Waas Daniela, Juraschitz Marc, Chen Yen-Fu Adam, Waltenberger Harald, Hammerschmidt Wolfgang, Zeidler Reinhard, Molinaro Sonja, Gärtner Kathrin
Eximmium Biotechnologies GmbH, Munich, Germany.
Microcoat Biotechnologie GmbH, Bernried, Germany.
J Extracell Vesicles. 2025 Aug;14(8):e70115. doi: 10.1002/jev2.70115.
Extracellular vesicles (EVs) hold great promise as circulating biomarkers for diagnostic and therapeutic approaches. Thus, many research groups world-wide investigate important aspects of EVs including their biology and medical significance. For this, a large number of procedures and protocols has been established making it difficult to almost impossible to compare and replicate results. Consequently, diagnostic tests remain problematic to interpret, mainly because the use of reliable reference EVs as a qualified standard has not yet gained widespread acceptance. Beyond doubt, such reference EVs are key to assess EV preparations quantitatively and to establish robust quality control processes to ensure overall quality and validity of data. To further advance the establishment of such controls, we designed and generated a new class of reference EVs expressing horseradish peroxidase (HRP) to facilitate simple and reliable EV tracing during isolation and standardization of EV purification and downstream analysis processes. HRP EVs can be quantified easily and with unmatched sensitivity either directly via measuring HRP activity or indirectly via immunodetection of HRP on the EV surface. We demonstrate that HRP EVs allow the reliable quantification of absolute EV numbers in biological or medical samples to normalize clinical specimens in liquid biopsies.
细胞外囊泡（EVs）作为用于诊断和治疗方法的循环生物标志物具有巨大潜力。因此，全球许多研究团队都在研究EVs的重要方面，包括其生物学特性和医学意义。为此，已经建立了大量的程序和方案，这使得几乎不可能比较和重复结果。因此，诊断测试的结果仍然难以解释，主要是因为使用可靠的参考EVs作为合格标准尚未得到广泛认可。毫无疑问，此类参考EVs是定量评估EV制剂以及建立稳健的质量控制流程以确保数据的整体质量和有效性的关键。为了进一步推进此类对照的建立，我们设计并生成了一类新的表达辣根过氧化物酶（HRP）的参考EVs，以便在EV纯化及下游分析过程的分离和标准化过程中实现简单可靠的EV追踪。HRP EVs可以通过直接测量HRP活性或间接通过免疫检测EV表面上其HRP来轻松且以无与伦比的灵敏度进行定量。我们证明，HRP EVs能够可靠地定量生物或医学样品中的绝对EV数量，从而对液体活检中的临床标本进行标准化。
