ixcell_PBMC_RSV_phage_Pan0
Briefly, total RNA was extracted from ~10 million PBMCs for single-stranded cDNA synthesis using SuperScriptTM IV (Invitrogen) with random hexamer and oligo(dT) 12-18 primers. Antibody heavy chain (HC) and light chain (LC) variable regions were obtained by polymerase chain reaction (PCR) using mixed HuJ reverse primers and separate HuV forward primers (87). To generate HC-LC fragments, overlap PCR was performed in 25  50 l reactions (10 cycles) with 50 ng of gel-purified HC and 50 ng of gel-purified LC (equal amounts of  and  LC) without primer. Subsequent PCR was performed in 50  50 l reactions (15 cycles) with SfiI-F and SfiI-R primers using 100 ng of gel-purified HC-LC as a template to obtain full-length scFv inserts. The scFv inserts and phagemid vector, pAdLTM-20c (Antibody Design Labs), were then digested with SfiI and purified through gel extraction. The scFv DNA (256 ng) was ligated into the phagemid vector (400 ng) using the T4 Ligase Kit (New England BioLabs) in 25  40 l reactions and incubated at 16C overnight. After purification, the phagemids were electroporated into phage-competent TG1 bacterial cells (Lucigen) with the MicroPulsuerTM system (Bio-Rad). Specifically, 1 l of phagemid and 25 l of competent cells were incubated in a 0.1 cm cuvette for electroporation with the preset program at 1.8 kV. The transformed bacteria were spread on 2YT agar plates supplemented with 100 g/ml carbenicillin and 2% (w/v) glucose, which were then incubated at 37C overnight. Bacteria were then scraped from the plates for phage culture with the helper phage CM13 (Antibody Design Labs) for biopanning. The purified UFCR1-P2-NQ trimer was conjugated with DynabeadsTM M-270 beads before biopanning. A total of four biopanning steps were performed, incorporating up to 5 washes in each step to eliminate phages that were either not bound or showed low affinity for the RSV-F trimer. Lastly, plasmids from each biopanning step were extracted from 50 ml of the bacterial suspension and used for the subsequent deep sequencing and bioinformatics analyses of scFv libraries.