Reactivity of platelet‐activating and nonplatelet‐activating anti‐ PF 4/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 ( aPF 4/H Abs) to rule out heparin‐induced thrombocytopenia. EIA s 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 aPF 4/H Abs bind more strongly to PF 4/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 EIA s detecting aPF 4/H Abs. Methods We investigated first the conformational change of PF 4 when binding to heparin under various pH and salt conditions using circular dichroism spectroscopy, and then the binding of aPF 4/H Abs to PF 4/H complexes by EIA. Results Maximal conformational change of PF 4 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 aPF 4/H Abs. Conclusion Changing physicochemical conditions may become an approach to better discriminate the signal of platelet‐activating and nonactivating PF 4/H Abs in antigen tests.