oCtoBeR 2010 - American Association for Clinical Chemistry

they received insulin therapy (7). IAAs are
diverse, and in general these high-affinity
autoantibodies are more predictive of T1D
and share certain characteristics, including
appearance at a young age, association with
HLA DRB1*04, subsequent progression to
multiple autoantibody positivity, binding
to human insulin A chain residues 8-13,
and binding to proinsulin.
IAAs are usually the first autoantibodies
to appear in young children who develop
T1D and can persist for many years before
the appearance of T1D clinical symptoms.
While they are considered one of the most
important autoantibodies for predicting
T1D in young children, current assays produce
highly variable results, and only a few
clinical laboratories consistently perform
the assay with high sensitivity and specificity
for the disease.
GAD65 autoantibodies. The next major
autoantigen to be characterized was a 65kDa
isoform of glutamic acid decarboxylase
(GAD65) (8). Cloning of the GAD65
gene enabled researchers to synthesize the
protein with radioisotopically labeled amino
acids in an in vitro transcription/translation
reaction and to use the product as the
antigen in radiobinding assays (RBAs). In
the early stages of GAD65 autoimmunity,
the epitopes recognized by GADA are primarily
in the middle region of the protein,
but later they may include regions at the
middle/C-terminal region of the protein.
Autoantibodies to GAD65 are particularly
important in late onset autoimmunity,
such as that seen in LADA patients.
IA-2 or ICA512 autoantibodies. In 1995,
researchers characterized two tryptic digest
fragments of islet antigens from individuals
with T1D. One, a 40-kDa fragment
from the intracellular portion of a tyrosine
phosphatase-like protein (PTPRN gene), is
now referred to as IA-2ic or ICA512ic (9).
Although IA-2 autoantibodies (IA-2A)
can be detected early in the course of autoimmunity,
they often appear after other
autoantibodies and have a higher positivepredictive
value for T1D than GADA.
Researchers have also characterized the
other 37-kDa tryptic fragment as IA-2β or
phogrin. Since almost all autoantibodies
that react with IA-2β also react with IA-2,
clinical labs typically do not use IA-2β autoantibodies
as a first line test. However, it
may be useful for identifying individuals at
high risk of disease progression.
ZnT8 autoantibodies. There is growing interest
in autoantibodies to a member of the
zinc transporter protein family, ZnT8, for
autoimmune diabetes testing. Researchers
discovered ZnT8 autoantibodies by screening
for highly expressed proteins in islet
β-cells of the pancreas (10). ZnT8 is one of
a large family of zinc transporter proteins
that is associated with the membrane of
secretory granules of islet β-cells. The zinc
within these granules forms a complex with
insulin to develop storage crystals. Human
ZnT8 exists in three major polymorphic
forms with amino acid differences at position
325: arginine, tryptophan, or glutamine.
The arginine and tryptophan forms
are the most common, and the glutamine
form is rare. Sera from some T1D patients
recognize ZnT8 epitopes not affected by
the polymorphism at position 325. Their
sera react with all polymorphic antigen
forms, but some patients’ sera are specific
to the polymorphic antigen. The latter sera
are from T1D patients who are homozy-
gous for either the arginine or tryptophan
polymorphism. Other patients may have
autoantibodies that are specific for the glutamine
polymorphism, but very few T1D
patients are homozygous for this polymorphism
because it is not common.
Biochemical Assays and Standardization
ICAs measured by indirect immunofluorescence
are still among the most sensitive
markers of T1D; however, they have been
largely replaced by biochemical autoantibody
assays for the major specific protein
antigens described above. While most labs
use various forms of fluid phase RBAs for
biochemical autoantibody assays, some
commercial ELISAs now perform as well or
better than RBAs in detecting GAD65 (11)
and IA-2ic (unpublished data).
RBAs generally begin with the synthesis
of 35 S-methionine labeled protein antigens.
For assay, the laboratorian incubates the
test serum with labeled antigen and precipitates
the resultant autoantibody-bound
antigen with Protein-A Sepharose. Each serum
sample is usually tested in duplicate on
a 96-well plate. The laboratorian must then
wash the plates to remove unbound labeled
antigen and measure the radioactive label.
For the IAA assay, commercial 125 I-labeled
insulin is generally used as the substrate.
Standardizing these assays is challenging.
To improve comparability of measurements
among laboratories, the World
Health Organization (WHO) adopted a serum
reference standard for GADA and IA-
2A assays. These standards have assigned
values of 250 units/mL for each autoantibody
(12). Progress on an international
standard for IAA, however, has been slow
because of difficulties obtaining suitable
IAA-positive sera. Standards for ZnT8 autoantibodies
are also being developed.
Given the need for standardized autoantibody
testing for diabetes, the Immunology
of Diabetes Society (IDS) and the U.S.
Centers for Disease Control and Prevention
(CDC) established the Diabetes Autoantibody
Standardization Program (DASP) in
2000 to improve comparability and to act
as a mechanism to evaluate new autoantigens
and test methodologies. The goal of
table 1
common autoantibody Tests
Test Abbreviations Description Comments
glutamic acid
decarboxylase
autoantibodies
gada protein antigen found in
neuroendocrine cells. 65Kd
isoform in islet β-cells.
islet antigen-2 ia-2a islet β-cell insulin granule
membrane protein. also
found in other neuroendocrine
cells.
insulin
autoantibodies
Zinc transporter 8
autoantibodies
iaa autoantibodies targeted to
(pro)-insulin. Highly specific
for β-cells.
Znt8 autoantibodies targeted to
β-cell membrane protein
of insulin storage granules.
predominantly in β-cells.
these tests are used to distinguish between t1d and diabetes due to other causes.
Modified from Lab Tests Online (www.labtestsonline.org). Reprinted with permission.
the organization is to improve detection
and diagnosis of autoimmune diabetes by:
1) providing technical support, training,
and information about the best methods;
2) providing proficiency testing to evaluate
laboratory performance; 3) supporting development
of highly sensitive and specific
measurement technologies; and 4) developing
reference materials.
Since its inception, DASP has conducted
six international workshop in which
laboratories assay blinded samples from 50
patients with new onset T1D and up to 100
controls. This format provides an evaluation
of the sensitivity and specificity of each
test and enables DASP to assess implementation
of assay methods and to document
any improvement in performance. Among
the major accomplishments to date, DASP
demonstrated that the performance of
ELISAs for measuring T1D autoantibodies
can equal that of RBAs and validated
ZnT8 as the fourth major T1D autoantigen.
Other activities include: validation of
IA-2β autoantibody assays; evaluation of
the stability of the WHO GADA and IA-2
autoantibody standard; validation of affinity
measurements to improve IAA test performance
(13); and evaluation of standard
method protocols for GADA and IA-2A.
Laboratories that have participated in
multiple DASP workshops have improved
the quality of their autoantibody assays. In
2009, participating labs found the majority
of new-onset patient samples were positive
for multiple autoantibodies, including all
four major autoantigens, followed by patient
samples positive for the combination
of GADA, IA-2, and ZnT8 autoantibodies.
DASP is also looking at new technology
to measure autoantibodies. Several
DASP 2009 autoantibody assays used a new
non-radioactive assay format, the luciferase
immunoprecipitation system (LIPS), that
looks promising.
To further improve quantitative agreement
of assays for GAD65 and IA-2ic, a
harmonization effort led by NIH research
consortia labs and DASP committee members
developed a standard method protocol
(15), which is now available to all DASP participants.
The National Institute of Diabetes
the most commonly used test. autoantibodies
detected in about 70–80% of newly
diagnosed t1d patients. often persist after
diagnosis.
antibodies to intracellular portion detected
in about 60–70% of t1d children at onset.
levels often fall soon after diagnosis.
detected in about 70% of t1d children (and
90% of those age