Method and Kit for Rapid Measurement of Autoantibody Activity with Respect to TSH Receptor

Patent metadata

Field	Value
Publication number	US-2021318305-A1
Application number	US-201917272752-A
Country	US
Kind code	A1
Filing date	Sep 2, 2019
Priority date	Sep 5, 2018
Publication date	Oct 14, 2021
Grant date	—

Abstract

An object of the present invention is to provide a method and a kit that can accurately measure autoantibody activity against a TSH receptor in a short time without pretreating a blood sample (blood specimen). The autoantibody activity against a TSH receptor in a blood sample can be 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 a method comprising 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. This method eliminates the need of pretreatment of a blood sample (specimen), can complete measurement itself in a short time (e.g., within 3 hours) by convenient means, and further eliminates the need of a special apparatus except for a commercially available apparatus measuring light emission. Therefore, the method is sufficiently available in testing laboratories in hospitals.

First claim

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 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.

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), the step (p) of 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 Gαs.

7 . A kit for measurement of autoantibody activity against a thyroid stimulating hormone (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.

8 . The kit for measurement according to claim 7 , wherein the autoantibody activity is a stimulatory activity or inhibitory activity.

9 . The kit for measurement according to claim 7 , wherein the mammal cell is a human embryonic kidney cell-derived cell line.

10 . The kit for measurement according to claim 7 , wherein the mammal cell is a GNAS gene- and GNAL gene-knockout mammal cell expressing exogenous Gαs.

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

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

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

14 . The method according to claim 11 , wherein the mammal cell is a human embryonic kidney cell-derived cell line.

15 . The method according to claim 2 , further comprising, before the step (a), the step (p) of 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 3 , further comprising, before the step (a), the step (p) of 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.

17 . The method according to claim 4 , further comprising, before the step (a), the step (p) of 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.

18 . The method according to claim 11 , further comprising, before the step (a), the step (p) of 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.

19 . The method according to claim 12 , further comprising, before the step (a), the step (p) of 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.

20 . The method according to claim 13 , further comprising, before the step (a), the step (p) of 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

• G01N2333/4719

  G-proteins · CPC title

• G01N2333/726

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

• C12Q1/66

  involving luciferase · CPC title

• G01N33/76

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