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).