AR222, AR249, AS274, AS702 and AS708 antibodies recognize the spike S protein from SARS-CoV-2 by immunofluorescence

Abstract

The recombinant antibodies AR222, AR249, AS274, AS702 and AS708 detect by immunofluorescence 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). Five recombinant antibodies (AR222, AR249, AS274, AS702 and AS708) successfully detect by immunofluorescence the S protein from SARS-CoV-2 (UniProt P0DTC2) expressed in Vero-B4 cells.

Materials & Methods

Antibodies: ABCD_AQ806, ABCD_AR222, ABCD_AR249, ABCD_AS273, ABCD_AS274, ABCD_AS702 and ABCD_AS708 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 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)
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

Antigen: Vero-B4 adherent cells (growing in DMEM, Gibco 11960044, supplemented with 10% FBS), were transiently transfected 24 h 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). Transfected cells were then seeded on multi-test glass slides (Thermo Fisher 15546375), and used to detect the viral protein. Non-transfected cells were used as a negative control.

Protocol: Transfected Vero-B4 cells were fixed with ice-cold Acetone/Methanol (ratio 1:1) for 10 min, and slides rehydrated for 10 min in PBS + 0.1% Tween20 (w/v) (PBS-T). Cells were then blocked in PBS-T + 0.2% BSA (w/v) for 30 min, and then incubated with the anti-S antibodies (final concentration 5 mg/L in PBS-T + BSA) for 1 h. After 3 washes with PBS-T, cells were incubated for 30 min in PBS-T + BSA with secondary goat anti-mouse IgG conjugated to AlexaFluor-488 (1:400, Molecular Probes, A11029). After 3 washes with PBS-T, slides were briefly rinsed with dH₂O, and mounted with Möwiol (Hoechst) + 2.5% (w/v) DABCO (Fluka 33480). Pictures were taken using a Zeiss LSM700 confocal microscope, with a 63x Neofluar oil immersion objective.

Results

AR222, AR249, AS274, AS702 and AS708 antibodies specifically detected a signal in Vero-B4 cells transfected with the SARS-CoV-2 S protein (Fig. 1); AQ806 was used as a positive control (Marchetti et al., 2020). The distribution observed is consistent with a presence mostly in the early secretory pathway (endoplasmic reticulum and Golgi apparatus). The specificity of the signal was verified by the absence of staining in non-transfected cells (Fig. 1).

Figure 1. Antibodies AQ806, AR222, AR249, AS274, AS702 and AS708 specifically labeled Vero-B4 cells expressing the SARS-CoV-2 S protein. No labeling was seen in non-transfected cells. Scale bar: 20 µm.

Acknowledgments

This work was co-sponsored by NASA TRISH contract #NNX16AO69A/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.