AI334 and AQ806 antibodies recognize the spike S protein from SARS-CoV-2 by ELISA

Authors

  • Philippe Hammel
  • Anna Marchetti
  • Wanessa C Lima
  • Kelvin Lau
  • Florence Pojer
  • David Hacker
  • Pierre Cosson

DOI:

https://doi.org/10.24450/journals/abrep.2020.e186

Abstract

We tested 10 recombinant antibodies directed against the spike S protein from SARS-CoV-1. Among them, antibodies AI334 and AQ806 detect by ELISA the spike S protein from SARS-CoV-2.

Introduction

The spike (S) glycoprotein (UniProt #P0DTC2) mediates 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). Here we describe the ability of two recombinant antibodies (AI334 and AQ806) to detect by ELISA the soluble ectodomain of the S protein from SARS-CoV-2.

Materials & Methods

Antibodies: ABCD_AA831, ABCD_AF167, ABCD_AF618, ABCD_AH286, ABCD_AH287, ABCD_AH971, ABCD_AH974, ABCD_AI334, ABCD_AQ601, ABCD_AQ602, and ABCD_AQ806 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 scFv 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). 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 5 days.

A BCD Clone Epitope Reference Yield ( mg /L)
AA831 80R S1/RBD Sui et al., 2004 100
AF167 S230.15 S1/RBD Rockx et al., 2008 80
AF618 S227.14 S1/RBD Rockx et al., 2008 80
AH286 m396 S1/RBD Prabakaran et al., 2006 120
AH287 F26G19 S1/RBD Berry et al., 2004 <5
AH974 CR3013 S1 van den Brink et al., 2005 120
AI334 CR3022 S1 ter Meulen et al., 2006 50
AQ601 3C7 S1/RBD Coughlin et al., 2007 50
AQ602 4D4 S1 Coughlin et al., 2007 <5
AQ806 VHH-72 S1/RBD Wrapp et al., 2020a 100
AH971 CR3009 N protein van den Brink et al., 2005 150
Table 1. : Clone number, epitope, reference and production yields for the antibodies used in this study.

Antigen: The prefusion ectodomain (residues 1-1208) of the SARS-CoV-2 S protein, with a KV->PP substitution at residues 986/987, a RRAR->GSAS substitution at residues 682-685, and C-terminal T4 fibritin trimerization motif, protease cleavage site, TwinStrepTag and 8xHisTag (PDB #6VSB; Wrapp et al., 2020b), was transiently transfected into 25x108 suspension-adapted ExpiCHO cells (Thermo Fisher) using 1.5 mg plasmid DNA and 7.5 mg of PEI MAX (Polysciences) in 500 mL ProCHO5 medium (Lonza). Incubation with agitation was continued at 31°C and 4.5% CO2 for 5 days. The clarified supernatant was purified in two steps: via a Strep-Tactin XT column (IBA Lifesciences) followed by Superose 6 10/300 GL column (GE Healthcare) to a final concentration of 180 µg/ml in PBS.

Protocol: S protein (10 µg/ml, 50 µl/well in PBS 0.5% (w/v) BSA, 0.1% (w/v) Tween20) was immobilized on streptavidin-coated ELISA plates (Pierce #15124) for 30 min. Each well was rinsed three times with 100 μl of washing buffer (PBS + 0.5% (w/v) BSA + 0.05% (w/v) Tween20), then incubated for 1 hour with 50 µl of each antibody-containing supernatant diluted in washing buffer (Fig. 1). After rinsing 3 times (100 µl washing buffer), wells were incubated with horseradish peroxidase-coupled goat anti-mouse IgG (Bio-Rad #170-6516, dilution 1:1000, 50 μl per well) for 30 min. After 3 rinses, Tetramethylbenzidine (TMB) substrate (Sigma #T5569) was added (50 μl per well). The reaction was stopped by the addition of 25 μl of 2 M H2SO4. The absorbance (OD) was measured at 450 nm, and the absorbance at 570 nm was subtracted.

Results

We tested 10 antibodies, originally developed against the SARS-CoV-1 S protein, for detection of the SARS-CoV-2 S protein by ELISA. From these, only two (AI334 and AQ806) bound in a concentration-dependent manner to the SARS-CoV-2 protein (Fig. 1). Two other antibodies, used as negative control (AH917 against the SARS-CoV-1 N protein and RB168 against an amoeba protein), also did not bind the S protein. AI334 and AQ806 antibodies have recently been shown to bind the RBD of the SARS-CoV-2 spike protein (Yuan et al., 2020; Wrapp et al., 2020a).

Figure 1. Specific binding of AI334 and AQ806 antibodies to the SARS-CoV-2 S protein, as detected by ELISA. On the Y axis, ELISA signal (in arbitrary units). On the X axis, the antibody dilution (1:100, 1:1’000 and 1:10’000). ‘Spike’ refers to the binding to the spike S protein; ‘Ctr’ refers to the binding to biotinylated BSA.

Acknowledgments

We would like to thank Prof. Jason McLellan (University of Texas, Austin) for providing the Spike expressing construct; and Laurence Durrer and Soraya Quinche (Protein Production and Structure Core Facility, EPFL) for the help with the mammalian cell culture.

Conflict of interest

Pierre Cosson and Wanessa C. Lima are editors of the Antibody Reports journal.

References

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Published

2020-05-11

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Article

How to Cite

1.
Hammel P, Marchetti A, Lima WC, Lau K, Pojer F, Hacker D, Cosson P. AI334 and AQ806 antibodies recognize the spike S protein from SARS-CoV-2 by ELISA. Antib. Rep. [Internet]. 2020 May 11 [cited 2024 May 21];3(3):e186. Available from: https://oap.unige.ch/journals/abrep/article/view/186

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