JPH031959B2 - - Google Patents

Description

[Detailed Description of the Invention] a. Industrial Application Field The present invention does not recognize human protein C in the absence of calcium ions (Ca ⁺⁺ ), but recognizes it in the presence of calcium ions (Ca ⁺⁺ ). Monoclonal antibodies, hybridomas that produce them,
The present invention relates to a method for producing the antibody and a method for separating human protein C. b. Prior Art Protein C is a vitamin K-dependent plasma protein, that is, a protein containing γ-carboxyglutamic acid, and is activated by thrombin in the presence of thrombomodulin on the surface layer of vascular endothelial cells [Esmon,
C.T & Owen, WG: Proc.Natl.Acad.Sci.USA.
78:2249-2251 (1981)] becomes activated protein C (APC). Activated protein C is a type of serine protease, and is a type of serine protease that acts as an oxygen supplement for the blood coagulation system, such as factor V (FV.FVa) and factor (F,
Fa) and has a strong anticoagulant effect [Suzuki.K.
et.al.: J.Biol.Chem. 258 :1914-1920 (1983),
Vehar, GA & Davie, EW:
Biochemistry.19:401-409 (1980)] and releases plasminogen activator from the blood vessel wall, promoting the fibrinolytic system [Comp, P.
C. & Esmon, CT: J. Clin. Invest. 8 : 1221-1228
(1981)]. Furthermore, it has been reported that protein C deficiency causes severe thrombosis [Griffin, JHet
al: J.Clin.Invest., 68 , 1370-1373 (1980),
Bertina, RMet al: Thromb.Haemostas., 48
1-5 (1982)] Protein C has been shown to be an important regulator of the blood coagulation and fibrinolytic system. Therefore, if we can clarify the mechanism of action of protein C, measure the amount of antigen and activity of protein C in the blood, and understand their trends, this will be extremely important in the fields of basic and clinical medicine. It is thought that it has a significant meaning. On the other hand, monoclonal antibodies are specific for a single antigenic determinant, and have the advantage of being able to stably produce antibodies with the same specificity. It has recently become widely used. In particular, functional analysis of antigen proteins,
For molecular analysis, finding antibodies that recognize sites involved in the function of antigenic proteins or special structural sites can be a powerful means. The structure of human protein C has a molecular weight of approximately 41,000
The H chain and the L chain with a molecular weight of 21,000 are linked by an S-S bridge, and the H chain has a serine protease active site, and the amino terminal of the L chain contains nine calcium ion-binding amino acids, That is, γ−
Gla containing carboxyglutamic acid (Gla) residues
It is known that the domain exists. Blood coagulation factors with Gla domains include protein C, factor (prothrombin), factor
Both factors are known to undergo Gla-dependent conformational changes in the presence of calcium ions (Ca ⁺⁺ ), and this mechanism plays an important role in the expression of the blood coagulation system. It is known that there are Previously, it has been reported that a monoclonal antibody against human protein C was created by Suzuki et al.
(1984)], this antibody is used to measure the antigen amount and activity of human protein C.
There has been no report yet on a monoclonal antibody that recognizes human protein C that undergoes a structural change in the presence of calcium ions (Ca ⁺⁺ ). Therefore ^, the present inventors focused on the fact that protein C undergoes a steric structural change in the presence of calcium ions (Ca ⁺⁺ ).
The present invention was achieved as a result of research into monoclonal antibodies that specifically recognize protein C, which undergoes a structural change in the presence of. c. Structure of the invention That is, the present invention does not recognize human protein C in the absence of calcium ions (Ca ⁺⁺ ), and does not recognize human protein C in the presence of calcium ions (Ca ⁺⁺ ). This is a monoclonal antibody against human protein C that specifically recognizes human protein C. Another aspect of the present invention is a hybridoma that produces a monoclonal antibody against the human protein C, and a method for producing the monoclonal antibody against the human protein C from a product produced by the hybridoma. Still another aspect of the present invention is that it does not recognize human protein C in the absence of calcium ions (Ca ⁺⁺ ) and does not recognize human protein C in the absence of calcium ions (Ca ⁺⁺ ).
The monoclonal antibody against human protein C, which specifically recognizes human protein C in the presence of the adsorbent bound to an insoluble carrier,
Human protein from a human protein C-containing mixture, characterized in that the human protein C-containing mixture is brought into contact with the human protein C-containing mixture in the presence of calcium ions (Ca ⁺⁺ ) to bind human protein C to the adsorbent. This is a separation method for C. Hybridoma cells producing the monoclonal antibodies of the present invention can be prepared by the method of Koehler and Milstein [Ko¨hler & Milstein, Nature: 256 495-497
(1975)].
That is, after immunizing a mouse with human protein C, the mouse's spleen cells are fused with mouse myeloma cells, and the resulting hybridoma cells are immobilized on a microtiter plate.
Antibodies that react with protein C are systematically tested and selected. At this time, a test in the presence of calcium ions (Ca ⁺⁺ ) and a test in the absence of calcium ions are conducted at the same time, and hybridomas that are positive only in the former are selected to identify hybridomas that synthesize and secrete the target antibody. can be isolated. The monoclonal antibodies of the present invention are obtained from products produced by such novel hybridoma cells,
It acts monospecifically on a particular antigenic determinant on the human protein C molecule in the presence of calcium ions (Ca ⁺⁺ ). The monoclonal antibody of the present invention is very advantageous when used for the purification of human protein C due to its characteristics. That is, the monoclonal antibody of the present invention is immobilized on an insoluble carrier, and plasma (prepared so as not to coagulate in the presence of calcium ions) or other human protein C is added in a solution containing calcium ions (Ca ⁺⁺ ). Human protein C is adsorbed and separated from raw materials containing , or their crude extracts, crude purified products, and solutions, and after washing in the presence of calcium ions (Ca ⁺⁺ ), the solution is converted to a material containing no calcium ions (e.g.
solution in which EDTA is present), human
Protein C can be eluted. According to this method, proteins are purified under mild conditions without exposing them to extreme conditions (e.g., 0.2M glycine hydrochloride or 8M urea) as in conventional purification of antigenic proteins using insolubilized antibodies. It can be performed. In addition, human protein C and other γ-carboxyglutamic acid-containing proteins do not contain Gla.
It is known that there are abnormal molecules that do not undergo Gla-dependent structural changes, and it is thought that the use of the monoclonal antibody of the present invention will make it possible to measure these abnormal molecules. That is, human protein C antigen in plasma and other samples can be detected by immunological means (e.g. EIA, RIA) using antibodies that recognize human protein C, regardless of the presence or absence of calcium ions (Ca ⁺⁺ ). If human protein C in plasma or other samples is measured by immunological methods (EIA, RIA) in the presence of calcium ions using the monoclonal antibody according to the present invention, the measured value can be From the difference, the amount of abnormal human protein C can be determined. Next, a specific method for producing monoclonal antibodies according to the present invention will be explained in detail. A. Isolation and purification of antigen; Human protein C used as antigen was prepared by the method of Suzuki et al. [Suzuki.K.et al, J.Biol.Chem. 258 :
1914-1920 (1983)] was isolated and purified from human plasma. B. Immunization of mice with human protein C; female Balb/C mice can be used, but mice of other strains may also be used.
The immunization regimen and the concentration of human protein C should then be chosen so that a sufficient amount of antigen-stimulated lymphocytes are formed. For example, mice are administered 50 μg of human protein C intraperitoneally three times at two-week intervals, followed by an additional 30 μg intravenously. A few days after the final immunization, spleen cells are removed for fusion. C Cell fusion: The spleen of the mouse immunized as above is removed aseptically and a single cell suspension is prepared therefrom.
By using these spleen cells with mouse myeloma cells from an appropriate line and an appropriate fusion promoter,
Fuse the cells. The preferred ratio of spleen cells to myeloma cells ranges from about 20:1 to about 2:1.
It is appropriate to use 0.5-1.5 ml of fusion medium for approximately 10 ⁸ splenocytes. Mouse myeloma cells used for cell fusion are well known, but in the present invention, P3-X63-Ag8
−U1 cells (P3−U1) [Yelton, DE et al,
Current.Topics in Microbiology and
Immunology, 81 1 (1978)] was used. As a preferred fusion promoter, for example, polyethylene glycol having an average molecular weight of 1000 to 4000 can be advantageously used, but other fusion promoters known in the art can also be used. In the present invention, polyethylene glycol with an average molecular weight of 1540 was used. D. Selection of fused cells; selection that does not favor unfused mouse myeloma cells by adding a mixture of unfused spleen cells, unfused mouse myeloma cells, and fused hybridoma cells in a separate container (e.g., a microtiter plate) Dilute with culture medium and culture for sufficient time (about 1 week) to kill unfused cells. The medium is
those that do not support unfused mouse myeloma cells;
(e.g. HAT medium) is used. In this selective medium, unfused myeloma cells die.
Since these unfused spleen cells are non-neoplastic cells,
It dies after a certain period of time (one week). Cells fused to these cells have the tumor properties of the parent cells of myeloma and the properties of the parent splenocytes, so they can survive in the selective medium. E. Confirmation of human protein C antibodies in each container; thus, after hybridoma cells are detected, their culture supernatants are collected and screened for antibodies against human protein C by Enzyme Linked Immunosobent Assay. do. At this time, culture supernatant,
Add a certain concentration to the enzyme-labeled antibody solution and washing solution.
By conducting both measurements with and without CaCl ₂ and selecting hybridomas that _were positive only for the former, human
Hybridomas that do not recognize protein C but produce and secrete antibodies that recognize human protein C in the presence of calcium ions can be selected. F. Cloning of hybridoma cells that produce the antibody of interest and production of the antibody When hybridoma cells that produce the antibody of interest are cloned using an appropriate method (e.g., limiting dilution method), antibodies can be produced in two different ways. . According to the first method, monoclonal antibodies produced by hybridoma cells can be obtained from the culture supernatant by culturing hybridoma cells in an appropriate medium for a certain period of time. According to a second method, hybridoma cells can be injected into the peritoneal cavity of isogenic or semi-isogenic mice. Monoclonal antibodies produced by the hybridoma cells can be obtained from the blood and ascites of the host animal after a certain period of time. G Human protein C from human protein C-containing mixture
Separation of protein C: As mentioned above, the monoclonal antibody of the present invention does not recognize human protein C in the absence of calcium ions (Ca ⁺⁺ ), and does not recognize human protein C in the presence of calcium ions (Ca ⁺⁺ ). Since it has the property of specifically recognizing human protein C, this property can be used to extract human protein C from a mixture containing human protein C (such as human plasma). can be easily separated. Therefore, first, a monoclonal antibody against human protein C is immobilized or bound to an insoluble carrier to obtain an adsorbent. The insoluble carrier used in this case may be one that is commonly used as a base material for measurement reagents or measurement kits using monoclonal antibodies. For example, agarose, polyacrylamide, cellulose, dextran, maleic acid polymer, or a mixture thereof is preferably used as the material. These inert carriers can be in 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, which is generally used for measuring or separating plasma or its fractionated components. Using the above adsorbent, a human protein C-containing mixture was added to it, and calcium ions (Ca ⁺⁺ ) were added to it.
When the monoclonal antibody immobilized on the adsorbent and human protein C are brought into contact with each other in the presence of
As a result, human protein C binds to the adsorbent. In this manner, human protein C can be separated and removed. Alternatively, human protein C can be bound to the adsorbent as described above, the residual mixture preferably washed away, and then the human protein C bound to the adsorbent can be substantially freed of calcium ions (Ca ⁺⁺ ). When it comes into contact with or is washed with a liquid that does not contain human protein C, human protein C detaches from the adsorbent, and by obtaining it, human protein C
can be isolated. Thus, according to the separation method of the present invention, human protein C is removed from a mixture containing human protein C, human protein C is separated and purified from the mixture, and human protein C is removed from the mixture. Measuring the content can be accomplished with extremely simple operations. 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 was injected into female Balb/C mice (4
Two mice (age) were immunized four times at 14-day intervals. The first immunization was dissolved in PBS. 50 μg human
PC was mixed with an equal volume of Complete Freund's adjuvant and the emulsion was administered intraperitoneally (0.5 mg/
head), the second and third doses were 50 μg of human
PC on the front incomplete adjuvant (Freund's)
(incomplete adjuvant) and administered intraperitoneally. Final immunization: 30μg human PC in PBS
The solution was administered to mice through the tail vein. Spleen cells from mice immunized 3 days after the final immunization were used for cell fusion. Spleen cells from immunized mice and myeloma cells (P3U1) from syngeneic mice were mixed at a ratio of about 2:1 to about 15:1, and mixed with Ko¨hler using 50% polyethylene glycol 1540 (Wako) as a fusion promoter. Cell fusion was performed according to the method of Milstein. After fusion, the cells are 1
10% FCS- to give a cell concentration of ×10 ⁶ cells/ml
The suspension was suspended in RPMI-1640 medium, and 100 μl was dispensed per well into a 96we11s microplate (Coster). The fused cells were grown in a _CO2 incubator (5%
Cultured in CO ₂ , 37°C), and cultured in a medium containing hypoxanthine, aminoprithene, and thymidine (HAT medium).
Change the medium and grow in HAT medium.
A hybridoma consisting of spleen cells and myeloma cells was screened. Antibodies in the hybridoma culture supernatant were detected by ELISA using a microtiter plate coated with the antigen human PC. An alkaline phosphatase-labeled rabbit anti-mouse IgG antibody was used as the second antibody, and 5mMCaCl ₂ was added to one culture supernatant to see the difference in binding to human and PC in the presence and absence of calcium ions. did
TBS (0.02MTris/HCl, 0.14MNaCl, PH7.4) was added to the other side. Furthermore, _5mMCaCl2 was added to the second antibody dilution solution and washing solution.
TBS, 0.05% Tween20, 0.02% _NaN3 or
TBS, 0.05% Tween20, 0.02% _NaN3 was used. Colony formation was observed in 523 wells out of a total of 541 wells seeded with fused cells. Among these, 44 wells were positive for antibody production in the presence of calcium ions and 32 in the absence of calcium ions, as shown in Table 1 below. Cloning by limiting dilution method was repeated twice for 12 of these antibody-producing positive wells, and 13 clones were obtained. The obtained clones were suspended in 90% FCS-10% DMSO and stored in liquid nitrogen. The monoclonal antibodies produced by each clone were grown intraperitoneally in Balb/C mice and purified from the ascites using a protein A-Sepharose B column. [Table] [Table] Example 2 (Properties of purified IgG) The IgG of each clone purified from mouse ascites was examined for subclass, influence on human/PC activity, L chain, or binding to L chain. Subclasses were determined by the Ouchterlony method using anti-mouse antisera specific for each class. The effect on human PC activity was determined by adding IgG to human PC at a molar ratio of 1:5 and incubating it at 4°C overnight to activate human PC by thrombin-thrombomodulin complex. This was determined by measuring the decomposition activity of the next synthesized substrate. As this synthetic substrate, [Here, Vel represents D-type optically active valine, Leu represents L-type optically active leucine, and Arg represents L-type optically active argine. S-2266 manufactured by KabiVitrum AB (Sweden) was used. L
The binding to the chain and H chain was determined by electrophoresing human PC under reducing conditions and using a nitrocellulose membrane and HRP label.
Determination was performed by immunoblotting using Goat anti-mouse IgG. The results obtained for each property are shown in Table 2 below. All calcium ion-dependent antibodies are L
It was chain-binding and had no effect on human PC activity. [Table] Example 3 (Influence of calcium ions) The influence of calcium ions on the reaction between human PC and purified IgG was studied. Calcium ion-dependent antibodies B12 and 10E12 in ELISA method coated with human PC
TBS _- Tween (0.02M Tris/
HCl, 0.14M NaCl, PH7.4 0.05% Tween20,
When diluted with 0.02% NaN ₃ ) or TBS-Tween and reacted with human PC, the amount of binding was measured by color development using alkaline phosphatase _- labeled rabbit anti-mouse IgG. showed binding to human PC that was dependent on antibody concentration, but when diluted with buffer without CaCl ₂ , no binding was observed even at high antibody concentrations. The results are shown in Table 3 below. In addition, calcium ion-independent antibody 6B10
The results of a similar study on the influence of calcium ions using -1 and 10H11 are also shown in Table-3. [Table] Example 4 (Influence of calcium ion concentration) Using the calcium ion-dependent antibodies 7B12 and 10E12 from Example 3, their concentrations were kept constant (1 μg/ml), and the calcium ion concentration in the antibody solution was When the CaCl2 concentration was varied, the binding to human PC increased as the _CaCl2 concentration increased, and reached approximately 1mM.
saturation was reached at a concentration of The results are shown in Table 4 below. Also, calcium ion-independent antibodies
The results of similarly examining the influence of changes in CaCl ₂ concentration using 6E2 are shown in Table 5 below. From the results in Table 5, it can be seen that with antibodies in the absence of calcium ions, the binding to human PC remains almost constant without affecting the calcium ion concentration. Note that the measurement was performed using TBS containing a predetermined concentration of _CaCl2 .
Prepare a 1μg/ml IgG solution with Tween, add 100μ
This was done in addition to wells coated with human PC. TBS-Tween containing various concentrations of CaCl ₂ was also used to wash the wells after incubation. [Table] [Table] [Table] Example 5 (1) Immobilization of antibody on an insoluble carrier; 0.5 g of dry gel of bromcyan-activated Sepharose 4B (manufactured by Pharmacia Huain Chemicals) was placed on a G3 glass filter. 100ml
Swell with 1mM HCl, wash, and add a coupling buffer (containing 0.5M NaCl).
Washed with 0.1M NaHCO ₃ PH8.3). Immediately after the coupling buffer was removed by suction, the gel was suspended in 2 ml of an antibody (6H2) coupling buffer solution (3 mg/ml) and gently shaken at 4°C overnight. The gel was then transferred into 1M ethanolamine-HCl (PH 8.0, 2 ml) and shaken at room temperature for 2 hours to block remaining active groups. After blocking, transfer the antibody-conjugated Sepharose gel to 0.5M on a glass filter.
0.1M acetic acid buffer PH4.0 containing NaCl, and
0.1M boric acid buffer PH- containing 0.5M NaCl
8.0 was used alternately for washing. When the absorbance of the liquid at 280 nm becomes 0.01 or less,
Contains 5mM CaCl ₂ and 1mM Benzamidine
Equilibrated with 0.05M Tris/HClPH7.4 and packed into the column. Anti-human prepared in this way
Affinity chromatography was performed using a Sepharose 4B column conjugated with PC monoclonal antibody (6H2). (2) Adsorption and elution of protein C to antibody-bound Sepharose 4B; Add 8 ml of 1M BaCl ₂ solution to 100 ml of plasma and incubate at 4°C.
The mixture was stirred for 1 hour. Collect the precipitate by centrifugation
0.1M with 5mM BaCl ₂ , 5mM benzamidine
After washing with NaCl, dissolve the precipitate in 15 ml of 0.2 M EDTAPH7.4 containing 5 mM benzamidine.
A barium adsorption fraction was obtained. This barium adsorption fraction was mixed with 0.05M Tris containing 1mM benzamidine/
Dialyzed against HClPH7.4 to a final concentration of 5mM
CaCl solution was added and applied to an antibody (6H2) binding column equilibrated with 0.05M Tris/HCl containing 5mM _CaCl2 and 1mM benzamidine. 5mM
Washed with 0.05M Tris/HCl containing CaCl ₂ , 1mM benzamidine and 1M NaCl, and washed with 50mM EDTA.
and 0.05M Tris/containing 1mM benzamidine
When eluted with HCl, a single peak containing PC was obtained. PC in the antibody Sepharose 4B elution fraction was purified approximately 52 times as compared to the barium adsorption fraction, and the recovery rate was approximately 48.2%.

Claims (1)

[Claims] 1. Does not recognize human protein C in the absence of calcium ions (Ca ⁺⁺ ) and is specific for human protein C in the presence of calcium ions (Ca ⁺⁺ ) A monoclonal antibody against human protein C that recognizes human protein C. 2 A hybridoma made by fusing mouse spleen cells immunized with human protein C with mouse myeloma cells, which does not recognize human protein C in the absence of calcium ions (Ca ⁺⁺ ) and does not recognize human protein C. In the absence of calcium ions (Ca ⁺⁺ ), the product of a hybridoma that produces a monoclonal antibody against human protein C that specifically recognizes human protein C in the presence of calcium ions (Ca ⁺⁺ ) A monoclonal antibody characterized by isolating a monoclonal antibody against human protein C that does not recognize human protein C but specifically recognizes human protein C in the presence of calcium ions (Ca ⁺⁺ ). Method for producing antibodies.