AS739, AT693, AU197 and AU734 antibodies label the spike S protein from SARS-CoV-2 by western blot

Abstract

The recombinant antibodies AS739, AT693, AU197 and AU734 detect by western blot 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 western blot. Four antibodies (AS739, AT693, AU197 and AU734) detected the S protein from SARS-CoV-2; one (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: Vero-B4 adherent cells (growing in DMEM, Gibco 11960044, supplemented with 10% FBS) were transiently transfected 48 hours before the experiment with a vector coding for the full-length SARS-CoV-2 S protein (BEI Resources, NR-52310, pCAGGS vector containing the full-length SARS-CoV-2/Wuhan-Hu-1 S glycoprotein coding sequence). Non-transfected cells were used as a negative control.

Protocol: 5x10⁵ transfected Vero-B4 cells were pelleted and lysed in PBS containing 0.5% (v/v) Triton X-100. Nuclei were pelleted by centrifugation (5 min at 12’000 g) and the supernatant was recovered and mixed with reducing or non-reducing sample buffer (20.6% (w/v) sucrose, 100 mM Tris pH 6.8, 10 mM EDTA, 0.1% (w/v) bromophenol blue, 4% (w/v) SDS, +/- 6% (v/v) ß-mercaptoethanol). Each sample was migrated (200 V, 30 min) in a 4-20% acrylamide gel (SurePAGE Bis-Tris, Genscript M00655), and transferred to a nitrocellulose membrane using a dry transfer system for 10 min (iBlot gel transfer device, Invitrogen IB1001EU). The membranes were blocked 2 h in PBS containing 0.1% (v/v) Tween20 and 7% (w/v) milk and washed three times for 5 min in PBS + 0.1% (v/v) Tween20. The membranes were then incubated with the recombinant anti-S antibodies (dilution 1:10 in PBS-Tween-milk) for 30 min at room temperature and washed three times for 5 min. The membranes were then incubated for 30 min with horseradish peroxidase-coupled goat anti-rabbit IgG (Sigma-Aldrich A8275, dilution 1:3000 in PBS-Tween-milk) and washed 5 times for 5 min in PBS-Tween. The signal was revealed by enhanced chemiluminescence (ECL) (Millipore) using a PXi-4 gel imaging system (Syngene).

Results

Antibodies AS739, AT693, AU197 and AU734 recognized by western blot the SARS-CoV-2 spike S protein expressed in Vero-B4 transfected cells and migrated in non-reducing conditions (Fig. 1). No signal was detected in non-transfected cells, or in reducing conditions (Fig. 1). AU753 did not recognize the S protein; this is possibly due to the fact that this antibody is poorly produced. Three bands were observed with the antibodies AS739 and AU197: (i) a ~120 kDa band, presumably corresponding to the full-length spike protein; (ii) a higher molecular weight band, likely a spike oligomer; and (iii) a smaller band at ~70 kDa, presumably corresponding to the processed S1 subunit of the spike protein. Antibodies AT693 and AU734 only recognized the high-molecular form of the protein. A weak signal was obtained with the antibody AT693.

Figure 1. Specific binding of AS739, AU197, AU734 and AT693 antibodies to the SARS-CoV-2 S protein in Vero-B4 transfected (T) cells in non-reducing (NR) conditions, but not in reducing (R) conditions. No band was observed in non-transfected (NT) cells. AU753 did not recognize the S protein by western blot.

Conflict of interest

The authors declare no conflict of interest.