Determination of anti-PF4/heparin complex antibodies in the fluid phase (P4549)

Abstract

Antibodies against the human platelet factor 4/heparin complex (PF4/H) are found in the immune-mediated adverse drug reaction heparin-induced thrombocytopenia. These antibodies are directed against PF4 within the complex due to conformational changes of PF4. To study the humoral immune response to PF4/H in greater detail, C57BL/6 mice were immunized with murine (mu) PF4/H by retro-orbital injection. Serum was obtained, and analyzed by ELISA either with muPF4/H directly plate-bound (solid phase), or in a fluid phase assay with muPF4/H that contained 10% biotinylated muPF4. The two approaches differed notably in their specificity and sensitivity to detect anti-muPF4/H antibodies. The solid phase ELISA could not distinguish between antibodies against plate-bound muPF4 alone and plate-bound muPF4/H. Only incubation with excess heparin allowed to distinguish these antibodies. In contrast, the fluid phase ELISA permitted the direct distinction between anti-muPF4 and anti-muPF4/H antibodies. The fluid phase system was also successfully applied to detect muPF4/H-specific B cells by ELISpot. Thus, the fluid phase ELISA for the detection of anti-muPF4/H antibodies was superior to the solid phase assay. Furthermore, our data suggest that surface binding of PF4 might induce conformational changes similar to those induced by heparin binding. Finally, fluid phase ELISAs might be preferable for the detection of antibodies that bind to conformation-dependent epitopes.