Production of a Monoclonal Antibody to the Nucleocapsid Protein of SARS-CoV-2 and Its Application to ELISA-Based Detection Methods with Broad Specificity by Combined Use of Detector Antibodies

Abstract

The coronavirus disease 2019 pandemic, elicited by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is ongoing. Currently accessible antigen-detecting rapid diagnostic tests are limited by their low sensitivity and detection efficacy due to evolution of SARS-CoV-2 variants. Here, we produced and characterized an anti-SARS-CoV-2 nucleocapsid (N) protein-specific monoclonal antibody (mAb), 2A7H9. Monoclonal antibody 2A7H9 and a previously developed mAb, 1G10C4, have different specificities. The 2A7H9 mAb detected the N protein of S clade, delta, iota, and mu but not omicron, whereas the 1G10C4 antibody recognized the N protein of all variants under study. In a sandwich enzyme-linked immunosorbent assay, recombinant N protein bound to the 1G10C4 mAb could be detected by both 1G10C4 and 2A7H9 mAbs. Similarly, N protein bound to the 2A7H9 mAb was detected by both mAbs, confirming the existence of dimeric N protein. While the 1G10C4 mAb detected omicron and mu with higher efficiency than S clade, delta, and iota, the 2A7H9 mAb efficiently detected all the strains except omicron, with higher affinity to S clade and mu than others. Combined use of 1G10C4 and 2A7H9 mAb resulted in the detection of all the strains with considerable sensitivity, suggesting that antibody combinations can improve the simultaneous detection of virus variants. Therefore, our findings provide insights into the development and improvement of diagnostic tools with broader specificity and higher sensitivity to detect rapidly evolving SARS-CoV-2 variants.

A mouse xenograft long‐term replication yields a SARS‐CoV‐2 Delta mutant with increased lethality

“…S‐2000), each for 15 min at room temperature. The samples were then incubated for 2 h at room temperature with biotinylated anti‐SARS‐CoV‐2 nucleocapsid (N) antibody (1G10C4 mAb, 1 μg/slide), 25 biotinylated anti‐SARS‐CoV‐2 S antibody (Sino Biological, Catalog no. 40591‐T62, 1 μg/slide), rabbit anti‐ACE2 polyclonal antibody (MyBioSource, Catalog no.…”

In vitro and in vivo suppression of SARS‐CoV‐2 replication by a modified, short, cell‐penetrating peptide targeting the C‐terminal domain of the viral spike protein

“…SARS‐CoV‐2 N protein‐specific mAbs (1G10C4) were biotinylated using an EZ‐Link TM Sulfo‐NHS‐Biotinylation Kit (Catalog No. 21425, Thermo Fisher Scientific) according to the manufacturer's instructions 31 . Each tissue section slide was incubated with biotinylated mAbs at room temperature for 2 h and then with HRP‐streptavidin (Vector Laboratories, Catalog No: PK‐6100) for 30 min.…”

“…30 The slides were blocked with avidin blocker, biotin blocker (Vector Laboratories, to the manufacturer's instructions. 31 Each tissue section slide was incubated with biotinylated mAbs at room temperature for 2 h and then with HRP-streptavidin (Vector Laboratories, Catalog No: PK-6100) for 30 min. The enzyme-substrate reaction was then developed using 3, 3ʹ-diaminobenzidine (DAB; Thermo Fisher Scientific).…”

Analysis of spike protein variants evolved in a novel mouse model of persistent SARS-CoV-2 infection

“…Each slide was then blocked with avidin blocker, biotin blocker (Vector Laboratories), and normal horse serum (Vector Laboratories). Then, each slide was incubated with biotinylated anti-SARS-CoV-2 N monoclonal antibody (1G10C4 mAb, 1 μg/slide) 44 , rabbit anti-SARS-CoV-2 S polyclonal antibody (Sino Biological, cat. no.…”