JP6637290B2 - Monoclonal antibody specific to CK19, hybridoma producing the same, cancer detection kit, method for detecting cancer and method for determining metastasis of cancer - Google Patents

Description

  The present invention relates to a monoclonal antibody specific to cytokeratin 19 (CK19) and a hybridoma producing the same. Furthermore, the present invention relates to a kit for detecting cancer using the monoclonal antibody, a method for detecting cancer, and a method for determining metastasis of cancer.

  Currently, sentinel lymph node diagnosis is performed as a method for diagnosing metastasis of breast cancer. Sentinel lymph nodes are the lymph nodes that are considered the closest to the cancer and are the first lymph nodes to receive lymphatic flow from the cancer. It is believed that the first metastasis to the sentinel lymph nodes occurs when cancer spreads. The sentinel lymph node measures the flow of lymph from the lump site by radioactive material, staining, or the like, and is determined to be the earliest lymph node. By diagnosing metastasis of the cancer in the sentinel lymph node, it can be determined whether or not to perform axillary lymph node dissection.

  As a sentinel lymph node diagnosis, a method is known in which after local anesthesia or general anesthesia is performed, a tissue is collected, and a thin section is observed with a microscope to find epithelial cells likely to be derived from breast cancer. In this method, a specimen is transported from the operating room to the pathology room, the lymph nodes are split, and the cut surface is stamped for cytology, the lymph nodes are rapidly frozen, and the cut surface is sectioned into hematoxylin and eosin. Is performed and a pathologist reviews the presence or absence of metastasis. This method includes (A) transporting a specimen to a pathological room, (B) preparing a thin section and staining with hematoxylin and eosin, (C) performing cytodiagnosis as necessary, (D) performing histological diagnosis, And many other processes are required. For this reason, there is a problem that it takes about 20 to 40 minutes even in a facility where a pathologist is full-time to complete the examination, and several hours in a facility where the pathologist is not full-time. In addition, the steps (A) to (D) require specialized techniques or expertise concerning cytodiagnosis and pathological diagnosis. In addition, although there is a difference between facilities, if only the section in which the lymph node is cut is sliced, there is a possibility that a false negative is obtained for a minute metastasis or the like. In fact, the diagnosis was negative during the operation, but the positive diagnosis was sometimes made positive after the operation.

  As another method, an OSNA method (One-Step Nucleic acid Amplification assay) in which lymph node solubilization and gene amplification by the RT-LAMP method are performed in one step is known (for example, Non-Patent Document 1).

  However, even with the OSNA method, it takes about 30 to 50 minutes for the test result to become clear, so that a further reduction in the time required for performing the procedure during surgery is desired. In addition, small hospitals require expensive equipment (such as a gene amplifying machine) and staff to operate the equipment. Furthermore, in the case of the OSNA method, it is necessary to dissolve a sample, so that there is a problem that a pathological test cannot be performed in parallel on the same sample.

  As described above, there has been a demand for the development of a simple cancer detection method that does not require expensive equipment and that can rapidly detect cancer metastasis without using specialized staff such as a pathologist.

  As a method for solving such a problem, there has been proposed a method of detecting lymph node metastasis of cancer by using a dot blot method using an antibody specific to a cancer cell (Patent Document 1).

  In the method of Patent Document 1, dot blot analysis using an antibody specific to cytokeratin is performed to determine whether or not cytokeratin is contained in a lymph node sample separated from a patient with epithelial cell cancer outside the living body. It is evaluated by. If the amount of cytokeratin detected in the lymph node sample exceeds the amount of cytokeratin contained in the normal lymph node sample, metastasis of epithelial cells to the lymph node may be present. We evaluate that there is.

Patent No. 5610125

Intra-operative rapid diagnostic method based on CK19 mRNA expression for the detection of lymph node metastases in breast cancer.Visser M, Jiwa M, Horstman A, Brink AA, Pol RP, van Diest P, Snijders PJ, Meijer CJ. Int J Cancer . 2008 Jun 1; 122 (11): 2562-7.

  However, also in the method of Patent Document 1, it takes 40 minutes or more to determine the presence or absence of cancer and metastasis. Further, in the case of the method of Patent Document 1, there is a problem that it is not always easy to ensure reproducibility.

  The present invention has been made in view of the above circumstances, is a low-cost and short-time, with high accuracy, a cancer detection kit capable of detecting cancer, a method for detecting cancer and a method for determining metastasis of cancer The challenge is to provide Another object of the present invention is to provide a monoclonal antibody used in such a cancer detection kit and cancer detection method, and a hybridoma that produces the monoclonal antibody.

The present invention provides the following monoclonal antibodies, hybridomas, cancer detection kits, cancer detection methods, and cancer metastasis determination methods.
<1> site a monoclonal antibody specific for keratin 19, accession number NITE B P-02135 or accession number NITE B P-02136 monoclonal antibody hybridoma produced by,.
<2> accession number NITE B P-02135, or accession number NITE B P-02136 hybridomas.
<3> a chromatographic medium including a labeled antibody in which a first antibody that reacts with cytokeratin 19 with an antigen-antibody and a labeling substance is bound, and an antibody capture unit on which a second antibody that reacts with cytokeratin 19 with an antigen-antibody is immobilized A kit for detecting cancer by immunochromatography, comprising:
A monoclonal antibody that first antibody hybridoma accession number NITE B P-02136 is produced, and a detection kit for cancer second antibody hybridoma accession number NITE B P-02135 is a monoclonal antibody produced.
<4> A method for detecting cancer by immunochromatography using the cancer detection kit, comprising the following steps:
(1) a step of developing a mixture containing the sample derived from the lymph node of the subject and the labeled antibody in the antibody capturing unit; and (2) the complex of cytokeratin 19 and the labeled antibody in the sample is A method for detecting cancer, comprising a step of determining that a cancer cell is contained in the sample when detected by the antibody capturing unit.
<5> A method for determining cancer metastasis by immunochromatography using two types of a low-sensitivity detection kit and a high-sensitivity detection kit having different detection sensitivities as the cancer detection kit of the above <3>, comprising: Process:
(1) a step of developing a mixed solution containing the sample derived from the lymph node of the subject and the labeled antibody in the antibody capturing unit; and (2) using a low-sensitivity detection kit and a high-sensitivity detection kit to detect cytokeratin 19 in the sample. When a complex of the and the labeled antibody is detected in the antibody capturing unit, it is determined that there is a macrometastasis exceeding a size that is a reference for performing lymph node dissection,
When the complex is not detected in the antibody capturing portion in the low-sensitivity detection kit, and the complex is detected in the antibody capturing portion in the high-sensitivity detection kit, a size serving as a reference for performing lymph node dissection. Micrometastasis less than, 0.2mm or less Isolated Tumor Cells (ITCs), it is determined that at least one of the contamination of epithelial components is present,
A method for determining cancer metastasis, comprising a step of determining that cancer metastasis does not exist when the complex is not detected by the antibody capturing unit in the low-sensitivity detection kit and the high-sensitivity detection kit.
<6> The method for determining metastasis of cancer according to <5>, wherein the size of the metastasis serving as a reference for performing lymph node dissection is 2 mm.
<7> The labeled antibody of the low sensitivity detection kit is labeled with a metal colloid having a particle size of 20 to 60 nm, and the labeled antibody of the high sensitivity detection kit is a metal colloid particle of the labeled antibody of the low sensitivity detection kit. A method for determining metastasis of cancer, characterized by being labeled with a metal colloid having a diameter larger than a diameter and a particle diameter of 40 to 80 nm.

  According to the cancer detection kit and detection method of the present invention, cancer such as breast cancer can be detected accurately at low cost and in a short time. Therefore, for example, during surgery, assess the presence or absence of cancer metastasis, and based on the results, quickly determine the surgical strategy, such as whether to remove the lymph nodes or how much to dissect. Can be. Further, according to the cancer detection kit and the detection method of the present invention, the presence or absence of cancer metastasis can be evaluated without consuming a specimen (tissue piece), and other tests (cytology, pathological examination) can be performed on the same specimen. Can be done in parallel.

FIG. 1 is a schematic diagram illustrating an embodiment of the cancer detection kit of the present invention.

  The present inventors focused on cytokeratin 19 (hereinafter sometimes referred to as “CK19”) which is abundant in breast cancer, and detected cancer using a novel monoclonal antibody that specifically binds to CK19. I created a kit.

  Hereinafter, the cancer detection kit of the present invention will be described.

  The cancer detection kit of the present invention utilizes immunochromatography, and comprises a labeled antibody in which a first antibody that reacts with CK19 as an antigen-antibody and a labeling substance are bound, and a second antibody that reacts with CK19 as an antigen-antibody. And a chromatographic medium provided with the phased antibody capture unit.

  The types of cancer detectable by the cancer detection kit of the present invention are not particularly limited, and include, for example, breast cancer, lung cancer, stomach cancer, colon cancer, esophagus cancer, thyroid cancer, gynecological cancer, skin cancer, head and neck cancer, and the like. Can be exemplified. With the use of the cancer detection kit of the present invention, it is possible to determine the presence or absence of metastasis to cancer tissue with a small amount of tissue biopsy sample, thereby minimizing the magnitude of invasion when collecting the tissue. Can be suppressed. Therefore, the cancer detection kit and cancer detection method of the present invention are particularly advantageous for examining the presence or absence of metastasis of breast cancer to axillary lymph nodes.

  FIG. 1 is a schematic view illustrating an embodiment of the cancer detection kit of the present invention.

  As shown in FIG. 1, the cancer detection kit 1 of this embodiment includes a sample addition pad 2, a labeled antibody holding pad 3, and a chromatograph along a sample developing direction (the direction of arrow Y in FIG. 1). The medium 4 and the absorption pad 5 are arranged in this order.

(Sample addition pad)
The material of the sample addition pad 2 is not particularly limited, and examples thereof include materials having uniform properties such as cellulose filter paper, glass fiber filter paper, polyurethane, polyacetate, cellulose acetate, nylon, polyester nonwoven fabric, and cotton cloth. The sample addition pad 2 not only receives the added sample, but also has a function of filtering insoluble matter particles and the like in the sample. In addition, in order to prevent CK19 in the sample from non-specifically adsorbing to the sample addition pad 2 at the time of analysis and to prevent the accuracy of the analysis from decreasing, the sample addition pad 2 prevents non-specific adsorption in advance. Processing can also be performed.

(Labeled antibody holding pad, labeled antibody)
Examples of the material for the labeled antibody holding pad 3 include cellulose filter paper, glass fiber filter paper, and nonwoven fabric. The labeled antibody holding pad 3 can be formed by directly applying and drying a suspension containing the labeled antibody 6 on the chromatographic medium 4 on which the antibody capturing section 7 is formed. Further, the suspension containing the labeled antibody 6 is applied and dried on another porous substance, for example, cellulose filter paper, glass fiber filter paper, nylon non-woven fabric, etc., to form the labeled antibody holding pad 3, and the antibody capturing section 7 is formed. It can also be arranged so as to be connected to the chromatographic medium 4 that has been made by a capillary.

  Note that the labeled antibody holding pad 3 is not limited to this embodiment, and the labeled antibody 6 can be dispersed in a developing solution constituting a mobile phase.

  In the labeled antibody 6, the first antibody A1 and the labeling substance L are bound.

Here, the first antibody A1 is a hybridoma deposited at the National Institute of Technology and Evaluation (NITE) Patent Microorganisms Depositary [Accession number: NITE BP-02136 (hybridoma deposited in Japan ( Accession number: NITE P -02136, entrusted Date: October 5, 2015), on October 21, 2016, was transferred according to an international deposit under the Budapest Treaty)] is a monoclonal antibody produced. As described later, this monoclonal antibody is a novel antibody discovered by the present inventors as the most suitable labeled antibody 6 (first antibody A1) for a kit for detecting cancer using immunochromatography. Hybridoma (accession number: NITE B P-02136) which produces the monoclonal antibody culture of, in vitro or mouse, can preferably be carried out in vivo, such as a mouse ascites. In this case, the monoclonal antibodies can be obtained from the culture supernatant of the hybridoma and the ascites of the mouse, respectively.

  In general, an enzyme or the like can be used as the labeling substance L, but it is preferable to use an insoluble carrier because it is suitable for visually determining the detection result.

  As the insoluble carrier as the labeling substance L, noble metals of gold, silver, and platinum, and iron, lead, copper, cadmium, bismuth, antimony, tin, or mercury can be used. More preferably, gold, silver, and platinum are used. Noble metals can be used. Further, as the labeling substance L, a metal colloid label such as a platinum colloid, a gold colloid, a silver colloid, an iron colloid, or an aluminum hydroxide colloid can be preferably exemplified. Further, as the labeling substance L, a metal sulfide label such as each sulfide of iron, silver, lead, copper, cadmium, bismuth, antimony, tin, or mercury can be preferably exemplified. As the labeling substance L, for example, one or more of these metal colloid labels and / or metal sulfide labels can be used. Among them, platinum colloid, gold colloid, and silver colloid are particularly preferable. preferable.

  In addition, in order to obtain a strong color tone by detection, in the case of metal colloids such as platinum colloid, gold colloid, and silver colloid, the average particle size is preferably 1 to 500 nm, more preferably 10 to 150 nm, More preferably, it is 20 to 100 nm. The detection sensitivity can be further improved by designing the average particle diameter of the metal colloid in the range described later.

  Note that the binding between the first antibody A1 and the metal colloid can be performed by a conventionally known method such as physical adsorption or chemical bonding. For example, when the first antibody A1 is sensitized to gold colloid, the first antibody A1 is added to a solution in which gold particles are dispersed in a colloidal form, and the solution is physically adsorbed. It can be prepared by blocking the particle surface which is a bond.

  Further, as the labeling substance L, latex particles made of a synthetic polymer can be used.

  Latex particles can be obtained by polymerizing or copolymerizing various monomers. Examples of the monomer include polymerizable unsaturated aromatics such as styrene, chlorostyrene, α-methylstyrene, divinylbenzene, and vinyltoluene, for example, polymerization of (meth) acrylic acid, itaconic acid, maleic acid, fumaric acid, and the like. Unsaturated carboxylic acids, for example, methyl (meth) acrylate, ethyl (meth) acrylate, n-butyl (meth) acrylate, 2-hydroxyethyl (meth) acrylate, glycidyl (meth) acrylate, ethylene Polymerizable unsaturated carboxylic esters such as glycol-di- (meth) acrylate, tribromophenyl (meth) acrylate, (meth) acrylonitrile, (meth) acrolein, (meth) acrylamide, N-methylol- ( (Meth) acrylamide, methylenebis (meth) acrylamide, Unsaturated carboxylic acid amides such as tadiene, isoprene, vinyl acetate, vinylpyridine, N-vinylpyrrolidone, vinyl chloride, vinylidene chloride, and vinyl bromide, polymerizable unsaturated nitriles, vinyl halides, conjugated dienes, etc. Can be mentioned. These monomers are appropriately selected depending on the surface properties, specific gravity, and the like required as the labeling substance L, and one kind may be used alone or two or more kinds may be used in combination. Since latex particles are usually white and cannot be used as such as the labeling substance L, for example, they are dyed with oil-soluble dyes, particularly, latex particles in an aqueous medium by emulsion of a solution of oil-soluble dyes with an oily organic solvent. By doing so, it is possible to obtain a desired color at a desired density.

(Chromatographic medium, antibody capture unit)
The chromatographic medium 4 is an inert substance made of a microporous substance exhibiting a capillary phenomenon, does not react with the labeled antibody 6 (the first antibody A1) or the like, and is used in a short time. The material is not particularly limited as long as it has a developing speed capable of obtaining sufficient sensitivity in the judgment.

  Specifically, examples of the chromatographic medium 4 include ceramic fine particles such as silica, titania, zirconia, ceria, and alumina or fine particles of an organic polymer, polyurethane, polyester, polyethylene, polyvinyl chloride, polyvinylidene fluoride, nylon, and nitrocellulose. Alternatively, a fibrous or non-woven fibrous matrix composed of a cellulose derivative such as cellulose acetate, a membrane, a filter paper, a glass fiber filter paper, a cloth, and cotton can be exemplified. Even if the fine particles themselves are not porous, the fine particles can function as the chromatographic medium 4 by forming voids between the fine particles in a filled state. Among these, the chromatographic medium 4 is preferably a nitrocellulose membrane, a cellulose membrane, an acetylcellulose membrane, a polysulfone membrane, a polyethersulfone membrane, a nylon membrane, a glass fiber filter, a nonwoven fabric, or a cloth.

  The chromatographic medium 4 has an antibody capturing section 7 on which a second antibody A2 that reacts with CK19 as an antigen-antibody is immobilized at an arbitrary position.

The second antibody A2 is a hybridoma deposited at the National Institute of Technology and Evaluation (NITE) Patent Microorganisms Depositary Center [Accession number: NITE BP-02135 (hybridoma deposited in Japan ( Accession number: NITE P-02135, entrusted Date: October 5, 2015), on October 21, 2016, it was transferred according to an international deposit under the Budapest Treaty)] is a monoclonal antibody produced. As described later, this monoclonal antibody is a novel antibody discovered by the present inventors as a capture antibody (second antibody A2) optimal for a kit for detecting cancer using immunochromatography. Hybridoma (accession number: NITE B P-02135) which produces the monoclonal antibody culture of, in vitro or mouse, preferably can be performed in vivo, such as a mouse ascites, on the respective monoclonal antibodies of hybridoma culture, It can be obtained from Qing and mouse ascites.

  The method for forming the antibody capturing portion 7 is not particularly limited. For example, the second antibody A2 can be directly immobilized on a part of the chromatographic medium 4 by physical or chemical bonding. As another method, the second antibody A2 is physically or chemically bound to insoluble fine particles such as latex particles, and the insoluble fine particles are trapped in a part of the chromatographic medium 4 to be indirectly immobilized. You can also.

  As a method of direct immobilization, physical adsorption may be used, or covalent bonding may be used. Generally, when the chromatographic medium 4 is a nitrocellulose membrane or a mixed nitrocellulose ester membrane, physical adsorption can be performed. In the covalent bond, cyan bromide, glutaraldehyde, carbodiimide, or the like can be generally used to activate the chromatographic medium 4.

  In the method of indirect immobilization, the particle size of the insoluble fine particles can be selected to be a size that can be captured by the chromatographic medium 4 but cannot move, and preferably the average particle size is about 10 μm or less. Particles. As these particles, various types used for an antigen-antibody reaction are known, and these known particles can be used in the present invention. For example, organic polymers such as organic polymer latex particles obtained by an emulsion polymerization method such as polystyrene, styrene-butadiene copolymer, styrene-methacrylic acid copolymer, polyglycidyl methacrylate, and acrolein-ethylene glycol dimethacrylate copolymer. Fine particles of a substance, fine particles such as gelatin, bentonite, agarose, and cross-linked dextran; inorganic oxides such as silica, silica-alumina, and alumina; and inorganic particles in which a functional group is introduced into an inorganic oxide by a silane coupling treatment or the like. .

  In the cancer detection kit of the present invention, it is preferable that the second antibody A2 is directly immobilized on the chromatographic medium 4 from the viewpoint of ease of sensitivity adjustment and the like. The form of the antibody capturing section 7 is not particularly limited, but may be fixed as a circular spot, a line perpendicular to the developing direction of the chromatographic medium 4, a number, a character, a symbol, or the like.

  Further, after the second antibody A2 is immobilized on the chromatographic medium 4, if necessary, the chromatographic medium 4 may be immobilized on the chromatographic medium 4 by a known method in order to prevent a decrease in the accuracy of analysis due to non-specific adsorption. A blocking process can be performed. Generally, proteins such as bovine serum albumin, skim milk, casein, and gelatin are suitably used for the blocking treatment. After such a blocking treatment, if necessary, one or two or more surfactants such as Tween 20, Triton X-100, and SDS can be washed in combination.

  Further, the form and size of the chromatographic medium 4 are not particularly limited, and can be appropriately designed in consideration of actual operability and ease of observing reaction results. In order to make the operation easier, it is preferable to provide a support made of plastic or the like on the back surface of the chromatographic medium 4 on which the antibody capturing section 7 is formed.

(Absorbing pad)
The absorption pad 5 is a site where the added sample or developing solution is physically absorbed by the chromatographic movement, and also absorbs and removes the unreacted labeling substance L which is not insolubilized in the detection unit of the chromatographic medium 4. Further, the absorption pad 5 has a role of keeping the chromatographic movement of the added sample constant.

  Examples of the material of the absorbent pad 5 include cellulose filter paper, non-woven fabric, cloth, cellulose acetate, and the like.

(Other)
The chromatographic medium 4 can be provided with a control section 8 which is a region where the control antibody A3 is immobilized. As the control antibody, an antibody that does not have reactivity with CK19 and has reactivity with the labeled antibody 6 (first antibody A1) can be used as appropriate. Mouse IgG rabbit polyclonal antibody, anti-mouse IgG goat polyclonal antibody and the like can be exemplified.

The developing solution is a liquid constituting a mobile phase in the immunochromatography method, and moves along with the sample and the labeled antibody 6 on the chromatographic medium 4 which is a stationary phase. Any developing solution may be used.
As described above, the cancer detection kit of the present invention comprises the first antibody A1 that reacts with CK19 as an antigen-antibody and the labeled antibody 6 in which the labeling substance L is bound, and the second antibody A2 that reacts with CK19 as an antigen-antibody. And a chromatographic medium 4 provided with an antibody capturing part 7 which has been converted into a cancer. The first antibody A1 is hybridoma accession number NITE B P-02136 is a monoclonal antibody produced, and a second antibody A2 is the hybridoma accession number NITE B P-02135 is a monoclonal antibody produced.

  The cancer detection kit of the present invention utilizes immunochromatography using the novel monoclonal antibody as described above, so it is low-cost, and in a short time of about 5 to 10 minutes, with high accuracy, cancer metastasis. Can be detected.

  Next, an embodiment of the cancer detection method of the present invention will be described.

The method for detecting cancer according to the present invention is a method for detecting cancer by immunochromatography using the above-described kit for detecting cancer according to the present invention. Description of the cancer detection kit of the present invention is omitted.
The method for detecting cancer of the present invention comprises the following steps:
(1) a step of spreading a mixed solution containing a sample derived from the subject's lymph node and a labeled antibody to the antibody capturing unit; and (2) a case where a complex of CK19 and the labeled antibody in the sample is detected by the antibody capturing unit. And determining that the sample contains cancer cells.

  The subject to be subjected to the method for detecting cancer of the present invention is a mammal, preferably a human.

  The sample analyzed by the cancer detection method of the present invention can be exemplified by a lysate of a lymph node derived from a subject. In particular, the lymph nodes are preferably sentinel lymph nodes. Sentinel lymph node means a lymph node where cancer cells first reach the lymph stream. Sentinel lymph nodes can be easily injected by injecting a radioactive substance, dye, or other labeling substance into the dermis immediately above the tumor or the areola (in the case of breast cancer), and selecting the lymph node to which this labeling substance reached the earliest. Can be determined. Methods for determining sentinel lymph nodes are described, for example, in Guidelines for the Practice of Breast Cancer Surgery, 2008 edition, Japan Breast Cancer Society / ed., Kinbara Publishing Co., Ltd. In breast cancer, usually one of the ipsilateral axillary lymph nodes is a sentinel lymph node.

  Lysates of lymph nodes can be prepared by a conventionally known method. For example, the lymph nodes are washed with a physiological buffer (PBS, TBS, liquid medium, etc.), the parenchymal cells in the lymph nodes are released from the supporting structure (interstitium) of the lymph nodes, and the released parenchymal cells are dissolved in a solubilizing buffer. It can be solubilized with a liquid or the like. In this case, since the content of the cancer cell-derived component in the lysate is higher than in the case of simply imprinting the cells on the lymph node cleavage surface, it is expected that the detection sensitivity of lymph node metastasis of carcinoma will increase.

  The sample can be used by diluting a tissue collected from a subject with an appropriate developing solution. As such a developing solution, for example, a solvent such as water, a physiological saline solution, or a buffer solution used in a usual immunological analysis method can be appropriately used.

  In the step (1) of the method for detecting cancer according to the present invention, a mixed solution containing a sample derived from the lymph node of the subject and the labeled antibody 6 is developed on the antibody capturing section 7.

  Specifically, for example, when a predetermined amount of a sample is added to the sample addition pad 2, the sample moves to the labeled antibody holding pad 3 by capillary action. In the labeled antibody holding pad 3, the labeled antibody 6 is rapidly dissolved in the developing solution and can be freely moved, and the mixed solution containing the sample and the labeled antibody 6 is transferred from the labeled antibody holding pad 3 to the chromatographic medium 4 by the capillary. It moves by the phenomenon and expands to the antibody capturing part 7 side.

  At this time, when CK19 is contained in the sample, CK19 and the labeled antibody 6 are specifically bound by an antigen-antibody reaction to form a complex. This antigen-antibody reaction can be performed in the same manner as a normal antigen-antibody reaction.

  Then, when this complex is developed from the labeled antibody holding pad 3 to the chromatographic medium 4 by capillary action and reaches the antibody capturing section 7 formed on the chromatographic medium 4, the complex of CK19 and the labeled antibody 6 is formed. Is captured by specifically binding to the second antibody A2 of the antibody capturing section 7.

  In step (2), when the complex of CK19 and the labeled antibody 6 in the sample is detected by the antibody capturing unit 7, it is determined that the sample contains cancer cells.

  When the complex of CK19 and the labeled antibody 6 is captured by specifically binding to the second antibody A2 of the antibody capturing section 7, the labeled substance L of the captured labeled antibody 6 is concentrated in the antibody capturing section 7. Therefore, the presence of CK19 in a sample can be qualitatively and quantitatively analyzed visually or using an appropriate instrument.

  When the control part is provided in the chromatographic medium 4 of the cancer detection kit of the present invention, the labeled antibody 6 not bound to CK19 binds to the control antibody in the control part. Thereby, it can be confirmed that the sample and the labeled antibody 6 have developed on the chromatographic medium 4.

  On the other hand, when CK19 is not contained in the sample, no complex with the labeled antibody 6 is formed, and no specific binding of the antibody capturing unit 7 with the second antibody A2 occurs. In this case, since the labeled antibody 6 binds to the control antibody in the control section, only concentration of the labeling substance L in the control section can be confirmed.

  Even when CK19 is contained in the sample, the labeled antibody 6 similarly binds to the control antibody in the control section even when the content is below the preset detection limit. Only the concentration of L can be confirmed.

  As described above, the method for detecting cancer according to the present invention utilizes immunochromatography using the novel monoclonal antibody as described above. Presence or metastasis can be detected. For this reason, for example, using a tissue separated from a patient in the course of a cancer excision operation, while the excision operation is being performed, cancer metastasis can be performed by the cancer detection kit and detection method of the present invention. The possibility of existence can be determined. Therefore, it is possible to proceed with the operation of removing the cancer while confirming whether or not the cancer has metastasized. Further, according to the cancer detection kit and the detection method of the present invention, the presence or absence of cancer metastasis can be evaluated without consuming a specimen (tissue piece), and other tests (cytology, pathological examination) can be performed on the same specimen. Can be done in parallel.

  Therefore, this determination result can be used as one of the indices when determining a treatment policy or a tissue dissection area. That is, for example, from a patient such as breast cancer, lung cancer, stomach cancer, colorectal cancer, esophageal cancer, thyroid cancer, gynecological cancer, skin cancer, head and neck cancer, lymph node tissue near the primary cancer site is collected by biopsy, and Since the possibility of cancer lymph node metastasis can be determined by the cancer detection kit and detection method of the present invention, a determination for determining a policy such as whether to remove a lymph node or how much to dissect it is made. Can be material.

  Further, an embodiment of the method for detecting cancer metastasis of the present invention will be described. The description of the contents described as the above embodiment is partially omitted.

  In this embodiment, two detection kits (low sensitivity and high sensitivity) having different sensitivities are used as the detection kit for cancer of the present invention.

Also in this embodiment, the first antibody A1 labeled antibody 6 is a monoclonal antibody hybridoma accession number NITE B P-02136 is produced, and, second antibody A2 include the hybridoma of accession number NITE B P-02135 Is a monoclonal antibody produced.

  The low-sensitivity detection kit uses, for example, a labeled antibody prepared using a metal colloid solution containing metal particles (preferably gold particles) having an average particle diameter of 20 to 60 nm, preferably 30 to 50 nm, as a labeling substance. can do. The concentration of the metal colloid solution when preparing the labeled antibody in the low-sensitivity detection kit is not particularly limited, for example, a label prepared using a gold colloid solution having a maximum absorbance of 0.8 to 1.2, preferably a maximum absorbance of 1.0. Antibodies can be used.

  The high-sensitivity detection kit uses, for example, a labeled antibody prepared using a metal colloid solution containing metal particles (preferably gold particles) having an average particle size of 40 to 80 nm, preferably 50 to 70 nm as a labeling substance. can do. Further, the concentration of the colloidal gold solution when preparing the labeled antibody in the low-sensitivity detection kit is not particularly limited, for example, a labeled antibody prepared using a gold colloid solution having a maximum absorbance of 1.8 to 2.2, preferably a maximum absorbance of 2.0. Can be used. When preparing a labeled antibody for a high-sensitivity detection kit, use a gold colloid having a larger particle size and a higher concentration than the gold colloid for preparing a labeled antibody for a low-sensitivity detection kit. As a result, the sensitivity of the high-sensitivity detection kit can be increased by a factor of 4 to 6 times, and in practice, by a factor of approximately 5 compared to the low-sensitivity detection kit.

  In this embodiment, it is preferable that the high sensitivity detection kit has 4 to 6 times the detection sensitivity of the low sensitivity detection kit. The method for producing a high-sensitivity detection kit and a low-sensitivity detection kit having different detection sensitivities is not limited to the above method, and various methods capable of adjusting the efficiency of the antigen-antibody reaction can be employed. . Specifically, for example, when it is desired to lower the detection sensitivity, a low-sensitivity detection kit in which the detection sensitivity is reduced to a desired detection sensitivity can be prepared by setting the amount of the antibody (the amount of the labeled antibody) low. On the other hand, when it is desired to increase the detection sensitivity, a method of increasing the amount of the antibody (the amount of the labeled antibody) while considering the saturation of the amount of the antibody can be exemplified.

  The method for adjusting the detection sensitivity described above illustrates a method suitable for the immunochromatography of the present invention. Therefore, in the case of a known detection sensitivity adjustment method applied to a dot blot method or the like as described in Patent Document 1, for example, it may be impossible to sufficiently adjust the detection sensitivity.

  And in this embodiment, by adjusting the sensitivity of the low-sensitivity detection kit and the high-sensitivity detection kit, for example, when both the low-sensitivity detection kit and the high-sensitivity detection kit are positive (when CK19 is detected) It can be determined that there is a metastasis of a cancer (eg, breast cancer) larger than 2 mm that requires lymph node dissection.

  In addition, for example, when the low-sensitivity detection kit is negative (CK19 is not detected) and the high-sensitivity detection kit is positive, metastasis of cancer (breast cancer) of 2 mm or less that does not require lymph node dissection or 0.2 mm It can be determined that the following Isolated Tumor Cells (ITCs) or epithelial components are present.

  Furthermore, when both the low-sensitivity detection kit and the high-sensitivity detection kit are negative, it can be determined that there is no metastasis of cancer (breast cancer).

  In this way, by combining the low-sensitivity detection kit and the high-sensitivity detection kit, it is possible to distinguish between macro and metastatic metastases of cancer, and to determine whether lymph nodes should be removed or how much should be dissected. Can be determined.

As described above, it is common to perform lymph node dissection when cancer metastasis greater than 2 mm is present, but the size of the metastasis serving as a criterion for performing lymph node dissection is For example, it can be determined in a range of about 2 mm to 5 mm. Therefore, in this embodiment, by adjusting the sensitivity of the low-sensitivity detection kit and the high-sensitivity detection kit according to the size of the metastasis, which serves as a criterion for performing lymph node dissection in the clinic, It is possible to accurately determine metastasis that exceeds the limit (metastasis that requires lymphadenectomy) or metastasis that is equal to or smaller than the reference size (metastasis that does not require lymphadenectomy).
That is, this method for detecting cancer metastasis is a method for determining cancer metastasis by immunochromatography using two types of low sensitivity detection kits and high sensitivity detection kits having different detection sensitivities as cancer detection kits. , The following steps:
(1) a step of developing a mixed solution containing the sample derived from the lymph node of the subject and the labeled antibody in the antibody capturing unit; and (2) using a low-sensitivity detection kit and a high-sensitivity detection kit to detect cytokeratin 19 in the sample. When a complex of the and the labeled antibody is detected in the antibody capturing unit, it is determined that there is a macrometastasis exceeding a size that is a reference for performing lymph node dissection,
When the complex is not detected in the antibody capturing portion in the low-sensitivity detection kit, and the complex is detected in the antibody capturing portion in the high-sensitivity detection kit, a size serving as a reference for performing lymph node dissection. Micrometastasis less than, 0.2mm or less Isolated Tumor Cells (ITCs), it is determined that at least one of the contamination of epithelial components is present,
When the complex is not detected by the antibody capturing unit in the low-sensitivity detection kit and the high-sensitivity detection kit, the method includes a step of determining that cancer metastasis does not exist.

  The cancer detection kit and cancer detection method of the present invention are not limited to the above embodiments. For example, the cancer detection kit of the present invention may be in various forms of immunochromatography conventionally used, and may be appropriately designed in various forms according to the purpose, detection accuracy, and the like.

  Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited to the following Examples.

<Example 1> Preparation of cytokeratin protein for immunization cDNA of cytokeratin 19 (Homo sapiens keratin 19, KRT19, Accession No. NM_002276.4) was purchased from Origene (Catalog No. RC209707). This was similarly incorporated into Origene's vector, pEX-N-His-GST, to construct a His-GST fusion cytokeratin 19 expression vector. This was introduced into Escherichia coli for transformation. By culturing this transformant, His-GST fusion cytokeratin 19 protein was obtained in an insoluble fraction called Inclusion body. This fraction was solubilized with a PBS solution (pH 8.0) containing 6 M urea, and a purified product of the protein was obtained using Ni-NTA.

<Example 2> Preparation of cytokeratin 19 protein for screening When selecting antibody-producing hybridomas, it is necessary to exclude antibody-producing hybridomas that mainly recognize the GST portion in the protein used for immunization. Then, as a screening cytokeratin 19 protein expression vector, a vector purchased from Origene, pCMV6-AC-Myc-His-CK19, in which cytokeratin 19 cDNA was incorporated into pCMV6-AC-Myc-His, was constructed. When this was transfected into Free Style 293 cells, protein expression was observed in the cytoplasm. Therefore, the cells were solubilized with a PBS solution (pH 8.0) containing 6 M urea, and the protein was purified with Ni-NTA. Subsequently, the urea concentration was gradually lowered by dialysis, and refolding was performed to obtain a soluble Myc-His fusion cytokeratin 19 protein.

<Example 3> Confirmation of activity of screening cytokeratin 19 protein
The Myc-His fusion cytokeratin 19 protein was modified with Biotin using 20-fold molar concentration of Sulfo-NHS-LC-Biotin. The reactivity of this biotinylated protein was examined using a commercially available anti-cytokeratin 19 antibody clone Ks19.1 which has already been used in the measurement system for cytokeratin 19 fragment (Cyfra 21.1), and it was confirmed that this could be used for screening. It was confirmed.

Example 4 Preparation of Monoclonal Antibody The protein solution obtained in Example 1 was mixed with an equal amount of TiterMax Gold (TiterMax USA, Inc.) to prepare an emulsion, which was used as an immunogen. The monoclonal antibody was prepared by the method described in K. Watanabe et al., Vasohibin as an endothelium-derived negative feedback regulator of angiogenesis, J. Clin. Invest. 114 (2004), 898-907. That is, to a 5-week-old female Balb / c mouse or A / J mouse, 50 μg of the protein was subcutaneously or intraperitoneally divided and administered in a single dose per administration. After 5 immunizations, the spleen was excised from the mouse that had undergone the final boost (4 days before cell fusion), and splenocytes were prepared. Subsequently, cell fusion between splenocytes and myeloma cells (P3U1) was performed. Repeat dilution and culture, and measure antibody titer and affinity each time to obtain 10 high affinity monoclonal antibodies (1H1, 2G12, 2D5, 3A6, 3E10, 4E2, 7A9, 8C3, 14E9, 8A9) Was obtained.

(1) Method of measuring antibody titer The antibody titers of the antiserum and the hybridoma supernatant were evaluated by the DELFIA method. That is, to a 96-well microplate on which a goat anti-mouse IgG antibody was immobilized, an antiserum or a hybridoma supernatant was added, and further, a biotinylated Myc-His fusion cytokeratin 19 protein and Eu-labeled streptavidin were mixed and stirred, followed by stirring at room temperature. The reaction was allowed to proceed for 2 hours or overnight at 4 ° C. After the reaction, the plate was washed three times with a washing solution (a physiological saline solution containing 0.01% Tween 20 and 0.1% ProClin 150), 100 μL of an enhancing reagent was added, and after stirring, the mixture was allowed to stand for 5 minutes, and the time-resolved fluorescence at 615 nm was measured using ARVO MX. (Perkin Elmer). A sample was determined to be positive if it showed a signal intensity that was more than three times the signal obtained when no antiserum or hybridoma was added.

(2) Affinities and Subclasses of Monoclonal Antibodies Table 1 shows the affinities and subclasses of the obtained 10 monoclonal antibodies (1H1, 2G12, 2D5, 3A6, 3E10, 4E2, 7A9, 8C3, 14E9, 8A9).

<Example 5> Confirmation of ELISA combination In order to determine a combination of a labeled antibody and a capture antibody suitable for immunochromatography, all the acquired antibodies (1H1, 2G12, 2D5, 3A6, 3E10, 4E2, 7A9, 8C3, 14E9, For 8A9), a sandwich ELISA was performed. This allows not only a good combination of antibodies to be identified, but also a combination of overlapping epitopes, an antibody having similar specificity, and an antibody whose activity is reduced by modification.

  All the established monoclonal antibodies were biotin-modified using Sulfo-NHS-LC-Biotin, and used as detection antibodies. As an antigen, cytokeratin 19 protein was used as an immunogen.

The sandwich ELISA assay conditions are as follows. First, an antibody solid phase plate on which each of the 10 types of purified antibodies is immobilized is prepared. An assay buffer (50 mM PBS (pH 7.4), 1% BSA, 0.05% proclin 150) and a cytokeratin 19 protein solution are added thereto, and the mixture is left at 4 ° C. overnight. After washing three times with the assay buffer, a biotin-labeled monoclonal antibody is added and reacted at room temperature for 2 hours. After washing three times with the assay buffer, peroxidase (HRP) -labeled streptavidin is added, and the mixture is left at room temperature for 1 hour. Wash again with assay buffer three times, add tetramethylbenzidine (TMB), allow to stand at room temperature for 10 minutes, add H ₂ SO ₄ solution and measure absorbance (450 nm).

  Table 2 shows the results. The vertical column indicates the solid phase antibody, the horizontal column indicates the biotinylated antibody, and the numbers indicate the approximate absorbance.

  As shown in Table 2, it can be seen that 3A6, 4E2, and 8C3 lose their reactivity due to biotinylation. In addition, for example, it can be inferred that a combination such as 3E10 and 14E9 that does not exhibit absorbance is an antibody having a similar epitope. Table 3 below shows five relatively reactive antibodies extracted from the combinations thus obtained.

<Example 6> Preparation of detection kit (test strip) for immunochromatography Antibodies were prepared for five clones (1H1, 2D5, 3A6, 3E10, 14E9) which were relatively highly reactive in Example 5, and used for immunochromatography. The detection kit was provided for examination.

(Judgment paper)
Each of the five antibodies was diluted to 1 mg / mL to prepare a coating solution. The coating solution is applied to the membrane and dried to form an antibody capturing section (determination line) on which five kinds of antibodies (second antibodies) are immobilized. Then, a blocking treatment is performed, and a determination paper (chromatographic medium 4 in FIG. 1) is formed. ) Were produced.

(1) Preparation of determination paper A nitrocellulose acetate membrane (High Flow Plus (HFC135) manufactured by Millipore) was fixed on a desk, and an anti-mouse IgG antibody (1 mg / mL, diluted with 10 mM phosphate buffer) solution was used using a dispenser. The control part 8 was formed by coating in a line. In addition, a solution of five kinds of antibodies (second antibody) (1 mg / mL, diluted with 10 mM phosphate buffer) was applied to the nitrocellulose acetate membrane in a line using a dispenser to form the antibody capturing part 7. did. The nitrocellulose membrane is impregnated in 50 mM phosphate buffer (pH 7.4) containing 1% by weight of skim milk, 2% by weight of sucrose, and 0.02% by weight of SDS, taken out, dried, and allowed to stand at room temperature. It was stored in a bag containing a desiccant until it was used, to obtain a chromatographic medium 4 in which the antibody capturing part 7 and the control part 8 shown in FIG. 1 were formed.

(Reagent paper)
Using five kinds of antibodies, a labeled antibody (labeled primary antibody) labeled with colloidal gold was prepared under the same conditions, impregnated into a support, dried, and coated with reagent paper (labeled antibody holding pad 3 in FIG. 1). ) Were produced.

(2) Preparation of reagent paper (2-1) Preparation of labeled primary antibody 2 mL of 50 mM Tricine buffer (pH8.15) was added to 20 mL of a commercially available colloidal gold solution having an average particle diameter of 40 nm (OD ₅₃₀ = about 1.0). And stirred. Thereafter, 1.6 mL of antibody diluted to 0.1 mg / mL with 2 mM Tricine buffer (pH 8.15) was added thereto, followed by stirring, and then allowed to stand for 30 minutes. Subsequently, 2.4 mL of 10% by weight bovine serum albumin (pH 9.0) was added, and the mixture was stirred for 10 minutes. The resulting mixture was dispensed into 1.5 mL tubes, and centrifuged at 6100 × g at 8 ° C. for 10 minutes. Performed and purified. The pellet of the precipitated labeled primary antibody was resuspended in distilled water to 1 mL and stored at 4 ° C. until use.

(2-2) Preparation of Reagent Paper The labeled primary antibody obtained in the above (2-1) was used as a 150 mM tricine buffer solution (pH 8.20) containing 1.5% by weight of bovine serum albumin and 2% by weight of sucrose. It was diluted using 15). At this time, the dilution was performed so that the labeled primary antibody was 5% by volume. 1 mL of the obtained solution was impregnated into glass fiber filter paper, dried, and stored in a bag containing a desiccant at room temperature.

(Test strip)
Twenty types of test strips were prepared by combining each of five types of determination paper and reagent paper.

(3) Preparation of a test piece and an analysis device A milk-white polyester mount having one side subjected to adhesive processing is fixed on a desk with the adhesive side up, and the determination paper (chromatographic medium 4) prepared in the above (1) is attached. Was. Subsequently, the reagent paper (labeled antibody holding pad 3) prepared in the above (2) was adhered, and the chromatographic medium 4 and the labeled antibody holding pad 3 were connected in a state of having an overlap of about 2 mm. Further, a sample addition pad 2 made of a polyester nonwoven fabric was attached so as to partially cover the labeled antibody holding pad 3. In addition, filter paper was attached to form an absorbent pad 5.

<Example 7> Judgment test using test strip (1) Test 1
(I) CK19-His was used as the antigen. The antigen solution diluted to 10 ng / mL with PBS (pH 7.4) was used as a sample solution. The concentration of the sample solution was adjusted to the concentration as CK19.
(Ii) 100 μL of the sample solution was added to the test tube, and the test strip was inserted into the test tube.
(Iii) After 10 minutes, the degree of coloring of the antibody capturing portion (determination line) was visually determined.

(2) Criteria
The degree of coloring of the antibody capturing portion (determination line) was visually determined according to the following criteria.

++: Strongly positive (strongly colored, clear judgment line is confirmed)
+: Positive (clear judgment line can be confirmed)
±: Weak positive (very thin judgment line can be confirmed)
Tr: Trace of judgment line is recognized (coloring is weaker than weak positive)
-: Negative (No judgment line is confirmed)
(3) Results The results are shown in Table 4.

  As shown in Table 4, CK19-His 10 ng / mL was detectable in 16 combinations of 20 prepared test strips. As for the combinations capable of detecting CK19, results almost equivalent to those in Table 3 were obtained.

  Among them, as capture antibody (second antibody) x labeled antibody (first antibody), 1H1 x 3E10, 1H1 x 14E9, 2D5 x 14E9, 3A6 x 14E9, 2D5 x 3E10, 3E10 x 1H1, 14E9 x 1H1, 14E9 x Good results were shown with eight combinations of 2D5.

(4) Test 2
The detection of the antigens MCF7 Lysate 2.5 ng / mL and 5 ng / mL (prepared with a cell lysate) was attempted for the eight combinations extracted in Test 1. The test method and criteria were the same as in Test 1.

(5) Results The results are shown in Table 5.

  As shown in Table 5, it was confirmed that the sensitivity of condition No. 2 (capture antibody (second antibody) × labeled antibody (first antibody): 1H1 × 14E9) was the best.

The hybridoma producing the monoclonal antibody (1H1) has been deposited at the National Institute of Technology and Evaluation (NITE) Patent Microorganisms Depositary Center [Accession number: NITE BP-02135 (Hybridoma deposited in Japan ( Accession number: NITE) P-02135, entrusted Date: October 5, 2015), on October 21, 2016, was transferred according to an international deposit under the Budapest Treaty)]. Similarly, a hybridoma producing a monoclonal antibody (14E9) has been deposited at the National Institute of Technology and Evaluation (NITE) Patent Microorganisms Depositary Center [Accession number: NITE BP-02136 (Hybridoma deposited in Japan (Accession No. number: NITE P-02136, entrusted Date: October 5, 2015), on October 21, 2016, was transferred according to an international deposit under the Budapest Treaty)].

<Example 8> Sensitivity test The test of Example 7 for the combination of antibodies selected in (4) Test 2 of Example 7 (capture antibody (second antibody) x labeled antibody (first antibody): 1H1 x 14E9) In the same manner as in Examples 1 and 2, the sensitivity test was performed by changing the concentration of the antigen (CK19-His, MCF7 lysate).

  The results are shown in Tables 6 and 7.

Table 6, as shown in Table 7, the test strip (capture antibody (second antibody) × labeled antibody (the first antibody): 1H1 (Accession No. NITE B P-02135) × 14E9 ( accession number: NITE B P -02136)) was confirmed to be detectable at 0.1 ng / mL for CK19-His and 5 ng / mL for MCF7 lysate (weak positive (±)).

<Example 9> immunochromatographic diagnosis of lymph node metastasis by (1) the test strip used in Example 8 (capture antibody (second antibody) × labeled antibody (the first antibody): 1H1 (Accession No. NITE B P-02135) × 14E9 (accession number: NITE B P-02136) on), using clinical specimens (protein extract) of 141 cases, it was evaluated for the presence or absence of metastasis of cancer (breast cancer).

In addition, the protein extract was prepared by the following procedure.
(I) The lymph node removed by the operation is cut into a 2 mm width with a scalpel blade, and the scalpel blade is mixed by inverting 20 times with 2 mL of Phosphate Buffered Saline (PBS) and washed.
(Ii) The invaded lymph node is mixed by inverting 20 times with the same PBS, washed, and the removed lymph node is submitted for pathological diagnosis.
(Iii) The lymph node washing solution is centrifuged (5200 × g) for 1 minute, and the supernatant is discarded.
(Iv) Add 40 μL of cell lysate to the remaining cell clumps, disrupt the cell clumps and wait 3 minutes. The following composition reagent was used for the cell lysate.
0.1% NP40 / PBS (pH 7.4) / Protease inhibitor
PBS: 8.1 mM Na ₂ HPO ₄ , 1.47 mM KH ₂ PO ₄ , 2.68 mM KCl, 137 mM NaCl
(V) Inject the cell lysate into the filter and centrifuge (5200 × g) for 15 seconds. The filter is removed to obtain a protein extract.

  (2) Table 8 shows the results. In Table 8, positive and negative test strips are described as "SDB positive" and "SDB negative", respectively. In the pathological determination, a tissue section of a lymph node is observed under a microscope, and it is determined that cancer cells are positive when they are confirmed and negative when cancer cells are not confirmed.

  As shown in Table 8, according to this test strip, the sensitivity was 80.7% (46/57 × 100), the specificity was 100% (84/84 × 100), and the agreement rate was 92.2% (130 / 141 × 100).

  Note that “sensitivity” is the probability of correctly determining what should be determined to be positive as positive, and “specificity” is the probability of correctly determining a negative one as negative, and “matching rate”. Means the ratio of coincidence between the pathological judgment and the judgment by the test strip.

  On the other hand, in clinical practice, when cancer cells are present in a tissue section and macrometastasis larger than 2 mm is confirmed, axillary lymph nodes around the sentinel lymph nodes are dissected (metastasis positive). In the clinical setting, axillary lymph node dissection is not performed (metastatic negative) because micrometastasis or isolated tumor cells (ITCs) of 2 mm or less do not affect the prognosis.

  In this example, 9 out of 11 cases that were determined to be positive in the pathological determination and were determined to be negative in this test strip were less than the single micrometastasis that was determined to be clinically negative for metastasis, and the remaining Two cases had macrometastasis, which was clinically considered positive.

  There are 44 cases of macrometastasis that are clinically determined to be positive for metastasis, and this test strip can detect 95.5% (42/44 x 100) of macrometastasis that is positive for clinically determined metastasis. Was confirmed.

Test strips used in <Example 10> sensitive test strips Preparation and evaluation of sensitivity Examples 8 and 9 (capture antibody (second antibody) × labeled antibody (the first antibody): 1H1 (Accession No. NITE B P-02135 ) × 14E9 (accession number: the NITE B P-02136)), to prepare a test strip that employs a housing type horizontally.

(1) Preparation of determination paper (chromatographic medium 4 in FIG. 1) A nitrocellulose membrane (immunoporeRP (pore size: 8 μm) manufactured by GE Healthcare Japan) was fixed on a desk, and an anti-mouse IgG antibody (1 mg / mL, 2% by weight) was fixed. The control portion 8 was formed by applying a solution (diluted with a 10 mM phosphate buffer containing sugar) using a dispenser so as to form a line. Further, a nitrocellulose membrane to complement捉抗body: a (second antibody 1H1 (Accession No. NITE B P-02135) diluted with 10mM phosphate buffer containing 1 mg / mL, 2 wt% sucrose) solution, using a dispenser The antibody capturing portion 7 was formed by coating in a line. This nitrocellulose membrane is impregnated and dried in a 50 mM phosphate buffer (pH 7.4) containing 1% by weight of skim milk, 2% by weight of sucrose, and 0.02% by weight of SDS, and dried at room temperature. Saved until use.

(2) Preparation of reagent paper (labeled antibody holding pad 3 in FIG. 1) (2-1) Preparation of labeled primary antibody (labeled antibody) 20 mL of a commercially available gold colloid solution having an average particle size of 60 nm (OD530 = about 2.0) Was added with 4 mL of 50 mM Tricine buffer (pH 8.15) and stirred. Then, was added and stirred first antibody 14E9 (accession number NITE B P-02136) 1.0mL was diluted to 0.1 mg / mL in 2mM Tricine buffer (pH8.15), then allowed to stand at room temperature. Subsequently, 2.0 mL of 20 mM Tris buffer containing 0.1% by weight casein was added and stirred, and the resulting mixture was dispensed into 1.5 mL tubes and purified by centrifugation at 3400 xg at 8 ° C for 10 minutes. did. The pellet of the precipitated labeled primary antibody was resuspended in distilled water to 1 mL and stored at 4 ° C. until use.

(2-2) Preparation of Reagent Paper The labeled primary antibody obtained in the above (2-1) was prepared using a 300 mM tricine buffer (pH8.15) containing 1.5% by weight of bovine serum albumin and 2% by weight of sucrose. ). At this time, the dilution was performed so that the labeled primary antibody was 5.5% by volume. 1 mL of the obtained solution was impregnated into glass fiber filter paper, dried, and stored in a bag containing a desiccant at room temperature.

(3) Preparation of Test Piece and Analytical Apparatus A milk-white polyester mount having one side subjected to adhesive processing was fixed on a desk with the adhesive side up, and the chromatographic medium 4 prepared in the above (1) was bonded. Subsequently, a protective cover made of a transparent polypropylene film was attached with the adhesive side facing down. Subsequently, the labeled antibody holding pad 3 prepared in the above (2) was attached, and the chromatographic medium 4 and the labeled antibody holding pad 3 were continuously connected in a state of having an overlap of about 2 mm. Furthermore, the sample addition pad 2 made of a polyester non-woven fabric was attached to one end so as to partially cover the labeled antibody holding pad 3. In addition, filter paper was attached to form an absorbent pad 5.

  The laminated sheet was sequentially cut into strips so as to cross each member at right angles, and the cut test pieces were filled in a housing case and stored in a bag with a desiccant at room temperature until used in an analytical test.

(1) Test An antigen (MCF7 lysate) diluted to a predetermined concentration with a cell lysate was used as a sample solution. The sample liquid concentration was adjusted by adjusting the concentration as CK19. The cell lysate used the following composition reagent.

0.1% NP40 / PBS (pH7.4) / Protease inhibitor
PBS: 8.1 mM Na ₂ HPO ₄ , 1.47 mM KH ₂ PO ₄ , 2.68 mM KCl, 137 mM NaCl
Then, the prepared sample liquid was added to a sample addition pad of a test strip, developed on a chromatographic medium, and after 10 minutes, the degree of coloring of the antibody capturing portion (determination line) was visually determined.

(2) Criteria
The degree of coloring of the antibody capturing portion (determination line) was visually determined according to the following criteria.

+: Positive (clear judgment line can be confirmed)
±: Weak positive (very thin judgment line can be confirmed)
Tr: Trace of judgment line is recognized (coloring is weaker than weak positive)
-: Negative (No judgment line is confirmed)

(3) Results The results are shown in Table 9.

  As shown in Table 9, it was confirmed that in this test strip, a test solution of MCF7 lysate 1 ng / mL could be detected as weakly positive (±). That is, it was confirmed that the sensitivity of this test strip was improved about 5 times as compared with the test strip of Example 8 (Table 7).

<Example 11> Diagnosis of lymph node metastasis using a highly sensitive test strip For the test strip prepared in Example 10, metastasis of cancer (breast cancer) using 139 clinical samples (protein extract) as in Example 9 Was evaluated. The protein extract was prepared in the same manner as in Example 9.

  Table 10 shows the results.

  As shown in Table 10, according to this test strip, the sensitivity was 90.9% (51/55 × 100), the specificity was 91.7% (77/84 × 100), and the matching rate was 92.1% (128 / 139 × 100).

  In addition, four specimens that were determined to be positive in the pathological judgment and negative in this test strip were one case of isolated tumor cells (ITCs) (180 μm), one case of 500 × 200 μm single micrometastasis, and one case of 500 μm One case had micrometastasis and one case had 1500 μm single-shot micrometastasis.

  In addition, among the 51 cases that were determined to be positive in the pathological judgment and were positive in the test strip, 8 cases of micrometastasis that were judged to be metastatic negative in clinical cases, and macro metastases that were judged to be metastatic positive in clinical cases were 8 cases. , 43 cases. Therefore, this test strip had a sensitivity of 100% (43/43 × 100) for macrometastasis that was judged to be metastasis-positive in clinical practice.

<Example 12> Judgment of the size of metastatic lesions using two types of test strips (detection kits) having different sensitivities Test strips used in Examples 8 and 9 (hereinafter referred to as "low-sensitivity test strips") and Examples It was examined whether to estimate the size of the metastatic lesion by using two types of test strips, the test strips used in 10 (hereinafter, referred to as “high-sensitivity test strips”).

  Specifically, when both the low-sensitivity test strip and the high-sensitivity test strip are positive, it is determined that a macro transition larger than 2 mm exists (SDB Macro). When the low-sensitivity test strip is negative and the high-sensitivity test strip is positive, it is judged that micrometastasis of 2 mm or less or Isolated Tumor Cells (ITCs) or contamination of epithelial components of 0.2 mm or less (SDB micro or less). If both the low-sensitivity test strip and the high-sensitivity test strip are negative, it is determined that there is no metastasis (SDB micro or less).

  In other words, by combining two qualitative reactions, it is possible to determine whether metastasis below the micrometastasis, which does not require axillary lymph node dissection, or metastasis negative, can be determined by clinically performing axillary lymph node dissection. The test was performed.

  Table 11 shows the results.

  As shown in Table 11, by using two types of test strips having different sensitivities, the sensitivity was 95.3% (41/43 × 100), the specificity was 95.8% (92/96 × 100), and the matching rate was 95.7% ( 133/139 × 100).

  That is, it was confirmed that by using two types of test strips having different sensitivities, it was possible to accurately discriminate between macro metastasis and micro metastasis of cancer.

DESCRIPTION OF SYMBOLS 1 Detection kit 2 Sample addition pad 3 Labeled antibody holding pad 4 Chromatographic medium 5 Absorption pad 6 Labeled antibody 7 Antibody capture part 8 Control part A1 First antibody A2 Second antibody A3 Control antibody L Labeled substance

Claims (7)

A monoclonal antibody specific for cytokeratin 19, accession number NITE B P-02135 or accession number NITE B P-02136 monoclonal antibody hybridoma produced by,. Accession number NITE B P-02135, or accession No. NITE B P-02136 hybridoma. A labeled antibody in which a first antibody reacting with an antigen antibody with cytokeratin 19 and a labeling substance are bound, and
A chromatographic medium comprising an antibody capture unit on which a second antibody that reacts with cytokeratin 19 as an antigen antibody is immobilized,
A kit for detecting cancer by immunochromatography, comprising:
Wherein the first antibody hybridoma accession number NITE B P-02136 is a monoclonal antibody produced, and the second antibody hybridoma accession number NITE B P-02135, characterized in that a monoclonal antibody produced Cancer detection kit. A method for detecting cancer by immunochromatography using the cancer detection kit according to claim 3, comprising the following steps:
(1) developing a mixed solution containing a sample derived from the lymph node of the subject and the labeled antibody in the antibody capturing unit; and (2) forming a complex of cytokeratin 19 and the labeled antibody in the sample with the antibody A method for detecting cancer, comprising a step of determining that a cancer cell is contained in the sample when detected by the capture unit. A method for determining metastasis of cancer by immunochromatography using two low sensitivity detection kits and two high sensitivity detection kits having different detection sensitivities as the cancer detection kit according to claim 3, comprising the following steps:
(1) a step of developing a mixed solution containing the sample derived from the lymph node of the subject and the labeled antibody in the antibody capturing unit; and (2) using a low-sensitivity detection kit and a high-sensitivity detection kit to detect cytokeratin 19 in the sample. When a complex of the and the labeled antibody is detected in the antibody capturing unit, it is determined that there is a macrometastasis exceeding a size that is a reference for performing lymph node dissection,
When the complex is not detected in the antibody capturing portion in the low-sensitivity detection kit, and the complex is detected in the antibody capturing portion in the high-sensitivity detection kit, a size serving as a reference for performing lymph node dissection. Micrometastasis less than, 0.2mm or less Isolated Tumor Cells (ITCs), it is determined that at least one of the contamination of epithelial components is present,
A method for determining cancer metastasis, comprising a step of determining that cancer metastasis does not exist when the complex is not detected by the antibody capturing unit in the low-sensitivity detection kit and the high-sensitivity detection kit.   6. The method for determining metastasis of cancer according to claim 5, wherein the size of metastasis serving as a reference for performing lymph node dissection is 2 mm.   The labeled antibody of the low-sensitivity detection kit is labeled with a metal colloid having an average particle diameter of 20 to 60 nm, and the labeled antibody of the high-sensitivity detection kit is based on the particle size of the metal colloid of the labeled antibody of the low-sensitivity detection kit. 7. The method for determining metastasis of cancer according to claim 5, wherein the label is large and is labeled with a metal colloid having an average particle size of 40 to 80 nm.