This is a summary, written by members of the CITF Secretariat, of:

Fu Z, Rais, Y, Drabovich AP. Immunoprecipitation-targeted proteomics assays facilitate rational development of SARS-CoV-2 serological diagnostics. medRxiv. 25 Oct 2021. doi: 10.1101/2021.10.25.21265408v1

The results and/or conclusions contained in the research do not necessarily reflect the views of all CITF members.

In a not-yet peer-reviewed preprint, CITF-funded investigator Dr. Andrei Drabovich and colleagues from the University of Alberta, designed and evaluated the performance of serological diagnostics, used to measure the presence of SARS-CoV-2 antibodies. The particular assays studied, known as immunoprecipitation – parallel reaction monitoring (IP-PRM) and immunoprecipitation – selected reaction monitoring (IP-SRM), use a combination of two common laboratory techniques, ELISA and mass spectrometry and proved to be highly specific and sensitive in their detection of antigen-antibody combinations.  

Limitations of current serological diagnostics

While serological immunoassays that exploit antigen-antibody binding (e.g., ELISAsAn enzyme-linked immunosorbent assay (ELISA) is a laboratory technique used to detect the presence of antigens in a biological sample. An ELISA, like other types of immunoassays, relies on antibodies to detect a target antigen (e.g., viral proteins) using highly specific antibody-antigen interactions.) are well suited to identify SARS-CoV-2 proteins and anti-SARS-CoV-2 antibodies, they are unable to provide a comprehensive overview of the antibody response. Limitations of such tests, which can affect their accuracy, include, among other things, semi-quantitative as opposed to precise quantitative measurements, lack of standardization to enable comparison across assay platforms, potential cross-reactivity with irrelevant targets, and it could be laborious to differentiate between antibody subclasses (i.e., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

On the other hand, an analytical technique called mass spectrometry, by which proteins are identified by analysis of their fragments following processing, is an alternative approach to the identification and quantification of SARS-CoV-2 proteins and anti-SARS-CoV-2 antibodies. It too, however, comes with a set of limitations, including relatively poor test sensitivity Diagnostic sensitivity is a measure of a test’s capacity to correctly diagnose a positive result. and false negatives.

Designing a new, combined, serological testing approach

In the hopes of creating a more accurate and highly informative diagnostic test, Dr. Andrei Drabovich and colleagues from the University of Alberta have combined the two techniques: a serological immmunoassay with high analytical sensitivity and a mass spectrometry-based assay with near-absolute selectivity, or ability to distinguish between very similar protein molecules. These combinatory assays are called immunoprecipitation parallel reaction monitoring (IP-PRM) and immunoprecipitation selected reaction monitoring (IP-SRM).

In their preprint, Dr. Drabovich and colleagues describe how the IP-PRM assay measured a synthetic version of the nucleocapsid protein of SARS-CoV-2 (the RNA-binding protein that is essential for viral replication) in human blood with a limit of detectionA limit of detection is defined as the lowest quantity or concentration of a molecule that can be reliably detected. of 313 pg/mL. Likewise, their IP-SRM assay facilitated the simultaneous quantification of numerous antigen-antibody subclass combinations. Indeed, this assay revealed that the SARS-CoV-2 receptor-binding domain (RBD) antigen linked to the IgG1 antibody subclass had the highest diagnostic specificity and sensitivity.

The authors propose that the combination of immunoassays with mass spectrometry provides many advantages over the use of either method alone. Further development of these techniques may enable improvement of existing serological diagnostic tests for SARS-CoV-2, novel mutant strains, and other viruses and bacteria, as well as providing a means for comprehensive investigation of various antigen-antibody subclass combinations, which assist in understanding the breadth of an individual’s immune response.