Rigid monoclonal antibodies improve detection of SARS-CoV-2 nucleocapsid protein

Monoclonal antibodies (mAbs) are the basis of treatments and diagnostics for pathogens and other biological phenomena. We conducted a structural characterization of mAbs against the N-terminal domain of nucleocapsid protein (NP^NTD) from SARS-CoV-2 using small-angle X-ray scattering and transmission electron microscopy. Our solution-based results distinguished the mAbs' flexibility and how this flexibility affects the assembly of multiple mAbs on an antigen. By pairing two mAbs that bind different epitopes on the NP^NTD, we show that flexible mAbs form a closed sandwich-like complex. With rigid mAbs, a juxtaposition of the antigen-binding fragments is prevented, enforcing a linear arrangement of the mAb pair, which facilitates further mAb polymerization. In a modified sandwich enzyme-linked immunosorbent assay, we show that rigid mAb-pairings with linear polymerization led to increased NP^NTD detection sensitivity. These enhancements can expedite the development of more sensitive and selective antigen-detecting point-of-care lateral flow devices, which are critical for early diagnosis and epidemiological studies of SARS-CoV-2 and other pathogens.