JPH0736019B2 - Method for quantifying human thrombomodulin and its degradation products by a novel anti-human thrombomodulin monoclonal antibody - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a novel human thrombomodulin monoclonal antibody and an enzyme immunoassay method using a monoclonal antibody thereof, and quantifies human thrombomodulin and its degradation products present in blood. By diagnosing a vascular endothelial disorder.

[Background technology]

Endothelial cells are cells that cover the lumen of blood vessels and are in direct contact with blood. They produce extremely many physiologically active substances, antithrombotic properties, selective permeability of substances, maintenance of vascular tone, etc. It has been clarified that when homeopathy is added to the vascular endothelium, homeostasis of the living body is lost and various pathological conditions occur (Toshihiko Nagasawa, Proceedings of the 85th Annual Meeting of the Medical Society of Japan). 2 (1989)).

The onset of arteriosclerosis is said to originate from damage to the vascular endothelium. This ischemic disease of the brain and heart due to arteriosclerosis is the leading cause of death in Europe and the United States, and is increasing even in Japan. Further, diabetic nephropathy, diabetic retinopathy, and other complications of diabetes based on vascular disorders are considered to be a major factor in determining the life expectancy of diabetic patients. In addition, systemic lupus erythematosus (SL
E), rheumatoid arthritis, various collagen diseases such as scleroderma have not yet been elucidated for their causes, but they are all suggested to be involved in immune abnormalities such as autoimmune phenomena, and also for inflammation. It is known that the place is a vascular connective tissue and therefore exhibits vascular disorders.

Conventionally, as one of the means for diagnosing various collagen diseases, there is a method for examining the presence of rheumatoid factor and antinuclear antibody, and the presence or absence of immune abnormality such as increase or decrease in serum complement value.
The parameter indicating the degree of vascular disorder in collagen disease is
Not reported so far.

Other diseases thought to involve vascular endothelial damage in the pathogenesis, such as hemolytic uremic syndrome, pregnant kidney, Kawasaki disease, renal dysfunction when large amounts of cyclosporine are used to control rejection of renal transplants, Extremely many.

Therefore, if there is a method for simply and quantitatively detecting the degree of vascular endothelium damage, the onset factor of the disease that is considered to be involved in the vascular endothelial damage, the mechanism of pathogenesis, the preventive method,
It is thought to play an important role in the search for treatment methods.

Blood vessels consist of vascular endothelial cells that are in direct contact with blood and muscle fibers and connective tissues that cover them.To solve the above, blood vessel-derived endothelial cells derived from vascular disorders are used. Measuring the components is considered valid. However, vascular endothelial damage cannot be detected by conventional blood tests using a coagulation system, and in recent years, studies using vascular endothelial cell culture systems to create damage models and vascular endothelium-derived Although researches on various substances have progressed and are attracting attention, a method for accurately determining the degree of vascular endothelial damage has not been established yet.

Among the substances derived from vascular endothelium, thrombomodulin is one of the substances that have recently received special attention.

Human thrombomodulin, a vascular endothelial cell membrane protein, is a single-chain glycoprotein with a molecular weight of about 78,000 daltons. Based on its cDNA structure, the mature protein consists of 557 amino acids, and the immature protein immediately after synthesis has a signal peptide consisting of 18 amino acids. To do. The mature protein is arranged so that the NH ₂ terminal side is extracellular and the COOH terminal side is cytoplasmic. The extracellular region has an NH ₂ terminal domain and is composed of 6 continuous growth factor-like structures to which thrombin binds. Site, epidermal growth factor (EGF) -like domain,
There is a sugar chain binding domain. Thrombomodulin is
It binds to thrombin at a ratio of 1: 1 and functions as a cofactor that increases the activation of protein C by thrombin by about 2,000 times, and is involved in the protein C coagulation control system. Further, thrombomodulin inhibits thrombin's procoagulant action (fibrinogen coagulation activity, platelet activation, factor V activation, etc.). Therefore, thrombomodulin is an important coagulation regulator on the vascular endothelium that transforms the function of hirombin from procoagulant to anticoagulant, and is the lumen of arteries, veins, capillaries, and lymph vessels in almost all tissues except the brain. It exists on the surface of syncytiotrophoblasts in the placenta and placenta, and is considered to be deeply involved in the inhibition of coagulation of body fluids.

Thrombomodulin has been shown to be present in blood and urine (Ishii H. and Majerus PW, J. Cli
n.Invest., 76, 2178-2181 (1985)). Thrombomodulin, which is present in blood, is a hemolytic protein with a heterogeneous molecular weight, and its origin is not released by the secretory reaction from vascular endothelial cells, but by proteases during the process of vascular endothelial cell destruction. It is considered to have been decomposed and released into the blood. Therefore, if these thrombomodulins present in blood and their degradation products (hereinafter abbreviated as blood thrombomodulins) can be quantified, the quantified value can serve as an index indicating the degree of vascular endothelial damage, and thus sensitivity, accuracy and There is a strong demand for the development of a simple and easy measurement method.

As a method for quantifying blood thrombomodulin, a radioimmunoassay using an anti-thrombomodulin polyclonal antibody (IshiiH. And Majerus PW, J.Cl.
in.Invest., 76,2178-2181 (1985)), an enzyme immunoassay using an anti-thrombomodulin monoclonal antibody (Ishii et al., Clinical Pathology, 37, 266-271 (1989), Ki.
mura S. et al., J. Biochem., 105, 478-483 (1989)) have been reported, but the absolute values differ depending on the measurement system, and in each measurement system, each measurement value is related to the disease. When divided into groups, the variations are large, and it cannot be said that they actually reflect the degree of vascular endothelial damage.

[Disclosure of Invention]

In immunoassays such as radioimmunoassays and enzyme immunoassays, the specificity of the antibody used,
The sensitivity and accuracy depend on the binding site, stability, etc. In particular, blood thrombomodulin is not a protein having a uniform molecular weight, and therefore it is very important to generate an appropriate antibody in order to obtain stable data. Therefore, as a result of various studies based on this point, the present inventor has an effect of inhibiting the action of thrombomodulin that promotes the activation of protein C by thrombin in an anti-human thrombomodulin monoclonal antibody that affects the physiological activity of human thrombomodulin ( Hereinafter, using an anti-human monoclonal antibody having a protein C activation-damaging effect),
By performing enzyme-linked immunosorbent assay, among blood thrombomodulin, the substance that binds to the monoclonal antibody is quantified, and based on the comparison of the quantified value with the quantified value in blood of a healthy person, vascular endothelial damage can be rapidly detected. We have succeeded in providing a simple and quantitative diagnosis method.

In particular, as the monoclonal antibody used in the above method,
Two types of monoclonal antibodies: a monoclonal antibody that inhibits the binding of thrombin to thrombomodulin (hereinafter referred to as a thrombin binding inhibitory action) and an anti-human monoclonal antibody that does not have a thrombin binding inhibitory action but a protein C activation inhibiting action. When an enzyme-linked immunosorbent assay based on the sandwich method is performed using an antibody, it is excellent in accuracy and sensitivity as a method for diagnosing vascular endothelial damage, and by measuring a quantitative value in the patient's blood over time, It was found to be useful for follow-up.

The present invention provides (A) an anti-human thrombomodiurin monoclonal antibody that does not inhibit the binding of thrombin to thrombomodulin, and that inhibits the action of thrombomodulin that promotes protein C activation by thrombin. Human thrombomodulin and its degradation present in blood using two different types of monoclonal antibody, an antibody and an anti-human thrombomodulin monoclonal antibody having an action of inhibiting the binding of thrombin to thrombomodulin (B). The present invention provides a method for quantifying human thrombomodulin and its degradation products in blood, which comprises quantifying the product by an enzyme immunoassay method based on the Sangertian method. The present invention provides an enzyme-linked immunosorbent assay using an anti-human thrombomodulin monoclonal antibody having a protein C activation inhibitory effect,
Among thrombomodulin in blood, a substance that binds to the above-mentioned monoclonal antibody is quantified, and based on comparing the quantified value with the quantified value in blood of a healthy person, a method for diagnosing vascular endothelial damage is provided. Further, as the monoclonal antibody used in the above method, two types of monoclonal antibodies are used, one of which is an anti-human thrombomodulin monoclonal antibody having a thrombin binding inhibitory action, and the other of which is a thrombin binding inhibitor. It is intended to provide a method for diagnosing vascular endothelial damage, which is an anti-human thrombomodulin monoclonal antibody having no action but an inhibitory action on protein C activation.

Hereinafter, the present invention will be described in more detail with reference to Examples of the present invention. In addition, this Example is only one example, and, for example, as an enzyme immunoassay method, a first antibody solid phase method, a two antibody method, and an Emit method (Enzyme multiplexed immunoassay tech
nique; EMIT), enzyme channeling immunoassay, enzyme activity modifier-labeled immunoassay, liposome membrane-enzyme immunoassay, sandwich method, immunoenzyme metric assay method, enzyme activity enhanced immunoassay method, and proxymar linkage immunoassay method. There are non-competitive methods such as law, which can be arbitrarily selected and used.

Further, in the above-mentioned measurement method, various solid phase carriers such as polystyrene, polycarbonate, polypropylene, or polyvinyl balls that passively and well adsorb antigens and antibodies, such as balls, microplates, sticks, test tubes, etc. The materials and usage forms of can be used appropriately.

The labeling enzyme includes peroxidase, alkaline phosphatase, β-D-galactosidase, and the like, and the method for measuring the enzyme activity thereof includes a colorimetric method, a fluorescence method, a bioluminescence method, an optical luminescence method, and the like. Therefore, these enzymes and enzyme activity measuring methods can be appropriately combined and used. On the other hand, as the antibody to which the enzyme label is attached, the DE
It is also possible to use an IgG fraction purified by anion exchange gel such as AE-Sephacel, and further a specific binding moiety Fab ′ obtained by digestion with pepsin followed by reduction.

Further, as the sample to be measured, any of serum, plasma and the like can be used.

Example 1 Preparation of anti-human thrombomodulin monoclonal antibody (a) Preparation of antigen-human thrombomodulin Using human placenta as a material, J. Biol. Chem., 259, 12246-1225.
Human thrombomodulin was extracted and solubilized with a detergent according to the method of Salem et al., 1 (1984).

Next, affinity-chromatography-like method similar to the method for purifying thrombomodulin from bovine lung of Suzuki et al. Described in Biochim.Biophys.Acta, 882, 343-352 (1986).
And thrombomodulin was purified from the solution containing this thrombomodulin using gel filtration method. That is, DIP-thrombin-immobilized thrombin inactivated by diisopropylfluorophosphate on agarose
After affinity-chromatography was performed twice using agarose, gel filtration using an Ultrogel AcA44 column (LKB) was performed to isolate and purify.

Purified human thrombomodulin was prepared according to J. Cell. Biol., 17,
According to the method of Blobell & Dobberstein described in 835-851 (1975), when examined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE),
In the non-reduced state, it showed a single band with a molecular weight of about 78,000 daltons, and in the reduced state, it showed a single band of 105,000 daltons.

(B) Preparation of antibody-producing cells Two 6-week-old Balb / c female trout were first immunized with human thrombomodulin purified in (a) above in Freund's complete adjuvant. That is, for each mouse
50 μg of human thrombomodulin was intraperitoneally administered as a 0.5 ml solution. Then, on the 15th day, human thrombomodulin was boosted to 50 μg dissolved in 12.5 mM Tris-hydrochloric acid buffer solution (pH 7.5) containing 62.5 mM sodium chloride and 0.031% lubrol. Furthermore, as the final immunization, intraperitoneally administered on the 49th day (50 μg / 450 μl; 20 mM Tris-hydrochloric acid buffer solution containing 100 mM sodium chloride and 0.05% lubulol, pH
(Dissolved in 7.5), and after 3 days, the mouse spleen was removed to prepare splenocytes.

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

RPMI1640 medium: RPMI No.1640 (Flow Lab., Inc.), sodium bicarbonate (24mM), sodium pyruvate (1m
M), penicillin G potassium (50 U / ml), streptomycin sulfate (50 μg / ml) and amikacin sulfate (100 μg)
/ ml) and adjust the pH to 7.2 with dry ice, 0.2μ
m Toyo membrane filter was used to remove bacteria.

NS-1 medium: Fetal bovine serum (MABioproducts) that had been sterilized and filtered in the RPMI1640 medium was added to a concentration of 15% (v / v).

PEG 4,000 solution: Polyerylene glycol in RPMI 1640 medium
A 4,000 (PEG 4,000, Merck & Co., Inc.) 50% (w / w) serum-free solution was prepared.

8-azaguanine-resistant myeloma cell NS-1 (P3-NS1
-1) Fusion with Seleced Methodin Cellular Immuno
logy (ed.BBMishell and SMShiigi), WHFreeman
and Company (1980), 351-372 and the method of oi et al.

(2) Nucleated splenocytes prepared in (b) above (live cell ratio 100
%) And myeloma cells (live cell ratio 100%) were fused at a ratio of 5: 1. Separately the spleen cells and myeloma cells
It was washed with RPMI1640 medium. It was then suspended in the same medium and mixed at the above ratio for fusion. Using a conical styrene resin test tube (Iwaki Glass) with a capacity of 50 ml, 40 ml
Was centrifuged at 400 xg for 10 minutes in the RPMI1640 medium to completely suck out the supernatant. To the precipitated cells, PEG4,000 solution heated at 37 ℃
2.1 ml is added dropwise over 1 minute with gentle agitation, and an additional 1 minute to resuspend and disperse the cells. Then 37 ° C
2.1 ml of warm RPMI1640 medium was added dropwise for 1 minute. After this operation was repeated once more, 14.7 ml of the same medium was added dropwise over 2 to 3 minutes with constant stirring to disperse the cells. this
After centrifugation at 400 xg for 10 minutes, the supernatant was completely removed by suction.

Next, 21 ml of 37 ° C. warmed NS-1 medium was immediately added to the precipitated cells, and a large clump of cells was dispersed by careful pipetting with a 10 ml pipette. Furthermore, the same medium 42
Dilute by adding ml to a 96-well microwell made of polystyrene (Iwaki Glass), 6.0 × 10 ⁵ per well / 0.1 m
l of cells were added. The 96-well microwell used at this time was added with 0.2 m of NS-1 medium as a pretreatment and kept warm in a carbon dioxide incubator (37 ° C.) overnight, and the medium was removed by suction at the time of use. 7% of the above microwell containing cells
The cells were cultured in carbon dioxide / 93% air at a temperature of 37 ° C and a humidity of 100%.

(D) Selective growth of hybridoma by selective medium (1) The medium used is as follows. HAT medium:
Hypoxanthine (100 μM), aminopterin (0.4 μM) and thymidine (16 μM) were further added to the NS-1 medium described in (c) above.

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

(2) On the next day (1st day) after the start of the culture in (c) above, 2 drops (about 0.1 ml) of HAT medium was added to the cells with a Pasteur pipette. Replace half of the medium (0.1 ml) with fresh HAT medium on days 2, 3, 5, 8, and 11 and replace half of the medium with fresh H2O on day 14.
Replaced with T medium. After that, every 3 to 4 days, half of the medium was replaced with fresh HT medium. Sufficient hybridoma growth is usually observed within 2-3 weeks. Regarding the hybridoma growing wells, positive wells were checked by the solid phase-antibody binding test (ELISA) method described in (e) below. Next, HT medium containing 10 'mouse thymocytes as a feeder
Using 1 ml added to polystyrene 24-cell wells (Iwaki Glass), the entire contents of each positive hybridoma detected above was transferred. This was subjected to culture at 37 ° C. for about 1 week in the presence of 7% carbon dioxide gas as in the above (c). During this time, once or twice, add 0.5 ml of supernatant from each well to fresh HT.
The medium was replaced with 0.5 ml. When the hybridomas were sufficiently grown, the positivity was confirmed again by the ELISA method, and each of them was subjected to cloning by the limiting dilution method described in (f) below. The residual liquid used for cloning was transferred to a polystyrene 25 cm ² tissue culture flask (Iwaki Glass) to prepare a sample for cryopreservation.

(E) Search for hybridomas producing anti-human thrombomodulin antibody by solid phase-antibody binding test (ELISA) A method slightly modified from the method of Rennard et al. Described in Anal. Biochem. 104, 205-214 (1980) was used. This method is suitable for detecting hybridoma antibodies. A 96-well microtitration plate (Flow Lab., Inc.) was coated with 50 ng of human thrombomodulin and then the uncoated area was 1
Blocked with% bovine serum albumin (BSA). A part of the supernatant of the hybridoma growing well obtained in (d) above was added thereto 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, hydrogen peroxide and o-phenylenediamine, which are the substrates, are added and the degree of brown color produced is qualitatively judged visually, or the absorbance at 492 nm is measured using a microplate reader (MPR-A4, Tosoh Corporation). Was measured.

(F) Cloning Since two or more hybridomas may grow in each well after the operation of (d), cloning was performed by the limiting dilution method to obtain monoclonal antibody-producing hybridomas. A cloning medium containing 10 ⁷ mouse thymocytes as a feeder per 1 ml of NS-1 medium was prepared, and 5 wells, 1 well and 0.5 wells were added to 36 wells, 36 wells and 24 wells of 96-well microwells.
Hybridomas were added. On the 5th and 12th days, about 0.1 ml of NS-1 medium was added to each well. ELISA was performed for a group in which sufficient growth of hybridomas was observed 14 to 15 days after the start of cloning, and the number of colony formation negative wells was 50% or more. When all the tested wells were not positive, the number of colonies in the antibody-positive wells was confirmed, and 4 to 6 wells in which one colony was confirmed were selected and recloned. Finally, 6 monoclonal antibody-producing hybridoma strains against human thrombomodulin were obtained.

(G) In Vitro and In Vivo Proliferation of Monoclonal Antibody Proliferation of the monoclonal antibody was carried out according to a conventional method. That is, each of the obtained hybridomas was cultured in an appropriate culture medium such as NS-1 medium (in vitro growth), and 10
It was possible to obtain monoclonal antibodies 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 mouse)
In addition, 0.5 ml per animal of the tumor formation promoter pristane (2,
6,10,14-Tetramethylpentadecane, Aldrich Chemica
l) was administered intraperitoneally. After 1 to 3 weeks, 1 × 10 ^{7 of} each hybridoma was intraperitoneally administered, and
After 2 weeks, ascites containing 4 to 7 mg / ml of moclonal antibody produced in vivo could be obtained.

(H) Isotype of Monoclonal Antibody Each ascites fluid obtained in (g) above was added to a microtitration plate coated with human thrombomodulin according to the above-mentioned ELISA method. After washing with phosphate buffer (pH 7.1) containing 0.15 M sodium chloride (PBS), an isotype-specific rabbit anti-mouse IgG antibody (Zym
ed Lab. Inc.) was added. After washing with PBS, horseradish peroxidase-labeled goat anti-rabbit IgG (H + L) antibody was added, and detection was performed using hydrogen peroxide and 2,2'-azinodi (3-ethylbenzothiazoline sulfate) as substrates. The results are summarized in Table 1 below. Of the obtained anti-human thrombomodulin monoclonal antibodies, 5 were immunoglobulin chains γ1 / κ and 1 was γ
It had 2a / κ.

(I) Purification of Monoclonal Antibody Each ascites fluid obtained in (g) above was treated with affigel protein.
A MAPS-II kit (Bio-Rad) was used for purification.

(J) Affinity Constant of Monoclonal Antibody to Human Thrombomodulin The affinity constant of each monoclonal antibody obtained in (i) above to human thrombomodulin was determined by an ELISA method in the presence of 5 mM CaCl ₂ . That is, the above ELI
Microtitration plates were coated with human thrombomodulin according to the SA method and blocked with 1% BSA. Next, each of the monoclonal antibodies obtained in (i) above was dissolved in a buffer containing 5 mM CaCl ₂ and adjusted to 500 ng / ml, and serially diluted, and 100 μl of each was diluted with the microtitration plate prepared above. Was added to each well and incubated at room temperature for about 1 hour. Hereinafter, the secondary antibody reaction and the color reaction were carried out according to the above-mentioned ELISA method (e), and the affinity constant (Kd) was determined from the obtained measured values according to a conventional method. The results are shown in Table 2.

Example 2 Selection of Antibody Used for Quantification of Thrombomodulin in Blood (a) Examination of Protein C Activation Inhibitory Effect A solution prepared by mixing a thrombomodulin solution and an antibody solution is prepared and used for protein C activity measurement. Thus, the effect of each monoclonal antibody on the protein C activation promoting action of thrombomodulin was examined.

That is, a thrombomodulin fragment having a molecular weight of 50,000 (elastase fragment) was prepared by elastase treatment according to the method of Kurosawa et al. Described in J. Biol. Chem., 262, 2206-2212, (1987). This elastase fragment has a thrombomodulin protein C activation promoting action and is soluble in water.
This elastase fragment was dissolved (500 ng / ml) in 0.1 M phosphate buffer (pH 7.5). Using this elastase fragment solution and the buffer solution (0 to 12 μg / ml) of each monoclonal antibody prepared in Example 1 (j), the molar ratio of the antibody to thrombomodulin was 0,1 /
A mixed solution was prepared for each of 4, 1/2, 1/1, 2/1 and 4/1. Using 10 ml each of this mixed solution, protein C (activity was measured according to the method of Suzuki et al. Described in Clinical Pathology, 61 (Special Issue), 63 (1985).
(Clone 21-3H1), IgG (clone 21-4G3), IgG
(Clone 21-6F7) and IgG (clone 21-9H1
2) was found to reduce the protein C activation promoting action of thrombomodulin (Table 3).

(B) Examination of thrombin binding inhibitory effect By treating the immobilized thrombomodulin fragment with an antibody solution and then allowing thrombin to act,
The effect of each monoclonal antibody on the binding of thrombin to thrombomodulin was examined.

That is, after coating a 96-well microtitration plate (Flow L-ab. Inc.) with the elastase fragment solution (500 ng / ml) prepared in Example 2 (a), the uncoated portion was blocked with 1% BSA. did. Next, the concentration of each monoclonal antibody obtained in Example 1 (i)
50 μl of phosphate buffer solution (pH 7.5) adjusted to 0, 0.25, 0.5, 1, 2 and 10 μg / ml was added to each well of the coated plate and reacted at 37 ° C. for 6 hours. . Next, the antibody solution was removed, the plate was washed twice with 0.1% BSA-containing Tris-HCl buffer (pH 7.5), and 50 μl of a thrombin solution (500 ng / ml physiological saline) was added to each well of the plate. In addition, the mixture was reacted at 37 ° C for 30 minutes. The thrombin solution was removed, and 0.1% BSA-containing Tris-HCl buffer (pH
After washing twice with 7.5), thrombin assay using chromogenic synthetic substrate (Nobuo Sakuragawa, Medical Technology, 13,704-
713 (1985), the thrombin activity was measured.

That is, a substrate-polybrene solution (S-2238: 0.8 mM) prepared with a reagent manufactured by Kabi was added to each well of the plate.
After adding 100 μl each and reacting at 37 ° C. for 5 minutes, 50 μl of a 50% acetic acid solution was added to stop the reaction. After stopping the reaction,
The absorbance at 405 nm was measured using the microplate reader. This absorbance is proportional to the amount of thrombin bound to the elastase fragment of thrombomodulin coated on the plate. From this measurement result, IgG (clone 21-3H1) and IgG (clone 21-3H1)
-4G3) was found to strongly inhibit the binding of thrombin to thrombomodulin (Table 3).

From the results of (a) and (b), IgG (clone 21-3H
1), IgG (clone 21-4G3), IgG (clone 21-
6F7) and IgG (clone 21-9H12) are anti-human thrombomodulin monoclonal antibodies that affect the physiological activity of human thrombomodulin, and were found to have a protein C activation inhibitory effect. In addition, IgG (clone 21-3H1) and IgG (clone 21-4G3)
Was found to have a strong thrombin binding inhibitory effect. In addition, the binding properties of these monoclonal antibodies to the elastase fragment of thrombomodulin were examined using the immunoblotting method.
(Clone 21-3H1), IgG (clone 21-4G3), IgG
(Clone 21-6F7) and IgG (clone 21-9H12)
Were found to have binding properties to this elastase fragment.

Example 3 Quantification of thrombomodulin in human blood (a) Preparation of enzyme-labeled monoclonal antibody (1) Preparation of Fab ′ fraction Each purified monoclonal antibody (IgG) obtained in Example 1 (i) was added with 0.1 M sodium chloride. 0.1M acetate buffer (pH
4.2) and the solution was digested with pepsin as described below. That is, 2% (w / w) of pepsin was added to IgG in the fraction and digested at 37 ° C for 24 hours. To the digest, add 2M Tris solution to adjust the pH to 7.
The reaction was stopped by adjusting to 0, and the F (ab ') ₂ fraction was fractionated by gel filtration using a Ultrogel AcA 44 column equilibrated with 0.1 M phosphate buffer (pH 7.0).

Next, this F (ab ') ₂ fraction was dialyzed against 0.1 M phosphate buffer (pH 6.0) containing 5 mM ethylenediaminetetraacetic acid (EDTA) to give a final concentration of 10 mM aminoethanethiol (MEA). Added and reduced at 37 ° C for 1.5 hours, then containing 5 mM EDTA
The gel was filtered using a Ultrogel AcA 44 column equilibrated with 0.1 M phosphate buffer (pH 6.0) to collect the Fab ′ fraction.

(2) Maleimide-labeled horseradish peroxidase (PO
D) Preparation of fractions In addition to the procedure in (1) above, PO
D was labeled with maleimide. That is, POD was dissolved in 0.1M phosphate buffer (pH 7.0) at a concentration of 10 mg / ml, and the POD was
On the other hand, a 25-fold molar amount of N- (ε-maleimidocaproyloxy) succinimide (EMCS) was added as a dimethylformamide solution and reacted at 30 ° C. for 30 minutes. this
Gel filtration was performed on a Sephadex G-50 column equilibrated with 0.1 M phosphate buffer (pH 6.0) to collect a maleimide-labeled POD fraction.

(3) Preparation of Fab′-POD complex fraction Fractions prepared in (1) above are equimolar to Fab ′ in the fraction obtained in (2) above as maleimide-labeled POD. Both fractions were mixed so that the final concentration of Fab ′ and maleimide-labeled POD was 100 μM.
It diluted with 0.1 M phosphate buffer (pH 6.0) containing 5 mM EDTA.
After reacting this mixed solution at 4 ° C. for 20 hours, unreacted thiol groups were blocked with N-ethylmaleimide in a molar amount 10 times that of Fab ′. This was gel-filtered using a Ultrogel AcA 44 column equilibrated with 0.1 M phosphate buffer (pH 6.5), and Fa
After collecting the b'-POD complex fraction, 0.1% BSA and 0.001%
Chlorhexidine was added and stored at 4 ° C.

(B) Quantification of thrombomodulin in human blood by one-step sandwich method Among anti-human thrombomodulin monoclonal antibodies that affect the physiological activity of human thrombomodulin, a monoclonal antibody having an inhibitory effect on protein C activation,
It was performed using a combination with a monoclonal antibody having a thrombin binding inhibitory effect.

That is, according to the method of Ishikawa et al. Described in J. Immunoassay, 4, 209-327 (1983), the purified monoclonal antibody obtained in Example 1 (i) was added to 0.1M sodium azide-containing 0.1M phosphate buffer solution ( pH7.5) and the concentration was adjusted to 0.1 mg / ml. Add polystyrene balls (diameter 6.5 mm, Percision Plasti
c Ball) was soaked, and polystyrene balls were coated with the antibody. Next, the antibody immersion liquid was collected, and the polystyrene balls were washed with 10 mM phosphate buffer (pH 7.0) containing 0.1% BSA, 0.1M sodium chloride and 0.001% chlorhexidine,
Stored at 4 ° C.

As a labeled sample, thrombomodulin purified from human placenta using affinity chromatography with an anti-human thrombomodulin monoclonal antibody (clone 21-4G3) -bound Sepharose 4B column (hereinafter referred to as anti-TM antibody column) was used. That is, Example 1
The anti-human thrombomodulin monoclonal antibody (clone 21-4G3) obtained in (i) was immobilized at a ratio of 5 mg per 1 ml of CNBr-activated Sepharose 4B (Pharmacia). This was packed in a column (1 × 5 cm) to obtain an anti-TM antibody column. On the other hand, according to the method of Example 1 (a), human thrombomodulin was extracted and solubilized using a surfactant, and this solution was added with 20 mM Tris-HCl buffer containing 0.1% sodium chloride containing 0.5% Triton-X ( pH
7.5) (TBS) equilibrated with anti-TM antibody column, 0.5
After thoroughly removing unbound protein with TBS containing% Triton-X, human thrombomodulin bound to the column was removed.
Elution with 8M urea solution. The eluate was collected and subjected to gel filtration using a Ultrogel AcA 44 column (1.5 × 100 cm; 168 ml) equilibrated with TBS containing 0.01% Lubrol. The fraction with high absorbance at A ₂₈₀ was collected and concentrated to concentrate human thrombomodulin. Was purified. When this purified human thrombomodulin was examined by SDS-PAGE described in Example 1 (a), it showed a migration pattern similar to that of the purified human thrombomodulin obtained in Example 1 (a). This purified human thrombomodulin was treated with 0.1 M sodium chloride and 1
64 ng / m with 10 mM Tris buffer (pH 7.0) containing 10% BSA
l solution was prepared and serially diluted to 50 μm each.
1 of each was used as a standard sample.

On the other hand, as a sample sample, 50 μl each of serum of a healthy person and serum of a patient with collagen disease was used.

Each of the above samples was placed in a test tube and prepared in (a).
Fab′-POD complex fraction (300-1000 ng / ml), 1% BSA,
It was dissolved in 300 μl of 30 mM phosphate buffer (pH 7.0) containing 0.1 M sodium chloride and 10 mM EDTA. Next, each of these test tubes was added with one of the above-prepared monoclonal antibody-bonded polystyrene balls, and the mixture was allowed to stand at room temperature for 1 hour. Then, 50 mM sodium chloride-containing 5 mM phosphate buffer (pH
It was washed in 7.0). Next, POD substrate dissolved in 0.1M acetate buffer (pH 5.5), that is, 0.025% tetramethylbenzidine (TMBZ) was added in 300 μl aliquots, and 0.0075% hydrogen peroxide solution was added in 300 μl aliquots at room temperature. After standing for a minute, the reaction was stopped by adding 800 μl of 1.75N sulfuric acid. Shimadzu Microflow UV-Visible Spectrophotometer (UV-
730), the absorbance of the reaction mixture at a wavelength of 450 nm was measured, and the human thrombomodulin concentration (hereinafter, abbreviated as TM concentration) corresponding to the absorbance of the specimen sample was read from the calibration curve prepared from the standard sample.

IgG, an anti-human thrombomodulin monoclonal antibody that affects the physiological activity of human thrombomodulin
(Clone 21-3H1), IgG (clone 21-4G3), IgG
(Clone 21-6F7) and IgG (clone 21-9H12)
Any of these could be used as the solid phase carrier in this example. However, when IgG (clone 21-3H1) was used as the solid phase antibody and IgG (clone 21-4G3) was used as the labeling antibody, IgG (clone 21-4G3) was used as the solid phase antibody, and IgG (clone When 21-3H1) was used as the labeling antibody, measurement was impossible. IgG (clone 21
Fig. 1 shows a standard curve obtained by using -4G3) as a solid phase antibody and IgG (clone 21-9H12) as a labeling antibody. As shown in FIG. 1, A ₄₅₀ increased with increasing concentration of the human thrombomodulin standard sample, and the quantification sensitivity was 1 ng per 1 ml of the sample.

(C) Quantification of Thrombomodulin in Blood in Collagen Disease Patients The amount of thrombomodulin in blood in normal subjects and five types of collagen disease patients was quantified by the enzyme immunoassay method described in (b) above. In other words, 50 samples of serum from healthy subjects (79 samples) and serum from patients with collagen disease (54 samples) were used as sample samples.
The TM concentration was measured using each μl. As a result, as shown in 1 to 3 of Table 4, the TM concentration (mean ± SD) in the serum of healthy subjects was 2.96 ± 0.71 ng / ml. On the other hand, in patients with collagen disease, 26 of SLE patient serum, 5 of mixed connective tissue patient serum, 9 of rheumatoid arthritis patient serum,
TM concentrations (mean ± SD) in 5 serum samples of progressive systemic sclerosis and 9 serum samples of Sjogren's syndrome were 6.98 ± 4.55 ng / ml, 5.98 ± 3.53 ng / ml, 7.86 ± 4.95 ng, respectively.
/ ml, 6.04 ± 4.63 ng / ml, 7.57 ± 6.14 ng / ml. All of these values are TM concentrations in the serum of healthy individuals (2.96 ± 0.
71 ng / ml).

Next, FIG. 2 shows an example of the results of observing the change in TM concentration in the pathological state of patients with collagen disease using the enzyme immunoassay method shown in (b) above. The patient shown in FIG. 2 is an SLE patient, and is a serum complement value (complement peptide component of the third component of complement) in serum, which is conventionally used for the follow-up observation of this pathological condition and is representative of an immune disorder. : C ₃ c and complement fourth component: C ₄ ) were measured simultaneously. (○) in Fig. 2 is TM
Concentration, (Δ) indicates the amount of C ₃ c, and (□) indicates the amount of C ₄ . As shown in FIG. 2, an inverse correlation was observed between the TM concentration in the patient serum and the C ₃ c amount and C ₄ amount indicating the complement value in the patient serum. As shown here, in the observation of pathological conditions in the treatment of patients with collagen disease, in addition to the conventionally used parameters showing immunological abnormalities, by quantifying blood thrombomodulin, the blood vessels at the lesion site can be detected. It is expected that the degree of disability can be known and the therapeutic effect can be improved.

(D) Quantification of thrombomodulin in blood in patients with renal disease Using the enzyme immunoassay method described in (b) above, 4
Blood thrombomodulin was quantified in three types of kidney disease patients. That is, as a sample specimen, serum of kidney disease patients (34
The TM concentration was measured using 50 μl of each of the samples) and compared with the TM concentration in the serum of a healthy person obtained in (c) above. As a result, as shown in Tables 1 and 5 of Table 4, as compared with the TM concentration of 2.96 ± 0.71 ng / ml (mean ± SD) in serum of healthy subjects, in patients with renal disease, diabetic nephropathy patients were compared. TM concentration (mean ± SD) in 10 serum samples, 11 serum samples for membranous nephropathy patients, 10 serum samples for nephrotic syndrome and 3 serum samples for amyloidosis was 7.06 ± 7.19 ng / m, respectively.
l, 6.37 ± 2.39ng / ml, 9.24 ± 5.39ng / ml, 8.83 ± 1.17ng /
ml was significantly higher.

[Brief description of drawings]

FIG. 1 shows that the standard curve obtained in Example 3 (b), that is, IgG (clone 21-4G3) was used as a solid phase antibody,
FIG. 2 is a diagram showing a standard curve of human thrombomodulin in a one-step sandwich method using (clone 21-9H12) as a labeling antibody, and FIG. 2 shows the pathological condition of patients with collagen disease obtained in Example 3 (c). FIG. 3 is a diagram showing an example of changes in serum TM concentration and complement value over time.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Misako Nagai 40 Kandashinmachi, Takaoka City, Toyama Prefecture Employment Promotion House No. 206 (72) Inventor Kyoko Mikawaya 1-29-10 Takaido, Suginami-ku, Tokyo ( 56) References JP-A 64-47391 (JP, A)

Claims (1)

[Claims] 1. An anti-human thrombomodulin having (A) an action that does not inhibit the binding of thrombin to thrombomodulin and that inhibits the action of thrombomodulin that promotes protein C activation by thrombin. Using two different types of monoclonal antibodies, the monoclonal antibody and the anti-human thrombomodulin monoclonal antibody (B) that inhibits the binding of thrombin to thrombomodulin, and human thrombomodulin present in blood and its A method for quantifying human thrombomodiurin and its degradation products in blood, which comprises quantifying the degradation products by an enzyme immunoassay method based on the Sangerci method.