Institute for Immunology and Transfusion Medicine, University Medicine of Greifswald, Greifswald, Germany.
ZIK HIKE - Center for Innovation Competence, Humoral Immune Reactions in Cardiovascular Diseases, University of Greifswald, Greifswald, Germany.
J Thromb Haemost. 2019 Jul;17(7):1113-1119. doi: 10.1111/jth.14455. Epub 2019 May 17.
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.
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.
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.
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.
Changing physicochemical conditions may become an approach to better discriminate the signal of platelet-activating and nonactivating PF4/H Abs in antigen tests.
在低 pH 值和低盐浓度下：PF4 与肝素复合时发生最大构象变化。改变理化条件可能成为更好地区分血小板激活和非激活 PF4/H Abs 在抗原检测中信号的一种方法。
酶免疫吸附测定（EIA）广泛用于检测人抗血小板因子 4/肝素抗体（aPF4/H Abs）以排除肝素诱导的血小板减少症。EIA 不能区分临床相关的、血小板激活的和非相关的、非血小板激活的 Abs，并且只有~50%的通过 EIA 检测呈阳性的患者血清中含有激活血小板的抗体。最近，我们已经表明，血小板激活的 aPF4/H Abs 比非血小板激活的抗体更强烈地结合到 PF4/H 复合物上。已知抗原抗体相互作用取决于由 pH 值、热和离子强度控制的静电相互作用。我们测试了 pH 值和离子强度的变化是否可以提高检测 aPF4/H Abs 的 EIA 的特异性。
我们首先使用圆二色光谱法研究了 PF4 在各种 pH 值和盐浓度下与肝素结合时的构象变化，然后通过 EIA 研究了 aPF4/H Abs 与 PF4/H 复合物的结合。
在低 pH 值和低盐浓度下，PF4 与肝素复合时的最大构象变化被鉴定出来。在 50mmol/L NaCl、pH 6.0 条件下进行的大量血清 EIA 测试显示出提高检测血小板激活 aPF4/H Abs 特异性的潜力。
改变理化条件可能成为更好地区分血小板激活和非激活 PF4/H Abs 在抗原检测中信号的一种方法。
