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Intact Virus and Exosome Enrichment |
For functional studies of viruses, such as growth rate and infectivity, live intact virus is required. Viruses can be present in very low concentrations in samples such as cell culture media and wastewater. To perform downstream analyses, it is often necessary to purify and concentrate the virus from the sample matrix. A range of isolation and enrichment methods including ultracentrifugation and filtration can be used. These are methods that require expensive instruments, are time consuming, or may result in low yield.
Dynabeads intact virus enrichment magnetic beads offers a short and simple virus isolation method of negatively charged viruses that also maintain an active reverse transcriptase (RT) enzyme. The same magnetic beads can be used to isolate exosomes from any sample in a short and simple enrichment approach that reduces the risk of low yield and does not affect the integrity of the exosomes.
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See our selection guide for intact virus and exosome enrichment
On this page
- Methods of intact virus enrichment
- Intact exosome enrichment
- How to enrich viruses and exosomes with Dynabeads intact virus enrichment beads
- Automation of intact virus and exosome enrichment with Kingfisher instruments
- Selection guide for intact virus enrichment magnetic beads
- Videos for virus enrichment
- Ordering information
Methods of intact virus enrichment
Viruses and exosomes can be easily purified and concentrated into a smaller volume by either precipitation with Invitrogen Virus Precipitation Reagent followed by centrifugation, or by using Dynabeads Intact Virus Enrichment magnetic beads. Each method of enrichment has specific benefits so you can choose the option that best fits your needs.
Both methods of virus enrichment are suitable for live or inactivated virus, exosomes, virus-like particles (VLPs), other enveloped viruses and vesicles present in cell culture media, virus transport media, and wastewater samples.
Virus enrichment by magnetic beads with intact virus enrichment reagent
Magnetic bead-based methods involve exposing the virus-containing sample to micron-sized magnetic beads possessing a strong anion exchange surface. After mixing for ten minutes, the virus-bound beads are collected via magnet, washed, and eluted in a small volume to yield concentrated virus. Virus purification by Dynabeads Intact Virus Enrichment magnetic beads is a rapid, convenient, and automatable way to concentrate virus-containing solutions.
Benefits of using intact virus enrichment
- Fast—intact virus in ~20 minutes
- Functional—the intact virus can be used in any functional assay
- Release—release virus from the beads in 10 min
Concentrated intact virus can be used in:
- Functional studies
- Protein analysis (e.g., western blot)
- Nucleic acid research (e.g., RT-qPCR)
Learn more about isolating SARS-CoV-2 from wastewater samples using Dynabeads magnetic beads
Figure 1. Enrichment of heat-inactivated SARS-CoV-2 virus from (A) VTM, (B) CCM, and (C) wastewater. For enrichment and nucleic acid (NA) detection of SARS-CoV-2 genes (N, orf1ab, and S) by qPCR, heat-inactivated SARS-CoV-2 virus was spiked into VTM, CCM, or wastewater followed by a 10-minute enrichment using Dynabeads Intact Virus Enrichment beads. Extraction of NAs was performed using the Applied Biosystems™ MagMAX Viral/Pathogen II (MVP II) Nucleic Acid Enrichment Kit (VTM and CCM) or MagMAX Microbiome Ultra Nucleic Acid Isolation Kit (wastewater) after virus enrichment, followed by downstream analysis using the Applied Biosystems™ TaqPath COVID-19 Combo Kit. The results demonstrated that the qRT-PCR performance of the beads matched the sensitivity of the qRT-PCR performance of the MagMAX Viral/Pathogen II kit used as a positive control for VTM, CCM, and wastewater (Figure 6)—Ct values were within 2 cycles compared to the control.
Figure 2. Enrichment of adenovirus, influenza A virus, enterovirus, and norovirus from wastewater, and detection by qRT-PCR. For enrichment and NA detection of other viruses, inactivated adenovirus, influenza A virus, norovirus, and enterovirus were spiked into 10 mL of wastewater. The viruses were enriched within 10 minutes of using Dynabeads Intact Virus Enrichment beads, followed by RNA isolation using the MagMAX Microbiome Ultra kit. The results demonstrated that the Dynabeads Intact Virus Enrichment beads can isolate other negatively charged viruses besides SARS-CoV-2. The enrichment efficiency matched the sensitivity of the PBS control.
Figure 3. Enrichment of infectious virus with Dynabeads Intact Virus Enrichment reagent (virus plaque assay). Dynabeads Intact Virus Enrichment beads were added to samples containing influenza virus A (INFV A, strain H1N1), respiratory syncytial virus (RSV), zika virus (ZIKV), and ebola virus glycoprotein VSV-based pseudo-virus (VSV-EBOV) for 10 minutes for virus capture. The enrichment was assessed for virus titers or protein analysis. Different dilutions of bead-bound virus samples were prepared for plaque assay analysis (INFV, RSV and ZIKV) as shown in "A". The virus titers were 3.3 x 105 PFU/mL for INFV A, 2.6x104 PFU/mL for RSV and 5.0x105 PFU/mL for Zika. Luciferase assay was used to calculate virus titer for VSV-EBOV (3.6x104 RLU/mL). The presence of influenza A nucleoprotein, RSV fusion protein and Zika virus NS1 protein in samples enriched with the Dynabeads Intact Virus Enrichment beads were confirmed by western blot analysis as shown in "B".
Virus enrichment by precipitation with intact virus precipitation reagent
Addition of Invitrogen Virus Precipitation Reagent to a dilute sample results in less-soluble components (i.e., virus) being forced out of solution. The precipitated virus is collected after a brief centrifugation—no ultracentrifugation is required. This virus purification process is simple, well-suited to a large volume of starting material, and requires only about three hours with minimal hands-on time.
Figure 4: The intact virus precipitation protocol is simple. Add the reagent to the sample and incubate for 2 hours. This is followed by a short centrifugation of 30 minutes after which the supernatant is removed. The remaining pellet contains the enriched virus and is ready for use in downstream applications.
Benefits of using intact virus precipitation reagent
- Simple—3-step virus enrichment
- Fast—isolate in <3 hours with short hands-on time
- Flexible—dilute virus samples can be enriched from any sample volume range
- Straight to PCR—no further NA extraction is needed prior to PCR
Intact exosome enrichment
Effortlessly enrich your intact exosomes with the intact virus enrichment kit in 10 minutes with an option to release the exosomes from the beads in just 10 minutes. This short and simple enrichment approach reduces the risk of low yield and does not affect the integrity of the exosomes.
Figure 5.Exosome isolation workflow using a manual (A) or automated (B) method
Figure 6. Isolation of exosomes from cell culture with Dynabeads Intact Virus Enrichment beads. (A) Exosomes were isolated with 10 µL or 25 µL of beads and analyzed for CD81 by western blot. (B) Exosomes were isolated with 10 µL or 25 µL of beads, followed by release. The eluates and unreleased exosomes were analyzed for CD81 by western blot. Std = Invitrogen SeeBlue Plus2 Pre-stained Protein Standard. Exosomes isolated and released from Dynabeads Intact Virus Enrichment beads demonstrated the presence of the exosome marker CD81 in a western blot. Increasing the number of beads used for isolation increased the CD81 signal in the western blot (A). Exosomes were efficiently released from the beads by adding 0.25 M KI in 20 mM triethanolamine to the bead-bound exosomes for 10 minutes at room temperature (B).
Figure 7. Evaluation of exosome size, concentration and phenotype using Spectradyne ARC particle analyzer. Exosomes were isolated with the positively charged Dynabeads Intact Virus Enrichment beads, followed by release with 0.25 M KI in 20 mM triethanolamine. Size and concentration were measured with the Spectradyne nCS1 Particle Analyzer (A–C) according to the manufacturer’s instructions. CD81-positive subpopulations of exosomes were quantified with the Spectradyne ARC Particle Analyzer according to the manufacturer’s instructions. (A) High-quality size and concentration data from 65–400 nm exosomes. (B) High-quality size and concentration data from 250–2,000 nm exosomes. (C) Comparable size and concentration data across the 65–2,000 nm size range. (D) Size, concentration, and phenotyping of exosomes (CD81).
Figure 8. Flow cytometry analysis of isolation efficiency show efficient isolation of exosomes in sample. (A) Exosomes were isolated from spiked samples (in PBS) with 10 µL or 25 µL of Dynabeads Intact Virus Enrichment beads. The isolated exosomes were then discarded. The remaining exosomes in the supernatant were isolated with Exosome-Human CD81 Flow Detection Reagent and stained with a PE-labeled anti-CD81 detection antibody, and finally analyzed by flow cytometry. (B) Exosomes present in the sample prior to isolation with Dynabeads Intact Virus Enrichment beads. Exosomes left in the sample after isolation of exosomes with (C) 10 µL or (D) 25 µL of beads. The gray histograms represent the isotype control (B–D). This signal decrease demonstrates that the beads have depleted approximately 80% of the exosomes that were present in the samples.
Figure 9. Flow cytometry analysis of exosomes released from Dynabeads Intact Virus Enrichment beads demonstrates high recovery. (A) Exosomes were isolated from spiked samples (in PBS) with 10 µL or 25 µL of beads. The isolated exosomes were released from the beads with 0.25 M of KI and recaptured with Exosome-Human CD81 Flow Detection Reagent. The bead-bound exosomes were stained with PE-labeled anti-CD81 detection antibodies and finally analyzed by flow cytometry. (B) Exosomes present in the sample prior to Dynabeads magnetic beads isolation. Exosomes released after isolation with (C) 10 µL or (D) 25 µL of beads. Gray histograms represent the isotype control (B–D). This demonstrates that the recovery of the released exosomes from the beads was high.
Figure 10. Ultrastructural analysis of exosomes isolated with Dynabeads Intact Virus Enrichment beads. Exosomes were isolated with strong anion exchange magnetic beads and processed for (A, B) transmission electron microscopy or (C) transmission electron microscopy in combination with immunolabeling and negative-stain analysis of released exosomes. (A, B) Exosomes on the surface of magnetic beads (arrowhead in B points to a vesicle structure). (C) Released exosomes labeled for CD81 (arrowhead points to a labeled vesicle). Ultrastructural analysis of the isolated exosomes was performed by electron microscopy. The intact vesicles were observed to be surrounding the magnetic beads (Figure 6A). At higher magnification, the vesicular membranes of the structures were easily observed (Figure 6B). For identification, electron microscopy in combination with immunolabeling were used. After isolation, the vesicles were released from the beads and subjected to immunolabeling followed by negative-stain electron microscopy. The vesicles released were CD81-positive (Figure 6C), supporting the exosome nature of the isolated vesicles.
How to enrich intact viruses and exosomes with intact virus enrichment magnetic beads
The proximity of the beads to the targets in the solution translates directly to short incubation times and therefore fast protocols. Here we take advantage of the negative charge of vesicles combined with the rapid binding kinetics of the Dynabeads™ magnetic beads. Positively charged Dynabeads™ Intact Virus Enrichment beads bind to negatively charged exosomes, viruses, or proteins within 10 minutes. Following capture, viruses, exosomes (or other negatively charged vesicles) are released from the beads in 10 minutes by adding an anion with a stronger relative affinity than the bound vesicle.
Figure 11. Isolation principle. (A) The positively charged Dynabeads Intact Virus Enrichment beads are near the negatively charged exosomes, enabling rapid binding kinetics and a fast isolation protocol. (B) For isolation of negatively charged exosomes or viruses, positively charged Dynabeads Intact Virus Enrichment beads protected with Cl– ions are used. Exosomes added to the Dynabeads Intact Virus Enrichment beads will replace the Cl– ions and bind to the bead surface. An anion with higher relative affinity can subsequently be added to replace the exosomes and thus release them into the sample.
Automate your sample preparation using Dynabeads magnetic beads with protocols for Kingfisher instruments
The short and easy intact virus and exosome enrichment can be further simplified by using KingFisher Sample Purification Systems. The automated enrichment method allows larger numbers of samples to be processed with high reproducibility, reduced hands-on time, and minimal error rates in only 10–20 minutes.
Learn more about KingFisher instruments for automated protocols
KingFisher virus concentration protocols
Selection guide for intact virus enrichment magnetic beads and intact virus precipitation reagent
 Intact Virus Precipitation Reagent |  Dynabeads Intact Virus Enrichment | |
| Benefits |
|
|
| Best for | Large-volume samples |
|
| No. of samples/run | 1–24 (Determined by centrifuge size/capacity) |
|
| Total processing time | ~3 hours (or overnight incubation, if preferred) | 20–30 minutes |
| Automation compatibility | No | Yes |
| Cat. No. | 10720D | 10700D |
Virus purification videos
Virus purification using Invitrogen Intact Virus Precipitation Reagent
Virus purification using Invitrogen Dynabeads Intact Virus Enrichment
Ordering information for virus enrichment products
Customized Dynabeads for OEM supply
Do you need to commercialize an assay or service function based on any of these virus enrichment products? Are you looking to customize a Dynabeads product for your future commercial needs? Browse our custom bead offerings with Dynabeads Plus then tell us how we can help you!
Virus and exosome enrichment related resources
Resources
For Research Use Only. Not for use in diagnostic procedures.
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