- Recombinant virus comparison
- Product finder
- Transduction posters
- Lenti-X FAQs
- Lentiviral workflow
- Lentiviral products guide
- Lentivirus biosafety
- Lentiviral tips
- Lentiviral vectors
- Lenti-X packaging
- High-throughput lentivirus production
- Lenti-X Concentrator
- Lentiviral titration
- Lenti-X GoStix Plus video protocols
- Lenti-X GoStix Plus FAQs
- Rapid lentivirus titration by p24 ELISA
- Lentiviral particles
- Lentiviral particles—fluorescent
- Lentiviral particles webinar
- Lentiviral particles FAQs
- Adeno-associated virus
Lenti-X GoStix Plus FAQs
What are the benefits of using Lenti-X GoStix Plus to measure lentiviral titer?
Lenti‑X GoStix Plus cassettes provide a fast and simple method for obtaining an accurate viral titer. Traditional methods for measuring titer such as qPCR, ELISA, or functional assays can be fairly lengthy and cumbersome, and are therefore not ideal for monitoring your supernatant to identify an optimal harvest time, or for testing multiple samples. Lenti‑X GoStix Plus enable you to obtain a titer in 10 minutes by simply adding 20 µl of supernatant to the GoStix cassette and then scanning the cassette with your smartphone camera after a brief incubation. The app also records the titer so that you can email yourself the result for record keeping. The chart below compares the time required to measure lentivirus titer with Lenti‑X GoStix Plus relative to traditional methods.
What is p24?
Recombinant lentiviruses generated for research use contain major structural proteins, including envelope proteins, matrix proteins, and virus core proteins (Figure 2). p24 is a structural protein that comprises a majority of the capsid (or shell) of lentiviral particles, and is encoded by the gag gene (along with the nucleocapsid proteins, p6 and p7, and the matrix protein, p17).
Lenti‑X GoStix Plus and methods such as p24 ELISAs are designed to specifically measure the amount of p24 capsid protein present in lentiviral supernatants, because levels of p24 correlate directly with infectious viral titer.
What is a GoStix Value (GV) titer?
The Lenti‑X GoStix App scans the GoStix cassette for the test and control bands, measures the intensity of each band (Figure 3), and then performs a calculation that generates a metric which we refer to as the GoStix Value (GV). The intensity of the test band on the cassette increases with the amount of p24 protein in your lentiviral supernatant, which in turn correlates with increasing infectious units (IFU). The GV is correlated to infectious titer in the same way that ng p24 or genome copies (gc) correlate to infectious titer when using traditional p24 ELISAs or qRT-PCR assays, respectively, to determine titer (Figures 4–6, see subsequent questions). And just like ng p24 or gc, the GV that is best for each experimental application will vary depending on several factors including the target cells, the MOI required, and the lentiviral vector and packaging system used.
How accurately do GV titers correlate to infectious units (IFU)?
Data presented in Figures 4 and 5 (below) demonstrate a strong correlation between GV and IFU across varying concentrations of sample and varying lentiviral packaging systems.
Where do I download the app?
You can download the Lenti‑X GoStix App from the App Store or Google Play. Search for Lenti‑X GoStix.
Which devices are supported by the Lenti-X GoStix App?
The app works on all Apple iPhones from 5 onwards using the iOS 9 operating system or newer. Android devices running Android v4.4 and later are also supported.
How do I correlate GV to IFU/ml or ng p24?
It is as simple as doing a side-by-side comparison. The data presented below compare ELISA- and Lenti-X GoStix Plus-based titers.
What factors can impact a GoStix titer?
When using Lenti‑X GoStix Plus (or any other method, e.g., p24 ELISA, or qRT-PCR) to compare titers between different experiments, the following variables must be consistent: lentiviral packaging systems, lentiviral vectors, transfection methods, harvest times, and scanning devices. If any one of these parameters is changed between experiments, we recommend that you re-correlate the GV to IFU.
How can harvest time affect the GoStix Value?
The GoStix Value (GV) is proportional to the amount of p24 protein produced, which in turn correlates with the length of the time to harvest. Therefore, as demonstrated in Figure 7 (below), a longer harvest time will result in a larger GV, assuming that other conditions (e.g., packaging system, transfection method, etc.) are kept constant. The data presented below also demonstrate that the ratio between GV and IFU/ml varies depending on the harvest time, which is why harvest time must be kept constant to enable calculation of IFU/ml from GV, and for comparison across experiments.
What happens if I do not wait 10 minutes for the signal to develop?
The test will not give consistent results if the full development time is not observed and you will not be able to accurately compare your result to previously tested samples. A warning within the app will appear if the Skip button is pressed before 10 minutes have elapsed on the timer.
Can Lenti-X GoStix Plus be used even in the absence of a smartphone?
Absolutely, however, without scanning the intensity of the band, the result will be qualitative (yes, my prep is good; or no, my prep is not ready) rather than a quantitative titer.
Why do I NOT need to create a standard curve for Lenti-X GoStix Plus?
For other commonly used titration kits such as p24 ELISAs and pRT-PCR assays, you always need to compare your test sample to a standard curve that you generate. This is not required for Lenti‑X GoStix Plus because the standard curve for each lot of Lenti‑X GoStix Plus is generated at Takara Bio USA, Inc. and loaded onto the app when you open it and connect your mobile device to the internet. In addition to saving you time and money, this also ensures maximum accuracy in GV readings between lots and experiments.
Why do I have to enter the lot number of my kit into the app and where is the lot number found?
In order to generate accurate data, the app requires that you enter the lot number because different lots of Lenti‑X GoStix Plus yield slightly different standard curves (similar to an ELISA). The lot number can be entered by scanning the QR code located on the GoStix cassette pouch, or it can be entered manually (Figure 9).
Does my mobile device need to be connected to the internet?
When you first use the app, you will need to connect your device to the internet so that the standard curves for available lots of the kit can be downloaded into the app. The app uses the lot-specific standard curve to calculate the GV. When you purchase a new lot of GoStix, make sure to reconnect your device to the internet so that the standard curves for new lots can applied to your app.
How do I know if my titer is outside the range of the GoStix?
For accurate results, it is important that the intensity of the test band is less than that of the control band. The closer this ratio is to 1, the closer the sample is to exceeding the standard curve. If the ratio is close to 1, perform a 1:2 dilution of your sample and apply the dilution to a fresh cassette, then indicate the dilution factor in the app when scanning, as indicated by the green arrow in the screenshot below.
Is any data saved in the app?
Yes. Your results will be saved in the “Results history” in the main menu, provided that you do not delete the app. To avoid losing data, we recommend emailing yourself the data by pressing the Share button at the bottom of each data set page, as indicated by the green arrow in the screenshot below.
Takara Bio USA, Inc.
United States/Canada: +1.800.662.2566 • Asia Pacific: +1.650.919.7300 • Europe: +33.(0)1.3904.6880 • Japan: +81.(0)77.565.6999
FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES. © 2020 Takara Bio Inc. All Rights Reserved. All trademarks are the property of Takara Bio Inc. or its affiliate(s) in the U.S. and/or other countries or their respective owners. Certain trademarks may not be registered in all jurisdictions. Additional product, intellectual property, and restricted use information is available at takarabio.com.