AS739, AT693 and AU734 antibodies recognize the spike S protein from SARS-CoV-2 by ELISA

Abstract

The recombinant antibodies AS739, AT693 and AU734 detect by ELISA the spike S protein from SARS-CoV-2.

Introduction

The spike S glycoprotein (UniProt P0DTC2) mediates the attachment of coronaviruses to the host ACE2 receptor (through the Receptor-Binding Domain [RBD] in the S1 subunit) and fusion with the host cell membrane (through the S2 subunit) (Yan et al., 2020). Five recombinant antibodies recognizing the S1 domain of the S protein from SARS-CoV-2 (AS739, AT693, AU197, AU734 and AU753) were tested for their ability to recognize the S protein by ELISA. Three antibodies (AS739, AT693 and AU734) detected the S protein from SARS-CoV-2; two others (AU197 and AU753) did not.

Materials & Methods

Antibodies: ABCD_AS739, ABCD_AT693, ABCD_AU197, ABCD_AU734 and ABCD_AU753 antibodies (ABCD nomenclature, http://web.expasy.org/abcd/) were produced by the Geneva Antibody Facility (http://unige.ch/medecine/antibodies/) as mini-antibodies with the antigen-binding portion fused to a rabbit IgG Fc. The synthesized scFv sequences (GeneArt, Invitrogen) correspond to the sequences of the variable regions joined by a peptide linker (GGGGS)₃ (see Table 1 for clone names and references). HEK293 suspension cells (growing in FreeStyle™ 293 Expression Medium, Gibco 12338) were transiently transfected with the vector coding for the scFv-Fc of each antibody. Supernatants (see Table 1 for individual yields) were collected after 4 days.

Table 1. Clone number, epitope, reference and production yields for the antibodies used in this study.

ABCD	Clone	Epitope	Reference	Yield (mg/L)
AS739	S309	S1/RBD	Pinto et al., 2020	100
AT693	BD-23	S1/RBD	Cao et al., 2020	80
AU197	2B04	S1/RBD	Alsoussi et al., 2020	30
AU734	2-43	S1/RBD	Liu et al., 2020	40
AU753	Mab362	S1/RBD	Ejemel et al., 2020	10

Antigen: The prefusion ectodomain (residues 1-1208) of the SARS-CoV-2 S protein, with a KV->PP substitution at residues 986/987, a RRAR->GSAS substitution at residues 682-685, and C-terminal T4 fibritin trimerization motif, protease cleavage site, TwinStrepTag and 8xHisTag (PDB 6VSB; Wrapp et al., 2020), was transiently transfected into 25x10⁸ suspension-adapted ExpiCHO cells (Thermo Fisher) using 1.5 mg plasmid DNA and 7.5 mg of PEI MAX (Polysciences) in 500 mL ProCHO5 medium (Lonza). Incubation with agitation was continued at 31°C and 4.5% CO₂ for 5 days. The clarified supernatant was purified in two steps: via a Strep-Tactin XT column (IBA Lifesciences) followed by Superose 6 10/300 GL column (GE Healthcare) to a final concentration of 180 µg/ml in PBS.

Protocol: The whole procedure was carried out at room temperature. Biotinylated BSA (10 µg/mL) or S protein (10 µg/mL) were incubated in a streptavidin-coated 8-well plate (50 µl/well) (Pierce 15120) for 30 min. Each well was rinsed three times with 100 μl of washing buffer (PBS + 0.1% (w/v) BSA + 0.05% (w/v) Tween20), then incubated for 1 h with 50 µl of antibody-containing supernatant diluted in washing buffer as indicated (Fig. 1). After rinsing 3 times (100 µl washing buffer), wells were incubated with horseradish peroxidase-coupled goat anti-rabbit IgG (Sigma A8275, dilution 1:1000, 50 μl per well) for 30 min. After 5 rinses, Tetramethylbenzidine (TMB) substrate (Sigma T5569) was added (50 μl per well). The reaction was stopped by the addition of 25 μl of 2 M H₂SO₄. The absorbance (OD) was measured at 450 nm.

Results

Antibodies AS739, AT693 and AU734 bound in a concentration-dependent manner to the SARS-CoV-2 spike S protein, but not to the BSA negative control (Fig. 1). AU197 and AU753 did not recognize the S protein by ELISA; for AU753, this is possibly due to the fact that this antibody is poorly produced.

Figure 1. AS739, AT693 and AU734 bound specifically to the SARS-CoV-2 S protein, but not to the BSA control (shown only for AT693; AS739, AU197, AU734 and AU753 background curves were superimposed), as detected by ELISA.

Conflict of interest

The authors declare no conflict of interest.