10-minute, quantitative lentiviral titer test—Lenti-X GoStix Plus

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Titration with Lenti-X GoStix Plus takes only ten minutes, which is significantly shorter than titration times required for conventional methods

Most commonly used methods of lentiviral vector titration and their associated timelines (in hours). Lenti-X GoStix Plus: a lateral flow-based method for the detection of lentiviral p24 in supernatants. qRT-PCR: quantitation of viral RNA genomes by qRT-PCR. ELISA: measurement of the amount of p24 capsid protein. PCR: detection of integrated DNA proviruses by qPCR. IFU (FACS):determination of the percentage of infected cells via FACS analysis of RFP/GFP positive cells. IFU (Selection): infectious units determined by quantifying the number of drug resistant colonies in transduced populations.

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p24 production, GoStix Value, and IFU/ml depend on harvest time

p24 production, GoStix Value, and IFU/ml are dependent on harvest time. ZsGreen1 lentivirus preparations were made in duplicate in accordance with the manufacturer's instructions using Lenti-X Single Shots and harvested at the times indicated. At each timepoint, samples were analyzed for GV using Lenti-X GoStix Plus and virus dilutions were titrated on HT1080 cells to determine IFU/ml. In addition to showing that GV (i.e., p24 accumulation) and IFU/ml are dependent on harvest time, these data demonstrate that the ratio between GV and IFU/ml changes over time.

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Lenti-X GoStix Plus-based titers correlate with transduction titers across varying concentrations

Lenti-X GoStix Plus-based titers correlate with transduction titers across varying concentrations. Lenti-X ZsGreen1 lentivirus was prepared using the Lenti-X Packaging Single Shots lentiviral packaging system. The sample preparation was serially diluted and added to Lenti-X GoStix Plus cassettes and densitometric analysis was performed. In addition, each dilution was titrated on HT1080 cells to determine actual IFU/ml. The IFU/ml for the unknown samples was calculated using a GV to IFU/ml ratio determined from a reference virus produced using the same packaging system and the same harvest time as the unknown sample. The IFU/ml calculated from the Lenti-X GoStix Plus was then plotted against the actual IFU/ml.

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The Lenti-X GoStix App quantifies titer by measuring the relative intensities of test and control bands on the GoStix cassette

The Lenti-X GoStix App uses a smartphone camera to determine the relative intensities of the test (T) and control (C) bands and uses the ratio to calculate the titer.

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Lenti-X GoStix Plus-based titers correlate with transduction titers across varying packaging systems.

Lenti-X GoStix Plus-based titers correlate with transduction titers across varying packaging systems. ZsGreen1 lentivirus was prepared using three different commercially available lentiviral vector and packaging systems. Samples for all preparations were diluted and added to Lenti-X GoStix Plus cassettes and analyzed. In addition, each dilution was titrated on HT1080 cells to determine actual IFU/ml. The IFU/ml for each unknown sample was calculated using a GV to IFU/ml ratio determined from a reference virus produced using the same packaging system and the same harvest time as the unknown sample.

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Lenti-X GoStix Plus-based titers perform comparably to ELISA-based titers

Lenti-X GoStix Plus-based titers can be used in a similar fashion as ELISA-based titers. Lenti-X ZsGreen1 lentivirus was prepared using the Lenti-X Packaging Single Shots lentiviral packaging system. Percentages of cells transduced (% transduced) were determined on HT1080 cells followed by FACS analysis. In addition, the viral p24 value was assessed using the Lenti-X p24 Rapid Titer Kit and the GoStix Value was determined using Lenti-X GoStix Plus. Transductions were performed on HT1080 cells using a range of MOIs and the resulting transduction efficiencies were plotted against the associated ELISA and GoStix Value equivalents. Fluorescent images of the transduced cells were taken at 48 hours post-transduction.

Back

Titration with Lenti-X GoStix Plus takes only ten minutes, which is significantly shorter than titration times required for conventional methods

Most commonly used methods of lentiviral vector titration and their associated timelines (in hours). Lenti-X GoStix Plus: a lateral flow-based method for the detection of lentiviral p24 in supernatants. qRT-PCR: quantitation of viral RNA genomes by qRT-PCR. ELISA: measurement of the amount of p24 capsid protein. PCR: detection of integrated DNA proviruses by qPCR. IFU (FACS):determination of the percentage of infected cells via FACS analysis of RFP/GFP positive cells. IFU (Selection): infectious units determined by quantifying the number of drug resistant colonies in transduced populations.

Back

p24 production, GoStix Value, and IFU/ml depend on harvest time

p24 production, GoStix Value, and IFU/ml are dependent on harvest time. ZsGreen1 lentivirus preparations were made in duplicate in accordance with the manufacturer's instructions using Lenti-X Single Shots and harvested at the times indicated. At each timepoint, samples were analyzed for GV using Lenti-X GoStix Plus and virus dilutions were titrated on HT1080 cells to determine IFU/ml. In addition to showing that GV (i.e., p24 accumulation) and IFU/ml are dependent on harvest time, these data demonstrate that the ratio between GV and IFU/ml changes over time.

Back

Lenti-X GoStix Plus-based titers correlate with transduction titers across varying concentrations

Lenti-X GoStix Plus-based titers correlate with transduction titers across varying concentrations. Lenti-X ZsGreen1 lentivirus was prepared using the Lenti-X Packaging Single Shots lentiviral packaging system. The sample preparation was serially diluted and added to Lenti-X GoStix Plus cassettes and densitometric analysis was performed. In addition, each dilution was titrated on HT1080 cells to determine actual IFU/ml. The IFU/ml for the unknown samples was calculated using a GV to IFU/ml ratio determined from a reference virus produced using the same packaging system and the same harvest time as the unknown sample. The IFU/ml calculated from the Lenti-X GoStix Plus was then plotted against the actual IFU/ml.

Back

The Lenti-X GoStix App quantifies titer by measuring the relative intensities of test and control bands on the GoStix cassette

The Lenti-X GoStix App uses a smartphone camera to determine the relative intensities of the test (T) and control (C) bands and uses the ratio to calculate the titer.

Back

Lenti-X GoStix Plus-based titers correlate with transduction titers across varying packaging systems.

Lenti-X GoStix Plus-based titers correlate with transduction titers across varying packaging systems. ZsGreen1 lentivirus was prepared using three different commercially available lentiviral vector and packaging systems. Samples for all preparations were diluted and added to Lenti-X GoStix Plus cassettes and analyzed. In addition, each dilution was titrated on HT1080 cells to determine actual IFU/ml. The IFU/ml for each unknown sample was calculated using a GV to IFU/ml ratio determined from a reference virus produced using the same packaging system and the same harvest time as the unknown sample.

Back

Lenti-X GoStix Plus-based titers perform comparably to ELISA-based titers

Lenti-X GoStix Plus-based titers can be used in a similar fashion as ELISA-based titers. Lenti-X ZsGreen1 lentivirus was prepared using the Lenti-X Packaging Single Shots lentiviral packaging system. Percentages of cells transduced (% transduced) were determined on HT1080 cells followed by FACS analysis. In addition, the viral p24 value was assessed using the Lenti-X p24 Rapid Titer Kit and the GoStix Value was determined using Lenti-X GoStix Plus. Transductions were performed on HT1080 cells using a range of MOIs and the resulting transduction efficiencies were plotted against the associated ELISA and GoStix Value equivalents. Fluorescent images of the transduced cells were taken at 48 hours post-transduction.