Quantitative PCR and plaque assay are powerful virological techniques used to measure the load of defective or infectious virus in mouse and human. However, these methods display limitations such as cross contamination and long run-time. Here, we describe a novel technique termed as semi-functional quantitative flow cytometry (SFQF) for the accurate estimation of the quantity of infectious lymphocytic choriomeningitis virus (LCMV). LCMV titration method using flow cytometry was previously developed but has technical shortcomings, owing to the less optimized parameters such as cell overgrowth, plate scale, and detection threshold. Therefore, we first established optimized conditions for SFQF assay using LCMV nucleoprotein (NP)-specific antibody to evaluate the threshold of the virus detection range in the plaque assay. We subsequently demonstrated that the optimization of the method increased the sensitivity of virus detection. We revealed several new advantages of SFQF assay, which overcomes some of the previously contentious points, and established an upgraded version of the previously reported flow cytometric titration assay. This method extends the detection scale to the level of single cell, allowing extension of its application for in vivo detection of infected cells and their phenotypic analysis. Thus, SFQF assay may serve as an alternative analytical tool for ensuring the reliability of LCMV titration and can be used with other types of viruses using target-specific antibodies.
Bibliographical noteFunding Information:
This study was supported by the Basic Science Research Program (2015R1A2A1A10056084) through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning. This study was also supported by the Korean Health Technology R&D Project (HI14C2680 and HI15C0493) through the Korean Health Industry Development Institute (KHIDI) funded by the Ministry of Health & Welfare.
© 2017. The Korean Association of Immunologists.
All Science Journal Classification (ASJC) codes
- Immunology and Allergy
- Infectious Diseases