(A) Western blot analysis with Gal-deficient IgA1 (Mce) as antigen demonstrated binding of serum IgG from 2 IgAN patients but only minimal binding of IgG from 2 healthy controls to the IgA1 heavy chain. After removal of sialic acid, IgG binding increased, as it did for binding to HAA. N+, treated with neuraminidase; N-, not treated with neuraminidase. (B) To test glycan-specific IgG binding to GalNAc, these IgA1 proteins were used: lane 1, Gal-deficient IgA1 (Mce); lane 2, dd-IgA1; lane 3, enzymatically regalactosylated dd-IgA1; and lane 4, enzymatically resialylated dd-IgA1. dd-IgA1 bound the greatest amount of HAA, with enzymatically galactosylated or sialylated dd-IgA1 binding very little. IgG from an IgAN patient bound to these antigens in a fashion similar to that for HAA. (C and D) Component chains of Gal-deficient IgA1 (Mce) were separated by SDS-PAGE under reducing conditions and electroblotted. The membrane was then treated with HAA to assess whether blockade with this GalNAc-specific lectin can inhibit IgG binding. The intensity of each band was quantified by densitometry. The binding of serum IgG from an IgAN patient to Gal-deficient IgA1 was reduced by 66% after treatment with HAA. Conversely, blocking with serum IgG from an IgAN patient reduced the binding of HAA to Gal-deficient IgA1 by 60%. Binding of anti-human IgA (heavy-chain specific) confirmed equivalent loading. Representative results from 3 experiments are shown in A–C; lanes were run on the same gel but were noncontiguous.