Multiplex serological assay for establishing serological profiles of polymorphic, closely related peptide antigens.

Profiling of serological responses to establish the landscape of antibody specificities in individuals exposed to pathogens or vaccines is crucial for (a) revealing humoral immune correlates of protection; (b) uncovering markers of pathogen exposure; and (c) identifying antigens and epitopes associated with disease protection. Establishing the antigenic profile of serological responses requires either expensive microarrays or labor- and time-intensive ELISA assays. Multiplex assay platforms are increasingly being evaluated for their usefulness for high-throughput testing of sera or plasma. The methodology described here utilizes a plate-based assay that allows the simultaneous detection of up to ten antigens per well in a 96 well format using an electrochemiluminescence immunoassay (ECLIA).•The newly developed protocol outlines high-throughput profiling of serological responses using a multiplex testing platform with subsequent computational analysis.•The protocol is a modification of the basic assay development manual from the manufacturer of the MESO QuickPlex SQ 120 instrument (MSD, Gaithersburg, MD) and can be used for synthetic peptides as well as full length proteins.•The protocol can be applied to map serological responses to pathogens or pathogen-derived antigens to establish serological profiles in search for biomarkers or immune correlates.