Multiplexed measurement of binding- and neutralizing antibodies to SARS-CoV-2 variants in 12.000 post-vaccine sera

Abstract

Diagnostic assays currently used to monitor the efficacy of COVID-19 vaccines measure levels of antibodies to the receptor-binding domain of ancestral SARS-CoV-2 (RBDwt). However, the predictive value for protection against new variants of concern (VOCs) has not been firmly established. Here, we used bead-based arrays and flow cytometry to measure binding of antibodies to spike proteins and receptor-binding domains (RBDs) from VOCs in 12,000 sera. Effects of sera on RBD-ACE2 interactions were measured as a proxy for neutralizing antibodies. The samples were obtained from healthy individuals or patients on immunosuppressive therapy who had received two to four doses of COVID-19 vaccines and from COVID-19 convalescents. The results show that anti-RBDwt titers correlate with the levels of binding- and neutralizing antibodies against the Alpha, Beta, Gamma, Delta, Epsilon and Omicron variants. The benefit of multiplexed analysis lies in the ability to measure a wide range of anti-RBD titers using a single dilution of serum for each assay. The reactivity patterns also yield an internal reference for neutralizing activity and binding antibody units per milliliter (BAU/ml). Results obtained with sera from vaccinated healthy individuals and patients confirmed and extended results from previous studies on time-dependent waning of antibody levels and effects of immunosuppressive agents. We conclude that anti-RBDwt titers correlate with levels of neutralizing antibodies against VOCs and propose that our method may be implemented to enhance the precision and throughput of immunomonitoring.

Immunogenicity and Safety of Standard and Third‐Dose SARS–CoV‐2 Vaccination in Patients Receiving Immunosuppressive Therapy

“…Assessments of immunogenicity were performed at the Department of Immunology at Oslo University Hospital. The samples were first screened for antibodies to RBD at the full‐length spike protein by using an in‐house bead‐based method, with seroconversion defined as an anti‐RBD antibody level ≥5 AU ( 25 , 26 ). Measurement of the World Health Organization international standard for anti‐RBD antibody showed that the screening assay has a lower detection limit of 1 binding antibody unit per milliliter (BAU/ml) and an upper dynamic range of ~100 BAU/ml.…”

“…Measurement of the World Health Organization international standard for anti‐RBD antibody showed that the screening assay has a lower detection limit of 1 binding antibody unit per milliliter (BAU/ml) and an upper dynamic range of ~100 BAU/ml. For quantification of antibody levels, most patient samples and a representative selection of control samples (Supplementary Table 1 ) were thereafter analyzed using a second assay, with a dynamic range of 300–10,000 BAU ( 25 ). In this assay, effects of sera on binding of angiotensin‐converting enzyme 2 to RBDs from SARS–CoV‐2 variants were measured as a proxy for neutralizing antibody activity ( 25 ).…”

“…For quantification of antibody levels, most patient samples and a representative selection of control samples (Supplementary Table 1 ) were thereafter analyzed using a second assay, with a dynamic range of 300–10,000 BAU ( 25 ). In this assay, effects of sera on binding of angiotensin‐converting enzyme 2 to RBDs from SARS–CoV‐2 variants were measured as a proxy for neutralizing antibody activity ( 25 ).…”

Fourth dose of the SARS-CoV-2 vaccine in kidney transplant recipients with previously impaired humoral antibody response

“…Based on in vitro neutralizing assays in our lab, and in line with Dimeglio et al, we have defined 200 BAU/ml as a cut‐off for neutralizing activity against ancestral SARS‐CoV‐2 and 11 000 BAU/ml as a cut‐off for neutralization against the Omicron‐variant. 2 , 3 Observational data after dose 3 registered in the Norwegian Renal Registry indicate >200 BAU/ml as clinically “protective” against the delta variant of concern. Neutralizing effect following dose 4 is presented in Figure 1 .…”

“…The dot plots show accumulated data for 188 patients, and each dot corresponds to a different serum. Bead‐based arrays were incubated with sera diluted at 1:100 and then labeled with fluorochrome‐conjugated anti‐human IgG or recombinant ACE2 (Tran et al 2 ).…”