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Development of a highly specific and sensitive VHH-based sandwich immunoassay for the detection of the SARS-CoV-2 nucleoprotein.

ABSTRACT: The current COVID-19 pandemic illustrates the importance of obtaining reliable methods for the rapid detection of SARS-CoV-2. A highly specific and sensitive diagnostic test able to differentiate the SARS-CoV-2 virus from common human coronaviruses is therefore needed. Coronavirus nucleoprotein (N) localizes to the cytoplasm and the nucleolus and is required for viral RNA synthesis. N is the most abundant coronavirus protein, so it is of utmost importance to develop specific antibodies for its detection. In this study, we developed a sandwich immunoassay to recognize the SARS-CoV-2 N protein. We immunized one alpaca with recombinant SARS-CoV-2 N and constructed a large single variable domain on heavy chain (VHH) antibody library. After phage display selection, seven VHHs recognizing the full N protein were identified by ELISA. These VHHs did not recognize the nucleoproteins of the four common human coronaviruses. Hydrogen Deuterium eXchange-Mass Spectrometry (HDX-MS) analysis also showed that these VHHs mainly targeted conformational epitopes in either the C-terminal or the N-terminal domains. All VHHs were able to recognize SARS-CoV-2 in infected cells or on infected hamster tissues. Moreover, the VHHs could detect the SARS variants B.1.17/alpha, B.1.351/beta, and P1/gamma. We propose that this sandwich immunoassay could be applied to specifically detect the SARS-CoV-2 N in human nasal swabs.

SUBMITTER: Gransagne M

PROVIDER: S-EPMC8526496 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Validation and standardization of accurate serological assays are crucial for the surveillance of the coronavirus disease 2019 (COVID-19) pandemic and population immunity. We describe the analytical and clinical performance of an in-house fluorescent multiplex immunoassay (FMIA) for simultaneous quantification of antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleoprotein and spike glycoprotein. Furthermore, we calibrated IgG-FMIA against World Health Organization (WHO) International Standard and compared FMIA results to an in-house enzyme immunoassay (EIA) and a microneutralization test (MNT). We also compared the MNT results of two laboratories. IgG-FMIA displayed 100% specificity and sensitivity for samples collected 13 to 150 days post-onset of symptoms (DPO). For IgA- and IgM-FMIA, 100% specificity and sensitivity were obtained for a shorter time window (13 to 36 and 13 to 28 DPO for IgA- and IgM-FMIA, respectively). FMIA and EIA results displayed moderate to strong correlation, but FMIA was overall more specific and sensitive. IgG-FMIA identified 100% of samples with neutralizing antibodies (NAbs). Anti-spike IgG concentrations correlated strongly (ρ = 0.77 to 0.84, P < 2.2 × 10-16) with NAb titers, and the two laboratories' NAb titers displayed a very strong correlation (ρ = 0.95, P < 2.2 × 10-16). Our results indicate good correlation and concordance of antibody concentrations measured with different types of in-house SARS-CoV-2 antibody assays. Calibration against the WHO international standard did not, however, improve the comparability of FMIA and EIA results. IMPORTANCE SARS-CoV-2 serological assays with excellent clinical performance are essential for reliable estimation of the persistence of immunity after infection or vaccination. In this paper we present a thoroughly validated SARS-CoV-2 serological assay with excellent clinical performance and good comparability to neutralizing antibody titers. Neutralization tests are still considered the gold standard for SARS-CoV-2 serological assays, but our assay can identify samples with neutralizing antibodies with 100% sensitivity and 96% specificity without the need for laborious and slow biosafety level 3 (BSL-3) facility-requiring analyses.

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Development of a highly specific and sensitive VHH-based sandwich immunoassay for the detection of the SARS-CoV-2 nucleoprotein.

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