Bioassay method for detecting physiologically active substance

Patent metadata

Field	Value
Publication number	US-9316588-B2
Application number	US-201113997489-A
Country	US
Kind code	B2
Filing date	Dec 22, 2011
Priority date	Dec 24, 2010
Publication date	Apr 19, 2016
Grant date	Apr 19, 2016

Abstract

Provided are a procedurally simple and safe method and kit for measuring the concentration in a biological sample of a physiologically active substance which binds to a receptor that causes a change in intracellular cAMP concentration. By providing a composition including genetically-modified cells which show co-expression of a receptor that causes a change in intracellular cAMP concentration, a cyclic nucleotide-responsive calcium channel, and aequorin luminescent protein, it is possible to measure how much of a physiologically active substance which binds to said receptor is included in a biological sample.

First claim

The invention claimed is:

1. A transformed cell, wherein the cell expresses Gs-coupled GPCR, wherein the GS-coupled GPCR is a GLP-1 receptor or a vasopressin V2 receptor,
a modified CNG calcium channel having the amino acid sequence represented by SEQ ID NO: 2, and
a modified apoaequorina having the amino acid sequence represented by SEQ ID NO: 3.

2. A kit for measuring the amount of an active form of GLP-1 or the amount of AVP in a biological sample, comprising a cell according to claim 1 .

3. The kit according to claim 2 , wherein the kit is for diagnosing diabetes insipidus or SIADH, for determining either a hypotonic or hypertonic fluid infusion to be selected or for determining the effect of a GLP-1-related therapeutic drug.

4. The kit according to claim 2 , wherein the cell is a frozen cell.

5. The kit according to claim 2 , further comprising a luminescent substrate for the calcium sensitive protein.

6. The kit according to claim 5 , wherein the luminescent substrate for the calcium sensitive protein is coelenterazine or a coelenterazine derivative.

7. The kit according claim 2 , wherein the kit comprises the transformed cell expressing:
a vasopressin V2 receptor,
a modified CNG calcium channel having the amino acid sequence represented by SEQ ID NO: 2, and
a modified apoaequorin having the amino acid sequence represented by SEQ ID NO: 3.

8. The kit according to claim 2 , wherein the kit comprises the transformed cell expressing:
a GLP-1 receptor,
a modified CNG calcium channel having the amino acid sequence represented by SEQ ID NO: 2, and
a modified apoaequorin having the amino acid sequence represented by SEQ ID NO: 3.

9. The transformed cell according to claim 1 , wherein the cell is a frozen cell.

10. The transformed cell according to claim 1 , wherein the cell expresses a vasopressin V2 receptor.

11. The transformed cell according to claim 10 , wherein the vasopressin V2 receptor has the amino acid sequence represented by SEQ ID NO: 1.

12. The transformed cell according to claim 1 , wherein the cell expresses a GLP-1 receptor.

13. The transformed cell according to claim 12 , wherein the GLP-1 receptor has the amino acid sequence represented by SEQ ID NO:8.

Assignees

• Araki Naohiro
• Iida Mitsuru
• Machida Kiyotaka
• Otsuka Pharma Co Ltd

Inventors

• Araki Naohiro
• Iida Mitsuru
• Machida Kiyotaka

Classifications

• G01N33/6872 Primary

  Intracellular protein regulatory factors and their receptors, e.g. including ion channels · CPC title

• G01N2333/726

  G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH · CPC title

• G01N2800/042

  Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism · CPC title

• G01N2500/10

  involving cells · CPC title

• G01N21/6486 Primary

  Measuring fluorescence of biological material, e.g. DNA, RNA, cells (G01N21/6428 takes precedence) · CPC title