Ferritin-binding proteins (FBPs) in bovine serum were characterized by ferritin immunoassay, ferritin-binding activity, and immunoblotting. Serum ferritin, but not tissue ferritin, was precipitated by centrifugation at 14000 x g for 30 min, and bovine spleen ferritin added to bovine serum was precipitated by centrifugation at 1650 x g for 20 min. Two FBPs (FBP1 and FBP2) were purified from bovine serum by sequential chromatography on bovine spleen ferritin-Sepharose 4B affinity and Sephacryl S-300 columns. FBP1 separated into 82 kDa- and 26 kDa-bands on SDS-PAGE, while FBP2 separated into 55 kDa- and 26 kDa-bands. FBP1 and FBP2 were identified as IgM and IgG, respectively, by immunoblotting with alkaline phosphatase-labeled antibodies specific for bovine IgM, IgG, and IgA heavy chains. Given these results, we suggest that bovine FBPs are autoantibodies (IgM and IgG) to ferritin and that circulating ferritin exists as an immune complex.
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