ELISA, InCell ELISA and Western Blot; alternative to the plaque reduction assay for in vitro determination of HSV susceptibility to acyclovir
- ELISA, InCell ELISA and Western Blot; alternative to the plaque reduction assay for in vitro determi...
Background and Aims: Despite the fact that The Plaque Reduction Assay (PRA) has been proposed a standard method for in vitro determination of HSV susceptibility to acyclovir (ACV) by The National Committee for Clinical Laboratory Standards and a range of methods have been developed as tools for the determination of susceptibility of HSV to antiviral agents, there are several drawbacks associated with them, such as labor-intensive, time-consuming, high cost, and questionable reproducibility. The goal of this study was to test the usability of the ELISA, InCell ELISA and Western Blot (WB) techniques to improve capability for the determination of susceptibility of HSV-1 to antiviral agents. In addition to this aim of the study, we also investigated PancI and T98G cell lines in addition to Vero for determination of susceptibility of HSV- 1. Materials and Method: For WB, extracted proteins from the cells infected with HSV-1 and treated with different concentration of ACV were used in SDS-PAGE and followed with immunoblot using HSV-1 specifi antibody. Two ELISA methods were tested: in the fist ELISA protein extracts obtained for the WB used to coat the 96 well high binding ELISA plates and standard ELISA protocol followed. In the second ELISA protocol that is also called InCell ELISA, cell growing in the 96 cell culture plates were treated for stabilization and permeabilization and then ELISA protocol followed. PRA, WB, and ELISAs were tested in Vero, PancI and T98G cell lines and compared for usability control cells. Results: This study showed that both WB and ELISAs detected HSV-1 presence in 24 hours after the virus infection which is 1 or 2 days earlier than the PRA result. WB result showed that it is more sensitive compared to PRA, such as PRA could not detected HSV-1 presence in 2 mg ACV, WB could detected HSV-1 survived in 2 mg ACV concentration. The study also showed that cytotoxic effects of the HSV-1 is faster in both PancI and T98G compared to the Vero cell line. Conclusion: The study showed that WB is found as the most sensitive method. Therefore, it may be more useful especially for determination of borderline resistant HSV-1 strains. Both ELISA tests are useful for in vitro determination of HSV susceptibility to ACV and more advantageous in terms of time and plating the susceptibility curve, and identifiation of IC50 value may be more accurate compared to that of PRA. We also noticed that cytotoxic effects of the HSV-1 is faster in both PancI and T98G compared to the Vero cell line.