Ferritin-binding proteins (FBPs) in horse serum were characterized by immunoblotting and ferritin-binding experiments. FBPs purified from horse serum by horse spleen ferritin-Sepharose 4B affinity chromatography were separated into two fractions by Sephacryl S-300 gel chromatography: FBP 1 and FBP 2. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, FBP 1 separated into 79.0- and 25.3-kDa bands, and FBP 2 separated into 62.7- and 25.3-kDa bands. Immunoblotting analysis using antibodies specific for horse IgM and IgA heavy chains and IgG F(c) fragment showed that the 79.0- and 62.7-kDa bands were IgM and IgG heavy chains, respectively. After forming complexes of horse FBPs with horse spleen ferritin, rabbit anti-horse ferritin antibody was used to form immune complexes against ferritin, allowing co-precipitation and subsequent identification by monoclonal antibodies to horse immunoglobulin (IgM, IgA, IgGa and IgGb). Horse serum was positive for IgM, IgGa, IgGb and IgA complexes and affinity-purified FBPs for IgM and IgGb complexes. FBP 1 was identified as an IgM complex that forms with ferritin but FBP 2 did not form any complexes with ferritin. These results suggest that circulating horse serum ferritin is in the form of IgM, IgG (IgGa and IgGb) and IgA complexes.
View full abstract