3.4 ZnT8/GAD65 application in immunoassay for the simultaneous
detection of ZnT8A and GADA
Considering the results achieved by SDS-PAGE and WB, and indirect ELISA,
we decided to apply the recombinant chimera in the development of
low-cost immunoassays, such as ELISA, for the detection of ZnT8A and
GADA simultaneously, either to confirm autoimmune diabetes in cases of
doubtful diagnosis or for detection in routine screening of individuals
at risk for the pathology. To achieve this aim, we selected 49 sera
samples from diabetic patients collected in our laboratory during the
routine detection of humoral markers.
The assay developed herein is based on the double interaction of
specific antibodies with the chimeric molecule. On the one hand,
ZnT8/GAD65 is immobilized on the plastic surface of the well (solid
phase), which interacts with one paratope of the antibody, leaving the
other paratope free to interact with the biotin-labeled chimera
(ZnT8/GAD65-biotin) in a fluid phase.
This assay presented a wide dynamic range (-2.00 to 48.00 SDs). The
specificity was 98.04 %, this is 100% minus the percentage of false
positives calculated with healthy control individuals, n = 51. By means
of receiver operating characteristic curves (ROC) where the effect of
different cut-off values were evaluated in terms of specificity and
sensitivity, the performance of the test was optimized (Figure 5). This
method had high accuracy to distinguish between samples from healthy
individuals and diabetic patients as shown by the area under the ROC
curve (AUC = 0.9488) (Carter et al., 2016). Accordingly, the cut-off
value was stablished at 2 SDs. Thus, 37 (75.51 %) out of 49 patients
sera analysed were detected as positive for ZnT8A and/or GADA, with SDs
ranging from −0.814 to 47.99 and a median of 5.222 (Figure 6). To
calculate the coefficient variation, a positive GADA and ZnT8A serum
from a diabetic patient was employed. The intra‐assay coefficient
variation was 6% and the inter‐assay coefficient variation was 4.7%.
Taken together, the results showed herein demonstrate that it was
possible to express a chimera molecule containing the immunodominant
regions of the two major DM autoantigens by a simple and low-cost
expression system, with high yield and purity. This recombinant chimera
retains the immunoreactive conformation of the epitopes that are
recognized by their specific antibodies, so it can be use in
immunoassays for the simultaneous detection of highly prevalence ZnT8A
and GADA. This immunoassay is useful either to confirm autoimmune
diabetes or for detection in routine screening of individuals at risk of
autoimmune DM.
In this sense, since the FDA has recently approved a MAb therapy to
delay DM debut (Teplizumab), there is a need for development of low
complexity methods for prediction in order to establish prevention
protocols. Herein we describe the development of a screening tool
appropriate for autoantibodies detection in risk groups. This is a
single assay with high prevalence of detection, then if it is necessary,
GADA or ZnT8A can be discriminated by a confirmation assay.
Moreover, it is important to note that cellular auto-aggression is the
cause of the pathology, therefore its study is of the utmost importance
for understanding the underlying autoimmune process that leads to the
onset of the disease and this knowledge it will be helpful in the
development of tolerance induction strategies. Regarding this, it is our
intention to use ZnT8/GAD65 as antigen in the evaluation of the cellular
immune response in diabetic patients or individuals at risk, and to
characterize the associated inflammatory context.