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US 12,366,570 B2Granted
Cell-based ZNT8 assay
AssigneeThe Johns Hopkins University
InventorsDax Fu, Chengfeng Merriman
Life of this patent
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Abstract
The present invention relates to the fields of immunology and diabetes. More specifically, the present invention provides methods and compositions directed to the use of antibodies to quantify cellular pancreatic zinc transporter 8. In certain embodiment, the present invention provides methods and compositions directed to the use of antibodies to screen for modulators of the pancreatic zinc transporter, ZnT8. In one embodiment, a method comprises the steps of (a) permeabilizing human beta cells present in a substrate; (b) contacting the cells with a test agent; and (c) measuring the amount of zinc transporter 8 (ZnT8) using at least one anti-ZnT8 antibody or antigen-binding fragment thereof.
Claims19
A method comprising:
- (a) permeabilizing human beta cells present in a substrate;
- (b) contacting the cells with a test agent;
- and
- (c) measuring the amount of zinc transporter 8 (ZnT8) using at least one anti-ZnT8 antibody or antigen-binding fragment thereof, and
- wherein the at least one anti-ZnT8 antibody or antigen-binding fragment thereof comprises a Fab, and
- wherein the Fab comprises SEQ ID NO:32 and SEQ ID NO:37.
3 more elements
A method comprising:
- (a) permeabilizing human beta cells present in a substrate;
- (b) contacting the cells with a test agent;
- and
- (c) measuring the amount of zinc transporter 8 (ZnT8) using at least one anti-ZnT8 antibody or antigen-binding fragment thereof, and
- wherein the at least one anti-ZnT8 antibody or antigen-binding fragment thereof comprises a Fab, and
- wherein the Fab comprises:
- (a) heavy chain complementary determining regions (CDRs) 1, 2, and 3, wherein the heavy chain CDR1 comprises SEQ ID NO:33, the heavy chain CDR2 comprises SEQ ID NO:34, and the heavy chain CDR3 comprises SEQ ID NO:35;
- and
- (b) light chain CDRs 1, 2, and 3, wherein the light chain CDR1 comprises SEQ ID NO:38, the light chain CDR2 comprises SEQ ID NO:39, and the light chain CDR3 comprises SEQ ID NO:40.
6 more elements
A method comprising:
- (a) permeabilizing human beta cells present in a substrate;
- (b) contacting the cells with a test agent;
- and
- (c) measuring the amount of zinc transporter 8 (ZnT8) using at least one anti-ZnT8 antibody or antigen-binding fragment thereof, and
- wherein the at least one anti-ZnT8 antibody or antigen-binding fragment thereof comprises (a) a first Fab comprising SEQ ID NO:32 and SEQ ID NO:37;
- and a second Fab comprising SEQ ID NO:52 and SEQ ID NO:57.
3 more elements
A method comprising:
- (a) permeabilizing human beta cells present in a substrate;
- (b) contacting the cells with a test agent;
- and
- (c) measuring the amount of zinc transporter 8 (ZnT8) using at least one anti-ZnT8 antibody or antigen-binding fragment thereof, and
- wherein the at least one detectably labeled anti-ZnT8 antibody or antigen-binding fragment thereof comprises:
- (a) a first Fab comprising:
- (i) heavy chain complementary determining regions (CDRs) 1, 2, and 3, wherein the heavy chain CDR1 comprises SEQ ID NO:33, the heavy chain CDR2 comprises SEQ ID NO:34, and the heavy chain CDR3 comprises SEQ ID NO:35, and
- (ii) light chain CDRs 1, 2, and 3, wherein the light chain CDR1 comprises SEQ ID NO:38, the light chain CDR2 comprises SEQ ID NO:39, nd the light chain CDR3 comprises SEQ ID NO:40;
- and
- (b) a second Fab comprising:
- (i) heavy chain complementary determining regions (CDRs) 1, 2, and 3, wherein the heavy chain CDR1 comprises SEQ ID NO:53, the heavy chain CDR2 comprises SEQ ID NO:54, and the heavy chain CDR3 comprises SEQ ID NO:55, and
- (ii) light chain CDRs 1, 2, and 3, wherein the light chain CDR1 comprises SEQ ID NO:58, the light chain CDR2 comprises SEQ ID NO:59, and the light chain CDR3 comprises SEQ ID NO:60.
9 more elements
A method comprising:
- (a) permeabilizing human beta cells present in a substrate;
- (b) contacting the cells with a test agent;
- and
- (c) measuring the amount of zinc transporter 8 (ZnT8) using at least one anti-ZnT8 antibody or antigen-binding fragment thereof, and
- wherein the at least one anti-ZnT8 antibody or antigen-binding fragment thereof comprises SEQ ID NO:2 and SEQ ID NO:7;
- SEQ ID NO:12 and SEQ ID NO:17;
- SEQ ID NO:22 and SEQ ID NO:27;
- SEQ ID NO:32 and SEQ ID NO:37;
- SEQ ID NO:42 and SEQ ID NO:47;
- or SEQ ID NO:52 and SEQ ID NO:57.
7 more elements
A method of identifying a modulator of ZnT8 comprising the steps of:
- (a) contacting human beta cells with a test agent;
- and
- (b) detecting a change in the amount of ZnT8 in the cell as compared to the amount of ZnT8 in a cell not contacted with the test agent, wherein the detecting step utilizes at least one anti-ZnT8 antibody or antigen-binding fragment thereof,
- wherein the at least one anti-ZnT8 antibody or antigen-binding fragment thereof comprises SEQ ID NO:2 and SEQ ID NO:7;
- SEQ ID NO:12 and SEQ ID NO:17;
- SEQ ID NO:22 and SEQ ID NO:27;
- SEQ ID NO:32 and SEQ ID NO:37;
- SEQ ID NO:42 and SEQ ID NO:47;
- or SEQ ID NO:52 and SEQ ID NO:57.
6 more elements
A method of identifying a modulator of ZnT8 comprising the steps of:
- (a) contacting human beta cells with a metabolic or cytokine stressor;
- (b) contacting the cells with a test agent;
- and
- detecting a change in the amount of ZnT8 in the cell as compared to the amount of ZnT8 in a cell contacted with the stressor but not contacted with the test agent, wherein the detecting step utilizes at least one anti-ZnT8 antibody or antigen-binding fragment thereof,
- wherein the at least one anti-ZnT8 antibody or antigen-binding fragment thereof comprises SEQ ID NO:2 and SEQ ID NO:7;
- SEQ ID NO:12 and SEQ ID NO:17;
- SEQ ID NO:22 and SEQ ID NO:27;
- SEQ ID NO:32 and SEQ ID NO:37;
- SEQ ID NO:42 and SEQ ID NO:47;
- or SEQ ID NO:52 and SEQ ID NO:57.
7 more elements
A method comprising:
- (a) permeabilizing human beta cells present in a substrate;
- (b) contacting the cells with a test agent;
- and
- (c) measuring the amount of zinc transporter 8 (ZnT8) using at least one anti-ZnT8 antibody or antigen-binding fragment thereof, and
- wherein the at least one anti-ZnT8 antibody or antigen-binding fragment thereof comprises:
- (a) (i) heavy chain CDRs 1, 2, and 3, wherein the heavy chain CDR1 comprises SEQ ID NO:3, the heavy chain CDR2 comprises SEQ ID NO:4, and the heavy chain CDR3 comprises SEQ ID NO:5, and
- (a) (ii) light chain CDRs 1, 2, and 3, wherein the light chain CDR1 comprises SEQ ID NO:8, the light chain CDR2 comprises SEQ ID NO:9, and the light chain CDR3 comprises SEQ ID NO:10;
- or
- (b) (i) heavy chain CDRs 1, 2, and 3, wherein the heavy chain CDR1 comprises SEQ ID NO:13, the heavy chain CDR2 comprises SEQ ID NO:14, and the heavy chain CDR3 comprises SEQ ID NO:15, and
- (b) (ii) light chain CDRs 1, 2, and 3, wherein the light chain CDR1 comprises SEQ ID NO:18, the light chain CDR2 comprises SEQ ID NO:19, and the light chain CDR3 comprises SEQ ID NO:20;
- or
- (c) (i) heavy chain CDRs 1, 2, and 3, wherein the heavy chain CDR1 comprises SEQ ID NO:23, the heavy chain CDR2 comprises SEQ ID NO:24, and the heavy chain CDR3 comprises SEQ ID NO:25, and
- (c) (ii) light chain CDRs 1, 2, and 3, wherein the light chain CDR1 comprises SEQ ID NO:28, the light chain CDR2 comprises SEQ ID NO:29, and the light chain CDR3 comprises SEQ ID NO:30;
- or
- (d) (i) heavy chain CDRs 1, 2, and 3, wherein the heavy chain CDR1 comprises SEQ ID NO:33, the heavy chain CDR2 comprises SEQ ID NO:34, and the heavy chain CDR3 comprises SEQ ID NO:35, and
- (d) (ii) light chain CDRs 1, 2, and 3, wherein the light chain CDR1 comprises SEQ ID NO:38, the light chain CDR2 comprises SEQ ID NO:39, and the light chain CDR3 comprises SEQ ID NO:40;
- or
- (e) (i) heavy chain CDRs 1, 2, and 3, wherein the heavy chain CDR1 comprises SEQ ID NO:43, the heavy chain CDR2 comprises SEQ ID NO:44, and the heavy chain CDR3 comprises SEQ ID NO:45, and
- (e) (ii) light chain CDRs 1, 2, and 3, wherein the light chain CDR1 comprises SEQ ID NO:48, the light chain CDR2 comprises SEQ ID NO:49, and the light chain CDR3 comprises SEQ ID NO:50;
- or
- (f) (i) heavy chain CDRs 1, 2, and 3, wherein the heavy chain CDR1 comprises SEQ ID NO:53, the heavy chain CDR2 comprises SEQ ID NO:54, and the heavy chain CDR3 comprises SEQ ID NO:55, and
- (f) (ii) light chain CDRs 1, 2, and 3, wherein the light chain CDR1 comprises SEQ ID NO:58, the light chain CDR2 comprises SEQ ID NO:59, and the light chain CDR3 comprises SEQ ID NO:60.
19 more elements
A method of identifying a modulator of ZnT8 comprising the steps of:
- (a) contacting human beta cells with a test agent;
- and
- (b) detecting a change in the amount of ZnT8 in the cell as compared to the amount of ZnT8 in a cell not contacted with the test agent, wherein the detecting step utilizes at least one anti-ZnT8 antibody or antigen-binding fragment thereof, and
- wherein the at least one anti-ZnT8 antibody or antigen-binding fragment thereof comprises:
- (a) (i) heavy chain CDRs 1, 2, and 3, wherein the heavy chain CDR1 comprises SEQ ID NO:3, the heavy chain CDR2 comprises SEQ ID NO:4, and the heavy chain CDR3 comprises SEQ ID NO:5, and
- (a) (ii) light chain CDRs 1, 2, and 3, wherein the light chain CDR1 comprises SEQ ID NO:8, the light chain CDR2 comprises SEQ ID NO:9, and the light chain CDR3 comprises SEQ ID NO:10;
- or
- (b) (i) heavy chain CDRs 1, 2, and 3, wherein the heavy chain CDR1 comprises SEQ ID NO:13, the heavy chain CDR2 comprises SEQ ID NO:14, and the heavy chain CDR3 comprises SEQ ID NO:15, and
- (b) (ii) light chain CDRs 1, 2, and 3, wherein the light chain CDR1 comprises SEQ ID NO:18, the light chain CDR2 comprises SEQ ID NO:19, and the light chain CDR3 comprises SEQ ID NO:20;
- or
- (c) (i) heavy chain CDRs 1, 2, and 3, wherein the heavy chain CDR1 comprises SEQ ID NO:23, the heavy chain CDR2 comprises SEQ ID NO:24, and the heavy chain CDR3 comprises SEQ ID NO:25, and
- (c) (ii) light chain CDRs 1, 2, and 3, wherein the light chain CDR1 comprises SEQ ID NO:28, the light chain CDR2 comprises SEQ ID NO:29, and the light chain CDR3 comprises SEQ ID NO:30;
- or
- (d) (i) heavy chain CDRs 1, 2, and 3, wherein the heavy chain CDR1 comprises SEQ ID NO:33, the heavy chain CDR2 comprises SEQ ID NO:34, and the heavy chain CDR3 comprises SEQ ID NO:35, and
- (d) (ii) light chain CDRs 1, 2, and 3, wherein the light chain CDR1 comprises SEQ ID NO:38, the light chain CDR2 comprises SEQ ID NO:39, and the light chain CDR3 comprises SEQ ID NO:40;
- or
- (e) (i) heavy chain CDRs 1, 2, and 3, wherein the heavy chain CDR1 comprises SEQ ID NO:43, the heavy chain CDR2 comprises SEQ ID NO:44, and the heavy chain CDR3 comprises SEQ ID NO:45, and
- (e) (ii) light chain CDRs 1, 2, and 3, wherein the light chain CDR1 comprises SEQ ID NO:48, the light chain CDR2 comprises SEQ ID NO:49, and the light chain CDR3 comprises SEQ ID NO:50;
- or
- (f) (i) heavy chain CDRs 1, 2, and 3, wherein the heavy chain CDR1 comprises SEQ ID NO:53, the heavy chain CDR2 comprises SEQ ID NO:54, and the heavy chain CDR3 comprises SEQ ID NO:55, and
- (f) (ii) light chain CDRs 1, 2, and 3, wherein the light chain CDR1 comprises SEQ ID NO:58, the light chain CDR2 comprises SEQ ID NO:59, and the light chain CDR3 comprises SEQ ID NO:60.
18 more elements
A method of identifying a modulator of ZnT8 comprising the steps of:
- (a) contacting human beta cells with a metabolic or cytokine stressor;
- (b) contacting the cells with a test agent;
- and
- (c) detecting a change in the amount of ZnT8 in the cell as compared to the amount of ZnT8 in a cell contacted with the stressor but not contacted with the test agent, wherein the detecting step utilizes at least one anti-ZnT8 antibody or antigen-binding fragment thereof, and
- wherein the at least one anti-ZnT8 antibody or antigen-binding fragment thereof comprises:
- (a) (i) heavy chain CDRs 1, 2, and 3, wherein the heavy chain CDR1 comprises SEQ ID NO:3, the heavy chain CDR2 comprises SEQ ID NO:4, and the heavy chain CDR3 comprises SEQ ID NO:5, and
- (a) (ii) light chain CDRs 1, 2, and 3, wherein the light chain CDR1 comprises SEQ ID NO:8, the light chain CDR2 comprises SEQ ID NO:9, and the light chain CDR3 comprises SEQ ID NO:10;
- or
- (b) (i) heavy chain CDRs 1, 2, and 3, wherein the heavy chain CDR1 comprises SEQ ID NO:13, the heavy chain CDR2 comprises SEQ ID NO:14, and the heavy chain CDR3 comprises SEQ ID NO:15, and
- (b) (ii) light chain CDRs 1, 2, and 3, wherein the light chain CDR1 comprises SEQ ID NO:18, the light chain CDR2 comprises SEQ ID NO:19, and the light chain CDR3 comprises SEQ ID NO:20;
- or
- (c) (i) heavy chain CDRs 1, 2, and 3, wherein the heavy chain CDR1 comprises SEQ ID NO:23, the heavy chain CDR2 comprises SEQ ID NO:24, and the heavy chain CDR3 comprises SEQ ID NO:25, and
- (c) (ii) light chain CDRs 1, 2, and 3, wherein the light chain CDR1 comprises SEQ ID NO:28, the light chain CDR2 comprises SEQ ID NO:29, and the light chain CDR3 comprises SEQ ID NO:30;
- or
- (d) (i) heavy chain CDRs 1, 2, and 3, wherein the heavy chain CDR1 comprises SEQ ID NO:33, the heavy chain CDR2 comprises SEQ ID NO:34, and the heavy chain CDR3 comprises SEQ ID NO:35, and
- (d) (ii) light chain CDRs 1, 2, and 3, wherein the light chain CDR1 comprises SEQ ID NO:38, the light chain CDR2 comprises SEQ ID NO:39, and the light chain CDR3 comprises SEQ ID NO:40;
- or
- (e) (i) heavy chain CDRs 1, 2, and 3, wherein the heavy chain CDR1 comprises SEQ ID NO:43, the heavy chain CDR2 comprises SEQ ID NO:44, and the heavy chain CDR3 comprises SEQ ID NO:45, and
- (e) (ii) light chain CDRs 1, 2, and 3, wherein the light chain CDR1 comprises SEQ ID NO:48, the light chain CDR2 comprises SEQ ID NO:49, and the light chain CDR3 comprises SEQ ID NO:50;
- or
- (f) (i) heavy chain CDRs 1, 2, and 3, wherein the heavy chain CDR1 comprises SEQ ID NO:53, the heavy chain CDR2 comprises SEQ ID NO:54, and the heavy chain CDR3 comprises SEQ ID NO:55, and
- (f) (ii) light chain CDRs 1, 2, and 3, wherein the light chain CDR1 comprises SEQ ID NO:58, the light chain CDR2 comprises SEQ ID NO:59, and the light chain CDR3 comprises SEQ ID NO:60.
19 more elements
Dependencies are inferred from the claim text.