JP2022134528A - Cancer detection method, cancer testing method, and kit used therefor - Google Patents

Abstract

To provide a cancer detection method which allows for specifically and accurately detecting prostate cancer using a novel biomarker as an indicator.SOLUTION: A method of detecting prostate cancer is provided, the method comprising a measurement step of measuring the level of α2-3 sialylated CEA in a sample.SELECTED DRAWING: None

Description

TECHNICAL FIELD The present invention relates to a cancer detection method, a cancer examination method, and a kit used therefor, and more specifically, a method for detecting prostate cancer, a method for examining prostate cancer, and a kit used for these methods. Regarding.

Biomarkers such as tumor markers are useful for detection of diseases such as malignant tumors, guidelines for deciding treatment policies, and monitoring markers for evaluating therapeutic effects, and have been extensively studied in recent years. Examples of such biomarkers include CEA (Carcinoembryonic Antigen). CEA was established in 1965 by Gold P. and Freedman S. O. It is a glycoprotein with a molecular weight of about 200,000, and is also known as CD66e or CEACAM5. The protein portion of CEA is a single-chain polypeptide consisting of about 700 amino acids, and one molecule of CEA has 28 N-type glycosylation sites, most of which are bound by complex-type sugar chains. has been reported to contain 50% or more sugar chains (Non-Patent Document 1). CEA is detected in the blood of healthy people (meaning those who do not have cancer, the same shall apply hereinafter), but it is also found in gastrointestinal cancers, especially in cancer patients such as colon cancer, pancreatic cancer, and stomach cancer. It is known that its concentration is increased in cancer, and cases of lung cancer, uterine cancer, ovarian cancer, fetal cancer, etc. showing high blood concentration have been reported.

Therefore, CEA has been conventionally used as a monitoring marker for detecting these cancers, as a guideline for determining treatment policy, and for determining therapeutic efficacy. "Lumipulse Presto (registered trademark) CEA" is a kit for measuring CEA in a sample by the sandwich method that captures CEA present in the sample and binds it with a labeled anti-CEA antibody (labeled substance). Manufactured and sold by Revio Corporation.

For example, the amount of PSA (Prostate Specific Antigen) in blood has conventionally been used as an index for detection of prostate cancer, a guideline for determining treatment policy, and determination of therapeutic effect. Furthermore, it has been reported that GalNAc (N-acetylgalactosamine) is significantly increased in PSA of prostate cancer patients compared to healthy subjects (Non-Patent Document 2, etc.). In addition, for example, Patent Document 1 discloses the amount of free PSA in a sample and a sugar chain (α2-3 A method for determining prostate cancer from the ratio of the amount of PSA containing sialic acid) is described. However, since the amount of PSA increases not only in patients with prostate cancer but also in patients with prostatic hyperplasia, when the cut-off value is set in the low concentration range, the prostatic cancer hit rate tends to be low. is an issue. In addition, there is also the problem that it cannot be used as a monitoring marker in prostate cancer patients whose PSA level has stopped increasing due to hormone therapy or the like.

WO2019/221279

Katsuko Yamashita et al., Journal of Japanese Society for Molecular Tumor Markers, Vol. 19, 2003, p. 1-2 Fukushima et al., Glycobiology, 2010, vol. 20, no. 4, p. 452-460

As described above, although the sugar chain content in CEA reaches 50%, its sugar chain structure has not been fully clarified yet. Potential as a biomarker is expected.

In addition, as described above, CEA is detected in small amounts even in the blood of healthy individuals. and positive prediction or discrimination. However, such a conventional method for measuring CEA uses only one type of anti-CEA antibody, and thus the specificity for each cancer type is still insufficient. Furthermore, in prostate cancer patients, it is rare for the measured value of CEA to rise due to the morbidity of prostate cancer. The present inventors have found that it is insufficient to discriminate between and with higher accuracy.

The present invention has been made in view of the above problems, and uses a new biomarker as an index to detect cancer with high accuracy and specifically detect prostate cancer. The object is to provide a kit for use in the method.

For the purpose of searching for sugar chains as new tumor markers that could not be measured with existing antibodies, the present inventors, in addition to anti-CEA antibodies, have developed a water-soluble carrier consisting of a water-soluble polymer and immobilized on the water-soluble carrier. A complex (blocked labeled lectin) comprising the labeled substance and the lectin was also used for screening. As a result, the amount of molecules detected when MAM was used as a lectin, that is, the amount of CEA with α2-3 sialylated lectin-binding sugar chains (α2-3 sialylated CEA) was found to be It was found to significantly increase in prostate cancer patients compared to men. Therefore, by measuring the amount of α2-3 sialylated CEA, prostate cancer was specifically detected, and CEA was measured only with an anti-CEA antibody in prostate cancer patients and healthy subjects or patients with prostatic hyperplasia. The inventors have found that it is possible to make a determination with higher accuracy than in the case, and have completed the present invention.

The aspects of the present invention obtained from such findings are as follows.
[1]
A method for detecting prostate cancer, comprising a measuring step of measuring the amount of α2-3 sialylated CEA in a sample.
[2]
A method for examining prostate cancer, comprising a measuring step of measuring the amount of α2-3 sialylated CEA in a sample derived from a subject.
[3]
A method for screening a subject predicted to have prostate cancer, comprising a measuring step of measuring the amount of α2-3 sialylated CEA in a sample derived from the subject; The method according to [2], characterized by comprising a screening step of screening subjects using the amount of acid-added CEA as an index.
[4]
The method according to any one of [1] to [3], wherein in the α2-3 sialylated CEA, α2-3 sialic acid is a MAM-linked sugar chain that binds to MAM. .
[5]
The measuring step is a step of contacting the sample with a first probe molecule capable of specifically binding to CEA and a second probe molecule capable of specifically binding to α2-3 sialic acid. A method according to any one of [1] to [4], characterized in that:
[6]
The method of [5], wherein the first probe molecule is an antibody capable of specifically binding to CEA.
[7]
The method according to [5] or [6], wherein the second probe molecule is a lectin capable of specifically binding to α2-3 sialic acid.
[8]
the measuring step is a step of contacting the sample with a capturing body and a labeling body;
The capture body comprises a water-insoluble carrier and either one of a first probe molecule and a second probe molecule immobilized on the water-insoluble carrier, and
wherein the label comprises a labeling substance and the other of the first probe molecule and the second probe molecule;
The method according to any one of [5] to [7], characterized in that
[9]
The capture body comprises a water-insoluble carrier and a first probe molecule immobilized on the water-insoluble carrier, and
The label comprises a water-soluble carrier, a labeling substance immobilized on the water-soluble carrier, and a second probe molecule, and the second probe molecule is a lectin capable of specifically binding to α2-3 sialic acid. is a blocked labeled lectin that is
The method according to [8], characterized by:
[10]
A kit for use in the method according to any one of [5] to [9], comprising: a first probe molecule capable of specifically binding to CEA; and a bindable second probe molecule.
[11]
The kit of [10], wherein the first probe molecule is an antibody capable of specifically binding to CEA.
[12]
The kit of [10] or [11], wherein the second probe molecule is a lectin capable of specifically binding to α2-3 sialic acid.
[13]
a capture body comprising a water-insoluble carrier and either one of a first probe molecule and a second probe molecule immobilized on the water-insoluble carrier; and
a label comprising a labeling substance and the other of the first probe molecule and the second probe molecule;
The kit according to any one of [10] to [12], comprising:
[14]
The capture body comprises a water-insoluble carrier and a first probe molecule immobilized on the water-insoluble carrier, and
The label comprises a water-soluble carrier, a labeling substance immobilized on the water-soluble carrier, and a second probe molecule, and the second probe molecule is a lectin capable of specifically binding to α2-3 sialic acid. is a blocked labeled lectin that is
The kit according to [13], characterized in that:

According to the present invention, there are provided a cancer detection method and a cancer examination method capable of specifically detecting prostate cancer with high accuracy using a new biomarker as an index, and a kit used for these methods. becomes possible.

2 is a graph showing CEA measurement results for a healthy subject group and a prostate cancer group. 2 is a graph showing measurement results of CEA/MAM in a healthy subject group and a prostate cancer group. FIG. 10 is a graph showing the measurement results of CEA in the prostatic hyperplasia group and the prostatic cancer group. FIG. FIG. 10 is a graph showing the measurement results of CEA/MAM in the prostatic hyperplasia group and the prostatic cancer group; FIG. 4 is a graph showing the relationship between measured values of CEA and measured values of CEA/MAM; 4 is a graph showing the relationship between PSA measurements and CEA/MAM measurements. 4 is a graph showing measurement results of CEA/WFA in a healthy subject group and a prostate cancer group.

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will now be described in detail with reference to its preferred embodiments.

<Cancer detection method, cancer examination method>
The cancer detection method of the present invention is a method for detecting prostate cancer, and is a method comprising a measuring step of measuring the amount of α2-3 sialylated CEA in a sample. Further, the cancer testing method of the present invention is a method of testing for prostate cancer, and is a method comprising a measuring step of measuring the amount of α2-3 sialylated CEA in a sample derived from a subject.

[cancer]
In the present invention, "cancer" includes epithelial malignant tumors (cancer) and non-epithelial malignant tumors (sarcoma). The cancer to be detected or tested in the method of the present invention is prostate cancer. Prostate cancer according to the present invention refers to cancer that develops in the prostate, such as prostate cancer, prostate small cell carcinoma, prostate ductal carcinoma, prostate sarcoma, prostate squamous cell carcinoma, prostate adenosquamous cell carcinoma, and prostate basal cell. cancer, prostatic mucinous carcinoma, prostatic signet ring cell carcinoma. Among these, prostate cancer is preferable as the prostate cancer according to the present invention.

[α2-3 sialylated CEA]
In the present invention, “α2-3 sialylated CEA” means a CEA molecule containing a sugar chain α2-3 sialic acid (α2,3-linked sialic acid) attached thereto. is also referred to as “α2-3 sialic acid-containing CEA”. In the present invention, "CEA" refers to carcinoembryonic antigen, which is a glycoprotein. CEA is one of the molecules constituting the CEACAM (carcinoembryonic antigen-associated adhesion molecule) family involved in various processes such as cell adhesion, proliferation, differentiation, and tumor suppression, and is labeled as "CD66e" or "CEACAM5 (carcinoembryonic antigen CD66e or CEACAM5 and CEA are used synonymously herein. CEA typically consists of a protein portion, which is a single-chain polypeptide consisting of about 700 amino acids, and a sugar chain portion, which is bound thereto and consists of multiple sugar chains.

The α2-3 sialylated CEA according to the present invention contains at least α2-3 sialic acid as the sugar chain moiety. In the present invention, “α2-3 sialic acid” refers to a sugar chain (α2-3Gal sialic acid) in which the 2-position carbon of sialic acid is glycoside-bonded to the 3-position carbon of galactose (Gal), which is a monosaccharide. In one molecule of α2-3 sialylated CEA, a plurality of α2-3 sialic acids may be present. may be combined with The α2-3 sialylated CEA according to the present invention preferably contains α2-3 sialic acid as a MAM-linked sugar chain that binds to MAM, and the terminal sialic acid residue is 2 from the end of the sugar chain. It is more preferable to contain a sugar chain (α2-3Gal sialic acid) linked to the second galactose residue as an α2,3-linked sugar chain (α2-3Gal sialic acid). It is more preferably contained as a terminal α2-3 sialic acid (α2-3Galβ1-4GlcNAc sialic acid) of the glycosylation site.

The average mass of such α2-3 sialylated CEA (average mass calibrated with a gel filtration chromatography (GFC) marker, hereinafter the same) is not particularly limited, but is 100,000 to 300,000 Da. preferably 180,000 to 200,000 Da. The content of the sugar chain moiety in one molecule of α2-3 sialylated CEA is not particularly limited, but is preferably 40 to 60% by mass.

[Subject]
In the present invention, the term "subject" refers to a subject, preferably a person, on whom the cancer examination method of the present invention is performed. A subject according to the present invention may be a healthy subject for the purpose of screening or the like, or a subject suffering from prostate cancer but without subjective symptoms. In addition, for the purpose of determining prognosis, determining treatment policy, confirming therapeutic effect, confirming recurrence, etc. There may be. In addition, in the present invention, the term “healthy subject” refers to a subject who does not have cancer (that is, prostate cancer) to be examined. It should be noted that whether or not a subject truly suffers from prostate cancer is determined (definite diagnosis) by a biopsy of prostate tissue collected from the subject.

[sample]
As the "sample" used in the cancer detection method and cancer examination method of the present invention (in this specification, these are sometimes collectively referred to as "the method of the present invention"), α2-3 sialylated There is no particular limitation as long as the sample can contain CEA. In general, blood samples such as serum, plasma, and whole blood collected from cancer test subjects such as the subject; urine, sputum, saliva, sweat, cerebrospinal fluid, digestive fluid, semen, lymph body fluid specimens other than blood such as ascites; mucous membrane specimens such as oral mucosa, pharyngeal mucosa and intestinal mucosa; and various biopsy specimens. Moreover, the sample may be a cultured cell or a cell culture solution. The sample according to the present invention is preferably a blood sample, more preferably serum (also referred to as "serum sample").

These samples may be diluted or suspended with a diluent as necessary, or may be pretreated as appropriate. Examples of the diluent include buffers such as phosphate buffer, Tris buffer, Good's buffer, borate buffer, acetate buffer, citrate buffer, glycine buffer, succinate buffer, and phthalate buffer. liquid. Examples of the pretreatment include pulverization, freezing, heating, concentration, fractionation, desalting, and the like; addition of pH adjusters, stabilizers, preservatives, preservatives, surfactants, and the like; purification, and the like. These may be used alone or in combination of two or more. The purification treatment is not particularly limited, but includes, for example, column treatment; α2-3 sialylation by adsorbing contaminants with a substance such as an antibody that adsorbs contaminants in a sample immobilized on a water-insoluble carrier. Treatment to remove from a sample containing CEA; Treatment to capture CEA containing α2-3 sialylated CEA with an anti-CEA antibody immobilized on a water-insoluble carrier, remove contaminants by washing or the like, and then liberate, etc. is mentioned.

[Measurement process]
The method of the present invention includes a measuring step of measuring the amount of α2-3 sialylated CEA in the sample. The method for measuring the amount of α2-3 sialylated CEA is not particularly limited as long as it is a method capable of measuring the amount of α2-3 sialylated CEA. Any suitable combination can be employed. For example, methods using probe molecules capable of specifically binding to α2-3 sialylated CEA; high performance liquid chromatography isotope dilution mass spectrometry (LC-IDMS); inductively coupled plasma optical emission spectrometry (ICP-OES); Inductively Coupled Plasma Mass Spectrometry (ICP-MS); Atomic Absorption Spectrometry (AAS); HPLC; FPLC; NMR; IR; FTIR; and a combination thereof, but are not limited to these.

Among them, the measuring step according to the present invention is preferably a method using a probe molecule capable of specifically binding to α2-3 sialylated CEA. Examples of such probe molecules include antibodies; binding proteins such as protein A, protein G and protein L; avidins such as avidin D and streptavidin; lectins; be done. In the present invention, the term "antibody" includes not only complete antibodies, but also antibody fragments (eg, Fab, Fab', F(ab') ₂ , Fv, single-chain antibodies, diabodies, etc.) and variable regions of antibodies. Also included are low-molecular-weight antibodies conjugated with

The probe molecule capable of specifically binding to α2-3 sialylated CEA includes a probe molecule capable of specifically binding to CEA (referred to as a “first probe molecule” in the present invention) and α2-3 sialic acid A combination with a specifically binding probe molecule (referred to as a “second probe molecule” in the present invention) is preferred.

[First probe molecule]
In the present invention, unless otherwise specified, the term "capable of binding specifically to CEA" refers to sites other than α2-3 sialic acid (more preferably, sialic acid α2-3Galβ1-4GlcNAc sugar chain) in CEA. It shows that it can specifically bind to The “site other than α2-3 sialic acid” may be the whole or part of the protein moiety, or the whole or part of the sugar chain moiety other than α2-3 sialic acid. It may be a combination of two or more. Examples of the first probe molecule capable of specifically binding to CEA include antibodies capable of specifically binding to CEA (anti-protein antibody having a recognition site for the protein portion of CEA, an anti-protein antibody having a recognition site for the sugar chain portion of CEA, and antibodies whose recognition sites are the protein portion and sugar chain portion of CEA.Herein, sometimes referred to as "anti-CEA antibody"), anti-CEACAM family antibody (CEA and other CEACAM Antibodies whose recognition sites are family molecules), among which anti-CEA antibodies are preferred, and anti-CEA protein antibodies whose recognition sites are at least part of the CEA protein portion are more preferred, and α2- Antibodies that do not interfere with the binding of trisialic acid to the second probe molecule are preferred.

The anti-CEA antibody is not particularly limited as long as it has the ability to bind to CEA, and may be a polyclonal antibody or a monoclonal antibody, but from the viewpoint of homogeneity and stability, a monoclonal antibody is preferred. preferable. Anti-CEA antibodies can be produced by appropriately adopting and improving conventionally known production methods, and commonly available methods may be used as appropriate.

As the anti-CEA antibody according to the present invention, for example, when the following lectin is used as the second probe molecule, oxidation treatment is performed in order to prevent the lectin from recognizing the antibody sugar chain and lowering the detection sensitivity. , glycosidase treatment, protease treatment, etc. to remove or destroy lectin-binding sugar chains; antibody molecules from which regions containing glycosylated amino acids have been removed; or genetic recombination expressing antibody genes Antibodies are preferably produced under conditions that do not cause glycosylation by using Escherichia coli or cells or by restricting nutritional conditions in culturing antibody-producing cells.

[Second probe molecule]
Examples of the second probe molecule capable of specifically binding to α2-3 sialic acid include, for example, an antibody capable of specifically binding to α2-3 sialic acid α2-3 sialic acid-binding artificial proteins such as Lectenz (registered trademark), lectins capable of specifically binding to α2-3 sialic acid (e.g., canine endu lectin (MAM/MAL/MAA-II ), canine cochlea lectins such as canine cochlea lectin (MAH/MAA-I); willow pine mushroom lectin (ACG); jackfruit lectin (Jacalin)). Preferred, more preferred is canine endulectin, and particularly preferred is MAM. MAM (Maackia amurensis lectin; also referred to as MAL or MAA-II) is a protein that recognizes the sugar chain structure of α2-3 sialic acid (particularly sialic acid α2-3Galβ1-4GlcNAc) and exhibits binding activity. It is a leguminous lectin derived from the seeds of a plant, the dog pagoda. The lectin may be a modified lectin into which a mutation has been introduced or an artificially synthesized lectin for the purpose of increasing the specificity of sugar chain recognition activity. Moreover, you may use suitably what is generally distribute|circulating.

When using the first probe molecule and the second probe molecule, the sample is brought into contact with the first probe molecule and the second probe molecule in the measurement step. As a result, both α2-3 sialic acid and the portion of CEA other than α2-3 sialic acid are recognized, and α2-3 sialylated CEA containing both can be detected and measured. The contact between the sample and the first probe molecule and the contact between the sample and the second probe molecule may be at the same time or at different times. may come first.

[Labeling substance]
In the present invention, the amount of α2-3 sialylated CEA is measured using a probe molecule (preferably, the first probe molecule and/or the second probe molecule) capable of specifically binding to α2-3 sialylated CEA. It is preferably carried out by detecting a signal generated by a labeling substance attached to a molecule or to a molecule (eg, secondary antibody or protein A) that recognizes these molecules. The amount of α2-3 sialylated CEA can be measured by measuring the amount of the detected signal and, if necessary, semi-quantifying or quantifying it. The above-mentioned "signal" includes coloration (color development), quenching, reflected light, luminescence, fluorescence, radiation from radioactive isotopes, etc., and in addition to those that can be confirmed with the naked eye, measurement methods and devices according to the type of signal This includes items that can be verified. In the present invention, the amount of α2-3 sialylated CEA to be measured may be semi-quantified or quantified using a standard sample or the like. may be the amount of α2-3 sialylated CEA.

As the labeling substance according to the present invention, those used as labeling substances in known immunoassay methods and methods based thereon can be used without particular limitation. For example, enzymes; luminescent substances such as acridinium derivatives; fluorescent substances such as europium; fluorescent proteins such as allophycocyanin (APC) and phycoerythrin (R-PE); radioactive substances such as ¹²⁵ I; low molecular weight labeling substances such as rhodamine isothiocyanate (RITC); gold particles; avidin; biotin; latex; dinitrophenyl (DNP); A combination of the above may also be used. For example, when an enzyme is used as the labeling substance, various measurements can be performed by adding a chromogenic substrate, a fluorescent substrate, a chemiluminescent substrate, or the like as the substrate. Examples of the enzyme include, but are not limited to, horseradish peroxidase (HRP), alkaline phosphatase (ALP), β-galactosidase (β-gal), glucose oxidase, and luciferase.

[Sandwich method]
Measurement methods using the first probe molecule and the second probe molecule include immunological measurement methods such as the sandwich method, competitive method, and immunoturbidimetric method, and measurement methods based on these principles. There are no particular restrictions. Such measurement methods include, for example, generally ELISA, digital ELISA, CLEIA (chemiluminescent enzyme immunoassay), CLIA (chemiluminescence immunoassay), ECLIA (electrochemical immunoassay), RIA (radioimmunoassay). ) using microplates, particles, etc. as carriers; immunochromatography; surface plasmon resonance analysis; detection methods based on fluorescence resonance energy transfer;

As the measurement method according to the present invention, the sandwich method is preferable from the viewpoint of the possibility of constructing a measurement system with higher sensitivity and specificity. Hereinafter, a more specific aspect of the measuring process according to the present invention will be described by taking the sandwich method as an example.

As an aspect when using the sandwich method,
the measuring step is a step of contacting the sample with a capturing body and a labeling body;
The capture body comprises a water-insoluble carrier and either one of a first probe molecule and a second probe molecule immobilized on the water-insoluble carrier, and
the label comprises a labeling substance and the other of the first probe molecule and the second probe molecule;
Aspect (hereinafter sometimes referred to as "first aspect") can be mentioned. In the first aspect, the first probe molecule and the second probe molecule may be provided in either the capturing body or the labeling body, respectively. One is included in the label. By capturing both α2-3 sialic acid and CEA in this way, α2-3 sialylated CEA can be captured and detected with high accuracy and ease.

Other aspects of the sandwich method are not limited to those described above. For example, the label comprises a first labeling substance and either one of a first probe molecule and a second probe molecule. a second labeled body comprising a first labeled body, a second labeled substance, and the other of the first probe molecule and the second probe molecule, and the capture body is a non- An aspect (hereinafter sometimes referred to as a “second aspect”) that is a capturing body comprising a water-soluble carrier and probe molecules immobilized on the water-insoluble carrier may be employed. In the second aspect, the first labeling substance and the second labeling substance produce signals different from each other. In this case, the probe molecules provided in the capture body include probe molecules capable of specifically binding to α2-3 sialylated CEA (including first probe molecules and second probe molecules); and/or a probe molecule capable of specifically binding to a second probe molecule.

As such a sandwich method, there is a two-step forward sandwich method (reaction between capture body and α2-3 sialylated CEA in the sample, α2-3 sialylated CEA bound to the capture body and method), reverse sandwich method (preliminarily reacting a labeled substance with α2-3 sialylated CEA in a sample, and reacting the resulting complex with a capturing substance), and a one-step method. (Method in which reactions of α2-3 sialylated CEA in a sample, a capturing entity, and a labeling entity are simultaneously performed in one step), and any of these can be employed.

For example, in the forward sandwich method, first, the sample and the capture body are brought into contact, and binding between the probe molecule of the capture body and α2-3 sialylated CEA (for example, binding of the first probe molecule and CEA ) to trap α2-3 sialylated CEA in the trapping body (primary reaction: trapping step). Next, the labeled substance is brought into contact with the α2-3 sialylated CEA captured by the capturing substance, and the probe molecule of the labeled substance binds to the α2-3 sialylated CEA (for example, the second probe molecule and α2-3 sialic acid) (secondary reaction: labeling step). This reaction forms a complex containing the capture entity-α2-3 sialylated CEA-label. After removing unbound sample and label by washing as necessary (washing step), a signal derived from the labeling substance is measured by a predetermined method according to the labeling substance (measurement step).

(trapping body)
The "capture body" according to the present invention is a complex comprising a water-insoluble carrier and a probe molecule capable of specifically binding to α2-3 sialylated CEA immobilized on the water-insoluble carrier, , a conjugate in which the water-insoluble carrier and the probe molecule are bound directly or indirectly. The probe molecules provided in the capture body according to the first aspect are preferably either one of the first probe molecules and the second probe molecules. In this case, the probes provided in the capture body The molecule may be either the first probe molecule or the second probe molecule, but when a lectin is used as the second probe molecule, the capture The probe molecule provided in the body is preferably a first probe molecule capable of specifically binding to CEA, more preferably an anti-CEA antibody.

<Water-insoluble carrier>
The water-insoluble carrier contained in the capturing body is a water-insoluble substance that mainly supports the probe molecule and functions as a carrier for immobilization. In the present invention, the term "water-insoluble substance" refers to a substance that is insoluble in water at normal temperature and pressure (solubility in water is 0.001 g/mL or less, preferably 0.0001 g/mL or less, the same shall apply hereinafter). .

As the material of such a water-insoluble carrier, those commonly used in immunoassays and measurements based thereon can be used, and are not particularly limited. (Meth)acrylate, polymethyl methacrylate, polyimide, nylon, etc.), gelatin, glass, latex, silica, metals (gold, platinum, etc.), and metal compounds (iron oxide, cobalt oxide, nickel ferrite, etc.) At least one selected from In addition, the material of the water-insoluble carrier may be a composite material of these substances or a composite material of these substances and other substances. It may be an organic-inorganic composite material comprising one kind of organic polymer and at least one kind of metal compound selected from the group consisting of iron oxide (such as spinel ferrite), cobalt oxide, and nickel ferrite. Further, the water-insoluble carrier includes a carboxy group, an epoxy group, a tosyl group, an amino group, a hydroxyl group, an isothiocyanate group, an isocyanate group, an azide group, an aldehyde group, a carbonate group, an allyl group, an aminooxy group, and a maleimide group. , the surface of which is modified with an active group such as a thiol group.

In the present invention, the shape of the water-insoluble carrier is not particularly limited, and may be, for example, a plate, fiber, film, particle, etc. However, from the viewpoint of reaction efficiency, it is a particle. From the viewpoint of automation and shortening of the time, magnetic particles are more preferable. As such a water-insoluble carrier, conventionally known ones can be used as appropriate, and commercially available ones can also be used as appropriate.

<Structure and manufacturing method of capturing body>
In the capture body, the content of the probe molecule is not particularly limited, and can be appropriately adjusted according to the ease of binding between the probe molecule and α2-3 sialylated CEA. The mass of the probe molecule with respect to 100 parts by mass of the water-insoluble carrier (preferably particles) (the sum of the two or more when the water-insoluble carrier is a combination of two or more) (in a combination of two or more probe molecules, In some cases, the total thereof) is preferably 0.1 to 10 parts by mass, more preferably 1 to 5 parts by mass.

The capture body can be produced by immobilizing the probe molecule on the water-insoluble carrier. As such a production method, a conventionally known method or a method analogous thereto can be appropriately employed, and the probe molecule may be directly or indirectly immobilized on the water-insoluble carrier.

In the case of direct immobilization, for example, the water-insoluble carrier and/or the probe molecule include a carboxy group, an epoxy group, a tosyl group, an amino group, a hydroxy group, an isothiocyanate group, an isocyanate group, an azide group, an aldehyde group, By using those having active groups such as carbonate groups, allyl groups, aminooxy groups, maleimide groups, thiol groups, etc., or by imparting the active groups as necessary, and binding them together, the probe molecules are converted into the It can be immobilized directly to a water-insoluble carrier.

In the case of indirect immobilization, for example, a linker that binds to the probe molecule is immobilized to the water-insoluble carrier, and the probe molecule is bound to the linker to bind the probe molecule to the water-insoluble carrier. Can be fixed indirectly. The linker is not particularly limited. hydrazide) and the like. Alternatively, the probe molecule may be modified in some way, a substance that captures the modified portion may be immobilized on the water-insoluble carrier, and the probe molecule may be immobilized on the water-insoluble carrier. For example, a representative example of the modifying moiety is biotin, and a representative example of a substance that captures the modifying moiety is streptavidin, but the present invention is not limited to these.

The ratio of the water-insoluble carrier and probe molecule to be subjected to these reactions can be appropriately selected so as to achieve the preferred range of the above ratio in the capturing body. In addition, if necessary, for the purpose of preventing non-specific adsorption to the probe molecule or water-insoluble carrier, a suitable blocking agent (eg, bovine serum albumin, gelatin, etc.) may be added to the water-insoluble carrier. Blocking may be performed. Furthermore, as such a trapping body, a commercially available one may be used as appropriate.

(Label)
The "label" according to the present invention is a complex comprising a labeling substance and a probe molecule capable of specifically binding to α2-3 sialylated CEA, wherein the labeling substance and the probe molecule are directly linked. A directly or indirectly bound conjugate. Among the first probe molecule and the second probe molecule, the probe molecule provided in the label according to the first aspect is preferably the other molecule of the molecules provided in the capture body. In this case, The probe molecule provided in the label may be either the first probe molecule or the second probe molecule. From the viewpoint of being used as a probe molecule in preparation for a capturing body, it is preferably a second probe molecule capable of specifically binding to α2-3 sialic acid. Lectins are more preferred, and MAM is particularly preferred.

(blocked labeled lectin)
In the present invention, the labeling substance includes a labeled antibody comprising the labeling substance and the antibody (anti-CEA antibody, anti-α2-3 sialic acid antibody, etc.), a labeled lectin comprising the labeling substance and a lectin, and a block Examples include tagging lectins, which may be used alone or in combination of two or more. Among these, a blocked labeled lectin is preferable as the labeled substance in the present invention, particularly in the first aspect. In the present invention, the term "blocked labeled lectin" refers to a complex comprising a water-soluble carrier made of a water-soluble polymer, and a labeling substance and a lectin immobilized on the water-soluble carrier, wherein the water-soluble carrier and the label It is a conjugate in which a substance and a lectin are bound directly or indirectly. In the present invention, the blocked labeled lectin comprises a lectin capable of specifically binding to α2-3 sialic acid as a second probe molecule. In this case, it is preferable that the probe molecule provided in the capturing body is the first probe molecule. The lectin is as described above, preferably MAM. In addition, the labeling substance is as described above, including its preferred embodiments.

In the blocked labeled lectin, it is sufficient that the labeling substance and the lectin are supported on the water-soluble carrier. Even if both the substance and the lectin are bound to the other two, even if the lectin is bound to the water-soluble carrier via the labeling substance, the labeling substance is bound to the water-soluble carrier via the lectin. may In general, the affinity at each binding point between a lectin and a lectin-binding sugar chain structure is weak. to form Therefore, by using this, multiple binding points are generated in one complex, and the affinity as a whole is improved, making it possible to detect the target α2-3 sialic acid with high sensitivity.

<Water-soluble carrier>
The water-soluble carrier contained in the blocked labeled lectin mainly functions as a carrier for carrying the labeling substance and the lectin, and is composed of a water-soluble polymer. The water-soluble polymer constituting the water-soluble carrier according to the present invention (hereinafter referred to as "first water-soluble polymer") is particularly a water-soluble polymer capable of immobilizing and supporting the labeling substance and lectin. There are no restrictions. In the present invention, the term "water-soluble polymer" means that the solubility in water at normal temperature and normal pressure exceeds 0.01 g/mL, preferably 0.05 g/mL or more, more preferably 0.1 g/mL or more. A polymer compound is shown.

As the first water-soluble polymer according to the present invention, the weight-average molecular weight (weight-average molecular weight in terms of polystyrene by gel permeation chromatography (GPC), hereinafter the same) is from the viewpoint of sensitivity of measurement and water solubility. From this point of view, it is preferably 6,000 to 4,000,000, more preferably 20,000 to 1,000,000.

Further, the first water-soluble polymer according to the present invention has an average mass of 70,000 to 1,000,000 Da from the viewpoint of obtaining a blocked labeled antibody with a more preferable average particle size. and more preferably 150,000 to 700,000 Da.

In addition, as the blocked labeled lectin, one blocked labeled lectin may contain, as the first water-soluble polymer, a plurality of types of water-soluble polymers having different weight-average molecular weights. Furthermore, as the blocked labeled lectin, a high molecular weight blocked labeled lectin in which the weight average molecular weight of the first water-soluble polymer is 200,000 or more, and a high molecular weight blocked labeled lectin in which the weight average molecular weight of the first water-soluble polymer is 100, 000 (more preferably 100,000 or less) with a low molecular weight blocked labeled lectin, and the weight average molecular weight of the first water-soluble polymer is 200,000 to 700,000 (further A high molecular weight blocked labeled lectin, preferably 250,000 to 500,000), and a first water-soluble polymer having a weight average molecular weight of 20,000 to 100,000 (more preferably 50,000 to 70,000) ) with a low-molecular-weight blocked labeled lectin. When the high-molecular-weight blocked labeled lectin and the low-molecular-weight blocked labeled lectin are combined as the blocked-labeled lectin, their mass ratio (mass of high-molecular-weight blocked labeled lectin:mass of low-molecular-weight blocked labeled lectin) is preferably 10:1 to 1:10, more preferably 5:1 to 1:5, even more preferably 3:1 to 1:3.

Examples of the first water-soluble polymer according to the present invention include dextran, aminodextran, Ficoll (trade name), dextrin, agarose, pullulan, various celluloses (eg, hemicellulose, ligrin, etc.), chitin, chitosan, and the like. polysaccharide; β-galactosidase; thyroglobulin; hemocyanin; polylysine; or a combination of two or more. Among these, the first water-soluble polymer according to the present invention is inexpensive and available in large quantities, and is relatively easy to chemically process such as addition of functional groups and coupling reactions. is preferably at least one selected from the group consisting of polysaccharides and modifications thereof, and at least one selected from the group consisting of dextran and aminodextran, and modifications thereof More preferred, dextran is even more preferred.

<Construction and production method of blocked labeled lectin>
In the blocked labeled lectin, the content of the labeling substance is not particularly limited, and can be appropriately adjusted according to the measurement mechanism and the like. It is preferable to set the number of molecules of the labeling substance to be bound to one molecule as large as possible. For example, when the labeling substance is an enzyme, the mass of the first water-soluble polymer is a combination of two or more, the same shall apply hereinafter) The mass of the labeling substance per 100 parts by mass (the sum of them when the labeling substance is a combination of two or more) is 100 to 1, It is preferably 000 parts by mass, more preferably 300 to 800 parts by mass.

In the blocked labeled lectin, the lectin content is not particularly limited, but is set so that the number of lectin molecules that bind to one molecule of the first water-soluble polymer is as large as possible in order to further improve the measurement sensitivity. For example, the mass of the lectin relative to 100 parts by mass of the first water-soluble polymer (when the lectins are a combination of two or more, the total thereof) is 100 to 2,000 parts by mass. preferably 300 to 1,500 parts by mass.

The blocked labeled lectin preferably has a weight average molecular weight of 1,000,000 to 10,000,000, preferably 1,500,000 to 5,000, per molecule of the blocked labeled lectin. 000 is more preferred. When the weight-average molecular weight is 1,000,000 or more, the measurement sensitivity tends to be higher. be.

The blocked labeled lectin can be produced by immobilizing the labeled substance and lectin on the water-soluble carrier. As such a production method, a conventionally known method or a method analogous thereto can be appropriately adopted, and the labeling substance and lectin (hereinafter collectively referred to as “supported substance” in some cases) are directly immobilized on the water-soluble carrier. may be fixed or indirectly fixed.

As a method for directly fixing the to-be-supported substance to the water-soluble carrier, for example, the first water-soluble polymer constituting the to-be-supported substance and/or the water-soluble carrier has a carboxy group, an epoxy group, or a tosyl group. , an amino group, a hydroxy group, an isothiocyanate group, an isocyanate group, an azide group, an aldehyde group, a carbonate group, an allyl group, an aminooxy group, a maleimide group, a thiol group, an active group such as a pyridyl disulfide group, or the above A method of using a water-soluble polymer having these active groups as the substance to be supported and/or the water-soluble carrier and binding them together for immobilization can be exemplified. As the material to be supported and the first water-soluble polymer to which the active groups are attached, commercially available products may be used as they are, or the active groups may be added to the surface of the material to be supported and the water-soluble polymer under appropriate reaction conditions. may be introduced and prepared. As an example, thiol groups can be introduced using commercially available reagents such as S-acetylmercaptosuccinic anhydride and 2-iminothiolane hydrochloride. Further, introduction of a maleimide group to an amino group on the first water-soluble polymer constituting the material to be supported and/or the water-soluble carrier can be performed by, for example, N-(6-maleimidocaproyloxy) succinimide or N It can be carried out using commercially available reagents such as -(4-maleimidobutyryloxy)succinimide. Introduction of a pyridyl disulfide group can be performed by, for example, N-Succinimidyl 3-(2-pyridyldithio)propionate (SPDP), N-{6-[3-(2-Pyridyldithio)propionamido]hexanoyloxy}sulfosuccinimide, sodium sulfosuccinimide, SPDP) and other commercially available reagents can be used. A pyridyl disulfide group can also be introduced by reducing it to a thiol group after introduction.

Methods for indirectly immobilizing the substance to be supported on the water-soluble carrier include, for example, oligopeptides containing polyhistidine, polyethylene glycol, cysteine and/or lysine, linker molecules having the active group (for example, the above capture method of fixing via a linker such as those mentioned in the method for producing the body). The selection and size of the linker can be appropriately set in consideration of the strength of binding to the substance to be supported, steric hindrance due to immobilization of the substance to be supported on the water-soluble carrier, and the like.

In the method for producing the blocked labeled lectin, the labeling substance and the lectin may be immobilized on the water-soluble carrier at once, or they may be immobilized separately and sequentially. From the viewpoint of easiness in controlling the amounts of substances and lectins, it is preferable to immobilize one of them on the water-soluble carrier before immobilizing the other. In addition, the blocked labeling lectin is obtained by immobilizing the labeling substance and the lectin on separate water-soluble carriers, and the labeling substance (blocked labeling substance) immobilized on one water-soluble carrier and the other water-soluble carrier. It can also be produced by binding to a lectin (blocked lectin) immobilized on a protein directly or via the linker or the like.

The method for producing the blocked labeled lectin is not particularly limited. For example, as described in Examples below, the labeling substance is an enzyme, and the first water-soluble polymer is a polysaccharide or glycoprotein. , first, the first water-soluble polymer is oxidized with an oxidizing agent such as sodium periodate to give an aldehyde group, reacted with hydrazine hydrochloride, and then treated with dimethylamine borane (DMAB). hydrazinization by reacting with a reducing agent such as On the other hand, the enzyme is also oxidized with an oxidizing agent such as sodium periodate to impart an aldehyde group to its sugar chain. Next, the hydrazine residue and the aldehyde group provided above are reacted to form a hydrazone bond to obtain the first water-soluble polymer-enzyme conjugate. The resulting first water-soluble polymer-enzyme conjugate is treated with a crosslinker having N-hydroxysuccinimide and a maleimide group at each end (e.g., SM(PEG) ₄ , SMCC, etc.) to introduce a maleimide group. do. On the other hand, a lectin is thiolated with a thiolating reagent to give a thiol group, or in the case of a lectin having a disulfide bond in its molecule, a thiol group is obtained by reduction. Finally, the first water-soluble polymer (water-soluble carrier)-enzyme-lectin is formed by binding the maleimide group introduced into the first water-soluble polymer-enzyme conjugate with the thiol group provided to the lectin. can be covalently bonded. According to this method, a lectin is obtained by binding two or more molecules of the first water-soluble polymer via an enzyme. The ratios of the first water-soluble polymer, the labeling substance, and the lectin subjected to these reactions can be appropriately selected so as to achieve the preferred range of each content in the blocked labeling lectin.

(capturing step)
In the capturing step, the method for contacting the sample with the capturing body is not particularly limited, and a conventionally known method or a method based thereon can be employed as appropriate. In the case of , the method of injecting the sample into this (probe molecule-immobilized plate), or when the water-insoluble carrier is a particle, the capturing body (probe molecule-immobilized particle) is added to the sample. method.

In the reaction between the sample and the capturing body in the capturing step, the content (final concentration) of the capturing body in the reaction solution containing the capturing body and the sample (when the capturing body is a combination of two or more is the total of them, the same applies hereinafter) is not particularly limited, and is not particularly limited because it is appropriately adjusted according to the type of sample, concentration, etc., but from the viewpoint of efficient capture in a short time, for example, It is preferably 0.01 to 1.5% by mass, more preferably 0.05 to 1% by mass, even more preferably 0.1 to 0.5% by mass.

In addition, the conditions for the capture step are not particularly limited, and can be adjusted as appropriate. It can be carried out for about 10 minutes, preferably about 30 seconds to 5 minutes, but is not limited to these conditions.

(labeled step)
In the reaction between the labeled product and α2-3 sialylated CEA in the labeling step, the content (final concentration) of the labeled product in the reaction solution containing the labeled product and α2-3 sialylated CEA (labeled product is a combination of two or more, the sum of them, the same applies hereinafter) is not particularly limited, and is not particularly limited because it is adjusted appropriately according to the type of sample, concentration, etc., but excessive From the viewpoint of the possibility of generating a high background signal when used, for example, it is preferably 0.001 to 10 μg / mL, more preferably 0.01 to 5 μg / mL, 0.1 to 1 μg / More preferably in mL.

In addition, other conditions for the labeling step are not particularly limited, and can be adjusted as appropriate. to 6.5, the heating can be performed for about 3 minutes to 120 minutes, preferably about 5 minutes to 10 minutes, but the conditions are not limited to these.

(Washing step)
The sandwich method includes, after the capturing step and/or the labeling step, α2-3 sialylated CEA bound to the capture body and other substances not bound to the capture body (not captured). It is preferable to further include a washing step to separate and remove contaminants. The method for removing the contaminants is not particularly limited, and conventionally known methods or methods based thereon can be employed as appropriate. Examples include a method of removing the liquid phase (supernatant) from above, and a method of recovering the particles by centrifugation or magnet collection and removing the liquid phase (supernatant) in the case of the probe molecule-immobilized particles. In addition, in the cleaning step, the injection and removal of the cleaning liquid may be repeated as necessary. Examples of the washing solution include neutral (preferably pH 6 to 9) known buffers (sodium phosphate buffer, MES, Tris, CFB, MOPS, PIPES, HEPES, tricine buffer, bicine buffer, glycine buffer, etc.). and may contain a stabilizing protein such as BSA, a surfactant, or the like.

When the sandwich method is used as the measurement step according to the present invention, the sample may be diluted with a diluent as described above. may be used by suspending it in the particle suspension medium (particle liquid), and further, another reaction buffer may be appropriately added to the reaction system between the sample and the capturing body and/or the labeling body. may These particle suspension media and reaction buffers are not particularly limited. buffer, glycine buffer, etc.), and may be independently added with a stabilizing protein such as BSA, serum, or the like.

Further, when the blocked labeled lectin is used as the label, a water-soluble polymer (hereinafter referred to as "second different from the first water-soluble polymer constituting the water-soluble carrier in that it is a free water-soluble polymer that does not carry the labeling substance and lectin), free lectin ( It differs from the lectin contained in the blocked labeled lectin in that it is neither immobilized on the water-soluble carrier nor the labeling substance.

Examples of the second water-soluble polymer include those mentioned as the first water-soluble polymer. good. Moreover, it may be the same type of polymer as the first water-soluble polymer. Among these, the second water-soluble polymer is preferably at least one selected from the group consisting of polysaccharides and modifications thereof, from the viewpoint that the measurement sensitivity tends to be further improved, and dextran and aminodextran, and at least one selected from the group consisting of modifications thereof, more preferably dextran.

Further, the weight average molecular weight of the second water-soluble polymer is preferably 500,000 to 5,000,000, more preferably 1,000,000 to It is more preferably 3,000,000, and even more preferably 1,500,000 to 2,500,000.

The amount of the second water-soluble polymer is not particularly limited, but the content of the second water-soluble polymer in the reaction solution containing the sample, the blocked labeled lectin, and the second water-soluble polymer ( When the second water-soluble polymer is a combination of two or more types, the total thereof) is preferably 0.01 to 10 w/v%, and preferably 0.5 to 3 w/v%. More preferred (w/v %: weight/volume (g/mL) percentage, same below).

Examples of the free lectin include those exemplified above as the lectin, and one of them may be used alone, or two or more of them may be used in combination. Moreover, it may be of the same kind as the lectin contained in the blocked labeled lectin. Among these, the free lectin is particularly preferably of the same type as the lectin contained in the blocked labeled lectin, from the viewpoint of improving reactivity and suppressing background.

The amount of the free lectin is not particularly limited, but the content of the free lectin (free When the lectin is a combination of two or more, the total thereof) is preferably 1 to 10,000 parts by mass, more preferably 10 to 5,000 parts by mass.

(measurement step)
In the measuring step, a signal is measured according to the labeling substance. For example, when the labeling substance is an enzyme, a signal (for example, color development or luminescence) generated by adding a chromogenic or luminescent substrate corresponding to the enzyme and reacting is measured. As a result, the amount of α2-3 sialylated CEA in the sample can be detected as a signal amount. The amount of CEA added can be quantified.

[Cancer detection method]
According to the cancer detection method of the present invention, the amount of α2-3 sialylated CEA measured in the above-described measurement step is used as an index to detect the presence of prostate cancer in the sample or the subject from which the sample is derived. It is possible to detect the presence or absence of Such a cancer detection method can be used for research purposes such as drug discovery, as well as for the cancer examination method of the present invention.

[Cancer test method]
In the cancer testing method of the present invention, the amount of α2-3 sialylated CEA in a sample derived from a subject is measured, and the presence or absence of prostate cancer is detected using this as an index, whereby in the subject, It is possible to predict whether or not a person is afflicted with prostate cancer, determine whether or not he or she is afflicted with the cancer, or evaluate the degree of progression or severity of the cancer.

That is, specific aspects of the cancer testing method of the present invention include, for example, the following aspects:
A method for screening a subject predicted to have prostate cancer, comprising a measuring step of measuring the amount of α2-3 sialylated CEA in a sample derived from the subject; A screening method, comprising a screening step of screening subjects using the amount of acid-added CEA as an index;
A method for discriminating the presence or absence of prostate cancer in a subject, comprising a measuring step of measuring the amount of α2-3 sialylated CEA in a sample derived from the subject, and the measured α2-3 sialylated CEA. and a discrimination step of discriminating a subject using the amount of CEA as an index;
A method for evaluating the degree of progression or severity of prostate cancer in a subject, comprising a measuring step of measuring the amount of α2-3 sialylated CEA in a sample derived from the subject, and the measured α2-3 sialyl and an evaluation step of evaluating the subject using the amount of acid-added CEA as an index;
is mentioned.

[Screening method]
In the present invention, "screening" means selection of subjects who may have prostate cancer. Specifically, in the present invention, the screening method includes predicting that the subject is highly likely to be afflicted with prostate cancer (including recurrence). includes a method of selecting from In the screening method, depending on the purpose, for example, subjects may be screened at a level at which a medium possibility can be expected.

More specifically, for example, two groups, a group consisting of persons truly affected by the cancer (positive group) and a group consisting of persons not affected by the cancer (normal group: negative group). , and the subjects are sorted out from the normal group (negative group) by predicting that they will be included in the positive group. As a criterion for prediction in this case, for example, a negative concordance rate (percentage of subjects predicted to be in the normal group by the screening that are truly in the normal group) is preferably 95% or more.

[Distinction method]
In the present invention, "differential" means distinguishing prostate cancer from other diseases or conditions to determine whether or not a subject is affected by the cancer. In addition, it includes not only the determination of the presence or absence of the disease, but also the evaluation of the degree of the possibility of the disease (evaluation such as high/moderate/low). In the present invention, the discrimination method specifically includes a method for determining whether or not a subject suffers from prostate cancer or whether there is a high possibility of having prostate cancer, regardless of the presence or absence of symptoms. , for example, a method for determining whether the cancer that the subject is suffering from is prostate cancer, or a method for determining that the cancer is likely to be the cancer; It also includes a method for determining whether or not prostate cancer has recurred, or a method for determining that there is a high possibility that the cancer has recurred.

More specifically, for example, two groups, a group consisting of persons truly affected by the cancer (positive group) and a group consisting of persons not affected by the cancer (normal group: negative group). Then, it is determined whether the subject is included in the positive group or the negative group. Such differentiation can be applied to assist doctors in diagnosing prostate cancer. As a discrimination criterion in this case, for example, it is preferable that the negative concordance rate (percentage of subjects who are discriminated as the normal group by the discrimination actually belong to the normal group) is 95% or more.

〔Evaluation method〕
In the present invention, the evaluation method includes, for example, a method of evaluating the progression or severity of prostate cancer that a subject is suffering from; a method for providing an index for; and a method for determining the therapeutic effect of the cancer.

More specifically, for example, the amount of α2-3 sialylated CEA in a group consisting of those who are not affected by cancer or those with low progression or severity of cancer, and the α2- The amount of 3-sialylated CEA is compared, and if the amount in the subject is higher, the degree of progression or severity can be evaluated as high, and if the amount in the subject is lower, the degree of progression or severity can be evaluated as low. In addition, the amount of α2-3 sialylated CEA at the time of the cancer discovery or before treatment in the subject is compared with the current amount of α2-3 sialylated CEA, and if the amount is increased, the therapeutic effect can be evaluated as low, and if it decreases, the therapeutic effect can be evaluated as high.

(cutoff value)
In the selection step of the screening method and the determination step of the discrimination method, for example, the amount of α2-3 sialylated CEA measured in the measurement step is compared with a predetermined cutoff value, and the α2-3 A subject whose amount of sialylated CEA is higher than the cutoff value is predicted or discriminated as having (or having a high possibility of having) prostate cancer.

In the present invention, the "cutoff value" is a predetermined value that serves as a reference for determination based on the amount of α2-3 sialylated CEA, and is a boundary for determining the positive group and the negative group. Indicates a value. Such a cut-off value is appropriately set depending on the purpose of the cancer testing method of the present invention, the method for measuring the amount of α2-3 sialylated CEA, the properties of the subject and sample, the dilution conditions, and the like. , is not particularly limited. For example, by setting the cut-off value to a relatively low value, the detection sensitivity is high, that is, patients in the early stages of cancer can be picked up to some extent, and early detection becomes possible. On the other hand, since a small amount of α2-3 sialylated CEA is detected even in samples derived from healthy subjects, setting the cutoff value to a relatively high value enables the screening and the discrimination to be performed with higher accuracy. becomes possible.

As an example of the cutoff value, for example, the sample is a serum specimen, diluted to 1/10 by volume, anti-CEA antibody-immobilized particles are used as the capturing agent, and blocked labeled MAM is used as the labeling agent. When the amount of α2-3 sialylated CEA measured by a sandwich immunoassay using is indicated by the emission intensity (count) of light having a maximum absorption at a wavelength of 463 nm with ALP as the labeling substance and AMPPD as the substrate (of Example 1 case) includes, but is not limited to, 210,000 to 320,000 counts, preferably 270,000 to 300,000 counts. It should be noted that the cut-off value determined in the above range can be any value within the range depending on the purpose of the cancer testing method of the present invention, the method for measuring the amount of α2-3 sialylated CEA, and the properties of the subject and sample. or one point is selected and applied.

According to such a cancer examination method of the present invention, it is possible to provide information for determining a treatment policy specific to prostate cancer, distinguishing it from other cancers. In addition, early detection and early treatment intervention of prostate cancer can be achieved by identifying subjects who are suffering from prostate cancer (including recurrence) or who are likely to have it, and conducting further examinations and definitive diagnoses. becomes possible. Furthermore, since it has been reported that information on the sugar chain structure derived from cancer serves as an index of cancer invasiveness, information on cancer malignancy and prognosis can be obtained by the cancer testing method of the present invention. It is also possible to obtain The method of the present invention is also a method of assisting a doctor's diagnosis of prostate cancer, or a method of providing information for a doctor's diagnosis of prostate cancer.

Furthermore, the method of the present invention is also suitable as a method for combining with conventional CEA measurement methods and other monitoring marker measurement methods, thereby further improving the detection sensitivity and diagnostic accuracy of prostate cancer. becomes possible.

<Kit>
The kit of the present invention is a kit for use in the cancer detection method or cancer examination method of the present invention, and comprises a first probe molecule capable of specifically binding to CEA and a and a second probe molecule capable of binding to. The first probe molecule and the second probe molecule are as described above, including their preferred embodiments.

In addition, the kit of the present invention includes a capture body comprising a water-insoluble carrier and either one of a first probe molecule and a second probe molecule immobilized on the water-insoluble carrier; and It is also preferred to provide a label comprising a labeling substance and the other of the first probe molecule and the second probe molecule. The capturing body and the labeling body are as described above, including their preferred embodiments.

In the kit of the present invention, the first probe molecule, the second probe molecule, the capturing body, and the labeling body are each independently solid (powder) or liquid dissolved in a buffer solution. may be When liquid, the concentrations of the first probe molecule, the second probe molecule, the capturing body, and the labeling body in each solution are not particularly limited, but are each independently, for example, 0.01 to 10 μg/ It is preferably mL, more preferably 0.1 to 5.0 μg/mL, even more preferably 0.5 to 3.0 μg/mL.

The kit of the present invention may further comprise components to be provided in conventional immunoassay methods such as ELISA, CLEIA, immunochromatography, and similar methods. For example, when the above sandwich method is used as the principle of the measurement method according to the present invention, magnetic beads or a plate for immobilizing the probe molecule, a sensor chip, the standard sample (each concentration), a control reagent, It may further include at least one selected from the group consisting of the particle suspension medium, reaction buffer, washing solution, second water-soluble polymer, and free lectin. Moreover, when the labeling substance is an enzyme, it may further contain a substrate, a reaction stopping solution, and the like necessary for detection and quantification of the labeling substance.

Furthermore, the kit of the present invention may optionally include the diluent, a pretreatment liquid for pretreatment of the sample; a cartridge for dilution; and a pretreatment reaction stopping solution or neutralizing solution. . Moreover, when immunochromatography is employed as the sandwich method, a device including a zone carrying the capturing body and/or the labeling body may be further included. The device can include other components suitable for immunochromatography, such as a developing pad and an absorbent pad. Furthermore, the kit of the present invention may further include instructions for use of the kit.

EXAMPLES The present invention will be described in more detail below based on examples and comparative examples, but the present invention is not limited to the following examples. In each example and comparative example, "%" means weight/volume (w/v: g/mL) percentage unless otherwise specified.

(Example 1) Measurement of MAM-linked glycosylated CEA (CEA/MAM) contained in a serum specimen using blocked labeled lectin (MAM) (1) Preparation of hydrazinated dextran 4.8 mL of 0.1 M phosphorus 240.0 mg of dextran (manufactured by CarboMer) having a molecular weight of 500 k was added to an acid buffer (pH 7.0) and dissolved by stirring in a dark place at 25° C. for 30 minutes. Then, 2.664 mL of 150 mM NaIO ₄ and 0.536 mL of deionized water were added and stirred in the dark at 25° C. for 30 minutes. Then, using a PD-10 column (manufactured by GE Healthcare, Sephadex G-25 packed column: hereinafter simply "Sephadex G-25"), the buffer was exchanged with 0.1 M sodium phosphate buffer (pH 6.0), 20.0 mL of solution was obtained. To the resulting solution was added 5.04 g of NH ₂ NH ₂ .HCl and stirred at 25° C. in the dark for 2 hours. 800 mg of DMAB (dimethylamine borane) was added and further stirred at 25° C. in the dark for 2 hours. Using an RC50K (regenerated cellulose with a molecular weight of 50,000) dialysis membrane, dialysis with 4 L of ion-exchanged water was performed in a dark place for 3 hours, and then allowed to stand overnight at 4°C. Buffer exchange was performed by gel filtration (Sephadex G-25) using 0.1 M sodium phosphate buffer (pH 6.0) to obtain 85.0 mL of solution. The concentration of dextran was adjusted to 1.0 mg/mL to obtain a solution of hydrazinated dextran.

(2) Preparation of dextran-enzyme conjugate 30.0 mL of 10 mg/mL alkaline phosphatase (ALP-50, manufactured by Oriental Yeast Co., Ltd.) was subjected to gel filtration (Sephadex) using 0.1 M sodium phosphate buffer (pH 6.0). G-25), buffer exchange was performed to prepare 90.6 mL of a 3.0 mg/mL solution. Then 45.3 mL of 27 mM NaIO ₄ was added and stirred for 3 min at 25° C. in the dark. Then, buffer exchange was performed by gel filtration (Sephadex G-25) using 0.1 M sodium phosphate buffer (pH 6.0) to prepare a 0.5 mg/mL solution. 1.0 mg/mL hydrazide dextran prepared in (1) of Example 1 was added so that the hydrazide group (amino group) concentration was 25 μM, and the mixture was stirred at 25° C. in the dark for 16 hours. 85 mg of DMAB was added and stirred in the dark at 25° C. for 2 hours. Then, 10.1 mL of 1.5 M Tris buffer (pH 9.0) was added and stirred for 2 hours in the dark at 25°C. An ultrafiltration module (Pellicon XL50, manufactured by Merck Millipore) was attached to Labscale TFF System (manufactured by Merck Millipore), concentrated to 15 mL, and subjected to gel filtration (Superdex 200 pg ) to obtain 14 mL of a 3.0 mg/mL dextran-enzyme conjugate solution.

(3) Maleimide-PEGylation of dextran-enzyme conjugate 0.1 M sodium phosphate buffer (pH 7.0) was added to the dextran-enzyme conjugate prepared in (2) of Example 1 to give 2 mg/mL dextran-enzyme. 750 μL of conjugate was prepared. To this, 8.35 μL of 250 mM SM(PEG) ₄ (SM(PEG) ₄ manufactured by Thermo Fisher Scientific) dissolved in DMSO was added, mixed, and mixed by inversion in a dark place at 25° C. for 1 hour. After the reaction, the buffer was exchanged with 0.1 M sodium phosphate buffer (pH 6.3) containing 20 mM EDTA/2Na and 0.5% CHAPS using a PD-10 column (Sephadex G-25). After buffer exchange, the maleimide-PEGylated dextran-enzyme conjugate was concentrated using a centrifugal filter (Merck, Amicon Ultra 50K) to adjust the final concentration to 2 mg/mL.

(4) Thiolation of Lectin 6 mg of canine endurance lectin (MAM; manufactured by J-Chemical) was dissolved in 3 mL of 0.1 M sodium phosphate buffer (pH 7.0) to obtain a 2 mg/mL MAM solution. Add 100 μL of 0.5 M EDTA.2Na (pH 8.0) to 2.5 mL of MAM solution and mix, then add 75 μL of 10 mg/mL 2-iminothiolane hydrochloride solution and incubate at 25° C. in the dark. Mixed by inversion for 1 hour. After the reaction, the buffer was exchanged with 0.1 M sodium phosphate buffer (pH 6.3) containing 20 mM EDTA/2Na and 0.5% CHAPS using a PD-10 column (Sephadex G-25). MAM after buffer exchange was adjusted to 650 μg/mL.

(5) Coupling Maleimide-PEGylated dextran-enzyme conjugate obtained in (3) of Example 1 was added to 2 mL of thiolated MAM adjusted to 650 μg/mL obtained in (4) of Example 1. 500 μL of solution (2 mg/mL) was added and mixed by inversion in the dark at 25° C. for 1 hour to couple MAM and dextran-enzyme conjugate. After the reaction, 25 μL of 200 mM 3-Mercapto-1,2-propanediol was added and mixed by inversion for 30 minutes at 25° C. in the dark. After that, 50 μL of 200 mM 2-Iodoacetamide was added and mixed by inversion for 30 minutes in the dark at 25°C. After the reaction, the solution was concentrated using a centrifugal filter (Merck, Amicon Ultra 50K), passed through a φ0.22 μm filter, and subjected to gel filtration chromatography (column: Superose 6 Increase 10/300 GL, buffer: 0.000). 1 M MES, 0.5 M NaCl, 1 mM MgCl ₂ , 0.1 mM ZnCl ₂ , 5 mM Glucose, 0.05% CHAPS, pH 6.8) to give a final concentration of 382.1 μg/mL blocked labeled lectin (MAM ) (dextran-enzyme-MAM conjugate) solution was obtained in 2.0 mL.

(6) Preparation of Measurement Reagent 6 mL of 20 mM sodium periodate solution (20 mM NaIO ₄ , 100 mM NaOAc, 150 mM NaCl, pH 5.5) was added to 24 mg of anti-CEA antibody-bound magnetic particles (manufactured by Fujirebio). The particles were mixed by mixing under a light-shielding condition at 4° C. for 30 minutes by inversion mixing to oxidize the sugar chains of the antibody bound to the particles. The oxidized particles were washed three times with 6 mL of 0.1 M sodium phosphate buffer (pH 6.0). The washed particles were replaced with 0.1 M sodium phosphate buffer (pH 6.0) containing 10 mM glycine, and mixed by inversion for 1 hour at 25°C in the dark to remove aldehyde groups generated by oxidation of sugar chains of antibodies. blocked with glycine. Furthermore, 100 μL of 10 mg/mL DMAB was added to the antibody-bound particle solution after the reaction, and mixed by inversion for 30 minutes in the dark at 25° C. to stabilize the unstable bond between the aldehyde group derived from the antibody sugar chain and glycine. let me After the reaction, the particles were washed three times with 0.6 mL of a buffer containing 2% BSA (50 mM MES, 1 mM EDTA, 150 mM NaCl, 2% BSA, 0.1% ProClin 300, pH 6.0). BSA was physically adsorbed by mixing by inversion for 16 hours at ℃. The antibody-bound particles to which BSA was physically adsorbed were washed three times with a storage buffer (50 mM Tris, 2% BSA, 150 mM NaCl, 1 mM EDTA, 0.1% ProClin 300, pH 7.2), and stored in the same buffer at 4°C. saved. The obtained oxidized anti-CEA antibody-bound magnetic particles were diluted with a 50 mM Tris-based solution so that the concentration of the antibody-bound particles was 0.005% to prepare an oxidized anti-CEA antibody-bound particle solution. did.

In addition, the blocked labeled lectin (MAM) obtained in (5) of Example 1 was diluted to a concentration of 0.5 μg/mL in a 50 mM MES-based solution to prepare a labeled body fluid.

(7) Measurement of CEA and MAM-linked glycosylated CEA (CEA/MAM) contained in serum specimens 10 serum specimens collected from healthy subjects (group of healthy subjects: 1 to 10 healthy subjects) and from patients with benign prostatic hyperplasia For 10 serum specimens (prostate hypertrophy group: prostatic hyperplasia 1 to 10) and 15 serum specimens collected from prostate cancer patients (prostate cancer group: prostate cancer 1 to 15), respectively, a sample diluent (Fujirebio Co., Ltd.) (manufactured) to a concentration of 1/10 by volume.

For each diluted sample, Lumipulse (registered trademark) L-2400 (manufactured by Fujirebio) was used to measure MAM-linked glycosylated CEA (CEA/MAM) contained in the serum sample. That is, 50 μL of each diluted sample solution was mixed with 50 μL of the oxidized anti-CEA antibody-bound particle liquid prepared in (6) of Example 1, and reacted at 37° C. for 8 minutes. Next, the magnetic particles were collected and washed five times with Lumipulse (registered trademark) washing solution (manufactured by Fujirebio). Next, 50 μL of the labeled body fluid prepared in (6) of Example 1 was added to each well, and reacted at 37° C. for 8 minutes. Next, the magnetic particles were collected, washed five times, and then AMPPD (3-(2′-spiroadamantane)-4-methoxy-4-(3′-phosphoryloxy)phenyl-1,2-dioxetane disodium 50 μL of LUMIPULSE (registered trademark) substrate solution (manufactured by Fujirebio) containing salt) was added and reacted at 37° C. for 4 minutes. The luminescence intensity (count) of light having a maximum absorption at a wavelength of 463 nm, which is emitted by the decomposition of AMPPD by the catalytic action of alkaline phosphatase of blocked labeled lectin (MAM) bound to magnetic particles, was measured using Lumipulse (registered trademark). ) L-2400 (manufactured by Fujirebio Co., Ltd.) was used for measurement, and the measurement results were obtained.

In addition, the CEA contained in each sample diluted above was obtained using Lumipulse Presto (registered trademark) CEA (Fujirebio) equipped with anti-CEA antibody-binding particles and alkaline phosphatase (ALP)-labeled anti-CEA antibody. Similarly, it was measured by Lumipulse (registered trademark) L-2400 (manufactured by Fujirebio Co., Ltd.). The measurement results were output as the luminescence intensity (count) of the substrate (AMPPD) in the same manner as described above. The measurement results for each specimen are shown in Table 1 below. The results shown are the average values of duplicate measurements.

(8) Comparison of measured values among healthy subjects, prostatic hyperplasia, and prostate cancer groups Regarding the results of Table 1, examine whether there is a statistically significant difference between the healthy subjects group and the prostate cancer group. did. FIG. 1 shows the CEA measurement results for the healthy subject group and the prostate cancer group, FIG. 2 shows the CEA/MAM measurement results for the healthy subject group and the prostate cancer group, and FIG. 3 shows the CEA measurement results for the prostatic hyperplasia group and the prostate cancer group. , and FIG. 4 shows the measurement results of CEA/MAM in the prostatic hyperplasia group and the prostatic cancer group, respectively.

When tested by the Wilcoxon test, no significant difference was observed between the healthy subject group and the prostate cancer group in the CEA measurement value (Fig. 1, p = 0.1018). A significant difference was observed for the measured value of /MAM (Fig. 2, p = 0.0247). Similarly, between the prostatic hyperplasia group and the prostatic cancer group, no significant difference was observed in the CEA measurements (Fig. 3, p = 0.9337), whereas the CEA/MAM measurements A significant difference was observed for (Fig. 4, p = 0.0006).

Also, FIG. 5 shows the relationship between the CEA measured value and the CEA/MAM measured value. As shown in FIG. 5, no clear correlation was shown between measured CEA and CEA/MAM (regression line: CEA/MAM (count)=257173.58-10.643388 x CEA (counts), ^R2 = 0.015112, ANOVA p-value = 0.4817). For this reason, the measured value of CEA/MAM does not simply represent the abundance of CEA, but CEA (recognized by an anti-CEA antibody capable of specifically binding to sites other than MAM-linked sugar chains). were shown to be different biomarkers.

From the above, it was shown that the measurement of CEA/MAM serves as a novel index for discriminating between healthy subjects and prostate cancer patients, or between prostatic hyperplasia patients and prostate cancer patients.

(9) Comparison of CEA/MAM Measured Values and PSA Measured Values First, 10 serum specimens collected from patients with benign prostatic hyperplasia similar to those used in (7) of Example 1, serum collected from patients with prostate cancer For 15 samples, the amount of PSA contained in the samples was measured. Measurement was carried out for each sample without dilution, using Lumipulse Presto (registered trademark) PSA (manufactured by Fujirebio) equipped with anti-PSA antibody-binding particles and alkaline phosphatase (ALP)-labeled anti-PSA antibody, Lumipulse (registered trademark) ) Measured by L-2400 (manufactured by Fujirebio). The measurement results were output as PSA concentrations (ng/mL). The measurement results for each sample are shown in Table 2 below.

Next, the results in Table 2 were tested for the ability to detect prostate cancer patients using a common cutoff value of 4 ng/mL for PSA measurements and testing positive samples above the cutoff value. As a result, 4 out of 15 specimens in the prostate cancer group, or 26.7%, were determined to be positive at the cut-off value. In addition, in the prostatic hyperplasia group, 1 out of 10 specimens was determined to be positive at the cutoff value, which was a false positive.

On the other hand, for the CEA/MAM measurement values (Table 1), the cutoff value was calculated from the measurement values of the healthy subject group, and the ability to detect prostate cancer patients was tested when samples exceeding the cutoff value were considered positive. The cut-off value was calculated as 299,216 counts or less by adding the value obtained by multiplying the standard deviation of the measured values of the healthy subject group by 2 to the mean of the measured values of the healthy subject group. As a result, 8 out of 15 specimens in the prostate cancer group were determined to be positive at this cut-off value, accounting for 53.3%. In addition, all 20 samples from the healthy subject group and the prostatic hyperplasia group were determined to be negative.

Furthermore, FIG. 6 shows the relationship between the measured values of PSA and the measured values of CEA/MAM. As shown in FIG. 6, the results of this time did not show a clear correlation between the measured values of PSA and the measured values of CEA/MAM (regression line: CEA/MAM (count) = 233853 .68-4056.1952×PSA (ng/mL), R ² =0.008851, ANOVA p-value=0.6546).

From the above, it was shown that the detectability (sensitivity, specificity) of prostate cancer by measuring CEA/MAM is sufficiently high compared to PSA, and that it can be used as a diagnostic index independent of PSA.

(Comparative Example 1) Measurement of sugar chain-added CEA by blocking labeled lectin using lectin other than MAM Blocking by binding Nodafuji lectin (WFA, manufactured by VECTOR) that recognizes N-acetylgalactosamine instead of MAM A labeled lectin (WFA) was prepared. That is, except that WFA was used instead of MAM and the dextran was changed to one with a molecular weight of 250 k (CarboMer), the same method as (1) to (5) of Example 1 was performed, and finally 373 A 2.0 mL solution of 0.9 μg/mL blocked labeled lectin (WFA) (dextran-enzyme-WFA conjugate) was obtained. The size of dextran does not affect the specificity of lectin sugar chains.

In the same manner as in (6) to (7) of Example 1, except that the obtained blocked enzyme lectin (WFA) was used instead of the blocked enzyme lectin (MAM), ( 10 serum specimens collected from healthy subjects similar to those used in 7) (healthy subject group: healthy subjects 1 to 10), and 15 serum specimens collected from prostate cancer patients (prostate cancer group: prostate cancer 1 to 15 ), WFA-linked glycosylated CEA (CEA/WFA) contained in each sample was measured. The measurement results for each sample are shown in Table 3 below.

Regarding the results in Table 3, it was examined whether there was a statistically significant difference between the healthy subject group and the prostate cancer group. FIG. 7 shows the CEA/WFA measurement results for the healthy subject group and the prostate cancer group. Wilcoxon's test showed no significant difference in CEA/WFA measurements between the healthy and prostate cancer groups (Fig. 7, p = 0.4540). From the above, it was shown that the measurement of CEA/MAM, which measures the combination of CEA and MAM-linked sugar chains, is a new specific indicator for discriminating between healthy subjects and prostate cancer patients.

INDUSTRIAL APPLICABILITY According to the present invention, a novel biomarker is used as an indicator to provide a highly sensitive cancer detection method and a cancer examination method capable of specifically detecting prostate cancer, and a kit used for these methods. becomes possible.

Claims (14)

A method for detecting prostate cancer, comprising a measuring step of measuring the amount of α2-3 sialylated CEA in a sample. A method for examining prostate cancer, comprising a measuring step of measuring the amount of α2-3 sialylated CEA in a sample derived from a subject. A method for screening a subject predicted to have prostate cancer, comprising a measuring step of measuring the amount of α2-3 sialylated CEA in a sample derived from the subject; 3. The method according to claim 2, further comprising a selection step of selecting subjects using the amount of acid-added CEA as an index. 4. The method according to any one of claims 1 to 3, wherein in the α2-3 sialylated CEA, the α2-3 sialic acid is a MAM-linked sugar chain that binds to MAM. The measuring step is a step of contacting the sample with a first probe molecule capable of specifically binding to CEA and a second probe molecule capable of specifically binding to α2-3 sialic acid. A method according to any one of claims 1 to 4, characterized in that. 6. Method according to claim 5, characterized in that the first probe molecule is an antibody capable of specifically binding to CEA. Method according to claim 5 or 6, characterized in that the second probe molecule is a lectin capable of specifically binding to α2-3 sialic acid. the measuring step is a step of contacting the sample with a capturing body and a labeling body;
The capture body comprises a water-insoluble carrier and either one of a first probe molecule and a second probe molecule immobilized on the water-insoluble carrier, and
wherein the label comprises a labeling substance and the other of the first probe molecule and the second probe molecule;
A method according to any one of claims 5 to 7, characterized in that The capture body comprises a water-insoluble carrier and a first probe molecule immobilized on the water-insoluble carrier, and
The label comprises a water-soluble carrier, a labeling substance immobilized on the water-soluble carrier, and a second probe molecule, and the second probe molecule is a lectin capable of specifically binding to α2-3 sialic acid. is a blocked labeled lectin that is
9. A method according to claim 8, characterized in that: A kit for use in the method according to any one of claims 5 to 9, comprising a first probe molecule capable of specifically binding to CEA and capable of specifically binding to α2-3 sialic acid and a second probe molecule. 11. Kit according to claim 10, characterized in that the first probe molecule is an antibody capable of specifically binding to CEA. Kit according to claim 10 or 11, characterized in that the second probe molecule is a lectin capable of specifically binding to α2-3 sialic acid. a capture body comprising a water-insoluble carrier and either one of a first probe molecule and a second probe molecule immobilized on the water-insoluble carrier; and
a label comprising a labeling substance and the other of the first probe molecule and the second probe molecule;
Kit according to any one of claims 10 to 12, characterized in that it comprises a The capture body comprises a water-insoluble carrier and a first probe molecule immobilized on the water-insoluble carrier, and
The label comprises a water-soluble carrier, a labeling substance immobilized on the water-soluble carrier, and a second probe molecule, and the second probe molecule is a lectin capable of specifically binding to α2-3 sialic acid. is a blocked labeled lectin that is
14. The kit according to claim 13, characterized in that:

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