JPH0260267B2 - - Google Patents

Description

[Detailed description of the invention]

a. Industrial Application Field The present invention relates to a method for measuring the activity of human protein C. More specifically, using a monoclonal antibody against human protein C,
The present invention relates to a method for measuring the activity of human protein C in a sample containing 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,
CT & 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 that degrades factor factor (F.Fa) and coenzyme of the blood coagulation system, 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. 68 :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. is considered to have important 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 approximately 21,000 are connected 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 that have a Gla domain, including protein C, factor (prothrombin), factor 1, and factor all undergo Gla-dependent steric structural changes in the presence of calcium ions (Ca ⁺⁺ ). It is known that this mechanism plays 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 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 ⁺⁺ ).
We conducted research on a monoclonal antibody that specifically recognizes protein C, which undergoes a structural change in the presence of calcium ions (Ca ⁺⁺ ). We have discovered a monoclonal antibody against human protein C that specifically recognizes human protein C in the presence of calcium ions (Ca ⁺⁺ ). The present inventors conducted further research and found that using this novel monoclonal antibody against human protein C, the activity of human protein C in samples containing human protein C could be easily and accurately measured. The present invention was achieved by discovering that the present invention can be obtained. c. Structure of the Invention In other words, the present invention provides a method that (i) 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 ⁺⁺ ); A sample containing human protein C to be measured is placed on an adsorbent in which a monoclonal antibody against human protein C, which specifically recognizes protein C, is bonded to the surface of an insoluble carrier in the presence of calcium ions (Ca ⁺⁺ ). (ii) then adding the thrombin-thrombomodulin complex, (iii) subsequently inactivating the thrombin, and (iv) quantifying human protein C activity on the adsorbent using a synthetic substrate. A method for measuring human protein C activity, characterized in that: The monoclonal antibody of the present invention does not inhibit the activity of human protein C when it recognizes it. According to the method of the present invention, protein C activity in a sample containing human protein C (for example, human plasma) can be easily and accurately measured, so it is useful for discovering and treating patients with protein C deficiency. I hope it will be helpful. The monoclonal antibody against human protein C used in the method of the present invention is new and was discovered by the present inventors, and an application has already been filed regarding the monoclonal antibody itself and the hybridoma that produces it. [Special request 1982]
−254186 (filed on December 3, 1982), JP-A-61
No. 134399 (published on June 21, 1985), title of the invention "Monoclonal antibody, hybridoma, method for producing monoclonal antibody, and method for separating human protein C"]. Therefore, the monoclonal antibodies used in the method of the present invention can be obtained by the method described in the specification, and the details thereof and the method of the present invention will be explained below. The hybridoma cells producing the monoclonal antibodies used in the method of the present invention are prepared according to the method of Kohler and Milstein [Kohler & 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 resulting hybridoma cells are immunized with antibodies that react with human protein C immobilized on a microtiter plate. , systematically examined 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 used in the present invention are obtained from products produced by such novel hybridoma cells and are directed against specific antigenic determinants on human protein C molecules in the presence of calcium ions (Ca ⁺⁺ ). Acts monospecifically. 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 is immobilized on an insoluble carrier, and the monoclonal antibody is immobilized in a solution containing calcium ions (Ca ⁺⁺ ) containing plasma (prepared so as not to coagulate in the presence of calcium ions) or other human protein C. Human protein C is adsorbed and separated from raw materials, their crude extracts, crude purified products, and solutions, and after washing in the presence of calcium ions (Ca ⁺⁺ ), the solution is purified with a solution that does not contain calcium ions (for example, in the presence of EDTA). human protein C can be eluted by replacing the solution with According to this method, unlike the conventional purification of antigen proteins using insolubilized antibodies,
Purification can be carried out under mild conditions without exposing the protein to extreme conditions (eg, 0.2M glycine-HCl or 8M urea). 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 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) using the monoclonal antibody in the presence of calcium ions, the difference in the measured values can be determined. , the amount of abnormal human protein C can be determined. Next, a specific method for producing monoclonal antibodies used in the method of the present invention will be described 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)] and 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, a mouse
50 μg of human protein C is administered 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. The spleen cells are fused with mouse myeloma cells from an appropriate line using an appropriate fusion promoter. A 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 about 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 having 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 a sufficient time (about 1 week) to kill unfused cells. The medium should be one that does 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-tumor cells, they die after a certain period of time (one week). Cells fused to these cells have both 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, the culture supernatant is collected and human protein C antibody is detected.
Enzyme immunoassay (Enzyme immunoassay) for antibodies against
Screening using Linked Immunosobent Assay). At this time, measurements are performed both under conditions where a certain concentration of CaCl ₂ is added to the culture supernatant, enzyme-labeled antibody solution, and washing solution, and under conditions where CaCl ₂ is not added, and only the former is positive. By selecting a hybridoma, it does not recognize human protein C in the absence of calcium ions,
Human protein C in the presence of calcium ions
Hybridomas that produce and secrete antibodies that recognize 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. Ru. 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 ⁺⁺ ). So human
Since it has the property of specifically recognizing protein C, this property can be used to easily extract human protein C from a mixture containing human protein C (such as human plasma). Can be 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. Furthermore, it is advantageous to use a plate having a large number of concave depressions (for example, commonly known as a microtiter plate), which is generally used for measuring or separating plasma or its fractionated components. Using the adsorbent, human protein C was added to it.
When the containing mixture is brought into contact with the presence of calcium ions (Ca ⁺⁺ ), the monoclonal antibody immobilized on the adsorbent and human protein C bond, and as a result, human protein C is attached to the adsorbent. join to. In this manner, human protein C can be separated and removed. Alternatively, the human protein C bound to the adsorbent may be bound to the adsorbent, the residual mixture preferably washed away, and the human protein C bound to the adsorbent then substantially freed of calcium ions (Ca ⁺⁺ ). When it comes into contact with or is washed with a liquid that does not contain the adsorbent, human protein C is separated from the adsorbent, and by obtaining this, human protein C can be isolated. Thus, by using the monoclonal antibody of the present invention, human protein C can be removed from a mixture containing human protein C, human protein C can be separated and purified from the mixture, and human protein C in the mixture can be removed. Measuring the content can be accomplished with extremely simple operations. H Measurement of human protein C activity; A sample containing human protein C is brought into contact with the adsorbent on which the monoclonal antibody is bound on the surface of an insoluble carrier as described above, and at this time calcium ions (Ca ⁺⁺ ), human protein C binds to the adsorbent. Next, a thrombin-thrombomodulin complex is added to this adsorbent in the presence of calcium ions (Ca ⁺⁺ ) to activate human protein C bound to the adsorbent. In this case, purified bovine thrombin can be used as thrombin, and thrombomodulin isolated from rabbit lung can be used. Thereafter, thrombin is inactivated using, for example, a mixed solution of antithrombin, heparin, and aprotinin. Next, human protein C activity on the adsorbent is measured using a synthetic substrate. The synthetic substrate used in this case is degraded by human protein C activity,
It is sufficient that the activity of human protein C can be determined, for example, by color development; examples thereof include: [Here, Val is D-type optically active valine, Leu
represents optically active leucine in the L form, and Arg represents optically active arginine in the L form. S-2266 manufactured by Kabi Vitrum AB (Sweden) was used. ] [Here, PyorGlu represents pyroglutamine, Pro represents proline, and Arg represents arginine, all of which have L-type optical activity. S-2366 manufactured by Kabi Vitrum AB (Sweden) was used]. The present invention will be described in detail below with reference to Examples. In the following examples, protein C may be abbreviated as PC. Experimental example 1 Purified human PC was introduced into female Balb/C mice (4
Two mice (age) were immunized four times at 14-day intervals. The first immunization was dissolved in PBS. 50μg human
Mix PC with an equal amount of Complete Freund's adjuvant;
The emulsion was administered intraperitoneally (0.5
mg/head), and for the second and third times, 50 μg of human PC was mixed with Freund's incomplete adjuvant.
It was also administered intraperitoneally. For the final immunization, 30 μg of human PC was administered as a PBS solution through the mouse 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 Kohlet and Milstein's spleen cells were mixed with 50% polyethylene glycol 1540 (Wako) as a fusion promoter. Cell fusion was performed according to the method. Cells after fusion are 1x
10% FCS to achieve a cell concentration of 10 ⁶ cells/ml←
It was suspended in RPMI-1640 medium and dispensed into a 96-well microplate (Coster) in an amount of 100μ per well. The fused cells were incubated in a _CO2 incubator (5%
Cultured in CO ₂ , 37°C), and cultured in a medium containing hypoxanthine, aminopterin, 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 (0.02M Tris/HCl, 0.14M NaCl, PH7.4)
I also added TBS to the other side. Furthermore, 5mM _CaCl2 was added to the dilution solution of the second antibody and the washing solution.
TBS, 0.05% Tween 20, 0.02% _NaN3 or TBS, 0.05% Tween 20, 0.02% _NaN3 were used. Out of a total of 541 wells seeded with fused cells, colony formation was observed in 523 wells, and among these, antibody-producing positive wells were found in 44 wells in the presence of calcium ions and 32 in the absence of calcium ions. It was hot. 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 4B column.

[Table] Experimental Example 2 (Properties of purified IgG) The IgG of each clone purified from mouse ascites was examined for subclass, influence on human PC activity, and binding to L chain or H 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. This synthetic substrate is [Here, Vel is D-type optically active valine, Leu
represents L-form optically active leucine, and Arg represents L-form optically active argine. S-2266 manufactured by Kabi Vitrum AB (Sweden)] was used. The binding to L chain and H chain was determined by electrophoresing human PC under reducing conditions and using nitrocellulose membrane and
Determination was performed by immunoblotting using HRP-labeled Goat anti-mDuse 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] Experimental Example 3 (Influence of Calcium Ions) The influence of calcium ions on the reaction between human PC and purified IgG was investigated. Calcium ion-dependent antibodies 7B12 and 10E12 in human PC coated ELISA method
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. Human and PC depend on antibody concentration.
However, when diluted with buffer without CaCl ₂ , no binding was observed even at high antibody concentrations. The results are summarized in Table 3 below.
It was shown to. 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】

[Table] Experimental 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 CaCl 2 concentration was varied, the binding to human PC increased as the CaCl ₂ concentration increased, and approximately
Saturation was achieved at a concentration of 1mM. The results are shown in Table 4 below. Furthermore, the effects of changes in CaCl ₂ concentration were similarly investigated using the calcium ion-independent antibody 6E2, and the results 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 1 (Measurement of human protein C activity) Buffer solution (15mM Na ₂ CO ₃ , 35mM NaHCO ₃ ,
Concentration 20μg/dissolved in 5mM CaCl ₂ PH=9.6)
ml of each of the monoclonal antibodies 7B12 and 10E12, which specifically recognize human protein C, were left on a microtiter plate overnight at 4°C to be immobilized. The above buffer containing 2% bovine serum albumin was added to this, and the solution was left at 37°C for 2 hours, followed by a washing solution (50mM Tris buffer, 0.15NaCl, 5mM
It was washed five times with CaCl ₂ , 0.05% Tween 20). Next, healthy human plasma or patient plasma diluted with the above buffer solution to various concentrations was added to the wells, and the wells were left at 37°C for 4 hours. After that, it was washed 5 times with the washing solution, and further washed with a buffer solution (0.1% bovine serum albumin, 50mM Tris buffer, 0.15M NaCl, 5mM
100 µ of a complex formed from bovine thrombin (2 units/ml) and rabbit lung thrombomodulin dissolved in CaCl ₂ PH 7.4) was added and allowed to stand at 37°C for 1 hour. Furthermore, antithrombin (5 units/ml), heparin (1 unit/ml), and aprotinin (10 units/ml) dissolved in a buffer solution (50mM Tris buffer, 0.1M NaCl, 5mM CaCl ₂ PH8.0) were added to this. 50μ of the mixture was added and left at 37°C for 10 minutes. After that, a synthetic substrate solution (S-
2266 or S-2366) Add 50μ ELISA
405nm with ANALYZER [ETY-96 manufactured by Toyo Sokki Co., Ltd.]
The change in absorbance per minute at the wavelength of was measured. The results are shown in FIG. 1 for a calibration curve using S-2266 as a synthetic substrate, and in FIG. 2 for a calibration curve using S-2366 as a synthetic substrate. In addition, when the amount of protein C activity was measured according to the method of the present invention using normal human plasma, protein C-deficient heterozygous patient plasma, and protein C-deficient homozygous patient plasma as specimens, the amount of protein C antigen was known. The results shown in Figure 3 were obtained. From these figures, it is understood that the relationship between the amount of human protein C activity and the change in absorbance is approximately linear. Therefore, by using a monoclonal antibody that does not inhibit human protein C activity and recognizes human protein C only in the presence of calcium ions (Ca ⁺⁺ ), it is possible to easily determine the amount of protein C activity in a sample. can be measured. Experimental Example 6 (1) Immobilization of antibody on an insoluble carrier; Dry gel of bromcyan-activated Sepharose 4B (manufactured by Pharmacia Huain Chemicals)
0.5g to 1mM in 100ml on G3 glass filter
Swell with HCl, wash, and add a coupling buffer (0.1M _NaHCO3 containing 0.5M NaCl).
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 washed with 1M ethanolamine-HCl (PH
8.0, 2 ml) and shaken at room temperature for 2 hours to block remaining active groups. After blocking, the antibody-conjugated sepharose gel was placed on a glass filter in 0.1M acetate buffer containing 0.5M NaCl PH.
0.1M boric acid buffer containing 4.0, and 0.5M NaCl
Washing was performed alternately with pH 8.0. When the absorbance of the liquid at 280 nm becomes 0.01 or less,
Contains 5mM CaCl ₂ and 1mM Benzamine
Equilibrate with 0.05M Tris/HCl PH7.4 and fill the column. Anti-human PC prepared in this way
Sepharose conjugated to monoclonal antibody (6H2)
Affinity chromatography was performed using a 4B column. (2) Adsorption and elution of protein C to antibody-bound Sepharose 4B; 8 ml of 1M BaCl ₂ solution was added to 100 ml of plasma and stirred at 4°C for 1 hour. Centrifuge and collect the precipitate to 5mM
Wash with BaCl ₂ , 0.1M NaCl containing 5mM benzamidine, then 15ml containing 5mM benzamidine.
The precipitate was dissolved with 0.2M EDTA PH7.4 to obtain a barium adsorption fraction. This barium adsorption fraction
0.05M Tris/HCl PH with 1mM benzamidine
7.4, added _CaCl2 solution to a final concentration of 5mM, and applied to an antibody (6H2) binding column equilibrated with 0.05M Tris _/ HCl containing 5mM CaCl2 and 1mM benzamidine. 5mM _CaCl2 ,
0.05M with 1mM benzamidine and 1M NaCl
Wash with Tris/HCl, 50mM EDTA and 1mM
When eluted with 0.05M Tris/HCl containing benzamidine, a single peak containing PC was obtained.
PC in the antibody Sepharose 4B elution fraction was about 52 times more purified than the barium adsorption fraction, and the recovery rate was about 48.2%.

[Brief explanation of drawings]

FIG. 1 and FIG. 2 respectively show the relationship between the plasma content in the sample and the change in absorbance (human protein activity) in Examples of the present invention. In addition, Figure 3 shows the relationship between the amount of human protein C antigen and protein C activity. In Figure 3, the results for group A (3 cases) are normal human plasma, and the results for group B (6 cases). The results for group C (one case) show the results for heterozygote patient plasma and the results for homozygote patient plasma.

Claims (1)

[Claims] 1 (i) 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 ⁺⁺ ) A sample containing human protein C to be measured is contacted in the presence of calcium ions (Ca ⁺⁺ ) to an adsorbent in which a monoclonal antibody against human protein C that specifically recognizes human protein C is bound on the surface of an insoluble carrier. (ii) then adding a thrombin-thrombomodulin complex, (iii) subsequently inactivating thrombin, and (iv) measuring human protein C activity on the adsorbent using a synthetic substrate. A method for measuring human protein C activity, characterized by: