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

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.

Table 1. Clone number, epitope, reference and production yields for the antibodies used in this study.

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

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

The authors declare no conflict of interest.