JPH05172811A - Method of quantifying homo sapiens tissue factor - Google Patents

Abstract

PURPOSE:To perform quantitative measurement of Homo sapiens tissue factors in blood, which is considered to be effective for preview of thrombus formation and early diagnosis of a thrombus case such as a DIC, with a two-antibody immunity measurement using monochronal antibodies by unique and still convenient operation. CONSTITUTION:Two different monochronal antibodies which are uniquely reacted on Homo sapiens tissue factors are prepared to quantify Homo sapiens tissue factors in antibodies with a sandwich two-antibody immunity measurement easing these by unique and still convenient operation. Not only the measurement of free Homo sapiens tissue factors in blood plasma but also the measurement of Homo sapiens tissue factors existing in blood corpuscle cells can be made in a unique and still convenient manner by applying this method after giving interfacial active agent treatment to the examined.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for quantifying human tissue factor in blood.

[0002]

Tissue factor is a protein that triggers the blood coagulation cascade reaction. That is, when tissue factor enters the blood, it forms a complex with blood coagulation factor VII (VII) present in the blood, and the complexed VII is activated, which is blood coagulation factor IX (IX). , Blood coagulation factor X (X)
Activate. Activated IX activates X and activated X activates prothrombin to thrombin.
Thrombin generated as a result of such a cascade reaction is
Blood is clotted by converting soluble fibrinogen into insoluble fibrin. Tissue factors that initiate these blood coagulation reactions are usually not present in the blood, although [Th
omas A. Drake et al., Am. J. Pathol. 134, 1087-1092, (198
9)], If tissue factor comes out into the blood for some reason, it is considered that the blood is coagulated in the blood vessel to form a thrombus. In this case, if the amount of tissue factor in blood can be quantified with high sensitivity, prediction of thrombus formation and early diagnosis of thrombosis, particularly prediction / diagnosis of diffuse intravascular coagulation (DIC) becomes possible.

In general, there are two types of tissue factors that appear in the blood, namely, those present in cells such as blood cells and those present in the form free from cells. In the former, leukemia cells and There are tissue factors present in LPS-stimulated monocytes, and the latter include tissue factors contained in broken fragments of the above cells, tissue factors released from leukemia cells by shedding [R. Bona et al., Thromb. Res. 48, p487-500 (198
7)], tissue factors that flow into the blood from damaged tissue.

In the case of quantifying these tissue factors, there is a method for separating the tissue factors existing in the cells and measuring the coagulation activity thereof, and for the tissue factor existing free from the cells, Separates plasma from blood,
There is a method to measure its coagulation activity [K. Iijima et al., Thro.
mb. Haemostas 62, p69 (1989)]. However, in the method for measuring the coagulation activity of these tissue factors, the operation is complicated and the coagulation mechanism of blood is very complicated. There is also a problem that it is not possible to obtain proof that this method for measuring activity measures the coagulation activity derived from tissue factor.

[0005]

It is desired to develop a method for specifically and highly sensitively and easily measuring human tissue factor, which is considered to be useful for predicting thrombus formation such as DIC and early diagnosis of thrombosis. ing.

[0006]

[Means for Solving the Problems] In such a situation,
The present inventors have succeeded in producing a monoclonal antibody that specifically reacts with tissue factor, and utilizing this,
We have completed a two-antibody immunoassay that allows specific, simple, rapid, and highly sensitive determination of tissue factor in blood.

First, the monoclonal antibody of the present invention and its preparation method will be outlined. The monoclonal antibody according to the present invention recognizes human tissue factor. The monoclonal antibody immunizes a mouse with human tissue factor to fuse the resulting spleen cells with mouse myeloma cells,
It can be prepared by selecting cells that react with human tissue factor from the resulting fused cells (hybridomas) and culturing the cells.

Regarding the preparation of this hybridoma, Ko
It is based on the method of hler and Milstein [Nature 256, p495 (1975)]. BALB / c mice, BA
F1 mice of LB / c mouse and other mouse are used. Immunization is carried out for one mouse (6 to 8 weeks old, 20 to 30 g) with 20 to 200 μg of the antigen protein every 2 to 3 weeks 3 to 6 times. The breeding of mice and the collection of spleen cells follow conventional methods. As myeloma cells, MOPC-21NS /
1 [Nature, 256, p495 (1975)], SP2 / 0-Agl4 [Nature,
277, p131 (1979)], S194 / 5, XXO.8U.l [J. Exp. Me
d., 148, p313 (1978)] and the like are preferably used. Spleen cells and myeloma cells are mixed at a ratio of 1: 1 to 10: 1,
Fusion is NaCl (about 0.85%), dimethyl sulfoxide (10 ~
20% (v / v)) and a polyethylene glycol having a molecular weight of 1,000 to 6,000 and a phosphate buffer (pH 7.2 to 7.4). Fusion is performed by incubating a mixture of both cells at 35-37 ° C for 1-3 minutes. The selection of fused cells (hybridomas) was based on hypoxanthine (1.3-1.4 mg).
/ Dl), aminopterin (18-20 μg / dl), thymidine (375-4000 μg / dl), streptomycin (50-100)
μg / ml), penicillin (50-100 units / ml), glutamine (3.5-4.0g / l), fetal calf serum (10-20%) in basal medium, and select as 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.

When the hybridoma is cultured in the same manner as in the culture of normal animal cells, the antibody of the present invention can be obtained in the medium as a result. 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 / ml).
l), RPMI-1640 medium containing fetal bovine serum (10-20%)
Using 10 to 20 ml, 35 to 37 in the presence of 5% CO ₂ in the flask.
The antibody is secreted and accumulated in the culture medium by culturing at 3 ° C. for 3 to 7 days. Moreover, the antibody of the present invention can be accumulated in ascites by transplanting the hybridoma into the abdominal cavity of a nude or BALB / c mouse treated with blistane and allowing the hybridoma to grow. That is, 0.5 to 1 ml of blistane is inoculated into the abdominal cavity of these mice, and then 5 to 10 ⁶ to 1 × 10 ⁷ hybridomas are transplanted into the abdominal cavity at 2-3 weeks. Ascites usually accumulates after 7 to 10 days and is collected. Monoclonal antibodies in the culture and ascites are accumulated by means of affinity chromatography using Affigel Protein A MAPS-II kit (Bio-Rad). The immunoglobulin subclass of the obtained monoclonal antibody was identified by an in-gel precipitation reaction and was found to be IgG1-κ.

The hybridomas HTF-K14 and HTF-K180 which produce the monoclonal antibody of the present invention were deposited under the contract number 11945 (FER) at the Institute of Microbiology, Institute of Industrial Science and Technology (MICRO).
M-P11945) and accession number 11946 (FERM-P11946).

By constructing a sandwich double antibody immunoassay utilizing the monoclonal antibody, it becomes possible to easily quantify the tissue factor in blood and overcome the complexity of the conventional assay for tissue factor. Further, it becomes possible to further enhance the specificity in the measurement.

The construction of the sandwich double antibody immunoassay is not particularly limited, and existing immunoassay techniques can be applied. Examples of labeling means include enzyme immunoassay (EIA) and radioimmunoassay. Assay (RIA), Fluorescent Immunoassay (FIA) and Luminescent Immunoassay (LI)
A method such as A) can be suitably selected.

The immunoassay method of the present invention can measure not only tissue factor released in blood but also tissue factor existing in cells. In that case, the sample to be measured is treated with a surfactant to solubilize the tissue factor present in the cells, and the tissue factor is easily present in the cells by subjecting it to the immunoassay of the present invention. It becomes possible to quantify the total tissue factor. As the surfactant, usual ones are used, but Triton X-100 and the like are mentioned as a preferable example thereof. In general, in an immunoassay method such as the present invention, treatment of a sample to be measured with a surfactant may lower the measurement sensitivity, but immunization using the monoclonal antibody of the present invention In the measuring method, no such obstacle was confirmed. Hereinafter, the present invention will be described along with examples to deepen the understanding thereof, but the present invention is not limited to these examples.

[0014]

[Example 1] Preparation of human tissue factor Human tissue factor (TF) was prepared by treating human placenta with acetone and drying acetone powder (acetone powder) in TBS.
(PH7.5) / 5mM EDTA, washed twice, then 2% Triton X-
TF was extracted with 100/40 mM Tris (pH8.2) and the final concentration
The plus of CaCl ₂ 5 mM VIIa- agarose column (5mg
VIIa / ml gel, 0.9 × 15cm), TBS (pH7.5) /0.5
% Luburol / 5mM CaCl ₂ after washing, TBS / 0.05% Luburol / 1M
Prepared by eluting with NaCl / 2.5 mM EDTA.

(1) Mouse Immunization A group of BALB / c mice aged 8 to 12 weeks was used. Each group was immunized with human tissue factor prepared by the method described in the above preparation method. Immunization shall be performed by inoculating 3 times by intraperitoneal route and then 1 time by intravenous route, on day 0 in the presence of Freund's complete adjuvant, and on day 14 in the presence of Freund's incomplete adjuvant for 28 days. Eyes were inoculated with 20 μg of antigen human tissue factor in the presence of Freund's incomplete adjuvant and on the 45th day in the absence of adjuvant.

(2) 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 ratio of 1 to 5 and centrifuged (1200 rpm / 5
After removing the supernatant and removing the cell debris that had precipitated, the mixture was added to 1 ml of the mixed solution [polyethylene glycol-4000 (2 g), MEM (2 ml), dimethyl sulfoxide] while stirring. After incubating at 37 ° C. for 5 minutes, MEM was slowly added so that the total amount of the solution was 50 ml. After centrifugation (900 rpm / 5 minutes), the supernatant was removed and the cells were loosely loosened. Normal medium (RPMI-1640
100 ml of medium supplemented with 10% fetal calf serum) was added, and the cells were gently suspended using a measuring pipette.

Dispense the suspension into a 24-well culture plate (1 m
l / hole), temperature 37 ° C in an incubator containing 5% carbon dioxide
The cells were cultured for 24 hours. Next, 1 ml / well of HAT medium [hypoxanthine (1 × 10 ⁻⁴ M) was added to normal medium) and thymidine (1.5 × 10 5).
^-3 M) and aminopterin (4 × 10 ^-7 M)], and
It was cultured for 24 hours. Then, every 24 hours for 2 days, 1 ml of the culture supernatant was replaced with the same amount of HT medium (except aminopterin from HAT medium), and the cells were cultured for 10 to 14 days in the same manner as above. For each hole in which the fused cells (about 300 cells) grown in a colony 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. .. After culturing in HT medium for 3 to 4 days, a part of the culture supernatant was collected and the target hybridoma was selected by the screening method described below.

(3) Hybridoma Screening Hybridomas for the purpose of selection were selected by combining the following EIA method and Western blotting method.

EIA method Human tissue factor (protein concentration: 0.5 μg / ml) prepared as described above was added to a 96-well microtest plate at 50 μl / well, and incubated at 37 ° C. for 1 hour for immobilization. 4% BSA (bovine serum albumin) solution
250 μl was added and incubated in the same manner to carry out masking. The hybridoma obtained by the cell fusion method and the culture supernatant of the hybridoma after cloning were added to the human tissue factor-immobilized plate thus prepared.
After incubating at 37 ℃ for 1 hour, wash 3 times with PBS,
A peroxidase-labeled anti-mouse immunoglobulin antibody solution (manufactured by Kappel, diluted 1000 times) was added at 100 μl / well. 37
After incubating at ℃ for 1 hour, wash 5 times with PBS,
After that, OPDA substrate solution is added and expressed by a conventional method.
The absorbance was measured at a wavelength of 492 nm. Hybridoma clones that react with human tissue factor were thus selected.

Western blotting method Human tissue factor was electrophoresed using 12% SDS-PAGE, the gel was placed on a nitrocellulose membrane to transfer the human tissue factor onto the membrane, and the membrane was cut to a width of 0.4 to 0.5 cm. did. Each fragment was immersed in the hybridoma culture supernatant and incubated at 37 ° C. for 2 hours. Then, the strips were washed three times with PBS, and then kept warm for 2 hours in a 1: 750 dilution of biotin-labeled anti-mouse IgG (TAGO). The strips were washed 3 times with PBS, dipped in horseradish peroxidase-conjugated avidin (manufactured by Sigma) (1000-fold dilution), and kept warm for 1 hour. PB
After washing 3 times with S, expression was performed with a coloring reagent (Bio-Rad) using 4-chloro-1-naphthol, and a hybridoma showing a coloring band of human tissue factor was selected and cloned.
The hybridoma clones after cloning were also selected by the same method.

By the above selection method, 67 clones of hybridoma producing a monoclonal antibody reactive with human tissue factor were obtained. The relationship between the affinity of these produced monoclonal antibodies for human tissue factor and the epitope by the criss cross method was investigated, and the optimal combination of monoclonal antibodies for quantifying the amount of human tissue factor antigen was examined by the two antibody method. As a result, the monoclonal antibody used in the assay method of the present invention recognizes different epitopes among the epitopes existing in the peptide region from the 175th amino acid to the 219th amino acid from the amino terminus of human tissue factor peptide. Two
It has been found that certain monoclonal antibodies are suitable. Furthermore, as such a monoclonal antibody,
2 produced by hybridomas HTF-K14 and HTF-K180
It was found that the combination of different types of monoclonal antibodies enables the most sensitive measurement.

(4) Production of Monoclonal Antibody The hybridoma HTF-K14 (5 × 10 ⁶ cells / mouse) obtained in the above-mentioned example was intraperitoneally administered to 8-week-old BALB / c female mice treated with blistane. Ascites disease developed after 10 to 21 days. Ascites fluid was collected from the mouse and centrifuged at 3000 rpm for 5 minutes to remove solid formation, followed by Affigel Protein A MA.
It was purified by affinity chromatography using a PS-II kit (manufactured by Bio-Rad).

[0023]

Example 2 Measurement of human tissue factor using the monoclonal antibody of the present invention
Method The monoclonal antibody (HTF-K14) of the present invention was added to a 96-well microtest plate at a concentration of 40 μg / ml in an amount of 50 μl / well, and incubated at 37 ° C. for 1 hour for immobilization. Furthermore, 4% BSA (bovine serum albumin) solution 25
Masking was performed by adding 0 μl and incubating in the same manner. The sample was added to the solid-phased plate prepared in this way and incubated at 37 ° C for 1 hour, after which 0.1% Tween20 was added.
Was washed 3 times with PBS / PBS and 0.5n of the monoclonal antibody (HTF-K180) of the present invention labeled with HRP by the maleimide method was used.
100 μl / well was added at a concentration of g / ml. After incubating at 37 ° C. for 1 hour, the plate was washed 5 times with 0.1% Tween 20 / PBS, then OPDA substrate solution was added, color was developed by a conventional method, and the absorbance was measured at a wavelength of 492 nm. The calibration curve measured by mixing the tissue factor purified according to the above method with TBS is shown in FIG. In addition, when the same concentration of tissue factor used in the preparation of this calibration curve was mixed with human plasma and measured in the same manner,
The same result as the calibration curve shown in FIG. 1 was obtained.

When the tissue factor existing in the cells is measured, an equal amount of 2% Tri is added to Ret-1 cells producing the tissue factor.
Ton X-100 / TBS / 5 mM EDTA was added to the sample, and the sample was subjected to the measuring method of the present invention. The results are shown in FIG. As a result, it is understood that the tissue factor existing in the cells in the blood can be accurately detected and quantified even when the amount thereof is very small.

From these results, it was confirmed that human tissue factor in plasma can be measured up to a concentration of 1 ng / ml according to the present measuring method, and the usefulness of the double antibody measuring method using the monoclonal antibody of the present invention is proved. It was

[Brief description of drawings]

FIG. 1 shows a calibration curve of human tissue factor measured by the two antibody immunoassay method of the present invention in Example 2.

[Fig. 2] Fig. 2 shows the measurement results when the sample treated with 2% Triton in Example 2 was measured by the double antibody immunoassay method of the present invention.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location // C12N 5/20 15/06 C12P 21/08 8214-4B (C12P 21/08 C12R 1:91 )

Claims (4)

[Claims] 1. The amount of human tissue factor in a sample is directly measured by a sandwich double antibody immunoassay using two different monoclonal antibodies specific for human tissue factor. Measuring method. 2. The measuring method according to the above (1), which comprises an operation of treating a sample to be measured with a surfactant before performing the measuring method according to the above (1). 3. The above-mentioned two monoclonal antibodies are antibodies that recognize different epitopes existing in the peptide region from the 175th amino acid to the 219th amino acid from the amino terminus of human tissue factor peptide. The measuring method described in 1). 4. The above-mentioned (1), wherein the two different monoclonal antibodies are antibodies produced from HTF-K14 of Micro Engineering Research Co. No. 11945 and HTF-K180 of Micro Engineering Research Co. No. 11946. The measurement method described.