4 CONCLUSIONS
GAD65 and ZnT8 are the major autoantigens in autoimmune DM. As this pathology has a long asymptomatic preclinical period where the autoimmune process is silent, the detection of these autoantibodies (GADA and/or ZnT8A) in diabetic patients and the individuals at risk, is the first evidence that the pancreatic beta cells are being destroyed. That is why both autoantibodies became a promising tool as humoral immune markers for the early diagnosis of autoimmune DM.
As it was mentioned before, GADA and ZnT8A are one of the earliest detectable islet cell autoantibodies, and are considered highly predictive for the development of autoimmune DM. Therefore, the serological testing of these antibodies is extremely important to establish an adequate preventive treatment, which allows avoiding an abrupt onset of ketoacidosis and a strong metabolic imbalance. RBA is the reference method for the detection of both markers, yet this method is costly, and environmentally inappropriate. So, it is necessary to have low-cost immunoanalytical strategies that are feasible to be carried out in laboratories of medium and low complexity.
In autoimmune DM, the autoantibodies involved mainly recognize discontinuous conformational epitopes of the autoantigens. For this reason, in the development of immunoanalytical methodologies for the detection of these autoantibodies, it is necessary to have a recombinant antigen production system that guarantees both correct folding and high expression yield. In this sense and given that the proposed chimera constitutes a high molecular weight protein, difficult to be expressed with correct folding in E. coli , the eukaryotic expression system such as baculovirus-insect cells represents an interesting strategy for the achievement of our aim. ZnT8 and GAD65 could be successfully combined into a single molecule where each antigen retained its immunoreactivity. This allowed us to develop a one-step screening assay for the simultaneous detection of GADA and/or ZnT8A, useful for the identification of positive individuals at risk of DM or at onset of the disease.