优化Western blot检测人体细胞中低水平组织因子的方法

IF 3.4 3区医学 Q2 HEMATOLOGY

Research and Practice in Thrombosis and Haemostasis Pub Date : 2025-08-01 DOI:10.1016/j.rpth.2025.103016

Megan V. Perkins , Ana T.A. Sachetto , Nigel Mackman

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引用次数: 0

摘要

组织因子(TF)/活化因子VII复合物是凝血系统的主要激活剂。TF由多种细胞表达，包括活化的单核细胞和肿瘤细胞。TF表达升高可引起不同疾病的血栓形成，包括败血症、病毒感染和癌症。我们之前已经描述了一种通过Western blotting分析高表达细胞中人TF的方法。目的建立一种在低表达细胞中检测人TF的方法。方法检测兔多克隆人源性TF抗体NBP2-15139 （Novus Biologicals）、山羊多克隆人源性TF抗体AF2339 （R&D Systems）和兔单克隆人源性TF抗体ab252918（克隆EPR22548-240； Abcam）在低表达细胞系中检测TF的能力。我们还使用Abcam抗体检测了TF在脂多糖刺激的外周血单个核细胞中的表达。结果检测结果显示，阻断条件、检测方法、一抗和二抗等因素均影响其敏感性。R&；D和Abcam抗体在评估TF表达方面都比Novus抗体更具特异性；而在低表达细胞系中，Abcam抗体对TF的评价效果最好。用Abcam抗体检测脂多糖刺激的人外周血单个核细胞中的TF。结论在建立检测低丰度抗原（如TF）的方法时，应考虑Western blotting的每一步。

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Optimization of a Western blot protocol for the detection of low levels of tissue factor in human cells

Background

The tissue factor (TF)/activated factor VII complex is the major activator of the coagulation system. TF is expressed by a variety of cells, including activated monocytes and tumor cells. Increased TF expression can cause thrombosis in different diseases, including sepsis, viral infections, and cancer. We have previously described a method for analyzing human TF in high-expressing cells by Western blotting.

Objectives

The goal of this study was to establish a method for detecting human TF in low-expressing cells.

Methods

We examined the ability of 3 different antibodies to detect TF in low-expressing cell lines: rabbit polyclonal anti-human TF antibody NBP2-15139 (Novus Biologicals), goat polyclonal anti-human TF antibody AF2339 (R&D Systems), and rabbit monoclonal anti-human TF antibody ab252918 (clone EPR22548-240; Abcam). We also used the Abcam antibody to measure TF expression in lipopolysaccharide-stimulated peripheral blood mononuclear cells.

Results

We found that sensitivity was affected by various factors, including the blocking conditions, the detection method, and the primary and secondary antibodies. Both the R&D and Abcam antibodies were more specific in assessing TF expression than the Novus antibody; however, the Abcam antibody was the best of the 3 in evaluating TF in low-expressing cell lines. We detected TF in lipopolysaccharide-stimulated human peripheral blood mononuclear cells using the new method with the Abcam antibody.