Purification of p30 mAb and p72 mAb
The ascitic fluid of both mAbs was centrifuged at 12,000 rpm for 20 min
at 4 °C to remove the precipitate, and the supernatant was collected. An
equal volume of saturated ammonium sulfate solution (50% saturation)
was added, mixed, and incubated for 4–6 h at 4 °C, and the supernatant
was discarded following centrifugation. The precipitate was dissolved in
PBS (0.01 M, pH 7.4). The ammonium sulfate was removed by dialysis, and
the sample was subjected to affinity chromatography using a HiTrap
Protein G affinity column first eluted with 10× the column volume of
0.02 M Na3PO3 buffer (pH 7) to wash the
column. Protein samples were added at a speed of 1 mL/min using a
syringe, followed by 5–10-fold column volume of
Na3PO3 buffer wash and final elution
with 2–5-fold column volumes of 0.1 M glycine-HCl (pH 2.7). The eluate
was collected and concentrated by dialysis at 4 °C.