Cross-reactivity of antilepirudin antibodies with bivalirudin. Sera of 43 patients who developed antihirudin antibodies during treatment with lepirudin were incubated with lepirudin- or bivalirudin-coated microtiter plates as described under “Patients, materials, and methods.” To demonstrate binding specificity, sera were preincubated with either buffer or lepirudin in high concentrations (400 μg/mL) or bivalirudin in high concentrations (1 mg/mL), respectively. (A) Twenty-one sera contained antibodies that bound to lepirudin (lane 1) but not to bivalirudin (lane 4). Binding to lepirudin was strongly inhibited by high concentrations of lepirudin (lane 2) but only very weakly by high concentrations of bivalirudin (lane 3). Only 2 sera contained antibodies whose binding to lepirudin was inhibited by more than 30% by high concentrations of bivalirudin. (B) Twenty-two of 43 sera contained antibodies that bound to lepirudin (lane 1) and to bivalirudin (lane 4). When lepirudin was coated to microtiter plates, a higher OD occurred compared with bivalirudin-coated microtiter plates. Antibody binding to lepirudin was inhibited by high concentrations of lepirudin (lane 2), whereas high concentrations of bivalirudin had only a minor impact on antibody binding to lepirudin (lane 3). Conversely, when bivalirudin was coated, lepirudin in high concentrations inhibited antibody binding (lane 5) to a similar extent as did high concentrations of bivalirudin (lane 6).