JPS61272663A - Detection of cancer-associated antigen - Google Patents

Abstract

PURPOSE:To detect a cancer-associated antigen with good sensitivity by crushing cells to liberate the cancer-associated antigen or the antigen in the cells and detecting the liberated antigen. CONSTITUTION:A pancrea structure lump 1 is cleaned with a Hanks soln. and is transferred into a petri disch 2. The lump is then cut to fragments by a surgeon's knife or scissors. The tissue fragments 3 thereof are transferred into a flask 4 contg. a disperse or trypsine liquid 5 and the liquid is shaken. The enzyme liquid 5 is removed by centrifugal sepn. or filtration, by which the suspension 6 of the cells is obtd. Antigen CEA8a exists on the film surface of the cancer cells 7 and the antigen is produced in the cells as well if the cells are cancerized. An antigen CEA antibody 9 is added to the liquid 6 to induce the antigen antibody reaction with all of the CEA on the film. The antibody 9 is preliminarily labeled by FITC10. The unreacted FITC-anti-CEA antibody is then removed. The cells 7 are crushed by a blender and the antigen is dissolved in a nonionic surface active agent soln. The soln. is passed through a solid phase 12 bound with an anti-rabbit IgG antibody 11 and the fluorescence of the solid phase 12 is measured.

Description

[Detailed Description of the Invention] [Field of Application of the Invention] The present invention relates to a method for detecting cancer-related antigens in cancer cells, and is particularly suitable for detecting antigens expressed on the membrane surface of cells or antigens inside cells. Concerning methods.

[Background of the invention]

As described in "Tumor Markers", immunological methods such as detection of cancer-related antigens are being considered in the diagnosis of various cancers in 1 Karada no Kagaku J Ha i-00 (1981). In particular, for colorectal cancer, carcinoembryonic antiseptics
yonic antjgen (hereinafter abbreviated as CEA), carbohydrate andidiene 19-9 (carboh
ydrate antjgen 19-9, hereinafter CA
19-9), tissue polypeptide antiseptics (abbreviated as 19-9), tissue polypeptide
For pancreatic cancer, CEA, biphreatic oncophytal antiseptics (pancreatic cancer), etc.
jc oncofel;alantjgen), CA1
．． 9-9, etc., in hepatocellular carcinoma, α-fetoprotein (α-
fetoprotein), CEA, etc. are considered cancer-related antigens in gastric cancer, and the concentration of these antigens in the blood is currently used as an index for cancer diagnosis. These antigens are produced within cancer cells, and the amount released into the blood depends on the size of the cancer tissue. Therefore, it is difficult to detect early cancers with small cell clusters even if the antigen concentration in the blood is measured once it has risen.

In addition, as a method for detecting antigens bound to cells, there is a method in which cell surface antigens are stained with fluorescently labeled antibodies and detected using a flow cytometer (eds. ”). This method detects only antigens on the cell surface and cannot detect antigens inside the cells.

Therefore, a method that can detect cancer-related antigens produced by cancer cells with high sensitivity is desired.

[Purpose of the invention]

An object of the present invention is to provide a method for efficiently and quantitatively measuring cancer-related antigens produced by cancer cells.

[Summary of the invention]

Antigens produced within cancer cells are secreted outside the cells, but
Naturally, it also exists on the cell membrane and inside the cell. Generally, in early stage cancer, the amount of antigen released into the blood is very small. Also, depending on the antigen, it is produced within the cell,
It has also been pointed out that there are cancer cells that do not secrete extracellular substances.

Therefore, rather than antigen secreted outside the cell, cell membrane
Detecting the amount of antigen present inside cells and/or cells is advantageous for early detection of cancer.

Generally, antibodies do not invade inside cells, so it is difficult to detect antigens present inside cells as they are. However, if cancer-related antigens on the membrane surface and/or inside the cells are released by disrupting the cells, they can be easily detected by antigen-antibody reactions.

[Embodiments of the invention]

Taking pancreatic cancer as an example of the present invention, a method for quantitatively detecting CEA, which is a cancer-related antigen, will be explained.

Example 1 FIG. 1 is a schematic diagram of a means for preparing cells to be used as a sample.

First, a pancreatic tissue mass 1 cut out from the body was prepared using Hank's solution (
c MF-thanks). This tissue mass 1 is transferred to a petri dish 2 and thoroughly chopped using a scalpel or scissors (FIG. 1 (1)). The finely chopped tissue pieces 3 are transferred to a disperser or a flask 4 containing tolizine (enzyme) solution 5, and shaken at 37°C for a certain period of time (see Fig. 1).
C)). Thereafter, the enzyme solution 5 is removed by centrifugation or filtration to obtain a cell suspension 6 (FIG. 1(d)).

FIG. 2 is a schematic diagram showing a method for measuring ambiguous antigens bound to cells. When a cell has become cancerous, the antigen CE A 8 a is present on the membrane surface of the cancer cell 7 .

Furthermore, antigen 8b is also produced within the cells. Anti-CEA that specifically reacts with CE A 8 a in cell suspension 6
Antibody (rabbit) 9 is added to cause an antigen-antibody reaction with all CEA on the membrane (FIG. 2(a)).

This anti-CEA antibody 9 is labeled with fluorescein isothiocyanate (FITC) 10. Next, unreacted FTTC-anti-CEA antibody is removed by centrifugation or filtration (Figure 2(b)). Membrane-bound cEA (8a
), the cells 7 are crushed using a blender, and the antigen portion is further dissolved with a 0.5% solution of a nonionic surfactant such as NP-40 (Figure 2 (C)).
). FITC-anti-CEA antibody- by passing this solution through a solid phase 12-1- such as a membrane bound with an antibody that specifically reacts with FTTC-anti-CEA antibody, for example, anti-heron IgG antibody (goat) 11. The CEA complex is fixed (FIG. 2(d)). At this time, the solid phase is the same as fluorescently labeled with FTTC, and by taking out this same phase and measuring the fluorescence, the amount of antigen on the membrane surface of the pancreatic tissue mass 1 can be quantified. For example, Ar laser light of 488 nm is irradiated in the same phase, and the fluorescence intensity of 520 to 540 nm is measured. The amount of antigen can be quantified by comparing this fluorescence intensity with a previously calibrated relationship between fluorescence intensity and antigen amount.

Example 2 In FIG. 2(a) of Example 1, the antigen-antibody reaction occurs only against the antigen 8a on the cell surface, and against the antigen 8a present inside the cell.
It has no effect on b. Therefore, in Figure 2(d),
Antigen 8b inside the cell was not fixed in the same phase, and all -
Dissolved in the L purifying solution.

Therefore, by collecting this 2-separate liquid and measuring the antigen 8b in the liquid, it is possible to quantify the amount of antigen 8b that was inside the cells. An example in which the antigen 8b is CEA, which is the same as the antigen 8a on the membrane surface, will be explained. Figures 3 and 4 show antigen CE
FIG. 2 is a schematic diagram showing a method for measuring sensitivity of A8b. In Figure 3,
- FTTC-labeled anti-CEA antibody is added to the clarified solution in the same manner as in FIG. 2 to cause an antigen-antibody reaction. Next, C
In order to immobilize only E A 8 b, a solid phase 12 bound to an anti-CEA antibody (goat) that specifically reacts with CEA was used.
−[− to pass through. In this way, CEA8 b is immobilized on the solid phase. Contrary to FIG. 73, as shown in FIG. 4, CE A 8 b is first immobilized on the solid phase 12, and then
The same effect can be obtained even if an antigen-antibody reaction is caused with the 1'C-anti-Cl (A antibody).The method for placing the antigen is the same as in Example 1.

Example 3 In Examples 1 and 2, a method was described in which the antigen CEA was quantified separately into the amount of CEΔ8a found in the cell membrane-L and the amount of CEABb present inside the cell. Cell membrane-
When detecting human and internal CEA all at once without separating them, put the cancer tissue piece 3 in a blender and 0.5% NP.
-40 liquid is used to crush and dissolve CEA. After this, CI=: A can be quantified in the same manner as in Example 2.

Example 4 Examples 1, 2 and 3 show cases where the antigen on the cell membrane and the antigen inside the cell membrane are the same. However, when a cell becomes cancerous, sugar chains on the membrane surface may change and become cancer-specific antigens. Furthermore, one cancer cell produces not just one type of antigen, but many types. For example, the pancreatic syringe produces CEA and CA19-9. Therefore, the membrane-L antigen to be detected does not necessarily match the antigen inside the cell. However, even in this case, the amount can be quantified in the same manner as in Examples 1 and 2 by applying antibodies that specifically react with each.

In Examples (1 to 4), quantitative measurements are performed after cells are crushed and antigens are released. After the antigen is released, the process is almost the same as the quantitative measurement of antigen in blood. Therefore, there is an effect that a detection system for antigens on the surface and inside of cells can be incorporated into a conventional blood antigen fixation device very easily and at low cost. Moreover, this makes it possible to perform the immobilization of antigens in blood and the quantification of antigens in cells using a single apparatus.

According to this example, antigens on the membrane and internal antigens of cancer cells can be independently quantified. Furthermore, antigens that are secreted only inside cells (not secreted outside cells) can also be efficiently measured. Furthermore, since the antigen portion is dissolved and measured, the amount of antigen can be quantified regardless of whether the cells are colored cells or cells that contain fluorescent substances inside.

In addition, since all reactions with reagents are specific reactions called antigen-antibody reactions, detection sensitivity is theoretically high.

In this embodiment, a pancreatic cancer-related test has been described, but the test can be similarly performed for colon cancer, hepatocellular carcinoma, stomach cancer, lung cancer, etc.

Further, in cases where the cells are floating cells from the beginning, such as in malignant lymphoma, the adjustment as shown in FIG. 1 is unnecessary.

〔Effect of the invention〕

According to the present invention, cancer-related antigens bound to cells can be efficiently and directly quantified, so even early cancers with low antigen production can be detected with high sensitivity.

Furthermore, since it is possible to quantify only the antigens on the cell membrane or only the antigens inside the cells, there is an effect that more information can be obtained when diagnosing cancer. Furthermore, since antigens in cancer cells that do not release antigens outside the cells can be quantified, this method has the effect of expanding the usefulness of diagnosis.

[Brief explanation of the drawing]

Figure 1 is a schematic diagram of the procedure for preparing cancer cells, and Figure 2 is a cell membrane.
Figure 3 is a schematic diagram showing the method for quantitative measurement of antigens inside cells. Figure 4 is a schematic diagram showing the quantitative measurement method for antigens inside cells. Tissue mass, 2...
・Petri dish, 3. Tissue piece, 4. Flask, 5.
・Enzyme solution, 6... Cell suspension, 7... Cancer cells, 8a
...Antigen (CEA) on the cell membrane, 8b...Antigen (CEA) inside the cell, 9...Anti-CEA antibody, Figure 1 (C) 'fJ2 Figure (b, )

Claims (1)

[Claims] 1. A method for detecting a cancer-related antigen bound to a cell using an antigen-antibody reaction, in which the antigen on the membrane surface of the cell and/or the antigen inside the cell is released by crushing the cell. , a cancer-related antigen detection method characterized by detecting the released antigen.