Expi293f expression system

Antibody expression and purification were performed as described (7 (link)). Briefly, MO11 was expressed by the co-transfection of the antibody plasmids described above using the Expi293F Expression System (Thermo Fisher Scientific, Waltham, MA), and the antibody was purified by affinity purification with rProtein A Sepharose (Cytiva, Marlborough, MA).

cDNAs for heavy and light chains of Ra62 antibody [21 (link)] were subcloned into the pcDNA3.4 expression vector using PCR and In-Fusion Reaction (Clontech). Ra62 antibody was expressed using the Expi293F Expression System (Thermo Fisher Scientific), according to the manufacturer’s instruction. The antibody secreted in culture medium was purified by protein G Sepharose 4 Fast Flow (GE Healthcare), and then buffer was exchanged to PBS by PD-10 column (GE Healthcare). Antibody concentration was determined by extinction coefficient method [23 (link)] with NanoDrop spectrophotometer (Thermo Fisher Scientific). Purified antibody was frozen and stored at -30°C. Preparations of Ra62 antibody conjugated Sepharose (Ra62 antibody Sepharose) was conducted using NHS-activated Sepharose 4FF (GE Healthcare), according to the manufacturer’s instructions. Two mg of Ra62 antibody was immobilized on 1 mL of Sepharose. Ra62 antibody Sepharose was stored at 4°C.

cDNAs for the anti-AGIA antibody heavy and light chains [17 (link)] were subcloned into the pcDNA3.4 expression vector using PCR and In-Fusion Reaction. Anti-AGIA antibody was expressed using the Expi293F Expression System (Thermo Fisher Scientific) according to the manufacturer’s instruction. The antibody secreted in culture medium was purified by protein G sepharose 4 Fast Flow (GE Healthcare), and then buffer exchange was conducted using a PD-10 column. Purified antibody was frozen and stored at -20°C.
Preparations of antibody conjugated sepharose beads was conducted using NHS-activated Sepharose 4FF (GE Healthcare). Anti-AGIA antibody sepharose was stored at 4°C. HRP-conjugated antibody was prepared using the HRP Conjugation Kit (Abcam). Anti-AGIA antibody-HRP was stored at -30°C.

Proteins of the pre-fusion stabilized spike ectodomain of each SARS-CoV-2 variant, RBD (spike 334–528), and the ectodomain of human ACE2 (1-614) with mouse Fc were prepared as described (7 (link), 23 (link)). Briefly, the pCAGGS plasmid harboring each gene was transfected to HEK293T cells by polyethyleneimine, and the supernatant was collected 4–5 days later. The Expi293F expression System (Thermo Fisher Scientific) was also used according to the manufacturer’s protocol. As all of the gene constructs included C-terminal His₆ sequences, the expressed proteins were purified by nickel-charged nitriloacetic acid agarose (Qiagen, Hilden, Germany).

Full-length, coding sequences (CDS) of SmTrx1 (Smp_008070.1) and SmVAL28 (Smp_176160) were obtained from Wormbase Parasite (parasite.wormbase.org). To prevent incorporation of mammalian glycans into the final protein, Asn-Gln substitutions were made at predicted sites of N-linked glycosylation (using NetNGlyc 1.0 (http://www.cbs.dtu.dk/services/NetNGlyc/)) at AA71 of SmTrx1 and AA6 of SmVAL28. Sequences were then codon-optimized for Homo sapiens prior to gene synthesis (GeneArt, Thermo Fisher Scientific). A C-terminal 6-His tag was incorporated into the target sequences for downstream purification. The SmTrx1 and SmVAL28 sequences were then cloned into the pSecTAG2A expression vector (Thermo Fisher Scientific) and sequenced by dideoxy chain-termination sequencing (LGTC sequencing facility, LUMC) to ensure in-frame insertion of correct sequences. The constructs were then transfected into Exp293F cells as per the manufacturer’s instructions for the Expi293F expression system (Gibco).