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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.

SUBMITTER: Hodge CD

PROVIDER: S-EPMC8043170 | biostudies-literature |

REPOSITORIES: biostudies-literature

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

Project description: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 (SAXS). Our solution-based results distinguished the mAbs' flexibility and how this flexibility impacts 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 Fabs is prevented, enforcing a linear arrangement of the mAb pair, which facilitates further mAb polymerization. In a modified sandwich ELISA, we show the 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 (LFA), key for early diagnosis and epidemiological studies of SARS-CoV-2 and other pathogens.

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Monoclonal Antibodies against Nucleocapsid Protein of SARS-CoV-2 Variants for Detection of COVID-19.

Project description:Mitigation strategies of the coronavirus disease 2019 (COVID-19) pandemic have been greatly hindered by the continuous emergence of SARS-CoV-2 variants. New sensitive, rapid diagnostic tests for the wide-spectrum detection of viral variants are needed. We generated a panel of 41 monoclonal antibodies against the SARS-CoV-2 nucleocapsid protein (NP) by using mice hybridoma techniques. Of these mAbs, nine exhibited high binding activities and were applied in latex-based lateral flow immunoassays (LFIAs). The LFIAs utilizing NP-mAb-7 and -40 had the best sensitivity and lowest limit of detection: 8 pg for purified NP and 625 TCID50/mL for the authentic virus (hCoV-19/Taiwan/4/2020). The specificity tests showed that the NP-mAb-40/7 LFIA strips did not cross-react with five human coronavirus strains or 20 other common respiratory pathogens. Importantly, we found that 10 NP mutants, including alpha (B.1.1.7), beta (B.1.351), gamma (P.1), and delta (B.1.617.2) variants, could be detected by NP-mAb-40/7 LFIA strips. A clinical study (n = 60) of the NP-mAb-40/7 LFIA strips demonstrated a specificity of 100% and sensitivity of 90% in infected individuals with cycle threshold (Ct) values < 29.5. These anti-NP mAbs have strong potential for use in the clinical detection of SARS-CoV-2 infection, whether the virus is wild-type or a variant of concern.

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Highly specific monoclonal antibodies and epitope identification against SARS-CoV-2 nucleocapsid protein for antigen detection tests.

Project description:The ongoing coronavirus disease 2019 (COVID-19) pandemic is a major global public health concern. Although rapid point-of-care testing for detecting viral antigen is important for management of the outbreak, the current antigen tests are less sensitive than nucleic acid testing. In our current study, we produce monoclonal antibodies (mAbs) that exclusively react with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and exhibit no cross-reactivity with other human coronaviruses, including SARS-CoV. Molecular modeling suggests that the mAbs bind to epitopes present on the exterior surface of the nucleocapsid, making them suitable for detecting SARS-CoV-2 in clinical samples. We further select the optimal pair of anti-SARS-CoV-2 nucleocapsid protein (NP) mAbs using ELISA and then use this mAb pair to develop immunochromatographic assay augmented with silver amplification technology. Our mAbs recognize the variants of concern (501Y.V1-V3) that are currently in circulation. Because of their high performance, the mAbs of this study can serve as good candidates for developing antigen detection kits for COVID-19.

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Development of SARS-CoV-2 Nucleocapsid Specific Monoclonal Antibodies.

Project description:The global COVID-19 pandemic has caused massive disruptions in every society around the world. 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 a major component of the viral replication processes, integral to viral particle assembly, and is a major diagnostic marker for infection and immune protection. Currently available antibody reagents targeting the nucleocapsid protein were primarily developed against the related SARS-CoV virus and are not specific to SARS-CoV-2 nucleocapsid protein. Therefore, in this work we developed and characterized a series of new mouse monoclonal antibodies against the SARS-CoV-2 nucleocapsid protein. The anti-nucleocapsid monoclonal antibodies were tested in ELISA, western blot, and immunofluorescence analyses. The variable regions from the heavy and light chains from five select clones were cloned and sequenced, and preliminary epitope mapping of the sequenced clones was performed. Overall, the new antibody reagents described here will be of significant value in the fight against COVID-19.

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Single-Domain Antibodies for the Detection of SARS-CoV-2 Nucleocapsid Protein.

Project description:The goal of this work was to develop recombinantly expressed variable domains derived from camelid heavy-chain antibodies known as single-domain antibodies (sdAbs) directed against the SARS-CoV-2 nucleocapsid protein for incorporation into detection assays. To achieve this, a llama was immunized using a recombinant SARS-CoV-2 nucleocapsid protein and an immune phage-display library of variable domains was developed. The sdAbs selected from this library segregated into five distinct sequence families. Three of these families bind to unique epitopes with high affinity, low nM to sub-nM KD, as determined by surface plasmon resonance. To further enhance the utility of these sdAbs for the detection of nucleocapsid protein, homobivalent and heterobivalent genetic fusion constructs of the three high-affinity sdAbs were prepared. The effectiveness of the sdAbs for the detection of nucleocapsid protein was evaluated using MagPlex fluid array assays, a multiplexed immunoassay on color-coded magnetic microspheres. Using the optimal bivalent pair, one immobilized on the microsphere and the other serving as the biotinylated recognition reagent, a detection limit as low as 50 pg/mL of recombinant nucleocapsid and of killed virus down to 1.28 × 103 pfu/mL was achieved. The sdAbs described here represent immune reagents that can be tailored to be optimized for a number of detection platforms and may one day aid in the detection of SARS-CoV-2 to assist in controlling the current pandemic.

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Development and characterization of monoclonal antibodies recognizing nucleocapsid protein of multiple SARS-CoV-2 variants

Project description: Not available

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Preparation of highly specific monoclonal antibodies against SARS-CoV-2 nucleocapsid protein and the preliminary development of antigen detection test strips.

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Project description: Not available

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Epitopes recognition of SARS-CoV-2 nucleocapsid RNA binding domain by human monoclonal antibodies.

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