mAb14C12 blocks C2 domain recognition by inhibitory Abs in plasma samples taken from patients with hemophilia A inhibitor. Plasma samples of unrelated patients with hemophilia A (A-F), or of the patient from whom mAbBO2C11 was cloned (G), were preincubated with 50, 150, or 450 μg/mL mAb14C12 before addition of 35S-methionine–labeled C2 domain, to establish how much mAb14C12 was needed to block the binding of C2 to inhibitory antibodies. IgG Abs from plasma were then precipitated by protein-A Sepharose, and residual radioactivity was counted on the pellet. Maximum blocking of C2 binding (100%) was obtained by adding 450 μg mAb14C12 to the FVIII-mAbBO2C11 mixture. Three of 6 plasma samples (B,D,F) show reactivity with mAb14C12 equivalent to that of the mAbBO2C11 source patient (G). The experiment was run only once and in single determination because of a shortage of plasma material. A control experiment (not shown) was carried out in which human mAbBO2C11 was used instead of polyclonal antibodies, which resulted in 100% neutralization.
