Poster Title

1
Characterization of ZnT8 Monoclonal
Antibodies Christina Miller, Suparna Sarkar,
Catherine Lee, Jay Walters, Janet Wenzlau, Julie
Wu, Howard Davidson, John HuttonBarbara Davis
Center for Diabetes, University of Colorado
School of Medicine, Aurora, CO
Results
Introduction
Type 1 Diabetes (T1D) is a multifactorial disease
characterized by an autoimmune attack on
pancreatic ß cells in the islets of Langerhans
(1). Previous studies have identified in humans
with T1D autoantibodies reactive to ß cell
antigens glutamate decarboxylase (GAD), protein
tyrosine phosphatase (IA2), insulin, islet
cytoplasm (IC), and most recently zinc
transporter 8 (ZnT8) (2). The presence of these
autoantibodies serve as a predictor of diabetes
and the associated antigens are potential targets
for therapy. The ZnT8 autoantibody is detected
in 60-80 of individuals with new-onset T1D (3).
The function of ZnT8 in pancreatic ß cells is to
transport zinc into insulin granules in order to
stabilize insulin for storage. Insulin is stored
as a solid hexamer bound to two Zn2. Since zinc
is also released with insulin upon granule
secretion, it is likely that it is used for
paracrine/autocrine communication with nearby
pancreatic cells (4). Therefore, ZnT8 plays a
crucial role in ß cell function through zinc
homeostasis. However, there is still little known
about the autoantigen. Developing and optimizing
monoclonal antibodies for ZnT8 could be very
useful for purifying the protein in order to
study ZnT8s characteristics.
Objectives

• Overall goal to further characterize mouse ZnT8
  monoclonal antibodies that previously were found
  to be specific for ZnT8 in both mouse and human
  pancreatic islet tissue (5). Specific goals of
  this project were to
• compare the reactivity of the monoclonal
  antibodies to ZnT8 in wild type mouse and knock
  out mouse pancreatic tissue
• determine if of the antibodies are specific to
  rat ZnT8 in insulinoma cells
• identify any co-localization of ZnT8 with other
  subcelluar compartment markers
• characterize the ZnT8 antibodies specificity in
  western blots

Methods
Immunohistochemistry pancreas tissue of mice
(both wild type and knock out for SLC8A30) were
incubated in 1ZnT8 mouse monoclonal antibodies
(17H2, 10D2, 7G4, and 4D2), insulin (guinea pig),
and glucagon (rabbit). Tissues were then
incubated in 2antibodies tagged with fluorescent
dyes and imaged at 20x magnification. Cell
Cultures INS-1 cells were starved in no glucose
media for 30 minutes and then treated with 2.2 mM
glucose, 5.6 mM, or 20.0 mM glucose for 2 hours.
Some cell cultures were fixed with
paraformaldehyde (4) for staining, and others
were harvested for western blotting. MIN-6 and
COS-7 cells were grown with normal glucose
media. Immunocytochemistry fixed INS-1 cells
were incubated in 1ZnT8 mouse monoclonal
antibodies (17H2, 10D2, 7G4, and 4D2) and
subcellular marker antibodies insulin (guinea
pig), phogrin (rabbit) and TGN38 (sheep). Cells
were then incubated in 2antibodies tagged with
fluorescent dyes and imaged at 60x oil
magnification. Transfection COS-7 cells were
transfected with DNA constructs of mouse, rat,
and human (R and W) ZnT8. Western Blot protein
from transfected COS-7 cells, INS-1 cells (only
5.6 mM glucose) and MIN-6 cells was loaded into
an SDS-page gel electrophoresis. The gel was
transferred to a PVDF membrane and immunoblot
with the 1ZnT8 mouse monoclonal antibodies
(17H2, 10D7, 7G4, 4D2). The membrane was
incubated in a 2antibody and exposed on x-ray
film.