La Trobe

An anion exchange membrane sensor detects EGFR and its activity state in plasma CD63 extracellular vesicles from patients with glioblastoma

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journal contribution
posted on 2024-06-28, 06:29 authored by NH Maniya, S Kumar, JL Franklin, JN Higginbotham, Andrew ScottAndrew Scott, Hui GanHui Gan, RJ Coffey, S Senapati, HC Chang
We present a quantitative sandwich immunoassay for CD63 Extracellular Vesicles (EVs) and a constituent surface cargo, EGFR and its activity state, that provides a sensitive, selective, fluorophore-free and rapid alternative to current EV-based diagnostic methods. Our sensing design utilizes a charge-gating strategy, with a hydrophilic anion exchange membrane functionalized with capture antibodies and a charged silica nanoparticle reporter functionalized with detection antibodies. With sensitivity and robustness enhancement by the ion-depletion action of the membrane, this hydrophilic design with charged reporters minimizes interference from dispersed proteins, thus enabling direct plasma analysis without the need for EV isolation or sensor blocking. With a LOD of 30 EVs/μL and a high relative sensitivity of 0.01% for targeted proteomic subfractions, our assay enables accurate quantification of the EV marker, CD63, with colocalized EGFR by an operator/sample insensitive universal normalized calibration. We analysed untreated clinical samples of Glioblastoma to demonstrate this new platform. Notably, we target both total and “active” EGFR on EVs; with a monoclonal antibody mAb806 that recognizes a normally hidden epitope on overexpressed or mutant variant III EGFR. Analysis of samples yielded an area-under-the-curve (AUC) value of 0.99 and a low p-value of 0.000033, surpassing the performance of existing assays and markers.

Funding

This work was partially supported by the NIH Commons Fund, through the Office of Strategic Coordination/Office of NIH Director, 1UH3CA241684-01 (HCC & SS) and 1UH3CA241685 (RJC).

History

Publication Date

2024-06-03

Journal

Communications Biology

Volume

7

Issue

1

Article Number

677

Pagination

11p.

Publisher

Springer Nature

ISSN

2399-3642

Rights Statement

This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit: http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2024