Zheng Jianlu, El Harakeh Mira Diab, Seo Deokwon, Cabral Horacio, Nguyen Thi-Huong, Yagi Shunsuke, Kim Jinsang, Sugihara Kaori
Institute of Industrial Science, The University of Tokyo, Tokyo 153-8505, Japan.
Department of Materials Science and Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States.
Anal Chem. 2025 Aug 12;97(31):16851-16858. doi: 10.1021/acs.analchem.5c01934. Epub 2025 Jul 30.
Heparin-induced thrombocytopenia (HIT) is a serious side effect that occurs in patients undergoing heparin therapy. The known risk factor is the presence of antibodies created against platelet factor 4 and heparin complexes (PF4/heparin) in the blood, which activate platelet Fc receptors (FcγRIIA). Although immunoassays have been developed for HIT diagnosis, their specificity remains low (∼50%) due to the binding of nonpathogenic antibodies to the same antigen (PF4/heparin). As a result, a lack of rapid, highly sensitive, and selective diagnostic tests poses challenges for HIT treatment. In this study, we used monoclonal HIT-like KKO antibodies as a model and demonstrated a rapid biosensor based on quartz crystal microbalance (QCM) that effectively distinguishes the pathogenic (KKO) from nonpathogenic (RTO) antibodies within 10 min. Based on our key finding, KKO and RTO present clear binding affinity differences against FcγRIIA: RTO binds to FcγRIIA while KKO can bind to FcγRIIA only in the presence of PF4/heparin. The determined negative zeta potentials of proteins confirmed that the observed affinity differences toward FcγRIIA result from specific binding rather than nonselective electrostatic interactions. This highlights the potential use of FcγRIIA as an antigen instead of traditional PF4/heparin complexes. Although only a monoclonal HIT-like antibody was tested, the use of FcγRIIA to distinguish the binding patterns of KKO and RTO could be extended to the detection of human HIT antibodies.
肝素诱导的血小板减少症（HIT）是接受肝素治疗的患者中出现的一种严重副作用。已知的风险因素是血液中存在针对血小板因子4与肝素复合物（PF4/肝素）产生的抗体，这些抗体可激活血小板Fc受体（FcγRIIA）。尽管已经开发出用于HIT诊断的免疫测定法，但由于非致病性抗体与相同抗原（PF4/肝素）结合，其特异性仍然较低（约50%）。因此，缺乏快速、高度灵敏且具有选择性的诊断测试给HIT治疗带来了挑战。在本研究中，我们使用类HIT单克隆KKO抗体作为模型，展示了一种基于石英晶体微天平（QCM）的快速生物传感器，该传感器能够在10分钟内有效区分致病性（KKO）和非致病性（RTO）抗体。基于我们的关键发现，KKO和RTO对FcγRIIA呈现出明显的结合亲和力差异：RTO可与FcγRIIA结合，而KKO仅在存在PF4/肝素时才能与FcγRIIA结合。所测定的蛋白质负zeta电位证实，观察到的对FcγRIIA的亲和力差异是由特异性结合而非非选择性静电相互作用导致的。这凸显了将FcγRIIA用作抗原而非传统PF4/肝素复合物的潜在用途。尽管仅测试了一种类HIT单克隆抗体，但使用FcγRIIA区分KKO和RTO的结合模式可扩展至检测人类HIT抗体。
