SEPARATION OF POLYSACCHARIDE-SPECIFIC HUMAN IMMUNOGLOBULIN-G SUBCLASSES USING A PROTEIN-A SUPEROSE COLUMN WITH A PH GRADIENT ELUTION SYSTEM

Protein A Superose was employed to separate affinity-purified anticarb ohydrate antibodies according to immunoglobulin G (IgG) subclass. Sepa ration was achieved with a novel buffer system (disodium phosphate-sod ium acetate-sodium chloride-glycine), which allowed the generation of a linear pH gradient from pH 8 to 3. Protein A-bound anti-carbohydrate antibodies were eluted as three peaks, two of them mainly containing IgG2 and one consisting of highly enriched IgG1. The enriched antibody preparations retained their functional activity. This separation proc edure can be considered as an alternative to the preparation of IgG su bclasses with subclass-specific monoclonal antibodies and could be emp loyed whenever contamination with immune complexes has to be avoided.