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.