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 |
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.