Among several strategies used for Herpes simplex virus (HSV) detection inbiological specimens, standard plaque assay (SPA) remains the most reliablemethod to evaluate virus infectivity and quantify viral replication. However, it is a manual procedure, thereby affected by operator subjectivity, and it may beparticularly laborious for multiple sample analysis. Here we describe aninnovative method to perform the titration of HSV type 1 (HSV-1) in differentsamples, using the "In-Cell WesternTM" Assay (ICW) from LI-COR, a quantitativeimmunofluorescence assay that exploits laser-based scanning of near infrared(NIR). In particular, we employed NIR-immunodetection of viral proteins tomonitor foci of HSV-1 infection in cell monolayers, and exploited an automateddetection of their fluorescence intensity to evaluate virus titre. Thisinnovative method produced similar and superimposable values compared to SPA, butit is faster and can be performed in 96 well plate, thus allowing to easily andquickly analyze and quantify many samples in parallel. These features make ourmethod particularly suitable for the screening and characterization of antiviral compounds, as we demonstrated by testing acyclovir (ACV), the main anti-HSV-1drug. Moreover, we developed a new data analysis system that allowed to overcome potential bias due to unspecific florescence signals, thus improving datareproducibility. Overall, our method may represents a useful tool for bothclinical and research purposes.
A Novel Method to Titrate Herpes Simplex Virus-1 (HSV-1) Using Laser-Based Scanning of Near-Infrared Fluorophores Conjugated Antibodies
Limongi D;
2017-01-01
Abstract
Among several strategies used for Herpes simplex virus (HSV) detection inbiological specimens, standard plaque assay (SPA) remains the most reliablemethod to evaluate virus infectivity and quantify viral replication. However, it is a manual procedure, thereby affected by operator subjectivity, and it may beparticularly laborious for multiple sample analysis. Here we describe aninnovative method to perform the titration of HSV type 1 (HSV-1) in differentsamples, using the "In-Cell WesternTM" Assay (ICW) from LI-COR, a quantitativeimmunofluorescence assay that exploits laser-based scanning of near infrared(NIR). In particular, we employed NIR-immunodetection of viral proteins tomonitor foci of HSV-1 infection in cell monolayers, and exploited an automateddetection of their fluorescence intensity to evaluate virus titre. Thisinnovative method produced similar and superimposable values compared to SPA, butit is faster and can be performed in 96 well plate, thus allowing to easily andquickly analyze and quantify many samples in parallel. These features make ourmethod particularly suitable for the screening and characterization of antiviral compounds, as we demonstrated by testing acyclovir (ACV), the main anti-HSV-1drug. Moreover, we developed a new data analysis system that allowed to overcome potential bias due to unspecific florescence signals, thus improving datareproducibility. Overall, our method may represents a useful tool for bothclinical and research purposes.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
