Development of a SARS-CoV-2 nucleocapsid specific monoclonal antibody

Abstract

To help fight COVID-19, new molecular tools specifically targeting critical components of the causative agent of COVID-19, SARS-Coronavirus-2 (SARS-CoV-2), are desperately needed. The SARS-CoV-2 nucleocapsid protein is critical for viral replication, integral to viral particle assembly, and a major diagnostic marker for infection and immune protection. Currently the limited available antibody reagents targeting the nucleocapsid protein are not specific to SARS-CoV-2 nucleocapsid protein, and sequences for these antibodies are not publicly available. In this work we developed and characterized a series of new mouse monoclonal antibodies against the SARS-CoV-2 nucleocapsid protein, with a specific clone, mBG86, targeting only SARS-CoV-2 nucleocapsid protein. The monoclonal antibodies were validated in ELISA, Western blot, and immunofluorescence analyses. The variable regions from six select clones were cloned and sequenced, and preliminary epitope mapping of the sequenced clones was performed. Overall, these new antibody reagents will be of significant value in the fight against COVID-19.

Fig. 1

Production and characterization of truncated SARS-CoV-2 nucleocapsid protein. A) Sequence alignment of human coronavirus N proteins. The arrow denotes the start of the expressed proteins. B) Size exclusion chromatograph of nickel-column purified SARS-CoV-2 NP (133–419). C) SDS-PAGE gel of purified recombinant Coronavirus nucleocapsid proteins. Each recombinant nucleocapsid protein starts at the same tryptophan residue as SARS-CoV-2 AA-133.

Figure 2

Screening of Anti-Nucleocapsid Clones. A) Direct ELISA analysis of 920 clones picked from hybridoma fusion. B) Verification ELISA of top 18 anti-nucleocapsid monoclonal antibody clones and counter screening against bacterial lysate (BLP) and 6-His-tagged SARS-CoV-2 Spike RBD domain. Averages are presented following background subtraction.

Fig. 3

Western Blot Analysis of Anti-Nucleocapsid Monoclonal Antibody Clones Against Uninfected (U) or SARS-CoV-2 infected (I) Vero cells.

Fig. 4

Immunofluorescence Analysis of Anti-Nucleocapsid Monoclonal Antibody Clones. A) Relative reactivities of clones in SARS-CoV-2 infected Vero cells fixed with methanol or paraformaldehyde. B) representative images of uninfected and SARS-CoV-2 infected Vero cells (paraformaldehyde fixed) processed for immunofluorescence analysis with mBG17.

Fig. 5

Epitope Mapping Using N Protein Deletions. A) Western blot analysis of selected antibody reactivity against SARS-CoV-2 nucleocapsid protein deletions. Bacterial lysates of non-induced protein expression cultures (U) and induced protein expression cultures (I) were run side by side. B) Sequence alignments of NP AA133-179 and AA381-419 regions with heterologous human coronavirus nucleocapsid proteins.

Fig. 6

mBG Western Blot Reactivity to Linearized Recombinant Human Coronavirus N Proteins. Recombinant human coronavirus western blots show the reactivity of the selected top mBG antibodies. SDS-PAGE gels for mBG67 and mBG86 were loaded with 10 μg/well recombinant N protein while the other gels were loaded with 1 μg/well as mBG67 and mBG86 appear to bind weakly to linearized epitopes compared to mBG17, mBG21, and mBG22.

Fig. 7

Reactivity of mBG17 and mBG86 Against Captured Virion-Derived N Proteins. Virion-derived N protein serially diluted from 5.5 × 10⁵ PFU/ml stock and captured by anti-NP rabbit polyclonal antibody. mBG17 and mBG86 used in tandem with goat anti-mouse HRP-conjugated antibody to report on mBG effectiveness in capture ELISA.