Reactions of the recombinant PA, NA, and NB clonal antibodies. (A) PA and NA but not NB clonal antibodies recognize PF4/H and are inhibited by high-dose heparin (HD Hep). Quantity of antibodies used was 100 μg/mL, except for clones HIT1P4G8, HIT3P1A10, and HIT3P7D2, for which 200 μg/mL was used. Positive controls were plasma from patients HIT2 through HIT5. For convenience, only reactions of one NB clone are shown, but all other NB clones behaved comparably. (B) PA, but not NA or NB, clones (80 μg/mL) activate PF4-treated platelets (P-selectin expression) in reactions that were inhibited by the FcγRIIa-specific monoclonal antibody IV.3 and by HD Hep. Positive controls were HIT plasma from patients HIT2 and HIT3 and thrombin receptor-activating peptide (TRAP). Negative reactions shown for 3 NA and 2 NB clones are typical of all other clones in these categories. (C) PA, but not NA or NB, clones (80 μg/mL) bind physically to PF4-coated, but not uncoated, platelets in reactions that are inhibited by HD Hep. Negative reactions shown for 3 NA and 2 NB clones are typical of all other clones in these categories. (D) Seven PA and 22 NA clones did not differ significantly in their reactions against immobilized PF4/H. Antibodies were diluted threefold serially to produce final concentrations ranging from 300 to 0.14 μg/mL and were tested in PF4/H ELISA. Antibody binding strength (ordinate) was plotted as the area under the curve (AUC) in arbitrary units. Data shown in A through C are representative of at least 4 independent studies. Error bars represent mean ± SD of 2 replicates within each experiment. BSA, bovine serum albumin; N plasma 1, normal plasma 1; ns, not significant.