AI334, AQ806, AR222, AR249, AS274, AS702, AS708, RB590, RB591 and RB596 antibodies recognize the spike S protein from SARS-CoV-2 by western blot

Authors

  • Anna Marchetti
  • Frederic Zenhausern

DOI:

https://doi.org/10.24450/journals/abrep.2022.e725

Abstract

The recombinant antibodies AI334, AQ806, AR222, AR249, AS274, AS702, AS708, RB590, RB591 and RB596 detect by western blot 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). Ten recombinant antibodies (AI334, AQ806, AR222, AR249, AS274, AS702, AS708, RB590, RB591 and RB596) successfully detect by western blot the spike S protein from SARS-CoV-2 (UniProt P0DTC2) expressed in Vero-B4 cells.

Materials & Methods

Antibodies: ABCD_AI334, ABCD_AQ806, ABCD_AR222, ABCD_AR249, ABCD_AS274, ABCD_AS702, ABCD_AS708, ABCD_RB590, ABCD_RB591 and ABCD_RB596 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 or VHH 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)
AI334 CR3022 S1 ter Meulen et al., 2006 50
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
RB590 RB590 Cytosolic this work 50
RB591 RB591 Cytosolic this work 120
RB596 RB596 S2 Farrera-Soler et al., 2020 100
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 31966021, supplemented with 10% FBS), transiently transfected 2 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), were used to detect the full-length S protein. Non-transfected cells were used as a negative control.

Protocol: 5x106 cells were pelleted and lysed for 15 min in 100 L of ice-cold lysis buffer (25 mM Tris-HCl pH 7.4 + 0.5 % Triton X-100 + 120 mM NaCl) containing protease inhibitors. Lysate was centrifuged 15 min, 10’000 g at 4 °C to remove nuclei. One volume of reduced sample buffer was added to the lysate (20.6% (w/v) sucrose, 100 mM Tris pH 6.8, 10 mM EDTA, 0.1% (w/v) bromophenol blue, 4% (w/v) SDS, 6% (v/v) -mercaptoethanol) and boiled for 15 min at 95 oC. 10 L of each sample (2.5x105 cells) was migrated (150 V, 45 min) in a 4-20% acrylamide gel (Genscript, SurePAGE Bis-Tris, M00655), and transferred to a nitrocellulose membrane using a dry transfer system for 7 minutes (iBlot gel transfer device, Invitrogen IB23001). The membranes were blocked during 60 min in PBS containing 0.1% (v/v) Tween20 and 7% (w/v) milk, and washed once for 15 minutes in PBS + 0.1% (v/v) Tween20 (PBS-Tween). The membranes were then incubated overnight at RT with the anti-S antibodies (final concentration 5 mg/L in PBS-Tween). The membranes were then washed three times (15+15+10 min) in PBS-Tween, incubated for 1 hour with the horseradish peroxidase-coupled goat anti-mouse IgG (Biorad, 170-6516, dilution 1:3000) and washed three times (15 min) in PBS-Tween. The signal was revealed by enhanced chemiluminescence (ECL) (Amersham Biosciences) using a PXi-4 gel imaging systems (Syngene).

Results

AI334, AQ806, AR222, AR249, AS274, AS702, AS708, RB590, RB591 and RB596 antibodies specifically recognize the S protein in Vero-B4 transfected cells (Fig. 1). The ~180 kDa and the ~90 kDa bands correspond to the full-length and cleaved S proteins; higher molecular weight bands (>250 kDa) correspond to oligomerized (dimeric or trimeric) S proteins (Ou et al., 2020). The AI334 antibody also recognizes a non-specific band (~250 kDa) on non-transfected cells (Fig. 1).

Figure 1. Antibodies AI334, AQ806, AR222, AR249, AS274, AS702, AS708, RB590, RB591 and RB596 specifically recognize the spike S protein from SARS-CoV-2 (the asterisks (*) denote the main forms of the protein: cleaved, full-length, and oligomerized; shown only for AQ806).

Acknowledgments

This work was co-sponsored by NASA TRISH contract #NNX16A069A/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

Farrera-Soler L, Daguer JP, Sofia Badiola B, Winssinger N. The RB596 antibody recognizes a linear epitope from the spike S protein from SARS-CoV-2. Antibody Reports, 2020, 3:e232. doi:10.22450/ journals/abrep.2020.e232

Ou X, Liu Y, Lei X, et al. Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV. Nat Commun. 2020; 11:1620. PMID: 32221306

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.

ter Meulen J, van den Brink EN, Poon LL, et al. Human monoclonal antibody combination against SARS coronavirus: synergy and coverage of escape mutants. PLoS Med. 2006; 3:e237. PMID:16796401

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

2022-03-29

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Article

How to Cite

1.
Marchetti A, Zenhausern F. AI334, AQ806, AR222, AR249, AS274, AS702, AS708, RB590, RB591 and RB596 antibodies recognize the spike S protein from SARS-CoV-2 by western blot. Antib. Rep. [Internet]. 2022 Mar. 29 [cited 2024 May 22];5(1):e725. Available from: https://oap.unige.ch/journals/abrep/article/view/725

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