Cnbr sepharose

The anti-EgAgB VHH clone 1 (for simplicity anti-EgAgB clone 1) was expressed in 500 mL of liquid LB medium by IPTG induction as described above. After 4 hours induction, the culture was centrifuged at 6,000 x g at 4°C for 15 min. The supernatant was discarded and the cellular pellet was washed using LB medium (25 mL), centrifuged as mentioned above and suspended in PBS (10 mL). The cells were then lysed with 0.2 M Tris-HCl pH 8.0 containing 0.5 mM EDTA and 0.5 M sucrose. The lysed cells were then centrifuged at 17,000 x g at 4°C for 20 min and the supernatant was filtered through 0.45 μM pore size filters. VHH purification was performed on Ni-NTA columns in the ÄKTA purification system (General Electric Healthcare, Uppsala, Sweden) according to the manufacturer’s instructions. The purified anti-EgAgB clone 1 was covalently bound to CNBr-Sepharose (GE Healthcare) using a ratio of 10 mg protein/mL matrix and following the protocol recommended by the manufacturer. The efficiency of coupling was around 99% and was estimated by measuring the absorbance at 280 nm of the liquid phase after conjugation. To avoid any possible contamination between native and recombinant EgAgB samples, the obtained anti-EgAgB clone 1-Sepharose matrix was divided into two immunoaffinity columns (1.5 mL each).

Metabolic labeling using Easytag Express [³⁵S]-Met/Cys protein labeling, Perkin Elmer with 100 Ci/ml for 2 hr, and immunoprecipitation of Fcγ fragments using CNBr-Sepharose (GE Healthcare) was performed as described previously (Sprague et al., 2008 (link)). Generation and purification of human Fcγ fragments wtFc and nbFc are described elsewhere (Sprague et al., 2004 (link)). In brief, wild-type and mutant IgG Fc proteins were collected from CHO cell supernatants and purified using Ni²⁺-NTA affinity chromatography followed by pH sensitive FcRn-Sepharose column separation and subsequent size exclusion chromatography. B12 and B12-LALA were kind gifts from Ann Hessell (Hessell et al., 2007 (link)). Direct IgG precipitation was performed using Protein G Sepharose (Amersham). Samples were de-glycosylated using EndoH (NEB, as suggested by the supplier).

Anti-S-IgGs and anti-RBD-IgGs were obtained using S-Sepharose and RBD-Sepharose on an Akta Start chromatograph (GE Life Sciences, New York, NY, USA) and as described in [46 (link)]. The sorbents with immobilized S-protein and RBD were prepared according to the standard protocol using CNBr Sepharose (GE Life Sciences, New York, NY, USA) and our previously published works [46 (link),63 (link)]. An equimolar mixture of 10 IgG preparations was applied to 3 mL of RBD-Sepharose pre-equilibrated with 50 mM Tris-HCl, pH 7.5, containing 0.15 M NaCl (TBS). The fraction eluted with the acidic buffer was neutralized by adding 1/10 v/v 1.0 M Tris-HCl, pH 8.8, and then dialyzed against 20 mM Tris-HCl, pH 7.5. The IgG fraction not bound to RBD-Sepharose was applied to a 10 mL S-Sepharose column pre-equilibrated with TBS. The IgGs were eluted with a two-step gradient: 50 mM Tris-HCl, pH 7.5, containing 1.0 M NaCl and 0.1 M Gly-HCl, pH 2.6. The resulting fraction was also dialyzed against 20 mM Tris-HCl, pH 7.5.