- After overnight incubation at 30C, the number of plaques on
each dilution plate is counted and helper phage titer calculated
as plaque forming units (PFU)/mL by taking into consider-
ation the dilution factor and the volume of cells plated.
3.6 Immunized
Sheep Antibody Phage
Display Library Rescue
and Panning
The following protocol for library rescue and panning has been
adapted from MRC Centre for Protein Engineering Tomlinson
(Tomlinson & Winter) and Griffin1 library protocols.- Independently inoculate VH-Vλand VH-Vкphagemid library
glycerol stocks obtained in TG1 cells in Subheading 3.4,
step 12in 200 mL of pre-warmed 2TY medium containing
100 μg/mL ampicillin and 1% (wt/vol) glucose. In this way the
starting culture should have 10–100 copies of each antibody
clone from the library. The number of cells is estimated by
measuring the initial optical density of the culture postulating
that for an A 600 0.1, the number of cells in the culture is
~8 107 cells/mL. Cells are grown at 37C with shaking at
250 rpm until A 600 0.5. - M13KO7 helper phage is added to the cultures to a 1:20 ratio
(cell:helper phage) and cells are infected by incubating at 37C
without shaking followed by 1 h at 37C with shaking at
250 rpm. - To determine the helper phage rescue rate of the library, 10μL
of the infected culture is set aside and serially diluted in 2TY
medium, plated onto TYE agar plates containing 100μg/mL
ampicillin and 50μg/mL kanamycin, and incubated at 37C
overnight. The infection rate is determined by counting bacte-
rial colonies on the agar plates. - The two 200 mL cultures infected overnight with M13KO7
are then centrifuged at 2000gfor 10 min at 4C. - Cell pellets fromstep 4are suspended in 100 mL of 2TY
medium supplemented with 100μg/mL ampicillin and 50μg/
mL kanamycin, and incubated overnight at 30C with shaking
at 250 rpm. - For the precipitation of rescued phage, overnight cultures from
step 5are centrifuged at 4000gfor 30 min and the super-
natants containing phage particles are transferred to sterile
tubes. Add 20 mL of PEG-NaCl solution to the phage super-
natants, mix well, and incubate on ice for 2 h. Centrifuge for
30 min at 4000gat 4C and discard the supernatants. - Wash the phage pellets with 40 mL of H 2 O and centrifuge at
4000 gat 4C for 20 min. The supernatants containing the
phage are precipitated by mixing with 8 mL of PEG-NaCl and
30 min incubation on ice. Centrifuge at 4000gat 4C for
10 min, discard supernatant and resuspend phage pellets in
Monoclonal Antibodies Inactivating AHL Signal Molecules 343