integrin α6  (R&D Systems)

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 3. Branched DNA amplification improves EVPio assay performance. Schematics of oligonucleotide constructs tested for signal amplification: (A) linear strand with one fluorophore; (B) two- branch tree with four fluorophores; (C) and four-branch tree with eight fluorophores. Length ratios of oligos are not to scale. (D) SNR of biotin-streptavidin, linear, two-branch, and four-branch detection schemes for the CD9 detection of SW403 EVs at 9.1 × 1010 particles/ mL. Barcoded antibodies were first incubated and bound to the proteins expressed in immobilized SW403 EVs, followed by incubation and hybridization of pre-assembled oligo trees bearing Alexa Fluor 647 fluorophores. The two-branch amplification design gave signals closest to the benchmark biotin−streptavidin detection. Signal thresholds for each detection scheme, calculated as the NC (GAR) signal + 2 SD, are indicated by horizontal lines color-matched to the corresponding bars. Error bars are SE.

Article Snippet: The following trios of barcoded antibodies were used for triplexed detection: CD63-BC38, ITG α2 (MAB1233, R&D Systems)-BC64, and HSP70-BC23; CD9-BC51, ITG α6 (MAB1350, R&D Systems)-BC09, and Alix (MA1-83977, Invitrogen)-BC60; CD81-BC50, ITG β1 (MAB17781, R&D Systems)-BC27, and HSP90-BC60; CD82 (342102, Biolegend)-BC43, ITG β4 (MAB4060, R&D Systems)-BC79, and claudin-2 (32−5600, Thermo Fisher)-BC61.

Techniques: DNA Amplification, Construct, Amplification, Incubation, Hybridization

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 4. Combinatorial EVPio analysis of inner, cytosolic proteins and outer, membrane proteins and validation of multiplexing through multiplexed vs singleplexed analysis. (A) Fluorescence micrographs of inner protein HSP70 and outer proteins CD9 and CD63 detected on EVPio microarrays with CD63, CD9, CD81, and EpCAM capture spots, and negative controls (GFP) in HT29 EVs at 3.8 × 1010 particles/mL. Complete array images can be found in Figure S7. (B) Violin log plots of the averaged ratios for all four capture antibodies (CD63, CD9, EpCAM, and CD81) as well as a nonspecific control (GFP) of multiplexed over singleplexed signals for inner (HSP70) and outer (CD9 and CD63) detection antibodies and different amplification trees for HT29 EVs. Averaged multiplexed signals are comparable, and up to ∼2.5× higher than the singleplexed signals. Violin plots are used to show the distribution of the data, and overlay a boxplot (median, interquartile range) with a kernel density plot (probabilities).50 (C) Bar graphs of linear, two-branch, and four-branch trees for the signal amplification. Two-branch amplification gave the highest signals. Errors bars are SE.

Article Snippet: The following trios of barcoded antibodies were used for triplexed detection: CD63-BC38, ITG α2 (MAB1233, R&D Systems)-BC64, and HSP70-BC23; CD9-BC51, ITG α6 (MAB1350, R&D Systems)-BC09, and Alix (MA1-83977, Invitrogen)-BC60; CD81-BC50, ITG β1 (MAB17781, R&D Systems)-BC27, and HSP90-BC60; CD82 (342102, Biolegend)-BC43, ITG β4 (MAB4060, R&D Systems)-BC79, and claudin-2 (32−5600, Thermo Fisher)-BC61.

Techniques: Membrane, Biomarker Discovery, Multiplexing, Fluorescence, Control, Amplification

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 5. Multiplexed EVPio analysis of HT29 and SW403 EVs at 1.8 × 1010 particles/mL immobilized with four capture antibodies and a NC (GFP), with four separate 3-plex assays to detect 12 inner and outer proteins. Phenotyping results for (A) HT29 and (B) SW403 EVs. The values for SW403 EVs are expressed as the ratio of the SW403 to the HT29 signal, highlighting the differences between EVs from the two cell lines. Inner proteins are colored in red for emphasis. In HT29 EVs, the CD9+ EV subpopulations presented the highest overall signals, with ITG α6, CD63, ITG β1, and ITG β4 as notable detected markers. HSP70 and Alix were the strongest inner targets. SW403 EVs presented notably higher ITG α6, CD9, and CD81 signals in the EpCAM+ EV subpopulation. Grayed values represent the absence of signal.

Article Snippet: The following trios of barcoded antibodies were used for triplexed detection: CD63-BC38, ITG α2 (MAB1233, R&D Systems)-BC64, and HSP70-BC23; CD9-BC51, ITG α6 (MAB1350, R&D Systems)-BC09, and Alix (MA1-83977, Invitrogen)-BC60; CD81-BC50, ITG β1 (MAB17781, R&D Systems)-BC27, and HSP90-BC60; CD82 (342102, Biolegend)-BC43, ITG β4 (MAB4060, R&D Systems)-BC79, and claudin-2 (32−5600, Thermo Fisher)-BC61.

Techniques: