Adenovirus QC

Adenovirus QC

Quantitation

Calculate the corrected A260 and A280 absorbance measurements and corrected ratio
Corrected A260 = (A260 – A320)
Corrected A280 = (A280 – A320)
Corrected ratio = (Corrected A260 / Corrected A280)
A260 = the absorbance at 260 nm
A280 = the absorbance at 280 nm
A320 = the absorbance used for background and scatter correction

Record the dilution factor and particles/mL on Form 1. Calculate the particle count according to Maizel et al. in particles/mL as follows:
Virus concentration [ptc/mL] = 1.1×10¹² × corrected A260 × DF
Where:
1.1×10¹² = the number of virus particles per mL per 1 AU at 260 nm
DF = sample dilution factor

Plaque Assay - to determine infectivity titer

The standard protocol involves making 6 serial dilutions of the virus and infecting 2, 6-well plates of 293 cells with the virus. At ~16 hours postinfection, the cultures are overlayed with an agar/media mixture. The cells are observed daily for the formation of plaques. The plaques are counted and the applied to a formula that will give the infectious titer/ml. The protocol takes 10-14 days to complete.

Rapid Titer Assay - to determination of infectivity titer

the AdenoX Rapid Titer kit (Clontech) can be used to titer adenovirus in just a few days. This immunohistochemical assay involves making 3 serials dilutions of the virus and infecting 6 wells of a 12 well plate of 293 cells. At 48 hours post-infection, the cells are fixed and immunostained with an anti-hexon antibody. Invidual or clusters of stained cells are counted and the resulting figures are applied to a formula that will give th infectious titer/ml. This protocol takes 3 days to complete.

Assay for Replication Competent Adenovirus (RCA) - Cell-Based Assay

Preparations are judged to be free of replication competent adenovirus if they pass a test of incubation of 10^9 particles of recombinant Ad on A549 cells for 10 days. Appropriate positive and negative controls are run with each RCA test.

Assay for Replication Competent Adenovirus (RCA) - Quantitative Real-Time PCR

A new rapid RCA test using real time quantitative PCR using primer/probes within Ad region E1 has been developed.