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Although recombinant expression of RBD has been achieved in several eukaryotic systems, mammalian cells have proven particularly useful. The authors aimed to optimize RBD produced in HEK293\u20106E cells towards a stable homogeneous preparation and addressed its O\u2010glycosylation as well as the unpaired cysteine residue 538 in the widely used RBD (319\u2010541) sequence. The authors found that an intact O\u2010glycosylation site at T323 is highly relevant for the expression and maintenance of RBD as a monomer. Furthermore, it was shown that deletion or substitution of the unpaired cysteine residue C538 reduces the intrinsic propensity of RBD to form oligomeric aggregates, concomitant with an increased yield of the monomeric form of the protein. Bead\u2010based and enzyme\u2010linked immunosorbent assays utilizing these optimized RBD variants displayed excellent performance with respect to the specific detection of even low levels of SARS\u2010CoV\u20102 antibodies in convalescent sera. 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