JPS6222064A - Method for measuring viii-th factor clotting active antigen - Google Patents

Abstract

PURPOSE:To easily and quickly and economically make specific measurement of FVIII:CAg by using two monoclonal antibodies which recognize different antigen sites without reacting with serum components to measure the VIII-th factor clotting active antigen. CONSTITUTION:The FVIII:CAg is specifically measured by acting the 2nd anti- FVIII:CAg subjected to enzyme labeling, radioactive material labeling or fluorescent labeling to the FVIII:CAg conjugated with the solid phase antibody obtd. by bringing the 1st anti-FVIII:CAg monoclonal a antibody fixed to the solid phase into contact with a specimen of the human plasma, bodily fluid or soln. contg. the FVIII:CAg, etc. The antibody to be used may be an IgG antibody, intact antibody or antibody fragment such as F(ab')2 or Fab'. This method is simpler and more economical than a measuring method in which the IgG prepd. from a hemophilia A patient or anti-VIII:CAg polyclonal antibody is used.

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Field of Industrial Application The present invention relates to a novel method for measuring factor Ⅰ coagulation activity antigen, which is involved in blood coagulation ability.

(○) Conventional technology Factor Ⅰ in human plasma is known as anti-hemophilic factor (antihemophilic factor) because its coagulation activity is lacking in plasma of hemophiliac patients.
It is a coagulation factor called mophilic factor (AHF). This factor is considered to be a large molecular glycoprotein with two important biological activities:
One is coagulation activity (F■:C) that corrects abnormalities in the endogenous coagulation mechanism of hemophilia A patients, and the other is von W.
won Wi I lebrand factor activity (FVI) corrects platelet dysfunction in patients with lebrand disease.
R: WF). In addition to the above-mentioned biological activity, factor (1) also has antigenicity that can be measured immunologically. One of them is factor ■ coagulation activation antigen (F■: CAg).
This is measured by antibody neutralization reaction of F■:C protein, radioimmunoassay, etc. using alloantibodies or autoantibodies against the coagulation-activating antigen. The other is factor 1-related antigen (FVIR: A(J)), which is measured by immunoelectrophoresis or radioimmunoassay of FR:WF protein using a specific antibody against the related antigen.

Incidentally, hemophilia A is a congenital deficiency or abnormality of F■:C protein, and is known to be genetically inherited, and severe cases of congenital deficiency cause repeated severe deep bleeding. Currently, there is no root cause treatment for hemophilia A.
The mainstay of treatment is bleeding control by replenishing the factor that the patient lacks, namely F■:C protein, using fresh frozen plasma, cryoprecipitate, concentrated preparations, etc. In the diagnosis or treatment of hemophilia A, in order to know the degree of deficiency of F■:C protein and its molecular abnormality, it is necessary to know the extent of F■:C protein deficiency and its molecular abnormalities. :C
Measuring Ag is also considered clinically important.

Conventionally, the method for measuring coagulation activity F■:C of factor ■ is as follows:
The following one-step assay method and synthetic peptide substrate method are known.

(1) One-step measurement method: Test plasma is added to plasma of a known severe hemophilia patient (F■2C is 1% or less), and partial thromboplastin time (PTT) or activated partial thromboplastin time (APTT) is measured. ).

(2) Synthetic peptide substrate method 2 F
). For water droplets, test plasma is sufficiently supplemented with F■a, and F■:C is measured using 3-2222 as a substrate for FXa (Journal of the Japanese Society of Hematology 47, 646-648.
(see 1984). (1) The one-step assay requires plasma from a known hemophilia A patient, resulting in large measurement variations; (2) Synthetic peptide substrate method requires expensive FIXa, FX, etc. Although they have their drawbacks, they are commonly used as measurement methods.

On the other hand, the method for measuring coagulation activation antigen F■: CA is as follows:
The following two methods are known. (1): Blood coagulation I prepared from blood of a hemophilia A patient with a prisoner-inhibiting substance
(Method of measuring IG using a labeled antibody labeled with a radioactive substance or an enzyme (British Journal
al of Haen+atology 1981°47, 269-282 or Thrombosis
Re5search 31°707-718.1983). (2): F■: A method in which a polyclonal antibody or monoclonal antibody obtained by immunizing an animal with C protein is measured using a labeled antibody labeled with a radioactive substance or an enzyme. F■:CAg has the advantage of higher storage stability and easier preservation of specimens than F■:C, but currently used immunoassay methods include the following. There are drawbacks.

(l\) Problem F that the invention seeks to solve: CAo
Measurement method (1) is for hemophilia A with factor Ⅰ blocking substances.
Since patient-derived 1aG is used, it is difficult to obtain it continuously and in large quantities. In addition, F■: CAO measurement method (method using polyclonal antibodies in 2) is based on anti-F■: CAO bolic O-nal antibody can be obtained by immunizing animals with F■:C protein, but F■ used as an immunogen. : Since it is difficult to completely purify the C protein, there is a problem with the specificity of the antibodies obtained.

On the other hand, the method using the anti-F■:CA() monoclonal antibody does not require complete purification of the F■:C protein to obtain the monoclonal antibody, and furthermore, the method using the anti-F■:CAg monoclonal antibody can be applied to antibody-producing cells ( hybridoma)
It has the advantage of being easy to obtain since it can be produced in unlimited quantities.

However, it is known that the F■:C protein has some antigenic homology with celluloplasmin, a serum protein (Nature, 312°37
7, 1984), there is a risk that the antibody used for measuring F■:CAg may recognize a foreign protein unless it is a monoclonal antibody that recognizes a portion specific to the F■:C protein.

In view of the above circumstances, the present inventors have conducted intensive research to develop a method for specifically quantifying the coagulant-active antigen F■: CA(I). : Anti-F specific for C protein■:
CA! By using the If monoclonal antibody, F
■: It was confirmed that it was possible to specifically measure CAg, and the present invention was achieved.

That is, the present invention provides a method for measuring a factor (I) coagulation activity antigen in a specimen by an immunological method, in which a first monoclonal antibody having the property of recognizing the antigen but not reacting with serum components, and a second monoclonal antibody that recognizes a different antigenic site from that of the first monoclonal antibody.
This is a method for measuring factor coagulation activity antigen.

The outline of the method for producing the monoclonal antibody used in the present invention is as follows. F■: When a foreign animal is immunized with C protein as an antigen, antibodies against many of the antigenic determinants are produced by antibody-producing cell groups. These antibody-producing cells are fused with animal myeloma cells, which have unlimited proliferation potential, and the fused cells are cloned to select cells that produce the desired antibody. The selected cell group is
Infinitely, it is a group of cells that produce the antibody of interest, and each individual antibody produced is a specific antibody that corresponds to a specific site of F■:CAg. In the present invention,
Among them, anti-F■:CA(I monoclonal antibodies, such as M 10K 9 and 56Y18, which did not react with serum components and reacted only with F■:CA(+) were selected.

The antibody preferably used in the present invention is an I(+G antibody, and even if it is an intact antibody, F(ab'>2
It may also be an antibody fragment such as Fab' or Fab'.

The measurement method of the present invention uses a first anti-F■ immobilized on a solid phase:
CAa monoclonal antibody, human plasma.

Body fluid or F■: A substance that binds to a solid-phase antibody obtained by contacting a sample such as a solution containing CA(+).
It can be carried out by treating CA!It with a second anti-F:OAσ monoclonal antibody labeled with an enzyme, a radioactive substance, or a fluorescent substance. Anti-F: CA(I When a monoclonal antibody is used as a solid-phase antibody in the present invention, a known method can be used for immobilization, and a solid-phase carrier such as polystyrene, polyethylene, polyacrylate,
Balls, beads, etc. made of Teflon polyacetal, etc.
Gears and microplates are preferably used. on the other hand,
Anti-F■: When using CA(I monoclonal antibody as a labeled antibody, F(atl')z
Alternatively, if Fab' is made of w4, the measurement sensitivity will be improved.

In the method using enzymes as labeling agents (EIA), enzymes such as horseradish peroxidase, β-D-galactosidase, alkaline phosphatase, etc. are used as labeling agents, and in the method using radioactive substances (RIA), 1''r, ``H, etc. In the method using a fluorescent substance (FIA), fluorescein isothiocyanate and the like are used, but other materials may be used as long as the activity of the labeling agent can be measured. In the case of
[3-Ethylbenzthiazolinesulfonic acid (6)] ammonium salt-H202,0-phenylenediamine-H2
02,4-aminoantipyrine-H2O2 etc. are β-
Fluorescein as a substrate for D-galactosidase
Examples include g(β-D-galactopyranoside), o-nitrophenol-β-D-galactopyranoside, and the like.

In addition to these reagents, known reagents such as a solubilizer, a detergent, and a reaction terminator are used for the measurement.

(, t) Actions and Effects of the Invention The present invention uses anti-F■: CAt) monoclonal antibodies that are infinitely produced by antibody-producing cells.
1g made from hemophilia A patients with factor blocking substances
G or anti-F■: Compared to the measurement method using C, AQ polyclonal antibodies, it is clearly simpler and more economical, and F■
: It has the potential to be widely used in the measurement of CAo. In addition, in the present invention, the solid-phase antibody and the labeled antibody have different recognition sites, and F■:CAg
By using two types of anti-F■: CA(l monoclonal antibodies, measurement by the two-antibody sandwich method becomes possible;
This is a method for measuring CA(l).Furthermore, the monoclonal antibody of the present invention has no cross-reactivity with ceruloplasmin, which has high homology with F■:C protein, and F■:CA
It was shown to be specific to q.

Furthermore, since F■:C is extremely unstable, there is a problem in that it is difficult to preserve the specimen. However, as shown in Example 2, when the measurement method of the present invention is used, F■:C and F■:
Since CAo is very well correlated, blood coagulation ability can also be examined by measuring only F■:CA(l), which has high storage stability, instead of measuring F■:C. .

Hereinafter, the present invention will be explained in detail with reference to Examples.

(f) Examples Example 1 (1) Preparation of peroxidase-labeled wave F■: CAIJ monoclonal antibody Fab' (a) Anti-F■: CA(l monoclonal antibody (Mlo
9) 5q (1Rg/ll!, 0.IMNa
CR-0, 1M acetate buffer, pH 4,0)
Pepsin 0.25 IQ (o, sIRg, /m1.
0.1M acetate buffer.

After reacting overnight at 37°C, the reaction product was purified with Sephadex Q-150 equilibrated with Phosphorus W1-buffered saline (PBS) and purified with anti-F■: CA(l
Monoclonal antibody (MIOK 9) F (ab
') got 2.

(C1) Obtained F(absu22ryJ(2II!)
g/mI! , 0.1M Na1fI phosphate solution, 1
)) l 6.0), 511MEDTA-0, IM
0,1M dissolved in H6,0>MNa buffer (1)
Add 60 µm of mercaptoethylamine and incubate at 37°C for 90 µm.
After reacting for a minute, the reaction solution was mixed with 5771META-0,1
Msu'acidゝ°! 1 buffer solution 3°16・0) 1 equilibrium level L,
tc t.

It was purified using Fadex G-25 to obtain anti-F■:CA9 monoclonal O-nal antibody (MloK9) Fab'.

(c) Peroxidase 8Rg derived from horseradish with 0.1M phosphorus! tNa buffer (DH7,5) 1 a
d! 1. -Dissolved N-succinimidyl-3-(2-pyridylthio)propionate 1.1 (5I11 g/ae).

After adding ethanol) and reacting at 25°C for 30 minutes,
0.1M phosphorus MNa 'at! Purification was performed using Sephadex G-25 equilibrated with Solution E (DH6,0) to obtain pyridyl disulfide peroxidase.

) Anti-F■ prepared in (0): GAg monoclonal antibody (9 to Mlo) Fab' 1.2* (5,6
III/d, 5mM EDTA-0, 1M Nag phosphate
! ! i solution, l) 86.0) and (/previously prepared pyridyl disulfide-peroxidase 111 g (4W)
I/d O, IM Na phosphate! 1lti liquid, l)8
6.0) were mixed and reacted at 4°C for 3 days. After the reaction,
Peroxidase-labeled anti-F■:CAg monoclonal antibody Fab: (HRP-MloK 9-Fab') purified using Ultrogel ACA44 (manufactured by LKB) equilibrated with 0.1M Na phosphate buffer (1) 86.5)
I love you.

(2) Peroxidase labeled wave F■: CA(+ Preparation of monoclonal antibody 1aG (a) Anti-F■: CA(+ -E/ clot antibody (MloK9) 0.6IItg (0.6ruy/
ld, PBS) kl, vs. l-maleimidobenzoyl-N-hydroxysuccinimide ester 0.31
ftg(1■/d.

After adding DMF) and reacting at 25°C for 3 hours, 0.1
It was purified using Sephadex G-25 equilibrated with Mlo@Na buffer (pH 6.0) to obtain an anti-F:CAG monoclonal antibody (Mlo: 9) into which a maleimide group was introduced.

(0) Anti-F■ having a maleimide group prepared in (a):
CA++ monoclonal antibody 0. (For illg, S
-Ultrogel A containing SHM-introduced peroxidase 2.4#19 with acetylmercapto anphosuccinic acid, reacted at 4°C for 2 hours, and then equilibrated with PBs.
Purified with CA44 and peroxidase labeled wave F■:C
AIj monoclonal antibody I(]G()-IRP-
9-1 (IG) was obtained for Mlo.

(3) Anti-F■: Immobilization of CA (] monoclonal antibody on a ball or microplate.Anti-F■: CAg monoclonal antibody (S6Y18) was dissolved in PBS at a protein concentration of 100 μg/d.
Add a polystyrene ball to this, soak it overnight, and use anti-F: CALJ monoclonal antibody (S6Y18).
Fixed polystyrene balls were obtained. Also anti-F■: CAg
Monoclonal antibody (S6Y18) at a protein concentration of 100μ
g/IIdl in PBS, and then diluted with E
Add 100μ/well to IA microplate,
- The plate was left to stand day and night to obtain an anti-F■:CA (l anti-monoclonal antibody (S 6 Y 18)-immobilized microplate.

(4) Anti-F■: F■:GA(] in plasma and serum of healthy subjects by CAIJ monoclonal antibody sandwich method
Measurement of the anti-F■:GA (l monoclonal antibody (S6Y18)) obtained in (3) above on a fixed microplate.
200 μU of 1.5% BSA (bovine serum albumin)
/ well, left at room temperature for one hour, and then washed. Next, add 50μ of a 2-fold dilution of plasma and serum from a healthy person, add 9-Fab' solution to HRP-Mlo obtained in (1) above, or add 9I (
50 µm of IG solution was added, and the mixture was allowed to react at 4°C overnight. After washing, add ABTS O,5 in 0.1M phosphate citrate buffer (pH 4.5)
rItg/d and 2MH2O20,5tlB-/d) at 25°C for 30 minutes, and the absorbance at 420 nm was measured. In this way, pooled plasma from healthy individuals (20 people) was used as a standard material for the m-line test, and F■:CA(J) in human plasma and serum was quantified.
9-Fab',b to RP-Mlo; HRP-Mlo
9- to G) are shown in Figure 1, and the measured values of 14 people are shown in Figure 1.
Shown in the table.

From FIG. 1, it can be seen that the sensitivity is better when using Fab' than IqG. Also, Table 1 or “
These results show that the monoclonal antibody of the present invention shows no cross-reactivity with serum components.

Table 1 U: Unit where 1U is the amount of coagulation active antigen per 1d of pooled plasma from 20 healthy people.

Example 2 Correlation of F■:C and F■:CA(I) in healthy human plasma and hemophilia A patient plasma For 14 types of healthy human plasma and 5 types of hemophilia A patient plasma, F■:C is According to the one-step method, F■:CA(] was measured by the sand inch method according to the present invention, and F■:
C and F■: CA (l is a 1% risk rate) A significant correlation was observed (for hemophilia A patients, both were 0.) Example 3 F■: CA (] and cell Examination of cross-reactivity of plasmin Celluloplasmin 1 q/at!
■: Measured by Sanderch method using CAg monoclonal antibody, F■: CAg is 7.8×1O-3U
/rd or less. Furthermore, F■: CAo is 5000t
J/IItg, and the cross-reactivity rate is 1.56 x 10
→% or less, and no cross-reactivity was observed.

[Brief explanation of drawings]

Figure 1 shows factor ■ coagulation activation antigen (F■: CAo) R1
This is a calibration curve showing the relationship between intensity and absorbance.

Claims (1)

[Scope of Claims] 1. In a method for measuring a factor VIII coagulation activity antigen in a specimen by an immunological method, a first monoclonal antibody that recognizes the antigen but does not react with serum components; and a second monoclonal antibody that has the same properties but recognizes a different antigenic site than the first monoclonal antibody. 2. The first and/or second monoclonal antibody is an IgG
The method for measuring the factor VIII coagulation activity antigen according to claim 1, which is antibody F(ab')_2 or Fab'. 3. Factor VIII according to claim 1 or 2, wherein the first monoclonal antibody is immobilized on a solid phase, and the second monoclonal antibody is labeled with an enzyme, a radioactive substance, or a fluorescent substance. Method for measuring coagulation active antigen.