Antigens and Assays on Bead-Based Antigen Arrays.

A total of 367 protein fragments were used in this study, representing 196 unique Ensembl Gene IDs. The protocols for the design and expression of these antigens within the Human Protein Atlas framework were applied as previously described (37). In brief, using a whole-genome bioinformatics approach, antigens of 80- to 150-aa acid residues were designed in silico based on the principle of lowest sequence similarity to other human proteins and were expressed in Escherichia coli. In addition to the protein fragments, the antigen set included a full-length protein for the viral protein EBNA-1 (Tebu-Bio) and other control analytes, building a final set of 384 analytes. All antigens were coupled to carboxylated magnetic beads (MagPlex-C; Luminex Corp.) as recently described (5).

Assay conditions for the bead-based antigen arrays were applied as recently described (5). Briefly, plasma samples were diluted 1:150 in an assay buffer, and an R-phycoerythrin–conjugated Fcγ-specific goat anti-human IgG F(ab')2 fragment (Jackson ImmunoResearch) was used to detect bound human IgG in plasma. All measurements were done on the same FlexMap3D instrument (Luminex Corp.), and binding events were displayed as MFI values.