JPH07113806A - Measuring method of heparan sulphate endoglycosidase - Google Patents

Abstract

PURPOSE:To measure a quantity of heparan sulphate endoglycosidase connected with an antibody by a method wherein the antibody against heparan sulphate endoglycosidase is fixated, is brought into contact with a biological sample adjusting liquid having it and measured by surface plasmon resonance. CONSTITUTION:A polyclonal antibody is made by using heparan sulphate endoglycosidase that is purificated from a lung cancer cell of a mouse Lewis as an antigen to be fixated. The lung cancer cell is grafted to a branch of a right leg of a black mouse and after it passes on about 10 days, a blood and a tumour structure are sampled and the blood is used for a blood serum. The structure is added with a HEPES buffer solution having the same quantity as the structure, is homogenized and is subjected to the centrifugal separation so that an insoluble matter is removed. The biological sample adjusting liquid is diluted about 10 times with the HEPES buffer solution and is set in a BIA core bio-sensor device together with the fixated antibody so that a quantity of the material reacting with the antibody is measured by surface plasmon resonance.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to the detection and measurement of heparan sulfate glycosidases (HS endoglycosidase, general names heparitinase and heparanase).
Surface plasmon (SPR) using immobilized antibody against
The present invention relates to a novel immunoassay method characterized by being detected in.

[0002]

2. Description of the Related Art The above-mentioned measuring method is considered to be effective for detecting metastasis of cancer and malignancy. Tumor cell metastasis is established by continuous interaction between tumor cells and normal tissues and cells. These processes involve invasion from the primary tumor site into surrounding tissues,
The stages are invasion into blood vessels or lymph vessels, implantation and invasion into remote organs, and proliferation.

[0003] In each of these processes, in order for tumor cells to enter each organ, the basement membrane present in each organ must be destroyed. This basement membrane is composed of species IV collagen, laminin, heparan sulfate proteoglycan (HSPG) and fibronectin network, and HSPG plays a very important role in the network organization (Kanwar Et al., J. Cell Biol., 86, 688-693,
1980). HSPG is involved in the interaction with type IV collagen and other basement membrane constituent proteins and protects the basement membrane matrix from various proteolytic enzymes. Therefore, it is considered that destruction of HSPG existing in the basement membrane is essential for tumor cells to enter various organs.

From the above, Savion et al. (J. Cell Phy
s., 118, 169-178, 1984), Vlodasky et al. (Exp. Cell Re
s., 140, 149-159, 1982), Kramer et al. (J. Biol. Chem.,
257, 2678-2686,1982), Nakajima et al. (Science, 220, 611)
-613, 1983, J. Biol. Chem., 259, 2283-2290, 198
4), Yokota et al. (The 79th Annual Meeting of the Japanese Society of Pathology, vol 79, No.1,
139, 1990) has a very high correlation between HS endoglycosidase (heparitinase and heparanase) activity and tumor cell metastatic ability, Nakajima et al. (Int. J. Cancer, 45, 1088-1095, 19
90) and Katayama et al. (51st Annual Meeting of the Japanese Cancer Society, 225, 1992,
The Journal of the Japanese Pathological Society, vol 1, No 1, 188, 1993), tumor cells that have acquired metastatic potential can be detected by an immunoassay using an antibody against heparanase or heparitinase. It is reported to be very effective in the test.

[0005]

[Problems to be Solved by the Invention] HS Endoglycosider
For the measurement of zease (heparitinase or heparanase), activity measurement or immunoassay method is used. To measure activity,
The released reaction product is measured using basement membrane or immobilized HS as a substrate. In order to measure this reaction product, various methods of labeling the substrate with radioactive allotrope or fluorescent substance (O
ldberg et al., Biochemistry, 19, 5755-5762, 1980, Yaha.
lom et al., J. Clin. Invest., 74, 1842-1849, 1984, Nicholson et al., published patent publication, Sho 62-265998). In addition, the immunological method was to produce an antibody against HS endoglycosidase, which was conventionally used for ELISA.
And Western blot method, Nicholson et al. (Published patent publication, Sho 62-265998), Yokota et al. (79th Annual Meeting of the Japanese Pathological Society, v
ol 79, No 1, 139, 1990) and Katayama et al. (51st Annual Meeting of the Japanese Cancer Society, 225, 1992, Journal of Japanese Pathology Society, vol 1, No 1, 1
88, 1993).

[0006] The ELISA method using an HS endoglycosidase activity and an antibody against the enzyme is used in the HS in a biological sample preparation solution.
Although it is an effective method for measuring endoglycosidase content,
Until the results are obtained, special technical knowledge is required, the operation is very complicated, the measurement sensitivity is low, and it takes a long time.

[0007]

According to the present invention, an antibody against sulfated endoglycosidase is immobilized and a biological sample preparation solution which may contain heparan sulfate endoglycosidase is brought into contact with the antibody. The present invention proposes a method for measuring the amount of heparan sulfate endoglycosidase bound to the present antibody by surface plasmon resonance.

[0008]

Function: That is, the present invention provides SPR (Kenichi Sasai, Protein Nucleic Acid Enzyme, 37, 2977-2, which can be measured with high sensitivity of 50 pg to 60 ng.
(984, 1992) and proposed an immunoassay method using BIAc
ore Biosensor (Farmasia Co., Ltd.) was immobilized with an antibody against heparitinase in the sensor part, and a biological sample preparation solution that may contain heparitinase was set in the same device, and the amount of the substance that reacted with the antibody was set. Is detected by SPR.

[0009]

EXAMPLES The present invention will now be described with reference to examples. Preparation and Immobilization of Antibodies: Oosta from Mouth Luis Lung Cancer Cells
(J. Biol. Chem., 257, 11249-11255, 1982) using a heparitinase purified according to the method of (1) as an antigen, and a polyclonal antibody (Yokota et al., 79th Annual Meeting of the Japanese Pathological Society, vol 79,
No 1, 139, 1990) and monoclonal antibody (Katayama et al., No. 51).
Annual Meeting of the Japanese Cancer Society, 225, 1992, Journal of the Japanese Society of Pathology, v
ol 1, No 1, 188, 1993). BI these antibodies
It was immobilized on the CM dextran of the sensor part of the A core biosensor according to the method instructed by Falmatia.

Preparation of biological samples: a. Mouthlouis lung cancer cells (1 × 10 ⁶ cells) were transplanted into the right foot pad of C57 black mice, and blood and tumor tissues were collected daily for 28 days. Blood was made into serum based on a conventional method, and an equal amount of Hepes buffer was added to the tissue, the mixture was homogenized with Polytron, and insoluble matter was removed by centrifugation. b. Human serum is healthy as a normal group, males and females up to 28-60 years old 50
Humans, 50 male and female patients with cancer, and 10 post-operative male and female patients who had no recurrence for 10 years after surgery to remove cancer were used.

Measurement of heparitinase: The above-mentioned biological sample preparation solution was diluted 10 times with Hepes buffer and set on a BIA core biosensor device according to the method instructed by Pharmacia. No difference was observed in the results of the polyclonal antibody and the monoclonal antibody.
Table 1 shows the results of the mouse using the antibody, and Table 2 shows the results of the human serum.

[0012]

[Table 1]

[0013]

[Table 2]

[0014]

INDUSTRIAL APPLICABILITY The present invention uses an immobilized antibody against heparitinase for the detection and measurement of heparan sulfate endoglycosidase, and by detecting with surface plasmons,
The present invention provides a new and effective assay method for detecting metastasis and malignancy of cancer.

(72) Inventor Takashi Yokota 2-9-15-403 Hatanodai, Shinagawa-ku, Tokyo (72) Inventor Hiroshi Oami 3-16-4 Minamiogikubo, Suginami-ku, Tokyo

Claims (3)

[Claims] 1. Immobilizing an antibody against heparan sulfate endoglycosidase, contacting this antibody with a biological sample preparation solution that may contain heparan sulfate endoglycosidase, and binding to this antibody by surface plasmon resonance The method for measuring the amount of heparan sulfate glycosidase. 2. The method according to claim 1, wherein the antibody is a polyclonal antibody. 3. The method according to claim 1, wherein the antibody is a monoclonal antibody.