Antibodies to the PF4 c-terminus do not compete with HIT antibodies. (A) Binding of rabbit polyclonal anti-C-terminal antibodies to PF4/heparin complex. (⧫) Binding of rabbit antibody developed to C-terminal PF4 peptide complexed to heparin at various concentration to microtiter wells coated with PF4/heparin complex. (○) Same as above, but for antibody raised against the C-terminus only. (▴) Binding of rabbit antibody developed to C-terminal PF4 peptide complexed to heparin at various concentration to uncoated wells. (B) Competition of binding to PF4/heparin in ELISA by polyclonal anti-PF4 C-terminus and HIT antibodies. Polyclonal anti-PF4 C-terminus antibodies raised in rabbits were used at different concentrations and preincubated with immobilized WT PF4/heparin or NAP-2/heparin complexes attached to microtiter plates. HIT antibodies in a predetermined concentration giving 50% binding were added to the plates and residual binding was measured. As shown, no significant competition with HIT antibody binding was demonstrated by either rabbit antibody. (□) Polyclonal antibody raised against PF4/heparin and tested with PF4/heparin complexes on microtiter wells. (•) Polyclonal antibody raised against PF4 and tested with PF4/heparin complexes on microtiter wells. (★) Binding of the alkaline phosphatase antihuman IgG to the previously added rabbit serum in the absence of HIT antibody.