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

Abstract

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.

Production of SARS-CoV-2 N Protein-Specific Monoclonal Antibody and Its Application in an ELISA-Based Detection System and Targeting the Interaction Between the Spike C-Terminal Domain and N Protein

“…Several reports have described assays to detect the SARS-CoV-2 N protein using a monoclonal antibody ( Mertens et al, 2020 ; Ciotti et al, 2021 ; Hodge et al, 2021 ). Hodge et al (2021) induced polymerization of two monoclonal antibodies that recognize different epitopes on the N protein to improve its detection.…”

“…Several reports have described assays to detect the SARS-CoV-2 N protein using a monoclonal antibody ( Mertens et al, 2020 ; Ciotti et al, 2021 ; Hodge et al, 2021 ). Hodge et al (2021) induced polymerization of two monoclonal antibodies that recognize different epitopes on the N protein to improve its detection. Li et al developed magnetic nanobead-labeled anti-SARS-CoV-2 N protein monoclonal antibody to induce immunomagnetic enrichment and signal amplification.…”

“…Currently, most studies are being performed to detect SARS-CoV-2 using antibodies against SARS-CoV-2 N protein. For example, the COVID-19 Ag Respi-Strip assay (Coris BioConcept, Gembloux, Belgium) detects SARS-CoV-2 using antibodies against SARS-CoV-2 N protein ( Mertens et al, 2020 ; Hodge et al, 2021 ).…”

Neutralizing antibodies for the prevention and treatment of COVID-19

“…However, they can be used for other applications, such as diagnosis and epidemiology. For example, monoclonal antibodies specifically targeting the SARS-CoV-2 N protein increase the detection sensitivity of ELISAs, providing a tool for the early and accurate diagnosis based on clinical samples and for epidemiological studies of SARS-CoV-2 [ 79 , 80 ]. In addition, the NP-monoclonal antibody system and related test strips can be utilized for large-scale screening of COVID-19 samples [ 81 ].…”

Ultrasensitive Detection of SARS-CoV-2 Spike Proteins Using the Thio-NAD Cycling Reaction: A Preliminary Study before Clinical Trials

“…In many antigen tests for COVID-19, the nucleocapsid proteins are utilized as the target antigen protein of SARS-CoV-2 [14,15]. The nucleocapsid proteins of SARS-CoV-2, however, are hardly distinguishable from those of conventional coronaviruses and SARS-CoV [16][17][18]. Thus, to avoid false-positive test results, a combination of tests against both the nucleocapsid and spike antigen proteins is strongly recommended [19], because the spike protein, which is the reason for the name coronavirus, is coronavirus-specific [20,21].…”