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.