For screening, 100 μl of supernatant containing rescued phage from each monoclonal (scFv) was added to the blocked streptavidin-coated plate coated with biotinylated preS1-peptide (5 μg/ml) and incubated for 1 hour at 37 oC. Post washing, the bound phage was detected using 100 μl of anti-M13 HRP-conjugated antibody (GE lifesciences, 27942101) (1:2000) using TMB as substrate. For further characterization, the phage containing supernatant was precipitated using PEG/NaCl and used in ELISA in a concentration of 1 × 1012 phages/100 μl.
The scFv gene of selected scFvs was cloned into pAK400 vector and transformed into HB2151 E. coli cells for periplasmic expression. The scFv clone was then grown in 1L 2xYT/Chloramphenicol media and induced at an OD~0.8 with 1 mM IPTG (Amresco, 0487) for 15 hours at 20 °C, 250 rpm. The periplasmic extract was prepared using ‘Osmotic Shock method’60 (link). 100 μL of supernatant was added to the preS1-peptide coated streptavidin plate and incubated for 1 hour at 37 °C. Post washing, bound phages were detected using 100 μl of anti-his antibody (1:2000) (Cell Signaling Technologies, 3724) and anti-rabbit HRP-conjugated antibody (Cell Signalling Technologies, 70745) using TMB as substrate.
The scFv gene of selected scFvs was cloned into pAK400 vector and transformed into HB2151 E. coli cells for periplasmic expression. The scFv clone was then grown in 1L 2xYT/Chloramphenicol media and induced at an OD~0.8 with 1 mM IPTG (Amresco, 0487) for 15 hours at 20 °C, 250 rpm. The periplasmic extract was prepared using ‘Osmotic Shock method’60 (link). 100 μL of supernatant was added to the preS1-peptide coated streptavidin plate and incubated for 1 hour at 37 °C. Post washing, bound phages were detected using 100 μl of anti-his antibody (1:2000) (Cell Signaling Technologies, 3724) and anti-rabbit HRP-conjugated antibody (Cell Signalling Technologies, 70745) using TMB as substrate.