Separation of polysaccharide-specific human immunoglobulin G subclasses using a protein A superose column with a pH...

Protein A Superose was employed to separate affinity-purified anticarbohydrate antibodies according to immunoglobulin G (IgG) subclass. Separation was achieved with a novel buffer system (disodium phosphate-sodium 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 procedure can be considered as an alternative to the preparation of IgG subclasses with subclass-specific monoclonal antibodies and could be employed whenever contamination with immune complexes has to be avoided.