The major finding in our study was that variant-specific in vitro transcribed and translated long ZnT8 (268–369) proteins primarily displaced the corresponding specific ZnT8Ab variant, although the reciprocal permutation experiment showed displacement as well. These data suggest that the 325 variant is part of a conformation-dependent ZnT8Ab epitope, but one which is not exclusively controlled by the amino acid at
this position. A major finding was also that a 15-mer ZnT8 peptide was insufficient to define the conformation-dependent Selleck Sunitinib epitope. Even though the short synthetic peptides appeared to increase autoantibody binding to some extent (Fig 3 lower panels), this may be the results from non-specific
binding. Selleck Palbociclib Therefore, a competing peptide would require a certain length as the short ZnT8 (318–331) peptide variants did not compete with any of the variant-specific patient sera. On the other hand, competition with the long ZnT8W proteins revealed distinctly different patterns with the unique human sera selected for this study. It was noted that the ZnT8 Triplemix RBA, detecting conformation-dependent antibodies rather than the typical ELISA linear epitopes, was positive in only 6 of 12 mice. Indeed, only one mouse (M3-W in Fig. 2) showed ZnT8tripleAb reactivity that could be readily diluted. However, in ELISA, this mouse did not show the highest end-point titers against the short ZnT8 (R/W/Q) linear peptides (Fig. 2). Lack of serum precluded experiments to establish whether epitope-specific conformational antibodies, not detectable in ELISA, were generated in the MRIP mice. Further studies immunizing mice with longer peptides
will be needed in attempts to generate single amino acid-specific antibodies. Such antibodies have been possible to generate against other antigens in the past [25-27]. The lack of recognition to the short ZnT8 peptides in the human ZnT8Ab sera supports previous studies that the ZnT8Ab are conformation dependent [4, 13]. However, it has previously not been reported that the Kd values are higher for proteins with the same epitope as the patient sera. Our data in Table 2 demonstrate lower Kd values, which correspond to higher dilutions of the variant-specific in vitro transcription translation long ZnT8 (268–369) proteins tested on its variant-specific patient serum. We believe it is an important finding that binding of ZnT8RAb-specific sera could be displaced with long cold ZnT8W protein and vice versa. This finding underscores the conclusion that a single amino acid is unable to solely control the epitope specificity. Other amino acids outside the immediate polymorphic 325 site would therefore be important to autoantibody epitope. Previously proposed residues were R332, E333, K336 and K340 (Fig.