JPH0827282B2 - Immunological assay method for human protein C, assay reagent and kit thereof - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for immunologically measuring human protein C, a measuring reagent, and a kit thereof. For more details,
In the absence of calcium ion (Ca ⁺⁺ ), either one does not recognize human protein C and
⁺⁺ ) is a monoclonal antibody against human protein C that specifically recognizes human protein C that has undergone a three-dimensional structural change depending on the Gla domain, and the other one is active human protein C. Not recognizing, human
An immunological assay for physiologically active human protein C using a monoclonal antibody against human protein C, which is a monoclonal antibody against human protein C that recognizes protein C,
The present invention relates to a measurement reagent and a kit therefor.

b. Conventional Technology Human protein C plays an important role in the body as a regulator of coagulation / fibrinolysis system. Protein C is a serine protease precursor, and is activated by thrombin bound to thrombomodulin on the surface layer of vascular endothelial cells to become active protein C. This active protein C has serine protease activity and exhibits strong anticoagulant action and fibrinolytic action [Kisiel et al: Biochem.
istry, 16 , 5824-5831 (1977), Comp, PC & Esmon, CT:
J. Clin, Invest., 68 , 1221-1228 (1977)].

As used herein, "human protein C" means inactive serine protease precursor (referred to simply as "P
"C" may be abbreviated), and "active human protein C" means active protein C activated by thrombin (this may be simply referred to as "APC").

Human PC synthesized as a single chain molecule in the liver is
The Glu residue near the amino terminal side undergoes a γ-carboxylation reaction in the presence of vitamin K, is converted into a Gla (γ-carboxyglutamic acid) residue, and is then secreted into the blood.

Most of it in the blood is an L chain with a molecular weight of 21000 and a molecular weight of 41000.
It has a Gla domain containing 9 Gla residues at the amino terminus of the L chain.

Gla is a calcium-binding amino acid, and PC undergoes a conformational change by binding to a calcium ion (Ca ⁺⁺ ) in the Gla domain. This change is known to be important in the expression of activity in vivo [Esmon, NLet, al: J. Biol. Chem. 258, 5548-5553 (19
83)].

However, when vitamin K is deficient or when vitamin K antagonists (warfarin, dicoumarol, etc.) are administered, the γ-carboxylation reaction does not proceed, so Gla, which should constitute the Gla domain, remains Gla, which is a Gla-deficient PC.
(PIVKA-PC: Protein induced by vitamin K absence o
r antagonists) are secreted into the blood. Since this Gla-deficient PC does not undergo a Gla-dependent conformational change in the presence of calcium ions, it does not exhibit physiological activity. In recent years, DIC has been used for the purpose of understanding the coagulation state in the living body.
(Generalized intravascular coagulation), liver injury, and after surgery, immunological measurements of human and PC have been performed, and decrease in PC antigen level correlates with DIC, liver injury, and thrombotic tendency after surgery. It is becoming clear [D'Angelo, SV, et a
l: J. Clin. Invest., 77 , 416-425 (1986)].

Polyclonal antibodies against PC are often used for immunoassays for PC, but in this case, PI
The amount of antigen including VKA-PC will be measured.

Also, in the case of severe DIS patients, it is reported that ACP bound to PC inhibitor is present in the blood [Marlar, R.
D. et al: Blood, 66, 59-63, (1985)], but in the immunoassay for PC using a polyclonal antibody, the complex of APC and PC inhibitor, which had already lost its activity, was detected. Will be measured together.

Therefore, in order to infer the coagulation state in vivo from the change in the amount of PC antigen, and to try to use the knowledge obtained from it for the control of coagulation, PIVKA-PC or APC-PC inhibitor complex was not measured. In addition, PC capable of physiologically expressing activity
Only the specific needs to be measured.

c. Structure of the invention The inventors of the present invention have conducted extensive research on monoclonal antibodies to PC, and have found the following two monoclonal antibodies and have already proposed them (JP-A-1-134399 and JP-A-61-283868). And Japanese Patent Application No. 62-9337
Publication No. 7 (filed on April 17, 1987): Title of the invention "monoclonal antibody and separation / purification method using monoclonal antibody").

In the absence (a) calcium ion (Ca ⁺⁺⁾ did not recognize human protein C, the human protein C sterically structural change depending on the Gla domain in the presence of calcium ions (Ca ⁺⁺⁾ A monoclonal antibody against human protein C that specifically recognizes it.

(B) A monoclonal antibody against human protein C that does not recognize active human protein C but recognizes human protein C.

The present inventors have taken advantage of the specific properties of these two monoclonal antibodies to obtain PIVKA-PC lacking a Gla residue.
It was found that only PC capable of physiologically expressing activity can be measured accurately and with good reproducibility without measuring APC or APC or APC-PC inhibitor complex.

That is, according to the present invention, one is calcium ion (Ca ⁺⁺ ).
Does not recognize PC in the absence of calcium ion (Ca ⁺⁺ )
In the presence, it is a monoclonal antibody against PC that specifically recognizes PC that has undergone a three-dimensional structural change depending on the Gla domain, and the other one does not recognize APC and PC that recognizes PC.
PC characterized by being a monoclonal antibody against
It is an immunological assay method for PC capable of expressing physiological activity in a document using a monoclonal antibody against, a reagent for immunological assay and a kit therefor.

In the present invention, in order to accurately and reproducibly measure PC capable of expressing physiological activity, an antibody bound to an insoluble carrier (hereinafter referred to as an immobilized antibody) and a labeled antibody are reacted so as to sandwich the PC. Then, a sandwich method is used in which the produced sandwich-type complex is detected by the labeling substance of the labeled antibody.

This sandwich method can exert activity in physiology
In the measurement of PC, first, the immobilized antibody and the target PC
After reacting with and completely removing unreacted substances by washing, a labeled antibody is added to form a sandwich type complex consisting of immobilized antibody-PC-labeled antibody (two-step method) Alternatively, immobilized antibody, labeled antibody and PC
Method of forming sandwich type complex at once by mixing and reacting at the same time (1 step method or Simu
ltaneous Assay Method) can be used.

In the present invention, APC does not recognize as an immobilized antibody and P
Using a monoclonal antibody that recognizes C, it does not recognize PC in the absence of calcium ion (Ca ⁺⁺ ) as a labeling substance, and changes in three-dimensional structure depending on the Gla domain in the presence of calcium ion (Ca ⁺⁺ ). received it is possible to use an antibody that specifically recognizes PC, also did not recognize the PC with calcium ions (Ca ⁺⁺⁾ absence as immobilized antibody Conversely, calcium ion (Ca ⁺⁺⁾ presence Then, an antibody that specifically recognizes PC that has undergone a three-dimensional structural change depending on the Gla domain can be used, and a monoclonal antibody that does not recognize APC but recognizes PC can be used as a labeled antibody.

In any of the above cases, "calcium ion (Ca
⁺⁺ ) Does not recognize PC in the absence of calcium ion (Ca
⁺⁺ ) In the presence of "Gla domain-dependent three-dimensional structural change, the monoclonal antibody that specifically recognizes PC" is calcium ion-dependent, so when reacting this antibody, calcium ion (Ca ^{+ +)} coexist is necessary. Thus the addition of calcium ions into an immune reaction buffer, is preferable to the dissolved calcium ions to the wash solution.

When measuring PC in a sample such as plasma, the antibody and plasma sample come into contact with each other in the presence of calcium ions.Therefore, in order to suppress activation of the coagulation system due to the presence of calcium ions, anticoagulation such as heparin It is preferable to carry out the reaction in the presence of an agent.

Examples of the insoluble carrier used in the measurement reagent of the present invention include:
It may be any of those usually used for immunological measurement, for example, polystyrene, polyethylene, polypropylene, polyvinyl chloride, polyester, polyacrylic acid ester, nylon, polyacetal, synthetic resin such as fluororesin, cellulose, agarose, etc. Polysaccharides, glass, metals, etc.

The shape of the insoluble carrier is, for example, a tray shape,
Various shapes such as spherical shape, fibrous shape, rod shape, disk shape, container shape, cell, test tube and the like can be used.

As the labeling substance, it is advantageous to use an enzyme, a fluorescent substance, a luminescent substance, a radioactive substance, or the like. Enzymes are peroxidase, alkaline phosphatase, β-D-galactosidase, etc., and fluorescent substances are fluorescein isothiocyanate, Phycobillip.
rotein, etc., luminescent substances such as isoluminol and lucigenin, and radioactive substances such as ¹²⁵ I, ¹³¹ I, ¹⁴
C, ³ H, etc. can be used, but these are not limited to those exemplified, and other substances can be used as long as they can be used in the immunological assay.

When the labeling substance is an enzyme, a substrate and, if necessary, a color former are used to measure its activity. When peroxidase is used as a substrate, H ₂ is used as a substrate.
O ₂ is used, and a 2,2′-azinodi- [3-ethylbenzthiazolinesulfonic acid] ammonium salt (ABT is used as a color developing agent.
S), 5-aminosalicylic acid, o-phenylenediamine, 4-
Aminoantipyrine, 3,3 ′, 5,5′-tetramethylbenzidine, etc., o-nitrophenyl phosphate as a substrate when alkaline phosphatase is used as an enzyme, and fluorescein as a substrate when β-D-galactosidase is used as an enzyme. -Di- (β-D-galactopyranoside), 4-methylumbelliteryl-β-D-galactopyr
anoside, o-nitrophenyl-β-D-galactopyranoside
Etc. can be used.

The present invention also includes a kit of reagents in addition to the above-mentioned assay method and reagents. As the preferred specific examples, (1) APC does not recognize the PC is a PC that recognize immobilized antibody (2) calcium ion (Ca ⁺⁺⁾ absence without recognition, calcium ion (Ca ⁺⁺⁾ In the presence of a labeled antibody that specifically recognizes PC that has undergone a three-dimensional structural change depending on the Gla domain, or the opposite combination (1) in the absence of calcium ion (Ca ⁺⁺ ). An immobilized antibody that does not recognize PC and specifically recognizes PC that has undergone a three-dimensional structural change depending on the Gla domain in the presence of calcium ions (Ca ⁺⁺ ), and (2) does not recognize APC but recognizes PC. If necessary, (3) a detection reagent for the labeled antibody is used as a main component, and if necessary for the measurement, a known agent such as a solubilizer, a detergent, a reaction terminator, etc., may be used in addition to these reagents. By immunological method consisting of reagents, A reagent kit for measuring the human protein C capable of expressing the activity physiology of standing.

As mentioned above, the antibodies used in the assay method, assay reagent and kit of the present invention have been found by the present inventors,
Previously filed a patent application (JP-A-61-134399, JP-A-
61-283868 and Japanese Patent Application No. 62-93377 (filed on April 17, 1987): title of invention "monoclonal antibody and separation / purification method using monoclonal antibody"). The above-mentioned monoclonal and the method for producing the same of the present invention are described in detail in the above-mentioned patent application specification, but the contents will be briefly described below.

A. Isolation / purification of antigen; Human protein C used for antigen is the method of Suzuki et al. [Su
zuki.K.et.al, J.Biol.Chem. 258 : 1914-1920 (1983)]
Is isolated and purified from human and plasma by.

B. Immunization of mice with human protein C; female Balb / C mice can be used, but other systems (St
rain) mouse may be used. At that time, the immune plan,
And the concentration of human protein C should be chosen such that lymphocytes are formed upon receiving a sufficient amount of antigen stimulation. For example, mice are immunized with 50 μg of human protein C three times in the abdominal cavity at intervals of 2 weeks, and then 30 μg is intravenously administered. Spleen cells are removed for fusion a few days after the final immunization.

C. Cell fusion; The spleen of the mouse immunized as described above is aseptically removed, and a single cell suspension is prepared therefrom. The spleen cells are fused with mouse myeloma cells from an appropriate line by using an appropriate fusion promoter. The preferred ratio of spleen cells to myeloma cells is in the range of about 20: 1 to about 2: 1. Use of 0.5-1.5 ml of fusion medium for about 10 ⁸ spleen cells is suitable.

Mouse myeloma cells used for cell fusion are well known, but in the present invention, P3-X63-Ag8-U1 cells (P3-U1) are used.
[Yelton, DE et al, Current.Topics in Microbiology
and Immunology, 81 (1978)] was used.

As a preferable fusion promoter, for example, an average molecular weight of 10
A polyethylene glycol of 00 to 4000 can be used advantageously, but other coalescing agents known in the art can also be used. In the present invention, polyethylene glycol having an average molecular weight of 1540 was used.

D. Selection of fused cells: a mixture of unfused spleen cells, unfused mouse myeloma cells and fused hybridoma cells in a separate container (eg microtiter plate) does not support unfused mouse myeloma cells Dilute with selective medium and incubate for a time sufficient to kill unfused cells (about 1 hour). The medium used is one that does not support unfused mouse myeloma cells (eg, HAT medium). In this selective medium, the unfused myeloma cells die. Since these unfused spleen cells are non-neoplastic cells, they die after a certain period of time (1 week). The cells fused to these cells can survive in the selective medium because they have both the tumorigenicity of parental cells of myeloma and the properties of parental splenocytes.

E. Confirmation of human protein C antibody in each container; Thus, after the hybridoma cells were detected, the culture supernatant thereof was collected, and an enzyme immunoassay for the antibody against human protein C (Enzyme Linked Immunosobent A) was performed.
screen). At this time, the culture supernatant,
By carrying out both the measurement under the condition of adding a constant concentration of CaCl ₂ to the constituent labeled antibody solution and the washing solution, and the measurement under the condition of not adding CaCl ₂ , by selecting a hybridoma that is positive only for the former, In the absence of calcium ions, human protenin C is not recognized, and in the presence of calcium ions, antibodies recognizing human protein C and secreting hybridomas can be selected.

F. Cloning of hybridoma cells producing the desired antibody and production of the antibody; When the hybridoma cells producing the desired antibody are cloned by an appropriate method (for example, limiting dilution method), 2
Produced in two different ways. According to the first method, the hybridoma cells are cultured for a certain period of time in an appropriate medium, whereby the monoclonal antibody produced by the hybridoma cells can be obtained from the culture supernatant. According to the second method, the hybridoma cells can be injected into the abdominal cavity of a mouse having an isogenic or semiisogenic gene. The monoclonal antibody produced by the hybridoma cells can be obtained from the blood and ascites of the host animal after a certain period of time.

 The present invention will be described in detail below with reference to examples.

Example 1 Measurement of physiologically active PC by enzyme immunoassay (EIA); (1) Immobilization of antibody Japanese Patent Application No. 62-93377 (filed on April 17, 1987: title of invention) "Monoclonal antibody and separation / purification method using monoclonal antibody") APC described in Example 3 described in the specification was not recognized, and a monoclonal antibody (10H11) that recognizes PC was used as an immobilized antibody.

0.01 M phosphate buffered saline (pH 7.4) (hereinafter abbreviated as PBS) is added to anti-PC-monoclonal antibody (10H11) to prepare an antibody solution having a protein concentration of 10 μg / ml. Next, add polystyrene balls (diameter 6 mm) to this and soak it for one day and night. Then, separate the balls and further soak them for one day and night in PBS containing 1% bovine serum albumin (BSA) to obtain antibody-immobilized balls. It was

(2) Preparation of oxygen-labeled antibody PC is not recognized in the absence of calcium ion (Ca ⁺⁺ ) obtained by the method described in JP-A-61-134399, but calcium ion (Ca ⁺⁺ ) is present. Below, a monoclonal antibody (6H2) that specifically recognizes PC that has undergone a three-dimensional structure change in the Gla domain was labeled with horseradish peroxidase (HRP) according to a conventional method, and HRP
A labeled antibody was obtained.

(3) Preparation of calibration curve The method of Suzuki et al. [Suzuki K. et al, J. Biol. Chem., 258 , 19
14-1920 (1983)] isolated and purified from human plasma
To the PC was added 0.05M Tris buffer (pH7.4) containing 0.01M CaCl ₂ (hereinafter abbreviated as TBS) to obtain 100ng / ml, 500ng / ml, 25ng / ml.
ml, 12.5 ng / ml dilution series was prepared. Next, 200 μl of each of these solutions was placed in a test tube, and 1 each of the antibody-immobilized balls of (1) above and TBS of the HRP-labeled antibody of (2) above were placed in it.
200 μl of each solution was added and reacted at 37 ° C. for 1 hour.
Then, after washing with physiological saline containing 0.01M CaCl ₂ , the substrate solution for HRP (0.0M in 0.1M phosphate / citrate buffer (pH 4.0) was used.
Containing 5w / v% tetramethylbenzidine and 0.005MH ₂ O ₂ ) 4
00 μ was added, and the mixture was reacted at 7 ° C. for 30 minutes to develop color. Furthermore, 1.0m of an aqueous solution containing 0.1% NaF and 2% acetic acid.
After adding l to stop the reaction, the absorbance at a wavelength of 650 nm was measured, and the absorbance and the PC concentration were plotted to obtain a calibration curve.
The calibration curve obtained is shown in FIG.

In addition, in FIG. 1, the PC concentration is shown in terms of plasma concentration in order to use it as a calibration curve of a system in which a plasma sample is diluted 100 times and measured.

Example 2 Measurement of plasma sample As a plasma sample, 10 healthy subjects, 12 myocardial infarction patients (administered warfarin powder), 6 administered warfarin,
The PC concentration in plasma was measured for 2 samples of angina patients (administered warfarin powder) and 8 samples administered with warfarin.

First, take 20μ of each of these plasma samples and use 2U of heparin.
After adding 1 ml of TBS containing 1 ml / ml and diluting 50 times, 200 μl of each dilution was put into a test tube, and 1 each of the antibody-immobilized balls prepared in Example 1 and T of HRP-labeled antibody
200 μl of each BS solution was added and reacted at 37 ° C. for 1 hour. Then, the reaction solution and the antibody-immobilized ball are separated, and the antibody-immobilized ball is washed with 0.01 M CaCl ₂ -containing physiological saline, and then the substrate solution for HRP (0.1 M phosphate / citrate buffer (pH 4.0) is used. 0.05w
/ v% tetramethylbenzidine and 0.005MH ₂ O ₂ included) 400
μ was added thereto, and color reaction was allowed to proceed at 37 ° C. for 30 minutes. Furthermore,
Stop solution containing 0.1% NaF and 2% acetic acid 1.
After stopping the enzymatic reaction by adding 0 ml each, the absorbance at a wavelength of 650 nm was measured, and the concentration of protein C in plasma was read from the calibration curve prepared by the method of Example 1, and the results are shown in the following table and Table 2. Summarized in the figure. The low PC value of the warfarin-treated patient sample is considered to be due to the production of PIVKA-PC.

[Brief description of drawings]

FIG. 1 is a diagram showing a calibration curve showing the relationship between the absorbance and the PC concentration, and FIG. 2 shows the measurement results of the PC concentration in various plasmas in Example 2 of the present invention.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Ryoichi Hasegawa 2-1, Hinodecho, Iwakuni-shi, Yamaguchi Teijin Ltd. Biomedical Research Institute (72) Yoichi Sakata 1-13-39, Hanagaki-cho, Oyama-shi, Tochigi Prefecture (72) Inventor Nobuo Aoki 4-20-2-304 Hongo, Bunkyo-ku, Tokyo (56) Reference JP-A-60-120825 (JP, A) JP-A-61-133861 (JP, A) JP-A-61 -134399 (JP, A) JP-A-61-283868 (JP, A)

Claims (5)

[Claims] 1. In the absence of calcium ion (Ca ⁺⁺ ), human protein C is not recognized and calcium ion (C
a ⁺⁺ ) in the presence of a human-specifically recognizing human protein C undergoing a three-dimensional structural change dependent on the Gla domain
By using two kinds of monoclonal antibodies, a monoclonal antibody against protein C and a human antibody C that does not recognize active human protein C but recognizes human protein C, physiological activity in the data A method for immunologically measuring human protein C which can be expressed. 2. In the immunoassay reagent by the sandwich method, one of the antibody bound to the insoluble carrier and the labeled antibody does not recognize human protein C in the absence of calcium ion (Ca ⁺⁺ ), Calcium ion (Ca ⁺⁺ )
In the presence, it is a monoclonal antibody against human protein C that specifically recognizes human protein C that has undergone a three-dimensional structural change depending on the Gla domain, and the other one does not recognize active human protein C, Human protein C characterized by being a monoclonal antibody against human protein C that recognizes human protein C
An immunological assay reagent for human protein C capable of expressing physiological activity in a sample using a monoclonal antibody against. 3. The second insoluble carrier is a plastic container, plastic beads, glass beads or metal particles.
The measuring reagent according to the item. 4. The measuring reagent according to claim 2, wherein the labeled antibody is an antibody labeled with an enzyme, a radioisotope or a fluorescent substance. 5. An antibody bound to an insoluble carrier and a labeled antibody, wherein these antibodies specifically recognize different antigen-determining sites of human protein C, and one of them is calcium ion. In the absence of (Ca ⁺⁺ ), human protein C was not recognized and calcium ion (Ca +
⁺⁺ ) In the presence of human, which specifically recognizes human protein C undergoing a three-dimensional structural change dependent on the Gla domain
A monoclonal antibody against protein C, the other one is a monoclonal antibody against human protein C that does not recognize active human protein C but recognizes human protein C. ) A kit for immunological measurement, which further comprises (c) a substrate for measuring enzyme activity and a reaction terminator when using a detergent and an enzyme-labeled antibody.