Maxisorp immunotubes

Libraries were rescued and selected twice using Nunc Maxisorp immunotubes as previously described (26 (link)). For the first round of panning, tubes were washed five times with PBS containing 0.1% Tween 20 (PBS-T) and five times with PBS; for pan 2, the number of washes with and without Tween 20 was doubled. Following each round of panning, two 96-well plates of individual colonies were picked with a QPix2 XT (Genetix, San Jose, CA, USA) and grown for periplasmic protein extraction. Binding to HSA (and HEL control) was evaluated by ELISA using 50% of the crude periplasmic protein extract (27 (link)). VNARs were detected via their HA tag using a high-affinity mAb HRP conjugate (clone 3F10; Roche, Basel, Switzerland). All samples were processed with a Perkin Elmer MiniTrak robotic liquid handling system (Waltham, MA, USA).

Selection was performed using Bradyrhizobium strains SUTN9-2 and DOA9 as targets. Two Maxisorp immuno tubes (Nunc, Denmark) were coated separately with 20 μg of boiled Bradyrhizobium strains, SUTN9-2 and DOA9, in sodium carbonate buffer at 4°C overnight. After that, the immuno tubes were washed three times with phosphate-buffered saline (PBS) and blocked with PBS containing 2% (wt/vol) skim milk (2% MPBS) for 2 h at room temperature. The tubes were washed three times with phosphate-buffered saline (PBS) before adding 10 μl (10¹¹ PFU/μl) of phage library in 290 μl of 2% (wt/vol) MPBS and then incubated at room temperature for 2 h. Unbound phage was washed away with five times PBS supplemented with 0.1% (vol/vol) Tween 20 (PBST) and followed by 5 times PBS. Phage antibodies against each Bradyrhizobium strain was eluted with 1 μg/ml of trypsin buffer, followed by 0.1 M glycine-HCl, pH 2.0. To obtain individual phage clones, the eluted phage was infected with E. coli TG1 and incubated at 37°C for 30 min without shaking. Then, infected cells were subjected to 10-fold-serial dilution (10¹-10⁴) and spread on 2x YT agar plates supplemented with 100 μg/ml ampicillin and 1% (wt/vol) glucose. The agar plates were incubated overnight at 37°C. Each single bacterial colony represents individual phage clone. One round of selection was carried out for both strains.