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.