Development and certification of SARS-CoV-2 S-RBD protein reference material via dual isotope dilution mass spectrometry.

The persistent evolution of SARS-CoV-2 and its sustained global public health impacts necessitate enhanced diagnostic precision and efficiency. Although certified reference materials (CRMs) are fundamental for ensuring methodological reliability and cross-platform consistency, CRMs with metrological traceability specifically targeting the SARS-CoV-2 spike protein receptor-binding domain (S-RBD) remain unavailable. To bridge this gap, two isotope dilution mass spectrometry (IDMS)-based quantification methods were developed for S-RBD protein analysis at the amino acid and peptide levels. These methods enabled systematic characterization of the reference material's homogeneity, stability, and measurement uncertainty. The final CRM demonstrates a certified S-RBD protein concentration of 90.4 ± 8.5 µg/g (k = 2), with metrological traceability to the International System of Units (SI), thereby establishing a technical framework for standardizing S-RBD detection assays and quality control in biomedical product development.