Simultaneous evaluation of antibodies that inhibit SARS-CoV-2 variants via multiplex assay

Abstract

The SARS-CoV-2 Receptor Binding Domain (RBD) is both the principal target of neutralizing antibodies, and one of the most rapidly evolving domains, which can result in the emergence of immune escape mutations limiting the effectiveness of vaccines and antibody therapeutics. To facilitate surveillance, we developed a rapid, highthroughput, multiplex assay able to assess the inhibitory response of antibodies to 24 RBD natural variants simultaneously. We demonstrate how this assay can be implemented as a rapid surrogate assay for functional cell-based serological methods to measure the SARS-CoV-2 neutralising capacity of antibodies at the ACE2-RBD interface. We describe the enhanced affinity of RBD variants N439K, S477N, Q493L, S494P and N501Y to the ACE2 receptor, and demonstrate the ability of this assay to bridge a major gap for SARS-CoV-2 research; informing selection of complementary monoclonal antibody candidates and the rapid identification of immune escape to emerging RBD variants following vaccination or natural infection.

Heterologous SARS-CoV-2 IgA neutralising antibody responses in convalescent plasma

“…1d & e). Thus, most SARS-CoV-2 convalescent plasma induced antibodies that recognise RBDWT and have the capacity to inhibit the binding of RBD to ACE2, as previously reported (Lopez et al, 2021).…”

“…RBDWT antibody isotype titres (IgG, IgM and IgA) have previously been correlated to the level of plasma neutralisation or ACE2 binding inhibition (Gasser et al, 2021; Lopez et al, 2021; Sterlin et al, 2021). Here the cohort was broken down by isotype seropositivity and the ability to block ACE2 engagement with RBDWT to observe the surface level contribution of the isotypes to neutralisation in this cohort (fig.…”

“…To examine the ability of subject plasma to neutralise SARS-CoV-2, we used a multiplex RBDWT-ACE2 binding inhibition assay, that has been demonstrated to highly correlate with gold standard live microneutralization assays (Lopez et al, 2021). Almost all convalescent subjects (95.12%) generated significant inhibition of ACE2 binding to RBDWT (median=73.23%, with ACE2 binding inhibition <20% considered to be below the limit of dection (Lopez et al, 2021)) (fig. 1d).…”

Comparative Magnitude and Persistence of Humoral SARS-CoV-2 Vaccination Responses in the Adult Population in Germany

“…While we have used an ACE2-RBD competition assay to measure ACE2 binding inhibition as opposed to classic virus neutralization assays, this assay analyses neutralizing antibodies as seen by its similar performance to VNT ( 24 , 29 ). ACE2 inhibition assays instead of a VNT have also already been used successfully by other groups to determine neutralizing antibody activity ( 52 ). Methodically, MULTICOV-AB and the ACE2-RBD competition assay are also complementary and are measured using a single initial sample dilution which further reduces variability between their results.…”

SARS-CoV-2 Multi-Antigen Serology Assay

“…As each antigen is covalently coupled to a specific bead set, antibody levels to multiple antigens can be assessed simultaneously in single plasma, serum, or saliva samples. Luminex-based serology assays have been effectively used to measure antigen-specific antibodies to several pathogens, including malaria, HIV and now also respiratory viruses [10][11][12][13][14][15][16][17][18][19]. The advantage of this assay on a MAGPIX system is that a small sample volume of as little as 1 µL can be used for the detection of up to 50 antigens using magnetic beads (and up to 500 antigens on the Flexmap).…”