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pI-dependent isolation of antibody isoforms by semipreparative isoelectric focusing
A Gangopadhyay1, C A Saravis, A D van den Abbeele
1Department of Radiology (Division of Nuclear Medicine), Harvard Medical School, Boston, MA 02115, USA.
A procedure based on isoelectric point (pI) was developed to separate immunoreactive antibody isoforms. A polyclonal IgG, rabbit anti-human serum albumin (R-HSA), was subjected to free-flow isoelectrophoresis using a semipreparative isoelectric focusing apparatus that fractionates proteins by pI. Twenty fractions were collected and their pH, protein content, and immunoreactivity determined. The development of a pH gradient and separation of proteins took place within 3 hours with about 93% protein recovery. The protein concentration of the individual fractions varied. Isoelectric focusing of fractions in agarose slab gels confirmed the clear separation of antibody isoforms. Enzyme-linked immunosorbent assay demonstrated significantly higher immunoreactivity (P < or = 0.03) of the majority of the antibody isoform fractions compared with native R-HSA IgG. The procedure is capable of isolating immunoreactive antibody isoform fractions from immunologically irrelevant and low-affinity antibodies.
A procedure based on isoelectric point (pI) was developed to separate immunoreactive antibody isoforms. A polyclonal IgG, rabbit anti-human serum albumin (R-HSA), was subjected to free-flow isoelectrophoresis using a semipreparative isoelectric focusing apparatus that fractionates proteins by pI. Twenty fractions were collected and their pH, protein content, and immunoreactivity determined. The development of a pH gradient and separation of proteins took place within 3 hours with about 93% protein recovery. The protein concentration of the individual fractions varied. Isoelectric focusing of fractions in agarose slab gels confirmed the clear separation of antibody isoforms. Enzyme-linked immunosorbent assay demonstrated significantly higher immunoreactivity (P < or = 0.03) of the majority of the antibody isoform fractions compared with native R-HSA IgG. The procedure is capable of isolating immunoreactive antibody isoform fractions from immunologically irrelevant and low-affinity antibodies.