AS739, AT693 and AU734 antibodies label the spike S protein from SARS-CoV-2 by immunofluorescence

Authors

  • Cyril Guilhen cyril.guilhen@unige.ch UNIGE
  • Ezgi Gozlugol
  • Margaux Gosetto
  • Nina Payot
  • Khatiba Khatibi
  • Emma Jaques
  • Marie N. Schmid
  • Julien Ollivier
  • Alexandre P. Vaudano
  • François Prodon
  • Maxime Volery
  • Clément Poncet
  • Nylsa Chammartin
  • Célia Lazzarotto
  • Sara Da Fonte
  • Anthony Nemeth
  • Clément Bindschaedler
  • Zacharie El Matribi
  • Serkan Berkcan
  • Ezia Oppliger
  • Daniel Gil
  • Ali Sassi

DOI:

https://doi.org/10.24450/journals/abrep.2021.e305

Abstract

The recombinant antibodies AS739, AT693 and AU734 detect by immunofluorescence the spike S protein from SARS-CoV-2.

Introduction

The spike S glycoprotein (UniProt P0DTC2) mediates the attachment of coronaviruses to the host ACE2 receptor (through the Receptor-Binding Domain [RBD] in the S1 subunit) and fusion with the host cell membrane (through the S2 subunit) (Yan et al., 2020). Five recombinant antibodies recognizing the S1 domain of the S protein from SARS-CoV-2 (AS739, AT693, AU197, AU734 and AU753) were tested for their ability to recognize the S protein by immunofluorescence. Three antibodies (AS739, AT693 and AU734) detected specifically the S protein from SARS-CoV-2; two others (AU197 and AU753) did not.

Materials & Methods

Antibodies: ABCD_AS739, ABCD_AT693, ABCD_AU197, ABCD_AU734 and ABCD_AU753 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 rabbit IgG 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). HEK293 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 (see Table 1 for individual yields) were collected after 4 days.

ABCD Clone Epitope Reference Yield ( mg /L)
AS739 S309 S1/RBD Pinto et al., 2020 100
AT693 BD-23 S1/RBD Cao et al., 2020 80
AU197 2B04 S1/RBD Alsoussi et al., 2020 30
AU734 2-43 S1/RBD Liu et al., 2020 40
AU753 Mab362 S1/RBD Ejemel et al., 2020 10
Table 1. Clone number, epitope, reference and production yields for the antibodies used in this study.

Antigen: Vero-B4 adherent cells (growing in DMEM, Gibco 11960044, supplemented with 10% FBS) were transiently transfected 3 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). Non-transfected cells were used as a negative control.

Protocol: Transfected Vero-B4 cells were fixed with methanol at -20 °C for 2 min. Fixed cells were washed once in PBS and once in PBS + 0.2% (w/v) BSA (PBS-BSA) during 10 min, and then incubated with the anti-S antibodies (final concentration 5 mg/L in PBS-BSA) for 30 min. After 3 washes (10 min) with PBS-BSA, cells were incubated for 30 min in PBS-BSA with secondary goat anti-rabbit IgG conjugated to AlexaFluor-488 (1:400, Molecular Probes A11034). 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 LSM800 confocal microscope, with a 63x Neofluar oil immersion objective.

Results

Antibodies AS739, AT693 and AU734 specifically detected a signal in Vero-B4 cells expressing the SARS-CoV-2 S protein (Fig. 1). The absence of staining in non-transfected cells indicated the specificity of the signal observed. The distribution observed is consistent with a presence mostly in the early secretory pathway (endoplasmic reticulum and Golgi apparatus). AU753 did not recognize the S protein; this is possibly due to the fact that this antibody is poorly produced. A non-specific signal was observed with the AU197 antibody in non-transfected cells.

Figure 1. AS739, AT693 and AU734 specifically labeled transfected Vero-B4 cells expressing the SARS-CoV-2 S protein. AU753 did not recognize the S protein. No labeling was seen in non-transfected cells except with the antibody AU197, which gives a non-specific signal in these conditions. Scale bar: 20 µm.

Conflict of interest

The authors declare no conflict of interest.

References

Alsoussi WB, Turner JS, Case JB, et al. A potently neutralizing antibody protects mice against SARS-CoV-2 infection. J Immunol. 2020; 205:915-22. PMID: 32591393

Cao Y, Su B, Guo X, et al. Potent neutralizing antibodies against SARS-CoV-2 identified by high-throughput single-cell sequencing of convalescent patients’ B cells. Cell 2020; 182:73-84. PMID: 32425270

Ejemel M, Li Q, Hou S, et al. A cross-reactive human IgA monoclonal antibody blocks SARS-CoV-2 spike-ACE2 interaction. Nat Commun. 2020; 11:4198. PMID: 32826914

Liu L, Wang P, Nair MS, et al. Potent neutralizing antibodies against multiple epitopes on SARS-CoV-2 spike. Nature 2020; 584:450-6. PMID: 32698192

Pinto D, Park Y-J, Beltramello M, et al. Cross-neutralization of SARS-CoV-2 by a human monoclonal SARS-CoV antibody. Nature 2020; 583: 290-5. PMID: 32422645

Yan R, Zhang Y, Li Y, Xia L, Guo Y, Zhou Q. Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2. Science 2020; 367:1444-1448. PMID: 32132184

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Published

2021-01-14

Section

Article

How to Cite

1.
Guilhen C, Gozlugol E, Gosetto M, Payot N, Khatibi K, Jaques E, Schmid MN, Ollivier J, Vaudano AP, Prodon F, Volery M, Poncet C, Chammartin N, Lazzarotto C, Da Fonte S, Nemeth A, Bindschaedler C, El Matribi Z, Berkcan S, Oppliger E, Gil D, Sassi A. AS739, AT693 and AU734 antibodies label the spike S protein from SARS-CoV-2 by immunofluorescence. Antib. Rep. [Internet]. 2021 Jan. 14 [cited 2024 May 24];4(1):e305. Available from: https://oap.unige.ch/journals/abrep/article/view/305

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