Purification of Fab Fragment

Purification of the Fab Fragment-The crude papain digest was fractionated by size-exclusion chromatography on an AcA 44 column (LKB, Bromma, Sweden) (100 X 2.5 cm) with 0.1 M sodium acetate buffer, pH 5.5, at a flow rate of 0.5 ml/min. The fractional eluate was analyzed both by absorbance measurements at 280 nm and by nonreducing sodium dodecyl sulfate-gel electrophoresis. In nonreducing gels, the Fab fragment appears as a band at 43 kDa whereas the Fc fragment shows up as a 20-22 kDa band. The Fab fragment containing fractions were combined, concentrated, and dialyzed against 10 mM Tris/acetate buffer, pH 7.8, which was the starting buffer for the following anion exchange chromatography. The 17/9 Fab was purified by ion exchange chromatography on DEAE-cellulose (DE52, Whatman, Springfield Mill, Maidstone, Kent, United Kingdom). A salt gradient from 0-0.25 M sodium acetate in 10 mM Tris/acetate, pH 7.8, was used for developing a 20-ml column with a flow rate of 1.5 ml/min. The total gradient volume was 200 ml. Under these conditions the Fab fragment elutes as one major peak with a shoulder very early in the gradient. Contaminating Fc fragments are eluted at much higher salt concentrations. The Fab fragment, eluting in the major front peak, was concentrated and dialyzed against 0.1 M sodium acetate buffer, pH 5.5, 0.02% sodium azide, for crystallization. The purification procedures were monitored by analytical scale size exclusion HPLC as well as sodium dodecyl sulfate-gel electrophoresis. Final analysis of the purified Fab by sodium dodecyl sulfategel electrophoresis and isoelectric focusing gels revealed only one band. The Fab binding activity for peptide remained unchanged from that of the intact IgG as judged from peptide binding studies in ELISA. The amino acid sequence of the antibody Fab was determined by sequencing the mRNA from the hybridoma. The mRNA was isolated using standard methods (Chirgwin et al., 1979)