https://eln.excellgen.com/index.php?action=history&feed=atom&title=Adenovirus_QCAdenovirus QC - Revision history2026-10-18T16:58:27ZRevision history for this page on the wikiMediaWiki 1.44.0https://eln.excellgen.com/index.php?title=Adenovirus_QC&diff=8154&oldid=prevPaul.xu at 20:41, 6 February 20132013-02-06T20:41:59Z<p></p>
<p><b>New page</b></p><div>= Adenovirus QC =<br />
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== Quantitation<br> ==<br />
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Calculate the corrected A260 and A280 absorbance measurements and corrected ratio<br>Corrected A260 = (A260 – A320)<br>Corrected A280 = (A280 – A320)<br>Corrected ratio = (Corrected A260 / Corrected A280)<br>A260 = the absorbance at 260 nm<br>A280 = the absorbance at 280 nm<br>A320 = the absorbance used for background and scatter correction<br><br>Record the dilution factor and particles/mL on Form 1. Calculate the particle count according to Maizel et al. in particles/mL as follows:<br>Virus concentration [ptc/mL] = 1.1×10<sup>12</sup> × corrected A260 × DF<br>Where:<br>1.1×10<sup></sup><sup>12</sup> = the number of virus particles per mL per 1 AU at 260 nm<br>DF = sample dilution factor<br><br><br />
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== Plaque Assay - to determine infectivity titer ==<br />
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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. <br />
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== Rapid Titer Assay - to determination of infectivity titer ==<br />
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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. <br />
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== Assay for Replication Competent Adenovirus (RCA) - Cell-Based Assay ==<br />
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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. <br />
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== Assay for Replication Competent Adenovirus (RCA) - Quantitative Real-Time PCR ==<br />
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A new rapid RCA test using real time quantitative PCR using primer/probes within Ad region E1 has been developed. <br> <br />
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[[Category:Protocols]]</div>Paul.xu