N_wt/N_Ala for antibody (3F6.B1.4B1) selection

Binding

Frequency of Occurrence

unitless

ELISA , Phage Display , Shotgun-Scanning,Binding Selection Assay

None

None

None

None

None

None

Phage from the library were cycled through rounds of binding selection with hGHbp or anti-hGH monoclonal antibody 3F6.B1.4B1 coated on 96-well Nunc Maxisorp immunoplates as the capture target. Phage were propagated in E. coli XL1-blue with the addition of M13-VCS helper phage (Stratagene). After one (antibody sort) or three (hGHbp sort) rounds of selection, individual clones were grown in a 96-well format in 500ml of 2YT
broth supplemented with carbenicillin (10 µg/ml) and M13-VCS (1E10 phage per ml). The culture supernatants were used directly in phage ELISAs to detect phage-displayed hGH variants that bound to either hGHbp or anti-hGH antibody 3F6.B1.4B1 immobilized on a 96-well Maxisorp immunoplate.