JPH0670792A - Monoclonal antibody reactive with protein recognizing phosphatidyl serine - Google Patents

Abstract

PURPOSE:To provide an anti-idiotype antibody prepared by using an antibody specifically recognizing phosphatidyl serine as an antigen without using a PA- recognizing protein such as protein kinase C or blood coagulation factor V as an antigen. CONSTITUTION:A monoclonal antibody reactive with a protein recognizing phosphatidylserine.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention has a reactivity with a PS recognition protein which recognizes phosphatidylserine (PS) and further requires PS for its activation, particularly protein kinase C or blood coagulation factor V. It relates to a monoclonal antibody.

[0002]

2. Description of the Related Art Protein kinase C is 1986-19.
It is known as a protein having a molecular weight of about 80,000, which has been cloned one after another in 1987. Protein kinase C specifically requires phosphatidylserine or phosphatidylinositol upon activation. Protein Kinner
The structure of zeC is the C1 and C2 regulatory domains and the C3 kinase.
It is believed that there is a phospholipid and diacylglycerol domain in the C1 domain and a calcium ion binding domain in the C2 domain.

A large amount of protein kinase C is present in the brain, and a purification method from rat brain and the like has been established (J. Bio.
Chem., 257, 13341-13348 etc.). As a result of immunization with protein kinase C as an antigen, a large number of monoclonal antibodies have been produced, and their recognition sites have been predicted (J. Biol. Chem., 262, 2291-2297 etc.). Although much information has been obtained on the function of protein kinase C using these antibodies, unfortunately the phospholipid recognition pattern of protein kinase C has not been examined on the strict recognition site and recognition mode at the molecular level. This has not been done, and the role and functional analysis of protein kinase C in vivo has not been completed yet.

When blood coagulation factor V is also activated, PS
Is specifically required. But PS
Although the recognition site has been predicted, the exact recognition site and recognition pattern at the molecular level have not been studied.

[0005]

[Problems to be Solved by the Invention] Protein kinase C
Alternatively, an antibody that can be a useful tool in analyzing the phospholipid recognition site or recognition mode of blood coagulation factor V is desired.

[0006]

As the above-mentioned antibody, for example, when the antibody is prepared, phosphatidylserine is specifically recognized without using PA recognizing protein such as protein kinase C or blood coagulation factor V as an antigen. There is an anti-idiotype antibody prepared by using the antibody as an antigen. Such an anti-idiotype antibody has a structure (internal image) similar to that of PS.
It has a possibility of cross-reacting with the PS recognition site of the PS recognition protein.

The present inventors have prepared anti-idiotypic antibodies using PS-specific antibodies as antigens and examined their cross-reactivity with protein kinase C, which is a PS recognition protein, or blood coagulation factor V. We have succeeded in producing antibodies having cross-reactivity with these proteins and completed the present invention.

That is, the present invention is a monoclonal antibody reactive with a phosphatidylserine recognition protein. The present invention will be described in detail below.

(1) Antigen The monoclonal antibody of the present invention is prepared using a monoclonal antibody that makes PS specific as an antigen. Examples of the monoclonal antibody used as such an antigen include PS4A7 (J. Immunology, which is an antibody established by Umeda and Igarashi, which specifically recognizes PS up to its stereoisomeric structure. , 1989,143,2273-22
79) can be exemplified. The monoclonal antibody of the present invention is P
It may have a structure similar to S (internal image).

(2) Immunization and establishment of hybridoma Hybridoma can be prepared according to a known method by immunizing an animal with an antigen and then removing splenocytes. As an example, the monoclonal anti-PS antibody PS4A7 antigen is used and immunized into the abdominal cavity of an animal with Freund's complete adjuvant. A few weeks later, booster immunization is similarly performed with Freund's incomplete adjuvant, followed by final immunization with an antibody, and several days later, splenocytes are excised from the animal to prepare antibody-producing cells. The splenocytes prepared above can be fused with myeloma cells using a conventional cell fusion technique (Nature, 256, 495-497, 1975) to prepare hybridomas.

(3) Primary Screening The target hybridoma is selected by the ELISA method (Met
h. Enzymol. 70, 419-439, 1980), and can be carried out by analyzing the hybridoma culture supernatant. For example, when the hybridoma is prepared as described above, the hybridoma producing an antibody that inhibits the binding of the anti-PS antibody PS4A7 to PS is selected as the primary screening, and the limiting dilution method is selected. Thus, hybridoma can be established.

(4) Secondary Screening Hybridoma culture supernatant selected in the primary screening is used to select those having binding properties to protein kinase C or blood coagulation factor V. However, secondary screening can be performed by examining detailed reactivity thereafter.

(5) Recovery and purification of antibody A hybridoma is prepared as described above, and the hybridoma is cultured in a growth medium according to a conventional method.
Alternatively, the antibody can be produced by injecting the hybridoma into the abdominal cavity of an experimental animal (mouse, rat, etc.) in which the cells can grow and culturing in vivo. The antibody is obtained from the culture supernatant when the hybridoma is grown in the growth medium, or from the ascites of the animal when it is grown in the abdominal cavity of the experimental animal, by a conventional method (for example, ammonium sulfate fractionation method). It is possible to recover the monoclonal antibody of the present invention. Furthermore, gel filtration method, affinity-chromatography-
It is also possible to obtain a purified product having a high antibody titer by using the above or a combination thereof.

(6) Binding of Monoclonal Antibody to Protein Kinase C The binding of the prepared monoclonal antibody to protein kinase C was carried out using a system in which protein kinase C was immobilized. It can be carried out by measuring the residual activity of protein kinase C after the above-mentioned ELISA method or immunoprecipitation method. As a specific method, the method described in the examples of the present invention can be exemplified.

(7) Blood coagulation of monoclonal antibody V
Binding to Factor The binding of the produced monoclonal antibody to blood coagulation factor V was determined by using a system in which blood coagulation factor V was immobilized, and the blood coagulation factor V after the above-mentioned ELISA method or immunoprecipitation method. It can be carried out by measuring the residual activity.
As a specific method, the method described in the examples of the present invention can be exemplified.

[0016]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Example 1 Monoclonal anti-PS antibody PS4A7 producing hybrid-
The mouse was cultured in a serum-free medium, and the monoclonal antibody was recovered and purified from the culture supernatant. After adding an equal volume of saturated ammonium sulfate solution (pH 7.4) to the culture supernatant and collecting immunoglobulin as a precipitate, a single band was obtained by SDS polyacrylamide gel electrophoresis by gel filtration using high performance liquid chromatography. It was purified until

PS4A7 has a protein concentration of 1: 2 (PS4A7: KLH) using KLH (keyhole limpet hemocyanin) and glutaric aldehyde as carriers.
Was used as an antigen. The antigen was dissolved in phosphate buffer to 1 mg / ml, mixed with Freund's complete adjuvant, and 20 per BALB / c mouse.
Immunized intraperitoneally with 0 μl. Two weeks later, the same antigen was boosted with an equal amount of Freund's incomplete adjuvant, and two weeks later, the same antigen was not immunized with the adjuvant, and an equal final immunization was performed. Three days later, splenocytes were obtained, and myeloma cell line P3-X63-Ag653 was fused with cells in DMEM (Dulbebeco's denaturing medium) medium in the presence of 50% polyethylene glycol to prepare a hybridoma.

Screening of the hybridoma was carried out by applying the method of Umeda et al. (J. Immunology, 1989, 143, 2273-2279). That is, the hybridoma culture supernatant was mixed with biotin-labeled PS4A7, reacted at room temperature for 2 hours, coated with PS and blocked with bovine serum albumin, a 96-well microtiter plate (manufactured by Daynateck). And allowed to react for 2 hours. Then PS4A7 bound to PS with peroxidase-labeled steptavidin
Was detected. Here, the hybridoma that inhibited the binding of PS4A7 to PS was monoclonalized by limiting dilution, and the culture supernatant was purified according to a conventional method to examine the reactivity.
In addition, according to this embodiment, 8B8, 8F7, 9D12, 1
0A8, 10F4, 11A2, 11B4, and 12E1
Eight hybridomas producing eight monoclonal antibodies of 1 were prepared.

Example 2 The reactivity of the eight types of monoclonal antibodies prepared in Example 1 with protein kinase C by the ELISA method was examined by the following method. 50 ng of purified protein kinase C derived from rat brain was coated on a 96-well microtiter plate, blocked with bovine serum albumin, and 10 mM Tris-HCl containing 1% bovine serum, 150 m
It was reacted with a monoclonal antibody diluted with M sodium chloride solution (TBS) for 2 hours at room temperature. Subsequently, protein kinase C was treated with a biotin-labeled anti-mouse immunoglobulin antibody and peroxidase-labeled steptoavidin.
The antibody bound to was detected. Specifically, o-phenylenediamine was added as an enzyme substrate to cause an enzymatic reaction, and then the absorbance at 492 nm was measured. The results are shown in Fig. 1.

As a result, as shown in FIG. 1, antibodies 8F7 and 11A2 showed strong reactivity, and 11B4 had weak reactivity.

Example 3 The inhibitory effect of phospholipids on the binding of antibody 8F7 to protein kinase C found in Example 2 was examined by the following method. As in Example 2, protein kinase C was coated on a microtiter plate and blocked with bovine serum albumin to obtain PS and phosphatidylcholine (P
C), a phosphatidylethanolamine (PE) or a phosphatidylinositol (PI) liposome.
50 μl of 8-125 μM concentration was added to each well and reacted for 2 hours. Next, 5 μg of antibody 8F7 at 0.6 μg / ml was added.
0 μl was added to each well and the reaction was continued for 2 hours. In the same manner as in Example 2 below, the antibody bound to protein kinase C was detected. The results are shown in Figure 2.

As a result, as shown in FIG. 2, the binding of antibody 8F7 to protein kinase C was almost completely inhibited by PS at a phospholipid concentration of about 31 μM. This strongly suggests that antibody 8F7 binds to the PS binding site of protein kinase C.

Example 4 Reactivity to blood coagulation factor V by ELISA was examined in the same manner as in Example 2. That is, purified cattle blood coagulation V
50ng of the factor was coated on a microtiter plate,
After blocking with bovine serum albumin, it was reacted with a monoclonal antibody diluted with TBS containing 1% bovine serum for 2 hours at room temperature. Subsequently, an antibody bound to blood coagulation factor V (FV) was detected with a biotin-labeled anti-mouse immunoglobulin antibody and peroxidase-labeled steptavidin. The detection method was the same as that described in Example 2. Also, as a control, activated blood coagulation No. X
Factor (FXa) and Prothrombin (Prothrom)
Bin) was used to perform the same measurement. The results are shown in Fig. 3.

As a result, as shown in FIG. 3, antibody 10F
4, 11A2 and 12E11 showed strong reactivity to blood coagulation factor V, and showed no cross-reactivity with activated blood coagulation factor X and prothrombin as controls. Further, the reactivity of 8F7, which showed a strong reactivity with protein kinase C, to blood coagulation factor V was very weak.

[0026]

INDUSTRIAL APPLICABILITY According to the present invention, there is provided a monoclonal antibody characterized by having a binding property to protein kinase C which is a PS recognition protein or blood coagulation factor V. This monoclonal antibody has a binding property with respect to a phosphatidylserine recognition site of protein kinase C or blood coagulation factor V or a part of the recognition site. Therefore,
The monoclonal antibody provided by the present invention has a great effect on the study of the interaction between protein kinase C or blood coagulation factor V and PS. Further, the antibody of the present invention is useful for searching PS recognition proteins and identifying PS recognition motifs shared by PS recognition proteins, and has a great effect on production of PS recognition protein agonists or antagonists. .

[Brief description of drawings]

FIG. 1 shows the results of Example 2, showing the reactivity of the eight kinds of antibodies prepared in Example 1 with protein kinase C.

FIG. 2 shows the results of Example 3 and shows the inhibitory effect of various phospholipids on the binding between the antibody 8F7 and protein kinase C obtained in Example 1.

FIG. 3 shows the results of Example 4, showing the reactivity of the eight kinds of antibodies obtained in Example 1 with blood coagulation factor V.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location G01N 33/53 L 8310-2J 33/573 A 9015-2J 33/577 B 9015-2J (C12P 21 / 08 C12R 1:91)

Claims (5)

[Claims] 1. A monoclonal antibody having reactivity with a phosphatidylserine recognition protein. 2. The monoclonal antibody according to claim 1, wherein the phosphatidylserine recognition protein is protein kinase C. 3. The monoclonal antibody according to claim 2, which inhibits the phosphorylation activity of protein kinase C. 4. The monoclonal antibody according to claim 1, wherein the phosphatidylserine recognition protein is blood coagulation factor V. 5. The monoclonal antibody according to claim 4, which inhibits the activity of blood coagulation factor V.