RB572, RB574 and RB576 antibodies recognize the membrane M protein from SARS-CoV-2 by immunofluorescence

Abstract

The recombinant antibodies RB572, RB574 and RB576 detect by immunofluorescence the spike S protein from SARS-CoV-2.

Introduction

The SARS-CoV membrane (M) protein is the most abundant structural protein, driving virus assembly and budding into the lumen of the endoplasmic reticulum-Golgi intermediary compartment (ERGIC) and interacting with the E, S and N proteins (Siu et al., 2008). Here we describe the ability of three recombinant antibodies (RB572, RB574 and RB576) to successfully detect by immunofluorescence the M protein from SARS-CoV-2 (UniProt P0DTC5) expressed in Vero-B4 cells.

Materials & Methods

Antibodies: ABCD_RB572, ABCD_RB574 and ABCD_RB576 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 VHH portion fused to a mouse IgG2A Fc (Hammel and Zenhausern, 2020). HEK 293T suspension cells (growing in FreeStyle™ 293 Expression Medium, Gibco 12338) were transiently transfected with the vector coding for the VHH-Fc of each antibody. Supernatants (50-100 mg/L) were collected after 4 days.

Antigen: Vero-B4 cells (growing in DMEM, Gibco 31966, supplemented with 10% FBS), cultured on glass coverslips (Menzel-Gläser, 22x22 mm) and transiently transfected 2 days before the experiment with an expression vector coding for the full-length SARS-CoV-2 M protein, were used to detect the viral protein. Cells transfected with a vector coding for full-length SARS-CoV-2 E protein were used as a negative control.

Protocol: Two different fixation steps were used. Transfected Vero-B4 cells were either (i) fixed in PBS + 4% paraformaldehyde (w/v) (Applichem, A3013) for 30 min, blocked with PBS + 40 mM ammonium chloride (NH₄Cl) (Applichem, A3661) for 5 min, and then permeabilized in PBS + 0.2% saponin (w/v) (Sigma, S7900) for 3 min; or (ii) fixed with methanol at -20 ^oC for 3 min. Fixed cells were washed once in PBS and once with PBS + 0.2% (w/v) BSA (PBS-BSA) during 5 min, and then incubated with the anti-M antibodies (final concentration 5 mg/L in PBS + BSA) for 1 h. After 3 washes (10 min) with PBS-BSA, cells were incubated for 30 min in PBS-BSA with secondary goat anti-mouse IgG conjugated to AlexaFluor-488 (1:400, Molecular Probes, A11029). After 3 washes (10 min) with PBS-BSA, cells were mounted on slides (Menzel-Gläser, 76x26 mm) 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

RB572, RB574 and RB576 antibodies specifically detected a signal in Vero-B4 cells expressing the SARS-CoV-2 M protein, but not in cells expressing the control E protein (Fig. 1). The specificity of the signal could be verified by the absence of staining in non-transfected cells (Fig. 1, arrowheads). A specific signal was detected only when cells were fixed with methanol (Fig. 1, MeOH column), but not when paraformaldehyde was used (Fig. 1, PAF column).

Figure 1. Antibodies RB572, RB574 and RB576 specifically labeled Vero-B4 cells expressing the SARS-CoV-2 M protein (‘+M protein’), but not cells expressing the control E protein (‘Ctr’) in methanol-fixed cells (MeOH column). Arrowheads indicate unlabeled non-transfected cells. The same antibodies do not show a specific signal in cells expressing the M protein (‘+M protein’) compared to control cells (‘Ctr’) in paraformaldehyde-fixed cells (PAF column). Scale bar: 20 µm.

Acknowledgments

This work was co-sponsored by NASA TRISH contract #NNX16AO69A/CAT0001.

Conflict of interest

The authors declare no conflict of interest.