AI334, AQ806, AR222, AR249, AS274, AS702, AS708, RB590, RB591 and RB596 antibodies recognize the spike S protein from SARS-CoV-2 by western blot

Abstract

The recombinant antibodies AI334, AQ806, AR222, AR249, AS274, AS702, AS708, RB590, RB591 and RB596 detect by western blot the spike S protein from SARS-CoV-2.

Introduction

The spike (S) glycoprotein mediates attachment of coronaviruses to the host ACE2 receptor and fusion with the host cell membrane (Yan et al., 2020). Ten recombinant antibodies (AI334, AQ806, AR222, AR249, AS274, AS702, AS708, RB590, RB591 and RB596) successfully detect by western blot the spike S protein from SARS-CoV-2 (UniProt P0DTC2) expressed in Vero-B4 cells.

Materials & Methods

Antibodies: ABCD_AI334, ABCD_AQ806, ABCD_AR222, ABCD_AR249, ABCD_AS274, ABCD_AS702, ABCD_AS708, ABCD_RB590, ABCD_RB591 and ABCD_RB596 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 mouse IgG2A Fc. The synthesized scFv or VHH 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). HEK 293T suspension cells (growing in FreeStyle™ 293 Expression Medium, Gibco 12338) were transiently transfected with the vector coding for the scFv-Fc or VHH-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)
AI334	CR3022	S1	ter Meulen et al., 2006	50
AQ806	VHH-72	S1/RBD	Wrapp et al., 2020	50
AR222	Sb#14	S1/RBD	Walter et al., 2020	60
AR249	Sb#45	S1/RBD	Walter et al., 2020	100
AS274	H4	S1/RBD	Wu et al., 2020	20
AS702	CV24	S1	Seydoux et al., 2020	20
AS708	CV30	S1/RBD	Seydoux et al., 2020	20
RB590	RB590	Cytosolic	this work	50
RB591	RB591	Cytosolic	this work	120
RB596	RB596	S2	Farrera-Soler et al., 2020	100

Antigen: Vero-B4 adherent cells (growing in DMEM, Gibco 31966021, supplemented with 10% FBS), transiently transfected 2 days 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), were used to detect the full-length S protein. Non-transfected cells were used as a negative control.

Protocol: 5x10⁶ cells were pelleted and lysed for 15 min in 100 L of ice-cold lysis buffer (25 mM Tris-HCl pH 7.4 + 0.5 % Triton X-100 + 120 mM NaCl) containing protease inhibitors. Lysate was centrifuged 15 min, 10’000 g at 4 °C to remove nuclei. One volume of reduced sample buffer was added to the lysate (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) -mercaptoethanol) and boiled for 15 min at 95 ^oC. 10 L of each sample (2.5x10⁵ cells) was migrated (150 V, 45 min) in a 4-20% acrylamide gel (Genscript, SurePAGE Bis-Tris, M00655), and transferred to a nitrocellulose membrane using a dry transfer system for 7 minutes (iBlot gel transfer device, Invitrogen IB23001). The membranes were blocked during 60 min in PBS containing 0.1% (v/v) Tween20 and 7% (w/v) milk, and washed once for 15 minutes in PBS + 0.1% (v/v) Tween20 (PBS-Tween). The membranes were then incubated overnight at RT with the anti-S antibodies (final concentration 5 mg/L in PBS-Tween). The membranes were then washed three times (15+15+10 min) in PBS-Tween, incubated for 1 hour with the horseradish peroxidase-coupled goat anti-mouse IgG (Biorad, 170-6516, dilution 1:3000) and washed three times (15 min) in PBS-Tween. The signal was revealed by enhanced chemiluminescence (ECL) (Amersham Biosciences) using a PXi-4 gel imaging systems (Syngene).

Results

AI334, AQ806, AR222, AR249, AS274, AS702, AS708, RB590, RB591 and RB596 antibodies specifically recognize the S protein in Vero-B4 transfected cells (Fig. 1). The ~180 kDa and the ~90 kDa bands correspond to the full-length and cleaved S proteins; higher molecular weight bands (>250 kDa) correspond to oligomerized (dimeric or trimeric) S proteins (Ou et al., 2020). The AI334 antibody also recognizes a non-specific band (~250 kDa) on non-transfected cells (Fig. 1).

Figure 1. Antibodies AI334, AQ806, AR222, AR249, AS274, AS702, AS708, RB590, RB591 and RB596 specifically recognize the spike S protein from SARS-CoV-2 (the asterisks (*) denote the main forms of the protein: cleaved, full-length, and oligomerized; shown only for AQ806).

Acknowledgments

This work was co-sponsored by NASA TRISH contract #NNX16A069A/CAT0001. The following reagent was obtained through BEI Resources, NIAID, NIH: Vector pCAGGS containing the SARS-related Coronavirus 2, Wuhan-Hu-1 Spike glycoprotein gene, NR-52310.

Conflict of interest

The authors declare no conflict of interest.