A detailed description of the SERA assay has been published26 (link). For this study, serum or plasma was incubated with a fully random 12-mer bacterial display peptide library (1 × 1010 diversity, 10-fold oversampled) at a 1:25 dilution in a 96-well, deep well plate format. Antibody-bound bacterial clones were selected with 50 µL Protein A/G Sera-Mag SpeedBeads (GE Life Sciences, cat#17152104010350) (IgG) or by incubation with a biotinylated anti-human IgM antibody (Jackson ImmunoResearch, cat# 709-066-073) final assay dilution 1:100, followed by a second incubation with 50 µl Dynabead MyOne Streptavidin T1 conjugated magnetic beads (IgM) (Thermo-Fisher 65602). The selected bacterial pools were resuspended in growth media and incubated at 37 °C shaking overnight at 300 RPM to propagate the bacteria. Plasmid purification, PCR amplification of peptide-encoding DNA, barcoding with well-specific indices was performed as described26 (link). Samples were normalized to a final concentration of 4 nM for each pool and run on the Illumina NextSeq500. Every 96-well plate of samples processed for this study contained healthy control run standards to assess and evaluate assay reproducibility and possible batch effects.
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