AR222, AR249, AS274, AS702 and AS708 antibodies recognize the spike S protein from SARS-CoV-2 by immunofluorescence

Authors

  • Anna Marchetti
  • Frederic Zenhausern

DOI:

https://doi.org/10.24450/journals/abrep.2020.e228

Abstract

The recombinant antibodies AR222, AR249, AS274, AS702 and AS708 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). Five recombinant antibodies (AR222, AR249, AS274, AS702 and AS708) successfully detect by immunofluorescence the S protein from SARS-CoV-2 (UniProt P0DTC2) expressed in Vero-B4 cells.

Materials & Methods

Antibodies: ABCD_AQ806, ABCD_AR222, ABCD_AR249, ABCD_AS273, ABCD_AS274, ABCD_AS702 and ABCD_AS708 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 mouse IgG2A 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). HEK 293T suspension cells (growing in FreeStyle™ 293 Expression Medium, Gibco 12338) were transiently transfected with the vector coding for the scFv-Fc or VHH-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 50
AR222 Sb#14 S1/RBD Walter et al., 2020 60
AR249 Sb#45 S1/RBD Walter et al., 2020 100
AS274 H4 S1/RBD Wu et al., 2020 20
AS702 CV24 S1 Seydoux et al., 2020 20
AS708 CV30 S1/RBD Seydoux et al., 2020 20
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 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

AR222, AR249, AS274, AS702 and AS708 antibodies specifically detected a signal in Vero-B4 cells transfected with the SARS-CoV-2 S protein (Fig. 1); AQ806 was used as a positive control (Marchetti et al., 2020). 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 AQ806, AR222, AR249, AS274, AS702 and AS708 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.

References

Marchetti A, Hammel P, Zenhausern F. AI334, AQ806 and RB596 antibodies recognize the spike S protein from SARS-CoV-2 by immunofluorescence. Antib. Rep. 2020, 3:e219. doi:10.24450/journals/abrep.2020.e219

Seydoux E, Homad LJ, MacCamy AJ, et al. Analysis of a SARS-CoV-2-infected individual reveals development of potent neutralizing antibodies with limited somatic mutation. Immunity 2020; 53:1-8. PMID: 32561270.

Walter JD, Hutter CAJ, Zimmermann I, et al. Sybodies targeting the SARS-CoV-2 receptor-binding domain. Preprint. bioRxiv 2020; 2020.04.16.045419. doi:10.1101/2020.04.16.045419

Wrapp D, De Vlieger D, Corbett KS, et al. Structural basis for potent neutralization of betacoronaviruses by single-domain camelid antibodies. Cell 2020; 181:1004-1015. PMID:32375025

Wu Y, Wang F, Shen C, et al. A noncompeting pair of human neutralizing antibodies block COVID-19 virus binding to its receptor ACE2. Science 2020; 368:1274-1278. PMID:32404477

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

2020-07-13

Section

Article

How to Cite

1.
Marchetti A, Zenhausern F. AR222, AR249, AS274, AS702 and AS708 antibodies recognize the spike S protein from SARS-CoV-2 by immunofluorescence. Antib. Rep. [Internet]. 2020 Jul. 13 [cited 2024 May 19];3(4):e228. Available from: https://oap.unige.ch/journals/abrep/article/view/228

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