Fig 1: Overview of experimental framework for EV detection using Simoa and size exclusion chromatography (SEC).(a) Different methods of EV isolation can be directly compared to assess yield and purity by measuring the three tetraspanins (CD9, CD63, and CD81) and albumin. (b) Single immuno-complexes are formed by binding the target tetraspanin protein on EVs to a magnetic bead conjugated to a capture antibody and a biotin-labeled detection antibody. Detection antibodies are labeled with a streptavidin-conjugated enzyme. The beads are then loaded into individual wells of a microwell array where each well matches the size of the magnetic bead limiting a maximum of one bead per well. Wells with the full immuno-complex (on wells) produce a fluorescent signal upon conversion of substrate, unlike wells with beads lacking the immuno-complex (off wells”). (c) EV and free proteins such as albumin in a biofluid sample are separated by SEC. Free proteins elute from the column in later fractions than EVs because free proteins are smaller than the pore size of the beads while EVs are larger and are excluded from entering the beads. EV, extracellular vesicle.
Fig 2: Separation of extracellular vesicles (EVs), lipoproteins, and free proteins from plasma using density gradient centrifugation.Levels of CD9, CD63, CD81, albumin, and ApoB-100 were measured by Simoa in individual 1 ml fractions (collected from the top) after density gradient centrifugation of plasma using an iodixanol gradient. Error bars represent the standard deviation of two replicates of each measurement. Figure 3—source data 1.Simoa data (protein concentrations) for different density gradient centrifugation fractions.
Fig 3: Comparison of novel columns for extracellular vesicle (EV) isolation from plasma using electron microscopy and Simoa.(A) Schematic of the columns being compared: size exclusion chromatography (SEC) column comprised of 10 ml Sepharose CL-6B, dual-mode chromatography (DMC) columns comprised of 10 ml Sepharose CL-6B SEC resin atop 2 ml Fractogel cation exchange resin, Tri-Mode Chromatography (TMC) columns comprised of 10 ml Sepharose CL-6B SEC resin atop 2 ml 2:1 ratio of 2 ml Fractogel cation exchange resin to Capto Core 700 multimodal chromatography resin. (B) Electron microscopy of EVs isolated from plasma using SEC (left), DMC (middle), or TMC (right) columns. EVs indicated with red arrows (among background of lipoproteins). (C) EV recovery is calculated for EV isolation from plasma for SEC (fractions 7–10), DMC (fractions 9–12), or TMC (fractions 9–12). Simoa measurements in the designated fractions for CD9, CD63, and CD81 are taken as a ratio relative to measurements of these proteins from diluted plasma and these three ratios are then averaged to calculate recovery. (D) Purity of EVs with respect to free proteins is determined by dividing relative EV yield (the average of the ratios of CD9, CD63, and CD81) by relative levels of albumin in each condition. (E) Purity of EVs with respect to lipoproteins is determined by dividing relative EV yield (the average of the ratios of CD9, CD63, and CD81) by relative levels of ApoB-100 in each condition. Error bars represent the standard deviation of four column measured on different days with two technical replicates each. Figure 4—source data 1.Simoa data (protein concentrations) for TMC columns with different ratios of resins in the bottom layer. Figure 4—source data 2.Simoa data (protein concentrations) for different fractions of SEC and DMC columns. Figure 4—source data 3.Simoa data (protein concentrations) comparing SEC (fractions 7-10), DMC and TMC columns (fractions 9-12).
Fig 4: Size exclusion chromatography (SEC) of plasma using different resins.(A) Levels of CD9, CD63, CD81, ApoB-100, and albumin were measured by Simoa after SEC of 1 ml plasma in each fraction using either Sepharose CL-2B, Sepharose CL-4B, or Sepharose CL-6B resin. (B) Extracellular vesicle (EV) yield is calculated in fractions 7–10 for Sepharose CL-2B, Sepharose CL-4B, or Sepharose CL-6B by averaging the ratios of CD9, CD63, and CD81. (C) Purity of EVs with respect to lipoproteins or free proteins is calculated by dividing relative EV yield (the average of the ratios of CD9, CD63, and CD81) by levels of ApoB-100 (top) or albumin (bottom). Error bars represent the standard deviation of four columns measured on different days with two technical replicates each. Figure 2—source data 1.Simoa data (protein concentrations) for fractions of SEC columns with different resins. Figure 2—source data 2.Simoa data (protein concentrations) for SEC column with different number of washes.
Fig 5: Development and validation of automated device for running size exclusion chromatography (SEC) columns in parallel.(A) CAD image of semi-automated SEC stand designed to hold eight columns at once with sliding collection tube holder that allows liquid to drip either into 2 ml collection tubes, or to waste. (B) Photograph of stand connected to a Tecan Cavro syringe pump controlled by a Raspberry Pi. (C) Simoa comparison of CD9, CD63, CD81, ApoB-100, and albumin when SEC was performed on 16 samples of 1 ml plasma using either manual SEC (8 samples) or SEC on the automated device (8 samples). Each point is the average of two Simoa measurements (technical replicates). Figure 5—source data 1.Simoa data (protein concentrations) comparing manual and automated SEC EV isolation (fractions 7-10).
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