JP2007298420A - Cancer detection method and detection kit - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cancer detection method, and a detection kit used therefor. <P>SOLUTION: The cancer detecting method includes at least a step of making an "antibody coupled to 2-O-desulfated akaran sulphate" and/or an "antibody coupled to akaran sulphate" contact with a sample derived from a biotissue. The "antibody coupled to 2-O-desulfated akaran sulphate" is preferably not substantially couple to akaran sulphate, and preferably not substantially coupled to N-acetyl heparozane, and preferably not substantially coupled to heparine derived from a pig bowel. The "antibody coupled to alakane sulphate" is preferably not substantially coupled to heparane sulphate derived from a bovine kidney, preferably not substantially coupled to heparine derived from the pig bowel, and preferably not substantially coupled to heparane sulphate derived from Engelbreth-Holm-Swarm sarcoma of a mouse. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a cancer detection method and a detection kit.

The abbreviations used in the application documents and their significance are as follows.
GAG: Glycosaminoglycan HA: Hyaluronic acid HEP: Heparin HS: Heparan sulfate EHS-HS: HS from mouse Angelbreth-Home-Swarm sarcoma
Bi-GAG: Biotin-labeled GAG
Bi-HEP derivative: biotin-labeled HEP derivative GlcNS: N-sulfated glucosamine GlcNAc: N-acetylglucosamine IdoA: iduronic acid IdoA (2S): 2-O-sulfated iduronic acid

NAH: N-acetylheparosan

AS: Acalane sulfate Bi-AS: Biotin-labeled AS
RA-AS: Reductive amination AS
PDP-AS: 2-pyridyl disulfide propionylated AS
SH-AS: Thiopropionylated AS

ACH: 2-O-desulfated AS
Bi-ACH: Biotin-labeled ACH
RA-ACH: reductive aminated ACH
PDP-ACH: 2-pyridyl disulfide propionylated ACH
SH-ACH: Thiopropionylated ACH

NH ₂ -HEP: N- desulfated HEP
NAc-HEP: N-desulfated / N-acetylated HEP

2DSH: 2-O-desulfated HEP
NH ₂ -6SH: (2-O · N) -desulfated HEP
6SH: (2-O · N) -desulfated / N-acetylated HEP

6DSH: 6-O-desulfated HEP
NAc-6DSH: N-acetylated 6DSH
NSH: (2-O · 6-O) -desulfated HEP
NAc-NSH; N-acetylated NSH
NH ₂ -2SH: (6-O · N) -desulfated HEP
2SH: (6-O · N) -desulfated / N-acetylated HEP

NH ₂ -CDSH: complete desulfurization oxidation HEP
CDSH: Completely desulfated / N-acetylated HEP

Ch: Chondroitin CS: Chondroitin sulfate CS-A (S): Shark-derived chondroitin sulfate A
CS-A (W): Whale-derived chondroitin sulfate A
CS-B: Chondroitin sulfate B
CS-C: Chondroitin sulfate C
CS-D: Chondroitin sulfate D
CS-E: Chondroitin sulfate E

KS: Keratan sulfate

FITC: Fluorescein isothiocyanate

HRP: Horseradish peroxidase

BSA: bovine serum albumin KLH: hemocyanin PDP-KLH: 2-pyridyl disulfide propionylated KLH

FCS: Fetal calf serum PBS: Phosphate buffered saline TMB: Tetramethylbenzidine SPDP: N-succinimidyl-3- [2-pyridyldithio] propionic acid EDC: 1-ethyl-3- (3-dimethylaminopropyl) Carbodiimide ELISA: Enzyme-labeled antibody assay

AS is a kind of GAG isolated from African Maimai (scientific name: Achatina fulica). AS is a polysaccharide having a repeating structure of a disaccharide (-[IdoA (2S) -GlcNAc]-) composed of GlcNAc and IdoA (2S) as a basic sugar chain structure, and has a structure very similar to HS and HEP. (Non-patent Document 1). ACH is known as a compound obtained by chemically desulfating AS (Non-patent Document 2). MW3G3 is known as an antibody that binds to AS (Non-patent Document 3). However, “an antibody that binds to ACH” is not known. In addition, it is not known that these antibodies are used for cancer detection.

Yon S. Yeong S. Kim et al., The Journal of Biological Chemistry, (USA), 1996, 271, 20, p. 11750-11755 M.M. M. Ishihara et al., The Journal of Biochemistry, 1997, Vol. 121, No. 2, p. 345-349 Jeddy B. Gerdy B. ten Dam et al., The Journal of Biological Chemistry, (USA), 2004, 279, p. 38346-38352

  An object of the present invention is to provide a novel method for detecting cancer and a detection kit that can be used therefor.

  As a result of diligent studies to solve the above problems, the present inventors have found that anti-ACH antibody and anti-AS antibody surprisingly bind to human-derived cancer cells, and based on this finding, It came to provide the method which can detect cancer, and the detection kit which can be used for it.

That is, the present invention includes a method for detecting cancer (hereinafter referred to as “the method of the present invention”) comprising at least a step of contacting an “antibody that binds to ACH” and / or “an antibody that binds to AS” with a sample derived from a living tissue. )I will provide a.
The “antibody that binds to ACH” is preferably one that does not substantially bind to AS. The “antibody that binds to ACH” is preferably one that does not substantially bind to NAH. The “antibody that binds to ACH” is preferably one that does not substantially bind to HEP derived from porcine intestine. The “antibody that binds to ACH” is preferably one that does not substantially bind to bovine kidney-derived HS. The “antibody that binds to ACH” is preferably one that does not substantially bind to EHS-HS.
The “antibody that binds to ACH” is preferably a monoclonal antibody. This “antibody that binds to ACH” is formed by cell fusion between a lymphocyte derived from a mammal immunized with a substance formed by chemically binding a protein and ACH as an antigen and a myeloma cell derived from the mammal. It is preferably a monoclonal antibody produced by a hybridoma. The lymphocytes and myeloma cells are preferably derived from a mouse.
The immunoglobulin class of the “antibody that binds to ACH” is preferably IgM. The “antibody binding to ACH” is preferably an antibody produced by a hybridoma whose accession number is FERM P-20828 at the National Institute of Advanced Industrial Science and Technology (AIST).
The “antibody that binds to AS” is preferably one that does not substantially bind to bovine kidney-derived HS. The “antibody that binds to AS” is preferably one that does not substantially bind to HEP derived from porcine intestine. The “antibody that binds to AS” is preferably one that does not substantially bind to EHS-HS. The “antibody that binds to AS” is preferably one that does not substantially bind to KS derived from bovine cornea. The “antibody that binds to AS” is preferably one that does not substantially bind to HA. The “antibody that binds to AS” is preferably one that does not substantially bind to NAH.
The “antibody that binds to AS” is preferably a monoclonal antibody. This “antibody that binds to AS” is formed by cell fusion of a lymphocyte derived from a mammal immunized with a substance obtained by chemically binding a protein and AS as an antigen and a myeloma cell derived from a mammal. It is preferably a monoclonal antibody produced by a hybridoma. The lymphocytes and myeloma cells are preferably derived from a mouse.
The immunoglobulin class of the “antibody that binds AS” is preferably IgM or IgG. In addition, this “antibody that binds to AS” has an accession number of FERM P-20823, FERM P-20824, FERM P-20825, FERM P-20826, or FERM P- at the National Institute of Advanced Industrial Science and Technology. Preferably, the antibody is produced by the hybridoma that is 20827.

  The “biological tissue” is preferably a vertebrate biological tissue. The “cancer” is preferably colon cancer or squamous cell carcinoma.

  The present invention also provides a cancer detection kit (hereinafter referred to as “the kit of the present invention”) comprising at least “an antibody that binds to ACH” and / or “an antibody that binds to AS” as a constituent component.

  The method of the present invention and the kit of the present invention are extremely useful because cancer can be detected simply, rapidly and inexpensively.

<1> Method of the Present Invention The method of the present invention is a method for detecting cancer comprising at least a step of contacting an “antibody that binds to ACH” and / or “an antibody that binds to AS” with a sample derived from a living tissue.
(1) Antibody binding to ACH The “antibody binding to ACH” used herein is not particularly limited as long as it is an antibody binding to ACH. The “antibody that binds to ACH” is preferably one that does not substantially bind to AS, one that does not substantially bind to NAH is also preferred, and one that does not substantially bind to HEP derived from porcine intestine is also preferred. Those that do not substantially bind to bovine kidney-derived HS are also preferred, and those that do not substantially bind to EHS-HS are also preferred. Among these, those satisfying all these properties are preferable.
In the present specification, “substantially does not bind” does not mean that one molecule does not bind, and it is weak even if binding cannot be detected or binding can be detected. This means that it is negligible for those skilled in the art in the technical field to which it belongs. For example, when the binding property to ACH is 100%, it can be said that “substantially does not bind” when the binding property to a certain substance is 5% or less. Therefore, ACH55 shown in the examples described later binds to ACH, but does not substantially bind to any of AS, NAH, HEP derived from porcine intestine, HS derived from bovine kidney, and EHS-HS. be able to.
The “antibody that binds to ACH” may be either a polyclonal antibody or a monoclonal antibody, but may be a monoclonal antibody from the viewpoint of ensuring continuous productivity and antibody homogeneity. preferable.

  Such an “antibody that binds to ACH” can be produced by a known method using ACH or a modified product thereof as an antigen. Among these, it is preferable to use a substance obtained by chemically binding a protein and ACH as an antigen. As this “protein”, KLH or BSA is preferably employed.

When a monoclonal antibody is employed as the “antibody that binds to ACH”, for example, a mammal is immunized using a “substance formed by chemically binding a protein and ACH” as an antigen, and lymphocytes are collected from the animal. Then, this may be cell-fused with a myeloma cell derived from a mammal to form a hybridoma, and an antibody may be collected from the hybridoma. The “lymphocyte” and “myeloma cell” are preferably derived from a mouse.
The immunoglobulin class of “an antibody that binds to ACH” is not particularly limited, but is preferably IgM.
The most preferable “antibody that binds to ACH” is an antibody produced by a hybridoma whose accession number is FERM P-20828 at the National Institute of Advanced Industrial Science and Technology (AIST).
(2) Antibody that binds to AS The “antibody that binds to AS” used herein is not particularly limited as long as it is an antibody that binds to AS. The “antibody that binds to AS” is preferably one that does not substantially bind to HS derived from bovine kidney, and also preferably one that does not substantially bind to HEP derived from porcine intestine, and also substantially binds to EHS-HS. Also preferred are those that do not substantially bind to KS derived from bovine cornea, those that do not substantially bind to CS and HA, and those that do not substantially bind to NAH. Among these, those satisfying all these properties are preferable.
The meaning of “not substantially bonded” is the same as described above. For example, when the binding property to AS is 100%, when the binding property to a certain substance is 5% or less, it can be said that “substantially does not bind”. Therefore, AS22 shown in the Examples described later binds to AS, but substantially includes any of HS derived from bovine kidney, HEP derived from porcine intestine, EHS-HS, KS derived from bovine cornea, HA, and NAH. It can be said that they are not combined.
In addition, the “antibody that binds to AS” may be either a polyclonal antibody or a monoclonal antibody, but may be a monoclonal antibody from the viewpoint of ensuring continuous productivity and antibody homogeneity. preferable.

  Such an “antibody that binds to AS” can be produced by a known method using AS or a modified product thereof as an antigen. Among them, it is preferable to use a substance obtained by chemically binding a protein and AS as an antigen. As this “protein”, KLH or BSA is preferably employed.

When a monoclonal antibody is employed as an “antibody that binds to AS”, for example, a mammal is immunized with a “substance formed by chemically binding a protein and AS” as an antigen, and lymphocytes are collected from the animal. Then, this may be cell-fused with a myeloma cell derived from a mammal to form a hybridoma, and an antibody may be collected from the hybridoma. The “lymphocyte” and “myeloma cell” are preferably derived from a mouse.
The immunoglobulin class of “an antibody that binds to AS” is not particularly limited, but is preferably IgM or IgG.
The most preferable "antibody that binds to AS" has an accession number of FERM P-20823, FERM P-20824, FERM P-20825, FERM P-20826, or FERM at the National Institute of Advanced Industrial Science and Technology. It is an antibody produced by any hybridoma that is P-20827.

  Such “antibodies that bind to ACH” and “antibodies that bind to AS” may be purified as immunoglobulins or unpurified (for example, hybridoma culture supernatant, ascites, antiserum itself) Etc.), but those purified as immunoglobulins are preferred. In this specification, the term “purification” refers to so-called partial purification (purification to the extent that the fraction of the target substance to be purified in the fraction is not substantially pure but to a large extent). It is used as a concept that includes purification (purification until substantially pure). For example, when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), if a substance other than immunoglobulin is not detected or very little is detected, “purification as immunoglobulin” It can be said that

  Immunoglobulin purification methods include salting out with sodium sulfate, ammonium sulfate, etc., low temperature alcohol precipitation and selective precipitation fractionation with polyethylene glycol or isoelectric point, electrophoresis, DEAE (diethylaminoethyl) -derivative, CM (carboxymethyl). ) -Ion exchange chromatography using ion exchangers such as derivatives, affinity chromatography using protein A or protein G, hydroxyapatite chromatography, immunoadsorption chromatography with immobilized antigen, gel filtration and ultracentrifugation The law etc. can be mentioned.

“Antibodies that bind to ACH” and “antibodies that bind to AS” include those that completely retain the immunoglobulin molecular structure, as well as proteases that do not degrade the antigen-binding site (Fab) (for example, plasmin, pepsin, A fragment containing Fab may be processed by papain or the like. Examples of the fragment containing Fab of the antibody include Fabc, (Fab ′) _{2 and the} like in addition to Fab.

“Antibodies that bind to ACH” and “antibodies that bind to AS” can be determined by genetic engineering if the nucleotide sequences of the genes encoding these antibodies or the amino acid sequences of these antibodies are determined. Or a chimeric antibody (for example, a chimeric antibody containing the Fab part of “an antibody that binds to ACH” or “an antibody that binds to AS”). As long as the above-mentioned fragments or chimeric antibodies containing Fabs of “antibody that binds to ACH” or “antibody that binds to AS” also bind to ACH or AS, “antibody that binds to ACH” in this application document And “an antibody that binds to AS”. Whether or not the produced antibody binds to ACH or AS can be easily determined by the method described in Examples described later.
(3) Contact with biological tissue-derived sample The method of the present invention includes at least the step of bringing the above-mentioned “antibody binding to ACH” and / or “antibody binding to AS” into contact with a biological tissue-derived sample. It is out.

The antibody to be brought into contact with the sample derived from a biological tissue may be either one or both of “an antibody that binds to ACH” and “an antibody that binds to AS”.
Further, an antibody other than these antibodies may be further contacted. Examples of such an antibody include “an antibody that binds to NAH”.

  When multiple types of antibodies are brought into contact with a sample derived from a biological tissue, depending on the purpose, these multiple types of antibodies may be mixed and brought into contact with a single sample derived from the biological tissue. One type of each antibody may be brought into contact with a plurality of samples derived from the living tissue.

The method of “contacting” an antibody with a sample derived from a biological tissue is not particularly limited as long as the antibody molecule comes into contact with the molecule contained in the sample derived from the biological tissue.
The “biological tissue” in the present invention is not particularly limited as long as it is a tissue derived from a living body, but is preferably a vertebrate living tissue. Examples of vertebrates include fish, amphibians, reptiles, birds, mammals and the like, and mammals are preferred. Of these, human biological tissue is more preferable.
Further, the “sample derived from biological tissue” is not particularly limited as long as it is a sample that can be obtained from such biological tissue. Examples of such biological tissue-derived samples include biological tissue itself, cells derived from the biological tissue, fractions containing cell membranes of the cells, molecules extracted from the biological tissue (for example, sugar chain molecules), and the like. Can be illustrated. Among these, cells derived from the living tissue are preferable.
(4) Detection of cancer “An antibody that binds to ACH” and / or “An antibody that binds to AS” is brought into contact with a sample derived from such a biological tissue, and the biological tissue, cell, cell membrane fraction, biological tissue in the sample. Cancer can be detected by detecting the binding of the antibody to a molecule (for example, a sugar chain molecule) extracted from. That is, if these antibodies bind to these in a sample derived from a living tissue, it can be detected that the living tissue is cancerous.
Detection of antibody binding can be performed by a known immunological technique. For example, a substance that can be finally detected as some special signal is bound to an antibody (or an antibody that binds to the antibody (secondary antibody)), and a sample derived from a biological tissue (biological tissue, cell, If the signal remains in the sample after contact with a cell membrane fraction, a molecule extracted from a living tissue (such as a sugar chain molecule) and washing, the antibody is bound to the sample. Judgment can be made.

“Substances that can be finally detected as some special signal” include, for example, enzymes (peroxidase, alkaline phosphatase, β-galactosidase, luciferase, acetylcholinesterase, glucose oxidase, etc.), radioisotopes ( ¹²⁵ I, ¹³¹ I, ³ H), fluorescent dyes (FITC, 7-amino-4-methylcoumarin-3-acetic acid (AMCA), dichlorotriazinylaminofluorescein (DTAF), tetramethylrhodamine isothiocyanate (TRITC), lisaminerhodamine B (Lissamine) Rhodamine B), Texas Red, Phycoerythrin (PE), Umbelliferone, Europium, Phycocyanin, Tricolor, Cyanine, etc., Chemiluminescent substances (Luminol, etc.), Haptens (Dinitrofluorobenzene, Adenosine) one Phosphate (AMP), 2,4-dinitroaniline, etc.), metal particles (ferritin particles, gold colloid particles, etc.), specific binding pairs (biotin and avidins (streptavidin, etc.), lectins and sugar chains, agonists and agonists And any one substance such as HEP and antithrombin III (ATIII)).

  Such signal detection should be appropriately set by a person skilled in the art from known methods according to the type and state of the sample used and the type of “substance that can be finally detected as some special signal”. Can do. For example, when a specimen of a biological tissue slice is used as a sample, it can be detected with a microscope. The pigment can be observed with the naked eye, the density of metal particles, the radioactivity count, the fluorescence intensity, the fluorescence polarization, and the emission intensity. Etc. may be observed and measured. In addition, when a cell suspension is used as a sample, a technique such as a microscope or flow cytometry can be employed. In addition, when a cell membrane fraction or a molecule (for example, a sugar chain molecule) extracted from a biological tissue is used as a sample, a technique such as an ELISA method can be employed.

The method of the present invention further includes other steps as long as it includes at least the step of “contacting an“ antibody that binds to ACH ”and / or“ an antibody that binds to AS ”with a sample derived from biological tissue” as described above. May be included.
The type of “cancer” to be detected in the method of the present invention is not particularly limited, but is preferably colon cancer or squamous cell carcinoma.
The “detection of cancer” in the present invention is a concept that includes not only detection of cancer, but also detection (differentiation) of the type of cancer.
For example, since “an antibody that binds to ACH” and “an antibody that binds to AS” easily binds to a sample derived from a living tissue of colorectal cancer or squamous cell carcinoma among cancers, When bound, it can be detected that “the living tissue is cancer” and can also be detected (differentiated) that the biological tissue is colon cancer or squamous cell carcinoma.
In addition, the type of cancer can be detected (differentiated) by bringing a plurality of types of antibodies into contact with a plurality of samples derived from the same living tissue one by one. In this case, the accuracy of detection (discrimination) can be further increased. For example, by contacting a plurality of samples derived from the same living tissue with an “antibody that binds to ACH” and “an antibody that binds to AS”, respectively, and examining the binding pattern of these antibodies, the accuracy can be improved. High detection (differentiation). For example, as shown in the Examples described later, to a plurality of samples derived from a certain biological tissue, AS17 antibody (antibody that binds to AS), AS22 antibody (antibody that binds to AS), AS25 antibody (antibody that binds to AS), When each of ACH55 (an antibody that binds to ACH) is brought into contact, only the AS22 antibody and the ACH55 antibody bind, and the AS17 antibody and the AS25 antibody do not bind, and the biological tissue is detected as colon cancer ( Can be identified). In addition, as shown in the examples described later, “an antibody that binds to NAH” (for example, NAH46 antibody) binds to cancer tissue, and by further combining this antibody, the accuracy of detection of such cancer can be further increased. Can do.
Further, “cancer detection (differentiation)” here is a concept that includes detection (differentiation) of the possibility. That is, for example, when at least “an antibody that binds to ACH” and / or “an antibody that binds to AS” binds to a sample derived from a living tissue, “the living tissue may be cancerous”. In addition to being detectable, it is also possible to detect (differentiate) that the biological tissue may be colon cancer or squamous cell carcinoma.

The method of the present invention also includes concepts such as detection and analysis of the chemical structure of “sugar chain recognized by“ an antibody that binds to ACH ”and“ an antibody that binds to AS ”” expressed in cancer tissue. For example, the ACS22 antibody described below has NAc-HEP (one of the characteristics is that it retains N-acetyl group, 6-O sulfate group and 2-O sulfate group) and NAc-6DSH (N-acetyl group). N-sulfate group and 2-O sulfate group are one of the features). Therefore, when the ACS22 antibody is brought into contact with a sample derived from a living tissue, if this antibody binds, cancer can be detected, and the cancer tissue has N-acetyl group, 6-O sulfate group and 2- It can be detected and analyzed that a sugar chain holding an O sulfate group or a sugar chain holding an N-acetyl group, an N-sulfate group and a 2-O sulfate group is expressed.
<2> Kit of the Present Invention The kit of the present invention is a cancer detection kit comprising at least “an antibody that binds to ACH” and / or “an antibody that binds to AS” as a constituent component.

  The description of “an antibody that binds to ACH” and “an antibody that binds to AS”, which are components of the kit of the present invention, is the same as in the above <1>. The kit of the present invention may contain only one of such “an antibody that binds to ACH” and “an antibody that binds to AS”, or may contain both.

  The kit of the present invention can be used according to the above-described method of the present invention. Method for contacting the antibody constituting the kit of the present invention with a sample derived from biological tissue, the meaning of “biological tissue” or “sample derived from biological tissue”, a method for detecting cancer, and the type of “cancer” to be detected The meaning of “cancer detection” and the like are all the same as in the above <1>.

  The kit of the present invention is not particularly limited as long as it contains at least “an antibody that binds to ACH” and / or “an antibody that binds to AS” as a constituent component, and a detection reagent for a labeling substance can be added as a constituent component. .

  In addition to these components, a cleaning solution, an enzyme reaction stop solution, and the like may be included. Furthermore, the kit of the present invention may contain a positive control (QC control) for keeping the execution level between measurement batches at a constant level.

  These components can be stored in separate containers.

  Hereinafter, the present invention will be described in detail by way of examples.

Preparation of antibody binding to ACH (Reference Example 1) Preparation of AS and ACH AS was prepared from African Maimai (scientific name: Achatina fulica) according to the method of Kim, YS et al. (Method described in Non-Patent Document 1 above). did. Using the obtained AS as a raw material, ACH was prepared according to the method of Ishihara, M. et al. (Method described in Non-Patent Document 2 above).

Reference Example 2 Preparation of PDP-KLH The introduction of 2-pyridyl disulfide structure into KLH was performed according to the method of Carlsson, J. et al. (Biochem. J., 173, 723 (1978)).

That is, 60 mg of KLH (manufactured by Sigma) was dissolved in 0.1 M phosphate buffer (pH 7.5) -0.1 M NaCl so as to have a final concentration of 2.5 mg / ml. To this solution, 5 mM SPDP (manufactured by Sigma) ethanol solution was added and mixed so that the final concentration was 0.238 mM, and kept at room temperature for 30 minutes. After dialyzing against distilled water to remove excess SPDP, the mixture was lyophilized to give 59.4 mg of PDP-KLH.

Reference Example 3 Preparation of ACH-BSA conjugate via uronic acid ACH and BSA (manufactured by Bayer) were each added to 0.1 M MES buffer (pH 5.5) so that the final concentration was 10 mg / ml. It melt | dissolved and obtained the ACH solution and the BSA solution. After mixing 300 μl of the ACH solution and 150 μl of the BSA solution and adding 400 μg of EDC (PIERCE), the mixture was kept at room temperature for 20 hours with stirring. The obtained solution after the reaction was dialyzed overnight against distilled water and then freeze-dried to obtain 3.5 mg of ACH-BSA conjugate via uronic acid.

Reference Example 4 Preparation of Bi-GAG and Bi-HEP Derivatives AS and ACH used were those prepared in Reference Example 1. HA derived from pig skin (hereinafter simply referred to as “HA”), CS-A (W), CS-A (S), CS-B, CS-C, CS-D, CS-E, bovine kidney HS (hereinafter simply referred to as “HS”) and bovine cornea-derived KS (hereinafter simply referred to as “KS”) were manufactured by Seikagaku Corporation. NAH was prepared from a culture of E. coli K5 according to the method described in JP-A-2004-18840. HEP derived from porcine intestine (hereinafter simply referred to as “HEP”) was purchased from Scientific Protein Laboratories. EHS-HS was prepared by the method described in Japanese Patent Publication No. 7-53756.

Further, various HEP derivative _{(NH 2 -HEP, NAc-HEP} , 6DSH, NAc-6DSH, NH 2 -6SH, 6SH, NH 2 -2SH, 2SH, NSH, NAc-NSH, NH 2 -CDSH, CDSH) is It was prepared by using various desulfation reactions and / or N-acetylation reactions alone or in combination by the method shown in FIG. In the figure, 6-O, 22-O, and N-desulfation of HEP were performed according to the method of Takano et al., Shibuya et al. And Ayotte, L. et al. (Takano, R. et al., J. Carbohydr. Chem. 14, 885 (1995), Takano, R. et al., Carbohydr. Lett. 3, 71 (1998), Kariya, Y. et al., J. Biochem., 123, 240 (1998), Ayotte, L. et al., Carbohydr. Res., 145, 267 (1986)).

  Moreover, N-acetylation followed the method of Danishefsky, I. et al. (Danishefsky, I. et al., Methods Carbohydr. Res., 5, 407 (1965)).

  When 6-O desulfation is carried out according to the method of Takano et al., Some N-desulfation also occurs as a side reaction. Therefore, part of the obtained 6DSH and NSH undergoes N-acetylation, and NAc-6DSH and NAc- NSH was prepared.

The above various GAGs and various HEP derivatives, and AS and ACH prepared in Reference Example 1 were dissolved in 0.1 M MES buffer (pH 5.5) so that the final concentration was 10 mg / ml, and various GAG solutions and Various HEP derivative solutions were obtained. 25 μl each of biotin-LC-hydrazide (manufactured by PIERCE) prepared to 20 mM with dimethyl sulfoxide (manufactured by Wako Pure Chemical Industries, Ltd.) was added to each 1 ml of these various GAG solutions and various HEP derivative solutions. Subsequently, 12.5 μl of an EDC solution adjusted to 100 mg / ml with 0.1 M MES buffer (pH 5.5) was added. After stirring this well, it was reacted at room temperature (15 ° C. to 25 ° C.) for 20 hours. After completion of the reaction, the reaction product was treated with a dialysis membrane (trade name: Cellu Sep H1 (manufactured by Funakoshi Co., Ltd.), cutoff: molecular weight 1,000 or less) and Dulbecco's phosphate buffered saline (pH 7.2-7.5, Without using divalent ions such as calcium ions; hereinafter referred to as “PBS (−)”), and free biotin is sufficiently removed to obtain various Bi-GAG and various Bi-HEP derivatives. Obtained. After completion of dialysis, the Bi-GAG concentration and the Bi-HEP derivative were adjusted to 5 ng / ml and stored frozen.

(Reference Example 5) Preparation of Streptavidin-immobilized Microplate Streptavidin (Vector) was diluted to 20 µg / ml with PBS (-), and Maxisorp (registered trademark) 96-well microplate (Nunk) was used. 50 μl was added to each well. The plate was stored for 18 hours at 4 ° C. so that streptavidin was uniformly immobilized on the plate and then washed twice with PBS (−). Subsequently, ApplieDuo (registered trademark, manufactured by Seikagaku Corporation) was used as a blocking agent, and the portion not coated with streptavidin was blocked by the following method. That is, 5 times dilution of ApplieDuo (registered trademark) using a phosphate buffer solution (pH 7.2 to 7.5: hereinafter referred to as "PB") containing 0.05% proclin 300 (registered trademark, manufactured by SUPELCO) as a preservative. A liquid (hereinafter referred to as “blocking liquid”) was prepared, and 250 μl of this liquid was added to each well and allowed to stand at room temperature for 2 hours. After standing, the blocking solution was sufficiently removed and dried at 37 ° C. for 2 hours to obtain a desired streptavidin-immobilized microplate. The obtained plate was enclosed in an aluminum laminate bag together with a desiccant and stored refrigerated.

(Reference Example 6) Preparation of various GAG-immobilized microplates and various HEP derivative-immobilized microplates 1) Preparation of ACH-immobilized microplates ACH-BSA conjugate (50 ng) prepared in Reference Example 3 Block Ace (registered trademark) added to Maxisorp (registered trademark) 96-well microplate, kept at 4 ° C. for 18 hours, and diluted 4-fold with PBS (−) containing 0.05% proclin 300 (registered trademark) as a preservative (Trademark; manufactured by Dainippon Pharmaceutical Co., Ltd.). After allowing to stand at room temperature for 1 hour, a desired ACH-immobilized microplate was obtained. This ACH-immobilized microplate was used for verification of antibody titer in serum in Reference Example 8 described later.
2) Preparation of various Bi-GAG solid-phase microplates and various Bi-HEP derivative solid-phase microplates The final concentrations of various Bi-GAGs and various Bi-HEP derivatives described in Reference Example 4 above were 1 μg / ml. So that it was dissolved in ApplieDuo (registered trademark) solution diluted 20 times with PBS (-) containing 0.05% proclin 300 (registered trademark) (hereinafter, this solution was referred to as "various Bi-GAG solutions" and "various Bi -"HEP derivative solution"). Each well of the streptavidin-immobilized microplate prepared in Reference Example 5 was mixed with PBS (−) (hereinafter referred to as 300 μl of 0.05% procrine 300 (registered trademark) and 0.05% polyoxyethylene (20) sorbitan monolaurate). Washed 4 times with "wash buffer". Dispense 100 μl of each Bi-GAG solution and each Bi-HEP derivative solution into each well, leave it at room temperature for 30 minutes, and then wash each well 4 times with washing buffer to obtain the desired various Bi-GAG solid-phased microplates and various Bi-HEP derivative solid-phased microplates were obtained. These plates were used in the cloning in Reference Example 8 described later and the reactivity test in Reference Example 10 described later.

Reference Example 7 Preparation of ACH Antigen 1) Preparation of RA-ACH 4.5 mg of ACH prepared in Reference Example 1 was dissolved in 160 μl of 2M aqueous ammonium chloride solution. To this solution, 12 mg of sodium cyanoborohydride was added, and a reductive amination reaction was performed at 70 ° C. for 2 days. To the solution after the reaction, 5 mg of sodium cyanoborohydride was added, and the reaction was further performed for 2 days under the same conditions as described above. After cooling the solution after the reaction in an ice bath, 32 μl of acetic acid was added to completely stop the reaction. RA-ACH was recovered by a solvent precipitation method using twice the amount of ethanol. The obtained precipitate was washed with ethanol and then freeze-dried to obtain 2.1 mg of RA-ACH freeze-dried product.
2) Preparation of 2-pyridyl disulfide propionylated ACH 2.1 mg of RA-ACH prepared in 1) above was dissolved in 1 ml of 0.1 M NaCl-0.1 M phosphate buffer (pH 7.5). After adding 80 μl of 5 mM SPDP ethanol solution to this solution, it was allowed to stand overnight at room temperature to carry out 2-pyridyl disulfide propionylation reaction (PDP reaction). In order to remove excess SPDP, dialysis was performed using distilled water, followed by lyophilization to obtain 1.7 mg of lyophilized PDP-ACH.
3) Preparation of SH-ACH 1.7 mg of PDP-ACH prepared in the above 2) was dissolved in 160 μl of 0.1M NaCl-0.1M sodium acetate buffer (pH 4.5). Dithiothreitol was added to this solution to a final concentration of 25 mM, and a reduction reaction was performed at room temperature for 60 minutes. SH-ACH was recovered by a solvent precipitation method using twice the amount of ethanol. The obtained precipitate was washed with ethanol and then freeze-dried to obtain 1.3 mg of SH-ACH freeze-dried product.
4) Preparation of ACH-KLH conjugate via disulfide bond 1.3 mg of SH-ACH prepared in 3) above and 0.65 mg of PDP-KLH prepared in Reference Example 2 were added to 0.1M NaCl-0.1M phosphate buffer ( pH 7.5) Dissolved in 1 ml, conjugation reaction was performed at room temperature for 2 hours. In order to remove pyridyl-2-thione produced during the reaction, the solution after the above reaction was dialyzed overnight against distilled water and then freeze-dried to obtain 1.5 mg of a freeze-dried ACH-KLH conjugate. It was. The obtained lyophilized product was used as an ACH antigen in Reference Example 8 described later.

(Reference Example 8) Establishment of a hybridoma cell line that produces an antibody that reacts with ACH 1) Immunization of a mouse 1 mg of the ACH antigen obtained in 4) of Reference Example 7 was dissolved in a small amount of distilled water. Was mixed with 2 ml of TiterMAX Gold (registered trademark; manufactured by Sigma) to prepare an antigen solution. As animals to be immunized, four BALB / C mice (6-week-old females; manufactured by Charles River, Japan) were used. 100 μl / animal of the above antigen solution was subcutaneously administered 2 or 3 times every 2 weeks. When the serum antibody titer reached a sufficient value, 100 μl / animal of ACH antigen solution without adjuvant was administered as the final immunization. Three days after the final immunization, the immunized mouse was euthanized and the spleen was removed.

In the above, the antibody titer in the serum was verified by the following method. That is, using the ACH-immobilized microplate prepared in 1) of Reference Example 6 and alkaline phosphatase-labeled anti-mouse IgG + M + A antibody (hereinafter referred to as “ALP-anti-mouse Ig”), the antibody titer in serum was verified by ELISA. did. That is, 50 μl of serum diluted 1000-fold with PBS (−) was dispensed onto an ACH-immobilized microplate and incubated at 37 ° C. for 1 hour. Subsequently, the plate was washed 4 times with PBS (−), and 50 μl of an ALP-anti-mouse Ig solution diluted 1000-fold with 10% Block Ace (registered trademark) / PBS (−) was dispensed into each well. Furthermore, after washing 4 times with PBS (−), 50 μl of a substrate solution (ALP Rose; manufactured by Shino Test Co., Ltd.) was dispensed into each well and left at room temperature for 20 minutes. Further, 50 μl of a coloring reagent (manufactured by Sinotest Co., Ltd.) was added, and the absorbance at 495 nm was measured using 660 nm as a background correction.
2) Creation of hybridoma The immunized lymphocytes obtained from the spleen removed in 1) and mouse myeloma P3U1 cells (manufactured by Shima Institute) were mixed at a ratio of 4: 1 to 5: 1. After mixing, cell fusion was performed by co-centrifuging in 50% polyethylene glycol 1500 (Roche). The myeloma cells used for the cell fusion were those grown in a HAT medium containing 8-azaguanine from one week before cell fusion. After cell fusion, cells grown in HAT medium were used for selection of the following clones.
3) Selection and evaluation of clones 3-1) Cloning The limiting dilution method was adopted for cloning. That is, the cells were diluted with HAT medium so that the number of cells was 1 or less per well, and this was seeded on a 96-well microplate. This was cultured according to a conventional method to obtain a culture supernatant. The antibody titer of the culture supernatant was evaluated by ELISA using the Bi-ACH solid-phased microplate prepared in 2) of Reference Example 6, and clones were selected. The above cloning steps were performed at least twice. As a result of the above, one clone was selected and obtained.
3-2) Evaluation of clones In order to confirm that the activity of the clones obtained in 3) -1 above is maintained, the clones are cultured on a culture scale of a 24-well plate, and the resulting culture supernatant is obtained. The Bi-ACH solid-phased microplate prepared in 2) of Reference Example 6 and the HRP-labeled goat anti-mouse immunoglobulin antibody (hereinafter referred to as “HRP anti-mouse Ig”; manufactured by Dako) This was performed by the ELISA method used. Details are shown below.
[Evaluation of antibody titer of clone]
100 μl of the culture supernatant was dispensed onto a Bi-ACH solid-phased microplate previously washed 4 times with a washing buffer and allowed to stand at room temperature for 1 hour. Further, after washing 4 times with the washing buffer, the HRP antibody diluted 2000 times with the reaction buffer (ApplieDuo (registered trademark) solution diluted 20 times with PBS (-) containing 0.05% Procrine 300 (registered trademark)). 100 μl of mouse Ig was dispensed into each well. The plate was allowed to stand at room temperature for 1 hour, then washed 4 times with a washing buffer, 100 μl of TMB solution (HRP substrate solution; manufactured by BIOFX) was added to each well, and an enzyme reaction was performed at room temperature for 30 minutes. . After completion of the reaction, 100 μl of a coloring reagent (manufactured by BIOFX) was added to each well, and absorbance at 450 nm was measured using 630 nm as a background correction. The reaction buffer was used as a negative blank. As a result, it was confirmed that the clone produced an anti-ACH antibody. Since the clone number of the established hybridoma was ACH55, the antibody produced by this hybridoma was named ACH55 antibody. The above hybridoma was entrusted to the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology on March 1, 2006, with the deposit number FERM P-20828. As a result of examining the immunoglobulin class of the antibody according to a conventional method, it was confirmed that the immunoglobulin class of the ACH55 antibody was IgM.

(Reference Example 9) Preparation of anti-ACH monoclonal antibody 1) Production of anti-ACH monoclonal antibody As a method for producing the target anti-ACH monoclonal antibody, the mouse ascites method was employed. That is, 3 hybridomas (clone number: ACH55) 5 × 10 ⁶ established in 3) of Reference Example 8 were treated with pristane (2,6,10,14-Tetrametylpentadecane: Tokyo Chemical Industry Co., Ltd.) in advance. BALB / C mice (15 weeks old female) were injected into the abdominal cavity. Between 10 and 20 days after the injection, the ascites of the mouse was collected in several times to obtain a total of about 10 ml of ascites.
2) Purification of anti-ACH monoclonal antibody The ascites fluid obtained in 1) above was dialyzed overnight against adsorption buffer 2 (20 mM phosphate buffer (pH 7.5) containing 0.5 MK ₂ SO ₄ ). The dialyzed solution was filtered through a membrane filter (pore size: 0.45 mm), and the resulting filtrate was HiTrap IgY Purification HP column (5 ml) previously equilibrated with adsorption buffer 1 (20 mM phosphate buffer (pH 7.0)). Applied to Amersham Bioscience), and the column was washed with the adsorption buffer 1. When the absorbance at 280 nm of the washing solution passed through was almost zero, 20 mM phosphate buffer (pH 7.5) was passed through the column to elute the ACH55 antibody. The eluted ACH55 antibody was recovered by salting out with NH ₄ SO ₄ (50% saturation). The resulting precipitate was dialyzed against PBS (-) to obtain a dialyzed internal solution containing 2.2 mg of purified ACH55 antibody.

(Reference Example 10) Reactivity Test of Anti-ACH Monoclonal Antibody 1) Reactivity of ACH55 Antibody to Various GAGs 1) -1 Method In order to produce microplates with different amounts of Bi-GAG immobilized, solidification was performed. Bi- produced by the same method as 2) of Reference Example 6 by changing the final concentration of Bi-GAG in the Bi-GAG solution to be used to 0.001, 0.004, 0.012, 0.037, 0.111, 0.333, and 1.000 μg / ml. Each GAG-immobilized microplate was washed 4 times with washing buffer. Thereafter, PBS (-) containing 0.05% procrine 300 as an additive and ApplieDuo (registered trademark; final dilution ratio 20 times; manufactured by Seikagaku Corporation) as an additive (hereinafter referred to as "reaction solution A") 100 μl of each test solution consisting of ACH55 antibody adjusted to a concentration of 0.06 μg / ml using the above, and allowed to stand at room temperature for 60 minutes for antigen-antibody reaction. After completion of the reaction, each well was washed 4 times with a washing buffer, and 100 μl of HRP anti-mouse Ig solution diluted 2000 times with reaction solution A was added as a secondary antibody solution, and this was allowed to stand at room temperature for 60 minutes. And antigen-antibody reaction was carried out. After completion of the reaction, the plate was washed 4 times with a washing buffer, and 100 μl of TMB solution (HRP substrate solution, manufactured by BIOFX) was added as an HRP substrate and allowed to react at room temperature for 30 minutes for color development. Subsequently, 100 μl each of a reaction stop solution (BIOFX) was added to the plate to stop the reaction, and the absorbance at a wavelength of 450 nm (control wavelength: 630 nm) increased by TMB decomposition was measured using well reader SK-603 (registered trademark; Measured by Chemical Industry Co., Ltd.). The reactivity of the antibody was determined based on the absorbance when the above measurement was performed using a Bi-GAG solid-phased microplate prepared using each concentration of Bi-GAG, instead of Bi-GAG solution. -Prepared by the same method as Reference Example 62) except that ApplieDuo (registered trademark) solution diluted 20 times with PBS (-) containing 0.05% procrine 300 (registered trademark) without GAG was used. Evaluation was performed by an absorbance difference (hereinafter simply referred to as “absorbance difference”) obtained by subtracting the absorbance (blank value) when measurement was performed according to the above-described measurement method using a control measurement microplate.
1) -2 Result The result at the time of using NAH, AS, ACH, HS, HEP, and EHS-HS as GAG of a Bi-GAG solid-phased microplate is shown in FIG.

  The ACH55 antibody reacted strongly against ACH even when the Bi-ACH concentration was 0.01 μg / mL (0.001 μg / well) (absorbance difference = approximately 1.0), while AS, HEP, HS, NAH And it did not react to EHS-HS even when the Bi-GAG concentration was increased to 1.0 μg / mL (0.1 μg / well) (FIG. 2).

In addition, the ACH55 antibody is used in the same manner as in the case of HEP described above, HA, various CS (CS-A (W), CS-A (S), CS-B, CS-C, CS-D, CS-E) and There was no reaction to KS.
2) Reactivity of ACH55 antibody to various HEP derivatives The reactivity to various HEP derivatives was evaluated. As HEP derivative of Bi-HEP derivative-coated _{microplate, NH 2 -HEP, NAc-HEP} , 6DSH, NAc-6DSH, NH 2 -6SH, 6SH, NH 2 -2SH, 2SH, NSH, NAc-NSH, NH _2- CDSH and CDSH were used. In this test, the concentration of the solution containing the Bi-HEP derivative used for immobilization was fixed at 1 μg / ml (0.1 μg / well). The results are shown in FIG.

  The ACH55 antibody reacted strongly against CDSH, and reacted extremely weakly with 2SH, 6SH and NAc-NSH (FIG. 3).

  ACH reacted strongly with the ACH55 antibody has an iduronic acid unit (-[IdoA-GlcNAc]-) as the main constituent disaccharide, and thus the epitope of the ACH55 antibody is considered to be composed of the iduronic acid unit. In addition, NAH which the antibody did not react with is a uronic acid C5-epimer of ACH consisting of glucuronic acid units (-[GlcA-GlcNAc]-). It was suggested that it was essential. Furthermore, since no reactivity is observed with AS (the hydroxyl group at the 2-position of iduronic acid residue is sulfated), sulfation of iduronic acid (IdoA (2S)) is a reaction to the antigen of ACH55 antibody. It was suggested to inhibit sex.

CDSH is a polysaccharide mainly composed of glucuronic acid units and iduronic acid units, and the abundance ratio of iduronic acid units is as high as 60% or more. Therefore, there is no contradiction in the result of CDSH reacting with the antibody. In addition, since this reactivity disappeared by N-deacetylation (NH ₂ -CDSH) and N-sulfation (NSH) of the GlcNAc residue, the amino group of the glucosamine residue is also acetylated. It was suggested to be important in the recognition of antibody antigens.

The reason why the antibody ACH55 that does not react with AS reacts weakly with 2SH, 6SH, or NAc-NSH is because HEP undergoes multiple modifications such as N-desulfation, 2-O desulfation, and 6-O desulfation. It was assumed that the acid unit was manifested in the HEP derivative.

Preparation of antibody binding to AS (Reference Example 11) Preparation of AS-BSA conjugate via uronic acid AS was used instead of ACH, and the same procedure as in Reference Example 3 was performed. Finally, 3.5 mg of AS-BSA conjugate via uronic acid was obtained.

Reference Example 12 Preparation of Bi-GAG and Bi-HEP Derivatives The same procedure as in Reference Example 4 was performed using AS instead of ACH.

Reference Example 13 Preparation of Streptavidin-Solid-Phase Microplate The same procedure as in Reference Example 5 was performed.

(Reference Example 14) Production of various GAG solid-phased microplates and various HEP derivative solid-phased microplates 1) Production of AS solid-phased microplates Using AS-BSA (50 ng) prepared in Reference Example 11, It carried out like 1) of the reference example 6. This AS-immobilized microplate was used to verify the antibody titer in serum in Reference Example 16 described later.
2) Preparation of various Bi-GAG solid-phased microplates and various Bi-HEP derivative solid-phased microplates.

(Reference Example 15) Preparation of AS antigen 1) Preparation of RA-AS 4 mg of AS prepared in Reference Example 1 was weighed and dissolved in 160 μl of 2M aqueous ammonium chloride solution. To this solution, 12 mg of sodium cyanoborohydride was added, and a reductive amination reaction was performed at 70 ° C. for 2 days. To the solution after the reaction, 5 mg of sodium cyanoborohydride was added, and the reaction was further performed for 2 days under the same conditions as described above. After cooling the solution after the reaction in an ice bath, 32 μl of acetic acid was added to completely stop the reaction. RA-AS was recovered by a solvent precipitation method using twice the amount of ethanol. The obtained precipitate was washed with ethanol and then freeze-dried to obtain 3.3 mg of RA-AS freeze-dried product.
2) Preparation of PDP-AS 3.3 mg of RA-AS prepared in 1) above was dissolved in 1 ml of 0.1M NaCl-0.1M phosphate buffer (pH 7.5). After adding 80 μl of 5 mM SPDP ethanol solution to this solution, it was allowed to stand at room temperature overnight to carry out 2-pyridyl disulfide propionylation reaction (PDP reaction). In order to remove excess SPDP, dialysis was performed using distilled water, followed by lyophilization to obtain 3.8 mg of a lyophilized product of PDP-AS.
3) Preparation of SH-AS 2.0 mg of PDP-AS prepared in 2) above was weighed and dissolved in 160 μl of 0.1M NaCl-0.1M sodium acetate buffer (pH 4.5). Dithiothreitol was added to this solution to a final concentration of 25 mM, and a reduction reaction was performed at room temperature for 60 minutes. SH-AS was recovered by a solvent precipitation method using twice the amount of ethanol. The obtained precipitate was washed with ethanol and then freeze-dried to obtain 1.5 mg of SH-AS freeze-dried product.
4) Preparation of AS-KLH conjugate via disulfide bond: 1.5 mg of SH-AS prepared in 3) above and 0.75 mg of PDP-KLH prepared in Reference Example 2 were mixed with 0.1M NaCl-0.1M phosphate buffer ( pH 7.5) Dissolved in 1 ml and conjugated at room temperature for 2 hours. In order to remove pyridyl-2-thione produced during the reaction, the solution after the above reaction was dialyzed overnight against distilled water and then freeze-dried to obtain 1.9 mg of a freeze-dried AS-KLH conjugate. It was. The obtained freeze-dried product was used as an AS antigen in Reference Example 16 described later.

(Reference Example 16) Establishment of hybridoma cell line producing antibody reacting with AS 1) Immunization of mouse 1 mg of AS antigen dissolved in a small amount of distilled water and 2 ml of TiterMAX Gold (registered trademark; manufactured by Sigma) The mixture was mixed to prepare an antigen solution. As animals to be immunized, four BALB / C mice (6-week-old females; manufactured by Charles River, Japan) were used. 100 μl / animal of the antigen solution was administered subcutaneously 2 or 3 times every 2 weeks. When the serum antibody titer reached a sufficient value, 100 μl / AS antigen solution containing no adjuvant was administered as the final immunization. Three days after the final immunization, the immunized mouse was euthanized and the spleen was removed.

In the above, the antibody titer in the serum was verified by the following method. That is, the antibody titer in serum was verified by ELISA using the AS solid-phased microplate prepared in 1) of Reference Example 14 and ALP-anti-mouse Ig. Specifically, 50 μl of serum diluted 1000-fold with PBS (−) was dispensed onto an AS-immobilized microplate and incubated at 37 ° C. for 1 hour. Subsequently, the plate was washed 4 times with PBS (−), and 50 μl of ALP-anti-mouse Ig solution diluted 1000-fold with 10% Block Ace (registered trademark) / PBS (−) was dispensed into each well. Furthermore, after washing 4 times with PBS (−), 50 μl of a substrate solution (ALP Rose; manufactured by Sinotest Co., Ltd.) was dispensed into each well and allowed to stand at room temperature for 20 minutes. Further, 50 μl of a coloring reagent (manufactured by Sinotest Co., Ltd.) was added, and the absorbance at 495 nm was measured using 660 nm as background correction.
2) Creation of hybridoma The immunized lymphocytes obtained from the spleen removed in 1) and mouse myeloma P3U1 cells (manufactured by Shima Institute) were mixed at a ratio of 4: 1 to 5: 1. After mixing, cell fusion was performed by co-centrifuging in 50% polyethylene glycol 1500 (Roche). The myeloma cells used for the cell fusion were those grown in a HAT medium containing 8-azaguanine from one week before cell fusion. After cell fusion, cells grown in HAT medium were used for selection of the following clones.
3) Selection and evaluation of clones 3-1) Cloning The limiting dilution method was adopted for cloning. That is, the cells were diluted with HAT medium so that the number of cells was 1 or less per well, and this was seeded on a 96-well microplate. This was cultured according to a conventional method to obtain a culture supernatant. The antibody titer of the culture supernatant was evaluated by ELISA using the Bi-AS solid-phased microplate prepared in Reference Example 14 2), and clones were selected. The above cloning steps were performed at least twice. As a result of the above, six clones were selected and obtained.
3-2) Evaluation of clones In order to confirm that the activity of each clone obtained in 3) -1 above is maintained, the clone is cultured on a culture scale of a 24-well plate, and the obtained culture supernatant The antibody titer of the solution was evaluated by ELISA using Bi-AS solid-phased microplate prepared in 2) of Reference Example 14 and HRP anti-mouse Ig. Details are shown below.
[Evaluation of antibody titer of clone]
100 μl of the culture supernatant was dispensed onto a Bi-AS solid-phased microplate that had been washed 4 times with a washing buffer in advance, and allowed to stand at room temperature for 1 hour. Further, after washing 4 times with a washing buffer, the reaction buffer (ApplieDuo (registered trademark; manufactured by Seikagaku Corporation) solution diluted 20-fold with PBS (-) containing 0.05% Procrine 300 (registered trademark)) was used. 100 μl of HRP anti-mouse Ig diluted 2000 times was dispensed to each well. The plate was allowed to stand at room temperature for 1 hour, then washed 4 times with a washing buffer, 100 μl of TMB (HRP substrate solution; manufactured by BIOFX) was added to each well, and an enzyme reaction was performed at room temperature for 30 minutes. After completion of the reaction, 100 μl of a coloring reagent (manufactured by BIOFX) was added to each well, and absorbance at 450 nm was measured using 630 nm as a background correction. The reaction buffer was used as a negative blank. As a result, it was confirmed that each clone produced an anti-AS antibody. Since the clone numbers of the established hybridomas were AS17, AS22, AS25, AS38 and AS48, the antibodies produced by this hybridoma were named AS17 antibody, AS22 antibody, AS25 antibody, AS38 antibody and AS48 antibody, respectively. The above hybridoma is registered with the Patent Organism Depositary of the National Institute of Advanced Industrial Science and Technology on March 1, 2006, with a deposit number of FERM P-20823, FERM P-20824, FERM P-20825, FERM P-20826, or FERM. Each was commissioned as P-20827. As a result of examining the immunoglobulin class of the antibody and its subclass according to a conventional method, it was confirmed that the AS17 antibody was classified as IgG2a, the AS25 antibody and AS38 antibody were classified as IgG1, and the AS22 antibody and AS48 antibody were classified as IgM. .

Reference Example 17 Preparation of Anti-AS Monoclonal Antibody 1) Production of Anti-AS Monoclonal Antibody As a method for producing the target anti-AS monoclonal antibody, the mouse ascites method was employed. That is, 5 × 10 ^{6 of} each hybridoma (AS17, AS22, AS25, AS38, AS48) established in 3) of Reference Example 16 was previously stored in advance as pristane (2,6,10,14-Tetrametylpentadecane: Tokyo Chemical Industry). Co.) Injected into the peritoneal cavity of 3 treated BALB / C mice (female 15 weeks old). Between 10 and 20 days after the injection, the ascites of the mouse was collected in several times to obtain a total of about 10 ml of ascites.
2) Purification of anti-AS monoclonal antibody A method for purifying IgG type antibodies (AS17 antibody, AS25 antibody and AS38 antibody) is shown below. That is, the ascites obtained by 1) above was dialyzed overnight against the adsorption buffer 1 (20 mM phosphate buffer (pH 7.0)). The dialyzed internal solution was filtered through a membrane filter (pore diameter: 0.45 mm), and the obtained filtrate was applied to a HiTrap Protein G HP column (5 ml; manufactured by Amersham Biosciences) equilibrated with the adsorption buffer 1 in advance. The column was washed with the same buffer. When the absorption at 280 nm of the flow-through became almost zero, 0.1 M glycine buffer (pH 2.7) was passed through the column to elute AS17 antibody, AS25 antibody and AS28 antibody, respectively. Each solution containing each antibody was collected and dialyzed sufficiently against PBS (-). The dialysis internal solution was appropriately adjusted to an appropriate concentration by ultrafiltration concentration or the like as necessary, and these were used as purified antibodies. The amounts of purified AS17 antibody, AS25 antibody and AS38 antibody were 30.8 mg, 4.6 mg and 11.5 mg, respectively.

The purification method of IgM type antibodies (AS22 antibody and AS48 antibody) is shown below. That is, the ascites obtained in 1) above was dialyzed overnight against adsorption buffer 2 (20 mM phosphate buffer (pH 7.5) containing 0.5 MK ₂ SO ₄ ). After the dialysis internal solution was filtered through a membrane filter (pore diameter: 0.45 mm), the filtrate was applied to a HiTrap IgY Purification HP column (5 ml; manufactured by Amersham Biosciences) that had been equilibrated with the adsorption buffer 1 in advance. The column was washed. When the absorption at 280 nm of the flow-through solution was almost zero, 20 mM phosphate buffer (pH 7.5) was passed through the column to elute the AS22 antibody and AS48 antibody, respectively. The eluted AS22 antibody and AS48 antibody were recovered by salting out (NH ₄ SO ₄ : 50% saturation). The precipitate was dialyzed against PBS (−), and the resulting dialyzed internal solution was used as a purified antibody. The amount of purified AS22 antibody and AS48 antibody was 2.7 and 2.2 mg, respectively.

(Reference Example 18) Reactivity test of each anti-AS monoclonal antibody The reactivity of each anti-AS monoclonal antibody (purified antibody) to various GAGs and various HEP derivatives was verified.
1) Reactivity test of each purified antibody, 1-AS reactivity test 1) -1 method Bi-AS solution used for immobilization to prepare microplates with different amounts of Bi-GAG immobilization Bi-AS solid-phase microplate prepared by changing the final concentration of Bi-AS to 0.001, 0.004, 0.012, 0.037, 0.111, 0.333, 1.000 μg / ml and the same method as in Reference Example 14 2) Was washed four times with each washing buffer, and then each well was supplemented with ApplieDuo (registered trademark; final dilution factor 20 times, manufactured by Seikagaku Corporation) as an additive and PBS containing 0.05% proclin 300 as a preservative ( -) (Hereinafter referred to as “reaction solution A”), 0.025 mg / ml (AS17 antibody), 0.07 mg / ml (AS22 antibody), 0.006 mg / ml (AS25 antibody), 0.006 mg / ml ( AS38 antibody), 0.1 mg / ml (AS48 antibody) Adding each test solution containing each purified antibody by 100 [mu] l, which was allowed to stand 60 minutes at room temperature, allowed to antigen-antibody reaction. After completion of the reaction, each well was washed 4 times with a washing buffer, and 100 μl each of HRP anti-mouse Ig (Dako) solution diluted 2000 times with reaction solution A was added as a secondary antibody solution, and this was added at room temperature. The reaction was allowed to stand for minutes for antigen-antibody reaction. After completion of the reaction, the plate was washed 4 times with a washing buffer, and 100 μl of TMB solution (HRP substrate solution; manufactured by BIOFX) was added as a substrate for HRP, and reacted at room temperature for 30 minutes to develop color. Subsequently, the reaction was stopped by adding 100 μl each of a reaction stop solution (manufactured by BIOFX) to the plate, and then the absorbance at a wavelength of 450 nm (control wavelength: 630 nm) increased by TMB decomposition was measured using well reader SK-603 (registered trademark; Measured by Chemical Industry Co., Ltd.). The reactivity of the antibody was determined based on the absorbance when the above measurement was performed using a Bi-GAG solid-phased microplate prepared using each concentration of Bi-GAG, instead of Bi-GAG solution. -Prepared by the same method as Reference Example 62) except that ApplieDuo (registered trademark) solution diluted 20 times with PBS (-) containing 0.05% procrine 300 (registered trademark) without GAG was used. Evaluation was performed by an absorbance difference (hereinafter simply referred to as “absorbance difference”) obtained by subtracting the absorbance (blank value) when measurement was performed according to the above-described measurement method using a control measurement microplate.
1) -2 Results The results are shown in FIG. The relative sensitivity of AS22 antibody and AS48 antibody to AS was 100 times stronger than that of AS17 antibody, AS25 antibody and AS38 antibody. When the Bi-AS concentration was 1.0 μg / ml (0.1 μg / well), all antibodies showed strong reactivity (absorbance difference; ≧ 1).
2) Reactivity test of each purified antibody Part 2-Reactivity test for various GAGs 2) -1 Method In addition to fixing the concentration of various Bi-GAGs used to 1.0 μg / ml (0.1 μg / well), the above 1) The reactivity of each purified antibody against various GAGs and various HEP derivatives was evaluated in the same manner as in 1.

2) -2 Results FIG. 5 shows the results of the reactivity of each purified antibody against various GAGs.

  Similar to 1) above, each antibody reacted strongly with AS. It was confirmed that none of the antibodies reacted with HS and HEP. As for the AS17 antibody, AS22 antibody, AS25 antibody and AS38 antibody, other GAGs, that is, ACH, EHS-HS, KS, HA, NAH, CS-A (W), CS-A (S), CS- It confirmed that it did not react substantially with all of B, CS-C, CS-D, CS-E, and Ch. On the other hand, the AS48 antibody is against EHS-HS, KS, HA, NAH, CS-A (W), CS-A (S), CS-B, CS-C, CS-D, CS-E, and Ch. Although it did not react, it was confirmed that it has about 55% reactivity with ACH.

From these results, it was suggested that 2-O sulfated iduronic acid (IdoA (2S)) residue is included in the epitope of these antibodies. Furthermore, in the HS and HEP molecules where each antibody did not show reactivity, α (1-4) bonds of IdoA (2S) exist, but most of them bind to GlcNS or O-sulfated GlcNS. Therefore, it was speculated that modification of the glucosamine residue was also an important negative factor for the reaction of these antibodies.
3) Reactivity test of each purified antibody Part 3-Reactivity test against various HEP derivatives In addition to using various Bi-HEP derivative solid-phased plates described in Reference Example 14 to aid in epitope analysis Then, by evaluating the reactivity according to the method described in 2) -1, it was verified how the modified state of the N-position of the glucosamine residue of the antigen affects the reactivity of each antibody. . Hereinafter, the state where the N-position of the glucosamine residue is “NH ₂ —” is referred to as “unmodified”. The results are shown in FIG.

  Both antibodies reacted with 2SH. The reactivity of the AS17 antibody, AS22 antibody, AS25 antibody, AS38 antibody and AS48 antibody were 172.2%, 104.9%, 76.4%, 31.7% and 104.8%, respectively. 2SH is mainly composed of a glucuronic acid unit (-[GlcA-GlcNAc]-) and a 2-O sulfated iduronic acid unit (-[IdoA (2S) -GlcNAc]-). Since the abundance ratio of acid units is as high as 60% or more, this result is consistent with the results of 1) and 2) above.

On the other hand, the AS17 antibody, AS25 antibody, and AS38 antibody did not react with NH ₂ -2SH. Most of the N position of the glucosamine residue of NH ₂ -2SH is unmodified. Therefore, it was suggested that the epitope of these antibodies contains an N-acetylated glucosamine residue.

  In addition, although the N-position of the N-position of the glucosamine residue of NAc-6DSH is acetylated, the AS17 antibody, AS25 antibody and AS38 antibody do not substantially react with NAc-6DSH. Despite the fact that most of the N-positions of the glucosamine residues of HEP were acetylated, these antibodies did not react substantially with NAc-HEP. It was suggested to be inhibited by N-sulfation and / or O-sulfation of residues.

The AS22 antibody and AS48 antibody did not substantially react with NH ₂ -HEP and 6DSH, but they had the same reactivity as AS with Nac-acetylated NAc-HEP and NAc-6DSH. showed that. Most of the N position of the glucosamine residue of NH ₂ -HEP is unmodified. Further, most of the N-position of the 6DSH glucosamine residue is sulfated, but part of the glucosamine residue is unmodified because N-desulfation occurs in part during the preparation process. Therefore, from the above results, it was suggested that the epitopes of the AS22 antibody and the AS48 antibody include a glucosamine residue modified in some way at the N position, particularly an N-acetylated glucosamine residue. . The reactivity with NAc-HEP and NAc-6DSH suggested that O-sulfation and N-sulfation of the glucosamine residue did not substantially affect the reactivity of the antibody, respectively.

  Table 1 shows the reactivity of these antibodies to various sugar chains.

In Table 1, “ACS” is AS, “Heparin” is HEP, “NH2-Heparin” is NH ₂ -HEP, “NAc-Heparin” is NAc-HEP, “Acetylated 6DSH” is NAc-6DSH. and the "Acetylated NSH" is NAc-NSH, the "NH2-CDSH" is _NH 2 -CDSH, "Acetylated NAH" means respectively the NAH.
In Table 1, “NH 2”, “N-Acetyl”, “NS”, “6S”, “2S” are amino group, N-acetyl group, N-sulfate group, 6-O in the sugar chain skeleton, respectively. A sulfate group and a 2-O sulfate group are shown. In Table 1, the symbol ◎, symbol ◯, symbol Δ, and symbol x indicate the abundance of the group in the sugar chain.
The symbol ◎ is an image that the group is present in the sugar chain, the symbol ◯ is the image that the group is present in the sugar chain, and the symbol △ is the group that is not present in the sugar chain. Or an unknown symbol, and a symbol x indicates an image that the group is not substantially present in the sugar chain. However, ◎, ○, and Δ are only images, and do not mean the absolute amount of the group, and do not necessarily mean the same level between symbols.
The numbers (from the left, 0.02, 0.05, 0.01, 0.005, 0.15, 0.05) immediately below the name of each antibody in Table 1 indicate the concentration (μg / ml) of each reacted antibody. These antibodies were reacted with various GAG biotin-labeled products (1 μg / ml) listed on the left to evaluate their reactivity.
From Table 1, for example, the ACS22 antibody has NAc-HEP (which is characterized by retaining 6-O sulfate group and 2-O sulfate group) and NAc-6DSH (N-sulfate group and 2-O sulfate). One characteristic is that the sulfate group is retained).

Flow cytometry HSC-4 strain (Anticancer Res., 2005 Nov-Dec; 25 (6B): 4053-4059) and HO-1-u-1 strain (Oncol. Rep., 2004) which are cell lines derived from squamous cell carcinoma Aug; 12 (2): 339-345) and HO-1N-1 strain (Cancer. Lett. 2000 Apr 14; 151 (2): 199-208), and LS174T strain (Clin. Cancer Res., 2006 Mar 1; 12 (5): 1606-1614) and C-1 strain (Cancer Res. 2001 Jun 1; 61 (11): 4620-4627) were used as samples derived from living tissue. As a control, COLO201 strain (Cancer Res. 1994 Jan 1; 54 (1): 272-275), which is a colon cancer-derived cell line, was used.

  In some experiments, these cells were used after being treated with heparitinase I (derived from Flavobacterium heparinum; manufactured by Seikagaku Corporation).

  As antibodies (primary antibodies), the above-mentioned antibodies, 10E4 and JM403 (both are anti-HS monoclonal antibodies), 3G10 (anti-Δ-HS monoclonal antibodies), NAH46 (anti-NAH monoclonal antibodies (“anti-HS antibodies”)) All of them were manufactured by Seikagaku Corporation.

  The primary antibody was added to the cell suspension to a final concentration of 5 μg / ml or 20 μg / ml and incubated at 4 ° C. for 30 minutes. Subsequently, the secondary antibody (FITC) was washed with PBS containing 2% final FCS, and then diluted 1/200 according to the recommended dilution factor (1/20 to 1/400) described in the accompanying instructions. A labeled anti-mouse immunoglobulin antibody (Chemicon; catalog number AQ326F)) was added and incubated at 4 ° C. for 30 minutes. Thereafter, the plate was washed with PBS containing FCS having a final concentration of 2%, and analyzed by flow cytometry. In addition, what was incubated only with the secondary antibody without using the primary antibody was used as a control. The results are shown in FIGS. In each figure, the uppermost graph on the left shows the population of cells subjected to analysis. In each figure, the horizontal axis of the graph other than the top on the left side shows the fluorescence intensity, and the vertical axis shows the number of cells. In each figure, the mountain-shaped solid line at the left side of the graph other than the top of the left side indicates the control. When the solid line peak is shifted to the right side of this control, the primary antibody is bound to the cell (the cell is stained with the primary antibody).

  7 to 20 show that all cancer-derived cell lines are stained with NAH46.

  Among them, HSC-4 and HO-1N-1 strains were stained with 10E4 and JM403 in addition to NAH46. This staining property hardly changed even when the cells were treated with heparitinase I. The C-1 strain was also stained with 10E4 and JM403 although it was slightly weaker than the HSC-4 and HO-1N-1 strains.

  The LS174T strain was stained with 10E4 and JM403 in addition to NAH46. Surprisingly, the LS174T strain was also stained with AS22 and ACH55. This staining property hardly changed even when the cells were treated with heparitinase.

From the above results, it was shown that cancer can be detected by bringing “an antibody that binds to ACH” and / or “an antibody that binds to AS” into contact with a sample derived from living tissue.

The kit of the present invention comprising the following components was prepared.
One purified AS22 antibody (primary antibody)
One purified ACH55 antibody (primary antibody)
3. One FITC-labeled anti-mouse immunoglobulin antibody (secondary antibody)
4). AS (standard product) 1 One ACH (standard product) 6. 1 cleaning solution (PBS)

It is a figure which shows the flow of the manufacturing method of various HEP derivatives. It is a figure which shows the reactivity with respect to various GAG of ACH55 antibody. Bi-GAG (μg / ml) on the horizontal axis indicates the final concentration of Bi-GAG in the Bi-GAG solution used in the preparation of the Bi-GAG solid-phased plate. It is a figure which shows the reactivity with respect to various HEP derivatives etc. of ACH55 antibody. It is a figure which shows the reactivity with respect to AS of AS17 antibody, AS22 antibody, AS25 antibody, AS38 antibody, and AS48 antibody. Bi-AS (μg / ml) on the horizontal axis indicates the final concentration of Bi-AS in the Bi-AS solution used in the preparation of the Bi-AS solid-phase plate. It is a figure which shows the reactivity with respect to various GAG of AS17 antibody, AS22 antibody, AS25 antibody, AS38 antibody, and AS48 antibody. It is a figure which shows the reactivity with respect to various HEP derivatives etc. of AS17 antibody, AS22 antibody, AS25 antibody, AS38 antibody, and AS48 antibody. It is a figure which shows the grade of the coupling | bonding of each antibody in COLO201 strain | stump | stock. The results of using the AS17 antibody, AS22 antibody, AS25 antibody, and ACH55 antibody as the primary antibodies in order from the second graph from the top to the bottom on the left are shown. In addition, the results of using 10E4, JM403, and NAH46 as primary antibodies in order from the top graph on the right side to the bottom are shown. The final concentration of the primary antibody was 5 μg / ml. It is a figure which shows the grade of the coupling | bonding of each antibody in COLO201 strain | stump | stock. The results of using the AS17 antibody, AS22 antibody, AS25 antibody, and ACH55 antibody as the primary antibodies in order from the second graph from the top to the bottom on the left are shown. In addition, the results of using 10E4, JM403, and NAH46 as primary antibodies in order from the top graph on the right side to the bottom are shown. The final concentration of the primary antibody was 20 μg / ml. It is a figure which shows the grade of the coupling | bonding of each antibody in HSC-4 stock | strain. The results of using the AS17 antibody, AS22 antibody, AS25 antibody, and ACH55 antibody as the primary antibodies in order from the second graph from the top to the bottom on the left are shown. In addition, the results of using 10E4, JM403, and NAH46 as primary antibodies in order from the top graph on the right side to the bottom are shown. The final concentration of the primary antibody was 20 μg / ml. It is a figure which shows the grade of the coupling | bonding of each antibody in HO-1-u-1 stock | strain. The results of using the AS17 antibody, AS22 antibody, AS25 antibody, and ACH55 antibody as the primary antibodies in order from the second graph from the top to the bottom on the left are shown. In addition, the results of using 10E4, JM403, and NAH46 as primary antibodies in order from the top graph on the right side to the bottom are shown. The final concentration of the primary antibody was 20 μg / ml. It is a figure which shows the grade of the coupling | bonding of each antibody in HO-1N-1 stock | strain. The results of using the AS17 antibody, AS22 antibody, AS25 antibody, and ACH55 antibody as the primary antibodies in order from the second graph from the top to the bottom on the left are shown. In addition, the results of using 10E4, JM403, and NAH46 as primary antibodies in order from the top graph on the right side to the bottom are shown. The final concentration of the primary antibody was 20 μg / ml. It is a figure which shows the grade of the coupling | bonding of each antibody in HSC-4 stock | strain (the result tested separately from FIG. 9). The results of using the AS22 antibody and the ACH55 antibody as primary antibodies in order from the second graph from the top to the bottom on the left are shown. In addition, the results of using 10E4, JM403, NAH46, and 3G10 as primary antibodies in order from the top graph on the right side to the bottom are shown. The final concentration of the primary antibody was 20 μg / ml. It is a figure which shows the grade of the coupling | bonding of each antibody in HSC-4 strain | stump | stock treated with heparitinase I. The results of using the AS22 antibody and the ACH55 antibody as primary antibodies in order from the second graph from the top to the bottom on the left are shown. In addition, the results of using 10E4, JM403, NAH46, and 3G10 as primary antibodies in order from the top graph on the right side to the bottom are shown. The final concentration of the primary antibody was 20 μg / ml. It is a figure which shows the grade of the coupling | bonding of each antibody in LS174T strain | stump | stock. The results of using the AS22 antibody and the ACH55 antibody as primary antibodies in order from the second graph from the top to the bottom on the left are shown. In addition, the results of using 10E4, JM403, NAH46, and 3G10 as primary antibodies in order from the top graph on the right side to the bottom are shown. The final concentration of the primary antibody was 20 μg / ml. It is a figure which shows the grade of the coupling | bonding of each antibody in LS174T strain | stump | stock treated with heparitinase I. The results of using the AS22 antibody and the ACH55 antibody as primary antibodies in order from the second graph from the top to the bottom on the left are shown. In addition, the results of using 10E4, JM403, NAH46, and 3G10 as primary antibodies in order from the top graph on the right side to the bottom are shown. The final concentration of the primary antibody was 20 μg / ml. It is a figure which shows the grade of the coupling | bonding of each antibody in HSC-4 stock | strain (the result tested separately from FIG.9 and FIG.12). The results of using the AS17 antibody, AS22 antibody, AS25 antibody, and ACH55 antibody as the primary antibodies in order from the second graph from the top to the bottom on the left are shown. In addition, the results of using 10E4, JM403, NAH46, and 3G10 as primary antibodies in order from the top graph on the right side to the bottom are shown. The final concentration of the primary antibody was 20 μg / ml. FIG. 14 is a diagram showing the degree of binding of each antibody in HSC-4 strain treated with heparitinase I (results of testing separately from FIG. 13). The results of using the AS17 antibody, AS22 antibody, AS25 antibody, and ACH55 antibody as the primary antibodies in order from the second graph from the top to the bottom on the left are shown. In addition, the results of using 10E4, JM403, NAH46, and 3G10 as primary antibodies in order from the top graph on the right side to the bottom are shown. The final concentration of the primary antibody was 20 μg / ml. It is a figure which shows the grade of the coupling | bonding of each antibody in C-1 stock | strain. The results of using the AS17 antibody, AS22 antibody, AS25 antibody, and ACH55 antibody as the primary antibodies in order from the second graph from the top to the bottom on the left are shown. In addition, the results of using 10E4, JM403, NAH46, and 3G10 as primary antibodies in order from the top graph on the right side to the bottom are shown. The final concentration of the primary antibody was 20 μg / ml. FIG. 15 is a diagram showing the degree of binding of each antibody in the LS174T strain (results tested separately from FIG. 14). The results of using the AS17 antibody, AS22 antibody, AS25 antibody, and ACH55 antibody as the primary antibodies in order from the second graph from the top to the bottom on the left are shown. In addition, the results of using 10E4, JM403, NAH46, and 3G10 as primary antibodies in order from the top graph on the right side to the bottom are shown. The final concentration of the primary antibody was 20 μg / ml. FIG. 16 is a diagram showing the degree of binding of each antibody in the LS174T strain treated with heparitinase I (results of testing separately from FIG. 15). The results of using the AS17 antibody, AS22 antibody, AS25 antibody, and ACH55 antibody as the primary antibodies in order from the second graph from the top to the bottom on the left are shown. In addition, the results of using 10E4, JM403, NAH46, and 3G10 as primary antibodies in order from the top graph on the right side to the bottom are shown. The final concentration of the primary antibody was 20 μg / ml.

Claims (25)

A method for detecting cancer, comprising at least a step of contacting “an antibody that binds to 2-O desulfated acalan sulfate” and / or “an antibody that binds to acaran sulfate” with a sample derived from a living tissue. The method according to claim 1, wherein the “antibody that binds to 2-O desulfated acalan sulfate” does not substantially bind to acaran sulfate. The method according to claim 1 or 2, wherein the "antibody that binds to 2-O desulfated acalane sulfate" does not substantially bind to N-acetylheparosan. The method according to any one of claims 1 to 3, wherein the "antibody that binds to 2-O desulfated acalan sulfate" does not substantially bind to porcine intestinal heparin. The method according to any one of claims 1 to 4, wherein the "antibody that binds to 2-O desulfated acalan sulfate" does not substantially bind to heparan sulfate derived from bovine kidney. 6. The antibody according to claim 1, wherein the “antibody that binds to 2-O desulfated acalan sulfate” does not substantially bind to heparan sulfate derived from mouse Angel Breath-Home-Swan tumor tissue. The method described. The method according to any one of claims 1 to 6, wherein the "antibody that binds to 2-O desulfated acalan sulfate" is a monoclonal antibody. “Antibody that binds to 2-O desulfated akaran sulfate” is a lymphocyte derived from a mammal immunized with a substance formed by chemically binding a protein and 2-O desulfated akaran sulfate, and derived from a mammal The method according to claim 7, which is a monoclonal antibody produced by a hybridoma formed by cell fusion with a myeloma cell. The method according to claim 8, wherein the lymphocytes and myeloma cells are derived from a mouse. The method according to any one of claims 1 to 9, wherein the immunoglobulin class of "an antibody that binds to 2-O desulfated akaran sulfate" is IgM. The "antibody that binds to 2-O desulfated acalan sulfate" is an antibody produced by a hybridoma whose receipt number at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology is FERM AP-20828. 11. The method according to any one of items 10. The method according to any one of claims 1 to 11, wherein the "antibody that binds to acalane sulfate" does not substantially bind to heparan sulfate derived from bovine kidney. The method according to any one of claims 1 to 12, wherein the "antibody that binds to acalane sulfate" does not substantially bind to heparin derived from porcine intestine. 14. The method according to any one of claims 1 to 13, wherein the "antibody that binds to acaran sulfate" does not substantially bind to heparan sulfate derived from mouse Angel Breath-Home-Swan tumor tissue. The method according to any one of claims 1 to 14, wherein the "antibody that binds to acalane sulfate" does not substantially bind to keratan sulfate derived from bovine cornea. The method according to any one of claims 1 to 15, wherein the "antibody that binds to acalane sulfate" does not substantially bind to hyaluronic acid. The method according to any one of claims 1 to 16, wherein the "antibody that binds to acalane sulfate" does not substantially bind to N-acetylheparosan. The method according to any one of claims 1 to 17, wherein the "antibody that binds to acalane sulfate" is a monoclonal antibody. “Antibodies that bind to acalane sulfate” are formed by cell fusion of lymphocytes derived from mammals immunized with a substance obtained by chemically binding protein and acalane sulfate and myeloma cells derived from mammals. The method according to claim 18, which is a monoclonal antibody produced by a hybridoma. The method according to claim 19, wherein the lymphocytes and myeloma cells are derived from a mouse. 21. The method according to any one of claims 1 to 20, wherein the immunoglobulin class of “an antibody that binds to acalane sulfate” is IgM or IgG. “Antibodies that bind to akaran sulfate” are registered in the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center with the receipt number FERM AP-20823, FERM AP-20824, FERM AP-20825, FERM AP-20826, or FERM AP-20827. The method according to any one of claims 1 to 21, which is an antibody produced by a hybridoma. The method according to claim 1, wherein the biological tissue is a vertebrate biological tissue. The method according to any one of claims 1 to 23, wherein the cancer is colon cancer or squamous cell carcinoma. A cancer detection kit comprising at least “an antibody that binds to 2-O desulfated acalan sulfate” and / or “an antibody that binds to acaran sulfate” as a constituent component.

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