JP7539369B2 - Pathological condition information generating method, pathological condition information generating system, HE4 sugar chain analysis kit and HE4 - Google Patents

Description

The present invention relates to a pathological information generation method, a pathological information generation system, an HE4 glycan analysis kit, and HE4. More specifically, the present invention relates to a pathological information generation method that enables diagnosis with reduced burden on patients and doctors by evaluating the malignancy of cancers such as ovarian cancer in a simple and non-invasive manner.

Ovarian cancer is the second most common gynecological cancer after breast cancer. Ovarian cancer is difficult to detect early because it has almost no symptoms in the early stages of onset, and symptoms are often already advanced by the time it is discovered. Therefore, the prognosis is poor, and it is the most fatal gynecological malignant tumor with the highest mortality rate among gynecological cancers.
The majority of ovarian tumors are benign, and it is important to distinguish between malignant and benign tumors in determining a treatment plan. In the diagnosis of ovarian cancer, in addition to a medical interview and a pelvic examination, tumor marker measurements, mainly CA125, transvaginal ultrasound examination, MRI (Magnetic Resonance Imaging) examination, and CT (Computed Tomography) examination are performed as necessary.

CA125 is the tumor marker most commonly used to diagnose ovarian cancer, but elevated values have also been observed in benign diseases such as endometriosis, menstruation, pregnancy, and pleural and peritoneal inflammatory diseases, and it cannot be said to have sufficient specificity for distinguishing between malignant and benign ovarian tumors. Therefore, there has been hope for the development and clinical application of more useful new markers and analytical methods.

On the other hand, human epididymis protein 4 (hereinafter abbreviated as "HE4"), which is detected in high concentrations in the serum of ovarian cancer patients and whose level increases as the disease progresses, belongs to the WAP (Whey Acidic Protein) family and is also called WFDC2 (WAP Four-Disulfide Core Domain Protein 2), and is a secretory glycoprotein with a molecular weight of approximately 25 kDa, which was given this name because it was discovered in epithelial cells in the distal epididymis.
Mature human HE4 has a structure in which two WAP domains are linked together, but the first or second WAP domain can be deleted by splicing, generating multiple isoforms with substitutions of 10, 22, or 28 amino acids.

HE4 is strongly expressed in serum in ovarian cancer and shows extremely high values in advanced cancer. HE4 is a highly specific tumor marker that is less elevated in benign gynecological diseases such as endometriosis than CA125, a mucin glycoprotein.
However, HE4 is expressed not only in ovarian cancer, but also in lung adenocarcinoma and pulmonary cystic fibrosis, and a correlation has been suggested between the size and invasiveness of the tumor and the expression of the HE4 isoform in which the first WAP domain is replaced by 22 amino acids. Increased HE4 concentrations are also found in tissues and serum in type I/II endometrial cancer, and in urine in transitional cell carcinoma and early/late stage ovarian cancer.

HE4 is also expressed in normal male and female reproductive tract epithelium, upper respiratory tract, salivary gland ducts, and breast, with variably expressed distal convoluted tubules, colon, and endometrium.
Therefore, even HE4 does not have sufficient specificity in distinguishing between malignant and benign ovarian tumors, and the development and clinical application of more useful new markers and analytical methods are still desired. In addition, there is a need to develop an analytical method for HE4 as a more effective tool for detecting potentially curable early malignant tumor states such as ovarian cancer (see, for example, Patent Document 1).

There are methods for distinguishing between malignant and benign tumors, such as pelvic examination, rectal examination, ultrasound (echo), CT scan, and MRI scan. However, in the case of ovarian cancer, it is difficult to distinguish between benign and malignant ovarian tumors using imaging tests or physical examinations, so a diagnosis must be confirmed by conducting a pathological examination.
Pathological examinations include cytology and histology. Cytology is used to check whether pleural fluid or ascites contains cancer cells. The basic method is to collect the fluid during surgery, but if ascites is found before surgery, a needle is inserted through the skin to collect the fluid for testing. Histology is used to examine tissue collected during surgery to determine whether it is benign, borderline malignant, or malignant, and to determine the tissue type, and it takes several weeks for the final results to be obtained. Both of these pathological examination methods are invasive, place a heavy burden on patients, and take time to obtain results.

Special table 2013-536442 publication

The present invention has been made in consideration of the above problems and circumstances, and the problem to be solved is to provide a pathological information generation method, a pathological information generation system, and an HE4 glycan analysis kit, etc., which enable diagnosis with reduced burden on patients and doctors by accurately evaluating the incidence, onset, or malignancy of cancers such as ovarian cancer in a rapid, simple and non-invasive manner.

In order to solve the above problems, the present inventors, in the course of investigating the glycans of HE4, which are associated with the onset of cancers such as ovarian cancer, discovered that HE4 having specific residues at the (non-reducing) end of the glycan has a significant correlation with specific cancers, leading to the present invention.
That is, the above-mentioned problems of the present invention are solved by the following means.

1. A pathology information generating method for generating information on cancer morbidity or onset, comprising:
a first step of obtaining information on the amount of HE4 having any one of four types of residues, i.e., β-N-acetylgalactosamine residues, α2,3-sialic acid residues, α2,6-sialic acid residues, or mannose residues, contained in a test sample;
A second step of generating information regarding the onset or occurrence of cancer in a subject related to the test sample based on the amount of HE4 having any one of the four types of residues;
A pathological condition information generating method comprising the steps of:

2. The method for generating pathological information described in paragraph 1, characterized in that the first step is a step of measuring information regarding the amount of HE4 having any one of the four types of residues by mass spectrometry or a method of contacting the test sample with a first substance having affinity for any one of the four types of residues.

3. The method for generating pathological information described in paragraph 1 or 2, further comprising a third step of obtaining the total amount of all HE4 contained in the test sample, regardless of whether the HE4 has the four types of residues.

4. A method for generating pathological information described in any one of paragraphs 1 to 3, characterized in that the third step is a step of measuring the total amount of HE4 by mass spectrometry, chromatography, or a method of contacting the test sample with a second substance having affinity for the polypeptide chain portion of HE4.

5. The method for generating pathological information described in paragraph 4, characterized in that the second step is a step of calculating the ratio of the amount of HE4 having any of the four types of residues to the total amount of all of the HE4 contained in the test sample.

6. A pathological condition information generating method as described in any one of paragraphs 1 to 5, characterized in that the second step is a step of generating information regarding the subject's cancer incidence or onset, such as information regarding whether the subject has cancer, the probability that the subject has cancer, the malignancy of the subject's cancer, or the stage of the subject's cancer.

7. A method for generating pathological information described in any one of paragraphs 1 to 6, characterized in that the second step is a step of generating information regarding whether or not the subject is suffering from ovarian cancer as information regarding the subject's cancer incidence or onset.

8. A pathology information generating method described in any one of paragraphs 3 to 7, characterized in that the first step is performed before the third step.

9. The first step is a step of measuring the amount of HE4 having any one of the four types of residues by a method of contacting the test sample with a first substance having affinity for any one of the four types of residues;
9. The method for generating pathological information described in claim 8, wherein the first substance is a lectin or an antibody having affinity for any of the four types of residues.

10. The method for generating pathological information described in paragraph 9, characterized in that the first substance is a lectin having affinity for the HE4 and having any of the four types of residues, and is any of the lectins ConA, Fujilectin, SSA, or MAM.

11. The first substance is immobilized on a support,
the third step is a step of measuring the total amount of all of the HE4 by a method of contacting the test sample with a second substance having affinity for a polypeptide chain portion of HE4;
11. The method for generating pathological information described in claim 9 or 10, wherein the second substance is an antibody having affinity for any of the four types of residues.

12. A method for generating pathological information described in any one of paragraphs 8 to 11, characterized in that the first step is a chromatographic step.

13. A pathology information generation method described in any one of paragraphs 3 to 7, characterized in that the third step is performed before the first step.

14. The second substance is immobilized on a support;
the first step is a step of measuring the amount of HE4 having any one of the four types of residues by a method of contacting the test sample with a first substance having affinity for any one of the four types of residues;
the first substance is a lectin having affinity for any of the four types of residues,
the first substance is labeled with a fluorescent substance;
The method for generating pathological information described in paragraph 13, characterized in that the first step is a step of measuring the intensity of fluorescence emitted from a fluorescent substance that labels the first substance bound to HE4 captured by the second substance, and calculating the amount of HE4 having any of the four types of residues based on the intensity of the fluorescence.

15. A method for generating pathological information as described in paragraph 13, characterized in that in the third step, the intensity of a peak obtained by mass spectrometry or chromatography is compared with the intensity of a predetermined reference peak.

16. A method for generating pathological information described in any one of paragraphs 1 to 15, characterized in that any one of the four types of residues, namely the β-N-acetylgalactosamine residue, the α2,3-sialic acid residue, the α2,6-sialic acid residue, or the mannose residue, is located at the (non-reducing) end of the glycan of HE4.

17. The pathological condition information generating method described in any one of paragraphs 1 to 16, wherein the cancer is ovarian cancer.

18. A pathology information generating system for generating information regarding cancer morbidity or onset, comprising:
a first step for acquiring information on the amount of HE4 having any one of four types of residues, i.e., β-N-acetylgalactosamine residue, α2,3-sialic acid residue, α2,6-sialic acid residue, or mannose residue, contained in a test sample;
A second step of generating information regarding the onset or occurrence of cancer in a subject related to the test sample based on the amount of HE4 having any one of the four types of residues;
A pathological condition information generating system comprising:

19. A pathology information generation system as described in paragraph 18, characterized in that it is a system for implementing the pathology information generation method as described in any one of paragraphs 1 to 17.

20. A HE4 glycan analysis kit comprising an HE4 having a glycan and an anti-HE4 antibody,
20. A HE4 glycan analysis kit, which is an analysis kit for carrying out the pathological information generation method according to any one of claims 1 to 17.

21. A HE4 glycan analysis kit comprising an HE4 having a glycan and an anti-HE4 antibody,
21. The HE4 glycan analysis kit according to item 20, characterized in that it comprises an anti-HE4 antibody and a substance having affinity for HE4 and having a β-N-acetylgalactosamine residue, an α2,3-sialic acid residue, an α2,6-sialic acid residue or a mannose residue at the (non-reducing) end of the glycan.

22. The HE4 glycan analysis kit described in paragraph 21, characterized in that the substance having affinity for HE4 is a lectin.

23. The HE4 glycan analysis kit according to any one of items 20 to 22, characterized in that it is configured by combining the lectin, an anti-HE4 antibody, and an analytical substrate.

24. The HE4 glycan analysis kit according to any one of claims 20 to 23, characterized in that the lectin is equipped with a fluorescent substance.

25. An HE4 glycan analysis kit according to any one of items 20 to 24, characterized in that it contains at least one type of lectin, Fujilectin or MAM, equipped with a fluorescent substance.

26. An HE4 glycan analysis kit according to any one of claims 20 to 25, further comprising a chromatography device.

27. The HE4 glycan analysis kit described in paragraph 26, further characterized in that it is configured to include a labeling reagent for chromatographic measurement.

28. HE4 as a biomarker for cancer testing, comprising:
HE4 is characterized in that it has any one of four types of residues, namely, a β-N-acetylgalactosamine residue, an α2,3-sialic acid residue, an α2,6-sialic acid residue, or a mannose residue, at the (non-reducing) end of the sugar chain.

By means of the present invention, it is possible to provide a pathological information generation method, a pathological information generation system, and an HE4 glycan analysis kit, etc., which enable diagnosis with reduced burden on patients and doctors by accurately evaluating the incidence, onset, or malignancy of cancers such as ovarian cancer in a rapid, simple, and non-invasive manner.

The expression mechanism or mechanism of action of HE4 having specific residues according to the present invention has not been clarified.
However, by measuring the amount of HE4 having a specific residue in a test sample, information regarding the prevalence, onset, or malignancy of cancer in a subject related to the test sample can be generated with high accuracy, making it possible to rapidly evaluate the prevalence, onset, or malignancy of cancer in a subject related to the test sample using a simple and non-invasive method, thereby reducing the burden on patients and doctors.
The pathological information generating method, pathological information generating system, HE4 glycan analysis kit, etc. of the present invention are characterized by their ability to obtain highly accurate information on whether or not subjects aged 50 or younger are suffering from ovarian cancer.

Schematic diagram showing the configuration of an SPFS measurement device FIG. 2 is an enlarged view of the sensor chip and its surroundings in the measuring device of FIG. 1. Schematic diagram showing an example of a HE4 glycan analysis kit FIG. 1 shows the quantitative results of HE4 (GalNAcHE4) having a β-N-acetylgalactosamine residue at the end of the sugar chain by the lectin column method using WFA as a lectin. ROC curve obtained by comparing cancer samples and non-cancerous samples (including healthy subjects and endometriosis patients) ROC curve obtained by comparing cancer patients under 50 years old with non-cancer samples (healthy subjects and endometriosis patients) ROC curve obtained by comparing grade 1 cancer samples with non-cancerous samples (healthy subjects and endometriosis patients) FIG. 1 shows the relationship between the amount of HE4 protein having α2,3-sialic acid and the grade of ovarian cancer.

The pathology information generating method of the present invention is a pathology information generating method that generates information regarding the occurrence or onset of cancer, and is characterized by having a first step of obtaining information regarding the amount of HE4 having any of four types of residues, i.e., β-N-acetylgalactosamine residue, α2,3-sialic acid residue, α2,6-sialic acid residue or mannose residue, contained in a test sample, and a second step of generating information regarding the occurrence or onset of cancer in a subject related to the test sample based on the amount of HE4 having any of the four types of residues.
This feature is a technical feature common to or corresponding to each of the following embodiments.

In an embodiment of the present invention, from the viewpoint of the manifestation of the effect and accuracy of the present invention, it is preferable that the first step is a step of measuring information regarding the amount of HE4 having any of the four types of residues by mass spectrometry or a method of contacting the test sample with a first substance having affinity for any of the four types of residues. Furthermore, from the viewpoint similar to the above, it is preferable to have a third step of obtaining the total amount of all HE4 contained in the test sample, regardless of HE4 having the four types of residues.

It is also preferable that the third step is a step of measuring the total amount of HE4 by mass spectrometry, chromatography, or a method of contacting the test sample with a second substance that has affinity for the polypeptide chain portion of HE4.

As an embodiment of the present invention, from the viewpoint of the manifestation of the effect and accuracy of the present invention, it is preferable that the second step is a step of calculating the ratio of the amount of HE4 having any of the four types of residues to the total amount of all of the HE4 contained in the test sample.
In addition, the second step is preferably a step of generating information on whether or not the subject is suffering from cancer, the probability that the subject is suffering from cancer, the malignancy of the subject's cancer, or the stage of the subject's cancer, as information on the subject's suffering from or onset of cancer. Furthermore, the second step is preferably a step of generating information on whether or not the subject is suffering from ovarian cancer, as information on the subject's suffering from or onset of cancer.

In one embodiment of the present invention, it is preferable that the first step is performed before the third step. Moreover, it is preferable that the first step is a step of measuring the amount of HE4 having any of the four residues by a method of contacting a first substance having affinity for any of the four residues with the test sample, and that the first substance is a lectin or an antibody having affinity for any of the four residues.
Furthermore, the first substance is a lectin having affinity for the HE4 and having any of the four types of residues, and is preferably any of the lectins ConA, Fujilectin, SSA, and MAM.

In addition, from the viewpoint of manifestation of the effect of the present invention, it is preferable that the first substance is immobilized on a support, the third step is a step of measuring the total amount of all of the HE4 by a method of contacting the test sample with a second substance having affinity for the polypeptide chain portion of HE4, and the second substance is an antibody having affinity for any of the four types of residues. Furthermore, it is preferable that the first step is a chromatographic step.

As another embodiment of the present invention, it is also preferable that the third step is performed before the first step. In addition, it is preferable that the second substance is immobilized on a support, the first step is a step of measuring the amount of HE4 having any of the four residues by a method of contacting a first substance having affinity with any of the four residues with the test sample, the first substance is a lectin having affinity with any of the four residues, the first substance is labeled with a fluorescent substance, and the first step is a step of measuring the intensity of fluorescence emitted from a fluorescent substance that labels the first substance bound to HE4 captured by the second substance, and calculating the amount of HE4 having any of the four residues based on the intensity of the fluorescence, from the viewpoint of accuracy, etc.

Furthermore, from the same viewpoint as above, in the third step, it is preferable to compare the intensity of the peak obtained by mass spectrometry or chromatography with the intensity of a predetermined reference peak.

In the present invention, it is preferable from the viewpoint of the effects of the present invention that any one of the four types of residues, β-N-acetylgalactosamine residue, α2,3-sialic acid residue, α2,6-sialic acid residue, or mannose residue, is at the (non-reducing) end of the glycan of HE4. It is also preferable that the cancer is ovarian cancer.

The pathology information generating system of the present invention is a pathology information generating system that generates information related to the prevalence or onset of cancer, and is characterized by having a first process unit that acquires information related to the amount of HE4 having any of four types of residues, namely β-N-acetylgalactosamine residues, α2,3-sialic acid residues, α2,6-sialic acid residues, or mannose residues, contained in a test sample, and a second process unit that generates information related to the prevalence or onset of cancer in a subject related to the test sample, based on the amount of HE4 having any of the four types of residues. Furthermore, an embodiment of the present invention is characterized by being a system for implementing the pathology information generating method.

The HE4 glycan analysis kit of the present invention is an HE4 glycan analysis kit including an HE4 having a glycan and an anti-HE4 antibody, and is characterized in that it is an analysis kit for carrying out the above-mentioned pathological condition information generation method of the present invention. The HE4 glycan analysis kit including an HE4 having a glycan and an anti-HE4 antibody is characterized in that it includes a substance having affinity for HE4 having a β-N-acetylgalactosamine residue, an α2,3-sialic acid residue, an α2,6-sialic acid residue or a mannose residue at the (non-reducing) end of the glycan, and an anti-HE4 antibody.

In an embodiment of the HE4 glycan analysis kit, from the viewpoints of accuracy, simplicity, etc., the substance having affinity for HE4 is preferably a lectin. Also, it is preferable that the kit is configured by combining the lectin, an anti-HE4 antibody, and an analytical substrate. Furthermore, it is preferable that the lectin is equipped with a fluorescent substance. Also, it is preferable that the kit contains at least one type of lectin, Fujilectin or MAM, equipped with a fluorescent substance.
In addition, as an embodiment, it is preferable that the configuration includes a chromatography device, and further, it is preferable that the configuration includes a labeling reagent for chromatographic measurement.

The HE4 of the present invention is an HE4 used as a biomarker for cancer testing, and is characterized in that it has any one of four types of residues at the (non-reducing) end of the glycan: a β-N-acetylgalactosamine residue, an α2,3-sialic acid residue, an α2,6-sialic acid residue, or a mannose residue.

The present invention, its components, and the modes and aspects for implementing the present invention will be described in detail below. In this application, the symbols "to" are used to mean that the numerical values before and after the symbol "to" are included as the lower and upper limits.

In this application, the term "detection target glycan" (also referred to as analyte, detection target, or specimen) refers to a glycan that is specifically possessed by a glycoprotein, which is an antigen that serves as a marker that is present at relatively high concentrations in the blood when a specific disease is developed, and which is intended to have as many detection lectins bound as possible in order to detect the analyte.
On the other hand, a "non-detection target glycan" refers to a glycan carried by a contaminant (such as a glycoprotein or glycolipid different from the analyte) that is present in a specimen together with the analyte, and which is intended to be prevented from binding to a detection lectin as much as possible because it interferes with the detection of the analyte.
In this specification, the term "detection method" naturally includes not only a method for qualitatively detecting an analyte, but also a method for quantitatively detecting and measuring an analyte (method for quantitatively measuring an analyte). Furthermore, the detection method is included in the steps of the pathology information generation method of the present invention.

[1] Overview of pathological condition information generating method The pathological condition information generating method of the present invention is a pathological condition information generating method that generates information regarding the occurrence or onset of cancer, and is characterized by having a first step of obtaining information regarding the amount of HE4 having any of four types of residues, i.e., β-N-acetylgalactosamine residue, α2,3-sialic acid residue, α2,6-sialic acid residue, or mannose residue, contained in a test sample, and a second step of generating information regarding the occurrence or onset of cancer in a subject related to the test sample based on the amount of HE4 having any of the four types of residues.
The components and methods are described in detail below.

<Detection target substance and detection target sugar chain>
The detection target substance according to the present invention is HE4 having a glycan, and the detection target glycan is a glycan having any one of four types of residues, i.e., a β-N-acetylgalactosamine residue, an α2,3-sialic acid residue, an α2,6-sialic acid residue, or a mannose residue, at the (non-reducing) end of the glycan.
These are selected as detection targets because screening tests conducted by the present inventors have confirmed that they have a high correlation with cancer, particularly ovarian cancer.

"Glycosylation" is a general term for molecules in which monosaccharides such as glucose, galactose, mannose, fucose, xylose, N-acetylglucosamine, N-acetylgalactosamine, and sialic acid, and their derivatives, are linked in a chain form via glycosidic bonds. Among biopolymers, glycans are extremely diverse, and it is becoming clear that they are deeply involved in various functions of naturally occurring organisms, such as intercellular signaling and regulating protein functions and interactions.

Glycosylation often occurs in vivo as complex carbohydrates bound to proteins and lipids. Examples of biopolymers with glycans include proteoglycans in the cell walls of plant cells that contribute to cell stabilization, glycolipids that affect cell differentiation, proliferation, adhesion, and migration, and glycoproteins that are involved in cell-cell interactions and cell recognition. The mechanisms by which the glycans contained in these biopolymers control advanced and precise biological reactions while acting as substitutes for, assisting, amplifying, regulating, or inhibiting the functions of the biopolymers are gradually becoming clear. If the relationship between such glycans and cell differentiation and proliferation, cell adhesion, immunity, and cell canceration can be clarified, it is expected that new developments will be achieved by closely linking this glycoengineering with medicine, cell engineering, and organ engineering.

<Substances with affinity for glycan-containing HE4>
As the substance having affinity for HE4 having a glycan according to the present invention, various substances can be used, but it is preferable that the substance is any lectin selected from the entire lectin group (1) to (4) below, or a glycan-recognizing antibody. Furthermore, it is preferable that the substance having affinity for HE4 having any of the four types of residues is any of lectins ConA, WFA, WFL, SSA, or MAM, and particularly any of ConA, WFA, or SSA.
In the present application, the term "lectin" refers collectively to proteins (glyco-binding proteins) that exhibit binding activity to the glycans of glycoproteins and glycolipids that constitute cell membranes. Details of lectins will be described later.

Lectins:
(1) Proteins that recognize high-mannose glycans:
Lectins such as Artocarpin, CRLL, Conarva, CCA, Heltuba, jacalin, Calsepa, MornigaM, UDA, PSA, LTA, LTL, NPA, HHA, HHL, GNL, GNA, AMA, Succinylae and ConA, and anti-high mannose type glycan antibodies.

(2) Proteins that recognize N-acetylgalactosamine residues:
Lectins such as BRL, TJA-II, RCA I, RCA120, BPA, BPL, SBA, WFA, VVA-G, VVA, VVL, PTLI, WBAI, PTLII, WBAII, RCAII, RCA60ricin, WFA, WFL, SBA, GSL, SBA, WSA, WJA and SIA, and anti-N-acetylgalactosamine residue antibodies such as RM2.

(3) Proteins that recognize sialic acid α2,6-residues:
Lectins such as SNA, EBL, BRL, BRA2, TJA-I, PPL, SNA, SAN-I, SSA, RCA120, RCAI and AMA, and anti-sialic acid α2,6-residue antibodies.

(4) Proteins that recognize sialic acid α2,3-residues:
Lectins such as MALII, MAH and MAM, and anti-sialic acid α2,3-residue antibodies.

In the present invention, the lectin that is particularly preferably used is a lectin that has affinity for the β-N-acetylgalactosamine residue (GalNAcβ1→R), α2,3 sialic acid residues, α2,6 sialic acid residues, mannose residues, etc. bound to the (non-reducing) terminus of the sugar chain of HE4.
For example, lectins having particular affinity for β-N-acetylgalactosamine residues include WFA, TJA-II, ECA, RCA120, and DSA.
Examples of lectins having affinity for α2,3 sialic acid residues include MAL and MAM, while examples of lectins having affinity for α2,6 sialic acid residues include SNA, SSA, and TJA-I, and examples of lectins having affinity for high-mannose include ConA and UDA.

TJA-II is a lectin extracted and purified from the tuberous roots of Triticum vulgare, and has a molecular weight of 64 kDa as determined by electrophoresis under non-reducing conditions. However, it is a dimer with disulfide bonds, and its molecular weights as determined by electrophoresis under reducing conditions are 32 kDa and 29 kDa.
WFA is a lectin extracted and purified from the seeds of Wisteria nodosa, and shows strong affinity to the β-N-acetylgalactosamine residue (GalNAcβ1→R) itself, as well as to GalNAcβ1→4Gal residues and GalNAcβ1→4GlcNAc residues.

In an embodiment of the present invention, it is particularly preferable that the substance having affinity for HE4 and having any of the four types of residues is any one of the following lectins: ConA, WFA, WFL, SSA or MAM, and more preferably, any one of ConA, WFA or SSA.

In the detection method according to the present invention, a lectin is used as a labeling lectin for labeling a detection target (analyte) having a detection target sugar chain.
Furthermore, lectins may be used as a masking glycan recognition molecule for masking the glycan to be detected that is contained in impurities. In the present invention, the glycan recognition molecule used for masking is not limited to lectins, and includes, for example, antibodies that recognize and specifically bind to the glycan to be detected as an epitope, but lectins are preferred from the viewpoints that, like labeling lectins, they are inexpensive and available in large quantities, have excellent protein stability, and can be stored for a long period of time.

Lectins bind with high binding ability to specific glycans according to their type, but may also bind to other glycans with low binding ability. Therefore, the detection method of the present invention is applied to the case where a lectin that binds with high binding ability to the detection target glycans of the analyte and with weak binding ability to the non-detection target glycans of the impurities is used as the labeling lectin. A glycan recognition molecule that binds with strong binding ability to the non-detection target glycans to which the labeling lectin binds with weak binding ability is used as the masking glycan recognition molecule. Such a masking glycan recognition molecule must be a glycan recognition molecule (e.g., a different type of lectin from the labeling lectin) that does not substantially bind or binds with weak binding ability to the detection target glycans to which the labeling lectin binds with strong binding ability.

<Test sample containing detection target substance>
In the detection method according to the present invention, a specimen that may contain the detection target substance (analyte) can be used as the test sample. The test sample (specimen) that may contain the detection target substance may be a specimen that actually contains the detection target substance, or may be a test sample (specimen) that does not actually contain the detection target substance.
Examples of such test samples include biological samples and biologically derived samples such as blood, serum, plasma, urine, cerebrospinal fluid, saliva, cells, tissues, ascites, or organs, or preparations thereof (e.g., biopsy specimens), etc. In particular, blood, serum, or plasma, which may contain cancer antigens/tumor markers, are suitable as test samples.

Liquid samples such as blood, serum, plasma, urine, cerebrospinal fluid, saliva, and ascites can be used after diluting with an appropriate buffer solution. Solid samples such as cells, tissues, and organs can be homogenized with an appropriate buffer solution having a volume about 2 to 10 times the volume of the solid sample, and the suspension or its supernatant can be used as is or after further dilution.

<Labeled body>
In the method of the present invention, in order to detect the sugar chain-containing HE4 (detection target antigen), it is preferable to use a label complexed with a lectin. For this purpose, it is preferable to use a labeled lectin in which the label and the lectin are complexed (bound).

Labels that can be used include those known to those skilled in the art, such as fluorescent dyes (fluorescent substances), enzymes/coenzymes, chemiluminescent substances, and radioactive substances.
Examples of fluorescent dyes (fluorophores) include fluorescent dyes of the fluorescein family (Integrated DNA Technologies), fluorescent dyes of the polyhalofluorescein family (Applied Biosystems Japan, Ltd.), fluorescent dyes of the hexachlorofluorescein family (Applied Biosystems Japan, Ltd.), fluorescent dyes of the coumarin family (Invitrogen Corporation), fluorescent dyes of the rhodamine family (GE Healthcare Biosciences, Ltd.), fluorescent dyes of the cyanine family, fluorescent dyes of the indocarbocyanine family, fluorescent dyes of the oxazine family, fluorescent dyes of the thiazine family, fluorescent dyes of the squaraine family, fluorescent dyes of the chelated lanthanide family, fluorescent dyes of the BODIPY (registered trademark) family (Invitrogen Corporation), fluorescent dyes of the naphthalenesulfonic acid family, fluorescent dyes of the pyrene family, fluorescent dyes of the triphenylmethane family, and Alexa Examples of such organic fluorescent dyes include the Fluor (registered trademark) dye series (Invitrogen Corporation).

Other examples of fluorescent dyes include rare earth complexes of Eu, Tb, etc. (e.g., ATBTA-Eu3+), fluorescent proteins such as blue fluorescent protein (BFP), cyan fluorescent protein (CFP), green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (DsRed) or Allophycocyanin (APC; LyoFlogen (registered trademark)), and fluorescent microparticles such as latex and silica.

When a sample derived from a blood specimen is subjected to analysis, it is desirable to use a fluorescent dye having a maximum fluorescence wavelength in the near-infrared region, such as Cy5 or Alexa Fluor 647, in order to minimize the effect of light absorption by iron derived from blood cell components in the blood.
Radioactive substances include radioisotopes (32P, 14C, 125I, 3H, 131I, etc.).

<Combination of detection target substance, labeling lectin, and masking glycan recognition molecule>
In the detection method of the present invention, it is also a preferred embodiment to use a combination of an object to be detected (analyte: antigen to be detected), a labeling lectin, and a masking sugar chain recognition molecule (lectin).
Taking into consideration the antigen to be detected, such as a cancer antigen/tumor marker, the contaminants that are likely to be contained in the sample together with the antigen to be detected (glycoproteins and glycolipids other than the antigen to be detected), and the type of glycan possessed by each of them, it is also possible to appropriately select and use in combination a labeling lectin that easily binds to the target glycan possessed by the former and a masking glycan recognition molecule (lectin) that easily binds to the non-target glycan possessed by the latter to which the lectin may simultaneously bind.

<Antigen capture substances such as HE4 antibodies>
In the present invention, a molecule that specifically recognizes and binds to HE4 (analyte: substance to be detected) and does not interfere with sugar chain recognition by lectin can be used as an antigen capture substance (ligand).
The antigen capture substance is not particularly limited as long as it is an appropriate substance corresponding to HE4, and examples of the antigen capture substance that can be used include antibodies, aptamers, synthetic peptides, PSA that has a binding ability to HE4, and enzymes such as trypsin and proteinase K. In particular, monoclonal antibodies against the antigen to be detected are preferable.

For example, when the antigen to be detected is a cancer antigen/tumor marker, it is appropriate to use an antibody (such as a monoclonal antibody) that specifically binds to that antigen as the antigen capture substance. For example, when the antigen is HE4, an anti-human HE4 antibody may be used.

The term "antibody" as used above includes not only complete antibodies but also any antibody fragment or derivative, and includes not only complete antibodies but also various types of antibodies such as Fab, F(ab')2, CDR, humanized antibodies, multifunctional antibodies, and single-chain antibodies (ScFv).

Capture Supports
In the detection method of the present invention, a support for immobilizing an antigen capture substance (ligand) (capturing a detection target thereon) can be used depending on the measurement system.
Examples of the support include insoluble supports such as insoluble polysaccharides, such as agarose and cellulose; synthetic resins, such as silicone resins, polystyrene resins, polyacrylamide resins, nylon resins and polycarbonate resins; and glass.
These supports are used in the form of beads (mainly spherical), plates (mainly planar), etc. As beads, magnetic beads, columns filled with resin beads, etc. can be used. As plates, multi-well plates (96-well multi-well plates, etc.), biosensor chips, etc. can be used. Planar supports such as plates are sometimes called substrates.

The antigen capturing substance (ligand) can be bound to the support by a commonly used method such as chemical binding, physical adsorption, etc. Commercially available supports can be suitably used for all of these supports.
In addition, an antigen capture substance (ligand) that is immobilized on a support and is in a state in which it is capable of capturing the detection target (analyte) in a sample being pumped and solidifying it on the support is sometimes called a solid-phase ligand.

(1.1) Embodiments of the pathology information generating method The pathology information generating method of the present invention is a pathology information generating method that generates information regarding the occurrence or onset of cancer, and is characterized by having a first step of acquiring information regarding the amount of HE4 having any of four types of residues, i.e., β-N-acetylgalactosamine residues, α2,3-sialic acid residues, α2,6-sialic acid residues, or mannose residues, contained in a test sample, and a second step of generating information regarding the occurrence or onset of cancer in a subject related to the test sample based on the amount of HE4 having any of the four types of residues.
Preferred aspects of each basic step will be described below.

(a) Step 1 Step 1 is a step of obtaining information regarding the amount of HE4 having any of four types of residues, namely, β-N-acetylgalactosamine residues, α2,3-sialic acid residues, α2,6-sialic acid residues, or mannose residues, contained in a test sample.
From the standpoint of accuracy, simplicity, efficiency, etc., it is preferable that the first step is a step of measuring information regarding the amount of HE4 having any of the four types of residues by mass spectrometry or a method of contacting the test sample with a first substance that has affinity for any of the four types of residues.

Examples of mass spectrometry that can be used include high performance liquid chromatography mass spectrometry (LC-MS), high performance liquid chromatography tandem mass spectrometry (LC-MS/MS), gas chromatography mass spectrometry (GC-MS), gas chromatography mass spectrometry (GC-MS/MS), capillary electrophoresis mass spectrometry (CE-MS), and inductively coupled plasma (ICP-MS). Examples of methods that can be used to contact a test sample with a first substance that has affinity for any of the four types of residues include chromatography (including lectin columns).

In this case, the first substance is preferably a lectin or antibody having affinity for any of the four types of residues.Furthermore, the first substance is preferably a lectin having affinity for the HE4 having any of the four types of residues, and is preferably any of the lectins ConA, Fujilectin, SSA, and MAM.
It is preferable that any one of the four types of residues, ie, the β-N-acetylgalactosamine residue, the α2,3-sialic acid residue, the α2,6-sialic acid residue, or the mannose residue, is located at the (non-reducing) terminal of the glycan of HE4.

(b) Step 2 The second step is a step of generating information regarding the prevalence or onset of cancer in a subject related to the test sample based on the amount of HE4 having any of the four types of residues.
It is also preferable that the second step is a step of calculating the ratio of the amount of HE4 having any of the four types of residues to the total amount of all HE4 contained in the test sample, regardless of whether the HE4 has the four types of residues.

It is also preferred that the second step is a step of generating information on whether or not the subject has cancer, the probability that the subject has cancer, the malignancy of the subject's cancer, or the stage of the subject's cancer, as information on the subject's cancer onset or onset. It is also preferred that the second step is a step of generating information on whether or not the subject has ovarian cancer, as information on the subject's cancer onset or onset.

(c) Third step In the pathological information generating method of the present invention, it is preferable from the standpoint of accuracy to further include a third step of obtaining the total amount of all HE4 contained in the test sample, regardless of whether the HE4 has the four types of residues.
The third step is preferably a step of measuring the total amount of HE4 by mass spectrometry or a method of contacting the test sample with a second substance having affinity for the polypeptide chain portion of HE4.
As the mass spectrometry, for example, the various mass spectrometry methods described above can be used. As a method for contacting a test sample with a second substance having affinity to the polypeptide chain portion of HE4, chromatography or immune reaction (including ELISA, Western blotting, gold colloid method, latex agglutination method, radioimmunoassay method, capillary electrophoresis method, enzyme activity luminescence method, etc.) using an antibody having affinity to the polypeptide chain portion of HE4 can be used.

It is also preferable that the third step is a step of measuring the total amount of all of the HE4 by a method of contacting the test sample with a second substance having affinity for the polypeptide chain portion of HE4, the second substance being an antibody having affinity for any of the four types of residues, the method of detecting the glycan of HE4 being a separation method using liquid chromatography, and the step of contacting the test sample containing HE4 having a glycan with a carrier having the function of capturing the glycan, and after a step of capturing the glycan on the carrier, releasing the glycan from the carrier that has captured the glycan.

Furthermore, it is preferable that the third step is a step of measuring the total amount of all the HE4 by mass spectrometry, and includes a step of separating the glycans released from the HE4 having the glycans by chromatography to obtain a glycan structure distribution, and a step of comparing the relative intensity of a peak in the glycan structure distribution with the relative intensity of a corresponding peak in a standard.

In the present invention, it is also preferable that the second substance is fixed to a support, the first step is a step of measuring the amount of HE4 having any of the four types of residues by a method of contacting a first substance having affinity for any of the four types of residues with the test sample, the first substance is a lectin having affinity for any of the four types of residues, the first substance is labeled with a fluorophore, and the first step is a step of measuring the intensity of fluorescence emitted from a fluorophore that labels the first substance bound to HE4 captured by the second substance, and calculating the amount of HE4 having any of the four types of residues based on the intensity of the fluorescence.

In addition, as an embodiment of the present invention, it is also preferable from the viewpoints of accuracy, simplicity, efficiency, etc. that the first step is performed before the third step. On the other hand, as another embodiment of the present invention, it is also preferable that the third step is performed before the first step.

i) When the first step is performed before the third step, the first step is a step of measuring the amount of HE4 having any of the four residues by a method of contacting a first substance having affinity for any of the four residues with the test sample, and the first substance is preferably a lectin or antibody having affinity for any of the four residues.Furthermore, the first substance is preferably a lectin having affinity for the HE4 having any of the four residues, and is preferably any of the lectins ConA, Fujilectin, SSA, and MAM.

From the viewpoint of manifestation of the effects of the present invention, it is also preferable that the first substance is immobilized on a support (e.g., a carrier for a lectin column), the third step is a step of measuring the total amount of all of the HE4 by a method of contacting the test sample with a second substance having affinity for the polypeptide chain portion of HE4, and the second substance is an antibody having affinity for any of the four types of residues. Furthermore, it is preferable that the first step is a chromatographic step.

ii) A case in which the third step is performed before the first step In this case, it is preferable from the viewpoint of accuracy and the like that the second substance is immobilized on a support, the first step is a step of measuring the amount of HE4 having any of the four types of residues by a method of contacting a first substance having affinity for any of the four types of residues with the test sample, the first substance is a lectin having affinity for any of the four types of residues, the first substance is labeled with a fluorophore, and the first step is a step of measuring the intensity of fluorescence emitted from a fluorophore that labels the first substance bound to HE4 captured by the second substance, and calculating the amount of HE4 having any of the four types of residues based on the intensity of the fluorescence.

Furthermore, from the same viewpoint as above, it is preferable that the third step is a step of measuring the total amount of all of the HE4 by mass spectrometry, and further includes a step of comparing the glycan structure distribution of all of the HE4 obtained by the mass spectrometry and the relative intensity of the peak in the glycan structure distribution with the relative intensity of the corresponding peak in a standard.
(1.2) Method for detecting HE4 glycan As described above, the method for detecting HE4 glycan of the present invention is preferably an embodiment in which, in the first step, information regarding the amount of HE4 having any of the four residues is measured by mass spectrometry or a method of contacting the test sample with a first substance having affinity for any of the four residues. Here, however, a method of contacting the test sample with a first substance having affinity for any of the four residues by contacting the lectin with a sample that may contain HE4 and detecting/measuring the amount of HE4 having affinity for the lectin will be described in detail.
More specifically, the following analytical methods (A) and (B) can be mentioned.
Analytical method (A): An analytical method in which HE4 having affinity for a specific lectin is separated from HE4 having no affinity for the lectin using the specific lectin, and the sugar chain of HE4 having affinity for the lectin or the total amount of HE4 is detected (determined) (hereinafter referred to as analytical method (A)).
Analytical method (B): An analytical method for determining the amount of HE4 having affinity for a specific lectin in a state in which the specific lectin and HE4 having affinity for the specific lectin are bound to the specific lectin.

<Analysis Method (A)>
The analytical method (A) is
(a) a step of discriminating between HE4 having affinity for the lectin of the present invention, for example, a lectin having affinity for β-N-acetylgalactosamine residues, and a sample that may contain HE4 (hereinafter also referred to as "discrimination step (a)"); and (b) a step of determining the amount of HE4 having affinity for the lectin in the sample (hereinafter also referred to as "determination step (b)").
Includes.

The method for separating HE4 having affinity for lectin from HE4 having no affinity for lectin in the separation step (a) is not particularly limited as long as it utilizes the affinity between lectin and HE4. For example, the method can be performed by binding lectin to a carrier, contacting a sample that may contain HE4 with the lectin bound to the carrier (hereinafter also referred to as a "lectin affinity column" or simply "lectin column"), and separating the HE4 bound to the lectin from the HE4 that does not bind to the lectin.

Carriers include, but are not limited to, any material to which lectins can be bound, such as sepharose, cellulose, agarose, dextran, polyacrylate, polystyrene, polyacrylamide, polymethacrylamide, copolymers of styrene and divinylbenzene, polyamide, polyester, polycarbonate, polyethylene oxide, hydroxypropylmethylcellulose, polyvinyl chloride, polymethylacrylate, polystyrene and polystyrene copolymers, polyvinyl alcohol, polyacrylic acid, collagen, calcium alginate, latex, polysulfone, silica, zirconia, alumina, titania, and ceramics.

The shape of the carrier is not particularly limited, and carriers in the form of particulate beads, plates, gels, etc. can be used. For example, when a lectin affinity column is used to separate HE4 that has affinity for lectins from HE4 that has no affinity for the lectins, the carrier is preferably in the form of a gel.

Lectin affinity columns can be prepared according to standard methods. For example, lectin columns can be prepared using CNBr-activated Sepharose 4B and coupling according to the manufacturer's recommended protocol. The amount of lectin bound to the Sepharose gel is preferably 2 to 10 mg/mL.

The method of separating HE4 having affinity for the lectin from HE4 having no affinity for the lectin using a lectin affinity column can be carried out according to a conventional method for separating glycoproteins using a lectin affinity column.
The lectin affinity column is equilibrated with a buffer solution before the introduction of a sample that may contain HE4. The equilibration buffer solution may be, for example, 0.1% bovine serum albumin (BSA)-containing phosphate buffer or 0.1% bovine serum albumin (BSA)-containing Tris-HCl buffer.

After equilibrating the column, a sample that may contain HE4 is added and allowed to stand for a certain period of time to allow contact between the lectin and HE4. The contact time is not particularly limited and can be appropriately determined depending on the type of lectin and the affinity of HE4, but considering the speed and efficiency of binding, it is usually performed for 15 to 30 minutes.
The temperature at which the lectin is contacted with HE4 is not particularly limited and can be appropriately determined depending on the type of lectin and its affinity with HE4. Taking into consideration the prevention of freezing of the column and the prevention of non-specific binding of proteins that have no affinity for the lectin, the temperature can be 0°C to 40°C, and preferably 0°C to 30°C.
For example, the temperature at which TJA-II and HE4 are contacted is not particularly limited, but is usually preferably 4° C. to 10° C. Similarly, the temperature at which WFA and HE4 are contacted is not particularly limited, but is usually preferably 4° C. to 10° C.

Next, a binding fraction having affinity for lectin (hereinafter also referred to as a "binding fraction") and a non-binding fraction having no affinity for lectin (hereinafter also referred to as a "non-binding fraction") are separated.
The fraction not bound to the lectin can be obtained by adding a washing buffer to the column and collecting the non-binding fraction. The washing buffer is not limited as long as it is capable of eluting the non-binding components without dissociating the binding between the lectin and HE4, but for example, the buffer used for the equilibration can be used. The volume of the washing buffer can be appropriately determined depending on the type of lectin and its affinity with HE4, but is preferably 3 to 7 times, and more preferably about 5 times, the column volume.

The lectin-bound fraction can be obtained by adding an elution buffer to the column and collecting the eluted fraction. The elution buffer contains a hapten sugar, which allows HE4 bound to the lectin to be separated from the lectin. The hapten sugar can be selected appropriately depending on the specificity of the lectin, but in the case of TJA-II, lactose or the like can be used as the hapten sugar, and the eluted fraction can be collected using, for example, phosphate buffer containing 10 mM lactose and 0.1% bovine serum albumin (BSA).

In the case of WFA, GalNAc or the like can be used as the hapten sugar, and for example, an eluted fraction can be collected using a phosphate buffer containing 10 mM GalNAc and 0.1% bovine serum albumin (BSA). The volume of the elution buffer can be appropriately selected, but is preferably 3 to 7 times, and more preferably about 5 times, the column volume. The elution temperature is not particularly limited, but can be 0 to 40°C, preferably 2 to 25°C, and more preferably 4 to 20°C.
For example, the temperature at which HE4 is eluted from TJA-II is not particularly limited, but is preferably room temperature, and the temperature at which HE4 is eluted from WFA is also not particularly limited, but is preferably room temperature.

The determination of the amount of HE4 having affinity for the lectin in the determination step (b) includes:
(1) Determination by measuring the amount of HE4 having affinity for the separated lectin;
(2) Determination by measuring the amount of HE4 in the sample before fractionation and measuring the amount of HE4 that has affinity for the fractionated lectin, or (3) Determination by measuring the amount of HE4 in the sample before fractionation and measuring the amount of HE4 that has no affinity for the fractionated lectin.

The determination (1) by measuring the amount of HE4 with affinity to the separated lectin can be performed by quantitatively or semi-quantitatively measuring the amount of HE4 in the fraction bound to the lectin. In other words, the determination is made by measuring the absolute amount of HE4 with affinity to the lectin contained in the patient's blood.

The determination (2) by measuring the amount of HE4 in the sample before fractionation and the amount of HE4 with affinity to the fractionated lectin can be performed by comparing the amount of HE4 in the sample before fractionation (or the total amount of HE4 in the fraction bound to the lectin and the fraction not bound to the lectin) with the amount of HE4 in the fraction bound to the lectin.
Specifically, the amount of HE4 with affinity to lectin can be determined by calculating the ratio of the amount of HE4 in the fraction that binds to the lectin to the amount of HE4 in the sample before separation (or the total amount of HE4 in the fraction that binds to the lectin and the fraction that does not bind to the lectin), and can be calculated, for example, by any of the following formulas.
HE4 binding rate = (HE4 amount in bound fraction/total amount of HE4 in bound fraction and non-bound fraction) x 100%
HE4 binding rate=(HE4 amount in bound fraction/HE4 amount in sample before fractionation)×100%

Furthermore, the determination (3) by measuring the amount of HE4 in the sample before fractionation and the amount of HE4 that has no affinity for the fractionated lectin can be performed by comparing the amount of HE4 in the sample before fractionation (or the total amount of HE4 in the fraction that binds to the lectin and the fraction that does not bind to the lectin) with the amount of HE4 in the fraction that does not bind to the lectin.
Specifically, the amount of HE4 having affinity for lectin can be determined by subtracting the amount of HE4 in the fraction not binding to the lectin from the amount of HE4 in the sample before fractionation (or the total amount of HE4 in the fraction binding to the lectin and the fraction not binding to the lectin). For example, it can be calculated by any of the following formulas.
Lectin affinity HE4 amount = HE4 amount in sample before fractionation - HE4 amount in non-binding fraction Lectin affinity HE4 amount = total amount of HE4 in binding fraction and non-binding fraction - HE4 amount in non-binding fraction

In the determinations (1) and (2) above, the HE4 bound to the lectin is separated from the lectin and its amount is measured, whereas in the determination of (3) above, the amount of HE4 bound to the lectin is not measured when determining the amount of HE4 in the sample before separation and the amount of HE4 in the unbound fraction, so that the determination can be made without separating the HE4 bound to the lectin.
It is preferable to measure the amount of HE4 in all fractions, both bound and unbound, but it is also possible to determine in advance which fractions contain HE4, measure these fractions, and analyze HE4.

In the determination step (b), the method for measuring HE4 to determine the amount of HE4 having affinity for lectin is not particularly limited as long as it is a method that can quantitatively or semi-quantitatively determine HE4, but a method for measuring total HE4 or a method for measuring free HE4 can be used.
The method for measuring total HE4 or free HE4 can be carried out according to standard methods, such as immunological techniques using antibodies or fragments thereof (e.g., enzyme immunoassay, latex agglutination immunoassay, chemiluminescence immunoassay, fluorescent antibody method, radioimmunoassay, immunoprecipitation, immunohistochemical staining, or Western blot, etc.), and it is also possible to use commercially available PSA measurement kits.

Analysis of the examples described below has demonstrated that a lectin affinity column using MAM or WFA that can be used in the HE4 analysis method of the present invention is capable of recovering approximately 100% (at least 97% or more) of HE4 in a sample.

<Analysis Method (B)>
The analysis method (B) is a method of directly detecting HE4, which has affinity for lectins, by lectins. Specifically, lectin blotting by electrophoresis or lectin blotting by dot blotting can be mentioned. Both lectin blotting can be performed according to a standard method, but in the case of lectin blotting by electrophoresis, a sample that may contain HE4 is electrophoresed, HE4 is transferred to a nitrocellulose membrane or a PVDF membrane, and used as a sample membrane. In the case of lectin blotting by dot blotting, a sample that may contain HE4 is adsorbed to a nitrocellulose membrane or a PVDF membrane by a dot blotting device, and used as a sample membrane. The sample membrane is blocked with a blocking buffer and contacted in a solution containing a biotin-labeled lectin, for example, biotin-labeled WFA or biotin-labeled TJA-II. Then, it is contacted with avidin labeled with a chromogenic enzyme or a luminescent enzyme, for example, HRP, and a chromogenic or luminescent solution is contacted, and the resulting signal is detected.

Furthermore, as a method (B) for directly detecting HE4, which has affinity for lectins, using lectins, immunoblot and enzyme immunoassay can be modified. Specifically, a monoclonal or polyclonal antibody against HE4 is immobilized on a nitrocellulose membrane or an ELISA plate, and blocked with a blocking buffer. A sample that may contain HE4 is brought into contact with a nitrocellulose membrane or an ELISA plate, and then contacted with a biotin-labeled lectin, for example, biotin-labeled WFA or biotin-labeled TJA-II. Thereafter, avidin labeled with a chromogenic enzyme or a luminogenic enzyme, for example, HRP, is bound, and a signal can be detected using a chromogenic or luminescent solution.

The test sample used in the HE4 analysis method of the present invention can be any of the above-mentioned test samples, but blood, serum, plasma, ascites or an ovarian biopsy specimen is preferred, with blood, serum or plasma being particularly preferred.
This is because the content or ratio of HE4 having β-N-acetylgalactosamine residues and/or α2,3 sialic acid residues and α2,6 sialic acid residues in the blood, serum, or plasma of healthy individuals is different from that of HE4 having these sugar chains in cancer patients.

Liquid samples such as urine, blood, serum, plasma, cerebrospinal fluid, saliva, and ascites can be used in the analysis method (A) or analysis method (B) after diluting with an appropriate buffer solution depending on the respective analysis method. Solid samples such as cells, tissues, or organs can be homogenized with an appropriate buffer solution having a volume about 2 to 10 times the volume of the solid sample, and the suspension or its supernatant can be used as is or after further dilution in the analysis method (A) or analysis method (B).

For example, when a lectin affinity column is used in the analysis method (A), the liquid sample, or the suspension or supernatant of a solid sample can be diluted with an appropriate buffer before use. The dilution ratio is not particularly limited as long as the binding between the lectin and HE4 is not inhibited, but is preferably 2 to 400 times, more preferably 2 to 300 times, and most preferably 4 to 200 times.
The volume of the sample applied to the lectin affinity column is preferably 40% or less of the bed volume of the column, more preferably 30% or less, and most preferably 20% or less. For example, when a lectin affinity column with a bed volume of 1 mL is used, the volume is preferably 400 μL or less, more preferably 300 μL or less, and most preferably 200 μL or less.

<Sandwich type assay>
The sandwich assay of the present invention is a sandwich assay for quantifying HE4 as a detection target substance using a labeled lectin and a capture substance immobilized in a measurement area, and includes a process for suppressing binding of the labeled lectin to the same glycan as the detection target substance, which is carried by impurities nonspecifically adsorbed to the measurement area.
The "measurement region" is an area (space) in which the intensity of the fluorescence emitted therefrom is measured, as will be described in detail later, and refers to an area including the immobilized capture substance, the support on which it is carried, and the surface of the member on which they are placed.

The treatment for suppressing the binding of the labeled lectin to the same glycan as the substance to be detected, which is possessed by impurities nonspecifically adsorbed to the measurement region, is not particularly limited, and can be modified according to the type of impurity (glycoprotein or glycolipid), the substance to be detected, the capture substance, the labeled lectin, the measurement system, etc.
In this specification, the present invention is mainly described in relation to an embodiment in which the impurity is HE4 having the same glycan as the substance to be detected; however, the explanation can also be applied to cases in which the impurity is a substance other than a glycoprotein having the same glycan as the substance to be detected, such as a glycolipid, so that the present invention can be carried out so as to achieve the desired action and effect.

<SS bond cleavage agent>
In a typical embodiment of the present invention, the impurity nonspecifically adsorbed to the measurement region is a glycoprotein having the same glycan as the detection target substance. In this case, the treatment for suppressing the binding of the labeled lectin to the glycan is preferably a treatment for releasing a domain containing the same glycan as the detection target substance by contacting the glycoprotein as the impurity with a substance that has the effect of cleaving disulfide bonds (referred to as an "SS bond cleaving agent" in this specification).

There is no particular limitation on the substance that has the effect of cleaving the disulfide bonds of impurities (SS bond cleaving agent), but a suitable example is a reducing agent, i.e., a compound that has the effect of reducing the -S-S- crosslinked structure to two sulfhydryl groups (thiol groups), -SH and -HS-.

The reducing agent is not particularly limited, and can be selected from various reducing agents used to cleave disulfide bonds in proteins. Examples of such reducing agents include lower oxygen acid salts such as sulfurous acid, hyposulfurous acid, phosphorous acid, and hypophosphorous acid, alkali hydroxides, and thiol salts that serve as nucleophiles. Specifically, for example, sodium metabisulfite, thioglycerol, sodium toluenethiosulfinate, 2-mercaptoethanol, dithiothreitol (DTT), 2-mercaptoethylamine hydrochloride, 2-aminoethylisothiouronium bromide hydrobromide, and tris(2-carboxyethyl)phosphine can be used, and among these, sodium metabisulfite is particularly preferred.
In addition to the above-mentioned compounds, disulfide reductase can also be used as the SS bond cleavage agent.

<Column Chromatography>
The analytical (measurement) method using column chromatography includes (a) a step of purifying HE4 from a sample derived from a specimen, (b) a step of preparing an HE4 derivative from the HE4 purified in the step (a), (c) a step of labeling the HE4 derivative obtained in the step (b), and (d) a step of analyzing the glycan structure on HE4 by detecting signals due to high-mannose glycans, N-acetylgalactosamine residues, sialic acid α2,6-residues, and sialic acid α2,3-residues using chromatography such as high performance liquid chromatography or capillary electrophoresis on the labeled HE4 derivative obtained in the step (c).

For example, step (1) includes a step of removing albumin from a serum sample using protein A agarose (PIERCE) or the like, and a step of purifying the HE4 protein using beads equipped with an anti-HE4 antibody or the like. Step (b) includes a step of cleaving the sugar chain and the HE4 protein recovered in step (a) using proteinase K, thermolysin, or the like.
In step (c), the HE4 derivative prepared in step (b) is labeled with a compound that binds to the aldehyde group of the sugar chain, such as BOA, PA, 2-AA, or 2-AB.
A method in which the lectin affinity column (also referred to as a "lectin column") is attached to a high performance liquid chromatography apparatus and used for measurement is also preferred.

<Mass spectrometry>
The method further comprises a step of analyzing the HE4 derivative prepared in the same manner as in steps (a) and (b) of the column chromatography method by mass spectrometry.
As the mass spectrometry, LC-MS, MALDI-ToF-MS, GC-MS, etc. can be used. Mass spectrometry can be performed in the following manner. In mass spectrometry, a sample to be measured is ionized, and then various ions generated are sent to a mass spectrometer, and ion intensity is measured for each mass-to-charge ratio m/z, which is the ratio of the mass number m and the valence z of the ion. The mass spectrum obtained as a result is composed of peaks of measured ion intensity (ion peaks) for each mass-to-charge ratio m/z value.

In this way, mass analysis of the ionized sample itself is called MS1. In a tandem mass spectrometer capable of multi-stage dissociation, an ion peak having a specific mass-to-charge ratio m/z value is selected from the ion peaks detected in MS1 (the selected ion species is called the parent ion), and the ion is further dissociated and decomposed by collision with gas molecules, etc., and the generated dissociated ion species are mass analyzed to obtain a mass spectrum in the same way. Here, dissociating a parent ion in n stages and mass analyzing the dissociated ion species is called MSn+1. In this way, in a tandem mass spectrometer, the parent ion is dissociated in multiple stages (1 stage, 2 stages, ..., n stages), and the mass number of the ion species generated in each stage is analyzed (MS2, MS3, ..., MSn+1).

<Surface Plasmon Enhanced Fluorescence Spectroscopy (SPFS) Method>
As a measurement method used in the detection method of the present invention, a surface plasmon-field enhanced fluorescence spectroscopy (SPFS) method is preferably used. SPFS is a method in which, when a metal thin film formed on a dielectric member is irradiated with excitation light at an angle at which attenuated total reflection (ATR) occurs, the evanescent wave transmitted through the metal thin film is enhanced several tens to several hundreds of times due to resonance with the surface plasmon, and the fluorescent signal is measured by efficiently exciting a fluorescent material that labels an analyte (analyte to be analyzed) captured near the metal thin film. Since such SPFS has extremely high sensitivity compared to general fluorescent labeling methods, it is possible to quantify an analyte even when only a very small amount of the analyte is present in a sample.

<SPFS measuring member>
A measurement component for SPFS generally has a laminated structure comprising a sensor chip in which a field (measurement area) is formed for forming a sandwich-type immune complex and performing fluorescence measurement by SPFS, and a component for constructing a flow path or well capable of holding various solutions (samples containing analytes, labeled ligand solutions, other reaction reagents, etc.) used for forming a sandwich-type immune complex, etc., on the measurement area.

The sensor chip basically comprises a transparent support for introducing excitation light to the back surface of the metal thin film, a metal thin film formed on the transparent support for generating surface plasmon resonance, and a reaction layer formed on the metal thin film for capturing the analyte on the sensor surface, and may further comprise, if necessary, a spacer layer formed between the metal thin film and the reaction layer for preventing metal quenching of fluorescence caused by the fluorophore being too close to the metal thin film.

The area where the reaction layer is formed corresponds to the measurement area. The reaction layer may be formed over the entire bottom surface of the flow channel or well to form the measurement area, or the reaction layer may be formed over only a portion of the bottom surface (with a desired pattern, if necessary). The area of the measurement area can be adjusted taking into consideration the irradiation area of the excitation light, which is generally irradiated as laser light. For example, if the spot diameter of the excitation light is about 1 mmφ, the assay area is usually designed to have an area of at least several mm square.

When the SPFS system is a "flow-type" system that delivers various solutions through a closed flow-channel, a "flow cell" with holes for forming a flow-channel is placed on the sensor chip, and if necessary, a "top plate" with a liquid delivery inlet and liquid delivery outlet is placed on top of the flow cell at positions corresponding to the holes in the flow cell, and these are fixed in close contact to construct a measurement member. The surface of the sensor chip at the position corresponding to the holes in the flow cell forms the bottom of the flow channel, and the measurement area is formed there. In the case of a flow-channel type, various liquids can be delivered from the liquid delivery inlet into the flow channel and discharged from the liquid delivery outlet using a liquid delivery means including, for example, a pump or a tube, and reciprocating or circulating liquid delivery can also be performed as necessary. Conditions such as the delivery speed and delivery (circulation) time can be appropriately adjusted while taking into consideration the amount of sample, the concentration of the analyte in the sample, the size of the flow channel or well, the state of the reaction layer (density of the solid-phased ligand, etc.), the performance of the pump, etc.

On the other hand, when the SPFS system is a "well type" that stores various solutions in a space larger than the above-mentioned flow path, a measurement member is constructed by mounting and fixing a "well member" having a through hole for forming a well on the sensor chip. In the case of a well type, various liquids can be added to and removed from the well using, for example, a pipette-shaped member.

The flow cell can be made of, for example, sheet-shaped polydimethylsiloxane (PDMS). The top plate is made of a transparent material that allows the measurement of fluorescence emitted from the measurement area, and can be made of, for example, plate-shaped polymethylmethacrylate (PMMA). Alternatively, the flow cell and top plate can be made of plastic that has been molded or photolithographically shaped to the desired shape.

There are no particular limitations on the means for tightly attaching and fixing the flow cell or well member onto the sensor chip, and generally, physical pressure can be applied from above and below. If necessary, an adhesive, matching oil, transparent adhesive sheet, etc., with the same optical refractive index as the transparent support may be used.

<SPFS measurement device>
The measurement method according to the present invention can be carried out using a general SPFS measurement device. The SPFS measurement device is basically a device in which the SPFS measurement member is detachable, and the fluorescent A light source for irradiating excitation light (preferably laser light) of an appropriate wavelength according to the body, a prism (a transparent support having a flat surface) for making the excitation light incident at a predetermined angle on the back surface of the metal thin film of the sensor chip, When using a substrate-type sensor chip, a receiver that receives light reflected by a thin metal film and measures its intensity, a lens for collecting the fluorescence emitted from the phosphor, and a The detector includes a detector for detecting the wavelength of the excitation light and the fluorescence, and various filters for transmitting only light having a predetermined wavelength and cutting off other light.

For more specific aspects, reference can be made to various documents, such as JP 2010-145272 A, JP 2011-80935 A, JP 2008-102117 A, and Japanese Patent No. 3,562,912.

A more detailed example of the configuration of the SPFS measurement device will be described below.
FIG. 1 is a schematic diagram illustrating an outline of a quantitative measurement device for explaining the quantitative measurement method of the present invention for an analyte, and FIG. 2 is a partially enlarged view of the quantitative measurement device of FIG.

As shown in Figures 1 and 2, the quantitative measuring device 10 of the present invention is equipped with a sensor chip 16 having a prism-shaped dielectric member 12 whose vertical cross-sectional shape is approximately trapezoidal and a metal film 14 formed on the horizontal upper surface 12a of the dielectric member 12, and this sensor chip 16 is loaded into the sensor chip loading section 18 of the quantitative measuring device 10.

In addition, as shown in the figure, a light source 20 is arranged on one of the lower side surfaces 12b of the dielectric member 12, and incident light 22 from this light source 20 is incident on the side surface 12b of the dielectric member 12 from the outside below the dielectric member 12, and is irradiated through the dielectric member 12 toward the metal film 14 formed on the upper surface 12a of the dielectric member 12.

In addition, a polarizing filter can be provided between the light source 20 and the dielectric member 12 to convert the laser light irradiated from the light source 20 into P-polarized light, which efficiently generates surface plasmons on the metal film 14.

In addition, on the other side 12c below the dielectric member 12, as shown in Figure 1, a light receiving means 26 is provided for receiving metal film reflected light 24, which is the incident light 22 reflected by the metal film 14.

The light source 20 is provided with an incident angle adjustment means (not shown) that allows the incident angle α1 of the incident light 22 emitted from the light source 20 with respect to the metal film 14 to be appropriately changed. On the other hand, the light receiving means 26 is also provided with a movable means (not shown) and is configured to reliably receive the metal film reflected light 24 in synchronization with the light source 20 even when the reflection angle of the metal film reflected light 24 changes.

The sensor chip 16, the light source 20, and the light receiving means 26 constitute an SPR measurement section 28 that performs SPR (Surface plasmon resonance) measurement in the quantitative measurement device 10 of the present invention.
Moreover, above the sensor chip 16, there is provided a light detection means 32 for receiving fluorescence 30 emitted by excited fluorescent substances as described below.

Between the sensor chip 16 and the light detection means 32, for example, a cut filter or a condenser lens may be provided.
The sensor chip 16, the light source 20, and the light detection means 32 constitute an SPFS measurement section 34 that performs SPFS measurement in the quantitative measurement device 10 of the present invention.

In addition, the light receiving means 26 and the light detection means 32 are each connected to the quantitative calculation means 40, and the amount of metal film reflected light 24 received by the light receiving means 26 and the amount of fluorescence 30 received by the light detection means 32 are configured to be transmitted to the quantitative calculation means 40.

In addition, in the sensor chip 16 of this embodiment, a microchannel 36 is formed on the upper surface 14a of the metal film 14. A sensor section 38 is provided in a part of this microchannel 36, in which a molecule (ligand) that specifically binds to the antigen (analyte) to be detected is immobilized.

[2] Pathological information generating system The pathological information generating system of the present invention is a pathological information generating system that generates information regarding the occurrence or onset of cancer, and is characterized in having: a first process unit that acquires information regarding the amount of HE4 having any of four types of residues, i.e., β-N-acetylgalactosamine residues, α2,3-sialic acid residues, α2,6-sialic acid residues, or mannose residues, contained in a test sample; and a second process unit that generates information regarding the occurrence or onset of cancer in a subject related to the test sample based on the amount of HE4 having any of the four types of residues.
A preferred embodiment of the present invention is a system for carrying out the above-mentioned pathology information generating method of the present invention.

[3] HE4 Sugar Chain Analysis Kit The HE4 sugar chain analysis kit of the present invention is an HE4 sugar chain analysis kit comprising an HE4 having a sugar chain and an anti-HE4 antibody, and is characterized in that it is an analysis kit for carrying out the above-mentioned pathological condition information generation method of the present invention. Also, the HE4 sugar chain analysis kit comprising an HE4 having a sugar chain and an anti-HE4 antibody is characterized in that it comprises a substance having affinity for HE4 having a β-N-acetylgalactosamine residue, an α2,3-sialic acid residue, an α2,6-sialic acid residue or a mannose residue at the (non-reducing) end of the sugar chain, and an anti-HE4 antibody.

In one embodiment, from the viewpoint of manifesting the effects of the present invention, it is preferable that the substance having affinity for HE4 is any lectin selected from the entire lectin group (1) to (4). It is also preferable that the substance having affinity for HE4 is two or more lectins selected from multiple groups among the lectin groups (1) to (4).

From the viewpoint of performing accurate measurements, the HE4 glycan analysis kit of the present invention is preferably configured to combine the lectin, an anti-HE4 antibody, and an analytical substrate. It is also preferable that the lectin is equipped with a fluorescent substance. Furthermore, it is preferable that the kit contains at least one type of lectin, WFA or MAM, equipped with a fluorescent substance.

In an embodiment, from the viewpoints of accuracy and simplicity, it is preferable that the configuration includes a chromatography device. It is also preferable that the configuration includes a labeling reagent for chromatographic measurement.

Figure 3 shows a schematic diagram of an example of an HE4 glycan analysis kit of the present invention. As shown in Figure 3, one embodiment of the HE4 glycan analysis kit 50 of the present invention has at least a sample holding well 51, a sample recovery well 52, a drug well 53, and a sample chip 54 connected and integrated, and is capable of detecting injected liquid such as a sample while delivering it. For example, the detection cartridge disclosed in International Publication No. 2017/130369 can be used as a reference.

In addition, the drug recovery well 53 can hold within the well a substance having affinity for HE4, which has a β-N-acetylgalactosamine residue, an α2,3-sialic acid residue, an α2,6-sialic acid residue or a high mannose residue at the end of the sugar chain, or a substance having affinity for the HE4 peptide, such as an HE4 antibody. Both the substance having affinity for the HE4 sugar chain and the HE4 antibody may be provided within the drug well.
The HE4 sugar chain analysis kit of the present invention can be preferably used as a kit for carrying out the above-mentioned method for detecting the HE4 sugar chain of the present invention.

[4] Cancer Testing Method The pathology information generating method of the present invention is a pathology information generating method that generates information regarding the occurrence or onset of cancer, and is characterized by comprising a first step of obtaining information regarding the amount of HE4 having any of four types of residues, i.e., β-N-acetylgalactosamine residue, α2,3-sialic acid residue, α2,6-sialic acid residue, or mannose residue, contained in a test sample, and a second step of generating information regarding the occurrence or onset of cancer in a subject related to the test sample, based on the amount of HE4 having any of the four types of residues.

The pathological condition information generating method of the present invention is a cancer testing method for detecting HE4 glycans to diagnose or predict the onset of cancer, characterized in that a subject evaluates whether a patient is suffering from ovarian cancer based on the quantitative value of a glycan of HE4 having a glycan with affinity to a specific lectin, or the ratio of the quantitative value of the glycan to the quantitative value of total HE4. Also, characterized in that the method evaluates the malignancy or stage of cancer in an ovarian cancer patient.

By analyzing the amount of HE4 having affinity for lectin in a sample using the above-mentioned method for analyzing HE4 sugar chains, the possibility of ovarian cancer and the degree of malignancy can be differentiated.
By measuring the amount of HE4 having affinity to lectins by the HE4 analysis method and comparing it with the amount of HE4 having affinity to lectins in blood or the like collected from a healthy person, it is possible to determine whether or not the patient has ovarian cancer. More specifically, when the amount of HE4 having affinity to lectins present in the patient's sample is significantly greater than the amount of HE4 having affinity to lectins present in a healthy person's sample, it is possible to determine that the patient has ovarian cancer.

The following examples and comparative examples are provided to provide a more detailed explanation of the present invention, but are not intended to limit the scope of the present invention.

Example 1: Quantification of GalNAcHE4 by lectin column method In this Example 1, a WFA column was prepared using WFA as a lectin, and the amount of HE4 having a β-N-acetylgalactosamine residue at the end of the glycan (abbreviated as "GalNAcHE4") was measured by the HE4 glycan analysis method of the present invention for patients diagnosed with ovarian cancer and non-cancer specimens, that is, healthy individuals and endometriosis patients.

(Preparation of WFA column)
1.5 mL of WFA agarose gel (J-Chemical, MAM concentration 3 mg/mL) was added to Pierce (registered trademark) Disposable Columns (Thermo Fisher Scientific) to prepare a WFA column packed with a Sepharose solution bound to WFA at a density of 0.5 mg per mL.

(Measurement of the amount of HE4 sugar chain)
2 mL of the WFA Sepharose solution was equilibrated with phosphate buffer containing 0.1% bovine serum albumin (BSA) at 4°C. 1 to 50 μL of serum sample was diluted with phosphate buffer to make 200 μL, which was applied to the column and held for 30 minutes. The column was then washed with 5 volumes of washing buffer (phosphate buffer containing 0.1% BSA) and fractionated into 1 mL portions to obtain the WFA-unbound fraction. After the column was returned to room temperature, it was eluted with 5 volumes of elution buffer (phosphate buffer containing 10 mM lactose and 0.1% BSA) in 1 mL portions to obtain the WFA-bound fraction.

The total HE4 amount in the serum sample before fractionation using the WFA column, the WFA non-binding fraction, and the WFA binding fraction was measured using ARCHITECT i1000SR (Abbott).
The HE4 recovery rate from the WFA column was always 97% to 100%. Table I shows the total He4 amount before fractionation, the WFA binding ratio (GalNAcHE4 ratio), and the grade of ovarian cancer patients.

The WFA binding rate was calculated according to the following formula.
WFA binding rate = (HE4 amount in WFA-bound fraction/(HE4 amount in WFA-bound fraction+HE4 amount in WFA-unbound fraction) x 100%
The WFA binding rate indicates the percentage of terminal GalNAc glycan HE4 relative to the total HE4 in the sample. Note that total HE4 represents the concentration of HE4 protein including all glycan structures.
The results of the above measurements are shown in Table I and FIG.

From the results shown in Table I and Figure 4, it is clear that the content ratio of glycans having GalNAc termini in total HE4 decreases as the grade of ovarian cancer increases, particularly by comparing the average values of the content ratio.
It is clear that the sugar chain structure content of HE4 protein is changed in ovarian cancer patients, and that in diagnosing ovarian cancer, grade diagnostic information in ovarian cancer specimens can be obtained from the content ratio of GalNAcHE4 in total HE4.

[Example 2]: Quantification of HE4 with various sugar chains by lectin column method (cancer cell culture)
Ovarian cancer cells (cell number JCRB1046, cell name OVSAHO) purchased from the JCRB Cell Bank were cultured.
The OVSAHO cell suspension was added to 20 mL of RPMI 1640 Medium 11875-093 to a concentration of 2.7 to 4.2 x ¹⁰ cells/mL, added to Nunc cell culture treated EasYFlask, and cultured at 36.7°C under 5% CO2 for 3 days. The cell culture supernatant was collected to prepare ovarian cancer cell culture supernatant.

(Preparation of lectin column)
Instead of the WFA agarose used in Example 1, a lectin agarose in which ConA, AAL MAM, or SSA was bound to agarose was used to prepare a lectin column in the same manner as in the preparation of the lectin column using WFA agarose.
(Measurement of the amount of various HE4 sugar chains)
The specimens (samples) used for testing the various lectin columns prepared above were the culture supernatant of the above-cultured ovarian cancer cells and serum from healthy women, and were measured using the lectin columns in the same manner as in Example 1. The results are shown in Table II.

As can be seen from the results shown in Table II, the glycan structures of HE4 secreted from healthy individuals and HE4 secreted from ovarian cancer cells are different, and the ratio of HE4 containing β-N-acetylgalactosamine (GalNAc), high mannose, and fucose residues to the total amount of HE4 decreases with canceration.

In particular, it is clear that the content ratio of HE4 having a sugar chain of β-N-acetylgalactosamine residues changes, and the content ratios (secretion amounts) of α2,3-sialic acid and α2,6-sialic acid increase.
As can be seen from the above results, HE4 having a glycan with a β-N-acetylgalactosamine residue shows a relatively more significant change in content ratio than HE4 having a glycan with other types of residues, making it particularly useful in distinguishing between cancer and non-cancerous.

Example 3: Measurement by surface plasmon excitation enhanced fluorescence spectroscopy In this Example 3, measurements were made by surface plasmon excitation enhanced fluorescence spectroscopy ("SPFS") as follows.

(Fabrication of Plasmon Sensor)
A 1 mm thick transparent flat glass substrate "S-LAL10" (manufactured by Ohara Corporation, refractive index [nd] = 1.72) was plasma cleaned using a plasma dry cleaner "PDC200" (manufactured by Yamato Scientific Co., Ltd.). A chromium film was first formed on one side of the plasma cleaned substrate by sputtering, and a gold film was then formed on the surface by sputtering. The thickness of the chromium film was 1-3 nm, and the thickness of the gold film was 44-52 nm.

The substrate thus obtained was then immersed in 10 mL of an ethanol solution of 10-carboxy-1-decanethiol adjusted to 25 mg/mL for 24 hours to form a self-assembled monolayer (SAM) on the surface of the gold film. The substrate was removed from the ethanol solution, washed with ethanol and isopropanol in that order, and then dried using an air gun.
Next, the substrate on which the SAM was formed was immersed for 1 hour in PBS containing 1 mg/mL of carboxymethyldextran [CMD] having a molecular weight of 500,000, 0.5 mM N-hydroxysuccinimide [NHS], and 1 mM water-soluble carbodiimide [WSC] to immobilize CMD on the SAM, and the unreacted succinate ester was hydrolyzed by immersing in a 1N aqueous NaOH solution for 30 minutes.

A 0.5 mm thick sheet-like silicone rubber spacer with a 2 mm x 14 mm hole was placed on the surface of the obtained substrate (surface on which gold film + SAM + solid-phase layer having a three-dimensional structure were formed in this order), and the substrate was positioned so that this surface was on the inside of the flow path (however, the silicone rubber spacer was not in contact with the liquid being delivered), and a 2 mm thick polymethyl methacrylate plate was placed and pressed onto the substrate from the outside of the flow path to cover it, and the flow path and the polymethyl methacrylate plate were fixed with screws.

Ultrapure water was circulated for 10 minutes, followed by PBS for 20 minutes, at room temperature and at a flow rate of 500 μL/min using a peristaltic pump. Next, 5 mL of PBS containing 50 mM NHS and 100 mM WSC was circulated for 20 minutes, and then 2.5 mL of anti-HE4 monoclonal antibody (3C24; manufactured by hytest) solution was circulated for 30 minutes to immobilize the primary antibody on the solid-phase layer having a three-dimensional structure. Finally, a PBS-buffered saline solution containing 1% bovine serum albumin (BSA) was circulated for 30 minutes to perform a nonspecific adsorption prevention treatment, thereby producing a plasmon sensor.

(Preparation of fluorescently labeled MAM)
MAM (J110, J Chemical) was fluorescently labeled using Alexa Fluor™ 647 Protein Labeling Kit (Invitrogen). The procedure followed the protocol attached to the kit. In order to remove unreacted lectin and fluorescent dye, the reaction product was purified using an ultrafiltration membrane (Nihon Millipore) to obtain an Alexa Fluor 647-labeled MAM solution. The absorbance was measured to determine the concentration of Alexa Fluor 647-labeled MAM in the solution.
The fluorescently labeled MAM was added to a 3% BSA (manufactured by Boval) and PBS (manufactured by Nippon Gene Co., Ltd.) solution so that the concentration of the fluorescently labeled MAM became 5 μg/mL.

(Preparation of blood samples)
Serum samples from patients suspected of having cancer and serum samples from patients who had been definitively diagnosed with cancer were each diluted 3- to 54-fold with phosphate buffer solution to prepare serum specimens.

(Measurement by SPFS)
First washing step: Tris-buffered saline containing 0.05% by mass of Tween (registered trademark) 20 was circulated for 10 minutes.
Blank measurement step: With the flow path filled with Tris-buffered saline, a laser beam with a wavelength of 635 nm was adjusted in photon amount by an optical filter, and irradiated onto the metal thin film from the back side of the plasmon excitation sensor through a prism. The intensity of the light that passed through a filter that cuts off wavelengths other than the fluorescent components was measured by a CCD image sensor (Texas Instruments, Inc.) with a 20x objective lens installed above the measurement area. This measured value is a noise value that does not include the fluorescence emitted by the fluorescent substance that labeled the MAM, and is therefore used as the blank value.
First reaction step: The prepared serum sample was circulated for 30 minutes through the flow channel in which the anti-HE4 antibody immobilized region was formed.

Second washing step: Tris-buffered saline containing 0.05% by mass of Tween (registered trademark) 20. Second reaction step: The above-prepared labeled MAM solution was circulated for 10 minutes.

Third washing step: Tris-buffered saline containing 0.05% by weight of Tween (registered trademark) 20 was circulated for 10 minutes.

Signal measurement process: With the flow path filled with Tris-buffered saline, a laser beam with a wavelength of 635 nm was adjusted in photon amount by an optical filter, and irradiated onto the metal thin film from the back side of the plasmon excitation sensor through a prism. The intensity of the light that passed through a filter that cuts off wavelengths other than the fluorescent components was measured (signal value) by a CCD image sensor (Texas Instruments, Inc.) with a 20x objective lens installed above the measurement area. This measurement value is the fluorescence emitted by a fluorescent substance that labels MAM bound to a glycan (sia2,3-glycan) having α2,3-sialic acid residues (hereinafter abbreviated as "sia2,3") present on HE4 bound to the HE4 antibody, and the quantitative value of the amount of sia2,3-glycan (relative fluorescence intensity value; unit: pW) was calculated by multiplying the value obtained by subtracting the amount of fluorescence obtained in the blank measurement process from the amount of fluorescence obtained in the signal measurement process by the dilution factor of the sample. The numerical values shown in the Example (sia2,3-HE4) columns of Tables III and IV are the corresponding fluorescence intensity values.

(Calculation method of ROC curve)
The ROC curves obtained from the results shown in Tables III and IV are shown in Figures 5, 6 (A, B, C) and 7 (A, B, C).
The ROC curves obtained by comparing (1) cancer samples and non-cancerous samples (including healthy subjects and endometriosis patients), (2) cancer patients aged 50 or younger and non-cancerous samples (healthy subjects and endometriosis patients), and (3) grade 1 cancer samples and non-cancerous samples (healthy subjects and endometriosis patients) are shown in Figures 5 to 7. The areas under the curve (ROC-AUC) of the ROC curves determined based on these figures and other information are summarized in Table V.

The ROC curve (also known as the "recipient operating characteristic curve") shown here is a curve that is drawn by plotting the false positive rate on the horizontal axis and the sensitivity on the vertical axis, and varying the threshold from large to small as a parameter. At first, it is difficult to capture both positive and negative cases, so both take small values, but the sensitivity gradually increases, and the false positive rate increases after a delay. Eventually, both the sensitivity and false positive rate reach 100%. If the biomarker is effective, the ROC curve will move away from the 45-degree slope to the upper left. In other words, the area under the ROC curve (ROC-AUC) represents the performance of the biomarker.

From the results shown in Tables III to V and Figures 5 to 7, the ROC curve of the α2,3-sialic acid terminal HE4, which is the biomarker according to the present invention, exceeds 0.7, and therefore it is understood to be an excellent biomarker. In particular, in the diagnosis of cancer patients and non-cancer patients aged 50 or younger, the area under the ROC curve based on the evaluation results of α2,3-sialic acid HE4 is the largest compared to total HE4 and ROMA, and therefore it is understood that the α2,3-sialic acid according to the present invention is an excellent biomarker.
As shown in Table V, the area under the ROC curve (ROC-AUC) exceeded 0.7, and the sensitivity and specificity at the optimal threshold were high, indicating the high diagnostic performance of the biomarker of the present invention.

8 shows the relationship between the amount of HE4 protein having terminal α2,3-sialic acid residues and the grade of ovarian cancer (healthy subjects, G1, G2, and G3) obtained from the measurement results in Example 3. The Y axis is plotted as the logarithm of ΔS value, which is the quantitative value of α2,3-sialic acid.
The results show that the higher the grade of ovarian cancer, the higher the amount of HE4 with terminal α2,3-sialic acid residues. The glycan structure content of the HE4 protein changes, and it is found that in the diagnosis of ovarian cancer, the amount of HE4 with glycans containing α2,3-sialic acid residues correlates with the grade of ovarian cancer.

Conclusion
Based on the above results, it was demonstrated that HE4 having any of the four types of residues, i.e., β-N-acetylgalactosamine residues, α2,3-sialic acid residues, α2,6-sialic acid residues, or mannose residues, can be a biomarker for ovarian cancer.
Therefore, the present invention can provide a pathology information generating method and a pathology information generating method system for generating information on the onset or onset of cancer, including ovarian cancer. That is, HE4, which is expressed in association with ovarian cancer, can be effectively detected by a simple and inexpensive method, enabling early detection, diagnosis, and treatment of ovarian cancer. In addition, the method of the present invention can detect ovarian cancer in a minimally invasive manner using patient blood, making it possible to detect ovarian cancer simply and quickly.

The present invention can be used in the medical field.

10 Quantitative measurement device 12 Dielectric member 12a Top surface 12b Side surface 12c Side surface 14 Metal film 14a Top surface 16 Sensor chip 18 Sensor chip loading section 20 Light source 22 Incident light 24 Metal film reflected light 26 Light receiving means 28 SPR measurement section 30 Fluorescence 32 Light detection means 34 SPFS measurement section 36 Micro flow channel 38 Sensor section 40 Quantitative calculation means 50 HE4 sugar chain analysis kit 51 Sample holding well 52 Sample recovery well 53 Drug well 54 Sample chip

Claims (23)

A method for analyzing glycans of HE4 for diagnosing the onset or onset of ovarian cancer, comprising:
The method includes a first step of obtaining information about the amount of HE4 having any one of four types of residues, i.e., β-N-acetylgalactosamine residues, α2,3-sialic acid residues, α2,6-sialic acid residues, or mannose residues, contained in a test sample ; and
An analytical method, wherein the test sample is blood, serum, or plasma . The analytical method according to claim 1, wherein the first step is a step of measuring information regarding the amount of HE4 having any of the four types of residues by mass spectrometry or a method of contacting the test sample with a first substance having affinity for any of the four types of residues. The analytical method according to claim 1 or 2, further comprising a second step of calculating a ratio of the amount of HE4 having any of the four types of residues to the total amount of all HE4 contained in the test sample. The analytical method according to any one of claims 1 to 3 , further comprising a third step of obtaining the total amount of all HE4 contained in the test sample, regardless of whether the HE4 has the four types of residues. The analytical method according to any one of claims 1 to 4, wherein the third step is a step of measuring the total amount of HE4 by mass spectrometry , chromatography, or a method of contacting the test sample with a second substance having affinity for the polypeptide chain portion of HE4 . The method according to claim 4 or 5 , wherein the first step is carried out before the third step. the first step is a step of measuring the amount of HE4 having any one of the four types of residues by a method of contacting the test sample with a first substance having affinity for any one of the four types of residues;
The analytical method according to claim 6 , wherein the first substance is a lectin or an antibody having affinity for any of the four types of residues. The analytical method according to claim 7 , wherein the first substance is a lectin having an affinity for the HE4 and having any of the four types of residues, and is any one of ConA, Fujilectin, SSA, and MAM lectins . The first substance is immobilized on a support;
the third step is a step of measuring the total amount of all of the HE4 by a method of contacting the test sample with a second substance having affinity for a polypeptide chain portion of HE4;
The analytical method according to claim 7 or 8 , wherein the second substance is an antibody having affinity for any of the four types of residues . The analytical method according to any one of claims 6 to 9 , wherein the first step is a chromatographic step. The method according to claim 4 or 5 , wherein the third step is carried out before the first step. the second substance is immobilized on a support;
the first step is a step of measuring the amount of HE4 having any one of the four types of residues by a method of contacting the test sample with a first substance having affinity for any one of the four types of residues;
the first substance is a lectin having affinity for any of the four types of residues,
the first substance is labeled with a fluorescent substance;
The analytical method according to claim 11, wherein the first step is a step of measuring the intensity of fluorescence emitted from a fluorophore that labels the first substance bound to HE4 captured by the second substance , and calculating the amount of HE4 having any of the four types of residues based on the intensity of the fluorescence . 12. The analytical method according to claim 11 , wherein in the third step, the intensity of the peak obtained by mass spectrometry or chromatography is compared with the intensity of a predetermined reference peak . The analytical method according to any one of claims 1 to 13 , wherein any one of the four types of residues, i.e., the β-N-acetylgalactosamine residue, the α2,3 - sialic acid residue, the α2,6-sialic acid residue, or the mannose residue, is located at a (non-reducing) terminal of the glycan of HE4. A pathological condition information generating system for generating information regarding ovarian cancer morbidity or onset,
a first step for acquiring information on the amount of HE4 having any one of four types of residues, i.e., β-N-acetylgalactosamine residue, α2,3-sialic acid residue, α2,6-sialic acid residue, or mannose residue, contained in a test sample;
A second step of generating information on the onset or occurrence of ovarian cancer in a subject related to the test sample based on the amount of HE4 having any one of the four types of residues;
and
A pathological condition information generating system, wherein the test sample is blood, serum, or plasma . An HE4 glycan analysis kit for carrying out the analysis method according to any one of claims 1 to 14 , comprising HE4 having a glycan and an anti-HE4 antibody. The analytical kit according to claim 16 , comprising a substance having affinity for HE4 and having a β-N-acetylgalactosamine residue, an α2,3-sialic acid residue, an α2,6-sialic acid residue or a mannose residue at the (non-reducing) end of the glycan, and an anti-HE4 antibody. The analytical kit according to claim 17 , wherein the substance having an affinity for HE4 is a lectin. The analytical kit according to any one of claims 16 to 18 , which is configured by combining the lectin, an anti-HE4 antibody, and an analytical substrate. The analytical kit according to any one of claims 16 to 19 , wherein the lectin is equipped with a fluorophore. 21. The analytical kit according to any one of claims 16 to 20 , comprising at least one lectin of Fuji lectin or MAM equipped with a fluorophore. The analytical kit of any one of claims 16 to 21 , further comprising a chromatography device. 23. The analytical kit of claim 22 , further comprising a labeling reagent for chromatographic measurements.

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