JP2005106631A - Detection method for cancer marker in cancer cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a detection method for a cancer marker capable of easily detecting the cancer marker in protein solution acquired by solubilizing a cell collected from a patient, and acquiring a detection result safely and quickly with high sensitivity and high efficiency (high throughput). <P>SOLUTION: In this detection method of the cancer marker, a trapping substance which is a substance for trapping the cancer marker in the protein solution acquired by solubilizing the cell is immobilized on the substrate surface, and the cancer marker which is a protein or a peptide is specifically trapped by an interaction with the trapping substance, and it is discriminated whether an origin cell of the marker is a cancer cell or not. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  Cancer cells may have specific proteins that are rarely present in a healthy human body in addition to common proteins that exist in everyone. They are collectively referred to as cancer markers (or tumor markers). The present invention relates to a detection method for capturing a cancer marker and identifying whether the marker-derived cell is a cancer cell using a substance that specifically captures the cancer marker.

In cancer screening, there is a biopsy test as a method for accurately grasping the medical condition of a patient to be treated. That is, it is a method for examining a living tissue of a patient by using an endoscope or an injection needle for diagnosis of disease or diagnosis after surgery. This is because the cells are collected from the patient's tissue, so the burden on the patient is greater than that of blood tests. Furthermore, the presence or absence of a protein that serves as an indicator of disease is examined from cell samples collected in the diagnostic room or operating room, and in order to obtain a result, the diagnostic result cannot be confirmed until the next day or later.
"Clinical Surgery", Medical School 57, 735-738 (2002)

An object of the present invention is to detect a cancer marker in a cancer cell, in which a diagnostic result is obtained during surgery, and when the result is malignant, the malignant tissue can be immediately removed by extending the diagnostic state and the burden on the patient can be reduced. Is to provide.
Another object of the present invention is to provide a method for detecting a cancer marker in a cancer cell that can be diagnosed by collecting as little tissue as possible, and that can similarly reduce the burden on the patient while minimizing the wound.

(1) A capture substance, which is a substance that captures a cancer marker in a protein solution obtained by solubilizing cells on a surface of a substrate, is immobilized, and a cancer marker that is a protein or peptide is coated by interaction with the capture substance. A method for detecting a cancer marker that specifically captures as a captured substance and identifies whether the cell derived from the marker is a cancer cell;
(2) The method for detecting a cancer marker according to (1), wherein the captured product includes any one of a protein, a peptide, and a nucleic acid,
(3) The method for detecting a cancer marker according to (2), wherein the protein that is a captured substance is an antibody,
(4) Cancer markers are CEA, BFP, TPA, IAP, AFP, PIVKA-II, CA19-9, CA50, DUPAN-2, Span-1, NCC-ST
-439, elastase 1, SLX, CA125, CA130, CA602, STN, CA72-4, CA54 / 61, SCC antigen, NSE, CA15-3, BCA225, PAP, PSA, γ-Sm (1), (2 ) Or the method for detecting a cancer marker according to any one of (3),
(5) The method for detecting a cancer marker according to any one of (1), (2), (3), and (4), wherein an object to be captured interacts with an anticancer marker antibody,
(6) The method for detecting a cancer marker according to (5), wherein the anti-cancer marker antibody is labeled with biotin, fluorescent, or enzyme.
(7) The method for detecting a cancer marker according to (6), wherein the anti-cancer marker antibody labeled with biotin and fluorescent label, or enzyme-labeled avidin or streptavidin are bound.
It is.

  According to the method of the present invention, detection of a cancer marker in a protein solution obtained by solubilizing a cell collected from a patient is easy, safe, highly sensitive, quick and highly efficient (high throughput), and a detection result can be obtained. it can.

The detection method used in the present invention is characterized in that a captured substance is immobilized on a part of the surface of a solid phase substrate and a cancer marker can be detected.
The detection method of the present invention includes a substrate preparation process, a substrate surface modification process, a captured substance fixing process, a blocking process, a captured / captured substance interaction process, and a detection process.
Although it can be used even if the substrate surface treatment step is omitted, it is preferable to use it in order to immobilize the captured substance more firmly.

(Substrate material)
Glass, metal, etc. can be usually used as the material for the substrate, but as the material for the solid phase substrate used in the present invention, plastic is used from the viewpoint of ease of surface treatment and mass productivity, and particularly thermoplastic. A resin is preferred. As a thermoplastic resin, a thing with little fluorescence generation amount is preferable. For example, it is preferable to use linear polyolefins such as polyethylene, polypropylene, polypentene, polycarbonate, polystyrene, polyamide, saturated cyclic polyolefin, fluorine-containing resin, etc. It is more preferable to use a cyclic polyolefin. Here, the saturated cyclic polyolefin refers to a polymer having a cyclic olefin structure or a saturated polymer obtained by hydrogenating a copolymer of a cyclic olefin and an α-olefin.
For the purpose of efficiently reacting on the substrate, a flow path may be provided in the substrate.

(Substrate surface modification)
Various methods can be used as a method for modifying the surface of the substrate used in the present invention. When an aldehyde group is introduced, the captured substance is covalently bonded on the substrate and fixed more firmly. preferable. As a method for introducing an aldehyde group, a method in which a polyfunctional aldehyde is reacted after introduction of an amino group is preferably used. Examples of amino group introduction means include treatment with an amino group-containing silane coupling agent, plasma treatment under a nitrogen atmosphere, and coating of an amino group-containing polymer substance. From the viewpoint of simplicity of treatment and uniformity. The treatment with an amino group-containing silane coupling agent is preferred. As the polyfunctional aldehyde, glutaraldehyde is preferable.

(Immobilization of captured matter)
In order to enhance the reactivity with the aldehyde group, it is preferable to introduce an amino group in advance in the captured product used in the present invention. When the captured product is a nucleic acid, the amino group may be introduced at the molecular chain end or side chain of the nucleic acid, but is preferably introduced at the molecular chain end. In the case of proteins and polypeptides, since amino groups are provided, it is not necessary to introduce amino groups. Immobilization is usually performed by applying a treatment appropriately after spotting a solution in which a physiologically active substance is dissolved on a substrate.
In order to increase the reaction efficiency, when using a substrate with a flow channel, the captured material may be fixed after modifying the flow channel as described above, but the captured material is fixed externally to a microsphere or the like. Then, it may be introduced into the flow path.

(Blocking process)
Blocks the capture target from non-specific adsorption on the substrate surface before immobilizing the captured product and allowing the captured product and the captured product to interact with each other. Called. The substance used for blocking in the present invention is preferably at least one selected from the group consisting of skim milk, albumin, antibody, and Fc part of the antibody. These substances are prepared to 0.1 to 10% by weight with pure water or tris acid buffer solution, and a substrate on which a physiologically active substance is immobilized is immersed in the solution, and reacted with a functional group on the substrate surface for 1 to 3 hours. To do.
When using a substrate having a flow path, the solution flows into the flow path.

(Interaction process of captured / acquired objects)
A protein obtained by solubilizing a cell to be informed of the presence or absence of a target cancer marker, that is, a solution to be captured, is dropped onto a substrate on which the captured matter is immobilized and blocked.
When using a substrate having a flow path, the solution flows into the flow path.
Cancer markers include CEA, BFP, TPA, IAP, AFP, PIVKA-II, CA19-9, CA50, DUPAN-2, Span-1, NCC
-ST-439, elastase 1, SLX, CA125, CA130, CA602, STN, CA72-4, CA54 / 61, SCC antigen, NSE, CA15-3, BCA225, PAP, PSA, γ-Sm and the like.

(Detection process)
As described above, a fluorescent labeling substance may be bound to the capture target and detected by a detector. However, when the capture target and the fluorescent labeling substance are bound, the capture target undergoes a structural change and is specific to the capture target. May not be combined.
Therefore, an aqueous solution of an anti-cancer marker antibody such as an anti-CEA antibody or an anti-CA19-9 antibody that specifically binds to the capture target is dropped onto the substrate on which the capture target bound to the capture target is immobilized. It is preferable to bind the captured substance to the anticancer marker antibody.
More preferably, the anti-cancer marker antibody is labeled with biotin, a fluorescent label, or an enzyme.
If it is fluorescently labeled, the amount of fluorescence can be measured as it is, and if it is enzyme-labeled, the substrate is dropped on the substrate to develop color and detect the color development amount.
Furthermore, if it is biotin-labeled, fluorescence-labeled avidin or streptavidin and the biotin group bind specifically, so that the amount of fluorescence may be detected after binding.

  When the reactivity between the captured substance and the labeled anti-cancer marker antibody is low, an unlabeled anti-cancer marker antibody is used, and the anti-cancer is labeled with biotin, fluorescence or enzyme in the next step After binding to the marker antibody, detection may be performed in the same manner as described above.

(Example 1)
The saturated cyclic polyolefin resin was processed into a slide glass shape (dimensions: 76 mm × 26 mm × 1 mm). After the surface was hydrophilized, it was immersed in a 2% aqueous solution of an amino group-containing alkylsilane and then heat treated to introduce amino groups on the surface. This was immersed in a 1% glutaraldehyde aqueous solution to react the surface amino groups with glutaraldehyde to introduce aldehyde groups.
Next, a sandwich method was performed on the substrate. In detail, first, the primary antibody and anti-CA19-9 antibody were spotted on the substrate by an automatic spotter, and then allowed to stand in an environment at room temperature of 4 ° C. for 24 hours. Thereafter, the substrate was immersed in a 25 mM Tris buffer solution in which skim milk was dissolved to a concentration of 5% for blocking, and allowed to stand at room temperature for 2 hours. Thereafter, the CA19-9 aqueous solution was subjected to an antigen-antibody reaction with the primary antibody. Thereafter, an antigen-antibody reaction was carried out with an anti-CA19-9 antibody labeled with biotin as a secondary antibody. Finally, it was reacted with Cy5-labeled streptavidin.
Similarly, a protein solution obtained from cancer cells and a protein solution obtained from normal cells were reacted in place of the aforementioned CA19-9 aqueous solution.
In the case of the CA19-9 aqueous solution and the protein solution obtained from cancer cells, the effluent was fluorescently colored, but the protein solution obtained from normal cells was not colored.

(Example 2)
The saturated cyclic polyolefin resin was processed into a slide glass shape (dimensions: 76 mm × 26 mm × 1 mm). Similarly, the resin was processed into a slide glass shape having a flow path on one surface.
The two sheets were superposed and joined with a hot press so that the surface provided with the flow path was on the inside.
On the other hand, after immersing polystyrene beads having a diameter of 20 μm in an anti-CEA antibody aqueous solution kept at 4 ° C. for 24 hours, the substrate was placed in a 25 mM Tris buffer solution in which bovine serum albumin was dissolved to a concentration of 3%. It was immersed and allowed to stand at room temperature for 2 hours.
The treated beads were fixed in the flow path, and a CEA aqueous solution was introduced so as to come into contact with the beads. Then, in the same manner, peroxidase-labeled anti-CEA antibody was introduced, and then hydrogen peroxide solution and 3,3 ', 5,5'-tetramethylbenzidine (3,3', 5,5'-Tetramethylbenzidine) The mixed solution of was poured.
Similarly, a protein solution obtained from cancer cells and a protein solution obtained from normal cells were introduced instead of the CEA aqueous solution described above.
In the case of the CEA aqueous solution and the protein solution obtained from cancer cells, the effluent developed color, but the protein solution obtained from normal cells did not develop color.

  The method for detecting a cancer marker of the present invention is easy to detect, safe, highly sensitive, quick and highly efficient (high throughput), and can determine whether a patient is positive for cancer even during surgery. .

Claims (7)

A captured substance that is a substance that captures a cancer marker in a protein solution obtained by solubilizing cells on the surface of the substrate is fixed, and a cancer marker that is a protein or a peptide is captured as an object to be captured by interaction with the captured substance. A method for detecting a cancer marker, which specifically captures and identifies whether or not a cell derived from the marker is a cancer cell. The method for detecting a cancer marker according to claim 1, wherein the captured substance contains any one of a protein, a peptide, and a nucleic acid. The method for detecting a cancer marker according to claim 2, wherein the protein as a captured substance is an antibody. Cancer markers are CEA, BFP, TPA, IAP, AFP, PIVKA-II, CA19-9, CA50, DUPAN-2, Span-1, NC
From the group consisting of C-ST-439, elastase 1, SLX, CA125, CA130, CA602, STN, CA72-4, CA54 / 61, SCC antigen, NSE, CA15-3, BCA225, PAP, PSA, and γ-Sm The method for detecting a cancer marker according to any one of claims 1 to 3, wherein the method is at least one selected. The method for detecting a cancer marker according to any one of claims 1, 2, 3 and 4, wherein an object to be captured is allowed to interact with an anticancer marker antibody. 6. The method for detecting a cancer marker according to claim 5, wherein the anti-cancer marker antibody is labeled with biotin, fluorescent or enzyme. The method for detecting a cancer marker according to claim 6, wherein the anti-cancer marker antibody labeled with biotin is combined with a fluorescent label, or avidin or streptavidin labeled with an enzyme.