Redefining serological diagnostics with immunoaffinity proteomics

Abstract

Serological diagnostics is generally defined as the detection of specific human immunoglobulins developed against viral, bacterial, or parasitic diseases. Serological tests facilitate the detection of past infections, evaluate immune status, and provide prognostic information. Serological assays were traditionally implemented as indirect immunoassays, and their design has not changed for decades. The advantages of straightforward setup and manufacturing, analytical sensitivity and specificity, affordability, and high-throughput measurements were accompanied by limitations such as semi-quantitative measurements, lack of universal reference standards, potential cross-reactivity, and challenges with multiplexing the complete panel of human immunoglobulin isotypes and subclasses. Redesign of conventional serological tests to include multiplex quantification of immunoglobulin isotypes and subclasses, utilize universal reference standards, and minimize cross-reactivity and non-specific binding will facilitate the development of assays with higher diagnostic specificity. Improved serological assays with higher diagnostic specificity will enable screenings of asymptomatic populations and may provide earlier detection of infectious diseases, autoimmune disorders, and cancer. In this review, we present the major clinical needs for serological diagnostics, overview conventional immunoassay detection techniques, present the emerging immunoassay detection technologies, and discuss in detail the advantages and limitations of mass spectrometry and immunoaffinity proteomics for serological diagnostics. Finally, we explore the design of novel immunoaffinity-proteomic assays to evaluate cell-mediated immunity and advance the sequencing of clinically relevant immunoglobulins.

The proteomic toolbox for identification, quantification, and characterization of polyclonal antibodies

“…Our approach to identification and quantification of polyclonal antibodies is presented in Fig. 4 and was discussed in detail in our previous studies 14,18,41 . We previously demonstrated IA-MS as an experimental approach for rational design and development of SARS-CoV-2 serological diagnostics 14 .…”

“…We presented a proteomic toolbox for the identification, quantification, and characterization of the endogenous polyclonal antibodies. We demonstrated our approaches with the human antibodies generated against RSV, but the proposed methods could be extended to the investigation of animal antibodies and the numerous antigens of infectious diseases and non-infectious disorders 41 . The presented toolbox will facilitate the in-depth characterization of polyclonal antibodies and pave the way to quantitative approaches in serological testing and precision immunology.…”

“…Determination of the accurate masses may enable straightforward VH-VL chain matching for the mixtures of monoclonal antibodies, and potentially simple mixtures of polyclonal antibodies. We previously demonstrated the numerous applications of shotgun and targeted mass spectrometry to identify and quantify human proteins [16][17][18][19][20][21][22][23][24][25][26][27][28][29][30] , discover protein biomarkers [31][32][33][34][35][36][37][38][39][40][41][42][43][44] , and investigate the functional roles of human proteins [45][46][47][48][49][50][51][52][53][54] . While our previous studies nearly exclusively utilized trypsinbased proteomic approaches, the availability of novel high-quality proteases extended the variety of experimental tools available for proteomic studies.…”

Identification and Quantification of Human Relaxin Proteins by Immunoaffinity-Mass Spectrometry

“…Proteomics by mass spectrometry is a promising tool to identify, quantify, and characterize human proteins. − We previously demonstrated numerous applications of shotgun and targeted mass spectrometry to identify and quantify human proteins, − discover protein biomarkers, − and investigate the functional roles of human proteins. − The emerging proteomic assays combining the high analytical sensitivity of immunoassays with the nearly absolute analytical selectivity of mass spectrometry − could resolve issues related to the identification and quantification of structurally similar protein isoforms and proteins, including human relaxins. As independent assays, IA-SRM could detect and resolve the long-standing issues and cross-reactivities of ELISA, provide reference values and ranges of proteins in healthy populations, and independently verify and validate novel disease biomarkers. − …”

Identification and quantification of human relaxin proteins by immunoaffinity-mass spectrometry

“…We previously demonstrated numerous applications of shotgun and targeted mass spectrometry to identify and quantify human proteins [53][54][55][56][57][58][59][60][61][62][63] , discover protein biomarkers [64][65][66][67][68][69][70][71][72] , and investigate the functional roles of human proteins [73][74][75][76][77][78][79][80] . The emerging proteomic assays combining the high analytical sensitivity of immunoassays with the nearly absolute analytical selectivity of mass spectrometry [81][82][83] could resolve the issues related to the identification and quantification of structurally similar protein isoforms and proteins, including human relaxins. As independent assays, IA-SRM could detect and resolve the long-standing issues and crossreactivities of ELISA.…”