AQ806, AS739, AT693, AU197 and AU734 antibodies recognize the spike S protein from SARS-CoV-2 by flow cytometry

Authors

  • Margaux Gosetto University of Geneva
  • Ezgi Gozlugol
  • Nylsa Chammartin
  • Célia Lazzarotto
  • Julien Ollivier
  • Ezia Oppliger
  • Marie N. Schmid
  • Clément Bindschaedler
  • Ali Sassi
  • Cyril Guilhen Cyril.Guilhen@unige.ch
  • Anthony Nemeth
  • Khatiba Khatibi
  • Daniel Gil
  • Clément Poncet
  • Maxime Volery
  • Sara Da Fonte
  • Emma Jaques
  • Zacharie El Matribi
  • Serkan Berkcan
  • Nina Payot
  • Alexandre P. Vaudano
  • Jean-Pierre Aubry-Lachainaye

DOI:

https://doi.org/10.24450/journals/abrep.2021.e278

Abstract

The recombinant antibodies AQ806, AS739, AT693, AU197 and AU734 detect by flow cytometry 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). Six recombinant antibodies recognizing the S1 domain of the S protein from SARS-CoV-2 (AQ806, AS739, AT693, AU197, AU734 and AU753) were tested for their ability to recognize the S protein by flow cytometry. Five antibodies (AQ806, AS739, AT693, AU197 and AU734) detected the S protein from SARS-CoV-2; one (AU753) did not.

Materials & Methods

Antibodies: ABCD_AQ806, 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)
AQ806 VHH-72 S1/RBD Wrapp et al., 2020 100
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: 106 transfected cells were pelleted and fixed with 250 µL of fixation solution (BD Biosciences 554714). After 20 min of incubation on ice, cells were pelleted and washed twice with 1 mL of Perm/Wash buffer (BD Biosciences 554714). Cells were then incubated for 30 min with the recombinant antibodies diluted at 1 mg/L in Perm/Wash buffer. After two washes in Perm/Wash buffer, cells were incubated for 20 min with the secondary goat anti-rabbit IgG conjugated to Alexa Fluor 488 (1:400 in Perm/Wash buffer; Molecular Probes #A11034). After three washes in Perm/Wash buffer, cells were resuspended in 500 µL of Perm/Wash buffer and analyzed with a flow cytometer (BD AccuriTM C6).

Results

Antibodies AQ806, AS739, AT693, AU197 and AU734 detected the SARS-CoV-2 spike S protein in Vero-B4 transfected cells. No signal was detected in non-transfected cells (Fig. 1). AU753 did not recognize the S protein by flow cytometry; this is possibly due to the fact that this antibody is poorly produced.

Figure 1. Bi-parametric representation of flow cytometry analysis depicting Forward Scatter (FSC) and Alexa Fluor 488 signal. AQ806, AS739, AT693, AU197 and AU734 antibodies labeled Vero-B4 transfected cells (T) overexpressing the SARS-CoV-2 spike S protein. No signal was detected in non-transfected cells (NT). AU753 did not recognize the S protein by flow cytometry. Transfection efficiency was estimated to be around 30%.

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

Wrapp D, Wang N, Corbett KS, et al. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science 2020; 367:1260-1263. PMID: 32075877

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

Downloads

Published

2021-01-14

Section

Article

How to Cite

1.
Gosetto M, Gozlugol E, Chammartin N, Lazzarotto C, Ollivier J, Oppliger E, Schmid MN, Bindschaedler C, Sassi A, Guilhen C, Nemeth A, Khatibi K, Gil D, Poncet C, Volery M, Da Fonte S, Jaques E, El Matribi Z, Berkcan S, Payot N, Vaudano AP, Aubry-Lachainaye J-P. AQ806, AS739, AT693, AU197 and AU734 antibodies recognize the spike S protein from SARS-CoV-2 by flow cytometry. Antib. Rep. [Internet]. 2021 Jan. 14 [cited 2024 May 24];4(1):e278. Available from: https://oap.unige.ch/journals/abrep/article/view/278

Most read articles by the same author(s)

<< < 1 2 3 > >>