JP2518602B2 - Immunological assay reagent and kit using monoclonal antibody against human protein S - Google Patents

Description

Detailed Description of the Invention

[0001]

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

More specifically, free human protein S
The present invention relates to an immunological assay reagent for human protein S by a sandwich method using a monoclonal antibody that specifically recognizes and binds to and a kit thereof.

[0003]

2. Description of the Related Art Protein S is a vitamin K-dependent protein similar to protein C. DiScipi in 1977
o, R. G. Hermodon, M .; A. , Yat
es, S.S. G. and Davie, E .; W. : Bio
chemistry. , 16 ; 698-706, (19
77)]].

Protein S is 100 mg in human plasma
/ L is a single-chain glycoprotein having a molecular weight of 69,000 (human). Further, its structure is very similar to that of other vitamin K-dependent factors, and it has about 10 γ-carboxyglutamic acids (Gla) at the NH ₂ terminus. Protein S exists in two forms in blood, one is free protein S, and this free one acts as a cofactor for activated protein C. The other exists non-covalently with the high molecular weight C4b binding protein (C4bp) which is a regulator of the complement system. The ratio of protein S bound to this free is said to be approximately 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 [Nelsestue].
n, G.N. L. , Kisiel, w. and Disci
pio, R .; G. : Biochemistry, 17 ;
2134-2138, (1978)]. When cells are damaged or activated, protein S in the presence of Ca ²⁺
It is considered that the Gla-domain of the protein binds to phospholipids, and further that C4bp forms a complex with this protein S to bind to it and exert its function [Dahlba.
ck, B. : Semin. Thromb. Haemos
tas. , 10 ; 139-148 (1984)].

Regarding the functions of protein S and protein C related to coagulation and fibrinolysis system, it has been clarified from recent studies that they have an extremely important function with respect to their control mechanism, and their physiological significance also relates to thrombosis. Is attracting attention. It has been reported that congenital deficiency of protein S may cause thrombosis [Comp, P.
C. Nixon, R .; R. Cooper, M .; R.
and Esmon, C.I. T. : J. Clin. Inv
est. , 74 ; 2082-2088, (198
4)].

Therefore, if it is possible to clarify the mechanism of action of protein S, and to measure the amount of protein S in the blood, the amount of antigen in the blood, and grasp the trend, it is possible to apply it to the fields of basic medicine and clinical medicine. It is considered to have a very important meaning in.

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

As a conventionally known method for measuring protein S, the Laurel method using an antiserum against protein S,
IRMA (immunor using polyclonal antibody)
audiometric assay) and EIA (en
Zyme immunoassay), but the measured values obtained by these methods include both free protein S and protein S forming a complex with C4bp. Become. In the former method, it is extremely difficult to stably obtain a large amount of antiserum having a certain activity due to the use of animal antiserum, and it is necessary to correct the activity with a standard substance before use. There was In addition, there is a drawback that it takes a long time for immune diffusion. The latter method requires purification of antibody from antisera, and
It has a drawback that it is difficult to obtain a stable antibody constantly.

[0010]

SUMMARY OF THE INVENTION Accordingly, the present inventors
As a result of repeated research on a monoclonal antibody against human protein S, a monoclonal antibody specifically recognizing and binding to free protein S, and C4
A monoclonal antibody that selectively recognizes and binds to the bp-protein S complex was found, and a hybridoma cell producing these monoclonal antibodies could be created, which has already been proposed.

The present inventors utilize the newly found specific action of the above monoclonal antibody to selectively and accurately select free protein S and C4bp-protein S complex in solution. The present invention has been reached by finding out to measure.

[0012]

That is, the present invention is the following inventions.

(1) In the immunological assay reagent by the sandwich method, the antibody bound to the insoluble carrier and the labeled antibody are monoclonal antibodies that specifically recognize different antigen determining sites, and either one of the monoclonal antibodies The antibody is a human antibody characterized by not recognizing the complex of human complement system regulatory factor C4bp and protein S, and being capable of specifically and selectively recognizing and binding free protein S. An immunological assay reagent for human protein S using a monoclonal antibody against S.

(2) An antibody bound to an insoluble carrier and a labeled antibody, which are monoclonal antibodies that specifically recognize different antigen-determining sites of human protein S, and either one of them. Is a human complement system regulatory factor C4bp and protein S
It is a monoclonal antibody that does not recognize the complex with and recognizes and binds free protein S specifically and selectively. It is labeled with (a) a lysing agent, (b) a detergent and an enzyme. When an antibody is used, (c) a kit for immunological measurement, which comprises a substrate for measuring enzyme activity and a reaction terminator thereof. The present invention will be described in more detail below.

[0015] Generally, a method for producing an antibody bound to two different positions of an antigen and measuring the presence or absence of the antigen or the amount thereof is called a sandwich method, for example, a wide (Wid) method.
e) "Radioimmunoassay (Radioimunoas
"Say Methods""199-206 (197)
0).

The immunoassay reagent for human protein S of the present invention uses two types of monoclonal antibodies that recognize different antigen-determining sites of human protein S, and one of them is A monoclonal antibody capable of specifically recognizing free human protein S is used.

Thus, according to the present invention, there is no difference in the quality of the reagents and the solution state is constantly and accurately (for example, in plasma).
It becomes possible to measure human protein S. Further, since human protein S is directly measured, it can be measured accurately and in a short time without being affected by other contaminants.
Therefore, according to the present invention, there are provided a reagent and a kit therefor capable of accurately and rapidly measuring human protein S which did not exist in the past.

Next, the measuring reagent and the method for measuring the content of human protein S according to the present invention will be specifically described.

Method for measuring the content of human protein S: A monoclonal antibody against human protein S (first antibody) is immobilized on an appropriate insoluble carrier (for example, a plastic container) (hereinafter referred to as "immobilized antibody"). The surface of the insoluble carrier is then coated with a suitable substance (eg bovine serum albumin) to avoid non-specific binding between the insoluble carrier and the reagent or sample material to be measured.

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

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

Structure of measurement reagent : Thus, the measurement reagent of the present invention is mainly composed of an immobilized antibody in which a first antibody is bound to an insoluble carrier and a labeled second antibody. In order to use this reagent efficiently and conveniently, various auxiliary agents may be included in addition to these antibodies to form a kit. Examples of such an auxiliary agent include a solubilizer for dissolving a solid reagent, a detergent used for washing the insolubilized carrier, and an enzyme activity for measuring the enzyme activity when an enzyme is used as a labeling substance of the antibody. Examples include kits that are usually used as a kit of immunological measurement reagents such as a substrate and its reaction terminator.

Examples of the insoluble carrier used in the measuring reagent of the present invention include polymers such as polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluoroenzyme, crosslinked dextran, and polysaccharide, and other paper, glass, metal. , Acarose, and combinations thereof.

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.

As the labeling substance, it is advantageous to use a radioactive substance, an enzyme or a fluorescent substance. Radioactive substances such as ¹²⁵ I, ¹³¹ I, ¹⁴ C and ³ H, and enzymes such as alkaline phosphatase, peroxidase, β
-D-galactosidase, etc., and fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate, etc., can be used as the structural substance, but these are not limited to the exemplified ones and can be used in immunological measurement methods You can use other ones if

As described above, the antibodies used in the assay reagents and kits of the present invention, that is, "monoclonal antibodies that specifically recognize free protein S", were first discovered by the present inventors, and patent applications have already been filed. (Application on December 15, 1986: title of invention "monoclonal antibody, hybridoma, method for producing monoclonal antibody and method for separating human protein S").

The above-mentioned monoclonal antibody of the present invention and the method for producing the same are described in detail in the above-mentioned patent application specification, but the contents will be briefly described below. <Monoclonal antibody and method for producing the same>

A. Isolation / purification of the antigen; human protein S used for the antigen is the method of Bajaj et al. [Bajaj S
P, Rapaport SI, Maki SL, Bro
wn SF: Prep. Biochem. 13 , 19
1, (1983)] and isolated and purified from human plasma.

[0029]B. Exclusion of mice with human protein S
PlagueFemale Balb / C mice can be used but other
Strain mice may also be used. That
At this time, the immunization plan and the concentration of human protein S are sufficient
Selected to form lymphocytes stimulated by
Should be. For example, 50 μg of human protein in mouse
After immunizing the abdominal cavity 3 times with S every 2 weeks, 30 μg
Is administered intravenously. Spleen for fusion a few days after the final immunization
Take out the cells.

C. Cell fusion : The spleen of a mouse immunized as described above is aseptically removed, and a single cell suspension is prepared therefrom. The spleen cells are fused with mouse myeloma cells from an appropriate line by using an appropriate fusion promoter. The preferred ratio of spleen cells to myeloma cells is about 2.
It is in the range of 0: 1 to about 2: 1. The use of 0.5-1.5 ml of fusion medium is suitable for about 10 ⁸ spleen cells.

Mouse myeloma cells used for cell fusion are well known, but in the present invention, P3-X63-Ag8-U is used.
1 cell (P3-U1) [Yelton, D .; E. FIG. et
al, Current. Topics in Micr
obilogy and Immunology, 8
11 (1978)] was used.

As a preferred fusion accelerator, for example, polyethylene glycol having an average molecular weight of 1000 to 4000 can be advantageously used, but other fusion accelerators 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 ; unfused spleen cells in a separate container (eg microtiter plate),
A mixture of unfused mouse myeloma cells and fused hybridoma cells is diluted with a selective medium that does not support unfused mouse myeloma cells and cultured for a time sufficient to kill unfused cells (1 week drug). . A medium (eg, HAT medium) that is drug resistant (eg, 8-azaguanine resistant) and does not support unfused mouse myeloma cells is used. In this selective medium, the unfused myeloma cells die. Since these unfused spleen cells are non-neoplastic cells, they die after a certain period of time (1 week). The cells fused to these have the neoplastic properties of the parental cells of myeloma and the properties of the parental spleen cells, and therefore can survive in the selective medium.

E. For human protein S in each container
Confirmation of antibody ; Thus, after the hybridoma cell was detected, the culture supernatant thereof was collected, and an enzyme immunoassay (Fnzyme Li) was performed for an antibody against human protein S.
Screening with an Inked Immunosorbent Assay).

F. Hybridoma that produces the desired antibody
Cloning of cells ; When a hybridoma cell producing an antibody of interest is cloned by an appropriate method (for example, limiting dilution method), the antibody is produced by two different methods. According to the first method, the hybridoma cells are
By culturing in an appropriate medium, the monoclonal antibody produced by the hybridoma cells can be obtained from the culture supernatant. According to the second method, the hybridoma cells can be injected into the abdominal cavity of a mouse having an isogenic or semiisogenic gene. The monoclonal antibody produced by the hybridoma cell can be obtained from the blood and ascites of the host animal after a certain period of time.

G. Free monoclonal antibody
Binding to S. S. The monoclonal antibody thus obtained was diluted to an appropriate concentration, reacted with free protein S immobilized on a microtiter plate, and enzyme-labeled (for example, alkaline phosphatase). By detecting with an anti-mouse antibody, the binding properties of various monoclonal antibodies to free protein S are examined.

In the measuring reagent of the present invention, such a monoclonal antibody is used as either the first antibody or the second antibody. That is, the monoclonal antibody can be used as an immobilized antibody by binding to an insoluble carrier, or can be used as a labeled antibody with a labeling substance.

As described above, according to the present invention, human protein S
It is possible to measure the free protein S in a sample (eg, human plasma) containing C and the C4bp-protein S complex separately and accurately and easily.

The present invention is described in detail below with reference to examples. Example (Measurement of human protein S) Monoclonal antibody against human protein S (2B9
F12) was coated on a microtiter plate at a concentration of 15 μg / ml. 1% bovine serum albumin (B
After blocking with a phosphate buffer (PBS) containing (SA), a washing solution (containing 0.05% Tween 20, 0.
It was washed 3 times with 5% BSA-PBS. Next, purified protein S and human patient sample plasma were diluted with PBS to an appropriate concentration, and reacted with the monoclonal antibody adsorbed on the plate solid phase at 37 ° C. for 2 hours. 3 with cleaning liquid
After washing and washing, monoclonal antibody against human protein S labeled with peroxidase enzyme (2B9C10)
After adding the solution and reacting at 37 ° C. for 2 hours, washing with the washing solution three times, the substrate (ABTS) solution was added, and ELISA AN
The change in absorbance (ΔA415 nm / min) per minute was measured with an ALYZER (Toyo Sokki Co., Ltd., ETY-96). A calibration curve was prepared by plotting the concentration of purified protein S and the value of change in absorbance. The results are shown in the attached FIG. The absorbance change value of protein S concentration is 50
There is a linear relationship up to about ng / ml, and by using this calibration curve, the amount of human protein S can be accurately calculated with high sensitivity.

The amount of protein S in the plasma of human patient samples was calculated using this calibration curve and is shown in FIG. In FIG. 3, the amount of protein S in the plasma of a healthy person is 100%,
%it's shown. )

Reference Example (Measurement of Human C4bp-Protein S Complex) Monoclonal antibody against human protein S (2E1) that specifically binds to human C4bp-protein S complex
2C7) was coated on a microtiter plate at a concentration of 20 μg / ml. 1% bovine serum albumin (B
After blocking with a phosphate buffer (PBS) containing (SA), a washing solution (containing 0.05% Tween 20, 0.
It was washed 3 times with 5% BSA-PBS. Next, healthy human plasma and patient sample plasma were diluted with PBS to an appropriate concentration, and reacted with the monoclonal antibody adsorbed on the plate solid phase at 37 ° C. for 2 hours. After washing three times with a washing solution, a polyclonal antibody solution against human C4b binding protein (C4bp) labeled with alkaline phosphatase was added, and the mixture was reacted at 37 ° C for 2 hours. After washing 3 times with the washing solution, the substrate solution (1 mg / ml) was added and ELISA A
NALYZER (Toyo Sokki Co., Ltd., ETY-96) 1
The change in absorbance (ΔA405 nm / min) per minute was measured. Normal human plasma was diluted and human C4bp in plasma was diluted.
-The amount of protein S complex and the value of change in absorbance were plotted to prepare a calibration curve. The results are shown in the attached FIG.
There is a direct relationship between the human C4bp-protein S complex concentration in normal human plasma and the absorbance change value, and it can be seen that the amount of human C4bp-protein S complex can be accurately calculated with high sensitivity by using this calibration curve. .

The amount of human C4bp-protein S complex in the plasma of human patient samples was calculated using this calibration curve and is shown in the attached FIG. In FIG. 3, the human C4bp-protein S complex in the plasma of a healthy person is defined as 100%, and the percentage is displayed.

As described above, by measuring the amounts of free protein S and C4bp-protein S complex in human plasma by using the monoclonal antibody against human protein S, various disease states can be diagnosed.

[Brief description of drawings]

FIG. 1 shows the relationship between the concentration of human protein S and changes in absorbance in Examples of the present invention.

FIG. 2 shows the relationship between the amount of human C4bp-protein S complex and changes in absorbance.

FIG. 3 shows actual measured values of human protein S or human C4bp-protein S complex in plasma of human patient samples.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Yataro Ichikawa 4-32 Asahigaoka, Hino City, Tokyo Teijin Limited Central Research Laboratory

Claims (2)

(57) [Claims] 1. In the immunoassay reagent by the sandwich method, the antibody bound to the insoluble carrier and the labeled antibody are monoclonal antibodies that specifically recognize different antigen determining sites of human protein S, and either one of them. One of the monoclonal antibodies is a monoclonal antibody that does not recognize the complex of human complement system regulatory factor C4bp and protein S, but can recognize and bind free protein S specifically and selectively. An immunological assay reagent for human protein S using a monoclonal antibody against human protein S. 2. An antibody bound to an insoluble carrier and a labeled antibody, wherein these antibodies are monoclonal antibodies that specifically recognize different antigen-determining sites of human protein S, and one of the monoclonal antibodies is Is a monoclonal antibody that does not recognize the complex of human complement system regulatory factor C4bp and protein S, and specifically and selectively recognizes and binds free protein S,
When (a) a lysing agent, (b) a detergent and an enzyme-labeled antibody are used for this, (c) an immunological assay comprising a substrate for measuring the enzyme activity and its reaction terminator Kit for.