1 INTRODUCTION
There are several organ-specific autoimmune diseases, type 1 Diabetes Mellitus (DM 1) is one of them, in which the target organ is the pancreas. This pathology is caused by the autoaggression of LT CD8+ and CD4+ to insulin-producing pancreatic beta cells that leads to their destruction. Because of this destruction, autoantibodies against different beta cells structures are elicited, and these are the first detectable signal of an underlying autoimmune process. There are four main disease-related autoantibodies that have been shown to predict clinical DM1 (Knip, 2002) these are: insulin/proinsulin autoantibodies (IAA/PAA), autoantibodies to the 65 kD isoform of glutamic acid decarboxylase (GADA), autoantibodies to the protein tyrosine phosphatase-related IA-2 molecule (IA–2A) and autoantibodies to the islet-specific cation efflux transporter ZnT8 (ZnT8A). The combined presence of these autoantibodies in serum is the best predictive sign of both the loss of immune tolerance and the clinical manifestation of the pathology.
During the progression of the disease, an inflammatory environment called insulitis is created; this is produced by the infiltration of immune cells into the pancreas. In this context, the exposure of the islet antigens of the pancreas in the HLA class I increases, triggering and accelerating the development of diabetes (Coppieters et al., 2012; Willcox et al., 2009).
Although up to now it has not been possible to attribute a clear role for the humoral immune response in the etiopathogenesis of autoimmune DM, the early detection of the markers IAA/PAA, GADA, IA-2A and ZnT8A makes it possible to support differential diagnosis of the several types of DM, such as Immuno Mediated Autoimmune Diabetes in Adults (Adler et al., 2021; Valdez et al., 2022). A link was found between the number of positive markers in asymptomatic individuals within risk groups (direct relatives of diabetic patients), and the probability of developing the disease over a given period (Achenbach et al., 2007). In addition, prospective and predictive studies have shown that these markers are associated with the self-injurious cellular response of beta cells and anticipate the appearance of the first symptoms of diabetes by months or even years (Primavera, Giannini, & Chiarelli, 2020).
GADA (Baekkeskov et al., 1982) and ZnT8A (Wenzlau et al., 2007; Wenzlau, Hutton, & Davidson, 2008; Wenzlau et al., 2008) represent a high individual and combined prevalence in onset patients with DM1. The association of both markers would be an interesting rational combination for the search of autoimmunity in the infant and adolescent population, as well as in those patients in whom latent autoimmune diabetes of adults (LADA) is suspected (Trabucchi et al., 2012). The screening of GADA and ZnT8A could be an appropriate alternative to identify diabetic subjects with underlying autoimmunity, helping to reach to a correct diagnosis and guaranteeing the start of an early and adequate treatment. This leads to the delay in the appearance of chronic complications of the disease, which strongly impacts in benefits for the patient, as well as the reduction of the socioeconomic costs associated with the pathology.
The presence of diabetes-associated autoantibodies predicts progression to future insulin dependence after diagnosis of diabetes. In this sense, according to a UKPDS (UK Prospective Diabetes Study), at least 50% of patients with autoimmune DM with onset in adulthood required insulin treatment 6 years after diagnosis (Turner et al., 1997). An important feature in this group of patients is that they have functioning beta cells at the time of diagnosis, indicating that therapeutic strategies should be implemented urgently in order to improve their metabolic control as well as to preserve insulin secretory capacity (Steffeset al., 2003). It should be noted that before deciding on a therapeutic route, the estimation of C-peptide and diabetes-associated autoantibodies are highly relevant for the establishment of a personalized therapy since they are the fundamental characteristics of the disease. Likewise, the identification of an inflammatory context by determining associated cytokines and autoreactive cells against pancreatic beta cell antigens could contribute to improving this diagnosis and thus to the establishment of an adequate therapy.
Herein, we describe the expression of a chimera molecule including immunodominant regions of the antigens ZnT8 and GAD65 by using the baculovirus-insect cells system. This recombinant protein was used for the development of a high sensitivity and specificity bridge ELISA (b-ELISA) for the detection of ZnT8A and/or GADA, in a one-step screening assay. This test would be useful for screening the population at risk for DM1, as well as for the search for autoimmunity in cases of suspected LADA.