Proteome-Wide Serology Identifies Secreted IgD against RSV Fusion Glycoprotein as a Signature of Broadly Elevated Antibody Response

Serological evaluation of antibody response using conventional indirect immunoassays cannot capture the full depth, diversity, and functional complexity of human polyclonal antibodies. Here, we developed mass spectrometry-based Proteome-Wide Serology (ProWiSe) assays for the identification and absolute quantification of antigen-specific human antibodies across isotypes, subclasses, and variable-region repertoires. Using ProWiSe, we measured the depth and diversity of serum antibodies to multiple antigens of respiratory syncytial virus (RSV), a pathogen associated with repeated reinfections and skewed immune response. IgG1 antibodies targeting the fusion glycoprotein F (RSVF) dominated the antibody response, with a background-corrected median level of 1,700 ng/mL across 214 healthy maternal serum samples from the general population. ProWiSe identified reproducible co-enrichment of complement component 1q complexes, revealed the usage of several dozen IGHV variable-region germline genes in the anti-RSVF polyclonal response, and suggested limited IGHV repertoire diversity in some individuals. In addition to IgG1, we discovered a complex antibody response characterized by significantly elevated levels of nearly all human immunoglobulin isotypes and subclasses, including previously unexplored IgG4 and IgD. The identification of high-affinity IgD against prefusion and postfusion RSVF, but not against other antigens studied, was the most unexpected observation, occurring in 52% of the healthy maternal serum samples. ProWiSe unambiguously identified the secreted IgD isoform P01880-1 and supported the IgD paradox of preferential {lambda} light-chain usage in secreted IgD. Detectable anti-RSVF IgD levels ([≥]0.11 ng/mL) correlated with broadly elevated antibody responses across all other isotypes and subclasses, except IgE. ProWiSe assays offer a versatile platform for quantitative serological diagnostics, exploration of depth, diversity, and dynamics of polyclonal antibody response, and discovery of rare antibody isotypes.