Effect of pH and ionic strength on the binding strength of anti-PF4/polyanion antibodies
Effect of pH and ionic strength on the binding strength of anti-PF4/polyanion antibodies
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DOI：
10.1007/s00249-017-1240-8
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发表时间：
2017-07
期刊：
影响因子：
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通讯作者：
Thi‐Huong Nguyen;A. Greinacher
中科院分区：
文献类型：
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作者：
Thi‐Huong Nguyen;A. Greinacher
抗趋化因子血小板因子4（PF 4）的抗体与肝素复合可引起血栓前药物不良反应肝素诱导的血小板减少症（HIT）。HIT的实验室诊断的当前困境是，这些抗体中只有约50%是致病性的，但是用于临床实验室诊断的广泛可用的酶免疫吸附测定（EIA）不能区分非致病性和致病性（血小板活化）抗体。非常希望改善HIT的诊断。在这里，我们使用单分子力谱来确定条件下，非致病性抗体显示出不同的反应模式相比，致病性抗体。在生理条件下（pH 7.4，150 mM NaCl），非致病性抗体结合PF 4/肝素复合物的结合力弱于致病性抗体。在宽范围的pH或离子强度下测量的结合力表明，非致病性抗体不受环境变化的显著影响，而致病性抗体受环境变化的显著影响。基于这些抗体在500 mM盐下的不同行为，我们在该条件下进行了PF 4/肝素EIA，并观察到非致病性抗体的光密度略微降低，而致病性抗体的光密度急剧降低。我们的研究结果表明，抗PF 4/肝素EIA临床相关抗体的特异性可以通过改变测试条件来提高。
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.