Development of immuno-affinity mass spectrometry assays  for the detection and quantification of viral antigens and antibodies
Creators: Dara, Delaram
Publisher: name: University of Alberta Library
Year: 2022

<p>Currently, immunoassays are the gold standard tools for the detection and measurement of proteins in biological samples. These methods have a high level of sensitivity but are prone to limitations, such as non-specific binding and limited specificity due to cross-reactivity. This research aimed at addressing these limitations. Multiplexing sensitive and highly specific immunoaffinity-mass spectrometry (IA-MS) assays were developed for the detection and quantification of immunoglobulins (Igs) generated against severe acute respiratory syndrome coronavirus (SARS-CoV-2), and the human endogenous retrovirus-K (HERV-K) envelope proteins. </p>

<p>For the first project, the developed IA-MS assays were applied for the multiplexing detection and quantification of antibody subclasses and isotypes (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, and IgE). Proteotypic peptides unique to the constant regions of various antibody subclasses and isotypes were selected, and stable-isotope labeled peptide internal standards were designed and optimized, enabling the differential quantification of human Igs. For immunoprecipitation (IP), the recombinant receptor binding domain (RBD) of the spike glycoprotein was used as a bait to capture human antibodies specifically targeting the RBD domain. Following IP, ultra performance liquid chromatography was used for the separation of peptides following by selected reaction monitoring (SRM) assay for the detection and quantification of antibody subclasses and isotypes. Initially, 29 confirmed positive plasma samples and 12 pre-pandemic and confirmed negative serological samples were used to examine the sensitivity and specificity of the developed assay. There was a statistically significant increase in median concentrations of the anti-RBD IgG1, IgG3, total IgG, IgA1, total IgA and IgM in COVID-19 positive versus COVID-19 negative samples. Additionally, there was 100% diagnostic specificity at 100% sensitivity when measuring anti-RBD IgG1 median concentrations to detect COVID-19 positive patients from negative patients. Subsequently, a larger sample size was used evaluate the developed IA-SRM assay (82 positive plasma, and 142 pre-pandemic serum and plasma). The IgG1 cut-off for diagnosing SARS-CoV-2 positive versus negative patients was validated to be 407 ng/mL. IgG1 was found to be the most representative antibody in serological samples for distinguishing positive from negative patients. Additionally, with the multiplexing capacity of SRM assays and the assessment of both IgG1 and IgM antibodies, the sensitivity of developed assay increased from 89.1% to 97.62%. </p>

<p>An IA-SRM assay was developed for the differential quantification of HERV-K env (envelope) proteins in MCF-7, MDA-MB-231 and LNCaP cancer cell lines as well as human placenta tissues. Two commercial antibodies (HERM 1811-5 and ERVK-7) targeting highly conserved protein sequences were used to capture HERV-K env proteins. HERV-K env proteins, however, were not detected in these cell lines nor the placenta tissues. Synthetic peptides containing the epitope region of these two antibodies were used to assess antibody affinity and revealed that the HERM 1811-5 and ERVK-7 antibodies potentially did not bind to the corresponding epitope peptides. </p>

<p>Overall, the developed IA-SRM assays showcase the potential to facilitate diagnosis of infectious diseases. In such cases, specific peptides or antibodies can be used for the capturing and enrichment of target proteins to then be detected with a high level of sensitivity and specificity using liquid chromatography coupled to mass spectrometry.</p>