Figure 4.

Treatment of therapeutic Abs—Rituximab and Trastuzumab—with ferrous ions results in induction of antigen-binding polyreactivity. (A) ELISA assay was used to evaluate the reactivity of native and Fe²⁺ ion-exposed Rituximab or Trastuzumab. The Abs at 1 mg/mL (6.7 μM) were treated with 500 μM final concentration of FeSO₄. After native and iron ion-treated Abs were serially diluted 3350—3.27 nM and incubated with the indicated proteins and LPS from E. coli immobilized on ELISA plates. Each symbol represents the averaged binding intensity obtained from triplicate samples ± SD. (B) Immunoblot analyses of reactivity of native and ferrous ion-exposed Rituximab and Trastuzumab to antigens from total lysate from Bacillus anthracis. Abs were treated at 1 mg/mL (6.7 μM) with 500 μM FeSO₄. Next native and treated Abs were diluted to 100, 33 and 11 μg/mL and incubated with the immobilized antigens using the Immunetics miniblot system.