Bulk immunoassays for analysis of extracellular vesicles

Figures & data

Figure 1. Schematic overview of bulk immunoassays for EV characterization. Western Blot (WB) performs EV lysis, gel electrophoresis (EF) of all protein content, and transfer EF to a membrane for subsequent immuno-labeling. Micro-Nuclear Magnetic Resonance (µNMR) selects EV sizes by filtration, and labels EV with immunomagnetic particles for subsequent detection of the magnetic susceptibility of the sample. Amplified Luminescent Proximity Homogeneous Assay (ALPHA) detects transfer of singlet oxygen from the donor to the acceptor if they are in close proximity, i.e. bound to an EV. Surface Plasmon Resonance (SPR) captures EV on an immuno-functionalized surface, and detects the resulting change in refractive index. The other immunosorbent assays are all variations of the sandwich Enzyme Linked Immuno Sorbent Assay (ELISA), where capture of EV on a functionalized surface is complemented by detection of a different antigen, or epitope. The reporter molecules can be enzymes (ELISA), fluorescent molecules (FLISA, Fluorescent Immuno Sorbent Assay), or phosphorescent molecules (TRFIA, Time-Resolved Fluorescent ImmunoAssay). To overcome the mass transport limitations near a surface, electrohydrodynamic-induced nanoshearing (ac-EHD) generates additional flow near the surface, while the Integrated Microfluidic Exosome Analysis Platform (IMEAP) mobilizes the surface itself by means of immunomagnetic beads.

Table 1. Comparison of bulk immunoassays for extracellular vesicles (EV).

	In gel	In Suspension		Immunosorbent
Method	WB	µNMR	ALPHA	SPR	ELISA	FLISA	TRFIA	ac-EHD	IMEAP
Limit of detection (EV/mL)	10^12 ⁰	10^7 †	10^10 ‡	10^7 *	10^10 *	10^10 †	10^9 ⁰	10^9 ‡	10^8 ‡
Dynamic range^◊	10^2	10^2	10^3	10^6	10^3	-	10^5	-	10^4
Sample volume (μL)	10-1000	1-100	5	20-150	100	1-10	100 ⁰	500	30
Targets on same EV (n)	1	1	2	1	2	2	2	2	2
Parallel experiments	10	3	96	96	96	96	96	96	1
Analysis duration (h)	3	5	3	2	2	24	8	4	2
Articles (n)	49	1	1	5	18	2	1	1	1
Data references	[27]	[13]	[14]	[16, 17]	[16, 20]	[21]	[23]	[24]	[26]

Source: * Directly provided, † calculated from minimal number of EV /sample volume, ‡ relative to ELISA with sensitivity of 10¹⁰/mL, ⁰ Inquiry with author, ^◊ maximum/minimum detected signal

WB = western blot, µNMR = micro-nuclear magnetic resonance, ALPHA = Amplified Luminescent Proximity Homogeneous Assay, SPR = surface plasmon resonance, ELISA = enzyme-linked immunosorbent assay, FLISA = Fluorescent immunosorbent assay, TRFIA = time-resolved fluorescence immunoassay, ac-EHD = alternating current electrohydrodynamic-induced nanoshearing, IMEAP = integrated microfluidic exosome analysis platform

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