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There are currently major challenges in EV isolation of small amounts, i.e. microliter-scale, of human serum while minimizing contamination of blood proteins and lipoprotein particles. One solution we have developed involves a column-based CD9 antibody-immobilized HPLC immunoaffinityImmunoaffinity (CD9-HPLC-IAC) technology for EV isolation from serum for downstream proteomicProteomics analysis. The CD9-HPLC-IAC method can achieve EV isolation from 40 μL of serum in 30 min with a yield of 8.0 × 109 EVs. Mass spec analysis yielded 482 proteins identified in EVs from patient serum using liquid chromatography tandem mass spectrometry (LC–MS/MS). Among the detected proteins were common exosomal markers such as CD63, CD81, CD82, Alix, and TSG101. In the EV protein content, the top 10 serum proteins were significantly decreased by using the CD9-HPLC-IAC method compared to ultracentrifugationUltracentrifugation (UC) and size exclusionSize exclusion chromatography (SEC). In addition, contamination from alipoproteins and albumin were minimized. This technology could in principle be extended to other important surface protein markers for detection of cancer or other diseases.

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A Column-Based CD9-HPLC Immunoaffinity Isolation Method of Extracellular Vesicles from Serum

  • 章节

  • 发表日期：

    2025年

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  • Jie Zhang,
  • Jianhui Zhu,
  • Komal Abhange,
  • David M. Lubman

摘要

There are currently major challenges in EV isolation of small amounts, i.e. microliter-scale, of human serum while minimizing contamination of blood proteins and lipoprotein particles. One solution we have developed involves a column-based CD9 antibody-immobilized HPLC immunoaffinityImmunoaffinity (CD9-HPLC-IAC) technology for EV isolation from serum for downstream proteomicProteomics analysis. The CD9-HPLC-IAC method can achieve EV isolation from 40 μL of serum in 30 min with a yield of 8.0 × 109 EVs. Mass spec analysis yielded 482 proteins identified in EVs from patient serum using liquid chromatography tandem mass spectrometry (LC–MS/MS). Among the detected proteins were common exosomal markers such as CD63, CD81, CD82, Alix, and TSG101. In the EV protein content, the top 10 serum proteins were significantly decreased by using the CD9-HPLC-IAC method compared to ultracentrifugationUltracentrifugation (UC) and size exclusionSize exclusion chromatography (SEC). In addition, contamination from alipoproteins and albumin were minimized. This technology could in principle be extended to other important surface protein markers for detection of cancer or other diseases.