Shark vNAR Library Screening for anti-TGF-β

A phage display was performed to select a specific vNAR, starting with a naive vNAR library of H. francisci shark in the pCOMb3X plasmid previously generated^{37 (link)}. After reamplification, phages were obtained against rhTGF-β cytokine (Peprotech, 100-21) resuspended in 10 mM citric acid, pH 3.0, according to manufacturer instructions. Two wells of a 96-well plate coated with rhTGF-β (5 μg/mL) and incubated for 1 h at 37 °C. Wells were blocked with 150 μL of PBS-BSA 3% for 1 h at 37 °C. Then, 50 μL of phages were added and incubated at 37 °C for 1 h. Then, the washing steps are gradually increased to 7 for round 1, 14 for round 2, and 21 for round 3 to increase the stringency. These washes raise 150 μL of TBS-Tween 0.05% (TBST) per well five times and are allowed to stand 5 min between each wash. After the wash rounds, 50 μL of trypsin 10 μg/mL was added in 1% BSA, followed by 30 min at 37 °C incubation. The wells were washed by raising the solution volume vigorously ten times and using the eluted phages to infect a culture of 2 mL of E. coli strain ER2537 (OD_600nm = 1), followed by incubation of 15 min at room temperature. Finally, transferring the culture to a 50 mL tube containing 6 mL SB medium and 1.6 μL carbenicillin (100 mg/mL, Sigma, C1389) and incubated for 1 h at 37 °C at 250 pm. The output titration count was obtained with 2 μL of the initial 8 mL culture and diluted in 200 μL of SB medium, plating 10 μL and 100 μL in LB carbenicillin plates. To the input result, a culture of 2 mL of ER2537 cells was grown at an OD_600nm = 1. Then 50 μL was infected with 1 μL of a 1:10^–8 dilution of phages obtained after each panning round and incubated for 15 min at room temperature, finally plated onto LB agar plates with carbenicillin (100 μg/mL). The plates were incubated overnight (ON) at 37 °C. After incubation in standard conditions, the input and output titers were obtained by multiplying the number of colonies by the culture volume and dividing by the plating volume³⁸ .
After the 1 h incubation of the 8 mL culture, 2.4 μL of carbenicillin (100 mg/mL) was added, and the tube was incubated for another hour and transferred to a 500 mL flask. Next, 1 mL of helper phage VCSM13 phage VCSM13 (Stratagene, 200251), 91 mL of SB medium, and 46 μL of carbenicillin (100 mg/mL) were added to the flask and incubated for 2 h at 37 °C and 250 rpm. Then, 140 μL of kanamycin (Sigma, 60615) was added at 50 mg/mL and incubated for 12 to 16 h. This protocol was repeated in each round, except the next rounds used only one well with an immobilized cytokine and increased washed steps of 7, 14, and 21. Finally, a colony PCR screening selects clones with the vNAR sequence.