Why is the virus titer I measured different from that provided by VectorBuilder?
Discrepancies in the virus titer measured by different people usually arise either from differences in the titration methods used or due to variations between measurements caused by virus degradation. VectorBuilder employs different titration methods for different types of virus. For detailed information on how we determine viral titer at VectorBuilder please refer to the Vector FAQ entry “How is viral titer determined in VectorBuilder?”.
If the results for virus titer measurement performed by your lab differs significantly from the results supplied by VectorBuilder, it may be due to differences in the analysis methods used. For instance, while at VectorBuilder we use the more popular p24 ELISA method for determining lentivirus titer, some labs prefer using a qPCR-based approach for directly measuring lentiviral RNA since it is more cost-effective. Some labs also prefer more straight forward approaches such as assays based on FACS or quantification of colony-forming units. While each method has its own advantages and disadvantages, the values obtained for lentivirus titer can differ significantly from one method to another.
In cases where similar types of titration assays are compared, there may still be discrepancies in titer measurements due to other factors such as use of different experimental models, differences in reagents used, or issues with the sensitivity of the laboratory equipment used. Some common examples include inappropriately using polybrene with lentivirus (polybrene might be toxic to certain cell types), or measuring viral titer using cell lines or species which are not efficiently infected by the virus being tested. Another potential issue is that viral titer can be underestimated when the reagents or the detection equipment (e.g. microscopes) are not sensitive enough for identifying single virus integration events. Occasionally, small technical details of the titer measurement protocol can also have a significant impact on the viral titer values. For instance, AAV titer is usually determined using a qPCR-based approach to quantify the copy number of the viral genome in the virus stock, and the choice of the targeting location for the qPCR primers is critical for an accurate estimation of AAV titer. In our AAV titration method, the qPCR primers are designed to target the AAV ITRs which are the most commonly chosen sites in the field. The viral titer obtained using ITR-specific primers would differ from the titer values obtained using primers targeting other locations such as the region between the ITRs. This is due to a difference in the amplification kinetics of the viral ITRs versus the region between the ITRs, arising from the presence of secondary structures in the ITRs. Similarly, primer annealing conditions such as annealing temperature and annealing efficiency have also been found to impact AAV titer values.
Besides titration method, the sensitivity of virus stocks to storage and handling conditions can also contribute to discrepancies in titer measurements. Enveloped viruses tend to quickly lose infectivity if they undergo repeated freeze-thaw cycles or are stored above -80°C. Buffer components and conditions which damage nucleic acids or affect protein structures such as detergents and nucleases can also compromise virus infectivity. Additionally, purity of the viral preps is known to have an impact on virus stability. Although, AAV and adenovirus in general have greater stability compared to lentivirus, unpurified AAV and adenovirus however have been found to be susceptible to degradation by proteases and therefore, may consequently show lower titer after repeated freeze-thaw.
When planning to utilize viral vectors in your research, we highly recommend testing and optimizing the vectors in your own experimental model, as this will give the best indication of how each vector system will perform under the experimental conditions specific to your studies.