JP4411438B2 - Asialo GM1-expressing cell detection reagent, cell detection method and cell classification method using the same, and aging measurement method - Google Patents

Description

  The present invention relates to an asialo GM1-expressing cell detection reagent, and relates to a cell detection method, a cell classification method, and an aging measurement method using the reagent.

Asialo GM1 (asialoganglioside) is a kind of glycolipid, in which sialic acid residues are removed from ganglioside (a general term for sphingoglycolipids containing sialic acid as a constituent sugar).
It has been elucidated that this asialo GM1 is expressed in cells involved in cancer diseases, autoimmune diseases, neurological diseases and the like, specifically, in those diseases. Then, it is considered to use Asialo GM1 as a marker for testing various diseases. For example, a method of detecting asialo GM1 and quantitatively measuring it for use in cancer diagnosis has been proposed (see Patent Document 1), and an inspection method for use in intestinal tract disorders has also been proposed (Patent Document 1). Reference 2).

  The measurement method described in Patent Document 1 is an immunoassay using an anti-asialo GM1 antibody, and is used for cancer diagnosis by detecting and quantifying asialo GM1 that increases as cells become cancerous. In the test method described in Patent Document 2, the contents of asialo GM1 and fucosyl asialo GM1 in the feces of mice are measured by a thin-layer chromatography immunostaining method using anti-asialo GM1 antibody and anti-fucosyl asialo GM1 antibody. I am testing for intestinal disorders.

By the way, in order to diagnose the degree of disease progression (pathological condition), it is involved in the disease based on the degree of differentiation and shape change of cells, for example, classification of tumor disease (cancer disease) by Papanicolaou staining. It is known to classify cells that do.
Japanese Patent Laid-Open No. 3-115863 Japanese Patent Laid-Open No. 6-25542

  However, neither of the methods described in Patent Documents 1 and 2 is intended to detect cells expressing asialo GM1, but simply measures the content of asialo GM1, and based on that content, cancer or intestinal tract. They are just diagnosing the fault. In other words, a method for detecting asialo GM1, which is said to be expressed in cells involved in various diseases (for example, NK cells, NKT cells, etc.) has not been developed. There was a problem that knowledge could not be obtained.

  In addition, the methods as described in Patent Documents 1 and 2 above are diagnosis / tests only for tumor diseases (cancer diseases) or intestinal tract disorders, and other diseases, particularly autoimmune diseases and neurological diseases, can be rapidly performed. Even the simple diagnosis method has not been developed yet. Furthermore, both of these methods described in Patent Documents 1 and 2 use monoclonal antibodies, and are polyclonal antibodies that can be expected to bind to various antigen recognition sites on the antigen surface and strongly identify the antigen. It is not a diagnostic / inspection method using

  Furthermore, the conventional cell classification method such as classification by Papanicolaou staining has a problem that it cannot classify cells for more types of diseases and cannot determine the degree of disease progression (pathological condition). It was.

  Therefore, in order to solve the conventional problems, the present invention provides an asialo GM1-expressing cell detection reagent that can easily detect cells expressing asialo GM1 using a polyclonal antibody in order to solve the conventional problems. By using this asialo GM1-expressing cell detection reagent, it is possible to efficiently detect and identify asialo GM1-expressing cells, and various diseases such as autoimmune diseases, neurological diseases, infectious diseases, tumors A new detection method that can also diagnose diseases and the like, and by efficiently classifying cells based on the detection results, cells involving Asialo GM1 can be classified, and the progression of various diseases involving the cells It is also an object to provide a new cell classification method that can determine the degree (pathological condition).

  In order to solve the above-mentioned problems, the present invention firstly comprises, as an active ingredient, an anti-asialo GM1 polyclonal antibody that specifically recognizes and binds to asialo GM1 expressed in cells. And

In addition, the present invention secondly relates to a cell detection method using the first invention, wherein at least the following steps:
<1> a step of bringing an anti-asialo GM1 polyclonal antibody into contact with cells to be detected contained in a specimen;
<2> contacting the anti-asialo GM1 polyclonal antibody with a labeled antibody that recognizes and binds to the anti-asialo GM1 polyclonal antibody as a secondary antibody;
<3> adding a detection substance for detecting the labeled antibody;
<4> a step of fixing the cells; and <5> a step of detecting the presence or absence of asialo GM1 expression in the cells based on the presence or absence of binding of the anti-asialo GM1 polyclonal antibody by a flow cytometer or immunohistochemical staining;
Third, the cell to be detected is a cell derived from a disease, and fourth, the disease is at least one of an autoimmune disease, a neurological disease, an infectious disease and a tumor disease. Fifth, the labeled antibody is at least one of a fluorescent substance labeled antibody, a biological binding component labeled antibody, and an enzyme labeled antibody.

Furthermore, the present invention sixthly relates to a cell detection method using the first invention, wherein the anti-asialo GM1 polyclonal antibody is labeled and modified at the Fc site, and includes at least the following steps:
<1> a step of bringing a label-modified anti-asialo GM1 polyclonal antibody into contact with cells to be detected contained in a specimen;
<2> a step of adding a detection substance for detecting the label-modified anti-asialo GM1 polyclonal antibody;
<3> a step of fixing the cells; and <4> a step of detecting the presence or absence of asialo GM1 expression in the cells by a flow cytometer or immunohistochemical staining;
Fifth, the labeling modification is characterized by being due to at least one of a fluorescent substance, a biological binding component and an enzyme.

  Seventh, the cell to be detected is a cell derived from a disease, and eighth, the disease is at least one of an autoimmune disease, a neurological disease, an infectious disease, and a tumor disease, 9 is characterized in that the label modification is caused by at least one of a fluorescent substance, a biological binding component and an enzyme.

  Furthermore, according to the tenth aspect of the present invention, at least one of the degree of differentiation, the degree of activity, and the degree of shape change of asialo GM1-expressing cells detected by any one of the second to ninth inventions is determined. The cells are classified based on the determination result.

  In the eleventh aspect of the present invention, an increase or decrease in the expression of asialo GM1 is measured based on the asialo GM1-expressing cells detected by any one of the second to ninth inventions, and aging is performed based on the measurement result. It is characterized by measuring the degree.

  According to the first invention of the present application, it is possible to easily detect cells that express asialo GM1.

  Further, according to the second to ninth inventions, asialo GM1-expressing cells can be detected and identified efficiently, and various diseases can also be diagnosed.

  Furthermore, according to the tenth invention, by efficiently classifying cells, it is possible to classify cells in which asialo GM1 is involved, and to determine the degree of progression (pathological condition) of various diseases in which the cells are involved. be able to.

  According to the eleventh aspect, by efficiently measuring the degree of aging of cells, it is possible to measure and judge the degree of aging of organs and tissues constituted by cells, and consequently living organisms.

  The present invention has the features as described above, and the embodiment thereof will be described in detail below.

  The present invention relates to cells related to various diseases such as autoimmune diseases, neurological diseases, infectious diseases and tumor diseases (specifically, natural killer cells (NK cells) which are immunocompetent cells, and some Natural killer T cells (NKT cells) as well as certain types of T cells, etc.), including asialo GM1 (asialoganglioside) that specifically recognizes and binds as an active ingredient, It is an asialo GM1-expressing cell detection reagent.

And this invention can detect the cell which expresses Asialo GM1 using this Asialo GM1 expression cell detection reagent. Specifically, at least the following steps:
<1> For example, a specimen prepared by collecting blood, urine, tissue or the like from a living body such as a human, monkey, mouse, rat, sheep, horse, etc., for example, and adjusting the dilution by pretreatment as necessary A step of contacting an anti-asialo GM1 polyclonal antibody with a cell to be detected contained in the cell;
<2> contacting the anti-asialo GM1 polyclonal antibody with a labeled antibody that recognizes and binds to the anti-asialo GM1 polyclonal antibody as a secondary antibody;
<3> adding a detection substance for detecting the labeled antibody;
<4> a step of fixing the cells with a fixing solution containing paraformaldehyde or the like; and <5> the presence or absence of expression of asialo GM1 in the cells based on the presence or absence of binding of the anti-asialo GM1 polyclonal antibody. Detecting with a meter or immunohistochemical staining;
As a result, the asialo GM1-expressing cells can be detected and identified efficiently.

  At this time, the detection target cell is a disease-derived cell, so that various diseases can be diagnosed as described above. In particular, it is possible to efficiently diagnose autoimmune diseases such as rheumatoid arthritis, neural diseases such as neuralgia and Guillain-Barre syndrome, infectious diseases caused by viruses and bacteria, and tumor diseases such as cancer. The diseases that can be diagnosed are not particularly limited as long as the asialo GM1-expressing cells are involved.

  Various types of labeled antibodies can be used, and fluorescent substance labeled antibodies, biological binding component labeled antibodies, enzyme labeled antibodies, and the like can be used. Specifically, for example, fluorescein isothiocyanate (FITC) -labeled antibody, phycoerythrin (PE) -labeled antibody, biotin-labeled antibody and peroxidase-labeled antibody are preferable from the viewpoint of ease of handling, detection sensitivity, and the like. Depending on the type of antibody, a detection substance is appropriately used. As a detection substance, for example, streptavidin is used in the case of a biotin-labeled antibody, and o-phenylenediamine or the like is used as a substrate in the case of a peroxidase-labeled antibody. Of course, the labeled antibody may be an antibody labeled with a fluorescent protein such as green fluorescent protein or red fluorescent protein, a radioactive substance, or a lanthanoid such as samarium, europium, or terbium, and is not particularly limited. It is not something.

In the detection method of the present invention, as another embodiment, detection can be performed using an anti-asialo GM1 polyclonal antibody that has been labeled and modified at the Fc site. The process at that time is basically the same as the above detection method, but differs in that it is not necessary to use a “secondary antibody”. Specifically, at least the following steps:
<1> a step of bringing a labeled modified anti-asialo GM1 polyclonal antibody into contact with a cell to be detected contained in a specimen collected from a living body such as a human, monkey, mouse, rat, sheep, or horse;
<2> a step of adding a detection substance for detecting the label-modified anti-asialo GM1 polyclonal antibody;
<3> a step of fixing the cells; and <4> a step of detecting the presence or absence of asialo GM1 expression in the cells by a flow cytometer or immunohistochemical staining;
In this step, asialo GM1-expressing cells can be detected.

  And the cell of the detection target at this time can diagnose various diseases by making it a cell derived from a disease similarly to the said detection method. In particular, it is possible to efficiently diagnose autoimmune diseases such as rheumatoid arthritis, neural diseases such as neuralgia and Guillain-Barre syndrome, infectious diseases caused by viruses and bacteria, and tumor diseases such as cancer.

  As for the label modification, as in the case of the labeled antibody, a fluorescent substance labeled antibody, a biological binding component labeled antibody and an enzyme can be used. Specifically, for example, fluorescein isothiocyanate, phycoerythrin, biotin Peroxidase and the like are preferable from the viewpoint of ease of handling, detection sensitivity, and the like, and the labeled modified anti-asialo GM1 polyclonal antibody can be efficiently detected. Of course, it can be labeled and modified with fluorescent proteins such as green fluorescent protein and red fluorescent protein, radioactive substances, and lanthanoids such as samarium, europium and terbium, and is not particularly limited. Moreover, a detection substance is appropriately used by these label modifications. For example, streptavidin is used for labeling modification with biotin, and o-phenylenediamine is used as a substrate for labeling modification with peroxidase, whereby the substrate can be colored and detected.

  The “flow cytometer” in the detection method of the present invention will be described. The flow cytometer uses a fluorescent label by performing relative labeling of individual cells, differences in internal structure, and fluorescent labeling. And the type of fluorescence can be measured to identify the cell and analyze the abundance ratio of various cells constituting the cell group in a short time. The detection method of the present invention utilizes such characteristics and efficiently detects asialo GM1-expressing cells by using an asialo GM1-expressing cell detection reagent.

  In the detection method of the present invention, asialo GM1-expressing cells can also be efficiently detected by utilizing “immunological tissue staining”. This “immunological tissue staining” will be explained. In general, immunohistological staining is a technique for detecting the localization of a target protein in and outside cells and tissues using a specific binding reaction called an antigen-antibody reaction. is there. There are roughly two methods, the “direct method” and the “indirect method”. The direct method is a method in which an enzyme or a fluorescent substance is directly bound to a primary antibody that is a specific antibody against an antigen for detection. The indirect method is a method of visualizing using a secondary antibody that is an antibody against the primary antibody. The present invention can be used by either the direct method or the indirect method. In immunohistochemical staining, a pathological section is usually prepared from a biological sample such as an organ, and this section is used as a sample. By using immunohistological staining in the detection method of the present invention, the tissue of Asialo GM1 is used. Search of internal distribution is also possible.

  In the above detection method, in each step, phosphate buffer or Tris buffer is used as a washing solution as necessary to prevent nonspecific binding of the antibody or to wash unbound excess antibody. You may perform washing | cleaning operation using a liquid etc. In addition, surfactants such as Tween-20 (registered trademark), Triton-X (registered trademark), sodium dodecyl sulfate (SDS), and skim milk and bovine serum albumin (BSA) as blocking agents May further be included.

  In the above detection method, in the case of detection by color development of the substrate, for example, it can be detected by an absorbance meter, a spectrophotometer, a fluorescence meter, a multi-label counter, or the like. In the case of a coloring system using peroxidase or the like, a coloring terminator may be added as necessary to stop the coloring reaction of the substrate. This color terminator is appropriately selected according to the color development reaction system of the labeling substance and the substrate. For example, in the case of a coloring reaction system in which the labeling substance is peroxidase and the substrate is o-phenylenediamine, coloring can be stopped using hydrochloric acid.

  The anti-asialo GM1 polyclonal antibody in the present invention is obtained by collecting blood from animals such as rats, mice, rabbits, sheep, goats, etc. sensitized with purified asialo GM1, and subjecting the serum to separation and salting with ammonium sulfate. The γ globulin fraction is obtained by dialysis against a phosphate buffer (PBS) and using an antigen affinity column or the like. Thus, by producing an anti-asialo GM1 polyclonal antibody, a large number of IgG molecules having different antigen recognition sites are contained, and antigens can be strongly identified by binding to various antigen recognition sites.

  In general, monoclonal antibodies have the following problems compared to polyclonal antibodies. That is, (1) the monoclonal antibody may be able to obtain only a weakly binding antibody, and the detection sensitivity may be lowered. In addition, since (2) some monoclonal antibodies show a cross-reaction, in this case, it can be expected that even a highly binding antibody lacks accuracy. Furthermore, (3) the production of monoclonal antibodies requires a lot of time and cost, and asialo GM1 as an immunogen is a self-component for normal mice. Production was difficult. In order to solve such problems, the anti-asialo GM1 antibody in the present invention uses a polyclonal antibody.

  The present invention further determines the asialo GM1-expressing cells detected by the cell detection method described above by determining at least one of the degree of differentiation, the degree of activity, and the degree of shape change. It can also be classified. For example, asialo GM1-expressing cells can include natural killer cells (NK cells) and natural killer T cells (NKT cells) which are immunocompetent cells, and these NK cells and NKT cells can be detected and differentiated. Can be efficiently classified based on the degree of activity, the degree of activity, and the degree of shape change. And various cells in which Asialo GM1 is involved can be classified, and the degree of progression (pathological condition) of many types of diseases in which the cells are involved can also be determined.

  And this invention measures the increase / decrease in the expression of asialo GM1 based on the asialo GM1 expression cell detected by the cell detection method demonstrated above, and measures the aging degree of a cell based on this measurement result It also makes it possible. By measuring the degree of aging of cells efficiently by the method for measuring the degree of aging of cells having such characteristics, it is possible to measure and judge the degree of aging of organs and tissues constituted by cells, and consequently living organisms. .

  Examples will be described below, and the present invention will be described in more detail. Of course, the invention is not limited by the following examples.

Example 1: Detection of asialo GM1 using a flow cytometer (1) Eight-week-old female C578L / 6 mice, or the same-week-old, same-sex, syngeneic thymectomized mice (or athymic mice: nude mice) ) Liver, spleen, intestine, lymph node, bone marrow, and thymus.
(2) Leukocyte cells were separated from these organs using a cell separation mesh, Percoll and the like. Each cell was stored in RPMI 1640 medium containing 100% fetal bovine serum (containing 100 μg / mL streptomycin, 100 U / mL penicillin, 5 × 10 ⁻⁸ M 2-mercaptoethanol) at a concentration of 1 × 10 ⁶ cells / mL. And prepared for staining.
(3) Each cell prepared in a 96-well V-bottom plate (manufactured by Nunk) was dispensed by 1-2 × 10 ⁵ cells / well, centrifuged, and the cells were collected. After removing the supernatant, 50 μL / well of a blocking reagent was added, and the cells were suspended by pipetting.
(4) Next, incubation was performed at 4 ° C. for 15 minutes to complete the blocking reaction. After centrifuging and removing the supernatant, in order to detect asialo GM1-positive cells (asialo GM1-expressing cells) such as natural killer cells (NK cells) and natural killer T cells (NKT cells) Asialo GM1 polyclonal antibody (manufactured by Wako Pure Chemical Industries, Ltd.) is used as an active ingredient, PE (phycoerythrin) -labeled anti-CD3ε antibody (BD PharMingen), and biotin-labeled anti-NK1.1 antibody (BD PharMingen) The mixed solution containing 50 μL / well was added, and the cells were suspended by pipetting, followed by incubation at 4 ° C. for 15 minutes.
(5) Then, 100 μL / well of 0.1% sodium nitride and 0.1% bovine serum albumin-containing phosphate buffered physiological saline (hereinafter referred to as staining buffer) was added and centrifuged, and the supernatant was removed.

In order to remove and wash excess unreacted labeled antibody, staining buffer 150 μL / well was added, and the cells were suspended by pipetting, followed by centrifugation and removal of the supernatant.
(6) After the washing operation, in order to detect the anti-asialo GM1 polyclonal antibody, 50 μL / well of FITC-labeled anti-rabbit IgG antibody was added. After floating by pipetting, the cells were incubated at 4 ° C. for 15 minutes. The staining buffer was added at 100 μL / well and centrifuged, and the supernatant was removed. In order to remove excess unreacted labeled antibody, 150 μL / well of staining buffer was added, and the cells were washed by pipetting, followed by centrifugation and supernatant removal.
(7) In order to detect a biotin-labeled antibody, 50 μL / well of streptavidin-conjugated Cychrome (SA-Cychrome; manufactured by BD PharMingen) was added, and the cells were washed by pipetting for 15 minutes at 4 ° C. After incubation, a biotin-streptavidin complex formation reaction was performed. After incubation, 100 μL / well of staining buffer was added and centrifuged, and the supernatant was gradually removed. In order to wash away and remove excess unreacted SA-Cychrome, 150 μL / well of staining buffer was added, and the cells were suspended by pipetting, followed by centrifugation and supernatant removal.
(7) Then, 200 μL / well of 1% paraformaldehyde-containing phosphate buffer was added to fix the cells, pipetted, and the suspended cells were collected, and this was collected using a flow cytometer (manufactured by BD Biosciences). ), Asialo GM1, CD3 and NK1.1 on the cell surface were detected.
(8) As a result, as shown in FIGS. 1 (A), (B), (C) and FIGS. 2 (A), (B):
<1> The expression of asialo GM1 on NK cells, NKT cells, and T cells varies greatly depending on the origin of the organ,
<2> The expression of asialo GM1 on NK cells is greatly affected by the thymus,
<3> There is a positive correlation between the expression of asialo GM1 and the expression of NK1.1, and <4> until now, it was thought that asialo GM1 was expressed only in NK cells. Asialo GM1 is also expressed on some NKT cells and certain T cells,
As a result, asialo GM1-expressing cells could be detected by the present invention.

  In FIGS. 1 and 2, “liver” indicates the liver, “BM” indicates the bone marrow, and “spleen” indicates the spleen. In FIG. 2, “C57BL / 6” represents a normal mouse having a thymus, and “nu / nu” represents a nude mouse having an athymus.

From this result, it was possible to easily detect cells that express asialo GM1 by using the asialo GM1-expressing cell detection reagent of the present invention. Moreover, considering that the expression of asialo GM1 in cells varies greatly depending on the origin of the organ, asialo GM1 is efficiently involved by measuring the degree of differentiation, the degree of activity, and the degree of shape change of asialo GM1-expressing cells. Cells can be classified, and the degree of progression (pathological condition) of various types of diseases involving the cells can be determined.
Example 2: Examination of aging of mice and changes in expression of asialo GM1 (1) 8 weeks of age with respect to changes in aging of mice and expression of asialo GM1 under the same procedures, reagents and experimental conditions as in Example 1 Adult mice, 7-month-old and 9-month-old old mice were used. In addition, since the thymus usually shrinks with aging, the thymus is almost disappeared in an aged living individual (a mouse in this embodiment), and the thymus is absent.
(2) As shown in FIGS. 3A and 3B, the results are:
<1> With the aging of mice, the expression of asialo GM1 in certain T cells increases,
<2> In addition, the expression of asialo GM1 on NK cells increases with aging, and <3> the expression of asialo GM1 that increases with aging is greatly affected by the thoracic line. In the absence of increased expression of Asialo GM1,
I was able to confirm. That is, it was confirmed that the expression of asialo GM1 was enhanced when the thymus atrophy with the aging of living organisms.

  In FIG. 3, “CD3-NK1 +” indicates a natural killer cell, “CD3 + NK1 +” indicates a natural killer T cell, and “CD3 + NK1-” indicates a T cell. In addition, 8-week-old normal mice (C57BL / 6) were used as control mice in old normal mice (C57BL / 6: 7 months old, 9 months old) and old nude mice (nu / nu).

  From this result, the asialo GM1-expressing cell detection reagent comprising the anti-asialo GM1 antibody according to the present invention as an active ingredient is applied not only to removal of NK cells in vivo as in the prior art, but also to cell classification, disease diagnosis, and the like. be able to.

  Furthermore, this result shows that asialo GM1 can be a barometer for aging of the living body, and by using the asialo GM1-expressing cell detection reagent of the present invention, an organ composed of cells simply and efficiently. It shows that it is also possible to measure the degree of aging of tissues, tissues, and eventually living organisms.

It is the figure which showed the expression frequency of asialoGM1 on various cells which exist in each organ (Liver: liver, BM: bone marrow, Splene: spleen) of a normal mouse (C57BL / 6: 8 weeks old), (A) Indicates natural killer (CD3-NK1 +) cells, (B) indicates natural killer T (CD3 + NK1 +) cells, and (C) indicates T (CD3 + NK1-) cells. Natural killer (CD3−3) present in each organ of normal mice with thymus (C57BL / 6: 8 weeks old) and nude mice without thymus (nu / nu No. 1, No. 2: 8 weeks old) It is the figure which showed the expression frequency of asialo GM1 on a NK1 +) cell, (A) is Liver: liver, (B) is BM: Bone marrow, and Splene: Spleen. Various kinds of aged normal mice with thymus (C57BL / 6: 7 months old, 9 months old) and various aged nude mice without thymus (nu / nu No.1, No.2, No.3) It is the figure which showed the expression frequency of asialoGM1 on a cell (Natural killer (CD3-NK1 +) cell, Natural killer T (CD3 + NK1 +) cell, T (CD3 + NK1-) cell), (A) is an old normal mouse, (B ) Shows an aged nude mouse. The 8-week-old mice are controls for old normal mice and old nude mice, respectively.

Claims (6)

A method for separating and detecting natural killer cells (NK cells), natural killer T cells (NKT cells) and T cells that express asialo GM1 from cell populations individually prepared from different organs, comprising at least the following: Process:
<1> contacting the cell population with an anti-asialo GM1 polyclonal antibody;
<2> contacting the anti-asialo GM1 polyclonal antibody with a labeled antibody that recognizes and binds to the anti-asialo GM1 polyclonal antibody as a secondary antibody;
<3> adding a detection substance for detecting the labeled antibody;
<4> a step of fixing the cells; and <5> a cell to which the anti-asialo GM1 polyclonal antibody is bound is analyzed by a flow cytometer or immunohistochemical staining. Sorting and detecting cells and T cells;
A cell detection method comprising the steps of: Labeled antibody, fluorescent substance-labeled antibody, the method of cell detection claim 1 is at least one of the biological binding components labeled antibodies and enzyme-labeled antibody. A method for separating and detecting natural killer cells (NK cells), natural killer T cells (NKT cells) and T cells that express asialo GM1, respectively, from cell populations prepared separately from different organs, comprising at least the following: Process:
<1> A step of contacting a cell population with a labeled modified anti-asialo GM1 polyclonal antibody having a labeled modification at its Fc site;
<2> a step of adding a detection substance for detecting the label-modified anti-asialo GM1 polyclonal antibody;
<3> a step of fixing the cells; and <4> a cell to which the anti-asialo GM1 polyclonal antibody is bound is analyzed by a flow cytometer or immunohistochemical staining. Sorting and detecting cells and T cells;
A cell detection method comprising the steps of: The cell detection method according to claim 3 , wherein the label modification is performed by at least one of a fluorescent substance, a biological binding component and an enzyme. Based on the difference in the expression pattern of the asialo GM1-expressing cells detected by the cell detection method according to any one of claims 1 to 4 , the asialo GM1-expressing cells are NK cells, NKT cells or T cells derived from any organ. A cell classification method characterized by classifying whether or not there is. The expression level of asialo GM1 in at least one of NK cells, NKT cells and T cells detected by the cell detection method according to any one of claims 1 to 4 is higher than the expression level of asialo GM1 in the cells of young individuals. And determining that the individual from which the cell is derived is an aging individual.