Column-based Technology for CD9-HPLC Immunoaffinity Isolation of Serum Extracellular Vesicles

Area of Science:

• Biochemistry
• Proteomics
• Nanotechnology

Background:

• Serum-derived extracellular vesicles (EVs) are valuable biomarker sources.
• Challenges exist in isolating small-volume EVs from serum while minimizing protein and lipoprotein contamination.

Purpose of the Study:

• To develop and validate a novel CD9-antibody-immobilized high-performance liquid chromatography immunoaffinity chromatography (CD9-HPLC-IAC) technology.
• To enable efficient EV isolation from microliter-scale serum volumes for downstream proteomic analysis.

Main Methods:

• Development of a column-based CD9-HPLC-IAC system for EV isolation.
• Utilized a postcolumn cleaning step with a 50 kDa molecular weight cut-off filter for purification.
• EVs were analyzed using liquid chromatography tandem mass spectrometry.

Main Results:

• Isolated 8.0 × 10^9 EVs from 40 μL of serum in 30 min.
• Identified 482 proteins, including common exosomal markers (CD63, CD81, Alix, TSG101).
• Significantly reduced contamination by top 10 serum proteins and apolipoproteins compared to ultracentrifugation and SEC.

Conclusions:

• The CD9-HPLC-IAC method offers efficient isolation of EVs from microscale serum volumes.
• This technique minimizes protein contamination, enhancing proteomic characterization accuracy.
• Provides a promising tool for biomarker discovery from limited serum samples.