The rabbit Ab variable regions of heavy and kappa chains were PCR amplified using previously described primers and PCR conditions (Table S4 ) (McCoy et al., 2016 (link)). PCR products were purified and cloned into an expression plasmid adapted from the pFUSE-rIgG-Fc and pFUSE2-CLIg-rK1 vectors (InvivoGen) using the Gibson Assembly® Master Mix (NEB) under ampicillin selection following the manufacturer’s protocol. Ab variable regions were sequenced by Sanger sequencing.
Ab heavy and light plasmids generated through B cell sorting were co-transfected at a 1:1 ratio into HEK293F cells (Thermofisher) using PEI Max 40K (linear polyethylenimine hydrochloride, Polysciences, Inc.). Ab supernatants were harvested four days following transfection and purified using protein G affinity chromatography following the maunfacturers protocol (GE Healthcare).
Ab heavy and light plasmids generated through B cell sorting were co-transfected at a 1:1 ratio into HEK293F cells (Thermofisher) using PEI Max 40K (linear polyethylenimine hydrochloride, Polysciences, Inc.). Ab supernatants were harvested four days following transfection and purified using protein G affinity chromatography following the maunfacturers protocol (GE Healthcare).
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