Amplification produces large amounts of high-titer, high quality virus assayed for PFU titer and RCA.
If you have an adenovirus that you constructed in-house or obtained just a small amount from a collaborator, trust us to scale-up production. Amplification is a CRITICAL step for your experiments. Rather than setting up the cells, assays and protocols to amplify and purify the virus in-house, send your materials to us and we can make a high-titer, high-quality and tested prep for you. You could be doing experiments instead of struggling to make the tools.
Seed stock to purified particles in 10 days.
Viral amplification basics
A host cell line, commonly HEK 293 cells, is cultured to just sub-confluency and then infected with the adenovirus vector at an appropriate multiplicity of infection (MOI). Following infection, the cells are incubated under optimal conditions to allow viral replication. As the virus replicates within the host cells, cytopathic effects (CPE) become evident, indicating successful virus amplification and it's time to harvest the cells and begin purification.
We are experts in virus amplification
We do a few serial amplifications to obtain large scale lysates for purification on CsCl gradients. We can often start with low titer lysates from the initial virus passage, plaques or other seed stocks. Transgene expression does affect virus amplification and total yield. We initially grow your virus on flat stock. Our suspension cultures are reserved for very large scale amplifications >2e13 pts/prep, or if your virus does not grow well.
CsCl gradient of adenovirus particles.
Incomplete virus particles (upper band) are separated from intact particles l(ower band noted with yellow star). The lower band is then aspirated by needle puncture leaving the upper band behind.
Plaque purifications - what are they?
Plaque purification is used to isolate individual viral particles from a mixed population. The virus may have revertants or non-expressers, or may need several rounds of plaquing to satisfy FDA requirements. The process begins by infecting a monolayer of host cells such as HEK 293s with a limiting dilution of the virus sample. In 7 to 10 days, a single replicating virus forms an individual plaque in the cell monolayer. Individual plaques are then picked and used to infect fresh host cell cultures. We work closely with your lab should plaquing be necessary or required for your specific project. Lysates from the plaques should be functionally tested by your lab.The plaque purified virus can then either be plaqued a second or third time (project specific) and then scaled for final CsCl purification.
Viral purity is serious business at Antibodies Inc.
We take the purity of our viruses very seriously. Although the RAPAd® system's novel backbone eliminates the presence of non-recombinant virus in the prep, we still screen every prep for possible E1a sequences and the presence of RCA in cell-based assays. Check out more details on our Quality Control page here.
We also test incoming materials for RCA. If a virus seed stock is sent to us for amplification that contains RCA, we can plaque purify and remake the prep for you!
Our purification process
Our purification protocol includes 2 rounds of CsCl gradients, dialysis against A195 buffer (or the buffer of your choice), and then the final formulation. Our typical concentration is 1e12 pts/mL in 1 mL aliquots. The purified particles are suitable for direct injection for in-vivo experiments or cell culture. We can formulate the virus at higher concentrations and in smaller aliquots at the time of manufacture. This is generally used for in-vivo experiments that require high virus load in low volumes, and eliminates any freeze-thaw cycles on the finished product.
Ready to Get Started?
For questions or a quote from one of our scientists, submit your project details below. We will respond within two business days. If you require immediate assistance, call us at (800) 824-8540.
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