title: pI-dependent isolation of antibody isoforms by semipreparative isoelectric focusing.
abstract: 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.
authors.affiliation: Department of Radiology (Division of Nuclear Medicine), Harvard Medical School, Boston, MA 02115, USA.
authors.forename: A
authors.initials: A
authors.lastname: Gangopadhyay
authors.forename: C A
authors.initials: CA
authors.lastname: Saravis
authors.forename: A D
authors.initials: AD
authors.lastname: van den Abbeele
authors.forename: A I
authors.initials: AI
authors.lastname: Kassis
mesh_terms: D000818:Animals; D000906:Antibodies; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007074:Immunoglobulin G / Q000302:isolation & purification*; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D011817:Rabbits; D012685:Sepharose; D012709:Serum Albumin / Q000276:immunology*
chemical_list: D000906:Antibodies; D007074:Immunoglobulin G; D012709:Serum Albumin; D012685:Sepharose