JPH0636741B2 - Method for separating human protein C - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION a. INDUSTRIAL APPLICABILITY The present invention is not recognized by the absence of calcium ions (Ca + ⁺⁾ against human protein C, and hybridomas which produce the monoclonal antibodies recognizing the presence of calcium ions (Ca + ⁺⁾ The present invention relates to a method for separating human protein C.

b. Prior Art Protein C is a vitamin K-dependent plasma protein or γ
-A carboxyglutamic acid-containing protein, which is activated by thrombin in the presence of thrombomodulin on the surface of vascular endothelial cells [Esmon, C, T & Owen, W. G: Pro
c.Natl.Acad.Sci. USA. 78 : 2249-2251 (1981)] Activated protein C (APC). Activated protein C is a type of serine protease, which decomposes coenzymes of the blood coagulation system, factor V (FV.FVa) and factor VIII (FVIII, FVIIIa), and has a strong anticoagulant action [S
uzuki.K. et. al. : J. Biol. Chem. 258 : 1914-192
0 (1983). Vehar, G .; A. & Davie, E. W. : Bioc
hemistry. 19 : 401-409 (1980)] and release plasminogen activator from the blood vessel wall to promote the fibrinolytic system [Comp, P. et al. C. & Esmon, C.I. T:
J. Clin. Invest. 68 : 1221-1228 (1981)].

Furthermore, protein C deficiency has also been reported to exhibit severe thrombosis, [Griffin, J. et al. H. et al: J. C
lin. Invest., 68 , 1370-1373 (1980), Bertina, R .;
M. et al: Thromb. Haemostas., 48 1-5 (1982)]
Protein C has been shown to be an important regulator of the blood coagulation and fibrinolysis system.

Therefore, if the mechanism of action of protein C can be clarified, and the amount of antigen and activity of protein C in blood can be measured and its trends can be grasped, it is very important in the fields of basic medicine and clinical medicine. It is thought to have meaning.

On the other hand, a monoclonal antibody is specific to a single antigenic determinant and can stably produce an antibody having the same specificity. Therefore, the function and structure of an antigen protein can be analyzed or immunoassay (EAI, RIA) can be performed. Recently, it has become widely used. In particular, for functional analysis and molecular analysis of an antigen protein, finding an antibody that recognizes a site involved in the function of the antigen protein or a special structural site can be a powerful means.

In the structure of human protein C, an H chain having a molecular weight of about 41000 and an L chain having a molecular weight of about 21000 are linked by an SS crosslink,
It has a serine protease active site in the H chain and L-
At the amino terminus of the chain, nine calcium ion-binding amino acids, namely γ-carboxyglutamic acid (Gla)
It is known that there is a Gla domain containing residues. Blood coagulation factors having a Gla domain include Factor II (prothrombin), Factor VII, including protein C.
Both factor IX and factor X are calcium ions (Ca ^{+
In the} presence of ⁺ ), it is known to cause a Gla-dependent three-dimensional structural change, and this mechanism is known to play an important role in the expression of the blood coagulation system.

Previously, it has been reported that a monoclonal antibody against human protein C was produced by Suzuki et al. [Koji Suzuki et al .: “Blood and Vascular”, 15: 171-174 (1984)]. measurement of the amount of antigen human protein C, have been used to measure the activity, calcium ions (Ca ^{+ +)}
No report has yet been made on a monoclonal antibody that recognizes human protein C that has undergone a structural change in the presence thereof.

The present invention found that, paying attention to the protein C undergoes a three-dimensional structural change in the presence of calcium ions (Ca + ^+), calcium ion (Ca + ⁺⁾ presence specifically the protein C undergoes a conformational change in the The present invention has been achieved as a result of research on monoclonal antibodies that recognize specifically.

c. Configuration of the Invention Namely, the present invention is to provide a calcium ion (Ca + ⁺⁾ of human protein C in the presence of and calcium ions not recognized in the absence with respect to human protein C (Ca + ⁺⁾ Human protein C that specifically recognizes
A hybridoma that produces a monoclonal antibody against

Yet another of the present invention, specific for human protein C in the presence of calcium ions (Ca + ⁺⁾ in the absence and calcium ions not recognized for human protein C (Ca + ⁺⁾ Recognizes human protein C
Monoclonal antibodies adsorbent obtained by binding to an insoluble carrier for the human protein C-containing mixture is contacted in the presence of calcium ions (Ca + ^+), that allowed to bind human protein C in the adsorption body A method for separating human protein C from a characteristic mixture containing human protein C.

The hybridoma cells producing the monoclonal antibody of the present invention can be prepared by the method of Kohler and Milstein [Khler &
Milstein, Nature: 256 495-497 (1975)]. That is, after immunizing a mouse with human protein C, the spleen cells of this mouse are fused with mouse myeloma cells, and the obtained hybridoma cells react with human protein C immobilized on a microtiter plate. Systematically inspected and selected. At this time, calcium ions (Ca ^+
+ ) The test in the presence of calcium ion and the test in the absence of calcium ion are simultaneously carried out to select hybridomas showing positive only in the former case, whereby a hybridoma that synthesizes and secretes the target antibody can be isolated.

The monoclonal antibody obtained from the product produced by such a novel hybridoma cell is calcium ion (C
a ^{+ +} ) acts monospecifically for a particular antigenic determinant on a human protein C molecule.

The characteristics of the nomoclonal antibody obtained by the present invention are very advantageous when used for the purification of human protein C. That is, a monoclonal antibody obtained by the present invention to an insoluble carrier immobilized, calcium ions (Ca + ⁺⁾ (as prepared as not solidified in the presence of calcium ions) plasma in a solution exists, or, other human material containing protein C or crude extract thereof, a crude product and adsorb human protein C from a solution, the separated calcium ions (Ca + ⁺⁾ after washing in the presence, the solution containing no calcium ions ( For example, human protein C can be eluted by substituting it with a solution containing EDTA). According to this method, unlike the conventional purification of an antigen protein with an insolubilized antibody, purification is performed under mild conditions without exposing the protein to extreme conditions (for example, 0.2 M glycine hydrochloride or 8 M urea). It can be performed.

It is known that human protein C and other .gamma.-carboxyglutamic acid-containing proteins contain an abnormal molecule which does not contain Gla and does not undergo a Gla-dependent structural change. It is considered that this abnormal molecule can be measured by using. That is, calcium ion (C
a ^{+ +} ), the presence or absence of an antibody that recognizes human protein C is used for immunological means (eg EIA, R).
Human protein C in plasma and other samples by IA)
The amount of antigen is measured, and the monoclonal antibody according to the present invention is used to detect human protein C in plasma or other samples.
Immunoassay in the presence of calcium ions (EIA, R
If measured by IA), the amount of abnormal human protein C can be grasped from the difference between the measured values.

Next, a specific method for producing the monoclonal antibody obtained by the present invention will be described in detail.

A. Isolation / purification of antigen; Human protein C used for antigen is the method of Suzuki et al. [Suz
uki.K. et al, J.M. Biol. Chem. 258 : 1914-1920 (198
3)] isolates and purifies from human plasma.

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 that have received a sufficient amount of antigen stimulation. For example, 50 μg of human protein C is intraperitoneally administered to mice three times at 2-week intervals, and then 30 μg is intravenously administered. Spleen cells are removed for fusion a few days after the final immunization.

C. Cell fusion; Aseptically remove the spleen of a mouse immunized as described above,
From there, a single cell suspension is prepared. 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.
The use of 0.5-1.5 m 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 (P
3-U1) [Yelton, D .; E et al, Current. Top
ics in Microbiology and Immunology, 81 1 (1978)
Reference] was used.

As a preferable fusion promoter, for example, an average molecular weight of 1000
Advantageously, ~ 4000 polyethylene glycols can be used, but other fusion promoters 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; selection medium that does not support unfused mouse myeloma cells with a mixture of unfused spleen cells, unfused mouse myeloma cells and fused hybridoma cells in a separate container (eg, a microtiter plate) And incubate for a sufficient time (about 1 week) to kill unfused cells. A medium that does not support unfused mouse myeloma cells (eg, HAT medium) is used. 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 have the neoplastic properties of the parental cells of myeloma and the properties of spleen cells, and therefore can exist in the selective medium.

E. Confirmation of human protein C antibody in each container; Thus, after the hybridoma cells were detected, the culture supernatant thereof was collected and the enzyme immunoassay (EnzymeLinked Immunosoben) for the antibody to human protein C was collected.
t assay). At this time, a certain concentration of C was added to the culture supernatant, the enzyme-labeled antibody solution and the washing solution.
By carrying out both the measurement under the condition that aC ₂ was added and the condition under which no CaC ₂ was added, and selecting a hybridoma that showed positive only to the former, human protein was detected in the absence of calcium ion. It is possible to select a hybridoma that does not recognize C but produces and secretes an antibody that recognizes human protein C in the presence of calcium ions.

F. Cloning of Hybridoma Cells Producing the Antibody of Interest and Production of the Antibody When the hybridoma cells producing the antibody of interest are cloned by an appropriate method (eg, limiting dilution method), the antibody is produced by two different methods. According to the first method, by culturing the hybridoma cells in an appropriate medium for a certain period of time, the monoclonal antibody produced by the hybridoma cells can be obtained from the content 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.

G. Separation of human protein C from human protein C-containing mixture; monoclonal antibody obtained by the present invention, as described above, in the absence of calcium ions (Ca + ⁺⁾ not recognized for human protein C because and in the presence of calcium ions (Ca + ⁺⁾ have the property of recognizing specifically against human protein C,
Utilizing this property, human protein C can be easily separated from a mixture containing human protein C (eg, human plasma).

Therefore, first, the monoclonal antibody against human protein C is immobilized or bound to an insoluble carrier to obtain an adsorbent. The insoluble carrier used at this time may be one commonly used as a base material of a measurement reagent or a measurement kit using a monoclonal antibody. For example, agarose, polyacrylamide, cellulose, dextran, maleic acid polymer or a mixture thereof is preferably used as the material. The form of these inert carriers can be various forms such as powder, granules, pellets, beads, films and fibers. Further, it is advantageous to use a plate (well) having a large number of concave depressions that are generally used for measuring or separating plasma or its fractional components.

Using the adsorbent, the human protein C-containing mixture to and brought into contact in the presence of calcium ions (Ca + ^+), and the monoclonal antibody immobilized on adsorption body and human protein C is bound to As a result, human protein C binds to the adsorbent. By doing so, human protein C can be separated and removed.

The Human Protein C is bound to the adsorbent and the above-mentioned manner, if possible a mixture of the remainder is removed by washing, then essentially the calcium human protein C bound to the adsorbent ion (Ca + ⁺⁾ Human protein C is isolated from the adsorbent by contact with or washing with a liquid that does not contain human protein C, and human protein C can be isolated.

Thus, according to the above-mentioned separation method of the present invention, removal of human protein C from a mixture containing human protein C, separation and purification of human protein C from the mixture, removal of human protein C from the mixture, The measurement of the content can be achieved by an extremely simple operation.

The present invention will be described in detail below with reference to examples. In the following examples, protein C may be abbreviated as PC.

Example 1 Purified human / PC female Balb / C mice (4 weeks old)
Two animals were immunized four times at 14-day intervals.

The first immunization was dissolved in PBS. An equal volume of Freund's complete adjuvant (Complete Fr)
enud's adjuvant), and the emulsion was administered intraperitoneally (0.5 mg / head).
Similarly, 50 μg of human PC was mixed with Freund's incomplete adjuvant and intraperitoneally administered. For the final immunization, 30 μg of human PC was administered from the tail vein of the mouse as a PBC solution. Spleen cells of immunized mice were used for cell fusion 3 days after the final immunization.

Spleen cells of immunized mouse and myeloma cells (P3U1) of syngeneic mouse were mixed at a ratio of about 2: 1 to 15: 1, and the mixture was 50%.
Cell fusion was performed according to the method of Khler and Milstein using polyethylene glycol 1540 (Wako) as a fusion accelerator. The fused cells were suspended in 10% FCS-RPMI-1640 medium at a cell concentration of 1 × 10 ⁶ cells / m 2
100 wells per wells microplate (Coster)
Dispensed by μ.

The fused cells were in a CO ₂ incubator (5% CO ₂ , 37
C.), the medium is replaced with a medium containing hypoxatin, aminopterin and thymidine (HAT medium), and HA
After growing in T medium, spleen cells and myeloma cells were screened for hybridomas.

Antibodies in the culture supernatant of hybridomas were prepared using a microtiter plate coated with the antigen human / PC.
It was detected by the ISA method. As the second antibody, an alkaline phosphatase-labeled rabbit anti-mouse IgG antibody was used. To observe the difference in binding with human PC in the presence or absence of calcium ions, one culture supernatant contained 5 mM CaC.
TBS with ₂ added (0.02M Tris / HC, 0.14M
Na C, pH 7.4) and TBS was added to the other. Furthermore, 5 mM for the diluent of the second antibody and the washing solution
Ca C ₂ added TBS, 0.05% Tween 20, 0.02% N
a N ₃ or TBS, 0.05% Tween 20, 0.02% Na
Using the N _3.

Formation of colonies was observed in 523 wells out of a total of 541 wells in which the fused cells were seeded. Of these, the number of antibody production positive wells was 44 in the presence of calcium ions and 32 in the absence of calcium ions as shown in Table 1 below.

Twelve of these antibody production positive wells were cloned twice by the limiting dilution method to obtain 13 clones. The resulting clone is 90% F
Suspended in CS-10% DMSO and stored in liquid nitrogen.

The monoclonal antibody produced by each clone was obtained by growing the clone in the abdominal cavity of Balb / C mouse and purifying it from the ascites using a protein A-Sepharose 4B column.

Example 2. (Characteristics of purified IgG) IgG of each clone purified from mouse ascites was examined for binding to subclass, human / PC activity, and binding to L chain or H chain.

The subclass was determined by the Ouchterlony method using anti-mouse antiserum of each class specificity. The effect on human / PC activity was to add IgG to human / PC at a molar ratio of 1: 5 and incubate overnight at 4 ° C. to activate human PC by thrombin-thrombodulin complex.
The activity was measured by measuring the decomposition activity of the following synthetic substrate. As this synthetic substrate [Here, Vel-D type optically active valine, Leu is L type optically active leucine, and Arg is L type optically active algin. S manufactured by KabiVitrum AB (Sweden)
−2266 was used] was used. L-chain and H-chain binding properties were determined by subjecting human PC to electrophoresis under reducing conditions and performing immunoblotting using a nitrocellulose membrane and HRP-labeled Goat anti-mouse IgG.

The results obtained for each property are shown in Table 2 below. All the calcium ion-dependent antibodies were L chain-binding and did not affect the activity of human PC.

Example 3. (Effect of Calcium Ion) The effect of calcium ion on the reaction between human PC and purified IgG was examined.

5 mM of calcium ion-dependent antibodies 7B12 and 10E12 in ELISA coated with human PC
CaC ₂ added TBS-Tween (0.02M Tris / HC
, 0.14M Na C, pH7.4 0.05% Tween20, 0.02
% Na N ₃ ) or TBS-Tween and diluted with human
It was 5 m when the amount of binding was measured by the reaction with PC and the color development using alkaline phosphatase labeled rabbit anti-mouse IgG.
When it was diluted with a buffer containing M Ca C ₂ , binding with human PC depending on the antibody concentration was shown, but when it was diluted with a buffer without addition of Ca C ₂ , the antibody concentration was increased. No binding was observed. The results are shown in Table 3 below.

The calcium ion-independent antibodies 6B10-1 and 10H1
The results of investigating the effect of calcium ions using 1 were also shown in Table 3 below.

Example 4. (Influence of Calcium Ion Concentration) Calcium Ion Dependent Antibody 7B in Example 3
12 and 10E12 were used and their concentrations were kept constant (1 μg / m
), When the calcium ion concentration in the antibody solution was changed, the binding to human / PC increased as the Ca C ₂ concentration increased and became saturated at a concentration of about 1 mM. The results are shown in Table 4 below. Table 5 below shows the results of investigating the effect of changes in Ca C ₂ concentration in the same manner using the calcium ion-independent antibody 6E2.
From the results shown in Table 5, it can be seen that the calcium ion-absent antibody shows almost constant binding to human PC without affecting the calcium ion concentration.

The measurement was performed using TBS- containing a predetermined concentration of Ca C _2.
Prepare 1 μg / mIgG solution with Tween and add 100 μ
It was performed by adding it to the well coated with human / PC. After washing the wells after incubation, each concentration of Ca C
TBS-Tween containing ₂ was used.

Example. 5 (1) Immobilization of antibody on insoluble carrier; Bromcyan-activated Sepharose 4B (Pharmacia
0.5 g of dry gel (manufactured by Fine Chemicals Co., Ltd.)
Swell and wash with 100 mM 1 mM HC on a glass filter, and then use coupling buffer (0.5 M
It was washed with 0.1 M NaHCO ₃ pH 8.3 containing Na C). After the coupling buffer was removed by suction, the gel was immediately added to 2 m of the coupling buffer solution (3 mg / m) of the antibody (6H2) and suspended, and the mixture was gently shaken overnight at 4 ° C. The gel was then transferred into 1 M ethanolamine-HC (pH 8.0, 2 m) and shaken at room temperature for 2 hours to block residual active groups. After blocking, the antibody-coupled Sepharose gel was placed on a glass filter, and 0.1 M acetate buffer pH 4.0 containing 0.5 M Na C was added.
0.1M borate buffer pH 8.0 containing 0.5M Na C
Were used alternately to wash. When the absorbance at 280 nm of the filtrate became 0.01 or less, 5 mM Ca C ₂ ,
And Tris / HC containing 1 mM benzamidine
Equilibrated to pH 7.4 and packed on the column. Anti-human PC monoclonal antibody (6H2) thus prepared
Affinity chromatography was performed using a bound Sepharose 4B column.

(2) adsorption to the antibody-conjugated Sepharose 4B of protein C, eluted; and stirred for 1 hour at 4 ° C. was added a 1M Ba C ₂ solution 8m plasma 100 m. The precipitate was collected by centrifugation and 5 mM Ba
0.1 M Na C containing C _2, 5 mM benzamidine
After washing with, 0.2M containing 15m of 5mM benzamidine
The precipitate was dissolved with EDTA pH 7.4 to obtain a barium adsorption fraction. The barium adsorption fraction was dialyzed against 0.05 M Tris / HC pH 7.4 containing 1 mM benzamidine to give a final concentration of 5
Add Ca C ₂ solution to make 5 mM C
0.05 M Tris / containing aC ₂ and 1 mM benzamidine
It was applied to an antibody (6H2) binding column equilibrated with HC. Washed with 0.05 M Tris / HC containing 5 mM Ca C ₂ , 1 mM benzamidine and 1 M Na C, containing 50 mM EDTA and 1 mM benzamidine
When eluted with 0.05 M Tris / H C, a single peak containing PC was obtained. The PC in the fraction eluted with the antibody Sepharose 4B was purified about 52 times as much as the fraction adsorbed with barium, and the recovery rate was about 48.2%.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Nobuo Aoki 4-20-2-304 Hongo, Bunkyo-ku, Tokyo (56) References JP-A-60-120825 (JP, A)

Claims (1)

[Claims] In the absence of 1. A calcium ion (Ca ^{+ +),} specifically for human protein C in the presence of and calcium ions not recognized for human protein C (Ca + ⁺⁾ monoclonal antibodies to recognize human protein C in adsorbent obtained by binding to an insoluble carrier, brought into contact with the presence of calcium ions (Ca + ⁺⁾ to human protein C-containing mixture, human to adsorption body
A method for separating human protein C from a mixture containing human protein C, which comprises binding protein C.