title: Comparison of flow cytometry and laser scanning cytometry for the intracellular evaluation of adenoviral infectivity and p53 protein expression in gene therapy.
abstract: The determination of recombinant adenoviral (rAd) infectivity and p53 protein expression is important for the evaluation of rAd vectors containing the p53 gene (rAd-CMV-p53) for gene therapy. We have previously reported that rAd5-CMV-p53 vectors can be assessed for infectivity and concomitant p53 protein expression in single- and two-color assays using intracellular staining methodology and flow cytometric analysis. We have compared the flow cytometry-based assays for rAd infectivity (hexon protein) and p53 protein expression with the new slide-based laser scanning cytometry (LSC). We report that LSC analysis of rAd-CMV-p53-infected human 293 cells correlated very well with flow cytometric analysis across a wide range of viral infectivity for both infectivity assessment (r2 = 0.97) and p53 protein expression (r2 = 0.96). Absolute values for infective titer and p53 protein expression titer from an rAd5-CMV-p53 production batch were similar and within experimental error with the two different analytical methods. Finally, bivariate format analysis of rAd-CMV-p53-infected cells revealed comparable results between LSC and flow cytometric analysis. LSC is a reliable and useful tool for the intracellular staining for adenoviral hexon protein expression for determining infectivity and for p53 protein expression from an expressed p53 transgene.
authors.affiliation: Biotechnology-Bioanalytical, Schering-Plough Research Institute, Kenilworth, New Jersey, USA.
authors.forename: M L
authors.initials: ML
authors.lastname: Musco
authors.forename: S
authors.initials: S
authors.lastname: Cui
authors.forename: D
authors.initials: D
authors.lastname: Small
authors.forename: M
authors.initials: M
authors.lastname: Nodelman
authors.forename: B
authors.initials: B
authors.lastname: Sugarman
authors.forename: M
authors.initials: M
authors.lastname: Grace
mesh_terms: D000260:Adenoviruses, Human / Q000254:growth & development*; D015415:Biomarkers; D002213:Capsid / Q000032:analysis; D036022:Capsid Proteins; D002461:Cell Line, Transformed; D005434:Flow Cytometry / Q000379:methods*; D015316:Genetic Therapy; D006801:Humans; D007834:Lasers; D016159:Tumor Suppressor Protein p53 / Q000032:analysis*; D014764:Viral Proteins / Q000032:analysis
chemical_list: D015415:Biomarkers; D036022:Capsid Proteins; D016159:Tumor Suppressor Protein p53; D014764:Viral Proteins; C018315:hexon capsid protein, Adenovirus