Standard cloning procedures, determination of colony-forming units and
plaque-forming units, and immunoblot after PAGE were carried out
according to Sambrook et al.
Construction of the packaging cell line.
[M13KO7DeltapIII]. The M13 pIII gene fused to promoter P/A1/04/03
(ref. 17) was inserted into the multiple cloning site of the
lambdaattP plasmid pLDR9 (ref. 18). The origin of replication and the
kanamycin resistance gene were cut out and the remaining
lambdaattP-pIII nonreplicative DNA was religated. Escherichia coli
DH5alpha containing plasmid pLDR8 (ref. 18) were transformed with the
lambdaattP-pIII nonreplicative DNA and plated on agar plates
containing 25 mug/ml ampicillin. After incubation overnight at 42°C,
the colonies obtained were replica-plated and ampicillin-resistant
clones were identified, named E. coli DH5alpha/pIII. They were
subsequently transformed with M13KO7DeltapIII, resulting in the
packaging cell line E. coli DH5alpha/pIII [M13KO7DeltapIII].
Production of phage.
To obtain M13KO7DeltapIII helper phage,
M13KO7DeltapIII DNA was electrotransfected into E. coli K802
containing pNDI (ref. 15). To obtain hyperphage, M13KO7DeltapIII DNA
was electrotransfected into E. coli DH5alpha/pIII. Phage were produced
by cultivation in the presence of 0.5 mM
isopropyl-beta-D-thiogalactoside (IPTG) at 30°C for 24 h with shaking
at 200 r.p.m. Escherichia coli Top10F' or E. coli Xl1blue carrying the
pSEXphOx(Yol) phagemid or a scFv library in pSEX81, respectively, were
infected with a helper phage preparation at a OD600 of 0.1 at a
multiplicity of infection of 20 as described.
Phage quantification by ELISA.
Dilution series of phage were coated in
100 mM NaHCO3, pH 8.6 in MaxiSorb ELISA plates (Life Technologies,
Karsruhe, Germany) for 16 h at 4°C. Blocking was done with 2% skim
milk powder in PBS (137 mM NaCl, 3 mM KCl, 8 mM Na2HPO4, 1 mM KH2PO4,
pH 7.3) for 2 h at room temperature. Phage were detected with the
monoclonal mouse antibody B62-FE2 (Progen, Heidelberg, Germany)
specifically binding an epitope on the pVIII major coat protein of the
M13 phage. M13KO7 phage of known colony-forming units were used for
Approximately 1010 phage were applied per lane on a 10%
polyacrylamide gel. Blocking was done with 2% skim milk powder in PBS
for 2 h at room temperature. Immunostaining was done with the mouse
mAb anti-g3p (MoBiTec, Göttingen, Germany) recognizing the pIII coat
protein of M13KO7, visualized with 3,3', 5,5'-tetramethylbenzidene
(TMB) substrate (Promega, Madison, WI).
Antigen-binding phage ELISA.
Dilution series of BSA-phOx antigen in
100 mM NaHCO3, pH 8.6, were coated in MaxiSorb ELISA plates (Life
Technologies) for 16 h at 4°C. After blocking of unspecific binding
with 2% skim milk powder in PBS for 2 h at room temperature, 2 times
109 single-chain phage diluted in 2% skim milk powder in PBS were
applied to each well for 1 h at room temperature. After washing six
times with 400 mul PBS per well, the bound phage were detected with
mAb B62-FE2 as described above.
A human scFv fragment library in pSEX81 with a calculated
maximal complexity of 2 times 107 was generated from peripheral
lymphocyte preparations as described21. Tetanus toxin obtained from
Virotech (Rüsselsheim, Germany) was coated to six ELISA wells (Life
Technologies) at a concentration of 0.1 mug/ml in 100 mM NaHCO3, pH
8.6, for 16 h at 4°C. The wells were blocked with 2% skim milk in PBS for 2 h at room temperature, after which 1011 phage of the library
were applied to each well and incubated at 4°C overnight. After five
washes with PBS/0.1% Tween and five with PBS, bound phage were eluted
with 1 mug/ml trypsin (Life Technologies) in PBS for 15 min at room
temperature. Eluted phage were used for the infection of 20 ml of E.
coli XL1blue at a OD600 of 0.4. Infected bacteria were grown on
Luria–Bertani agar plates containing glucose and ampicillin, scratched
from the plates, and used for the production of antibody phage for the
next round of panning as previously described.