AI334, AQ806 and RB596 antibodies recognize the spike S protein from SARS-CoV-2 by immunofluorescence

Abstract

The recombinant antibodies AI334, AQ806 and RB596 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). Three recombinant antibodies (AI334, AQ806 and RB596) successfully detect by immunofluorescence the S protein from SARS-CoV-2 (UniProt P0DTC2) expressed in Vero-B4 cells.

Materials & Methods

Antibodies: ABCD_AI334, ABCD_AQ806 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 scFv portion fused to a mouse IgG2A Fc. The synthesized scFv sequences (GeneArt, Invitrogen) of antibodies AI334 and AQ806 correspond to the sequences of the variable regions of the clones CR3022 (ter Meulen et al., 2006) and VHH-72 (Wrapp et al., 2020), respectively. Antibodies RB596 was raised via phage display against the SARS-CoV-2 S protein (Hammel et al., 2020). HEK 293T 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 (30-100 mg/L) were collected after 5 days.

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

AI334, AQ806 and RB596 antibodies specifically detected a signal in Vero-B4 cells transfected with the SARS-CoV-2 S protein (Fig. 1). 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 AI334, AQ806 and RB596 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.