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Tech Note

Simplify adenovirus production with a simple and rapid titer test

Post-transfection—Determine if infectious virus was produced, even before cytopathic events are observed

Post-amplification—Ensure high titer by harvesting amplified adenovirus at the optimal time

Introduction Results Conclusions

Introduction  

The Adeno-X GoStix protocol takes as little as 30 seconds to indicate the presence of adenovirus particles in your post-transfection or post-amplification supernatant. Adeno-X GoStix are designed to instantly confirm the presence of adenovirus in cell culture supernatants or lysates. The test detects adenoviral hexon proteins and can be used to determine if infectious virus was produced post-transfection, or to select the best time to harvest your virus. The included Hexon Control provides confirmation of GoStix test function (Figure 1).

Figure 1. The Adeno-X GoStix workflow. The Adeno-X GoStix protocol takes only a couple of minutes to indicate the presence of adenovirus particles. First, supernatant from transfected cells is placed on the cassette, followed by 4 drops of chase buffer. If the viral titer in your supernatant is >2 x 106 IFU/ml you will detect a band in the test window within minutes.

Results  

Early detection of adenovirus during rescue

Adeno-X GoStix were used to confirm virus production during the rescue procedure. PacI-digested adenovirus expressing ZsGreen1 was transfected into HEK 293 cells, which were monitored for the emergence of cytopathic effects by phase and fluorescence microscopy. Twenty microliters of culture medium was applied to the Adeno-X GoStix on each day. Although no virus was detected at the start of the experiment, at four days post-transfection, Adeno-X GoStix were able to detect virus in culture supernatants containing plaques that were only weakly perceptible under phase microscopy and confirmed by fluorescence microscopy (Figure 2).

Figure 2. Early detection of adenoviral titers using Adeno-X GoStix. Viral titers were detected 4 days post-transfection with GoStix, which coincided with the early development of cytopathic effects and ZsGreen1 expression. Panel A: On Day 0, no viral titers were detected with GoStix. Panel B: On Day 4, viral titers were detected with GoStix, which coincided with appearance of cytopathic effects and fluorescence.

Rapid adenovirus detection during amplification

Adenovirus expressing ZsGreen1 was amplified in HEK293 cells 48 hr post-infection; and each day thereafter, the supernatant and the cell pellet were harvested and titers were quantified by counting fluorescent cells in an infectious unit (IFU) assay (Figure 3, Panel A). The increase in virus level in the cell pellets was mirrored by a proportional increase in the amount of virus collected from the culture supernatant. The amount of virus in the supernatant remained at <10% of the total virus output. The supernatant titer serves as a clear indicator of total yields. In addition to IFU determination, 20 μl of each sample was applied to an Adeno-X GoStix cassette. The increase in band intensity paralleled the increase in infectious unit titer (Figure 3, Panel B).

Figure 3. Rapid detection of adenoviral titer during amplification. Supernatant and pellet were collected each day post-infection to measure viral titer. As indicated by the graph, the supernatant titer serves as a clear indicator of total viral yields (Panel A). In addition to IFU determination, 20 μl of each sample was applied to an Adeno-X GoStix cassette. The increase in band intensity paralleled the increase in infectious unit titer (Panel B).

Conclusions  

Adeno-X GoStix save time and effort during adenovirus production by providing a simple and rapid test for the status of adenovirus rescue even before cytopathic events are observed. Each test is sensitive enough to detect adenovirus in a small amount of crude supernatant, and is also used to ensure maximum yields during the adenovirus amplification process. The presence of a strong band in the cassette window ensures that high-titer adenovirus is harvested at the peak time.



Related Products

Cat. # Product Size License Quantity Details
632270 Adeno-X™ GoStix™ 20 Tests USD $410.00

License Statement

ID Number  
89 This product is protected by U.S. Patent 9,945,850 and additional pending patent. For further license information, please contact a Takara Bio USA licensing representative by email at licensing@takarabio.com.

Adeno-X GoStix are designed to instantly confirm the presence of adenovirus in cell culture supernatants or lysates. The test detects adenoviral hexon proteins and can be used to determine if infectious virus was produced post-transfection, or for selecting the best time to harvest your virus. The included Hexon Control provides confirmation of GoStix test function.

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Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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Early detection of adenovirus during rescue

Early detection of adenovirus during rescue

Early detection of adenovirus during rescue. Panel A. The Adeno-X GoStix protocol takes only a couple of minutes to indicate the presence of adenovirus particles. Supernatant from transfected cells is placed on the cassette, followed by chase buffer. Panel B. Adeno-X GoStix were used to confirm virus production during the rescue procedure. PacI-digested adenovirus expressing ZsGreen1 was transfected into HEK 293 cells, which were monitored for the emergence of cytopathic effects by phase and fluorescence microscopy (middle row). 20 μl of culture medium was applied to the Adeno-X GoStix on each day (bottom row). Four days post-transfection, Adeno-X GoStix were able to detect virus in culture supernatants containing plaques that were only weakly perceptible under phase microscopy and confirmed by fluorescence microscopy.

Back

Rapid adenovirus detection during amplification

Rapid adenovirus detection during amplification

Rapid adenovirus detection during amplification. Panel A. Adenovirus expressing ZsGreen1 was amplified in HEK293 cells. 48 hr postinfection, and each day thereafter, the supernatant and the cell pellet were harvested, and titers were quantified by counting fluorescent cells in an infectious unit (IFU) assay. The increase in virus level in the cell pellets was mirrored by a proportional increase in the amount of virus collected from the culture supernatant. The amount of virus in the supernatant remained < 10% of the total virus output. Panel B. Supernatant serves as a clear indicator of total yields. In addition to IFU determination, 20 μl of each sample was applied to an Adeno-X GoStix. The increase in band intensity paralleled the increase in virus production observed by infectious unit titer.

Back

Use Adeno-X GoStix to instantly confirm the presence of adenovirus in cell culture supernatants or lysates

Use Adeno-X GoStix to instantly confirm the presence of adenovirus in cell culture supernatants or lysates
Use Adeno-X GoStix to instantly confirm the presence of adenovirus in cell culture supernatants or lysates. The test, which detects adenoviral hexon proteins, requires just 20 µl of adenoviral supernatant to determine if infectious virus was produced posttransfection, or to select the best time to harvest your virus following amplification. The included Hexon Control (bottom) confirms GoStix test function.

Back

Constructing recombinant adenovirus with In-Fusion Cloning technology

Constructing recombinant adenovirus with In-Fusion Cloning technology

Constructing recombinant adenovirus with In-Fusion Cloning technology. DNA sequences can be rapidly transferred as PCR products to any pAdenoX vector using the In-Fusion cloning method. In this example, your gene of interest is amplified with 15 bp extensions that are homologous to the ends of the linearized adenoviral vector. The PCR product is then purified and mixed with the linearized adenoviral vector of choice in the In-Fusion reaction. Following the reaction, a portion of the mixture is transformed into E. coli (Stellar Competent Cells) and screened. Once a PCR-positive clone is identified, the recombinant pAdenoX vector is amplified, purified, and subsequently linearized with the restriction enzyme PacI, then transfected into Adeno-X 293 cells for viral rescue and amplification. Adeno-X GoStix can be used to determine the status of adenovirus rescue.

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What to expect when measuring viral titer with Lenti-X or Adeno-X GoStix

What to expect when measuring viral titer with Lenti-X or Adeno-X GoStix

What to expect when measuring viral titer with Lenti-X or Adeno-X GoStix. The control band (C) will always be detected, the test band shows (T) positive only you if there is enough virus in your sample.

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632270: Adeno-X GoStix

632270: Adeno-X GoStix


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