JP2001041959A - System and kit for detecting cancer cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To simply and specifically detect cancer cells with a marker substance by bringing a liquid containing the cancer cell and a substance with affinity to cancer cells brought into contact with each other, and detecting the cancer cells coupled to the substance. SOLUTION: Not only is a substance itself (ligand) having an affinity to a cancer cell used as a carrier, but the ligand is preferably immumobilized on the carrier to specifically combine the cancer cell. Beads, plates or the like are easily available as carriers. For the ligand is preferably used a monoclonal antibody, which specifically couples by an electrostatic interaction or the like to a cell cortex substance, a polyclonal antibody, or the like, that is, an anti- insulin-form growth stimulator receptor antibody or the like, which will not interact with normal cells such as white blood cells, red cells, etc. The amount of the ligand group is preferably 1 fmol-10 mol per 1 cm2 surface area of the antibody. The carrier is added to a liquid containing the cancer cell to be combined with the cancer cell. The detect the combined cancer cell, a method using a combining substance of a marker compound which is easy to measure with a high sensitivity such as peroxidase or the like is preferred.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a system and a kit for detecting cancer cells in a liquid.

[0002]

2. Description of the Related Art Early detection of metastasis and recurrence during and after cancer treatment is one of the important issues in cancer treatment. Conventionally, methods for examining and diagnosing cancer include a method of observing and determining the cell morphology of a diseased tissue from a pathological specimen, and a sandwich ELISA method using a polyclonal antibody or a monoclonal antibody against a tumor marker.
Although these methods have greatly contributed to the diagnosis and treatment of cancer, it is difficult to determine whether they are benign or malignant, and the correlation between cancer progression and the concentration of tumor markers in blood is still unknown and not necessarily specific. It is difficult to say that there is also organ specificity, so at present it only plays an auxiliary role as a diagnostic method. In addition, these methods have a decisive disadvantage that cancer recurrence and metastasis cannot be predicted and diagnosed. In order to compensate for this drawback, in recent years, attempts have been made to detect cancer cells in tissue or blood by PCR using a tumor marker gene as an index, and a tumor marker gene has been detected at a high rate in the blood of a malignant patient. The presence of cancer cells in the blood has been suggested. However, since this method uses a PCR method, there is a possibility that false positives are common, and it is not only difficult to quantify, but also it is not possible to discriminate between genes in cancer cells and genes released outside the cells. There is. Further, there is a time and effort required to select a tumor marker depending on the type of cancer. As described above, a detection / diagnosis / test system capable of detecting many cancer cells with one marker in order to detect cancer cells in a body fluid has not been found so far.

[0003]

SUMMARY OF THE INVENTION Accordingly, the present inventors
In view of the above-mentioned problems, as a result of studying a method for detecting cancer cells regardless of their type, a substance having a strong affinity for a substance expressed on the surface of many cancer cells (hereinafter referred to as a ligand) was immobilized. The present inventors have found that cancer cells in a body fluid can be directly detected using the carrier thus obtained, and have reached the present invention. That is,
An object of the present invention is to provide a cell detection system in which cancer cells are adsorbed to a carrier having a ligand immobilized irrespective of their type, and the adsorbed cancer cells are detected simply and specifically with a labeling substance.

[0004]

That is, the present invention comprises at least a step of contacting a carrier containing a liquid containing cancer cells with a carrier and a step of detecting the cancer cells bound to the carrier. It is a detection system.

[0005] The present invention is also a cancer cell detection kit for detecting a cancer cell in a body fluid by bringing a human or animal body fluid into contact with a substance having an affinity for the cancer cell. A cancer cell detection kit wherein the substance is at least one selected from saccharides, polypeptides, oligopeptides, peptides, glycoproteins, glycopeptides, and vitamin acids.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.

As a substance (ligand) having affinity for cancer cells used in the cancer cell detection system and the cancer cell detection kit of the present invention, a substance (ligand) that directly binds to cancer cells can be used only as a carrier itself. Instead, it is also desirable that the ligand is immobilized on a carrier to specifically bind the target cancer cells. In this case, the carrier is not limited to any material as long as it does not dissolve when it comes into contact with the liquid and can immobilize the ligand. Beads, plates, and tubes are preferably used because they are easily available.

[0008] A ligand is a substance having an affinity for a substance expressed on the surface of a cancer cell, and is specific to a cell surface substance by an electrostatic interaction, a hydrophobic interaction, a Van der Waals interaction, or the like. Anything that can be combined with is acceptable. Specific binding includes monoclonal antibodies, polyclonal antibodies, fragments containing an antigen recognition site of an antibody, cell surface receptors, fragments containing a ligand binding site of a receptor, sugar chains, polypeptides,
Examples include oligopeptides, amino acids, polynucleotides, oligonucleotides, nucleotides, lipids, and the like. In addition, synthetic organic compounds and ligands of synthetic polymer compounds can be used. Examples of ligands include anti-integrin antibodies, anti-CD44 antibodies, anti-MUC-1 antibodies, anti-cytokeratin antibodies, anti-epidermal growth factor antibodies, anti-insulin-like growth factor antibodies, antibodies such as anti-insulin-like growth factor receptor antibodies, or Part of the antibody containing the antigen recognition site, polypeptide or oligopeptide such as collagen, RG
Oligopeptides containing a D sequence, glycoproteins such as fibronectin, polysaccharides such as hyaluronic acid and phosphomannan, oligosaccharides such as mannose-6-phosphate pentamer, monosaccharides such as mannose-6-phosphate, retinoic acid, etc. Vitamin acids and the like. Anti-insulin-like growth factor receptor antibody and mannose-6-phosphate pentamer are preferably used because they do not interact with normal cells such as leukocytes and erythrocytes. If the number of ligands is too small, sufficient detection sensitivity cannot be obtained, and if the number is too large, the ligands may overlap each other and hinder each other. Therefore, the amount of the ligand group, the carrier surface area 1 cm ² per 1fmol~10mol are preferable, 1 Pmol～1
mol is more preferred. The ligand to be immobilized is not limited to one type, and a plurality of ligands may be immobilized.

The carrier is desirably subjected to a treatment for preventing cells and substances other than cancer cells from being non-specifically adsorbed. For example, the carrier may be coated with serum albumin or gelatin, or a hydrophilic monomer may be used. Alternatively, a method of surface graft polymerization of a polymer, a plasma treatment method, or the like is preferably used.

In order to bind cancer cells to these carriers, a carrier is added to a liquid containing cancer cells, and the cancer cells are brought into contact with the carriers. At this time, the number of cells in the liquid may be arbitrary, and the liquid may be used as it is, or may be diluted with physiological saline or the like before use. Conditions for binding the cancer cells to the carrier are arbitrary, but it is preferable that the cancer cells be contacted at a temperature around body temperature for 1 hour or more in order to reliably bind living cells,
Shaking is preferred to further increase contact opportunities.

The detection of cancer cells bound to the carrier may be carried out in the liquid as it is. However, in order to suppress false positives, once the carrier having the cancer cells bound thereto is removed from the liquid and washed with physiological saline or the like. It is preferable to do so.

The method of detecting cancer cells bound to a carrier may be any method, and a method using a substance bound to a labeled compound, which is a method generally used in enzyme immunoassays and radioimmunoassays, is preferably used. The labeling compound may have high detection sensitivity and be easily measured,
There is no particular limitation. Examples thereof include enzymes such as peroxidase and alkaline phosphatase, fluorescent reagents such as fluorescein isothiocyanate and tetramethylrhodamine isothiocyanate, and compounds containing radioisotopes such as ³ H, ¹⁴ C, ³¹ P, and ¹²⁵ I. In addition, it is also possible to destroy cancer cells bound to the carrier and detect them by utilizing the amount of substances contained in the cells and the enzymatic activity. Upon detection,
The cancer cells may be left bonded to the carrier or may be peeled off. The detached cells can be cultured again and used for detection.

As the substance to which the labeling compound is bound, a substance that specifically binds to a cancer cell or a substance that has a specific affinity for a substance that specifically binds to a cancer cell can be used. For example, the former is a monoclonal antibody, a polyclonal antibody, a fragment containing an antigen recognition site of an antibody, a cell surface receptor, a fragment containing a ligand binding site of a receptor, a sugar chain, a polypeptide, an oligopeptide, an amino acid, a polynucleotide, an oligonucleotide, a nucleotide. , Lipid, avidin, biotin and the like, and the latter is preferably a secondary antibody against a monoclonal antibody. In addition to these, ligands of synthetic organic compounds and synthetic polymer compounds can be used.

[0014]

The present invention will be described below in more detail with reference to Examples and Comparative Examples, but the present invention is not limited thereto.

Examples 1 and 2 Forty polystyrene beads containing 1 nmol of amino groups per bead were immersed in 15 ml of polyethylene glycol diglycidyl ether and stirred at 25 ° C. for 3 days. The beads were washed with methanol, immersed in 15 ml of methanol, added with 1 ml of ethylenediamine, and stirred at 25 ° C. for 1 day. The beads were washed with methanol and air-dried (the beads obtained here are abbreviated as A hereinafter).

A20 pieces were immersed in 7 ml of 0.2 M phosphate buffer (pH = 7.0), and 10 mg of mannose-6-phosphate pentamer and 1.8 mg of tetramethylammonium borohydride were added.
At room temperature, washed with water, dried and washed with mannose-6-
Phosphoric acid pentamer-immobilized beads were prepared (hereinafter, referred to as immobilized beads A).

A20 pieces were immersed in 7 ml of 0.2 M phosphate buffer (pH = 7.0), 2 mg of mannose-6-phosphate and 1.8 mg of tetramethylammonium borohydride were added, and reacted at 4 ° C. overnight, And dried to prepare mannose-6-phosphate immobilized beads (hereinafter, referred to as immobilized beads B).

The above-mentioned immobilized beads A (Example 1) and immobilized beads B (Example 2) were each used as a human breast cancer-derived cell line.
It was immersed in PBS (phosphate buffer containing 0.9% sodium chloride) in which MCF-7 was suspended, and allowed to stand at 37 ° C. for 1 hour. The beads
After washing with PBS, the plate was immersed in PBS in which an anti-CD44 antibody labeled with fluorescein isothiocyanate was dispersed.
After standing for 0 minutes, the beads were washed with PBS. Excitation wavelength 495n
When the fluorescence intensity was measured at m and a detection wavelength of 520 nm, fluorescence was observed in each of Examples 1 and 2.

Examples 3 and 4 The same ligand-immobilized beads A (Example 3) and immobilized beads B (Example 4) as used in Examples 1 and 2 were obtained by using the human breast cancer-derived cell line MCF- 7 was suspended in Dulbecco's modified minimum essential medium (10% FCS D-MEM) containing 10% fetal bovine serum and shaken at 37 ° C. for 1 hour. The beads were mixed with Hanks' balanced salt solution (HBS) containing 0.25% bovine serum albumin (BSA).
After washing with S), the plate was immersed in PBS in which an anti-CD44 antibody labeled with fluorescein isothiocyanate was dispersed, and incubated at room temperature for 1 hour.
After standing for a period of time, the beads were washed with HBSS containing 0.25% BSA. When fluorescence intensity was measured at an excitation wavelength of 495 nm and a detection wavelength of 520 nm, fluorescence was observed in Examples 3 and 4, respectively.

Examples 5 and 6 The same ligand-immobilized beads A (Example 5) and immobilized beads B (Example 6) as used in Examples 1 and 2 were obtained by using the human breast cancer-derived cell line MCF- 10% FCS D-MEM with 7 suspended
And shaken at 37 ° C. for 1 hour. 0.25% B of the beads
After washing with HBSS containing SA, the plate was immersed in PBS in which an anti-cytokeratin (CK) antibody was dispersed, and allowed to stand at room temperature for 1 hour. The beads were washed with HBSS containing 0.25% BSA, 250 ml of an anti-mouse immunoglobulin G (IgG) antibody solution labeled with HRP was added, and the mixture was allowed to stand at room temperature for 1 hour. 250 ml was added. After allowing to stand at room temperature for 15 minutes, the reaction was stopped by adding 250 ml of 1 N sulfuric acid, and 450 ml was measured with a spectrophotometer.
When the absorbance at nm was measured, strong absorption was observed in each of Examples 5 and 6 as compared with the control.

Examples 7 and 8 The same ligand-immobilized beads A (Example 7) and immobilized beads B (Example 8) as used in Examples 1 and 2 were obtained from leukocytes collected from healthy human subjects, respectively. Was suspended in PBS and suspended at 37 ° C. for 1 hour. After washing the beads with PBS, the beads were immersed in PBS in which an anti-CD44 antibody labeled with fluorescein isothiocyanate was dispersed, allowed to stand at 37 ° C. for 30 minutes, and then the beads were washed with PBS. When the fluorescence intensity was measured at an excitation wavelength of 495 nm and a detection wavelength of 520 nm, no fluorescence was observed in each of Examples 7 and 8.

Examples 9 and 10 The same ligand-immobilized beads A (Example 9) and immobilized beads B (Example 10) as used in Examples 1 and 2 were obtained from leukocytes collected from healthy human subjects, respectively. 10% FCS suspended
It was immersed in D-MEM and shaken at 37 ° C. for 1 hour. The beads
After washing with HBSS containing 0.25% BSA, the beads were immersed in PBS in which an anti-CD44 antibody labeled with fluorescein isothiocyanate was dispersed, and allowed to stand at room temperature for 1 hour.
Washed with HBSS. When the fluorescence intensity was measured at an excitation wavelength of 495 nm and a detection wavelength of 520 nm, no fluorescence was observed in each of Examples 9 and 10.

Examples 11 and 12 The same ligand-immobilized beads A (Example 11) and immobilized beads B (Example 12) as used in Examples 1 and 2 were used.
10% FCS DM in which each mouse fibroblast cell line L929 was suspended
It was immersed in EM and shaken at 37 ° C. for 1 hour. 0.25
After washing with HBSS containing% BSA, the plate was immersed in PBS in which an anti-CD44 antibody labeled with fluorescein isothiocyanate was dispersed, and allowed to stand at room temperature for 1 hour.
Washed with SS. When the fluorescence intensity was measured at an excitation wavelength of 495 nm and a detection wavelength of 520 nm, no fluorescence was observed in each of Examples 11 and 12.

Examples 13 and 14 The same ligand-immobilized beads A (Example 13) and immobilized beads B (Example 14) as used in Examples 1 and 2 were used.
10% F in which leukocytes suspended from healthy humans were respectively suspended
It was immersed in CS D-MEM and shaken at 37 ° C. for 1 hour. After washing the beads with HBSS containing 0.25% BSA, the beads were immersed in PBS in which an anti-CK antibody was dispersed, and allowed to stand at room temperature for 1 hour. 0.25% beads
After washing with BSA-containing HBSS, 250 ml of an anti-mouse IgG antibody solution labeled with HRP was added and left at room temperature for 1 hour. After washing with HBSS containing 0.25% BSA, 250 ml of a coloring reagent was added. 15 at room temperature
After standing for 1 minute, the reaction was stopped by adding 250 ml of 1N sulfuric acid, and the absorbance at 450 nm was measured with a spectrophotometer. In each of Examples 13 and 14, a difference was observed in the absorption as compared with the control. Did not.

Examples 15 and 16 The same ligand-immobilized beads A (Example 15) and immobilized beads B (Example 16) as used in Examples 1 and 2 were used.
10% FCS DM in which each mouse fibroblast cell line L929 was suspended
It was immersed in EM and shaken at 37 ° C. for 1 hour. 0.25
After washing with HBSS containing% BSA, the plate was immersed in PBS in which anti-CK antibody was dispersed, and allowed to stand at room temperature for 1 hour. Contains 0.25% BSA beads
After washing with HBSS, the anti-mouse IgG antibody solution
0 ml was added, and the mixture was allowed to stand at room temperature for 1 hour. After washing with HBSS containing 0.25% BSA, 250 ml of a coloring reagent was added. After standing at room temperature for 15 minutes, the reaction was stopped by adding 250 ml of 1N sulfuric acid, and the absorbance at 450 nm was measured with a spectrophotometer. Was not seen.

[0026]

According to the present invention, cancer cells contained in a liquid can be easily and specifically detected irrespective of their types.

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (reference) G01N 33/574 G01N 33/574 D

Claims (10)

[Claims] 1. A cancer cell detection method comprising at least a step of contacting a liquid containing cancer cells with a substance having an affinity for the cancer cells, and a step of detecting the cancer cells bound to the substance. system. 2. A cancer cell detection kit for detecting a cancer cell in a body fluid by bringing a human or animal body fluid into contact with a substance having an affinity for cancer cells, wherein the substance having an affinity for the cancer cell is a saccharide. A cancer cell detection kit, which is at least one selected from the group consisting of a polypeptide, an oligopeptide, a peptide, a glycoprotein, a glycopeptide, and a vitamin acid. 3. The cancer cell detection kit according to claim 2, wherein the saccharide contains a phosphorylated saccharide. 4. The kit for detecting a cancer cell according to claim 3, wherein the saccharide contains mannose-6-phosphate. 5. The method according to claim 4, wherein the saccharide is a mannose pentamer terminated with mannose-6-phosphate.
4. The cancer cell detection kit according to item 1. 6. The kit according to claim 2, wherein the polypeptide is an antibody or a part thereof. 7. The kit for detecting a cancer cell according to claim 2, wherein the vitamin acid is retinoic acid. 8. The cancer cell detection kit according to claim 2, wherein the carrier for immobilizing a substance having an affinity for cancer cells is at least one selected from beads, plates and tubes. 9. The method according to claim 9, wherein the body fluid is blood, plasma, serum, lymph,
The cancer cell detection kit according to claim 2, wherein the kit is at least one selected from urine, ascites, pleural effusion, saliva, and bone marrow fluid. 10. The substance according to claim 1, wherein the substance used for detection is at least one selected from a polypeptide, an oligopeptide, a peptide, a saccharide, a vitamin acid, avidin, and biotin. Cancer cell detection kit.