JPS6265693A - Monoclonal anti-human blood coagulation factor xi antibody and determination of human coagulation factor xi using same - Google Patents

Abstract

PURPOSE:To obtain a monoclonal antibody having specificity to human coagula tion factor XI by immunizing a mouse with human blood coagulation factor XI and fusing the spleen cell of the immunized mouse with a cell originated from a mouse tumor line, thereby forming a hybridoma. CONSTITUTION:A monoclonal antibody having high specificity to human coagula tion factor XI can be produced by selecting the objective cell from a number of antibody-producing cells obtained by immunizing a mouse with human coagula tion factor XI and cloning by using the cell as a hybridoma. The active-type coagulation factor XI (factor XIa) can be directly determined by combining the anti-human coagulation factor XI antibody with anti-alpha1-antitrypsin antibody and determining the obtained composite of active-type coagulation factor XI and alpha1-antitrypsin antibody.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a novel monoclonal anti-human coagulation factor X antibody and a method for quantifying a factor in an activated human coagulation system using the same.

[Background of the invention]

Coagulation factor X is the most important factor involved at the initiation point of the blood coagulation cascade reaction. That is, in the presence of prekallikrein and high-molecular-weight kininogen, factor
Acts on factors and activates them. Thus the active type
Once factor XIa (factor
The factors are activated and a chain reaction (cascade reaction) proceeds according to the order shown in the diagram below, and finally stable fibrin is produced and the blood coagulation reaction is completed.

(Davie, E, W et al Advance
es in Enzymology 48277 Hs
7s)) (Blood Coagulation Cascade Reaction) Stable fibrin factor X is a glycoprotein with a molecular weight of 180,000 and is composed of two polypeptide subunits with approximately equal molecular weight. Chapter ■, IX, X, XI.

■Each factor is an enzyme that has serine at its active center and has similar amino acid sequences. Furthermore, there are similarities with thrombin and prekallikrein. In addition, in terms of biological activity, in addition to the blood coagulation system, platelet cheg + ota
xis (chemotaxis) and a reaction that activates factor It is involved in many important physiological reactions, such as:

In this way, fluctuations in the activity of coagulation factor

It is believed to be involved in various diseases that cause destruction of blood cells, damage to blood vessel walls, and abnormal blood flow. It has also been revealed that in essential hypertension and diabetes, the amount of factor X and plasma prekallikrein increases, and the coagulation system is accelerated. Therefore, as an indicator for clinically examining vascular lesions, it is of great significance for clinical diagnosis 1-1 to detect the activation of the coagulation system, particularly the activation of the coagulation factor Y- in the early phase of the coagulation reaction.

Conventionally, 1111 constant U of coagulation factors has been measured by activated partial thromboplastin time (aPTT) using factor-deficient plasma that is deficient in each factor, but abnormalities in the concentration or activity of factor X alone have been selected. could not be detected.

In addition, in recent years, a method for measuring factor On the other hand, immunochemical measurement methods that utilize antigen-antibody reactions are still not common.

[Purpose of the invention]

The present invention was made in view of the above-mentioned circumstances, and
Providing a monoclonal anti-human coagulation factor (III) antibody having high specificity for human coagulation factor
The purpose of this invention is to provide a new and highly accurate immunological assay method for fixed factor (I).

[Structure of the invention]

The present invention is produced from hybridomas formed by fusion of cells from mouse tumor lines and splenocytes from mice previously immunized with human blood coagulation factor X.
Medium clonal anti-human coagulation factor X antibody having specificity for human coagulation factor X and activated human coagulation factor X using the same
This invention relates to an immunochemical method for quantifying factor (XIa).

That is, the present inventors found that in a large number of antibody-producing cells obtained by immunization with human factor X, there were antibodies that could distinguish the unique structure of human coagulation factor X from other similar coagulation factors. We assumed that there were cells that produced the protein, and after extensive research, we selected the target cells and cloned them as hybridomas. As a result, we obtained a monoclonal antibody with high specificity for human coagulation factor X. , we have completed the present invention.

Furthermore, for the purpose of directly quantifying activated coagulation factor , Cl1n Inve
st.

Adopting α1 antitrypsin (α, AT), which was revealed by the research of
By using a combination of anti-human coagulation factor X antibody and anti-α1 antitrypsin antibody, we established a method for measuring activated coagulation factor We have completed a direct fixation method for intrinsic factor.

The novel monoclonal antibody specific for human coagulation factor X according to the present invention has a strong affinity for activated factor X as well as for native human coagulation factor X. Since these monoclonal P1 antibodies do not inhibit the enzyme activity of activated factor X, it is presumed that none of them recognize the active center of the enzyme.

These clonal antibodies of the present invention can be used by enzyme-linked immunosorbent assay (
It can be effectively used for quantifying human coagulation factor X and activated factor X by EIA), radioimmunoassay (RIA), fluorescence immunoassay (FIA), and the like. In addition, activated coagulation factor Factor (factor XIa)-α
, when quantifying antitrypsin complex, it has excellent specificity and good sensitivity, so activated coagulation factor X (XIa)-α, antitrypsin complex can be effectively quantified, This makes it possible to quantify activated coagulation factor X more effectively.

The anti-α1-antitrypsin antibody having specificity for α1-antitrypsin used in the present invention may be purified from antisera produced in known animals or may be a monoclonal antibody, and is not particularly limited. It's not something you can do.

The activated human coagulation factor III of the present invention using two types of antibodies (
Factor XIa)-α1 antitrypsin complex (XIa-
In the method of quantifying X1a-α, AT complex), for example, one is bound to a solid phase (insoluble carrier) and then reacted with a sample containing X1a-α, AT complex, which is then pre-labeled with a labeling substance. A possible method is the Sand-Germany method, in which the other antibody is reacted with the other antibody. It goes without saying that the labeling substances used here are enzymes in EIA, radioisotopes in RIA, and fluorescent substances in FIA.

In EIA using the monoclonal antibody of the present invention, examples of enzymes used as labeling substances include, but are not limited to, β-galactosidase, alkaline phosphatase, peroxidase, and the like.

In addition, as the insoluble carrier used in this EIA, EIA
Commonly used synthetic polymer compounds such as polystyrene, polyvinyl chloride, polypropylene, and polyethylene, as well as inorganic substances such as silica gel, are used without exception, and these are usually beads, tubes, disk pieces, and fine particles (latex particles). It is also well known that it is used in the form of microplates and the like.

In addition, the reagents and operating methods used in this EIA may be appropriately selected and carried out in accordance with the reagents and operating methods used in EIA that are known per se.

In addition, RIA, FIA using the mesoclonal antibody of the present invention
The method for quantifying the human coagulation factor f- also uses reagents similar to those used in RIA and FIA, which are known per se, except that the mesoclonal anti-human coagulation factor X antibody of the present invention is used as the antibody. This can be done according to the operating method of RIA and FIA which are known per se.

The first step to achieve the purpose of the present invention is to create a new monoclonal hybridoma that produces a new monoclonal antibody. Consists of.

1. Immunization 2. Cell fusion 3. Hybridoma selection and monocloning This will be explained step by step below.

In the case of immunization, the methods for preparing antiserum or immunization can be applied as they are. However, since the hybridomas are selected with I11 ability in the later step [-], it is not necessary to use pure human I-clotting factor as the antigen, which is essential for preparing the antiserum. The immunized animal is determined by the tumor cell line used for cell fusion, but is generally a rat,
A mouse is often used. Ba1b/ is an established mouse tumor cell line that does not produce immunoglobulin.
c is often used. For the initial immunization, it is preferable to administer 300 g of antigen υl to each mouse together with an adjuvant. Immunity is 2-4i! ! ! The final immunization is performed at intervals, and when no adjuvant is used, the antigen dissolved in physiological saline or the like is injected intraperitoneally or intravenously. Also, when using an adjuvant, it is injected intraperitoneally. Two to four days after the final immunization, the lymph nodes or spleen are removed, and the obtained lymphocytes are subjected to cell fusion. Furthermore, the immunization process does not have to be an in vivo immunization as described in L, but is effective even if it is an in vivo immunization. in vit
Immunization of ro is carried out as follows.

That is, a lymph node or spleen is removed, and the obtained lymphocytes are suspended in a culture solution and cultured in an incubator for 18 days.

The lymphocytes are optionally stimulated with antigen. The antigen stitch at this time is usually 50. g or less is sufficient, and the number of stimulations is once a day, and a total of three times or less is sufficient. Lymphocytes from 3 days to 3 weeks after the start of culture are used for cell fusion.

All known techniques can be applied to the steps of cell fusion, hybridoma selection, and monocloning.

A common method is shown below.

Initially, the tumor cell line used for cell fusion was M.
PC-11, P3-X63-8AZ, etc. were introduced, but since these produce their own immunoglobulin, recently P3-
X63-8AZ-Ul, P3-NS-1, etc. are commonly used. During cell fusion, there are 5 to 2 lymphocytes compared to tumor cells.
Use 0 times more amount. MEM medium, McCoy medium, RP
Tumor cells and lymphocytes washed with MI 1840 medium or isotonic sap, etc. are mixed and then centrifuged to form a pellet. After loosening the pellets, cells are fused with HVJ (Sendai virus) or polyethylene glycol (PEG), but in general, a 40-60% solution of PEG with an average molecular weight of 1,000-s and ooo, which is easy to handle, is .5-2I11/use. colchicine, dimethyl sulfoxide (DMSO), poly-L to promote fusion.
-Arginine etc. may be added, but it is not essential.

After performing the fusion reaction with the PEG solution for about 1-10 minutes, add MEM medium, RPM I 1840 medium, etc. for 10-50 minutes.
m/ gradually to stop the fusion reaction. Centrifuge after stopping.
Remove L supernatant. Add MEM medium or RPM I 1840 medium containing 5 to 20% fetal bovine serum (Fe2).
Lymphocytes were placed in a 4-well culture plate at 1×lθ per well.
Pour 1 m/minute to obtain ~5 x 106 pieces. Or 8
Lymphocytes per 1 hole in a 6-well culture plate ~ 2 x 105
Dispense every 0.2 d so that the amount is 1.

It is preferable to add feeder cells to both. As feeder cells, rat thymocytes, splenocytes, mouse thymocytes, splenocytes, etc. are used, and the concentration is 0.5.
-2 x 106/d. Next, Hyboxanthin I X lo'M, Aminopterin 4XIO
M, RPM I 1 containing thymidine 1.8X 10'M
840 medium (or MEM medium), ie, HAT medium. Dispense to replace the HAT medium: - Generally, the next day [1] Add a volume of clay equal to the volume 14 dispensed at the time of fusion to the culture plate, and then replace the clog with HAT medium the next day. Then 2-31
Replace half V with HAT medium every 1 hour. 10-14 after fusion
At F1, half of the HAT medium without 7-minopterin is replaced with a beaten HT medium, and from 1 to 3 days later, the "t'ld" of the medium is replaced with a normal HAT-free medium every 1 to 3 days.

Various analyzes of the cell culture supernatant from wells where hybridomas are actively proliferating I), such as RIA, plaque method, agglutination reaction,
Hybridomas producing the antibody of interest are selected by ELISA or the like. Once you have obtained a hybridoma, perform cloning. The cloning method is F A CS (Fl
uorescent Activated Ce1lS
There are several methods, such as using a microorganism (orter), a method of picking up colonies using 5 of Agar, and the commonly used limiting dilution method. Cloning is carried out at cell concentrations such that colonies are formed from one hybridoma.

In limit box dispensing, the number of cells per well of an 86-well plate is 0.
Do this so that there are no more than 6 pieces.

No matter which method is used, cloning is repeated twice to obtain a wide clone. Once a clone has been identified, the antibody can be cultured in 1n culture or in vitro.
It is produced by culturing in vivo. inma 1t
Antibodies produced by RO are not contaminated with other antibodies, but have low antibody titers. On the other hand, antibodies produced in vivo are slightly contaminated with antibodies from the host, but the antibody titer is much higher than that of 1nM 1tro. The method used to produce antibodies is arbitrary.

The present invention will be explained in more detail with reference to Examples below.
The present invention is not limited in any way by these Examples.

Example 1 Preparation of a monoclonal anti-Human coagulation factor antibody and a hybridoma producing it (1) Immune human coagulation factor Freund's Complete Adjuvant 0.1a/emulsion Ba
The mixture was injected intraperitoneally into 1b/c mice (male, 4 weeks old).

That 4i! ! 1, lomM) Lys buffer (pH 7,2
) 20 gg of human coagulation factor X dissolved in 0.1 a/ml was administered intraperitoneally.

(2) Cell fusion Three days after the final immunization, the spleen of the mouse was removed. 108 splenocytes and 107 MS-1 cells were incubated with 0.5 g of polyethylene glycol, 0.5 a/ml of MEM medium, and DMSO.
o, 15a! The cells were incubated for 1 minute at room temperature for fusion in a solution of Cells in RPMI 184 with 15% FC3
After suspending in 0 medium, dispense into 5 86-well plates,
Culture was started in a C02 incubator. The next day, 2 drops of HAT medium was added per well.

Then, the hand-sewn medium was replaced with HAT medium every 3 days.

Culture supernatants two weeks after fusion were used for detection of antibody production.

(3) Selection of hybridomas In order to select hybridomas producing anti-human factor X antibody, each of the cell culture supernatants from the above 86 wells was analyzed by ELISA.

First, in an 86-well plate, 5 factors were added to purified H.
．． The antigen was fixed on the plate by dispensing the antigen at a concentration of 0.1a/g/- and leaving it at 37°C for 2 hours.

Contains 0.05% Tween 20 (polyoxyethylene rubitan monolaurate, Kao Soapball brand name)
After washing three times with omM phosphate buffer p)I 7.4 (hereinafter referred to as washing solution), 1% BSA (bovine serum albumin) was added to avoid nonspecific adsorption of proteins in the culture-1 supernatant. The solution was dispensed into 0.2 d portions and allowed to stand at 37°C for 2 hours. After washing three times with washing solution, cell culture-L
The supernatant was dispensed into 0.1 aliquots and allowed to stand at 37°C for 2 hours. HAT medium was used as a negative control. Next, after washing three times with a washing solution, peroxidase-labeled anti-mouse immunoglobulin solution o and tag were dispensed and left at 37°C for 2 hours. After washing three times with washing solution, 0.4% orthophenylenediamine solution 0. After dispensing Iaff and reacting at room temperature for 30 minutes, 6N sulfuric acid
．． The reaction was stopped by adding 0.05d. D.

Measured at 490 nm. 2 times more than the negative control
Hybridomas growing in the culture supernatant exhibiting the following were selected as anti-human coagulation factor II antibody-producing hybridomas.

(4) Monocloning Cloning was performed by the limiting dilution method. The cells were dispensed so that 0.5 hybridomas per well of an 86-well plate. This operation was repeated twice to complete monocloning. Eight clones were selected by ELISA and used for primary antibody production.

(5) Production of monoclonal antibodies: 5 x 106 hybridomas of 8 types
Ba1b/c mice (male,
It was administered intraperitoneally to a child (8 weeks old), and ascites was collected about 2 weeks later.

The ascites was subjected to ammonium sulfate fractionation at 50% saturation, and further column chromatography using DEAE separator was performed to obtain monoclonal antibodies of the present invention 1-1.2-1.4-
1, 8-3, 7-1, 8-1, 9-3, 10
Eight types of -1 were obtained.

Example 2 Antibody titer and specificity of monoclonal antibodies 5olid using a vinyl chloride 86-well microplate
It was analyzed using a phase radioimmunoassa7υ. First, purified coagulation factor X (F, XT) was added to an 86-well microplate at 1.5 μs/lO.
The antigen was immobilized on the plate by standing at 4° C. overnight. Tween20 (polyoxyethylene sorbitan monolaurate, Kao soap ■trade name) 0.0
After washing three times with 5% 10mM phosphate buffer pH 7.4 (hereinafter referred to as washing solution), in order to avoid non-specific adsorption, 0.2al of 1% BSA (bovine serum albumin) solution was dispensed. The mixture was allowed to stand at room temperature for 3 hours. After washing three times with washing solution, each car was treated with clonal antibody enrichment solution (0.01gg/II
/~100 gg/d) 0.02 ml was dispensed and allowed to stand at room temperature for 3 hours to perform an immune reaction. Add to this reaction solution 0.02 d of 125I-labeled anti-mouse IgG goat antibody (
about 40,000 cpm) and allowed to react at room temperature for 3 hours. After thorough washing with a washing solution, the radioactivity remaining in the microplate was measured using a gamma counter.

Figure 1 shows the monoclonal antibody of the present invention (No. 2-1°4).
-1.7-1 and 1O-1) and coagulation factor X, measured from radioactivity.

In addition, in order to examine the specificity of monoclonal antibodies, L
The same 5 solid phase RIA method was performed using various coagulation factors and coagulation system inhibitors as antigens instead of the coagulation factor As a result, all of the monoclonal antibodies obtained in the present invention specifically bind to factor X, including factor X, activated factor X (factor X fragment), plasma prekallikrein,
It was found that it did not react with polymeric kininogen, protein C1α1-antitrypsin, antithrombin ■, and corn trypsin inhibitor (a specific inhibitor of factor X fragments).

Example 3 IgG subclass of each monoclonal antibody anti-mouse IgG1. IgG2a, IgG2b, 1gG
3 Microophthalony method (Duc) was performed using rabbit serum.
hterlony &Nilsson; Handbo
ok of Experimental
The IgG subclass of each monoclonal antibody was examined using the 19.1978 (Japanese). Both monoclonal antibodies were found to be rgcl.

Example 4 Epitope analysis of each monoclonal antibody Epitope analysis of 6 monoclonal antibodies was performed using 5DS-polyacrylamide electrophoresis (SDS-PAGE) and Weste
This was performed using enzyme immunostaining using rn Blotting. Using a slab gel with a gel concentration of 5% to 10%,
Coagulation factor X (F, XI) activated factor X (F, XIa
), and the reduced forms ■ and Xla, which were obtained by reducing them with 1% 2-mercaptoethanol;
The complex of α1-antitrypsin and Lae+5m1i
(Nature, 227.680-E185.197
5DS-PAGE was performed for about 1.5 hours at a current of 15 mA according to method 0)+7). The gel after electrophoresis is tow
bin et al.'s method (Proc, Natl, Acad,
Sci, 78.4350-4354.11179)
Transfer was performed to a nitrocellulose membrane according to . The protein bands transferred onto nitrocellulose were reacted with each monoclonal antibody, further reacted with a peroxidase-labeled rabbit anti-mouse IgG antibody as a secondary antibody, washed, and then subjected to a diaminobenzidine reaction. Detected.

As a result, each monoclonal antibody has a native clotting factor (F, XI) (molecular weight 180,000).
and its active form (F, XIa), but when reduced with 2-mercaptoethanol, the reduced form F,
Molecular weight, 80,000)
It was found that it did not react with Ia. Furthermore, it was found that each monoclonal antibody also reacted with a complex of active F, X[a and α1-antitrypsin. Therefore, the epitopes of the mesoclonal antibody of the present invention are F, XI and F,
It is presumed to have a certain ζ-form structure held by disulfide bonds common to Ia, and is thought to be a structural part different from the enzyme active center.

Example 5 Activated coagulation factor X (F, XIa)-α,-
Quantification of antitrypsin (α, -AT) complex (quantification of activated coagulation factor The cells were transplanted using an ELISA method using a combination of a monoclonal anti-human coagulation factor antibody and a commercially available anti-α1-antitrypsin antibody.

First, reagents for the assay were prepared.

Polystyrene beads were immersed overnight at 4°C in a solution of medium clonal anticoagulant factor 1 antibody No. 7-1 (50 Hiroshi 8/d) of the present invention. lomM phosphate saline (hereinafter referred to as PBS)
After washing with 5% BSA solution at 4° C. overnight.

On the other hand, peroxidase-labeled anti-α1-antitrypsin antibodies are available from known authors (Eiji Ishikawa, Tadashi Fuchiai, Kiyoshi Miyai, eds.
It was prepared by conjugating the Fab' fragment of the antibody and peroxidase using the maleimide method using enzyme immunoassay (Igaku Shuin 1982). The standard activated coagulation factor X-α1-antitrypsin complex (F, XIa-a
, -AT) were prepared by activating purified F and XI with purified factor Ⅰ fragment and further adding purified α1-AT. The ELISA method is to react a PBS solution (2004) containing standard or sample plasma with anticoagulant factor , peroxidase-labeled anti-α1-antitrypsin antibody solution (300g)
and was reacted at 4°C for 18 hours. After the reaction, the beads were thoroughly washed, and the peroxidase activity bound to the beads was reacted for 30 minutes at room temperature using O-phenylenediamine-H2O2 as a substrate solution. After stopping the reaction with IN o2so4, the absorbance at 492 nm was immediately measured.

FX[a-α, -AT complex content was measured using a standard sample with known content by the method described in -1-, and the calibration curve created was
As shown in the figure. Note that the horizontal axis is shown in terms of the amount of FXIa. As is clear from the calibration curve, activated coagulation factor (FXIa) can be measured down to 0.1 ng/assay by the quantitative method of the present invention.

By the quantitative method of the present invention, it was found that the amount of the coagulation factor in the plasma of patients with DIC (disseminated intraluminal coagulation) was several times higher than that of normal subjects.

〔Effect of the invention〕

As described above, the present invention provides a novel monoclonal anti-human coagulation factor X antibody having high specificity for human coagulation factor By using the monoclonal antibody of the present invention in combination with an anti-α1-antitrypsin antibody, a method for quantifying activated human coagulation factor This invention has a remarkable effect on the ability to provide blood vessel lesions such as arteriosclerosis, and it also makes it possible to directly quantify coagulation factors using an immunochemical method, which can be used as an indicator for clinically examining vascular lesions such as arteriosclerosis. It is a great invention that contributes to this industry.

[Brief explanation of drawings]

FIG. 1 shows the Rakuchu clonal antibody of the present invention (in Example 2).
2-1.4-1.7-1 and 1O-1) and the coagulation factor determined from radioactivity, and the horizontal axis shows the antibody concentration (m/a/). , the vertical axis is the radioactivity (
cpll) respectively. Figure 2 shows the calibration curve obtained in Example 5, which connects the points obtained by plotting the absorbance obtained for each concentration of activated sterile factor X on the horizontal axis along the vertical axis. be.

Claims (9)

[Claims] (1) specificity for human coagulation factor A monoclonal anti-human coagulation factor X I antibody. (2) The monoclonal anti-human coagulation factor X I antibody according to claim 1, wherein the cells from the mouse tumor line belong to P3-X63. (3) The monoclonal anti-human coagulation factor X I according to claim 1 or 2, which has specificity for human blood coagulation factor X I and does not react with any other human blood coagulation factors.
factor antibody. (4) Claims 1 or 2 have specificity for human blood coagulation factor X I and further specifically react to activated factor X I (factor X I a). Monoclonal anti-human coagulation factor X I antibody as described in . (5) Cells from mouse tumor lines and human coagulation X
A hybridoma formed by fusion with splenocytes from a mouse previously immunized with factor I and producing a monoclonal anti-human coagulation factor X I antibody with specificity for human coagulation factor X I. (6) The hybridoma according to claim 5, wherein the cells from the mouse tumor line belong to P3-X63. (7) Specific for human coagulation factor XI, produced by a hybridoma formed by fusion of cells from a mouse tumor line and splenocytes from a mouse previously immunized with human blood coagulation factor XI. 1. A method for quantifying activated human coagulation factor X I (factor X I a), which comprises using a monoclonal anti-human coagulation factor X I antibody. (8) Using a monoclonal anti-human coagulation factor X I antibody with specificity for human coagulation factor X I, a complex of activated human coagulation factor X I (factor X Ia) and α_1 antitrypsin Activated human coagulation factor X I (X I
Quantification method of a factor). (9) Claims that use a combination of a monoclonal anti-human coagulation factor X I antibody that has specificity for human coagulation factor X I and an anti-α_1 antitrypsin antibody that specifically reacts with α_1 antitrypsin. 9. The method for quantifying activated human coagulation factor X I (factor X Ia) according to item 8.