JP3359391B2 - Patch for tumor test and test method using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a patch for testing and a testing method using the same. More specifically, the present invention relates to a test patch that can be used for testing a tumor and a test method using the same.

[0002]

2. Description of the Related Art Conventionally, various methods have been used for diagnosing a disease. For example, in a blood biochemical test, blood is invasively collected and analyzed by analyzing blood components, various diagnostic markers, cells, and the like. In a pathological test, a biological tissue test such as cancer tissue is invasively performed. Inspections are performed by examining the presence or absence of abnormalities at the cell level, and analyzing urine, stool, saliva, etc. collected by urine collection, stool collection, saliva storage, and the like. In these methods, doctors, nurses, and laboratory technologists collect blood, etc. with a syringe, cut out biological tissue with a sharp scalpel, and collect urine, etc. with the cooperation of patients in special containers. That is the current situation.

[0003] Incidentally, tumors, particularly malignant melanomas, have a very poor prognosis and are extremely high in invasive destructive ability and metastatic ability. Once metastasized or recurred, the treatment is extremely difficult. Is waiting. This definitive diagnosis of malignant melanoma is performed by examining the presence or absence of a tumor marker such as melanoma cells in a surgically excised tissue. As the inspection method, a stamp fluorescence method using a stamp / smear created from a lesion cleavage plane immediately after an operation and an immunostaining method using a lesion tissue are used. Various melanoma marker antibodies (anti-melanoma antibodies) are used for tissue staining.

[0004] However, such surgical resection of the tissue is undesirable because it not only causes pain and fear to the patient, but also increases the risk of tumor metastasis. In addition, since a sample must be collected by a skilled doctor at a specific place, it is not suitable for a screening method in a mass examination or the like. Although methods for measuring dopa such as 5-S-cysteinyldopa and tumor markers in serum and urine have been studied, any of these methods can be used to progress the disease at the stage when the symptoms are worsened. Although it serves as an index of the disease state such as the degree and presence or absence of metastasis,
It is not suitable as an early diagnosis method. Under such circumstances, the development of a method that can safely and early diagnose a tumor without much of the high skill and experience required for conventional tumor examinations is awaited. .

On the other hand, conventionally, as a patch, a medical patch containing a drug such as a salicylic acid-based anti-inflammatory analgesic or a drug for treating angina such as nitroglycerin, a patch test for an irritation test and an allergic drug Patches for detecting allergens for identifying sensitizing substances such as dermatitis are known. JP-A-63-106558 discloses that after a stratum corneum is adhered to an adhesive substance, the adhesive substance is adhered to a transparent plate coated with an adhesive, and then immersed in an organic solvent to dissolve and remove the adhesive substance. Thus, a method for preparing a stratum corneum specimen comprising fixing the stratum corneum to the transparent plate is disclosed. Further, Japanese Patent Application Laid-Open No.
No. 3358 discloses a method for examining the state of the stratum corneum using the above stratum corneum specimen, and the examination carried out there is mainly an examination for measuring the physical properties of the stratum corneum. Further, Japanese Patent Application Laid-Open No. Hei 4-121664 by the same applicant discloses that the stratum corneum is peeled off with an adhesive film, transferred, then washed with an organic solvent to extract lipids contained in the stratum corneum, and the lipids are analyzed. A method for doing so is disclosed.

However, Japanese Patent Application Laid-Open No. 63-1065 discloses
In the method according to Nos. 58 and 63-113358, it is necessary to dissolve the adhesive layer with an organic solvent or the like in order to fix the exfoliated keratinocytes. In addition, those extracted by organic solvents such as lipids in the stratum corneum will not be present in the stratum corneum specimen, and the stratum corneum should be examined in the same state as it was on the skin Is impossible.
Further, according to the method disclosed in JP-A-4-121664, it is impossible to analyze a substance not extracted by an organic solvent. An example of diagnosing a disease by measuring tissue, cells or other substances peeled and fixed with an adhesive tape or the like or substances secreted from tissues or cells absorbed and fixed to a carrier, for example, by an immunological method or the like. Did not exist until now.

[0007]

An object of the present invention is to eliminate the above-mentioned disadvantages of the prior art, that is, it does not require surgical resection of tissue or the like in the early stage of tumors, especially malignant melanoma, and Provided is a method for collecting a living tissue, a cell, etc. without causing pain, fear, discomfort, etc. to a patient and promptly diagnosing a tumor, and a patch required for carrying out the method. It is in.

[0008]

Means for Solving the Problems In order to achieve the above object, the present inventors have conducted intensive studies on a method using a patch for diagnosing a tumor. As a result, the support layer, the adhesive,
A living tissue, a cell, or the living tissue or the cell, provided on the support layer, including an imparting agent and a softening agent;
An adhesive layer for peeling and fixing a substance secreted from the
Exfoliated and fixed biological tissue , cells or cells
Serial living tissue or with the test patch, characterized in that it comprises a carrier for adsorbing and fixing the quality ones secreted from the cells, tumor-associated antigens peeled fixed or adsorbed fixed to the patch, EIA method using melanoma antibody for tumor marker or tumor-related substance , using labeled particles
The present inventors have found that the disadvantages of the prior art can be eliminated by performing the measurement using an immunoassay or a fluorescence or luminescence immunoassay , and completed the present invention.

That is, the present invention relates to a support layer, an adhesive,
A living tissue, a cell, or the living tissue or the cell provided on the support layer containing a tackifier and a softener ;
An adhesive layer for peeling and fixing a substance secreted from the
The living tissue peeled and fixed by the adhesive layer,
Secreted from the cell or the living tissue or the cell
And a carrier for adsorbing and fixing the substance to be tested. The present invention further peeling fixed or adsorbed fixed biological tissue by a tumor test patch of the cell or the living tissue or
The substance secreted from the cells , using a melanoma antibody
EIA method, immunoassay using labeled particles or fluorescence
Or a method for examining a tumor characterized by measurement by a luminescence immunoassay .

The support layer used in the patch for inspection of the present invention is not particularly limited, but may be woven or non-woven fabric such as paper, cloth, cotton cloth, vinyl chloride, polyethylene, polyolefin, polyester, polyurethane, polyvinyl alcohol, chloride. A plastic film such as vinylidene polyamide and ethylene-vinyl acetate copolymer or a metal foil such as aluminum is preferably used. The thickness of the support layer is not particularly limited, but is preferably about 1 μm to 5000 μm, particularly preferably about 10 μm to 600 μm.

The main components of the pressure-sensitive adhesive constituting the pressure-sensitive adhesive layer of the patch of the present invention include, for example, natural rubber or a derivative thereof, SBR-based rubber, NBR-based rubber, butyl rubber, polyisobutylene, polyalkyl acrylate A mixture of one or more of elastic materials such as, but not limited to, polyalkyl vinyl ether, silicone rubber, SIS rubber, and synthetic isoprene can be used. Also, tackifiers are used as components of the adhesive.
However , as the tackifier, for example, rosin, hydrogenated rosin and esters thereof, polyterpene resin, petroleum resin, oil-soluble phenol resin and the like are preferably used. Further, as a component of the pressure-sensitive adhesive, a softening agent such as polybutene, liquid polyisobutylene, liquid paraffin and animal and vegetable oils is used , and a filler such as zinc oxide and crystalline calcium sulfate and an antioxidant can also be used. . The adhesive strength of the adhesive of the patch of the present invention can be changed by changing the mixing ratio of the above-mentioned components such as the elastic body and the tackifier.

The pressure-sensitive adhesive used in the patch of the present invention can be applied on the support layer to form a pressure-sensitive adhesive layer. In this case, the coating method may be any known method. Although the thickness of the adhesive layer is not particularly limited, it is about 0.1 μm to 5000 μm, particularly about 10 μm to 400 μm.
It is preferably μm. It is desirable that the adhesive layer is stable and does not dissolve in organic solvents such as water, alcohol and formaldehyde.

[0013] Further, it is used in the patch of the present invention.
That carriers are, for example, is dispersed in the adhesive layer can be used, it is preferable to be the ones quality secreted from the release fixed tissue or cells by the adhesive layer is capable of adsorbing stationary. The amount of these carriers dispersed in the adhesive layer is 10% or less, particularly 3%, based on the total volume of the adhesive layer.
It is preferably about 5%. Responsible body that is used in the adhesive patch of the present invention, like the adhesive layer, water, an alcohol,
Desirably, it is stable and does not dissolve in an organic solvent such as formaldehyde. The carrier is not limited to these, but, for example, cellulose, cellulose derivatives such as nitrocellulose, paper, cloth, glass fiber, porous gel, porous fiber matrix, starch-based substance, ceramic, polyethylene frit and the like are preferably used. You. Further, the carrier can be present in the pressure-sensitive adhesive of the present invention as a film-like form.

[0014] One or more of the support, the adhesive layer and the carrier, particularly all of them, are transparent or translucent to facilitate observation of fixed biological tissues, cells and the like. Is preferred. The form of the patch for inspection of the present invention may be in any form, for example, a rectangle, a circle, an ellipse, a square, a heart, a jaw, and the like, but is not limited thereto. The size and the color are not particularly limited as long as they have practicality.

Using a plurality of the patch for tumor examination of the present invention, living tissues, cells, etc. can be peeled off several times to collect living tissues, cells, etc. in multiple layers. In this case, it is convenient to print, engrave, or color the number or symbol on the surface of each patch so that the number of peelings can be identified, which is convenient. In addition, a plurality of patches of the present invention can be laminated and used. In that case, it is also possible to provide an appropriate separating sheet between the patches and the patch, and after one peeling is completed, remove the separating sheet and repeat the operation of peeling with the next patch. .
Further, one or a plurality of the patch for tumor test of the present invention can be used by being adhered and fixed to a polyhedron having a size that can be grasped by hand. Also in this case, in order to identify the number of times of peeling, a number, a symbol, or the like can be provided on the patch surface by printing, engraving, coloring, or the like.

Next, an inspection method according to the present invention will be described. The test patch of the present invention, peeling fixed or adsorbed fixed the living tissue, other cells, that exists on the skin surface
Object substance is tumor-associated antigens, tumor markers or a tumor-related substances, EIA method These were used melanoma antibody,
Immunoassay using labeled particles or fluorescence or luminescence
Diagnosis of tumors is possible by measuring by the epidemiological assay . Examples of tumors that can be detected by the patch and the test method of the present invention include skin cancer, oral cancer, and rectal cancer. Examples of skin cancer include malignant melanoma, basal cell carcinoma, squamous cell carcinoma, and the like, and examples of oral cancer include tongue cancer, buccal mucosa cancer, and laryngeal cancer.

[0017] The tumor-associated antigens, tumor markers <br/> or tumor-related substances, melanoma cells, melanoma marker (NKI / C3), melanoma marker (PAL
/ M ₁ ), melanoma markers (S-100α and β), carcinoembryonic antigen (CEA), neuroblastoma (CE7),
Neuroblastoma (AD2), marigulin, α-fetal protein (A
FP), pepsinogen, basic fetal protein (BFP),
Pancreatic cancer fetal antigen (POA), fetal prealbumin (E
PA), carbohydrate antigen (CA19-9), pancreatic cancer-related antigen (CA50), cancer antigen (CSLEX-1), pancreatic cancer-related antigen (serial SSEA-1), pancreatic cancer-related antigen (Du-p)
an-2), pancreatic antigen (NCC-ST-439), carbohydrate antigen (serial Tn), pancreatic antigen (CA72-4), cancer antigen (KM-01), pancreatic cancer-related antigen (Span-1),
Carbohydrate antigen (CA125), cancer antigen (CA15-
3), squamous cell carcinoma (SCC), seminoprotein (γ-S
n), prostate specific antigen (PA), ferritin, tissue polypeptide antigen (TPA), immune acidic protein (IAP), immunosuppressive acidic protein, prostate acidic protein (PAP), neuron-specific enolase (NSE), enzymes, amino acids (Particularly Ala, Glu), mucus containing mucopolysaccharide, dopa, dopamine, hormones and the like.

For the detection of a melanoma marker, various melanoma antibodies can be used in consideration of specificity and sensitivity. Any melanoma antibody may be used as long as it has high malignant melanoma specificity. The test patch of the present invention, in which tissues, cells, and the like are peeled and fixed by adsorption or fixed by adhering to a diseased part, is obtained by the EIA method using the antibody, the immunological method using various labeled particles (latex, colloidal gold, etc.). By using the measurement method and the fluorescence or luminescence immunoassay, the presence or absence of a tumor marker in a tissue or a cell can be detected in the form of cell staining. Also, 5-
In the case of S-cysteinyl dopa, after the test patch of the present invention is treated with formaldehyde gas, it can be detected by the presence or absence of fluorescence using a fluorescence microscope. In that case, the determination can be made qualitatively based on the color intensity and the number of stained cells. In addition, any known detection method other than the above method can be used to measure tissues, cells, and the like, which have been fixed by peeling or adsorption by the patch of the present invention.

[0019]

The present invention will be described in more detail with reference to the following examples. Example 1 A vinyl chloride film having a width of 21 cm × a length of 29.7 cm and a thickness of 100 μm was used as a support layer, on which a synthetic styrene isoprene copolymer rubber, rosin and liquid paraffin were added in 100 parts:
A pressure-sensitive adhesive obtained by blending in a conventional manner at a ratio of 170: 50 was applied to a thickness of about 90 μm to form a pressure-sensitive adhesive layer, thereby preparing a patch for inspection of the present invention.

Example 2 Nitrocellulose was blended in the same pressure-sensitive adhesive as in Example 1 at a rate of 5% based on the total volume thereof, and was spread on the same support layer as in Example 1 by about 90 μm. It was applied in a thickness to prepare a patch for inspection of the present invention.

Example 3 The adhesive force was changed by changing the thickness of the adhesive layer and the ratio of the synthetic styrene isoprene copolymer rubber (A), rosin (B) and liquid paraffin (C) as shown in Table 1 below. Except for the change, the test patch of the present invention was prepared in the same manner as in Example 1.

[0022]

[Table 1] Thickness of adhesive layer ratio-------------------------------------------------------- −−−−−−−−−−−−−−−−−−−−−−−−−− Comparative control 90 100 170 50 1 Patch 1 100 100 200 50 4.3 Patch 2 150 100 200 50 5.5 Patch Agent 3 200 100 200 50 6.1 ----------------------------------------------------- Comparative control (salicylic acid-based as active ingredient) Relative ratio of the adhesive force of each patch when 400 g / 18 mm (180 degree peel test) is defined as 1.

Example 4 Using the patch for examination prepared in Example 3 (each having a rectangular shape of 1.5 × 2.5 cm), the sole of a healthy person (3 persons) and the inner part of the forearm (4 persons) And the stratum corneum was peeled off. This operation was repeated three times using each of the three patches. The weight of the stratum corneum peeled off from each patch was measured. The results are shown in Table 2 below.

[0024]

[Table 2] Stratum corneum exfoliation weight (mg / 1.5 x 2.5 cm)---------------------------------------------------------------------------------------- −−−−−−−−−−−−−−−−−−−−−−−−− 1st 2nd 3rd 1st 2nd 3rd −−−−−−−−−−−−−−−−− −−−−−−−−−−−−−−−−−−−−−− Comparative control 160 (N = 1) 170 (N = 1) 180 (N = 1) 80 (N = 1) 80 (N = 1) 0 (N = 1) Patch 1 610 ± 30 440 ± 38 290 ± 15 228 ± 15 208 ± 14 185 ± 21 Patch 2 663 ± 113 453 ± 32 347 ± 43 240 ± 30 155 ± 25 198 ± 16 Patch 3 530 ± 174 360 ± 105 273 ± 46 218 ± 17 190 ± 19 185 ± 7 --- --- --- --- --- --- --- --- --- --- --- --- --- −−−−−−−

As is evident from the results shown in Table 2, even when the adhesive strength increased after Patch 1, the difference in the weight of the exfoliated stratum corneum was slight. In consideration of the pain of the skin at the time of peeling, it is considered that the adhesive force of the patch 1 is sufficient.

Example 5 The test patch 1 prepared in Example 3 was applied to the palms and forearms of five healthy 24- to 39-year-old adult men.
The stratum corneum tissue was exfoliated. This operation was performed five times for each person using the five patches 1 described above, and five layers of tissues were collected. In addition, the shape of the adhesive layer for peeling is 1.5 × in diameter.
It was an ellipse of 3.0 cm. The presence of melanoma cells and melanoma markers in the detached samples was examined by peroxidase-labeled immunostaining. They also asked about pain during peeling. Table 3 shows the results.

[0027]

[Table 3] Existence of cells exfoliated in healthy people and pain at the time of exfoliation Stainability of cells Subject --------- Fifth layer exfoliation Initial 1st layer 2nd layer 3rd layer 4th layer 5th layer Pain ------------------------------------------- AO------Slight YS-----Slight HO- − − − − Slight KY − − − − − Slight SO − − − − − − Slight −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− −

As is evident from the results shown in Table 3, no melanoma cells or melanoma markers were detected from the exfoliated tissues of five subjects. In addition, the pain at the time of the fifth peeling by the patch was weak.
In addition, when the same peeling test was performed by changing the adhesive force of the patch in various ways, it was possible to reduce pain.

Example 6 The adhesive of patch 1 described in Example 3 was applied to a transparent 100 μm
It was applied on a m-thick polypropylene film to prepare a substantially transparent patch for inspection.

Example 7 Human normal keratinocytes and cancerous cells HMV-1 were fixed to the patch for inspection prepared in Example 6. After blocking the patch with BSA, anti-human melanoma marker antibodies (PAL-M1 and S-100α ·
β, mouse IgG1, 1 μg / ml) for 1 hour. Thereafter, a peroxidase-labeled anti-mouse IgG antibody (rabbit, 1000-fold) was reacted for 1 hour. The coloring is
The reaction was carried out by adding a 1.25 mg / ml solution of aminoethylcarbazole in ethanol and a 0.0086% hydrogen peroxide solution in a ratio of 1: 9. After 60 minutes, a 1% sulfuric acid solution was added to stop the reaction, and the staining at the cell level was observed with a microscope. As a result, the human melanoma-derived cell line (MHV-1) stained dark reddish brown, but did not stain normal human keratinocytes (NHEK).

Example 8 The test patch prepared in Example 6 was applied to the affected skin of a malignant melanoma patient and a benign tumor patient, respectively.
A lesion tissue sample was collected. As a comparative control, a patch was applied to the soles and hands of healthy persons, and a stratum corneum sample was collected. These samples were reacted with saturated formaldehyde gas in a vessel with a relative humidity of 60%. The reaction is
The test was carried out by allowing the container containing the sample to stand at 80 ° C. for 10 minutes. After the reaction, the fluorescence of the tissue fixed to the patch for examination was observed with a fluorescence microscope. As a result, in the affected tissue of melanoma patients, compared to benign tumors and healthy people, strong fluorescence was observed, the patch for testing of the present invention,
It was confirmed to be useful for diagnosis of malignant melanoma.

[0032]

The patch for testing provided by the present invention is useful for diagnosing tumors. When the patch for inspection of the present invention is used, a sample can be collected extremely easily without requiring surgical resection or puncture suction of the skin surface and without causing pain to the patient. Therefore, mass examination of tumors such as skin cancer, oral cancer, and rectal cancer is also possible. Further, it is possible to reduce recurrence cases which are considered to be caused by a surgical procedure or the like. In addition, the use of the test patch of the present invention makes it possible to confirm the presence or absence of a tumor-related antigen, a tumor-related marker or other tumor-related substance even in a very early stage tumor, which is difficult to determine, thereby enabling early diagnosis of the tumor. Becomes possible. Furthermore, according to the patch for examination of the present invention, the tissue or the like on the skin surface is directly peeled and fixed to the patch, or a substance or other substance secreted from such a tissue or the like is adsorbed and fixed to a carrier, and Can be used directly as a sample, so that a tumor can be diagnosed quickly and easily as compared with the prior art, and is economically useful.

──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Nobuchira Hirashima 408-Tashiro Daikancho, Tosu City, Saga Prefecture Hisamitsu Pharmaceutical Co., Ltd. (72) Inventor Shuhei Imayama 5-7-15-1 Kodo, Nishi-ku, Fukuoka City, Fukuoka Prefecture (56) reference Patent flat 4-121664 (JP, a) JP Akira 63-113358 (JP, a) JP Akira 64-53165 (JP, a) (58 ) investigated the field (Int.Cl. ⁷ A61B 10/00 G01N 33/50

Claims (6)

(57) [Claims] 1. A support layer comprising an adhesive, a tackifier and a softener.
A living tissue or cell provided on the support layer containing an agent, or a substance secreted from the living tissue or the cell;
An adhesive layer for peeling the quality and fixed to the adhesive layer
The living tissue, the cells or
Absorbs substances secreted from the living tissue or cells.
A patch for tumor examination , comprising a carrier for fixation and fixation . 2. The adhesive is a synthetic styrene isoprene copolymer.
Rubber, tackifier is rosin, softener is liquid paraffin
The patch for tumor examination according to claim 1. 3. The patch according to claim 1, wherein at least one of the support layer, the adhesive layer, and the carrier is transparent or translucent. 4. The living tissue, the cell or the living body
Substances secreted from tissues or said cells, tumor-associated antigen, patch for tumor testing according to claim 1, wherein the or tumor marker is a tumor-related substances. Wherein said tumor-associated antigen, wherein the tumor marker or a pre Symbol tumor-related substances, melanoma cells, melanoma marker (NKI / C3), melanoma marker (PA
L / M ₁ ), melanoma markers (S-100α and β), carcinoembryonic antigen (CEA), neuroblastoma (CE7),
Neuroblastoma (AD2), marigulin, α-fetal protein (A
FP), pepsinogen, basic fetal protein (BFP),
Pancreatic cancer fetal antigen (POA), fetal prealbumin (E
PA), carbohydrate antigen (CA19-9), pancreatic cancer-related antigen (CA50), cancer antigen (CSLEX-1), pancreatic cancer-related antigen (serial SSEA-1), pancreatic cancer-related antigen (Du-p)
an-2), cancer antigen (NCC-ST-439), carbohydrate antigen (serial Tn), cancer antigen (CA72-4), cancer antigen (KM-01), pancreatic cancer-related antigen (Span-1),
Carbohydrate antigen (CA125), cancer antigen (CA15-
3), squamous cell carcinoma (SCC), seminoprotein (γ-S
n), prostate specific antigen (PA), ferritin, tissue polypeptide antigen (TPA), immune acidic protein (IAP), immunosuppressive acidic protein, prostate acidic protein (PAP), neuron-specific enolase (NSE), enzymes, amino acids The patch according to claim 4 , wherein the patch is selected from the group consisting of mucus containing mucopolysaccharide, dopa, dopamine and hormones. 6. A biological tissue, cell or cell that has been peeled off and fixed by the patch for tumor examination according to any one of claims 1 to 5.
The biological tissue or substances secreted from the cells or the body tissue which is sucked and fixed by the support, said cell
Or a substance secreted from the living tissue or the cell
Use the quality, EIA method using a melanoma antibody, labeled particles
A method for examining a tumor, which is measured by a conventional immunoassay or a fluorescence or luminescence immunoassay .