Comparison of flow cytometry and laser scanning cytometry for the intracellular evaluation of adenoviral infectivity and p53 protein expression in gene therapy.
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.
Musco
M L
ML
Biotechnology-Bioanalytical, Schering-Plough Research Institute, Kenilworth, New Jersey, USA.
Cui
S
S
Small
D
D
Nodelman
M
M
Sugarman
B
B
Grace
M
M
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
D015415:Biomarkers; D036022:Capsid Proteins; D016159:Tumor Suppressor Protein p53; D014764:Viral Proteins; C018315:hexon capsid protein, Adenovirus