Lentiviral Titer

BioInnovatise Viral Vector Team

Updated May 13, 2024

How To Determine Lentiviral Titer:

There are several methods to determine the concentration of viral particles in your lentivirus packaging production. Each method has its own strengths and weaknesses, and the choice of method depends on factors such as accuracy requirements or time constraints. These are some commonly used methods to determine lentivirus titer.

qPCR (Quantitative Polymerase Chain Reaction): qPCR measures the copy number of lentiviral genomic RNA or vector DNA present in lentiviral preparations. It provides a quantitative measure of lentiviral genome copies and can be more accurate than p24 ELISA for determining lentiviral titer.

RT-qPCR (Real Time – Quantitative Polymerase Chain Reaction): RT-PCR amplifies specific RNA sequences using reverse transcriptase enzymes to convert RNA into complementary DNA, followed by PCR amplification of the cDNA. It is used for detecting and quantifying RNA molecules, such as lentiviral RNA genomes or RNA transcripts produced by transduced cells. These assays for lentiviral RNA are also highly sensitive and can detect low copy numbers of viral RNA molecules, providing a measure of the infectious viral particles present in lentiviral preparations. Our team uses RT-qPCR in our lentivirus packaging production QC.

P24 ELISA (Enzyme-Linked Immunosorbent Assay): This method quantifies the amount of the viral capsid protein p24 present in lentiviral preparations. It is a simple and widely used method for estimating lentiviral titer. It indirectly measures viral particles and may not accurately reflect the functional infectivity of the lentivirus.

Infectious Titer Assay: This assay measures the ability of lentiviral particles to infect and transduce target cells. Common methods include flow cytometry-based assays, where target cells are transduced with lentivirus encoding a fluorescent reporter gene (e.g., GFP), and the percentage of transduced cells is quantified. We also use flow cytometry-based assays in our lentivirus packaging production QC.

TCID50 (Tissue Culture Infectious Dose 50): TCID50 is a viral titration method based on dilution series of lentiviral preparations inoculated onto target cells in tissue culture plates. The endpoint dilution that results in 50% of wells showing cytopathic effects or transduction is calculated to determine the viral titer. TCID50 provides a measure of functional infectivity but requires multiple dilutions and observations, making it labor-intensive.

Lenti-X GoStix Plus Lentivirus Rapid Titer Detection Kit: This is a rapid lateral flow-based assay for detecting and quantifying lentiviral titers directly from the supernatant without the need for cell culture. It provides a semi-quantitative measure of lentiviral titer within 10 minutes, making it convenient for quick titer estimation. However, it may not be as accurate as other quantitative methods such as RT-qPCR.

Lentivirus Packaging Transfection Process Overview Lentivirus Packaging Protocol

The above diagram illustrates the lentivirus packaging process at BioInnovatise, where our team uses a HEK293T cell line for lentivirus packaging manufacturing. Our viral vector team uses a validated RT-qPCR assay to measure the viral titer in all lentivirus productions.

Why Is A High Titer Important?

A high titer is important for your research application. A high titer of lentivirus is essential for achieving efficient transduction, reducing toxicity, scalability, consistency, and successful in vivo applications, all of which are critical for the success of lentivirus based experiments and therapeutic interventions. However, a lentivirus titer can in fact be too high. Overtitered lentivirus can be cytotoxic to target cells, increase the chances of off-target effects where the lentiviral vector integrates into unintended genomic loci or alters the expression of unintended genes, and even trigger unwanted immune responses that may not occur in lower titer lentivirus.

Ensuring the proper lentivirus titer requires having a well designed and appropriate lentivirus transfection protocol.

Determining lentiviral titer is important to ensuring your research gets the results it deserves while minimizing unnecessary risk. If you are unsure what titer level is necessary for your research, contact our team.

Learn about our quick turnaround lentivirus packaging services.

Want to learn more about the latest in lentivirus based research? Our colleagues at ScienceDirect and Genetic Engineering & Biotechnology News continuously collect and publish the latest information on lentivirus-based research.

Real-time polymerase chain reaction validation equipment used for lentivirus titering RT-qPCR