Circulatory antigen of Heymann nephritis. II. Isolation of a 70,000 MW antigen from normal rat serum which cross-reacts with Heymann nephritis antigen.

Antigens cross-reactive with crude Heymann antigen, FXIA and purified antigen, gp600, were isolated from rat serum by immunoaffinity chromatography using polyclonal anti-gp600 antibodies. The eluted antigens predominantly contained a 70,000 molecular weight (MW) polypeptide. An antiserum was raised against this preparation which, when tested against rat serum by Protein A immunoblotting, showed that the antibodies were directed to 70,000 MW antigens. By immunodiffusion in gel, the antiserum produced three precipitin lines against the antigen preparation, three lines against FXIA and a single line against gp600. All three lines of the serum antigen were continuous with the three lines of FXIA showing complete identity of the isolated serum antigens with FXIA. One of the three common lines of the serum antigen and FXIA was in continuity with the line of gp600 showing cross-reaction of gp600 with the isolated 70,000 MW antigen. Testing of Western blots of gp600 and FXIA against this antiserum by protein A immunoblotting showed reaction with the 70,000 MW subunit of gp600 and FXIA indicating cross-reaction of the serum antigen with 70,000 MW antigen of FXIA and gp600. The antiserum reacted with structures in the glomerulus by indirect immunofluorescence. Patchy brush border staining was visible. When injected into an isolated perfused kidney, these antibodies localized in fine granular pattern along the glomerular capillary wall by direct immunofluorescence. Ultrastructural immunogold technique revealed that the 70,000 MW antigen was located in the brush border and in the GBM-endothelial interface. These results demonstrate that the 70,000 MW antigen present in rat serum is also present in the glomerular capillary wall.