Abstract
ABSTRACT
Transformation of B cells by Epstein-Barr Virus (EBV) is used by the CCR at Coriell Institute for Medical Research to produce renewable cell lines and DNA to further genomic and proteomic studies of inherited and complex diseases alike. Optimal and efficient transformation requires an accurate assessment of the titer of the virus in each virus preparation. However, current methods to determine EBV titer assess transformation efficiency using large-scale biological assays ending in the establishment of lymphoblastoid cell lines (LCLs). This method determines the virus dilution capable of producing LCL outgrowth but does not determine virion number and optimal dilution will vary with the preparation. Therefore, we developed a real-time PCR method to detect and quantify EBV DNA. In addition to quantifying the titer of the viral supernatants, we evaluated both the biological activity of the viral dilutions as measured by LCL outgrowth and the use of diluted EBV viral stocks to transform previously frozen and untransformed lymphocytes using days to cryopreservation as a comparative endpoint. To this end, we determined that there was no difference in time to cryopreservation/transformation of lymphocytes exposed to undiluted or diluted viral stocks. The Real-Time PCR assay provides a method to assess the quality and virion number of new lots of virus without the need for large-scale and time-consuming biological assays. With this information, we are able to determine quickly and efficiently the titer of new virus preparations and to optimize and extend the usage of existing virus stocks.