recover the supernatant containing released protein, and filter
through a 0.30μm filter.- For purification of scAbs, IMAC Sepharose 6 Fast Flow resin
(Binding Capacity 5–10 mg/mL, GE Healthcare) is added to a
poly-prep chromatography column and the resin supernatant is
allowed to drain. After capping the bottom of the column,
wash once with 5 volumes of PBS and twice with 5 volumes
of sterile H 2 O. - Charge the resin with nickel by adding 5 gel volumes of 0.1 M
NiSO 4 and incubate on a rotator for 1 h at room temperature.
Drain the column and wash with 5 gel volumes of PBS contain-
ing 10 mM imidazole. The activated Ni-sepharose column has
a binding capacity of 5–10 mg of protein per mL of resin. The
column thus prepared can be used immediately for the purifi-
cation of antibody fragments or stored at 4C by leaving 2 gel
volumes of PBS in the column until required. - Add scAb fragments obtained fromstep 25to the column
prepared insteps 26and 27. Mix gently and incubate over-
night at 4C by gentle rotation. Drain the column, collect the
flow-through and pass it again through the column two times.
Wash with 5 mL of PBS and 5 mL of 20 mM imidazole to
remove any nonspecific protein. Cap the column and gently
resuspend the resin in 1 mL of 200 mM imidazole. Allow the
resin to settle for 5 min and drain. Collect the eluent contain-
ing antibody fragments in a 1.5 mL microfuge tube. Repeat
this elution step four times and collect separate fractions. - Dialyze separately each eluted fraction in 5 L of PBS for 6 h at
4 C with gentle stirring. Change PBS and continue dialysis at
4 C overnight. Collect dialyzed scAb fragments in 1.5 mL
microfuge tubes and store at 4C. - The purified antibodies can be analyzed by SDS-
polyacrylamide gel electrophoresis and western analysis. After
blotting and hybridization, the presence of the purified anti-
bodies is revealed with HRP-conjugated secondary antibody
and developed with an appropriate luminescent substrate fol-
lowing manufacturer’s instructions. - The HuCκdomain fused downstream of the scFv region in the
expression vector pIMS147 facilitates quantification of anti-
body fragments by comparison with a human whole IgG stan-
dard using Capture ELISA. Coat an Immulon 4 96-well flat
bottomed ELISA plate with 100μL of a 1:1000 dilution of
anti-human IgG (C-kappa specific) antibody (Sigma) and incu-
bate at 37C for 1 h. - Wash three times with PBST and three times with PBS, add
200 μL of MPBS for blocking and incubate at 37C for 1 h.
Repeat washing steps and add double dilutions of crude or
350 Soumya Palliyil