Identification of target antigens of self-reactive IgG in intravenous immunoglobulin preparations

Intravenous immunoglobulin (IVIg) contains a wide range of self-reactive immunoglobulins (Ig) G. Acidic pH is known to increase the reactivity of purified IgG with self-antigens. We describe here the target antigens of IgG autoantibodies in IVIg and analyze the influence of acidic pH on IgG reactivities. We used 2-DE and immunoblotting with protein extracts of human umbilical vein endothelial cells (HUVEC) and HEp-2 cells. Two IVIg preparations obtained by ethanol fractionation [one with an acidic pH step (acidic-IVIg) and one with beta-propiolactone (propiolactone-IVIg)] and a pool of sera from 12 healthy individuals were tested. Serum IgG of 3 healthy individuals and IgG purified from the same sera with elution at pH 2.8 were also tested individually. Finally, propiolactone-IVIg was acidified at pH 2.8. IgG obtained with a step at low pH recognized many more target spots than IgG obtained without acidic pH. Our findings demonstrate that an acidic pH step artificially enlarges the repertoire of self-reactive IgG. Thus, protein spots recognized by IgG in propiolactone-IVIg represent the core set of self-antigens targeted by IVIg. Overall, 96 proteins were identified by MS. Fourteen were recognized in both extracts including glycolysis proteins such as a-enolase, RNA processing and cytoskeletal proteins such as lamin-A/C.