Fig. 1: Selecting VHHs against purified P. aeruginosa proteins.
From: Bacterial cell surface characterization by phage display coupled to high-throughput sequencing
a Schematic of experiment. Flagellin and pilin were purified from P. aeruginosa using standard methods. In each case, a mock purification was carried out on isogenic mutants lacking the antigen of interest; this “antigen–” sample was handled and diluted similarly to the “antigen+” sample. Wells of a 96-well plate were coated with purified antigen and phage display panning was performed for 4 rounds. Phage were first applied to the antigen– (counter-selection) wells, then transferred to the antigen+ (selection) wells. b Clonal ELISAs. After the final round of selection, eluted phage from the antigen+ wells was plated and 15 clones were picked and amplified; phage-displayed VHHs were tested for binding to the antigen of interest (orange circles), or the mock, antigen− prep (blue circles) by ELISA. “Ab YU573” and “Ab YU586” are positive controls using polyclonal rabbit antiserum raised against the indicated antigen. “No 1°” is a negative control where buffer replaces the primary antibody. n = 3 independent replicates per sample. c Cell-based ELISA using phage-displayed VHHs: P. aeruginosa cells were fixed to a plate with methanol and dried, then phage-displayed VHHs were tested for binding by ELISA as in panel (b). n = 3 independent replicates per sample. d Bacterial dot blot using rVHHs: VHHs were cloned into a mammalian expression vector, expressed, and purified. P. aeruginosa cells were serially diluted and spotted onto dry nitrocellulose membranes. Total protein was estimated by Ponceau S staining (right column). Blots were then blocked, probed with rVHH or polyclonal antisera, appropriate secondary antibody, and developed with enhanced chemiluminescence (left column). e Flow cytometry of P. aeruginosa cells fixed with methanol, then stained with rVHHs or polyclonal antiserum and PE-conjugated secondary antibody. Orange traces show antigen-positive cells and blue traces show antigen-negative cells. Source data for panels (b, c) are provided as a Source Data file. Raw data for panel (e) is available at https://doi.org/10.5281/zenodo.12826667. BSA bovine serum albumin.
