Fig. 3.

Novel IgG binds to a protein Mr-55-60kDa that is confined to the neuronal cytoskeleton. (a) Using PC12 lysate as a source of antigen, proteins were separated, immunoblotted, and probed with patient or normal human sera. A common band (∼55kDa by reference to molecular weight standards) was revealed by IgG in patients' sera (lanes 1-4), but not by IgG in control human serum (lanes 5-7).

To verify specificity, patient IgG was affinity purified on the putative antigenic band and a control band. Eluates from the putative antigenic band (b) and control band (c) were reapplied to the composite mouse tissue substrate slide, and compared to the original immunostaining pattern of whole patient serum IgG on enteric nerve fibers (d). An identical staining pattern was observed in eluates from putative antigenic band. To determine the subcellular distribution of the antigen, PC12 lysates were fractionated by differential detergent extraction. Proteins in each fraction were resolved electrophoretically, transblotted, and probed with patient sera (e). IgG bound exclusively to a protein in the cytoskeletal fraction.