JPH07117535B2 - Immunological assay method for human protein S, assay reagent and kit used therefor - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for immunologically measuring human protein S in a solution state, a measuring reagent and a kit used therefor.

More specifically, an immunoassay method for human protein S, which can specifically and highly sensitively measure only free protein S without measuring the complex of human complement system regulatory factor C4bp and protein S , A measuring reagent and a kit used therefor.

(B) Prior art Protein S is a vitamin K-dependent protein similar to protein C, and was isolated from cattle and humans by DiScipio et al. In 1977 [DiScipio, RG, Hermodson, MA, Yates, SGand.
Davie, EW; Biochemistry, 16 , 698-706 (1977)]. Human protein S is contained in plasma at about 10 mg /
It is a single-chain glycoprotein with a molecular weight of 69,000. Also the structure, other vitamin K is very similar to the structure of the dependent factor, NH ₂ terminus about 10 γ- carboxyglutamic acid (G
la). Human protein S exists in two forms in blood, one of which is free protein S, and this free one acts as a cofactor of activated protein C. The other is polymer C4b, which is a regulator of the complement system.
It exists non-covalently with the binding protein (C4bp). This "free protein S" and "combined protein S"
The ratio is about 1: 1.

The importance of the function of protein S in the C4bp-protein S complex lies in its very strong affinity with the negatively charged surface of phospholipids [Nelsestuen, GL, Kisiel, W. and DiSc.
ipio, RG: Biochemistry, 17 ; 2134-2138, (1978)].
When cells are damaged or activated, the Gla-domain of protein S binds to phospholipids in the presence of Ca ²⁺ , and C4bp forms a complex with this protein S and binds to its function. It is believed to be effective [Dahlback, B .:
Semin. Thromb. Haemostas., 10 ; 139-148 (1984)].

Regarding the functions of protein S and protein C related to coagulation and fibrinolytic system, it has been clarified from recent studies that they have an extremely important role in the control mechanism, and their physiological significance has been noted in relation to thrombosis. ing. Congenital deficiency of protein S has been reported to cause thrombosis [Comb, PC, Nixon, RR, Cooper, MRan
d Esmon, CT: J.Clin.Invest., 74 ; 2082-2088, (198
Four)].

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

As a conventionally known method for measuring protein S, the Laurel method using an antiserum against protein S, IRMA (immunoradiometric assay) using a polyclonal antibody, and EI
A (enzyme immunoassay) can be mentioned, but the measurement values obtained by these methods include both free protein S and protein S forming a complex with C4bp. become.

Therefore, in the conventional method for measuring only free protein S, it was necessary to remove the complex of C4bp and protein S by precipitating the complex by treating the sample with an aqueous solution of polyethylene glycol or the like. However, this measuring method has the disadvantage that the operation is extremely complicated.

In addition, a monoclonal antibody is specific for a single antigenic determinant and can stably produce an antibody having the same specificity. Therefore, analysis of the function and structure of an antigen protein or immunoassay (EIA, RIA ) In recent years
It has become widely used in general. Particularly in the functional analysis and molecular analysis of the 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.

Also, in the method for measuring free human protein S, two types of monoclonal antibodies that distinguish and recognize free protein S and protein S forming a complex are used, and free human protein S with extremely high specificity is obtained. A method for measuring the amount of human protein S antigen and the like has been performed (Japanese Patent Application No. 61-298881).

(C) Problems to be Solved by the Invention However, in the method for measuring free human protein S using two kinds of monoclonal antibodies, it is not possible to react with the antigen because of the characteristic of the monoclonal antibody that it does not react with a site other than the specific site of the antigen. In some cases, the affinity of E. coli is not necessarily sufficiently high, and an immunological assay method for protein S capable of measuring free protein S with higher sensitivity has been desired.

(D) Means for Solving the Problems In view of the problems of the prior art, the inventors of the present invention can easily and highly sensitively measure free human protein S in a solution state, As a result of diligent studies to develop a measurement reagent and kit, C4bp and protein S
A combination of a monoclonal antibody that does not recognize a complex with and can specifically recognize and bind free human protein S, and a polyclonal antibody that specifically recognizes human protein S and has a high affinity for an antigen As a result, they have found that free specificity of human protein S with high specificity and high sensitivity can be easily measured, and have reached the present invention.

That is, the present invention is: 1. When performing immunological measurement of human protein S using an antibody bound to an insoluble carrier and a labeled antibody, a polyclonal antibody that specifically recognizes human protein S is used as either one of the antibodies. A human being characterized by using, as the other antibody, a monoclonal antibody capable of specifically recognizing and binding free human protein S without recognizing the complex of human complement system regulatory factor C4bp and protein S An immunological assay method for protein S, 2. comprising an antibody bound to an insoluble carrier and a labeled antibody,
One of the antibodies is a polyclonal antibody that specifically recognizes human protein S, the other antibody does not recognize the complex of human complement system regulatory factor C4bp and protein S,
A measuring reagent for immunological measurement of human protein S, which is a monoclonal antibody capable of specifically recognizing and binding free human protein S, and 3. a body bound to an insoluble carrier and a labeled antibody, One antibody is a polyclonal antibody that specifically recognizes human protein S, and the other antibody does not recognize the complex of human complement system regulatory factor C4bp and protein S, and specifically recognizes free human protein S. When using a measurement reagent for immunological measurement of human protein S, which is a monoclonal antibody capable of binding with the antibody, and (a) a lysing agent, (b) a detergent, and an enzyme-labeled antibody, c)
A kit for immunological measurement of human protein S, which comprises a substrate for measuring enzyme activity and its reaction terminator.

In general, a method of measuring the presence or absence of an antibody or the amount thereof using an antibody that binds to two different sites of an antigen is called a sandwich method, and for example, Wide “Radioimmunoassay Methods” 199- 206 (1970).

In the immunological assay method for human protein S of the present invention, a monoclonal antibody and a polyclonal antibody that specifically recognize human protein S are used as two kinds of antibodies that bind to two different sites of an antigen. As an antibody, a monoclonal antibody that does not recognize the complex of human C4b binding protein (C4bp), which is one of the human complement system regulatory factors, and human protein S, and specifically recognizes and binds free human protein S As the polyclonal antibody, an antibody component of anti-human protein S antiserum that specifically recognizes and binds to human protein S is used.

Next, the immunological assay method for human protein S according to the present invention, the assay reagent and kit used therefor will be specifically described.

Immunological measurement method for human protein S: A polyclonal antibody (first antibody) against human protein S is immobilized on an appropriate insoluble carrier (for example, a plastic container) (hereinafter referred to as "immobilized antibody"). Then, the surface of the insoluble carrier is coated with a suitable substance (for example, bovine serum albumin) in order to avoid non-specific binding between the insoluble carrier and the reagent or sample to be measured.

The thus obtained insoluble carrier on which the first antibody is immobilized is brought into contact with a sample for a certain period of time and temperature to react. During this period, the immobilized antibody (first antibody) is bound to the human protein S in the specimen sample. Then, after washing with an appropriate washing solution, a solution (eg, an aqueous solution) of a monoclonal antibody (second antibody) against human protein S labeled with an appropriate labeling substance (eg, enzyme) is added to the human protein bound to the immobilized antibody in an insoluble carrier. S is contacted with S for a certain time and temperature to react with the second antibody. This is washed with an appropriate washing solution, and then the amount of the labeling substance labeled on the second antibody present on the insoluble carrier is measured.

The reaction may be carried out by simultaneously mixing an analyte sample containing an immobilized antibody, a labeled antibody and human protein S, and contacting these three at the same time for a fixed time and temperature.

Thus, the amount of free human protein S in the sample can be calculated from the value.

Configuration of Measurement Sample and Kit The measurement reagent for immunological measurement of human protein S comprises the antibody bound to the insoluble carrier described above and a labeled antibody.

In addition, a kit for immunological measurement of human protein S
To wash the above-mentioned measurement reagents and auxiliary agents for efficiently and conveniently using these measurement reagents, for example, a solubilizer for dissolving a solid reagent or a liquid sample, an antibody bound to an insoluble carrier In the case of using the detergent used for the above, and the antibody labeled with the enzyme, a substrate for measuring the enzyme activity and its reaction terminator, and other commonly used kits for immunological measurement are used. Can be mentioned. The measurement method, the measurement reagent, or the measurement kit of the present invention includes a non-specific reaction for high-sensitivity reduction.
Among them, surfactants, body fluids or protein solutions, skim milk, and the like, skim milk can be added.

Examples of the insoluble carrier used in the immunological assay method for human protein S of the present invention include, for example, polymers such as polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluororesin, crosslinked dextran, and polysaccharide. Examples include paper, glass, metal, agarose, and combinations thereof.

Further, the shape of the insoluble carrier may be various shapes such as tray shape, spherical shape, fibrous shape, rod shape, plate shape, container shape, cell, and test tube.

Further, it is advantageous to use a radioactive substance, an enzyme or a fluorescent substance as the labeling substance of the labeled antibody. ¹²⁵ I, ¹³¹ I, ¹⁴ C, ³ H, etc. as radioactive substances, alkaline phosphatase, peroxidase, β-D-galactosidase, etc. as enzymes, and fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate as fluorescent substances. Etc. can be used, but these are not limited to the exemplified ones, and other ones can be used as long as they are used in the immunological measurement method.

Examples of the polyclonal antibody of the present invention include those obtained as an antibody component of an anti-human protein S antiserum obtained by immunizing an animal with human protein S as an antigen by a conventionally known method. Among them, goat anti-human protein S-
Polyclonal antibody, rabbit anti-human protein S-polyclonal antibody and the like are preferable.

Regarding the monoclonal antibody used in the present invention and a method for producing the same, Japanese Patent Application No. 61-296766 (filed on Dec. 15, 1986: "Monoclonal Antibody, Hybridoma, Production of Monoclonal Antibody" filed earlier) Methods and methods for separating human protein S ") are described in detail in the patent application.

(E) Effect of the Invention According to the present invention, free human protein S in a solution state (for example, in plasma) can be easily measured with high specificity and high sensitivity without being affected by contaminants.

Further, the present invention provides a reagent and a kit, which have not existed in the past, that can accurately and rapidly measure free human protein S even in the presence of a complex of C4bp and protein S.

(F) Examples Hereinafter, the present invention will be described in detail with reference to Examples. % In the examples means% by weight.

Reference Example 1 Anti-Human Protein S (PS) Monoclonal Antibody and Production and Purification Two female Balb / C mice (4 weeks old) were immunized with purified human protein S four times at 14-day intervals. The first immunization was dissolved in PBS. 50 μg of human protein S in an equal amount of Freund's complete adjuvant (Complete Freund's ad
juvant), the emulsion was intraperitoneally administered (0.5 mg / head), and the second and third times were also mixed with 50 μg of human protein S with Freund's incomplete adjuvant. It was also administered intraperitoneally. The final immunization is 30 μg of human protein S
Was further administered as a PBS solution through the tail vein of the mouse. Spleen cells of immunized mice were used for cell fusion 3 days after the final immunization.

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 50% polyethylene glycol 1540 (Wako Pure Chemical Industries, Ltd.) was used as a fusion accelerator. As a result, cell fusion was performed according to the method of Kohler and Milstein. The cells after fusion were suspended in 10% FCS-RPMI-1640 medium at a cell concentration of 1 × 10 ⁶ cells / ml, and 96 wells
100μ per well in microplate (Coster)
I wrote each one.

The fused cells were cultured in a CO ₂ incubator (5% CO ₂ , 37 ° C), the medium was exchanged with a medium containing hypoxanthine, aminopterin and thymidine (HAT medium), and the cells were grown in the HAT medium and the spleen was grown. A hybridoma consisting of cells and myeloma cells was screened.

For the antibody in the culture supernatant of the hybridoma, a microtiter plate coated with the antigen human protein S was used.
It was detected by the ELISA method. As the second antibody, an alkaline phosphatase-labeled rabbit anti-mouse IgG antibody was used, and the antigen PS
Was tested for binding to. Colonies were formed in 487 wells out of a total of 494 wells in which the fused cells were seeded, and 94 wells of antibody production showing binding to the antigen PS were 94 wells.

Cloning by the limiting dilution method was repeated twice for 4 wells of these antibody production positive wells to obtain 6 clones. 90% of the obtained clones
Suspended in FCS-10% DMSO and stored in liquid nitrogen.

Clone the monoclonal antibody produced by each clone
Balb / C mice were grown in the abdominal cavity and purified from the ascites using a Protein A-Sepharose 4B column.

Reference Example 2 Properties of Purified Monoclonal Antibody IgG of each clone purified from mouse ascites was examined for binding to class and human protein S.

The class of mouse monoclonal antibody was determined by the Ouchterlony method using anti-mouse antiserum of each class specificity.

The results are shown in Table 2 below.

The binding property to human protein S was obtained by reacting human protein S immobilized on a microtiter plate with a monoclonal antibody diluted to an appropriate concentration,
Goat anti-mouse labeled with alkaline phosphatase
It was evaluated by detecting with IgG.

As a result, the binding strength of the 6 kinds of monoclonal antibodies to human protein S was 2B9F12 to 2B9C10>3C3G8> 3C4.
It was found that G4> 2B9G3 >> 2E12C7.

Reference Example 3 Reactivity to Human C4B and Protein S Complex The purified 6 types of monoclonal antibodies described above were coated on a microtiter plate at a concentration of 10 μg / ml to prepare a 1% solution.
After blocking with BSA, human normal human plasma diluted to an appropriate concentration was added, and C4bp-protein S complex in plasma was reacted with the monoclonal antibody. Next, add an alkaline phosphatase-labeled anti-C4bp antibody,
The binding properties of various types of monoclonal antibodies to the C4bp-protein S complex were detected and examined.

As a result, the monoclonal antibody 2E12C7 showed very weak binding to free protein S, but highly specific binding to C4bp-protein S complex pair. C4bp-protein S
The strength of binding to the complex is 2E12C7 >> 2B9F10-2B9C
It was found that 12>3C3G8>3C4G4> 2B9G3.

As shown in Reference Examples 2 and 3, C4bp and protein S
2B as a monoclonal antibody capable of specifically recognizing and binding free human protein S without recognizing the complex with
9F12 and 2B9C10 were obtained.

Example 1 (1) Preparation of antibody-immobilized beads Polystyrene beads (diameter 6 mm) were used as goat anti-human protein S antibody (polyclonal antibody: American Diagnost).
ica) 0.01 M phosphate buffered saline (PBS) solution of pH 7.4 having a concentration of 20 μg / ml, left at 4 ° C. for 1 day, washed with PBS, and then 0.5% cow The antibody-immobilized beads were obtained by carrying out post-coating treatment by allowing the mixture to stand in a serum albumin (BSA) aqueous solution at a temperature of 4 ° C. for one day.

(2) Preparation of horseradish peroxidase-labeled monoclonal antibody 1.0 ml of 1.0 mg / ml PBS solution of the monoclonal antibody (2B9F12) that specifically recognizes free human protein S was added to 1.0 ml of N solution.
10-mg / ml dimethylformamide solution of-(m-maleimidobenzoic acid) -N-succinimide ester (MBS) 50
After adding μ and reacting for 30 minutes at a temperature of 25 ° C., using a column packed with Sephadex G-25, gel filtration is performed with 0.1 M phosphate buffer (pH 6.0) to give a maleimidated monoclonal antibody. And unreacted MBS were separated.

Meanwhile, horseradish peroxidase (HRP)
To 2.0 ml of a 1.0 mg / ml PBS solution of N-succinimidyl-
3- (2-Pyridylthio) propionate (SPDP) 10
After adding a mg / ml ethanol solution and reacting at 25 ° C for 30 minutes, a column packed with Sephadex G-25 was used.
The product was purified by gel filtration with 0.01 M acetate buffer (pH 4.5), and the fraction containing pyridyl disulfide HRP was collected and concentrated about 10 times under ice cooling in a collodion bag. It then contains 0.85% NaCl and 0.1 M dithiothreitol.
After adding 1 ml of 0.1 M acetate buffer (pH 4.5) and stirring the mixture at 25 ° C. for 30 minutes to reduce the pyridyl disulfide group introduced into the HRP molecule, a Sephadex G-25 column was used.
Gel with 1M phosphate buffer (pH 6.0) and thiolate H
A fraction containing RP was obtained.

Next, the obtained maleimidated monoclonal antibody and thiolated HRP were mixed, concentrated to a protein concentration of 4 mg / ml under ice-cooling using a collodion bag, and allowed to stand at 4 ° C for 1 day, then Ultrogel AcA44 ( An HRP-labeled monoclonal antibody was obtained by gel filtration with PBS using a column packed with LKB, France.

(3) Measurement of human protein S 0,50,100,200,400 ng / ml of bead immobilized with goat anti-human protein S antibody and purified human protein S
PBS solution (pH 7.4) containing 0.1% BSA at each concentration of 200
μ and 0.1% BS containing HRP-labeled monoclonal antibody
A containing PBS solution (pH7.4) 200μ and each test tube (n = 2)
And incubated at 37 ° C for 1 hour.

Next, the solution in the test tube was removed by suction, and the tube was washed twice with PBS, and the test tube was replaced. Then, 0.1M phosphoric acid-citrate containing 0.02% tetramethylbenzidine hydrochloride and 0.005% hydrogen peroxide was added. Add 400μ each of acid buffer (pH4.0) to each test tube, incubate at 37 ℃ for 30 minutes, and then add 0.1% NaF and 2% acetic acid as a reaction terminator.
The enzymatic reaction was stopped by adding 1 ml to each test tube.

Then, the absorption intensity of this solution at a wavelength of 650 nm was measured using a spectrophotometer, and this was plotted with the human protein S concentration to obtain a calibration curve for measuring the concentration of human protein S, which has concentration dependence. (See FIG. 1).

To measure the concentration of human protein S in plasma samples, test 200 μ of 50-fold diluted normal mixed human plasma with 0.1% BSA-containing PBS solution (pH 7.4) together with antibody-immobilized beads and 200 μ of HRP-labeled monoclonal antibody solution. In addition to the tube, the immunoreaction and color reaction were performed in the same manner as above, and then the absorptiometry was measured with a spectrophotometer. The human protein S concentration was calculated by converting this value into the plasma concentration using the calibration curve in FIG. 1. As a result, the plasma concentration was 10.4 μg / ml.

Example 2 Rabbit anti-human protein S antibody immobilized by the same method as in Example 1 except that rabbit anti-human protein S antibody (polyclonal antibody: Diagnostica Stago) was used in place of goat anti-human protein S antibody. 200 μ of 0.5% BSA-containing PBS solution (pH 7.4) containing 1 bead and purified human protein S at concentrations of 0, 50, 100, 200, 400 ng / ml, and HRP-labeled mouse prepared in the same manner as in Example 1. Contains 0.5% BSA containing anti-human protein S-monoclonal antibody
200 μl of PBS solution (pH 7.4) was added to each test tube (n = 2) and incubated at a temperature of 37 ° C. for 1 hour.

Thereafter, the same treatment as in Example 1 (3) was performed, and 400 μM each of 0.1 M phosphoric acid-citrate buffer solution (pH 4.0) containing 0.02% tetramethylbenzidine hydrochloride and 0.005% hydrogen peroxide was added to each test tube. In addition, after incubating at 37 ° C. for 30 minutes, 1 ml of an aqueous solution containing 0.1% NaF and 2% acetic acid as a reaction terminator was added to each test tube to stop the enzymatic reaction.

Then, the absorption intensity of this solution was measured at a wavelength of 650 nm using a spectrophotometer and plotted against the concentration of human protein S. The absorption intensity at the baseline was 0.15, which was slightly high but showed good concentration dependence. A calibration curve was obtained.

Example 3 Measurement of Human Protein S in the Presence of C4bp-Protein S-Complex A sample containing human protein S at a concentration of 4.0 and 8.0 μg / ml, respectively, was dissolved in PBS containing 0.1% BSA (pH 7.4). Dahlback [Dahlback, B., Seminars in Thrombos
is &Hemostasis;10; 139-148 (1984)], and the C4bp-protein S-complex was added so that the concentration before dilution was 60,120,240,360,480 µg / ml, respectively.

Next, using each of these solutions as a sample, the concentration of human protein S was measured in the same manner as in Example 1.

As a result, the concentration of human protein S in the sample was the same as the concentration in the absence of C4bp-protein S-complex within the measurement error range.
-It was observed that free human protein S alone was selectively measured without measuring the complex (see Figure 2).

Comparative Example 1 In the preparation of antibody-immobilized beads in Example 1, mouse anti-human protein S-monoclonal antibody (2B9C10) was used instead of goat anti-human protein S-polyclonal antibody.
0, 50, 100, 2 were prepared by the same method as in Example 1 except that the mouse anti-human protein S-monoclonal antibody-immobilized beads each and purified human protein S were used.
It was prepared in the same manner as in Example 1 with 200 μ of PBS solution (pH 7.4) containing 0.1% BSA containing each concentration of 00,400 ng / ml.
PBS solution containing 0.1% BSA (pH7.4) containing HRP-labeled mouse anti-human protein S-monoclonal antibody (2B9F12)
200 μl was added to each test tube (n = 2) and incubated at a temperature of 37 ° C. for 1 hour.

Thereafter, the same treatment as in Example 1 (3) was performed, and each 400 μm of 0.1M phosphoric acid-citrate buffer solution (pH 4.0) containing 0.02% tetramethylbenzidine hydrochloride and 0.005% hydrogen peroxide was tested. After adding to the tube and incubating for 30 minutes at a temperature of 37 ° C., 1 ml of an aqueous solution containing 0.1% NaF and 2% acetic acid as a reaction terminator was added to each test tube to stop the enzymatic reaction.

Next, the absorption intensity at a wavelength of 650 nm was measured for this solution using a spectrophotometer, and this was plotted against the concentration of human protein S to obtain a calibration curve for protein S measurement (see FIG. 3).

As is clear from comparison with FIG. 1 of the present invention, this calibration curve has a high absorbance at the base line of 0.2 and a protein S concentration of 200.
The absorbance at ng / ml shows a relatively low value of 0.38.
Since the slope of the calibration curve is extremely gentle, the measurement sensitivity of the protein S measurement becomes insufficient, and it is judged that the measurement system is not preferable. It is considered that this is because the antigen affinity of the monoclonal antibody for protein S is not sufficiently high.

[Brief description of drawings]

FIG. 1 shows a calibration curve for human protein S measurement in the measurement method of the present invention (Example 1). FIG. 2 shows the results of the measurement of human protein S in the measurement method of the present invention (Example 3) in the presence of C4bp-protein S-complex. -○ in the figure
− Indicates human protein S concentration of 8.0 μg / ml in sample, − ● −
Represents a human protein S concentration of 4.0 μg / ml in the sample. FIG. 3 shows a calibration curve for measuring human protein S in the measuring method (Comparative Example 1) using a mouse anti-human protein S monoclonal antibody instead of the polyclonal antibody of the present invention.

Front page continuation (72) Inventor Hideaki Suzuki 2-7-18 Sakuramachi, Koganei-shi, Tokyo (56) References JP-A-59-163565 (JP, A) JP-A-63-151856 (JP, A) BLOOD, Vol. 67, No. 6, (1986), p. 1583-1590

Claims (4)

[Claims] 1. When carrying out an immunological measurement of human protein S using an antibody bound to an insoluble carrier and a labeled antibody,
A polyclonal antibody that specifically recognizes human protein S is used as one of the antibodies, and the other antibody does not recognize the complex of human complement system regulatory factor C4bp and protein S, and is specific for free human protein S. A method for immunologically measuring human protein S, which comprises using a monoclonal antibody capable of specifically recognizing and binding. 2. The antibody bound to an insoluble carrier is a goat and / or
Or the rabbit anti-human protein S-polyclonal antibody, and the labeled antibody is a mouse anti-human protein S-monoclonal antibody. 3. An antibody bound to an insoluble carrier and a labeled antibody, one of which is a polyclonal antibody that specifically recognizes human protein S, and the other antibody is C4bp which is a human complement system regulatory factor. Human protein S, which is a monoclonal antibody capable of specifically recognizing and binding free human protein S without recognizing a complex of protein S
Reagents for immunological measurement of. 4. An antibody bound to an insoluble carrier and a labeled antibody, one of which is a polyclonal antibody specifically recognized by human protein S, and the other antibody is C4bp which is a human complement system regulatory factor. Human protein S, which is a monoclonal antibody capable of specifically recognizing and binding free human protein S without recognizing a complex of protein S
And a measuring reagent for immunological measurement of (a) a lysing agent,
(B) When using a detergent and an enzyme-labeled antibody, (c) a kit for immunological measurement of human protein S, which comprises a substrate for measuring enzyme activity and a reaction terminator thereof in combination. .