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\begin{document}

\title{Expression of a ZnT8/GAD65 chimera in insect cells useful for Autoimmune
Diabetes diagnosis}



\author[1]{Aldana Trabucchi}%
\author[1]{Silvina Bombicino}%
\author[1]{Adriana Victoria Sabljic}%
\author[1]{Juan Marfia}%
\author[1]{Alexandra Targovnik}%
\author[1]{Ruben Iacono}%
\author[1]{Maria Miranda}%
\author[1]{Silvina Valdez}%
\affil[1]{Universidad de Buenos Aires Facultad de Farmacia y Bioquimica}%


\vspace{-1em}



  
  \date{May 26, 2024}


\begingroup
\let\center\flushleft
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\selectlanguage{english}
\begin{abstract}
The combined presence of autoantibodies to the 65 kD isoform of glutamic
acid decarboxylase (GADA) and to the islet-specific cation efflux
transporter ZnT8 (ZnT8A) in serum is the best predictive sign of the
loss of immune tolerance and the clinical manifestation of autoimmune
diabetes mellitus (DM). 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. 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,
yielding 30 mg/L culture medium. This recombinant chimera retains the
immunoreactive conformation of the epitopes that are recognized by their
specific antibodies, so it was used for the development of a high
sensitivity (75.51 \%) and specificity (98.04 \%) bridge ELISA for the
detection of highly prevalence ZnT8A and/or GADA, in a one-step
screening assay. This immunoassay is useful either to confirm autoimmune
diabetes or for detection in routine screening of individuals at risk of
autoimmune DM. As DM is a slow progress disease, remaining asymptomatic
for a long preclinical period, serological testing is of importance to
establish a preventive treatment.%
\end{abstract}%



\sloppy


\textbf{Expression of a ZnT8/GAD65 chimera in insect cells useful for
Autoimmune Diabetes diagnosis}

Aldana Trabucchi\textsuperscript{1,2}; Silvina Sonia
Bombicino\textsuperscript{1,2}, Adriana Victoria
Sabljic\textsuperscript{1,2}; Juan Ignacio Marf\selectlanguage{ngerman}ía\textsuperscript{1,2},
Alexandra Marisa Targovnik\textsuperscript{3,4}, Ruben Francisco
Iacono\textsuperscript{1,2}, María Victoria
Miranda\textsuperscript{3,4}, Silvina Noemí Valdez\textsuperscript{1,2}.

\textsuperscript{1}Universidad de Buenos Aires (UBA), Facultad de
Farmacia y Bioquímica, Departamento de Microbiología, Inmunología,
Biotecnología y Genética, Cátedra de Inmunología, Buenos Aires,
Argentina

\textsuperscript{2}Consejo Nacional de Investigaciones Científicas y
Técnicas (CONICET) Universidad de Buenos Aires, Instituto de Estudios de
la Inmunidad Humoral ``Prof. Ricardo A. Margni'' (IDEHU), Buenos Aires,
Argentina

\textsuperscript{3}Universidad de Buenos (UBA), Facultad de Farmacia y
Bioquímica, Departamento de Microbiología, Inmunología, Biotecnología y
Genética, Cátedra de Biotecnología, Buenos Aires, Argentina

\textsuperscript{4}Consejo Nacional de Investigaciones Científicas y
Técnicas (CONICET) Universidad de Buenos Aires, Instituto de
Nanobiotecnología (NANOBIOTEC), Buenos Aires, Argentina

\textbf{Corresponding Author}

Aldana Trabucchi, PhD.

Cátedra de Inmunología, Facultad de Farmacia y Bioquímica, Universidad
de Buenos Aires

Junín 956, 4to piso (C1113AAD) Buenos Aires, Argentina

Tel.: 54 11 5287 4413

E-mail: atrabucchi@ffyb.uba.ar

\textbf{Short running title: ZnT8/GAD65 chimera for Autoimmune Diabetes
diagnosis}



\textbf{ABSTRACT}

The combined presence of autoantibodies to the 65 kD isoform of glutamic
acid decarboxylase (GADA) and to the islet-specific cation efflux
transporter ZnT8 (ZnT8A) in serum is the best predictive sign of the
loss of immune tolerance and the clinical manifestation of autoimmune
diabetes mellitus (DM). 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. 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,
yielding 30 mg/L culture medium. This recombinant chimera retains the
immunoreactive conformation of the epitopes that are recognized by their
specific antibodies, so it was used for the development of a high
sensitivity (75.51 \%) and specificity (98.04 \%) bridge ELISA for the
detection of highly prevalence ZnT8A and/or GADA, in a one-step
screening assay. This immunoassay is useful either to confirm autoimmune
diabetes or for detection in routine screening of individuals at risk of
autoimmune DM. As DM is a slow progress disease, remaining asymptomatic
for a long preclinical period, serological testing is of importance to
establish a preventive treatment.

\textbf{KEY WORDS}

Autoimmune Diabetes Mellitus, ZnT8/GAD65 chimera, insect cells,
autoantibodies, ELISA

\textbf{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.

\textbf{2 MATERIALS AND METHODS}

\textbf{2.1 Recombinant virus construction}

The design of the chimeric molecule contained the coding sequence of the
C-terminal domain of human ZnT8, which consisted of a dimer that
includes amino acids 268--369 {[}R325{]} and amino acids 268--369
{[}W325{]}, together with full-length human GAD65. Also, a
histidine-hexapeptide (His\textsubscript{6}) tail at the N-terminus was
added. This coding sequence was synthesized by GenScript Corporation
(Piscataway, NJ, USA; www.GenScript.com). Codon optimization for insect
cell expression was done.

The chimera cassette was directly cloned under the polyhedrin (polh)
promoter using the EcoR1 and XbaI site, into the pFastBac\selectlanguage{english} Dual vector
(Thermo Fisher Scientific), previously modified with the enhanced green
fluorescent protein (EGFP) cDNA under the p10 promoter (Targovnik et
al., 2019). The final construction pFBD\selectlanguage{english}-p10\selectlanguage{english}-EGFP\selectlanguage{english}-polh\selectlanguage{english}-ZnT8/GAD65 is
shown in Figure 1.

The recombinant baculoviruses were obtained using the Bac to Bac\selectlanguage{english}
baculovirus expression system (Thermo Fisher Scientific), following the
manufacturer's instructions, as we have previously described (Smith et
al., 2022). Minor modifications were done in order to generate the
desired virus. Once the bacmids were purified from \emph{Escherichia
coli} DH10Bac\selectlanguage{english} strain (Thermo Fisher Scientific), and before
transfecting Sf9 cells, the sequences were verified by Sanger sequencing
(GenBiotech). The transfection was evaluated by the green fluorescence
generated under UV light (Figure 2). After 4 days of infection, the
culture supernatant was recovered. The recombinant virus was named
Acpolh\selectlanguage{english}-ZnT8/GAD65.

Finally, amplification was performed at a multiplicity of infection
(MOI) of 0.02 in Sf9 cells seeded in T\selectlanguage{english}-25 flasks at 27degC. The amplified
Acpolh\selectlanguage{english}-ZnT8/GAD65 was titrated by plaque assay (O'Reilly et al., 1994).
This high\selectlanguage{english}-titer Acpolh\selectlanguage{english}-ZnT8/GAD65 was the viral stock used for protein
production in insect cell.

The Sf900 II insect cell culture medium was from
Invitrogen\textsuperscript{TM} (Gaithersburg) and the antibiotic and
antimycotic solutions (Anti-Anti 100X) and the fetal bovine serum were
from GIBCO (Thermo Fisher Scientific).

\textbf{2.2 Recombinant ZnT8/GAD65 expression and purification from cell
cultures}

For the ZnT8/GAD65 expression, independent Sf9 (serial passage 30)
suspension cultures

in log-phase at a cell density of 4 x 10\textsuperscript{7} cells in 30
mL (2 x 10\textsuperscript{6} cell/mL) were infected with
Acpolh\selectlanguage{english}-ZnT8/GAD65 MOI 1. After 4 days of incubation in the dark at 27
degC, in continuous agitation, cells were centrifuged. The recombinant
chimera was recovered following the protocols of lysis and purification
previously described by our group (Trabucchi et al., 2019).

For the determination of total protein concentration Bradford microassay
(Bradford, 1976) was performed using Coomassie Plus\selectlanguage{english} Protein Assay
(Thermo Fisher Scientific).

The recombinant ZnT8/GAD65 chimera was stored with a mixture of 50\% v/v
glycerol, Tween 20 and protease inhibitors, and kept at -20degC until
used.

\textbf{2.3} \textbf{Sodium dodecyl sulphate-polyacrylamide gel
electrophoresis and western blot analysis}

The protocols followed for SDS-PAGE and WB analysis were the same as
Faccinetti et al. (2017) and Trabucchi et al. (2019). Briefly, protein
fractions were separated by 10\% SDS-PAGE under reducing conditions,
followed by Coomassie Brilliant Blue R-250 staining. For detection by
WB, once the protein bands were transferred onto nitrocellulose
membranes, the expression of the chimera was evidenced by either a mouse
monoclonal antibody to GAD65, a rabbit policlonal antibody to ZnT8 or a
mouse monoclonal anti-histidine antibody (BD Pharmingen). Bound
antibodies were visualized by incubation with peroxidase-conjugated goat
antibodies to mouse IgG or with peroxidase-conjugated goat antibodies to
rabbit IgG, respectively. Protein bands were visualized by the addition
of \selectlanguage{greek}α-\selectlanguage{english}chloronaphthol (Sigma-Aldrich) and 10 vol.
H\textsubscript{2}O\textsubscript{2}.

\textbf{2.4 Identification of the recombinant ZnT8/GAD65 protein
expressed in Sf9 cells by indirect ELISA.}

The identification of purified chimera was evaluated by means of
indirect enzyme linked immunosorbent assay (ELISA) using mouse
monoclonal antibodies against GAD65 and a rabbit serum against ZnT8. For
this purpose, polystyrene microplates (Maxisorp, NUNC) were coated ON at
4 \selectlanguage{ngerman}°C with 0,1 ug of purified ZnT8/GAD65 chimera per well, washed three
times with phosphate buffered saline (PBS: 1.5 mM
KH\textsubscript{2}PO\textsubscript{4}, 8.1 mM
Na\textsubscript{2}HPO\textsubscript{4}, 140 mM NaCl, 2.7 mM KCl, pH
7.4), blocked for 1.5 h with 200 \selectlanguage{greek}μ\selectlanguage{english}L of blocking solution (3\% skim milk
in PBS) per well, and washed five times with PBS containing 0.05\% Tween
20 (PBS-T). Samples diluted 1/100 in 3\% skim milk in PBS-T (PBS-MT)
were evaluated five times, and microplates were incubated for 1 h. After
another round of 5 washes with PBS-T the bound specific antibodies were
detected by the addition of peroxidase-conjugated goat antibodies to
mouse IgG or with peroxidase-conjugated goat antibodies to rabbit IgG
diluted 1/3000 in PBS-MT. Following washing (five times with PBS-T plus
one final wash with PBS), the chromogenic substrate was added
(3,3',5,5'-tetramethyl-benzidine/H2O2 mixture; Single Component TMB
Peroxidase EIA Substrate Kit, BioRad), and plates were incubated for 15
min in the dark. The color reaction was stopped with 4N
H\textsubscript{2}SO\textsubscript{4}. The oxidized substrate was
measured at 450 nm with an ELISA plate reader MultiskanFC (Thermo
Scientific Labsystems). The blank control was made by replacing serum
samples with PBS-MT. Results were expressed as specific absorbance (A =
the mean absorbance of each sample minus the mean absorbance of the
blank control).

\textbf{2.5 ZnT8/GAD65 application in immunoassay for the simultaneous
detection of ZnT8A and GADA}

\textbf{2.5.1 Sera collection}

Sera from diabetic patients (n = 49) were selected among the samples
collected in our laboratory during the routine detection of
autoantibodies (Servicios Tecnol\selectlanguage{ngerman}ógicos de Alto Nivel, STAN-CONICET).

Control sera were obtained from 51 healthy subjects without personal or
family history of autoimmune disease.

This work was performed with the approval of the Ethical Committee of
José de San Martín Clinical Hospital, Buenos Aires, Argentina.

All experiments were done in accordance with the relevant guidelines and
regulations.

Written informed consent was obtained from all participants.

\textbf{2.5.2 Biotinylation of the chimera ZnT8/GAD65}

Biotinylation of the purified chimera was done as we have previously
described (Trabucchi et al., 2019, Trabucchi et al, 2022). Two hundred
eighty micrograms (280 ug) of desalted protein were incubated with
excess of sulfo-NHS-biotin (Pierce Biotechnology), and free biotin was
then removed.

ZnT8/GAD65-biotin was stored until use at -20 °C with the addition of
50\% v/v glycerol, Tween 20 and protease inhibitors.

\textbf{2.5.3 Bridge ELISA (b-ELISA)}

The protocol employed was based on that previously described (Villalba
et al., 2007; Trabucchi et al., 2019; Trabucchi et al., 2022), with
minor modifications. Briefly, polystyrene microplates were coated ON at
4 °C with 0.1 ug of purified ZnT8/GAD65 chimera per well. After washing
with PBS, adding 200 \selectlanguage{greek}μ\selectlanguage{english}L of blocking solution per well for 1.5 h, and
washing five times with PBS-T, 50 ul samples were added in duplicate.
After 1 h of incubation at room temperature, plates were washed again
and 56.0 ng of ZnT8/GAD65-biotin per well were added. One hour later,
another round of washing steps were done, and the bound
ZnT8/GAD65-biotin was detected by the addition of
Streptavidin-Horseradish Peroxidase (Jackson ImmunoResearch
Laboratories, Inc.). Finally, microplates were washed five times with
PBS-T plus one final washing step with PBS. The color reaction was
elicited by the addition of the chromogenic substrate incubated for 15
minutes in the dark and was then stopped with 4N
H\textsubscript{2}SO\textsubscript{4}. The oxidized substrate was
measured at 450 nm with an ELISA plate. The blank control was made by
replacing serum samples with PBS-MT. Results were calculated as specific
absorbance and expressed as standard deviation scores: SDs = (A-Ac)/SDc,
where Ac is the mean specific absorbance of 25 healthy control sera and
SDc its standard deviation. The cut-off value of the assay was set at
SDs = 2.

\textbf{2.6 Statistical analysis}

The normal distribution of the data was analyzed by the D'Agostino \&
Pearson Omnibus normality test. To select the cut-off with maximum
sensitivity and specificity, ROC curves were made by plotting these
parameters against the corresponding cut-off values. Statistical
significance was assessed by parametric tests: Student's t-test for
unpaired samples with Welch correction; or non-parametric tests:
Mann-Whitney U test for unpaired data, when applicable. Calculations
were performed using GraphPad Prism version 6.01 for Windows (GraphPad
Software, San Diego California, USA, www.graphpad.com). A value of p
\textless{}0.05 was considered statistically significant.

\textbf{3 RESULTS AND DISCUSSION}

\textbf{3.1 Generation of the Recombinant Baculovirus Acpolh-ZnT8/GAD65}

For the construction of the chimera, we based ourselves in the knowledge
of the immunodominant regions of both proteins. As previously described
by Rickert et al. (2001), GAD65 has nine conformational epitopes
throughout its entire structure so we decided to use full length GAD65.
As for ZnT8, it is known that C-terminal domain (amino acids 268--369)
is the region most recognized by specific autoantibodies (Wenzlau et
al., 2007). Also, this region contains a polymorphism in amino acid 325.
In this sense, we decided to use a heterodimeric variant of C-terminal
domain containing R or W in 325 position, based on that we have
previously demonstrated that this heterodimeric construction showed
higher signal levels and increased dynamic range in RBA for ZnT8A
detection (Faccinetti et al., 2016).

The cassette containing a dimer of ZnT8 and full length GAD65, fused to
a His-tag was cloned under the control of the polyhedrin promoter, hence
pFBD-polh-ZnT8/GAD65 was obtained. The N-terminal His-tag was added to
facilitate the purification of the recombinant chimera by
Ni\textsuperscript{2+}--NTA Immobilize Metal Affinity Chromatography.
The expression vector used in this work has another distinctive feature,
which is the employment of EGFP, cloned under the p10 promoter, as a
reporter gene useful for visualizing viral infection. The corresponding
bacmids were produced with these plasmids. The Acpolh-ZnT8/GAD65 virus
was finally obtained by transfection of the bacmids and amplification in
Sf9 cells.

\textbf{3.2 Recombinant ZnT8/GAD65 expression and purification from cell
cultures}

The recombinant chimera was expressed in Sf9 cells with a MOI of 1 based
on previous experiences (Trabucchi et al., 2019). After 4 days of
infection, we efficiently produced ZnT8/GAD65, with a yield of 30 mg of
chimera per litre of culture medium, 80\% pure. SDS-PAGE analyses of
total cell lysates and the different purification steps showed one band
of approximately 80 kDa, the expected molecular weight for the
full-length engineered chimera (Figure 3). Moreover, the analysis by WB
allowed identifying the recombinant chimera using specific antibodies to
His\textsubscript{6}, to GAD65 or to ZnT8.

Even though the purity of the expressed protein obtained is not
extremely high, it is suitable for our intended use, the development of
highly specific and sensitive immunoassays for the detection of
autoantibodies.

\textbf{3.3 Identification of the recombinant ZnT8/GAD65 protein
expressed in Sf9 cells by indirect ELISA}

Identification of the ZnT8 and GAD65 domains in the recombinant chimera
was evaluated by indirect ELISA using ZnT8/GAD65 as the immobilized
antigen that interacted with polyclonal rabbit ZnT8 antibodies and
monoclonal mouse GAD65 antibodies. As it is shown in Figure 4, both
domains were correctly recognized by their specific antibodies,
demonstrated by the significant difference of the signals obtained with
the specific antibodies with respect to the normal controls. This
encouraged us to go ahead with the design of a bridge ELISA for the
detection of autoantibodies in individuals at risk or diabetic patients
with doubtful classification.

\textbf{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.

\textbf{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 \emph{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.

\textbf{Acknowledgments}

We would like to thank the Hemotherapy Division at the Jos\selectlanguage{ngerman}é de San
Martín Clinical Hospital (Buenos Aires, Argentina) for collecting and
providing the sera of diabetic patients and healthy human individuals.

This work was supported in part by grants from FONCYT Program of the
National Agency for Science and Technology Promotion (ANPCYT)
PICT-2017-1436 and PICT-2016-1296, National Research Council (CONICET)
PIP 11220200102551CO, the University of Buenos Aires, Buenos Aires,
Argentina (UBA 20020190100184BA and 20020170200186BA) and the Argentine
Diabetes Society (SAD, Subsidio de la Sociedad Argentina de Diabetes,
área básica).

\textbf{CONFLICT OF INTERESTS}

Aldana Trabucchi, Silvina Sonia Bombicino, Alexandra Marisa Targovnik,
María Victoria Miranda and Silvina Noemí Valdez and are career
researchers of the Consejo Nacional de Investigaciones Científicas y
Técnicas de Argentina (CONICET). Adriana Victoria Sabljic is a research
fellow of UBA. Juan Ignacio Marfia and Ruben Francisco Iacono are
research assistants of CONICET.

The authors declare that there are no conflict of interests.

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\textbf{Figure Legends}

\textbf{Figure 1.} Recombinant baculovirus (Acpolh-ZnT8/GAD65) for the
expression of ZnT8/GAD65 chimera under the polh promoter. The chimera
includes two sequences of the C-terminal región of ZnT8 (residues
268-369) with arginine (R) or triptophan (W) in the residue 325, and
full length GAD65. The N-terminal includes a histidine
(His\textsubscript{6}) tag.

\textbf{Figure 2.} Fluorescence microscopic observation of Sf9 cells
infected with the baculovirus expressing ZnT8/GAD65. The green
fluorescence is due to the expression of GFP (green fluorescence
protein) as an infection control. (a) negative control (uninfected
cells). (b) infected cells.

\textbf{Figure 3.} Expression and purification of ZnT8/GAD65 in
Sf9-cells. (a) SDS-PAGE (10.0\% T 6.0\% C, 1.5 mm, under reducing
conditions, stained with Coomassie Brilliant Blue R-250); (b) WB
revealed with monoclonal antibodies to GAD65 as primary antibody; (c) WB
revealed with policlonal antibodies to ZnT8 as primary antibody; (d) WB
revealed with monoclonal antibodies to His\textsubscript{6} as primary
antibody. Samples: 1. Molecular weight markers, 2. Pellet, 3. Total
soluble fraction, 4. Unbound material, 5. Wash step, 6-9. Consecutive
eluates of purified ZnT8/GAD65. Arrows indicate the electrophoretic
mobility of ZnT8/GAD65.

\textbf{Figure 4.} Indirect ELISA for the characterization of the
recombinant ZnT8/GAD65 chimera. NRS: Normal Rabbit Serum used as
control, a-ZnT8: hiperinmune rabbit serum against human ZnT8, TAF: Total
Ascitic Fluid from mouse used as control, a-GAD65: monoclonal mouse
antibodies against human GAD65. (***p \textless{} 0.0001).

\textbf{Figure 5.} Analysis of b-ELISA performance resulting from the
study of 51 sera from normal control individuals and 49 sera from
diabetic patients. (a) Sensitivity curve (o) and specificity (×) as a
function of the possible cut\selectlanguage{english}-off values. The vertical dashed line
indicates the cut\selectlanguage{english}-off value with the optimized sensitivity and
specificity parameters (cut\selectlanguage{english}-off = 2.0). (b) ROC curve analysis of
b-ELISA, AUC is included.

\textbf{Figure 6.} GADA and ZnT8A results obtained by b-ELISA in sera
from healthy control individuals and sera from diabetic patients.
Results are expressed as SDs. The cut\selectlanguage{english}-off value (SDs \textgreater{} 2.0)
is indicated by a dotted line and medians for each population are
indicated as a full line (***p \textless{} 0.0001).\selectlanguage{english}
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