Figure Legends
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
(His6) tag.
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.
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 His6 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.
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 < 0.0001).
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‐off values. The vertical dashed line
indicates the cut‐off value with the optimized sensitivity and
specificity parameters (cut‐off = 2.0). (b) ROC curve analysis of
b-ELISA, AUC is included.
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‐off value (SDs > 2.0)
is indicated by a dotted line and medians for each population are
indicated as a full line (***p < 0.0001).