MMP-1 specifically.

In the quantification method of the present invention, an immunological measurement method is used, and the solid phase carrier in that case is a ball made of polystyrene, polycarbonate, polypropylene, or polyvinyl that well adsorbs proteins such as antibodies. Various materials and forms such as microplates, sticks, microparticles or test tubes can be arbitrarily selected and used. On the other hand, as the antibody to which the labeled substance is added, DEAE-Sep is used after the antibody-containing substance is fractionated with ammonium sulfate.
An anion exchanger-purified IgG fraction such as hacel, and a specific binding portion Fab ′ obtained by reduction after digestion with pepsin can be used. Examples of labeled substances in these cases include enzymes (peroxidase, alkaline phosphatase, β-D-galactosidase, etc.), chemical substances, fluorescent substances, radioisotopes and the like.

Diseases in which collagenase activity is enhanced, for example,
The human active MMP-1-TIMP-1 complex or human active MMP-1-TIMP-2 complex in the serum or synovial fluid of patients with arthropathy, cancer, metastatic cancer, etc. It can be applied to the diagnosis or monitoring of the above-mentioned disease group by quantifying using each of the quantification methods described above. Examples of the present invention will be shown below.

[0016]

【Example】

Example 1 Preparation of antigen (a) Preparation of human normal skin fibroblast-derived pro-MMP-1 1) Cell culture MEM Eagle medium: Minimum Essen
tial Medium Eagle (modify
d) with Earle's salts (Flow
Lab. Sodium bicarbonate (24 mM) is added to
The pH was adjusted to 7.2 with 1N sodium hydroxide or 1N hydrochloric acid, and sterilized with 0.2 μm Toyo membrane filter. When using, add non-essential amino acid,
This was M Eagle medium.

Human normal skin fibroblast CCD-41SK
(ATCC No. CRL1505) was cultured at 37 ° C. for 3 to 4 days in the presence of 5% carbon dioxide using MEM Eagle medium containing 10% fetal calf serum (FCS).
The culture supernatant was removed, and 0.1 mM sodium chloride-containing 10 mM
An appropriate amount of phosphate buffer solution (pH 7.4) (PBS) was added and gently shaken to wash. Next, PBS containing 0.125% trypsin and 0.01% sodium ethylenediaminetetraacetate (EDTA) was added, and the cells were gently shaken to remove the cells from the culture flask (within 1 minute), and then 10% FC was added.
An appropriate amount of S-containing MEMeagle medium was added. After centrifugation,
The supernatant was removed, and an appropriate amount of 10% FCS-containing MEMeagle medium was added to suspend the cells. Next, about 2 × 10 ⁵ cells / ml of cells were added to another culture flask, and the cells were cultured at 37 ° C. for 3 to 4 days in the presence of 5% carbon dioxide.

2) Cell Stimulation The cultured cells obtained in 1) were treated with interleukin 1α (IL
Cell stimulation is performed by acting -1α). Decant the culture solution cultivated in 1) above, then about 100 ml
MEM Eagle medium was added, and lactalbumin hydrolyzate (Gibco) and IL-1α (Genzym) were added.
e) was added to a final concentration of 0.2% and 10 U / ml, respectively. 7-1 at 37 ° C in the presence of 5% carbon dioxide
After leaving still for 0 days, the culture supernatant was collected and used as a material for purifying human pro MMP-1.

3) Purification of human pro-MMP-1 The culture supernatant prepared in 2) above was subjected to ultrafiltration (Toyo Roshi U.
It was concentrated with HP-76) and dialyzed against 50 mM Tris-hydrochloric acid buffer (pH 7.4) containing 50 mM sodium chloride. The solution after dialysis is treated with heparin-Sepha.
rose CL-6B column (Pharmacia F
ine Chemicals), 50 mM Tris-HCl buffer containing 0.1 M sodium chloride (pH 7.
After washing with 4), the adsorbed protein was washed with 50 M Tris-hydrochloric acid buffer containing 0.5 M sodium chloride (pH 7.
It was eluted in 4). The elution fraction was 0.15 M sodium chloride, 5 mM calcium chloride, 0.01% polyoxyethylene lauryl alcohol ether (Bridge-35),
It was dialyzed against 50 mM Tris-HCl buffer (pH 7.5, buffer A) containing 0.02% sodium azide. Next, Sephacryl-S-200 (Pharmci
a Fine Chemicals), buffer A
Was used for gel filtration. The fraction having human MMP-1 activity was dialyzed against 20 mM Tris-HCl buffer (pH 7.5, buffer B) containing 50 mM sodium chloride, 1 mM calcium chloride and 0.01% Bridge-35. Furthermore, this fraction was mixed with Green A Actigel-
The mixture was applied to an ALD column (Stergene), and the adsorbed protein was eluted by applying a gradient from the sodium chloride concentration of buffer B to 50 mM to 1M. The human pro-MMP-1 thus obtained was subjected to polyacrylamide gel electrophoresis (SD) containing sodium dodecyl sulfate.
S-PAGE), molecular weight 57 kDa and 52 kDa
Band was shown.

(B) Purification of TIMP-1 1) Purification of bovine TIMP-1 derived from bovine dental pulp J. Biochem. 96, 395-404, 1984
Con A-Sepharose, Ultroge from a culture solution obtained by culturing the root pulp of bovine non-erupted tooth in MEM Eagle medium (Nissui Pharmaceutical Co., Ltd.) according to the method of the present invention described in 1.
Collagenase inhibitors were purified using lAcA44 and DE-52 cellulose columns. Purified inhibitors are described in J. Mol. Biol. 80, 579-5
99 (1973) and the method of Laemmli et al., The molecular weight was about 32 kDa when examined by SDS-PAGE.
Showed a single band.

2) Purification of human placenta-derived human TIMP-1 J. Immunol. Meth. 127, 103-10
Human TIMP-1 was purified from human placenta according to the method of Kodama et al.

(C) Purification of Human TIMP-2 Derived from Human Placenta Placenta was cut into small pieces, 1 mM calcium chloride, 0.1 M sodium chloride, 0.05% bridge-35, 1 mM N-ethylmaleimide, 5 mM EDTA, 5 mM hydrochloric acid. 20 mM Tris-HCl buffer containing benzamidine (pH 7.
4) was added and stirred, and the mixture was left standing for 30 minutes. Then, 10,000 rpm with a high-speed cooling centrifuge (Hitachi)
The mixture was centrifuged at 4 ° C. for 50 minutes to obtain a supernatant. The same operation was repeated once more, and the supernatants were pooled. next,
This supernatant was applied to anti-TIMP-2 monoclonal antibody (clone No. 67-4H11) -bound Sepharose 4
After adsorbing to B column, 1 mM calcium chloride,
0.1 M sodium chloride, 0.005% bridge-35
The antibody-bound Sepharose 4B gel was washed on a glass filter with 20 mM Tris-hydrochloric acid buffer (pH 7.4, buffer C) contained. Anti-T used here
IMP-2 monoclonal antibody (clone No. 67-
4H11, Research Institute for Microfabrication, FERMP-12690)
Is a human TIMP-2 polypeptide (YRGAAPPK
QEFLDIED) and keyhole limpet hemocyanin (KLII) as a carrier protein were bound as immunogens and prepared according to the method described in Example 2.

Further, buffer C and 1 mM calcium chloride,
0.1 M sodium chloride, 0.005% bridge-35
After alternately washing with 0.1 M acetate buffer (pH 5.5) containing the gel, the gel was repacked in the column and the adsorbed protein was adjusted to 1 mM.
Calcium chloride containing 0.005% Bridge-35.
Elute with 1 M glycine-hydrochloric acid buffer (pH 2.5) and immediately elute with 1 mM calcium chloride, 0.005% bridge-3.
It was neutralized with 5M-containing 3M Tris-HCl buffer (pH 7.5). Next, this elution fraction was subjected to ultrafiltration (Toyo Filter Paper UHP
-43) and concentrated with Ultrogel AcA54
Gel filtration with (LKB). A for each fraction
₂₈₀ was measured and the TIMP-2 containing fractions were collected. SD
The resulting human TIMP-2 was purified on S-PAGE into a single band with a molecular weight of approximately 24 kDa.

Example 2 Preparation of Anti-Human MMP-1 Monoclonal Antibody (a) Immunization Method and Preparation of Spleen Cells CCD-Purified by the method described in Example 1, (a)
41 SK cell-derived human pro MMP-1, 187 μg,
8 weeks old Balb / with complete Freund's adjuvant
c It was intraperitoneally administered to female mice and used as the initial immunization. After 15 days, human Bro MMP-1, 3 dissolved in 10 mM phosphate buffer (pH 7.0) containing 0.15 M sodium chloride was used.
1 μg was intraperitoneally administered to the first immunized mouse to boost the immunization. 38 days later, human proM
MP-1,27 μg was intravenously administered and the same 8 μg was intraperitoneally administered to give the final immunization. Three days later, the spleen was removed and a splenocyte suspension was prepared.

(B) Cell fusion 1) Material RPMI 1640 medium: RPMI 1640 (Fl
ow Lab. ) To sodium bicarbonate (24 mM), sodium pyruvate (1 mM), potassium penicillin G (50 U / ml), streptomycin sulfate (50 μg)
/ Ml) and amikacin sulfate (100 μg / ml) were added, pH was adjusted to 7.2 with dry ice, and sterilization filtration was performed with a 0.2 μm Toyo Membrane filter.

NS-1 medium: FCS which had been sterilized and filtered was added to the RPMI 1640 medium so as to have a concentration of 15% (v / v).

PEG 4,000 solution: RPMl 164
Polyethylene glycol 4,000 (PEG
4,000, Merck & Co. ) 50% (w / w)
To prepare a serum-free solution.

8-Azaguanine-resistant myeloma cell SP
2 (SP2 / 0-Ag14) fusion is Select
ed Method in Cellular Imm
unology (eds. BB Misshella
nd S. M. Shiigi), W.W. H. Freema
n and Company (1980), 351 to 3
The method of Oi et al.

2) Cell fusion method Nucleated spleen cells (live cell ratio 100) prepared in the above (a)
%) And myeloma cells (live cell ratio 100%) 5: 1
Fused at a rate of. Spleen cells and myeloma cells were washed separately in RPMI 1640 medium as described above, then suspended in the same medium and mixed at the above ratio for fusion. Using a polypropylene centrifuge tube (Iwaki Glass) with a capacity of 250 ml, 400 xg in 40 ml of RPMI 1640 medium,
After centrifugation for 10 minutes, the supernatant was completely aspirated. To the precipitated cells, 6.0 ml of a PEG 4,000 solution heated at 37 ° C. was added dropwise over 1 minute with gentle stirring, and further stirred for 1 minute to resuspend and disperse the cells. Next, RPMI heated at 37 ℃
6.0 ml of 1640 medium was added dropwise for 1 minute. After repeating this operation once more, 42.0 ml of the same medium was added to
The cells were dispersed by dropwise addition for 3 minutes with constant stirring. This was centrifuged at 400 × g for 10 minutes, and the supernatant was completely removed by suction. Next, 60 ml of 37 ° C. warmed NS-1 medium was rapidly added to the precipitated cells, and 10 ml of a large lump of cells was added.
Using a pipette, carefully pipette to disperse. Further, 120 ml of the same medium was added to dilute, and 6.0 × 10 ⁵ cells / 0.1 ml of cells were added to a 96-well microwell made of polystyrene (Iwaki Glass). The above-mentioned microwell containing the cells was cultured in 7% carbon dioxide / 93% air at a temperature of 37 ° C. and a humidity of 100%.

(C) Selective Growth of Hybridomas by Selective Medium 1) Medium Used HAT medium: The NS-1 medium described in (b) above was further supplemented with hypoxanthine (100 μM) aminopterin (0.4 μm).
μM) and thymidine (16 μM).

HT medium: It has the same composition as the above HAT medium except that aminopterin was removed.

2) Selection of hybridoma On the next day (1st day) after the start of the culture in (b) above, 2 drops (about 0.1 ml) of HAT medium was added to the cells with a Pasteur pipette. Half of the medium (0.1 m
l) was replaced with fresh HAT medium and on day 14 half of the medium was replaced with fresh HT medium. Half of the medium was replaced with fresh IIT medium every 3-4 days thereafter. Usually about 2
Sufficient hybridoma growth is observed in a week. For all wells in which hybridomas grew, positive wells were checked by the solid phase-antibody binding test method (ELISA) described in (d) below. Next, 1 ml of HT medium containing 10 ⁷ mouse sternal cells was added to a polystyrene 24-well cell well (Iwashiro Glass) as a feeder, and the entire contents of each positive hybridoma detected above were transferred. In the same manner as in (b) above, in the presence of 7% carbon dioxide gas,
The cells were cultured at 37 ° C for about 1 week. In the meantime, 0.5 ml of the supernatant in each well was replaced with 0.5 ml of fresh HT medium once or twice. ELISA when hybridoma is fully grown
Reconfirm the positive by the method, and for each of the following (e)
Cloning was performed by the described limiting dilution method. In addition,
The residual liquid after cloning is made of polystyrene 25 cm
² Transferred to a tissue culture flask (Iwaki Glass) and prepared a sample for cryopreservation.

(D) Anti-human MMP by ELISA method
-1 Search for antibody-producing hybridoma Anal. Biochem. 104, 205-214
The method of Rennard et al. Described in (1980) was slightly modified. This method is suitable for detecting hybridoma antibodies. 96-well microtitration plate (FlowLab.) With 50 ng of human pro-MM
Coated with P-1, then the uncoated part was blocked with 1% bovine serum albumin (BSA). A portion of the supernatant of the hybridoma growth well obtained in (c) above was added to this 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 substrate, hydrogen peroxide and o-phenylenediamine were added, and the degree of brown produced was measured by a microplate reader (MPR-A
(4, Tosoh) was used to measure and determine the absorbance at 492 nm.

(E) Cloning After the operation of (c) above, there may be two or more hybridomas growing in each well, so cloning is performed by the limiting dilution method to obtain a monoclonal antibody-producing hybridoma. get. A cloning medium containing 10 ⁷ mouse thymocytes as a feeder per 1 ml of NS-1 medium was prepared, and 5 wells per well in 36 wells, 36 wells and 24 wells of 96-well microwells.
And 0.5 hybridomas were added. The fifth day,
On day 12, all wells were supplemented with about 0.1 ml of NS-1 medium. A sufficient amount of hybridoma growth was observed 14 to 15 days after the start of cloning, and an ELISA method was performed on a group in which the colony formation negative well was 50% or more. When all the tested wells are not positive, the number of colonies in the antibody-positive wells is confirmed, and 4 to 6 wells in which one colony is confirmed are selected and recloned.
Finally, as shown in Table 1, monoclonal antibody-producing hybridomas against human proMMP-1 were obtained.

(F) Proliferation of monoclonal antibody in vitro and in vivo Proliferation of the monoclonal antibody is carried out by a conventional method. That is, each of the obtained hybridomas was cultured (in vitro growth) in an appropriate culture medium such as NS-I medium, and 10 to 10
It was possible to obtain a monoclonal antibody at a concentration of 100 μg / ml. On the other hand, in order to obtain a large amount of antibody, an animal syngeneic with the origin of splenocytes and myeloma cells (Balb / c
To each mouse, 0.5 ml of a tumor formation promoter pristane (2,6,10,14-tetramethylpentadecane, Aldrich Chem.) Was intraperitoneally administered to each mouse. After 1 to 3 weeks, 1 × 10 ^{7 of} each hybridoma was similarly intraperitoneally administered, and 1 to 2 weeks after that, ascites containing 4 to 7 mg / ml of the monoclonal antibody produced in vivo could be obtained. .

(G) Heavy chain and light chain of monoclonal antibody According to the above-mentioned ELISA method, the culture supernatant of each monoclone obtained in (e) above was added to a microtitration plate coated with human pro MMP-1. It was
After washing with PBS, an isotype-specific rabbit anti-mouse Ig antibody (Zymed Lab.) Was added. After washing with PBS, horseradish peroxidase-labeled goat anti-rabbit IgG (H + L) antibody was added, and each heavy chain and hydrogen peroxide and 2,2'-azino-di (3-ethylbenzothiazoline sulfate) were used as substrates. The light chain was determined. The results are summarized in Table 1.

(H) Purification of Monoclonal Antibodies Each ascites fluid obtained in (f) above was fractionated with 40% saturated ammonium sulfate, and then an IgG1 class antibody of 0.
The protein A Affigel (Bio-Rad) column equilibrated with 1.5 M glycine-NaOH buffer (pH 8.9) containing 5 M sodium chloride was adsorbed, washed with the above washing solution, and then 0.1 M citrate buffer (pH 5. Purified by eluting with 0).

Example 3 Selection of Anti-Human MMP-1 Monoclonal Antibody (a) Preparation of Materials CCD purified by the method of Example 1, paragraph (a) above.
-41SK cell-derived human pro MMP-1
It was used as a sample for immunoblotting to select a monoclonal antibody against MP-1.

Further, as a sample for immunoblotting for selecting a monoclonal antibody against human active MMP-1, Biochemistry 17, 23 was used.
31-2337, Stricklin described in 1978
In accordance with the method described above, human active M in which CCD-41 SK cell-derived human proMMP-1 purified by the method described in Example 1, item (a) above was activated by trypsin.
MP-1 (molecular weight: 46 kDa / 42 kDa) was used.

(B) Immunoblotting After subjecting the sample prepared in Example 3, (a) to SDS-PAGE, Cell Engineering 1 & 2, 1061-1068 (19)
Western blotting was performed according to the method of Tabe described in 83), and immunoblotting was performed by a direct method using the enzyme-labeled antibody (Fab′-POD complex) prepared in Example 4 described later.

As a result, the anti-human MMP-1 listed in Table 1
Among the monoclonal antibodies, clone no. 78-52
C12 antibody reacts only with human proMMP-1, other clones are 74-18B9, and 78-32E.
Except for the antibody No. 1, all of them reacted specifically with human pro MMP-1 and human active MMP-1. Furthermore, among the clones that react specifically, the highly reactive clone is 78-4B.
The antibodies were two clones of 11, 78-12G8.

Example 4 Preparation of Enzyme-Labeled Antibody (a) Preparation of Fab′-POD Complex 1) Preparation of Fab ′ Immunoassay 4,209-327,1
Mouse anti-human pro-MMP-1 Fab'-POD conjugates were prepared according to the method of Ishikawa et al. Monoclonal antibody (IgG; clone No. 78) having reactivity with human pro-MMP-1.
-4B11, Research Institute for Micro Engineering, FERMP-13114)
Was dialyzed against 0.1 M citrate buffer (pH 3.5), 0.25% (w / w) pepsin was added to the IgG amount, and digested at 37 ° C. for 18 hours. The digestion reaction was stopped by adding a 2 M Tris solution to the digested solution to adjust the pH to 7.0, and a 0.1 M phosphate buffer solution (pH 7.
Gel filtration was carried out using an Ultrogel AcA54 column equilibrated in (0) to collect the F (ab ′) ₂ fraction. Then F
The (ab ′) ₂ fraction was dialyzed against 0.1 M phosphate buffer (pH 6.0) containing 5 mM EDTA, and the final concentration was 10 μm.
Aminoethanethiol (MEA) was added so that M was obtained, and the mixture was reduced at 37 ° C for 1.5 hours. After that, 5 mM E
Gel filtration was performed on an Ultrogel AcA54 column equilibrated with 0.1 M phosphate buffer (pH 6.0) containing DTA, and the Fab ′ fraction was collected.

2) Preparation of maleimide-labeled peroxidase (POD) POD was dissolved in 0.1 M phosphate buffer (pH 7.0) to a concentration of 10 mg / ml, and the molar amount was 25 times the POD amount. N- (ε-maleimidocaproyloxy) succinimide (EMCS) was added as a dimethylformamide solution, and the mixture was reacted at 30 ° C. for 30 minutes. The reaction solution was subjected to gel filtration on a Sephadex G-25 column equilibrated with 0.1 M phosphate buffer (pH 6.0) to collect a maleimide-labeled POD fraction.

3) Preparation of Fab'-POD complex To Fab 'prepared in 1) above, equimolar amount of maleimide-labeled POD prepared according to the method described in 2) above was added, and Fab' and maleimide-labeled. The POD was diluted with 0.1 M phosphate buffer (pH 6.0) containing 5 mM EDTA so that the final concentration of POD was 100 μM. After leaving this mixed solution at 4 ° C. for 20 hours, it was mixed with Fab ′ at 10
Unreacted thiol groups were blocked with a double molar amount of N-ethylmaleimide. Add this to 0.1M phosphate buffer (pH
After gel filtration with an Ultrogel AcA54 column equilibrated in 6.5) and collection of Fab'-POD complex fractions, BSA and chlorhexidine were added to 0.1% and 0.001% respectively, Stored at 4 ° C.

(B) Preparation of IgG-POD complex 1) Preparation of SH group-labeled IgG Mouse anti-human TIMP-2 IgG (clone No. 67)
-4H11) was dialyzed against 0.1 M phosphate buffer (pH 6.5) and 100 against the IgG contained in the solution.
Double molar S-acetylmercaptosuccinic anhydride was added as a dimethylformamide solution, and the mixture was incubated at 30 ° C. for 30 minutes. Next, 100 μl of 0.1 M Tris-hydrochloric acid buffer solution (pH 7.0), 10 μl of 0.1 M EDTA solution (pH 6.0) and 100 μl of 1 M hydroxylamine solution (pH 7.0) were added, and after standing at 30 ° C. for 5 minutes. 0.1 M phosphate buffer containing 5 mM EDTA (pH
Gel filtration with Sephadex G-25 equilibrated with 6.0), SH group labeled mouse anti-human TIMP-2 Ig
Got G.

2) Preparation of IgG-POD complex To 1 mol of the SH group-labeled IgG prepared in 1) above, about 5 mol of the maleimide-labeled POD obtained in (a) -2) above was added, and the mixture was kept at 4 ° C. for 20 minutes. Let stand for hours. This mixed solution was adjusted to 0.
Ultr equilibrated with 1M phosphate buffer (pH 6.5)
Gel filtration was carried out using an ogel AcA44 column to collect a mouse anti-human TIMP-2 IgG-POD complex fraction. BSA and chlorhexidine were added to 0.1% and 0.001%, respectively, and stored at 4 ° C.

Example 5 Human active MMP-1-TIM
Preparation of Ps Complex (a) Activation of Human Pro-MMP-1 CCD purified by the method described in Example 1, item (a) above.
Human pro-MMP-1 derived from −41 SK cells was activated by 4-aminophenylmercuric acetate (APMA).

50 mM Tris-HCl buffer containing 0.1 M sodium chloride and 10 mM calcium chloride (pH 7.
5) human pro-MMP-1 dissolved in 5) had a final concentration of 1 mM
AMA (Aldrich Chem.)
Was added and incubated at 37 ° C. for 3 hours. Then, the reaction was stopped by transferring to 4 ° C. SDS-P
The reaction was confirmed by AGE (12.5% gel, in the presence of 2-mercaptoethanol) (Fig. 1). APMA
Addition of human pro MMP-1 (molecular weight 57 kDa
/ 52kDa) (lane 1) is fully human active MM
It was confirmed that P-1 (molecular weight 46 kDa / 42 kDa) was activated (lane 2).

(B) Preparation of Human MMP-1-TIMPs Complex The human active MMP-1 prepared in the above (a) was purified by the method described in the above Example 1, (b) -2). Human placenta-derived TIMP-1 or the above-mentioned Example 1,
Human placenta-derived TIMP-purified by the method of (c)
2, the molar ratio of human active MMP-1: human TIMP-
1 or human TIMP-2 = 1: 2 was added, and the mixture was reacted overnight at 4 ° C. Then, it applied to the zinc chelate column (SigmaChem.). This zinc chelate column was saturated with zinc ions with 1 mM zinc chloride, washed well with distilled water, and then washed with 0.15 M sodium chloride, 1 mM calcium chloride, 0.05% bridge-35.
It was equilibrated with a contained 25 mM borate buffer solution (pH 8.0). The adsorption protein of the zinc chelate column is 0.8M sodium chloride, 0.05% bridge-3.
Elution was carried out with 5 mM 50 mM borate buffer (pH 4.7). During this reaction, human TIMP-1 added in excess
Alternatively, human TIMP-2 has been identified in a corresponding amount in the flow-through fraction of the zinc chelate column, and thus the adsorbed fraction does not include free human TIMP-1 or human TIMP-12. SDS-PAGE (1
In the presence of 2.5% gel and 2-mercaptoethanol, as shown in FIG. 2), human active MMP-1 and human TIMP-1 were present in the human active MMP-1-TIMP-1 complex (lane 2). ), And human active MMP-1-TIMP-2
In the complex, human active MMP-1 and human TIMP-
The presence of 2 (lane 3) was confirmed. Lane 1 is human active MMP-1 only.

Example 6 Quantification of human latent MMP-1 and human active MMP-1 (a) Preparation method of monoclonal antibody binding carrier J. Immunoassay 4,209-327 (1
983) according to the method of Ishikawa et al.
Mouse anti-human pro MMP-1 IgG (clone no.
78-12G8, Research Institute for Microcomputer Research, FERM P-131
15) were each dissolved in 0.1M acid buffer (pH 7.0) containing 0.1% sodium azide to obtain 100 μg / ml.
The concentration was adjusted to (A ₂₈₀ = 0.15). 100 μl of the monoclonal antibody solution was added to each well of a 96-well microplate, or polystyrene balls (φ6.5 mm, Ichiko) were dipped in the antibody solution,
It was left still at 24 ° C for 24 hours. Next, the monoclonal antibody solution was removed, and each well was washed twice with 10 mM phosphate buffer (pH 7.0, buffer D) containing 0.1 M sodium chloride, and then 1
It was immersed in buffer solution D containing% BSA and stored at 4 ° C.

(B) One-step sandwich EIA method 1) Plate method 1% BSA, 0.1M sodium chloride, 50 mM Tris-hydrochloric acid buffer solution containing 10 mM calcium chloride (pH 7.
20 μl of a sample containing purified human pro-MMP-1 diluted with buffer solution E) as a standard or containing human pro-MMP-1 was added to a 96-well vinyl plate (Falcon). Next, 1000 'of Fab' (clone No. 78-4B11) -POD conjugate prepared in Example 4, (a) was used.
It was diluted with buffer solution E so that the concentration became ng / ml, and 100 μl of each was added to the vinyl plate and mixed. Add this mixture to the antibody-binding plate prepared in (a) above.
0 μl was added, the mixture was reacted at room temperature for 1 hour, and washed with buffer D four times. Next, 2 mg / m 2 dissolved in 0.1M citric acid-phosphate buffer solution (pH 4.9) containing 0.02% hydrogen peroxide.
100 μl of l-o-phenylenediamine per well
In addition, after reacting at room temperature for 30 minutes, 100 μl of 2N sulfuric acid was added to stop the reaction. Microplate reader (MPR-A4, Tosoh) and _{A 492} of the reaction mixture measured was used to determine the human pro MMP-1 amount in the sample from the calibration curve. The above operation method is shown in Table 2 and the calibration curve is shown in FIG.
●-). In the calibration curve, human pro MMP-1
Standard solution 0.5-32 ng / ml (10-640 pg / w
The linearity was observed up to the concentration of (ell).

2) Ball Method Purified human pro-MM diluted with 10 mM phosphate buffer (pH 7.0, buffer F) containing 1% BSA, 0.1 M sodium chloride, 1 mM magnesium chloride, 1 μM zinc chloride.
50 μl of each of the specimens containing P-1 or human proMMP-1 was added to a plastic tube (12 × 75 mm). Next, 500 ng of Fab ′ (clone No. 78-4B11) -POD complex prepared in Example 4, (a) was used.
It was diluted with the buffer solution F so that the concentration became / ml, and 300 μl of each was added to the above plastic tubes and mixed. The antibody-bound ball prepared in the above (a) was added to this mixed solution, reacted at 17 to 27 ° C. for 1 hour, and washed with buffer solution 3 times. Next, 2 mg / m 2 dissolved in 0.1M citric acid-phosphate buffer solution (pH 4.9) containing 0.02% hydrogen peroxide.
300 μl of lo-phenylenediamine per tube
In addition, after reacting for 30 minutes at 17 to 27 ° C., 1 ml of 1.33N sulfuric acid was added to stop the reaction. A of this reaction mixture
₄₉₂ Micro Flow Spectrophotometer UV
730 (Shimadzu Corporation), and the amount of human proMMP-1 in the sample was determined from the calibration curve. The above operating method is shown in Table 2, and the calibration curve is shown in FIG. 3 (-▲-). In the calibration curve, human pro MMP-1 standard solution 1.0 to 64 ng /
Linearity was observed up to a concentration of ml (50 to 3,200 pg / well).

(C) Simultaneous reproducibility test and sensitivity A simultaneous reproducibility test was carried out on the standard solution and the sample (serum of healthy person) according to the method of the above item (b). The absorbance of the standard solution and the CV value of the serum measurement value were both 10% or less in the plate method (Table 3A) and 10% or less in the ball method (Table 3B). In addition, the average (Mean) and standard deviation (SD) when the absorbance of the standard antigen 0 mg / ml was measured 8 times were calculated, and Mean +
When the standard solution concentration corresponding to 2SD is defined as the sensitivity, the sensitivity is about 0.29 ng / ml (5.8 pg in the plate method).
/ Well), and about 0.83 ng / m by the ball method
1 (41.5 pg / well).

Example 7 Human active MMP-1-TIM
Quantitation of P-1 complex (a), method for preparing monoclonal antibody-bound carrier According to the method described in Example 6, (a), mouse anti-human pro MMP-1 IgG (clone No. 78-12G).
8) A binding plate was prepared.

(B) One-step Gandwich EIA method Human active MMP-prepared in Example 5 as a standard substance
Fab 'prepared by following the method described in Example 4, item (a), wherein 1-TIMP-1 complex was used as an enzyme-labeled antibody.
The procedure of Example 6, (b) -1) was repeated except that (Clone No. 7-6Cl) -POD complex was used, and the human active MMP-1-TIM in the sample was analyzed from the calibration curve.
The amount of P-1 was determined. The anti-TIMP-1 monoclonal antibody used here (clone No. 7-6Cl, Accession No. FERM BP-3468, Microindustry Research Institute) was prepared as in Example 1,
An antibody against bovine TIMP-1 derived from bovine dental pulp purified by the method of (b) -1), which is prepared according to the method described in Example 2. This antibody is human TI
It also showed cross-reactivity with MP-1 (Kodama et al., Co
llagen Rel. Res. 7,341-350,
1987). The operation method is shown in Table 2 as a plate method, and the calibration curve is shown in FIG. In the calibration curve, human active MM
P-1-TIMP-1 composite standard solution 0.1-100 ng
Linearity was observed up to a concentration of / ml (2-2,000 pg / well).

(C) Simultaneous reproducibility and sensitivity A simultaneous reproducibility test was carried out on the standard solution and the sample (serum of healthy person) according to the method of the above-mentioned item (b) (Table 4). 0n
The absorbance and the CV value of the serum measurement values of the standard solutions excluding the g / ml standard solution were all 10% or less. Further, when the sensitivity is calculated in the same manner as in Example 6 (c), it is 0.1 ng.
/ Ml (2.1 pg / well).

Example 8 Human Active MMP-1-TIM
Quantification of P-2 Complex (a) Method for Preparing Monoclonal Antibody-Binding Carrier According to the method described in Example 6, (a), mouse anti-human pro MMP-1 IgG (clone No. 78-12G).
8) A binding plate was prepared.

(B) One-step sandwich EIA method Human active MMP-prepared in Example 5 as a standard substance
The 1-TIMP-2 complex was also used as an enzyme-labeled antibody for IgG prepared in Example 4, item (b) (clone No. 6).
7-4H11) -POD complex was used except that the method described in Example 6, (b) -1) was used, and the amount of human active MMP-1-TIMP-2 in the sample was determined from the calibration curve. I asked. The operation method is shown in Table 2 as a plate method, and the calibration curve is shown in FIG. In the calibration curve, human active MMP-1
-TIMP-2 composite standard 3.9-250 ng / ml
Linearity was observed up to a concentration of (78 to 5,000 pg / Well).

(C) Simultaneous reproducibility and sensitivity A simultaneous reproducibility test was carried out on the standard solution and the sample (serum of healthy person) according to the method of the above item (b) (Table 5). Absorbance of standard solution and CV value of serum measurement value are both 10%
It was below. Further, when the sensitivity was calculated in the same manner as in the item (c) of Example 6, it was 8.2 ng / ml (164 pg / wel
l).

Example 9 Quantification of Human MMP-1 and Others in Human Serum Healthy human serum and rheumatoid arthritis (RA) were used as samples.
(A) Human MMP-in each serum using patient serum
1, (b) human active MMP-1-TIMP-1 complex and (c) human active MMP-1-TIMP-2 complex were quantified respectively (Table 6).

(A) Quantification of human MMP-1 According to the method described in Example 6, (b) -1). As a result, the human MMP-1 concentration in the serum of healthy subjects (15 cases) was 8.0 ± 7.1 ng / ml (Mean ± SD),
It is 18.7 ± 15.5 in the serum of RA patients (11 cases).
It was ng / ml, and a significant difference was observed between both samples.

(B) Human active MMP-1-TIMP
-1 Quantitation of complex The method described in Example 7, item (b) was followed. As a result, it was 0.22 ± 0.04 ng / ml in the serum of a healthy person, and R
A patient's serum is 0.45 ± 0.49 mg / ml,
Human active MMP-1-TIMP-1 between both samples
A significant difference was observed in the amount of complex.

(C) Human active MMP-1-TIMP
-2 Quantification of complex The method described in Example 8, item (b) was followed. As a result, it was 10.1 ± 6.80 ng / ml in the blood consumption of healthy subjects, and R
The serum of patient A was 62.1 ± 105 ng / ml, and a significant difference in the amount of human active MMP-1-TIMP-2 complex was observed between both samples.

Example 10 Human Active Form M in Human Synovial Fluid
Quantification of MP-1-TIMP-1 complex and others Synthetic fluid and osteoarthritis (O
A) The amount of (a) human active MMP-1-TIMP-1 conjugate and (b) human active MMP-1-TIMP-2 complex in each synovial fluid is determined using the synovial fluid of the patient. (Table 7).

(A) Human active MMP-1-TIMP
-1 Quantitation of complex The method described in Example 7, item (b) was followed. As a result, R
Human active MMP-1-in the synovial fluid of patients A (18 cases)
The concentration of TIMP-1 complex is 4.4 ± 5.9 ng / ml, which is 0.75 in the synovial fluid of OA patients (20 cases).
It was ± 1.1 ng / ml, and a significant difference was observed between both samples.

(B) Human active MMP-1-TIMP
-2 Quantification of complex The method described in Example 8, item (b) was followed. As a result, R
47.5 ± 36.3 ng / m for joint fluid of patient A
1 and 10.2 ± 10.
3 ng / ml, human active form M between both samples
A significant difference was observed in the amount of MP-1-TIMP-2 complex.

[0067]

[Table 1]

[0068]

[Table 2]

[0069]

[Table 3]

[0070]

[Table 4]

[0071]

[Table 5]

[0072]

[Table 6]

[Brief description of drawings]

FIG. 1 SDS-PA after activation of human pro-MMP-1
GE's Cozy Brilliant Blue R-250 (CB
It is a figure which shows the result of B, Nacalai Tesque) staining.

FIG. 2 is a diagram showing the results of CBB staining of SDS-PAGE after preparation of human active MMP-1-TIMPs complex.

FIG. 3 Standard curve of human pro-MMP-1 (vertical axis is 492)
Absorbance in nm, horizontal axis shows pro-MMP-1 concentration, ng
/ Ml).

FIG. 4 is a standard curve of human active MMP-1-TIMP-1 complex (abscissa on the vertical axis is absorbance at 492 nm, horizontal axis is human active MMP-1-TIMP-1 complex concentration, ng /
(ml) is a graph showing.

FIG. 5: Standard curve of human active MMP-1-TIMP-2 complex (absorbance at 492 nm on the vertical axis, human active MMP-1-TIMP-2 concentration on the horizontal axis, ng / ml)
It is a graph which shows.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Nobuko Mohri 445 No. 2 Yoshisaku, Toyama City, Toyama Prefecture (72) Inventor Chie Sakai No. 3799 Toba, Kosugi-cho, Imizu-gun, Toyama Prefecture (72) Inventor Kazushi Iwata 190 Izumiri Higashi-cho, Takaoka City, Toyama Prefecture (72) Takayuki Yamashiro, Inventor 1000-102 Sakano Shinmachi, Takaoka City, Toyama Prefecture (72) Inventor Hideaki Tokai, 11-11 Yokotahonmachi, Takaoka City, Toyama Prefecture Yasuo Nagai Toyama 1297-14 Nomura, Takaoka City, Prefecture (4th) (72) Inventor Shinichi Yoshida 4-13-16 Nakajima, Toyama City, Toyama Prefecture

Claims (3)

[Claims] 1. A human active type prepared from human active type collagenase and TIMP-1 using a monoclonal antibody specifically binding to human active type collagenase and a monoclonal antibody specifically binding to TIMP-1. Using collagenase-TIMP-1 complex as a standard substance,
A method for quantifying a human active collagenase-TIMP-1 complex, which comprises immunologically measuring the human active collagenase-TIMP-1 complex in a human body fluid. 2. A human active type prepared from human active type collagenase and TIMP-2 using a monoclonal antibody specifically binding to human active type collagenase and a monoclonal antibody specifically binding to TIMP-2. Using collagenase-TIMP-2 complex as a standard substance,
A method for quantifying a human active collagenase-TIMP-2 complex, which comprises immunologically measuring the human active collagenase-TIMP-2 complex in a human body fluid. 3. A method for diagnosing rheumatoid arthritis disease by quantifying human active collagenase-TIMP-1 complex or human active collagenase-TIMP-2 complex in blood or synovial fluid. And the quantification method described in 2.