Method and kit for detecting stem cell

Patent metadata

Field	Value
Publication number	US-10539553-B2
Application number	US-201515548921-A
Country	US
Kind code	B2
Filing date	Dec 21, 2015
Priority date	Mar 17, 2015
Publication date	Jan 21, 2020
Grant date	Jan 21, 2020

Abstract

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.

First claim

The invention claimed is:

1. A method for detecting a stem cell by detecting podocalyxin contained in a culture supernatant or a lysate of cells, the method comprising steps of:
contacting the culture supernatant or the lysate,
a lectin capable of binding to a sugar chain represented by (Formula 1) below:

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,
or (Formula 2) below:

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 the lectin is:
a protein comprising the amino acid sequence of SEQ ID NO: 1 or
a protein comprising an amino acid sequence in which one amino acid is deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 1 and being capable of binding to a sugar chain represented by the (Formula 1) or (Formula 2), and
an antibody capable of binding to keratan sulfate, wherein the antibody is an antibody produced by hybridoma R-10G (accession number: FERM BP-11301),
to form a complex composed of the lectin, podocalyxin, and the antibody; and

detecting the complex, wherein the detection of the complex is indicative of the presence of the stem cell.

2. The method according to claim 1 , wherein the antibody is an antibody capable of binding to low-sulfated keratan sulfate.

3. The method according to claim 1 , wherein for the antibody, the epitope comprises Gal-GlcNAc (6S) or a tandem repeat thereof.

4. The method according to claim 1 , wherein the culture supernatant or the lysate contains serum.

5. The method according to claim 1 , comprising steps of:
contacting the culture supernatant or the lysate with the lectin to form a first complex composed of the lectin and podocalyxin contained in the culture supernatant or the lysate; and
contacting the first complex with the antibody to form a second complex composed of the lectin, podocalyxin, and the antibody.

6. The method according to claim 5 , wherein the lectin is bound to an insoluble support.

7. A method for detecting a stem cell by detecting a sugar chain represented by (Formula 1) below:

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,
or (Formula 2) below:

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, contained in a culture supernatant or a lysate of cells,
the method comprising steps of:
contacting the culture supernatant or the lysate,
a lectin capable of binding to the sugar chain, wherein the lectin is:

a protein comprising the amino acid sequence of SEQ ID NO: 1 or
a protein comprising an amino acid sequence in which one amino acid is deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 1 and being capable of binding to a sugar chain represented by the (Formula 1) or (Formula 2), and
an antibody capable of binding to keratan sulfate, wherein the antibody is an antibody produced by hybridoma R-10G (accession number: FERM BP-11301),
to form a complex comprising the lectin, the sugar chain, and the antibody; and

detecting the complex, wherein the detection of the complex is indicative of the presence of the stem cell.

8. The method according to claim 7 , further comprising a step of determining the presence or absence or abundance of the stem cell contained among the cells based on the presence or absence or a detected amount of the complex.

9. The method according to claim 7 , further comprising a step of determining the differentiation status of the cells based on the presence or absence or a detected amount of the complex.

10. A method for detecting podocalyxin contained in a culture supernatant or a lysate of cells, the method comprising steps of:
contacting the culture supernatant or the lysate,
a lectin capable of binding to a sugar chain represented by (Formula 1) below:

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,
or (Formula 2) below:

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 the lectin is:
a protein comprising the amino acid sequence of SEQ ID NO: 1 or
a protein comprising an amino acid sequence in which one amino acid is deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 1 and being capable of binding to a sugar chain represented by the (Formula 1) or (Formula 2), and
an antibody capable of binding to keratan sulfate, wherein the antibody is an antibody produced by hybridoma R-10G (accession number: FERM BP-11301),
to form a complex composed of the lectin, podocalyxin, and the antibody; and

detecting the complex, wherein the detection of the complex is indicative of the presence of podocalyxin.

11. A kit for detecting a stem cell contained among cells by detecting podocalyxin contained in a culture supernatant or a lysate of the cells, the kit comprising:
a lectin capable of binding to a sugar chain represented by (Formula 1) below:

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,
or (Formula 2) below:

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 the lectin is:
a protein comprising the amino acid sequence of SEQ ID NO: 1 or
a protein comprising an amino acid sequence in which one amino acid is deleted, substituted, inserted, or added in the amino acid sequence of SEQ ID NO: 1 and being capable of binding to a sugar chain represented by the (Formula 1) or (Formula 2); and
an antibody capable of binding to keratan sulfate, wherein the antibody is an antibody produced by hybridoma R-10G (accession number: FERM BP-11301).

12. The kit according to claim 11 , wherein the antibody is an antibody capable of binding to low-sulfated keratan sulfate.

13. The kit according to claim 11 , wherein for the antibody, the epitope comprises Gal-GlcNAc (6S) or a tandem repeat thereof.

14. The kit according to claim 11 , wherein the lectin is bound to an insoluble support.

15. The method according to claim 1 , further comprising a step of determining the presence or absence or abundance of the stem cell contained among the cells based on the presence or

Assignees

• Aist
• Fujifilm Wako Pure Chemical Corp

Inventors

• Tateno Hiroaki
• Warashina Masaki
• Fukuda Masakazu
• Hirayasu Kazunari

Classifications

• G01N33/53

  Immunoassay; Biospecific binding assay; Materials therefor · CPC title

• G01N33/5758

  involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites · CPC title

• C07H5/04 Primary

  to nitrogen · CPC title

• G01N33/5073 Primary

  Stem cells · CPC title

• G01N33/57484

  Physics · mapped topic