Effect of pH and ionic strength on the binding strength of anti-PF4/polyanion antibodies.

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.

Reactivity of platelet-activating and nonplatelet-activating anti-PF4/heparin antibodies in enzyme immunosorbent assays under different conditions.

Essentials At low pH and low salt concentrations: Maximal conformational change of PF4 upon complexation with heparin occurs. Changing physicochemical conditions may become an approach to better discriminate the signal of platelet-activating- and nonactivating PF4/H Abs in antigen tests. BACKGROUND: Enzyme immunosorbent assays (EIA) are widely used to detect human antiplatelet factor 4/heparin antibodies (aPF4/H Abs) to rule out heparin-induced thrombocytopenia. EIAs cannot differentiate between clinically relevant, platelet-activating, and nonrelevant, nonplatelet-activating Abs and only ~50% of patients' sera testing positive by EIA contain antibodies that activate platelets. Recently, we have shown platelet-activating aPF4/H Abs bind more strongly to PF4/H complexes than nonplatelet-activating antibodies. Antigen-antibody interactions are known to depend on electrostatic interactions governed by pH, heat, and ionic strength. We tested whether changes in pH and ionic strength can improve the specificity of EIAs detecting aPF4/H Abs. METHODS: We investigated first the conformational change of PF4 when binding to heparin under various pH and salt conditions using circular dichroism spectroscopy, and then the binding of aPF4/H Abs to PF4/H complexes by EIA. RESULTS: Maximal conformational change of PF4 on complexation with heparin was identified at low pH and low salt concentrations. EIA tested with a large number of sera at 50 mmol/L NaCl, pH 6.0 shows a potential to increase the specificity for the detection of platelet-activating aPF4/H Abs. CONCLUSION: Changing physicochemical conditions may become an approach to better discriminate the signal of platelet-activating and nonactivating PF4/H Abs in antigen tests.