Analytical characterization of the SARS-CoV-2 EURM-017 reference material.

Background: Current serological methods for SARS-CoV-2 lack adequate standardization to a universal standard reference material. Standardization will allow comparison of results across various lab-developed and commercial assays and publications. SARS-CoV-2 EURM-017 is human sera reference material containing antibodies directed against SARS-CoV-2 proteins, S1/S2 (full-length spike [S]), S1 receptor-binding domain (S1 RBD), S1, S2, and nucleocapsid (N) protein. The goal of this study was to characterize five antigen-specific serum fractions in EURM-017 for standardization of serology assays.

Methods: Five antigen-specific serum fractions were affinity purified, quantified, and PRNT titers compared. Standardization methods were established for two anti-S1 RBD (IgG and Total Ig) and one N protein assay. For the anti-S1 RBD assays, standardization involved determining assay index values for serial dilutions of S1-RBD anti-sera. Index values for the anti-S1 RBD IgG assay and PRNT titers were determined for 44 symptomatic COVID-19 patient sera. The index values were converted to EURM-017 ug/mL.

Results: Anti-sera protein content was as follows: S1 (17.7 µg/mL), S1 RBD (17.4 µg/mL), S1/S2 (full-length S) (34.1 µg/mL), S2 (29.7 µg/mL), and N protein (72.5 µg/mL). S1 anti-serum had the highest neutralization activity. A standardization method for S1 RBD anti-serum and an anti-S1 RBD IgG assay yielded the linear equation (y = 0.75x-0.10; y = index, x=µg/mL anti-serum). Patient sample index values for the S1-RBD IgG assay correlated well with PRNT titers (Pearson r = 0.84). Using the equation above, patient index values were converted to standardized µg/mL.

Conclusions: Standardization of different lab-developed and commercial assays to EURM-017 antigen-specific anti-sera will allow comparison of results across studies globally due to traceability to a single standard reference material.