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

Kim, Dongbum; Kim, Minyoung; Baek, Kyeongbin; Kang, Mijeong; Choi, Jun-Kyu; Maharjan, Sony; Akauliya, Madhav; Lee, Younghee; Kwon, Hyung‐Joo

doi:10.3389/fmicb.2021.726231

Cited by 16 publications

(23 citation statements)

References 27 publications

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“…For some proteins, multiple bands were detected, e.g., nsp16 was detected on the predicted size of 35-40 kDa (arrowhead Figure 1B), with several additional bands of lower molecular weight, as observed by others [14]. Similarly, additional bands of lower molecular weight observed for the Nucleocapsid protein are also reported in publications [15,16] or datasheets of other antibody producers (Proteintech cat.no. 1B3C3, Cell Signaling cat.no.…”

Section: Resultssupporting

confidence: 67%

Collection of Monoclonal Antibodies Targeting SARS-CoV-2 Proteins

Matešić

Brlić

Roviš

et al. 2022

Viruses

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In early 2020, the COVID-19 pandemic sparked a global crisis that continues to pose a serious threat to human health and the economy. Further advancement in research is necessary and requires the availability of quality molecular tools, including monoclonal antibodies. Here, we present the development and characterization of a collection of over 40 new monoclonal antibodies directed against different SARS-CoV-2 proteins. Recombinant SARS-CoV-2 proteins were expressed, purified, and used as immunogens. Upon development of specific hybridomas, the obtained monoclonal antibody (mAb) clones were tested for binding to recombinant proteins and infected cells. We generated mAbs against structural proteins, the Spike and Nucleocapsid protein, several non-structural proteins (nsp1, nsp7, nsp8, nsp9, nsp10, nsp16) and accessory factors (ORF3a, ORF9b) applicable in flow cytometry, immunofluorescence, or Western blot. Our collection of mAbs provides a set of novel, highly specific tools that will allow a comprehensive analysis of the viral proteome, which will allow further understanding of SARS-CoV-2 pathogenesis and the design of therapeutic strategies.

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Section: Resultssupporting

confidence: 67%

Collection of Monoclonal Antibodies Targeting SARS-CoV-2 Proteins

Matešić

Brlić

Roviš

et al. 2022

Viruses

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“…The binding affinity of the purified HCoV-OC43 N protein-specific monoclonal antibody was measured by ELISA as described previously ( 35 ). Briefly, streptavidin (2 μg/well) was coated in 96-well immunoplates (Thermo Fisher Scientific), and then recombinant HCoV-OC43 N-Bio-His 6 protein (3 μg/well) was added into each well along with serially diluted (1:5) monoclonal antibody (clone 1C7D7 mAb) in PBST.…”

Section: Methodsmentioning

confidence: 99%

“…SigmaPlot was used to determine the EC 50 value ( 27 ). To evaluate the specificity of the monoclonal antibody, recombinant MERS-CoV N protein-Bio-His 6 and SARS-CoV-2 N protein-Bio-His 6 ( 35 ) were analyzed as a control.…”

Section: Methodsmentioning

confidence: 99%

“…The interaction of HCoV-OC43 Spike CD and HCoV-OC43 N protein was measured by ELISA as described previously ( 35 ). Briefly, HCoV-OC43 N protein-specific monoclonal antibody (clone 1C7D7 mAb, 3 μg/well) was coated in 96-well immunoplates overnight at 4°C, then blocked with PBST containing 1% BSA.…”

Section: Methodsmentioning

confidence: 99%

“…To analyze the interaction of HCoV-OC43 Spike CD and HCoV-OC43 N protein, we designed a “bait and prey” ELISA, described previously ( 35 ). Briefly, streptavidin (2 μg/well) was coated in 96-well immunoplates then blocked with PBST containing 1% BSA.…”

Section: Methodsmentioning

confidence: 99%

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Targeting the Interaction Between Spike Protein and Nucleocapsid Protein for Suppression and Detection of Human Coronavirus OC43

Kim

Kang

et al. 2022

Front. Immunol.

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Human coronavirus OC43 (HCoV-OC43) is the coronavirus most associated with “common colds”, infections of the upper respiratory tract. Previously, we reported that direct interactions of nucleocapsid (N) protein and C-terminal domain of Spike protein (Spike CD) are essential for replication of SARS-CoV-2 and MERS-CoV. Thus, we developed a novel ELISA-based strategy targeting these specific interactions to detect SARS-CoV-2 and MERS-CoV. Here, we investigated whether the same principles apply to HCoV-OC43. We discovered that the S protein of HCoV-OC43 interacts with N protein and that cell penetrating Spike CD peptide inhibits virus protein expression and replication of HCoV-OC43. The interaction between HCoV-OC43 S and N proteins were recapitulated with a recombinant HCoV-OC43 Spike CD fusion protein and a recombinant HCoV-OC43 N fusion protein in vitro. By producing an anti-HCoV-OC43 N protein-specific monoclonal antibody, we established a virus detection system based on the interaction between recombinant Spike CD and N protein of HCoV-OC43. We suggest that the interaction between Spike CD and N protein is conserved in coronaviruses and therefore could be a target for therapeutics against both novel coronavirus and its variants.

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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

Kim

Baek

et al. 2023

Journal of Medical Virology

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Peptides are promising therapeutic agents for COVID‐19 because of their specificity, easy synthesis, and ability to be fine‐tuned. We previously demonstrated that a cell‐permeable peptide corresponding to the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) Spike C‐terminal domain (CD) inhibits the interaction between viral spike and nucleocapsid proteins that results in SARS‐CoV‐2 replication in vitro. Here, we used docking studies to design R‐t‐Spike CD(D), a more potent short cell‐penetrating peptide composed of all D‐form amino acids and evaluated its inhibitory effect against the replication of SARS‐CoV‐2 S clade and other variants. R‐t‐Spike CD(D) was internalized into Vero cells and Calu‐3 cells and suppressed the replication of SARS‐CoV‐2 S clade, delta variant, and omicron variant with higher potency than the original peptide. In hemizygous K18‐hACE2 mice, intratracheal administration of R‐t‐Spike CD(D) effectively delivered the peptide to the trachea and lungs, whereas intranasal administration delivered the peptide mostly to the upper respiratory system and stomach, and a small amount to the lungs. Administration by either route reduced viral loads in mouse lungs and turbinates. Furthermore, intranasally administered R‐t‐Spike CD(D) mitigated pathological change in the lungs and increased the survival of mice after infection with the SARS‐CoV‐2 S clade or delta variant. Our data suggest that R‐t‐Spike CD(D) has potential as a therapeutic agent against SARS‐CoV‐2 infection.

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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

Cited by 16 publications

References 27 publications

Collection of Monoclonal Antibodies Targeting SARS-CoV-2 Proteins

Collection of Monoclonal Antibodies Targeting SARS-CoV-2 Proteins

Targeting the Interaction Between Spike Protein and Nucleocapsid Protein for Suppression and Detection of Human Coronavirus OC43

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

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