National Institute of Advanced Industrial Science and Technology

Wako Pure Chemical Industries, Inc.

Nissan Chemical Corporation

Shizuoka University

Kyoto Institute of Technology

Fujifilm Wako Pure Chemical Corporation

J-oil Mills, Inc.

Hiroaki Tateno

Jun Hirabayashi

Masakazu Fukuda

Makoto Asashima

Yasuko Onuma

Yuzuru Ito

Masaki Warashina

Hisashi Narimatsu

Takashi Sato

Yasunori Chiba

Hiroyuki Kaji

Masanori Goto

Yuka Kobayashi

Hirokazu Kawagishi

Atsushi Kuno

Yoichi Kumada

Junko Katayama

Kazunari Hirayasu

Kazutaka Matoba

Hideo Dohra

Tomoko Watanabe

Provided is a highly sensitive and less expensive lectin-immobilized base material (for example, a lectin plate), such as lectin-immobilized base material having stable qualities and being able to be sufficiently washed after a target sugar chain-containing antigen binds thereto. Further provided is a method for immobilizing lectin to a base material therefor. Particularly provided are: a method whereby a lectin-peptide fusion, in which a peptide capable of adsorbing to a base material surface such as a polystyrene (PS) tag is fused with the N-terminal side or C-terminal side of lectin capable of recognizing a target sugar chain, is immobilized on the peptide side to a base material; and a lectin-immobilized base material produced by this method. By using the lectin-immobilized base material, a target sugar chain-containing antigen can be highly sensitively and evenly measured and, moreover, target sugar chain-containing cells, etc. can be separated (concentrated and harvested).

Method for immobilizing lectin

Provided is a method for detecting a stem cell based on an undifferentiated sugar chain marker having a specific sugar chain structure, wherein the stem cell is detected by detecting podocalyxin contained in a culture supernatant or a lysate of cells by a 'lectin-antibody sandwich method' using a combination of a lectin and an antibody and having high sensitivity, the method including steps of: contacting the culture supernatant or the lysate, a lectin capable of binding to a sugar chain represented by (Formula 1) or (Formula 2), and an antibody capable of binding to keratan sulfate to form a complex composed of the lectin, podocalyxin and the antibody; and detecting the complex. wherein R1 represents an OH group or any sugar chain and R2 represents an OH group or any sugar chain, protein, lipid, or another molecule. wherein R1 represents an OH group or any sugar chain and R2 represents an OH group or any sugar chain, protein, lipid, or another molecule.

Method and kit for detecting stem cell

Amonomeric lectin polypeptide is provided. Themonomeric lectin polypeptide includes any one of the amino acid sequences selected from the group consisting of: (1) the amino acid sequence represented by SEQ ID NO: 2; (2) the amino acid sequence defined in (1) above, except that one to 20 amino acids at positions other than Cys272 position is/are deleted, substituted, inserted, or added; and (3) the amino acid sequence defined in (1) or (2) above, further having an N-terminus deletion of one to 30 amino acids, in which Cys272 is alkylated, and the polypeptide is capable of specifically binding to a GalNAc terminal sugar chain.

Modified lectin derived from Wisteria floribunda

An object of the present invention is to provide a method for introducing a target substance into undifferentiated cells, and a carrier therefor, whereby the target substance can be specifically introduced into the undifferentiated cells by contacting the undifferentiated cells with an rBC2LCN-target substance fusion product in which rBC2LCN lectin is fused to the target substance. Particularly, an rBC2LCN-toxin fusion product in which rBC2LCN lectin is fused to a toxin functioning in cells or its domain having the ability to kill cells functions as an agent for eliminating undifferentiated stem cells and can be administered into a medium after inducing the differentiation of the stem cells to reliably kill only the stem cells in an undifferentiated state.

Method of introducing a toxin or dye into mammalian pluripotent cells using rBC2LCN

[Problem] The purpose of the present invention is to stably supply high-quality and highly uniformagglutinin (WFA) that recognizes biologically important sugar-chain markers, to elucidate the sugar-chain recognition activity in detail, and to furthermore increase the specificity of the sugar-chain recognition activity. [Solution] The present invention involves the development of a technique for cloning genes for codingagglutinin (WFA) and producing recombinant WFA having the same sugar-chain recognition activity as natural WFA from transformed bacteria. Natural WFA is reduced to thereby manufacture a reduced WFA monomer for specifically recognizing terminal GalNAc residue. A recombinant monomer WFA for recognizing LDN (GalNAc&#x3b2;1, 4GlcNAc) sugar chain, which is important as a diagnostic marker among sugar chains having a terminal GalNAc residue, is manufactured by introducing cysteine mutation to recombinant WFA or by C-terminal-side amino acid deletion.

An object of the present invention is to provide a method for evaluating a differentiation status of cells using a culture supernatant of stem cells. Provided is 'undifferentiation sugar chain marker' composed of the sugar chain structure 'Fuc&#x3b1;1-2Gal&#x3b2;1-3GlcNAc' or &#x201c;Fuc&#x3b1;1-2Gal&#x3b2;1-3GalNAc&#x201d; and capable of sensitively determining the undifferentiated state of stem cells using a culture supernatant of stem cells. Also found is BC2LCN lectin or a modified product thereof capable of sensitively recognizing the &#x201c;undifferentiation sugar chain marker&#x201d; as excellent &#x201c;probe for detecting the undifferentiation sugar chain marker&#x201d; capable of determining the undifferentiation status of cells using a culture supernatant.

Undifferentiated cell detection method and complex carbohydrate detection method

Provided are a method for accurately evaluating the differentiation status of stem cells by selectively staining only stem cells in an undifferentiated state, and a method for positively isolating only stem cells in an undifferentiated state. Specifically provided is a method for determining differentiation of a cell comprising a step of contacting a test cell with a probe comprising protein (A) or (B) below and a step of detecting the presence of binding of the probe to the test cell. The method for determining differentiation of a cell is capable of detecting the presence or absence of an undifferentiated stem cell in test cells by using a probe that specifically reacts with undifferentiated stem cells and detecting the presence of bonding to the test cell. (A) A protein comprising an amino acid sequence shown in SEQ ID NO: 1 and recognizing a sugar chain structure of 'Fuc&#x3b1;1-2Gal&#x3b2;1-3GlcNAc' and/or 'Fuc&#x3b1;1-2Gal&#x3b2;1-3GalNAc;' and (B) a protein comprising an amino acid sequence showing 80% or more similarity to the amino acid sequence shown in SEQ ID NO: 1 and recognizing a sugar chain structure of &#x201c;Fuc&#x3b1;1-2Gal&#x3b2;1-3GlcNAc&#x201d; and/or &#x201c;Fuc&#x3b1;1-2Gal&#x3b2;1-3GalNAc.&#x201d;

Cell differentiation assay method, cell isolation method, method for producing induced pluripotent stem cells, and method for producing differentiated cells

Disclosed is a novel lectin which can bind specifically to an L-fucose &#x3b1;1&#x2192;6 sugar chain. Also disclosed is use of the lectin. The L-fucose &#x3b1;1&#x2192;6 specific lectin of the present invention is characterized in that: (1) the lectin is extracted from a basidiomycete or an ascomycete; (2) the lectin has a molecular weight by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of 4,000 to 40,000; and (3) the lectin has an affinity to an L-fucose &#x3b1;1&#x2192;6 sugar chain, the affinity being represented by an association constant of 1.0&#xd7;10Mor more (at 25 degrees C.). The lectin can be used for detecting an L-fucose &#x3b1;1&#x2192;6 sugar chain specifically, and is effective for the purification of an L-fucose &#x3b1;1&#x2192;6 sugar chain or a non L-fucose &#x3b1;1&#x2192;6 sugar chain.

L-fucose &#x3b1;1&#x2192;6 specific lectin

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Ibaraki, Japan

Hiroaki Tateno