Immuno-qPCR: a simple and powerful tool for high-throughput EV surface protein profiling across diverse biofluids

Introduction:
The molecular cargo of extracellular vesicles (EVs) is reflective of their cellular origin, making EVs a promising source of biomarkers for various diseases. Traditionally, biomarker research has focused on nucleic acids rather than proteins, largely due to the limitations of existing methods for protein detection. Current methods often lack the sensitivity to accurately quantify low-abundance proteins in complex biological fluids, they are difficult to scale up, and they may require specialized equipment, limiting their practicality for routine clinical use. To address these challenges, we developed an immuno-qPCR method, which significantly enhances sensitivity while offering a cost-effective and high-throughput approach for detecting surface proteins on EVs derived from clinical samples.

Methods:
To adapt immuno-qPCR for EV surface protein detection, we evaluated different protein immobilization strategies. The final optimized protocol employed magnetic beads to capture EVs, followed by surface protein detection using commercially available antibodies conjugated to DNA tags. These DNA tags were then amplified and quantified through qPCR using specific primers. In a proof-of-concept study, we compared the performance of our immuno-qPCR method to conventional flow cytometry-based detection in clinical serum samples from patients with atopic dermatitis.

Results:
The use of magnetic beads, both general protein-binding and exosome-specific, allowed for efficient capture of EVs, with the exosome-specific beads providing an additional advantage of increased specificity and faster workflow. When tested on a cohort of 16 atopic dermatitis patients and 16 healthy volunteers, our optimized immuno-qPCR method simultaneously detected 10 different surface proteins and demonstrated superior sensitivity compared to conventional flow cytometry-based detection. Notably, for one of the tested proteins, CD25, we detected a significant difference in levels between patients and healthy volunteers, which was not uncovered with flow cytometry. As CD25-positive immune cells have been previously shown to be enriched in the blood of atopic dermatitis patients, this finding further validates our approach.

Conclusions:
The immuno-qPCR method offers a highly sensitive, scalable, and versatile platform for the detection of EV surface proteins. This approach holds great promise for biomarker discovery and non-invasive diagnostics, with potential applications extending beyond blood and atopic dermatitis.