Background and aims SARS-CoV-2 antibody assays are relevant in managing the COVID-19 pandemic, providing valuable data on the immunization status of the population. However, current serology tests are highly variable, due to their different characteristics and to the lack of reference materials. The aim of the World Health Organization (WHO) first International Standard (IS) for anti-SARS-CoV-2 immunoglobulin is to harmonize humoral immune response assessment after natural infection or vaccination, and recommend reporting the results for binding activity in Binding Antibody Units (BAU). Materials and methods This study analyzed six commercial quantitative anti-SARS-CoV-2 S-protein assays in a head-to-head comparison, using the manufacturers' conversion factors for the WHO IS to obtain BAU/mL values. Results Our data showed good alignment up to 1000 BAU/mL, then began to disperse, exhibiting some discrepancies. Moreover, correlations among methods varied with Cohen’s Kappa ranging from 0.580 to 1.00, with the lowest agreement values for kits using different target antigens or different antibody isotypes, making it clear that the laboratory report should include this information. Values expressed as BAU/ml showed a reduced between-assays variability compared to AU/ml (median coefficients of variation 0.38 and 0.68, respectively; p<0.001). Conclusion On the basis of these data at present anti-SARS CoV-2 serological assays’ results are not interchangeable, and, more importantly, individual immune monitoring should be performed with the same method.

Background: Several commercial surrogate Virus Neutralization Tests (sVNTs) have been developed in the last year. Neutralizing anti-SARS-CoV-2 antibodies through interaction with Spike protein Receptor Binding Domain (S-RBD) can block the virus from entering and infecting host cells. However, there is a lack of information about the functional activity of SARS-CoV-2 antibodies that may be associated with protective responses. For these reasons, to counteract viral infection, the conventional virus neutralization test (VNT) is still considered the gold standard. The aim of this study was to contribute more and detailed information about sVNTs' performance, by determining in vitro the anti-SARS-CoV-2 neutralizing antibody concentration using four different commercial assays and then comparing the obtained data to VNT. Methods: Eighty-eight samples were tested using two chemiluminescence assays (Snibe and Mindray) and two ELISA assays (Euroimmun and Diesse). The antibody titers were subsequently detected and quantified by VNT. Results: The overall agreement between each sVNT and VNT was 95.45% for Euroimmun and 98.86% for Diesse, Mindray and Snibe. Additionally, we investigated whether the sVNTs were closer to the gold standard than traditional anti-SARS-CoV-2 antibody assays S-RBD or S1 based, finding a higher agreement mean value for sVNTs (98.01 ± 1.705% vs 95.45 ± 1.921%; p < 0.05). Furthermore, Spearman's statistical analysis for the correlation of sVNT versus VNT showed r = 0.666 for Mindray; r = 0.696 for Diesse; r = 0.779 for Mindray and r = 0.810 for Euroimmun. Conclusions: Our data revealed a good agreement between VNT and sVNTs. Despite the VNT still remains the gold standard, the sVNT might be a valuable tool for screening wider populations.

A large number of immunoassays have been developed to detect specific anti-SARS-CoV-2 antibodies; however, not always they are functional to neutralize the virus. The reference test for the anti-spike neutralizing antibodies (nAbs) ability to counteract the viral infection is the virus neutralization test (VNT). Great interest is developing on reliable serological assays allowing antibodies concentration and antibody protective titer correlation. The aim of our study was to detect nAbs serum levels in paucisymptomatic, symptomatic and vaccinated subjects, to find a cut-off value able to protect from virus infection. nAbs serum levels were detected by a competitive automated immunoassay, in association to VNT with the SARS-CoV-2 original and British variant strains. The median nAbs concentrations were: 281,3 BAU/ml for paucisymptomatics; 769,4 BAU/ml for symptomatics; 351,65 BAU/ml for the vaccinated cohort; 983 BAU/ml considering only the second dose vaccinated individuals. The original strain VNT analysis showed 1:80 median neutralization titers in paucisymptomatic and vaccinated subjects; 1:160 in symptomatic patients; 1:160 in the second dose groups. The British variant VNT analysis showed lower neutralization titers in paucisymptomatic and vaccinated groups (1:40); the same titer in symptomatic patients (1:160); the second dose group confirmed the original strain titer (1:160). In conclusion, our data showed optimal correlations with a proportional increase between neutralizing activity and antibody concentration, making nAbs detection a good alternative to virus neutralization assays, difficult to carry out in routine laboratories. Finally, ROC curve analysis established a cut-off of 408,6 BAU/ml to identify subjects with a low risk of infection.

Background: SARS-CoV-2 T-cells are crucial for long-term protection against reinfection. The aim was to demonstrate the Interferon-gamma Release Assay (IGRA) test could be useful for vaccination monitoring. Methods: In a prospective cohort of 98 vaccinated healthcare workers for SARS-CoV-2, we selected 23 people in low-antibodies (Group 1, N = 8), high-antibodies (Group 2, N = 9), and negative control groups (Group 3, N = 6). SARS-CoV-2-specific humoral and cellular responses were analyzed at 8 months after two doses of Pfizer BioNTech, evaluating anti-RBD (Receptor Binding Domain) and RBD-ACE2 (Angiotensin Converting Enzyme-2) blocking antibodies in sera through a Chemiluminescence Immunoassay (CLIA) and T-cells through the IGRA test in heparinized plasma. Moreover, lymphocyte subtyping was executed by a flow cytometer. Statistical analysis was performed. Results: The data confirmed that RBD and RBD-ACE2 blocking ACE2 antibody levels of Group 1 were significantly lower than Group 2; p < 0.001. However, T-cells showed no significant difference between Group 1 and Group 2. Conclusions: This work suggests the need for new strategies for booster doses administration.

No abstract