Characterization of a monoclonal antibody (ADA) to PRT/heparin complexes. (A) Isotype of ADA. ADA was confirmed to be an IgG3 subtype by an antigen-capture assay using isotype-specific antibodies as shown on the x-axis. (B) ADA specificity. ADA binding to PRT/heparin complexes, protamine alone (PRT), mouse PF4/heparin (mPF4/H) complexes, human PF4/heparin (hPF4/H) complexes, or albumin was measured by ELISA. Mean absorbance of triplicate wells at 450 nm is shown on the y-axis. (C) ADA binding to PRT/heparin complexes vs protamine alone. Serial dilutions of ADA were incubated in microtiter wells coated with PRT/heparin or protamine alone. Concentration of ADA is shown on the x-axis, and mean absorbance of triplicate wells is shown on the y-axis. Half-maximal binding of ADA to PRT/heparin complexes occurred at 1.44 µg/ml. (D) ADA binding to low molecular weight heparins (LMWHs). ADA binding to protamine/LMWH (enoxaparin or fondaparinux) complexes was determined by ELISA. Increasing concentration of enoxaparin or fondaparinux is shown along the x-axis.
