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Accurate titer determination is an essential component of viral vector quality control and a prerequisite for preclinical and clinical studies of relevant gene therapy products. Among them, biological titer is more accurate than physical titer in measuring the extent to which a virus can infect target cells. Creative Biogene has combined our expertise with a variety of advanced technologies to establish a sound solution for accurately characterizing the biological titer of viruses.
Biological titer includes infection titer and transduction titer. The purpose of determining infection titer is to determine whether the viral genome is transferred to the nucleus and replicates. In contrast, transduction titer is a gradient dilution of the virus that infects experimental cells and determines positivity by detecting the expression of the target gene (or transgene). The transduction titer is calculated based on the number of positive cells or the number of positive wells, combined with the dilution of the virus, and is measured in transduction units (TU). For lentiviral or retroviral vectors, the transduction titer is expressed as transduction units per milliliter (TU/mL). Adenovirus vector titers are typically expressed as phage-forming units per milliliter (PFU/mL) or infection units per milliliter (IFU/mL).
The viral plaque assay is one of the most widely used methods for determining infection titer. A permissive cell line is transduced with serial dilutions of virus, and cells are infected at very low multiplicity of infection (MOI) to ensure that the majority of transduced cells are infected by a single viral particle for accurate quantification. The virus multiplies in the infected cells, eventually leading to cell lysis, spread to neighboring cells, and ultimately the formation of visible pores (or plaques) on the cell monolayer. Total plaque counts at a specific dilution provide the number of plaque forming units (PFU) per milliliter.
The TCID50 measures infectious virus titer by quantifying the amount of virus required to kill or produce cytopathic effects in 50% of host cells.
The IFA shows good correlation with plaque assays and captures viruses that do not lyse cell membranes (incompatible with plaque assays) or do not exhibit detectable cytopathic effects (perform TCID50). IFA utilizes an antibody-based staining method to detect virus-infected cells. IFA results are expressed as focal formation units per milliliter (FFU), or IFU/ml. IFA is considered a faster and more sensitive method than traditional plaque assays or TCID50 to determine infectious titer.
Creative Biogene is committed to establishing accurate and rapid biological titer tests during the production of viral vectors to optimize process development while ensuring the quality of gene therapy products. Please feel free to contact us if you have a need, and our experienced specialists will be happy to assist you.
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