RB572, RB574 and RB576 antibodies recognize the membrane M protein from SARS-CoV-2 by immunofluorescence
The recombinant antibodies RB572, RB574 and RB576 detect by immunofluorescence the spike S protein from SARS-CoV-2.
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.
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).
This work was co-sponsored by NASA TRISH contract #NNX16AO69A/CAT0001.
Conflict of interest
The authors declare no conflict of interest.
Hammel P, Zenhausern F. RB571, RB572, RB573, RB574, RB575, RB576, RB577 and RB578 antibodies recognize a fragment of the membrane M protein from SARS-CoV-2 by ELISA. Antib. Rep. 2020, 3:e230. doi:10.24450/ journals/abrep.2020.e230
Siu YL, Teoh KT, Lo J, et al. The M, E, and N structural proteins of the severe acute respiratory syndrome coronavirus are required for efficient assembly, trafficking, and release of virus-like particles. J Virol. 2008; 82:11318-30. PMID:18753196
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