Antibody Conjugated Nanoparticle-based Quantification Using Dark Field Microscopy: A Procedure to Capture and Quantify Specific Exosomes from Body Fluids

1. Preparation of Nanoparticle Probes

NOTE: This assay utilizes Functionalized Gold Nanorods (AuNRs; 25 nm diameter x 71 nm length) that are covalently conjugated with neutravidin polymers (AV) and have a surface plasmon resonance peak that produces a red (641 nm peak) scattering signal upon DFM illumination.

1. Wash 40 µL of AuNR-AV (2.56 x 10¹¹ particles) three times with 200 µL PBS (pH 7.0) by centrifugation and aspiration (8,500 x g at 4 °C for 10 minutes), followed by a final centrifugation and aspiration step after which the AuNR-AV pellet is suspended in 40 µL PBS.
2. Mix this AuNR-AV suspension with 10 µL biotinylated antibody (0.5 mg/mL) specific for an antigen on the surface of the exosome subtype of interest and 150 µL of PBS and then mix at 4 °C for 2 h using a mixer to allow neutravidin-biotin binding to reach completion.
3. Wash the resulting antibody-conjugated AuNRs (AuNR-IgG) three times by centrifugation and aspiration (6,500 x g at 4 °C for 10 minutes) and then suspend them in 200 µL PBS and store them at 4 °C until use.
   NOTE: Sterile technique and short storage times must be used to avoid contamination and degradation of the AuNR-IgG. It is best to use antibody-conjugated AuNRs within 24 hours of their conjugation.

2. Preparation of EV Capture Slides

1. Dilute selected exosome capture antibodies to 0.025 mg/mL in PBS and add 1 µL/well of this dilution onto a multi-well protein A/G slide and then incubate this slide at 37 °C for 1 h in a humidified chamber to allow capture antibody binding to protein A/G immobilized on the slide.
2. Aspirate wells to remove unbound antibodies and wash them three times by the addition and aspiration of 1 µL/well of PBS. Then, load each well with 1 µL of blocking buffer (see Table of Materials) and incubate the slide for 2 h at 37 °C in a humidified chamber to block any remaining protein binding sites.
3. Aspirate wells to remove blocking buffer, wash wells three times by the addition and aspiration of 1 µL/well of PBS, and immediately use the blocked slides for exosome capture and analysis.

3. Standard Curve Preparation

1. To accurately quantify the absolute or relative abundance of a specific exosome subtype, the user must generate a standard curve with a pure exosome population that uniformly expresses the exosome surface biomarker of interest. This study analyses the abundance of exosomes expressing a metastasis-associated membrane protein, Ephrin A2 receptor, which has a reported relationship with pancreatic cancer stage and prognosis.
   NOTE: The human pancreatic cancer cell line PANC-1 and its exosomes are known to express this protein and isolated exosomes from this cell line were used to generate a standard curve to quantify the number of exosomes that express this protein in complex exosome samples.
2. Culture cells for 48 hours at 37 °C in serum-free culture media to allow exosome accumulation in the media, then isolate cell culture supernatants by centrifugation of suspension cultures or direct aspiration of culture media from adherent cell cultures.
3. Centrifuge the collected media at 2000 x g for 30 min to remove debris and recover the supernatant.
4. Filter the clarified culture supernatant through a 0.45 µm low protein binding filter unit of appropriate capacity (e.g., a 250 mL polyether sulfone vacuum filtration unit).
5. Concentrate the resulting filtrate by centrifugation at 3200 x g using a 100,000 nominal molecular weight limit filter system to a 250 µL final volume. Collect the retained volume from this filter, then wash the filter with 200 µL PBS, and combine this wash volume with the collected exosome sample volume.
6. Centrifuge this sample at 21,000 x g for 45 minutes and carefully recover the supernatant, taking care not to collect any precipitated material.
7. Centrifuge the recovered supernatant at 100,000 x g for 3 hours to precipitate the exosomes. Aspirate away the supernatant and collect the exosome pellet in 100 µL PBS.
8. Store the resulting exosome suspensions at 4 °C if used within 24 hours or at -80 °C for long-term storage.
   NOTE: Do not subject exosome samples to repeat freeze-thaw cycles.
9. Quantify an aliquot of the exosome suspension after mixing by direct measurement of exosome numbers (e.g., by nanoparticle tracking analysis or tuneable resistive pulse sensing or by measuring the protein concentration of exosome lysates by micro-bicinchoninic acid assay, or an equivalent method, as a means to approximate exosome quantity).
10. Generate a set of serial dilutions of the exosome suspension to allow comparison of nanoparticle signal to input exosome number or protein content.
11. Transfer 1 µL of each exosome standard to each of its replicate wells on the assay plate.
    NOTE: Standard curves can be used to calculate the slope of the correlation line between nanoparticle signal and exosome concentration to (1) evaluate assay performance and (2) determine the relative concentration of target exosomes in experimental samples.

4. Processing Human Plasma or Serum Samples

1. Collect plasma or serum samples by standard methods and store at -80 °C until needed for exosome analysis. Rapidly thaw samples in a room temperature water bath. Repeatedly mix the thawed samples by inversion to promote homogenous suspension.
   NOTE: Results from serum and plasma samples may not be equivalent since there is a significant release of exosomes during the clotting reaction.
2. Centrifuge plasma or serum samples at 500 x g for 15 min to precipitate protein aggregates and other debris. Transfer an aliquot of the plasma or serum sample to a fresh tube and add PBS to generate a 1:1 dilution. Mix the diluted sample by gentle vortexing or inversion, as appropriate. Transfer 1 µL of each plasma or serum suspension to each of its replicate wells on the assay plate.

5. Exosome Capture and Detection

1. Load wells of a blocked EV capture slide with 1 µL/well of exosome sample, using 8 replicates per sample, and incubate the slide overnight at 4 °C in a humidified chamber. Aspirate all sample wells and then add 1 µL/well of PBS to wash wells and remove unbound exosomes and other contaminants from the loaded exosome sample.
2. Load sample wells with 1 µL/well of a previously prepared AuNR-IgG suspension (see section 1 above) and incubate the slide for 2 h at 37 °C in a humidified chamber. Aspirate the nanoparticle solution and wash the slide in PBS supplemented with 0.01% Tween-20 (PBST) for 10 min using a mixer, then aspirate and wash all sample wells with deionized water for 10 min using a rotating mixer and air-dry for subsequent LMDFM imagery.
   NOTE: Inter-assay coefficients of variation (CVs) are assessed from eight replicates of the same sample. Samples that exhibit CVs >20% are considered non-informative and should be repeated, if there is sufficient sample.