qEVoriginal Gen 2 User Manual

1. Definitions and Writing Conventions

This guide uses specific symbols to highlight important information:

Terminology Used in this Manual

2. Safety and Hazards

Refer to the Safety Data Sheet at support.izon.com/safety-data-sheets for hazard classification and precautionary statements.

2.1 Hazards

qEV columns are laboratory products. If biohazardous samples are present, adhere to current Good Laboratory Practices (cGLPs) and local guidelines.

Chemical Hazards

! The qEV column contains < 0.1% sodium azide, which is potentially fatal if swallowed or in contact with skin. Review the following guidelines and precautions before each use:

Prevention

1. Do not get into eyes, on skin, or on clothing.
2. Wash skin thoroughly after handling.
3. Do not eat, drink, or smoke when using this product.
4. Avoid release of product into the environment.
5. Wear protective gloves and clothing; follow general laboratory precautions.

Response

1. IF SWALLOWED: immediately call a POISON CONTROL CENTRE/Doctor.
2. IF ON SKIN: Gently wash with plenty of soap and water and immediately call a POISON CONTROL CENTRE/Doctor.
3. Remove immediately any contaminated clothing and wash before reuse.
4. Collect any spillage and dispose of appropriately.

Disposal of Biohazardous Material

Adhere to local, regional, and national guidelines for the use and disposal of biohazardous material.

General Precautions

• Always wear laboratory gloves, coats, and safety glasses with side shields or goggles.
• Keep hands away from mouth, nose, and eyes.
• Completely protect any cut or abrasion before working with potentially infectious or hazardous material.
• Wash hands thoroughly with soap and water after working with potentially infectious or hazardous material before leaving the laboratory.
• Remove watches and jewellery before working at the bench.
• Contact lenses are not recommended due to potential complications during emergency eye-wash procedures.
• Before leaving the laboratory, remove protective clothing.
• Do not use a gloved hand to write, answer the telephone, turn on a light switch, or physically engage with people without gloves.
• Change gloves frequently.
• Remove gloves immediately when visibly contaminated.
• Do not expose materials that cannot be properly decontaminated to potentially infectious or hazardous material.
• Upon completion of tasks involving potentially infectious or hazardous materials, decontaminate the work area with an appropriate disinfectant or cleaning solution (1:10 dilution of household bleach is recommended).

Dispose of potentially contaminated materials in accordance with laboratory local, regional, and national regulations:

• Biological samples
• Reagents
• Used reaction vessels or other consumables that may be contaminated

2.2 Storage

Avoid rapid temperature changes, as they can introduce bubbles into the resin bed.

Store unused qEV columns at room temperature. Used qEV columns can be stored at room temperature if cleaned according to instructions and stored in 20% ethanol or 0.05% w/v sodium azide. If these solutions are unavailable, columns can be stored at +4 to +8 °C after use.

2.3 Disposal

Waste buffer should be disposed of safely. Sodium azide accumulation in copper pipes over time can result in an explosion.

3. Introduction to Size Exclusion Chromatography

3.1 Overview

qEV Size Exclusion Chromatography (SEC) columns separate particles by size using a porous, polysaccharide resin. As the sample passes through the column, smaller particles are delayed by entering the resin pores, while larger particles exit first. Sequential volumes are collected, with particles distributed by size.

The column is filled with a buffer (mobile phase) and the resin (stationary phase). Particles do not bind to the resin, so buffer composition does not significantly affect resolution.

Diagram Description: Figure 1 illustrates the SEC process. (A) shows a resin bead with an electron microscopic enlargement. (B) depicts sample molecules diffusing into the resin beads. (C) graphically describes separation, showing larger particles (blue) eluting before smaller particles (pink).

3.2 Intended Use

qEV columns isolate extracellular vesicles (EVs) from biological samples. qEVoriginal Gen 2 columns are equipped with RFID chips for use with the Automatic Fraction Collector (AFC). These columns are intended for research laboratories by professional personnel for research use only. They are not intended for diagnostic purposes or to make treatment decisions.

qEV columns isolate and purify vesicles from various biological samples, including:

• Serum
• Plasma
• Saliva
• Urine
• Cerebrospinal fluid
• Cell culture media

Note: Most 'raw' samples require preparation, such as centrifugation and concentration, before running on qEV columns and analysis with tunable resistive pulse sensing (TRPS). Visit support.izon.com for recommendations and protocols.

3.3 qEVoriginal Gen 2 Specifications

*Loading higher sample volumes results in lower purity, greater overlap between protein and EV elution peaks, and a higher protein peak within the PCV. Loading lower sample volumes results in a higher dilution factor. The optimal recommended sample volume for purity on the qEVoriginal Gen 2 is 0.5 mL.

**Values based off analysis of human plasma samples.

3.4 qEVoriginal Gen 2 Performance Characteristics

qEV/35 nm columns show higher recovery of particles larger than 60 nm compared to qEV/70 nm columns. Proteins typically elute slightly earlier on qEV/35 nm columns.

Initial measurement of EV concentration and protein contaminants in collected fractions is recommended, as different samples may exhibit varying elution profiles and purity.

Diagram Description: Figure 2 compares protein elution levels and EV concentration between qEVoriginal/35 nm and qEVoriginal/70 nm Gen 2 columns using 0.5 mL of human plasma. It shows the concentration of EVs and similarly sized particles (>60 nm) versus volume, alongside the percentage of total loaded protein.

3.5 qEVoriginal Gen 2 Elution Profile

For a 0.5 mL sample volume and 0.4 mL collection volumes, vesicle elution typically peaks at 1.2 mL after the buffer volume. Figure 3 illustrates the elution of vesicles from a qEVoriginal/35 nm Gen 2 column with 0.5 mL of human plasma.

The majority of EVs typically elute at 2.0 mL after the buffer volume. For higher purity, collect only the first 1.2 mL. This allows the user to balance recovery volume with purity.

Plasma protein elution is slower, predominantly occurring from 1.6-9.2 mL after the buffer volume. Vesicles recovered beyond 1.6 mL may contain higher protein contamination and be less suitable for downstream analysis due to lower purity.

Protein elution profiles can be analyzed using bicinchoninic acid (BCA) assay or other colorimetric protein assays.

Diagram Description: Figure 3 shows a typical elution profile for a qEVoriginal/35 nm Gen 2 column with 0.5 mL human plasma. It plots vesicle concentration and protein levels against elution volume, indicating that proteins elute later than EVs and similarly sized particles.

3.6 Choosing a Purified Collection Volume

The optimal PCV depends on the sample's elution profile and downstream applications. The following recommendations are based on data from qEVoriginal Gen 2 columns with 0.5 mL human plasma. Other sample types may show slight variations.

For more information on optimization scenarios, visit support.izon.com/how-can-ev-isolation-be-customised-using-gen-2-columns.

*This is the default buffer volume setting for the Automatic Fraction Collector and may need adjustment for EV recovery or maximum purity scenarios.

3.6.1 Optimised for EV Concentration

The highest concentration of EVs and similarly sized particles typically occurs between 0.8-1.2 mL after the defined buffer volume of 2.5 mL (Figure 2). This may vary, peaking between 0.4-0.8 mL or 1.2-1.6 mL. To maximize EV concentration, Izon recommends collecting the entire volume from 0.4-1.6 mL after the buffer volume. Set the adjusted buffer volume to 2.9 mL for this.

3.6.2 Optimised for EV Recovery

To collect the majority of EVs, Izon recommends collecting up to 2.8 mL after the defined buffer volume of 2.5 mL. Note that this volume may contain higher protein levels than recommended for applications like TRPS (protein concentration limit 200-300 µg/mL). Pooling samples can help mitigate this.

3.6.3 Optimised for EV Purity

To collect a significant number of EVs with high purity, collect only the first 1.2 mL after the defined buffer volume of 2.5 mL. This minimizes protein content while still collecting a substantial portion of the EVs.

4. Manual Operating Instructions

This section provides instructions for the manual use of qEV columns. For use with the Automatic Fraction Collector (AFC), refer to the AFC User Manual at support.izon.com.

4.1 Operational Recommendations

Follow these recommendations for optimal qEV column performance:

Centrifuge samples prior to loading onto the column.

To prevent clogging of column frits, filter or centrifuge biological samples to remove large particulate matter.

• Centrifuge samples at 1,500 × g for 10 minutes to remove cells and large particles.
• Gently transfer the supernatant to a new tube and centrifuge again at 10,000 x g for 10 minutes.
• For microvesicle isolation, use lower g-forces for the second centrifugation step.

Sample Concentration

Samples can be concentrated before or after isolation. Izon offers multiple column sizes to reduce the need for pre-analytical concentration. For concentration protocols:

• Concentrating some sample types (e.g., urine) may form precipitates and protein aggregates. Centrifuge concentrated samples at 10,000 x g for 10 minutes before loading onto a qEV column.
• ! Izon recommends using Amicon® Ultra Centrifugal filters (Merck C7715) according to manufacturer's instructions.
• ! Sample concentration via filtration after qEV purification may lead to the loss of some EVs on the membrane.

Column Treatment

Treating columns as single-use is advisable for nucleic acid analysis to prevent cross-contamination.

Buffer Preparation

• Ensure the sample buffer is prepared correctly. The flushing buffer should match the sample buffer temperature to maintain EV functionality. SEC can also be used for buffer exchange.
• Sample buffer temperature should be between 18-24 °C (65-75 °F).
• Degas sample buffers and ensure they are at room temperature to prevent air bubbles in the resin bed.
• ! Rapid temperature changes (e.g., moving columns from a cold room to room temperature buffer) can introduce air bubbles, leading to poorer separation.
• Use a buffer with an ionic strength of 0.15 M or greater to prevent unwanted ionic interactions.
• Use only freshly filtered (0.22 µm) buffer to avoid particulate contamination.
• qEV columns are supplied equilibrated in filtered PBS with < 0.1% w/v sodium azide.

4.2 Column Setup and Equilibration

1. Equilibrate the column and sample buffer to the operational temperature range of 18-24 °C.
2. ! Do not remove column caps until the column has reached operational temperature.
3. Carefully remove the top cap and attach the column upright to a stand. Alternatively, use Automatic Fraction Collectors (AFCs) or qEVoriginal racks available from store.izon.com.
4. Remove the bottom cap and allow buffer to flow through the column.

4.3 Column Flushing

1. Attach the buffer reservoir to the top of the column.
2. Flush the column with at least two column volumes of PBS buffer. This minimizes potential effects of sodium azide on downstream applications. If using a buffer other than PBS, equilibrate the column with at least three column volumes of the new buffer.
3. ? Only use freshly filtered (0.22 µm) buffer to avoid particulate contamination.

4.4 Sample Collection

1. To avoid clogging column frits, filter or centrifuge the biological sample to remove large particulate matter. Refer to Section 4.1 for more information.
2. Allow buffer to run through the column until flow stops when the buffer has entered the loading frit.
3. Load the prepared centrifuged sample volume onto the loading frit.
4. ! Avoid stopping column flow for extended periods during the run to ensure accurate EV separation.
5. Start collecting the buffer volume immediately (includes volume displaced by sample loading).
6. Allow the sample to enter the column until flow stops when the sample has entered the loading frit.
7. Top up the column with buffer and continue collecting the buffer volume.
8. Once the buffer volume is collected, continue collecting the Purified Collection Volume (PCV).
9. ? To collect accurate volumes, load only the required volume to the top of the column, wait for it to flow through, and repeat.

4.5 Column Cleaning and Storage

1. After collecting fractions, clean and sanitize the column to remove residual proteins. Rinse with 8.5 mL of 0.5 M NaOH immediately after fraction collection, then flush with 17 mL of buffer to return the column pH to normal before loading another sample.
2. Flushing with a large volume of buffer after fraction collection is insufficient for complete cleaning and may result in carry-over from previous samples.
3. If storing the column for future use, store in a bacteriostatic agent like PBS with 0.05% w/v sodium azide, or 20% ethanol.
4. Columns stored in 20% ethanol should be flushed with two column volumes of DI water after cleaning, followed by two column volumes of 20% ethanol for storage. Columns stored in buffer should be flushed with two column volumes of buffer.
5. ! Avoid adding 20% ethanol to buffer inside the column, as this can precipitate salt and damage the resin bed.
6. Columns containing a bacteriostatic agent can be stored at room temperature after use if cleaned as instructed. If appropriate storage solutions are unavailable, clean columns can be stored at +4 to +8 °C after use.

5. Resources

5.1 Protocols for EV Isolation from Common Sources

Visit support.izon.com for application notes and protocols for common EV samples. For assistance with sample preparation, contact Izon customer support via the contact tab on www.izon.com.

5.2 EV Analysis Using TRPS

Izon recommends TRPS analysis for determining particle size, concentration, and zeta potential. The TRPS Reagent Kit includes coating solutions for pore pre-coating, minimizing non-specific binding and ensuring stable, accurate size and concentration analysis.

For TRPS analysis of EVs, Izon recommends an initial dilution of 1/5 or 1/10 in electrolyte. Optimize dilution for an operating pressure of approximately 500 to 1500 particles per minute to avoid pore blockage.

Visit support.izon.com for more information on EV analysis with TRPS.

5.3 qEV Concentration Kit and qEV RNA Extraction Kit

To support downstream analyses, Izon offers the qEV Concentration Kit and qEV RNA Extraction Kit. The qEV Concentration Kit uses functionalized particles that bind irreversibly to EVs, enabling EV concentration for analysis by Western blot or ELISA. The qEV RNA Extraction Kit can be used independently or with the qEV Concentration Kit.