Quantitative measurement of IgG to SARS-CoV-2

Standardized quantitative analysis of the humoral immune response to SARS-CoV-2 antigens may be useful in estimating the extent and duration of immunity. The objective was to develop enzyme immunoassays (ELISA) for the quantification of human IgG antibodies against SARS-CoV-2 antigens.

Enzyme immunoassays have been developed based on monoclonal antibodies directed against human IgG and recombinant SARS-CoV-2 antigens (Spike-S1 and Nucleocapsid). Immunoglobulin references WHO 67/086 and WHO 20/136 SARS-CoV-2 were used for standardization. Sera from a study group of COVID-19 positive subjects (n=144), pre-pandemic controls (n=135) and individuals vaccinated with the BioNTech–Pfizer BNT162b2 vaccine (n=48) were analyzes. Sera from the study group were also tested using EuroImmun SARS-CoV-2-ELISA and S1-specific quantitative fluorescence enzyme immunoassay (FEIA) from Thermo Fisher.

The ELISA results were reproducible and traceable to international units due to their parallelism with the two WHO references. In the study group, the median anti-S1-IgG concentrations were 102 BAU mL−1, compared to 100 and 1457 BAU mL−1 in the vaccination group after the first and second vaccination, respectively. The ELISAs achieved an area under the curve (AUC) of 0.965 (S1) and 0.955 (Nucleocapsid) in the receptor operating characteristics (ROC) analysis, and a specificity of 1 (S1) and 0.963 (Nucleocapsid) and a sensitivity of 0.903 (S1) and 0.833 (nucleocapsid) at the maximum Youden index. In comparison, commercial assays (S1-FEIA, S1, and Nucleocapsid ELISA EuroImmun) achieved sensitivities of 0.764, 0.875, and 0.882 in the study group, respectively.

Quantitative ELISAs for measuring IgG binding to SARS-CoV-2 antigens have good analytical and clinical performance characteristics and units traceable to international standards.

Specific detection of the immune response to SARS-CoV-2 infection plays an important role in monitoring the spread of COVID-19 in the population.1-7 IgG antibodies in particular remain detectable after the end of disease or even after symptomless infections.8,9 Many immunoassays have been developed to detect IgG against SARS-CoV-2 antigens,10,11 some of which are characterized by high specificity and sensitivity.12- 16 The spike protein that binds to receptors on the host cell for virus entry and the nucleocapsid protein necessary for virus replication are recognized by human antibodies and serve as antigens for the serological detection of infection.

17,18 Normally, the nucleocapsid protein is present in cells at a higher copy number than that responsible for the high sensitivity of immunoassays based on this antigen due to its strong immunogenicity. The spike protein is located on the surface of the enveloped RNA virus and consists of trimers of two glycosylated subunits (S1 and S2). The S1 subunit contains the receptor binding region (RBD) and shows less homology to other coronaviruses than the S2 protein.20 Because antibodies with proven neutralizing activity bind to the spike protein and the RBD in particular,21-23 the spike protein is also the main antigenic target of vaccines.

After the first vaccination, antibody responses are elicited and then boosted with the second dose of vaccine to protein-binding IgG levels higher than in convalescent human sera.24,25 Specific IgG antibody titers decline over time but remain elevated for up to 6 months after full vaccination. vaccination.26, 27 The long-term persistence of SARS-CoV-2-specific antibodies is unclear, as is whether the presence of antibodies confers protective immunity against SARS-CoV-2