Journal: ACS sensors
Article Title: Extracellular Vesicle Antibody Microarray for Multiplexed Inner and Outer Protein Analysis.
doi: 10.1021/acssensors.2c01750
Figure Lengend Snippet: Figure 2. EVPio assay optimization for inner protein detection. (A) Illustration of the need for an AR step. (i) The inner proteins of captured EVs are bound by the lipid bilayer membrane and inaccessible to antibodies. (ii) Fixation cross-links inner proteins, while permeabilization allows antibodies to diffuse into EVs, but methylene cross-linking limits the accessibility to epitopes. Permeabilization can be used to allow antibodies to breach the membrane while keeping cytosolic proteins in place, but cross-links can limit the epitope accessibility. (iii) Heat-based AR treatment of EVs induces partial hydrolytic breakage of cross-links, allowing antibody binding of retained and immobilized proteins. (B) AR optimization experiments with different buffers/additives, temperatures and incubation times, and fluorescence detection signal for inner protein HSP90 and outer protein EGFR, with superposed heatmap. The red rectangle indicates the optimal condition that was chosen based on high HSP90 and high EGFR signals. Signal values are above the threshold of NC + 2SD unless grayed. A zero value indicates a negative or zero difference between the signal and its associated no EV control. (C) Fluorescence micrograph of the optimal AR condition: 1 min at 90 °C, in PBS. (D) Bar graph of HSP90 detection signal on spots with CD63 capture antibody and negative control spots with GAM antibody serving as NC. The threshold of NC + 2SD is illustrated as a horizontal line.
Article Snippet: 2022, 7, 3817−3828 3825 Invitrogen), and biotinylated EGFR antibodies (BAF231, R&D Systems) were used at the detection step at concentrations of 5, 5, and 1 μg/mL, respectively, followed by labeling with Alexa Fluor 647- conjugated goat anti-rabbit antibodies (A-21245, Invitrogen) and streptavidin (S21374, Invitrogen).
Techniques: Membrane, Binding Assay, Incubation, Fluorescence, Control, Negative Control
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