TCID50 End-Point Dilution and qPCR for Sensitive Determination
Although the clinical application of adeno-associated virus (AAV) vectors has been increasingly widespread, it is still necessary to develop appropriate quality control methods to accurately identify the vectors. In the quality standard, the titration of infectious particles is of great significance to the effectiveness of the vector. At Creative Biolabs, we provide quantitative polymerase chain reaction (qPCR) combined with TCID50 (median tissue culture infective dose) end-point dilution method for rAAV infectious titer testing.
Determination for AAV Vector Infectious Titration
More than 50 years after the discovery of AAV, interest in AAV as a vector for gene therapy has continued to grow. Since the quality attributes of AAV raw materials could be different depending on the manufacturing platform, it is very important to have accurate analytical methods for their characterization. Among the quality attributes, the infectious titer is very important to ensure the efficacy of the product. Traditionally, plaque analysis is a common method for most viruses, but AAV infection does not lead to cytopathic effects, so plaque detection cannot be used to determine the infectious titer of AAV. At present, the detection method based on TCID50 end-point dilution is widely used to determine the infectious titer of AAV and other virus samples. This method is to determine the dilution of 50% pore infection by continuously diluting the cells growing in the holes of 96-well tissue culture plate. Previous studies have shown that the combination of TCID50 detection and qPCR can greatly increase the sensitivity of AAV infection titers by 10 times.
Figure 1. A schematic representation of a TCID50 based end-point dilution assay. (Pankaj, 2013)
Method for AAV Vector Infectious Titration
TCID50 test is based on cell culture in vitro. The infectious titer (50% tissue culture dose/ml) of the original test item is calculated by repeated end-point dilution sequence. These methods use real-time PCR readings to determine whether viral replication occurs in infected cells. The final product of real-time qPCR is visualized after isolation.
The gene transfer vector of human parvovirus AAV type 2 (AAV-2) based on replication deficiency is a viable candidate for human use in vivo and in vitro. Based on the combination of real-time qPCR and TCID50 end-point dilution, we developed a simple and fast method for vector quantification. A HeLa-derived AAV-2 rep and cap expression cell line was used to grow in 96-well plate. In the presence of Ad type 5, the infection was carried out with 10-fold repetitive dilution of AAV vector series. After infection, qPCR was used to determine the genomic replication of the vector. The results show that the TCID50 and the qPCR-based detection method is highly sensitive.
Features
- TCID50 has the greatest value when a lack of experimental material or analysis costs excludes extensive replicate titration measurements.
- Compared with plaque analysis, the combination method of TCID50 and the qPCR-based detection has smaller relative error and higher sensitivity.
- According to the customer's needs, the experimental scheme can be determined to meet the different projects as much as possible, so that users can quickly and easily determine the virus concentration, combined with the current analysis methods to reduce constrictions.
We provide determination services for the physical titer of AAV (infectious titration). If you want to add other quality control services to your AAV order, please contact us for more information.
Reference
- Pankaj, K. (2013). Methods for rapid virus identification and quantification. Mater Methods. 3: 207.
