Li-Yu Chen, Uwe Schirmer, Peter F Zipfel, Doris Heinrich, Thi-Huong Nguyen
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引用次数: 0
Abstract
Accurate detection of heparin-induced thrombocytopenia (HIT) antibodies is crucial for diagnosing and managing thrombotic events. Conventional immunoassays, however, often lack specificity and require confirmatory testing with fresh human platelets. To address this limitation, we optimized our previously developed cell-based enzyme-linked immunosorbent assay (ELISA) for improved HIT detection under various experimental conditions. Platelet factor 4 was immobilized on breast cancer cells (MDA-MB-231) to capture monoclonal HIT-like (KKO) and non-HIT (RTO) antibodies, which served as models to evaluate assay performance under different pH levels, ionic strengths (NaCl), and fixation methods (ethanol, paraformaldehyde, glutaraldehyde). To identify the most suitable substrate, additional cancer cell lines (HCT-116, MCF-7, HepG2) were tested under live and fixed conditions, with selected conditions validated using human HIT sera. Optimal detection tested with monoclonal antibodies was achieved using 50 mM NaCl and 4% paraformaldehyde fixation. Notably, live MDA-MB-231 and HCT-116 cells demonstrated superior sensitivity and specificity compared to fixed cells. Furthermore, these cell lines enable the efficient detection of HIT antibodies using flow cytometry, a robust and platelet-free diagnostic method. Our findings establish live MDA-MB-231 and HCT-116 cells as highly promising platforms for clinical applications in HIT antibody detection.
准确检测肝素诱导的血小板减少(HIT)抗体是诊断和管理血栓事件的关键。然而,传统的免疫测定法往往缺乏特异性,需要用新鲜的人血小板进行确认试验。为了解决这一限制,我们优化了之前开发的基于细胞的酶联免疫吸附测定(ELISA),以改进在各种实验条件下的HIT检测。将血小板因子4固定在乳腺癌细胞(MDA-MB-231)上,捕获单克隆hit样抗体(KKO)和非hit样抗体(RTO),并将其作为模型,评估不同pH水平、离子强度(NaCl)和固定方法(乙醇、多聚甲醛、戊二醛)下的检测性能。为了确定最合适的底物,在活的和固定的条件下测试了额外的癌细胞系(HCT-116, MCF-7, HepG2),并使用人HIT血清验证了选定的条件。采用50 mM NaCl和4%多聚甲醛固定,单克隆抗体检测效果最佳。值得注意的是,与固定细胞相比,活的MDA-MB-231和HCT-116细胞表现出更高的敏感性和特异性。此外,这些细胞系能够使用流式细胞术高效检测HIT抗体,流式细胞术是一种强大的无血小板诊断方法。我们的研究结果表明,活的MDA-MB-231和HCT-116细胞在HIT抗体检测的临床应用中具有很高的前景。