Method and kit for rapid measurement of autoantibody activity with respect to TSH receptor

Patent metadata

Field	Value
Publication number	US-12480946-B2
Application number	US-201917272752-A
Country	US
Kind code	B2
Filing date	Sep 2, 2019
Priority date	Sep 5, 2018
Publication date	Nov 25, 2025
Grant date	Nov 25, 2025

Abstract

Provided herein is a method and kit that accurately measure autoantibody activity against a TSH receptor without pretreating a blood sample. The autoantibody activity against a TSH receptor in a blood sample is measured by using a kit for measurement of autoantibody activity against a TSH receptor in a mammal cell, comprising a mammal cell expressing both a cAMP biosensor and the TSH receptor, and a substrate capable of visualizing and/or quantifying the CAMP biosensor as constituents, and performing the steps of: (a) incubating a mammal cell expressing both a cAMP biosensor and the TSH receptor in the presence of a blood sample collected from a test subject; (b) measuring an activation level of the CAMP biosensor after the step (a); and (c) comparing the activation level measured in the step (b) with an activation level in a control to calculate the autoantibody activity.

First claim

The invention claimed is:

1 . A method for measuring autoantibody activity against a thyroid stimulating hormone (TSH) receptor in a mammal cell, comprising the following steps (a) to (c):
(a) incubating a mammal cell expressing both a luciferase containing CAMP binding region and the TSH receptor in the presence of a blood sample collected from a test subject and a substrate capable of visualizing or quantifying the luciferase containing cAMP binding region; (b) measuring an activation level of the luciferase containing cAMP binding region after the step (a); and (c) comparing the activation level measured in the step (b) with an activation level in a control to calculate the autoantibody activity.

2 . The method according to claim 1 , wherein the autoantibody activity is a stimulatory activity or inhibitory activity.

3 . The method according to claim 1 , wherein the method is completed within 3 hours.

4 . The method according to claim 1 , wherein the mammal cell is a human embryonic kidney cell-derived cell line.

5 . The method according to claim 1 , further comprising, before the step (a), subjecting the mammal cell to equilibration treatment in a solution for incubation, wherein in the step (a), the mammal cell and the blood sample are incubated in the solution for incubation, and the sum of a time required for carrying out the steps (a) and (b) and an equilibration treatment time is 2 hours or less.

6 . The method according to claim 1 , wherein the mammal cell is a GNAS gene- and GNAL gene-knockout mammal cell expressing exogenous Gas protein.

7 . The method according to claim 2 , wherein the method is completed within 3 hours.

8 . The method according to claim 2 , wherein the mammal cell is a human embryonic kidney cell-derived cell line.

9 . The method according to claim 3 , wherein the mammal cell is a human embryonic kidney cell-derived cell line.

10 . The method according to claim 7 , wherein the mammal cell is a human embryonic kidney cell-derived cell line.

11 . The method according to claim 2 , further comprising, before the step (a), subjecting the mammal cell to equilibration treatment in a solution for incubation, wherein in the step (a), the mammal cell and the blood sample are incubated in the solution for incubation, and the sum of a time required for carrying out the steps (a) and (b) and an equilibration treatment time is 2 hours or less.

12 . The method according to claim 3 , further comprising, before the step (a), subjecting the mammal cell to equilibration treatment in a solution for incubation, wherein in the step (a), the mammal cell and the blood sample are incubated in the solution for incubation, and the sum of a time required for carrying out the steps (a) and (b) and an equilibration treatment time is 2 hours or less.

13 . The method according to claim 4 , further comprising, before the step (a), subjecting the mammal cell to equilibration treatment in a solution for incubation, wherein in the step (a), the mammal cell and the blood sample are incubated in the solution for incubation, and the sum of a time required for carrying out the steps (a) and (b) and an equilibration treatment time is 2 hours or less.

14 . The method according to claim 7 , further comprising, before the step (a), subjecting the mammal cell to equilibration treatment in a solution for incubation, wherein in the step (a), the mammal cell and the blood sample are incubated in the solution for incubation, and the sum of a time required for carrying out the steps (a) and (b) and an equilibration treatment time is 2 hours or less.

15 . The method according to claim 8 , further comprising, before the step (a), subjecting the mammal cell to equilibration treatment in a solution for incubation, wherein in the step (a), the mammal cell and the blood sample are incubated in the solution for incubation, and the sum of a time required for carrying out the steps (a) and (b) and an equilibration treatment time is 2 hours or less.

16 . The method according to claim 9 , further comprising, before the step (a), subjecting the mammal cell to equilibration treatment in a solution for incubation, wherein in the step (a), the mammal cell and the blood sample are incubated in the solution for incubation, and the sum of a time required for carrying out the steps (a) and (b) and an equilibration treatment time is 2 hours or less.

Assignees

• Yamasa Corp
• Univ Tohoku

Inventors

• Kawasaki Atsushi
• Hoshina Motoki
• Aoki Junken
• Kawakami Kouki
• Inoue Asuka

Classifications

• G01N2333/726

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

• G01N2333/4719

  G-proteins · CPC title

• G01N33/76

  Human chorionic gonadotropin {including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors} · CPC title

• C12Q1/66

  involving luciferase · CPC title

• G01N33/564 Primary

  for pre-existing immune complex or autoimmune disease {, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9} · CPC title