Analysis of the transferrin-IgG complex and ferrokinetics measurements. (A) Identification of an IgG-transferrin complex in the serum of both patients by Western blot. A 5 μL sample of diluted serum (1:1000) or 1 μg of protein A–purified proteins from the 2 patients, from 1 healthy individual, and from 1 IgG-κ myeloma were boiled in 1% SDS loading buffer without (i, ii) or with (iii, iv) 5% β-mercaptoethanol, subjected to 1% SDS–7.5% polyacrylamide gel electrophoresis, and transferred on Hybond ECL Nitrocellulose Membrane (Amersham, Saclay, France). Following blocking with 3% nonfat milk, the membrane was incubated with polyclonal anti–human transferrin (i, iii) or anti–human IgG (ii, iv) antibodies. The purified fraction (lane 1) and the serum (lane 2) of patient 1 were run in parallel with the purified fraction (lane 3) or the serum (lane 4) from a control individual, the purified fraction (lane 5) and the serum (lane 6) of patient 2, and the purified fraction from a monoclonal IgG2 (lane 7). Human transferrin (lane 8) was used as a control. The transferrin-IgG complex is indicated by an arrow and free transferrin by an arrowhead. (B) Protein A chromatography. A 300 μL serum sample from patient 2 was applied on a (5 × 0.5 cm) column packed with protein A–Sepharose 4B and eluted as indicated. IgG, transferrin, and iron were measured in the various fractions. Transferrin was in both unbound (1 to 14) and bound fractions (15 to 29). IgG was only demonstrated in the bound fractions. (C) In vivo plasma clearance of ⁵⁹Fe. Radioactivity in the plasma was measured at different time intervals following injection of ⁵⁹Fe-labeled autologous plasma. The reference curve established from the ferrokinetics measurement obtained for 30 healthy controls⁷  shows that the normal half-life is 75 minutes (range, 70-110 minutes). The half-life of the radioactivity in the patient's plasma was 206 minutes, 2.75 times longer than normal half-life.