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

  1. Curtis D. Hodge
  2. Daniel J. Rosenberg
  3. Mateusz Wilamowski
  4. Andrzej Joachimiak
  5. Greg L. Hura
  6. Michal Hammel

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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 (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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    SciScore for 10.1101/2021.01.13.426597: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    No key resources detected.

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    However, a limitation of existing immunoassays in detecting antigens relative to RT-PCR is the lack of exponential amplification of signal when probes detect an antigen. Immunoassays mainly rely on antibody-antigen binding at a 1:1 ratio. An immunoassay diagnostic with a greater detection-to-capture antibody ratio will also have a greater signal-to-antigen ratio, effectively enhancing overall specific antigen detection. Despite the widespread use of antibodies in diagnostics and treatments, an understanding of structural properties that affect antibody-antigen interactions in their aqueous environment is currently insufficient to guide optimization for these purposes. Here, we describe how antibodies’ inherent flexibility leads to distinct antigen-binding arrangements that drive different functional outcomes in a SARS-CoV-2 detection ELISA. We show that we can rapidly assess new antibody-antigen interactions using SEC-MALS-SAXS to identify pairs that bind in a linear arrangement (Figure 3), which results in a more sensitive detection assay (Figure 4). A critical benefit of using the SEC-MALS-SAXS approach is its ease of use and the ability to study antibody-interactions in solution. It has previously been shown that other techniques that rely on grids (EM) or crystals often do not reflect the dynamic nature of antibodies in solution (Jay et al., 2018). A central finding in this work is the observation and rationalization of how mAb flexibility can impact larger assemblies of ...

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    About SciScore

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  2. Version published to 10.1101/2021.01.13.426597v1 on bioRxiv
  3. Version published to 10.1080/19420862.2021.1905978