Development and characterization of a new monoclonal antibody against SARS‐CoV‐2 NSP12 (RdRp)

Abstract

SARS‐CoV‐2 NSP12, the viral RNA‐dependent RNA polymerase (RdRp), is required for viral replication and is a therapeutic target to treat COVID‐19. To facilitate research on SARS‐CoV‐2 NSP12 protein, we developed a rat monoclonal antibody (CM12.1) against the NSP12 N‐terminus that can facilitate functional studies. Immunoblotting and immunofluorescence assay (IFA) confirmed the specific detection of NSP12 protein by this antibody for cells overexpressing the protein. Although NSP12 is generated from the ORF1ab polyprotein, IFA of human autopsy COVID‐19 lung samples revealed NSP12 expression in only a small fraction of lung cells including goblet, club‐like, vascular endothelial cells, and a range of immune cells, despite wide‐spread tissue expression of spike protein antigen. Similar studies using in vitro infection also generated scant protein detection in cells with established virus replication. These results suggest that NSP12 may have diminished steady‐state expression or extensive posttranslation modifications that limit antibody reactivity during SARS‐CoV‐2 replication.

1. INTRODUCTION

Since the initial outbreak in 2019, severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) causing coronavirus disease 2019 (COVID‐19) has been widely transmitted in a worldwide pandemic. ¹ , ² , ³ While vaccines against spike (S) protein effectively prevent or reduce disease severity, the virus continues to evolve immune escape variants. ⁴ , ⁵ , ⁶ , ⁷ , ⁸ Targeted small molecule therapies that specifically inhibit highly conserved and essential viral replication enzymes thus remain critical in disease control during the pandemic.

SARS‐CoV‐2 belongs to the family of enveloped, positive‐sense, single‐stranded RNA coronaviruses. ¹ , ⁹ , ¹⁰ On entering host cells, the virus will release and uncoat the large (>30 kb) genomic RNA that generates two large open reading frames (ORFs), ORF1a and ORF1b through frame‐shift recoding ¹¹ in addition to many individual ORFs encoding structural and accessory genes. First‐round translation of these two ORFs produces pp1a and pp1ab polyproteins, which are subsequently processed by proteolytic cleavage into individual nonstructural proteins (NSPs) required for viral replication and transcription. ¹² , ¹³ , ¹⁴

NSP12 is the catalytic subunit with RNA‐dependent RNA polymerase (RdRp) activity required for active sarbecovirus replication. ¹³ , ¹⁵ , ¹⁶ , ¹⁷ NSP12 itself possesses low RNA polymerase activity and requires interaction with cofactors NSP7 and NSP8 to promote polymerase activity. ¹⁸ As the central component of the replication‐transcription complex (RTC), NSP12 is an attractive antiviral target ¹⁹ , ²⁰ , ²¹ , ²² that can be inhibited by nucleoside analogs (e.g., remdesivir, molnupiravir). ²³ Inhibition of NSP12 activity not only blocks viral replication and reduces the systemic viral load but reduces pathogenesis associated with SARS‐CoV‐2 infection. ²⁴ , ²⁵ Pharmacologic studies of NSP12 inhibitors and basic studies of the RTC would be advanced by a specific monoclonal antibody to detect NSP12 protein.

Here we report the characterization of a rat monoclonal antibody, CM12.1, that specifically recognizes a SARS‐CoV‐2 NSP12 N‐terminal linear peptide motif and readily detects overexpressed NSP12 protein by immunoblotting and immunofluorescence assay (IFA). Antibody detection of NSP12 protein in human autopsy lung samples, however, was markedly less sensitive for detecting virus infection than antibodies against S structural protein, with most S‐positive cells staining negative for NSP12 expression. A small subset of SARS‐CoV‐2‐infected cells from COVID‐19 patients expressed detectable NSP12 protein, including goblet, club‐like, vascular endothelial cells, as well as immune cells including macrophages, monocytes, neutrophils, natural killer (NK) cells, B cells, and T cells as determined by cell‐lineage markers.

2. MATERIALS AND METHODS

2.1. Cells and viruses

293 and Vero E6 cells were maintained at 37°C, 5% CO₂ in Dulbecco's modified Eagle's medium (DMEM; Corning) with 10% fetal bovine serum (FBS). SARS‐CoV‐2 used for Vero E6 cell lysate immunoblotting was previously isolated and propagated from a patient sample, under an approved protocol. ²⁶ The NSP12 locus was sequenced and confirmed to be identical to the peptides used to generate the reported antibodies. SARS‐CoV‐2 used for K18‐hACE2 mice (Jackson Laboratory) inoculations was the 2019n‐CoV/USA‐WA1/2019 strain (Accession number: MN985325.1).

2.2. Plasmid construction

Genomic RNA of the USA‐WA1/2020 SARS‐CoV‐2 isolate was obtained from BEI resources (NR‐52285; ATCC), and from which the SARS‐CoV‐2 RNA, cDNA was synthesized using random hexamer primers and the iScript cDNA synthesis kit (BioRad). NSP12 was amplified from the cDNA and cloned into p3xFLAG‐CMV‐14 (Sigma) to express N‐terminally FLAG‐tagged NSP12 protein. However, an immunoblot with FLAG antibody failed to detect NSP12 protein expression in transfected 293 cells when the NSP12 cDNA from the viral RNA sequence was expressed from the nucleus. To achieve NSP12 protein expression, we synthesized codon‐optimized NSP12 cDNA (IDT) which was cloned into pcDNA6.2 (Invitrogen) and p3xFLAG‐CMV‐14 to generate untagged full‐length SARS‐CoV‐2 NSP12 (NSP12 FL) and FLAG‐tagged SARS‐CoV‐2 NSP12 (FLAG‐NSP12) expression vectors, respectively. Amino acid sequence of SARS‐CoV‐2 NSP12 (YP_009725307.1) was used to generate codon‐optimized NSP12 FL and FLAG‐NSP12.

2.3. Antibodies

Primary antibodies included antibodies to FLAG (1:1000, F1804; Sigma), SARS‐CoV‐2 Spike S1 (1:500, 40592‐T62; Sino Biological), NSP13 (1:800, NBP2‐89168; Novus), NSP8 (1:800, NBP2‐89180; Novus), MUC5AC (1:50, MA5‐12178; Invitrogen), MUC5B (1:50, 37‐7400; Invitrogen), CD34 (1:500, MA1‐10202; Invitrogen), CD144 (1:100, 14‐1449‐82; Invitrogen), CD68 (1:1000, 14‐0688‐82; Invitrogen), CD14 (1:250, 14‐0149‐82; Invitrogen), CD56 (1:50, AF2408; Invitrogen), ELA‐2 (1:50, MA1‐40220; Invitrogen), CD8a (1:100, 14‐0008‐82; Invitrogen), SARS‐CoV‐2 Spike S2 (1:1000, NB100‐56578; Novus Biologicals), β‐tubulin rhodamine (1:10 000, 12004166; BioRad).

Secondary antibodies included Goat anti‐Rat IgG (H + L) Highly Cross‐Adsorbed Secondary Antibody, Alexa Fluor Plus 555 (1:400, A48263; Invitrogen), Goat anti‐Rabbit IgG (H + L) Highly Cross‐Adsorbed Secondary Antibody, Alexa Fluor 488 (1:400, A‐11034; Invitrogen), Goat anti‐Mouse IgG (H + L) Highly Cross‐Adsorbed Secondary Antibody, Alexa Fluor 647 (1:400, A‐21236; Invitrogen).

2.4. Monoclonal antibody generation

Antibodies to NSP12 were commercially generated (GenScript) after peptide injection into rats using synthetic peptides AVAKHDFFKFRIDGDMV (aa 95‐111) or HNQDVNLHSSRLSFC (aa 355‐368). After preliminary hybridoma screening by indirect enzyme‐linked immunosorbent assay (ELISA), supernatants collected from positive clones were subjected to immunoblotting using 293 cells transiently transfected with codon‐optimized NSP12 constructs.

2.5. Immunoblotting

To examine the reactivities of rat antibodies generated against designed epitopes, 293 cells transiently expressing SARS‐CoV‐2 NSP12 were lysed in‐plate using radioimmunoprecipitation assay (RIPA buffer (50 mM Tris·HCl [pH 7.4], 150 mM NaCl, 0.5% Triton X‐100, 2 mM Na₃VO₄, 2 mM NaF) supplemented with protease inhibitors (Roche). Lysates were sonicated, mixed with sodium dodecyl sulfate (SDS) sample loading buffer, run on acrylamide gels, and transferred to nitrocellulose membranes. Membranes were blocked (1× tris‐buffer saline with 1% tween 20 (TBST) with 5% milk at room temperature for 30 min) and incubated with the rat anti‐SARS‐CoV‐2 NSP12 antibodies diluted in 1× TBST with 5% bovine serum albumin (BSA) overnight at 4°C. Membranes were then washed twice with 1× TBST and incubated with secondary antibody (goat anti‐rat IR800) for 1 h at room temperature. Excess secondary antibodies were washed three times using 1× TBST and imaged using a BioRad ChemiDoc.

For SARS‐CoV‐2‐infected cell lysates, Vero E6 cells were infected with SARS‐CoV‐2 from a stock previously propagated from primary infection. Two days postinfection (dpi), cells were lysed in NP‐40 lysis buffer (10 mM Tris‐HCl pH 8.0, 1 mM ethylenediaminetetraacetic acid (EDTA), 0.1% NP‐40, 50 mM NaCl, 8% sucrose, 2 mM MgCl2) supplemented with protease inhibitors. Infected and uninfected cells were lysed for 10 min, and insoluble debris were removed by centrifugation. Clarified lysates were treated with additional RIPA buffer (50 mM Tris‐HCl pH 7.4, 150 mM NaCl, 1% NP40, 0.5% Na‐deoxycholate, 0.1 mM EDTA, 0.1 mM egtazic acid, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, leupeptin, pepstatin) and boiled to further inactivate all infectious material. The lysate was mixed with an SDS sample loading buffer, run on 10% acrylamide gels, and transferred to nitrocellulose membrane. Membranes were blocked and incubated with CM12.1 (1:250) and anti‐Spike S2 (1 μg/ml) antibodies overnight at 4°C. Membranes were washed and incubated in secondary antibody (goat anti‐rat IR800 and goat anti‐rabbit IR680) and antitubulin rhodamine for 1 h at room temperature. Blots were washed and imaged using a BioRad ChemiDoc.

2.6. IFA

IFA was performed as previously described. ²⁷ In brief, slides were deparaffinized at 95°C for 10 min, followed by three washes of xylene for 5 min. Rehydration was performed by incubation in ethanol at 100%, 95%, and 75% for 10 min each. Citrate buffer (pH 6.0) was used for antigen retrieval in a microwave for 3 min at maximum power, followed by 15 min at 30% power, then cooled to room temperature. Slides were blocked for 1 h with 1× TBST with 5% BSA. Primary antibodies were incubated overnight at 4°C. Slides washed three times with 1× TBST were then incubated with secondary antibodies for 1 h at room temperature. Slides were treated with Vector TrueVIEW autofluorescence quenching (Vector Laboratories) for 5 min followed by incubation with 4′,6‐diamidino‐2‐phenylindole (DAPI) for 10 min. Slides were mounted with VECTASHIELD® Vibrance™ Antifade Mounting Medium (Vector Laboratories).

2.7. Paraffin embedding of cell pellets

Ten million 293 cells overexpressing FLAG‐NSP12 or untagged NSP12 were centrifuged at 1500 rpm for 5 min to form cell pellets, washed in PBS, and fixed with 10% formalin at 4°C overnight. After 16 h fixation, the buffer was aspirated and replaced with 70% ethanol without disturbing cell pellets. Fixed cell pellets were then paraffin embedded and sectioned by standard techniques.

2.8. Preparation of SARS‐CoV‐2 infected mice lung samples

9‐week‐old K18‐hACE2 mice (Jackson Laboratory) were inoculated with 2000 PFU of SARS‐CoV‐2 (2019n‐CoV/USA‐WA1/2019 strain) in a total volume of 50 μl DMEM intranasally after light anesthesia with ketamine/xylazine. At 5 dpi, mice were euthanized, and lungs were harvested and fixed in zinc formalin. All animal studies were approved by the Animal Care and Use Committee of the University of Iowa. All mouse experiments with SARS‐CoV‐2 were conducted in a Biosafety Level 3 (BSL3) Laboratory at the University of Iowa.

2.9. Human lung tissue samples

COVID‐19‐positive lung tissue samples were obtained from the Department of Pathology, Molecular and Cell‐Based Medicine, Icahn School of Medicine at Mount Sinai. COVID‐19‐negative lung tissue samples were obtained from Pitt Biospecimen Core. ²⁷

3. RESULTS

3.1. Generation of SARS‐CoV‐2 NSP12 monoclonal antibodies

To generate monoclonal antibodies against SARS‐CoV‐2 NSP12, two candidate epitope peptides in the N terminus (aa 95−111) and the linker region (aa 355‐368) of SARS‐CoV‐2 NSP12 were separately used to immunize rats (Figure 1A). These peptides have high homology to RdRp from other human CoV including SARS‐CoV (Supporting Information: Figure S1). After several rounds of confirmatory ELISA and immunoblotting, clones CM12.1 and CM12.2 directed against the N‐terminus and linker regions, respectively, were isolated and antibody supernatants were concentrated by ammonium sulfate fractionation. ²⁸

Figure 1.

Application of CM12.1 in immunoblotting and immunofluorescence assay. (A) Structural demonstration of epitope distributions in SARS‐CoV‐2. NSP12 epitopes (epitope 1: N terminus; epitope 2: linker region) are all exposed on the surface of the predicted protein structure, maximumly covering possible epitope regions. The SARS‐CoV‐2 RNA‐dependent polymerase complex formed by NSP12, NSP7, and NSP8 is shown (PDB ID: 6M71). ¹⁶ Figure was created using BioRender. (B) Immunofluorescence assay showing the detection of C‐terminally FLAG‐tagged NSP12 (FLAG‐NSP12) and full‐length NSP12 (NSP12 FL) on paraffin‐embedded formalin‐fixed cell pellet slides by CM12.1. EV, empty vector. (C) Illustration of the fragments’ distributions in SARS‐CoV‐2 NSP12. (D) Immunoblotting showing epitope mapping result of CM12.1. Whole‐cell lysates were harvested from 293 cells transfected with equal amounts of NSP12 fragments. Lanes 1: EV, 2: FLAG‐NSP12, 3: FLAG‐NSP12 1‐249 aa, 4: FLAG‐NSP12 1‐581 aa, 5: FLAG‐NSP12 131‐932 aa, 6: FLAG‐NSP12 582‐932 aa, 7: NSP12 FL. β‐tubulin served as loading control. (E) Vero E6 cells were infected with SARS‐CoV‐2 and lysed 2 days postinfection. Lysates from infected and uninfected cells were immunoblotted with CM12.1, anti‐Spike S2, and β‐tubulin.

3.2. In vitro CM12.1 immunofluorescence and immunoblotting

For IFA, cell pellets overexpressing either empty vector (EV), full‐length NSP12 with (FLAG‐NSP12), and without (NSP12 FL) a C‐terminal FLAG tag were fixed, embedded, and sectioned. CM12.1 (200 μg/ml) recognized both FLAG‐NSP12 and NSP12 FL‐expressing cells with minimal background fluorescence (Figure 1B).

The optimal dilution of CM12.1 for immunoblotting FLAG‐tagged NSP12 transiently expressed in 293 cells was determined using serial titration. Dilution to 1:1000 CM12.1 yielded specific bands at the correct molecular mass (100 kDa) with low background staining (Figure 1C). To confirm this antibody's specificity, C‐terminal FLAG‐tagged NSP12 fragments were generated spanning the entire ORF (Figure 1C) with expression determined by anti‐FLAG staining. As expected, FLAG‐NSP12 1‐249 aa and FLAG‐NSP12 1‐581 aa peptides encoding the N‐terminal epitope 1 were recognized by CM12.1, whereas FLAG‐NSP12 131‐932 aa and FLAG‐NSP12 592‐932 aa not encoding for epitope 1 were nonreactive (Figure 1D).

Testing for the specificity of the linker targeting CM12.2 antibody was performed similarly to that for CM12.1 (Supporting Information: Figure S2). CM12.2 detected FLAG‐NSP12 1‐581 aa, FLAG‐NSP12 131‐932 aa, and full‐length NSP12 containing epitope 2 but not FLAG‐NSP12 1‐249 aa or FLAG‐NSP12 582‐932 aa (Supporting Information: Figure S2A). CM12.2 also detected cells expressing NSP12 protein by IFA (Supporting Information: Figure S2B). The sensitivity of CM12.2 was higher than CM12.1 by IFA at similar dilutions, but also displayed higher background staining than CM12.1 and therefore was not used for tissue studies.

NSP12 was also detected in lysates from cells infected with SARS‐CoV‐2 by immunoblotting with CM12.1. Vero E6 cell lysates were subjected to sodium dodecyl sulfate‐polyacrylamide gel electrophoresis followed by immunoblotting with CM12.1 and anti‐Spike antibodies. A 100 kDa band corresponding to the molecular weight of NSP12 was detectable in lysates from infected cells and absent in lysates from negative control uninfected cells (Figure 1E).

3.3. SARS‐CoV‐2 NSP12 detection in infected mouse tissues

Mice infected with SARS‐CoV‐2 (2000 PFU/mice) were killed at 5 dpi and lung tissues fixed and paraffin‐embedded. Spike protein expression was readily detected in lung samples, indicative of SARS‐CoV‐2 infection; however, no NSP12 protein positivity was found in these samples using CM12.1 (Supporting Information: Figure S3).

3.4. CM12.1 detection of SARS‐CoV‐2 in COVID‐19 human autopsy lung samples

CM12.1 IFA was used to detect NSP12 expression in lung tissues from five COVID‐19 patients (Figure 2A). The distribution of NSP12‐positive cells in these tissues was generally scant but when positive had a cellular distribution consistent with viral protein expression. All NSP12‐positive cells were positive for spike protein S1, but the number of NSP12‐positive cells in any of the five cases were much lower than S1‐positive cells (Figure 2B). In general, we observed stronger S1 signal than NSP12 signal, suggesting a lower expression level of NSP12 protein than S1 protein or a lower sensitivity of CM12.1 than the S1 antibody.

Figure 2.

Application of CM12.1 in the detection of NSP12 in COVID‐19 patients. (A) Images of immunofluorescence detection of NSP12 using CM12.1 in COVID‐19 negative and positive lung tissues. 4′,6‐diamidino‐2‐phenylindole (DAPI) stained nuclei. (B) Images of immunofluorescence detection for NSP12 and SARS‐CoV‐2 spike protein S1 subunit (Spike) in COVID‐19 negative and positive lung tissues. DAPI stained nuclei. (C) Images of immunofluorescence detection for NSP12 and SARS‐CoV‐2 NSP13 protein in COVID‐19 negative and positive lung tissues. DAPI stained nuclei. (D) Images of immunofluorescence detection for NSP12 and SARS‐CoV‐2 NSP8 protein in COVID‐19 negative and positive lung tissues. DAPI stained nuclei.

To compare NSP12 staining with another pp1ab encoded protein, patient tissues were costained with an antibody recognizing NSP13. ¹⁴ Since both proteins are generated from the same polyprotein, they would be expected to have similar levels of positivity if low levels of expression are responsible for the low level of NSP12 staining. In addition, NSP12 and NSP13 form a complex involved in RNA synthesis, proofreading and RNA modification (Figure 2C). However, far more cells stained positive for NSP13 than NSP12 in autopsy lung tissues (Figure 2C). NSP8 (together with NSP7) is also a component of the core polymerase complex. ²⁹ As with NSP13, although we detected cells expressing both NSP8 and NSP12, far more cells stained positive for NSP8 alone than for NSP12 (Figure 2D).

3.5. NSP12 positivity for different cell types in COVID‐19 lung tissues

SARS‐CoV‐2 infects diverse cell types in lung tissues from severe COVID‐19 patients. ²⁷ We performed dual staining for NSP12 and different lung parenchyma cell lineage markers including AT1 and AT2 cells, goblet cells (MUC5AC), club‐like cells (MUC5B), and vascular endothelial cells (CD34 and CD144). NSP12 positivity was detected in goblet cells, club‐like cells (Figure 3A) and vascular endothelial cells (Figure 3B). NSP12 was not detectable in AT1 or AT2 cells even though these cells are believed to be primary targets for SARS‐CoV‐2 lung infection. The detection of NSP12 protein in these cells suggests that SARS‐CoV‐2 can infect and replicate in some epithelial cells and vascular endothelial cells.

Figure 3.

Multicolor immunofluorescence staining of SARS‐CoV‐2 NSP12, Spike and cellular markers in lung tissues from COVID‐19 patients. Images of immunofluorescence detection for (A) SARS‐CoV‐2 NSP12 (CM12.1), spike protein S1 subunit (Spike), and MUC5AC (goblet cells, pseudo color white) or MUC5B (club‐like cells, pseudo color white). Nuclei were stained with 4′,6‐diamidino‐2‐phenylindole (DAPI). (B) SARS‐CoV‐2 NSP12 (CM12.1), spike protein S1 subunit (Spike), and CD34 or CD144 (vascular endothelial cells, pseudo color white). Nuclei were stained with DAPI. (C) SARS‐CoV‐2 NSP12 (CM12.1), spike protein S1 subunit (Spike), and CD68 or CD14 (monocytes and macrophages, pseudo color white). Nuclei were stained with DAPI. (D) SARS‐CoV‐2 NSP12 (CM12.1), spike protein S1 subunit (Spike), and CD56 (NK cells, pseudo color white) or ELA‐2 (neutrophils, pseudo color white). Nuclei were stained with DAPI. (E) SARS‐CoV‐2 NSP12 (CM12.1), spike protein S1 subunit (Spike), and CD8 (CD8⁺ T cells, pseudo color white). Nuclei were stained with DAPI.

Lung tissues from severe COVID‐19 cases generally have extensive immune cell infiltrates, ²⁷ , ³⁰ which can harbor the SARS‐CoV‐2 virus as detected with antibodies to spike and nucleocapsid proteins. ²⁷ We detected NSP12 protein in a variety of infiltrating immune cells including monocytes and macrophages (CD68 and CD14), NK cells (CD56), neutrophils (ELA‐2), and CD8 T cells (Figure 3C,E).

4. DISCUSSION

Here we report the development and characterization of a rat monoclonal antibody CM12.1 recognizing SARS‐CoV‐2 NSP12 protein. NSP12 is the essential RNA polymerase for SARS‐CoV‐2 mRNA synthesis and genome replication. NSP12 inhibitors, such as remdesivir and molnupiravir, ³² , ³³ , ³⁴ block SARS‐CoV‐2 replication and prevent or reduce COVID‐19 symptoms.

CM12.1 recognizes an epitope in the SARS‐CoV‐2 NSP12 N‐terminus, and this antibody is both specific and sensitive for overexpressed protein from detection in multiple assays including immunoblotting and IFA (Figure 1). This antibody weakly detects native NSP12 expressed during in vitro infection and fails to detect NSP12 in virally infected mice. NSP12 is generated by protease cleavage from the ORF1ab polyprotein, and its synthesis should be concordant with other proteins such as NSP13 that are readily detectable in infected tissues. The most likely reasons for failure to detect this protein are increased turnover of NSP12, resulting in a low steady‐state expression in infected cells, or posttranslational modification (PTM) that destroys the epitope recognized by the antibody. Of note, the epitope recognized by CM12.1 is highly conserved among coronaviruses including three which have been associated with significant human disease (Figure S1). Although not formally tested in our study, this newly developed antibody may also be reactive to NSP12 of a range of other coronaviruses.

Among the few CM12.1 positive cells in human autopsy lung tissues, NSP12 protein expression was detected in parenchymal cells including goblet cells, club‐like cells and vascular endothelial cells, and numerous types of immune cells including monocytes, macrophages, NK cells, neutrophils (ELA‐2), and CD8 T cells. These results suggest that SARS‐CoV‐2 is capable of infecting and replicating in these cells, confirming its broad cell tropism in lung tissues from severe COVID‐19 patients. ²⁷ , ³⁵ , ³⁶

CM12.1, however, failed to detect NSP12 protein in lung samples from SARS‐CoV‐2 infected mice. It is possible that NSP12 is expressed at levels below our detectability in infected mouse tissues but the central requirement for NSP12 to SARS‐CoV‐2 replication makes this unlikely. More probable, NSP12 protein is either expressed at early time points after infection and thus not detected at 5 days, or PTMs of the protein render it undetectable with CM12.1.

In conclusion, we have developed a specific monoclonal antibody to SARS‐CoV‐2 NSP12 protein. This antibody can detect NSP12 protein in multiple assays and in lung samples from COVID‐9 patients, which could be useful for the scientific community to facilitate research on NSP12 and SARS‐CoV‐2.

AUTHOR CONTRIBUTIONS

Wen Meng and Siying Guo contributed equally to this work. Wen Meng, Siying Guo, Simon Cao, Masahiro Shuda, Yoko Shuda, Lindsey R. Robinson‐McCarthy, and Kevin R. McCarthy performed the experiments. Alberto E. Paniz Mondolfi, Clare Bryce, Zachary Grimes, Emilia M. Sordillo, and Carlos Cordon‐Cardo collected clinical specimens. Pengfei Li and Stanley Perlman designed the animal experiment and collected mice specimens. Wen Meng, Siying Guo, Hu Zhang, Haitao Guo, Shou‐Jiang Gao, Yuan Chang, and Patrick S Moore designed and contributed to data interpretation. Wen Meng, Siying Guo, and Lindsey R. Robinson‐McCarthy wrote the first draft of the manuscript. All authors critically reviewed the manuscript and approved the final manuscript for submission.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

1. ETHICS STATEMENT

The University of Pittsburgh institutional review board (IRB) determined that the study was not research involving human subjects (STUDY20050085). All experiments with SARS‐CoV‐2 were performed in a BSL3. All animal studies were approved by the University of Iowa Animal Care and Use Committee and meet stipulations of the Guide for the Care and Use of Laboratory Animals.

Supporting information

Figure S1.

Figure S2.

Figure S3.

Meng W, Guo S, Cao S, et al. Development and characterization of a new monoclonal antibody against SARS‐CoV‐2 NSP12 (RdRp). J Med Virol. 2022;95:e28246. 10.1002/jmv.28246

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.