Q: What is the difference between physical titer and infectious titer?
A: Viral titers are represented in two ways: either functional (infectious) titer measured in transduction units (TU/mL) or physical titer, measured in viral particles (VP/mL). Physical titer is a measurement of how much virus is present and is usually calculated based on the level of protein, such as p24, or viral nucleic acid. The functional titer is a measurement of how much virus can infect the cell and is typically 100-1000 fold lower than physical titer. Direct functional titer is a more accurate measurement for calculating MOI, but is more time consuming and sometimes not feasible. Physical titer is sufficient for most lentiviral experiments, and functional titer can be calculated from physical titer.
Q: Can you help me choose a Lentiviral Titer/Quantitation Kit from the kits you offer?
A: We offer three kits to quantify lentivirus:
- The Traditional p24 ELISA kit is the most commonly published method for measuring lentiviral titer. This kit is suitable for tittering native or purified recombinant virus. However, in crude (unpurified) lentiviral supernatant, significant concentrations of overexpressed p24 protein may be present that are not assembled into viral particles. This causes an extreme overestimation of lentiviral titer.
- Our Lentivirus Associated p24 ELISA kit specifically measures only viral-associated p24. In unpurified recombinant lentiviral preps, it is critical that the free p24 is removed to get an accurate titer. This kit uses two chemical polymers that form a complex with the virus, but not with free p24 protein. The viral complexes are then pelleted, dissolved, and disrupted to release p24 which is then detected by ELISA.
- If a quick test of physical titer is desired, choose our Lentivirus Rapid Quantitation Kit. This kit measures viral nucleic acid and is less sensitive than our Lentivirus Titer Kit, but it offers a quick hour protocol. This kit uses beads to capture and pull down the virus. After capture and denaturation, viral nucleic acid is quantified using a fluorescence plate reader.
Q: Can I use a cell line to titer my lentivirus?
A: If your lentivirus contains a reporter such as GFP, it is possible to measure the titer by infecting cells. We recommend using 293 or 293T cells which are easy to infect with lentivirus.
Q: How can my lentiviral titer be improved?
A: A typical lentiviral titer from crude supernatant is 1x10^6 TU/mL. Here are some suggestions to improve the titer:
- Transfection efficiency should be at least 80% If the efficiency is lower than this, the transfection conditions may require optimization.
- Because lentivirus contains LTR repeat sequences, recombination can happen after transformation, resulting in a plasmid of reduced size. We recommend using Invitrogen Stbl3 competent cells for plasmid amplification, which will minimize recombination. If these cells were not used to amplify the plasmids, perform a restriction digest to confirm that the plasmids have maintained the correct size.
- Healthy 293T or 293LTV cells at low passage number should be used for transfection.