Optimization of Serum Concentration and Determination of Linear Range

For serial dilution assays, the serum or plasma was diluted 1:50, 1:100, 1:200, or 1:400 before adding it to the slides and commencing with the hybridization assay, as described below. All prototype microarrays were developed measuring IgG responses using 20 μg/mL AlexaFluor (AF) 647-labeled anti-human IgG. Notably, we observe no significant difference in performance of our immunofluorescence assays with serum or plasma (data not shown) and consider the assay to be equally compatible with both.

Microarray slides were washed with PBST (PBS, 0.2% Tween-20, pH 7.4) at RT for 3 × 5 min with gentle agitation, then dried by centrifugation at 1200× g for 2 min. Individual arrays were isolated using ProPlate 24 plex multi-well chambers (GraceBio-Labs, Bend, OR, USA). Prior to assays, serum samples were incubated with 0.1% Triton X-100 for 1 h on ice to deactivate potential live virions, then diluted 1:50 in assay buffer (PBST, 0.1% BSA, 0.1% milk powder). Individual arrays were incubated with 50 μL diluted serum for 1 h at RT with gentle agitation, then briefly rinsed with PBST, after which the slides were removed from the gaskets, washed for 3 × 5 min in PBST and dried by centrifugation at 1200× g for 2 min.

Arrays were then incubated with detection antibody (20 μg/mL Cy3-labeled anti-human IgG in assay buffer) for 30 min at RT with gentle agitation. The wells were briefly rinsed with PBST, after which the slides were removed from the gaskets and washed for 3 × 5 min in PBST with gentle agitation and dried by centrifugation at 1200× g for 2 min.