Antibodies against the chemokine platelet factor 4 (PF4) in complex with heparin cause the prothrombotic adverse drug reaction heparin-induced thrombocytopenia (HIT). The current dilemma for laboratory diagnosis of HIT is that only about 50% of these antibodies are pathogenic, but the widely available enzyme immunosorbent assays (EIA), used for diagnosis in the clinical laboratory cannot differentiate between non-pathogenic- and pathogenic (platelet activating) antibodies. It would be highly desirable to improve diagnosis for HIT. Here, we use single-molecule force spectroscopy to identify conditions at which non-pathogenic antibodies show a different reactivity pattern compared to pathogenic antibodies. At physiological conditions (pH 7.4, 150 mM NaCl), non-pathogenic antibodies bound to PF4/heparin complexes with weaker binding forces than pathogenic ones. Binding forces measured over a wide range of pH or ionic strength show that non-pathogenic antibodies are not significantly affected by environmental changes, whereas pathogenic antibodies are. Based on the dissimilar behavior of these antibodies at 500 mM salt, we performed a PF4/heparin EIA at this condition and observed that optical density of non-pathogenic antibodies slightly reduced while it drastically reduced for pathogenic antibodies. Our results suggest that the specificity of anti-PF4/heparin EIAs for clinically relevant antibodies can be improved by changing test conditions.
Keywords: Binding force; Discrimination; EIA OD; Human PF4/P antibody; Ionic strength; pH.