Figure 3. Characterization of the Antibody Response to Reduced and Nonreduced Phospholipase A₂ Receptor (PLA₂R).

Panel A shows a Western blot in which equal amounts of human glomerular extract (HGE) and equal amounts of recombinant PLA₂R (rPLA₂R) were electrophoresed under reducing and nonreducing conditions. Western blotting was performed with the use of a reactive serum sample from a patient with membranous nephropathy (MN5) or a polyclonal anti-PLA₂R antibody and detected with appropriate secondary antibodies. Both the serum sample from the patient with membranous nephropathy and the polyclonal anti-PLA₂R antibody reacted with the native and recombinant PLA₂R in the nonreduced state, but the reduced proteins were reactive with only the anti-PLA₂R antibody. Panel B shows the IgG-subclass specificity to PLA₂R. HGE was blotted initially with serum samples from six patients with membranous nephropathy (MN1 through MN6), followed by sheep antibodies specific for each human IgG subclass (1 through 4), and was detected with peroxidase-conjugated antisheep IgG antibody. The arrowheads indicate the fully glycosylated native PLA₂R. The predominant IgG subclass that reacted with the native antigen was IgG4, with varying amounts of reactivity seen for IgG1, IgG2, and IgG3. Identical results were obtained with the use of recombinant PLA₂R instead of HGE (not shown).