A High Through-put Assay for Circulating Antibodies Directed against the S Protein of Severe Acute Respiratory Syndrome Corona virus 2

Svenja Weiss; Jeromine Klingler; Catarina Hioe; Fatima Amanat; Ian Baine; Erna Milunka Kojic; Jonathan Stoever; Sean Liu; Denise Jurczyszak; Maria Bermudez-Gonzalez; Viviana Simon; Florian Krammer; Susan Zolla-Pazner

The recombinant S and RBD proteins were produced as previously described 7 in Expi293F cells (ThermoFisher) by transfections of purified DNA using an ExpiFectamine Transfection Kit (ThermoFisher). The soluble version of the spike protein included the S protein ectodomain (amino acids 1-1213), a C-terminal thrombin cleavage site, a T4 foldon trimerization domain and a hexahistidine tag. The protein sequence was modified to remove the polybasic cleavage site (RRAR to A) and two stabilizing mutations (K986P and V987P, wild type numbering). The RBD (amino acids 319-541) also contained a hexahistidine tag. Supernatants from transfected cells were harvested on day three post-transfection by centrifugation of the culture at 4000 g for 20 minutes. Supernatant was then incubated with 6 mL Ni-NTA agarose (Qiagen) for one to two hours at room temperature. Next, gravity flow columns were used to collect the Ni-NTA agarose and the protein was eluted. Each protein was concentrated in Amicon centrifugal units (EMD Millipore) and re-suspended in phosphate buffered saline (PBS).