Iterative immunoprecipitation and phage pre-wash dramatically improve epitope-resolved serology by VirScan.

Accurate mapping of antibody epitope repertoires is essential for understanding infection, vaccination, and immune history. Phage immunoprecipitation sequencing (PhIP-seq), including the widely used VirScan platform, offers single - peptide resolution across the human virome; however, such measurements are sometimes beset with limitations stemming from weak signal-to-noise ratio, non-specific phage binding, and inconsistent peptide enrichment. With such methods, it is imperative to differentiate true antibody-antigen interactions from background noise. Here, we systematically evaluate the impact of key experimental variables on assay performance and identify two synergistic modifications that markedly improve epitope-level viral serology: (1) iterative rounds of immunoprecipitation and (2) serum pre-washing with wild-type phage. Across healthy donor sera, pooled HIV-seropositive sera, and influenza-vaccinated rabbit sera, the optimized workflow produces a substantial expansion of the enriched peptide population, significantly higher normalized peptide counts, and improved separation of viral epitopes from background noise while preserving global library representation. The protocol enables quantitative detection of HIV epitopes across a 100-fold dilution series, resolving immunodominant gp160 regions, and identifies strain-specific hemagglutinin epitopes elicited by influenza vaccination, including expected public stem-directed linear epitopes. These results provide a reproducible, generalizable workflow that enhances viral epitope discovery, serosurveillance sensitivity, and vaccine-response mapping. This optimized PhIP-seq framework strengthens the utility of VirScan in systems virology by enabling more accurate and quantitative inference of viral antibody repertoires.