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 cont aining the p53 gene (rAd-CMV-p53) for gene therapy, We have previously reported that rAd5-CMV-p53 vectors can be assessed for infectivity an d concomitant p53 protein expression in single- and two-color assays u sing 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 las er scanning cytometry (LSC), We report that LSC analysis of rAd-CMV-p5 3-infected human 293 cells correlated very well with flow cytometric a nalysis across a wide range of viral infectivity for both infectivity assessment (r(2) = 0.37) and p53 protein expression (r(2) = 0.96). Abs olute 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 f ormat analysis of rAd-CMV-p53-infected cells revealed comparable resul ts between LSC and flow cytometric analysis. LSC is a reliable and use ful tool for the intracellular staining for adenoviral. hexon protein expression for determining infectivity and for p53 protein expression from an expressed p53 transgene. Cytometry 33:290-296, 1998, (C) 1998 Wiley-Liss, Inc.