Evaluation of accuracy and precision of adenovirus absorptivity at 260 nm under conditions of complete DNA disruption.
A simple, accurate, and precise method to determine adenovirus particle concentration using 260-nm absorbance was developed as an enhancement to the method of Maizel et al. (1968, Virology 36, 115-125). This modified method ensures complete disruption of virus particles and viral DNA prior to absorbance measurements, therefore eliminating absorbance measurement errors due to hyperchromic shift and thus providing an extinction coefficient at 260 nm that is directly related to protein concentration as measured by the method of Lowry et al. (1951, J. Biol. Chem. 193, 265-275). Application of this modified method will reduce interlaboratory variability in determining adenovirus particle concentrations, as current practices reflected in the literature utilize varying sample preparation procedures and calculation methods which cause underestimation of adenovirus concentration by almost twofold. The sample pretreatment conditions used in this modified method entail incubation in 1% sodium dodecyl sulfate at 100 degrees C for 4 min and result in an adenovirus 260-nm absorptivity of 1.8 x 10(12) viral particles per milliliter per absorbance unit in a 1-cm-pathlength cell.
Sweeney
Joyce A
JA
Bioprocess and Bioanalytical Research, Merck Research Laboratories, Merck & Co., Inc., West Point, Pennsylvania, 19486, USA. Joyce_Sweeney@Merck.com
Hennessey
John P
JP
D000042:Absorption; D000260:Adenoviruses, Human / Q000737:chemistry*; D004279:DNA, Viral / Q000737:chemistry*; D006358:Hot Temperature; D006801:Humans; D009691:Nucleic Acid Denaturation; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D014764:Viral Proteins / Q000032:analysis; D014771:Virion / Q000737:chemistry / Q000302:isolation & purification*; D014773:Virology / Q000379:methods
D004279:DNA, Viral; D014764:Viral Proteins