Protein a agarose resin

TB31F was created by fusing the variable regions to the human IGHG*01 or IGLC2*02 constant regions and cloning into the pHL-sec plasmid (GenScript). Heavy and light chain plasmids were mixed in equal amounts and transfected into expi293F cells according to manufacturer instructions and cell-free supernatant was harvested after 4 days of expression.
Cell-free supernatant was batch incubated with protein A agarose resin (GoldBio) for 1 h at room temperature. Resin was collected and washed with 10 column volumes (CV) protein A IgG binding buffer (ThermoFisher Scientific). Protein was eluted with 10 CV IgG elution buffer (ThermoFisher Scientific) and neutralized with 1 CV 1 M Tris pH 9.0. Antibody was concentrated and buffer exchanged into PBS using an Amicon centrifugal filter (MilliporeSigma).

Antibodies Ab-217 and Ab-218 (anti-EBA-175-RII), N3 and N4 (anti-AMA1), and 8C2 were purified from hybridoma supernatants using Protein A agarose resin (Gold Biotechnology, St. Louis, MO). Antibodies were buffer exchanged into PBS after purification and sterile filtered for use in parasite cultures. The heavy and light chain variable segments of 9AD4 as deposited in PDB 4U0R (Wright et al., 2014 (link)) were grafted onto a mouse IgG2a backbone (AF466698.1) and a mouse kappa light chain backbone (AM745100.1), respectively, codon-optimized and cloned into mammalian expression vector pHLSec. A 1:1 ratio of 9AD4 heavy and light chain constructs were transiently transfected into 293 F cells using polyethylenimine (PEI) at a ratio of 1:3 DNA:PEI. Five days post transfection, cultures were harvested and the 9AD4 IgG was purified from the supernatant by Q sepharose resin (GE Healthcare, Marlborough, MA) followed by protein A resin. The purified protein was then buffer exchanged into PBS and sterile filtered for use in parasite cultures. The control Ab-DBP (α-PvDBP) Ab was generously provided by the lab of John Adams (University of South Florida).

Bulk anti-iRBD serum was generated by Covance. Purified recombinant EnvP(b)1 iRBD was used to immunize a single rabbit. Serum from the terminal bleed was tested for detection of EnvP(b)1 by Western blot. Crude rabbit serum was diluted 1:50 in buffer A. Bulk IgGs were captured by protein A agarose resin (GoldBio) at 4°C with rotating overnight. The resin was collected in a chromatography column, washed with a column volume of buffer A, and eluted with 0.1 M glycine buffer (pH 2.5), which was immediately neutralized by 1 M Tris (pH 8.0). Antibodies were then dialyzed against phosphate-buffered saline (PBS) (pH 7.4). To produce an EnvP(b)1 iRBD affinity resin, EnvP(b)1 was expressed and purified as described above. Cleaved iRBD was then concentrated, dialyzed against phosphate-buffered saline (PBS) (pH 7.4), and arrested to Pierce N-hydroxysuccinimide (NHS)-activated agarose beads per the manufacturer’s protocol. The reaction was then quenched by the addition of Tris (pH 7.5). Rabbit antibodies were then incubated with the iRBD resin at 4°C with rotating overnight. The iRBD resin was collected in a chromatography column, washed with a column volume of PBS (pH 7.4), and eluted with 0.1 M glycine buffer (pH 2.5), which was immediately neutralized by 1 M Tris (pH 8.0). Antibodies were then dialyzed against PBS (pH 7.4).