Immunoprecipitation-targeted proteomics assays facilitate rational development of SARS-CoV-2 serological diagnostics

Abstract

Current design of serological tests employs conservative immunoassay approaches and is often focused on convenience, speed of manufacturing, and affordability. Limitations of such serological tests include semi-quantitative measurements, lack of standardization, potential cross-reactivity, and inability to distinguish between antibody subclasses. As a result of cross-reactivity, diagnostic specificity of serological antibody tests may not be sufficiently high to enable screening of the general asymptomatic populations for the acquired immunity against low-prevalence infectious diseases, such as COVID-19. Likewise, lack of a single standard for assay calibration limits inter-laboratory and international standardization of serological tests. In this study, we hypothesize that combination of immunoaffinity enrichments with targeted mass spectrometry measurements would enable rational design of serology diagnostics of infectious diseases, such as COVID-19. The same instrumental platform allows for sensitive and specific measurements of viral protein antigens, as wells as anti-viral antibodies circulating in human serum. Our proof-of-concept immunoprecipitation - parallel reaction monitoring (IP-PRM) assays quantified NCAP_SARS2 protein with a limit of detection of 313 pg/mL in serum. In addition, a multiplex IP-selected reaction monitoring (IP-SRM) assay facilitated differential quantification of anti-SARS-CoV-2 antibody isotypes and subclasses in patient sera. Simultaneous evaluation of numerous antigen-antibody subclass combinations revealed a receptor-binding domain (RBD)-IgG1 as a combination with the highest diagnostic specificity and sensitivity. Anti-RBD IgG1, IgG3, IgM and IgA1 subclasses, but not IgG2, IgG4 and IgA2, were found elevated in COVID-19-positive sera. Synthetic heavy isotope-labeled peptide internal standards as calibrators revealed elevated anti-RBD IgG1 in positive (510-6700 ng/mL; 0.02-0.22% of total serum IgG1) versus negative sera (60 [interquartile range 41-81] ng/mL). Likewise, anti-RBD IgM was elevated in positive (190-510 ng/mL; 0.06-0.16% of total serum IgM) versus negative sera (76 [31-108] ng/mL). Further validation of immunoprecipitation-targeted proteomics assays as a platform for serological assays will facilitate standardization and improvement of the existing serological tests, enable rational design of novel tests, and offer tools for comprehensive investigation of antibody isotype and subclass cooperation in immunity response.