Anti m13hrp

Individual K91BK colonies on titration plates from the last panning were pick into 96-deep well plates containing 1.5 ml of 2×YT + 20 μg/ml tetracycline and grown overnight in a shaker at 37°C to produce scFv or VHH phage particles. Phages from 1 ml of supernatant were precipitated with 150 μl of the PEG/NaCl solution and resuspended in 200 μl of PBS.
Each well of Nunc Maxisorp 96-well microplates was coated with 100 μl of 5–10 μg/ml of each antigen. After overnight incubation at 4°C, plates were blocked with 2% MPBS for 1.5 h followed by three washed with PBS-T and three washes with PBS. All washings were performed on AquaMax 2000 plate washer (Molecular Devices, LLC). The selected phage preparation was diluted 1:2 in 4% MPBS before adding 100 μl into each well, and incubated for 1.5 h. The plates were washed three times with PBS-T, followed by three times with PBS, and incubated with 100 μl of a 1:4000 dilution of anti-M13-HRP (Sino Biological US, Inc., Wayne, PA) in 2% MPBS for 1 h. Plates were washed 4 times with PBS-T and four times with PBS. TMB substrate solution 100 μl (Thermo Fisher Scientific) was added to each well (for 2–30 min). After adding 100 μl of 2 M sulfuric acid stop solution, the absorbance was read at 450 nm, using SpectraMax iD5 microplate reader (Molecular Devices, LLC).

Phage ELISA was performed as described previously except that the 384-well plate was used instead of the 96-well plate [18 (link)]. Briefly, the wells of the 384-well ELISA plate (Nunc) were coated with neutravidin (Thermo Fisher) for 1 hr at room temperature. The wells were washed with PBS-T (PBS containing 0.1% Tween 20) and blocked with PBS containing 0.5% (w/v) BSA (Gemini Bio) for 1hr. After removing the blocking buffer, biotinylated antigens were added to each well and washed three times with PBS-T. The 5-fold dilution of the cell culture supernatants containing phage were added to the wells and incubated for 30 min. After washing the wells with PBS-T three times, anti-M13HRP (Sino Biological) was added to the wells. SIGMAFAST™ OPD (Sigma) was used as a substrate and 2 M HCl was used as a quenching solution. The absorbance at 490 nm was measured using a BioTek Epoch 2 plate reader (BioTek).