Toward precision serology: resolving human polyclonal antibody repertoires with proteomics

Introduction: Human polyclonal antibodies constitute a highly diverse and dynamic universe of clonotypes, isotypes, and subclasses circulating in serum and contributing to immune response. Traditionally, the analysis of human polyclonal antibodies has relied on indirect immunoassays which suffered from semi-quantitative measurements and lack of standardization. Recent advances in mass spectrometry (MS) and immunoaffinity proteomics, including our studies [1-4], facilitated selective, sensitive, and reproducible quantification of low-abundance proteins in serum and proximal fluids. Here, we will introduce a proteomic toolbox for the quantification and characterization of endogenous human polyclonal antibodies. We will demonstrate the complexity of the antibody response to viral antigens and cancer auto-antigens and discuss pathways toward Precision Serology.

Methods: The proteomic toolbox included shotgun, targeted and intact protein MS assays to identify, quantify and sequence human polyclonal antibodies [5]. High-throughput immunoenrichments combined with targeted MS and carefully designed standards enabled absolute quantification of antibody isotypes (IgG, IgA, IgM, IgE, IgD) and subclasses (IgG1-4, IgA1-2) circulating in patient sera (>100 samples/day). Restricted proteolytic cleavage generated Fab, Fd, and L fragments for intact protein MS. Alternative proteases enabled increased sequence coverage of repertoires of antibody variable chains.

Results: Significantly elevated levels of anti-SARS-CoV-2 RBD IgG1 (1.6 µg/mL), IgG3, IgM and IgA1 in patient sera (N=231) suggested a conventional Th1 immune response [4]. Respiratory Syncytial Virus antigens revealed a mixed Th1/Th2 immune response with elevated levels of IgG1 (2.6 µg/mL), IgM, IgA1, IgG2 and IgG4 in sera (N=69). The unexpected discovery of antigen-specific secreted IgD suggested a distinct immune response in certain infections. Profiling of the repertoire diversity of the heavy variable chains revealed the frequent use of only ~30 IGHV genes. In prostate cancer, a panel of prostate-specific antigens demonstrated distinct IgG1- and IgA1-mediated auto-antibody response in serum (N=50), with anti-PSA IgG1 found in 70% of cancer patients at 3-14 ng/mL.

Conclusions: Our proteomic toolbox will facilitate quantification and characterization of the endogenous human polyclonal antibodies, antibody sequencing, investigation of immunoglobulin subclass cooperation in immune response, precision approaches in serology diagnostics, and immunology studies of infectious diseases and cancer [6].

References: [1] Fu Z… Drabovich AP. Mol. Cell. Proteomics 2021, 20, 100075; [2] Zhang J… Drabovich AP. Mol. Cell. Proteomics 2023, 22, 100556; [3] Rais Y, Drabovich AP. J. Proteome Res. 2024, 10.1021/acs.jproteome.4c00027; [4] Fu Z… Drabovich AP. Anal. Chem. 2022, 94, 12990; [5] Wize T, Drabovich AP. bioRxiv 2023, 10.1101/2023.10.27.564451; [6] Walter J… Drabovich AP. Clin. Proteomics 2023, 20, 42.