JPH09302000A - Anti-tissue factor monoclonal antibody and determination of tissue factor coagulation activity using the monoclonal antibody - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new monoclonal antibody specifically reactive with a tissue factor, free from inhibitory action on the coagulation activity of tissue factor, enabling easy and accurate determination of the coagulation activity of tissue factor and useful for the prediction of thrombogenesis, early diagnosis of thrombosis, etc. SOLUTION: This new anti-tissue factor monoclonal antibody is specifically reactive with tissue factor(TF) and free from inhibitory action on the coagulation activity of tissue factor. The use of the anti-tissue factor monoclonal antibody enables easy and accurate determination of exclusively the tissue factor coagulation activity and is extremely effective for the prediction of thrombogenesis and the early diagnosis of thrombosis. The monoclonal antibody can be produced by administering a BALB/c mouse with a human tissue factor separated from human placenta together with an adjuvant to effect the immunization of the mouse, collecting the spleen cell after the final immunization, fusing the spleen cell with a myeloma cell, culturing the fused cell in HAT medium, selecting and cloning a strain capable of producing anti-tissue factor antibody and culturing the obtained strain.

Description

Detailed Description of the Invention

[0001]

The present invention relates to tissue factor (Tissue Fac
tor: hereinafter, it relates to an anti-TF monoclonal antibody that specifically reacts with TF) but does not inhibit the coagulation activity of TF. Further, the present invention relates to a method for measuring TF coagulation activity using the monoclonal antibody.

[0002]

Background of Development and Prior Art TF is an initiation factor of the blood coagulation cascade reaction. That is, when TF enters the blood, it forms a complex with blood coagulation factor VII (FVII) existing in the blood, and the complexed FVII is activated, which is further blood coagulation factor IX (FIX). And activate blood coagulation factor X (FX). Finally activated FX (FX
Prothrombin is activated to thrombin by a). Thrombin resulting from such a cascade reaction causes blood to clot by converting soluble fibrinogen into insoluble fibrin. The term "TF coagulation activity" as used herein means TF and FVII as described above.
It means a series of activation reactions of blood coagulation factors in the blood coagulation reaction initiated by the reaction with. When this TF comes out in the blood for some reason, it is considered that the blood is coagulated in the blood vessel to form a thrombus. Therefore, if the amount of TF antigen in blood and the coagulation activity (activity that initiates / progresses the blood coagulation reaction) can be measured with high sensitivity and accuracy, prediction of thrombus formation and early diagnosis of thrombosis, particularly disseminated intravascular coagulation syndrome, can be performed. Since it is useful for pathological analysis of coagulopathy centering on (DIC), TF is specifically, highly sensitively and accurately,
Moreover, it is desired to develop a method that allows easy measurement.

Under such circumstances, the present inventors have succeeded in producing a monoclonal antibody which specifically reacts with TF, and by utilizing this, the amount of TF antigen in blood was specifically determined. In addition, we have completed a two-antibody immunoassay that can be quantified with high sensitivity [eg, JP-A-5-72811, JP-A-6-
148182, Shin Nakamura et al., Laboratory test, 38, 925 (1994)]. However, the TF antigen detected by this assay method may include a TF antigen having no coagulation activity. Considering that it is desired to specifically measure only TF having a coagulation activity for the prediction of thrombus formation caused by TF and the early diagnosis of thrombosis, the T
The F antigen measurement method has a problem in terms of accuracy.

On the other hand, as a method for measuring TF coagulation activity in blood, a chromogenic synthetic substrate [C. Fukuda et al., Clin.
Chem., 35, 1897 (1989); Murakami et al., Clinical Pathology, 43, 137 (1)
995)] is known. For example, according to the method of Fukuda et al., Plasma and serum samples were inactivated by heating-Euglobulin fractionation treatment to inactivate protease inhibitors and coagulation factors, and then FVII and FX were added to activate the residual TF. The activity of FX (FXa) is determined by the chromogenic synthetic substrate (S-222
By measuring with 2), it becomes possible to measure the coagulation activity of TF. However, all of these methods require complicated pretreatment of the sample, and the operations are complicated.
Furthermore, since the coagulation mechanism of blood is extremely complicated, it is highly possible that the sample to be measured contains other factors that cause blood coagulation and conversely factors that cause fibrinolysis of blood. It was a problem whether the TF-derived coagulation activity was measured accurately and with good reproducibility by the activity measurement method described in 1.

[0005]

As described above, it is doubtful that the conventional TF activity measuring method accurately measures the TF activity. Therefore, if the coagulation activity derived only from TF can be accurately and easily measured with good reproducibility, it will open a great way to establish clinical diagnosis of various thrombosis.

[0006]

Under these circumstances, the present inventors have conducted extensive studies to solve the above-mentioned problems, and as a result, they react specifically with TF and coagulate TF. We have found an anti-TF monoclonal antibody characterized by not inhibiting the activity. Then, by using the antibody, a method for accurately and simply measuring only the desired TF coagulation activity was successfully established, and the present invention was completed. Specifically, after reacting with the test sample that may contain the antibody and TF, TF in the test sample is selectively bound to the antibody and then activated by direct or indirect action of TF. The coagulation factor to be activated,
T consisting of measuring the activity of activated coagulation factors
The present invention relates to a method for measuring the coagulation activity of F.

[0007]

The monoclonal antibody capable of specifically binding to TF can be obtained by the method of Kohler and Milstein [Nature, 256, 495].
(1975)], spleen cells obtained by immunizing a mouse with TF are fused with mouse myeloma cells, and cells that react specifically with TF are selected from the obtained fused cells (hybridomas), and the cells are cultured. It can be prepared by Here, as the TF used as an immunogen, a placenta-derived TF or a TF obtained by using a gene recombination technique is used.

BALB / C mice are used as the mice for immunization. Immunization is carried out for one mouse (6 weeks old) with 20 to 200 μg of TF antigen every 2 to 3 weeks 3 to 6 times, and the mice are bred and spleen cells are collected according to a conventional method. As myeloma cells, MOPC-21NS / 1 [Nature, 256, 495 (197
5)], SP2 / 0-Ag14 [Nature, 277, 131 (1979)], S194
/5,XXO.8U.1 [J. Exp. Med. 148, 313 (1978)] and the like are preferably used. 1 to 1 for spleen cells and myeloma cells
Mix at a ratio of 10 to 1, fusion is NaCl (about 0.85%),
Dimethyl sulfoxide (10-20% (v / v)) and molecular weight 1,
Perform in phosphate buffer (pH 7.2-7.4) containing 000-6,000 polyethylene glycol. Fusion is performed by incubating a mixture of both cells at 35-37 ° C for 1-5 minutes. Fused cells (hybridomas) were selected by hypoxatin (1.3-1.4 mg / dl) and aminopterin (18-20).
μg / dl), thymidine (375 to 4000 μg / dl), streptomycin (50 to 100 μg / ml), penicillin (50 to 100 units / ml), glutamine (3.5 to 4.0 g / l), fetal bovine serum (10)
Basal medium containing ~ 20%) is used to select growing cells. As the basal medium, RPMI-1640 medium, Eagle's MEM medium and the like which are generally used for culturing animal cells are used. Cloning of the fused cells is repeated at least 3 times by the limiting dilution method.

The antibody of the present invention can be obtained in the medium by culturing the hybridoma in the same manner as in the usual culture of animal cells. For example, 2 to 5 × 10 ⁶ hybridomas are treated with streptomycin (50 to 100 μg / ml), penicillin (50 to 100 units / ml), glutamine (3.5 to 4.0 g / l),
RPMI-1640 medium containing fetal bovine serum (10-20%) 10-
Using 20 ml, in a flask in the presence of 5% CO ₂ , at 35 to 37 ° C.,
By culturing for 3 to 7 days, the antibody is secreted and accumulated in the culture solution. Moreover, the antibody of the present invention can be accumulated in ascites by transplanting the hybridoma into the abdominal cavity of a pristane-treated nude mouse or BALB / C mouse and proliferating it. For example, 0.5 to 1 ml of pristane is inoculated into the abdominal cavity of these mice, and then 5 to 10 ⁶ to 1 × 10 ⁷ hybridomas are transplanted to the abdominal cavity in the second to third weeks. Ascites usually accumulates after about 7 to 10 days and is collected. Finally, the anti-TF monoclonal antibody can be prepared as a single component by purifying it from the culture and ascites by affinity chromatography using a gel to which Protein A is bound. The immunoglobulin subclass of the obtained monoclonal antibody was IgG1-κ as a result of confirmation by gel precipitation.

Not all of the anti-TF monoclonal antibodies thus obtained can be used for measuring the TF coagulation activity which is the object of the present invention. This is because the monoclonal antibody also includes an antibody that inhibits the coagulation activity of TF. Here, the coagulation activity of TF means that the blood coagulation reaction is initiated by the reaction between TF and FVII.
It is an activity to promote. Specifically, the coagulation activity is
It is an activity of forming a complex of TF and activated FVII (TF-FVIIa complex) by the reaction of TF and FVII, and further, the TF-FVIIa complex causes blood coagulation factor X (F
X) is an activity that activates X) directly or indirectly. Such a monoclonal antibody that inhibits the coagulation activity of TF cannot be used for the TF coagulation activity measurement of the present invention. Therefore, the anti-TF monoclonal antibody of the present invention can be obtained by selecting a monoclonal antibody that does not inhibit the TF coagulation activity from the above-mentioned anti-TF monoclonal antibodies. The hybridoma HTF-K234 that produces a monoclonal antibody having the above properties has been deposited at the Institute of Biotechnology, Institute of Industrial Science, with the deposit number FERM P-15484.

In order to measure TF coagulation activity, first, the anti-TF monoclonal antibody of the present invention is immobilized on a solid phase, and the immobilized monoclonal antibody is bound to TF in blood by an antigen-antibody reaction, followed by washing and the like. Excess protein etc. were removed by the operation of. Then, by adding a coagulation factor and measuring the activated coagulation factor activity, it becomes possible to specifically detect the coagulation activity of only TF with high sensitivity and accuracy.

As the solid phase, a solid phase generally used in immunoassays such as enzyme immunoassays and radioimmunoassays, for example, wells of microplates and polystyrene beads can be used.

The coagulation activity of TF is determined by adding a coagulation factor directly or indirectly activated by TF according to a conventional method,
It is determined by measuring the activity of activated coagulation factors. As a specific measuring method, for example, TF is immobilized on a solid phase via the anti-TF monoclonal antibody of the present invention, and then according to the method of Fukuda et al. [C. Fukuda et al., Clin.C.
hem., 35, 1897 (1989)], FVIIa and FX were added, and TF was added.
The activity of FX (FXa) activated from the -FVIIa complex can be directly measured by the chromogenic synthetic substrate (S-222).
By detecting using 2), it becomes possible to measure the coagulation activity of TF. In addition to the above coagulation factors,
After adding FIX or coagulation factors such as prothrombin, FIX
a or thrombin activity to measure TF
It is also possible to determine the coagulation activity of. T according to the invention
In the F activity measurement, it is possible to measure not only the activity of TF released in blood but also the activity of TF existing in cells. In that case, the sample to be measured is treated with a surfactant to solubilize the TF present in the cells, and the TF present in the cells can be easily present by using this in the TF activity measuring method of the present invention. The activity of TF can also be measured. Usual surfactants are used, for example, Triton X-1
00 and the like are given as preferable examples. Hereinafter, the present invention will be described along with examples in order to deepen the understanding thereof, but the present invention is not limited to these examples.

[0014]

【Example】

<< Embodiment 1. Preparation of Monoclonal Antibody Against Human Tissue Factor >> The hybridoma producing the monoclonal antibody of the present invention was prepared according to the method of Kohler and Milstein [Nature, 256, 495 (1975)]. (1) Preparation of Human Tissue Factor (TF) TF was prepared by treating human placenta with acetone and drying acetone powder, which was washed twice with TBS (pH 7.5) / 5 mM EDTA.
Extract TF with 2% Triton X-100 / 40mM Tris (pH8.2),
The final concentration of 5 mM CaCl ₂ was added to the FVIIa-agarose column (5 mg FVIIa / ml gel, 0.9 × 15 cm), washed with TBS (pH 7.5) /0.5% Luburol / 5 mM CaCl ₂ , and then T
Prepared by eluting with BS / 0.05% Luburol / 1M NaCl / 2.5mM EDTA.

(2) Immunization of mice A group of 6 to 12 weeks old BALB / C mice was used. Each group was immunized with the TF prepared above. Immunization was performed by inoculating 3 times by the intraperitoneal route and then once by the intravenous route. On the 1st day, in the presence of Freund's complete adjuvant,
20 μg of TF was inoculated on the day in the presence of Freund's incomplete adjuvant, on the 28th day in the presence of Freund's incomplete adjuvant, and on the 45th day in the absence of adjuvant.

(3) Cell fusion and culture of hybridoma Three days after the final immunization, spleen cells were collected from mice by a conventional method. Spleen cells were mixed with myeloma cells p3X63Ag8-U1 at a cell ratio of 5: 1 and centrifuged (1,200 rpm / 5m
After the supernatant was removed by in) and the precipitated cell mass was sufficiently loosened, it was added to 1 ml of the mixed solution [polyethylene glycol-4000 (2 g), MEM (2 ml), dimethyl sulfoxide] while stirring. After incubating for 5 minutes at 37 ° C., MEM was slowly added so that the total amount of the solution became 50 ml. After centrifugation (900 rpm / 5 min), the supernatant was removed and the cells were loosely loosened. To this, 100 ml of normal medium (RPMI-1640 medium plus 10% fetal calf serum) was added, and the cells were gently suspended using a measuring pipette. The suspension was dispensed into a 24-well culture plate (1 ml / well) and cultured at a temperature of 37 ° C. for 24 hours in an incubator containing 5% carbon dioxide gas. Next, 1 ml / well of HAT medium [hypoxanthine (1 × 10 ⁻⁴ M), thymidine (1.5 × 10 ⁻³ M) and aminopterin (4 × 10 ⁻⁷ M) were added to the normal medium, and further 24 Incubated for hours. After that, every 24 hours for 2 days, 1 ml of the culture supernatant was
The culture medium was replaced with HT medium (HAT medium except aminopterin), and the cells were cultured for 10 to 14 days in the same manner as above. For each well in which the fused cells grown in the form of colonies were observed, 1 ml of the culture supernatant was replaced with the same amount of HT medium, and then the same replacement was performed every 24 hours for 2 days. 3 in HT medium
After culturing for 4 days, a part of the culture supernatant was taken and the target hybridoma was selected by the screening method described below.

(4) Screening of hybridomas The following EIA method and prothrombin time (PT) method were used for selection of hybridomas for the purpose. The TF (protein concentration:
0.5 μg / ml) was added at 100 μl / well and incubated at 37 ° C. for 1 hour for immobilization. Further, 250 μl / well of 4% bovine serum albumin (BSA) solution was added, and incubated in the same manner for masking. To the TF-immobilized microplate thus prepared, 100 μl / well of the culture supernatant of the hybridoma obtained by the cell fusion method and the hybridoma after cloning was added, incubated at 37 ° C. for 1 hour, and then washed 5 times with PBS. , Peroxidase-labeled anti-mouse immunoglobulin antibody solution (manufactured by Kappel,
1000-fold dilution) was added at 100 μl / well. After incubating at 37 ° C. for 1 hour, the plate was washed 5 times with PBS, then orthophenylenediamine (OPDA) substrate solution was added to develop color, and the absorbance was measured at a wavelength of 490 nm. In this way, hybridoma clones that react with TF were selected. By the above selection method, a plurality of clones of hybridoma producing a monoclonal antibody that reacts with TF were obtained.

Next, the PT method was used to select an antibody finally used in the present invention, that is, a monoclonal antibody which does not inhibit TF coagulation activity even when forming an antigen-antibody complex with TF. . Thromboplastin C (international reagent) was used as the PT reagent. First, 1 volume of 3.2% sodium citrate and 9 volumes of blood of a healthy person were mixed to obtain citrate plasma. 0.1 ml of this citric acid plasma and 0.1 ml of TF-reactive monoclonal antibody (protein concentration: 50 μg / ml) were placed in a small test tube and heated in a constant temperature water bath at 37 ° C. for about 3 minutes. Incidentally, 0.1 ml of citrate plasma and 0.1 ml of PBS solution were taken in a test tube as a control. Next, 0.2 ml of TF / calcium reagent (included in the kit) kept at 37 ° C. was added, and at the same time, a stopwatch was started, and the time until fibrin was deposited was measured while gently shaking and tilting. This time,
If the monoclonal antibody that reacted with TF inhibits the coagulation activity of TF, prothrombin time will be longer than that of the control. On the other hand, if the monoclonal antibody that reacted with TF does not inhibit the coagulation activity of TF, the prothrombin time becomes almost equal to that of the control. As a result of selection by the above-mentioned PT method in order to select the antibody used in the present invention from the hybridomas producing the monoclonal antibody which reacts with TF obtained by the above-mentioned selection by the EIA method, one clone of the hybridoma HTF-K234 was found. It was selected as a particularly preferred antibody-producing cell (FIG. 1). The hybridoma HTF-K234 obtained was treated with pristane B.
ALB / C mouse was intraperitoneally administered and a large amount of monoclonal antibody was purified from ascites. The purification method was performed according to a conventional method using protein A gel (Pharmacia-LKB).

Example 2 Method for measuring TF coagulation activity using anti-TF monoclonal antibody >> Monoclonal antibody of the present invention (HTF-K23
100 μl of 4) was added at a concentration of 10 μg / ml, and the mixture was solidified by incubating at 37 ° C. for 1 hour. Next, 1% bovine serum albumin (BSA) solution was added at 300 μl / well, and incubated in the same manner for masking. 100 μl / well of the sample was added to the monoclonal antibody (HTF-K234) -immobilized plate thus prepared, and the plate was incubated at 37 ° C. for 1 hour and then washed 5 times with 0.1% Tween-20 / TBS. After this,
Phospholipid (25 μM), activated FVII (FVIIa, initial concentration: 0
~ 50 ng / ml) and FX (FX, initial concentration: 2 U / ml) were added at 75 µl / well each and incubated at 37 ° C.
The reaction was stopped with 0.2 M EDTA. After stopping the reaction, 25 μl of a chromogenic synthetic substrate (S-2222 (Daiichi Pure Chemicals); 2 mM) was added to measure the activity of the activated FX (FXa) formed, and K
The activity was measured by the inetics method. That is, the mass of the chromogenic group hydrolyzed by FXa was measured by the plate reader (Tm
ax: Molecular Device) to measure the absorbance at 405 nm, and the FXa activity can be determined by the correlation between the hydrolysis amount of the chromogenic substrate and the FXa activity amount. Thus, FXa activated by the TF / FVIIa complex
By measuring the activity, the coagulation activity of TF can be determined.

First, it was demonstrated that the activity of FXa can be measured by the assay method of the present invention. That is, the HTF-K2 of the present invention
34 antibody-immobilized plate (K234 immobilized plate),
Plates that have not been solid-phased (non-solid-phased plates) and plates (K180 solid-phased plates) that have immobilized antibodies (HTF-K180) that specifically react with TF but inhibit its coagulation activity Purified TF (110 ng / ml) was reacted according to the above method. After washing, FVIIa (50ng / ml) and FX (2U / m)
l) was added at 75 μl / well to examine how FXa was produced by changing the reaction time. The FXa activity is measured by a chromogenic synthetic substrate (S-2222:
(1st chemical). The result is shown in FIG. As a result, only the plate on which the monoclonal antibody HTF-K234 of the present invention was immobilized was subjected to FX in a time-dependent manner.
It was confirmed that the amount of a increased. On the other hand, the control plate showed no increase in the amount of FXa. From this, it was confirmed that the monoclonal antibody of the present invention (HTF-K234) does not inhibit the coagulation activity of TF even when an antigen-antibody reaction with TF forms an immune complex.

Next, the purified TF was mixed with TBS to prepare TF standard solutions having various concentrations (0.86 to 110 ng / ml), and FXa activity was measured according to the above method. As a control, non-immobilized plate and HTF-K1
The same reaction was performed using a plate on which 80 was immobilized. The incubation time of the plate with FVIIa and FX was 30 minutes. The calibration curve created based on the results of this measurement is shown in FIG. As a result, it was confirmed that only the plate on which the HTF-K234 antibody was immobilized had a calibration curve of significant TF activity. Also, when the same measurement was performed using a TF standard solution prepared by mixing purified TF with human plasma, the same result as the calibration curve shown in FIG. 3 was obtained. From this, it was confirmed that the TF activity can be measured by the TF activity measuring method using the anti-TF monoclonal antibody of the present invention.

[0022]

EFFECT OF THE INVENTION By using an anti-TF monoclonal antibody which reacts specifically with TF obtained by the present invention but does not inhibit the coagulation activity of TF, the coagulation activity of TF can be easily and accurately measured. Became. Therefore, the TF coagulation activity measurement method of the present invention is extremely effective for predicting thrombus formation and early diagnosis of thrombosis.

[Brief description of drawings]

FIG. 1 T of anti-TF monoclonal antibody by PT method
The result of having examined the F coagulation inhibitory activity is shown.

FIG. 2 shows the time course of the activity of FXa activated by TF in a measurement system using an anti-TF monoclonal antibody.

FIG. 3 shows TF concentration dependence of FXa activity activated by TF in a measurement system using an anti-TF monoclonal antibody.

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Noriaki Aoyama 1-6-1, Okubo, Kumamoto-shi, Kumamoto Foundation Forensic Chemistry and Serum Therapy Laboratory (72) Inventor Seiji Miyamoto 1-6, Okubo, Kumamoto-shi, Kumamoto No. 1 Foundation Institute for Human Chemistry and Serum Therapy

Claims (13)

[Claims] 1. An anti-T characterized by reacting specifically with tissue factor (TF) and not inhibiting the coagulation activity of TF.
F monoclonal antibody. 2. The monoclonal antibody according to claim 1, wherein the coagulation activity of TF is an activity of initiating / promoting a blood coagulation reaction by a reaction between TF and blood coagulation factor VII (FVII). 3. The complex of TF and activated FVII (TF-FVII) is obtained by the reaction of TF and FVII.
(a complex) The monoclonal antibody according to claim 1 or 2, which has an activity of forming a complex. 4. The coagulation activity of the TF is the TF-FVI.
The monoclonal antibody according to claim 3, which has an activity of directly or indirectly activating blood coagulation factor X (FX) by the Ia complex. 5. The monoclonal antibody according to claim 1, wherein the monoclonal antibody is an antibody produced by a hybridoma HTF-K234 (Institute of Biotechnology, Industrial Technology Institute, Accession No .: FERM P-15484). Monoclonal antibody. 6. An anti-TF monoclonal antibody according to claim 1, which reacts specifically with TF and does not inhibit the coagulation activity of TF, is immobilized on a solid phase, and contains the monoclonal antibody and TF. Alternatively, by reacting with a test sample that may contain, the monoclonal antibody specifically binds to TF, and after washing, at least one coagulation factor directly or indirectly activated by TF is added. A method for measuring TF coagulation activity, which comprises measuring the activity of a coagulation factor that has been added and activated. 7. The coagulation factor is FVII or activated FVII.
The measuring method according to claim 6, which is (FVIIa). 8. The method according to claim 6, wherein the coagulation factor is FVII or FVIIa and FX. 9. The method according to claim 6, wherein the coagulation factor is FVII or FVIIa, and blood coagulation factor IX (FIX). 10. The coagulation factor is FVII or FVIIa,
The measuring method according to claim 6, which is FIX or FX. 11. The coagulation factor is FVII or FVIIa,
FIX, FX and prothrombin are claims 6 to 10.
The measurement method according to any one of 1. 12. The activated coagulation factor to be measured is activated F
The method according to any one of claims 6 to 11, which is selected from IX, activated FX and thrombin. 13. The assay method according to claim 6, wherein the activity of the activated coagulation factor is detected using a chromogenic synthetic substrate.