JPH03200066A - Method for measuring activated human protein c - Google Patents

Abstract

PURPOSE:To exactly and immunologically measure the human protein C in blood in a short period of time with a high sensitivity by utilizing the monoclonal antibody specific for activated human protein C and human protein C inhibitor, etc. CONSTITUTION:The monoclonal antibody immobilized to the solid phase which specifically recognizes the activated human protein C, a sample, the monoclonal antibody labeled to specifically recognized the human protein C inhibitor, and the monoclonal antibody labeled to specifically recognize human a1 anti-trypsin are brought into contact and the label of the liberated or immobilizd labeled antibody is directly or indirectly detected to determine the product of immune reaction. A sandwich method is used for this immunoassay. Any known methods may be adopted without limitations for the process for producing the monoclonal antibodies, the method for immobilizing the solid phase of the activated human protein C antibody, the method for labeling the human protein C inhibitor antibody, etc.

Description

Detailed Description of the Invention (Field of Industrial Application) The present invention provides a monoclonal antibody specific for activated human protein C, a monoclonal antibody specific for human protein C inhibitor, and a monoclonal antibody specific for human α1 antitrypsin. This invention relates to a method for measuring activated human protein C using a monoclonal antibody.

(Prior Art) An important reaction for controlling blood coagulation is the decomposition reaction of coagulation factors by protease. This reaction is mainly caused by protein C.

Protein C is one of the Gla-containing coagulation factors (vitamin-dependent factors) and exists as a two-chain precursor in circulating blood. Protein C becomes activated protein C by liberating 12 amino acids from the amino terminus of the polymer chain by thrombin. This is the Va factor of the coagulation system and ■a
Decompose and inactivate the prisoner. In addition, activated protein C
also has the effect of suppressing the activity of blasminogen activator inhibitor and causing regression of fibrinolysis.

Human protein C inhibitor is one of the physiological inhibitors of activated protein C, a blood coagulation regulator. Purified protein C inhibitor in vitro:
It inhibits factor Xa, thrombin, factor XIa, plasma kallikrein, as well as fibrinolytic protease tissue plasminogen activator and urine-derived plasminogen activator (urokinase). However, when the blood coagulation process is carried out in vitro in a state in which protein C is normally hardly activated, little decrease in protein C inhibitor activity is observed. In addition, protein C in vivo
It is also known that variations in inhibitor concentration closely match variations in protein C concentration. Than these things,
Protein C inhibitors are thought to react best with protein C in vivo.

In addition, as another inhibitor of protein C,
α, antitrypsin has been reported to act (M, J, Heeb & J, H.

Gri　f　f　in, J, Biol, Chem.

263.11613. (1988)).

From the above, blood coagulation inhibitory activity can be detected by measuring activated protein C as a complex with its inhibitor. Furthermore, pathological diagnosis of diabetes mellitus, which is considered to be a pre-thrombotic state, and DIC becomes possible. The blood concentration of protein C is approximately 2 to 8 mg/I in healthy people.

As a measurement method, EIA using a monoclonal antibody against protein C is currently being performed, but it is not suitable for measuring activated protein C in a strict sense.

(Problems to be Solved by the Invention) The purpose of the present invention is to
The object of the present invention is to provide a method for immunologically measuring activated human protein C with high sensitivity.

(Means for Solving the Problems) The present inventors have made extensive studies regarding the above problems, and as a result, have arrived at the present invention.

That is, the present invention provides a method for measuring activated human protein C in a sample, comprising: (a) - a monoclonal antibody (1) immobilized on a solid phase that specifically recognizes activated human protein C; - a sample.・A labeled or unlabeled monoclonal antibody that specifically recognizes human protein C inhibitor (2) ・A labeled or unlabeled monoclonal antibody that specifically recognizes human α, antitrypsin (b) When monoclonal antibodies (2) and (3) not labeled in (a) are used, the immunoreaction products generated in (a), or the monoclonal antibodies (2) ) and (3), and (e) directly or indirectly detect the label of the free or immobilized labeled antibody to detect the immune reaction product. This is a method for measuring activated human protein C, which is characterized by quantifying activated human protein C.

The human protein C inhibitor measurement method of the present invention utilizes a monoclonal antibody specific to human protein C, a monoclonal antibody specific to human protein C inhibitor, and a monoclonal antibody specific to human α and antitrypsin. This method is an immunoassay method for human protein C, and the details thereof will be explained below.

In the method of the present invention, the monoclonal antibodies used can be prepared by methods known per se (G.

Kohler & C, Mi 1stein.

Nature, 256,495. (1975)).

By the above method, multiple types of monoclonal antibodies that specifically recognize human protein C1 human protein C inhibitor, human α, and antitrypsin were obtained. The binding constants of each antibody were within the range of ]07 to IOIIM-1. Using these monoclonal antibodies, it has become possible to perform an immunoassay method based on the Sand-Deutsch method that allows quantitative measurement of human protein C.

A known method can be used to immobilize the anti-activated human protein C antibody used in the method of the present invention on a solid phase. Examples of the solid phase include polystyrene, polyethylene, polyvinyl chloride, polycarbonate, Sepharose particles, and latex. , agarose, cellulose, polymethacrylate, etc. are used.

Furthermore, when an anti-human protein C inhibitor antibody and an anti-human α, antitrypsin antibody are labeled, the method of labeling and the method of detection thereof are not limited at all, and the labeling and detection can be performed by known methods. When using unlabeled anti-human protein C inhibitor antibodies and anti-human α1 antitrypsin antibodies, labeled antibodies that specifically recognize these antibodies or immune reaction products are used.

When using something that can be detected indirectly as a label, such as an enzyme, examples of the labeling substance include peroxidase,
β-D-galactosidase, alkaline phosphatase,
Enzymes such as urease, catalase, and β-glucuronidase are used. Also, examples of labels that can be detected directly, such as radioactive substances, include J+251. As a method using a fluorescent substance, for example, fluorescamine, fluorella centiocyanate, tetrarhodamine isothiocyanate, etc. are bound to a monoclonal antibody by a conventional method. However, the labeling substance should not be limited to the above-mentioned substances.

In addition to the above-mentioned substances, known reagents used in the measurement include substrates, solubilizers, buffers, detergents, reaction terminators, and the like.

There are no particular limitations on the order in which samples and antibodies are added.

The finally produced immunoreaction product, that is, the immobilized labeled antibody label or the free antibody label, may be detected and quantified.

(Effect) Activated human protein C forms a complex with human protein C inhibitor or human α1 antitrypsin coexisting in the sample. Therefore, by quantifying this complex, activated human protein C can be indirectly quantified. Human protein C inhibitor has a strong binding force to human protein C, but is present in small amounts in the blood, while human α and antitrypsin have a weak binding force to human protein C, but are present in large amounts in the blood. exists in

Therefore, activated human protein C-human protein C inhibitor complex and activated human protein C-human α
By measuring both antitrypsin and antitrypsin complexes, activated human protein C can be measured more precisely. Therefore, in the method of the present invention, this complex is sandwiched between an anti-activated protein C antibody and an anti-human protein C inhibitor antibody, or an anti-activated human protein C antibody and an anti-human α1 antitrypsin antibody, and then measured.

If human protein C inhibitor, α, and antitrypsin are not present in the sample, they should be added externally to form a complex and then measured using the method of the present invention. However, if the sample is human blood, since both are contained therein, there is no need to add them from the outside, and for this reason as well, it is preferable that the sample is human blood.

(Effects of the Invention) As is clear from the above explanation, according to the present invention, (1)
Activated human protein C concentration in blood is 1-200n
(2) Compared to conventional methods, activated human protein C can be measured in a short time with extremely simple operations, and in a more precise sense, a large number of samples can be measured with high sensitivity. is possible.

(Example) Detailed examples of the present invention will be described below. However, the present invention is not limited to these examples.

(Preparation of monoclonal antibodies) (A) Preparation of antigen-sensitized animal cells B a 1 using human protein C1 human protein C inhibitor or human α1 antitrypsin as an antigen.
b/c mice (♀) were each immunized.

For immunization, 100 μl of blood emulsified with Freund's complete adjuvant and 100 μg of antigen/mouse was administered into the abdominal cavity of the mouse. Two weeks later, a booster dose of 100 μg of antigen/
The mice were emulsified with incomplete Freund's adjuvant and 100 μl of the emulsified mixture was intraperitoneally administered to the mice. One week later, as final immunization, 100 μg/mouse of antigen was given to phosphate t1! Enhanced physiological saline (0.01% phosphate buffer containing 0.85% NaCl,
100 μl of the solution dissolved in pH 7.2 (hereinafter referred to as PBS) was administered intraperitoneally. Three days later, the lungs of the treated mice were aseptically removed. 15% calf fat serum (hereinafter referred to as 15%FCS)
DMEMI Om 1 containing (abbreviated as ) was aspirated with a syringe and a 27-gauge needle was attached. I put the lungs into a water-cooled date bag and injected them with a few needles. A syringe needle was inserted and reflux was performed to cause the lung cells to flow out into the tissue. The effluent was filtered through a nylon mesh, placed in a centrifuge tube, centrifuged at 1000 rpm for 10 minutes, and the supernatant was discarded. The red blood cells in the cell pellet were dissolved in a 0.15M ammonium chloride solution (0.01% containing 1mM ethylenediaminetetraacetic acid-disodium salt (hereinafter abbreviated as EDTA)).
The cells were hemolyzed with M carbonate buffer, pH 7.2) and centrifuged, and the cell pellet was centrifugally washed twice with DMEM to obtain lung cells.

(B) Preparation of myeloma cells SP210-Ag14 (hereinafter S
P210) was used.

The cells were cultured in DMEM containing 20 μg/ml 8-azaguanine and 15% FC3 until one week before cell fusion, and then DMEM containing 15% FCS was used until the day of cell fusion. Immediately before cell fusion, 5P210 was aseptically prepared in DMEM by repeating centrifugal washing twice at 11,000 rpm for 10 minutes.

(C) Cell fusion The lung cells prepared in section (A) above and the myeloma cells prepared in section (B) above were mixed at a ratio of 5=1 and centrifuged (1000r
pm, 10 min) and collected the cell pellet. The cell pellet was spread on the wall by tapping the centrifuge tube. 50% PEG (MERK
DMEM containing polyethylene glycol 4000)
0.5 ml of the solution was dropped little by little while rotating the centrifuge tube. After mixing by slowly rotating the centrifuge tube for 1 minute, DMEM that had been heated to 37° C. was added 10 times while rotating the centrifuge tube at a rate of 1 ml every 30 seconds. Next, slowly add 2ml of Fe2 and add 11000r
p, centrifuged for 10 minutes. The cell pellet was transferred to DMEM containing 15% FCS and 1X10-'M hypoxanthine, 4X10-'M aminopterin, 1.6X10-5M thymidine.
(hereinafter abbreviated as HAT medium) twice by centrifugal washing (10
00 rpm for 10 minutes). Transfer this culture solution to a 96-well plate (Falcon #3042) at 5×10
'200 μl was dispensed at a rate of 1 cell/well. The HAT medium was replaced with 100 μl/well every third day. After 3 weeks, lXl0-'M hypoxanthine,
DM with 1.6X10-'M thymidine and 15% FCS
EM (hereinafter abbreviated as HT medium) was used for medium exchange.

(D) Selection of hybridomas Cell colonies observed in 96-well plate lO0
After a day, solid-phase enzyme immunoassay was performed to examine the presence of specific antibodies in the culture supernatant.

Dispense 50 μl/well of 2 μg/ml of each antigen into a 96-well plate with a flat bottom (manufactured by Intermed), and
Leave at 7°C for 1.5 hours. Remove remaining solution in the wells and add 0.04% tween-
After washing three times with a solution containing 20 (hereinafter referred to as PBS-T), 300 μl of a PBS-T solution containing 0.1% bovine serum albumin (hereinafter referred to as BSA) was added to each well.
Blocking treatment was performed at ℃ for 1.5 hours. Next, 100 μm of the above culture supernatant was dispensed into each well and heated to 37°C for 1.5 μm.
Let it stand for a while. After washing these wells three times with PBST solution, peroxidase-labeled rabbit anti-mouse Ig
G antibody (manufactured by Jackson) 4000 times diluted at 50μm/
The solution was dispensed into each well and allowed to stand at 37°C for 1.5 hours. P.B.
After washing three times with S-T solution, substrate solution (1,2%
0,1M citrate buffer (pH 5,1) containing 2,2-azinodi=(3-ethylbenzthiazoline sulfate)-diammonium salt (ABTS) and 0.01% hydrogen peroxide (H20□)). 100 μm was added to each well. 3
Leave at room temperature for 0 minutes, then add 200mM oxalic acid solution to 100%
The enzymatic reaction was stopped by adding μl. The absorbance at 415 nm was measured, and it was found that hybridomas producing specific monoclonal antibodies were present in wells in which enzyme activity was observed. As described above, an antibody-producing hybridoma with a strong antibody titer was obtained.

(E) Preparation of conditioning medium In a syringe fitted with a 26 gauge needle, 10 ml of refrigerated 0.3
The 4M sucrose solution was blotted. A Ba1b/c mouse (male) was subjected to vertebrae dislocation, and the above solution was injected intraperitoneally in a sterile manner.

Within 5 minutes after injection, the intraperitoneal solution was collected using a water-cooled syringe with an 18-gauge needle attached to the left flank. Pour the recovered solution into an ice-cooled centrifuge tube, and proceed to step 11.
Centrifuged at 000 rpm for 5 minutes. After centrifugation, the supernatant was discarded, and 5% FC5-DMEM was added to the cell pellet, stirred, and placed in a push tube. 37℃, 5% carbon dioxide concentration, 95%
Cultured overnight in humidity. The culture supernatant was collected, filtered through a 0.22 μm membrane filter, and used as a conditioning medium.

(F) Cloning Hybridomas capable of producing antibodies were made into a single clone using the limiting dilution method. 1 containing 1 ml of the conditioning medium prepared in (E) above (AT medium 20
m1 was prepared. Adjust the hybridoma cells to be cloned into the above culture medium so that there is one cell in each well, and add 200 μl/well to a 96-well plate (Fal
con#3042). For wells where cell colonies are observed from around the 10th day of culture, see (D) above.
Antibody-producing hybridomas were selected according to the solid-phase enzyme immunoassay method described in Section 1, and cloning was repeated again to establish single hybridomas. Finally, 27 clones of /X hybridomas were established.

(G) Purification of antibody Bristane (2,6,10,14-tetramethylpentadecane) was administered into the abdominal cavity of 6-10 week old Ba1b/c mice (female) in an amount of 0.5 ml/mouse. Two weeks later, each specific antibody-producing hybridoma line obtained in the above (F) was intraperitoneally transplanted into mice at 2×10 6 cells/mouse for each clone. Ascites generated around the 10th day was injected with 1/20 volume of 0.2M-EDTA by inserting an 18-gauge needle into the abdominal cavity.
was dropped into a centrifuge tube containing 4 centrifuge tubes
Centrifugation was performed at 00 rpm for 10 minutes, and the supernatant was collected. The collected supernatant was crudely purified according to a 50% ammonium sulfate precipitation fractionation method, and added to PBS containing 0.05% sodium azide.
After dialysis against a solution, it was purified by ion exchange chromatography and gel filtration. The purity of the antibody was confirmed by the formation of two bands, one heavy chain and one light chain, in 12% 5DS-polyacrylamide electrophoresis under mercaptoethanol reduction.

(Measurement of human protein C by enzyme labeling method) (A) Immobilization of anti-human protein C antibody In each well of an untreated microtiter plate (96-well Nunc plate, manufactured by Intermed), O, 1M sodium carbonate buffer (
200 μg of a solution of 3 μg/ml mouse-derived anti-human protein C antibody dissolved in pH 9.6) was added and incubated overnight at 4°C. Next, the solution in each well was removed and washed three times with PBS-T, followed by 0.1% BS.
300 μl of a PBS-T solution in which A was dissolved was added to each well, subjected to blocking treatment at 4° C., and stored as is.

(B) Preparation of horseradish peroxidase (HRP) labeled antibody: 0.3M sodium bicarbonate buffer (pH 8
, 1) was added with 0.1 ml of 1% 1-fluoro-2,4-dinitrobenzene in ethanol solution (5 mg/m 1 ), and reacted at room temperature for 1 hour. Add 1.0 0.06M sodium periodate to the solution.
ml was added and reacted for 30 minutes. Unreacted sodium periodate was dissolved in 0.16M ethylene glycol 1. Oml
was added and removed, and then dialyzed against 0.01M sodium carbonate buffer (pH 9.5). Next, 5 mg of mouse-derived anti-human protein C inhibitor monoclonal antibody was added and reacted for 5 to 6 hours. 5 mg of sodium borohydride was added and left at 4°C overnight. After this, in order to remove unreacted sodium borohydride, 0.85
Dialysis was performed against 10 mM sodium phosphate buffer (pH 7.1) containing % sodium chloride at 4° C. overnight with stirring. The above reaction product was mixed with TSK-Gel G-30005W (
It was purified by high performance liquid chromatography using Tosoh Corporation (trade name) to obtain an HRPeA2 antibody. Similarly, a labeled antibody of anti-human α1 antitrypsin antibody was prepared in the same manner.

(C) Quantification of human protein C in blood (
The microtiter plate prepared by the method described in A) was brought to room temperature, washed with PBS-T solution, and 20 μm of standard blood containing a known amount of activated human protein C inhibitor complex was added to each well. . Next, the two HRP-labeled antibodies obtained in Example (B) were added to PBS.
-T solution and added 100 μl to each well. After incubating at room temperature for 3 hours, the solution was removed and the plate was washed three times with PBS-T solution. Besides, 1.2
200 μm of substrate solution consisting of 0.1 M citrate buffer (pH 4.1) containing 0.6% ABTS and 1% hydrogen peroxide (H20□) was added to each well and incubated for 30 min at room temperature.
After enzymatic reaction for 1 minute, 200mM oxalic acid solution was added to
The enzyme reaction was stopped by adding 0 μl. For each well of the microtiter plate, the absorption intensity at a wavelength of 415 nm and a reference wavelength of 492 nm was measured using an automatic microtiter plate reader (manufactured by Tosoh Corporation, MPR-A4, trade name).

The results are shown in Table 1.

Table 1 As is clear from Table 1, it was confirmed that human protein C in the sample could be quantified in the range of 1 to 200 ng/ml.

Claims (1)

[Claims] A method for measuring activated human protein C in a sample, comprising: (a) specifically recognizing activated human protein C;
A monoclonal antibody (1) immobilized on a solid phase, - a sample, - a labeled or unlabeled monoclonal antibody that specifically recognizes human protein C inhibitor (2), and human α_1 antitrypsin. Contact with a labeled or unlabeled monoclonal antibody (3) that specifically recognizes (b) (a).
When using 2) and (3), the immune reaction product generated in (a) or the monoclonal antibodies (2) and (3)
) and (c) directly or indirectly detecting the label of the free or immobilized labeled antibody to quantify the immune reaction product. Characteristic method for measuring activated human protein C.